Cryopreservation of bioprinted tissue fibers

Abstract

Aspects of the present disclosure include cryopreserved tissue fibers and multilayer structures comprising the fibers, wherein the fibers encapsulate dissociated cells, and wherein the fibers and / or the structures are cryopreserved prior to aggregation of the encapsulated cell population. Methods of making and using such fibers and structures are also provided.

Description

Cross-reference to related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 548,164, filed November 10, 2023, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] This invention relates to cryopreserved tissue fibers encapsulating cell populations, and methods for manufacturing and using the same, said cryopreserved tissue fibers having enhanced cell viability and (optionally) increased cell density. Background Technology

[0003] Implantable solid organ cell therapies typically require replacing 5% to 20% of the native epithelial (parenchymal) cell mass with transplanted cells to establish a sufficient functional threshold for therapeutic efficacy. To further enhance the lifespan and function of these cells, many treatments focus on mimicking organ density through cell aggregation. This increases cell-to-cell adhesion, crucial for maintaining the epithelial cell type, which in turn leads to improved physiologically relevant functions. However, this increased reliance on cell aggregation can also lead to other significant complications.

[0004] To cite just one example, cell therapies can be difficult to manufacture and store due to their lack of functional stability outside the in vivo environment. Without cryopreservation, they must be used within a very short timeframe after production, a factor that severely limits the availability and scalability of this technological approach. Unfortunately, effective cryopreservation, particularly for “solid” spheroids or aggregates, has not been adequately demonstrated and is currently not feasible on a sufficiently large scale. Conventional methods for cryopreserving spheroids typically result in significant cell death and functional decline. (Acker et al., Cryobiology June 1999; 38(4):363-71; Acker et al., Biophys J September 2001; 81(3): 1389-1397).

[0005] Furthermore, the formation of intracellular ice crystals must be prevented through the use of specialized cryoprotectants to avoid cell lysis. Permeation solutions such as dimethyl sulfoxide (DMSO) are typically used to permeate cells and reduce their water content, thereby minimizing the formation of potentially damaging ice crystals. In this regard, recent bioprinting work has incorporated DMSO directly into bioprinting materials in an attempt to ensure adequate contact and coverage. (Lee et al., Innovative Cryopreservation Process Using a Modified Core / Shell Cell-Printing with a Microfluidic System for Cell-Laden Scaffolds) ACS Applied Materials&Interfaces (2018). This would inevitably increase cellular exposure to DMSO; however, recent reports suggest that this fundamentally damages cells. See Verheijen et al. DMSO induces dramatic changes in human cellular processes and the epigenetic landscape in vitro. Sci Rep 9, 4641 (2019). Therefore, DMSO is clearly not inert, and incorporating it into the bioprinting process is particularly unwise.

[0006] Therefore, improvements in design, materials, and technology are still needed to provide tissue therapies that can be effectively cryopreserved while maintaining sufficient cell viability and functionality. Summary of the Invention

[0007] By providing cryopreserved core / shell tissue fibers, medical devices comprising said tissue fibers, and methods of manufacturing and using the same, the present invention successfully addresses the aforementioned challenges in the art. One aspect includes a cryopreserved tissue fiber comprising a continuously bioprinted core / shell fiber encapsulating a cell population for implantation, the core / shell fiber comprising a core and at least one shell, the cell population comprising at least 90%, at least 95%, at least 98%, at least 99%, or 100% dissociated cells embedded in a biocompatible matrix, wherein said fiber is cryopreserved prior to the aggregation of said dissociated cells.

[0008] The dissociated cells may be encapsulated in the core and / or the at least one shell. In one embodiment, the dissociated cells are encapsulated in the core. In another embodiment, the dissociated cells are encapsulated in the at least one shell. In yet another embodiment, the core / shell fiber comprises at least two shells, with the innermost shell comprising an annular layer encapsulating the cell population.

[0009] In one embodiment, the continuously bioprinted core / shell fiber comprises a core and at least one shell, optionally wherein the material strength of the core is less than that of the shell. In another embodiment, the core and the at least one shell comprise the same hydrogel material. Preferably, the hydrogel material is alginate. In yet another embodiment, the core and / or the at least one shell comprises chemically modified alginate.

[0010] In an embodiment, the biocompatible matrix further comprises a thickener, preferably wherein the thickener comprises fish gelatin. In an embodiment, the thickener is selected from the group consisting of or composed of: hydroxypropyl methylcellulose (HPMC), fish gelatin (e.g., low or high molecular weight), polyvinyl alcohol (PVA, e.g., 90 kDa PVA), polyethylene glycol (PEG), non-fish gelatin, poloxamer such as Pluronic (e.g., F126 or F68), xanthan gum, gellan gum, guar gum, gum arabic, or combinations thereof. In an embodiment, the thickener is present in an amount of about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5% of the biocompatible matrix. In an embodiment, the thickener is hydroxypropyl methylcellulose (HPMC), fish gelatin, non-fish gelatin, polyethylene glycol (PEG), guar gum, gum arabic, xanthan gum, or polyvinyl alcohol (PVA, e.g., 90 kDa PVA). In the implementation scheme, the HPMC is about 1%, 1.25%, or 1.5% of the biocompatible matrix, the fish gelatin is about 4%, 5%, or 6% of the biocompatible matrix, or the PVA is about 1%, 2%, or 3% of the biocompatible matrix.

[0011] In one embodiment, the biocompatible matrix does not contain DMSO. In another embodiment, the core / shell fibers are bioprinted without incorporating DMSO into the core or at least one shell.

[0012] In one embodiment, the cell population comprises primary cells from endocrine and exocrine glands selected from the group consisting of: pancreas, liver, kidney, parathyroid gland, thyroid gland, pineal gland, pituitary gland, thymus, adrenal gland, ovary, testis, tonsillar gland, enteroendocrine cells, stem cells, stem cell-derived cells of any of the aforementioned primary cell types, or cells engineered to secrete therapeutic agents. In another embodiment, the dissociated cells comprise HepG2, primary human hepatocytes (PHH), mesenchymal stem cells (MSCs), hepatocyte-like cells derived from embryonic stem cells or induced pluripotent stem cells, hepatocyte-like cells derived from liver stem cells, xenogeneic hepatocytes, genetically engineered stem cell-derived hepatocytes, stem cell-derived endothelial cells or primary endothelial cells, and combinations thereof. In yet another embodiment, the dissociated cells comprise HepG2, primary human hepatocytes (PHH), and / or mesenchymal stem cells (MSCs), and combinations thereof. In yet another embodiment, the dissociated cells comprise PHH and MSCs in a 3:1 ratio. In some embodiments, the dissociated cells include adrenal cells.

[0013] In another aspect, a composition / medical device is provided for implanting a cell population into a subject in need, the composition / medical device comprising a multilayer structure including continuous bioprinted tissue fibers according to any embodiment of this disclosure, wherein the multilayer structure is cryopreserved prior to dissociation of cell aggregation. In an embodiment, the composition / medical device comprises a multilayer grid structure as described herein.

[0014] In one embodiment, the multilayer structure includes at least one conformal coating. In another embodiment, the coating comprises a hydrogel with a material strength less than that of both the core and the at least one shell of the fiber. In yet another embodiment, the core, the at least one shell, and the coating comprise the same hydrogel material; preferably, the hydrogel material is alginate. In yet another embodiment, the solid core, the at least one shell, and / or the coating comprise chemically modified alginate.

[0015] In one embodiment, the biocompatible matrix comprises at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% v / v of dissociated cells in the physiological matrix material. In another embodiment, the cryopreserved tissue fibers are high-density tissue fibers, and the biocompatible matrix comprises at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, or at least about 85% v / v of dissociated cells in the physiological matrix material. In yet another embodiment, the biocompatible matrix comprises a cell-to-physiological matrix material ratio of at least about 2:1, preferably at least about 3:1, more preferably at least about 4:1.

[0016] In one embodiment, the cell population is encapsulated in the core, and the core diameter is between about 50 μm and about 250 μm, or between about 100 μm and about 200 μm, preferably between about 130 μm and about 200 μm, preferably between about 140 μm and about 190 μm, more preferably between about 150 μm and 175 μm, and even more preferably about 150 μm, about 155 μm, about 160 μm, about 165 μm, about 170 μm, or about 175 μm. In another embodiment, the shell thickness is between about 30 μm and about 100 μm, or between about 20 μm and about 200 μm, more preferably between about 45 μm and 70 μm, and even more preferably about 60 μm, about 65 μm, or about 70 μm. In the embodiments, the total fiber diameter is between about 200 μm and 500 μm, or between about 270 μm and about 330 μm, preferably between about 280 μm and about 320 μm, more preferably between about 290 μm and about 310 μm, and even more preferably between about 290 μm, about 300 μm or about 310 μm.

[0017] In one embodiment, the cell population is encapsulated within the annular layer, and the core diameter is between approximately 50 μm and 750 μm, or between approximately 300 μm and approximately 500 μm, preferably between approximately 350 μm and approximately 450 μm, more preferably about 350 μm, about 375 μm, about 400 μm, about 425 μm, or about 450 μm. In another embodiment, the thickness of the annular layer is between approximately 50 μm and approximately 300 μm, or between approximately 100 μm and approximately 300 μm, preferably between approximately 130 μm and approximately 200 μm, preferably between approximately 140 μm and approximately 190 μm, more preferably between approximately 150 μm and 175 μm, more preferably about 150 μm, about 155 μm, about 160 μm, about 165 μm, about 170 μm, or about 175 μm. In one embodiment, the tissue fibers further include a shell with a thickness between about 30 μm and about 120 μm, or between about 30 μm and 80 μm, more preferably between about 45 μm and 70 μm, and even more preferably about 60 μm, about 65 μm, or about 70 μm. In another embodiment, the total fiber diameter is between about 300 and 1400 μm, or between about 700 and 1200 μm, preferably between about 800 μm and about 1000 μm, and even more preferably about 800 μm, about 900 μm, or about 1000 μm.

[0018] In an embodiment, the structure comprises at least two, three, four, or five layers formed of continuous fibers, preferably wherein the structure comprises two, three, or four layers, and more preferably wherein the structure comprises four layers. In an embodiment, the core and / or the at least one shell are separated along the length of the fibers.

[0019] The invention also includes a method for implanting a cell population into a subject in need, the method comprising: a. thawing cryopreserved tissue fibers or compositions / medical devices of the present disclosure; and b. implanting the fibers or compositions / medical devices into the subject. In embodiments, the method may further include culturing the tissue fibers or compositions / medical devices in a fresh culture medium after thawing, prior to implantation into the subject, until the dissociated cells form aggregates in the tissue fibers.

[0020] An aspect of the present invention includes a method of manufacturing cryopreserved tissue fibers or implantable multilayer structures of the present disclosure, the method comprising: a) providing a bioprinting system, the bioprinting system comprising: a printhead including a plurality of microfluidic channels for selectively supplying a core material, at least one shell material, and a sheath fluid to a dispensing orifice; a receiving surface for receiving the material dispensed from the orifice; a positioning unit for positioning the orifice of the printhead in three-dimensional space, the positioning unit being operatively coupled to the printhead; and at least one dispensing member, the at least one dispensing member being... The dispensing component is used to dispense the fibers from the dispensing orifice; b) simultaneously print the core and at least one shell of the tissue fibers via the bioprinting system, wherein a cell population is encapsulated in the core and / or the at least one shell; and c) cryopreserve the bioprinted tissue fibers with a cryoprotectant; wherein the cell population comprises at least 95% dissociated cells embedded in a biocompatible matrix, and wherein the fibers are cryopreserved prior to the aggregation of the dissociated cells; preferably cryopreserved within 15 minutes to 3 hours of bioprinting, more preferably within 30, 45, or 60 minutes of bioprinting.

[0021] In one embodiment, the printing step includes distributing the continuously bioprinted core / shell fibers onto the receiving surface to form the multilayer structure, wherein the multilayer structure is cryopreserved prior to the aggregation of dissociated cells within the fibers. In another embodiment, the method further includes coating the multilayer (e.g., grid) structure with at least one conformal coating after printing is complete.

[0022] In one embodiment, the cryopreservation step includes adding a cryoprotectant to the printed fibers at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 minutes before freezing.

[0023] In an embodiment, the biocompatible matrix further comprises a thickener, preferably wherein the viscosity of the core material and the shell material is substantially the same. In an embodiment, the thickener is selected from the group consisting of or including: hydroxypropyl methylcellulose (HPMC), fish gelatin (e.g., low or high molecular weight), polyvinyl alcohol (PVA, e.g., 90 kDa PVA), polyethylene glycol (PEG), non-fish gelatin, poloxamer such as Pluronic (e.g., F126 or F68), xanthan gum, gellan gum, guar gum, gum arabic, or combinations thereof. In an embodiment, the thickener is present in an amount of about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5% of the biocompatible matrix. In an embodiment, the thickener is hydroxypropyl methylcellulose (HPMC), fish gelatin, or polyvinyl alcohol (PVA, e.g., 90 kDa PVA). In one embodiment, the HPMC is about 1%, 1.25%, or 1.5% of the biocompatible matrix, the fish gelatin is about 4%, 5%, or 6% of the biocompatible matrix, or the PVA is about 1%, 2%, or 3% of the biocompatible matrix. In another embodiment, the core material and the at least one shell material are bioprinted without DMSO.

[0024] In one embodiment, the receiving surface includes a manufacturing platform for supporting the core / shell fibers of the continuous bioprinting, the manufacturing platform including a frame defining a gap and including a plurality of posts on opposite sides of the frame for fixing and suspending the continuous bioprinting fibers during printing; and the printing step includes distributing the fibers around the plurality of posts to form a grid structure comprising at least two, three, four or five layers of the fibers.

