COMPOSITIONS AND PROCESSES for VASCULARIZATION OF KIDNEY TISSUES and IN-VITRO COMPOSITIONS THEREOF

Abstract

This disclosure provides compositions comprising engineered vascularized kidney tissues and methods of making vascularized glomerular kidney tissues. Also provided herein are in vitro kidneys comprising vascularized kidney tissues. Methods of making the compositions and in vitro kidneys and uses thereof are disclosed herein.

Description

Attorney Docket No. 224638-702601COMPOSITIONS AND PROCESSES FOR VASCULARIZATION OF KIDNEY TISSUES AND IN-VITRO COMPOSITIONS THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63 / 702,895, filed October 3, 2024, and titled “Compositions and Processes for Vascularization of Kidney Tissues and In-Vitro Compositions Thereof,” the entire contents of which are incorporated herein by reference.BACKGROUND

[0002] Kidney disease affects approximately 43 million Americans and is the 7th leading risk factor for mortality globally, with kidney disease rates continuing to rise. The kidney has many functions in the body including maintaining equilibrium of water and minerals, metabolism, excretion, regulating the endocrine system, promoting blood cell and bone formation, and maintaining blood pressure that regulates the cardiovascular system, and others. To improve survival in patients with kidney failure, dialysis is used to filter the patient’s blood by removing waste and excess water which temporarily ameliorates severe metabolic acidosis, hyperkalemia, intoxication, and life-threatening complications such as cardiac arrhythmias and fluid buildup in the lungs. However, dialysis is not a long-term solution for chronic kidney diseases, placing patients on wait lists for kidney replacement therapy with limited access to kidney transplant and dialysis machines. Therefore, there is a great unmet need for alternative sources for kidney replacement and dialysis therapies.BRIEF SUMMARY

[0003] In some aspects, the techniques described herein relate to an in vitro vascularized tissue including: a plurality of stem cell-derived glomeruli or precursors thereof; and a plurality of adipose-derived microvessel fragments (MVFs), wherein the plurality of MVFs are sufficient to form a network of microvessels (MVs) that penetrate or are penetrated by the plurality of stem cell-derived glomeruli at a density that is at least 10% greater than stem cell-derived glomeruli that have not been cultivated with the plurality of MVFs, wherein the density of penetrated stem cell- derived glomeruli or precursors thereof is measured based on an amount of penetrated stem cell- derived glomeruli or precursors thereof in a cross-sectional area of the in vitro vascularized tissue.Attorney Docket No. 224638-702601

[0004] In some aspects, the techniques described herein relate to an in vitro vascularized tissue, wherein the in vitro vascularized tissue includes at least one developing stem cell-derived glomerulus; and the plurality of MVFs or the network of MVs within the developing stem cell- derived glomerulus.

[0005] In some aspects, the techniques described herein relate to an in vitro vascularized tissue, wherein the in vitro vascularized tissue includes at least one formed stem cell-derived glomeruli; and the plurality of MVFs or the network of MVs within the formed stem cell-derived glomeruli.

[0006] In some aspects, the techniques described herein relate to an in vitro vascularized tissue, further including at least one kidney tissue component selected from a proximal tubule, a loop of Henle, a distal tubule, or a combination thereof.

[0007] In some aspects, the techniques described herein relate to an in vitro vascularized tissue, wherein an expression profile of endothelial cells in the stem cell-derived glomeruli or precursors thereof includes at least one of UEA1+, CD31+ or VEGFR2+.

[0008] In some aspects, the techniques described herein relate to an in vitro vascularized tissue, wherein an expression profile of podocytes in the stem cell-derived glomeruli or precursors thereof includes PODXL+.

[0009] In some aspects, the techniques described herein relate to an in vitro vascularized tissue, wherein an expression profile of renal tubule cells in the stem cell-derived glomeruli or precursors thereof includes EPCAM+.

[0010] In some aspects, the techniques described herein relate to an in vitro vascularized tissue, wherein an expression profile of proximal tubule cells in the stem cell-derived glomeruli or precursors thereof includes LTL+.

[0011] In some aspects, the techniques described herein relate to an in vitro vascularized tissue, wherein an expression profile of cells in the stem cell-derived glomeruli or precursors thereof vary along a z-direction.

[0012] In some aspects, the techniques described herein relate to an in vitro vascularized tissue, further including an extracellular matrix (ECM).

[0013] In some aspects, the techniques described herein relate to an in vitro vascularized tissue, wherein the ECM includes at least one of solubilized basement membrane preparation extracted from Engelbreth-Holm- Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibrin, fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, decellularized kidney ECM, or a combination thereof.Attorney Docket No. 224638-702601

[0014] In some aspects, the techniques described herein relate to an in vitro vascularized tissue, wherein the network of adipose-derived MVs or plurality of MVFs include at least a portion or segment of at least one artery, arteriole, capillary, venule, vein, or a combination thereof.

[0015] In some aspects, the techniques described herein relate to an in vitro vascularized tissue, wherein the network of adipose-derived MVs or plurality of MVFs are obtained from subcutaneous fat, perirenal fat, pericardial fat, omental fat, breast fat, epididymal fat, properitoneal fat, or a combination thereof.

[0016] In some aspects, the techniques described herein relate to an in vitro vascularized tissue, further including allogeneic components and autologous components.

[0017] In some aspects, the techniques described herein relate to an in vitro vascularized tissue, further including synthetic or semi -synthetic materials.

[0018] In some aspects, the techniques described herein relate to an in vitro vascularized tissue, further including a three-dimensional framework selected from at least one of a membrane, a mesh, a grid, a sponge, a foam, or a combination thereof.

[0019] In some aspects, the techniques described herein relate to an in vitro vascularized tissue, wherein the three-dimensional framework is selected from at least one of solubilized basement membrane preparation extracted from Engelbreth-Holm- Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibrin, fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, or a combination thereof.

[0020] In some aspects, the techniques described herein relate to an in vitro vascularized tissue including: a plurality of stem cell-derived glomeruli or precursors thereof; and a network of adipose-derived microvessels (MVs) or a plurality MVFs, wherein the network of adipose-derived MVs or plurality of MVFs are sufficient to obtain a branching index that is at least 10% greater than a plurality of stem cell-derived glomeruli that have not been cultivated with the network of adipose-derived MVs or the plurality MVFs, wherein the branching index is measured based on an amount of branch points in a cross-sectional area of the stem cell-derived glomeruli.

[0021] In some aspects, the techniques described herein relate to an in vitro vascularized tissue, wherein the in vitro vascularized tissue includes at least one developing stem cell-derived glomerulus; and the plurality of MVFs or the network of MVs within the developing stem cell- derived glomerulus.

[0022] In some aspects, the techniques described herein relate to an in vitro vascularized tissue, wherein the in vitro vascularized tissue includes at least one formed stem cell-derived glomeruli; and the plurality of MVFs or the network of MVs within the formed stem cell-derived glomeruli.Attorney Docket No. 224638-702601

[0023] In some aspects, the techniques described herein relate to an in vitro vascularized tissue including: a plurality of stem cell-derived glomeruli or precursors thereof; and a network of adipose-derived MVs or plurality of MVFs, wherein the network of adipose-derived MVs or plurality of MVFs are sufficient to obtain a total vessel length that is at least 10% greater than stem cell-derived glomeruli that have not been cultivated with the network of adipose-derived MVs or plurality of MVFs, the total vessel length is measured based on a linear extent of identified vessels in a cross-sectional area of the stem cell-derived glomeruli.

[0024] In some aspects, the techniques described herein relate to an in vitro vascularized tissue, wherein the in vitro vascularized tissue includes at least one developing stem cell-derived glomerulus; and the plurality of MVFs or the network of MVs within the developing stem cell- derived glomerulus.

[0025] In some aspects, the techniques described herein relate to an in vitro vascularized tissue, wherein the in vitro vascularized tissue includes at least one formed stem cell-derived glomeruli; and the plurality of MVFs or the network of MVs within the formed stem cell-derived glomeruli.

[0026] In some aspects, the techniques described herein relate to a method for vascularizing glomeruli in vitro, the method including: (a) contacting a plurality of stem cell-derived glomeruli or precursors thereof with a network of adipose-derived microvessels (MVs) or a plurality of MVFs; and (b) cultivating the contacted plurality of stem cell-derived glomeruli or precursors thereof in a cell culture medium, thereby allowing the network of adipose-derived MVs or plurality of MVFs to penetrate or be penetrated by the plurality of stem cell-derived glomeruli or precursors thereof at a density that is at least 10% greater than stem cell-derived glomeruli or precursors thereof that have not been cultivated with the network of adipose-derived MVs or plurality of MVFs, the density of penetrated stem cell-derived glomeruli or precursors thereof is measured based on an amount of penetrated stem cell-derived glomeruli or precursors thereof in a cross- sectional area of an in vitro vascularized tissue including the stem cell-derived glomeruli or precursors thereof.

[0027] In some aspects, the techniques described herein relate to a method, wherein the in vitro vascularized tissue includes at least one developing stem cell-derived glomerulus; and the plurality of MVFs or the network of MVs within the developing stem cell-derived glomerulus.

[0028] In some aspects, the techniques described herein relate to a method, wherein the in vitro vascularized tissue includes at least one formed stem cell-derived glomeruli; and the plurality of MVFs or the network of MVs within the formed stem cell-derived glomeruli.

[0029] In some aspects, the techniques described herein relate to a method, wherein the contacting includes incorporating the stem cell-derived glomeruli or precursors thereof and the network ofAttorney Docket No. 224638-702601 adipose-derived MVs or plurality of MVFs into a bioink, wherein the bioink includes a granular tissue, where the granular tissue includes stem cell-derived spherical aggregates, spheroids, organoids, embryoid bodies, or a combination thereof.

[0030] In some aspects, the techniques described herein relate to a method, wherein the contacting includes incorporating the stem cell-derived glomeruli or precursors thereof and the network of adipose-derived MVs or plurality of MVFs into a bioink, wherein the bioink includes a suspension culture.

[0031] In some aspects, the techniques described herein relate to a method, further including extruding the bioink at least one spatially defined location(s) on or within a three-dimensional framework.

[0032] In some aspects, the techniques described herein relate to a method, wherein the three- dimensional framework is selected from at least one of a membrane, mesh, a grid, a sponge, a foam, or a combination thereof.

[0033] In some aspects, the techniques described herein relate to a method, wherein the three- dimensional framework is selected from at least one of solubilized basement membrane preparation extracted from Engelbreth-Holm- Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibrin, fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, decellularized kidney ECM, or a combination thereof.

[0034] In some aspects, the techniques described herein relate to a method, wherein an expression profile of cells in the stem cell-derived glomeruli or precursors thereof includes at least one of UEA1+, CD31+ or VEGFR2+.

[0035] In some aspects, the techniques described herein relate to a method, wherein an expression profile of podocytes in the stem cell-derived glomeruli or precursors thereof includes PODXL+.

[0036] In some aspects, the techniques described herein relate to a method, wherein an expression profile of renal tubule cells in the stem cell-derived glomeruli or precursors thereof includes EPCAM+.

[0037] In some aspects, the techniques described herein relate to a method, wherein an expression profile of proximal tubule cells in the stem cell-derived glomeruli or precursors thereof includes LTL+.

[0038] In some aspects, the techniques described herein relate to a method, wherein an expression profile of cells in the stem cell-derived glomeruli or precursors thereof varies along a z-direction.

[0039] In some aspects, the techniques described herein relate to a method, wherein the cultivating includes exposure of the network of adipose-derived MVs or plurality of MVFs to flow and / or shear.Attorney Docket No. 224638-702601

[0040] In some aspects, the techniques described herein relate to a method, wherein the cell culture medium includes growth factors.

[0041] In some aspects, the techniques described herein relate to a method, wherein the growth factors include at least one of an agent selected from the group consisting of: CHIR99021, Noggin, Activin A, FGF1, FGF7, FGF9, BMP7, GDNF BMP4, Retinoic Acid, GDNF1, RSPO1, or a combination thereof.

[0042] In some aspects, the techniques described herein relate to a method, wherein the cultivating is performed on an extracellular matrix (ECM).

[0043] In some aspects, the techniques described herein relate to a method, wherein the ECM includes at least one of solubilized basement membrane preparation extracted from Engelbreth- Holm-Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibrin, fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, decellularized kidney ECM, or a combination thereof.

[0044] In some aspects, the techniques described herein relate to a method, wherein the network of adipose-derived MVs or plurality of MVFs include at least a portion or segment of at least one artery, arteriole, capillary, venule, vein, or a combination thereof.

[0045] In some aspects, the techniques described herein relate to a method, wherein the network of adipose-derived MVs or plurality of MVFs are obtained from subcutaneous fat, perirenal fat, pericardial fat, omental fat, breast fat, epididymal fat, properitoneal fat, or a combination thereof.

[0046] In some aspects, the techniques described herein relate to a method, further including allogeneic components and autologous components.

[0047] In some aspects, the techniques described herein relate to a method, further including synthetic or semi -synthetic materials.

[0048] In some aspects, the techniques described herein relate to a method, further including a three-dimensional framework selected from at least one of a membrane, a mesh, a grid, a sponge, a foam, or a combination thereof.

[0049] In some aspects, the techniques described herein relate to a method, wherein the three- dimensional framework is selected from at least one of solubilized basement membrane preparation extracted from Engelbreth-Holm- Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibrin, fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, decellularized kidney ECM, or a combination thereof.

[0050] In some aspects, the techniques described herein relate to a method, wherein the network of adipose-derived MVs or plurality of MVFs and cells in the plurality of stem cell-derived glomeruli or precursors thereof are in a ratio of 1 :500 or 1 : 1650.Attorney Docket No. 224638-702601

[0051] In some aspects, the techniques described herein relate to an in vitro vascularized kidney tissue including: a plurality of stem cell-derived kidney tissue components including: a plurality of glomeruli or precursors thereof, a plurality of podocytes, a plurality of proximal tubule cells, a plurality of renal tubule cells, a plurality of distal tubule cells, a loop of Henle, a plurality of endothelial cells, or a combination thereof; and a network of adipose-derived microvessels (MVs) or plurality of MVFs, in an amount sufficient to form a network of MVs that penetrate or are penetrated by the plurality of stem cell-derived kidney tissue components at a density that is at least 10% greater than a plurality of stem cell-derived kidney tissue components that have not been cultivated with the network of adipose-derived MVs or plurality of MVFs, the density of penetrated kidney tissue components is measured based on an amount of penetrated kidney tissue components in a cross-sectional area of the in vitro vascularized kidney tissue including the stem cell-derived kidney tissue components.

