Scalable bioreactor method for organoids using architected matrix microcarriers, constructs and uses thereof

Abstract

Described herein are methods for producing organoids at a large scale sufficient for tissue biomanufacturing that include combining cells and architected matrix microcarriers into a suspension in a stirred-tank (STR) bioreactor comprising culture medium. The method further includes culturing the cells and the matrix microcarriers and exposing the cells and the microcarriers to the fluidic shear stress (FSS) in the STR bioreactor to induce differentiation and / or maturation of the cells, and / or functional integration of the cells with other types of cells, if present in the STR. Described herein are also organoids and tissues produced by the described methods and their uses.

Description

SCALABLE BIOREACTOR METHOD FOR ORGANOIDS USING ARCHITECTED MATRIX MICROCARRIERS, CONSTRUCTS AND USES THEREOFRELATED APPLICATIONS

[0001] The present patent document claims the benefit of the filing date under 35 U.S.C. § 119(e) of Provisional U.S. Patent Application Serial No. 63 / 690,040, filed September 3, 2024, which is hereby incorporated by reference.FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under DK126023 and DK131821 awarded by National Institutes of Health (NIH). The government has certain rights in this invention.BACKGROUND

[0003] The development of engineered, patient-specific kidney tissue would meet an urgent clinical need for renal replacement therapy (McCormick, F., et al., J Am Soc Nephrol, 29, 2775-2776 (2018); Jansen, J., et al., Biotechnol Adv, 32, 1317-1327 (2014)) and serve as a model platform to expedite biomedical research (DesRochers, T. M., et al., Adv Drug Deliv Rev 0, 67-80 (2014)). The differentiation of human embryonic or induced pluripotent stem cells (hPSCs) into metanephric mesenchyme (MM)-derived kidney organoids containing nephron segments has rapidly advanced the field (Morizane, R. et al., Nat Biotechnol 33, 1193-1200 (2015); Takasato, M. et al., Nature 526, 564-568 (2015); Taguchi, A. et al., Cell Stem Cell, 14, 53-67 (2014)). However, MM-derived organoids generally lack ureteric bud (UB)-derived collecting ducts (CD) and a ureter through, which filtrate can exit the tissue, limiting drainage capacity (Takasato, M., et al., Nature 526, 564-568 (2015); Little, M.H. & Combes, A.N., Genes Dev, 33, 1319-1345 (2019)).

[0004] Several protocols have recently emerged for differentiating ureteric bud (UB) and collecting duct (CD) organoids under static conditions (Mae, S.-L, et al., Cell Reports, 32, 107963 (2020); Howden, S.E., et al., Cell Stem Cell, 28, 671-684. e6 (2021); Zeng, Z., et al., Nat Commun, 12, 3641 (2021); Shi, M., et al., Nat Biotechnol, 1-10(2022)). However, these organoids have yet to be produced at scales sufficient for tissue biomanufacturing .

[0005] Stirred-tank bioreactors (STRs) are currently the gold standard for scaling up cell culture because they efficiently distribute nutrients in large volumes, yet fluidic shear stress generated by stirring influences hPSC differentiation (Kropp, C., et al., Process Biochemistry, 59, 244-254 (2017)).

[0006] Understanding and harnessing fluidic forces during differentiation could enable control over UB maturation, UB-to-CD transition, and MM-CD integration.

[0007] Nascent kidney tubules in vivo are subject to fluid flow generated by ciliary beating in early development (Kramer-Zucker, A.G., et al., Development 132, 1907-1921 (2005)). Apical shear stress and circumferential stretch induced by tubular flow are known to drive collecting duct function in vivo (Liu, W., et al., American Journal of Physiology-Renal Physiology, 285, F998-F1012 (2003); Liu, W., et al., Am J Physiol Renal Physiol, 293, F227-235 (2007)).SUMMARY