[0025] An aspect of the invention includes a method of treating a subject, the method comprising implanting the composition / medical device of the present disclosure into the subject. In an embodiment, the subject is a person suffering from liver disease or ailment.

[0026] Incorporate by reference All publications, patents and patent applications mentioned in this specification are incorporated herein by reference as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference. Attached Figure Description

[0027] Figures 1A to 1C H&E staining of pre-aggregated fibers and solid fibers is shown. Figure 1A Fluorescence micrographs of the live / dead state of solid and pre-aggregated fibers on days 1, 3, and 7 after printing. Figure 1B), and albumin levels from solid and pre-aggregated fibers from two different donors ( Figure 1C ).

[0028] Figures 1D to 1G The relative albumin yield was shown. Figure 1D ), albumin production ( Figure 1E ), live / dead fluorescent staining images ( Figure 1F ) and photomicrographs of fibers ( Figure 1G ).

[0029] Figures 2A to 2B Fluorescence micrographs of the live / dead assays of solid and pre-aggregated fibers from two different donors are shown. Figure 2A ), and albumin and Cyp3A4 activity from solid and pre-aggregated fibers from two different donors ( Figure 2B ).

[0030] Figure 3A Bright-field microscopy images and fluorescence microscopy images of fibers depicting pre-aggregated PHH and MSC cells within the fiber core are presented.

[0031] Figure 3B Circular fibers were depicted, with a cell:fish gelatin ratio of 3:1 and 8.2 x 10⁻⁶ cells per milliliter. 6 Millions of PRHs and 4.1 x 10 6 The cell concentration of rat MSCs was printed, with the cells located in the first shell (ring region).

[0032] Figures 4A to 4D The live / dead assay was shown. Figure 4A ), plasma albumin level ( Figure 4B Images of surgical sites in NSG mice ( Figure 4C ) and plasma albumin levels ( Figure 4D ).

[0033] Figures 5A to 5B A bright-field image of the printed fibers is shown. Figure 5A ) and live / dead assay ( Figure 5B ).

[0034] Figures 6A to 6B The live / dead fluorescence staining of HepG2 solid fibers 3 days after thawing is shown. Figure 6A ) and live / dead fluorescent staining of HepG2 unfrozen control solid fibers ( Figure 6B ).

[0035] Figure 7 The Cyp3A4 activity and albumin production of HepG2 cells 5 days after printing are shown (Fig. 5C).

[0036] Figures 8A to 8C The solid fibers and aggregate rings printed using PHH+ MSCs or adrenocortical cells are shown. Figure 8A and Figure 8B ), and a cross-sectional view of bioprinted ring fibers containing dissociated PHH / MSCs ( Figure 8C ).

[0037] Figure 9 A representative image of solid fibers containing single cells in annular layers is shown.

[0038] Figures 10A to 10B Fluorescence micrographs of live / dead assays of fresh and cryopreserved fibers, as well as fibers printed with PHH+ MSCs or adrenocortical cells, in the form of solid fibers or aggregate rings, are shown. Figure 10A It is in color; Figure 10B (in grayscale).

[0039] Figures 11A to 11B The study shows albumin secretion levels in solid and aggregated ring forms printed using PHH+MSCs. Figure 11A ) and cortisol secretion levels in the form of solid and aggregated rings printed by adrenocortical cells ( Figure 11B ).

[0040] Figures 12A to 12B The relative albumin secretion levels were shown in solid and aggregated ring forms printed using PHH+MSCs. Figure 12A ) and relative cortisol secretion levels in the form of solid and aggregated rings printed by adrenocortical cells ( Figure 12B ). Detailed Implementation

[0041] 3D bioprinting is an additive manufacturing process in which synthetic tissue structures (optionally carrying cells) are laid up layer by layer to obtain multi-layered 3D structures. Various types of 3D bioprinting technologies have been developed, including extrusion (Panwar A et al., Molecules. (2016); 21: 685; Sakai S et al., Biofabrication. (2018); 10:045007; Han HW and Hsu SH, Neural Regener. Res. (2017); 12: 1595), inkjet (Gao G et al., Biotechnol. Lett. (2015); 37: 2349; Gao G and Cui X, Biotechnol. Lett. (2016); 38:203; Bsoul A et al., Lab Chip. (2016); 16: 3351), and laser-assisted (Sorkio A et al., Biomaterials. (2018); 171: 57; Pagès E et al., J. Nanotechnol. Eng.). Med. (2015); 6:021006; Catros S et al., In Vivo and In Situ Biofabrication by Laser-Assisted Bioprinting, Elsevier, Winston-Salem, USA. (2015)) and stereolithography (SLA) printing methods (Miri AK et al., Adv. Mater. (2018); 30: 1800242; Wang Z et al., ACS Appl. Mater. Interfaces. (2018); 10; 26859; Wang Z et al., Biofabrication. (2015); 7: 045009). Among these, extrusion is one of the most common methods, in which bio-ink is dispensed via one or more syringes to form a layer-by-layer scaffold from fibers.

[0042] The progress made has also facilitated the use of microfluidics-based 3D bioprinting systems (Beyer ST et al., 2013 Transducers Eurosensors XXVII 17). th Int. Conf. Solid-State Sensors, Actuators, Microsystems. IEEE, Piscataway, NJ (2013); pp. 1206-1209; BeyerST et al., The 17 thInt. Conf. on Miniaturized Systems for Chemistry and LifeSciences. (2013); pp. 176–178. With these systems and techniques, multiple materials (e.g., bio-inks, crosslinking agents, etc.) can flow through microchannels, allowing for precise control of one or more of the following: flow, switching, mixing, etc. When used in conjunction with sheath flow surrounding at least one internal material, microfluidic bioprinting can reduce shear stress during the printing process. Microfluidic-based 3D bioprinting can also advantageously allow material flows to intersect as they leave independent flow paths and enter a single flow path (e.g., a distribution channel), facilitating the generation of structures with a core surrounded by one or more shells.

[0043] This disclosure provides single cells for bioprinting that are dissociated and cryopreserved prior to aggregation, resulting in enhanced cell viability and function. This correspondingly enables significant improvements in the manufacturing workflow and allows for the advance manufacture and storage of therapeutic agents for future use. Global distribution is thus now feasible. Finally, cryopreservation enables the handling of emergency situations.

[0044] Furthermore, as demonstrated herein, the fibers printed according to this disclosure can achieve higher cell densities by eliminating the typical pre-aggregation steps employed in the prior art. Cell therapies under development typically employ cell aggregates encapsulated in biomaterials, where the biomaterials occupy a significantly larger volume than the cells. This can lead to significant size limitations, making the resulting implants difficult or impossible to use due to limited space at suitable surgical sites. According to the teachings of the invention herein, cells are printed as single-cell suspensions in a small amount of biomaterial, thereby significantly reducing the size of the resulting bioprinted device.

[0045] Finally, problems including, but not limited to, immune protection, adequate oxygen and nutrient access, and FBR avoidance can all be addressed based on the teachings of this invention.

[0046] definition For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa. If any definition set forth conflicts with any reference incorporated herein by reference, the definition set forth herein shall prevail. 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 disclosure pertains.

[0047] As used herein, the term "hydrogel" refers to a composition comprising a network or lattice of water and hydrophilic polymer chains.

[0048] As used herein, the terms "sheath fluid" or "sheath solution" refer to a fluid used to at least partially encapsulate or "wrap" a material as it passes through a fluid channel. In embodiments, the sheath fluid comprises an aqueous solvent, such as water or glycerol. In embodiments, the sheath fluid comprises a chemical or physical crosslinking agent. Non-limiting examples of crosslinking agents include divalent cations (e.g., Ca2+). 2+ Ba 2 + 、Sr 2+ (etc.), thrombin, and pH-altering chemicals such as sodium bicarbonate.

[0049] As used herein, the term "segmentation / division" refers to a discontinuous nature of a type of material and / or biomaterial included in the core or shell of the fibers disclosed herein, such as intentional gaps present in the deposition of that type of material and / or biomaterial along the length of the fiber. The spacing (e.g., length) between such segments / compartments may be regular (e.g., the spacing between regions of biomaterial may be substantially the same) or the spacing may be different.

[0050] As used herein, the term "solid core" refers to the core of the fiber of the present disclosure, which is composed of a particular material (e.g., a hydrogel cross-linked by a chemical cross-linking agent) such that the core does not include an inner cavity along the entire length of the fiber. This term is not intended to refer to a core that is completely impermeable along its length, as the solid core of the present disclosure allows certain fluids, molecules, and / or ionic substances to pass through the entire core.

[0051] As used herein, the term "biocompatible material" refers to a material in which biological material (including but not limited to cells) can be incorporated into and / or contacted with the biocompatible material, and wherein the biocompatible material does not adversely affect the ability of the biological material to perform one or more functions (e.g., cellular functions, including but not limited to the secretion of biologically related molecules, agonist / receptor binding, signal transduction, etc.).

[0052] As used herein, the term “immune protection” broadly refers to aspects of the fiber design of this disclosure that are intended to reduce, prevent, or eliminate host immune responses, including, for example, immune cell invasion of the fiber after it has been implanted into the body (e.g., a mammalian body).

[0053] As used herein, the term "agent" means any protein, nucleic acid molecule (including chemically modified nucleic acid molecules), antibody, small molecule, organic compound, inorganic compound, or other molecule of interest. Agents may include biologically related agents, therapeutic agents, diagnostic agents, pharmaceutical agents, chelating agents, cross-linking agents, etc. A therapeutic agent or pharmaceutical agent is one that, when administered to a subject alone or in combination with an adjunct compound in a manner consistent with this disclosure, induces a desired response (such as inducing a therapeutic or preventative effect). A biologically related agent is an agent that supports another biological process, such as an agent that supports cell viability.

[0054] As used herein, the term "cryoprotectant" refers to a composition intended to protect cells from damage during the cryopreservation process. In some embodiments, the cryoprotectant does not include dimethyl sulfoxide (DMSO). In some embodiments, the cryoprotectant may include DMSO. In some embodiments, DMSO-containing cryoprotectants may include CRYOSTOR® CS10, CRYOSTOR® CS5, CROSTOR CS2, PSC cryopreservation kit, Synth-a-Freeze™ cryopreservation medium, CELLBANKER® 1, CELLBANKER® 2, and STEM-CELLBANKER® GMP grade. In some embodiments, DMSO-free cryoprotectants may include CRYOSTOR® CSB, CrySOfree™ DMSO-free cryopreservation medium, STEM-CELLBANKER® DMSO-free GMP grade, and Cell-Vive™ CD DMSO-free cryopreservation solution.

[0055] As used herein, the term "dissociated cell" refers to a group of cells consisting primarily of single cells and small clusters of cells (e.g., fewer than 10, 12, or 15 cells), preferably wherein any cluster has a diameter of less than 20-50 micrometers. As is known in the art, dissociated cells have typically undergone enzymatic or mechanical digestion to separate the cells from each other. This contrasts with cells in solid organs or spheroids, where cells are connected to each other by direct cell-cell junctions or by being anchored together with an extracellular matrix. Spheroids and / or organoids containing more than 15 cells and / or exceeding 50 μm in size are not considered "dissociated" cell groups as envisioned herein. Dissociated cells can also be distinguished from aggregates by the presence of brighter areas between cells under phase-contrast microscopy and irregular cluster boundaries (confirming a lack of strong or extensive cell-cell adhesion between adjacent cells).

[0056] Cryopreserved fiber structure and its multi-layer grid structure One aspect of the present invention relates to a cryopreserved tissue fiber comprising a continuously bioprinted core / shell fiber encapsulating a cell population for implantation, the core / shell fiber comprising a core and at least one shell, the cell population comprising at least 90%, at least 95%, at least 98%, at least 99%, or 100% dissociated cells embedded in a biocompatible matrix, wherein the fiber is cryopreserved prior to the aggregation of the dissociated cells.

[0057] In the implementation scheme, the biocompatible matrix contains at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% v / v of dissociated cells in the physiological matrix material.

[0058] In the implementation, high-density tissue fibers are provided, which contain at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% v / v of dissociated cells in a physiological matrix material.

[0059] In the implementation scheme, the biocompatible matrix comprises a cell:physiological matrix material ratio of at least about 2:1, preferably at least about 3:1, and more preferably at least about 4:1.

[0060] Dissociated cells may be encapsulated in a core and / or at least one shell. In one embodiment, the dissociated cells are encapsulated in a core. In another embodiment, the dissociated cells are encapsulated in at least one shell. In yet another embodiment, the core / shell fiber comprises at least two shells, with the innermost shell comprising an annular layer encapsulating the cell population. For example, WO 2022 / 246550 describes fibers containing at least two shells, with the innermost shell comprising an annular layer, which is incorporated herein by reference in its entirety.

[0061] In one embodiment, the continuously bioprinted core / shell fiber comprises a core and at least one shell, optionally wherein the material strength of the core is less than that of the shell. In another embodiment, the core and at least one shell comprise the same hydrogel material. Preferably, the hydrogel material is alginate. In yet another embodiment, the core and / or at least one shell comprises chemically modified alginate.