[0052] In some aspects, the techniques described herein relate to an in vitro vascularized kidney tissue, wherein an expression profile of endothelial cells in the plurality of glomeruli or precursors thereof includes at least one of UEA1+, CD31+ or VEGFR2+.

[0053] In some aspects, the techniques described herein relate to an in vitro vascularized kidney tissue, wherein an expression profile of podocytes in the stem cell-derived kidney tissue components includes PODXL+.

[0054] In some aspects, the techniques described herein relate to an in vitro vascularized kidney tissue, wherein an expression profile of renal tubule cells in the stem cell-derived kidney tissue components includes EPCAM+.

[0055] In some aspects, the techniques described herein relate to an in vitro vascularized kidney tissue, wherein an expression profile of proximal tubule cells in the stem cell-derived kidney tissue components includes LTL+.

[0056] In some aspects, the techniques described herein relate to an in vitro vascularized kidney tissue, wherein an expression profile of cells in the stem cell-derived kidney tissue components vary along a z-direction.

[0057] In some aspects, the techniques described herein relate to an in vitro vascularized kidney tissue, further including an extracellular matrix (ECM).

[0058] In some aspects, the techniques described herein relate to an in vitro vascularized kidney tissue, wherein the ECM includes at least one of solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibrin, fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, decellularized kidney ECM, or a combination thereof.Attorney Docket No. 224638-702601

[0059] In some aspects, the techniques described herein relate to an in vitro vascularized kidney tissue, wherein the network of adipose-derived MVs or plurality of MVFs include at least a portion or segment of at least one artery, arteriole, capillary, venule, vein, or a combination thereof.

[0060] In some aspects, the techniques described herein relate to an in vitro vascularized kidney tissue, wherein the network of adipose-derived MVs or plurality of MVFs are obtained from subcutaneous fat, perirenal fat, pericardial fat, omental fat, breast fat, epididymal fat, properitoneal fat, or a combination thereof.

[0061] In some aspects, the techniques described herein relate to an in vitro vascularized kidney tissue, further including allogeneic components and autologous components.

[0062] In some aspects, the techniques described herein relate to an in vitro vascularized kidney tissue, further including synthetic or semi -synthetic materials.

[0063] In some aspects, the techniques described herein relate to an in vitro vascularized kidney tissue, further including a three-dimensional framework selected from at least one of a membrane, a mesh, a grid, a sponge, a foam, or a combination thereof.

[0064] In some aspects, the techniques described herein relate to an in vitro vascularized kidney tissue, wherein the three-dimensional framework is selected from at least one of solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibrin, fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, decellularized kidney ECM, or a combination thereof.

[0065] In some aspects, the techniques described herein relate to an in vitro vascularized tissue including: a plurality of stem cell-derived kidney tissue components including: a plurality of glomeruli or precursors thereof, a plurality of podocytes, a plurality of proximal tubule cells, a plurality of renal tubule cells, a plurality of distal tubule cells, a loop of Henle, a plurality of endothelial cells, or a combination thereof; and a network of adipose-derived microvessels (MVs) or plurality of MVFs, wherein the network of adipose-derived MVs or plurality of MVFs are in an amount sufficient to obtain a branching index that is at least 10% greater than stem cell-derived kidney tissue components that have not been cultivated with the network of adipose-derived MVs or plurality of MVFs, the branching index is measured based on an amount of branch points in a cross-sectional area of the stem cell-derived kidney tissue components.

[0066] In some aspects, the techniques described herein relate to an in vitro vascularized tissue including: a plurality of stem cell-derived kidney tissue components including: a plurality of glomeruli or precursors thereof, a plurality of podocytes, a plurality of proximal tubule cells, a plurality of renal tubule cells, a plurality of distal tubule cells, a loop of Henle, a plurality ofAttorney Docket No. 224638-702601 endothelial cells, or a combination thereof; and a network of adipose-derived microvessels (MVs) or plurality of MVFs, wherein the network of adipose-derived MVs or plurality of MVFs are in an amount sufficient to obtain a total vessel length that is at least 10% greater than stem cell-derived kidney tissue components that have not been cultivated with the network of adipose-derived MVs or plurality of MVFs, the total vessel length is measured based on a linear extent of identified vessels in a cross-sectional area of the stem cell-derived kidney tissue components.

[0067] In some aspects, the techniques described herein relate to a method for making an in vitro vascularized kidney tissue including: (a) combining a plurality of stem cell-derived glomeruli or precursors thereof with a plurality of stem cell-derived kidney tissue components selected from: a plurality of podocytes, a plurality of proximal tubule cells, a plurality of renal tubule cells, a plurality of distal tubule cells, a loop of Henle, a plurality of endothelial cells, or a combination thereof; (b) contacting the combination with a network of adipose-derived microvessels (MVs) or plurality of MVFs; and (c) cultivating the contacted combination in a cell culture medium such that the combination includes a network of adipose-derived MVs at a density that is at least 10% greater than a plurality of stem cell-derived kidney tissue components that have not been cultivated with the network of adipose-derived MVs or plurality of MVFs, the density of penetrated kidney tissue components is measured based on an amount of penetrated kidney tissue components in a cross-sectional area of the in vitro vascularized kidney tissue including the stem cell-derived kidney tissue components.

[0068] In some aspects, the techniques described herein relate to a method, wherein an expression profile of endothelial cells in the network of MVs include at least one markers selected from UEA1+, CD31+ or VEGFR2+.

[0069] In some aspects, the techniques described herein relate to a method, wherein an expression profile of podocytes in the network of MVs include PODXL+.

[0070] In some aspects, the techniques described herein relate to a method, wherein an expression profile of the renal tubule cells in the network of MVs include EPCAM+.

[0071] In some aspects, the techniques described herein relate to a method, wherein an expression profile of the proximal tubule cells in the network of MVs include LTL+.

[0072] In some aspects, the techniques described herein relate to a method, wherein the contacting includes incorporating the stem cell-derived kidney tissue components and the network of adipose- derived MVs or plurality of MVFs into a bioink, wherein the bioink includes a granular tissue, where the granular tissue includes stem cell-derived spherical aggregates, spheroids, organoids, embryoid bodies, or a combination thereof.Attorney Docket No. 224638-702601

[0073] In some aspects, the techniques described herein relate to a method, wherein the contacting includes incorporating the stem cell-derived kidney tissue components and the network of adipose- derived MVs or plurality of MVFs into a bioink, wherein the bioink includes a suspension culture.

[0074] In some aspects, the techniques described herein relate to a method, further including extruding the bioink at least one spatially defined location(s) on or within a three-dimensional framework.

[0075] In some aspects, the techniques described herein relate to a method, wherein the three- dimensional framework is selected from at least one of a membrane, mesh, a grid, a sponge, a foam, or a combination thereof.

[0076] In some aspects, the techniques described herein relate to a method, wherein the three- dimensional framework is selected from at least one of solubilized basement membrane preparation extracted from Engelbreth-Holm- Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibrin, fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, decellularized kidney ECM, or a combination thereof.

[0077] In some aspects, the techniques described herein relate to a method, wherein an expression profile of cells in the stem cell-derived glomeruli or precursors thereof varies along a z-direction.

[0078] In some aspects, the techniques described herein relate to a method, wherein the cultivating includes exposure of the network of MVs to flow and / or shear.

[0079] In some aspects, the techniques described herein relate to a method, wherein the cell culture medium includes growth factors.

[0080] In some aspects, the techniques described herein relate to a method, wherein the growth factors include at least one of an agent selected from the group consisting of: CHIR99021, Noggin, Activin A, FGF1, FGF7, FGF9, BMP7, GDNF BMP4, Retinoic Acid, GDNF1, RSPO1, or a combination thereof.

[0081] In some aspects, the techniques described herein relate to a method, wherein the cultivating is performed on an extracellular matrix (ECM).

[0082] In some aspects, the techniques described herein relate to a method, wherein the ECM includes at least one of solubilized basement membrane preparation extracted from Engelbreth- Holm-Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibrin, fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, decellularized kidney ECM, or a combination thereof.

[0083] In some aspects, the techniques described herein relate to a method, wherein the network of adipose-derived MVs or plurality of MVFs include at least a portion or segment of at least one artery, arteriole, capillary, venule, vein, or a combination thereof.Attorney Docket No. 224638-702601

[0084] In some aspects, the techniques described herein relate to a method, wherein the network of adipose-derived MVs or plurality of MVFs are obtained from subcutaneous fat, perirenal fat, pericardial fat, omental fat, breast fat, epididymal fat, properitoneal fat, or a combination thereof.

[0085] In some aspects, the techniques described herein relate to a method, wherein the cell culture medium includes allogeneic components and autologous components.

[0086] In some aspects, the techniques described herein relate to a method wherein the cell culture medium includes synthetic or semi-synthetic materials.

[0087] In some aspects, the techniques described herein relate to an in vitro composition including the in vitro vascularized tissue or the in vitro vascularized kidney tissue.

[0088] In some aspects, the techniques described herein relate to a composition, further including a cell culture medium.

[0089] In some aspects, the techniques described herein relate to a composition, further including growth factors.

[0090] In some aspects, the techniques described herein relate to a composition, wherein the growth factors include CHIR99021, Noggin, Activin A, FGF1, FGF7, FGF9, BMP7, GDNF BMP4, Retinoic Acid, GDNF1, RSPO1, or a combination thereof.

[0091] In some aspects, the techniques described herein relate to a composition, further including at least one metabolite.

[0092] In some aspects, the techniques described herein relate to a composition, wherein the at least one metabolite include: urea, creatinine, uric acid, ammonium phosphate, or a combination thereof.

[0093] In some aspects, the techniques described herein relate to a composition, further including an extracellular matrix.

[0094] In some aspects, the techniques described herein relate to a composition, wherein the extracellular matrix includes: a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibronectin, vitronectin, retronectin, elastin, hyaluronic acid, methylcellulose, a gelatin, solubilized ECM from decellularized kidney, or any combination thereof.

[0095] In some aspects, the techniques described herein relate to a composition, further including a diluent.

[0096] In some aspects, the techniques described herein relate to a composition, wherein the diluent includes a cryopreservation agent, a serum, or a suspension.Attorney Docket No. 224638-702601

[0097] In some aspects, the techniques described herein relate to a composition, further including a population of glomerular progenitor cells or a differentiated progeny thereof.

[0098] In some aspects, the techniques described herein relate to a composition, further including a population of human blood cells.

[0099] In some aspects, the techniques described herein relate to an in vitro composition including: an in vitro-differentiated kidney tissue including a population of glomerular tissues with a network of microvessels of a population of microvascular fragments (MVFs), wherein: the in vitro- differentiated kidney tissue that includes spatially-organized progenitors of a collecting duct or a differentiated population of cells thereof, and the in vitro-differentiated kidney tissue includes two or more markers selected from: LRP2, GATA3, MAFB, and CK8.

[0100] In some aspects, the techniques described herein relate to an in vitro composition, wherein the in vitro-differentiated kidney tissue includes two or more markers, wherein the two or more markers include: (i) MAFB and LRP2; (ii) MAFB and CK8; (iii) MAFB and GATA3; (iv) LRP2 and CK8; (v) LRP2 and GAT A3; or (vi) CK8 and GAT A3.

[0101] In some aspects, the techniques described herein relate to an in vitro composition, wherein the in vitro-differentiated kidney tissue includes three or more markers, wherein the three or more markers include: (i) MAFB, LRP2, and CK8; (ii) MAFB, LRP2, and GAT A3; (iii) MAFB, CK8, and GAT A3; or (iv) LRP2, CK8, and GAT A3.

[0102] In some aspects, the techniques described herein relate to an in vitro composition, wherein the in vitro-differentiated kidney tissue includes four or more markers, wherein the four or more markers include: MAFB, LRP2, CK8, and GAT A3.

[0103] In some aspects, the techniques described herein relate to an in vitro composition, wherein the population of glomerular tissues are derived from human stem cells.

[0104] In some aspects, the techniques described herein relate to an in vitro composition, wherein the population of MVFs are derived from human adipose tissue.

[0105] In some aspects, the techniques described herein relate to an in vitro composition, wherein the human stem cells are embryonic stem cells, induced pluripotent stem cells (iPSCs), or adult stem cells.

[0106] In some aspects, the techniques described herein relate to an in vitro composition, wherein a portion of a core of the glomerular tissues include epithelial cells.

[0107] In some aspects, the techniques described herein relate to an in vitro composition, wherein a portion of a core of the glomerular tissues include renal stromal cells.

[0108] In some aspects, the techniques described herein relate to an in vitro composition, wherein a portion of a core of the glomerular tissues include epithelial cells and renal stromal cells.Attorney Docket No. 224638-702601

[0109] In some aspects, the techniques described herein relate to an in vitro composition, further including a cell culture medium.

[0110] In some aspects, the techniques described herein relate to an in vitro composition, further including growth factors.

[0111] In some aspects, the techniques described herein relate to an in vitro composition, wherein the growth factors include CHIR99021, Noggin, Activin A, FGF1, FGF7, FGF9, BMP7, GDNF BMP4, Retinoic Acid, GDNF1, RSPO1, or a combination thereof.

[0112] In some aspects, the techniques described herein relate to an in vitro composition, further including at least one metabolite.

[0113] In some aspects, the techniques described herein relate to an in vitro composition, wherein the at least one metabolite include urea, creatinine, uric acid, ammonium phosphate, or a combination thereof.

[0114] In some aspects, the techniques described herein relate to an in vitro composition, further including an extracellular matrix.

[0115] In some aspects, the techniques described herein relate to an in vitro composition, wherein the extracellular matrix includes: a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibronectin, vitronectin, retronectin, elastin, hyaluronic acid, methylcellulose, a gelatin, or any combination thereof.

[0116] In some aspects, the techniques described herein relate to an in vitro composition, further including a diluent.

[0117] In some aspects, the techniques described herein relate to an in vitro composition, wherein the diluent includes a cryopreservation agent, a serum, or a suspension.

[0118] In some aspects, the techniques described herein relate to a method 85 to 115 in a bioreactor; and (b) forming a blood circuit between the subject's blood and the in vitro composition to remove excess waste and fluid, thereby treating the kidney disease.

[0119] In some aspects, the techniques described herein relate to a method 85 to 115, thereby treating the disease in the subject.

[0120] In some aspects, the techniques described herein relate to a method, wherein the disease is a kidney disease.

[0121] In some aspects, the techniques described herein relate to a method, wherein the kidney disease is a chronic kidney disease.