[0008] In one embodiment, described herein is a method for producing organoids at a large scale sufficient for tissue biomanufacturing, comprising: combining cells and architected matrix microcarriers into a suspension in a stirred-tank (STR) bioreactor comprising culture medium; culturing the cells and the architected matrix microcarriers; and exposing the cells and the architected matrix microcarriers to the fluidic shear stress (FSS) in the STR bioreactor to induce differentiation and / or maturation of the cells, and / or functional integration of the cells with other types of cells, if present in the STR. In the method, the architected matrix microcarriers are solid matrix fragments with a randomly architected geometry and / or architected, non-spherical geometry. The architected matrix microcarriers are capable of aggregating to each other to form multicarrier aggregates comprising the cells cultured between microcarriers’ interfaces and / or on the microcarriers’ surfaces exposed to the culture medium. In the method, once in the suspension, the cells deposit on the surface of the architected matrix microcarriers, adhere, proliferate, differentiate, and remodel the architected matrix microcarriers. The architected matrix microcarriers can comprise a basement membrane matrix (BsM). Thearchitected matrix microcarriers can comprise at least one of: Matrigel™, Geltrex™, poly L-lysine, gelatin, fibronectin, collagen I, collagen IV, fibrinogen, gelatin methacrylate, fibrin, silk, pegylated gels, collagen methacrylate, hyaluronic acid, laminins, synthetic adhesive peptides, or a combination thereof. The method may further comprise expanding cells originating from 2D culture. In the method, the cells may be selected from the group consisting of pluripotent stem cells, multipotent stem cells, progenitor cells, nephron progenitor cells, terminally differentiated cells, endothelial cells, endothelial progenitor cells, immortalized cell lines, or primary cells. In the method, the cells may be derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs). In the method, the cells may be ureteric bud (UB) cells isolated from UB organoids or collecting duct (CD) cells isolated from CD organoids. In the method, in certain embodiments, the cells may be derived from a patient. In the method, the culturing may be while imparting the FSS. The fluid perfusion may be at FSS from about 0.001 dyn / cm2to about 50 dyn / cm2. The culturing may be while imparting FSS for at least 1 day to a maximum of 600 days. In the method, wherein the fluidic shear stress influences the cell differentiation or maturation by at least one of: upregulating gene expression for markers of interest; down-regulating expression of sternness or off-target markers; increasing protein expression; changing cell population heterogeneity to include at least one other cell type; changing structural morphology of organoids and tissues; changing spatial orientation of cell types within organoids and tissues; increasing growth factor secretion; increasing exosome production; and / or changing functional transport across cell barriers. In the method, the cells may be exposed to FSS that is modulated. The modulating is by changing stir speed and / or medium viscosity. The method may further comprise exposing the cells to one or more of biological agents, a biological agent gradient, a pressure, a pressure gradient, oxygen tension, and an oxygen tension gradient to direct development, small molecules and proteins to promote differentiation, and / or functioning of the cells. In the method the cells are kidney cells, lung cells, gastrointestinal cells or vascular cells. In the method, the organoids are iPSC-derived ureteric bud organoids. The method may further comprise harvesting the organoids. The method may further comprise harvesting cellular-derived products from the medium, such as exosomes, growth factors, cytokines, or hormones.

[0009] Yet another embodiment relates to a tissue construct or an organoid produced by the described method.

[0010] Yet another embodiment relates to the tissue construct or an organoid produced by the described method for use in tissue replacement therapy.

[0011] Yet another embodiment relates to the tissue construct or an organoid produced by the described method for use in biomedical research.

[0012] Yet another embodiment relates to the tissue construct or an organoid produced by the described method for use in disease modeling.

[0013] Yet another embodiment relates to the tissue construct or an organoid produced by the described method for use in drug toxicity studies.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0015] Figure 1 depicts: A) schematic of directed differentiation using microcarriers and stirred bioreactors; B) Immunofluorescent maximum intensity projection of a microcarrier-derived UB organoid fixed at day 21 of differentiation.

[0016] Figure 2 depicts a schematic of a method for generating and validating UB organoids. Differentiation protocols in p-well format compared to established 96-well format increases yield and reduces culture time.

[0017] Figure 3 depicts scaling UB culture to stirred-tank bioreactors (STRs) using suspension culture models. A) Static culture compared to suspension culture models of different scales including flasks or plates on orbital shakers, spinner flasks, and STRs. Within suspension culture, matrix microcarriers can be included as fragments coated by cells or as droplets encapsulating cells. B) Nanostring RNA counting analysis comparing fragment-coated UB organoids to static embedded UB organoids (solid line = p<0.01, dashed line = p<0.05).

