Hydrogel microtubes for biomanufacturing and methods of manufacture and use thereof

Abstract

A hydrogel microtube for culturing or expanding cells is provided that includes, or is prepared from, one or more of a collagen protein; a blend of an alginate polymer and a collagen protein; and a peptide-functionalized alginate polymer and, optionally, an unfunctionalized alginate polymer, wherein hydrogel microtube includes a cavity configured to accommodate a plurality of cells. Also provided is a bioreactor system including such hydrogel microtubes, a method of culturing or expanding cells, a method producing synthetic tissue, a method producing a protein, viral particle, or extracellular vesicle, and a method of providing cell therapy using such hydrogel microtube. Further provided is an apparatus, a system of such apparatus, and a method for producing such hydrogel microtube.

Description

Attorney Docket No. 0073605-001131HYDROGEL MICROTUBES FOR BIOMANUFACTURING AND METHODS OF MANUFACTURE AND USE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of, and priority to, U.S. Provisional Application Serial No. 63 / 735,468, filed on December 18, 2024, and entitled “COLLAGEN AND COLLAGENALGINATE HYDROGEL MICROTUBES FOR BIOMANUFACTURING” and U.S. Provisional Application Serial No. 63 / 788,877, filed on April 15, 2025, and entitled “PEPTIDE-MODIFIED ALGINATE HYDROGEL MICROTUBES FOR CULTURING MAMMALIAN CELLS”, the entirety of each of which is incorporated herein by reference.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant Nos. CA235326, HD114044 and HL163711 awarded by the National Institutes of Health. The Government has certain rights in the invention.FIELD OF THE INVENTION

[0003] The present invention generally relates to biomanufacturing systems for culturing expanding, and manipulating cells, such as differentiating cells, reprogramming cells, as well as using the cultured cells to produce high-value biologies such as virus, extracellular vesicles (EVs), exosomes, and important proteins and tissues. In particular, the present invention is directed to hydrogel microtubes for biomanufacturing and methods of use and manufacture thereof.BACKGROUND

[0004] Mammalian cells have diverse applications. Stem cells, such as human pluripotent stem cells (hPSCs), can generate various tissue cells for regenerative medicine, disease modeling, drug screening, and toxicity testing. Immune cells, such as T cells and natural killer cells, are used to treat cancers. Mammalian cells are also widely utilized for producing recombinant proteins, viruses, and extracellular vesicles or exosomes for research and therapy. All these applications require large numbers of cells.

[0005] In vivo, human cells live in a three-dimensional (3D) microenvironment that provides important niche factors, supports interactions between cells and the extracellular matrix (ECM), provides robust nutrient and oxygen supply, and minimizes hydrodynamic stress. Current cell culture methods, however, often fail to replicate these conditions. For example, two-dimensional (2D) cell culture systems, such as flasks, lack the complexity of in vivo 3D microenvironment. These system typically operate based on a stiff substrate to produce only a limited number of cells per culture area.Attorney Docket No. 0073605-001131

[0006] Three-dimensional (3D) suspension culture systems and / or 3D bioreactors, such as stirred tank bioreactors and vertical wheels, have been developed to improve the scalability of cell culture methods. However, these systems face significant challenges, particularly uncontrolled cell aggregation. Aggregates exceeding 400 pm in diameter suffer from impaired transport of nutrients, oxygen, and growth factors, leading to slower proliferation, apoptosis, and undesired differentiation of cells. While agitation can mitigate aggregation, it also introduces shear forces, which can negatively impact cell survival, growth, and differentiation efficiency. As a result, 3D suspension cultures often exhibit high cell mortality, slow proliferation rates, and low volumetric yields. For instance, hPSCs in stirred-tank bioreactors typically undergo only a four-fold expansion over four days to produce approximately 2.0* 106cells / mL, and this process utilizes just 0.4% of the volume of a bioreactor.

[0007] As a result, current cell culture methods face formidable challenges in achieving robust and cost-effective large-scale cell production, especially for stem cells used in clinical applications. These challenges remain to be addressed.SUMMARY OF THE DISCLOSURE

[0008] An aspect of the present disclosure is a hydrogel microtube for culturing, expanding, differentiating, or manipulating cells. The hydrogel microtube includes, or is prepared from, one or more of (a) a collagen protein; (b) a blend of an alginate polymer and a collagen protein; and (c) a peptide-functionalized alginate polymer and, optionally, an unfunctionalized alginate polymer. The hydrogel microtube includes a cavity configured to accommodate a plurality of cells. In some embodiments, the hydrogel microtube has an inner diameter of at least 20 pm and no greater than 999 pm, preferably at least 100 pm and no greater than 600 pm. In some embodiments, the hydrogel microtube has a wall thickness of at least 1 pm and no greater than 500 pm, preferably at least 10 pm and no greater than 200 pm, more preferably at least 25 pm and no greater than 150 pm. In some embodiments, the hydrogel microtube has a substantially uniform wall thickness throughout its entire structure.

[0009] In some embodiments, the hydrogel microtube includes two closed ends. In some embodiments, the hydrogel microtube includes one open end and one closed end. In some embodiments, the hydrogel microtube includes two open ends.

[0010] In some embodiments, the hydrogel microtube has a circular, oval or polygonal cross- sectional area. Preferably, the cross-sectional area is circular or oval shaped. The microtube structure can include an elongated body that defines an inner channel therein that can extend between a firstAttorney Docket No. 0073605-001131 end of the microtube body and a second end of the microtube body. The inner channel can be the inner cavity of the microtube. The first and second ends of the microtube body can each be open or have a mouth defined therein that is in fluid communication with the inner channel.

[0011] The microtube can have a length and a diameter or width. The diameter can be an outer diameter of the body of the microtube, for example. The inner channel defined in the body of the microtube can also have an inner diameter or inner width that extend between opposed inner sides of the body of the microtube that defines the inner channel. The inner diameter, or inner width, is smaller than the outer diameter or outer width of the body of the microtube. The thickness of the wall(s) of the microtube that define the inner channel can make up the difference in size between the inner diameter and outer diameter in some embodiments, for example.

[0012] In some embodiments, the hydrogel microtube has a length of greater than 100 pm. In some embodiments, the hydrogel microtube has an aspect ratio, i.e., the ratio between the length of the hydrogel microtube and the inner diameter of the hydrogel microtube, of at least 1.0 (e.g., as substantially a spherical hydrogel microbubble) or at least 1.05, preferably at least 1.1, more preferably at least 2, further preferably at least 5, even further preferably at least 20.

[0013] In some embodiments, (b) the blend of the alginate polymer and the collagen protein has an alginate polymer-to-collagen protein ratio of 0.01 to 100 by mass, preferably 0.1 to 1.5 by mass, more preferably 0.25 to 0.75 by mass.

[0014] In some embodiments, the peptide-functionalized alginate polymer includes an arginineglycine-aspartic acid (RGD) peptide covalently linked to the alginate polymer. In some embodiments, the peptide-functionalized alginate polymer includes one or more of a peptide- functionalized alginate acid polymer, a peptide-functionalized sodium alginate polymer, a peptide- functionalized methacrylate-modified alginate polymer, and a peptide-functionalized vinyl sulfone- modified alginate polymer.

[0015] In some embodiments, the hydrogel microtube further includes a coating layer. In some embodiments, the coating layer includes one or more of an alginate acid polymer layer, a sodium alginate polymer layer, a methacrylate-modified alginate polymer layer, and a vinyl sulfone- modified alginate polymer layer, wherein the vinyl sulfone-modified alginate polymer is optionally peptide-functionalized.

[0016] In some embodiments, the hydrogel microtube further includes one or more extracellular matrix (ECM) proteins. In some embodiments, the one or more extracellular matrix (ECM) proteins includes laminin or fibronectin.Attorney Docket No. 0073605-001131

[0017] In some embodiments, the hydrogel microtube further includes one or more of polyethylene glycol and poly(vinyl alcohol).

[0018] Another aspect of the present disclosure is a bioreactor system including a plurality of the hydrogel microtubes described herein and a cell-compatible buffer or medium, wherein the plurality of the hydrogel microtubes is dispersed in the cell-compatible buffer or medium.

[0019] In some embodiments, the cell-compatible buffer or medium has a pH from about 7 to about 9, preferably about 7.4. In some embodiments, the cell-compatible buffer or medium includes or contains one or more multivalent ions, preferably one or more divalent ions. In some embodiments, the one or more divalent ions can include Mg2+, Ca2+, Zn2+, and / or Ba2+, preferably Ca2+.

[0020] Another aspect of the present disclosure is a method of culturing, expanding, differentiating, or manipulating cells. The method includes (i) extruding a cell solution and a hydrogel-forming solution into a cell-compatible buffer to produce the hydrogel microtube described herein. The method further includes (ii) suspending the hydrogel microtube including cells from the cell solution in a cell culture medium. The method further includes (iii) culturing, expanding, differentiating, or manipulating the cells under suitable conditions. The method further includes (iv) dissolving the hydrogel microtube to release the cells.

[0021] In some embodiments, the cell solution includes cells selected from the group consisting of embryonic stem cells; mammalian embryonic stem cells; human embryonic stem cells (hESCs); human induced pluripotent stem cells (iPSCs); mammalian induced pluripotent stem cells; mammalian naive pluripotent stem cells; mammalian tissue stem cells; human pluripotent stem cells (hPSCs); cells derived or differentiated from one or more of embryonic stem cells, mammalian embryonic stem cells, human embryonic stem cells (hESCs), human induced pluripotent stem cells (iPSCs), mammalian induced pluripotent stem cells, mammalian naive pluripotent stem cells, mammalian tissue stem cells, and human pluripotent stem cells (hPSCs); mammalian cells reprogrammed from other cell types; mammalian primary cells; human umbilical vein endothelial cells; primary tumor cells; cancer cells; immune cells; T cells; natural killer cells; mammalian cell lines; engineered human and mammalian cells; insect cells; plant cells; yeast and bacterial cells.Cells can be cultured as single cell type or as a mixture of different cell types or a mixture of cells from different donors.

[0022] In some embodiments, the cell solution includes without limitation embryonic stem cells; mammalian embryonic stem cells; human embryonic stem cells (hESCs); human inducedAttorney Docket No. 0073605-001131 pluripotent stem cells (iPSCs); mammalian induced pluripotent stem cells; mammalian naive pluripotent stem cells; mammalian tissue stem cells; human pluripotent stem cells (hPSCs); cells derived or differentiated from one or more of embryonic stem cells, mammalian embryonic stem cells, human embryonic stem cells (hESCs), human induced pluripotent stem cells (iPSCs), mammalian induced pluripotent stem cells, mammalian naive pluripotent stem cells, mammalian tissue stem cells, and human pluripotent stem cells (hPSCs); mammalian cells reprogrammed from other cell types; mammalian primary cells; human umbilical vein endothelial cells; primary tumor cells; cancer cells; immune cells; T cells; natural killer cells; mammalian cell lines; engineered human and mammalian cells; insect cells; plant cells; and yeast and bacterial cells, among others. Cells can be cultured as single cell type or as a mixture of different cell types or a mixture of cells from different donors.

[0023] In some embodiments, the method further includes, e.g., between step (i) and step (ii), coating the hydrogel microtube using a coating solution. In some embodiments, the method further includes, between step (iii) and step (iv), coating the hydrogel microtube using a coating solution. In some embodiments, the coating solution can include one or more of an alginate acid polymer, a sodium alginate polymer, a methacrylate-modified alginate polymer, and a vinyl sulfone-modified alginate polymer, wherein the vinyl sulfone-modified alginate polymer is optionally peptide functionalized, etc. In some embodiments, an alginate polymer with one or more other modifications not explicitly disclosed herein may be used. In some embodiments, a longer hydrogel microtube with cells are truncated into short tubular segments. In some embodiments, the coating can be applied during the cell culture and / or before the cell harvest.

[0024] In some embodiments, dissolving the hydrogel microtubes includes treating the hydrogel microtube using one or more of ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), and an alginate lyase.

[0025] In some embodiments, dissolving the hydrogel microtubes includes treating the hydrogel microtube using a collagenase, preferably Collagenase P.

[0026] In some embodiments, culturing or expanding the cells includes culturing or expanding the cells over a time period of at least 2 or 3 hours, preferably at least 6 hours, more preferably at least 12 hours, further preferably at least 24 hours.

[0027] In some embodiments, the cultured or expanded cells have a cell density of at least 5.0x 106cells per milliliter, at least O. l x lO8cells per milliliter, or at least l.OxlO8cells per milliliter, preferably at least 3. Ox 108cells per milliliter, more preferably at least 4.5x 108cells per milliliter,Attorney Docket No. 0073605-001131 further preferably at least 5.0* IO8cells per milliliter. In some embodiments, the cultured or expanded cells have a cell density of no greater than 0.1 * 108cells per milliliter, depending on the specific use case or user need.

[0028] In some embodiments, step (iii) of the method further includes truncating the hydrogel microtubes to release the cells.

[0029] Another aspect of the present disclosure is a method of producing synthetic tissue, preferably meat. The method includes (i) extruding a cell solution and a hydrogel-forming solution into a cell-compatible buffer to produce the hydrogel microtube described herein. The method further includes (ii) suspending the microtube including cells from the cell solution in a cell culture medium. The method further includes (iii) culturing or expanding the cells under suitable conditions. The method further includes (iv) optionally dissolving the hydrogel microtube to release the cells. The method further includes (v) producing the synthetic tissue using the released cells. In some embodiments, the cultured cells can be used as meat without dissolving the hydrogel microtubes, since the hydrogel microtubes are edible.

[0030] In some embodiments, dissolving the hydrogel microtubes includes treating the hydrogel microtube using one or more of ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), and an alginate lyase.

[0031] In some embodiments, dissolving the hydrogel microtubes includes treating the hydrogel microtube using a collagenase, preferably Collagenase P.

[0032] In some embodiments, culturing or expanding the cells includes culturing or expanding the cells over a time period of at least 2 or 3 hours, preferably at least 6 hours, more preferably at least 12 hours, further preferably at least 24 hours.

[0033] In some embodiments, the cultured or expanded cells have a cell density of at least 5.0x l06cells per milliliter, at least 0.1 * 108cells per milliliter, or at least 1.0* 108cells per milliliter, preferably at least 3.0* 108cells per milliliter, more preferably at least 4.5* 108cells per milliliter, further preferably at least 5.0* 108cells per milliliter. In some embodiments, the cultured or expanded cells have a cell density of no greater than 0.1 * 108cells per milliliter, depending on the specific use case or user need.

[0034] In some embodiments, the hydrogel microtube is a truncated hydrogel microtube, in accordance with details described throughout the present disclosure.

[0035] In some embodiments, the hydrogel microtube is a coated with a coating layer. In some embodiments, the coating layer includes one or more of an alginate acid polymer layer, a sodiumAttorney Docket No. 0073605-001131 alginate polymer layer, a methacrylate-modified alginate polymer layer, and a vinyl sulfone- modified alginate polymer layer, wherein the vinyl sulfone-modified alginate polymer is optionally peptide-functionalized.

[0036] In some embodiments, the method further includes coating the hydrogel microtube using a coating solution. In some embodiments, the coating solution includes one or more of an alginate acid polymer, a sodium alginate polymer, a methacrylate-modified alginate polymer, and a vinyl- sulfone-modified alginate, wherein the vinyl sulfone-modified alginate polymer is optionally peptide-functionalized.

[0037] In some embodiments, the method further includes, between step (iii) and step (iv), truncating the hydrogel microtube.

[0038] Another aspect of the present disclosure is a method of producing a biologic such as a protein, viral particle, or extracellular vesicle. The method includes (i) extruding a cell solution and a hydrogel-forming solution into a cell-compatible buffer to produce the hydrogel microtube described herein. The method further includes (ii) suspending the microtube including cells from the cell solution in a cell culture medium. The method further includes (iii) culturing or expanding the cells under suitable conditions to produce the protein, viral particle, or extracellular vesicle. The method further includes (v) optionally dissolving the hydrogel microtube to release the protein, viral particle, or extracellular vesicle. The method further includes (v) harvesting the protein, viral particle, or extracellular vesicle. In some embodiments, a protein, viral particle, or extracellular vesicle can be secreted into the cell culture medium. In some embodiments, a protein, viral particle, or extracellular vesicle can be retained in the hydrogel microtube. In some embodiments, both the protein, viral particle, or extracellular vesicle secreted into the cell culture medium and the protein, viral particle, or extracellular vesicle retained in the hydrogel microtube are collected.

[0039] In some embodiments, the hydrogel microtube is a truncated hydrogel microtube, in accordance with details described throughout the present disclosure.

[0040] In some embodiments, the hydrogel microtube is a coated with a coating layer. In some embodiments, the coating layer includes one or more of an alginate acid polymer layer, a sodium alginate polymer layer, a methacrylate-modified alginate polymer layer, and a vinyl sulfone- modified alginate polymer layer, wherein the vinyl sulfone-modified alginate polymer is optionally peptide-functionalized.

[0041] In some embodiments, the method further includes coating the hydrogel microtube using a coating solution. In some embodiments, the coating solution includes one or more of an alginateAttorney Docket No. 0073605-001131 acid polymer, a sodium alginate polymer, a methacrylate-modified alginate polymer, and a vinyl- sulfone-modified alginate, wherein the vinyl sulfone-modified alginate polymer is optionally peptide-functionalized.

[0042] In some embodiments, the method further includes truncating the hydrogel microtube to release the protein, viral particle, or extracellular vesicle.

[0043] In some embodiments, culturing or expanding the cells includes culturing or expanding the cells over a time period of at least 2 or 3 hours, preferably at least 6 hours, more preferably at least 12 hours, further preferably at least 24 hours.

[0044] Tn some embodiments, the cultured or expanded cells have a cell density of at least 5.0* 106cells, at least 0.1 * 108cells per milliliter, or at least 1.0* 108cells per milliliter, preferably at least 3. Ox 108cells per milliliter, more preferably at least 4.5x 108cells per milliliter, further preferably at least 5.0x l08cells per milliliter. In some embodiments, the cultured or expanded cells have a cell density of no greater than 0.1 x 108cells per milliliter, depending on the specific use case or user need.

[0045] Another aspect of the present is a method of providing cell therapy, the method including administering to a subject a therapeutically effective amount of cells cultured or expanded using, and isolated from, the hydrogel microtube described herein.

[0046] In some embodiments, the cells includes one or more members selected from the group consisting of embryonic stem cells; mammalian embryonic stem cells; human embryonic stem cells (hESCs); human induced pluripotent stem cells (iPSCs); mammalian induced pluripotent stem cells; mammalian naive pluripotent stem cells; mammalian tissue stem cells; human pluripotent stem cells (hPSCs); cells derived or differentiated from one or more of embryonic stem cells, mammalian embryonic stem cells, human embryonic stem cells (hESCs), human induced pluripotent stem cells (iPSCs), mammalian induced pluripotent stem cells, mammalian naive pluripotent stem cells, mammalian tissue stem cells, and human pluripotent stem cells (hPSCs); mammalian cells reprogrammed from other cell types; mammalian primary cells; human umbilical vein endothelial cells; primary tumor cells; cancer cells; immune cells; T cells; natural killer cells; mammalian cell lines; engineered human and mammalian cells; insect cells; plant cells; and yeast and bacterial cells.

[0047] In some embodiments, the cells include without limitation embryonic stem cells; mammalian embryonic stem cells; human embryonic stem cells (hESCs); human induced pluripotent stem cells (iPSCs); mammalian induced pluripotent stem cells; mammalian naive pluripotent stem cells; mammalian tissue stem cells; human pluripotent stem cells (hPSCs); cells derived orAttorney Docket No. 0073605-001131 differentiated from one or more of embryonic stem cells, mammalian embryonic stem cells, human embryonic stem cells (hESCs), human induced pluripotent stem cells (iPSCs), mammalian induced pluripotent stem cells, mammalian naive pluripotent stem cells, mammalian tissue stem cells, and human pluripotent stem cells (hPSCs); mammalian cells reprogrammed from other cell types; mammalian primary cells; human umbilical vein endothelial cells; primary tumor cells; cancer cells; immune cells; T cells; natural killer cells; mammalian cell lines; engineered human and mammalian cells; insect cells; plant cells; and yeast and bacterial cells, among others.

[0048] Another aspect of the present disclosure is an apparatus for preparing a hydrogel microtube. The apparatus includes an extruder having at least a first inlet and at least a second inlet and a housing in fluid connection with the extruder. The at least a first inlet is operable for introducing a cell solution into the extruder. The at least a second inlet includes, or is in fluid connection with, a plurality of feeding elements and operable for introducing a hydrogel-forming solution, through the plurality of feeding elements, into the extruder, in a plurality of directions symmetrically disposed with respect to the cell solution, thereby producing the hydrogel microtube containing a plurality of cells. In some embodiments, the plurality of feeding elements are symmetrically deployed with respect to the at least a first inlet.

[0049] Another aspect of the present disclosure is another apparatus for preparing a hydrogel microtube. The apparatus includes an extruder having at least a first inlet and at least a second inlet and a housing in fluid connection with the extruder. The at least a first inlet is operable for introducing a cell solution into the extruder. The at least a second inlet includes, or is in fluid connection with, a plurality of feeding elements and operable for introducing a hydrogel-forming solution, through the plurality of feeding elements, into the extruder, in a plurality of directions symmetrically disposed with respect to the cell solution. The housing has at least a third inlet and is configured to supply a cell-compatible buffer, and optionally one or more additional liquid flows, to the cell solution and the hydrogel-forming solution, thereby producing the hydrogel microtube containing a plurality of cells. In some embodiments, the plurality of feeding elements are symmetrically deployed with respect to the at least a first inlet.

[0050] In some embodiments, the extruder is configured to supply a core flow of the cell solution in a flow direction within a shell flow of the hydrogel-forming solution that is also passed in the flow direction and wherein the extruder is configured so that a sheath flow of the cell-compatible buffer is passed in the flow direction to surround the shell flow, which surrounds the core flow such that the shell flow is between the sheath flow and the core flow as the sheath flow, shell flow, andAttorney Docket No. 0073605-001131 core flow pass in the flow direction to form the hydrogel microtube containing the plurality of cells within an inner channel of the hydrogel microtube.

[0051] In some embodiments, the apparatus described herein further includes at least a syringe in fluid connection with the at least a first inlet, the at least a second inlet, and / or the at least a third inlet, such as without limitation via tubing or capillaries. In some embodiments, the at least a syringe is in fluid connection with the plurality of feeding elements. In some embodiments, the apparatus described herein further includes a syringe pump, wherein the at least a syringe is operated by the syringe pump.

[0052] In some embodiments, the cell solution or the cell -compatible buffer has a pH from about 7 to about 9, preferably about 7.4.

[0053] In some embodiments, the hydrogel-forming solution has a pH from about 2 to about 6, preferably from about 3 to about 5.

[0054] In some embodiments, the cell-compatible buffer includes one or more multivalent ions, preferably one or more divalent ions. In some embodiments, the one or more divalent ions includes Mg2+, Ca2+, Zn2+, and / or Ba2+, preferably Ca2+.

[0055] In some embodiments, the apparatus described herein further includes a cooling element configured to keep a temperature of the hydrogel-forming solution to no greater than 10 °C, preferably no greater than 4 °C.

[0056] In some embodiments, the apparatus described herein further includes a heating element configured to raise a temperature of the hydrogel microtube to about 37 °C. Other embodiments may utilize a heating element configured to raise the temperature of the hydrogel microtube to another suitable temperature in a range of 35-40 °C or 30-40 °C.

[0057] In some embodiments, the apparatus described herein further includes one or more temperature controllers.

[0058] In some embodiments, the apparatus further includes one or more ports each connected to a plurality of the first inlets or a plurality of the second inlets through one or more multifurcations, preferably one or more bifurcations.

[0059] In some embodiments, the apparatus further includes one or more ports each connected to a plurality of the first inlets, a plurality of the second inlets, or a plurality of the third inlets through one or more multifurcations, preferably one or more bifurcations.

[0060] Another aspect of the present disclosure is a system that includes a plurality of the apparatus described herein as a 2D array or 2D matrix.Attorney Docket No. 0073605-001131

[0061] In some embodiments, the system further includes at least one syringe having multiple outlets, wherein each outlet of the multiple outlets is in fluid connection with the at least a first inlet, the at least a second inlet, or the housing of at least one apparatus of the plurality of apparatus.

[0062] In some embodiments, the system further includes at least one syringe having multiple outlets simultaneously in fluid connection with the at least a first inlet of each apparatus of the plurality of apparatus.

[0063] In some embodiments, the system further includes at least one syringe having multiple outlets simultaneously in fluid connection with the at least a second inlet of each apparatus of the plurality of apparatus.

[0064] In some embodiments, the system further includes at least one syringe having multiple outlets simultaneously in fluid connection with the at least a third inlet of each apparatus of the plurality of apparatus.

[0065] In some embodiments, the system further includes at least one syringe having a single arm or outlet. In some embodiments, the system further includes at least one syringe having one or more multifurcations, preferably one or more bifurcations.

[0066] Another aspect of the present disclosure is a method of preparing a hydrogel microtube. The method includes (i) supplying a core flow of a cell solution though at least a first inlet of an extruder. The method further includes (ii) concurrently supplying a shell flow of a hydrogel-forming solution though at least a second inlet of the extruder, wherein the core flow is passed within and surrounded by the shell flow. The method may further include (iii) supplying a sheath flow of a cellcompatible buffer to surround the shell flow, such that the shell flow is between the sheath flow and the core flow as the sheath flow, shell flow, and core flow pass in a shared flow direction, thereby producing a hydrogel microtube containing a plurality of cells within an inner channel of the hydrogel microtube.

[0067] In some embodiments, the hydrogel-forming solution includes one or more of (a) a collagen protein; (b) a blend of an alginate polymer and a collagen protein; and (c) a peptide- functionalized alginate polymer and, optionally, an unfunctionalized alginate polymer, preferably one or more of (a) and (b).

[0068] In some embodiments, (b) the blend of the alginate polymer and the collagen protein has an alginate polymer-to-collagen protein ratio of 0.01 to 100 by mass, preferably 0.1 to 1.5 by mass, more preferably 0.25 to 0.75 by mass.Attorney Docket No. 0073605-001131

[0069] In some embodiments, the peptide-functionalized alginate polymer includes an arginineglycine-aspartic acid (RGD) peptide covalently linked to the alginate polymer.

[0070] In some embodiments, the peptide-functionalized alginate polymer includes one or more of a peptide-functionalized alginate acid polymer, a peptide-functionalized sodium alginate polymer, a peptide-functionalized methacrylate-modified alginate polymer, and a peptide-functionalized vinyl sulfone-modified alginate polymer.

[0071] In some embodiments, the cell solution includes one or more cells selected from the group consisting of embryonic stem cells; mammalian embryonic stem cells; human embryonic stem cells (hESCs); human induced pluripotent stem cells (iPSCs); mammalian induced pluripotent stem cells; mammalian naive pluripotent stem cells; mammalian tissue stem cells; human pluripotent stem cells (hPSCs); cells derived or differentiated from one or more of embryonic stem cells, mammalian embryonic stem cells, human embryonic stem cells (hESCs), human induced pluripotent stem cells (iPSCs), mammalian induced pluripotent stem cells, mammalian naive pluripotent stem cells, mammalian tissue stem cells, and human pluripotent stem cells (hPSCs); mammalian cells reprogrammed from other cell types; mammalian primary cells; human umbilical vein endothelial cells; primary tumor cells; cancer cells; immune cells; T cells; natural killer cells; mammalian cell lines; engineered human and mammalian cells; insect cells; plant cells; and yeast and bacterial cells. Cells can be cultured as single cell type or as a mixture of different cell types or a mixture of cells from different donors.

[0072] In some embodiments, the shell flow of the hydrogel-forming solution is applied in a plurality of directions symmetrically disposed with respect to the core flow of the cell solution.

[0073] In some embodiments, the sheath flow of cell-compatible buffer is applied in a plurality of directions symmetrically disposed with respect to the core flow of the cell solution and / or the shell flow of the hydrogel-forming solution.

[0074] In some embodiments, the cell solution or the cell-compatible buffer has a pH from about 7 to about 9, preferably about 7.4.

[0075] In some embodiments, the hydrogel-forming solution has a pH from about 2 to about 6, preferably from about 3 to about 5.

[0076] In some embodiments, the cell-compatible buffer includes one or more multivalent ions, preferably one or more divalent ions.

[0077] In some embodiments, the one or more divalent ions includes Mg2+, Ca2+, Zn2+, or Ba2+, preferably Ca2+.Attorney Docket No. 0073605-001131

[0078] In some embodiments, the hydrogel-forming solution is maintained at a temperature of no greater than 10 °C, preferably no greater than 4 °C, using a cooling element.

[0079] In some embodiments, the method further includes, after step (iii), raising a temperature of the hydrogel microtube to about 37 °C using a heating element.

[0080] In some embodiments, the method further includes, after step (iii), coating the hydrogel microtube using a coating solution.

[0081] In some embodiments, the coating solution includes one or more of an alginate acid polymer, a sodium alginate polymer, a methacrylate-modified alginate polymer, and a vinyl-sulfone- modified alginate, wherein the vinyl sulfone-modified alginate polymer is optionally peptide- functionalized.

[0082] Another aspect of the present disclosure is a method of coating a hydrogel microtube. The method includes (a) preparing the hydrogel microtube according the method described herein. The method further includes (b) dipping the hydrogel microtube in a coating solution for a time period of at least 1 second and no greater than 10 hours, preferably at least 1 minute and no greater than 30 minutes, more preferably at least 3 minutes and no greater than 15 minutes. The coating solution includes at least 0.1% by weight and no greater than 30% by weight, preferably at least 0.5% by weight and no greater than 1.5% by weight, of an alginate polymer. The alginate polymer includes one or more of an alginate acid polymer, a sodium alginate polymer, a methacrylate- modified alginate polymer, and a vinyl-sulfone-modified alginate, wherein the vinyl sulfone- modified alginate polymer is optionally peptide-functionalized. The method further includes (c) dipping the hydrogel microtube in a buffer containing or more divalent ions selected from Mg2+, Ca2+, Zn2+, and Ba2+, preferably Ca2+.

[0083] These and other aspects and features of nonlimiting embodiments of the present invention will become apparent to those skilled in the art upon review of the following description of specific nonlimiting embodiments of the invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] For the purpose of illustrating the invention, the drawings show aspects of one or more embodiments of the invention. However, it should be understood that the present invention is not limited to the precise arrangements and instrumentalities shown in the drawings.

[0085] FIGS. 1A-D depict various exemplary aspects of a setup for fabrication of collagen hydrogel microtubes (ColTubes). (A) Design and print of a micro-extruder described herein. (B) Design and print of a cooling box used for cooling collagen solution. (C) The setup for processingAttorney Docket No. 0073605-001131ColTubes, which includes three syringe pumps, the micro-extruder, the cooling box, a heating pad, and a HEPES buffer reservoir. (D) To fabricate ColTubes, a cell solution at room temperature (RT), an ice-cold collagen solution (pH = 3.0), and a HEPES buffer (RT, pH = 7.4) are pumped into inlet 1, 2, and 3 of the micro-extruder, respectively, to form coaxial core-shell-sheath laminar flows that are extruded into a heated HEPES buffer (37 °C, pH = 7.4). The shell collagen flow rapidly forms a hydrogel microtube as a result of the combined pH neutralization and temperature increase. PBS buffer or other cell-compatible buffers can be used in addition to, or instead of, the ELEPES buffer.

[0086] FIG. 2A depicts an exemplary schematic illustrating the engineering principles of the hydrogel microtubes described herein. (A) Cells are grown within hydrogel microtubes suspended in a culture medium. The microtubes protect cells from hydrodynamic stresses, restrict the cell mass to a radial diameter of <999 um, preferably <400 pm, provide free space for cell growth, and serve as a substrate for cell adhesion. The cell culture medium enters the microtubes through the nanopores in the microtube wall.

[0087] FIGS. 2B-C depict exemplary scanning electron microscopy (SEM) images of the hydrogel microtubes described herein. (B) The inner diameter (ID) varies from 300 to 500 pm, while maintaining a constant shell thickness of -100 pm. (C) The shell thickness is adjusted from 80 to 150 pm, while keeping the ID constant at -400 pm.

[0088] FIGS. 3A-B depict various exemplary confocal microscopy images comparing the nanostructures of ColTubes and bulk collagen hydrogels. (A) ColTubes and (B) bulk collagen hydrogels were fabricated using 4, 6, and 8 mg / mL collagen. For ColTubes, images include the entire microtube and the outer and inner surfaces.

[0089] FIGS. 4A-B depict exemplary scanning electron microscopy (SEM) images comparing the nanostructures of ColTubes and bulk collagen hydrogels. (A) ColTubes and (B) bulk collagen hydrogels were prepared using 4, 6, and 8 mg / mL collagen. For ColTubes (A), images include the entire microtube, inner surface, shell, and outer surface. For bulk hydrogels (B), both bulk and surface structures are shown.

[0090] FIGS. 5A-D depict exemplary confocal microscopy scanning electron microscopy (SEM) images of ColTubes Doped with Laminins. (A) Confocal microscopy images of the whole microtube, inner surface, and outer surface of ColTubes doped with laminin. (B) Confocal microscopy images of ColTubes without laminin doping. (C) SEM images showing the whole microtube, outer surface, inner surface, and shell of ColTubes doped with laminin. (D) SEM images of ColTubes without laminin doping.Attorney Docket No. 0073605-001131

[0091] FIGS. 6A-B depict exemplary phase-contrast and live / dead staining images of adherent HEK 293 cells (A) and suspension 293T 17SF cells (B) cultured in ColTubes.

[0092] FIG. 7A depicts exemplary phase-contrast images of H9 hESCs cultured in ColTubes at days 0, 1, 3, 5, and 7.

[0093] FIG. 7B depicts exemplary live / dead staining images of H9 cells inside the hydrogel microtubes and released from the hydrogel microtubes on day 7.

[0094] FIG. 7C depicts exemplary flow cytometry analysis showing that 99.6% of cells harvested on day 7 are Calcein AM-positive (live cells).

[0095] FIG. 7D depicts exemplary flow cytometry analysis confirming that majority of the cells on day 7 express pluripotency markers OCT4 and Nanog.

[0096] FIG. 8 A depicts an exemplary cardiomyocyte production protocol.

[0097] FIG. 8B exemplary results of differentiating H9 hESCs into cardiomyocytes in ColTubes. H9 hESCs were processed into ColTubes and expanded in E8 medium, followed by mesoderm induction for 1 day, cardiomyocyte differentiation from days 2 to 11, and metabolic enrichment from days 11 to 18. Phase-contrast and fluorescent images of cells in ColTubes on days 0, 1, 3, 5, 7, 11, 15, and 18 are shown. Cardiomyocytes are cTnT-positive.

[0098] FIG. 9A-D depict exemplary results illustrating that ColTubes exhibit fewer cell leakage events compared to AlgTubes. (A) Images showing no cell leakage from ColTubes. (B) Images showing significant cell leakage from AlgTubes, with large cell aggregates forming. (C) Quantification of cell leakage events over time for AlgTubes and ColTubes. (D) Leak rate comparison between AlgTubes and ColTubes. Leak rate is defined as the number of cells leaked into the medium divided by the total number of cells in the well.

[0099] FIG. 10A-C depict exemplary results of fabricating seminiferous tubules with ColTubes. (A) Phase-contrast images of ColTubes containing Leydig cells in the collagen shell and Sertoli cells inside the microtube at days 0, 1, and 3. (B) Live / Dead staining of the tubules on day 5 shows minimal cell death. (C) Confocal images showing Leydig cells in the collagen shell and Sertoli cells inside the microtube.