[0122] In some aspects, the techniques described herein relate to a method, wherein the kidney disease is selected from the group consisting of: atypical hemolytic uremic syndrome (aHUS),Attorney Docket No. 224638-702601Alport syndrome, amyloidosis, POLI -mediated kidney disease, cancer, cardiovascular kidney metabolic (CKM) syndrome, complement 3 glomerulopathy (C3G), cystinosis, diabetic kidney disease, end-stage renal failure, Fabry disease, focal segmental glomerulosclerosis (FSGS), glomerulonephritis (Glomerular Disease) Goodpasture syndrome, granulomatosis with polyangiitis (GPA), hemolytic uremic syndrome (HUS), Henoch-Schbnlein purpura (HSP), IgA nephropathy, interstitial nephritis, kidney failure, Lupus nephritis, minimal change disease, polycystic kidney disease, primary hyperoxaluria and oxalate, thrombotic thrombocytopenic purpura (TTP), and vasculitis of the kidney.

[0123] In some aspects, the techniques described herein relate to a method, wherein the subject has, is diagnosed with, or is suspected of having kidney failure.

[0124] In some aspects, the techniques described herein relate to a method, wherein the kidney failure is caused by at least one condition selected from the group consisting of: diabetes, high blood pressure, glomerulonephritis, polycystic kidney disease, lupus nephritis, IgA nephropathy, alcoholism, and nephrotoxicity.

[0125] In some aspects, the techniques described herein relate to a method, wherein the subject has, or is diagnosed with a kidney injury.

[0126] In some aspects, the techniques described herein relate to a method, wherein the kidney injury is caused by an infection, a pregnancy complication, a urinary tract obstruction, a kidney stone, or a physical injury.

[0127] In some aspects, the techniques described herein relate to a method, wherein the subject has, or is diagnosed with a congenital abnormality.

[0128] In some aspects, the techniques described herein relate to a method, wherein the subject has, or is diagnosed with a congenital abnormality, wherein the congenital abnormality includes renal agenesis, renal dysplasia, or renal hypoplasia.

[0129] In some aspects, the techniques described herein relate to a method, wherein the administering includes surgical transplantation of the in vitro composition in the subject.

[0130] In some aspects, the techniques described herein relate to a reagent including: fetal bovine serum (FBS), retinoic acid, R-spondin 1 (RSPO1) protein, glial-derived neurotrophic factor (GDNF), fibroblast growth factor 1 (FGF1), fibroblast growth factor 7 (FGF7), LDN193189, and solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma.

[0131] In some aspects, the techniques described herein relate to a reagent, wherein the FBS is present in an amount that is at least about 1% up to 20% volume by total volume of the reagent (v / v).Attorney Docket No. 224638-702601

[0132] In some aspects, the techniques described herein relate to a reagent, wherein the retinoic acid is present in an amount that is at least about 10 nanoMolar (nM) concentration up to 200 nM concentration.

[0133] In some aspects, the techniques described herein relate to a reagent, wherein the RSPO1 is present in an amount that is at least about 10 nanograms per milliliter (ng / mL) up to 200 ng / mL.

[0134] In some aspects, the techniques described herein relate to a reagent, wherein the GDNF is present in an amount that is at least about 0.1 nanograms per milliliter (ng / mL) up to 5 ng / mL.

[0135] In some aspects, the techniques described herein relate to a reagent, wherein the FGF1 is present in an amount that is at least about 10 nanograms per milliliter (ng / mL) up to 200 ng / mL.

[0136] In some aspects, the techniques described herein relate to a reagent, wherein the FGF7 is present in an amount that is at least about 3 nanograms per milliliter (ng / mL) up to 60 ng / mL.

[0137] In some aspects, the techniques described herein relate to a reagent, wherein the LDN193189 is present in an amount that is at least about 1 nanoMolar (nM) concentration up to 20 nM concentration.

[0138] In some aspects, the techniques described herein relate to a reagent, wherein the solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma is present in an amount of at least 5% up to 80% volume by total volume of the reagent (v / v).

[0139] In some aspects, the techniques described herein relate to a kit including: a first container including: a population of glomerular cells and a network of adipose-derived MVs or plurality of MVFs; and a second container including media, growth factors, and agents for making an in vitro kidney tissue.

[0140] In some aspects, the techniques described herein relate to a transplant composition including: a first container including: the in vitro vascularized tissue or the in vitro vascularized kidney tissue or a plurality thereof ; and a second container including: (a) an additional therapeutic agent; (b) at least one immunosuppressant; or (c) a combination thereof.

[0141] In some aspects, the techniques described herein relate to a method 138, thereby treating the kidney disease.

[0142] In some aspects, the techniques described herein relate to a method, wherein the second container of the transplant composition is administered to the subject prior to the first container.

[0143] In some aspects, the techniques described herein relate to a method, wherein the second container of the transplant composition is administered to the subject at a same time as the first container.Attorney Docket No. 224638-702601

[0144] In some aspects, the techniques described herein relate to a method, wherein the second container of the transplant composition is administered to the subject after the first container.

[0145] In some aspects, the techniques described herein relate to a method 1 to 16 or the in vitro vascularized kidney tissue into a kidney of the subject.

[0146] In some aspects, the techniques described herein relate to a method including: (a) transplanting the in vitro vascularized tissue or the in vitro vascularized kidney tissue into a subject having a kidney disease; and (b) administering to the subject at least one immunosuppressive agent, wherein the method increases a glomerular filtration rate (GFR) of the subject relative to the GFR of the subject prior to transplantation.

[0147] In some aspects, the techniques described herein relate to a method including: (a) transplanting the in vitro vascularized tissue or the in vitro vascularized kidney tissue into a subject having a kidney disease; and (b) administering to the subject at least one immunosuppressive agent, wherein the method increases reduces a level of a biomarker indicative of nephrotoxicity in the subject relative to the level of the biomarker indicative of nephrotoxicity in the subject prior to transplantation.

[0148] In some aspects, the techniques described herein relate to a method, wherein the method further includes surgically removing a diseased kidney from the subject.

[0149] In some aspects, the techniques described herein relate to a method, wherein the subject has a kidney disease.

[0150] In some aspects, the techniques described herein relate to a method, wherein the subject has a kidney injury.BRIEF DESCRIPTION OF THE DRAWINGS

[0151] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0152] FIGURE 1 shows a schematic representation of the incorporation of microvascular fragments (MVFs) into kidney tissues to form in vitro engineered kidney tissues made by the methods outlined in Example 1.

[0153] FIGURES 2A-2D show bioprinted kidney tissues made by the methods described in Example 1. FIG. 2A illustrates bioprinted nephron-rich kidney tissues, including podocytes (PODXL), renal tubule (EPCAM), proximal tubule (LTL), and endothelial cells (CD31). FIG. 2B illustrates bioprinted nephron-rich kidney tissue, with inclusion of human adipose-derivedAttorney Docket No. 224638-702601 microvascular fragments (MVFs). FIG. 2C illustrates a comparison of developing endothelial network in bioprinted nephron-rich kidney tissues with / without MVFs. All cultures under static conditions. FIG. 2D provides a quantitative analysis of vascular networks in bioprinted nephronrich kidney tissues. Angiotool graphical representation of analysis, with vessels outlined in red and branching points in blue.

[0154] FIGURE 3 illustrates bioprinted nephron-rich kidney tissues containing MVFs that have been cultured under perfusion and exposed to flow / shear stress. Podocytes (PODXL); renal tubule (EPCAM), proximal tubule (LTL), endothelial cells (UEA1).

[0155] FIGURE 4 illustrates reference raw data of vascularized kidney tissues by fluorescence microscopy, with morphological variation of microvascular network depending on focal plane within height of tissues (Z-dimension). Tissues contain MVFs and were exposed to flow / shear cues through culture in a millifluidic chip. Left to right illustrates different staining, z.e., podocytes (PODXL), human endothelial cells (UEA1), DNA (DAPI).

[0156] FIGURE S illustrates a high-resolution 3D confocal microscopy image of a vascularized kidney tissue. Triple axial sections shown: XY section (center), xz section (bottom), yz section (right). Podocytes (PODXL), human endothelial cells (UEA1).

[0157] FIGURE 6 illustrates bioprinted kidney tissues with segregation of vascular morphology in Z-dimension. Endothelial cell expansion is shown at a top of the bioprinted kidney tissue, capillary invasion of developing glomeruli is shown at a middle of the bioprinted kidney tissue, and a network of mature, pruned vessels with patent lumens is shown at a bottom of the bioprinted kidney tissue. Podocytes (PODXL), human endothelial cells (UEA1).

[0158] FIGURE 7 shows an image of vascularized bioengineered kidney tissue containing MVFs, nephron progenitor cell-derived glomeruli, renal tubules, and a Wolffian duct progenitor- derived tubular network engrafted onto underlying host kidney in a mouse model.

[0159] FIGURES 8A- 8C show that an in vztro-differentiated kidney tissue implanted on the kidney of a recipient mouse in vivo exhibits size-selective sieving of small (10 kDa Dextran) compounds and retention of large compounds (67 kDa Albumin) by the glomerulus of implanted bioengineered kidney tissue. FIG. 8A shows an image of a representative in vivo MPM image of implant glomerulus with its afferent arteriole, capillaries, bowman’s space, glomerular-tubular junction, and early proximal tubule. Plasma was labeled via iv injection of Alexa Fluor 680 conjugated albumin. FIG. 8B shows a representative quantification of the fluorescence intensity in the glomerular capillary (GC), Bowman’s space (BS) and the lumen of early proximal tubule (PT) segments over time. FIG. 8C shows a quantification of the relative difference in the presence (extrapolated by relative fluorescence intensity) of fluorescently-labeled albumin in Bowman’sAttorney Docket No. 224638-702601 space as compared to the presence of fluorescently-labeled 10 kDA Dextran in Bowman’s space. The fluorescence intensity of each molecule Bowman’s space is normalized to the fluorescence intensity in the glomerular capillaries (FBS / FGC).

[0160] FIGURES 9A-9C show images of Angiotensin Il-induced vasoconstriction of glomerular arterioles. FIG. 9A shows the vessel before administration (brackets). FIG. 9B shows the vessel after administration (brackets). FIG. 9C shows quantitative summary of the effects of angiotensin II on vascular and glomerular diameter of the afferent arteriole (AA), efferent arteriole (EA) and glomerulus (G). Angiotensin Il-induced vasoconstriction of afferent / efferent arterioles and glomerular mesangium, suggesting physiological activity and functional maturation of glomerular vasculature.

[0161] FIGURE 10 shows an image of the implanted bioengineered kidney with an arborized, dense vascular network which includes direct perfusion of nephron progenitor cell-derived glomeruli.

[0162] FIGURE 11 shows an image of implanted human microvessels within the bioengineered kidney tissues that formed connections to the host blood vessels, carry mouse blood and lead to human glomeruli. Podocytes (MAFB-GFP), plasma tracer (Albumin-680), human endothelial cells (UEA-1), mouse reticulocytes (CD71).DETAILED DESCRIPTION

[0163] Vascularization is the process by which new blood vessels form and organize within tissues, helping deliver more blood for growth, repair, or increased activity of an organ or tissue. The methods provided herein utilize isolated microvessel fragments grown in extracellular matrix gel, that can form functional microvascular networks and respond to growth signals even in the absence of blood flow. When these constructs are implanted into animals, they spontaneously connect with the host's blood vessels, mature into diverse vessel types, which can result in creating mixed, chimeric human-mouse vascular systems.

[0164] Provided herein are in vitro kidney tissues and in vitro kidneys for use in the treatment of a disease or condition in a subject. Further provided herein are compositions and methods of making, engineering, and using an in vitro kidney tissue, an in vitro kidney, compositions, systems, and kits. The in vitro kidneys and kidney tissues described herein are the first demonstration of kidney tissue vascularization from microvascular fragments (MVFs). The in vitro kidneys and kidney tissues have several advantages over primary kidney tissues, including reproducibility, scalability, safety, and can be made using human-derived kidney cells without being limited by donors.Attorney Docket No. 224638-702601

[0165] Generally, the glomeruli of the kidney are known to require perfusion in order for blood filtration and detoxification to occur. Therefore, the glomeruli of a bioengineered kidney must also be perfused for them to function properly and provide the intended renal function. Blood vessels must penetrate the glomerulus in order for blood to be properly filtered by the glomerulus and the filtrate generated (proto urine) drain into the associated proximal tubule. Further, implantation of bioengineered tissues such as kidney, is ultimately dependent on rapid vascularization to prevent tissue ischemia and, ultimately, necrosis and loss of function. As relates to bioengineered kidney tissues which contain developing nephrons, irrespective of point of maturation at the time of transplantation / implantation, it appears that pre-implantation vascular development and presence a vascular network is essential for persistence and durability of the implanted nephrons, comprising at least glomeruli, proximal and distal tubules, a loop of Henle, etc.

[0166] It is also known that microvascular fragments (MVFs) or subunits, isolated from adipose tissue, are capable of expansion, organization and maturation when maintained in 3D culture in a supportive hydrogel matrix. When these adipose-derived microvascular fragments have been maintained in culture for a period of time sufficient to allow for endothelial cell proliferation and microvascular network formation, they can then be transplanted into recipients (i.e., immunocompromised mice or rats) and the transplanted microvessels undergo spontaneous anastomosis (inosculation) with the surrounding host vasculature and become perfused with the host circulation. It is unclear how exactly this phenomenon occurs, but it is known to occur in a variety of host implant sites such as subcutaneous fat, skeletal muscle and the epicardial surface of the heart. It has also been reported that co-transplantation of adipose-derived microvessels with some targets of cell transplantation such as stem cell-derived cardiomyocytes or stem cell-derived islets, results in improved function and efficacy of the transplanted cells.

[0167] In the context of bioengineered kidney tissues containing stem cell-derived nephrons, endothelial cells, and stroma, methods to promote endothelial progenitor cell expansion and organization have been reported to increase the number of endothelial cells present within the tissue. However, those endothelial cells are largely unable to penetrate or invade the glomerulus and, instead, largely collect around the outside of the glomerulus. Because the blood vessels that form are not able to invade the glomerulus itself, those blood vessels may serve to improve overall tissue viability, but they will not help to improve tissue function or blood filtration. Thus, inefficient or incomplete vascularization of bioengineered kidney tissue results in low cell and tissue viability and / or compromised renal function. Innovative methods to promote tissue level vascularization, which include microvessel invasion of the glomerulus, are essential to the successful generation of bioengineered kidney tissue. By incorporating adipose-derivedAttorney Docket No. 224638-702601 microvessels into the bioengineered kidney tissue comprising stem cell-derived glomeruli, proximal tubule, renal tubule, endothelial cells and renal stroma, the inventors unexpectedly found that blood vessels were able to organize around, as well as invade, developing glomeruli. Unlike other methods aimed at improving vascularization, such as incorporation of exogenous endothelial cells (i.e. human umbilical vein endothelial cells), iPSC-derived endothelial cells, or growth factors (i.e. VEGF), which tend to result in a proliferation of endothelial cells without invasion of the glomeruli or can be limited in phenotypic and functional maturity, addition of adipose-derived microvessels resulted in an increase in vessel density and presence throughout the bioengineered renal tissue, including vascular invasion of the glomerulus. The methods discovered by the inventors provided herein create an in vv / ra-differentiated human kidney tissue that when transplanted into a subject, develops a network of exogenous and endogenous blood vessels that provide blood flow to the tissue, improve the structural integrity of the glomeruli, that improves the survival of the kidney tissue over a longer period of time, resulting in functional filtration or sieving of small molecules.