[0062] In embodiments, the biocompatible matrix further comprises a thickener, preferably said thickener comprising fish gelatin. In embodiments, the thickener may comprise hydroxypropyl methylcellulose (HPMC), fish gelatin (e.g., low or high molecular weight), polyvinyl alcohol (PVA, e.g., 90 kDa PVA), polyethylene glycol (PEG), non-fish gelatin, poloxamer such as Pluronic (e.g., F126 or F68), xanthan gum, gellan gum, guar gum, gum arabic, or combinations thereof. In embodiments, the thickener is present in amounts of about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the biocompatible matrix, or any range thereof. As an example and without limitation, PVA may be used at about 3%, HPMC at about 1% or 1.25%, low molecular weight fish gelatin at about 5%, PEG at about 1% to about 5%, non-fish gelatin at less than about 5%, poloxamer at about 5% to about 10%, and adhesive at about 1% (in the fiber core). In an embodiment, the biocompatible matrix does not contain DMSO. In an embodiment, the core / shell fiber is bioprinted without incorporating DMSO into the core or at least one shell.

[0063] In another aspect, a composition / medical device is provided for implanting a cell population into a subject in need, said composition / medical device comprising a multilayer structure including tissue fibers according to any embodiment of this disclosure, wherein said multilayer structure is cryopreserved prior to dissociation of cell aggregation. In an embodiment, the composition / medical device is a multilayer grid structure as described herein.

[0064] In one embodiment, the multilayer lattice structure includes at least one conformal coating. In another embodiment, the coating comprises a hydrogel containing a core and at least one shell with a material strength less than that of the fiber. In yet another embodiment, the core, at least one shell, and the coating comprise the same hydrogel material; preferably, said hydrogel material is alginate. In yet another embodiment, the solid core, at least one shell, and / or the coating comprises chemically modified alginate.

[0065] In the implementation scheme, the solid core comprises about 1.2% to about 1.8% alginate, preferably about 1.5% alginate.

[0066] In an embodiment, at least one shell comprises about 1.4% to about 3.0% alginate; preferably about 1.5% to about 2.5% alginate; more preferably about 1.8% to about 2.2% alginate.

[0067] In an embodiment, the coating comprises about 0.2% alginate to about 2% alginate, or about 0.25% alginate to about 1.5% alginate; preferably about 0.3% to about 1.0% alginate; more preferably about 0.4% to about 0.8% alginate.

[0068] In one embodiment, the at least one coating comprises a first inner coating and a second outer coating. In another embodiment, the first inner coating comprises a hydrogel with a material strength greater than that of the second outer coating. In another embodiment, the first inner coating comprises about 1.4% to about 3.0% alginate, preferably about 1.5% to about 2.5% alginate, more preferably about 1.8% to about 2.2% alginate. In another embodiment, the second outer coating comprises about 0.25% to about 1.5% alginate, preferably about 0.3% to about 1.0% alginate, more preferably about 0.4% to about 0.8% alginate.

[0069] In one embodiment, the core and / or at least one shell are separated along the length of the fiber.

[0070] In embodiments, a multilayer lattice structure formed from a single continuous fiber may include more than two layers, such as 3, 4, 5, 6, 7, 8, 9, or 10 layers, or even more than 10 layers in some examples. In embodiments, the lattice structure may be square, rectangular, elliptical, hexagonal, circular, etc. The size of the lattice structure includes dimensions suitable for implantation in a specific subject (e.g., human, dog, cat, rat, pig, etc.). In embodiments where the lattice structure is approximately square, the size of the structure may be between about 8 mm x 8 mm and about 150 mm x 150 mm. In embodiments where the lattice structure has a shape other than square, the area of ​​the lattice structure may range from about 64 mm². 2 Approximately 22,500 mm 2 Between, or any value between.

[0071] In implementations, the multilayer grid structure of this disclosure may include 1, 2, 3, 4, or 5 shells, and 1, 2, 3, 4, or 5 coatings. In some examples, the bioprinting fibers themselves may also be coated.

[0072] In one embodiment, the solid core, at least one shell, and one or more coatings are composed of a biocompatible material. Examples of biocompatible materials relevant to this disclosure include, but are not limited to, alginate, collagen, decellularized extracellular matrix, hyaluronic acid (HA), polyethylene glycol (PEG), fibroin, gelatin, gelatin methacrylate (GEL-MA), silk, chitosan, cellulose, polycaprolactone (PCL), polylactic acid (PLA), poly(oligomeric polyethylene glycol methacrylate) (POEGMA), or combinations thereof. In another embodiment, the solid core, at least one shell, and one or more coatings are composed of a hydrogel material. Hydrogel materials related to this disclosure may include, but are not limited to, alginates (e.g., SLG-100 alginate), chitosan, gel-MA, agarose, PEG, PCL, poly-L-lysine (PLL), triazole, fucoidan, poly(ethylene glycol) diacrylate (PEGDA), poly(ethylene glycol) tetraacrylate (PEGTA), poly(vinyl alcohol) (PVA), hyaluronic acid (HA), methacrylamide hyaluronic acid (HAMA), collagen, methacrylated collagen (ColMA), gelatin, gellan gum, fibrin (fibrinogen), and combinations thereof. In some embodiments, HA may be used to enhance / increase viscosity.

[0073] In the embodiments, the diameter of the solid core of the cryopreserved tissue fibers of this disclosure is between about 100 μm and about 300 μm, preferably between about 130 μm and about 200 μm, preferably between about 140 μm and about 190 μm, more preferably between about 150 μm and 175 μm, even more preferably about 150 μm, about 155 μm, about 160 μm, about 165 μm, about 170 μm or about 175 μm, for example about 130 μm, 140 μm, 150 μm, 155 μm, 160 μm, 165 μm, 170 μm, 175 μm, 180 μm, 190 μm, 200 μm or any value between these values.

[0074] In the embodiments, the thickness of at least one shell of the cryopreserved tissue fibers of the present disclosure is between about 30 μm and 100 μm, more preferably between about 40 μm and 70 μm, for example, about 30 μm, 40 μm, 50 μm, 60 μm, 70 μm or any value between therewith.

[0075] In embodiments, the core and / or shell of the continuous bioprinted fiber of this disclosure comprises multiple dissociated single cells, such as hepatocytes or other liver cells as an example. Depending on the species, the device of this disclosure may include a suitable number of cells.

[0076] Fill density The fill density of the lattice structure disclosed herein represents an important parameter determining diffusion flow and vascularization. For example, a lattice structure with excessively high fill density may impede / weaken diffusion flow through the host tissue and / or inward growth of the host tissue. Alternatively, a lattice structure with excessively low fill density may be undesirable in terms of structural stability, FF adhesion, ease of retrieval, or one or more other aspects. As discussed herein, fill density is referred to as the percentage fill density of a 3D lattice structure comprising one or more coatings. A lattice structure with fully filled fiber structures (i.e., no space) would therefore correspond to a 100% fill density, while a lattice structure with 90% unoccupied by any fiber structure would correspond to a 10% fill density.

[0077] In embodiments, the fill density of suitable multilayer grid structures of this disclosure is between about 10% and about 90%, for example between about 20% and about 80%, for example between about 30% and about 70%, for example between about 40% and about 60%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%, or any value between these. Preferably, the fill density is between about 50% and about 60%.

[0078] For the multilayer (e.g., grid) structures of this disclosure, the infill density is advantageously controlled by the manner in which the structure is manufactured. Specifically, as explained in more detail in International PCT Application No. PCT / CA2023 / 050667, using the manufacturing platform described therein, continuous fibers can be precisely printed into grids having 2, 3, 4, 5, or more layers. To generate the grid, continuous fibers can be bioprinted around a plurality of posts used to apply tension to the fibers during printing. For each layer, this advantageously maintains a uniform distance between the fibers in each row / column and the fibers corresponding to adjacent rows / columns, and ensures the linearity of the fibers between each opposing post during grid fabrication.

[0079] After fabrication of the grid structure, one or more conformal coatings can be applied to the entire exposed surface of the grid structure without damaging the structure, and even before (or during) effective fiber-to-fiber adhesion throughout the printed structure. Therefore, these conformal coatings can advantageously be used to impart stability and / or anti-FBR properties to the fibrous structure. The frame and the corresponding attached grid structure can be transported to perform post-printing processing steps, such as one or more coatings, while the grid structure remains attached to the frame. In this way, the fabrication platform can be advantageously used during printing, patterning, and / or post-printing processing of the apparatus of this disclosure.

[0080] In an implementation, the fill density can be a function of the number of columns on the manufacturing platform and their relationship to each other (e.g., the distance to adjacent columns and / or the number of columns on opposite and / or adjacent sides of the frame).

[0081] Material strength In embodiments, the solid core, at least one shell, and one or more coatings may comprise the same or different material strengths. For example, and without limitation, in a multilayer lattice structure consisting of a solid core, at least one shell, and at least one coating, the core may have a first material strength, the at least one shell may have a second material strength, and the at least one coating may have a third material strength. In this example, each of the first, second, and third material strengths may be the same. In other embodiments, the first, second, and third material strengths may be different, or may be different from the other two. In embodiments where the continuous fiber comprises two or more shells, each shell may comprise a different material strength. Additionally or alternatively, in some embodiments where the lattice structure comprises at least two coatings, each coating may comprise a different material strength. In exemplary embodiments, the multilayer lattice structure comprises a solid core, a shell, and a coating, wherein the material strength of the shell is greater than the material strength of the core, and wherein the material strength of the coating is less than the material strength of both the shell and the core.

[0082] Segmentation / Separation In an implementation, the multilayer lattice structure produced by the methods disclosed herein can be segmented / divided along at least a portion of the length of the continuous bioprinted fibers forming the lattice structure. Details regarding the production of the segmented / divided fiber structure are described in WO 2022 / 246550, the disclosure of which is expressly incorporated herein by reference in its entirety.

[0083] In one embodiment, the core may be segmented / divided along at least a portion of the continuous bioprinted fiber forming the grid structure. In another embodiment, at least one shell may be segmented / divided along at least a portion of the continuous bioprinted fiber forming the grid structure. In yet another embodiment, both the core and at least one shell may be segmented / divided along at least a portion of the continuous bioprinted fiber forming the grid structure. In yet another embodiment, one or more segments / compartments of the core and / or shell may include dissociated cells. It should be understood that the compartments may include the same or different materials (e.g., different hydrogel materials). Additionally, depending on the desired application, the shell may be segmented / divided in a similar manner, either additionally or alternatively.

[0084] The compartment size can be a function of one or more variables, including but not limited to fiber size (e.g., length and / or diameter), the number of shells and / or coatings, the type of material used in the continuous fiber generation process, coating composition, etc. In some embodiments, the fiber may consist of at least two segments / compartments including dissociated cells, wherein other segments adjacent to the at least two segments / compartments do not contain dissociated cells. For example, and without limitation, at least two segments including dissociated cells may be included in the core of the continuous bioprinted fiber of this disclosure. In another example, at least two segments including dissociated cells may be included in at least one shell. In embodiments, the length of the segment including dissociated cells may be greater than, equal to, or less than the length of the cell-free segment. In some embodiments, the spacing between the compartments / segments comprising biological material (e.g., cells) in the fiber of this disclosure may be between 1 and 5 mm, for example, spaced 1 mm, 2 mm, 3 mm, 4 mm, or 5 mm apart.

[0085] For example, other design considerations for septa may include optimizing oxygen and nutrient diffusion to improve viability and function, and allowing the core and / or shell of the fibrous structure to switch between different materials, cell types, and densities. Septa may also allow for the printing of fibrous structures with different therapeutic doses without altering the geometry of the fibrous structure.

[0086] The segments / compartments may include biological materials, such as dissociated cells with a specific density. In embodiments, the densities between compartments may be the same or different. In embodiments, the biological materials within the compartments may be the same or different. In embodiments, the density of the biological materials may be selected based on one or more of the following: a specific application (e.g., treatment of a specific disease / condition), cell viability determinants, materials that include biological materials (e.g., biocompatible materials).

[0087] In embodiments, one or more segments / compartments comprising biomaterial may be side-connected to segments comprising, for example, materials with immunoprotective properties. For illustrative purposes and without limitation, immunoprotective hydrogel materials may include, for example, functionalized alginates, including but not limited to methacrylated alginate, furan alginate, thiol alginate, maleimide alginate, and covalently clicked alginates (e.g., alginates blended with DMAPS-Alg and / or DMAPS-Hzd). For example, where the core of a lattice structure as disclosed herein comprises one or more segments containing biomaterial, one or more segments may be side-connected to other segments comprising immunoprotective materials as disclosed herein. In another example, where the shell of a lattice structure as disclosed herein comprises one or more segments containing biomaterial, one or more segments may be side-connected to other segments comprising immunoprotective materials as disclosed herein. Also within the scope of this disclosure is that, where the core of a bioprinted fiber comprises biomaterial, at least one shell and / or at least one coating may comprise a material with immunoprotective properties. In another embodiment, where the shell of a lattice structure comprises biomaterial, another shell and / or at least one coating may comprise a material with immunoprotective properties.

[0088] Input materials Aspects of the invention include the ability to print grid structures for advantageous use as input materials for biomaterials. As used herein, “biomaterial” refers to natural or synthetic substances that can be used to construct or replace tissues, such as human tissues with or without living cells. In the field of bioprinting, the term “biomaterial” is often synonymous with the term “bioink.” Many such materials have been described above and will be further elaborated below.

[0089] Input materials typically include at least one crosslinkable material, such as hydrogels, including but not limited to alginates (e.g., SLG-100 alginate), chitosan, PEGDA, PEGTA, hyaluronic acid (HA), HAMA, collagen, CollMA, gelatin, methacrylated gelatin (gelMA), agarose, gellan gum, fibrin (fibrinogen), PVA, etc., or any combination thereof, and non-hydrogels, including but not limited to PCL, PLGA, PLA, etc., or any combination thereof. In a preferred embodiment, the input material includes at least one hydrogel. Non-limiting examples of hydrogels include alginates, agarose, collagen, fibrinogen, gelatin, chitosan, hyaluronic acid-based gels, or any combination thereof. Various synthetic hydrogels are known and can be used in embodiments of the systems and methods provided herein. For example, in some embodiments, one or more hydrogels form at least a portion of the structural basis for a three-dimensional structure used for printing. In some embodiments, the hydrogel has the ability to support the growth and / or proliferation of one or more cell types, which may be dispersed within the hydrogel or added to the hydrogel after it has been printed in a three-dimensional configuration.