[0100] FIGS. 11 A-G depict various exemplary aspects of the setup for fabrication of collagenalginate hydrogel microtube (ColAlgTube) microbioreactors. (A) Design and printed version of the new micro-extruder. (B) Design and printed version of the cooling box used for temperature control. (C, D) The setup for processing ColAlgTubes includes two syringe pumps, a custom-made microextruder, a cooling box, and a HEPES buffer reservoir. To produce ColAlgTubes, a cell solution andAttorney Docket No. 0073605-001131 an ice-cold collagen-alginate solution (pH = 5.0) are pumped into the central and side channels of the micro-extruder, respectively, forming coaxial core-shell flows. These flows are extruded into a HEPES-CaCh buffer (pH = 7.4), which neutralizes the collagen-alginate solution. The shell flow forms a hydrogel microtube through collagen protein formation at pH = 7.4 and alginate crosslinking via Ca2+ions. (E) Cells are cultured within ColAlgTubes suspended in the culture medium inside a vessel. The microtubes protect cells from hydrodynamic stress and confine the cell mass to a radial diameter of no greater than 400 pm, ensuring efficient mass transport of nutrients and waste. These hydrogel microtubes create a cell-friendly microenvironment, allowing efficient cell interaction, growth, and diffusion of nutrients through the hydrogel shell. (F) The inner diameter (ID) is varied from 120 to 220 pm while maintaining a shell thickness of 50 pm. (G) The shell thickness is adjusted from 50 to 140 pm while keeping the ID constant at 170 pm.

[0101] FIGS. 12A-E depict exemplary scanning electron microscopy (SEM) images showing nanostructures of ColAlgTubes containing iPSCs prior to dissolution of the ColAlgTubes. (A) 1 mg / mL Collagen + 0.75% alginate hydrogel microtube. (B) 2 mg / mL collagen + 0.75% alginate hydrogel microtube. (C) 3 mg / mL Collagen + 0.75% alginate hydrogel microtube. (D) 0.75% alginate-only microtube. (E) 3 mg / mL collagen-only microtube.

[0102] FIGS. 13A-C depict exemplary scanning electron microscopy (SEM) images showing nanostructures of ColAlgTubes containing iPSCs after dissolution of the ColAlgTubes. (A) 2 mg / mL collagen + 0.75% alginate hydrogel microtube after dissolving alginate with EDTA. (B) 3 mg / mL Collagen + 0.75% alginate hydrogel microtube after dissolving alginate with EDTA. (C) 3 mg / mL Collagen + 0.75% alginate hydrogel microtube after degrading collagen with collagenase.

[0103] FIGS. 14A-B depict exemplary phase-contrast and live / dead staining images of HEK 293T cells cultured in ColAlgTubes (A) and AlgTubes (B) at various time points. In ColAlgTubes, HEK 293 T cells adhered to inner surface of the microtubes as outlined with the dash lines in (A), while in AlgTubes, HEK 293 T did not attach to the microtube and grew as unattached spheroids as outlined with the dash lines in (B).

[0104] FIGS. 15A depicts exemplary phase-contrast (Phi) and darkfield (Ph2) images of H9 hESCs cultured in ColAlgTubes on days 0, 1, 3, 5, 7, and 9. Cells adhered to inner surface of ColAlgTubes as shown by the dash lines.

[0105] FIGS. 15B depicts exemplary phase-contrast (Phi) and darkfield (Ph2) images of human iPSCs cultured in AlgTubes on days 0, 1, 3, 5, 8 and 9. Cells did not attach to the microtube and grew as unattached spheroids as shown by the dash lines.Attorney Docket No. 0073605-001131

[0106] FIGS. 15C-D depict exemplary live / dead staining images of day nine H9 cells within ColAlgTubes (C) and after release from ColAlgTubes (D).

[0107] FIG. 15E depicts exemplary flow cytometry analysis showing that the majority of cells harvested on day 9 express pluripotency markers OCT4 and Nanog.

[0108] FIGS. 16A-B depict exemplary results from differentiation of H9 hESCs into cardiomyocytes in ColAlgTubes. (A) Phase-contrast of H9 derived cardiomyocytes cultured in ColAlgTubes at days 0, 1, 5, 7, 9, 11, 15, and 18. (B) Immunostaining of cTnT marker for cardiomyocytes differentiated in ColAlgTubes.

[0109] FIGS. 17A-E depict exemplary results showing that ColAlgTubes exhibit fewer cell leakage events compared to AlgTubes. (A) Phase-contrast images of H9 hESCs expanded in AlgTubes and ColAlgTubes for 7 days. Arrows show cell leakage events in AlgTubes and ColAlgTubes, respectively. (B, C) Images showing significant cell leakage from AlgTubes, forming large cell aggregates (B), whereas minimal cell leakage is observed from ColAlgTubes (C). (D) Quantification of cell leakage events over time for AlgTubes and ColAlgTubes. (E) Comparison of leak rates between AlgTubes and ColAlgTubes. Leak rate is defined as the number of cells leaked into the medium divided by the total number of cells in the well. ***: p<0.001.

[0110] FIG. 18A depicts a scheme illustrating the reaction mechanism for making alginate- RGDs.[oni] FIGS. 18B-C depict various exemplary aspects of the setup for the fabrication of the AlgTubes. A cell suspension and an alginate solution is pumped into the central channel and side channel of the micro-extruder to form coaxial core-shell flows that are extruded through the nozzle into a CaCh buffer. The shell alginate flow is crosslinked by Ca2+ions to form an alginate hydrogel microtube instantly.

[0112] FIG. 18D depicts a scheme illustrating the cell microenvironment inside AlgTubes.

[0113] FIGS. 19A-D depict exemplary results of C2C12 expansion in AlgTubes. (A) Phase or dark-field pictures of C2C12 cells in AlgTubes on different days. (B) Live / Dead cell staining of C2C12 in AlgTubes on day 19. (C) Cell densities on day 0, day 10 and day 19. (D) C2C12 cells released from AlgTubes on day 19 and stained with MF20 and Propidium Iodide (PI).

[0114] FIGS. 20A-D depict exemplary results of C2C12 differentiation in AlgTubes. C2C12 were cultured for 7 days in AlgTubes before differentiation for 12 day. (A) C2C12 cells in AlgTubes on different days. (B) Live / Dead cell staining on day 19. Arrows: Myotubes. (C) Immunostaining onAttorney Docket No. 0073605-00113112 days. Cell fibers were fixed, cryosection and stained for myofibers (MF20) and DP Al (C) or stained for MyoD and PAX7 (D).

[0115] FIGS. 21A-C depict exemplary results of C2C12 and DI cells co-cultured in AlgTubes. (A) Cells in AlgTubes on different days. (B) Phase and fluorescent images of cells in AlgTubes. (C) Cells were released from AlgTubes on day 19 and stained for MF20 and nuclei.

[0116] FIGS. 22A-C depict exemplary results of C2C12 and DI co-differentiation in AlgTubes. C2C12 and DI cells were cultured for 7 days in AlgTubes before differentiation. (A) Phase or darkfield pictures of cells on different days. (B) Day 19 cell mass cryosections were immunostained for MF20, MyoD, and PAX7. (C) Quantifications under different conditions in AlgTubes on day 19.

[0117] FIGS. 23A-B depict exemplary results of QM7 cells expansion in AlgTubes. (A) Phase or dark-field pictures of QM7 cells in AlgTubes on different days. (B) Cell yields on day 11 and 18. ns: no significant difference.

[0118] FIGS. 24A-B depict exemplary results of live / dead cell staining of QM7 cultured in AlgTubes on day 11 (A) and day 18 (B). The first row of (A) and (B): released and digested cells on day 11 and day 18. Arrows: dense aggregates.

[0119] FIGS. 25A-C depict exemplary results of QM7 differentiation in AlgTubes. QM7 were first expanded for 6 days in AlgTubes before differentiation. (A) Phase or dark-field pictures of QM7 cells on different days. Day 18 cells were harvested for live / dead staining (B) and fixed for MF20, MyoD, and PAX7 immunostaining (C).

[0120] FIGS. 26A-B depict exemplary results of QM7 and 3T3 co-cultured in AlgTubes. (A) Phase or dark-field pictures of cells in AlgTubes on different days. (B) Live dead cell staining on day 18.

[0121] FIGS. 27A-D depict exemplary results of QM7 and 3T3 cells differentiation in AlgTubes. QM7 and DI cells were first expanded for 6 days in AlgTubes before differentiation. (A) Phase or dark-field pictures of cells in AlgTubes on different days. Day 18 cells were harvested for live / dead staining (B) and fixed for MF20 immunostaining (C). (D) Quantifications under different conditions on day 19.

[0122] FIGS. 28A-C depict exemplary results of X9 cells expansion in AlgTubes. (A) Phase or dark-field pictures of X9 cells on different days. (B) Cell quantification on day 0 and day 19. (C) Live / Dead cell staining on day 19.

[0123] FIG. 29A depicts a schematic illustrating an exemplary MSC isolation process.Attorney Docket No. 0073605-001131

[0124] FIG. 29B depicts exemplary scanning electron microscopy (SEM) images showing representative morphology of MSCs during isolation.

[0125] FIG. 29C depicts exemplary scanning electron microscopy (SEM) images showing differentiation of MSCs into adipocytes (FABP-4+) and osteocytes (osteocalcin+).

[0126] FIG. 29D depicts exemplary flow cytometry analysis of MSC surface markers. Negative markers include CD34, CD45, CD1 lb, CD79A, and HLA-DR.

[0127] FIG. 29E depicts exemplary size distribution of MSCs cultured in T-25 flasks at passages 4 (P4), 6 (P6), and 8 (P8).

[0128] FIG. 30A depicts representative images of MSC (P3) spheroids cultured for 3 days with varying initial cell numbers per spheroid.

[0129] FIG. 30B depicts exemplary results from quantification of spheroid diameter over the 3- day culture period.

[0130] FIG. 30C depicts representative images of individual MSCs following 2D and 3D spheroid culture.

[0131] FIGS. 30D-E depict exemplary cell size distributions of MSCs after 2D and 3D spheroid culture.

[0132] FIG. 30F depicts exemplary proportions of small (<15 pm) and large (>15 pm) MSCs after 2D and 3D spheroid culture. Spheroid culture duration in panels (C-F) was 72 hours.

[0133] FIG. 31 A depicts representative images showing morphological changes in MSC (P4) spheroids over a 7-day culture period.

[0134] FIG. 3 IB depicts representative images of individual MSCs cultured in 2D or in 25K- cell spheroids for varying durations.

[0135] FIG. 31C depicts exemplary data showing changes in the diameter of 25K-cell MSC 3D spheroids over 7 days.

[0136] FIG. 3 ID depicts exemplary data showing a comparison of mean MSC diameter in 2D versus 25K spheroid cultures over 7 days.

[0137] FIGS. 31E-F depict exemplary cell size distributions of MSCs within 25K spheroids across the 7-day culture period.

[0138] FIGS. 32A-D depict exemplary data showing effects of supplementing extracellular matrix proteins on MSC viability and size in spheroid culture. (A) Phase-contrast and dead cell staining (red) images of MSC (P4) spheroids cultured in EBM-2 + 10% FBS medium, with or without laminin (LN) and fibronectin (FN). Three representative spheroids are shown in eachAttorney Docket No. 0073605-001131 condition. The white arrow indicates the loosely associated cells. (B) Percentage of spheroids containing dead cell aggregates in the presence or absence of LN and FN. (C, D) MSC size distribution following 2D monolayer and 3D spheroid cultures. Spheroids were cultured for 48 hours.

[0139] FIGS. 33A-D depict exemplary data showing effects of chemically defined medium on MSC viability and size in spheroid culture. (A) Phase-contrast and dead cell staining (red) images of MSC (P6) spheroids cultured in various media. Three representative spheroids are shown in each condition. (B) MSC size distribution in 3D cultures across different media conditions. (C, D) Antiinflammatory capability of MSCs cultured under varying conditions. RAW-Dual™ reporter cells were stimulated with 100 ng / mL LPS and 10 ng / mL fFNy, then treated with MSCs. Dexamethasone (Dex, 1 pg / mL) was used as a positive control. Luciferase activity (C) and mouse IL-6 (D) were measured. Spheroid culture time was 48 hrs.

[0140] FIGS. 34A-F depict exemplary data showing effects of alternating 2D / 3D culture on MSC size and immunomodulatory function. (A) MSCs were cultured in flasks for four passages, with an additional 2-day spheroid culture following each passage. Shown are the mean cell diameters immediately after 2D culture and after subsequent spheroid culture. (B) MSC diameters from P5 to P8 using conventional 2D culture versus the alternating 2D / 3D method. P4 cells served as the starting population for both conditions. Diameters were measured after harvesting from 2D flasks. (C) Comparison of MSC sizes at P5-P8 between the two culture methods. (D) Comparison of doubling times at P5-P8 between the two methods. (E, F) Macrophages were activated with LPS and IFNy, then treated with either early-passage (P4) or late-passage (Pl 5) MSCs derived from conventional 2D culture or the alternating 2D / 3D protocol. Levels of mouse IL-6 and IL-10 were measured to assess immunomodulatory effects.

[0141] FIGS. 35A-H depict exemplary data based on RGD-modified alginate hydrogel microtubes (AlgTubes) for MSC culture. (A) Schematic of alginate modification with RGD peptides. (B, C) Chemistry underlying hydrogel microtube formation. (D, E) Process AlgTubes using a microextruder: a cell suspension and alginate solution are pumped into the central and side channels, respectively, creating coaxial core-shell flows. These are extruded through a nozzle into a CaCk buffer, where Ca2+ions crosslink the outer alginate shell, forming hydrogel microtubes instantly. (F) Illustration of growing MSCs within an AlgTube. (G, H) SEM images showing the porous structure of the AlgTubes.Attorney Docket No. 0073605-001131

[0142] FIGS. 36A-L depict exemplary data showing dynamic cell adhesion in RGD-modified AlgTubes. (A) MSCs were processed into RGD-modified AlgTubes. (B-D) Cells adhered to the inner surface and proliferated from day 0 to day 6. (E) On day 6, free RGD peptides were added to the culture medium, leading to cell contraction within 24 hours. (F) By 48 hours, cells had fully detached and formed spheroids. (G, H) Removal of free RGD peptides from the medium resulted in MSC reattachment to the AlgTube surface and resumed cell growth. (1-L) As a comparison, in the absence of free RGD peptides, day 6 MSCs continued to grow and eventually covered the inner surface of the hydrogel microtube.

[0143] FIGS. 37A-C depict various exemplary aspects pertaining to coating ColTubes with an alginate hydrogel. (A) Schematic illustration of fabricating alginate-coated collagen hydrogel microtubes (Alg-ColTubes). ColTubes were produced using a three-flow micro-extruder, then dipped in an alginate solution followed by immersion in a Ca2buffer to form a thin alginate hydrogel coating. (B) Coating thickness increases with longer coating times, while the alginate solution concentration is fixed (1.5% for B). (C) Coating thickness increases with higher alginate concentrations, while the coating time is fixed (15 min for C). In (B) and (C), collagen was labeled with a red fluorescent dye and alginate with a green fluorescent dye. Confocal imaging was used to visualize the coating.

[0144] FIGS. 38A-D depict exemplary data illustrating stability and cytocompatibility of alginate hydrogel coating in static culture. Human pluripotent stem cells (hPSCs) were cultured in uncoated ColTubes (A) or AlgColTubes with coating times of 5 min (B), 15 min (C), and 30 min (D). hPSCs exhibited healthy growth in all conditions with minimal cell death, as indicated by dead cell staining. The alginate hydrogel coating remained stable throughout the culture period. Images include phase contrast, dark-field, and phase overlays with alginate fluorescence. Cells were cultured statically without shaking.

[0145] FIGS. 39A-C depict exemplary data illustrating the effect of alginate coating in reducing surface adhesion of ColTubes. (A) ColTubes and Alg-ColTubes were suspended in PBS. ColTubes adhered to neighboring microtubes (red circles), whereas Alg-ColTubes did not. (B) When the plate was tilted, Alg-ColTubes settled freely, while ColTubes frequently adhered to the plate bottom, indicating strong interaction with the culture vessel. (C) ColTubes and Alg-ColTubes containing hPSCs were cultured in a 6-well plate. Additionally, single hPSCs were added to the medium to assess adhesion to microtube surfaces. Cells adhered to the outer surface of ColTubes. A 5-minAttorney Docket No. 0073605-001131 alginate coating reduced adhesion, while 15-min and 30-min coatings completely eliminated cell attachment. Cells were cultured statically without shaking.

[0146] FIGS. 40A-D depict exemplary data illustrating the stability and cytocompatibility of alginate hydrogel coating in static culture. Human pluripotent stem cells (hPSCs) were cultured in uncoated ColTubes (A) or AlgColTubes with coating times of 5 min (B), 15 min (C), and 30 min (D). hPSCs exhibited healthy growth in all conditions with minimal cell death, as indicated by dead cell staining. The alginate hydrogel coating remained stable throughout the culture period. Images include phase contrast, dark-field, and phase overlays with alginate fluorescence. Cells were cultured dynamic in a shaking incubator with 60 rpm.

[0147] FIGS. 41A-B depict exemplary data illustrating the effect of alginate coating in reducing surface adhesion of ColTubes under dynamic conditions. (A) ColTubes and Alg-ColTubes were suspended in PBS and shaken at 60 rpm. ColTubes adhered to neighboring microtubes (red circles), whereas Alg-ColTubes remained non-adherent. (B) When the plate was tilted, Alg-ColTubes settled freely, while ColTubes frequently adhered to the plate bottom, which indicates strong interaction with the culture vessel.

[0148] FIGS. 42A-E depict exemplary data based on truncated Alg-ColTubes for cell culture and product release. Cells were first processed into long ColTubes, which were then coated with an alginate hydrogel. These Alg-ColTubes support high-density cell growth. After culture, the microtubes were cut into short segments, exposing both ends to the surrounding medium. This configuration allows large proteins and particles — such as extracellular vesicles and viruses — to be released through the open ends (A). Without alginate coating, truncated ColTubes tended to adhere to each other. Additionally, some cells leaked out, attached to the outer surface, and promoted microtube aggregation (B). In contrast, alginate-coated microtubes retained cells within the truncated segments, preventing leakage and surface adhesion (C-E). This approach enables long-term cell culture in truncated Alg-ColTubes while facilitating continuous collection of cell-secreted products. The technology is particularly useful for producing bioparticles such as viruses and extracellular vesicles.

[0149] FIGS. 43A-C depict exemplary data showing Lentivirus production using HEK293 cells in truncated Alg-ColTubes. HEK293 cells were transfected with lentiviral packaging plasmids and cultured in ColTubes (A) or Alg-ColTubes (B), which were truncated after 2 days. Cells grew well in both formats. The virus titer in the medium from truncated Alg-ColTubes was significantly higherAttorney Docket No. 0073605-001131(C), indicating that open ends facilitate viral particle release into the medium. Parameters such as cell density and Alg-ColTube length can be further optimized to enhance particle release.

[0150] FIGS. 44A-C depict exemplary data showing production of Wnt3a-containing extracellular vesicles using L-Wnt cells in truncated Alg-ColTubes. L-cells engineered to express Wnt3a were cultured in ColTubes (A) or Alg-ColTubes (B). Both formats were truncated and cultured for several days, and cells grew well in both. Truncated ColTubes formed aggregates, and leaked cells adhered to the microtube surface (A). In contrast, truncated Alg-ColTubes did not form aggregates, and no cells adhered to the outer surface (B). Wnt3a proteins are known to associate with extracellular vesicles. The concentration of Wnt3a in the medium from truncated Alg-ColTubes was significantly higher (C), indicating that open ends facilitate extracellular vesicle release. Parameters such as cell density and Alg-ColTube length can be further optimized to enhance particle yield.

[0151] FIGS. 45A-H depict a conventional single-arm micro-extruder CAD design and 3D- printed models of a single-arm, 2-flow extruder for processing alginate hydrogel microtubes (A), a single-arm, 3-flow extruder for processing collagen hydrogel microtubes (B), a single-arm, 4-flow extruder (C), and a single-arm, 5-flow extruder (D). This configuration uses only one arm for the shell and sheath flows, resulting in hydrogel microtubes with asymmetrical walls. For example, in (E), the left wall is significantly thicker than the right wall. This asymmetry makes the thinner wall prone to breakage during cell culture, leading to cell leakage (F) and ultimately failure in cell production. Additional examples of hydrogel microtubes with asymmetrical walls, with and without cells, are shown in (G, H).

[0152] FIGS. 46A-D illustrate various aspects of a symmetrical multiple-arm micro-extruder design. (A) A 2-flow (core-shell) extruder featuring two symmetrical arms. The shell flow from the inlet is evenly divided into two streams by arms 1 and 2, positioned 180° apart. The core and shell inlets can be placed on any side of the extruder; in this example, the core inlet is on top, while the shell inlet is on the side. (B) Both the core and shell inlets are positioned on the top side of the extruder. The shell flow can be split into multiple streams, provided they are evenly spaced to maintain symmetry. For instance, (B) shows a three-arm extruder (arms separated by 120°), and (D) shows a four-arm extruder (arms separated by 90°).

[0153] FIG. 47 illustrates various aspects of a symmetrical multiple-arm micro-extruder for multi-flow configurations. This design principle extends to extruders with more than two flows, where all shell flows are divided into symmetrical arms. Shown here is an extruder with three flows:Attorney Docket No. 0073605-001131 one core flow, one shell flow, and one sheath flow. Both the shell and sheath flows are split into two arms to ensure a balanced distribution.

[0154] FIGS. 48A-E depict exemplary results illustrating the effect of the symmetrical-arm micro-extruder design in significantly enhancing microtube uniformity, microtubes produced with the asymmetrical single-arm extruder exhibit uneven wall thickness (A), whereas microtubes fabricated using symmetrical designs — such as the two-arm shell-flow extruder (B) or four-arm shell-flow extruder (C) — display uniform, symmetrical walls. The log ratio of left-to-right wall thickness for 50 microtubes fabricated with 1-, 2-, and 4-arm extruders is compared. Cells grown in these symmetrical microtubes have no leakage events (e). Statistical significance: p < 0.01, *p < 0.001, ****p < 0.0001.

[0155] FIGS. 49A-C depict exemplary micro-extruder arrays and matrices for high-throughput extrusion. (A) In the extruder array, unit extruders are arranged in a single direction. (B) In the extruder matrix, unit extruders are arranged along both the x- and y-axes. Each unit extruder can be configured as a 2-flow (core and shell), 3-flow (core, shell, and additional shell), or n-flow system. Shell flows may include a single arm or multiple symmetrical arms. For each unit extruder, the inlets are connected to syringes supplying the core flow solution and the shell flow solution (C).

[0156] FIGS. 50A-C depict exemplary high-output syringe designs for extruder arrays and matrices. Design 1: A syringe with multiple outlets, each connected to an extruder via tubing (A). Design 2: A syringe with a single main outlet that is bifurcated multiple times to create multiple suboutlets, each connected to an extruder via tubing (B). In both designs, a single syringe can supply flow solutions to multiple unit extruders (C).

[0157] FIGS. 51 A-E depict exemplary bifurcation high-throughput extruders. Each inlet flow is evenly split into two streams at the first bifurcation level, then into four streams at the second level. Additional bifurcation levels can be added as needed to achieve the desired number of flows.Bifurcation can occur in a single direction (a) or in both the x and y directions (B). This design accommodates multiple extruders with varying numbers of flows: two inlets for 2-flow extruders (a, B) and three inlets for 3-flow extruders (D, D) and n inlets for n-flow extruders. Shell flows may use a single arm or symmetrical multiple arms (A-D). In these designs, a single syringe can supply flow solutions to multiple unit extruders (E).

[0158] FIGS. 52A-C depict exemplary 1-to-n high-throughput extruders. Each inlet flow is evenly divided into n streams to achieve the desired number of flows. This design supports multiple extruders with varying configurations: two inlets for 2-flow extruders (A, B), three inlets for 3-flowAttorney Docket No. 0073605-001131 extruders (C, D), and n inlets for n-flow extruders. Shell flows can feature either a single arm or symmetrical multiple arms (A, B). In these designs, a single syringe can supply flow solutions to multiple unit extruders (C).

[0159] FIGS. 53A-E depict exemplary growth kinetics and pluripotency of hPSCs cultured in ColTubes at varying seeding densities. (A) hPSCs were seeded at 3*, 10*, or 20* 106cells / mL in ColTubes and cultured for 7 days. (B) Volumetric yield on day 7 for hPSCs seeded at different densities. (C) Fold expansion on day 7 indicated faster growth at lower seeding densities. (D-E) Flow cytometry analysis of Oct4 and Nanog confirmed that pluripotency was maintained across all seeding densities.

[0160] FIGS. 54A-E depict exemplary showing the impact of initial cell density on hPSC expansion and doubling time in ColTubes. (A) Phase-contrast images of hPSCs seeded at 3* 106cells / mL on day 0 and harvested on day 9. (B) Phase-contrast images of hPSCs seeded at 0.5* 106cells / mL on day 0 and harvested on day 13. (C) Volumetric yield at harvest for hPSCs seeded at 3* 106and 0.5 x lO6cells / mL in ColTubes. (D) Fold expansion at harvest for hPSCs seeded at 3* 106and 0.5 * 106cells / mL in ColTubes. (E) Doubling time at harvest for hPSCs seeded at 3 *106and 0.5 * 106cells / mL in ColTubes.

[0161] FIG.S 55A-E depict exemplary showing consistent expansion of multiple hPSC lines across passages in ColTubes. (A) Phase-contrast images of H9 hESCs, iPSCl, and iPSC2 cultured in ColTubes over five consecutive passages. (B) When seeded at 3 * 106cells / mL, all three hPSC lines consistently achieved yields of approximately 2.0* 108cells / mL. (C) Each line expanded ~70-fold per passage over 7 days. (D-E) More than 95% of cells expressed Oct4 and Nanog throughout the five passages, confirming maintenance of pluripotency.

[0162] FIGS. 56A-C depict exemplary data showing that H9 hESCs retain pluripotency and trilineage differentiation potential after long-term culture in ColTubes. (A) Immunostaining of pluripotency markers Nanog and Oct4 in passage 5 (P5) H9 hESCs cultured in ColTubes. (B) Flow cytometry analysis of Oct4 and Nanog expression in H9 hESCs after long-term culture in ColTubes, showing that >95% of cells expressed these markers. (C) Immunostaining of lineage-specific markers representing ectoderm (Nestin), mesoderm (Brachyury), and endoderm (Sox 17) in differentiated cells derived from P5 H9 hESCs cultured in ColTubes.

[0163] FIGS. 57A-C depict exemplary data showing that iPSCl retains pluripotency and trilineage differentiation potential after long-term culture in ColTubes. (A) Immunostaining of pluripotency markers Nanog and Oct4 in passage 5 (P5) iPSCl cultured in ColTubes. (B) FlowAttorney Docket No. 0073605-001131 cytometry analysis of Oct4 and Nanog expression in iPSCl after long-term culture in ColTubes, showing that >95% of cells expressed these markers. (C) Immunostaining of lineage-specific markers representing ectoderm (Nestin), mesoderm (Brachyury), and endoderm (Soxl7) in differentiated cells derived from P5 iPSCl cultured in ColTubes.

[0164] FIGS. 58A-C depict exemplary data showing that iPSC2 retains pluripotency and trilineage differentiation potential after long-term culture in ColTubes. (A) Immunostaining of pluripotency markers Nanog and Oct4 in passage 5 (P5) iPSC2 cultured in ColTubes. (B) Flow cytometry analysis of Oct4 and Nanog expression in iPSC2 after long-term culture in ColTubes, showing that >95% of cells expressed these markers. (C) Immunostaining of lineage-specific markers representing ectoderm (Nestin), mesoderm (Brachyury), and endoderm (Sox 17) in differentiated cells derived from P5 iPSC2 cultured in ColTubes.

[0165] FIGS. 59A-D depict an exemplary schematic and associated data describing differentiation of hPSCs into neural stem cells (NSCs) in ColTubes. (A) Schematic of the NSC differentiation protocol: hPSCs were expanded in ColTubes using E8 medium, followed by neural induction in E6 medium supplemented with LDN193189 and SB431542 for 7 days. (B) Flow cytometry analysis showing that 98.7% of cells in ColTubes were Pax6+and Nesting confirming successful generation of high purity H9-derived NSCs. (C) Immunostaining of H9-NSCs produced in ColTubes. (D) Phase-contrast images of H9-NSCs in ColTubes on days 0, 1, 3, 5, and 7 of differentiation.

[0166] FIGS. 60A-D depict an exemplary schematic and associated data describing differentiation of hPSCs into definitive endoderm (DE) in ColTubes. (A) Schematic of the DE differentiation protocol: hPSCs were expanded in ColTubes using E8 medium, followed by a 24- hour WNT activation with 3 pM CHIR and a subsequent 2-day BMP inhibition using LDN193189 in E5 medium. (B) Flow cytometry analysis showing that 80.2% of cells in ColTubes were SOX17+and FOXA2+, confirming efficient DE induction. (C) Immunostaining of H9-derived DE cells generated in ColTubes. (D) Phase-contrast images of H9-DEs in ColTubes on days 0, 1, 2, and 3 of differentiation.

[0167] FIGS. 61 A-B depict an exemplary schematic and associated data describing differentiation of H9-cTNT-GFP hPSCs into cardiomyocytes in ColTubes. (A) Schematic of the cardiomyocyte differentiation protocol: H9-cTNT-GFP cells (H9 hESCs engineered to carry a GFP reporter under the cardiac troponin T [cTnT] promoter) were expanded in ColTubes using E8 medium, followed by mesoderm induction for 1 day, cardiomyocyte differentiation from days 2 toAttorney Docket No. 0073605-00113111, and metabolic enrichment from days 11 to 18. (B) Phase-contrast and fluorescence images of H9-cTNT-GFP cardiomyocytes in ColTubes on days 0, 1, 3, 5, 7, 11, 15, and 18, showing GFP expression in cTnT-positive cells.

[0168] FIGS. 62A-C depict exemplary data showing differentiation of wild-type H9 hESCs into cardiomyocytes in ColTubes. (A) Phase-contrast images of H9-derived cardiomyocytes (H9-CMCs) in ColTubes on days 0, 1, 3, 5, 7, 11, 15, and 18 of differentiation. (B) Flow cytometry analysis showing that 98.8% of cells in ColTubes were cTnT+, confirming efficient cardiomyocyte generation. (C) Immunostaining of cardiomyocyte-specific markers in H9-CMCs produced in ColTubes.

[0169] FIGS. 63A-D depict exemplary data showing that both bovine and human ColTubes support robust hPSC expansion without compromising pluripotency. (A) Phase-contrast images of H9 hESCs cultured in bovine ColTubes (hydrogel tubes derived from bovine collagen) on days 0, 2, 4, 6, 8, 10, and 12. (B) Phase-contrast images of H9 hESCs cultured in human ColTubes (hydrogel tubes derived from human collagen) on days 0, 2, 4, 6, 8, 10, and 12. After 12 days, H9 cells expanded 79.4-fold in bovine ColTubes, reaching a volumetric yield of 2.38* 108cells / mL, and 41.6- fold in human ColTubes, reaching 1.25*108cells / mL. (C) Flow cytometry analysis of pluripotency markers (Oct4 and Nanog) for H9 cells cultured in bovine ColTubes. (D) Flow cytometry analysis of pluripotency markers for H9 cells cultured in human ColTubes.

[0170] FIGS. 64A-D depict exemplary data showing that bovine and human ColTubes support hPSC differentiation into neural stem cells (NSCs).(A) Phase-contrast images of H9-derived NSCs differentiated in bovine ColTubes on days 0, 1, 3, 5, and 7.(B) Phase-contrast images of H9-derived NSCs differentiated in human ColTubes on days 0, 1, 3, 5, and 7.(C) Flow cytometry analysis showing that 83.9% of cells in bovine ColTubes were Pax6+and Nesting confirming efficient NSC generation. (D) Flow cytometry analysis showing that 69.8% of cells in human ColTubes were Pax6+and Nesting indicating successful NSC differentiation.

[0171] FIG. 65 depicts exemplary scanning electron microscopy (SEM) images showing nanostructures of ColTubes from rat, bovine, and human sources exhibit similar features. The SEM images show the entire tube, outer surface, inner surface, and shell of ColTubes fabricated from different origins.

[0172] FIGS. 66A-E depict exemplary data showing that ColTubes Enable scalable production of hPSC-derived neural stem cells. (A) Phase-contrast images of H9 hPSCs cultured in 1.6 mL ColTubes suspended in a T-75 flask on days 0, 1, 3, and 5.(B) Phase-contrast images of H9-NSCsAttorney Docket No. 0073605-001131 differentiated in ColTubes on days 1, 3, 5, and 7, along with Live / Dead staining after tube dissolution. (C) Photograph of H9-NSCs in ColTubes suspended in a T-75 flask.(D) Flow cytometry analysis showing that 99.7% of cells in ColTubes are Pax6+and Nestin+H9-NSCs.(E) Immunostaining of H9-NSCs generated in ColTubes.

[0173] The drawings are not necessarily to scale and may be illustrated by phantom lines, diagrammatic representations, and fragmentary views. In certain instances, details that are not necessary for an understanding of the embodiments or that render other details difficult to perceive may have been omitted.DETAILED DESCRIPTION

[0174] To facilitate the understanding of this invention, a number of terms are defined below and throughout the disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. The terminology herein is used to describe specific embodiments of the invention, but their usage does not limit the invention, except as outlined in the claims. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0175] It is to be understood that any aspect and / or element of any embodiment of the method(s) described herein or otherwise may be combined in any way to form additional embodiments of the method(s), all of which are within the scope of the method(s).

[0176] Where a process is described herein, those of ordinary skill in the art will appreciate that the process may operate without any user intervention. In another embodiment, the process includes some human intervention (e.g., a step is performed by or with the assistance of a human).

[0177] As used herein, including the claims, the phrase “at least some” means “one or more” and includes the case of only one. Thus, e.g., the phrase “at least some ABCs” means “one or more ABCs” and includes the case of only one ABC.

[0178] As used herein, including the claims, the term “at least one” should be understood as meaning “one or more” and therefore includes both embodiments that include one or multiple components. Furthermore, dependent claims that refer to independent claims that describe features with “at least one” have the same meaning, both when the feature is referred to as “the” and “the at least one”.Attorney Docket No. 0073605-001131

[0179] As used herein, the term “portion” means some or all. Therefore, for example, “a portion of X” may include some of “X” or all of “X”. In the context of a conversation, the term “portion” means some or all of the conversation.

[0180] As used herein, including the claims, the phrase “using” means “using at least” and is not exclusive. Thus, e.g., the phrase “using X” means “using at least X”. Unless specifically stated by use of the word “only”, the phrase “using X” does not mean “using only X”.

[0181] As used herein, including the claims, the phrase “based on” means “based in part on” or “based, at least in part, on” and is not exclusive. Thus, e.g., the phrase “based on factor X” means “based in part on factor X” or “based, at least in part, on factor X”. Unless specifically stated by use of the word “only”, the phrase “based on X” does not mean “based only on X”.

[0182] In general, as used herein, including the claims, unless the word “only” is specifically used in a phrase, it should not be read into that phrase.

[0183] As used herein, including the claims, the phrase “distinct” means “at least partially distinct”. Unless specifically stated, distinct does not mean fully distinct. Thus, e.g., the phrase “X is distinct from Y” means that “X is at least partially distinct from Y” and does not mean that “X is fully distinct from Y”. Thus, as used herein, including the claims, the phrase “X is distinct from Y” means that X differs from Y in at least some way.