[0018] Figure 4 depicts generation of UB organoids using BsM microcarriers in STR bioreactors. A) Solid BsM is extruded into fragments and combined in suspension with single UB precursor cells at day 7 to generate cell-coated matrix fragments whichdifferentiate into UB organoids. B) UB precursor cells are first aggregated into spheroids, then individually encapsulated in BsM, and finally cultured in suspension to generate UB organoids.DETAILED DESCRIPTION OF THE DRAWINGS AND THE PRESENTLY PREFERRED EMBODIMENTS

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, compositions, devices and materials are described herein.

[0020] All patents, patent applications and publications, and other literature references cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

[0021] As used herein and in the appended claims, the singular forms “a,” “and,” and “the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a protein” includes a plurality of such proteins and reference to “the progenitor cell” includes reference to one or more progenitor cells known to those skilled in the art, and so forth.

[0022] The term “organoid” refers to an “embryoid body” whose cells have undergone a degree of differentiation. The term “embryoid body” refers to a plurality of cells containing pluripotent or multipotent stem cells formed into a three-dimensional sphere, spheroid, or other three-dimensional shape. It is acknowledged that the distinction between an organoid and embryoid body remains undefined, and the use of the terms should be considered interchangeable.

[0023] An organoid may be created by culturing at least one of: pluripotent stem cells, multipotent stem cells, progenitor cells, nephron progenitor cells, terminally differentiated cells, endothelial cells, endothelial progenitor cells, immortalized cell lines, or primarycells. In certain embodiments, the population of cells comprises at least one of human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs).

[0024] Methods of producing organoids were previously described in, for example, PCT Publication No. WO 2016 / 141137A1; PCT Publication No. WO 2022 / 010901A2; U.S. Pat. Pub. No. 2020 / 0289709; and U.S. Pat. Pub. No. 2020 / 0248147 Al, which are incorporated herein by reference.

[0025] Introduction

[0026] Described herein are methods that modulate cell exposure to fluidic forces to drive cellular differentiation, maturation, and functional integration with other renal cell types. For examples, described herein are methods that incorporate fluidic forces to drive ureteric bud (UB)-to-collecting duct (CD) organoid differentiation, maturation, and functional integration with other renal cell types.

[0027] Traditionally, UB organoids are differentiated first in 2D and then, at the CD1 17+ Wolffian duct-like stage or other UB precursor stage, aggregated and embedded in basement membrane matrix (BsM), such as Geltrex or Matrigel (Fig. 2). Figure 2 shows a schematic of a method for generating and validating UB organoids.Differentiation protocols in p-well format compared to established 96-well format increased yield and reduced culture time. While this is sufficient at the laboratory scale, culturing enough cells for one human kidney would require on the order of -100B cells. Scaling out would require -1000 plates, which is not practical to store or feed. Thus, adapting the culture to a stirred tank reactor greatly facilitates large scale manufacturing.

[0028] Methods

[0029] Rather than embedding in static BsM, the methods described herein include architected matrix microcarriers (e.g., BsM microcarriers) in a suspension culture, either as cell-coated fragments or droplets encapsulating aggregates (Fig 3A). This culture format not only enables scalable manufacturing of specific cell types, such as UB organoids, but also facilitates or modulates exposure of cells to fluidic shear stress during differentiation leading to upregulation in gene expression of collecting duct maturation markers (Fig 3B) compared to traditional static embedded methods.

[0030] Described herein is a method for producing organoids at a large scale sufficient for tissue biomanufacturing, comprising: combining cells and architected matrixmicrocarriers into a suspension in a stirred-tank (STR) bioreactor comprising culture medium, to modulate the cellular microenvironment within an STR. The method further includes culturing the cells and the matrix microcarriers and exposing the cells and the matrix microcarriers to the fluidic shear stress (FSS) in the STR bioreactor to induce differentiation and / or maturation of the cells, and / or functional integration of the cells with other types of cells, if present in the STR.