[0090] In the embodiments, the hydrogel can be cross-linked by a chemical cross-linking agent. For example, hydrogels containing alginate can be cross-linked in the presence of divalent cations such as calcium chloride (CaCl2), hydrogels containing chitosan can be cross-linked using polyvalent anions such as sodium tripolyphosphate (STP), hydrogels containing fibrinogen can be cross-linked in the presence of enzymes such as thrombin, and hydrogels containing collagen, gelatin, agarose, or chitosan can be cross-linked in the presence of heat or alkaline solutions.

[0091] In some embodiments, hydrogel fibers can be generated via a precipitation reaction achieved through solvent extraction from the input material upon exposure to a crosslinking agent material miscible with the input material. Non-limiting examples of input materials for forming fibers via precipitation reactions include collagen and polylactic acid (PLA). Non-limiting examples of crosslinking materials enabling the formation of precipitation-mediated hydrogel fibers include polyethylene glycol (PEG) and alginate. Crosslinking of the hydrogel increases its stiffness, thereby allowing the formation of a cured hydrogel in some embodiments.

[0092] In some embodiments, the hydrogel comprises alginate. When contacted with a divalent cation, alginate forms a cured colloidal gel (high water content gel or hydrogel). Any suitable divalent cation can be used to form a cured hydrogel with an input material containing alginate. In the alginate ion affinity series, Cd... 2+ >Ba 2+ >Cu 2+ >Ca 2+ Ni2+ >Co 2+ >Mn 2+ In, Ca 2+ The most characteristic and most commonly used method for forming alginate gels (Ouwerx, C. et al., Polymer Gels and Networks, 1998, 6(5):393-408). Studies have shown that calcium alginate gels synergistically bind Ca through poly-G blocks on adjacent polymer chains. 2+ Ion formation, the so-called "egg-box" model (ISP Alginates, Section 3: Algin-Manufacture and Structure, in Alginates: Products for Scientific WaterControl, 2000, International Specialty Products: San Diego, pp. 4-7). G-rich alginates tend to form thermally stable, strong, and brittle Ca gels, while M-rich alginates tend to form less thermally stable, weaker, but more elastic gels. In some embodiments, the hydrogel comprises depolymerized alginate.

[0093] In some embodiments, the hydrogel can be crosslinked using a free radical polymerization reaction to form covalent bonds between molecules. Free radicals can be generated by exposing a photoinitiator to light (typically ultraviolet light), or by exposing the hydrogel precursor to a chemical source of free radicals, such as ammonium persulfate (APS) or potassium persulfate (KPS), in combination with N,N,N,N-tetramethylethylenediamine (TEMED) as both an initiator and a catalyst. Non-limiting examples of photocrosslinkable hydrogels include methacrylated hydrogels, such as hyaluronic acid methacrylate (HAMA), gelatin methacrylate (GEL-MA), or poly(ethylene glycol) acrylate (PEG-acrylate) based hydrogels, which are used in cell biology due to their cell inertness. Polyethylene glycol diacrylate (PEG-DA) is commonly used as a scaffold in tissue engineering because the polymerization reaction occurs rapidly at room temperature, requires low energy input, has high water content, is elastic, and can be customized to include a variety of biomolecules.

[0094] In this implementation, the input material includes a non-biodegradable polymer. In an example, the input material may be a synthetic polymer, such as polyvinyl acetate (PVA). In this implementation, the input material may include hyaluronic acid (HA).

[0095] In some embodiments, the hydrogel comprises a chemically modified alginate. In examples, the chemically modified alginate comprises alginate functionalized with methacrylate groups, referred herein as "Alg-MA". In some embodiments, Alg-MA can be used for an immunoprotective shell via blending with zwitterionic alginate, referred herein as "Alg-zw". Due to the dual crosslinking capability of Alg-MA, in embodiments, Alg-MA can first be printed with Alg-zw via physical crosslinking. During printing, the fibers can be further irradiated to induce transfiber covalent crosslinking, resulting in FF adhesion. In some embodiments, the chemically modified alginate may comprise thiolated alginate.

[0096] In some embodiments, one or more synthetic components may be added to the hydrogel material. These synthetic components can be used to increase fiber-to-fiber adhesion and / or in vivo stability. In examples, the material may include an acrylated zwitterionic monomer (e.g., sulfobetaine methacrylate (SBMA)) and a crosslinking agent (e.g., poly(ethylene glycol) diacrylate (PEGDA)). In this example, the light-mediated crosslinking of the zwitterionic monomer with PEGDA makes the resulting crosslinked polymer matrix superhydrophilic and therefore less susceptible to foreign body reactions (see WO 2022 / 246550, the contents of which are expressly incorporated herein by reference in their entirety).

[0097] In some embodiments, the hydrogel material can be cross-linked via click chemistry. For example, copolymers comprising zwitterionic monomers and aldehyde motifs (e.g., [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (DMAPS)-aldehyde, referred to herein as "DMAPS-Ald") and zwitterionic monomers and hydrazide motifs (e.g., DMAPS-hydrazide, referred herein as "DMAPS-Hzd") can be used (see WO 2022 / 246550). The aldehyde readily reacts with the hydrazide to form a covalently cross-linked hydrogel. Due to the presence of zwitterionic monomers in the polymer backbone, these polymers can exhibit low protein binding properties. In embodiments, one of these polymers can be blended with the alginate in the shell. After printing, the structure can be immersed in a solution containing counteracting components, which in turn creates covalently cross-linked bridges between fibers, resulting in FF adhesion.

[0098] In the implementation scheme, the input material includes microparticles, where “microparticles” as used herein refers to immiscible particles in the range of about 0.1 μm to about 100 μm, typically composed of polymers, metals, or other inorganic materials. They may be symmetrical (e.g., spherical, cubic, etc.), but this is not required. Microparticles with an aspect ratio of 2:1 or greater may be considered as microrods or microfibers.

[0099] Input materials according to embodiments herein may include any of a wide variety of natural or synthetic polymers that support the viability of living cells, including, for example, alginate, laminin, fibroin, hyaluronic acid, poly(ethylene glycol)-based gels, gelatin, chitosan, agarose, or combinations thereof. In some embodiments, the subject bio-ink composition is physiologically compatible, i.e., conducive to cell growth, differentiation, communication, and various other cellular functions (e.g., insulin release). In some embodiments, the input material includes one or more physiological matrix materials or combinations thereof. “Physiological matrix material” refers to biological materials found in natural mammalian tissues. Non-limiting examples of such physiological matrix materials include: fibronectin, platelet-reactive proteins, glycosaminoglycans (GAGs) (e.g., hyaluronic acid, heparin sulfate, chondroitin 6-sulfate, dermatan sulfate, chondroitin 4-sulfate, or keratin sulfate), deoxyribonucleic acid (DNA), adhesion glycoproteins, and collagen (e.g., collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VI, or collagen XVIII). Such physiological matrix materials may contribute to, for example, pancreatic β-cell survival, proliferation, and / or insulin secretion within the lattice structure background of this disclosure (Riopel M and Wang R., Frontiers in Bioscience (Landmark Editing ) (2014)19(1):77-90); Nikolova G et al., Dev Cell. (2006); 10(3): 397-405; Johansson A et al., Diabetologia (2009); 52(11): 2385-94).

[0100] Additional fluid Aspects of the invention include one or more buffer solutions. Buffer solutions according to embodiments of the invention are miscible with the input material (e.g., hydrogel) and do not crosslink the input material. In some embodiments, the buffer solution comprises an aqueous solvent. Non-limiting examples of buffer solutions include polyvinyl alcohol, water, glycerol, propylene glycol, sucrose, gelatin, or any combination thereof.

[0101] The viscosity of the buffer solution according to embodiments of the invention can range from about 1 mPa·s to about 5,000 mPa·s, such as about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,250, 3,500, 3,750, 4,000, 4,250, 4,500, or 4,750 mPa·s. In some embodiments, the viscosity of the buffer solution can be adjusted to match the viscosity of one or more input materials.

[0102] Aspects of the invention include one or more sheath fluids. A sheath fluid according to an embodiment of the invention is at least partially used to encapsulate or “wrap” input material dispensed from a dispensing channel. In some embodiments, the sheath fluid comprises an aqueous solvent. Non-limiting examples of sheath fluids include polyvinyl alcohol, water, glycerin, propylene glycol, sucrose, gelatin, or any combination thereof. The viscosity of the sheath fluid according to embodiments of the invention can range from about 1 mPa·s to about 5,000 mPa·s, such as about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,250, 3,500, 3,750, 4,000, 4,250, 4,500, or 4,750 mPa·s. In some embodiments, the viscosity of the sheath fluid can be adjusted to match the viscosity of one or more input materials.

[0103] In some embodiments, the sheath fluid comprises a chemical crosslinking agent. In some embodiments, the chemical crosslinking agent comprises a divalent cation. Non-limiting examples of divalent cations include Cd. 2+ Ba 2+ Cu 2+ Ca 2+ Ni 2+ Co 2+ or Mn 2+ In a preferred embodiment, Ca is used. 2+ As a divalent cation. In some embodiments, the concentration of the divalent cation in the sheath fluid ranges from about 80 mM to about 140 mM, such as about 90, 100, 110, 120 or 130 mM.

[0104] cell population In embodiments, the cell population is selected from the group consisting of single-cell suspensions, cell aggregates, cell spheroids, cell organoids, or combinations thereof. Input materials according to embodiments of the invention may incorporate any mammalian cell type, including but not limited to stem cells (e.g., embryonic stem cells, adult stem cells, induced pluripotent stem cells), germ cells, endoderm cells (e.g., lung, liver, pancreas, gastrointestinal, or urogenital tract cells), mesodermal cells (e.g., kidney, bone, muscle, endothelial, or heart cells), and ectoderm cells (skin, nervous system, pituitary, or eye cells), stem cell-derived cells, or any combination thereof. In a preferred embodiment, at least one cell population comprises or is composed of hepatocytes.

[0105] Cells can be obtained from a donor (allogeneic), from a species different from the recipient (xenogeneic), or from the recipient (autologous). Specifically, in embodiments, cells can be obtained from a suitable donor such as a human or animal, or from a subject to whom the cells will be implanted. Mammal species include, but are not limited to, humans, monkeys, dogs, cattle, horses, pigs, sheep, goats, cats, mice, rabbits, and rats. In one embodiment, the cells are human cells. In other embodiments, cells can be derived from animals, such as dogs, cats, horses, monkeys, or any other mammal.

[0106] In some embodiments, the cell population comprises primary cells from endocrine and exocrine glands selected from the group consisting of: pancreas, liver, kidney, parathyroid gland, thyroid gland, pineal gland, pituitary gland, thymus, adrenal gland, ovary, testis, tonsillar gland, enteroendocrine cells, stem cells, stem cell-derived cells of any of the aforementioned primary cell types, or cells engineered to secrete therapeutic agents. In some embodiments, the cell population includes HepG2 cells, primary human hepatocytes (PHH), mesenchymal stem cells (MSCs), hepatocyte-like cells derived from embryonic stem cells or induced pluripotent stem cells, hepatocyte-like cells derived from liver stem cells, xenogeneic hepatocytes, genetically engineered stem cell-derived hepatocytes, stem cell-derived endothelial cells or primary endothelial cells, and combinations thereof. In some embodiments, the dissociated cells include adrenal cells. In some implementations, the adrenal cells may be selected from the group consisting of: primary adrenal cells, including primary adrenocortical cells derived from any cell layer (zona glomerulosa, zona fasciculata, and / or zona reticularis) of the adrenal cortex, primary adrenal medullary cells, adrenocortical-like cells derived from embryonic stem cells or induced pluripotent stem cells, adrenocortical-like cells derived from adrenal stem cells, adrenocortical cells derived from genetically engineered stem cells, NCI-H295R cells, and combinations thereof.

[0107] Because the hepatocytes within the solid fibers disclosed herein are protected from the host immune system, the hepatocytes can be derived from any suitable source, i.e., human or non-human. In embodiments, the hepatocytes are stem cells or progenitor cells, including induced pluripotent stem cells that differentiate into hepatocytes. Suitable populations of hepatocytes (including, but not limited to, mature hepatocytes, immature hepatocytes, fetal-like hepatocytes, hepatocyte-like cells, hepatic progenitor cells, and hepatic precursor cells) and methods for generating such populations are known in the art, see, for example, patents, patent publications, or patent applications: US20210189350A1, US20180030415A1, US8148151B2, US10683486B2, US20200308551A1, US2020 0157494A1, US20220233605A1, US9284576B2, US10227565B2, US10004826B2, US8569573B2, US9284531B 2. WO2020227711A1, US20190084944A1, US10081609B2, US20170191031A1, US7989204B2, US8574905B2.Publications: Si-Tayeb et al., (2010) Hepatology; Hannan et al., (2013) Nature protocols; Ogawa et al., (2013) Development; Magner et al., (2013) Stemcells; Zhu et al., (2014) Nature; Siller et al., (2015) Stem Cell Reports; Blackford et al., (2019) Stem cells translational medicine; Raggi et al., (2022) Stem Cell Reports; Blackford et al., (2023) Biomaterials; Carpentier et al., (2014) The Journal of clinical investigation; Carpentier et al., (2016) Stem cell research; Xia et al., (2017) Journal of hepatology; Uchida et al., (2021) Molecular therapy; Tilson et al., (2021) Hepatology; Duan et al., (2007) Stem cells; Bandi et al., (2019). Differentiation; Mallanna and Duncan (2013). Curr Protoc Stem Cell Biol., each of which is hereby incorporated in its entirety by reference. Stem cell-derived hepatocytes or kits for differentiating stem cells into hepatocytes are commercially available from suppliers including, but not limited to, Creative Bioarray (human iPSC-derived hepatocytes, catalog number CSC-00863L) and STEMCELL Technologies (STEMdiff™ Hepatocyte Kit, catalog number 100-0520).