[0184] It should be appreciated that the words “first”, “second”, and so on, in the description and claims, are used to distinguish or identify, and not to show a serial or numerical limitation.

[0185] Similarly, letter labels (e.g., “(A)”, “(B)”, “(C)”, and so on, or “(a)”, “(b)”, and so on) and / or numbers (e.g., “(i)”, “(ii)”, and so on) are used to assist in readability and to help distinguish or identify, and are not intended to be otherwise limiting or to impose or imply any serial or numerical limitations or orderings. Similarly, words such as “particular”, “specific”, “certain”, and “given”, in the description and claims, if used, are to distinguish or identify, and are not intended to be otherwise limiting.

[0186] As used herein, including the claims, the terms “multiple” and “plurality” mean “two or more,” and include the case of “two”. Thus, e.g., the phrase “multiple ABCs” means “two or more ABCs” and includes “two ABCs”. Similarly, e.g., the phrase “multiple PQRs” means “two or more PQRs” and includes “two PQRs”.

[0187] The present invention also covers the exact terms, features, values, and ranges, etc., in case these terms, features, values, and ranges, etc., are used in conjunction with terms such as “about”, “around”, “generally”, “substantially”, “essentially”, “at least”, etc. Thus, e.g., “about 3” orAttorney Docket No. 0073605-001131“approximately 3” shall also cover exactly 3, and “substantially constant” shall also cover exactly constant.

[0188] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0189] As used herein, unless stated otherwise, the terms “about” or “approximately” refer to a value that is within 10% above or below the value being described.

[0190] As used herein, including the claims, singular forms of terms are to be construed as also including the plural form and vice versa, unless the context indicates otherwise. Thus, it should be noted that as used herein, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. In other words, terms such as “a”, “an”, and “the” are not intended to refer to only a singular entity but include the general class of which a specific example may be used for illustration.

[0191] Throughout the description and claims, the terms “comprise”, “including”, “having”, “contain”, and their variations should be understood as meaning “including but not limited to” and are not intended to exclude other components unless specifically so stated.

[0192] As used herein, the terms “administration” or “administering” refer to a method of giving a dosage of a compound or pharmaceutical composition to a subject. A composition described herein may be administered to a subject by any one of a variety of manners or a combination of varieties of manners. For example, a composition may be administered orally, nasally, intraperitoneally, or parenterally, by intravenous, intramuscular, topical, or subcutaneous routes, or by injection into tissue.

[0193] As used herein, an “effective amount” or “therapeutically effective amount” is the amount of a composition of this disclosure which, when administered to a subject, is sufficient to effect treatment of a disease or condition in the subject. The amount of a composition of this disclosure which constitutes a “therapeutically effective amount” may vary depending on the composition, the condition and its severity, the manner of administration, and the age of the subject to be treated.Attorney Docket No. 0073605-001131

[0194] As used herein, the terms “treat”, “treating”, or “treatment” refer to administration of a compound or pharmaceutical composition for a therapeutic purpose. To “treat a disorder” or use for “therapeutic treatment” refers to administering treatment to a patient already suffering from a disease to ameliorate the disease or one or more symptoms thereof to improve the patient’s condition (e.g., by reducing one or more symptoms of a neurological disorder). The term “therapeutic” includes the effect of mitigating deleterious clinical effects of certain processes (i.e., consequences of the process, rather than the symptoms of processes). As nonlimiting examples, a treatment may include (i) preventing a disease or condition from occurring in a subject, in particular, when such subject is predisposed to the condition but has not yet been diagnosed as having it; (ii) inhibiting a disease or condition, i.e., arresting its development; (iii) relieving a disease or condition, i.e., causing regression of the disease or condition; or (iv) relieving the symptoms resulting from a disease or condition, i.e., relieving pain without addressing the underlying disease or condition.

[0195] As used herein, a “subject” includes, but is not limited to, humans and non-human vertebrates such as wild, domestic, and farm animals. The terms “subject” and “patient” may be used interchangeably throughout this disclosure. As a nonlimiting example, a subject or patient can include a human of any age, sex, gender, race, ethnicity, health record, etc., as deemed relevant and / or suitable by a person of ordinary skill in the art, upon reviewing the entirety of this disclosure. Additional nonlimiting examples of subjects or patients can include mammals or birds, such as without limitation, non-human primates, cats, dogs, cows, horses, rodents, pigs, sheep, goats, and poultry. The term subject can refer to any individual in need of treatment. While the present invention primarily describes treatment of human subjects, it should be noted that the scope of the present invention is not limited to human subjects.

[0196] It will be appreciated that variations to the embodiments of the invention can be made while still falling within the scope of the invention. Alternative features serving the same, equivalent, or similar purpose can replace features disclosed in the specification, unless stated otherwise. Thus, unless stated otherwise, each feature disclosed represents one example of a generic series of equivalent or similar features.

[0197] Use of exemplary language, such as “for instance”, “such as”, “for example” (“e.g.,”), and the like, is merely intended to better illustrate the invention and does not indicate a limitation on the scope of the invention unless specifically so claimed.

[0198] While the invention has been described in connection with what is presently considered to be the most practical and embodiments thereof are further described in theAttorney Docket No. 0073605-001131 examples below, it is to be understood that the invention is not to be limited to the disclosed embodiment, but on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0199] The following description sets forth various examples along with specific details to provide a thorough understanding of claimed subject matter. It will be understood by those skilled in the art, however, that claimed subject matter may be practiced without one or more of the specific details disclosed herein. Further, in some circumstances, well-known methods, procedures, systems, and / or components have not been described in detail in order to avoid unnecessarily obscuring claimed subject matter. The illustrative embodiments described in the detailed description and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented here. It will be readily understood that the aspects of the present disclosure, as generally described herein, can be arranged, substituted, combined, and designed in a wide variety of different configurations, all of which are explicitly contemplated and make part of this disclosure.Hydrogel Microtube

[0200] An objective of the present disclosure is to provide a hydrogel microtube for culturing or expanding cells. As used herein, a “hydrogel microtube” is a microtube that includes one or more hydrogels or hydrogel materials as its constituents. As used herein, a “microtube” is a micron-scale (in inner diameter) material having an elongated, tubular shape and a hollow cavity disposed inside. As used herein, a “hydrogel material” or “hydrogel” is a type of crosslinked hydrophilic polymers capable of expanding in aqueous solution and retaining water. Hydrogels are biphasic mixtures of a porous and permeable solid phase and typically at least 10% of water or another interstitial fluid. The solid phase of a hydrogel typically includes a water-insoluble, three-dimensional polymer network with an affinity for a polar molecule such as water. A hydrogel can be classified as a natural hydrogel or a synthetic hydrogel. A hydrogel can further be classified as a physical hydrogel, which is primarily based on non-covalent interactions, or a chemical hydrogel, which is primarily based on covalent bonds instead.

[0201] A microtube typically has a width, diameter, or lateral dimension between 1 pm and 1 mm (in inner diameter). The length or longitudinal dimension of a microtube is not particularly limited, and a microtube can be as long as permitted by the mechanical strength of its constituent(s) and / or the instrumental capability for its preparation. As used herein, a “longitudinal” direction of a microtube is the direction that extending from one terminal of the microtube to the other, whereas aAttorney Docket No. 0073605-001131“lateral” direction is the direction that is transverse (i.e., perpendicular to) the longitudinal direction. Accordingly, a “longitudinal dimension” of a microtube is the size of the microtube along its longitudinal direction, and a “lateral dimension” of a microtube is the size of the microtube along its lateral direction. In some embodiments, the lateral dimension can be an inner diameter of the body of the microtube, for example.

[0202] The length of the microtube can extend between its first end and its second end along a length of the body of the microtube. The first end can have an opening and the second end can have an opening as well. The openings of the first and second ends can be in fluid communication with an inner channel having an inner diameter defined by the elongated body of the microtube. The inner channel can extend along the length of the body and also have an inner lateral dimension extending between opposed inner sides of the body of microtube. The inner lateral dimension can be an inner diameter, for example. The cross-sectional area of the microtube can be circular, oval, or polygonal.

[0203] The hydrogel microtube described herein includes a cavity configured to accommodate a plurality of cells. In some embodiments, the hydrogel microtube described herein has an inner diameter of at least 20 pm and no greater than 999 pm, preferably at least 100 pm and no greater than 600 pm. It should be noted that the inner diameter of the hydrogel microtube can be equal to any value(s) within any of these respective ranges, including the endpoints of these ranges. As a preferred nonlimiting example, the hydrogel microtube described herein can have an inner diameter that is smaller than, or comparable to, the diffusion limit of human tissue, which is approximately 500 pm.

[0204] In some embodiments, the hydrogel microtube has a length or longitudinal dimension of greater than 100 pm, preferably greater than 500 pm, more preferably greater than 2 mm, further preferably greater than 5 mm, even further preferably greater than 1 cm. It should be noted that the length or longitudinal dimension of the hydrogel microtube is not particularly limited, in accordance with details described above in this disclosure.

[0205] In some embodiments, the hydrogel microtube has an aspect ratio, i.e., the ratio between the length of the hydrogel microtube and the inner diameter of the hydrogel microtube, of at least 1.00 or at least 1.05, preferably at least 1.1, more preferably at least 2, further preferably at least 5, even further preferably at least 20.

[0206] In some embodiments, the hydrogel microtube has a wall thickness of at least 1 pm and no greater than 999 pm, preferably at least 10 pm and no greater than 200 pm, more preferably at least 25 pm and no greater than 150 pm. It should be noted that the wall thickness of the hydrogelAttorney Docket No. 0073605-001131 microtube can be equal to any value(s) within any of these respective ranges, including the endpoints of these ranges.

[0207] The highly porous nature of hydrogel material provides for efficient mass transport; specifically, the porous wall of the hydrogel microtube permits nutrients from a cell culture medium to enter the hydrogel microtube to feed cells and allows for wastes and metabolites produced by the cells to leave the hydrogel microtube. As a result, the wall thickness of the hydrogel microtube is not particularly limited and can be any thickness deemed suitable or relevant by a person of ordinary skill in the art, in view of the entirety of the present disclosure.

[0208] In some embodiments, the hydrogel microtube has a substantially uniform wall thickness throughout its entire structure. In some embodiments, the hydrogel microtube has a wall thickness that varies by no greater than ±25%, ±20%, ±15%, ±10%, ±5%, with respect to the average wall thickness of the hydrogel microtube.

[0209] In some embodiments, the hydrogel microtube includes two closed ends. In some embodiments, the hydrogel microtube includes one open end and one closed end. In some embodiments, the hydrogel microtube includes two open ends.

[0210] In some embodiments, the hydrogel microtube has a circular or polygonal, preferably circular, cross-sectional area. In some preferred embodiments, the hydrogel microtube has concentric internal and external surfaces when viewed from its lateral direction.Materials

[0211] The hydrogel microtube described herein can be constructed using various materials, individually or in combination, as set forth below in several exemplary embodiments.

[0212] As a first embodiment, the hydrogel microtube described herein includes, consists of, consists essentially of, or is prepared from, a collagen protein. As used herein, a “collagen protein” is a protein or protein fiber including amino acid sequences that form a triple helix of elongated fibril. Collagen is the main structural protein in the extracellular matrix of the connective tissues of many animals. Collagen is also the most abundant protein in mammals, typically making up 25% to 35% of protein content. Collagen proteins are suitable for preparing the hydrogel microtube described herein particularly for its relatively high strength (e.g., compared to an unfunctionalized alginate polymer), which makes the resulting hydrogel microtube less likely to beak and prevents cells leakage therefrom. Further, collagen proteins can interact with and bind to cells more strongly than, e.g., an unfunctionalized alginate polymer, thereby promoting adhesion and preventing undesiredAttorney Docket No. 0073605-001131 leakage of cells. Furthermore, collagen proteins can be doped with other extracellular proteins such as laminin and fibronectin to create niches that can enhance cell viability, growth, and quality.

[0213] The collagen protein described herein can be derived from any suitable animal source. Nonlimiting examples of such animal source can include the skin, bone, joint cartilage, tendon, ligament, connective tissue, internal organ, blood vessel, etc., of an animal donor. Nonlimiting examples of such animal source further include human tissues, such as placenta and skin, or biowaste. The animal donor can include any type of animal donor (e.g., human, bovine, rat, rabbit, sheep, or pig, among others) deemed suitable or applicable by a person of ordinary skill in the art, in view of the entirety of the present disclosure.

[0214] As a second embodiment, the hydrogel microtube described herein includes, or is prepared from a blend of an alginate polymer and a collagen protein. The ratio between the alginate polymer and the collagen protein is not particularly limited and can include any ratio deemed suitable or applicable by a person of ordinary skill in the art, in view of the entirety of the present disclosure. In some embodiments, the blend of the alginate polymer and the collagen protein has an alginate polymer-to-collagen protein ratio of 0.01 to 100 by mass, preferably 0.1 to 1.5, more preferably 0.25 to 0.75 by mass. It should be noted that the alginate polymer-to-collagen protein ratio can be equal to any value(s) within any of these respective ranges, including the endpoints of these ranges. Further, the collagen protein described in the second embodiment can include any type of collagen protein described throughout the present disclosure.

[0215] As used herein, an “alginate polymer” is a polymer that includes, or is a derivative of, alginic acid or alginic acid polymer. As used herein, “alginic acid” or “alginic acid polymer” is a polysaccharide with homopolymeric blocks of ( l ^4)-linked -D-mannuronate (M) and a-L- guluronate (G) residues, respectively, covalently linked together in different sequences or blocks. The monomers of alginic acid can appear in homopolymeric blocks of consecutive G-residues (G- blocks), consecutive M-residues (M-blocks), or alternating M and G-residues (MG-blocks). Alginic acid can be found in natural sources such as brown algae. In some embodiments, the alginate polymer described herein includes one or more of an alginate acid polymer, a sodium alginate polymer, and a methacryl ate-modified alginate polymer.

[0216] The hydrogel microtube according to the second embodiment preferably includes an interpenetrating network of collagen nanofibers and alginate hydrogels, wherein collagen proteins form a dense nanofiber network interwoven with an alginate polymer mesh, thereby enhancing the mechanical properties of, while providing cell adhesion sites for, the hydrogel microtube.Attorney Docket No. 0073605-001131

[0217] As a third embodiment, the hydrogel microtube described herein includes, or is prepared from, one or more of a peptide-functionalized alginate polymer and, optionally, an unfunctionalized alginate polymer. As used herein, a “peptide-functionalized alginate polymer” is a modified form of an alginate polymer that includes a peptide sequence covalently linked to the alginate polymer. Accordingly, an alginate polymer without such peptide functionalization may be referred to as an “unfunctionalized alginate polymer”. The peptide-functionalized alginate polymer and the unfunctionalized alginate polymer can be combined in any mass or molar ratio deemed suitable or applicable by a person of ordinary skill in the art, in view of the entirety of the present disclosure, such as without limitation a ratio of 0.001 to 1,000, preferably a ratio of 0.01 to 100. It should be noted that the ratio between the peptide-functionalized alginate polymer and the unfunctionalized alginate polymer can be equal to any value(s) within any of these respective ranges, including the endpoints of these ranges.

[0218] In some embodiments, the peptide-functionalized alginate polymer includes one or more of a peptide-functionalized alginate acid polymer, a peptide-functionalized sodium alginate polymer, a peptide-functionalized methacrylate-modified alginate polymer, and a peptide-functionalized vinyl sulfone-modified alginate polymer, etc. In some embodiments, a peptide-functionalized alginate polymer with one or more other modifications not explicitly disclosed herein may be used.

[0219] The peptide-functionalized alginate polymer can include an alginate polymer functionalized by any type of peptide or amino acid sequence deemed suitable or relevant by a person or ordinary skill in the art, upon reviewing the entirety of the present disclosure. In some embodiments, the peptide-functionalized alginate polymer includes an arginine-glycine-aspartic acid (RGD) peptide covalently linked to the alginate polymer. This feature is particularly useful for improving adhesion of anchorage-dependent cells that otherwise do not bind strongly or effectively to a hydrogel microtube without such functionalization. Specifically, the RGD peptide can serve as an anchorage or binding site for integrin receptors, which are abundant on the surface of cells, thereby promoting adhesion of the cells onto the hydrogel microtube.

[0220] As a nonlimiting example, 20% to 30% of the -OH groups in an alginate polymer can be modified with divinyl sulfone (DVS) to produce a vinyl sulfone-functionalized alginate polymer (alginate-VS). RGD peptides containing a C-terminal cysteine can then be used to react with alginate-VS, via a click reaction under alkaline conditions, to produce an RGD-functionalized alginate polymer (alginate-RGD). 10% of the modified -OH groups can be reacted with RGDAttorney Docket No. 0073605-001131 peptides. These Alginate-RGDs can then be combined with unmodified alginate polymers to make a 2% alginate solution, which can then be used to produce hydrogel microtubes.

[0221] In some embodiments, the hydrogel microtube further includes a coating layer. In some embodiments, the coating layer includes one or more of an alginate acid polymer layer, a sodium alginate polymer layer, a methacrylate-modified alginate polymer layer, and a peptide-functionalized vinyl sulfone-modified alginate polymer layer, etc. In some embodiments, an alginate polymer with one or more other modifications not disclosed herein may be used as a coating layer. As a nonlimiting example, such coating layer can be applied to a hydrogel microtube including collagen, which may be sticky in its native form, to prevent or suppress microtube-to-microtube adhesion and / or the undesired binding between leaked cells and the outer surface of the hydrogel microtube. As a nonlimiting example, such coating layer can be applied by dipping or submerging a hydrogel microtube containing collagen in an alginate solution, and 2) dipping the hydrogel tubes treated with alginate in a solution containing a multivalent ion (e.g., Ca2+) to form the coating layer. The coating layer can be applied after processing the hydrogel microbes and / or at any time of cell culture.

[0222] The thickness of such coating layer is not particularly limited. As nonlimiting examples, the coating layer can be applied at a thickness of at least 20 nm and no greater than 100 pm, preferably at least 50 nm and no greater than 50 pm, more preferably at least 100 nm and no greater than 25 pm. It should be noted that the thickness of the coating layer can be equal to any value(s) within any of these respective ranges, including the endpoints of these ranges. In addition, the thickness of the coating layer can be adjusted by varying either the coating time or the concentration of the coating solution, in accordance with details described throughout the present disclosure.

[0223] In some embodiments, the hydrogel microtube further includes one or more extracellular matrix (ECM) proteins. In some embodiments, the one or more extracellular matrix (ECM) proteins includes laminin or fibronectin. As a nonlimiting example, collagen contains binding domains for ECM proteins, such as without limitation, fibronectin and laminin. Accordingly, one or more of these proteins can be incorporated into the hydrogel microtube described herein to support the adhesion and / or growth of specialized cell types.

[0224] In some embodiments, the hydrogel microtube further includes one or more of polyethylene glycol and poly(vinyl alcohol).Bioreactor System

[0225] Another objective of the present disclosure is to provide a bioreactor system. As used herein, a “bioreactor system” is a composition of matter, material, or apparatus containing suchAttorney Docket No. 0073605-001131 composition of matter or material, that is configured or adapted to produce one or more chemical species or organisms of interest from a biological source.

[0226] The bioreactor system includes a plurality of the hydrogel microtubes described herein and a cell-compatible buffer, wherein the plurality of the hydrogel microtubes is dispersed in the cell-compatible buffer.Buffer

[0227] As used herein, a “cell-compatible buffer” is a buffer or buffer solution that does not negatively impact the survival or growth of cells or microorganisms. A cell-compatible buffer can provide a stable chemical environment for the cells. In some embodiments, the cell-compatible buffer has a pH from about 7 to about 9, preferably about 7.4. It should be noted that the pH of the cell-compatible buffer can be equal to any value(s) within any of these respective ranges, including the endpoints of these ranges. In addition, the cell-compatible buffer described herein can include any type of buffer or buffer solution deemed suitable or relevant by a person of ordinary skill in the art, in view of the entirety of the present disclosure.

[0228] As used herein, a “buffer” or “buffer solution” is a solution or mixture that contains at least one pair of weak acid, HA, and its conjugate base, A’, in a molar ratio typically between 10:1 and 1 : 10, wherein the solution or mixture maintains a stable pH close to the pKaof the weak acid, against addition of acidic or basic chemical species. For simplicity, a buffer containing a pair of conjugate base and acid can be written as A7HA. As used herein, the “conjugate base” of a weak acid is a chemical species that contains exactly one fewer proton with respect to the weak acid. As used herein, the “pKa” of a weak acid is the negative log of the acid dissociation constant, Ka, of the weak acid. The pH of a buffer solution can be calculated using the Henderson Hasselbalch equation:

[0229] As a nonlimiting example, a buffer can include an acetate buffer (i.e., CHsCOONa / CHiCOOH). As another nonlimiting example, a buffer can include a borate buffer (i.e., Na2B4C>7 IOH2O / H3BO3). As another nonlimiting example, a buffer can include a bicarbonate buffer (i.e., NaHCO3 / H2CO3 or Na2CO3 / NaHCO , depending on the desired pH). As another nonlimiting example, a buffer can include a cacodylate buffer (i.e., NaC2HeAsO2 / HC2H6AsO2). As a nonlimiting example, a buffer can include phosphate buffer (i.e., Na^PC / HsPC 2I IPCWNal hPCh, or Na3HPO4 / Na2HPO4, depending on the desired pH).

[0230] As another nonlimiting example, a buffer can include a Good’s buffer. As used herein, “Good’s buffers” are a group of more than 20 conjugate acid / base pairs selected and described byAttorney Docket No. 0073605-001131Norman Good and colleagues for biochemical and biological research primarily during 1966-1980. For simplicity, only the conjugate acid may be shown for each conjugate acid / base pair. Nonlimiting examples of Good’s buffers include MES (C6H13NO4S), ADA (C6H10N2O5), PIPES (C8H18N2O6S2), ACES (C4H10N2O4S), MOPSO (C7H15NO5S), cholamine chloride hydrochloride (C5H16CI2N2), MOPS (C7H15NO4S), BES (C6HI5NO5S), TES (C6Hi5NO6S), HEPES (C8H18N2O4S), TAPSO (C7H17NO7S), POPSO (C10H22N2O8S2), HEPPSO (C9H20N2O5S), EPPS or HEPPS (C9H20N2O4S), Tri cine (C6H13NO5), Tris (C4H11NO3), glycinamide (C2H6N2O), glycylglycine or Gly-Gly (C4H8N2O3), Bicine (C6Hi3NO4), TAPS (C7H17NO6S), AMPSO (C7H17NO5S), CABS (C10H21NO3S), CHES (C8H17NO3S), CAPS (C9H19NO3S), and CAPSO (C9H19NO4S).

[0231] As another nonlimiting example, a buffer can include a phosphate-buffered saline (PBS) solution. As used herein, a “phosphate-buffered saline” or “PBS” is a commonly used buffer in biological research and pharmaceutical formulations that typically contains 137 mM NaCl, 2.7 mM KC1, 10 mM Na2HPO4, and 1.8 mM KH2PO4.

[0232] As another nonlimiting example, a buffer can include an ethanesulfonic acid solution. As used herein, an “ethanesulfonic acid” solution is a buffer solution that contains one or more ethanesulfonic acid derivatives. An ethanesulfonic acid solution can be a solution of chemical(s) such as HEPES, consistent with details described above.

[0233] As used herein, “HEPES”, also known as 4-(2-hydroxyethyl)piperazine-l -ethanesulfonic acid or by its IUPAC name as 2-[4-(2-hydroxyethyl)piperazin-l-yl]ethane-l-sulfonic acid, is a derivative of ethanesulfonic acid, with a formula of C8H18N2O4S, that contains a piperazine ring, - N(CH2CH2)2N-. HEPES is a zwitterionic sulfonic acid buffering agent. As another nonlimiting example, a buffer can include a morpholino-based solution.

[0234] As used herein, a “morpholino-based” solution is a buffer solution that includes one or more chemical species that contain a morpholine ring, -N(CH2CH2)2O. A morpholino-based solution can be a solution of chemical(s) such as MOPS, consistent with details described above. As used herein, “MOPS”, also known as 3-(N-morpholino)propanesulfonic acid or by its IUPAC name as 3- (morpholin-4-yl)propane-l -sulfonic acid, is a chemical species with a formula of C7H15NO4S.

[0235] It should be noted that the classification of buffers described herein may, in some cases, be arbitrary and overlapping, and some buffers may accordingly be categorized under multiple categories.

[0236] In some embodiments, the cell-compatible buffer further includes or contains one or more multivalent ions, preferably one or more divalent ions. In some embodiments, the one or moreAttorney Docket No. 0073605-001131 divalent ions can include Mg2+, Ca2+, Zn2+, and / or Ba2+, preferably Ca2+. In some embodiments, the multivalent or divalent ions can be present as a chloride, acetate, or nitrate salt, preferably a chloride salt. These multivalent / divalent ions can function as a crosslinker for one or more components (e.g., an alginate polymer) of the hydrogel microtube, thereby stabilizing the structure of the hydrogel microtube.

[0237] The multivalent or divalent ions can be present at a concentration of at least 1 nM and no greater than 200 mM, preferably at a concentration of at least 10 nM and no greater than 100 mM. It should be noted that the concentration of multivalent or divalent ions can be equal to any value(s) within any of these respective ranges, including the endpoints of these ranges.Method of Culturing, Expanding, Differentiating, or Manipulating Cells

[0238] Another objective of the present disclosure is to provide a method of culturing, expanding, differentiating, and / or manipulating cells.

[0239] At step (i), the method includes extruding a cell solution and a hydrogel-forming solution into a cell-compatible buffer to produce the hydrogel microtube described herein. This extrusion step can be performed using any apparatus or system, under any combination of parameters, in accordance with details described throughout the present disclosure.

[0240] At step (ii), the method further includes suspending the hydrogel microtube including cells from the cell solution in a cell culture medium. As used herein, a “cell culture medium” is a nutrient-rich mixture, typically an aqueous solution, that provides a stable, often a substantially sterile, environment to support the growth, survival, and / or replication of cells.

[0241] At step (iii), the method further includes culturing, expanding, differentiating, and / or manipulating the cells under suitable conditions. This step can be implemented in any manner, under any combination of parameters, in accordance with details described throughout the present disclosure. As used herein, a “suitable condition” is an environmental condition or factor suitable for the growth and / or replication of a cell. In some cases, a suitable condition can vary from one type of cell to another. A suitable condition can include without limitation a suitable temperature / temperature range, a suitable pressure / pressure range, a suitable pH or pH range, a suitable ionic strength / range of ionic strength, a suitable osmotic pressure or osmolarity / range of osmotic pressure or osmolarity, a suitable concentration / concentration range of one or more nutrients, a suitable level of metabolic waste / metabolites, among others. A person of ordinary skill in the art, upon reviewing the entirety of the present disclosure, will be able to identify suitable conditions specific to one or more cells described herein. In some embodiments, culturing orAttorney Docket No. 0073605-001131 expanding the cells includes culturing or expanding the cells over a time period of at least 2 or 3 hours, preferably at least 6 hours, more preferably at least 12 hours, further preferably at least 24 hours.

[0242] In some embodiments, the method further includes, e.g., between step (i) and step (ii) or between step (ii) and step (iii), coating the hydrogel microtube using a coating solution, in accordance with details described throughout the present disclosure. In some embodiments, the coating solution includes one or more of an alginate acid polymer, a sodium alginate polymer, a methacrylate-modified alginate polymer, a vinyl sulfone-modified alginate polymer, etc., in accordance with details described throughout the present disclosure. It should be noted that this coating step can be performed at any time throughout the production or use of the hydrogel microtube.

[0243] At step (iv), the method further includes dissolving the hydrogel microtube to release the cells. In some embodiments, e.g., when the hydrogel microtube includes an alginate polymer, dissolving the hydrogel microtubes includes treating the hydrogel microtube using one or more chelating agents, such as ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), and / or the like. Such chelating agents can selectively coordinate or bind to a multivalent or divalent ion, such as Ca2+, thereby destabilizing or dissembling an alginate polymer originally crosslinked by such multivalent or divalent ion. In some embodiments, e.g., when the hydrogel microtube includes an alginate polymer, dissolving the hydrogel microtubes includes treating the hydrogel microtube using an alginate lyase. In some embodiments, e g., when the hydrogel microtube includes a collagen protein, dissolving the hydrogel microtubes includes treating the hydrogel microtube using a collagenase, preferably Collagenase P. In some embodiments, a longer hydrogel microtube with cells are truncated into short tubular segments to facilitate the release of the cells.

[0244] In some embodiments, the cell solution includes cells selected from the group consisting of embryonic stem cells; mammalian embryonic stem cells; human embryonic stem cells (hESCs); human induced pluripotent stem cells (iPSCs); mammalian induced pluripotent stem cells; mammalian naive pluripotent stem cells; mammalian tissue stem cells; human pluripotent stem cells (hPSCs); cells derived or differentiated from one or more of embryonic stem cells, mammalian embryonic stem cells, human embryonic stem cells (hESCs), human induced pluripotent stem cells (iPSCs), mammalian induced pluripotent stem cells, mammalian naive pluripotent stem cells, mammalian tissue stem cells, and human pluripotent stem cells (hPSCs); mammalian cellsAttorney Docket No. 0073605-001131 reprogrammed from other cell types; mammalian primary cells; human umbilical vein endothelial cells; primary tumor cells; cancer cells; immune cells; T cells; natural killer cells; mammalian cell lines; engineered human and mammalian cells; insect cells; plant cells; and yeast and bacterial cells. Cells can be cultured as single cell type or as a mixture of different cell types or a mixture of cells from different donors.

[0245] In some embodiments, the cell solution includes without limitation embryonic stem cells; mammalian embryonic stem cells; human embryonic stem cells (hESCs); human induced pluripotent stem cells (iPSCs); mammalian induced pluripotent stem cells; mammalian naive pluripotent stem cells; mammalian tissue stem cells; human pluripotent stem cells (hPSCs); cells derived or differentiated from one or more of embryonic stem cells, mammalian embryonic stem cells, human embryonic stem cells (hESCs), human induced pluripotent stem cells (iPSCs), mammalian induced pluripotent stem cells, mammalian naive pluripotent stem cells, mammalian tissue stem cells, and human pluripotent stem cells (hPSCs); mammalian cells reprogrammed from other cell types; mammalian primary cells; human umbilical vein endothelial cells; primary tumor cells; cancer cells; immune cells; T cells; natural killer cells; mammalian cell lines; engineered human and mammalian cells; insect cells; plant cells; and yeast and bacterial cells, among others. Cells can be cultured as single cell type or as a mixture of different cell types or a mixture of cells from different donors.

[0246] In some embodiments, the cultured or expanded cells have a cell density of at least O. l x lO8cells per milliliter or at least l .Ox lO8cells per milliliter, preferably at least 3.0x l08cells per milliliter, more preferably at least 4.5 xlO8cells per milliliter, further preferably at least 5.0x l08cells per milliliter. In some embodiments, the cultured or expanded cells have a cell density of no greater than O. l x lO8cells per milliliter, depending on the specific use case or user need.Method of Producing Synthetic Tissue

[0247] Another aspect of the present disclosure is a method of producing synthetic tissue, preferably meat. At step (i), the method includes extruding a cell solution and a hydrogel-forming solution into a cell-compatible buffer to produce the hydrogel microtube described herein, in accordance with details described throughout the present disclosure.

[0248] At step (ii), the method further includes suspending the microtube including cells from the cell solution in a cell culture medium, in accordance with details described throughout the present disclosure.Attorney Docket No. 0073605-001131

[0249] At step (iii), the method further includes culturing or expanding the cells under suitable conditions, in accordance with details described throughout the present disclosure.

[0250] At step (iv), the method further includes dissolving the hydrogel microtube to release the cells, in accordance with details described throughout the present disclosure. In some embodiments, e.g., when the hydrogel microtube includes an alginate polymer, dissolving the hydrogel microtubes includes treating the hydrogel microtube using one or more chelating agents, such as ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), and / or the like. In some embodiments, e.g., when the hydrogel microtube includes an alginate polymer, dissolving the hydrogel microtubes includes treating the hydrogel microtube using an alginate lyase. In some embodiments, e.g., when the hydrogel microtube includes a collagen protein, dissolving the hydrogel microtubes includes treating the hydrogel microtube using a collagenase, preferably Collagenase P. In some embodiments, animal cells and tissues can be directly used or consumed (e.g., as meat) without dissolving the hydrogel microtubes, since the hydrogel microtubes are edible.

[0251] At step (v), the method further includes producing the synthetic tissue using the released cells, in accordance with details described throughout the present disclosure.

[0252] In some embodiments, the cultured or expanded cells have a cell density of at least O. l x lO8cells per milliliter or at least 1.0* 108cells per milliliter, preferably at least 3.0* 108cells per milliliter, more preferably at least 4.5 *108cells per milliliter, further preferably at least 5.0* 108cells per milliliter. In some embodiments, the cultured or expanded cells have a cell density of no greater than O. l x lO8cells per milliliter, depending on the specific use case or user need.

[0253] In some embodiments, the hydrogel microtube is a truncated hydrogel microtube.

[0254] In some embodiments, the hydrogel microtube is a coated with a coating layer. In some embodiments, the coating layer includes one or more of an alginate acid polymer layer, a sodium alginate polymer layer, a methacrylate-modified alginate polymer layer, and a vinyl sulfone- modified alginate polymer layer, wherein the vinyl sulfone-modified alginate polymer is optionally peptide-functionalized. These aspects can be implemented in accordance with details described throughout the present disclosure.

[0255] In some embodiments, the method further includes coating the hydrogel microtube using a coating solution. In some embodiments, the coating solution includes one or more of an alginate acid polymer, a sodium alginate polymer, a methacrylate-modified alginate polymer, and a vinyl- sulfone-modified alginate, wherein the vinyl sulfone-modified alginate polymer is optionallyAttorney Docket No. 0073605-001131 peptide-functionalized. These aspects can be implemented in accordance with details described throughout the present disclosure.

[0256] In some embodiments, the method further includes, between step (iii) and step (iv), truncating the hydrogel microtube, such as without limitation via mechanical shearing (e.g., using a blender), in accordance with details described throughout the present disclosure.Method of Producing a Protein. Viral Particle, or Extracellular Vesicle

[0257] Another objective of the present disclosure is to provide a method of producing a protein, viral particle (e.g., lentivirus), or extracellular vesicle.

[0258] At step (i), the method includes extruding a cell solution and a hydrogel -forming solution into a cell-compatible buffer to produce the hydrogel microtube described herein, in accordance with details described throughout the present disclosure.

[0259] At step (ii), the method further includes suspending the microtube including cells from the cell solution in a cell culture medium, in accordance with details described throughout the present disclosure.

[0260] At step (iii), the method further includes culturing or expanding the cells under suitable conditions to produce the protein, viral particle, or extracellular vesicle, in accordance with details described throughout the present disclosure. In some embodiments, culturing or expanding the cells includes culturing or expanding the cells over a time period of at least 2 or 3 hours, preferably at least 6 hours, more preferably at least 12 hours, further preferably at least 24 hours.

[0261] At step (iv), the method further includes optionally dissolving the hydrogel microtube to release the protein, viral particle, or extracellular vesicle, in accordance with details described throughout the present disclosure.

[0262] At step (v), the method further includes harvesting the protein, viral particle, or extracellular vesicle, e.g., from the cell-compatible buffer, in accordance with details described throughout the present disclosure.

[0263] In some embodiments, a protein, viral particle, or extracellular vesicle can be secreted into the cell culture medium. In some embodiments, a protein, viral particle, or extracellular vesicle can be retained in the hydrogel microtube. In some embodiments, both the protein, viral particle, or extracellular vesicle secreted into the cell culture medium and the protein, viral particle, or extracellular vesicle retained in the hydrogel microtube are collected.