[0031] The term “a large scale” as used in the context of the described method means sufficient for manufacturing volumes of cells required for cell-based therapies (generally >1 billion cells).

[0032] In certain specific embodiments, in the described method, the architected matrix microcarriers (e.g., BsM fragments) can be combined with UB precursor cells in suspension, allowing the cells to deposit on at least a portion of the architected matrix / microcarriers’ surface, proliferate, differentiate, and remodel the matrix / microcarriers (Fig 4A).

[0033] The architected matrix microcarriers may be solid matrix fragments with a randomly architected geometry and / or architected, non-spherical geometry. The non- spherical geometry described here may be, e.g., an irregularly- shaped matrix fragment that has a jagged surface. Non-spherical geometries could also include any number of polyhedral, cylindrical, toroid, and ellipsoid shapes. The term “architected” means predetermined by the fabrication and assembly method.

[0034] Once in the suspension, the cells can deposit on the surface of the architected matrix microcarriers, adhere, proliferate, differentiate, and remodel the microcarriers.

[0035] In certain embodiments, the architected matrix microcarriers are capable of aggregating to each other to form multi-carrier aggregates comprising the cells cultured between microcarriers’ interfaces and / or on the microcarriers’ surfaces exposed to the culture medium

[0036] In certain embodiments, the architected matrix microcarriers comprise a basement membrane matrix (BsM). The method of extruding BsM into fragments was adapted from the Muir, V.G., et al., “Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications,” J Vis Exp, 183, 10.3791 / 63867 (2022)).

[0037] The architected matrix microcarriers can include at least one of: Matrigel™, Geltrex™, poly L-lysine, gelatin, fibronectin, collagen I, collagen IV, fibrinogen, gelatin methacrylate, fibrin, silk, pegylated gels, collagen methacrylate, hyaluronic acid, laminins, synthetic adhesive peptides, or a combination thereof.

[0038] The cells used in the described method may be cells selected from the group consisting of pluripotent stem cells, multipotent stem cells, progenitor cells, nephron progenitor cells, terminally differentiated cells, endothelial cells, endothelial progenitor cells, immortalized cell lines, or primary cells.

[0039] Preferably, the cells are derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs).

[0040] In certain other embodiments, the cells are ureteric bud (UB) cells isolated from UB organoids or collecting duct (CD) cells.

[0041] In certain embodiments, the cells are derived from a patient.

[0042] In certain embodiments, about 10k-200M cells / mL are combined into the suspension.

[0043] In the described method, the culturing step may be while imparting the FSS.

[0044] Fluidic forces may be a powerful tool to drive UB-to-CD organoid differentiation, maturation, and functional integration with other renal cell types. In the described method, the fluid perfusion may be at FSS from about 0.001 dyn / cm2to about 50 dyn / cm2.

[0045] In the described method, the culturing step may be while imparting FSS for at least 1 day to a maximum of 600 days.

[0046] In the described method, the fluidic shear stress influences the cell differentiation or maturation by at least one of:

[0047] upregulating gene expression for markers of interest by, e.g., at least 1-fold; at least 2-fold; at least 3-fold or more;

[0048] down-regulating expression of sternness or off-target markers by at least 1- fold; at least 2-fold; at least 3-fold or more;

[0049] increasing protein expression by, e.g., at least 1-fold; at least 2-fold; at least 3- fold or more;

[0050] changing cell population heterogeneity to include at least one other cell type; at least two more cell types; at least three more cell types; or more;

[0051] changing structural morphology of organoids and tissues;

[0052] changing spatial orientation of cell types within organoids and tissues;

[0053] increasing growth factor secretion by, e.g., at least 1-fold; at least 2-fold; at least 3-fold or more;

[0054] increasing exosome production, e.g., by at least 1-fold; at least 2-fold; at least 3-fold or more; and / or

[0055] changing functional transport across cell barriers.

[0056] In the described method, the cells may be exposed to FSS that is modulated. Modulating the FSS may be by changing stir speed, medium viscosity or both. For example, the viscosity of the culture medium may be changed from 0.01 poise to 0.1 poise.