[0168] Briefly, described herein are (1) vascularized kidney tissues and methods of making the same; (2) methods of making and characterizing in vitro kidneys and vascularized in vitro kidneys; (3) transplant compositions; (4) systems; (5) kits and reagents; and (6) methods of treatment, dosing, and administration.Definitions

[0169] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art.

[0170] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof as used herein mean “comprising”.

[0171] Unless specifically stated or apparent from context, as used herein, the term “about” in reference to a number or range of numbers is understood to mean the stated number and numbers + / -20% thereof, or 20% below the lower listed limit and 20% above the higher listed limit for the values listed for a range.

[0172] The term “substantially” as used herein may refer to a value approaching 100% of a given value. In some embodiments, the term may refer to an amount that can be at least about 90%, 91%,Attorney Docket No. 224638-70260192%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of a total amount. In some embodiments, the term may refer to an amount that can be about 100% of a total amount.

[0173] The term “effective amount” or “therapeutically effective amount” may refer to a quantity of a composition, for example a composition comprising cells such as cells, that can be sufficient to result in a desired activity upon introduction into an isolated organ or portion thereof provided herein.

[0174] The term “function” and its grammatical equivalents as used herein may refer to a capability of operating, having, or serving an intended purpose. Functional may comprise any percent from baseline to 100% of an intended purpose. For example, functional may comprise or comprise about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or up to about 100% of an intended purpose. In some embodiments, the term functional may mean over or over about 100% of normal function, for example, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 600%, 700% or up to about 1000% of an intended purpose.

[0175] The terms “treatment” or “treating” and their grammatical equivalents may refer to the medical management of a subject with an intent to cure, ameliorate, stabilize, or prevent a disease, condition, or disorder. Treatment may include active treatment, that is, treatment directed specifically toward the improvement of a disease, condition, or disorder. Treatment may include causal treatment, that is, treatment directed toward removal of the cause of the associated disease, condition, or disorder. In addition, this treatment may include palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, condition, or disorder. Treatment may include preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of a disease, condition, or disorder. Treatment may include supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the disease, condition, or disorder. In some embodiments, a condition can be pathological. In some embodiments, a treatment may not completely cure, ameliorate, stabilize or prevent a disease, condition, or disorder.

[0176] The term “three-dimensional culture” may refer to a composition comprising a biocompatible matrix, scaffold, or the like. A three-dimensional culture may be liquid, gel, semisolid, or solid at 25° C. A three-dimensional culture may be biodegradable or non-biodegradable. Exemplary three-dimensional culture materials include polymers and hydrogels comprising collagen, fibrin, chitosan, MATRIGEL, decellularized kidney ECM, polyethylene glycol, dextrans including chemically crosslinkable or photocrosslinkable dextrans, hyaluronic acid, and the like or a combination thereof. In some embodiments, a three-dimensional culture comprises allogeneicAtorney Docket No. 224638-702601 components, autologous components, or both allogeneic components and autologous components. In some embodiments, a three-dimensional culture comprises synthetic or semi -synthetic materials. In some embodiments, a three-dimensional culture comprises a framework or support, such as a fibrin-derived scaffold. The term “scaffold” may refer to a wide variety of three- dimensional frameworks, for example, but not limited to a mesh, grid, sponge, foam, or the like.

[0177] The terms “engineered tissue”, “engineered tissue construct”, or “tissue engineered construct” as used herein may refer to a tissue or organ that is produced, in whole or in part, using tissue engineering techniques.

[0178] The term “microvascular fragment” or “microvessel” may refer to a segment or piece of vascular tissue, including at least a part or segment of at least one artery, arteriole, capillary, venule, or vein. A microvessel may comprise endothelial cells arranged in a tube surrounded by one or more layers of mural cells, such as smooth muscle cells or pericytes, and may further comprise extracellular matrix components, such as basement membrane proteins. Microvessels are known to typically range from 10-50 pm in diameter and 50-500 pm in length, though microvessels of alternative dimensions may be used herein as well. In some embodiments, the microvessel fragments are obtained from vascular tissue, for example, but not limited to, skin, skeletal muscle, cardiac muscle, the atrial appendage of the heart, lung, mesentery, or adipose tissue. In some embodiments, the adipose tissue microvessel fragments are obtained from, for example, but not limited to, subcutaneous fat, perirenal fat, pericardial fat, omental fat, breast fat, epididymal fat, properitoneal fat, and the like. The skilled artisan will appreciate that other fat deposits or any vascular-rich tissue or organ may serve as a source of microvessel fragments for use in the invention, for example, but not limited to, skin, muscle, including skeletal or cardiac muscle, lung, and mesentery. In certain embodiments, the microvessel fragments are obtained from adipose tissue harvested by liposuction or abdominoplasty.

[0179] The terms “vascularize”, “vascularizing”, or “vascularization” may refer to providing a functional or substantially functional vascular network to an organ or tissue, particularly an engineered tissue. A functional or substantially functional vascular network is one that perfuses or is capable of perfusing the tissue or organ to meet some or all of the tissue's or organ's nutritional needs, oxygen demand, and waste product elimination needs. A vascular tissue is a natural tissue that is rich in vascular elements, such as microvessels, for example, but without limitation, adipose tissue.

[0180] The terms “revascularize”, “revascularizing”, “neovascularization”, or “revascularization” may refer to revising an existing vascular network or establishing a new functional or substantially functional vascular network in a tissue or organ that has an avascular or hypovascular zone,Attorney Docket No. 224638-702601 typically due to disease, congenital defect, or injury. Additionally, the topical application of certain chemotherapeutic agents, for example, but not limited to, 5-flourouracil (5-FU), may also result in an ischemic or avascular zone. Such an avascular or hypovascular tissue or organ is often totally or partially dysfunctional or has limited function and may be in need of revascularization. Revascularizing such a tissue or organ may result in restored or augmented function.(1) Vascularized Kidney Tissues and Methods of Making the Same.

[0181] Described herein are in vitro vascularized kidney tissues. An in vitro vascularized kidney tissue includes at least a plurality of stem cell-derived glomeruli and a plurality of adipose-derived microvascular fragments (MVFs). In some embodiments, the MVFs comprise a plurality of human blood vessels, wherein the plurality of human blood vessels are derived from adipose tissue. In some embodiments, the MVFs comprise a plurality of primary human blood vessels, blood vessel segments, and adipose tissue.

[0182] In some embodiments, the adipose-derived MVFs are in an amount sufficient to form a network of microvessels that penetrate the plurality of stem cell-derived glomeruli at a vessel density such that at least 50% of the stem cell-derived glomeruli contain endothelial cells. In some embodiments, MVFs are in an amount sufficient to penetrate the stem cell-derived glomeruli to achieve a vessel density of endothelial cells of up to 10%, up to 20%, up to 30%, up to 40%, up to 50%, up to 60%, up to 70%, up to 80%, up to 90%, or up to 100% of the glomeruli.

[0183] In humans, each kidney contains approximately one million nephrons, which are each made up of a glomerulus and a renal tubule. The glomerulus filters waste and excess fluid from the blood, creating primary urine. The renal tubule returns some electrolytes back to the blood via reabsorption and removes waste. Reabsorption is either passive, due to diffusion, or active, due to pumping against a concentration gradient. Substances reabsorbed include: water, sodium chloride, glucose, amino acids, lactate, magnesium, calcium phosphate, uric acid, and bicarbonate. Secretion also occurs in the tubules and collecting duct. Substances secreted in the renal tubules and collecting duct include urea, creatinine, potassium, hydrogen, and uric acid. A countercurrent system in the renal medulla provides the mechanism for generating a hypertonic interstitium, which allows the recovery of solute-free water from within the nephron and returning it to the venous vasculature when appropriate. While glomerular cells have been differentiated from stem cells, current protocols and assays have not shown whether these cells can be vascularized from microvascular fragments to form a fully functioning kidney tissues comprising a glomerulus with a microvessel network, with markers of the glomerulus and each primary portion of the renal tubule (the proximal convoluted tubule, the loop of Henle, the distal convoluted tubule), connectedAttorney Docket No. 224638-702601 to the distal nephron and collecting duct. The inventors have recognized and appreciated that glomeruli when combined with MVFs by the methods described herein form a vascularized kidney tissue with a microvascular network with greater penetration than achieved by other methods.

[0184] In some embodiments, a plurality of stem cell-derived glomeruli are derived from stem cells, wherein the stem cells are induced pluripotent stem cells (iPSCs), embryonic stem cells, and adult stem cells. In some embodiments, a plurality of stem cell-derived glomeruli are derived from human iPSCs. In some embodiments, a plurality of stem cell-derived glomeruli are derived by differentiating any of the cells herein. In some embodiments, the differentiating includes methods of differentiating pluripotent stem cells into glomeruli as described, for example, in Lawlor, Vanslambrouck, Higgins et al., Nature Materials, 2021, the contents of which is incorporated herein by reference in its entirety. In some embodiments, the differentiating includes adaptations to methods of differentiating pluripotent stem cells into glomeruli. In some embodiments, prior to harvesting cultures, 2D monolayers of posterior intermediate mesoderm cells may be exposed to media with growth factors. In some embodiments, harvested cultures are prepared for bioprinting.

[0185] In some embodiments, an expression profile of cells, e.g., endothelial cells, in the stem cell-derived glomeruli comprises one or more of UEA1+, CD31+ or VEGFR2+. In some embodiments, an expression profile of cells in the stem cell-derived glomeruli comprises PODXL+. In some embodiments, an expression profile of cells in the renal tubule comprises EPCAM+. In some embodiments, an expression profile of cells in the proximal tubules comprises LTL+. In some embodiments, an expression profile of cells in the stem cell-derived glomeruli vary along a z-direction.

[0186] In some embodiments, an in vitro vascularized kidney tissue further includes an extracellular matrix (ECM). In some embodiments, the ECM comprises one or more of solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibrin, fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, decellularized kidney ECM, or the like or a combination thereof.

[0187] In some embodiments, the adipose-derived MVFs include at least a portion or segment of at least one artery, arteriole, capillary, venule, vein, or a combination thereof. In some embodiments, adipose-derived MVFs include mural cells, either formed as part of or separate from the adipose-derived MVFs.

[0188] In some embodiments, the adipose-derived MVFs are obtained from a fat source. In some embodiments, the fat source includes subcutaneous fat, perirenal fat, pericardial fat, omental fat, breast fat, epididymal fat, properitoneal fat, or the like.Attorney Docket No. 224638-702601

[0189] In some embodiments, an in vitro vascularized kidney tissue further includes allogeneic components. In some embodiments, an in vitro vascularized kidney tissue further includes autologous components. In some embodiments, an in vitro vascularized kidney tissue further comprises allogeneic components and autologous components.

[0190] In some embodiments, an in vitro vascularized kidney tissues, further comprise synthetic materials. In some embodiments, an in vitro vascularized kidney tissues, further comprise semisynthetic materials.

[0191] In some embodiments, an in vitro vascularized kidney tissue further includes a three- dimensional framework. In some embodiments, a three-dimensional framework is selected from one or more of a membrane, a mesh, a grid, a sponge, a foam, or the like. In some embodiments, the three-dimensional framework is selected from one or more of solubilized basement membrane preparation extracted from Engelbreth-Holm- Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibrin, fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, decellularized kidney ECM, or a combination thereof.

[0192] In some embodiments, MVFs and stem cell-derived kidney tissues (e.g., a plurality of glomeruli, a plurality of podocytes, a plurality of proximal tubule cells, a plurality of renal tubule cells, a plurality of distal tubule cells, a loop of Henle or a plurality of endothelial cells) or cells thereof in the in vitro vascularized kidney tissues are in a ratio of 1 : 1 to 1 :5, 1 :5 to 1 : 10, 1 :5 to 1 :15, 1 : 10 to 1 : 15, 1 : 10 to 1 :20, or 1 : 15 to 1 :20. In some embodiments, MVFs and stem cell- derived kidney tissues or cells thereof in the in vitro vascularized kidney tissues are in a ratio of 1: 100 to 1 :200, 1 :200 to 1 :300, 1 :300 to 1 :500, 1 :500 to 1 :1000, 1:500 to 1 : 1500, 1 :500 to 1 : 1650, 1 :500 to 1 :2000, 1 :250 to 1 : 1000, 1 :250 to 1 : 1500, 1 :250 to 1 : 1650, 1 :250 to 1 :2000, 1 :250 to 1 :2500, 1 : 100 to 1 :2000, 1 : 1000 to 1 :3000, or 1 : 100 to 1 :3000.

[0193] Described herein are methods of making in vitro vascularized kidney tissues.

[0194] In some embodiments, in vitro vascularized kidney tissues may be made by contacting a plurality of stem cell-derived glomeruli with a plurality of adipose-derived microvascular fragments (MVFs); and cultivating the contacted plurality of stem cell-derived glomeruli in a cell culture medium. In some embodiments, the in vitro vascularized kidney tissues may be made by contacting a plurality of stem cell-derived kidney tissues or nephron kidney tissues, e.g., a plurality of glomeruli, a plurality of podocytes, a plurality of proximal tubule cells, a plurality of renal tubule cells, a plurality of distal tubule cells, a loop of Henle, or the like or a combination thereof with a plurality of adipose-derived microvascular fragments (MVFs).

[0195] In some embodiments, MVF-contacted stem cell-derived glomeruli and / or other kidney tissues are cultivated such that MVFs affect various vascular parameters of the plurality of stemAttorney Docket No. 224638-702601 cell-derived kidney tissues. Such vascular parameters include, for example, vessel density, vessel length, lacunarity, or branching index.