[0264] In some embodiments, the hydrogel microtube is a truncated hydrogel microtube, in accordance with details described throughout the present disclosure.Attorney Docket No. 0073605-001131

[0265] In some embodiments, the hydrogel microtube is a coated with a coating layer. In some embodiments, the coating layer includes one or more of an alginate acid polymer layer, a sodium alginate polymer layer, a methacrylate-modified alginate polymer layer, and a vinyl sulfone- modified alginate polymer layer, wherein the vinyl sulfone-modified alginate polymer is optionally peptide-functionalized. These aspects can be implemented in accordance with details described throughout the present disclosure.

[0266] In some embodiments, the method further includes coating the hydrogel microtube using a coating solution. In some embodiments, the coating solution includes one or more of an alginate acid polymer, a sodium alginate polymer, a methacrylate-modified alginate polymer, and a vinyl- sulfone-modified alginate, wherein the vinyl sulfone-modified alginate polymer is optionally peptide-functionalized. These aspects can be implemented in accordance with details described throughout the present disclosure.

[0267] In some embodiments, the method further includes truncating the hydrogel microtube (e.g., via mechanical shearing, such as using a blender) to release the protein, viral particle, or extracellular vesicle, in accordance with details described throughout the present disclosure. In some embodiments, a longer hydrogel microtube with cells are truncated into short tubular segments to enhance cell growth and / or facilitate the release of the protein, viral particle, or extracellular vesicle.

[0268] As a nonlimiting example, hydrogel microtubes can be synthesized using collagen and coated with an alginate polymer. Cells adhere to the inner surface of the hydrogel microtubes and grow to reach a high density. The alginate polymer coating can be applied either before or after cells reach such high density. Afterwards, the microtubes can be cut into shorter segments, thereby exposing both ends to the medium. This configuration enables continuous release of large proteins and particles — such as extracellular vesicles and viruses — through the open ends.

[0269] In some embodiments, the cultured or expanded cells have a cell density of at least O. l x lO8cells per milliliter or at least l.Ox lO8cells per milliliter, preferably at least 3.0x l08cells per milliliter, more preferably at least 4.5xl08cells per milliliter, further preferably at least 5.0x l08cells per milliliter. In some embodiments, the cultured or expanded cells have a cell density of no greater than O. l x lO8cells per milliliter, depending on the specific use case or user need.Method of Providing Cell Therapy

[0270] Another objective of the present disclosure is to demonstrate a method of providing cell therapy, the method including administering to a subject a therapeutically effective amount of cells cultured or expanded using the hydrogel microtube described herein. Depending on the types ofAttorney Docket No. 0073605-001131 cultured cells, the method can be used to treat, alleviate symptoms of, or hinder disease progression of any medical condition deemed applicable by a person of ordinary skill in the art, in view of the entirety of the present disclosure.

[0271] In some embodiments, the therapeutically effective amount of cells includes one or more members selected from the group consisting of embryonic stem cells; mammalian embryonic stem cells; human embryonic stem cells (hESCs); human induced pluripotent stem cells (iPSCs); mammalian induced pluripotent stem cells; mammalian naive pluripotent stem cells; mammalian tissue stem cells; human pluripotent stem cells (hPSCs); cells derived or differentiated from one or more of embryonic stem cells, mammalian embryonic stem cells, human embryonic stem cells (hESCs), human induced pluripotent stem cells (iPSCs), mammalian induced pluripotent stem cells, mammalian naive pluripotent stem cells, mammalian tissue stem cells, and human pluripotent stem cells (hPSCs); mammalian cells reprogrammed from other cell types; mammalian primary cells; human umbilical vein endothelial cells; primary tumor cells; cancer cells; immune cells; T cells; natural killer cells; mammalian cell lines; engineered human and mammalian cells; insect cells; plant cells; and yeast and bacterial cells.

[0272] In some embodiments, the therapeutically effective amount of cells include without limitation embryonic stem cells; mammalian embryonic stem cells; human embryonic stem cells (hESCs); human induced pluripotent stem cells (iPSCs); mammalian induced pluripotent stem cells; mammalian naive pluripotent stem cells; mammalian tissue stem cells; human pluripotent stem cells (hPSCs); cells derived or differentiated from one or more of embryonic stem cells, mammalian embryonic stem cells, human embryonic stem cells (hESCs), human induced pluripotent stem cells (iPSCs), mammalian induced pluripotent stem cells, mammalian naive pluripotent stem cells, mammalian tissue stem cells, and human pluripotent stem cells (hPSCs); mammalian cells reprogrammed from other cell types; mammalian primary cells; human umbilical vein endothelial cells; primary tumor cells; cancer cells; immune cells; T cells; natural killer cells; mammalian cell lines; engineered human and mammalian cells; insect cells; plant cells; and / or yeast and bacterial cells, among others.

[0273] In some embodiments, the therapeutically effective amount of cells includes one or more multipotent stem cells, such as adult stem cells including hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). In some embodiments, the therapeutically effective amount of cells includes one or more tissue-specific stem cells, such as neural stem cells, epidermal stem cells, etc. It should be noted that the stem cells described herein can be derived from any type of tissue orAttorney Docket No. 0073605-001131 organ, such as without limitation, bone marrow, peripheral blood, adipose tissue, umbilical cord, placenta, amniotic fluid, etc. It should be further noted that the method described herein can be used to prepare any type of autogenic, allogenic, or xenogenic stem cells deemed suitable or applicable by a person of ordinary skill in the art, in view of the entirety of the present disclosure. Apparatus / System of Apparatus for Preparing a Hydrogel Microtube

[0274] Another objective of the present disclosure is to provide an apparatus for preparing a hydrogel microtube. Several embodiments of the apparatus are set forth below.

[0275] In a first embodiment, the apparatus includes an extruder 100 having at least a first inlet 104 and at least a second inlet 108 and a housing 112 in fluid connection with the extruder 100 (see FIGS. 45, 46A). The at least a first inlet 104 is operable for introducing a cell solution into the extruder 100. The at least a second inlet 108 includes, or is in fluid connection with, a plurality of feeding elements 116 (see FIG. 46A) and operable for introducing a hydrogel-forming solution, through the plurality of feeding elements 116, into the extruder 100, in a plurality of directions symmetrically disposed with respect to the cell solution, thereby producing the hydrogel microtube containing a plurality of cells. See FIGS. 46-D for additional details.

[0276] In a second embodiment, the apparatus includes an extruder 200 having at least a first inlet 204 and at least a second inlet 208 and a housing 212 in fluid connection with the extruder 200 (see FIGS. 45, 47). The at least a first inlet 204 is operable for introducing a cell solution (i.e., as a first flow) into the extruder 200. The at least a second inlet 208 includes, or is in fluid connection with, a plurality of feeding elements 216 (see FIG. 47) and operable for introducing a hydrogelforming solution (i.e., as a second flow), through the plurality of feeding elements 216, into the extruder 200, in a plurality of directions symmetrically disposed with respect to the cell solution. The housing 212 has at least a third inlet 220 and is configured to supply a cell-compatible buffer (i.e., as a third flow), and optionally one or more additional liquid flows, to the cell solution (the first flow) and the hydrogel -forming solution (the second flow), thereby producing the hydrogel microtube containing a plurality of cells. In some cases, the apparatus described herein and / or extruder 200 can be configured to accommodate more than three flows to prepare a hydrogel microtube having a more complex structure.

[0277] As used herein, an “extruder” is a machine used to shape a material by forcing it through a die to create a continuous profile. An extruder typically works by applying a pressure to a raw material, forming it into specific shapes as they are pushed through a nozzle or mold. The manufacturing process that involves the use of an extruder is accordingly termed “extrusion”.Attorney Docket No. 0073605-001131Similarly, the direction in which an extruder produces a shaped material is accordingly termed an “extrusion axis”. The extruder described herein can have a die of any cross-sectional profde and / or dimension in accordance with the lateral dimension of hydrogel microtube described herein.

[0278] As used herein, a “feeding element” is a structure or mechanism through which a chemical composition is added or supplied.

[0279] As used herein, the term “in fluid connection with” means that two or more objects or components are linked in a way that allows for the flow or transmission of a liquid, gas, and / or pressure therebetween.

[0280] As used herein, a “housing” is a structural element with a hollow interior that defines an internal space. The housing described herein can be constructed in any shape, geometry, and / or design deemed suitable or relevant by a person of ordinary skill in the art, such as without limitation a right or oblique circular cylinder, a right or oblique elliptical cylinder, or a right or oblique polygonal prism having a triangular, square, rectangular, pentagonal hexagonal, or octagonal base, among others. The dimension of the housing can be adjusted in any means to accommodate the rest of the apparatus and the operation thereof. As a nonlimiting example, the length of the housing can be increased or decreased to extend or shorten the time of buffer exchange.

[0281] Elements of the apparatus described herein can be constructed using any material or materials deemed suitable by a person of ordinary skill in the art, in view of the entirety of the present disclosure. Nonlimiting examples of such material or materials include quartz, glass, fused silica, and polymers such as polypropylene (PP), high-density polyethylene (HDPE), polycarbonate (PC), polylactic acid (PLA), polymethyl methacrylate (PMMA), polyimide (PI), polydimethylsiloxane (PDMS), etc.

[0282] Additionally, elements of the apparatus described herein can be constructed using any additive or subtractive manufacturing technique deemed suitable or applicable by a person of ordinary skill in the art, in view of the entirety of the present disclosure. As a nonlimiting example, one or more elements of the apparatus described herein can be constructed using one or more 3D printing techniques, such as without limitation stereolithography (SLA), fused deposition modeling (FDM), selective laser sintering (SLS), digital light processing (DLP), electron beam melting (EBM), and continuous liquid interface production (CLIP), among others.

[0283] In some embodiments, the extruder is configured to supply a core flow of the cell solution in a flow direction within a shell flow of the hydrogel -forming solution that is also passed in the flow direction and wherein the extruder is configured so that a sheath flow of the cell-compatibleAttorney Docket No. 0073605-001131 buffer is passed in the flow direction to surround the shell flow, which surrounds the core flow such that the shell flow is between the sheath flow and the core flow as the sheath flow, shell flow, and core flow pass in the flow direction to form the hydrogel microtube containing the plurality of cells within an inner channel of the hydrogel microtube.

[0284] Each of the cell solution (i.e., the core flow), the hydrogel-forming solution (i.e., the shell flow), and the cell-compatible buffer (i.e., the sheath flow) can be operated any flow rate deemed suitable or relevant by a person of ordinary skill in the art, in view of the entirety of the present disclosure. In some preferred embodiments, the cell solution, the hydrogel-forming solution, and the buffer solution form coaxial laminar flows.

[0285] For example, the apparatus can be configured so that an inner cell solution flow is introduced and passed through a conduit arrangement of the apparatus as an innermost flow. A shell flow can subsequently be introduced to surround this inner cell solution flow as a shell flow, such that the cell solution flow can be a core flow within the shell flow. The shell flow can be passed through in alignment with the core flow so that the shell flow has an annular cross-sectional shape that surrounds the inner core flow. A sheath flow can be introduced for passing along the outer sides of the shell flow in a further annular flow arrangement so that the sheath flow surrounds the sheath flow. This inter-spacing of the different flows all passing in the same flow direction with each other can facilitate formation of a hydrogel microtube having a cell culture within the inner channel of the formed tube structure. The inner channel of the microtube can define the microscope in which cells can be positioned within the protective hydrogel shell of the outer body of the microtube, for example. The body of the microtube can provide a hydrogel shell, such as a collagen hydrogel shell, that can isolate the cells within the inner channel from hydrodynamic stresses to help provide efficient mass transport and provide a suitable substrate for cell adhesion. The inner channel can allow the cells to interact with their chemical environment and with each other to maintain cell viability and a high cell growth rate, thereby providing a high volumetric yield as the cells are grown within the formed hydrogel microtube.

[0286] In addition, the dimension of the hydrogel microtube can be controlled by adjusting the flow rate of one or more of the cell solution, the hydrogel-forming solution, and the cell -compatible buffer. The cell-compatible buffer can not only neutralize the acidic hydrogel-forming solution but also provide a hydrodynamic focusing mechanism to control the diameter of the hydrogel microtube; increasing the flow rate of the buffer solution can reduce the diameter of the hydrogel microtube, and vice versa. The wall thickness of the hydrogel microtube can be adjusted by changing the flow rateAttorney Docket No. 0073605-001131 of the hydrogel-forming solution; a lower flow rate of the hydrogel-forming solution can reduce the wall thickness of the hydrogel microtube, and vice versa.

[0287] In some embodiments, the stream of the hydrogel-forming solution and / or the cellcompatible buffer are divided into two or more evenly distributed sub-flows by splitting the respective inlet channel into several arms.

[0288] In some embodiments, the plurality of feeding elements, and / or the plurality of directions in which the hydrogel-forming solution is introduced, is symmetrically disposed with respect to the at least a first inlet and / or the extrusion axis. In some embodiments, the plurality of feeding elements are evenly spaced from one another. As nonlimiting examples, the plurality of feeding elements, and / or the plurality of directions in which the hydrogel-forming solution is introduced, can include two feeding elements or directions 180° from one another, three feeding elements or directions 120° from one another, four feeding elements or directions 90° from one another, five feeding elements or directions 72° from one another, six feeding elements or directions 60° from one another, eight feeding elements or directions 45° from one another, n feeding elements or directions (360 / n)° from one another, etc. It should be noted that the number of feeding elements or the number of directions in which the hydrogel-forming solution is introduced is not particularly limited, and can include any number deemed suitable or applicable by a person of ordinary skill in the art, in view of the entirety of the present disclosure. The cell-compatible buffer can also be supplied in a similar fashion.

[0289] In some embodiments, the apparatus described herein further includes at least a syringe in fluid connection with the at least a first inlet, the at least a second inlet, and / or the housing, such as without limitation, via tubing or capillaries. In some embodiments, the at least a syringe is in fluid connection with the plurality of feeding elements. In some embodiments, the apparatus described herein further includes a syringe pump, wherein the at least a syringe is in fluid connection with and operated by the syringe pump.

[0290] In some embodiments, the cell solution or the cell-compatible buffer has a pH from about 7 to about 9, preferably about 7.4, in accordance with details described in the present disclosure. It should be noted that the pH of the cell-compatible buffer can be equal to any value(s) within any of these respective ranges, including the endpoints of these ranges.

[0291] In some embodiments, the hydrogel-forming solution has a pH from about 2 to about 6, preferably from about 3 to about 5. It should be noted that the pH of hydrogel -forming solution canAttorney Docket No. 0073605-001131 be equal to any value(s) within any of these respective ranges, including the endpoints of these ranges. Such acidic pH can prevent or suppress premature gelation of the hydrogel-forming solution.

[0292] In some embodiments, the cell-compatible buffer includes one or more multivalent ions, preferably one or more divalent ions. In some embodiments, the one or more divalent ions includes Mg2+, Ca2+, Zn2+, and / or Ba2+, preferably Ca2+, in accordance with details described throughout the present disclosure. These multivalent / divalent ions can trigger a crosslinking reaction of one or more constituents (e.g., alginate polymers) of the hydrogel-forming solution to form a hydrogel microtube.

[0293] In some embodiments, the apparatus described herein further includes a cooling element (e.g., an ice box) configured to keep a temperature of the hydrogel -forming solution to no greater than about 10 °C, preferably no greater than about 4 °C. The low temperature, in addition to the acidic pH, can prevent or suppress premature gelation of the hydrogel-forming solution.

[0294] In some embodiments, the apparatus described herein further includes a heating element (e.g., a heating pad) configured to raise a temperature of the hydrogel microtube to about 37 °C. The rise in temperature from, e.g., no greater than about 4 °C to about 37 °C can trigger the curing of one or more components (e.g., collagen protein) of the hydrogel-forming solution to form a hydrogel microtube. In some embodiments, the apparatus described herein further includes one or more temperature controllers.

[0295] In some embodiments, the apparatus further includes one or more ports 304 each connected to a plurality of the first inlets or a plurality of the second inlets through one or more multifurcations, preferably one or more bifurcations. See FIGS. 51A-B.

[0296] In some embodiments, the apparatus further includes one or more ports 404 each connected to a plurality of the first inlets, a plurality of the second inlets, or a plurality of the third inlets through one or more multifurcations, preferably one or more bifurcations. See FIGS. 51C-D. By incorporating these features, the apparatus described herein can have much higher throughput by producing multiple hydrogel microtubes simultaneously, using a single solution feed. Further, these features can reduce the batch-to-batch variation of hydrogel microtubes, as the hydrogel microtubes are produced in parallel. These embodiments may be referred to as bifurcation-based high- throughput extruders or 1-to-n high-throughput extruders throughout the present disclosure.

[0297] Another aspect of the present disclosure is a system that includes a plurality of the apparatus described herein as an array or matrix. In some embodiments, a plurality of the apparatus described herein can be deployed as a linear array, e.g., with elements forming a substantially straight line. In some embodiments, a plurality of the apparatus described herein can be deployed asAttorney Docket No. 0073605-001131 a 2D array or 2D matrix. As used herein, a “2D array” or “2D matrix” is a geometrical arrangement and / or topology among a plurality of elements within a substantially flat plane (i.e., a plane that is apparently flat to a naked eye). The 2D array or 2D matrix can be deployed in any means or layout deemed suitable or relevant by a person or ordinary skill in the art, in view of the entirety of the present disclosure, such as without limitation as a curved line, zig zag, circle, ellipse, or polygon, or in an oblique, rectangular / centered rectangular, square, or hexagonal lattice.

[0298] In some embodiments, the system further includes at least one syringe having multiple outlets, wherein each outlet of the multiple outlets is in fluid connection with the at least a first inlet, the at least a second inlet, or the housing of at least one apparatus of the plurality of apparatus.

[0299] In some embodiments, the system further includes at least one syringe having multiple outlets simultaneously in fluid connection with the first inlet(s) of each apparatus of the plurality of apparatus. In some embodiments, the system further includes at least one syringe having multiple outlets simultaneously in fluid connection with the second inlet(s) of each apparatus of the plurality of apparatus. In some embodiments, the system further includes at least one syringe having multiple outlets simultaneously in fluid connection with the housing and / or the at least a third inlet of each apparatus of the plurality of apparatus. These features, individually or in combination, can allow for high-throughput manufacturing of microtubes with reduced batch-to-batch variance.

[0300] In some embodiments, the system further includes at least one syringe having a single arm or outlet. In some embodiments, the system further includes at least one syringe having one or more multi furcations, preferably one or more bifurcations. This feature can minimize the number of syringes and syringe pumps required for large-scale extrusion. As nonlimiting examples, each inlet flow to the at least one syringe can be evenly divided into two streams at a first bifurcation level, then into four streams at the second bifurcation level, etc. Additional bifurcation stages can also be incorporated as needed to achieve the desired number of outputs.Method of Preparing a Hydrogel Microtube

[0301] Another objective of the present disclosure is to provide a method of preparing a hydrogel microtube. At step (i), the method includes supplying a core flow of a cell solution though at least a first inlet of an extruder. At step (ii), the method further includes concurrently supplying a shell flow of a hydrogel-forming solution though at least a second inlet of the extruder, wherein the core flow is passed within and surrounded by the shell flow. At step (iii), the method may further include supplying a sheath flow of a cell -compatible buffer to surround the shell flow, such that the shell flow is between the sheath flow and the core flow as the sheath flow, shell flow, and core flowAttorney Docket No. 0073605-001131 pass in a shared flow direction, thereby producing a hydrogel microtube containing a plurality of cells within an inner channel of the hydrogel microtube. These aspects can be implemented in accordance with details described throughout the present disclosure.

[0302] In some embodiments, the hydrogel-forming solution includes one or more of (a) a collagen protein; (b) a blend of an alginate polymer and a collagen protein; and (c) a peptide- functionalized alginate polymer and, optionally, an unfunctionalized alginate polymer, preferably one or more of (a) and (b). These aspects can be implemented in accordance with details described throughout the present disclosure.

[0303] Tn some embodiments, (b) the blend of the alginate polymer and the collagen protein has an alginate polymer-to-collagen protein ratio of 0.01 to 100 by mass, preferably 0.1 to 1.5 by mass, more preferably 0.25 to 0.75 by mass. These aspects can be implemented in accordance with details described throughout the present disclosure.

[0304] In some embodiments, the peptide-functionalized alginate polymer includes an arginine- glycine-aspartic acid (RGD) peptide covalently linked to the alginate polymer, in accordance with details described throughout the present disclosure.

[0305] In some embodiments, the peptide-functionalized alginate polymer includes one or more of a peptide-functionalized alginate acid polymer, a peptide-functionalized sodium alginate polymer, a peptide-functionalized methacrylate-modified alginate polymer, and a peptide-functionalized vinyl sulfone-modified alginate polymer, in accordance with details described throughout the present disclosure.

[0306] In some embodiments, the cell solution includes one or more cells selected from the group consisting of embryonic stem cells; mammalian embryonic stem cells; human embryonic stem cells (hESCs); human induced pluripotent stem cells (iPSCs); mammalian induced pluripotent stem cells; mammalian naive pluripotent stem cells; mammalian tissue stem cells; human pluripotent stem cells (hPSCs); cells derived or differentiated from one or more of embryonic stem cells, mammalian embryonic stem cells, human embryonic stem cells (hESCs), human induced pluripotent stem cells (iPSCs), mammalian induced pluripotent stem cells, mammalian naive pluripotent stem cells, mammalian tissue stem cells, and human pluripotent stem cells (hPSCs); mammalian cells reprogrammed from other cell types; mammalian primary cells; human umbilical vein endothelial cells; primary tumor cells; cancer cells; immune cells; T cells; natural killer cells; mammalian cell lines; engineered human and mammalian cells; insect cells; plant cells; and yeast and bacterial cells. The cells described herein can include any type of cells or cell combinations in accordance withAttomey Docket No. 0073605-001131 details described throughout the present disclosure. Cells can be cultured as single cell type or as a mixture of different cell types or a mixture of cells from different donors.

[0307] In some embodiments, the shell flow of the hydrogel-forming solution is applied (e.g., via a plurality of feeding elements) in a plurality of directions symmetrically disposed with respect to the core flow of the cell solution, in accordance with details described throughout the present disclosure.

[0308] In some embodiments, the sheath flow of cell-compatible buffer is applied (e.g., via a plurality of feeding elements) in a plurality of directions symmetrically disposed with respect to the core flow of the cell solution and / or the shell flow of the hydrogel -forming solution, in accordance with details described throughout the present disclosure.

[0309] In some embodiments, the cell solution or the cell-compatible buffer has a pH from about 7 to about 9, preferably about 7.4, in accordance with details described throughout the present disclosure.

[0310] In some embodiments, the hydrogel-forming solution has a pH from about 2 to about 6, preferably from about 3 to about 5, in accordance with details described throughout the present disclosure.

[0311] In some embodiments, the cell-compatible buffer includes one or more multivalent ions, preferably one or more divalent ions, in accordance with details described throughout the present disclosure.

[0312] In some embodiments, the one or more divalent ions includes Mg2+, Ca2+, Zn2+, or Ba2+, preferably Ca2+, in accordance with details described throughout the present disclosure.

[0313] In some embodiments, the hydrogel-forming solution is maintained at a temperature of no greater than 10 °C, preferably no greater than 4 °C, using a cooling element, in accordance with details described throughout the present disclosure.

[0314] In some embodiments, the method further includes, after step (iii), raising a temperature of the hydrogel microtube to about 37 °C using a heating element, in accordance with details described throughout the present disclosure.

[0315] In some embodiments, the method further includes, after step (iii), coating the hydrogel microtube using a coating solution, in accordance with details described throughout the present disclosure.

[0316] In some embodiments, the coating solution includes one or more of an alginate acid polymer, a sodium alginate polymer, a methacrylate-modified alginate polymer, and a vinyl-sulfone-Attorney Docket No. 0073605-001131 modified alginate, wherein the vinyl sulfone-modified alginate polymer is optionally peptide- functionalized, in accordance with details described throughout the present disclosure.

[0317] Another objective of the present disclosure is to provide a method of coating a hydrogel microtube. At step (a), the method includes preparing the hydrogel microtube according the method described herein. At step (b), the method includes dipping the hydrogel microtube in a coating solution for a time period of at least 1 second and no greater than 10 hours, preferably at least 1 minute and no greater than 30 minutes, more preferably at least 3 minutes and no greater than 15 minutes, in accordance with details described throughout the present disclosure. The coating solution includes at least 0.1% by weight and no greater than 30% by weight, preferably at least 0.5% by weight and no greater than 1.5% by weight, of an alginate polymer. The alginate polymer includes one or more of an alginate acid polymer, a sodium alginate polymer, a methacrylate-modified alginate polymer, and a vinyl-sulfone-modified alginate, wherein the vinyl sulfone-modified alginate polymer is optionally peptide-functionalized. At step (c), the method further includes dipping the hydrogel microtube in a buffer containing or more divalent ions selected from Mg2 1, Ca2 1, Zn21, and Ba2+, preferably Ca2+, in accordance with details described throughout the present disclosure.EXAMPLESExample 1 : Collagen Hydrogel microtube Microbioreactors for Cell and Tissue Manufacturing

[0318] The large-scale production of mammalian cells, particularly cells / stem cells for clinical applications, remains challenging with existing cell culture technologies. Current methods have issues such as excessive cell aggregation and significant shear stress-induced cell death, resulting in low cell yield, unacceptable batch-to-batch variation, high production costs, and difficulties in scaling up. We hypothesize that creating a cell-friendly microenvironment can significantly enhance cell culture efficiency. In this study, we developed a novel hydrogel microtube microbioreactor using collagen proteins (ColTubes) to test this hypothesis. First, we designed and fabricated an innovative micro-extruder to enable the large-scale production of ColTubes loaded with cells. Our results show that collagen proteins form a dense and robust nanofiber network capable of shielding cells from hydrodynamic stress while maintaining cell mass below 400 pm in diameter. The microtube shell contains abundant nanopores that allow the cell culture medium to permeate and nourish the cells. Additionally, the collagen proteins serve as a substrate for cell adhesion. This configuration ensures efficient mass transport and fosters a favorable microenvironment for cell growth. We show that ColTubes support high cell viability, rapid expansion, and impressiveAttorney Docket No. 0073605-001131 volumetric yields (5x l08cells / mL), offering substantial improvements over current methods. Furthermore, we demonstrate that ColTubes can be used to construct tubular tissue models. To our knowledge, ColTubes is a novel approach that has not been previously reported for cell manufacturing. ColTubes represents a scalable, cost-effective, and efficient solution for large-scale cell production.Introduction

[0319] Mammalian cells have diverse applications. Stem cells, such as human pluripotent stem cells (hPSCs), can generate various tissue cells for regenerative medicine, disease modeling, drug screening, and toxicity testing1. Immune cells, such as T cells and natural killer cells, are used to treat cancers2'6. Mammalian cells are also widely utilized for producing recombinant proteins, viruses, and extracellular vesicles for research and therapy7'9. All these applications require large numbers of cells1. For example, approximately 109cardiomyocytes or 1090 cells are needed to treat a patient with myocardial infarction or Type 1 diabetes10. Engineering a human liver or heart requires approximately IO10hepatocytes or cardiomyocytes11. Additionally, screening a one-millioncompound library would demand IO10cells10. Given the large patient population, the demand for cells is enormous.

[0320] However, large-scale cell production, especially for clinical purposes, is still very challenging1 10 12. In vivo, human cells live in a three-dimensional (3D) microenvironment that supports interactions between cells and the extracellular matrix (ECM), provides robust nutrient and oxygen supply, and minimizes hydrodynamic stress13'17. Current cell culture methods, however, often fail to replicate these conditions, resulting in low cell culture efficiencies and difficulties in scaling production. Two-dimensional (2D) cell culture systems, such as flasks, lack the complexity of in vivo 3D microenvironment. They provide a stiff substrate and can only produce a limited number of cells per culture area. They are not unsuitable for large-scale cell production1 10 12.

[0321] 3D suspension culture technologies, such as stirred-tank bioreactors, have been widely studied for scaling up cell production. However, they face challenges related to uncontrolled cell aggregation, particularly for stem cells with strong adhesion tendencies16,17. In such cultures, cells form large aggregates. It is known that the transport of nutrients, oxygen, and growth factors to cells at the core of aggregates larger than 400 pm becomes difficult, leading to slower proliferation, apoptosis, and undesired differentiation10,18. Although agitation can reduce aggregation and improve mass transport, it can also introduce shear forces that negatively impact cell survival and growth10,19,20. Consequently, 3D suspension cultures often exhibit high cell death, slow growth rates,Attorney Docket No. 0073605-001131 and low volumetric yields. For example, hPSCs typically expand less than 10-fold per passage to yield approximately 2.0>< 106cells / mL. The cell mass occupies just 0.4% of the culture volume21'23.

[0322] The complex hydrodynamic conditions in stirred-tank bioreactors-such as flow rate, shear stress, and chemical gradients-add further complications. These variables depend on multiple factors, including the bioreactor design, medium viscosity, and agitation speed, making precise control difficult1,10’12’19'24These factors lead to production variations. For instance, in recent studies to produce cardiomyocytes from hPSCs in stirred-tank bioreactors, the yields from three 100 mb batches varied from 40 to 100 million cells, with cardiomyocyte purity between 54% and 84%. Using a different hPSC line under the same conditions, the yields changed between 89 and 125 million cells, with purity varied from 28% to 88%25,26. The hydrodynamic complexity also complicates scaling up. Culture conditions optimized at a small scale may not translate effectively to larger scales, requiring extensive re-optimization, which is time-consuming and costly. To our knowledge, the largest demonstrated scale for hPSC-derived cardiomyocytes using stirred-tank bioreactors is approximately 3 * 109cells per batch, sufficient to treat only three patients27.

[0323] To address the scale-up challenge, we propose developing hydrogel microtube-based 3D microbioreactors. Previously, we demonstrated that growing cells in hollow hydrogel microtubes or microbioreactors made from alginate polymers (AlgTubes) prevents excessive cell aggregation, ensures efficient mass transport, and eliminates hydrodynamic stress. This approach resulted in high cell viability, rapid proliferation, and impressive volumetric yields. For example, we achieved up to 5* 108hPSCs per milliliter — approximately 250 times more than what is achieved in stirred-tank bioreactors28'37. These results highlight the transformative potential of hydrogel microtube microbioreactors for stem cell culture. However, AlgTubes do not support the growth of anchordependent stem cells, as they lack adhesion points. Additionally, AlgTubes break frequently, leading to cell culture inconsistencies. Furthermore, large proteins, virus, and extracellular vesicles produced by cells are often unable to effectively diffuse through the alginate hydrogel; therefore, it can be challenging to continuously produce these products using cells in AlgTubes.

[0324] Given the diverse phenotypes of mammalian cells, it is crucial to have multiple material options for fabricating hydrogel microtube microbioreactors. In this study, we developed a new type of hydrogel microtube-based microbioreactor using collagen proteins (ColTubes). These collagen hydrogel microtubes exhibit high cell culture efficiency comparable to alginate hydrogel microtubes. Additionally, they allow cells to adhere to the inner surface and they are much more resistant toAttorney Docket No. 0073605-001131 break. We believe collagen and alginate hydrogel microtube microbioreactors, together, will enable scalable, cost-effective, and efficient cell production for a broad range of applications.Methods

[0325] Collagen Extraction. Rat tails were soaked in 70% ethanol to remove debris, and the skin was stripped away using a scalpel and forceps. Tendons were then collected and washed three times with PBS. Subsequently, they were sterilized in 70% ethanol for 1 hour. Tendons were dissolved in 0.02 N acetic acid with continuous stirring at 4 °C for 48 hours. The resulting solution was centrifuged at 10,000 rpm for 60 minutes at 4 °C. The supernatant was dialyzed against 0.02 N acetic acid and collected as the collagen stock solution. Although we use rat tail collagen to demonstrate the preparation of ColAlgTubes in this study, it should be noted that the ColAlgTubes can be fabricated using collagens from various species and tissues, such as rat tails, bovine, and human skin, in accordance with details described throughout the present disclosure.

[0326] Fabrication of Collagen microtubes. A custom micro-extruder was designed using Fusion 360 (Autodesk) and fabricated using a stereolithography 3D printer (Form 3B+, Formlabs). The three inlets were connected to syringes mounted on syringe pumps (Fusion 200, Chemyx). Syringe 1, containing cells suspended in 1.5% methylcellulose (MC), was connected to the core flow channel of the micro-extruder and pumped at 30 pL / min. Syringe 2, loaded with collagen solution, was placed in a custom ice box, connected to the shell flow channel, and pumped at 180 pL / min. Syringe 3 containing 50 mM HEPES buffer was attached to the sheath flow channel and pumped at 2 mL / min. The extruder outlet was submerged in a 37 °C, 50 mM HEPES buffer maintained with a heating pad. Once the pumps were on, collagen microtubes were continuously generated.

[0327] Confocal Microscopy. To prepare bulk collagen hydrogel, the collagen solution was neutralized by adding a 10x neutralization buffer and incubated at 37 °C for 15 minutes. ATTO 488 NHS ester (Sigma) was used to label collagen proteins following the manufacturer's guidelines. Briefly, Bulk collagen and ColTubes were reacted with one pM ATTO 488 NHS ester in PBS for 15 minutes at room temperature, followed by three washes with PBS to remove excess dye. Confocal images were done using an Olympus FV3000 confocal microscope and a 60* objective.

[0328] Scanning Electron Microscope (SEM). Bulk collagen hydrogel and ColTubes were dehydrated through an ethanol series (25%, 50%, 70%, 85%, 95%, and 100%) with 5 minutes at each concentration, followed by critical point drying using a Leica EM CPD300 Critical Point Dryer. Samples were then sputter-coated with a 4.5 nm iridium layer using a Leica EM ACE600 Sputter Coater. SEM images were acquired with a Zeiss SIGMA VP-FESEM SEM.Attorney Docket No. 0073605-001131

[0329] Doping Collagen microtubes with Laminins. Recombinant Laminin 511 E8 fragments (iMatrix-511 SILK) were labeled with ATTO 594 NHS ester (Sigma) following the product instructions. The collagen solution was mixed with labelled laminins and processed into ColTubes to dope collagen microtubes with laminins.

[0330] Culturing hPSCs in ColTubes. For a typical cell culture, cells in 20 pL ColTubes were suspended in 2 mL E8 medium supplemented with 10 pM Y -27632 in a 6-well plate and incubated at 37 °C with 5% CO2 and 21% O2. The medium was changed daily. To passage cells, the medium was removed, and ColTubes were dissolved by incubating with 0.2 mg / mL Collagenase P for 15 minutes. The cell mass was collected, treated with Accutase at 37 °C for 10 minutes, and dissociated into single cells for subsequent culture.

[0331] Flow Cytometry and Live / Dead Cell Staining. Single cells were fixed with 4% paraformaldehyde (PF A) at room temperature for 15 minutes, followed by incubation with PBS containing 0.1% Triton X-100, 0.5% BSA, and primary antibodies at 4 °C overnight. After thorough washing, secondary antibodies were added and incubated for 2 hours at room temperature. Cells were washed three times with PBS containing 0.5% BSA before analysis using the Attune® NxT™ Acoustic Focusing Cytometer. Data analysis was conducted using FlowJo software. LIVE / DEAD® Cell Viability staining was performed according to the manufacturer's instructions.