[0057] In further embodiments, the described method may further comprise exposing the cells to one or more of biological agents, a biological agent gradient, a pressure, a pressure gradient, oxygen tension, and an oxygen tension gradient to direct development, small molecules and proteins to promote differentiation, and / or functioning of the cells.

[0058] As noted above, the described method is for producing organoids at a large scale sufficient for tissue biomanufacturing. The tissue may be kidney tissue, lung tissue, gastro-intestinal tissue or vascular tissue.

[0059] In certain further embodiments, the described method may further comprise harvesting the organoids.

[0060] In certain further embodiments, the described method may further comprise harvesting cellular-derived products from the culture medium. Non-limiting examples of the cellular-derived products include exosomes, growth factors, cytokines, or hormones.

[0061] In certain further embodiments, the described method may further comprise a step of expanding cells originating from 2D culture. Methods of expending cells in 2D culture are known to those skilled in the art.

[0062] In certain alternative embodiments, the described method includes microcarriers that form “droplets.” The “droplet” method is similar to the standard matrix embedding method (Zeng, Z., et al., Nat Commun, 12, 3641 (2021)) as organoids areencapsulated directly in the matrix. However, in this embodiment, organoids are made such that cells or cell aggregates are cast in their own matrix droplets, which are then cultured in suspension (Fig 4B).

[0063] Both the “fragment” and “droplet” methods described herein can use extracellular matrix proteins as microcarriers for cells (e.g., UB cells), enabling simultaneous cell-matrix interaction and fluidic shear stress during differentiation.

[0064] Tissue Constructs and Organoids and their Uses

[0065] Certain further embodiments relate to a tissue construct or an organoid produced by the methods described herein.

[0066] The tissue construct or organoid may be for use in tissue replacement therapy.

[0067] The tissue construct or organoid may be for use in biomedical research.

[0068] The tissue construct or organoid may be for use in disease modeling.

[0069] The tissue construct or organoid may be for use in drug toxicity studies.

[0070] EXAMPLES

[0071] Example 1

[0072] Method:

[0073] Human pluripotent stem cells (hiPSCs, BJFF.6) were first differentiated into ureteric bud (UB) precursor cells in 2D following an established protocol (Zeng, Z., et al. “Generation of patterned kidney organoids that recapitulate the adult kidney collecting duct system from expandable ureteric bud progenitors,” Nat Commun 12, 3641 (2021)).

[0074] The hiPSCs were dissociated using Accumax™ and seeded at 17,200 cells / cm2in mTeSR Plus medium containing lOpM Y-27632 onto a 2D substrate coated with 1% Matrigel™ in DMEM / F12. After 24 hours, the medium was replaced with basal medium (DMEM / F12, B-27 minus vitamin A [IX], penicillin / streptomycin [IX], GlutaMAX™ [IX], ITS [IX], MEM NEAA [IX]) containing lOnM LDN-193189 and 4.5pM CHIR- 99021. After 48 hours, the medium was replaced with basal medium containing 30nM LDN-193189, lOOnM TTNPB, 200ng / mL FGF2, and 200nM A83-01. After 24 hours, this same medium was replenished. After 24 hours, the medium was replaced with basal medium containing 30nM LDN-193189, lOOnM TTNPB, and 200ng / mL FGF2. After 24hours, this same medium was replenished. After 24 hours (day 0), the cells are identified as CD117+ UB precursor cells.

[0075] UB precursor cells were combined with microcarrier fragments in a 125mL Corning® stirred-tank bioreactor (STR) at day 0 (Fig. 1 A).

[0076] Fragments were generated by adding 5mL Geltrex™ to a 5mL syringe on ice, then incubating the syringe at 37°C for at least 30 minutes to allow for cross-linking. The solid matrix was then extruded using a protocol of Muir et al. (Muir, V. G., et al., “Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications,” J Vis Exp (2022) doi: 10.3791 / 63867) through a 20G nozzle (0.64mm I.D.) at ~1 drop / second into the STR containing 62.5mL human UB culture medium (hUBCM: basal medium, 200nM LDN-193189, lOOnM TTNPB, 200nM A83-01, IpM CHIR- 99021, 5pM SB202190, lOOnM JAKi, lOOng / mL Rspondinl, lOOng / mL GDNF, 50ng / mL EGF, and 50ng / mL FGF7) containing lOpM Y-27632. UB precursor cells were dissociated using Accumax™ and seeded into the STR at 50K-2M cells / mL. The suspension containing cells and microcarriers was then pipetted repeatedly to ensure even mixing of cells and separation of fragments. The STR was then cultured at 37°C, 5% CO2 with a stirring rate of 60 RPM.