[0196] In some embodiments, cultivation is performed to cause the MVFs to penetrate the kidney tissues to increase a desired vessel density of endothelial cells. In some embodiments, a vessel density refers to a percentage of a given area that is occupied by blood vessels. Kidney tissues are highly vascularized organs as the vasculature is essential for regulating blood flow, systemic blood pressure, urine concentration, and other kidney functions. Reduction in kidney tissue vasculature can result in reduced shear stress along the tissues as well, which can lead to vascular loss and / or tissue damage. Vessel density may be measured using imaging modalities using a contrast agent to fill blood spaces or label vessel cells, then quantifying the contrast or labeling to measure a vasculature. In some embodiments, a vessel density may be calculated based on a total vessel volume divided by a whole sample volume, which may be based on a delineated surface area of the sample. In some embodiments, MVFs are added in an amount that cultivation allows the MVFs to penetrate the kidney tissues to achieve a vessel density of endothelial cells of up to 10%, up to 20%, up to 30%, up to 40%, up to 50%, up to 60%, up to 70%, up to 80%, up to 90%, or up to 100% of the kidney tissues. In some embodiments, the MVFs are allowed to penetrate the kidney tissues so as to achieve a vessel density such that at least 50% of the stem cell-derived kidney tissues contain endothelial cells. In some embodiments, in vitro vascularized kidney tissues comprise at least 50% volume of the total volume (v / v) endothelial cells. In some embodiments, in vitro vascularized kidney tissues comprise at least 50% weight of the total weight (w / w) endothelial cells. In some embodiments, the MVFs are in an amount sufficient to penetrate the kidney tissues so as to achieve a vessel density of 10,000 to 25,000 vessels / mL, 25,000 to 50,000 vessels / mL, 50,000 to 75,000 vessels / mL, 75,000 to 100,000 vessels / mL, 100,000 to 150,000 vessels / mL, 150,000 to 200,000 vessels / mL, 200,000 to 250,000 vessels / mL, 250,000 to 500,000 vessels / mL, 500,000 to 1,000,000 vessels / mL, or more than 1,000,000 vessels / mL. In some embodiments, the MVFs are in an amount sufficient to penetrate the kidney tissues, such as, for example, glomeruli, proximal tubules, renal tubules, distal tubules, loop of Henle, or the like, so as to increase a vessel density in the kidney tissues by at least 1 to 10%, by at least 10 to 20%, by at least 20 to 30%, by at least 30 to 40%, by at least 40 to 50%, by at least 50 to 60%, by at least 60 to 70%, by at least 70 to 80%, by at least 80 to 90%, by at least 90-100%, or by more than 100% relative to kidney tissues that have not been cultivated with MVFs.

[0197] In some embodiments, cultivation is performed to cause the MVFs to penetrate the kidney tissues to increase a desired density of vascularized kidney tissue components. In some embodiments, the MVFs are in an amount sufficient to form a network of microvessels to penetrateAttorney Docket No. 224638-702601 the plurality of stem cell-derived glomeruli at a density that is at least 10% greater, at least 20% greater, at least 30% greater, at least 40% greater, at least 50% greater, at least 60% greater, at least 70% greater, at least 80% greater, at least 90% greater, or at least 100% greater than stem cell-derived glomeruli that have not been cultivated with the MVFs. Here, a density of penetrated glomeruli may be measured based on an amount of penetrated glomeruli in a cross-sectional area of the in vitro vascularized tissue comprising the stem cell-derived glomeruli.

[0198] In some embodiments, cultivation is performed to cause the MVFs to penetrate the kidney tissues to increase a desired density of vascularized kidney tissue components, such as, for example, glomeruli, proximal tubules, renal tubules, distal tubules, loop of Henle, or the like. In some embodiments, the MVFs are in an amount sufficient to form a network of microvessels that penetrate a plurality of stem cell-derived kidney tissue components at a density that is at least 10% greater, at least 20% greater, at least 30% greater, at least 40% greater, at least 50% greater, at least 60% greater, at least 70% greater, at least 80% greater, at least 90% greater, or at least 100% greater than stem cell-derived kidney tissue components that have not been cultivated with the MVFs. Here, a density of penetrated kidney tissue components may be measured based on an amount of penetrated kidney tissue components in a cross-sectional area of the in vitro vascularized tissue comprising the stem cell-derived kidney tissue components.

[0199] In some embodiments, the MVFs are in an amount sufficient to contact the kidney tissues so as to increase a vessel length (e.g., a total vessel length, length of lumen, etc.) in the kidney tissues. In some embodiments, a vessel length may be based on a linear extent of identified or visible vessels within a measured area (e.g., cross-sectional area), where the measured area may be across a range of focal lengths In some embodiments, the MVFs are in an amount sufficient to contact the kidney tissues so as to increase a vessel length in the kidney tissues by at least 1 to 10%, by at least 10 to 20%, by at least 20 to 30%, by at least 30 to 40%, by at least 40 to 50%, by at least 50 to 60%, by at least 60 to 70%, by at least 70 to 80%, by at least 80 to 90%, by at least 90-100%, or by more than 100% relative to kidney tissues that have not been cultivated with MVFs.

[0200] In some embodiments, the MVFs are in an amount sufficient to contact the kidney tissues, such as, for example, glomeruli, proximal tubules, renal tubules, distal tubules, loop of Henle, or the like, so as to increase a branching index in the kidney tissues. A branching index generally refers to a measurement of sprouting activity of a vascular network. In some embodiments, a branching index is calculated by dividing a number of branch points by a unit area. In some embodiments, the MVFs are in an amount sufficient to contact the kidney tissues so as to increase a branching index in the kidney tissues by at least 1 to 10%, by at least 10 to 20%, by at least 20Attorney Docket No. 224638-702601 to 30%, by at least 30 to 40%, by at least 40 to 50%, by at least 50 to 60%, by at least 60 to 70%, by at least 70 to 80%, by at least 80 to 90%, by at least 90-100%, or by more than 100% relative to kidney tissues that have not been cultivated with MVFs.

[0201] In some embodiments, the MVFs are in an amount sufficient such that the kidney tissues experience a reduction in lacunarity. In some embodiments, a lacunarity refers to a characterization in vessel non-uniformity, which may be done, via quantitative analyses (e.g., via imaging tools, etc.) by assessing a variation in foreground and background pixel mass densities across an area that is measured (e.g., an image). In some embodiments, lacunarity may further characterize disturbances in vessel organization, which may be effective in characterizing drug treated samples or pathological vasculature. In some embodiments, lacunarity may correlate with a stage of aggressiveness in a pathology. In some embodiments, the MVFs are in an amount sufficient to contact the kidney tissues so as to reduce a lacunarity of the kidney tissues by at least 1 to 10%, by at least 10 to 20%, by at least 20 to 30%, by at least 30 to 40%, by at least 40 to 50%, by at least 50 to 60%, by at least 60 to 70%, by at least 70 to 80%, by at least 80 to 90%, by at least 90- 100%, or by more than 100% relative to kidney tissues that have not been cultivated with MVFs.

[0202] In some embodiments, the MVFs polarize and segregate as a microvascular network within glomeruli or other components of the kidney tissues, e.g., podocytes, a plurality of proximal tubule cells, a plurality of renal tubule cells, a plurality of distal tubule cells, a loop of Henle. In some embodiments, the MVFs wrap around glomeruli. In some embodiments, MVFs are cultivated so as to form capillaries of vascular endothelium of glomeruli. In some embodiments, cultivating the MVFs and a plurality of kidney tissues allows for a formation of a microvascular network that invades at least a portion of the kidney tissues, surrounds and / or wraps around at least a portion of the kidney tissues, or both invades and surrounds and / or wraps around at least a portion of the kidney tissues. In some embodiments, the MVFs are in an amount sufficient to surround the kidney tissues, such as, for example, glomeruli, proximal tubules, renal tubules, distal tubules, loop of Henle, or the like, so as to increase an area of endothelial cells surrounding the kidney tissues by at least 1 to 10%, by at least 10 to 20%, by at least 20 to 30%, by at least 30 to 40%, by at least 40 to 50%, by at least 50 to 60%, by at least 60 to 70%, by at least 70 to 80%, by at least 80 to 90%, by at least 90-100%, or by more than 100% relative to kidney tissues that have not been cultivated with MVFs.

[0203] In some embodiments, contacting comprises incorporating the stem cell-derived glomeruli and the MVFs into a bioink. In some embodiments, contacting comprises incorporating one or more kidney tissue components selected from nephron kidney tissues, the nephron kidney tissues comprising a plurality of podocytes, a plurality of proximal tubule cells, a plurality of renal tubuleAttorney Docket No. 224638-702601 cells, a plurality of distal tubule cells, a loop of Henle, a plurality of endothelial cells, or a combination thereof, and the MVFs into a bioink. In some embodiments, contacting comprises incorporating stem cell-derived glomeruli and one or more kidney tissue components selected from a plurality of podocytes, a plurality of proximal tubule cells, a plurality of renal tubule cells, a plurality of distal tubule cells, a loop of Henle, a plurality of endothelial cells, or a combination thereof, and the MVFs into a bioink.

[0204] In some embodiments, a bioink comprises a granular tissue, where the granular tissue comprises stem cell-derived spherical aggregates, multi-cellular aggregates, spheroids, organoids, embryoid bodies, or a combination thereof. In some embodiments, the granular tissues comprises a slurry of stem cell-derived spherical aggregates, multi-cellular aggregates, spheroids, organoids, embryoid bodies, or a combination thereof In some embodiments, the stem cell-derived spherical aggregates, multi-cellular aggregates, spheroids, organoids, embryoid bodies, or combination thereof comprise nephron kidney tissues comprising stem cell-derived glomeruli, a plurality of podocytes, a plurality of proximal tubule cells, a plurality of renal tubule cells, a plurality of distal tubule cells, a loop of Henle, a plurality of endothelial cells, or a combination thereof. In some embodiments, the stem cell-derived spherical aggregates, multi-cellular aggregates, spheroids, organoids, embryoid bodies, or combination thereof comprise a population of MVFs.

[0205] In some embodiments, a bioink comprises a matrix. In some embodiments, a matrix comprises an extracellular matrix (ECM). In some embodiments, a matrix or ECM comprises one or more of solubilized basement membrane preparation extracted from Engelbreth-Holm- Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibrin, fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, decellularized kidney ECM, or the like or a combination thereof.

[0206] In some embodiments, a bioink comprises a granular tissue contacting at least a portion of a matrix. In some embodiments, a bioink comprises a granular tissue along at least a portion of a surface of a matrix. In some embodiments, a bioink comprises a granular tissue within an inner volume of at least a portion of a matrix. In some embodiments, a bioink comprises a granular tissue at varying densities along a surface of a matrix. In some embodiments, a bioink comprises a granular tissue at varying densities along an inner volume of a matrix.

[0207] In some embodiments, a bioink has been shaped into a three-dimensional geometry. In some embodiments, a three-dimensional geometry may include, for example, any that includes circles, squares, rectangles, triangles, polygons, and irregular geometries. In some embodiments, a bioink is shaped into a single or repeating pattern of functional units. In some embodiments, a bioink is shaped into a cylinder. In some embodiments, a bioink is shaped for input into aAttorney Docket No. 224638-702601 microfluidic system. In some embodiments, a bioink is shaped to be patterned into a blood vessel or network of blood vessels. In some embodiments, a bioink is shaped to be patterned into a blood vessel or network of blood vessels in a target tissue.

[0208] In some embodiments, a bioink comprises a suspension culture. In some embodiments, the suspension culture comprises adipose-derived MVFs, endothelial cells, and / or kidney cells (e.g., stem cell-derived glomeruli). In some embodiments, a cell density of the suspension culture ranges from between 1 to 100 million cells / mL, from between 100 to 200 million cells / mL, from between 200 to 350 million cells / mL, from between 350 to 500 million cells / mL, from between 500 to 750 million cells / mL, or from between 750 million to 1 billion cells / mL.

[0209] In some embodiments, a method making an in vitro vascularized kidney tissue further includes extruding a bioink at one or more spatially defined location(s) on or within a three- dimensional framework. In some embodiments, the bioink is extruded within a newly generated kidney tissue. In some embodiments, the three-dimensional framework is selected from one or more of a membrane, a gel, mesh, a grid, a sponge, a foam, or the like. In some embodiments, the three-dimensional framework is selected from one or more of solubilized basement membrane preparation extracted from Engelbreth-Holm- Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibrin, fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, decellularized kidney ECM, or a combination thereof.

[0210] In some embodiments, an expression profile of cells in the stem cell-derived glomeruli comprises one or more of a marker selected from UEA1+, CD31+ or VEGFR2+. In some embodiments, an expression profile of cells in the stem cell-derived glomeruli comprises PODXL+. In some embodiments, an expression profile of cells in the renal tubule comprises EPCAM+. In some embodiments, an expression profile of cells in the proximal tubules comprises LTL+.

[0211] In some embodiments, an expression profile of cells in the stem cell-derived glomeruli varies along a z-direction.

[0212] In some embodiments, cultivating the contacted plurality of stem cell-derived glomeruli in a cell culture medium includes exposing a network of microvessels to flow and / or shear.

[0213] In some embodiments, a cell culture medium includes growth factors. In some embodiments, growth factors include one or more of an agent selected from the group consisting of: CHIR99021, Noggin, Activin A, FGF1, FGF7, FGF9, BMP7, GDNF, BMP4, Retinoic Acid, GDNF1, RSPO1, FGF7, or a combination thereof.

[0214] In some embodiments, cultivating the contacted plurality of stem cell-derived glomeruli in a cell culture medium is performed on an extracellular matrix (ECM). In some embodiments,Attorney Docket No. 224638-702601ECM comprises one or more of solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibrin, fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, decellularized kidney ECM, or the like or a combination thereof.

[0215] In some embodiments, adipose-derived MVFs comprise at least a portion or segment of at least one artery, arteriole, capillary, venule, vein, or a combination thereof.

[0216] In some embodiments, adipose-derived MVFs are obtained from subcutaneous fat, perirenal fat, pericardial fat, omental fat, breast fat, epididymal fat, properitoneal fat, or the like.

[0217] In some embodiments, a method making an in vitro vascularized kidney tissue further includes cultivating stem cell-derived glomeruli and a plurality of adipose-derived microvascular fragments (MVFs) with one or more allogeneic components and / or autologous components.

[0218] In some embodiments, a method making an in vitro vascularized kidney tissue further includes cultivating stem cell-derived glomeruli and a plurality of adipose-derived microvascular fragments (MVFs) with one or more synthetic or semi -synthetic materials.