[0332] Fabricating Seminiferous Tubules. TM3 (Leydig) and TM4 (Sertoli) cells were cultured according to ATCC instructions. Briefly, cells were maintained in DMEM / F12 supplemented with 5% horse serum and 2.5% fetal bovine serum (FBS) at 37 °C and 5% CO2 with media changes every 2-3 days. TM3 cells were labeled with CellTrace™ CFSE, and TM4 cells were labeled with CellTrace™ Far Red (Fisher) following product instructions. TM4 and TM3 cells were mixed into the core and shell flow to process ColTubes.

[0333] Statistical Analysis. Data was analyzed using GraphPad Prism 8 statistical software and shown as mean ± standard error of the mean. P value was determined by one-way analysis of variance (ANOVA) for comparison between three or more groups or unpaired two-tailed t-tests for two groups. The significance levels are indicated by p-value, *: p<0.05, **: p<0.01, ***: p<0.001. Results

[0334] The Extrusion System for Processing Collagen Hydrogel microtubes (ColTubes).

[0335] A novel micro-extruder with three inlets and one outlet was designed for processing ColTubes (FIG. 1 A). The extruder was fabricated using a Formula 3B printer and clear resin. A cooling box was also designed and fabricated using 3D printing (FIG. IB). The ColTube processingAttorney Docket No. 0073605-001131 setup consists of three syringe pumps, the micro-extruder, the cooling box, a heating pad, and a conical microtube or container containing HEPES buffer (FIG. 1C). The three syringes contain the following solutions: (1) a cell solution at room temperature (RT, pH = 7.4), (2) an ice-cold collagen solution (pH = 3.0), and (3) a HEPES buffer (RT, pH = 7.4). The cooling box has a channel for holding syringe 2 (FIG. IB). Ice is loaded into the box to maintain the collagen solution in syringe 2 at a temperature below 4 °C. The collagen is dissolved in 0.02 N acetic acid to have a pH of 3.0, which, along with the low temperature, prevents premature collagen gelation.

[0336] To fabricate ColTubes, the three solutions are pumped into inlets 1, 2, and 3 of the micro-extruder, respectively, forming coaxial core-shell -sheath laminar flows that are extruded into the heated HEPES buffer (37 °C, pH = 7.4) (FIG. ID). The core, shell, and sheath flow contain the cells, collagen proteins, and HEPES buffer, respectively. The collagen solution is neutralized by the core solution and the HEPES buffer in both the sheath flow and the conical microtube. Additionally, the collagen solution is rapidly heated by the HEPES buffer in the conical microtube, forming a stable collagen microtube. PBS buffer or other cell-compatible buffers can be used in addition to, or instead of, the HEPES buffer.

[0337] In summary, our innovative micro-extruder design, combined with the cooling box and heating pad setup, enables the formation of stable core-shell-sheath coaxial laminar flows while achieving rapid pH and temperature changes in the collagen flow. These allow the rapid formation of microscale ColTubes for cell culture. To the best of our knowledge, there are no existing scalable technologies that can rapidly process microscale collagen hydrogel microtubes without compromising cell viability.

[0338] Engineering Principles and Control of ColTube Dimensions.

[0339] In our system, cells are cultured within ColTubes suspended in a culture medium. ColTubes are designed to overcome the limitations of current cell culture technologies to provide a physiologically relevant microenvironment for cells (FIG. 2A). The microtube shell is highly porous, allowing the medium to enter the microtube to feed cells. The microtube's inner diameter is less than 400 pm, so even if the whole microtube is filled with cells, the cell mass still has a radial diameter of less than 400 pm, less than the diffusion limit in human tissue — approximately 500 pm. Our design can ensure efficient nutrient and waste transport throughout the culture period. An inner diameter greater than 400 pm may be suitable for cell types with large diffusion limits or for applications where optimal mass transport is not critical. The hydrogel microtube also protects cells from hydrodynamic stresses within the culture vessel. The microspace created by the microtube can beAttorney Docket No. 0073605-001131 configured for achieving high cell viability, growth rate, and volumetric yield. Lastly, the collagen proteins can serve as a substrate for cell adhesion, which can be needed for growing anchordependent cells. Additionally, other extracellular matrix (ECM) proteins can be incorporated into the collagen matrix, as collagen contains binding domains for many ECM proteins. This feature can provide a highly physiologically relevant microenvironment for culturing certain stem cells that are otherwise difficult to grow in vitro due to the absence of in vzvo-like niches.

[0340] The ColTube dimension can be controlled by adjusting the core, shell, and sheath stream flow rates. The HEPES sheath flow not only neutralizes the acidic collagen shell solution but also acts as a hydrodynamic focusing mechanism to control the ColTube diameter. Increasing the sheath flow rate reduces the diameter. The shell thickness can be adjusted by changing the collagen shell flow rate-a lower shell flow rate results in a thinner microtube wall. FIGS. 2B and 2C show ColTubes with varied diameters and shell thicknesses.

[0341] Nanostructures of ColTubes.

[0342] ColTubes were labeled with ATTO 488 NHS ester and imaged using a confocal microscope (FIG. 3A). Bulk collagen hydrogels were also prepared and imaged for comparison (FIG. 3B). Collagen proteins formed a dense nanofiber network in ColTubes, similar to these in the bulk hydrogel, indicating the gelation mechanism in ColTubes and Bulk hydrogel were similar. The nanostructures of the outer surface and the microtube shell were similar, while some loose collagen nanofibers protruded inward at the inner surface. The collagen concentration in the tested range (3-8 mg / mL) had minimal influence on the nanofiber diameter, length, orientation, and the nanofiber network's porosity and pore size.

[0343] We used scanning electron microscopy (SEM) to investigate the detailed nanostructures. ColTubes were fixed with paraformaldehyde and dehydrated through an ethanol series, followed by metal sputtering and SEM imaging. The whole microtube, inner surface, outer surface, and shell were imaged (FIG. 4A). Bulk collagen hydrogels were used for comparison (FIG. 4B). Collagen nanofibers formed a dense network within the ColTube shell. Nanofibers were less densely packed at the inner and outer surfaces, with some protruding inward at the inner surface, consistent with confocal microscopy observations. No notable differences in nanostructures were observed between ColTubes and Bulk collagen hydrogels. Moreover, the collagen concentration had a minor impact on the hydrogel structures.

[0344] Although most mammalian cells can adhere to collagen proteins, certain stem cells require specific extracellular matrix (ECM) proteins for adhesion. For example, hPSCs are oftenAttorney Docket No. 0073605-001131 cultured on plates coated with laminins. To test whether ECM proteins can be incorporated and maintained in ColTubes, we labeled recombinant Laminin 511 protein with Alexa Fluor 594 NHS Ester (emitting red fluorescence). We mixed it with collagen to process ColTubes. The microtubes were soaked in PBS for three days to wash away soluble laminins. The microtubes were subsequently fixed with 4% PFA and labeled with Alexa Fluor™ 488 NHS Ester to visualize collagen and laminin in green fluorescence. Images showed that laminins were successfully incorporated and remained in ColTubes (FIG. 5A). Control ColTubes without laminins did not exhibit any red fluorescence signal (FIG. 5B). SEM analysis found that laminin doping did not alter ColTube nanostructures (FIG. 5C, D).

[0345] Culturing Cells in ColTubes.

[0346] HEK293 cells are widely utilized for producing protein and viral therapeutics. The original adherent HEK293 cells have also been adapted to suspension culture. We tested the suitability of ColTubes for culturing adherent (FIG. 6A) and suspension (FIG. 6B) HEK293 cells. After 24 hours, both cells adhered to the inner surface and expanded to form colonies attached to the microtubes. Over time, cells proliferated and filled the entire microtube. Live / Dead cell staining showed that most cells remained viable, and we could harvest >3 * 108cells from one milliliter of microspaces.

[0347] hPSCs, including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs), are ideal starting cells to prepare various human cells for treating diseases due to their unlimited proliferation capability and ability to differentiate into all human cell types. We cultured H9 hESCs in ColTubes in ColTubes. Single cells adhered to the inner surface within 24 hours. By day 3, cells formed small colonies attached to the microtube's inner surface, which grew into spheroids by day 5. By day 7, cells filled most of the microtubes (FIG. 7A). Live / Dead staining of cells before and after release from ColTubes showed minimal cell death (FIG. 7B). Flow cytometry confirmed that 99.6% of cells were viable (FIG. 7C). 97.9% and 96.1% of the cells expressed pluripotency markers, Nanog and Oct4, respectively, indicating that cells retained their pluripotency after culturing in ColTubes (FIG. 7D). Using ColTubes, we achieved over 4.5 * 108cells per milliliter of microspaces yield, which is approximately two orders of magnitude higher than what was achieved with stirred-tank bioreactors in our previous studies1,21,28.

[0348] We tested whether hPSCs could be differentiated into functional cells in ColTubes. H9 hESCs carrying a GFP reporter under the cTnT promoter were expanded in ColTubes in the E8 medium for 7 days. Without cell passaging, the expansion medium was switched to a mesodermAttorney Docket No. 0073605-001131 induction medium to differentiate hESCs into mesoderm progenitors. On day 2, the medium was changed to cardiac progenitor differentiation medium, followed by cardiomyocyte differentiation medium on day 7. Between days 11 and 18, cells were cultured in a metabolic enrichment medium (FIG. 8A). Most cells remained viable, and over 90% of the final cells were cTnT+ cardiomyocytes (FIG. 8B). We produced over 3>< 108cells per milliliter of hydrogel microtubes.

[0349] ColTubes Exhibit Fewer Cell Leakage Events Compared to AlgTubes.

[0350] Previously, we used alginate-based hydrogel microtubes (AlgTubes) for cell culture. However, AlgTubes break occasionally, leading to cell leakage and culture variation. As shown in FIG. 9B, cell leakage was observed using AlgTubes. Leaked cells formed large aggregates. In contrast, no cell leakage was observed in ColTubes (FIG. 9A). Quantification of leakage events (FIG. 9C) and the percentage of cells leaked into the medium on day 7 (FIG. 9D) confirmed that ColTubes exhibited no leakage events, whereas AlgTubes had.

[0351] Building Tubular Tissue Models with ColTubes.

[0352] In addition to cell production, ColTubes can be used to fabricate tissues for therapeutic applications and disease modeling. Human sperm is produced in seminiferous tubules within the testis, where Sertoli cells form a monolayer lining the inner surface. Leydig cells and other minor cell types reside in the surrounding interstitial tissue. To mimic this structure, we mixed Leydig cells with collagen to form the shell flow and suspended Sertoli cells in the core flow. The resulting ColTubes successfully contained Leydig cells within the microtube shell and Sertoli cells on the inner surface, resembling the in vivo seminiferous tubules (FIG. 10).

[0353] In summary, our results show that ColTube is a versatile platform for cell expansion, differentiation, and tissue modeling. ColTubes exhibit excellent mechanical properties, preventing cell leakage and ensuring culture integrity. Moreover, ColTubes allow cells to adhere, making ColTubes suitable for culturing both adherent and suspension cells.Discussion

[0354] Current 2D and 3D cell culture methods face significant challenges in achieving robust and cost-effective large-scale cell production, particularly for stem cells used in clinical applications38'40. Key issues include low cell yields, limited scalability, high costs, and significant culture variability. The fundamental problem is that these methods provide a cellular microenvironment that differs substantially from the natural in vivo 3D microenvironment. We hypothesize that replicating a biomimetic microenvironment can address these limitations and substantially improve cell culture efficiency28’31.Attorney Docket No. 0073605-001131

[0355] We also proposed that hydrogel microtube microbioreactors are a promising approach for creating a cell-friendly microenvironment28’31. To evaluate this approach, we previously developed a method to fabricate hydrogel microtubes using alginate polymers (AlgTubes)28'37. Alginates are widely available, inexpensive, biocompatible, and have been safely used in clinical applications. These polymers can be rapidly crosslinked with calcium ions to produce large quantities of AlgTubes through an extrusion process. After culture, AlgTubes can be dissolved with an EDTA solution to harvest the product. The microtubes are also transparent, facilitating real-time observation of cell growth28'37.

[0356] Using AlgTubes, we successfully cultured hPSCs over multiple passages with high consistency while maintaining pluripotency and chromosomal stability28. The cultures showed excellent viability, expansion, and yields (~5 x 108cells / mL of microspace), surpassing stirred-tank bioreactors by approximately 250-fold. Up to 4200-fold expansion per passage was achieved, far exceeding conventional 3D suspension cultures, which typically is less than 10-fold per passage. Additionally, hPSCs could be differentiated into various tissue cells, such as endothelial cells29-32, vascular smooth muscle cells35, neural stem cells30, and neurons33in AlgTubes, all achieving yields of 5* 108cells / mL. Adult cell types such as T cells could also be cultured in AlgTubes34. These results highlight the transformative potential of AlgTubes for high-efficiency, large-scale cell production.

[0357] However, given the diverse phenotypes of mammalian cells and their varying requirements for growth environments and substrates, AlgTubes alone is insufficient to support all cell types. There is a need to fabricate hydrogel microtubes using alternative materials. Most mammalian cells do not express receptors for alginates and therefore do not adhere to AlgTubes. The survival and proliferation of many cell types, such as mesenchymal stem cells and endothelial cells, require adhesion to a substrate. AlgTubes are only suitable for culturing anchor-independent cells, such as hPSCs and T cells. Additionally, AlgTubes occasionally break, leading to cell leakage and culture variability (FIG. 9). Furthermore, some cell culture media contain chelators that can extract Ca2+from the alginate hydrogel, causing AlgTubes to dissolve.

[0358] In this study, we successfully developed a second type of hydrogel microtube using collagen proteins. Current technologies are unable to efficiently process collagen proteins into stable microtubes. Previous research has shown that collagen solutions at pH 3.0 can form hydrogels when extruded into a buffer at pH 7.441. However, fabricating stable microtubes is significantly more complex than producing solid hydrogels and has not been achieved previously. By integrating aAttorney Docket No. 0073605-001131 cooling box, a three-flow micro-extruder, a heating pad, and a buffer exchange system (FIG. 1), we successfully developed a rapid method to produce ColTubes.

[0359] The resulting ColTubes had a dense nanofiber network that was stable in most cell culture media. They exhibited remarkable durability, with no breakages occurring during cell culture, thereby preventing cell leakage and ensuring consistent production (FIG. 9A). Furthermore, most cells express receptors for collagen, allowing them to adhere naturally (FIGS. 6, 7). Additionally, collagen contains binding domains for other ECM proteins, such as fibronectin and laminin. These proteins can be incorporated into ColTubes to support the growth of specialized cell types (FIG. 5). Our results showed that cells grew in ColTubes (FIGS. 6-8) as efficiently as they did in AlgTubes28'37.

[0360] Beyond cell culture, we also showed that ColTubes could be used to construct tissue models. The human body is rich in microscale tubular tissues, such as blood capillaries, lymphatic vessels, seminiferous tubules, and milk ducts. Many cancers, including various carcinomas, arise from epithelial transformations in tubular structures. In these tissues, endothelial or epithelial cells line the inner surfaces of the microtubes, while stromal cells form the shells and the interstitial regions between microtubes. ColTubes offer a platform for creating analogous tubular tissue models by placing endothelial or epithelial cells on the inner surfaces and stromal or interstitial cells within the microtube shell (FIG. 10). These engineered tissues have broad applications, including disease modeling, drug screening, and regenerative medicine.

[0361] ColTubes is a valuable tool for academic laboratories and biotechnology companies.ColTubes will enable labs to perform experiments that need large numbers of cells. For instance, 3D printing a human heart would need 1 x 1010cells, which requires a ~10 L stirred tank bioreactor to produce — a task that is difficult to complete in most research labs. The same number of cardiomyocytes can be made with 20 mL ColTubes, which can be readily done in a research lab.

[0362] ColTubes also have the potential to accelerate translational research. Taking the development of hPSC-derived cardiomyocytes for myocardial infarction treatment as an example42, early-stage research typically uses 2D flasks to generate cells for testing efficacy and safety in rodents43. Large-scale 3D suspension culture is then required to produce sufficient cells for large- animal studies and clinical trials44,45. Developing such a 3D bioprocess requires significant time and investment46. ColTubes offer a scalable platform suitable for all stages of the drug development pipeline. For instance, 2 mL of ColTubes can produce 109cells for small-animal studies, while 200 mL can yield 1011cells for large-animal studies and clinical trials. Further scaling up for industrial-Attorney Docket No. 0073605-001131 scale production can be readily achieved. By eliminating the need for multiple bioprocess transitions, ColTubes can significantly reduce the time, effort, and costs associated with developing cell-based therapeutics.

[0363] The high growth rate and cell density of ColTubes have a significant impact on industrial-scale cell production. For example, producing 1012hPSC-derived cardiomyocytes would require approximately 1,000 L of culture volume using stirred-tank bioreactors, based on cell density data from our lab and the literature1,21,28. In contrast, the same production can be achieved with just 2 liters of ColTubes. This dramatic reduction in culture volume not only makes large-scale production feasible and practical but also leads to substantial savings in labor, reagents, equipment, cGMP facility space, and overall manufacturing costs.

[0364] In conclusion, ColTubes offers a unique combination of physiologically relevant culture microenvironment, exceptional performance, and scalability, making them a promising solution for addressing the challenges of large-scale cell manufacturing. Recent advancements in biology have provided efficient protocols for differentiating hPSCs into various human cell types1,47. Future research should focus on integrating these protocols with ColTubes to enable the production of diverse human cell types at a low cost. By addressing the challenges of scalability, consistency, and cost, ColTubes have the potential to revolutionize cell culture across research, translational, and industrial applications.References cited in Example 1

[0365] 1 Lei Y, Schaffer D V. A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation. PNAS. 2013; 110: E5039-48.

[0366] 2. Chang Y, Cai X, Syahirah R, et al. CAR-neutrophil mediated delivery of tumormicroenvironment responsive nanodrugs for glioblastoma chemo-immunotherapy. Nat. Comimm. 2023; 14: 2266.

[0367] 3. Wang S, Yang Y, Ma P, et al. Comment CAR-macrophage : An extensive immune enhancer to fight cancer. eBioMedicine [Internet], 2022; 76: 103873.

[0368] 4. Villanueva MT. Macrophages get a CAR. Nat. Rev. Drug Discov. 2020; 20: 300.

[0369] 5. Vivier E, Rebuffet L, Nami-mancinelli E, Comen S, Igarashi RY, Fantin VR. Natural killer cell therapies. Nature. 2024; 626: 727.

[0370] 6. Cappell KM, Kochenderfer JN. Long-term outcomes following CAR T cell therapy : what we know so far. Nat. Rev. Clin. Oncol. 2023; 20: 359-71.Attorney Docket No. 0073605-001131

[0371] 7 Milone MC, Doherty UO. Clinical use of lentiviral vectors. Leukemia [Internet],2018; 32: 1529-41.

[0372] 8. Wang J, Gessler DJ. Adeno-associated virus as a delivery vector for gene therapy of human diseases. Sig. Trcmsduct. Target. Ther. 2024; 9: 78.

[0373] 9. Esmaeili H, Ghaleh G, Bolandian M, Dorostkar R, Jafari A, Farzaneh M. Biomedicine& Pharmacotherapy Concise review on optimized methods in production and transduction of lentiviral vectors in order to facilitate immunotherapy and gene therapy. Biomed. Pharmacother.[Internet], 2020; 128: 110276.

[0374] 10. Kropp C, Massai D, Zweigerdt R. Progress and challenges in large-scale expansion of human pluripotent stem cells. Process Biochem. 2016; 10842: 1-11.

[0375] 11. Badylak SF, Taylor D, Uygun K. Whole-organ tissue engineering: decellul arization and recellularization of three-dimensional matrix scaffolds. Annu. Rev. Biomed. Eng. 2011; 13: 27- 53.

[0376] 12. Jenkins MJ, Farid SS. Human pluripotent stem cell-derived products: Advances towards robust, scalable and cost-effective manufacturing strategies. Biotechnol. J. 2015; 10: 83-95.

[0377] 13. Thomson JA, Itskovitz-eldor J, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts. Science. 1998; 282: 1145-7.

[0378] 14. Wong CC, Loewke KE, Bossert NL, et al. Non-invasive imaging of human embryos before embryonic genome activation predicts development to the blastocyst stage. Nat. Biotechnol. 2010; 28: 1115-21.

[0379] 15. Kraehenbuehl TP, Langer R, Ferreira LS. Three-dimensional biomaterials for the study of human pluripotent stem cells. Nat. Methods. 2011; 8: 731-6.

[0380] 16. Chen KG, Mallon BS, Johnson KR, Hamilton RS, Mckay RDG, Robey PG.Developmental insights from early mammalian embryos and core signaling pathways that influence human pluripotent cell growth and differentiation. Stem Cell Res. 2014; 12: 610-21.

[0381] 17. Chen KG, Mallon BS, McKay RDG, Robey PG. Human pluripotent stem cell culture: considerations for maintenance, expansion, and therapeutics. Cell Stem Cell. 2014; 14: 13- 26.

[0382] 18. Hajdu Z, Mironov V, Mehesz AN, Norris RA, Markwald RR, Visconti RP. Tissue spheroid fusion-based in vitro screening assays for analysis of tissue maturation. J. Tissue. Eng. Regen. Med. 2010; 4: 659-64.Attorney Docket No. 0073605-001131

[0383] 19. Kinney MA, Sargent CY, Mcdevitt TC. The multiparametric effects of hydrodynamic environments on stem cell culture. Tissue Eng. Part B. 2011; 17: 249-62.

[0384] 20. Fridley KM, Kinney MA, McDevitt TC. Hydrodynamic modulation of pluripotent stem cells. Stem Cell Res. Ther. 2012; 3: 45.

[0385] 21. Lei Y, Jeong D, Xiao J, Schaffer D V. Developing defined and scalable 3D culture systems for culturing human pluripotent stem cells at high densities. Cell. Mol. Bioeng. 2014; 7: 172-183.

[0386] 22. Steiner D, Khaner H, Cohen M, et al. Derivation, propagation and controlled differentiation of human embryonic stem cells in suspension. Nat. Biotechnol. 2010; 28: 361-4.

[0387] 23. Serra M, Brito C, Correia C, Alves PM. Process engineering of human pluripotent stem cells for clinical application. Trends Biotechnol. 2012; 30: 350-8.

[0388] 24. Ismadi M, Gupta P, Fouras A, Verma P, Jadhav S. Flow characterization of a spinner flask for induced pluripotent stem cell culture application. PLoS One. 2014; 9: el06493.

[0389] 25. Jara-avaca M, Kempf H, Olmer R, et al. Controlling expansion and cardiomyogenic differentiation of human pluripotent stem cells in scalable suspension culture. Stem Cell Rep. 2014; 3: 1132-46.

[0390] 26. Chen VC, Ye J, Shukla P, et al. Development of a scalable suspension culture for cardiac differentiation from human pluripotent stem cells. Stem Cell Res. 2015; 15: 365-75.

[0391] 27. Raniga K, Nasir A, Vo NTN, et al. Strengthening cardiac therapy pipelines using human pluripotent stem cell-derived cardiomyocytes. Cell Stem Cell [Internet], 2024; 31 : 292-311.

[0392] 28. Li Q, Lin H, Du Q, et al. Scalable and physiologically relevant microenvironments for human pluripotent stem cell expansion and differentiation. Biofabrication. 2018; 10: 025006.

[0393] 29. Wang Z, Zuo F, Liu Q, et al. Comparative Study of Human Pluripotent Stem Cell-Derived Endothelial Cells in Hydrogel-Based Culture Systems. ACS Omega. 2021; 6: 6942-52.

[0394] 30. Lin H, Du Q, Li Q, et al. Hydrogel-Based Bioprocess for Scalable Manufacturing ofHuman Pluripotent Stem Cell-Derived Neural Stem Cells. ACSAppL Mater. Interfaces. 2018; 10: 29238-50.

[0395] 31. Wang O, Lei Y. Creating a cell-friendly microenvironment to enhance cell culture efficiency. Cell Gene Ther. Insights. 2019; 5: 341-50.

[0396] 32. Lin H, Du Q, Li Q, et al. Manufacturing human pluripotent stem cell derived endothelial cells in scalable and cell-friendly microenvironments. Biomater. Sei. 2019; 7: 373-88.Attorney Docket No. 0073605-001131

[0397] 33. Lin H, Li Q, Du Q, et al. Integrated generation of induced pluripotent stem cells in a low-cost device. Biomaterials. 2019; 189: 23-36.

[0398] 34. Lin H, Li Q, Wang O, et al. Automated expansion of primary human T cells in scalable and cell-friendly hydrogel microtubes for adoptive immunotherapy. Adv. Healthc. Mater. 2018; 5: el701297.

[0399] 35. Lin H, Qiu X, Du Q, et al. Engineered Microenvironment for Manufacturing HumanPluripotent Stem Cell-Derived Vascular Smooth Muscle Cells. Stem Cell Reports. 2019; 8: 84-97.

[0400] 36. Liu Q, Liu Z, Gu H, et al. Comparative study of differentiating human pluripotent stem cells into vascular smooth muscle cells in hydrogel -based culture methods. Regen. Ther. [Internet], 2023; 22: 39-49.

[0401] 37. Li Q, Lin H, Rauch J, et al. Scalable culturing of primary human glioblastoma tumor-initiating cells with a cell-friendly culture system. Sci. Rep. 2018; 8: 3531.

[0402] 38. Achieving large-scale, cost-effective, reproducible manufacturing of high-quality cells. A Technology Roadmap to 2025. Consortium, Natl. Cell Manuf. 2016.

[0403] 39. Baum E, Littman N, Ruffin M, Ward S, Aschheim K. Key tools and technology hurdles in advancing stem-cell therapies. California Institute for Regenerative Medicine, Alliance for Regenerative Medicine Catapult, Cell Therapy. 2013.

[0404] 40. National science and technology council: Advance manufacturing: a snapshot of priority technology areas across the federal government subcommittee for advanced manufacturing. 2016.

[0405] 41. Lee A, Hudson AR, Shiwarski DJ, et al. 3D bioprinting of collagen to rebuild components of the human heart. Science. 2019; 365: 482-7.

[0406] 42. Hartman ME, Dai D, La MA. Human pluripotent stem cells : Prospects and challenges as a source of cardiomyocytes for in vitro modeling and cell-based cardiac repair. Adv. DrugDeliv. Rev. 2016; 96: 3-17.

[0407] 43. Laflamme M a, Chen KY, Naumova A V, et al. Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nat. Biotechnol. 2007; 25: 1015-24.

[0408] 44. Burridge PW, Matsa E, Shukla P, et al. Chemically defined generation of human cardiomyocytes. Nat. Methods. 2014; 11: 855-60.

[0409] 45. Chong JJH, Yang X, Don CW, et al. Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts. Nature. 2014; 510: 273-7.Attorney Docket No. 0073605-001131

[0410] 46. Williams DJ, Archibald P, Bantounas I, et al. Comparability: manufacturing, characterization and controls, report of a UK regenerative medicine platform pluripotent stem cell platform workshop. Regen. Med. 2016; 11: 483-92.

[0411] 47. Lian X, Zhang J, Azarin SM, et al. Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt / |3-catenin signaling under fully defined conditions. Nat. Protoc. 2013; 8: 162-75.Example 2: Collagen Nanofiber Reinforced Alginate Hydrogel microtube Microbioreactors for Cell Culture

[0412] The large-scale production of mammalian cells is pivotal for various applications; however, current bioreactor technologies encounter significant technical and economic challenges. Scaling up cell cultures remains problematic due to excessive cell aggregation, shear stress-induced cell death, batch-to-batch inconsistencies, and limited scalability. We propose that engineering a cell-friendly microenvironment can enhance culture efficiency. Previously, we developed alginate hydrogel microtubes (AlgTubes) that significantly improved cell density and growth rates. Nevertheless, AlgTubes lack adhesion sites essential for anchorage-dependent cells and frequently break, causing cell leakage and production inconsistencies. To address these limitations, this study reinforced AlgTubes with collagen nanofibers, creating collagen-alginate hybrid hydrogel microtubes (ColAlgTubes). Utilizing a novel micro-extruder, we efficiently fabricate cell-loaded ColAlgTubes. Collagen formed a dense nanofiber network interwoven with the alginate mesh, enhancing the hydrogel's mechanical properties while providing cell adhesion sites. ColAlgTubes protected cells from hydrodynamic stress and maintained cell mass within a 400 pm diameter, ensuring efficient nutrient exchange and waste removal. This optimized microenvironment resulted in high cell viability, rapid proliferation, and exceptional yields of 5>< 108cells / mL, which is 200 times higher than conventional culture methods. With their scalability, cost-effectiveness, and efficiency, ColAlgTubes offers a transformative solution for large-scale cell production with broad applications in biotechnology, regenerative medicine, and therapeutic manufacturing.Introduction

[0413] Human and animal cells are integral to a broad spectrum of biomedical and industrial applications. Stem cells, such as human pluripotent stem cells (hPSCs) including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs), along with their differentiated progeny, are utilized in the treatment of various diseases and injuries1. They are also essential forAttorney Docket No. 0073605-001131 disease modeling, drug screening, and toxicity testing1. Immune cells, like T cells and natural killer cells, are used in cancer immunotherapy2'6. Additionally, mammalian cells are crucial for producing recombinant proteins and viruses, which are vital for research and clinical applications7'9.

[0414] These applications need large quantities of high-quality cells1. For example, treating a single patient requires approximately 109cardiomyocytes for myocardial infarction (MI) or 109P cells for Type 1 diabetes10. The demand is further amplified by the prevalence of degenerative diseases and organ failure, with 1-2.5 million people in the U.S. affected by Type 1 diabetes and around 8 million by MI10. Similarly, tissue engineering demands large numbers of cells, with approximately 1010hepatocytes or cardiomyocytes needed to construct an artificial human liver or heart11. Drug discovery efforts, such as screening a million-compound library, can also require 1010cells per screen10. Moreover, large quantities of mammalian cells, such as Chinese Hamster Ovary (CHO) cells and Human Embryonic Kidney 293 (HEK293) cells, are essential for producing therapeutic biologies, including monoclonal antibodies, enzymes, and viral particles7'9.

[0415] Despite the need for efficient and scalable cell production, existing methods remain inadequate, particularly for clinical applications1,10’12. Two-dimensional (2D) culture systems, such as flasks, are widely used but lack the complexity of natural cellular environments. They are also resource-intensive, requiring significant labor, space, and reagents, making them impractical for large-scale production. Three-dimensional (3D) suspension culture systems, such as stirred-tank bioreactors, have been developed to improve scalability13’14. However, these systems face significant challenges, particularly uncontrolled cell aggregation. Aggregates exceeding 400 pm in diameter suffer from impaired transport of nutrients, oxygen, and growth factors, leading to slower proliferation, apoptosis, and undesired differentiation. While agitation can mitigate aggregation, it also introduces shear forces, negatively impacting cell survival, growth, and differentiation efficiency10’15J6. As a result, 3D suspension cultures often exhibit high cell mortality, slow proliferation rates, and low volumetric yields. For instance, hPSCs in stirred-tank bioreactors typically undergo only a four-fold expansion over four days, yielding approximately 2.0* 106cells / mL, which utilizes just 0.4% of the bioreactor volume17'19.

[0416] The complexity of hydrodynamic conditions in stirred-tank bioreactors - such as flow dynamics, shear stress, and chemical gradients - adds further challenges. These factors depend on variables including bioreactor design, medium viscosity, and agitation speed, making precise control difficult1’10’12 15'20. The variable hydrodynamic conditions contribute to large cell production variation. For example, in cardiomyocyte production from hPSCs, yields from three -100 mbAttorney Docket No. 0073605-001131 batches of hESCs varied widely, ranging from 40 to 100 million cells, with cardiomyocyte purity fluctuating between 54% and 84%21-22. When using a different hPSC line under the same conditions, yields ranged from 89 to 125 million cells, with purity varying from 28% to 88%21,22. Scaling production from 100 mb to 1000 mL requires extensive re-optimization of agitation rates and culture protocols, underscoring the technical and economic challenges of achieving industrial-scale cell manufacturing21,22. Currently, the largest demonstrated hPSC suspension cultures are limited to volumes of just tens of liters10,23.

[0417] To overcome these limitations, we propose the development of hydrogel microtubebased 3D microbioreactors. Our previous studies demonstrated that culturing cells within hollow hydrogel microtubes or microbioreactors made from alginate polymers mitigate many of these challenges. These microtubes prevent excessive cell aggregation, enhance mass transport efficiency, and eliminate hydrodynamic stress, leading to high cell viability, rapid proliferation, and significantly improved volumetric yields. Notably, yields of up to 5* 1O8cells / mL have been achieved24’33. These findings highlight the transformative potential of hydrogel microtube microbioreactors for scalable, efficient, and cost-effective cell production24’33.

[0418] Despite their advantages, alginate hydrogel microtubes can have limitations. They are mechanically fragile, with a significant proportion breaking during cell culture, leading to cell leakage and production failures. Additionally, many cells do not grow well in alginate hydrogel microtubes because they cannot adhere to the alginate surface. There is a need for new, robust, and adhesive hydrogel microtube microbioreactors. This study introduces the fabrication and application of hybrid collagen-alginate microtubes for cell culture. Within these microtubes, collagen proteins form a nanofiber network that interpenetrates the alginate hydrogel mesh. The collagen nanofibers enhance the alginate hydrogel's structural integrity and serve as anchoring points for adhesiondependent cell growth. This advanced system can facilitate cost-effective large-scale cell culture for various biomedical and industrial applications.Methods

[0419] Collagen Extraction from Rat Tails. Rat tails were harvested and soaked in 70% ethanol for 30 minutes. The skin was carefully removed using a scalpel and forceps to isolate the tendons, which were then rinsed three times with PBS. To ensure sterilization, the tendons were submerged in 70% ethanol for at least one hour. After sterilization, they were soaked in 0.02N acetic acid and stirred at 4°C for 48 hours. The resulting viscous mixture was centrifuged at 10,000 rpm for 60 minutes at 4°C to remove debris. The supernatant was collected and dialyzed against 0.02N aceticAttorney Docket No. 0073605-001131 acid, yielding the collagen stock solution for further applications. Although we used rat tail collagen to demonstrate preparation of the ColAlgTubes in this study, it should be noted that ColAlgTubes can be fabricated using collagens from various species and tissues, such as rat tails, bovine, and human skin, among others.

[0420] Processing of ColAlgTubes. Rat tail collagen was adjusted to pH 5.0 using a neutralizing solution and then combined with alginate in a pH 5.0 NaCl solution to create a collagen-alginate mixture. A custom-built micro-extruder was used to fabricate ColAlgTubes. A 2% methylcellulose (MC) solution containing single cells was pumped into the central channel at a rate of 20 pL / min, while a pre-cooled syringe containing the collagen-alginate solution was placed in a homemade ice box and pumped into the side channel at 61 pL / min. The two solutions were co-extruded into a 50 mM HEPES + 100 mM CaCh buffer (pH 7.4), forming ColAlgTubes. After extrusion, the buffer was replaced with a cell culture medium to support cell growth.

[0421] Scanning Electron Microscope (SEM) Imaging. The nanostructures of ColAlgTubes were characterized using a Zeiss SIGMA VP-FESEM. Before imaging, samples were dehydrated through a graded ethanol series (25%, 50%, 70%, 85%, 95%, and 100%), with each step lasting 5 minutes. Critical point drying was performed using a Leica EM CPD300 Critical Point Dryer. The samples were sputter-coated with a 4.5 nm iridium layer to enhance conductivity using a Leica EM ACE600 Sputter Coater. SEM images were acquired at an accelerating voltage of 5 kV, with a working distance of 6 mm and magnifications ranging from 150x to 5,000*, allowing detailed visualization of the nanofiber structures.