[0077] After 24 hours (day 1), the suspension culture was gently centrifuged at 100g for 3 minutes to pellet the cell-coated fragments. The medium was removed and replaced with 62.5mL hUBCM (without Y-27632). The fragments were pipetted repeatedly for separation and then returned to culture at 37°C, 5% CO2, 60 RPM. The medium was refreshed every 2-3 days in this manner.

[0078] At day 21, UB organoids were identified by transcriptomic (RNA) and proteomic (immunofluorescence) analyses (Fig. IB).

[0079] UB organoids were then further differentiated to collecting duct (CD) organoids by replacing the medium with 62.5mL human CD culture medium (hCDM: basal medium, 3% KnockOut SR™, HU / mL Arg8-vasopressin, and lOOnM aldosterone) for an additional seven days.

[0080] Analysis

[0081] At day 28, CD organoids were identified by transcriptomic (RNA) and proteomic (immunofluorescence) analyses.

[0082] For transcriptomic analyses, 6-10 organoids were collected. Organoids were washed with DBPS- / -. Supernatant was removed and RNA was isolated using RNeasy Plus Mini Kit (Qiagen) according to manufacturer’s instructions. RNA concentration was assessed using both the Nanodrop 1000 spectrophotometer (NanoDrop Products, Thermo Scientific, Wilmington, DE) and Qubit (Life Technologies). A custom Kidney gene panel was designed by NanoString and produced by IDT. Probes contained 35-50 bp each. RNA quantification was performed by NanoString nCounter Elements™ with according reagents as per the manufacturer’s instructions. The total amount of 100 ng RNA was used per run. RNA was hybridized with probe pools, hybridization buffer and TagSet reagents in a total volume of 30 pl and incubated at 67 °C for 20 h.

[0083] After quantification, data from each experiment was analyzed by nSolver software using a custom advanced analysis. In this analysis, no low count data was omitted. ACTB, GAPDH, and TUBB were used as housekeeping genes.

[0084] For IF analyses, 6-10 organoids were collected and medium was removed. Prior to immunostaining, each sample was washed with DPBS+ / +and then fixed for 1 hr with 10% buffered formalin. The fixative was removed by 2 washes in PBS for ~2 hrs, and samples were then blocked overnight with 1% (vol / vol) donkey serum in DPBS+ / +with 0.125% (vol / vol) Triton X-100. Primary antibodies (Table 1) were incubated with the constructs for 2 days at 4 °C in a staining solution (0.5% (wt / vol) BSA and 0.125% (vol / vol) Triton X-100 in DPBS+ / +). Samples were washed with staining solution for 1 d. Secondary antibodies (Table 1) were incubated with the constructs for several hours in staining solution. Samples were counterstained with DAPI and then washed for at least several hours in PBS before imaging. Confocal images were acquired on a Zeiss LSM 710. Imaris was used to visualize 3D projections, stacks, and rendering. ImageJ was used for qualitative comparison.

[0085] Table 1: Antibodies used for immunostaining.

[0086] Results

[0087] Microcarrier-derived UB organoids experienced both cell-matrix interaction and fluidic shear stress during differentiation, leading to an upregulation in gene expression of key markers of ureteric epithelium and collecting duct (e.g., CALB1, WNT9B) and downregulation of sternness and precursor markers (e.g. SOX2, WNT11, RET).

[0088] Immunofluorescence staining qualitatively confirmed differential expression of these markers.

[0089] This method is adaptable to larger STR volumes.

Claims

CLAIMS1. A method for producing organoids at a large scale sufficient for tissue biomanufacturing, comprising: combining cells and architected matrix microcarriers into a suspension in a stirred- tank (STR) bioreactor comprising culture medium; culturing the cells and the architected matrix microcarriers; and exposing the cells and the architected matrix microcarriers to the fluidic shear stress (FSS) in the STR bioreactor to induce differentiation and / or maturation of the cells, and / or functional integration of the cells with other types of cells, if present in the STR.