[0219] In some embodiments, a method making an in vitro vascularized kidney tissue further includes cultivating the contacted plurality of stem cell-derived glomeruli on or within a three- dimensional framework selected from one or more of a membrane, a mesh, a grid, a sponge, a foam, or the like. In some embodiments, the three-dimensional framework is selected from one or more of solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibrin, fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, decellularized kidney ECM, or a combination thereof.

[0220] In some embodiments, following print, bioprinted tissues are maintained at an air-liquid interface. In some embodiments, media is refreshed for bioprinted tissues at predetermined time intervals. In some embodiments, bioprinted tissues are further cultivated. In some embodiments, bioprinted tissues are incorporated into microfluidic systems, including, for example, perfusion culture systems.

[0221] A cell culture medium provided herein can comprise a basal medium supplemented with one or more growth factors, metabolites, antioxidants, antigens, small molecules, and / or proteins that permit differentiation of a stem cell to glomeruli. In some embodiments, the cell culture medium comprises a basal differentiation medium (DM). In some embodiments, the cell culture medium comprises serum. In some embodiments, the serum is fetal bovine serum (FBS). In some embodiments, the cell culture medium is serum-free cell culture medium. In some embodiments,Attorney Docket No. 224638-702601 the cell culture medium comprises a basal medium, wherein the basal medium comprises Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (DMEM / F12).

[0222] In some embodiments, a cell culture medium is for at least one of nephron or glomerular differentiation. In some embodiments, a cell culture medium for at least one of nephron or glomerular differentiation is a medium known in the art. In some embodiments, the cell culture medium comprises an antigen. In some embodiments, the cell culture medium comprises a B27 supplement. In some embodiments, the cell culture medium comprises a retinoic acid receptor agonist. In some embodiments, the cell culture medium comprises retinoic acid. In some embodiments, the cell culture medium does not comprise retinoic acid. In some embodiments, the cell culture medium comprises insulin-transferrin-selenium (ITS). In some embodiments, the cell culture medium comprises non-essential amino acids (NEAA). In some embodiments, the cell culture medium comprises L-glutamine. In some embodiments, the cell culture medium comprises an antibiotic. In some embodiments, the cell culture medium comprises penicillin and / or streptomycin. In some embodiments, the cell culture medium comprises an antioxidant. In some embodiments, the cell culture medium comprises 2-mercaptoethanol. In some embodiments, the cell culture medium comprises: a Rho kinase (ROCK) inhibitor. In some embodiments, the Rho kinase (ROCK) inhibitor comprises: Y27632 (CAS No. 129830-38-2), Y30141 (CAS No. 199433-55-1), Y33075 (CAS No. 471843-75-1), Y39983 (CAS No. 203911-26-6), or the like or any combination thereof. In some embodiments, the cell culture medium comprises an activin receptor ligand (e.g., Activin A). In some embodiments, the cell culture medium does not comprise an activin receptor ligand (e.g., Activin A). In some embodiments, the cell culture medium comprises one or more growth factors selected from the group consisting of: CHIR99021, Noggin, Activin A, FGF1, FGF7, FGF9, BMP7, GDNF BMP4, Retinoic Acid, GDNF1, RSPO1, or a combination thereof. In some embodiments, Noggin is provided at a concentration ranging from about 5-25 ng / mL. In some embodiments, the cell culture medium comprises dorsomorphin in an amount ranging from about 100-500 nM. In some embodiments, the cell culture medium comprises: a transforming growth factor P receptor family ligand. In some embodiments, the growth factor comprises BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8, BMP9, BMP10, fibroblast growth factor (FGF) such as FGF2, FGF9 or the like, epidermal growth factor (EGF), hedgehog molecules, insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), VEGF, a WNT molecule, or the like or an inhibitor thereof. In some embodiments, the cell culture medium comprises an ALK inhibitor. In some embodiments, the ALK inhibitor comprises LDN193189 (4-[6-[4-(l-Piperazinyl)phenyl]pyrazolo[l,5-a]pyrimidin-3-yl]quinoline dihydrochloride, CAS No. 1435934-00-1). In some embodiments, the cell culture mediumAttorney Docket No. 224638-702601 comprises a WNT pathway activator. In some embodiments, the WNT pathway activator comprises a glycogen synthase kinase 3 inhibitor. In some embodiments, the cell culture medium does not comprise a glycogen synthase kinase 3 inhibitor (GSK-3). In some embodiments, the glycogen synthase kinase 3 inhibitor comprises a small molecule selected from the group consisting of: CHIR98014 (CAS No. 252935-94-7), CHIR98024 (CAS No. 556813-39-9), CHIR99021 (CAS No. 252917-06-9), CHIR99201, 2,4'-dibromoacetophenone, and dihydronarwedine. In some embodiments, the cell culture medium comprises A-77-01 (CAS No. 607737-87-1), EW-7197 (CAS No. 1352608-82-2), GW 788388 (CAS No. 452342-67-5), LDN- 193189, LDN-214117 (CAS No. 1627503 -67-6), SB-431542 (CAS No. 301836-41 -9), SB-202190 (CAS No. 152121-30-7), SB-505124 (CAS No. 694433-59-5), or SM-16 (CAS No. 614749-78- 9). In some embodiments, the cell culture medium comprises a fibroblast growth factor (FGF). In some embodiments, the FGF comprises FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF 10, FGF 16, FGF 17, FGF 18, FGF 19, FGF20, FGF21, FGF22, FGF23, or any combination thereof. In some embodiments, a cell culture medium comprises a glial cell line- derived neurotrophic factor (GDNF) or a tumor growth factor (e.g., TGF-beta 2).

[0223] The cells provided herein, for example, human iPSCs, human iPSC aggregates, glomerular progenitor cells, 3D aggregates, and / or glomerular cells provided herein, can be cultured under conditions that permit growth, maintenance, survival, and / or differentiation of the cells for use in generating an in vztro-differentiated kidney or in vitro kidney tissue provided herein. Cells can be cultured in an incubator or a bioreactor that maintains temperature, CO2 levels, oxygen levels, and humidity. In general, cells are cultured at between about 35 degrees Celsius to about 38 degrees Celsius, at approximately 5% CO2 level, and approximately 95% humidity, unless otherwise indicated. The cells provided here in can be cultured for a period of time that permits differentiation of one progenitor cell type to another cell type.

[0224] The cells provided herein can be in contact with an extracellular matrix that supports cellular structure, differentiation, growth, and survival. In some embodiments, the extracellular matrix is on a solid support, such as a cell culture dish or a tube. In some embodiments, the extracellular matrix is in suspension with the cells provided herein. In some embodiments, the extracellular matrix comprises: a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibronectin, vitronectin, retronectin, elastin, hyaluronic acid, methylcellulose, a gelatin, or any combination thereof.

[0225] In some embodiments, the human iPSCs or human iPSC aggregates are cultured in suspension culture at about 37 degrees C with about 5% CO2 and about 95% relative humidity forAttorney Docket No. 224638-702601 at least 8 hours, at least 12 hours, at least 20 hours, at least 24 hours, at least 36 hours, or at least 48 hours. In some embodiments, the human iPSCs or human iPSC aggregates are cultured at 38 degrees C with 5% CO2 and 95% relative humidity for at least 8 hours, at least 12 hours, at least 20 hours, at least 24 hours, at least 36 hours, or at least 48 hours.

[0226] In some embodiments, the human iPSC aggregates are cultured in a second cell culture medium to form in vztro-differentiated glomerular cells. In some embodiments, the cell culture medium comprises basal DM. In some embodiments, the cell culture medium comprises CHIR99021. In some embodiments, the cell culture medium comprises CHIR99021 and BMP4. In some embodiments, the cell culture medium comprises retinoic acid. In some embodiments, the cell culture medium comprises FGF9. In some embodiments, the cell culture medium comprises LDN193189. In some embodiments, the cell culture medium comprises SB431542. In some embodiments, the cell culture medium comprises retinoic acid, FGF9, LDN193189, and / or CHIR99021.

[0227] In some embodiments, the human iPSCs, human iPSC aggregates, glomerular progenitor cells, 3D aggregates, and / or glomerular cells are cultured at 35 °C with 5% CO2 and 95% relative humidity for at least 8 hours, at least 12 hours, at least 20 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 96 hours, at least 108 hours, at least 120 hours, at least 132 hours, at least 144 hours, at least 156 hours, or at least 168 hours. In some embodiments, the human iPSC aggregates or in vv / ra-differentiated glomerular progenitor cells are cultured at 36 °C with 5% CO2 and 95% relative humidity for at least 8 hours, at least 12 hours, at least 20 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 96 hours, at least 108 hours, at least 120 hours, at least 132 hours, at least 144 hours, at least 156 hours, or at least 168 hours. In some embodiments, the human iPSC aggregates or in vv / ra-differentiated glomerular progenitor cells are cultured at 37 °C with 5% CO2 and 95% relative humidity for at least 8 hours, at least 12 hours, at least 20 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 96 hours, at least 108 hours, at least 120 hours, at least 132 hours, at least 144 hours, at least 156 hours, or at least 168 hours. In some embodiments, the human iPSC aggregates or in vztro-differentiated glomerular progenitor cells are cultured at 38 °C with 5% CO2 and 95% relative humidity for at least 8 hours, at least 12 hours, at least 20 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 96 hours, at least 108 hours, at least 120 hours, at least 132 hours, at least 144 hours, at least 156 hours, or at least 168 hours.Attorney Docket No. 224638-702601

[0228] In some embodiments, cell aggregates comprising the in vv / ra-differentiated glomerular progenitor cells are collected from suspension culture and centrifuged. In some embodiments, the cell aggregates are centrifuged for at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, or at least 5 minutes. In some embodiments, the cell aggregates are centrifuged at a relative centrifugal force of at least about 50 g, at least about 60 g, at least about 70 g, at least about 80 g, at least about 90 g, at least about 100 g, at least about 110 g, at least about 120 g, at least about 130 g, at least about 140 g, at least about 150 g, at least about 200 g, at least about 300 g, at least about 400 g, or at least about 500 g.

[0229] In some embodiments, the cell aggregates are contacted with a proteolytic enzyme or a cell dissociation reagent to break up the cell aggregates into dispersed cells. In some embodiments, the proteolytic enzyme is trypsin, accutase, collagenase, prolinase, or a high purity recombinant fungal serine protease (e.g., TrypLE). In some embodiments, the cell aggregates are contacted with a proteolytic enzyme or a cell dissociation reagent for at least about 30 seconds, at least about 1 minute, at least about 2 minutes, at least about 3 minutes, at least about 4 minutes, at least about 5 minutes, at least about 6 minutes, at least about 7 minutes, at least about 8 minutes, at least about 9 minutes, at least about 10 minutes, or at least about 15 minutes. In some embodiments, a cell culture medium is added to the dispersed cells in contact with the proteolytic enzyme or cell dissociation reagent to stop single cell dissociation.

[0230] In some embodiments, the dissociated cells are enriched for or sorted for cell markers to obtain PODXL, NPHS1, MAFB, WT1, OLFM3 (early podocytes), ANXA1 (late podocytes), and COL4A3 cells. Methods of sorting and enriching for cells with specific marker include, for example, flow cytometry. In some embodiments, the cells are isolated and aggregated in a cell culture medium. In some embodiments the cell culture medium comprises retinoic acid, FGF9, LDN193189, CHIR99021, FGF1, Y27632, and / or Matrigel® (e.g., a solubilized basement membrane preparation extracted from Engelbreth-Holm- Swarm (EHS) mouse sarcoma, laminin, and collagen IV). In some embodiments, the CXCR4+ / CKIT+ cells are incubated at 37 degrees Celsius at 5% CO2 for at least 8 hours, at least 12 hours, at least 20 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 96 hours, at least 108 hours, at least 120 hours, at least 132 hours, at least 144 hours, at least 156 hours, or at least 168 hours.

[0231] In some embodiments, the 3D aggregates are contacted with a cell culture medium comprising: retinoic acid, CHIR99021, LDN193189, GDNF, FGF1, Y27632, Activin A, FGF9, BMP7, BMP4, Retinoic Acid, GDNF1, RSPO1, FGF7, or a combination thereof. In some embodiments, the 3D aggregates are incubated at 37 degrees Celsius at 5% CO2 for at least 8 hours,Attorney Docket No. 224638-702601 at least 12 hours, at least 20 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 96 hours, at least 108 hours, at least 120 hours, at least 132 hours, at least 144 hours, at least 156 hours, or at least 168 hours.

[0232] To promote the growth from the 3D aggregates and development of glomeruli with a microvessel network, the 3D aggregates can be moved from a suspension culture to a transwellbased cell culture. In some embodiments, the 3D aggregates are contacted with a cell culture medium and form vascularized glomerular kidney tissues. In some embodiments, the cell culture medium comprises an essential medium. In some embodiments, an essential medium is feeder- free and / or xeno-free. In some embodiments, a cell culture medium comprises: DMEM / F12, fetal bovine serum, penicillin, streptomycin, retinoic acid, R-spondin-1 (RSPO1), GDNF, FGF1, FGF7, LDN193189, FBS, FGF9, heparin, VEGF, Matrigel, or a combination thereof. In some embodiments, the 3D aggregates are incubated in the cell culture medium at 37 degrees Celsius at 5% CO2 for at least 8 hours, at least 12 hours, at least 20 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 96 hours, at least 108 hours, at least 120 hours, at least 132 hours, at least 144 hours, at least 156 hours, or at least 168 hours.

[0233] In some embodiments, kidney tissues are exposed to flow conditions that generate shear stress on the cells in the kidney tissues. In some embodiments, flow conditions can be implemented by applying fluid perfusion to the kidney tissues. In some embodiments, fluid perfusion is applied in closed-loop or open-loop flow systems.

[0234] In some embodiments, the fluid perfusion comprises a flow rate that is selected to create a desired shear stress. For a rectangular channel with a width w and height h, and a fluid with a viscosity r, the wall shear stress T and flow rate Q have the following relationship: T = 6r]Q / h2w. For a cylindrical channel with a radial distance r, and a fluid with a viscosity r, the wall shear stress T and flow rate Q have the following relationship: T = 4r|Q / r37t. In some embodiments, the flow rate is controlled to vary over time. In some embodiments, the flow rate is controlled to vary at different locations along the kidney tissues.