[0422] Collagen and Alginate Degradation. ColAlgTubes were incubated with 0.2 mg / mL Collagenase P at 37°C for 15 minutes to degrade collagen nanofibers. ColAlgTubes were incubated with 0.5 mM EDTA at room temperature for 15 minutes to dissolve the alginate hydrogel. Following degradation, the samples were prepared for SEM imaging as required.

[0423] Culturing hPSCs in Collagen Alginate microtubes. For a typical cell culture, 20 pL of cell solution in ColAlgTubes was suspended in 2 mL of E8 medium supplemented with 10 pM Y- 27632 in a 6-well plate. The culture was maintained in an incubator at 37 °C with 5% CO2 and 21% O2, and the medium was refreshed daily. For cell passaging, the medium was removed, and ColAlgTubes were dissolved using 0.2 mg / mL Collagenase P containing 0.5 mM EDTA for 20 minutes. The cell mass was collected by centrifugation at 100 g for 5 minutes, followed by treatment with Accutase at 37 °C for 10 minutes. The cells were then dissociated into single cells for subsequent culture or cryopreservation.Attorney Docket No. 0073605-001131

[0424] Cardiomyocyte differentiation. Human pluripotent stem cells (hPSCs) in ColAlgTubes were cultured in E8 medium supplemented with 10 pM Y-27632 until the diameter of the cell aggregates reached the inner diameter of the ColAlgTubes. On Day 0, the medium was replaced with E5 medium containing 10% lipid mix and 5 pM CHIR9902. After 24 hours, the medium was replaced with E5 medium containing 10% lipid mix and 3 pg / mL Heparin. From Day 2 to Day 4, the medium was refreshed daily with E5 medium containing 10% lipid mix, 3 pg / mL Heparin, and 3 pM IWR1. From Day 5 to Day 6, the medium was refreshed daily with E5 medium containing 10% lipid mix and 3 pg / mL Heparin. From Day 7 to Day 10, the medium was refreshed every other day with an E5 medium containing 10% lipid mix and 20 pg / mL insulin. From Day 11 to Day 18, the medium was refreshed every other day with DMEM medium (without glucose, glutamine, and pyruvate) supplemented with 5 mM Lactate. Cardiomyocytes were collected on Day 18 for analysis. For longterm culture, the medium was switched to a-MEM supplemented with 5% FBS and refreshed every other day.

[0425] Staining, Flow Cytometry, and Imaging. Single-cell suspensions were fixed in 4% paraformaldehyde (PF A) at room temperature for 15 minutes. The fixed cells were then incubated overnight at 4°C with primary antibodies in PBS containing 0.1% Triton X-100 and 0.5% BSA. After extensive washing, secondary antibodies were added and incubated for 2 hours at room temperature. The cells were washed three times with PBS supplemented with 0.5% BSA before analysis using the Attune® NxT™ Acoustic Focusing Cytometer (ThermoFisher). Flow cytometry data were processed using FlowJo software. LIVE / DEAD® Cell Viability staining was performed following the manufacturer's instructions for cell viability assessment. Phase-contrast and fluorescence imaging were conducted using a Zeiss Axio Observer Fluorescent Microscope.

[0426] Statistical Analysis. Data analysis was conducted using GraphPad Prism 8, with results presented as mean ± standard error of the mean (SEM). Statistical significance was determined using appropriate tests based on the type of comparison: One-way analysis of variance (ANOVA) was used for multiple group comparisons (three or more groups). Unpaired two-tailed t-tests were performed for comparisons between the two groups. Statistical significance was indicated as follows: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).Results

[0427] Fabrication of Collagen-Alginate Hybrid Hydrogel microtubes (ColAlgTubes)

[0428] This study aimed to develop ColAlgTubes as an innovative cell culture platform. Currently, no technology exists for rapidly fabricating hybrid ColAlgTubes for cell cultureAttorney Docket No. 0073605-001131 applications. To address this gap, we designed a specialized micro-extruder with dual inlets and a single outlet (FIG. 11 A). The extruder was constructed using a Formula 3B printer and clear resin. Additionally, we designed and 3D-printed a cooling box to maintain the collagen-alginate solution at a low temperature (FIG. 1 IB). The ColAlgTube extrusion setup comprises two syringe pumps, the micro-extruder, the cooling box, and a container filled with HEPES buffer (FIGS. 11C, D). Syringe1 is loaded with a room-temperature (RT) cell solution, while Syringe 2 contains an ice-cold collagen-alginate mixture (pH = 5.0). The cooling box features a designated channel to hold Syringe2 and is packed with ice, ensuring the collagen-alginate solution remains below 4°C (FIGS. 1 IB, C). Maintaining a low temperature and acidic pH prevents premature collagen gelation inside the syringe.

[0429] To fabricate ColAlgTubes, the two solutions are pumped into inlets 1 and 2, generating coaxial core-shell flows that are extruded into a HEPES buffer solution containing 100 mM CaCh at 37°C and pH 7.4 (FIGS. 11C, D). The core flow carries the cell solution, while the shell flow contains the collagen-alginate mixture. At the microscale, these solutions establish stable laminar flows. The acidic collagen solution in the shell flow is neutralized by the core cell solution and the HEPES buffer, triggering the rapid formation of a collagen nanofiber network. Simultaneously, the alginate in the shell undergoes crosslinking with Ca2+ions, forming a stable alginate network. This is the first time to rapidly process an interpenetrating collagen and alginate microtube for cell culture.

[0430] Engineering Principles and Control ColAlgTube Dimensions.

[0431] Similar to our previously developed AlgTubes, ColAlgTubes overcome the limitations of conventional cell culture techniques by providing a physiologically relevant microenvironment for cell growth (FIG. 1 IE). Cells are cultured within microscale ColAlgTubes, which remain suspended in the culture medium. The hydrogel microtubes create free spaces for cell-cell interactions and expansion while protecting cells from hydrodynamic stresses within the culture vessel. Additionally, the microtubes restrict cell mass to a radial diameter of less than 400 pm, aligning with human tissue's diffusion limit, ensuring efficient nutrient exchange and waste removal throughout the culture. Moreover, collagen nanofibers within the ColAlgTubes serve as adhesive substrates, supporting the growth of anchor-dependent cells.

[0432] The dimensions of ColAlgTubes can be tuned by adjusting the ratio of core and shell flows, as well as the diameter of the extruder nozzle. Wall thickness can be controlled by modifying the collagen-alginate shell flow rate, with lower flow rates producing thinner walls. FIGS. 1 IF and GAttorney Docket No. 0073605-001131 showcase ColAlgTubes with varying diameters and wall thicknesses, highlighting the adaptability of this system.

[0433] Nanostructures of ColAlgTubes

[0434] We hypothesize that (1) collagen and alginate form an interpenetrating network and (2) collagen nanofibers reinforce the alginate hydrogel, enhancing the mechanical strength of ColAlgTubes and increasing their resistance to force-induced breakage. We used scanning electron microscopy (SEM) to examine the nanostructures of ColAlgTubes. ColAlgTubes were fabricated with iPSC cells and cultured until the microtubes were partially filled. The microtubes were then fixed with paraformaldehyde (PF A), dehydrated through a graded ethanol series, metal-sputtered, and imaged using SEM. We captured images of the overall microtube structure, inner and outer surfaces, wall nanostructures, and encapsulated cells. The alginate concentration was maintained at 0.75%, while collagen concentrations varied from 1 to 3 mg / mL (FIGS. 12A-C). For comparison, pure alginate and pure collagen hydrogel microtubes were also prepared (FIGS. 12D, E).

[0435] In pure alginate hydrogel microtubes, a dense mesh-like structure was observed (FIG. 12D), while pure collagen hydrogel microtubes were composed of collagen nanofibers (FIG. 12E). The hybrid ColAlgTubes exhibited nanostructures similar to alginate hydrogel microtubes but contained small numbers of collagen nanofibers interwoven within the dense alginate matrix (FIGS. 12A-C). Increasing the collagen concentration from 1 mg / mL to 3 mg / mL resulted in more collagen nanofibers.

[0436] To further confirm the interpenetrating network, alginate was selectively dissolved using an EDTA solution, allowing clear visualization of the collagen nanofiber network (FIGS. 13A, B). Conversely, collagen nanofibers were degraded with collagenase, revealing the dense alginate network (FIG. 13C). These findings validate our hypothesis that ColAlgTubes consist of an interpenetrating network of collagen nanofibers and alginate hydrogels.

[0437] Culturing HEK293 Cells in ColAlgTubes

[0438] To assess the suitability of ColAlgTubes for cell culture, HEK293 cells (FIG. 14A) were processed into the microtubes. Within 24 hours, the cells adhered to the inner surface and began expanding into colonies. Over time, they proliferated further, eventually filling the entire microtube and forming a three-dimensional (3D) cell mass. Live / Dead cell staining confirmed that most cells remained viable. Over 4x 108cells / mL were successfully harvested. In contrast, when HEK293 cells were cultured in pure AlgTubes, they did not adhere to the microtube walls. Instead, they formed unattached spheroids (FIG. 14B). Their growth rate was significantly lower compared to thoseAttorney Docket No. 0073605-001131 cultured in ColAlgTubes, highlighting the importance of adhesion for HEK293 cell proliferation (FIG. 14B). These results confirm our hypothesis that incorporating collagen proteins into AlgTubes provides essential anchoring points for cell attachment and growth.

[0439] Culturing Human Pluripotent Stem Cells (hPSCs) in ColAlgTubes

[0440] H9 human embryonic stem cells (hESCs) were cultured within ColAlgTubes. Within 24 hours, the cells adhered to the inner surface of the microtubes. By day 3, small colonies had formed, which gradually developed into spheroids by day 5. By day 9, cell proliferation had expanded to fill most of the microtube (FIG. 15 A). Live / Dead staining performed before and after cell release indicated minimal cell death (FIGS. 1 C, D). The dissociated cell mass was subsequently fixed with paraformaldehyde (PF A) and stained for the pluripotency markers Nanog and Oct4. Flow cytometry analysis confirmed that 97.0% and 98.3% of the cells expressed Nanog and Oct4, respectively, demonstrating that hPSCs retained their pluripotency after culturing in ColAlgTubes (FIG. 1 E). Additionally, more than 4.5>< 108cells per milliliter of microspaces were harvested. In contrast, when hPSCs were cultured in pure AlgTubes (FIG. 15B), they did not adhere to the microtube and grew as unattached spheroids. This further supports the role of collagen proteins in providing anchoring points essential for cell attachment and growth.

[0441] Differentiating hPSCs into Cardiomyocytes in ColAlgTubes

[0442] To further explore the potential of ColAlgTubes, we differentiated hPSCs into cardiomyocytes. H9 hESCs were cultured in ColAlgTubes and expanded in Essential 8 (E8) medium for seven days. Without requiring passaging, the medium was switched to a mesoderm induction medium to initiate differentiation into mesoderm progenitors. On day 2, the medium was replaced with cardiac progenitor differentiation medium, followed by cardiomyocyte differentiation medium on day 7. From days 11 to 18, cells were maintained in a metabolic enrichment medium, with the majority remaining viable. Following treatment with EDTA and collagenase, fibrous cardiac tissues were successfully harvested (FIG. 16A). Further dissociation of these tissues using Accutase® yielded over 3* 1O8cardiomyocytes per milliliter of hydrogel microtubes. Immunostaining revealed that most cells expressed the cardiomyocyte marker cTnT, demonstrating that ColAlgTubes effectively support stem cell differentiation.

[0443] Reduced Cell Leakage in ColAlgTubes Compared to AlgTubes

[0444] Previously, we utilized alginate-based hydrogel microtubes (AlgTubes) for cell culture; however, frequent microtube breakages were observed (FIG. 17A). Since cells did not adhere to AlgTubes, a significant number of cells leaked into the medium through these breaks on days 5, 6,Attorney Docket No. 0073605-001131 and 7, leading to the formation of large cell aggregates (FIG. 17B). In contrast, ColAlgTubes demonstrated significantly greater structural integrity, exhibiting fewer breakages than AlgTubes. Additionally, due to their ability to support cell adhesion, cell aggregates remained attached to the microtubes and did not leak into the medium (FIGS. 17A, C). Quantitative analysis of leakage events (FIG. 17D) and the percentage of leaked cells on day 7 (FIG. 17E) further confirmed that ColAlgTubes had substantially lower cell leakage than AlgTubes.

[0445] In summary, we have demonstrated that ColAlgTubes can be a robust cell expansion and differentiation platform. The interpenetrating collagen-alginate network enhances mechanical stability, significantly reducing cell leakage compared to AlgTubes. Additionally, ColAlgTubes facilitate cell adhesion, making them suitable for culturing both adherent and suspension cells. Discussion

[0446] Current 2D and 3D cell culture methods face significant challenges in achieving efficient, scalable, and cost-effective large-scale cell production34’36. Key limitations include low cell yields, restricted scalability, high operational costs, and substantial culture variability34’36. A fundamental issue is that conventional systems, such as 2D flasks and 3D stirred-tank bioreactors, fail to accurately replicate the complex 3D microenvironments found in vivo34’36. In their native state, human cells exist within intricate 3D microenvironments that facilitate interactions with the extracellular matrix (ECM), support efficient nutrient and oxygen exchange, and minimize hydrodynamic stress13 14’37'39. We hypothesize that developing advanced culture technologies closely mimicking physiologically relevant microenvironments can overcome these challenges and significantly enhance culture efficiency24’27.

[0447] Hydrogel microtube microbioreactors represent a promising approach to creating cell- friendly environments suitable for large-scale culture (FIG. 1 IE)24,27. We previously developed a method for fabricating hydrogel microtubes using alginate polymers24’33. Alginate is abundant, cost- effective, biocompatible, and has an established clinical safety profile. It can be rapidly crosslinked with calcium ions, allowing scalable production of alginate hydrogel microtubes (AlgTubes) via extrusion. After culture, AlgTubes can be dissolved with EDTA to harvest cells. Their transparency also enables real-time monitoring24’33.

[0448] Using AlgTubes, we cultured human pluripotent stem cells (hPSCs) over multiple passages with high consistency, maintaining pluripotency and chromosomal stability24. Cultures achieved high viability and rapid expansion (e.g., a 1000-fold increase over 10 days), with volumetric yields of ~5* 108cells / mL, which is over 200-fold higher than stirred-tank bioreactors.Attorney Docket No. 0073605-001131Furthermore, hPSCs differentiated efficiently into endothelial cells25,28, vascular smooth muscle cells31, neural stem cells26, and neurons29, all reaching yields of 5* 108cells / mL. Adult cells, such as T cells, were also successfully cultured in AlgTubes. These findings highlight the transformative potential of hydrogel microtubes for high-efficiency, large-scale production27. Despite their success, AlgTubes have limitations. Mammalian cells lack receptors for alginate, preventing adhesion to microtube walls, which limits the culture of anchorage-dependent cells like mesenchymal stem cells. Additionally, AlgTubes are mechanically fragile and prone to breakage, leading to cell leakage and variability (FIGS. 17A, B). These limitations necessitate further advancements in hydrogel microtube design for more robust and versatile microbioreactor systems.

[0449] Research has shown that incorporating nanofibers into hydrogels is a promising strategy for enhancing mechanical properties and structural integrity40-42. Nanofibers, derived from biopolymers such as collagen and silk fibroin or synthetic materials like polycaprolactone (PCL), can form an interpenetrating network within the hydrogel matrix. This structural reinforcement significantly improves the hydrogel's tensile strength, elasticity, and stability. Additionally, nanofibers enhance cell adhesion, proliferation, and controlled drug release. By integrating nanofibers into hydrogel systems, researchers can develop advanced biomaterials with superior structural integrity and tunable mechanical properties, broadening their applications in regenerative medicine and biotechnology40-42.

[0450] Specific to alginate hydrogels, researchers have explored various nanofibers for reinforcement43. For instance, one study developed laminated composite scaffolds by combining alginate hydrogels with polycaprolactone (PCL) and gelatin electrospun mats44. These scaffolds demonstrated enhanced mechanical properties and controlled biodegradability, making them well- suited for cartilage tissue engineering. Another investigation created a three-dimensional composite scaffold of alginate hydrogels, PCL / gelatin nanofibers, and exosomes, effectively promoting regeneration in rat tympanic membrane perforations45. A recent review summarized various approaches for developing alginate composite hydrogels43.

[0451] However, incorporating nanofibers into alginate hydrogel microtubes presents significant challenges compared to embedding nanofibers within bulk hydrogels. To the best of our knowledge, this goal has not been achieved by others. In this study, we developed an innovative approach to fabricate interpenetrating collagen-alginate hydrogel microtubes. By integrating a cooling box, a two-flow micro-extruder, and a buffer exchange system (FIG. 11), we successfully established a method for processing collagen-alginate hybrid microtubes. Collagen proteins formed aAttorney Docket No. 0073605-001131 dense nanofiber network reinforcing AlgTubes (FIGS. 12, 3). As a result, ColAlgTubes exhibited remarkable durability, with significantly fewer breakages during cell culture, effectively preventing cell leakage (FIG. 17). Furthermore, since most cells express receptors for Collagen, they naturally adhered to the ColAlgTubes, enhancing cell attachment and growth (FIGS. 14, 15).

[0452] Our data showed that the cell growth rate, viability, and volumetric yield achieved with ColAlgTubes were comparable to those observed with AlgTubes24. The high growth rate and cell density attainable in this system have significant implications for large-scale cell production. By significantly increasing cell culture density, ColAlgTubes can substantially reduce culture volume, lowering labor requirements, reagent costs, equipment needs, facility space, and manufacturing expenses.Conclusion

[0453] In summary, ColAlgTubes represents a significant advancement in cell culture technology. Their ability to support high-yield cell growth and streamline large-scale manufacturing makes them a compelling alternative to conventional two-dimensional and three-dimensional culture systems. As advancements in stem cell differentiation protocols continue, future efforts should focus on integrating optimized differentiation processes into ColAlgTubes to enable high-yield production of diverse cell types, such as hPSCs-derived endothelial cells28, vascular smooth muscle cells31, red blood cells46'48, platelets49'52, beta cells, T cells, and neurons53'36. These cells can be used for preclinical and clinical applications and require large-scale production. Expanding ColAlgTubes applications to include other cell populations, such as adult stem cells and immune cells like T cells, will further increase their utility.References cited in Example 2

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[0468] 15. Kinney, M. A., Sargent, C. Y. & Mcdevitt, T. C. The multiparametric effects of hydrodynamic environments on stem cell culture. Tissue Eng. Part B 17, 249-262 (2011).

[0469] 16. Fridley, K. M., Kinney, M. A. & McDevitt, T. C. Hydrodynamic modulation of pluripotent stem cells. Stem Cell Res. Ther. 3, 45 (2012).

[0470] 17. Lei, Y., Jeong, D., Xiao, J. & Schaffer, D. V. Developing defined and scalable 3D culture systems for culturing human pluripotent stem cells at high densities. Cell. Mol. Bioeng. 7, 172-183 (2014).

[0471] 18. Steiner, D. et al. Derivation, propagation and controlled differentiation of human embryonic stem cells in suspension. Nat. Biotechnol. 28, 361-4 (2010).

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[0473] 20. Ismadi, M., Gupta, P., Fouras, A., Verma, P. & Jadhav, S. Flow characterization of a spinner flask for induced pluripotent stem cell culture application. PLoS One 9, el06493 (2014).

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[0476] 23. Kempf, H., Andree, B. & Zweigerdt, R. Large-scale production of human pluripotent stem cell derived cardiomyocytes. Adv. Drug Deliv. Rev. 96, 18-30 (2016).

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[0479] 26. Lin, H. et al. Hydrogel -Based Bioprocess for Scalable Manufacturing of HumanPluripotent Stem Cell-Derived Neural Stem Cells. ACS Appl. Mater. Interfaces 10, 29238-29250 (2018).

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[0484] 31. Lin, H. et al. Engineered Microenvironment for Manufacturing Human PluripotentStem Cell-Derived Vascular Smooth Muscle Cells. Stem Cell Reports 8, 84-97 (2019).

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[0486] 33. Li, Q. et al. Scalable culturing of primary human glioblastoma tumor-initiating cells with a cell-friendly culture system. Sci. Rep. 8, 3531 (2018).

[0487] 34. Achieving large-scale, cost-effective, reproducible manufacturing of high-quality cells. A Technology Roadmap to 2025. Consortium, Natl. Cell Manuf. (2016).Attorney Docket No. 0073605-001131

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[0498] 45. Chahsetareh, H. et al. Alginate hydrogel-PCL / gelatin nanofibers composite scaffold containing mesenchymal stem cells-derived exosomes sustain release for regeneration of tympanic membrane perforation. Int. J. Biol. Macromol. 262, 130141 (2024).

[0499] 46. Sivalingam, J. et al. A Scalable Suspension Platform for Generating High-DensityCultures of Universal Red Blood Cells from Human Induced Pluripotent Stem Cells. Stem Cell Reports 16, 182-197 (2021).

[0500] 47. Pavani, G. et al. Modeling primitive and definitive erythropoiesis with induced pluripotent stem cells. Blood Adv. 8, 1449-1463 (2024).Attomey Docket No. 0073605-001131

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[0509] 56. Montel-Hagen, A. et al. Organoid-Induced Differentiation of Conventional T Cells from Human Pluripotent Stem Cells. Cell Stem Cell 24, 376-389. e8 (2019).Example 3: Culturing Mammalian Cells in RGD Peptide-Modified Alginate Hydrogel Microtubes

[0510] Traditional livestock farming is resource-intensive and environmentally unsustainable, highlighting the need for alternative methods of meat production. Cell-cultured meat, produced through the in vitro expansion and differentiation of animal cells, offers a promising solution to supplement conventional meat sources. However, the emerging industry faces a significant challenge in achieving large-scale, cost-effective cell production. In this study, we developed a microbioreactor system based on RGD peptide-modified alginate hydrogel microtubes (AlgTubes) to address this challenge. AlgTubes provide a cell-friendly, three-dimensional microenvironment that supports efficient mass transport while minimizing shear stress, thereby significantly enhancing cell viability and yield. This system overcomes key limitations of traditional bioreactors, such as shear-Attorney Docket No. 0073605-001131 induced cell damage and cell aggregation. Using AlgTubes, we successfully expanded mouse (C2C12) and quail (QM7) myoblasts, achieving cell densities exceeding 1.0* 108cells / mL - an order of magnitude higher than those typically attainable in conventional stirred-tank bioreactors. Upon induction, the cells could be differentiated into mature myotubes. Co-culturing myoblasts with stromal cells further improved cell yield. By substantially increasing cell density, AlgTubes can significantly reduce the required culture volume, thereby lowering labor, reagent consumption, equipment usage, facility space, and overall manufacturing costs. This innovative system holds strong potential for enabling the large-scale, economically viable production of cultured meat. Introduction

[0511] According to data from the Food and Agriculture Organization, the global population is expected to reach 9 billion by 2050. 70% more food will be needed to feed this population. Meat is a significant component of the human diet. Currently, meat is obtained from natural animals or livestock raised on traditional farms or in modern factories. (Aiking, 2014; Ryschawy et al., 2019; Willett et al., 2019) Livestock farming requires large amounts of water and land. Additionally, farming causes significant environmental and animal welfare issues. (Aleksandrowicz et al., 2016; Gerber et al., 2015) Alternative approaches that can produce meat efficiently and in an environmentally friendly manner are highly wanted.

[0512] Plant-based meat has emerged as a promising alternative, offering both environmental and ethical advantages. (Ozsolak and Milos, 2023) These products are made from plant-derived ingredients such as soy, peas, and wheat, and are engineered to replicate the taste, texture, and nutritional profile of traditional meat. However, they have notable drawbacks. (Ozsolak and Milos, 2023) Plant-based meats are highly processed and may contain additives and high sodium levels, raising concerns about their long-term health impacts. (Ozsolak and Milos, 2023) Additionally, plant-based meats still fall short in replicating the exact flavor and texture of real meat. (Ozsolak and Milos, 2023)

[0513] Another promising alternative is cultured meat, also known as lab-grown meat, which is produced by cultivating animal cells in a controlled environment. (Bryant, 2020; Guan et al., 2025; Hong et al., 2021; Kang et al., 2021; Liu et al., 2025; Ma et al., 2024; Martins et al., 2024; Sanaki- Matsumiya et al., 2024; Stephanie Kawecki et al., 2024; Treich, 2021; Zhou et al., 2025; Zhu et al., 2023) The process typically involves isolating a small sample of animal cells, most commonly muscle stem cells, and expanding them in a nutrient-rich medium that supports their proliferation and differentiation into muscle tissue, which can then be harvested as meat. (Humbird, 2021;Attorney Docket No. 0073605-001131Negulescu et al., 2023) Despite its potential, cultured meat remains largely at the proof-of-concept stage, with several technological challenges that can be addressed to achieve commercial success. (Humbird, 2021; Negulescu et al., 2023) One of the most significant hurdles is achieving mass cell production. (Bellani et al., 2020; de Souza Vandenberghe et al., 2024) It is estimated that producing 1 kg of muscle tissue requires approximately 3 x 1012cells. Meeting the global demand for cultured meat will require cell production at a scale of hundreds of thousands of tons — a level that current cell culture technologies and bioreactor systems are not yet capable of achieving. (Humbird, 2021; Negulescu et al., 2023; Risner et al., 2021)

[0514] Culturing animal cells presents unique challenges due to their structural fragility - unlike bacteria or fungi, animal cells lack a cell wall and are highly sensitive to shear stress, (de Souza Vandenberghe et al., 2024; Manzoki et al., 2024) Furthermore, most cells used in cultured meat production are anchorage-dependent, necessitating physical support for growth, (de Souza Vandenberghe et al., 2024) This requirement often leads to the incorporation of microcarriers or scaffolds within the bioreactor system. In laboratories, animal cells are typically cultured in flasks as monolayers. However, these two-dimensional (2D) flasks can only yield a very limited number of cells per surface area and are only suitable for culturing cells at a small scale. Three-dimensional (3D) bioreactors are therefore needed for mass production. Animal cells are grown on the surface of microcarriers that are suspended in bioreactors, (de Souza Vandenberghe et al., 2024)

[0515] Several types of bioreactors have been explored for cultivated meat production, each offering distinct advantages and limitations. Among them, mechanically agitated bioreactors — particularly stirred tank reactors (STRs) — are the most widely studied. (Bodiou et al., 2025; de Souza Vandenberghe et al., 2024; Hanga et al., 2021; Norris et al., 2022; Tzimorotas et al., 2023; Yen et al., 2023; Yin et al., 2024) These systems are valued for their versatility, scalability, and precise control over culture parameters such as pH, dissolved gases, and temperature. However, STRs also present several significant limitations when applied to animal cell culture. (Bodiou et al., 2025; de Souza Vandenberghe et al., 2024; Hanga et al., 2021; Norris et al., 2022; Tzimorotas et al., 2023; Yen et al., 2023; Yin et al., 2024) First, although STRs are 3D systems, animal cells still grow as monolayers on the surface of microcarriers, making it challenging to achieve high volumetric yields. Second, due to strong cell-to-cell interactions, microcarriers tend to aggregate into large clumps, which impairs mass transport and negatively affects cell viability and growth rates. Third, the mechanical agitation inherent to STRs generates substantial shear forces that can damage fragile animal cells. As a result, typical cell densities in STRs are limited to approximately 106cells / mL,Attorney Docket No. 0073605-001131 meaning that the actual cell mass occupies less than 1% of the culture volume. Fourth, the efficiency of cell seeding onto microcarriers is suboptimal, with a significant proportion of cells failing to attach. Fifth, harvesting cells from microcarriers is a complex and costly process, typically involving enzymatic dissociation followed by the separation of cells from the microcarriers. Sixth, animal cells have a limited fold expansion per passage, necessitating multiple passages and the use of several seed bioreactors to reach the desired cell numbers, which significantly increases labor requirements and production costs. Lastly, although STRs with volumes up to 20,000 liters are used in the biopharmaceutical industry for culturing protein-producing cells such as CHO cells, growing animal cells for cultivated meat in large STRs remains challenging. Shear stress is not uniformly distributed in STRs, and as reactor size increases, a greater proportion of cells are damaged or killed by shear forces. Moreover, large bioreactors require aeration to supply sufficient oxygen; however, the gas bubbles themselves can cause additional cell death.

[0516] Pneumatically agitated bioreactors, including airlift and bubble column bioreactors, also have potential for cultured meat production, (de Souza Vandenberghe et al., 2024; Li et al., 2020; Manzoki et al., 2024) These systems utilize air bubbles for both agitation and aeration, resulting in lower shear stress and reduced cell death compared to mechanically agitated systems. However, aside from this advantage, they share many of the same limitations as STRs, including cell aggregation, limited volumetric yield, and difficulties in cell harvesting. Suspended cells often adhere to the surface of rising bubbles. When these bubbles reach the surface and burst, the resulting forces can damage cells. (Humbird, 2021)

[0517] In addition to these bioreactors, several specialized bioreactors have been explored. Wave bioreactors, which employ a rocking motion to mix media in disposable bags, provide low- shear environments and enhanced sterility, (de Souza Vandenberghe et al., 2024; Kim et al., 2025; Manzoki et al., 2024) Nevertheless, they are constrained by limited scalability and suboptimal homogenization efficiency. Hollow fiber bioreactors, which use semipermeable membranes to mimic vascular systems, can support high cell densities and continuous medium perfusion, (de Souza Vandenberghe et al., 2024; Manzoki et al., 2024; Nie et al., 2025) However, cells become entrapped within or between the hollow fibers, making cell harvesting extremely difficult. Moreover, scaling up these systems remains a significant challenge. Fluidized bed bioreactors suspend microcarriers in an upward fluid flow, providing effective mixing with low shear stress and maintaining high cell viability, (de Souza Vandenberghe et al., 2024; Manzoki et al., 2024) However, their working volumes are limited, and spatial heterogeneity can become problematic atAttorney Docket No. 0073605-001131 larger scales. Fixed or packed bed bioreactors, in which cells grow on immobilized matrices, are primarily used for the differentiation phase, (de Souza Vandenberghe et al., 2024; Manzoki et al., 2024) While they offer stable microenvironments conducive to cell maturation, they suffer from limitations in mass and heat transfer at scale and present considerable challenges for efficient cell harvesting.

[0518] In short, scaling up animal cell production using current bioreactor technologies remains a significant challenge, underscoring the need for novel and enabling cell culture systems. An ideal platform should support multilayer or 3D cell growth to enhance volumetric yield, minimize shear stress and cell aggregation, enable high seeding efficiency and facile cell harvest, support high expansion fold per passage to reduce passaging, and allow for easy scalability. Previously, our lab developed a new cell culture system that cultivates cells in hollow, microscale alginate hydrogel microtubes (AlgTubes). (Li et al., 2018a, 2018b; Lin et al., 2019a, 2019c, 2019b, 2018a, 2018b; Liu et al., 2023; Wang and Lei, 2019; Wang et al., 2021) It has all the aforementioned characteristics. When culturing human pluripotent stem cells, we have achieved up to a 4000-fold expansion per passage and a volumetric yield of 5 x 108cells / mL, which is -250 times the current state-of-the-art. Our modeling shows that high cell expansion per passage and high cell density have a profound impact on large-scale cell production (Li et al., 2018a, 2018b; Lin et al., 2019a, 2019c, 2019b, 2018a, 2018b; Liu et al., 2023; Wang and Lei, 2019; Wang et al., 2021), as they can significantly reduce the culture volume, labor, reagent costs, equipment needs, facility space, and overall manufacturing expenses. These results highlight the transformative potential of AlgTubes.

[0519] However, the original AlgTubes system does not support the growth of anchoragedependent cells, as mammalian cells lack surface receptors for alginate polymers. To extend the applicability of this transformative technology to cultured meat production, further engineering is required. In this study, we developed a simple method to conjugate arginine-glycine-aspartic acid (RGD) peptides, ligands for integrin receptors, to alginate polymers. The resulting second-generation AlgTubes have abundant RGD motifs, enabling robust cell adhesion. Using these modified AlgTubes, we successfully expanded mouse (C2C12) and quail (QM7) myoblasts, achieving cell densities exceeding 1.0* 108cells / mL - substantially higher than those attainable in conventional stirred-tank bioreactors. Upon induction, the myoblasts could be differentiated into mature myotubes within the microtubes. Our results demonstrate that this scalable platform holds a significant promise for addressing the scale-up challenge currently facing the cultivated meat industry. To the best of our knowledge, this is the first report of RGD-modified AlgTubes and their application in cell culture.Attorney Docket No. 0073605-001131Materials and Methods

[0520] Cell Lines and Materials: Mouse bone marrow stromal cell line (DI, CRL- 12424), mouse myoblast cell line (C2C12, CRL-1772), quail myoblast cell line (QM7, CRL-1962), mouse beige adipocyte cell line (X9, CRL-3282), mouse fibroblast cell line (3T3, CRL-1658) were purchased from ATCC and maintained as instructed by ATCC. Briefly, C2C12, DI, and 3T3 were cultured in DMEM and supplemented with 10% FBS. QM7 were cultured in medium 199 supplemented with 10% TPB and 10% FBS. X9 cells were cultured in DMEM / F12 supplemented with 15% FBS and 2.36 mM L-glutamine. All cell culture medium was refreshed every 2-3 days. To induce C2C12 and QM7 myotube formation, the culture medium was supplemented with 10% horse serum instead of fetal bovine serum.

[0521] Additionally, the following chemicals and reagents were also used for this study: sodium alginate (cat # 194-13321, 80~120cp, Wako Chemicals), sodium hyaluronate: (cat # HA 700K-5, Lifecore Biomedical), tryptose phosphate broth (TPB, cat # 18050039, Life Technologies), Dulbecco's Modified Eagle's Medium (DMEM, cat # 30-2002, ATCC), Medium 199 with Earle's BSS (cat # 12119F, Lonza), DMEM / F12 (cat # 30-2006, ATCC), Fetal Bovine Serum (FBS, cat # 10437028, Gibco), L-Glutamine (cat # 25030081, Gibco), propidium iodide (cat # 195458, MP Biomedicals, LLC), Vybrant multi-color cell-labeling kit (cat # V22889, Molecular Probes), MF20 antibody (cat # MAB4470, R&D systems), RGD peptide (Genscript), Alginate Lyase (cat # A1603, Sigma).

[0522] Modifying Alginates with RGD peptides: 2% alginate was dissolved in 0. IN NaOH and reacted with divinyl sulfone or DVS (1 :3 molar ratio between OH group and DVS) for 15 mins. Dialysis was done to remove excessive DVS. 20% to 30% of the OH groups in alginate polymers were modified with DVS. RGD peptides containing a C-terminal cysteine were reacted with alginate-VS under alkaline conditions to make alginate-RGD. 10% of the modified OH groups were reacted with RGD peptides. Alginate-RGDs were mixed with unmodified alginates to make a 2% alginate solution to process alginate hydrogel microtubes.