[0108] In this implementation, the hepatocytes are primary hepatocytes from any suitable source (i.e., human or non-human). Primary hepatocytes are commercially available from suppliers including, but not limited to, Thermo Fisher human hepatocytes (catalog numbers HMCPSQ and HMCPTS), KaLy Cells (platelet-compatible CryoHeps), Lonza (PHH cells, catalog number HUCP), and AnaBio (primary human hepatocytes). In this implementation, the hepatocytes may be packaged with other cell types, including, but not limited to, mesenchymal stem cells (MSCs). Mesenchymal stem cells (MSCs) are commercially available from suppliers including, but not limited to, RoosterBio (Xeno-Free RoosterVial™-hUC, catalog numbers C43002UC or C43001UC and RoosterVial™-hUC-20M-CC, catalog number C04020UC).

[0109] In some embodiments, the dissociated cells include HepG2 cells, primary human hepatocytes (PHH), mesenchymal stem cells (MSC), hepatocyte-like cells derived from embryonic stem cells or induced pluripotent stem cells, hepatocyte-like cells derived from liver stem cells, xenogeneic hepatocytes, genetically engineered stem cell-derived hepatocytes, stem cell-derived endothelial cells or primary endothelial cells, and combinations thereof. In some embodiments, the dissociated cells include HepG2 cells, primary human hepatocytes (PHH) and / or mesenchymal stem cells (MSC), and combinations thereof.

[0110] In some implementations, the dissociated cells comprise PHH and MSC in a ratio of 3:1.

[0111] In some embodiments, at least one biological material included in the lattice structure of this disclosure comprises a population of cells that express / secrete one or more endogenous bioactive agents (e.g., insulin, glucagon, ghrelin, pancreatic polypeptide, factor VII, factor VIII, factor IX, α-1 antitrypsin, angiogenic factors, growth factors, hormones, antibodies, enzymes, proteins, exogens, etc.). Endogenous bioactive agents discussed herein include those naturally produced by cells in their biological environment (e.g., insulin release in response to elevated glucose concentrations). Endogenous bioactive agents may constitute therapeutic agents in the context of this disclosure.

[0112] In some embodiments, the input material may include genetically engineered cells that secrete specific factors. Cell populations as discussed above may, in embodiments, include engineered cells that secrete specific factors (e.g., genetically engineered cells), which is within the scope of this disclosure. Cells may also be derived from established cell culture lines, or may be cells that have been genetically engineered and / or manipulated to achieve a desired genotype or phenotype. In some embodiments, tissue fragments may also be used, which may provide multiple different cell types within the same structure.

[0113] Genetic engineering techniques applicable to this disclosure may include, but are not limited to: recombinant DNA (rDNA) technology (Stryjewska et al., Pharmacologial Reports. 2013; 65: 1075); and cell engineering based on the use of targeted nucleases (e.g., broad-spectrum nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly spaced short palindromic repeat-associated nucleases Cas9 (CRISPR-Cas9), etc.) (Lim et al., Nature Communications . 2020; 11: 4043; Stoddard BL, Structure . 2011; 19(1): 7-15; Gaj et al., Trends Biotechnol . 2013; 31(7): 397-405; Hsu et al., Cell . 2014; 157(6): 1262; Miller et al., Nat Biotechnol . 2010; 29(2): 143-148); Cell engineering based on site-specific recombination using recombinase systems (e.g., Cre-Lox) (Osborn et al., 2010; 29(2): 143-148); Mol Ther . 2013; 21(6): 1151-1159; Hockemeyer et al., Nat Biotechnol . 2009; 27(9): 851-857; Uhde-Stone et al., RNA . 2014;20(6): 948-955; Ho et al., Nucleic Acids Res . 2015; 43(3): e17; Sengupta et al., Journal of Biological Engineering. 2017; 11(45): 1-9) etc. In some implementations, some combinations of the techniques for cell engineering mentioned above may be used.

[0114] This disclosure covers engineered cells capable of producing one or more therapeutic agents, including but not limited to proteins, peptides, nucleic acids (e.g., DNA, RNA, mRNA, siRNA, miRNA, nucleic acid analogs), peptide nucleic acids, aptamers, antibodies or fragments or portions thereof, antigens or epitopes, hormones, hormone antagonists, growth factors or recombinant growth factors and fragments and variants thereof, cytokines, enzymes, antibiotics or antimicrobial compounds, anti-inflammatory agents, antifungal agents, antiviral agents, toxins, prodrugs, small molecules, pharmaceuticals (e.g., drugs, dyes, amino acids, vitamins, antioxidants), or any combination thereof.

[0115] In embodiments, the cells of this disclosure may be modified to include at least one mechanism for providing local immunosuppression at the transplantation site, such as in the tissue fibers of this disclosure, upon transplantation into an allogeneic host. In examples, one or more cells may contain a set of transgenes, each transgene encoding a cytoplasmic, membrane-bound, or locally acting gene product, and its function may include, but is not limited to, mitigating the activation and function of antigen-presenting cells; mitigating the activity or cytolytic function of graft-attacked leukocytes; mitigating the cytolytic function of macrophages and the phagocytosis of allogeneic graft cells; inducing apoptosis in leukocytes attacking the graft; mitigating local inflammatory proteins; and preventing leukocyte-mediated apoptosis (WO2018 / 227286; Harding et al.). BioRxiv . 2019; DOI: 10.1101 / 716571 Lanza et al. Nature Reviews Immunology . 2019; 19: 723-733l; Harding et al., Cell Stem Cell .2020; 27(2): 198-199).

[0116] In the embodiments, the cells of this disclosure can be modified in a manner that controls cell proliferation. As an example, cells can be genetically modified at the cell division locus (CDL) to include a negatively selective marker and / or a gene expression system based on an inducible activator, thereby enabling the control of allowing, eliminating, and / or inhibiting the proliferation of genetically modified cells by adding or removing appropriate inducers (WO2016 / 141480; Liang et al.). Nature . 2018; 563(7733):701-704).

[0117] Appropriate growth conditions for mammalian cells are well known in the art (Freshney, RI (2000) Culture of Animal Cells, a Manual of Basic Technique. Hoboken NJ, John Wiley & Sons; Lanza et al. Principles of Tissue Engineering, Academic Press; 2nd ed., May 15, 2000; and Lanza & Atala, Methods of Tissue Engineering, Academic Press; 1st ed., October 2001). Cell culture media typically include essential nutrients and optional supplementary elements such as growth factors, salts, minerals, and vitamins, which can be selected depending on the cell type being cultured. Specific components can be selected to enhance cell growth, differentiation, secretion of specific proteins, etc. Generally, standard growth media include Durbeco modified Eagle medium, low glucose (DMEM), containing 110 mg / L pyruvate and glutamine, supplemented with 10% to 20% fetal bovine serum (FBS) or calf serum and 100 U / ml penicillin, which is as suitable as various other standard media known to those skilled in the art. Growth conditions will vary depending on the type of mammalian cells used and the desired tissue.

[0118] In some implementations, cell type-specific reagents can be advantageously used in subject input materials for the corresponding cell type. For example, ECM can be extracted directly from the tissue of interest, then dissolved and incorporated into the input material to generate a tissue-specific input material for printing the tissue. Such ECMs are readily available from patient samples and / or commercially available from suppliers such as zPredictA (rBone™, available at zpredicta.com / home / products).

[0119] Printing system In a preferred embodiment, the bioprinting system includes the technology described in WO2014 / 197999, WO2018 / 165761, WO2020 / 056517, WO2021 / 081672, WO2022 / 246550, and WO2023 / 108302, the disclosures of which are expressly incorporated herein by reference. As detailed therein, the disclosed bioprinting systems and their components enable multi-material switching and thus allow for changes in the composition (e.g., cell type and biomaterial composition) of one or more components of the synthesized tissue fibers along the length of the fibers during continuous printing. In an embodiment, the microfluidic-based bioprinting system is the RX1™ bioprinter (Aspect Biosystems, Vancouver, BC, Canada), or the RX2™ bioprinter system described in WO 2023 / 108302, the contents of which are incorporated herein by reference in their entirety.

[0120] In an exemplary embodiment of a preferred bioprinting system, the system includes a printhead comprising a dispensing channel, wherein one or more material channels and a core channel converge at a proximal end of the dispensing channel. The printhead may be configured to simultaneously dispense a buffer solution and / or sheath fluid with one or more crosslinkable materials. In some embodiments, the printhead is configured to maintain a constant mass flow rate in the dispensing channel. In this manner, the printhead may be configured to facilitate a smooth and continuous flow of one or more input materials (or a mixture of one or more input materials) along with the buffer solution and / or sheath fluid through the dispensing channel. In the use of such a printhead, the input material flowing through the dispensing channel may be crosslinked from within by fluid flowing through the core channel and / or from the outside by sheath fluid flowing through the downstream sheath fluid channel, as described in more detail in WO2020 / 056517. In some embodiments, the printhead includes one or more fluid concentration chambers in a tapered truncated shape and optionally one or more printhead adapters, as detailed in WO 2021 / 081672, WO 2022 / 246550, and WO 2023 / 108302. In some embodiments, the printhead is a DUO™ microfluidic printhead or a CENTRA™ microfluidic printhead (Aspect Biosystems, Vancouver, BC, Canada).

[0121] Other examples of bioprinting systems relevant in the context of this disclosure (e.g., those that may be modified or used in conjunction with the methods of this disclosure) include, but are not limited to, 3-D Bioplotter® (EnvisionTEC Inc., Dearborn, MI, USA), NovoGen Bioprinter® platform (Organovo®, San Diego, CA, USA), R-Gen 100 and R-Gen 200 (RegenHU, Villas-Saint-Pierre, Switzerland), Bioprinter Fabion and Fabion 2 (3D Bioprinting Solutions, Moscow, Russia), BioBot® Basic, BioAssemblyBot® 200 / 400 / 500 (Advanced Solutions, Louisville, KY, USA), BIO X™, BIO X6™, INKREDIBLE+™ (CellINK, Boston, MA, USA), Ourobotics Revolution (Ourobotics, Cork, Ireland), BioScaffolder 2.1 (GeSim, Radeberg, Germany), and Omega Bioprinter. (3Dynamic Systems, Bridgend, UK), Syn and Explorer (Bio3D, Singapore), Alevi 1 / 2 / 3 (Alevi by 3D Systems, Rock Hill, SC, USA), and Dr. Invivo4D6 (Rokit Healthcare, Seoul, South Korea).

[0122] Manufacturing method Aspects of the invention include: a) providing a bioprinting system comprising: a printhead including a plurality of microfluidic channels for selectively supplying a core material, at least one shell material, and a sheath fluid to a dispensing orifice; a receiving surface for receiving material dispensed from the orifice; a positioning unit for positioning the orifice of the printhead in three-dimensional space, the positioning unit being operatively coupled to the printhead; and at least one dispensing member for dispensing fibers from the dispensing orifice; b) simultaneously printing a core and at least one shell of tissue fibers via the bioprinting system, wherein a cell population is encapsulated in the core and / or at least one shell; and c) cryopreserving the bioprinted tissue fibers with a cryoprotectant; wherein the cell population comprises at least 95% dissociated cells embedded in a biocompatible matrix, and wherein the fibers are cryopreserved prior to the aggregation of the dissociated cells; preferably cryopreserved after 15 minutes to 3 hours of bioprinting, more preferably after 30, 45, or 60 minutes of bioprinting.

[0123] In one embodiment, the cryopreservation step includes adding a cryoprotectant to the printed fibers at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 minutes before freezing. In another embodiment, the core material also contains a tackifier, preferably wherein the viscosity of the core material and the shell material is substantially the same.

[0124] In some embodiments, the cryoprotectant does not include dimethyl sulfoxide (DMSO). In some embodiments, the cryoprotectant may include DMSO. In some embodiments, the DMSO-containing cryoprotectant is selected from the group consisting of: CRYOSTOR® CS10, CRYOSTOR® CS5, CROSTOR CS2, PSC cryopreservation kit, Synth-a-Freeze™ cryopreservation medium, CELLBANKER® 1, CELLBANKER® 2, and STEM-CELLBANKER® GMP grade. In some embodiments, the DMSO-free cryoprotectant is selected from the group consisting of: CRYOSTOR® CSB, CrySOfree™ DMSO-free cryopreservation medium, STEM-CELLBANKER® DMSO-free GMP grade, and Cell-Vive™ CD DMSO-free cryopreservation solution.

[0125] In one embodiment, the printing step may include distributing continuously bioprinted core / shell fibers onto a receiving surface to form a multilayer (e.g., grid) structure, wherein the multilayer structure is cryopreserved prior to the aggregation of dissociated cells within the fibers. In another embodiment, the receiving surface may include a fabrication platform for supporting the continuously bioprinted core / shell fibers, the fabrication platform including a frame defining a void and including a plurality of posts on opposite sides of the frame for securing and suspending the continuously bioprinted fibers during printing; and the printing step includes distributing fibers around the plurality of posts to form a grid structure comprising at least two, three, four, or five layers of fibers. In yet another embodiment, the method may further include coating the multilayer (e.g., grid) structure after printing is complete.