[0235] In some embodiments, the selected flow rate may generate shear stress anywhere from about 0.000001 dyn / cm2to about 100 dyn / cm2, from about 0.01 dyn / cm2to about 50 dyn / cm2, from about 0.01 dyn / cm2to about 10 dyn / cm2, from about 0.01 dyn / cm2to about 5 dyn / cm2, or from about 0.01 dyn / cm2to about 1 dyn / cm2In some embodiments, the selected flow rate may generate shear stress is about 0.1 up to 10 dyn / cm2. In some embodiments, the selected flow rate may generate shear stress is about 10 up to 20 dyn / cm2. The exposure to shear stress can be constant, continuous, or intermittent and can be for anywhere from 1 day to 200 days. In someAttorney Docket No. 224638-702601 embodiments, shear stress may also be pulsed to mimic blood pressure changes during regular heartbeats. The terms “constant” and “continuous” and “laminar” can be used interchangeably and refer to an uninterrupted and / or steady exposure to shear stress for a specified and extended period of time (e.g., from 1 to 200 days). The term “intermittent” refers to an interrupted or unsteady exposure to shear stress. In reference to the intermittent exposure, the kidney tissues can be exposed to shear stress in regular intervals, e.g., every 5 seconds, every 10 seconds, or every 15 seconds, etc., for a specified amount of time of exposure, e.g., for 1 second, for 2 seconds, for 3 seconds, for 4 seconds, for 5 seconds, etc., for a specified time period (e.g., from 1 to 200 days). In some embodiments, the kidney tissues can be exposed to shear stress in irregular intervals. The type of exposure to the shear stress can be pre-programmed.(2) Methods of Making and Characterizing In Vitro Kidneys and Vascularized In Vitro Kidneys.

[0236] Provided herein are methods of vascularizing glomerular kidney tissues and kidney tissues with MVFs provided herein. An exemplary protocol and timeline is provided in FIG. 1, which illustrates a schematic representation of the incorporation of microvascular fragments (MVFs) into kidney tissues to form in vitro engineered kidney tissues. As shown, hiPSCs are seeded, then contacted with one or more of Y27632, CHIR99021, or a combination thereof for any number of days, such as, for example, 1-3 days. Further examples of active agents (e.g., growth factors, metabolites, antioxidants, antigens, small molecules, and / or proteins) are provided herein that may be applicable to any number of steps in FIG. 1. In some embodiments, the contacting begins at DO. In some embodiments, as shown, the contacting is from 0 to 4 days.

[0237] As shown, from D1-D3, the hiPSCs may differentiate into a primitive streak, which is then contacted with one or more of heparin, FGF9, or a combination thereof for at least about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days. As shown, a primitive streak may be contacted from D4-D6 to form posterior intermediate mesoderm (PIM) cells. The PIM may then be admixed with a population of MVFs provided herein. In some embodiments, the MVFs are thawed. In some embodiments, the PIM cells provided herein are combined in suspension culture with MVFs. The combined cell and MVF populations can be centrifuged, resuspended, and loaded into a syringe for deposition and bioprinting. In some embodiments, CHIR99021 is pulsed for the contacting the bioink. Methods of cellular deposition and bioprinting cellular populations provided herein are discussed further below.

[0238] Once the bioink is prepared and printed, the composition is contacted with one or more of FGF9, heparin, VEGF, B-27, or a combination thereof to form a metanephric mesenchyme (MM) and promote growth of MVFs. In some embodiments, the contacting is performed for up to 1 day,Attorney Docket No. 224638-702601 up to 2 days, up to 3 days, up to 4 days, up to 5 days, up to 6 days, up to 7 days, up to 8 days, up to 9 days, or up to 10 days. As shown, the contacting may be performed at D7-D11, which are post-print days (PPD) 0-4 in the exemplary embodiment shown in FIG. 1.

[0239] After at least partial forming of an MM, the MM is contact with VEGF, B-27, or a combination thereof to allow for endothelial cell growth and tissue maturation. In some embodiments, the tissue maturation is performed for at least 5 days, at least 10 days, at least 15 days, or at least 20 days. In some embodiments, as shown in FIG. 1, the tissue maturation is performed at D12-25+, i.e., PPD 5-18+, which includes any number of days after D25 or PPD18. In some embodiments, the tissue maturation is performed in a bioreactor, a microfluidic system, or the like, which may include, for example, microfluidic chambers or constructs. In some embodiments, the tissue maturation is performed in a perfusion culture. In some embodiments, the tissue maturation is performed in an airfoil chip. More examples of bioreactors or microfluidic systems are described herein.Methods of Bioprinting and Arranging Kidney Tissues.

[0240] Provided herein are methods of bioprinting a kidney tissue provided herein. Bioprinting is a method of using cellular bioinks comprising either cells or a combination of cells with hydrogels deposited by a bioprinting system that spatially controls deposition of living cells in defined geometric patterns. Bioprinting can be used to establish the architecture of the glomerular and kidney tissues provided herein.

[0241] In some embodiments, the bioprinting system comprises a two-dimensional (2D) or three- dimensional (3D) prototype device for providing non-limiting examples of suitable bioprinting techniques. Such a device may include at least one or more controller(s) and one or more mechanical dispense tool(s). The mechanical dispense tool(s) is operably coupled with the controlled s) so as to allow for bioprinting in accordance with one or more instructions.

[0242] In some embodiments, each of the controller(s) comprise at least one processor and at least one non-transitory computer-readable medium. Data and / or instructions can be stored on the at least one non-transitory computer-readable medium. In some embodiments, the instructions comprise receiving one or more instructions for printing one or more 3D objects. In some embodiments, the one or more instructions comprise a file or a set of files that can be loaded into the controller(s). The file or set of files may correspond to one or more object(s) in 2D or 3D to be manufactured by the bioprinting system. The file or set of files may correspond to a plurality of slices for each of the one or more object(s). In some embodiments, the instructions can be determined by one or more computer model, such as, for example, a machine learning model.Attorney Docket No. 224638-702601

[0243] In some embodiments, bioprinting system is configured to print 2D or 3D structures according to computer-executable instructions from the computer device. In some embodiments, the instructions are predetermined based on one or more criteria. Such criteria may comprise, for example, one or more threshold(s) corresponding to signals obtained from sensor(s). In some embodiments, the one or more instructions are based on a user input.

[0244] The bioprinting system may further include a plurality of independently addressable printheads mounted on a 3-axis, motion-controlled gantry. In some embodiments, the mechanical dispense tool(s) comprise one or more syringe barrels in fluid communication with one or more outlet(s). In some embodiments, a diameter of the outlet comprises an inner diameter of about, 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 pm.

[0245] In some embodiments, the outlet(s) comprise syringe or dispense tip(s), capillary tube(s), or nozzle(s). Cellular bioinks, used interchangeably herein with cellular gels, can be stored in the syringe barrels, and can be extruded from the mechanical or pneumatic dispense tool(s) via the nozzle(s). The nozzle(s) can be of varying sizes, and may extrude the bioinks by applying air pressures corresponding to varying print speeds. In some embodiments, the applied air pressure ranges from 1-90 psi. In some embodiments, the print speeds range from 0.1-10 mm / sec. In some embodiments, the print speed comprises 0.6 mm / sec.

[0246] In some embodiments, cultivated cells are transferred to a syringe or dispense tip(s). In some embodiments, a syringe or dispense tip is of varying gauges, e.g., from 10 to 34 gauge syringes. In some embodiments, once loaded, a syringe or dispense tip is placed within a mechanical dispense tool.

[0247] In some embodiments, the instructions comprise translating the one or more mechanical dispense tool(s) to print the structures. To generate structures, such 3D kidney tissues, the printing is performed in a sequence or plurality of layers and / or functional units. In some embodiments, the functional units comprise any suitable geometry, such as, for example, circles, squares, rectangles, triangles, polygons, and irregular geometries. In some embodiments, the printing is performed so as to generate a repeating pattern of bioprinted functional units to form kidney tissues.

[0248] In some embodiments, the bioprinting system is designed to maintain cell viability throughout the printing process. In some embodiments, the bioprinting system comprises a printing chamber for maintaining culture conditions. In some embodiments, such conditions include temperature, humidity, gas partial pressures, or other conditions. In some embodiments, the temperature is maintained between about 4 degrees Celsius (° C) to about 45° C. In someAttorney Docket No. 224638-702601 embodiments, such conditions are monitored using sensors at different locations in the printing chamber.

[0249] In some embodiments, the syringe barrels are sized to hold between about 1 mL and about 200 mL. In some embodiments, the nozzle is sized to provide a minimum resolution between about 0.5 pL to about 10 pL. In some embodiments, the mechanical dispense tool comprises one or more sensor(s) for detecting and / or calibrating a location of the nozzle.

[0250] In some embodiments, the printing is performed on a variety of surfaces. The surface may comprise a membrane, such as, for example, a polyester membrane. The surfaces may comprise one or more well plate(s). In some embodiments, the well plate is a 96, 384, 1536-well plate or the like. In some embodiments, the surface comprises a membrane within each well of a well plate. In some embodiments, the well plate(s) comprise Transwell permeable supports.

[0251] In some embodiments, bioprinting comprises dispensing the one or more bioink(s) via the mechanical dispense tool(s). In some embodiments, the one or more bioink(s) comprise cells. Nonlimiting examples of bioinks with cells include cell solutions, cell suspensions, cell-comprising gels or pastes, cell concentrations, multicellular bodies (e.g., pre-formed cellular aggregates, spheroids, embryoid bodies, or the like), or combinations thereof. In some embodiments, the cells are of different types or combinations thereof. In some embodiments, the cells in the bioinks are based on the type of tissue to be printed. In some embodiments, the cells comprise pluripotent stem cells. In some embodiments, the cells comprise glomerular progenitor cells. In some embodiments, the one or more bioink(s) comprise stem cell-derived glomeruli.

[0252] In some embodiments, the one or more bioink(s) comprise adipose-derived microvascular fragments (MVFs). In some embodiments, MVFs may be incorporated into bioink at 100 to 1,000 MVFs / mL, at 1,000 to 10,000 MVFs / mL, at 10,000 to 100,000 MVFs / mL, at 100,000 to 500,000 MVFs / mL, at 500,000 to 1,000,000 MVFs / mL, or above 1,000,000 MVFs / mL. In some embodiments, cells may be incorporated into bioink at 100 to 1,000 cells / mL, at 1,000 to 10,000 cells / mL, at 10,000 to 100,000 cells / mL, at 100,000 to 500,000 cells / mL, at 500,000 to 1,000,000 cells / mL, at 1,000,000 to 10,000,000 cells / mL, at 10,000,000 to 100,000,000 cells / mL, or above 100,000,000 cells / mL. In some embodiments, the cells include pluripotent stem cells, posterior intermediate mesoderm cells, or other cells provided herein.

[0253] In some embodiments, hydrogels in the bioinks comprise extracellular matrix. In some embodiments, the extracellular matrix comprises a solubilized basement membrane or ECM preparation extracted from Engelbreth-Holm- Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibronectin, vitronectin, retronectin, elastin, hyaluronic acid, methylcellulose, a gelatin, chitosan, alginate, fibrin, decellularized kidney ECM, or anyAttorney Docket No. 224638-702601 combination thereof. In some embodiments, the extracellular matrix comprises a solubilized basement membrane preparation extracted from any of the above.

[0254] In some embodiments, the bioink(s) comprise media. In some embodiments, media is added to the printed structure after the bioprinting. In some embodiments, the printed structures are maintained in culture conditions upon being printed. In some embodiments, the media is dispensed from the mechanical dispense tool(s). In some embodiments, the media comprises basal Differentiation Medium (DM). In some embodiments, the media comprises any of the cell culture agents or supplements described herein.

[0255] In some embodiments, the printed structures are maintained at an air-liquid interface with media. In some embodiments, the media is refreshed at the air-liquid interface as designated time periods, such as, for example, every day, every other day, every two days, etc.

[0256] In some embodiments, the printing is performed at spatially defined locations.

[0257] In some embodiments, bioprinting with the bioprinting system can be combined with microfluidics to construct or culture the kidney tissues.Structural Characterization of In Vitro Kidneys and Tissues.

[0258] Provided herein are compositions comprising in vitro-Qn mQQ kidneys and kidney tissues and methods of making the same. In some embodiments, the compositions provided herein comprise a glomerular kidney tissue, microvascular fragments, or combinations thereof.

[0259] Provided herein are in vitro compositions comprising: an in iv' / ra-differentiated kidney tissue comprising a population of glomerular cells connected to a population of microvascular fragments (MVFs), wherein the in vztro-differentiated kidney tissue comprises glomeruli penetrated throughout by a microvessel network. In some embodiments, the glomeruli comprise one or more podocytes. In some embodiments, the glomeruli comprise two or more markers selected from: PODXL, NPHS1, MAFB, WT1, OLFM3 (early podocytes), ANXA1 (late podocytes), and COL4A3 (fully mature podocytes). In some embodiments, in vitro kidneys and kidney tissues comprise a marker selected from the group consisting of: calbindin 1 (CALB1), cluster of differentiation 13 (CD13), cadherin 1 (CDH1), CK8, cubulin (CUBN), cystatin C, death associated protein like 1 (DAPL1), E-cadherin (ECAD), empty spiracles homeobox 2 (EMX2), engrailed homeobox 2 (EN2), GATA3, hepatocyte nuclear factor 4 alpha (HNF4A), hepatocyte nuclear factor 1 beta (HNF1B), leucine rich repeat containing G protein-coupled receptor 5 (LGR5), leucine rich repeat containing G protein-coupled receptor 6 (LGR6), LIM homeobox 1 (LHX1), Lotus Tetragonolobus Lectin (LTL), LDL receptor related protein 2 (LRP2), LY6 / PLAUR domain containing 1 (LYPD1), MAFB, PAX2, paired box 8 (PAX8), platelet andAttorney Docket No. 224638-702601 endothelial cell adhesion molecule 1 (PECAM1), podocalyxin like (PODXL), RET1, serum creatinine (SCr), Six homeobox 1 (SIX1), SIX2, Special AT -rich sequence-binding protein (SATB2), SOX9, SRY-box transcription factor 17 (SOX17), parathyroid hormone 1 receptor (PTH1R), claudin 2 (CLDN2), tight junction protein 3 (TJP3), transcription factor 21 (TCF21), Wnt family member 4 (WNT4), WNT9, and WNT11, SALL1, EYA1, or the like.