[0523] Processing alginate hydrogel microtubes (AlgTubes): A custom-made micro-extruder was used to process AlgTubes. A hyaluronic acid (HA) solution containing single cells and an alginate / alginate-RGD solution were pumped into the central and side channels of the microextruder, respectively, to form coaxial core-shell flows that were extruded into a CaCh buffer (100 mM) to form AlgTubes. Subsequently, the CaCh buffer was replaced with cell culture medium. The core and shell flow rates can be adjusted to control the shell thickness, while the outer diameter ofAttorney Docket No. 0073605-001131 the hydrogel microtubes is determined by the extruder nozzle size. In typical experiments, both the core and shell flow rates were set to 120 pL / min. The extrusion was performed at room temperature inside a BSL-2 biosafety cabinet. Detailed methods for processing AlgTubes are described in previous publications. (Li et al., 2018a, 2018b; Lin et al., 2019a, 2019c, 2019b, 2018a, 2018b; Liu et al., 2023; Wang and Lei, 2019; Wang et al., 2021)

[0524] Culturing cells in AlgTubes: For a typical cell culture, 20 pL of cell solution in AlgTubes was suspended in 3 mL of medium in a 6-well plate. Cells were seeded at a density of 1- 2* 106cells / mL hydrogel microtube space. 2% alginate modified with 1 mM RGD peptide was used. The hydrogel microtube diameter was 200-300 pm with shell thickness around 30-70 pm. To passage cells, the medium was removed, and alginate hydrogels were dissolved with 0.5 mM EDTA and 100 pg / mL alginate lyase for 10 mins at 37 °C. Cell mass was collected by centrifuging at 100 g for 5 mins and treated with 0.25% trypsin-EDTA at 37 °C for 10 mins and dissociated into single cells. Digestion was neutralized by a complete cell culture medium. All cell culture experiments were performed in triplicate and repeated at least three times. Representative data are presented in the Results section.

[0525] Immunocytochemistry: Cells cultured on 2D were fixed with 4% paraformaldehyde (PF A) at room temperature for 15 mins, permeabilized with 0.25% Triton X-100 for 10 mins, and blocked with 5% donkey serum for 1 hr before incubating with primary antibodies in DPBS + 0.25% Triton X-100 + 5% donkey serum at 4 °C overnight. After washing, secondary antibodies were added and incubated at room temperature for 2 hrs, followed by incubating with 10 mM 4', 6- diamidino-2-phenylindole dihydrochloride (DAPI) for 10 mins. Cells were washed three times with DPBS before imaging using fluorescent microscopy. For 3D fibrous cell mass immunostaining, the cell mass was fixed with 4% PFA at 4 °C overnight. 40 pm thick tissue sections were obtained via cryosection. The sections were washed three times with DPBS and stained as 2D cell cultures. Alternatively, the cell mass was directly incubated with the primary antibody at 4 °C for 48 hrs after fixation and washing. After extensive washing, a secondary antibody was added and incubated at 4 °C for 24 hrs, followed by incubating with 10 mM DAPI for 10 mins and imaged with Fluorescent Microscopy.

[0526] Statistical analysis: The data are presented as the mean ± SEM. Data were analyzed using GraphPad Prism 8 statistical software and are presented as mean ± standard error of the mean. P value was determined by one-way analysis of variance (ANOVA) for comparison between three orAttorney Docket No. 0073605-001131 more groups or unpaired two-tailed t-tests for two groups. The significance levels are indicated by p- value, *: p<0.05, **: p<0.01, ***: p<0.001.Results

[0527] The RGD-AlgTube culture system

[0528] We modified alginate hydrogel microtubes (AlgTubes) with RGD peptides so that anchor-dependent cells could be cultured in AlgTubes. Briefly, the sodium alginate was dissolved in 0.1 NNaOH and reacted with divinyl sulfone to generate VS groups on the alginate polymers following published protocols. (Yu and Chau, 2012) The RGD peptides with free-SH groups then reacted with these VS groups via Michael addition reaction (FIG. 18A). Alginate-RGD was mixed with unmodified alginate to produce a 2% alginate solution with the final 1 mM RGD concentration, which was used to process AlgTubes. To process AlgTubes, a cell solution and an alginate-RGD solution were pumped into the central and side channels of a custom-made micro-extruder, respectively, to form a coaxial core-shell flow that was extruded through a nozzle and into a CaCL buffer (100 mM) (FIGS. 18B and 18C). Ca21ions crosslinked the shell alginate flow in seconds to form an alginate hydrogel microtube. Subsequently, cells were grown in the microtubes suspended in the cell culture medium.

[0529] The AlgTubes system is engineered to provide cells with a supportive microenvironment (FIG. 18D). First, RGD peptides enable cell attachment to the substrate, promoting proliferation.Second, the hydrogel shell permits the free passing of oxygen, nutrients, and macromolecules (up to 1,000 kDa), as shown in our previous studies. (Li et al., 2018a) Third, the hydrogel microtubes prevent the formation of large cell clumps. They confine the radial diameter of cell masses within the diffusion limit (-400 pm), thereby maintaining efficient mass transport throughout the culture period. The diffusion limit in 3D cell mass is typically less than 500 pm. (Li et al., 2018a, 2018b; Lin et al., 2019a, 2019c, 2019b, 2018a, 2018b; Liu et al., 2023; Wang and Lei, 2019; Wang et al., 2021) Fourth, the hydrogel microtubes shield cells from shear stresses present in the culture vessel. Fifth, they provide 3D microspaces for cells to grow. Previous studies have demonstrated that such free space can be needed for achieving high cell growth rates and yields. (Li et al., 2018a, 2018b; Lin et al., 2019a, 2019c, 2019b, 2018a, 2018b; Liu et al., 2023; Wang and Lei, 2019; Wang et al., 2021) Sixth, because cells are directly loaded into the microtubes and the cell viability is high, the seeding efficiency approaches 100%. Lastly, alginates are affordable, abundantly available, nontoxic to cells, and can be rapidly crosslinked with Ca2+ions to produce AlgTubes using scalable extrusion technology. (Yong and Mooney, 2012) The resulting AlgTubes are mechanically andAttorney Docket No. 0073605-001131 chemically stable for months, suitable for large-scale and long-term cultures. (Li et al., 2018a) AlgTubes can be easily dissolved with cell-compatible EDTA to release the cells, and they are optically transparent, allowing for real-time monitoring of cell growth. (Li et al., 2018a)

[0530] C2C12 expansion in AlgTubes

[0531] We cultured C2C12 in AlgTubes (FIG. 19). Day 0 image showed that the seeding density was very low (1 x 106cells / mL hydrogel microtube spaces). Only a few cells could be found in the amplified image on day 0. A low cell seeding density allows a large expansion fold per passage and is needed for industrial cell production. After 24 hrs, all cells attached to the inner surface of the hydrogel microtube with no or minor cell death. The loading efficiency is close to 100%. On day 4, cells formed a confluent monolayer. On day 7, multilayer cell masses were seen. The dark-field image on day 7 showed white cell mass, which indicates 3D multilayer cell masses. After 14 days, the dark-field images showed extensive 3D white cell masses, and some myotubes could be found in phase images. Some locations of the microtubes became bent (FIG. 19A) after 14 days, which indicates that the cell contraction force exceeded the strength of the hydrogel microtube.

[0532] Live / Dead cell staining on day 19 showed that most were live cells (FIG. 19B). The cell quantification shows that C2C12 cells reached 6.4 xlO7and l.l *108cells / mL hydrogel microtube space on day 10 and day 19, corresponding to a doubling time of 40 hrs and 67 hrs, respectively (FIG. 19C). The released day 19 cells had healthy morphology. There were some large cell aggregates (FIG. 19D), which might be the contracted myotubes. Immunostaining on myofiber (MF20) confirmed the existence of a few myotubes (FIG. 19D). These results are in line with the findings of the literature that some C1C12 cells spontaneously differentiate to form myotubes at high density, even in the expansion medium.

[0533] C2C12 differentiation in AlgTubes

[0534] Next, studies were performed to determine whether C2C12 cells could be differentiated into myotubes. Cells were expanded for 7 days in AlgTubes before initiating differentiation (FIG. 20). Myotubes significantly increased after six days of differentiation (FIG. 20A). Most cells were alive, as determined by the Live / Dead cell staining assay (FIG. 20B). After differentiation for 12 days, the volumetric yield reached 1.2x lO8 / mL. Immunostaining was applied to evaluate the formed myotubes. The staining confirmed the 3D cell masses in the hydrogel microtubes. There was some unfilled space at the microtube core (FIGS. 20C and 20D). A large percentage of cells, but not all, were MF20 positive (FIG. 20C). The myotubes were large and aligned along the hydrogel microtubes. Cells also expressed MyoD and PAX7, confirming the myo-cell lineage (FIG. 20D).Attorney Docket No. 0073605-001131

[0535] C2C12 and DI co-culture

[0536] Meat tissues contain not only myocytes, but also stromal cells (e.g., fibroblasts) and fat cells. It was further investigated whether myoblasts can be expanded and differentiated in the presence of stromal cells and whether the stromal cells can enhance cell viability and yield. C2C12 cells and mouse DI cells were co-cultured in AlgTubes (FIG. 21). DI cells are multipotent mouse bone marrow stromal precursors that have fibroblast-like characteristics. DI cells were pre-stained with the fluorescent cell-labeling dye DIO (green). C2C12 cells were seeded at l *106cells / mL, along with 1 * 105DI cells / mL in the hydrogel microtubes. After 24 hrs, the cells attached to the hydrogel microtubes and spread. By day 4, a confluent monolayer had formed on the inner surface. By day 7, significant cell growth resulted in the formation of multilayered cell masses (FIG. 21A). The presence of white cell masses in the dark-field image (FIG. 21 A) confirmed the formation of 3D multilayered structures. After 14 days of culture, extensive 3D cell masses were observed in the hydrogel microtubes, along with some myotubes. To prevent DI cells from dominating the culture, they were limited to 10% of the total cell population. Fluorescent imaging from day 0 to day 7 showed that DI cells did not exhibit a growth advantage over C2C12 cells (FIG. 21B). Cells maintained healthy morphologies when harvested and digested into single cells (FIG. 21C). Immunostaining for myofibers (MF20) confirmed the presence of small numbers of myotubes, similar to C2C12 monoculture.

[0537] It was further investigated whether C2C 12 cells could undergo differentiation in the presence of DI cells (FIG. 22). After 12 days of differentiation, immunostaining revealed that most cells were MF20-positive, which confirms that DI cells did not inhibit C2C12 differentiation (FIG. 22B). Cell counting showed that co-culture increased volumetric yield, particularly under differentiation conditions (FIG. 22C). In the expansion medium, C2C12 cells reached approximately l. l * 108cells / mL of hydrogel microtube space on day 19. Co-culturing with DI cells increased the yield to 1.3>< 108cells / mL. In the differentiation medium, C2C12 cells alone reached 1.2>< 108cells / mL on day 19, while co-culturing with DI cells boosted the yield to 1.6x l08cells / mL. Therefore, adding DI could improve the culture density, but not substantially. It should be noted that cell counting was performed using the Countess II cell counter. The actual yields are believed to be higher than the reported values, since the cell counter excluded large cell aggregates, which were frequently observed on day 19.

[0538] QM7 expansion in AlgTubesAttorney Docket No. 0073605-001131

[0539] To confirm the broad applicability of RGD-AlgTubes for culturing animal cells, myoblasts were cultured from a second species, quail (QM7) (FIG. 23). After 24 hrs, cells had attached to the hydrogel microtube walls and exhibited a fibroblast-like morphology. By day 3, cells had expanded significantly, though a confluent monolayer had not yet formed. By day 6, multilayered cell masses were observed (FIG. 23 A); however, some areas of the hydrogel's inner surface remained uncovered (FIG. 23 A). This behavior differed from C2C12 cells (FIG. 19A), which first formed a confluent monolayer before developing 3D cell masses. These findings suggest that quail myoblasts may have stronger cell-to-cell interactions but weaker adhesion to the hydrogel matrix than C2C12 cells. By day 9, dark-field images revealed extensive 3D white cell masses (FIG. 23 A). Dense cell aggregates became more prominent by day 12 (FIG. 23 A). Quantification showed that QM7 cell yields on days 11 and 18 were approximately 1.2* 108cells / mL (FIG. 23B).

[0540] Live / Dead staining on day 11 indicated that most released cells were viable, with only a few dead cells observed (FIG. 24A). Staining of the whole-cell fiber revealed that dead cells were primarily located within dense multilayered regions. By day 18, Live / Dead staining showed increased cell death, particularly within large cell aggregates (FIG. 24B). Despite continued cell proliferation between days 11 and 18, the overall yield remained similar due to the rise in cell death (FIG. 23B). Compared to C2C12 cells, QM7 cultures exhibited higher levels of cell death by day 18, likely due to their weaker adhesion to the hydrogel and tendency to form aggregates.

[0541] These results demonstrate that C2C12 and QM7 cells behave differently in AlgTubes. While C2C12 cells formed a confluent monolayer before developing 3D structures, quail cells preferentially formed 3D aggregates. To determine whether this difference was due to the AlgTubes or an intrinsic characteristic of the cells, C2C12 and QM7 cells were cultured and differentiated in traditional 2D culture dishes. By day 5, both cell types had formed myotubes. By day 11, C2C12 cells had generated more myotubes, whereas quail myotubes exhibited an aggregate morphology. These findings confirm that AlgTubes did not alter the intrinsic properties of the cells.

[0542] Quail cell differentiation in AlgTubes

[0543] QM7 cell differentiation was further examined in AlgTubes. Quail cells were first expanded for six days, reaching 80-90% confluence before initiating differentiation (FIG. 25). During differentiation, cells continued to proliferate and formed 3D cell masses. Myotubes became visible after three days of differentiation (FIG. 25A). Live / Dead staining revealed that dead cells were primarily located within large, dense aggregates (FIG. 25B). Immunostaining confirmed theAttorney Docket No. 0073605-001131 presence of myotubes aligned along the hydrogel microtubes. Additionally, the myotubes expressed MyoD and PAX7 (FIG. 25C).

[0544] QM7 and 3T3 cell co-culture

[0545] Like C2C12, we performed a co-culture of QM7 and DI cells, yielding comparable results. QM7 cells were further co-cultured with mouse 3T3 fibroblasts in AlgTubes (FIG. 26). After 24 hrs, cells attached to the hydrogel microtubes with minimal cell death. Dense aggregates developed by day 12 and continued to expand. By day 18, Live / Dead staining indicated dead cells primarily localized within large, dense aggregates (FIG. 26B).

[0546] Differentiation was initiated after six days of QM7 and 3T3 co-culture in AlgTubes. Myotubes appeared after six days of differentiation (FIG. 27 A), accompanied by the formation of dense aggregates. By day 18, Live / Dead staining still showed substantial cell death within these aggregates (FIG. 27B). Immunostaining confirmed that many cells were MF20-positive, verifying myotube formation. We quantified the cell yield under different conditions (FIG. 27C). Overall, QM7 cells in AlgTubes achieved a final yield of over 1.1 x lO8cells / mL. D I co-culture improved the yield to 1.8* 108cells / mL (FIG. 27D).

[0547] X9 expansion in AlgTubes

[0548] Adipocytes are a crucial component of meat composition. To assess whether the system described herein could support the production of pre-adipocytes or adipocytes, mouse pre-adipocyte X9 cells were cultured (FIG. 28). X9 cells were seeded at a low density (1 x 106cells / mL). After 24 hrs, most cells were attached to the hydrogel microtube walls, with minimal cell death. Unlike C2C12 and QM7 cells, X9 cells expanded very slowly. Cell numbers on day 3 remained similar to day 1, and a confluent monolayer had not yet formed even after 10 days. By day 13, most areas had reached confluence, though only a few multilayered cell masses were observed (FIG. 28A). Even after 19 days of culture, most of the hydrogel microtubes' inner surfaces remained covered by a single layer of X9 cells, with few 3D cell masses detected. The final yield of X9 cells was 3.5x l07cells / mL on day 19 (FIG. 28B). Live / Dead staining confirmed that most cells remained viable (FIG. 28C).Discussion

[0549] Cultured meat offers significant potential to meet the growing global demand for meat in a more sustainable and ethical manner. For this emerging industry to succeed, large-scale cell production needs to be achieved at a cost comparable to that of traditional meat products. (Bellani et al., 2020; de Souza Vandenberghe et al., 2024; Manzoki et al., 2024; Negulescu et al., 2023)Attorney Docket No. 0073605-001131However, current bioreactor technologies fall short in meeting both the scale and cost-efficiency requirements. AlgTubes, with their ability to support high cell densities (up to 108cells / mL), enable 100-fold expansion per passage, and support both stem cell proliferation and differentiation, present a promising solution.

[0550] Recent techno-economic modeling and assessments have highlighted that production batch size and cell culture density significantly influence production costs. (Humbird, 2021; Negulescu et al., 2023; Risner et al., 2021) One analysis modeled the production of 100 million kilograms of beef cells using bioreactors of varying sizes: 42,000-liter stirred tank reactors (STR), 211 ,000-liter STRs, and 262,000-liter airlift reactors (ALR). (Negulescu et al., 2023) The model assumes a cell culture density of 1 x 107cells / mL and a five-fold expansion per seed bioreactor (or passage), requiring five to six passages to reach the target cell quantity. The results show that the cost of goods (COGs) per kilogram of cultured meat is approximately $35 for the 42K STR, $25 for the 21 IK STR, and $17 for the 262K ALR. The cost reduction using large bioreactors is primarily attributed to the economies of scale, as larger bioreactors require significantly lower capital expenditures (CAPEX) and operating expenses (OPEX) per unit of product. (Negulescu et al., 2023)

[0551] Higher cell density allows for more cell mass per unit volume of culture medium or bioreactor, which is crucial for cost efficiency. (Humbird, 2021) Since culture medium is a significant cost driver in cell-based meat production, increasing yield per liter directly reduces the cost per kilogram of meat. Moreover, cultivating more cells within the same bioreactor volume reduces the number of bioreactors required to reach target production levels, significantly lowering both CAPEX and OPEX. Currently, animal cells are typically cultured as monolayers on microcarriers in stirred-tank bioreactors. These systems often lead to cell clumping and expose cells to considerable shear stress from agitation and aeration. As a result, cell culture densities remain low, typically around 106cells / mL or less, and culture volumes are limited. (Bellani et al., 2020; de Souza Vandenberghe et al., 2024; Manzoki et al., 2024; Negulescu et al., 2023) AlgTubes can shield cells from shear stress and prevent the formation of large aggregates. The microenvironment within AlgTubes supports the formation of 3D cell masses, enabling culture densities of up to l *108cells / mL, which is an order of magnitude higher than those typically achieved in STRs. The data presented herein showed that the microtubes were not fully filled (FIG. 20C). In previous studies culturing human pluripotent stem cells in AlgTubes, densities as high as 5 x 108cells / mL were achieved when the microtubes were filled, which suggests further potential for optimization.Attorney Docket No. 0073605-001131

[0552] The high expansion per passage offered by AlgTubes can also cut production costs. As demonstrated in a recent study (Negulescu et al., 2023), current STRs require cells to be passaged and transferred to larger bioreactors after only a 5-fold expansion, making the process labor- intensive and costly. In contrast, AlgTubes support up to 100-fold expansion per passage, drastically reducing the number of passages and seed bioreactors needed.

[0553] Moreover, AlgTubes enable both cell expansion and differentiation, further lowering production costs. Traditional STR-based processes separate these stages: cells are first expanded on microcarriers, then harvested and transferred to scaffolds in a second bioreactor for differentiation into muscle tissue. (Bellani et al., 2020; de Souza Vandenberghe et al., 2024; Manzoki et al., 2024; Negulescu et al., 2023) This multi-step workflow is complex and resource-intensive. AlgTubes streamline the process by enabling cells to proliferate and differentiate into muscle tissue without the need for additional scaffolds or transfers, thereby simplifying the bioprocess and reducing costs.

[0554] Additionally, AlgTubes can be dissolved in just five minutes using 0.5 mM EDTA — a biocompatible buffer — enabling efficient and gentle harvesting of the final product. This eliminates the need for costly enzymes and microcarrier separation steps associated with conventional STRs. (Bellani et al., 2020; de Souza Vandenberghe et al., 2024; Manzoki et al., 2024; Negulescu et al., 2023) The resulting fibrous microtissues can be consumed directly or used as modular building blocks to assemble larger tissue constructs, as demonstrated in our previous work. (Lin et al., 2017)

[0555] Although this study demonstrates the strong potential of AlgTubes for cultured meat production, it also has several limitations that should be addressed in future research. First, it was observed that cell growth rates in AlgTubes are slower compared to those in 2D flasks. Specifically, the doubling time of C2C12 cells in 2D flasks is approximately 24 to 40 hrs, whereas in AlgTubes it extends to around 48 hrs during the exponential growth phase (FIG. 19C). This difference may be attributed to the lower stiffness of the hydrogel matrix in AlgTubes compared to the rigid plastic surfaces of flasks, as previous studies have shown that cells generally proliferate more rapidly on stiffer substrates. (Yi et al., 2022) Additionally, we noticed that AlgTubes tend to bend during the later stages of culture (FIG. 19A). While the exact cause remains unclear, it is speculated that myoblasts may exert strong contractile forces that deform the microtubes. Future studies should systematically investigate how various engineering parameters of AlgTubes, such as microtube diameter, wall thickness, hydrogel stiffness, and RGD peptide concentration, affect cell growth rate and yield. Optimizing these parameters will be essential for maximizing both cell proliferation and culture density.Attorney Docket No. 0073605-001131

[0556] Second, the current data were obtained using small-scale cultures. Future research should focus on scaling up AlgTubes for industrial-scale cell production. Given their ability to protect cells from shear stress, AlgTubes are well-suited for use in scalable systems such as WAVE, bubble column, or airlift bioreactors, as suggested by our previous work with human pluripotent stem cells. (Li et al., 2018a) Third, the present study utilized established cell lines. Encouragingly, unpublished data from the inventors’ lab indicate that AlgTubes can support both cell types. Fourth, while we demonstrated that cells could grow as 3D masses within AlgTubes, the underlying biological mechanisms remain unclear and warrant further investigation. Lastly, the alginates used in the present system are modified with vinyl sulfone groups. Although AlgTubes are dissolved and alginates are removed during product harvesting, concerns remain regarding the potential food safety risks associated with residual vinyl sulfone groups. Future studies should develop methods to quantify any residual vinyl sulfone and establish effective cleaning procedures to ensure that residual levels remain below regulatory thresholds.Conclusion

[0557] In summary, the present example demonstrates, for the first time, a culture system capable of growing animal cells as 3D masses. AlgTubes outperform current state-of-the-art cell culture methods in terms of cell yield. By achieving cell densities exceeding 100 million cells per milliliter, AlgTubes can dramatically reduce culture volume, labor, reagent consumption, equipment requirements, facility space, and overall manufacturing costs.References cited in Example 3

[0558] 1. Aiking, H., 2014. Protein production: Planet, profit, plus people? American Journal ofClinical Nutrition 100, 483-489.

[0559] 2. Aleksandrowicz, L., Green, R., Joy, E.J.M., Smith, P., Haines, A., 2016. The impacts of dietary change on greenhouse gas emissions, land use, water use, and health: A systematic review. PLoS One 11, 1-16.

[0560] 3. Bellani, C.F., Ajeian, J., Duffy, L., Miotto, M., Groenewegen, L., Connon, C.J., 2020.Scale-Up Technologies for the Manufacture of Adherent Cells. Front. Nutr. 7, 575146.

[0561] 4. Bodiou, V., Cristini, N., De Cristofaro, L., Pareek, T., Rajagopal, V., Verrougstraete,L., Heinrich, J.M., Post, M.J., Moutsatsou, P., 2025. Process intensification of cultivated meat production through microcarrier addition strategy optimisation. Sci. Rep. 15, 1-12.

[0562] 5. Bryant, C.J., 2020. Culture, meat, and cultured meat J. Anim. Sci. 98, 1-7.Attorney Docket No. 0073605-001131

[0563] 6. de Souza Vandenberghe, L.P., de Mello, A.F.M., Biagini, G., de Mattos, P.B.G.,Piazenski, I.N., Candelario, J.P.M., Soccol, C.R., 2024. Bioreactors for Cultivated Meat Production. Cultivated Meat: Technologies, Commercialization and Challenges. 107-130.

[0564] 7. Gerber, P.J., Mottet, A., Opio, C.I., Falcucci, A., Teillard, F., 2015. Environmental impacts of beef production: Review of challenges and perspectives for durability. Meat Sci. 109, 2- 12.

[0565] 8. Guan, X., Pan, Z., Li, M., Shen, J., Sun, Y., Yu, C., Fei, Z., Ma, Z , Zhou, J., Chen, J.,2025. Production of cultivated meat with stably proliferated porcine muscle stem cells and edible scaffolds. Future Foods. 11 , 100618.

[0566] 9. Hanga, M.P., de la Raga, F.A., Moutsatsou, P., Hewitt, C.J., Nienow, A.W., Wall, I.,2021. Scale-up of an intensified bioprocess for the expansion of bovine adipose-derived stem cells (bASCs) in stirred tank bioreactors. Biotechnol. Bioeng. 118, 3175-3186.

[0567] 10. Hong, T.K., Shin, D.M., Choi, J., Do, J.T., Han, S.G., 2021. Current issues and technical advances in cultured meat production: A review. Food Sci. Anim. Resour. 41, 335-372.

[0568] 11. Humbird, D., 2021. Scale-up economics for cultured meat. Biotechnol. Bioeng. 118,3239-3250.

[0569] 12. Kang, D.H., Louis, F., Liu, H., Shimoda, H., Nishiyama, Y., Nozawa, H, Kakitani,M., Takagi, D., Kasa, D., Nagamori, E., Irie, S., Kitano, S., Matsusaki, M., 2021. Engineered whole cut meat-like tissue by the assembly of cell fibers using tendon-gel integrated bioprinting. Nat.Commun. 12, 5059.

[0570] 13. Kim, M., Harris, D.M., Cimpeanu, R., 2025. A simulation modeling framework for fluid motion and transport in a rocking bioreactor with application to cultivated meat production. ArXiv 2504.05421.

[0571] 14. Li, Q., Lin, H , Du, Q., Liu, K., Wang, O , Evans, C , Christian, H., Zhang, C., Lei,Y., 2018a. Scalable and physiologically relevant microenvironments for human pluripotent stem cell expansion and differentiation. Biofabrication. 10, 025006.

[0572] 15. Li, Q., Lin, H., Rauch, J., Deleyrolle, L.P., Reynolds, B.A., Viljoen, H.J., Zhang, C.,Zhang, C., Gu, L., Van Wyk, E., Lei, Y., 2018b. Scalable culturing of primary human glioblastoma tumor-initiating cells with a cell-friendly culture system. Sci. Rep. 8, 3531.

[0573] 16. Li, X., Zhang, G., Zhao, X., Zhou, J., Du, G., Chen, J., 2020. A conceptual air-lift reactor design for large scale animal cell cultivation in the context of in vitro meat production. Chem. Eng. Sci. 211, 115269.Attorney Docket No. 0073605-001131

[0574] 17. Lin, H., Du, Q , Li, Q., Wang, O., Wang, Z., Elowsky, C., Liu, K., Zhang, C., Chung,S., Duan, B., Lei, Y., 2019a. Manufacturing human pluripotent stem cell derived endothelial cells in scalable and cell-friendly microenvironments. Biomater. Sci. 7, 373-388.

[0575] 18. Lin, H, Du, Q., Li, Q., Wang, O., Wang, Z., Liu, K., Elowsky, C , Zhang, C., Lei,Y., 2018a. Hydrogel-Based Bioprocess for Scalable Manufacturing of Human Pluripotent Stem Cell- Derived Neural Stem Cells. ACS Appl Mater Interfaces. 10, 29238-29250.

[0576] 19. Lin, H, Li, Q., Du, Q., Wang, O., Wang, Z , Akert, L., Carlson, M.A., Zhang, C.,Subramanian, A., Zhang, C., Lunning, M., Li, M., Lei, Y., 2019b. Integrated generation of induced pluripotent stem cells in a low-cost device. Biomaterials. 189, 23-36.

[0577] 20. Lin, H., Li, Q., Lei, Y., 2017. Three-dimensional tissues using human pluripotent stem cell spheroids as biofabrication building blocks. Biofabrication. 9, 025007.

[0578] 21. Lin, H, Li, Q., Wang, O., Rauch, J., Harm, B., Viljoen, H.J., Zhang, C , Van Wyk,E., Zhang, C., Lei, Y., 2018b. Automated expansion of primary human T cells in scalable and cellfriendly hydrogel microtubes for adoptive immunotherapy. Adv. Healthc. Mater. el701297.

[0579] 22. Lin, H, Qiu, X., Du, Q., Li, Q., Wang, O., Akert, L., Wang, Z., Anderson, D., Liu,K., Gu, L., Zhang, C., Lei, Y., 2019c. Engineered Microenvironment for Manufacturing Human Pluripotent Stem Cell-Derived Vascular Smooth Muscle Cells. Stem Cell Reports 84-97.

[0580] 23. Liu, Q., Liu, Z., Gu, H., Ge, Y., Wu, X., Zuo, F., Du, Q., Lei, Y., Wang, Z., Lin, H,2023. Comparative study of differentiating human pluripotent stem cells into vascular smooth muscle cells in hydrogel-based culture methods. Regen. Ther. 22, 39-49.

[0581] 24. Liu, Y., Gao, A., Wang, T., Zhang, Y., Zhu, G., Ling, S., Wu, Z., Jin, Y., Chen, H.,Lai, Y., Zhang, R., Yang, Y., Han, J., Deng, Y., Du, Y., 2025. Growing meat on autoclaved vegetables with biomimetic stiffness and micro-patterns. Nat. Commun. 16, 161.

[0582] 25. Ma, T., Ren, R., Lv, J., Yang, R., Zheng, X., Hu, Y„ Zhu, G„ Wang, H., 2024.Transdifferentiation of fibroblasts into muscle cells to constitute cultured meat with tunable intramuscular fat deposition. Elife. 13, e98918.

[0583] 26. Manzoki, M.C., de Mello, A.F.M., Martinez-Burgos, W.J., da Silva Vale, A.,Biagini, G., Piazenski, I.N., Soccol, V.T., Soccol, C.R., 2024. Scaling-Up of Cultivated Meat Production Process. Cultivated Meat: Technologies, Commercialization and Challenges. 241-264.

[0584] 27. Martins, B., Bister, A., Dohmen, R.G.J., Gouveia, M.A., Hueber, R., Melzener, L.,Messmer, T., Papadopoulos, J., Pimenta, J., Raina, D., Schaeken, L., Shirley, S., Bouchet, B.P.,Attorney Docket No. 0073605-001131Flack, J.E., 2024. Annual Review of Animal Biosciences Advances and Challenges in Cell Biology for Cultured Meat. Annu. Rev. Anim. Biosci. 12, 345-368.

[0585] 28. Negulescu, P.G., Risner, D., Spang, E.S., Sumner, D., Block, D., Nandi, S.,McDonald, K.A., 2023. Techno-economic modeling and assessment of cultivated meat: Impact of production bioreactor scale. Biotechnol. Bioeng. 120, 1055-1067.

[0586] 29. Nie, M., Shima, A., Nie, M., 2025. Scalable tissue biofabrication via perfusable hollow fiber arrays for cultured meat applications. Trends Biotechnol. S0167-7799(25)00085-X.

[0587] 30. Norris, S.C.P., Kawecki, N.S., Davis, A.R., Chen, K.K., Rowat, A.C., 2022.Emul si on-templ ated microparticles with tunable stiffness and topology: Applications as edible microcarriers for cultured meat. Biomaterials. 287, 121669.

[0588] 31. Ozsolak, F., Milos, P.M., 2023. Plant-Based Meat Alternatives: Technological,Nutritional, Environmental, Market, and Social Challenges and Opportunities. Nutrients. 2 15, 452.

[0589] 32. Risner, D., Li, F., Fell, J.S., Pace, S.A., Siegel, J.B., Tagkopoulos, I., Spang, E.S.,2021. Preliminary techno-economic assessment of animal cell-based meat. Foods. 10, 3.

[0590] 33. Ryschawy, J., Dumont, B., Therond, O., Donnars, C., Hendrickson, J., Benoit, M.,Duru, M., 2019. Review: An integrated graphical tool for analysing impacts and services provided by livestock farming. Animal. 13, 1760-1772.

[0591] 34. Sanaki-Matsumiya, M., Villava, C., Rappez, L., Haase, K., Wu, J., Ebisuya, M.,2024. Self-organization of vascularized skeletal muscle from bovine embryonic stem cells. bioRxiv 2024.03.22.586252.

[0592] 35. Stephanie Kawecki, N., Chen, K.K., Smith, C.S., Xie, Q., Cohen, J.M., Rowat, A.C.,2024. Scalable Processes for Culturing Meat Using Edible Scaffolds. Annu. Rev. Food Sei. Technol. 15, 241-264.

[0593] 36. Treich, N., 2021. Cultured Meat: Promises and Challenges. Environ. Resour. Econ.(Dordr) 79, 33-61.

[0594] 37. Tzimorotas, D., Solberg, N.T., Andreassen, R.C., Moutsatsou, P., Bodiou, V.,Pedersen, M.E., Ronning, S.B., 2023. Expansion of bovine skeletal muscle stem cells from spinner flasks to benchtop stirred-tank bioreactors for up to 38 days. Front. Nutr. 10.

[0595] 38. Wang, O., Lei, Y., 2019. Creating a cell-friendly microenvironment to enhance cell culture efficiency. Cell Gene Ther. Insights 5, 341-350.Attorney Docket No. 0073605-001131

[0596] 39. Wang, Z , Zuo, F., Liu, Q., Wu, X , Du, Q., Lei, Y., Wu, Z , Lin, H, 2021.Comparative Study of Human Pluripotent Stem Cell-Derived Endothelial Cells in Hydrogel-Based Culture Systems. ACS Omega 6, 6942-6952.

[0597] 40. Willett, W ., Rockstrom, J., Loken, B., Springmann, M., Lang, T., Vermeulen, S.,Garnett, T., Tilman, D., DeClerck, F., Wood, A., Jonell, M., Clark, M., Gordon, L.J., Fanzo, J., Hawkes, C., Zurayk, R., Rivera, J. A., De Vries, W ., Majele Sibanda, L., Afshin, A., Chaudhary, A., Herrero, M., Agustina, R., Branca, F., Lartey, A., Fan, S., Crona, B., Fox, E., Bignet, V., Troell, M., Lindahl, T., Singh, S., Cornell, S.E., Srinath Reddy, K., Narain, S., Nishtar, S., Murray, C.J.L., 2019. Food in the Anthropocene: the EAT-Lancet Commission on healthy diets from sustainable food systems. The Lancet 393 , 447-492.

[0598] 41. Yen, F.C., Glusac, J., Levi, S., Zernov, A., Baruch, L., Davidovich-Pinhas, M.,Fishman, A., Machluf, M., 2023. Cultured meat platform developed through the structuring of edible microcarrier-derived microtissues with oleogel-based fat substitute. Nat. Commun. 14, 1-11.

[0599] 42. Yi, B., Xu, Q., Liu, W., 2022. An overview of substrate stiffness guided cellular response and its applications in tissue regeneration. Bioact. Mater. 15, 82-102.

[0600] 43. Yin, H., Wang, L., Hur, S.J., Liu, Y., Cong, P„ Liu, H., Jiang, X., Zheng, H., Xue,C., 2024. Cell-Cultured Fish Meat via Scale-Up Expansion of Carassius auratus Skeletal Muscle Cells Using Edible Porous Microcarriers and Quality Evaluation. J. Agric. Food Chem. 72, 16475- 16483.

[0601] 44. Yong, K., Mooney, DI, 2012. Alginate: properties and biomedical applications.Prog. Polym. Sci. 37, 106-126.

[0602] 45. Yu, Y., Chau, Y., 2012. One-Step “ Click “ Method for Generating Vinyl SulfoneGroups on Hydroxyl-Containing Water-Soluble Polymers. Biomacromolecules 13, 937-942.

[0603] 46. Zhou, X., Zheng, H., Wu, Y , Yin, H., Mao, X., Li, N., Guo, H., Chang, Y., Jiang,X., Ai, Q., Xue, C., 2025. Scalable production of muscle and adipose cell-laden microtissues using edible macroporous microcarriers for 3D printing of cultured fish fillets. Nat. Commun. 16, 1740.