2. The method of claim 1, wherein the architected matrix microcarriers are solid matrix fragments with a randomly architected geometry and / or architected, non-spherical geometry.

3. The method of claim 1 or claim 2, wherein the architected matrix microcarriers are capable of aggregating to each other to form multi-carrier aggregates comprising the cells cultured between microcarriers’ interfaces and / or on the microcarriers’ surfaces exposed to the culture medium.

4. The method of any of claims 1-3, wherein once in the suspension, the cells deposit on the surface of the architected matrix microcarriers, adhere, proliferate, differentiate, and remodel the architected matrix microcarriers.

5. The method of any of claims 1-4, wherein the architected matrix microcarriers comprise a basement membrane matrix (BsM).

6. The method of any of claims 1-5, wherein the architected matrix microcarriers comprise at least one of: Matrigel™, Geltrex™, poly L-lysine, gelatin, fibronectin, collagen I, collagen IV, fibrinogen, gelatin methacrylate, fibrin, silk, pegylated gels, collagen methacrylate, hyaluronic acid, laminins, synthetic adhesive peptides, or a combination thereof.

7. The method of any of claims 1-6, further comprising expanding cells originating from 2D culture.

8. The method of any of claims 1-7, wherein the cells are selected from the group consisting of pluripotent stem cells, multipotent stem cells, progenitor cells, nephron progenitor cells, terminally differentiated cells, endothelial cells, endothelial progenitor cells, immortalized cell lines, or primary cells.

9. The method of any of claims 1-8, wherein the cells are derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs).

10. The method of any of claims 1-7, wherein the cells are ureteric bud (UB) cells isolated from UB organoids or collecting duct (CD) cells isolated from CD organoids.

11. The method of any of claims 1-10, wherein the cells are derived from a patient.

12. The method of any of claims 1-11, wherein 10k-200M cells / mL are combined into the suspension.

13. The method of any of claims 1-12, wherein the culturing is while imparting the FSS.

14. The method of any of claims 1-13, wherein the fluid perfusion is at FSS from about 0.001 dyn / cm2to about 50 dyn / cm2.

15. The method of any of claims 1-14, wherein the culturing is while imparting FSS for at least 1 day to a maximum of 600 days.

16. The method of any of claims 1-15, wherein the fluidic shear stress influences the cell differentiation or maturation by at least one of: upregulating gene expression for markers of interest; down-regulating expression of sternness or off-target markers; increasing protein expression;changing cell population heterogeneity to include at least one other cell type; changing structural morphology of organoids and tissues; changing spatial orientation of cell types within organoids and tissues; increasing growth factor secretion; increasing exosome production; and / or changing functional transport across cell barriers.

17. The method of any of claims 1-16, wherein cells are exposed to FSS that is modulated.

18. The method of claim 17, wherein the modulating is by changing stir speed and / or medium viscosity.

19. The method of any of claims 1-18, further comprising exposing the cells to one or more of biological agents, a biological agent gradient, a pressure, a pressure gradient, oxygen tension, and an oxygen tension gradient to direct development, small molecules and proteins to promote differentiation, and / or functioning of the cells.

20. The method of any of claims 1-19, wherein the cells are kidney cells, lung cells, gastro-intestinal cells or vascular cells.

21. The method of any of claims 1-20, wherein the organoids are iPSC-derived ureteric bud organoids.

22. The method of any of claims 1-21, further comprising harvesting the organoids.

23. The method of any of claims 1-22, further comprising harvesting cellular-derived products from the medium.

24. The method of claim 23, wherein the cellular-derived products are exosomes, growth factors, cytokines, or hormones.

25. A tissue construct or an organoid produced by the method of any of claims 1-24.

26. The tissue construct or an organoid of claim 25 for use in tissue replacement therapy.

27. The tissue construct or an organoid of claim 25 for use in biomedical research.

28. The tissue construct or an organoid of claim 25 for use in disease modeling.

29. The tissue construct or an organoid of claim 25 for use in drug toxicity studies.

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