[0126] The general method of printing a first layer and (optionally) a second, third, etc., is described in detail above, as well as in, for example, International Patent Application Nos. PCT / CA2023 / 050668 and WO 2022 / 246550, each of which is incorporated herein by reference in its entirety. Broadly speaking, this process involves generating a multilayer structure from continuous fibers, preferably via a manufacturing platform, and optionally applying at least one coating to obtain an implantable device. Continuous fibers are typically generated in such a manner that, during printing, the continuous fibers are exposed to a sheath fluid containing a crosslinking agent that crosslinks the continuous fibers from the outside in; however, in other embodiments, crosslinking from the inside out (where the crosslinking agent is included as part of the core material) is also within the scope of this disclosure (see WO 2022 / 246550). It should also be understood that fibers comprising more than one sheath are also within the scope of this disclosure.

[0127] During fiber printing, sheath fluid is removed, for example, by flowing through a porous receiving surface. Following bioprinting, the method optionally further includes immersing the multilayer structure in a crosslinking solution (or otherwise applying the crosslinking solution, e.g., via spraying, or via the same dispensing member used for dispensing continuous fibers) to promote / continue uniform crosslinking throughout the printed lattice structure. Coating can also be applied, for example, by immersing the structure in a coating solution, dispensing the coating solution onto the finished structure using a microfluidic printhead (e.g., the same microfluidic printhead used for fabricating fibers), spraying the structure with the coating solution, etc.

[0128] In one embodiment, the coating solution comprises a crosslinkable material (e.g., alginate). In another embodiment, the coating solution comprises the same material as the material constituting the core and / or at least one shell of the coated structure. In yet another embodiment, the coating solution may comprise a material different from the material constituting the core and / or at least one shell. In another embodiment, a residual crosslinking agent (e.g., Ca) associated with the structure... 2+This facilitates the initial crosslinking of the material in the coating solution with the material constituting the shell. After coating the grid structure, the structure can be immersed in the crosslinking solution, or the crosslinking solution can be applied in other ways, such as by spraying or by dispensing via the same microfluidic printhead used for dispensing continuous fibers. Within the scope of this disclosure, different crosslinking solutions can be used, for example, in cases where the material used to manufacture at least one shell comprises a different material than the material used to manufacture the coating. In this way, the coating can be uniformly added to the entire multilayer structure produced by continuous bioprinting fibers. Although not specifically stated, within the scope of this disclosure, a second coating can be further applied in a manner similar to the first coating.

[0129] In embodiments where a single coating is applied, the coating may impart stability and / or fiber-to-fiber adhesion properties and / or anti-FBR properties to the resulting fiber structure. In embodiments where the fiber structure includes two coatings, the first coating may have specific desired properties (e.g., a material selected to enhance stability and / or FF adhesion), while the second coating may have additional / alternative properties (e.g., customized to have anti-FBR properties). In a preferred embodiment, the coating is a conformal coating that uniformly covers the entire exposed surface of the fiber structure.

[0130] In embodiments, the coating is composed of a hydrogel material, such as a hydrogel material comprising one or more of the following: alginate, zwitterionic alginate, SBMA, chitosan, PEGDA, PCL, PEG, poly-L-lysine (PLL), PEGTA, hyaluronic acid (HA), HAMA, collagen, CollMA, gelatin, gelMA, agarose, gellan gum, fibrin (fibrinogen), PVA, etc., or any combination thereof. In some examples, the coating is composed of functionalized alginate, i.e., alginate chemically modified to include one or more properties, including but not limited to immunoprotective properties that facilitate the fabrication of the lattice structure of this disclosure. Examples of functionalized alginate include, but are not limited to, methacrylated alginate, furan alginate, thiol alginate, maleimide alginate, and covalently clicked alginate (e.g., alginate blended with DMAPS-Alg and / or DMAPS-Hzd).

[0131] In some embodiments, the material strength of the coating is less than the material strength of one or more shells and / or cores. In embodiments where the structure includes two coatings, the material strength of the outer coating may be less than that of the inner coating, or vice versa. Alternatively, the outer and inner coatings may have substantially the same material strength, optionally wherein the outer and inner coatings are composed of different materials. In some embodiments, the core is solid, optionally wherein the core is segmented / divided along the length of the fibers. In other or alternative embodiments, at least one shell may be segmented / divided along the length of the fibers.

[0132] As described, conformal coatings can impart stability to fiber structures and / or impart properties that optimize the interface between the fiber structure and the host, such as anti-FBR properties and promotion of vascularization. Further details regarding the production of such conformal coatings are described in International Patent Application No. PCT / CA2023 / 050667, published as WO 2023 / 220816, the contents of which are expressly incorporated herein by reference in their entirety.

[0133] In the embodiments, the bioprinted fiber structure produced by the methods disclosed herein may also be segmented / divided along at least a portion of the length of the fiber (preferably a continuous fiber) constituting the bioprinted fiber structure. Details of producing segmented / divided bioprinted fiber structures are described in WO 2022 / 246550, the contents of which are expressly incorporated herein by reference in their entirety.

[0134] In some embodiments, a method first includes providing a design for a multi-layered structure, as disclosed herein, to be printed. The design may be created using commercially available CAD software. In some embodiments, the design includes information about specific materials to be assigned to specific locations within the structure to be printed (e.g., for heterogeneous structures comprising multiple materials).

[0135] Treatment The bioprinted 3D raster structure disclosed herein can be used in methods for treating diseases or ailments in subjects in need. An aspect of the invention includes a method for implanting a cell population into a subject in need, the method comprising: a. thawing cryopreserved tissue fibers or compositions / medical devices of the present disclosure; and b. implanting the fibers or compositions / medical devices into the subject. In embodiments, the method may further include culturing the tissue fibers or compositions / medical devices in fresh culture medium after thawing, prior to implantation into the subject, until dissociated cells form aggregates within the tissue fibers. In embodiments, thawing is performed using a suitable thawing device, such as ThawSTAR® CFT2, ThawSTAR CB, etc.

[0136] In the implementation plan, the disease or ailment is a disease or ailment of the liver.

[0137] The method involves: thawing cryopreserved tissue fibers or compositions / medical devices as described herein, the tissue fibers or compositions / medical devices comprising a therapeutically effective amount of dissociated cells (e.g., hepatocytes) encapsulated within the fibers and / or structures; and optionally, culturing the tissue fibers or compositions / medical devices in a fresh culture medium after thawing, prior to implantation into the subject, until the dissociated cells form aggregates within the tissue fibers. The therapeutically effective amount of cells may include a predetermined therapeutically effective number of cells. The therapeutically effective amount may also vary depending on, for example, the body size and physical condition of the individual being treated. For example, the therapeutically effective amount may include a certain number / quantity of hepatocytes per kilogram of body weight.

[0138] The method involves implanting fibers and / or structures as disclosed herein into a subject in need. In embodiments, the method includes implantation once or multiple times. For example, in embodiments, the method includes implanting thawed fibers or compositions / medical devices containing therapeutically effective amounts of cells into a subject in need approximately every 4 months, every 6 months, annually, every 2 years, every 3 years, every 4 years, every 5 years, or more. In embodiments, the encapsulated cell population survives for at least four months, or at least 6 months, or at least one year, or at least two years, or at least three years, or at least four years, or at least five years, or more after implantation. In embodiments, the subject may require only a single implantation. In embodiments, the composition / medical device may need to be replaced every 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 months, or every 1, 2, 3, 4, 5 years, or more.

[0139] In one implementation, a composition / medical device containing the cell population of interest is implanted into the greater omentum of a subject. The greater omentum (also known as the greater gastric mesentery, omentum majus, gastrocolic omentum, epiploon, or caul) is a large apron-like fold of the visceral peritoneum that descends from the stomach and extends posteriorly from the greater curvature of the stomach to ascend to the transverse colon, and then to the retroperitoneal wall. Therefore, this structure can be implanted into a pouch formed from the greater omentum through surgical intervention.

[0140] In the implementation scheme, minimally invasive surgical techniques (such as laparoscopic surgery) can be used to surgically implant the composition / medical device. In the implementation scheme, the composition / medical device is implanted into the abdominal or thoracic cavity via laparoscopy. In the implementation scheme, implantation is performed intraperitoneally, percutaneously, or subcutaneously.

[0141] In one embodiment, the composition / medical device is anchored or secured (e.g., by sutures) at the implantation site to maintain the structure near the implantation site. In another embodiment, the delivery of the therapeutic agent is location-independent and the biodistribution of the agent depends on the subject's vascular system or bodily fluids. In yet another embodiment, the structure is implanted percutaneously or subcutaneously under the skin of the abdomen, forearm, flank, back, buttocks, legs, etc., and remains substantially there until removal is required.

[0142] In some embodiments, the composition / medical device is retrievable after implantation. In some embodiments, anchoring or securing structures prevent the structure from migrating, moving, or traversing within the subject's body and facilitate retrieval. However, also within the scope of this disclosure, given the size of the composition / medical device, anchoring or otherwise securing the structure may not be necessary if retrieval remains easily facilitated. Retrieval may be desirable after the encapsulated cell population ceases or substantially ceases to function at a certain threshold, or after cell death exceeds or is expected to exceed a certain threshold. After retrieval, the structure can be replaced with a new structure to maintain the desired effect.

[0143] Within the scope of this disclosure, the implanted grating structure can be monitored periodically (e.g., weekly, bi-monthly, monthly) to ensure that the cells in the grating structure function as intended. For example, the structure may include one or more contrast agents to facilitate in vivo monitoring of fibrous device placement, implant location at a point in time post-implantation, implant integrity, adverse effects of non-target cell types, inflammation, and / or fibrosis. Suitable contrast agents include, but are not limited to, nanoparticles, nanocrystals, gadolinium, iron oxide, iron platinum, manganese, iodine, barium, microbubbles, fluorescent dyes, and other contrast agents known to those skilled in the art.

[0144] In vivo monitoring methods include, but are not limited to, confocal microscopy, two-photon microscopy, high-frequency ultrasound, optical coherence tomography (OCT), photoacoustic tomography (PAT), computed tomography (CT), magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), and positron emission tomography (PET). These, individually or in combination, can provide useful means for monitoring implanted lattice structures.

[0145] Example The following embodiments are provided to provide a complete disclosure and description to those skilled in the art regarding how to prepare and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors consider to be their invention. Efforts have been made to ensure the accuracy of the figures used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weights are average molecular weights, temperatures are degrees Celsius, and pressures are atmospheric pressure or close to atmospheric pressure.

[0146] Example 1. Bioprinting and evaluation of cell populations containing hepatocytes and elimination of pre-aggregation Using primary rat hepatocytes (PRH), solid fibers containing dissociated cells in the core of the core shell fiber and fibers containing pre-aggregated cells with aggregated spheroids in the core shell fiber were produced.

[0147] 2.4 x 10 7 Primary rat hepatocytes were thawed, resuspended in hepatocyte thawing medium (ThermoFisher), and centrifuged. 8 x 10⁸ cells were then... 6 The mesenchymal stem cells were thawed, resuspended in Rooster MSC medium (RoosterBio), and centrifuged. Thawing was performed using ThawSTAR CFR2 (Biolife Solutions). The medium was decanted, and the cells were resuspended in 1 mL of medium, combined, and centrifuged again. The cells were then resuspended in gelatin by adding 1 part gelatin (20% w / w stock solution) to 3 parts cell volume. The cells were then loaded into syringes for bioprinting.

[0148] A syringe containing 2% SLF-100 alginate (Novamatrix) dissolved in saline was inserted into the shell channel of the printhead. A syringe containing a crosslinking agent solution of 95 mM CaCl2, 5 mM BaCl2, and 3% PVA (90 kDa) was connected to the crosslinking agent channel of the printhead. A bottle containing 0.9% physiological saline was connected to the buffer channel of the printhead. Fibers containing 1 million cells were printed, and the process was repeated 24 times.

[0149] For pre-aggregated cell bioprinting, cells were thawed as described above and then used to form aggregates, each containing an average of 199 hepatocytes. Aggregation was performed using STEMCELL Technologies AggreWell plates according to the manufacturer's instructions. The cells were then cultured in William's E medium supplemented with FBS, ITS, and dexamethasone. The aggregates were harvested after 3 days and centrifuged. The precipitate was resuspended in 3% SLG-100 alginate and diluted to 1.5% (including cell volume).

[0150] Solid fibers (containing dissociated cells) were cultured in William's E medium containing FBS, ITS, and dexamethasone, then switched to medium without FBS. Pre-aggregated fibers were cultured in medium without FBS. The medium was changed every 2 days, and the medium was collected on days 1, 3, 5, and 7 to quantify albumin. Cyp3A4 activity was analyzed using the P450-GloCyp3A4 kit (Promega) according to the manufacturer's instructions. Secreted albumin in the conditioned medium was quantified by ELISA (Abcam catalog number ab235642 or ab179887) according to the manufacturer's instructions.

[0151] Histological analysis was performed 7 days after printing. The fibers were fixed in 10% formalin for 16 hours, followed by rinsing with 70% ethanol. The fibers were embedded in paraffin, sectioned, and stained with H&E using standard protocols.

[0152] The viability / death status of the fibers was determined by incubating them for 30 minutes in a medium supplemented with 4 μM calcein AM, 5 μg / mL DAPI, and 15 μg / mL propidium iodide, followed by imaging with a Zeiss AxioObserver.