[0604] 47. Zhu, G., Gao, D., Li, L., Yao, Y., Wang, Y., Zhi, M., Zhang, J., Chen, X., Zhu, Q.,Gao, J., Chen, T., Zhang, X., Wang, T., Cao, S., Ma, A., Feng, X., Han, J., 2023. Generation of three-dimensional meat-like tissue from stable pig epiblast stem cells. Nat. Commun. 14, 8163.Example 4: Alternating 2D and 3D Culture Reduces Cell Size and Extends the Lifespan of Placenta- Derived Mesenchymal Stem CellsAttorney Docket No. 0073605-001131

[0605] Mesenchymal stem cells (MSCs) hold great promise for treating a variety of human diseases; however, their clinical translation is hindered by challenges in large-scale expansion while preserving therapeutic potency and maintaining small cell size. Conventional 2D Culture on rigid substrates induces MSC senescence and enlargement, compromising their function and biodistribution. Here, we present an alternating 2D / 3D culture strategy that combines adherent monolayer expansion with transient spheroid formation to mitigate these limitations. Using placenta- derived MSCs, the present example demonstrates that spheroid culture significantly reduces cell size and enhances immunomodulatory function. Optimal spheroid conditions were identified, and extracellular matrix supplementation and chemically defined media further improved cell viability. The alternating 2D / 3D protocol slowed MSC enlargement and senescence over multiple passages while preserving anti-inflammatory activity. To address scalability, we developed RGD- functionalized alginate hydrogel microtubes (AlgTubes) that support dynamic transitions between adherent and spheroid states, enabling the implementation of the alternating culture strategy in a continuous and scalable format. This work demonstrates a promising strategy for MSC manufacturing.Introduction

[0606] Mesenchymal stem cells (MSCs) are multipotent stromal cells characterized by their capacity for self-renewal and differentiation into various mesenchymal lineages, including osteoblasts, chondrocytes, and adipocytes1. Initially identified in the bone marrow, MSCs have since been isolated from diverse tissues, including adipose tissue, umbilical cord, dental pulp, and placenta1. MSCs have garnered significant attention as therapeutics due to their exceptional safety profile and wide-ranging functions, including enhancing tissue repair, promoting angiogenesis, reducing fibrosis, cytoprotection, anti-inflammation, neutralizing reactive oxygen species (ROS), inhibiting NETosis, suppressing T cell activation, promoting Treg differentiation, and polarizing M2 macrophages1. For instance, MSCs have demonstrated the capability to reduce infarct size and improve cardiac function following myocardial infarction in animal models. MSC transplantation enhances angiogenesis, reduces fibrosis, and promotes cardiomyocyte survival2'9. In experimental autoimmune encephalomyelitis (EAE) mice, MSCs reduce central nervous system inflammation and enhance neurological recovery10'15. In rabbit and dog models of osteoarthritis, MSCs reduce cartilage degradation, alleviate pain, and improve joint function16'22. As of March 2025, more than 1,800 clinical studies involving MSCs and their secretome have been registered on clinicaltrials.gov, targeting over 920 medical conditions such as Acute Respiratory Distress Syndrome (ARDS), sepsis,Attorney Docket No. 0073605-001131Graft-versus-Host Disease (GvHD), stroke, spinal cord injury, myocardial infarction, multiple sclerosis, organ transplantation, rheumatoid arthritis, Crohn's, systemic lupus erythematosus, ulcerative colitis and COVID-1923’35. A meta-analysis of 55 randomized clinical studies involving 2696 patients finds that MSCs do not induce significant adverse effects36. No tumorgenicity and procoagulation risks are found36. Thirteen MSC-based therapies have been approved for clinical use worldwide.

[0607] However, MSC therapies face a significant challenge — the difficulty of producing large quantities of MSCs while preserving their functions and maintaining a small cell size. Currently, MSCs are expanded as a monolayer on rigid polymer substrates, including plastic flasks, polymer microcarriers in stirred tank bioreactors (STR), polymer scaffolds in packed bed (PB) bioreactors, and polymer hollow fibers in hollow fiber (HF) bioreactors37’40. In vivo, MSCs reside in a soft, three- dimensional (3D) niche rich in cell-cell and cell-matrix interactions, as well as autocrine and paracrine signaling1. Current cell culture technologies provide a starkly different two-dimensional (2D) stiff microenvironment. For instance, plastic flasks have a Young's modulus of -100,000 kPa, which is far stiffer than natural soft tissues41,42. Using current methods, MSCs rapidly undergo senescence, losing their replicative ability and therapeutic potency43,44. This reason may explain the discrepancy between compelling preclinical data and less effective clinical outcomes34,45’47. While preclinical studies use young, potent MSCs, clinical trials often rely on high-passage MSCs with impaired proliferation and functions.

[0608] MSCs also enlarge during in vitro expansion48’51. A determinant of MSC therapeutic efficacy is its in vivo biodistribution. After systemic administration, MSCs frequently encounter a “first-pass effect,” with most cells trapped in organs such as the lungs, liver, and kidney52,53. Cell size significantly influences this process; larger MSCs are more likely to become lodged in the microvasculature of these organs, impairing their ability to reach target tissues51. Moreover, oversized MSCs may cause microcirculation obstruction, ischemia, or stroke49,54,55. There is a need for new culture strategies that can efficiently expand MSCs without causing cell enlargement and loss of function.

[0609] Research finds that culturing MSCs as spheroids (referred to as spheroid culture) may overcome some of these limitations56’58. Spheroid Culture has been shown to mitigate senescence, preserving a youthful phenotype with smaller cell size, improved survival, increased secretion of trophic factors, and elevated expression of sternness-related genes48,50,59,60. However, due to their anchorage-dependent nature, MSCs do not proliferate effectively in spheroid culture, limiting theirAttorney Docket No. 0073605-001131 utility for large-scale MSC expansion. The present example presents an approach that combines the benefits of both 2D and 3D spheroid culture methods to grow placenta-derived MSCs. Briefly, MSCs are expanded as adherent monolayers in 2D flasks for several days. After each passage, MSCs are transitioned to a non-adherent environment for 24 to 72 hours to form 3D spheroids. For simplicity, this method may be referred to as the “alternating 2D / 3D culture protocol.” It is speculated that spheroid formation following 2D expansion can restore MSC size and function, thereby mitigating cell senescence and enlargement.Methods

[0610] MSC isolation: Full-term human placentas were obtained from ZenBio Inc. Briefly, placentas were washed and cut into approximately 0.5 cm3pieces, which were then partially digested with TrypLE Select solution (Gibco) at 37 °C for 30 minutes (FIG. 29A). Following digestion, 15- 20 tissue pieces were transferred to a 75 cm2tissue culture flask containing 9 mL of EBM-2 complete medium (EBM-2 supplemented with 10% fetal bovine serum and 1% Penicillin- Streptomycin). The flasks were placed in an incubator and left undisturbed for three days to allow the tissue to attach. After this period, the medium was replaced every three days until the cells reached confluence. These cells were designated as passage 0 (P0). P0 cells were either cryopreserved or sub-cultured. Details of MSC isolation and characterization can be found in previous publication61.

[0611] MSC surface marker characterization: MSCs were characterized using the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), which includes antibodies against the positive markers CD90, CD73, and CD105, as well as the negative markers CD45, CD34, CD 11b, CD79A, and HLA-DR. Additionally, the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) was used to assess expression of the positive markers CD44, CD 106, CD 146, and CD 166. Flow cytometric analysis was performed using the BD FACSCanto™ II System.

[0612] MSC differentiation: MSCs were evaluated for their differentiation potential using the Human Mesenchymal Stem Cell Functional Identification Kit (R&D Systems), following the manufacturer's instructions. After 21 days of induction, cells were fixed and stained with an FABP-4 antibody to identify adipocytes and an osteocalcin antibody to identify osteocytes.

[0613] MSC 2D culture: MSCs were seeded into T-25 flasks at a density of 8,000 cells / cm2in 4 mL of EBM-2 complete medium. The culture medium was replaced every three days. Once the cellsAttorney Docket No. 0073605-001131 reached approximately 90% confluency, they were harvested with 0.25% Trypsin and sub-cultured at the same seeding density.

[0614] MSC 3D spheroid culture: Dissociated single MSCs were seeded into 96-well clear, round-bottom, ultra-low attachment microplates (Coming) at densities ranging from 2,000 to 40,000 cells per well in 200 pL of EBM-2 complete medium or other indicated media. The medium was refreshed every three days. To promote spheroid formation, plates were centrifuged at 100 * g for 8 minutes. Spheroids were cultured for the indicated duration. To assess cell viability, propidium iodide was added to the medium and incubated for 30 minutes before imaging with fluorescence microscopy.

[0615] Alternating 2D / 3D Culture: MSCs harvested from 2D culture (25,000 cells per well) were seeded into 96-well clear, round-bottom, ultra-low attachment microplates to form spheroids. After 48 hours, the spheroids were dissociated into single cells, which were then seeded into 2D T- 25 flasks at a density of 8,000 cells / cm2in 4 mb of EBM-2 complete medium. The medium was changed every three days. Once the cells reached approximately 90% confluency, they were harvested and cultured again as spheroids for 48 hours before being replated onto 2D flasks.

[0616] MSC size quantification: Spheroids were harvested and washed twice with PBS. To dissociate them into single cells, 2 mL of 0.25% trypsin-EDTA (Coming) was added and incubated at 37 °C in a water bath for 10 minutes. The enzymatic reaction was stopped by adding 2 mL of EBM-2 complete medium. Cells were then centrifuged at 300 * g for 8 minutes and resuspended in EBM-2 complete medium. Images of the single cells were captured using a Zeiss fluorescence microscope. Cell size was analyzed using ImageJ software using the following procedure:

[0617] Import phase contrast images into ImageJ

[0618] Set the image scale via Analyze — Set Scale

[0619] Adjust threshold using Image — ► Adjust — Color Threshold

[0620] Measure cell area using Analyze — Analyze Particles, setting the circularity range to 0.70-1.00

[0621] Export the results to Excel and calculate cell diameter using the formula:

[0622] R = 2 x R: Cell diameter; S: Area value from Imag

[0623] P-Galactosidase Activity Assay: Senescence-associated P-galactosidase activity was assessed using the CellEvent Senescence Green Detection Kit (Thermo Fisher Scientific), according to the manufacturer's instructions. Cells cultured in 2D or alternating 2D / 3D spheroid conditions were dissociated into single-cell suspensions and seeded into flat-bottom 96-well plates, allowingAttorney Docket No. 0073605-001131 them to adhere overnight. Before staining, cells were washed with PBS and fixed with 2% paraformaldehyde (PF A) in PBS for 10 minutes at room temperature, protected from light. After an additional PBS wash containing 1% BSA, the CellEvent™ Working Solution was prepared by diluting the Senescence Green Probe (1 : 1000) into pre-warmed (37 °C) Senescence Buffer and added to each well (100 pL / well). Plates were sealed and incubated for 2 hours at 37 °C in the absence of CO2, protected from light. Following incubation, wells were washed three times with PBS, counterstained with DAPI, and imaged using a fluorescence microscope.

[0624] Macrophage culture: RAW 264.7 cells (RAW -Dual™ cells, InvivoGen) were cultured in DMEM supplemented with 4.5 g / L glucose, 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum (FBS), 100 pg / mL Normocin, and 1% Penicillin-Streptomycin. Cells were seeded at a density of 1.5 x 104cells / cm2, and the medium was replaced twice a week.

[0625] Macrophage inflammation assay: RAW 264.7 macrophage cells were stimulated in DMEM complete medium containing 100 ng / mL lipopolysaccharide (LPS; O111 :B4, Sigma) and 10 ng / mL murine interferon-gamma (IFN-y; Peprotech). MSCs were co-cultured with RAW 264.7 cells at a ratio of 1 : 10 MSC / RAW. After 24 hours, conditioned media were collected for cytokine analysis using enzyme-linked immunosorbent assays (ELISA). Additionally, the RAW-Dual cells are engineered to express a luciferase gene under the control of an ISG54 minimal promoter, in conjunction with five IFN-stimulated response elements (ISREs). It reports the activation of interferon regulatory factors (IRFs), which contribute to the inflammatory response. The luciferase level is measured using the commercial kit.

[0626] Modifying alginates with RGD peptides: A 2% (w / v) alginate solution (Cat. #194- 13321, 80-120 cP, Wako Chemicals) was prepared by dissolving alginate in 0.1 N NaOH. The solution was then reacted with divinyl sulfone (DVS) at a 1 :3 molar ratio of hydroxyl groups to DVS for 15 minutes. Excess DVS was removed by dialysis. Approximately 20-30% of the hydroxyl groups in the alginate polymer were successfully modified with DVS. To synthesize alginate-RGD, RGD peptides containing a C-terminal cysteine were conjugated to the DVS-modified alginate under alkaline conditions. About 10% of the modified hydroxyl groups were functionalized with RGD peptides. The resulting alginate-RGD was then blended with unmodified alginate to prepare a 2% (w / v) alginate solution, which was used to fabricate alginate hydrogel microtubes.

[0627] Processing alginate hydrogel microtubes (AlgTubes): A custom-made micro-extruder was used to process AlgTubes. A hyaluronic acid (HA) solution containing single cells and an alginate / alginate-RGD solution were pumped into the central and side channels of the microAttorney Docket No. 0073605-001131 extruder, respectively, to form coaxial core-shell flows that were extruded into a CaCh buffer (100 mM) to form AlgTubes. If necessary, AlgTubes were further soaked in 1 mM Poly(ethylene glycol) dithiol (HS-PEG-SH, Mw 3400) for 10 minutes at pH 8.0 to achieve secondary covalent crosslinking through the Michael addition reaction between -SH and -VS. Detailed methods for processing AlgTubes are described in our previous publications62'72.

[0628] Culturing cells in AlgTubes: For standard cell culture, 20 pL of cell-laden AlgTubes were suspended in 2 mb of culture medium in each well of a 6-well plate. Cells were seeded at a density of 1-2* 106cells / mL within the hydrogel microtube space. The microtubes were formed using 2% alginate modified with 1 mM RGD peptide. The resulting hydrogel microtubes had diameters ranging from 200 to 300 pm, with shell thicknesses of approximately 30-70 pm. To detach MSC from the AlgTubes, 1.2 mM free RGD peptides were added to the culture medium.

[0629] Statistical analysis: Experiments were performed in triplicate and repeated with MSCs from two donors. Representative data are presented in the Results section. Data were analyzed using GraphPad Prism 8 statistical software. P value was determined by one-way analysis of variance (ANOVA) for comparison between the means of three or more groups or unpaired two-tailed t-tests for two-group analysis. The signifi...

Claims

Attorney Docket No. 0073605-001131CLAIMS1. A hydrogel microtube for culturing, expanding, differentiating, or manipulating cells, having(i) an inner diameter of at least 20 pm and no greater than 999 pm, preferably at least 100 pm and no greater than 600 pm; and(ii) a wall thickness of at least 1 pm and no greater than 999 pm, preferably at least 10 pm and no greater than 200 pm, more preferably at least 25 pm and no greater than 150 pm; wherein the hydrogel microtube is prepared from one or more of(a) a collagen protein;(b) a blend of an alginate polymer and a collagen protein; and(c) a peptide-functionalized alginate polymer and, optionally, an unfunctionalized alginate polymer; and wherein the hydrogel microtube comprises a cavity configured to accommodate a plurality of cells.

2. The hydrogel microtube according to claim 1, wherein (b) the blend of the alginate polymer and the collagen protein has an alginate polymer-to-collagen protein ratio of 0.01 to 100 by mass, preferably 0.1 to 1.5 by mass, more preferably 0.25 to 0.75 by mass.

3. The hydrogel microtube according to claim 1, wherein the peptide-functionalized alginate polymer comprises an arginine-glycine-aspartic acid (RGD) peptide covalently linked to the alginate polymer.

4. The hydrogel microtube according to any one of claims 1-3, wherein the peptide- functionalized alginate polymer comprises one or more of a peptide-functionalized alginate acid polymer, a peptide-functionalized sodium alginate polymer, a peptide-functionalized methacrylate-modified alginate polymer, and a peptide-functionalized vinyl sulfone-modified alginate polymer.

5. The hydrogel microtube according to any one of claims 1-3, further comprising a coating layer.

6. The hydrogel microtube according to claim 5, wherein the coating layer comprises one or more of an alginate acid polymer layer, a sodium alginate polymer layer, a methacrylate- modified alginate polymer layer, and a vinyl sulfone-modified alginate polymer layer, wherein the vinyl sulfone-modified alginate polymer is optionally peptide-functionalized.Attorney Docket No. 0073605-0011317. The hydrogel microtube according to any one of claims 1-3, wherein the hydrogel microtube further comprises one or more extracellular matrix (ECM) proteins.

8. The hydrogel microtube according to claim 7, wherein the one or more extracellular matrix (ECM) proteins comprise laminin or fibronectin.

9. The hydrogel microtube according to any one of claims 1-3, further comprising one or more of polyethylene glycol and poly(vinyl alcohol).

10. The hydrogel microtube according to any one of claims 1-3, having a circular or polygonal, preferably circular, cross-sectional area.11 . The hydrogel microtube according to any one of claims 1-3, wherein the hydrogel microtube is a truncated hydrogel microtube.

12. The hydrogel microtube according to any one of claims 1-3, wherein at least one end of the hydrogel microtube is open.

13. The hydrogel microtube according to any one of claims 1-3, wherein both ends of the hydrogel microtube are open.

14. A bioreactor system comprising: a plurality of the hydrogel microtubes according to any one of claims 1-3; and a cell-compatible buffer or medium, wherein the plurality of the hydrogel microtubes is dispersed in the cell-compatible buffer or medium.

15. A bioreactor system according to claim 14, wherein the cell-compatible buffer or medium has a pEI from about 7 to about 9, preferably about 7.4.

16. The bioreactor system according to claim 14, wherein the cell-compatible buffer comprises one or more multivalent ions, preferably one or more divalent ions.

17. The bioreactor system according to claim 16, wherein the one or more divalent ions comprise Mg2+, Ca2+, Zn2+, or Ba2+, preferably Ca2+.

18. A method of culturing, expanding, differentiating, or manipulating cells, comprising:(i) extruding a cell solution and a hydrogel -forming solution into a cell-compatible buffer to produce the hydrogel microtube according to any one of claims 1-3;(ii) suspending the hydrogel microtube including cells from the cell solution in a cell culture medium;(iii) culturing, expanding, differentiating, or manipulating the cells under suitable conditions; and(iv) dissolving the hydrogel microtube to release the cells.Attorney Docket No. 0073605-00113119. The method according to claim 18, wherein the cell solution comprises cells selected from the group consisting of embryonic stem cells; mammalian embryonic stem cells; human embryonic stem cells (hESCs); human induced pluripotent stem cells (iPSCs); mammalian induced pluripotent stem cells; mammalian naive pluripotent stem cells; mammalian tissue stem cells; human pluripotent stem cells (hPSCs); cells derived or differentiated from one or more of embryonic stem cells, mammalian embryonic stem cells, human embryonic stem cells (hESCs), human induced pluripotent stem cells (iPSCs), mammalian induced pluripotent stem cells, mammalian naive pluripotent stem cells, mammalian tissue stem cells, and human pluripotent stem cells (hPSCs); mammalian cells reprogrammed from other cell types; mammalian primary cells; human umbilical vein endothelial cells; primary tumor cells; cancer cells; immune cells; T cells; natural killer cells; mammalian cell lines; engineered human and mammalian cells; insect cells; plant cells; and yeast and bacterial cells, wherein the cells are optionally cultured as single cell type, as a mixture of different cell types, or as a mixture of cells from different donors.

20. The method according to claim 18, further comprising coating the hydrogel microtube using a coating solution.

21. The method according to claim 20, wherein the coating solution comprises one or more of an alginate acid polymer, a sodium alginate polymer, a methacrylate-modified alginate polymer, and a vinyl sulfone-modified alginate polymer, wherein the vinyl sulfone-modified alginate polymer is optionally peptide functionalized.

22. The method of claim 18, wherein dissolving the hydrogel microtubes comprises treating the hydrogel microtube using one or more of ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), and an alginate lyase.

23. The method of claim 18, wherein dissolving the hydrogel microtubes comprises treating the hydrogel microtube using a collagenase, preferably Collagenase P.

24. The method according to claim 18, wherein culturing or expanding the cells comprises culturing or expanding the cells over a time period of at least 2 hours.

25. The method according to claim 18, wherein the cells have a cell density of at least 5* 106cells per milliliter, preferably at least 3.0* 108cells per milliliter, more preferably at least 4.5* 108cells per milliliter, further preferably at least 5. Ox 108cells per milliliter.

26. The method according to claim 18, wherein step (iii) further comprises truncating the hydrogel microtube into shorter segments.Attorney Docket No. 0073605-00113127. A method of producing synthetic tissue, comprising:(i) extruding a cell solution and a hydrogel -forming solution into a cell-compatible buffer to produce the hydrogel microtube according to any one of claims 1-3;(ii) suspending the microtube including cells from the cell solution in a cell culture medium;(iii) culturing or expanding the cells under suitable conditions;(iv) optionally dissolving the hydrogel microtube to release the cells; and(v) producing the synthetic tissue using the cells.

28. The method of claim 27, wherein dissolving the hydrogel microtubes comprises treating the hydrogel microtube using one or more of ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), and an alginate lyase.

29. The method of claim 27, wherein dissolving the hydrogel microtubes comprises treating the hydrogel microtube using a collagenase, preferably Collagenase P.

30. The method according to claim 27, wherein culturing or expanding the cells comprises culturing or expanding the cells over a time period of at least 2 hours.

31. The method according to claim 23, wherein the cells have a cell density of at least 5x 106cells per milliliter, preferably at least 3.0* 108cells per milliliter, more preferably at least 4.5* 108cells per milliliter, further preferably at least 5.0* 108cells per milliliter.

32. The method according to claim 27, wherein the hydrogel microtube is a truncated hydrogel microtube.

33. The method according to claim 27, wherein the hydrogel microtube is a coated with a coating layer.

34. The method according to claim 33, wherein the coating layer comprises one or more of an alginate acid polymer layer, a sodium alginate polymer layer, a methacrylate-modified alginate polymer layer, and a vinyl sulfone-modified alginate polymer layer, wherein the vinyl sulfone-modified alginate polymer is optionally peptide-functionalized.

35. The method according to claim 27, further comprising coating the hydrogel microtube using a coating solution.

36. The method according to claim 35, wherein the coating solution comprises one or more of an alginate acid polymer, a sodium alginate polymer, a methacrylate-modified alginate polymer, and a vinyl-sulfone-modified alginate, wherein the vinyl sulfone-modified alginate polymer is optionally peptide-functionalized.Attorney Docket No. 0073605-00113137. The method of claim 27, further comprising, between step (iii) and step (iv), truncating the hydrogel microtube.

38. A method of producing a protein, viral particle, or extracellular vesicle, comprising:(i) extruding a cell solution and a hydrogel -forming solution into a cell-compatible buffer to produce the hydrogel microtube according to any one of claims 1-3;(ii) suspending the microtube including cells from the cell solution in a cell culture medium;(iii) culturing or expanding the cells under suitable conditions to produce the protein, viral particle, or extracellular vesicle;(iv) optionally dissolving the hydrogel microtube, and(v) harvesting the protein, viral particle, or extracellular vesicle.

39. The method according to claim 38, wherein the hydrogel microtube is a truncated hydrogel microtube.

40. The method according to claim 38, wherein the hydrogel microtube is a coated with a coating layer.

41. The method according to claim 40, wherein the coating layer comprises one or more of an alginate acid polymer layer, a sodium alginate polymer layer, a methacrylate-modified alginate polymer layer, and a vinyl sulfone-modified alginate polymer layer, wherein the vinyl sulfone-modified alginate polymer is optionally peptide-functionalized.

42. The method according to claim 38, further comprising coating the hydrogel microtube using a coating solution.

43. The method according to claim 42, wherein the coating solution comprises one or more of an alginate acid polymer, a sodium alginate polymer, a methacrylate-modified alginate polymer, and a vinyl-sulfone-modified alginate, wherein the vinyl sulfone-modified alginate polymer is optionally peptide-functionalized.

44. The method of claim 38, further comprising truncating the hydrogel microtube to release the protein, viral particle, or extracellular vesicle.

45. The method according to claim 38, wherein culturing or expanding the cells comprises culturing or expanding the cells over a time period of at least 6 hours.

46. The method according to claim 38, wherein the cultured or expanded cells have a cell density of at least 5* 106cells per milliliter, preferably at least 3. Ox 108cells per milliliter, moreAttorney Docket No. 0073605-001131 preferably at least 4.5x IO8cells per milliliter, further preferably at least 5.0* 108cells per milliliter.

47. A method of providing cell therapy, comprising administering to a subject a therapeutically effective amount of cells cultured or expanded using the hydrogel microtube according to any one of claims 1-3.

48. The method of claim 47, wherein the cells comprise one or more members selected from the group consisting of embryonic stem cells; mammalian embryonic stem cells; human embryonic stem cells (hESCs); human induced pluripotent stem cells (iPSCs); mammalian induced pluripotent stem cells; mammalian naive pluripotent stem cells; mammalian tissue stem cells; human pluripotent stem cells (hPSCs); cells derived or differentiated from one or more of embryonic stem cells, mammalian embryonic stem cells, human embryonic stem cells (hESCs), human induced pluripotent stem cells (iPSCs), mammalian induced pluripotent stem cells, mammalian naive pluripotent stem cells, mammalian tissue stem cells, and human pluripotent stem cells (hPSCs); mammalian cells reprogrammed from other cell types; mammalian primary cells; human umbilical vein endothelial cells; primary tumor cells; cancer cells; immune cells; T cells; natural killer cells; mammalian cell lines; engineered human and mammalian cells; insect cells; plant cells; and yeast and bacterial cells.

49. An apparatus for preparing a hydrogel microtube, comprising: an extruder comprising at least a first inlet and at least a second inlet; and a housing in fluid connection with the extruder, wherein the at least a first inlet is operable for introducing a cell solution into the extruder; and the at least a second inlet is in fluid connection with a plurality of feeding elements and operable for introducing a hydrogel -forming solution, through the plurality of feeding elements, into the extruder, in a plurality of directions symmetrically disposed with respect to the cell solution, thereby producing the hydrogel microtube containing a plurality of cells.

50. An apparatus for preparing a hydrogel microtube, comprising: an extruder comprising at least a first inlet and at least a second inlet; and a housing in fluid connection with the extruder, whereinAttorney Docket No. 0073605-001131 the at least a first inlet is operable for introducing a cell solution into the extruder; the at least a second inlet is in fluid connection with a plurality of feeding elements and operable for introducing a hydrogel-forming solution, through the plurality of feeding elements, into the extruder, in a plurality of directions symmetrically disposed with respect to the cell solution; and the housing has at least a third inlet and is configured to supply a cell-compatible buffer, and optionally one or more additional liquid flows, to the cell solution and the hydrogel-forming solution, thereby producing the hydrogel microtube containing a plurality of cells.

51. The apparatus according to claim 49 or 50, further comprising at least a syringe in fluid connection with the at least a first inlet, the at least a second inlet, and / or the at least a third inlet.

52. The apparatus according to claim 49 or 50, wherein the at least a syringe is in fluid connection with the plurality of feeding elements.

53. The apparatus according to claim 49 or 50, further comprising a syringe pump, wherein the at least a syringe is operated by the syringe pump.

54. The apparatus according to claim 49 or 50, wherein the cell solution or the cell-compatible buffer has a pH from about 7 to about 9, preferably about 7.4.

55. The apparatus according to claim 49 or 50, wherein the hydrogel-forming solution has a pH from about 2 to about 6, preferably from about 3 to about 5.

56. The apparatus according to claim 49 or 50, wherein the cell-compatible buffer comprises one or more multivalent ions, preferably one or more divalent ions.

57. The apparatus according to claim 40, wherein the one or more divalent ions comprise Mg2+, Ca2+, Zn2+, or Ba2+, preferably Ca2+.

58. The apparatus according to claim 49 or 50, further comprising a cooling element configured to keep a temperature of the hydrogel-forming solution to no greater than 10 °C, preferably no greater than 4 °C.

59. The apparatus according to claim 49 or 50, further comprising a heating element configured to raise a temperature of the hydrogel microtube to about 37 °C.

60. The apparatus according to claim 49 or 50, wherein: the extruder is configured to supply a core flow of the cell solution in a flow direction within a shell flow of the hydrogel-forming solution that is also passed in the flow direction andAttorney Docket No. 0073605-001131 wherein the extruder is configured so that a sheath flow of the cell-compatible buffer is passed in the flow direction to surround the shell flow, which surrounds the core flow such that the shell flow is between the sheath flow and the core flow as the sheath flow, shell flow, and core flow pass in the flow direction to form the hydrogel microtube containing the plurality of cells within an inner channel of the hydrogel microtube.

61. The apparatus according to claim 49, further comprising one or more ports each connected to a plurality of the first inlets or a plurality of the second inlets through one or more multifurcations, preferably one or more bifurcations.

62. The apparatus according to claim 50, further comprising one or more ports each connected to a plurality of the first inlets, a plurality of the second inlets, or a plurality of the third inlets through one or more multifurcations, preferably one or more bifurcations.

63. A system comprising a plurality of apparatus according to claim 49 or 50 deployed as a 2D array or 2D matrix.

64. The system according to claim 63, further comprising at least one syringe having multiple outlets, wherein each outlet of the multiple outlets is in fluid connection with the at least a first inlet, the at least a second inlet, or the housing of at least one apparatus of the plurality of apparatus.

65. The system according to claim 63, further comprising at least one syringe having multiple outlets simultaneously in fluid connection with the at least a first inlet of each apparatus of the plurality of apparatus.

66. The system according to claim 63, further comprising at least one syringe having multiple outlets simultaneously in fluid connection with the at least a second inlet of each apparatus of the plurality of apparatus.

67. The system according to claim 63, further comprising at least one syringe having multiple outlets simultaneously in fluid connection with the at least a third inlet of each apparatus of the plurality of apparatus.

68. The system according to claim 63, further comprising at least one syringe having one or more multifurcations, preferably one or more bifurcations.

69. A method of preparing a hydrogel microtube, comprising:(i) supplying a core flow of a cell solution though at least a first inlet of an extruder;Attorney Docket No. 0073605-001131(ii) concurrently supplying a shell flow of a hydrogel-forming solution though at least a second inlet of the extruder, wherein the core flow is passed within and surrounded by the shell flow; and(iii) optionally supplying a sheath flow of a cell-compatible buffer to surround the shell flow, such that the shell flow is between the sheath flow and the core flow as the sheath flow, shell flow, and core flow pass in a shared flow direction, thereby producing a hydrogel microtube containing a plurality of cells within an inner channel of the hydrogel microtube.

70. The method according to claim 69, wherein the hydrogel-forming solution comprises one or more of(a) a collagen protein;(b) a blend of an alginate polymer and a collagen protein; and(c) a peptide-functionalized alginate polymer and, optionally, an unfunctionalized alginate polymer, preferably one or more of (a) and (b).

71. The method according to claim 69, wherein (b) the blend of the alginate polymer and the collagen protein has an alginate polymer-to-collagen protein ratio of 0.01 to 100 by mass, preferably 0.1 to 1.5 by mass, more preferably 0.25 to 0.75 by mass.

72. The method according to claim 69, wherein the peptide-functionalized alginate polymer comprises an arginine-glycine-aspartic acid (RGD) peptide covalently linked to the alginate polymer.

73. The method according to claim 69, wherein the peptide-functionalized alginate polymer comprises one or more of a peptide-functionalized alginate acid polymer, a peptide- functionalized sodium alginate polymer, a peptide-functionalized methacrylate-modified alginate polymer, and a peptide-functionalized vinyl sulfone-modified alginate polymer.

74. The method of claim 69, wherein the cell solution comprises one or more cells selected from the group consisting of embryonic stem cells; mammalian embryonic stem cells; human embryonic stem cells (hESCs); human induced pluripotent stem cells (iPSCs); mammalian induced pluripotent stem cells; mammalian naive pluripotent stem cells; mammalian tissue stem cells; human pluripotent stem cells (hPSCs); cells derived or differentiated from one or more of embryonic stem cells, mammalian embryonic stem cells, human embryonic stem cells (hESCs), human induced pluripotent stem cells (iPSCs), mammalian induced pluripotent stem cells, mammalian naive pluripotent stem cells, mammalian tissue stem cells,Attorney Docket No. 0073605-001131 and human pluripotent stem cells (hPSCs); mammalian cells reprogrammed from other cell types; mammalian primary cells; human umbilical vein endothelial cells; primary tumor cells; cancer cells; immune cells; T cells; natural killer cells; mammalian cell lines; engineered human and mammalian cells; insect cells; plant cells; and yeast and bacterial cells, wherein the cells are optionally cultured as single cell type, as a mixture of different cell types, or as a mixture of cells from different donors.

75. The method according to claim 69, wherein the shell flow of the hydrogel -forming solution is applied in a plurality of directions symmetrically disposed with respect to the core flow of the cell solution.

76. The method according to claim 69, wherein the sheath flow of cell-compatible buffer is applied in a plurality of directions symmetrically disposed with respect to the core flow of the cell solution and / or the shell flow of the hydrogel-forming solution.

77. The method according to claim 69, wherein the cell solution or the cell-compatible buffer has a pH from about 7 to about 9, preferably about 7.4.

78. The method according to claim 69, wherein the hydrogel-forming solution has a pH from about 2 to about 6, preferably from about 3 to about 5.

79. The method according to claim 69, wherein the cell-compatible buffer comprises one or more multivalent ions, preferably one or more divalent ions.

80. The apparatus according to claim 79, wherein the one or more divalent ions comprise Mg2+, Ca2+, Zn2+, or Ba2+, preferably Ca2+.

81. The apparatus according to claim 69, wherein the hydrogel-forming solution is maintained at a temperature of no greater than 10 °C, preferably no greater than 4 °C, using a cooling element.

82. The apparatus according to claim 69, further comprising, after step (iii), raising a temperature of the hydrogel microtube to about 37 °C using a heating element.

83. The method according to claim 69, further comprising, after step (iii), coating the hydrogel microtube using a coating solution.

84. The method according to claim 69, wherein the coating solution comprises one or more of an alginate acid polymer, a sodium alginate polymer, a methacrylate-modified alginate polymer, and a vinyl-sulfone-modified alginate, wherein the vinyl sulfone-modified alginate polymer is optionally peptide-functionalized.Attorney Docket No. 0073605-00113185. Use of the hydrogel microtube according to any one of claims 1-3 for culturing or expanding cells.

86. Use of the apparatus according to claim 49 or 50 for preparing a hydrogel microtube.

87. A method of coating a hydrogel microtube, comprising: a) preparing the hydrogel microtube according the method of claim 69; b) dipping the hydrogel microtube in a coating solution for a time period of at least 1 second and no greater than 10 hours, preferably at least 1 minute and no greater than 30 minutes, more preferably at least 3 minutes and no greater than 15 minutes; wherein the coating solution comprises at least 0.1% by weight and no greater than 30% by weight, preferably at least 0.5% by weight and no greater than 1.5% by weight, of an alginate polymer; and wherein the alginate polymer comprises one or more of an alginate acid polymer, a sodium alginate polymer, a methacrylate-modified alginate polymer, and a vinyl-sulfone- modified alginate, wherein the vinyl sulfone-modified alginate polymer is optionally peptide- functionalized; and c) dipping the hydrogel microtube in a buffer containing or more divalent ions selected from Mg2+, Ca2+, Zn2+, and Ba2+, preferably Ca2+.

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