[0153] For cryopreservation, within one hour of printing, the fibers are transferred to a controlled-rate cryostat in a CryoMed system. The cryopreservation protocol is as follows: maintain at 4°C until the sample reaches 5°C, decrease the chamber temperature by 1°C per minute until the sample reaches -4°C, decrease the chamber temperature by 10°C per minute until the chamber reaches -40°C, increase the chamber temperature by 1°C per minute until the chamber reaches -12°C, decrease the chamber temperature by 1°C per minute until the chamber reaches -40°C, and decrease the chamber temperature by 10°C per minute until the chamber reaches -90°C. The sample is then stored in liquid nitrogen.

[0154] Thaw the vials using a 37°C water bath until the solution is completely thawed, and then transfer them to a tissue culture dish by pouring. Next, aspirate the frozen culture medium and replace it with fresh medium for 1 minute. Then, aspirate the remaining medium and incubate according to the method described above.

[0155] For pre-aggregated fibers, there were 50 million PRH / mL, 100:50 PRH:MSC spheroids. For solid fibers, at a PRH:MSC ratio of 2:1, 75% v / v cells were present in the 5% fish gelatin core.

[0156] Figures 1A to 1C H&E staining of pre-aggregated fibers and solid fibers was shown on day 7 post-printing. Figure 1A ); Fluorescence micrographs of the live / dead state of solid and pre-aggregated fibers on days 1, 3, and 7 after printing ( Figure 1B This demonstrates that solid fibers have significantly less dead space than pre-aggregated fibers; and the albumin levels of solid and pre-aggregated fibers from two different donors ( Figure 1C This confirms that solid fibers have a much higher albumin yield. Figure 1D The relative albumin yield after cryopreservation was shown, thus confirming that more than 70% of albumin production capacity was retained compared to uncryopreserved solid fibers, indicating that tissue therapeutic agents can be manufactured well before being used in a clinical setting. Figure 1E The study shows the albumin production of cultured solid filaments, or solid filaments that were first cryopreserved and then cultured. Albumin production in cryopreserved cells was 70% higher than in non-cryopreserved cell types, confirming that not only MSCs but also hepatocytes can survive this process. Figure 1F The live / dead ratio of solid fibers, whether frozen or not, is shown. Figure 1G Micrographs of solid fibers, both cryopreserved and uncryopreserved, are shown, demonstrating aggregation after thawing. A moderate amount of latent heat of crystallization was observed, indicating that the samples were supercooled, leading to some intracellular ice formation. The fibers are soft but strong enough to be handled with forceps and exhibit no obvious cracks or deformation. Cell aggregation within the solid core fibers was observed in both fresh and frozen hepatocyte fibers.

[0157] These results confirm that the viability of PRH cells in solid fibers is higher than that in pre-aggregated PRH cells; albumin production is much higher in PRH cells within solid fibers compared to aggregates; and viability for PRH is lower than historical PHH data.

[0158] Primary human hepatocytes (PHH) were treated in the same way. For pre-aggregated fibrous cells, there were 50 million PHH cells / mL, with a 100:50 PHH:MSC spheroid ratio. For solid fibrous cells, there were 75% v / v cells in a 5% gelatin core at a PHH:MSC ratio of 2:1.

[0159] Figures 2A to 2BFluorescence micrographs of the live / dead assays of solid and pre-aggregated fibers from two different donors are shown. Figure 2A ), and albumin and Cyp3A4 activity from solid and pre-aggregated fibers from two different donors ( Figure 2B As shown in the figure, solid fibrous tissue therapeutic agents contain fewer dead spaces and aggregates after printing.

[0160] These results confirm that the activity in solid fibers is higher than that in pre-aggregated PRH cells; albumin production is much higher in PHH cells within solid fibers compared to aggregates; and printing with solid fibers significantly improves Cyp3A4 activity.

[0161] In addition, solid fibers are smaller in diameter than fibers with pre-aggregated cells.

[0162] Example 2. Bioprinting of tissue therapeutic agents In terms of the overall size of implants, increasing the density of tissue therapeutic agents has a major advantage. Efforts are being made to increase the cell density of the core of tissue therapeutic agents to a level similar to that of natural tissue. High-density tissue therapeutic agent construction can be achieved by generating fine fibers via bioprinting.

[0163] Hepatocytes are suspended in a cell-containing biomaterial and printed onto coaxial fibers with a cell-free shell. These fibers can optionally be patterned into three-dimensional shapes. Figure 3A As shown, this figure illustrates bright-field microscopy and fluorescence microscopy images of a fiber depicting cells within it, with cells distributed within the fiber's core. Cells can also be distributed within the core, as... Figure 3B As shown, the cell is located in the first shell (ring region).

[0164] Example 3. Bioprinting and in vivo evaluation of tissue therapeutic agents Standard care for many liver diseases, including acute liver failure (ALF) and urea cycle disorder (UCD), includes liver transplantation. While effective, this is invasive, requires long-term immunosuppression, and can lead to bleeding. Recent studies have demonstrated encouraging results in the implantation of encapsulated hepatocytes into the peritoneal (IP) space as a treatment for pediatric ALF. It remains unclear whether this procedure can be performed at less invasive surgical sites. The advantages of the subcutaneous (SubQ) space are its relative non-invasiveness and larger size, and the fact that the IP space can be contaminated with ascites in some liver diseases. This embodiment describes the implantation of liver BTTs containing primary human hepatocytes (PHH) and mesenchymal stem cells (MSCs) into both the IP and SubQ spaces of NSG mice. Additionally, we describe a method for manufacturing cryopreservable high-density BTTs, which would significantly alleviate supply chain constraints regarding any potential treatment.

[0165] Primary human hepatocytes (PHH) and mesenchymal stem cells (MSCs) can aggregate into spheroids and be three-dimensionally bioprinted into tissue therapeutic agents. These tissue therapeutic agents can be evaluated via biomarker measurements (such as one month post-implantation after subcutaneous or intraperitoneal implantation in mice) and live / dead analysis.

[0166] Specifically, PHH and MSCs are co-aggregated into spherical particles, which are then suspended in an alginate-based biomaterial. This produces a product containing 1 x 10⁻⁶ spherical particles. 6 One PHH and 1 x 10 6 A single MSC or cell-free tissue therapeutic agent was implanted into the IP or SQ space of immunodeficient NSG mice. Blood was collected within one month post-implantation, and biomarkers of liver function, including human serum albumin, were measured. Subsequent experiments were performed without cell pre-aggregation; instead, a single-cell bioprinting tissue therapeutic agent was used, wherein the single cells were able to aggregate after printing, and a subset of these fibers were cryopreserved and thawed to demonstrate long-term storage capability.

[0167] Tissue therapeutic agents were retrieved from NSG mice after 28 days. Calcein AM / propidium iodide activity assays were performed to determine cell viability in both the in vitro control tissue therapeutic agent and the retrieved ex vivo tissue therapeutic agent. Results were... Figure 4A The results show that this confirms that cells retained high viability when implanted into NSG mice via IP or SQ compared to the incubator-maintained control.

[0168] Figure 4B Plasma albumin concentrations (via ELISA) in NSG mice implanted with tissue therapies containing PHH or PHH and MSCs are shown. These results confirm that tissue therapies incorporating MSCs have a longer duration of function after implantation compared to tissue therapies without MSCs.

[0169] Figure 4C The surgical wound (indicated by the black arrow) is shown 3 days post-implantation for IP and SQ implantation in NSG mice. In either case, the wound closes; however, the wound is much smaller when SQ implantation is performed in mice. The outline of the tissue therapy agent is visible beneath the skin of the SQ mouse.

[0170] Figure 4D Plasma albumin concentrations in NSG mice implanted with tissue therapy via IP or SQ are shown, and similar plasma albumin levels between IP and SQ implantations are demonstrated, suggesting functional equivalence between the two implantation sites.

[0171] SQ surgery is relatively non-invasive. These results confirm that minimally invasive surgery for implanting hepatocyte therapy may have several benefits. The larger space available for SQ implantation allows for the implantation of larger tissue therapeutic agents and allows for more frequent monitoring and replacement. Reduced invasiveness also makes surgery an option for patients with non-life-threatening conditions or those who may not tolerate surgery. Data show that using fine fibers in the manufacture of tissue therapeutic agents can increase cell density and allow for the construction of smaller, more functional therapeutic agents. This new manufacturing method may also allow for the cryopreservation of BTT, which would greatly improve the ability to rapidly deliver treatment while a patient is ill. This is particularly relevant to conditions like ALF, in which the time from disease onset to death is finite.

[0172] Example 4. Cryopreservation of tissue fibers printed using HepG2 cells or PHH / MSC (3:1 ratio) This embodiment demonstrates the successful cryopreservation, thawing, and cell aggregation of bioprinted tissue fibers containing dissociated HepG2 single cells. The core-shell fiber is used as a representative example in this specific embodiment, but the concept is applicable to other fibers, such as loop fibers.

[0173] A 2% SLG-100 alginate solution was prepared by diluting the lyophilized powder in brine. The crosslinking agent material consisted of an aqueous solution of 95 mM CaCl2, 2 mM BaCl2, and 20% w / w PEG (8 kDa).

[0174] Core biomaterials were prepared by thawing HepG2 cells and precipitating them in a centrifuge. The cell precipitate was diluted to 75% with a 20% (w / w) high molecular weight fish gelatin solution at 37°C.

[0175] The core-shell fibers were printed using an Aspect Biosystems RX1 printer. A bilayer core-shell fiber was produced, with the outer shell containing 2% SLG-100 alginate and the core containing cellular biomaterials with dissociated cells. To allow oxygen and nutrients to diffuse into all cells, the core of the fiber (shown as a HepG2 cell) was 170 µm in diameter. Figure 5A As shown. Figure 5B Fluorescent staining of live and dead cells for HepG2 is shown.

[0176] The fibers can be held for up to 2 hours before cryopreservation, or allowed to culture and aggregate into tubular spheroid-like aggregates. The printed fibers for cryopreservation were transferred to cryoprotectants (Cryostor 10). To allow time for the cryoprotectant to diffuse throughout the fibers, the vials were incubated at room temperature for 10 minutes. HepG2 cells were then frozen in a -80°C freezer using Mr. Frosty cryoprotectants. After 16 hours, the cells were transferred to a liquid nitrogen atmosphere and stored for at least 2 days.

[0177] For thawing, the fibers are thawed by removing the vials from liquid nitrogen and immediately (<30 seconds) transferring them to a ThawStar cryotube thawing instrument. This device rapidly warms the vials to approximately 50% frozen and 50% thawed. After this cycle, the vials are transferred to a BSC, and using aseptic techniques, the liquid cryoprotectant is removed, followed by the addition of warm 37°C cell culture medium (EMEM with 10% FBS). The fibers are then transferred to cell culture plates with fresh medium. The fibers are allowed to incubate for 72 hours, after which the cells within the fiber core will begin to aggregate. Figures 6A to 6B Thawed fibers maintain high viability during the thawing process.

[0178] Figures 6A to 6B The live / dead fluorescence staining of HepG2 solid fibers 3 days after thawing is shown. Figure 6A ) and live / dead fluorescence staining of HepG2 unfrozen control solid fibers ( Figure 6B ).

[0179] Example 5. Cryopreservation of tissue fibers printed using PHH / MSC This embodiment demonstrates the successful cryopreservation, thawing, and cell aggregation of bioprinted tissue fibers containing dissociated primary human hepatocytes (PHH) and mesenchymal stem cells (MSC) in a 3:1 ratio.

[0180] method A 2% SLG-100 alginate solution was prepared by diluting the lyophilized powder in brine. The crosslinking agent material consisted of an aqueous solution of 95 mM CaCl2, 2 mM BaCl2, and 5% w / w PVA.

[0181] Dissociated primary human hepatocytes (PHH) and mesenchymal stem cells (MSC) (in a 3:1 ratio) were suspended in a 20% (w / w) high molecular weight fish gelatin solution to achieve a 3:1 volume:volume ratio (to a final gelatin concentration of 5%).

[0182] Core biomaterials were prepared by thawing cells and precipitating them in a centrifuge. The cell precipitate was diluted to 75% with a 20% (w / w) fish gelatin solution at 37°C. HepG2 cells or primary human hepatocytes (PHH) and mesenchymal stem cells (MSCs) in a 3:1 ratio were prepared.

[0183] Core-shell fibers were printed using an Aspect Biosystems RX1 printer at a flow rate of 40 cores, 100 shells, and 70 crosslinking agent. The nozzle of the printhead was placed in a water bath containing 125 mM CaCl2 dissolved in water. This produced bilayer core-shell fibers, with the outer shell containing 2% SLG-100 alginate and the core containing cellular biomaterials with dissociated cells.

[0184] Some fibers were cultured using standard conditions, while others were frozen. Frozen fibers were placed in cryotubes filled with NG5A Cryopreserv and cooled at 10°C per minute until the chamber reached -90°C, which was maintained until sample removal; then transferred to LN2 vapor. After more than 3 days, the fibers were thawed and cultured using standard conditions. In short, the fibers were cultured in William's E medium supplemented with 10% FBS, 1x ITS, and 10 μM dexamethasone. After 1 day, the medium was replaced with William's E medium containing 1x ITS and 10 μM dexamethasone. Thawing was performed by removing the vials from liquid nitrogen and immediately (<30 seconds) transferring them to the ThawStar cryotube thawing instrument. This device rapidly warms the vials to approximately 50% frozen and 50% thawed. After circulation, the vials were transferred to BSCs, and the liquid cryoprotectant was removed using aseptic techniques, followed by the addition of warm 37°C cell culture medium. The fibers were then transferred to cell culture plates with fresh medium. The fibers were incubated in the medium for 30 seconds, then replaced with fresh medium. The medium was William's E medium supplemented with 10% FBS, 1x ITS, and 10 μM dexamethasone. After one day, the medium was replaced with William's E medium containing 1x ITS and 10 μM dexamethasone. Cyp3A4 activity was analyzed using a Promega P450 Glo assay, and albumin was calculated using an ELISA. These fibers were compared to conventionally printed fibers containing pre-aggregated hepatocytes prior to printing, wherein the fibers were core-shell fibers with a larger diameter (1000 µm) and a core material of 1.5% alginate and a shell of 2% alginate.

[0185] result Bright-field microscopy, formalin fixation, paraffin embedding (FFPE), and subsequent H&E staining revealed that solid fibers aggregated after printing. Cell aggregation was observed after 3 days. Unfrozen cells were cultured and allowed to aggregate. Figure 7 ). Figure 7 The results of Cyp3A4 functional assays and albumin production assays are shown, comparing solid fibers with conventional (pre-aggregated globular fibers) on days 3 and 5. Functional assays revealed that solid fibers exhibited significantly higher albumin production over 5 days. On day 1, albumin production was 4800 ng / mL for pre-aggregated fibers, compared to 13,000 ng / mL for solid fibers. Cyp3A4 activity was also 6.3 times higher in solid fibers compared to pre-aggregated fibers. Following cryopreservation and thawing, albumin production after cryopreservation was 70% to 86% of that of uncryopreserved fibers within one week after thawing. These cryopreserved fibers also aggregated after thawing, as determined by bright-field imaging.

[0186] in conclusion Three main objectives were achieved. First, it has been demonstrated that cells can be arranged more tightly than previously expected, while exhibiting post-printing aggregation. Second, the fibers produced in this embodiment have shown to have higher functionality than fibers printed using pre-aggregated cells. Third, the fibers have shown to be cryopreservable with minimal loss of function. Taken together, these elements significantly improve the feasibility of creating off-the-shelf allogeneic cell therapies for liver diseases. Other treatment modalities may include allogeneic and autologous therapies.

[0187] Example 6. Cryopreservation of tissue fibers printed using PHH / MSCs or adrenocortical cells This embodiment demonstrates the successful cryopreservation, thawing, and cell aggregation of bioprinted tissue fibers containing dissociated primary human hepatocytes (PHH) and mesenchymal stem cells (MSC) in a 3:1 ratio, as well as the effectiveness of using pre-aggregated cells for bioprinting.

[0188] Single cells and aggregates of PHH / MSC or adrenocortical cells were bioprinted in the form of solid fibers (dissociated cells) and pre-aggregated rings, respectively. Figures 8A to 8C and Figure 9 Representative images of solid fibers containing PHH+MSCs or adrenal cells are shown. Figure 8A ), cell aggregates in the ring layer ( Figure 8B A cross-sectional view of a bioprinted ring fiber with a cell-free inner core, a cell-containing ring layer, and a cell-free outer shell. Figure 8C), and representative images of solid fibers containing single cells in the annular layer ( Figure 9 The annotations indicate the shell size (approximately 71.6–78.5 µm), the size of the cell-containing ring (approximately 270.4–292.7 µm), the size of the cellless core (approximately 424.8 µm), and the total fiber diameter (approximately 1.19 mm).

[0189] Fresh fibers were maintained in a medium at 37°C and 5% CO2. Cryopreserved fibers were frozen using a controlled-rate freezer and held in liquid nitrogen for 72 hours before thawing. To assess viability, the fibers were stained with calcein-AM (green) and propidium iodide (red). Figure 10A ; Figure 10B ).

[0190] For hepatocytes, solid fibers (containing dissociated cells) were cultured in William's E medium containing FBS, ITS, and dexamethasone, then switched to a medium without FBS. Pre-aggregated fibers were cultured in a medium without FBS. The medium was collected 24 hours after bioprinting or thawing for albumin quantification. (Acquired albumin secreted in conditioned medium was quantified by ELISA (Abcam catalog number ab235642 or ab179887) according to the manufacturer's instructions. For solid fibers containing adrenocortical cells, cortisol secretion was also quantified in 24-hour conditioned medium by ELISA (Abcam catalog number ab108665), and the adrenocortical cell medium was human adrenocortical cell medium (iXCells Biotechnologies).

[0191] result Figures 10A to 10B Fluorescent staining of live and dead cells in the form of solid fibers and aggregate rings is shown.

[0192] Figures 11A to 12B This shows the use of PHH + MSC ( Figure 11A and Figure 12A (targeting albumin) or adrenocortical cells ( Figure 11B and Figure 12B The albumin and cortisol secretion of solid fibers or aggregates (fresh or frozen) printed for cortisol. Figures 11A to 11B Expressed in nanograms per million cells per day, and for Figures 12A to 12B (Expressed as relative secretion).

[0193] in conclusion This data suggests that, compared to cryopreserved bioprinting of cell aggregates, solid fiber and solid ring fiber bioprinting methods allow for cryopreservation, while exhibiting significantly greater functionality and viability after thawing.

[0194] The embodiments and examples described above are intended to be illustrative rather than limiting. Those skilled in the art will recognize or be able to determine many equivalents of a particular compound, material, and procedure using only conventional experiments. All such equivalents are considered to be within the scope of and covered by the appended claims.

Claims

1. A cryopreserved tissue fiber comprising a continuously bioprinted core / shell fiber encapsulating a cell population for implantation, wherein the core / shell fiber comprises a core and at least one shell, wherein the cell population comprises at least 90%, at least 95%, at least 98%, at least 99%, or 100% dissociated cells embedded in a biocompatible matrix, and wherein the fiber is cryopreserved prior to the aggregation of the dissociated cells.

2. The cryopreserved tissue fibers of claim 1, wherein the cell population is encapsulated in the core and / or the at least one shell.

3. The cryopreserved tissue fibers of claim 2, wherein the core / shell fibers comprise at least two shells, the innermost shell comprising an annular layer encapsulating the cell population.

4. The cryopreserved tissue fibers as described in any of the preceding claims, wherein the biocompatible matrix does not contain DMSO, preferably wherein the core / shell fibers are bioprinted without incorporating DMSO into the core or the at least one shell.

5. The cryopreserved tissue fibers as described in any of the preceding claims, wherein the biocompatible matrix further comprises a thickener, preferably wherein the thickener comprises fish gelatin.

6. The cryopreserved tissue fiber as claimed in any of the preceding claims, wherein the core and the at least one shell comprise the same hydrogel material; preferably wherein the hydrogel material is an alginate.

7. The cryopreserved tissue fibers of claim 6, wherein the core and / or the at least one shell comprises chemically modified alginate.

8. Cryopreserved tissue fibers as described in any of the preceding claims, wherein the biocompatible matrix comprises at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% v / v of cells in a physiological matrix material.

9. The cryopreserved tissue fibers of claim 8, wherein the fibers are high-density fibers, and the biocompatible matrix comprises at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, or at least about 85% v / v of cells in a physiological matrix material.

10. The cryopreserved tissue fibers as claimed in any of the preceding claims, wherein the biocompatible matrix comprises a cell:physiological matrix material ratio of at least about 2:1, preferably at least about 3:1, more preferably at least about 4:

1.

11. The cryopreserved tissue fibers of claim 2, wherein the cell population is encapsulated in the core, and the core diameter is between about 100 μm and about 200 μm, preferably between about 130 μm and about 200 μm, preferably between about 140 μm and about 190 μm, more preferably between about 150 μm and 175 μm, and even more preferably about 150 μm, about 155 μm, about 160 μm, about 165 μm, about 170 μm, or about 175 μm.

12. The cryopreserved tissue fibers of claim 11, wherein the shell thickness is between about 30 μm and 80 μm, preferably between about 50 μm and 70 μm, more preferably about 60 μm, about 65 μm or about 70 μm.

13. The cryopreserved tissue fibers as claimed in claim 11 or 12, wherein the total fiber diameter is between about 270 μm and about 330 μm, preferably between about 280 μm and about 320 μm, more preferably about 290 μm to about 310 μm, and even more preferably about 290 μm, about 300 μm or about 310 μm.

14. The cryopreserved tissue fibers of claim 3, wherein the cell population is encapsulated in the annular layer and the core diameter is between about 300 μm and about 500 μm, preferably between about 350 μm and about 450 μm, more preferably about 350 μm, about 375 μm, about 400 μm, about 425 μm or about 450 μm.

15. The cryopreserved tissue fibers of claim 14, wherein the thickness of the annular layer is between about 100 μm and about 300 μm, preferably between about 130 μm and about 200 μm, preferably between about 140 μm and about 190 μm, more preferably between about 150 μm and 175 μm, and even more preferably about 150 μm, about 155 μm, about 160 μm, about 165 μm, about 170 μm, or about 175 μm.

16. The cryopreserved fiber as claimed in claim 14 or 15, wherein the cryopreserved fiber further comprises a shell, the thickness of which is between about 30 μm and 80 μm, more preferably between about 45 μm and 70 μm, and even more preferably about 60 μm, about 65 μm or about 70 μm.

17. The cryopreserved tissue fibers according to any one of claims 14 to 16, wherein the total fiber diameter is between about 500 and 1200 μm, preferably between about 700 μm and about 1000 μm, more preferably about 800 μm, about 900 μm or about 1000 μm.

18. The cryopreserved tissue fibers as described in any of the preceding claims, wherein the cell population comprises primary cells from endocrine and exocrine glands selected from the group consisting of: pancreas, liver, kidney, parathyroid gland, thyroid gland, pineal gland, pituitary gland, thymus, adrenal gland, ovary, testis, tonsillar gland, enteroendocrine cells, stem cells, stem cell-derived cells of any of the aforementioned primary cell types, or cells engineered to secrete therapeutic agents.

19. The cryopreserved tissue fibers of claim 18, wherein the cell population comprises HepG2, primary human hepatocytes (PHH), mesenchymal stem cells (MSC), hepatocyte-like cells derived from embryonic stem cells or induced pluripotent stem cells, hepatocyte-like cells derived from liver stem cells, xenogeneic hepatocytes, genetically engineered stem cell-derived hepatocytes, stem cell-derived endothelial cells or primary endothelial cells, and combinations thereof.

20. The cryopreserved fiber of claim 19, wherein the dissociated cells comprise PHH and MSC in a ratio of 3:

1.

21. A composition / medical device for implanting a cell population into a subject in need, the composition / medical device comprising a multilayer structure including tissue fibers according to any one of claims 1 to 20, wherein the composition is cryopreserved prior to the dissociated cell aggregation.

22. The composition / apparatus of claim 21, wherein the multilayer structure comprises at least one conformal coating.

23. The composition / apparatus of claim 22, wherein the multilayer structure comprises a first inner coating and a second outer coating; preferably wherein the first inner coating comprises a hydrogel with a material strength greater than that of the second outer coating.

24. The composition / device of any one of claims 21 to 23, wherein the core and / or the at least one shell are separated along the length of the fiber.

25. A method for implanting a cell population into a subject in need, the method comprising: a. Thawing the cryopreserved tissue fibers according to any one of claims 1 to 20, or the composition / device according to any one of claims 21 to 24; as well as c. Implant the fiber or the composition / device into the subject.

26. The method of claim 25, further comprising culturing the tissue fibers or the composition / device in a fresh culture medium after thawing, prior to implantation into the subject, until the dissociated cells form aggregates in the tissue fibers.

27. A method for manufacturing tissue fibers as described in any one of claims 1 to 20, the method comprising the following steps: a. Providing a bioprinting system, the bioprinting system comprising: a printhead including a plurality of microfluidic channels for selectively supplying a core material, at least one shell material, and sheath fluid to a dispensing orifice; a receiving surface for receiving the material dispensed from the orifice; a positioning unit for positioning the orifice of the printhead in three-dimensional space, the positioning unit being operatively coupled to the printhead; and at least one dispensing member for dispensing the fibers from the dispensing orifice; b. Simultaneously printing the core and the at least one shell of the tissue fibers via the bioprinting system, wherein a cell population is encapsulated in the core and / or the at least one shell; and c. Freeze-preserve the bioprinted tissue fibers using a cryoprotectant; The cell population comprises at least 95% dissociated cells embedded in a biocompatible matrix, and the fibers are cryopreserved prior to the aggregation of the dissociated cells; preferably cryopreserved within about 10 minutes to one hour of bioprinting, more preferably within about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes of bioprinting.

28. The method of manufacturing a composition / medical device according to any one of claims 2 to 24, as claimed in claim 27, wherein the printing step comprises dispensing the continuously bioprinted core / shell fibers onto the receiving surface to form a multilayer structure, and wherein the multilayer structure is cryopreserved prior to the aggregation of dissociated cells in the fibers.

29. The method of claim 27 or 28, wherein the core material and the at least one shell material are bioprinted without DMSO.

30. The method of any one of claims 27 to 29, wherein the cryopreservation step comprises adding a cryoprotectant to the printed fiber at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 minutes before freezing.

31. The method of any one of claims 27 to 30, wherein the core material further comprises a tackifier, preferably wherein the viscosity of the core material and the shell material is substantially the same.

32. The method of any one of claims 27 to 31, wherein the receiving surface includes a manufacturing platform for supporting the core / shell fibers of the continuous bioprinting, the manufacturing platform including a frame defining a gap and including a plurality of posts on opposite sides of the frame for securing and suspending the continuous bioprinting fibers during printing; and the printing step includes distributing the fibers around the plurality of posts to form a grid structure comprising at least two, three, four, or five layers of the fibers.

33. The method of any one of claims 27 to 32, further comprising coating the grid structure after printing is completed.

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