[0260] Provided herein are in vitro compositions comprising: an in rz / ZYz-differentiated kidney tissue comprising a population of stem cell-derived glomeruli comprising a network of microvessels of a plurality of MVFs, wherein: the in vztro-differentiated kidney tissue comprises a plurality of kidney tissue components such as a plurality of podocytes, a plurality of proximal tubule cells, a plurality of renal tubule cells, a plurality of endothelial cells, or a combination thereof. In some embodiments. In some embodiments, a plurality of MVFs are in an amount sufficient to form a network of microvessels that penetrate the plurality of stem cell-derived glomeruli at a vessel density such that at least 50% of the stem cell-derived glomeruli contain endothelial cells. In some embodiments, MVFs are in an amount sufficient to penetrate the stem cell-derived glomeruli to achieve a vessel density of endothelial cells of up to 10%, up to 20%, up to 30%, up to 40%, up to 50%, up to 60%, up to 70%, up to 80%, up to 90%, or up to 100% of the glomeruli. In some embodiments, MVFs are added in an amount that cultivation allow the MVFs to penetrate the kidney tissues to achieve a vessel density of endothelial cells of up to 10%, up to 20%, up to 30%, up to 40%, up to 50%, up to 60%, up to 70%, up to 80%, up to 90%, or up to 100% of the kidney tissues. In some embodiments, the MVFs are allowed to penetrate the kidney tissues so as to achieve a vessel density such that at least 50% of the stem cell-derived kidney tissues contain endothelial cells. In some embodiments, in vitro vascularized kidney tissues comprise at least 50% volume of the total volume (v / v) endothelial cells. In some embodiments, in vitro vascularized kidney tissues comprise at least 50% weight of the total weight (w / w) endothelial cells. In some embodiments, the MVFs are in an amount sufficient to penetrate the kidney tissues so as to achieve a vessel density of 10,000 to 25,000 vessels / mL, 25,000 to 50,000 vessels / mL, 50,000 to 75,000 vessels / mL, 75,000 to 100,000 vessels / mL, 100,000 to 150,000 vessels / mL, 150,000 to 200,000 vessels / mL, 200,000 to 250,000 vessels / mL, 250,000 to 500,000 vessels / mL, 500,000 to 1,000,000 vessels / mL, or more than 1,000,000 vessels / mL. In some embodiments, the MVFs are in an amount sufficient to penetrate the kidney tissues so as to increase a vessel density of endothelial cells in the kidney tissues by at least 1 to 10%, by at least 10 to 20%, by at least 20 to 30%, by at least 30 to 40%, by at least 40 to 50%, by at least 50 to 60%, by at least 60 to 70%, by at least 70 to 80%, by at least 80 to 90%, by at least 90-100%, or by more than 100% relative to kidney tissues that have not been cultivated with MVFs. In someAttorney Docket No. 224638-702601 embodiments, the MVFs are in an amount sufficient to form a network of microvessels that penetrate a plurality of stem cell-derived kidney tissue components at a density that is at least 10% greater, at least 20% greater, at least 30% greater, at least 40% greater, at least 50% greater, at least 60% greater, at least 70% greater, at least 80% greater, at least 90% greater, or at least 100% greater than stem cell-derived kidney tissue components that have not been cultivated with the MVFs. Here, a density of penetrated kidney tissue components may be measured based on an amount of penetrated kidney tissue components in a cross-sectional area of the in vitro vascularized tissue comprising the stem cell-derived kidney tissue components.

[0261]

[0262] In some embodiments, the MVFs are in an amount sufficient to contact the kidney tissues so as to increase a vessel length (e.g., a total vessel length, length of lumen, etc.) in the kidney tissues by at least 1 to 10%, by at least 10 to 20%, by at least 20 to 30%, by at least 30 to 40%, by at least 40 to 50%, by at least 50 to 60%, by at least 60 to 70%, by at least 70 to 80%, by at least 80 to 90%, by at least 90-100%, or by more than 100% relative to kidney tissues that have not been cultivated with MVF s. In some embodiments, the MVF s are in an amount sufficient to contact the kidney tissues so as to increase a branching index in the kidney tissues by at least 1 to 10%, by at least 10 to 20%, by at least 20 to 30%, by at least 30 to 40%, by at least 40 to 50%, by at least 50 to 60%, by at least 60 to 70%, by at least 70 to 80%, by at least 80 to 90%, by at least 90-100%, or by more than 100% relative to kidney tissues that have not been cultivated with MVFs. In some embodiments, the MVFs are in an amount sufficient to surround the kidney tissues so as to increase an area of endothelial cells surrounding the kidney tissues by at least 1 to 10%, by at least 10 to 20%, by at least 20 to 30%, by at least 30 to 40%, by at least 40 to 50%, by at least 50 to 60%, by at least 60 to 70%, by at least 70 to 80%, by at least 80 to 90%, by at least 90-100%, or by more than 100% relative to kidney tissues that have not been cultivated with MVFs.

[0263] In some embodiments, an expression profile of cells in a network of microvessels comprise one or more of UEA1+, CD31+ or VEGFR2+. In some embodiments, an expression profile of cells in the stem cell-derived glomeruli comprises PODXL+. In some embodiments, an expression profile of cells in the stem cell-derived glomeruli comprises EPCAM+. In some embodiments, an expression profile of cells in the stem cell-derived glomeruli comprises LTL+.

[0264] In some embodiments, the in vitro kidneys and kidney tissues provided herein comprise a glomerulus or a glomerular tissue. In some embodiments, the in vitro kidneys and kidney tissues provided herein comprise a glomerular marker comprising MAFB, WT1, nephrin, or podocin. In some embodiments, the in vitro kidneys and kidney tissues provided herein comprise a glomerularAttorney Docket No. 224638-702601 marker comprising PODXL, NPHS1, MAFB, WT1, OLFM3 (early podocytes), ANXA1 (late podocytes), and COL4A3.

[0265] In some embodiments, the in vitro kidneys and kidney tissues provided herein comprise a proximal tubule or a proximal tubule tissue. In some embodiments, the in vitro kidneys and kidney tissues provided herein comprises a proximal tubule marker comprising LRP2, LTL, CUBN, PTH1R, AQP1, CLDN2, TJP3, or CD13.

[0266] In some embodiments, the in vitro kidneys and kidney tissues provided herein comprise a tubular epithelium. In some embodiments, the in vitro kidneys and kidney tissues provided herein comprise a tubular epithelium marker comprising CK8, aquaporins, CD34, or WGA lectin.

[0267] In some embodiments, the in vitro kidneys and kidney tissues provided herein comprise a connecting segment. In some embodiments, the in vitro kidneys and kidney tissues provided herein comprise a connecting segment marker comprising GATA3 or AQP2.

[0268] In some embodiments, the in vitro kidneys and kidney tissues provided herein comprise a collecting duct. In some embodiments, the vitro kidneys and kidney tissues express aquaporins. The aquaporins are a family of membrane water channels expressed by collecting duct cells. In some embodiments, the in vitro kidneys and kidney tissues express aquaporin 1 (AQP1), aquaporin 2 (AQP2), aquaporin 3 (AQP3), aquaporin 4 (AQP4), or any combination thereof. In some embodiments, the in vitro kidneys and kidney tissues provided herein comprise a collecting duct marker comprising: GATA3, EPCAM, or ECAD.Functional characterization of in vitro kidneys and kidney tissues.

[0269] The in vitro kidneys and kidney tissues provided herein have functional parameters that can be characterized similar to a human kidney in vivo and are not observed in monolayer cultures of separate kidney cell populations. Functional parameters of the in vitro kidneys and kidney tissues provided herein include filtration of blood or solutions, formation of lumen, flow of fluid within the lumen, kidney metabolic functions, kidney morphological function, and ion exchange. Methods of measuring functional parameters can be performed using in vitro assays or performing urinalysis or blood analysis assays in vivo.

[0270] In some embodiments, the in vitro kidneys and kidney tissues provided herein are capable of having flow. Methods of measuring flow within a tissue include but are not limited to: diffuse correlation spectroscopy, ultrasound, Doppler techniques, venous occlusion plethysmography, permeability assays, flowthrough immunoassays, microscopy and imaging techniques. Flow rates, for example, can be measured in animal models that have received a kidney transplant using an optical Doppler velocimeter.Attorney Docket No. 224638-702601

[0271] In some embodiments, the in vitro kidneys and kidney tissues provided herein have cilia formation and movement in the core of the lumen. Cilia movement and formation can be tracked using methods such as microscopy.

[0272] In some embodiments, the in vitro kidneys and kidney tissues provided herein are capable of filtering urea. In some embodiments, the in vitro kidneys and kidney tissues provided herein are capable of filtering and / or secreting creatinine. In some embodiments, the in vitro kidneys and kidney tissues provided herein are capable of filtering and excreting uric acid. In some embodiments, the in vitro kidneys and kidney tissues provided herein are capable of transporting and excreting ammonia and / or ammonium phosphate. Ammonia is produced from glutamine (Gin) as a result of proximal tubule ammoniagenesis. Glutamine is transported across both the apical and basolateral plasma membranes and then transported into mitochondria. The enzyme glutaminase (GA) is the first step in ammoniagenesis, and glutamate dehydrogenase (GDH) results in the production of the second NH4+ molecule. Metabolism of a-ketoglutarate (aKG) leads to the production of the first of two HCO3- ions. Further metabolism in the cytoplasm results in the production of a second HCO3-. Thus, complete metabolism of each glutamine produces two NH4+ and two HCO3- ions. Methods of measuring the level or activity of urea, creatinine, uric acid, ammonium, and other metabolites include but are not limited to: enzymatic assays, immunosorbent assays, absorbance assays, in vivo animal model blood assays and urinalysis.

[0273] In some embodiments, the in vitro kidneys and kidney tissues provided herein produce renin. In some embodiments, the in vitro kidneys and kidney tissues provided herein produce erythropoietin. Renin and erythropoietin production can be measured by immunoassays (e.g., enzyme-linked immunosorbent assay, ELISA).

[0274] In some embodiments, the in vitro kidneys and kidney tissues provided herein are capable of ion exchange and ion transport. In some embodiments, the in vitro kidneys and kidney tissues provided herein express increased levels of kidney ion channels and ion transporters relative to a population of kidney cells that are not made by the methods provided herein. Non-limiting examples of kidney ion channels and kidney transporters that can be expressed by the in vitro kidneys and kidney tissues provided herein include: TRPC6 (Trpc6), TRPM6 (Trpm6), C1C-5 (CLCN5), CIC-Kb (CLCNKB), ROMK (KCNJ1), Kir4.1 (KCNJ10), ENaC (Scnnla), ENaC (Senn la), Poly cystin 2 (PKD2), and the Sodium / phosphate (Na / Pi) co-transporter. Ion channel and ion transport function can be measured by electrophysiological techniques (e.g., voltage clamp), microelectrode arrays, ion exchange chromatography, and ion transport assays.

[0275] In some embodiments, the in vitro kidneys and kidney tissues provided herein are capable of metabolic functions. In some embodiments, the in vitro kidneys and kidney tissues providedAttorney Docket No. 224638-702601 herein have albumin reabsorption function. Reabsorption of albumin occurs by cellular-mediated endocytosis in the glomerulus and the proximal tubule of the kidney. Albumin reabsorption by the in vitro kidneys and kidney tissues can be measured by immunoassays or labeling albumin with fluorescent tags or radioisotopes to track albumin movement through the kidney.(3) Transplant Compositions.

[0276] Provided herein are transplant compositions comprising the in vitro kidney tissues provided herein. In some embodiments, the kidney tissues and compositions comprising the kidney tissues provided herein further comprise a cell culture medium or diluents that maintain tissue survival and promote kidney tissue function. In some embodiments, the compositions provided herein further comprise growth factors or agents. In some embodiments, the growth factors or agents comprise CHIR99021, Activin A, GDNF, FGF1, FGF7, FGF9, BMP4, BMP7 Retinoic Acid, GDNF1, RSPO1, or any combination thereof.

[0277] In some embodiments, the compositions provided herein comprise one or more metabolites. In some embodiments, the compositions provided herein comprise urea, creatinine, uric acid, ammonium phosphate, or a combination thereof. In some embodiments, the compositions provided herein comprise an extracellular matrix. In some embodiments, the compositions provided herein comprise a cryopreservation agent, a serum, or a suspension.

[0278] In some embodiments, the compositions provided herein comprise a diluent or a saline solution. In some embodiments, the saline solution is normal saline solution (NSS), or Hank’s Balanced Salt solution (HBSS). In some embodiments, the compositions are formulated for administration of the compositions, kidneys, or kidney tissues to a subject.

[0279] Provided herein are transplant compositions comprising vascularized kidney tissues.

[0280] A transplant composition provided herein can further comprise agents that suppress an immune response in a subject. Transplantation can provoke the transplant recipient’s immune system to attack the transplant and reject the tissue. This side effect of transplantation can be lethal without pharmaceutical or genetic interventions. For example, immunosuppressants are drugs that are administered to a transplant recipient to reduce the risk of transplant rejection after a transplant by managing the immune system's response to the new transplant composition or graft. In some embodiments, the transplant composition provided herein further comprises one or more immunosuppressants. In some embodiments, the one or more immunosuppressants comprise a steroid, an anti-interleukin-2 antibody, a calcineurin inhibitor, an antibiotic, an anti-viral, an antifungal, an inosine monophosphate dehydrogenase inhibitor, a disease-modifying anti-rheumatic drug, an mTor inhibitor, or any combination thereof. Non-limiting examples ofAttorney Docket No. 224638-702601 immunosuppressants that can be used in combination with a composition or system provided herein include, for example, prednisone, prednisolone, penicillin, tetracycline, amoxicillin, azithromycin, tacrolimus, cyclosporine, fluconazole, nystatin, ketoconazole, clotrimazole, mycophenolate mofetil, apremilast, cyclophosphamide, hydroxychloroquine, leflunomide, methotrexate, mycophenolate, sulfasalazine, abatacept, belimumab, ixekizumab, rituximab, sailumab, secukinumab, tocilizumab, ustekinumab, azathioprine, rapamycin, ridaforolimus, deforolimus, everolimus, sirolimus, umirolimus, and zotarolimus. In some embodiments, the transplant composition comprises kidney tissues that have been genetically modified to prevent transplant rejection. In some embodiments, the transplant composition comprises kidney tissues that are hypoimmunogenic and evade immune rejection. Methods of generating genetically modified hypoimmunogenic cells and tissues can include CRISPR / Cas-mediated modifications or gene editing systems that reduce or remove major histocompatibility complex (MHC) class I or class II molecules from being expressed by the transplant composition. In some embodiments, the transplant composition further comprises a population of cells that induce transplant tolerance. For example, the transplant composition provided herein can further comprise alloantigen-specific T- regulatory cells.(4) Systems for culturing kidney and kidney tissues.

[0281] Provided herein are systems comprising a kidney or kidney tissue provided herein that permit filtering of a subject’s blood or permit the maintenance and survival of the kidney tissues provided herein. Such systems can be helpful for culturing biological materials such as cells, cellular aggregates, tissues and organoids provided herein, facilitating the growth and / or differentiation of such biological materials for downstream evaluation or treatment of subjects.

[0282] In some embodiments, the systems provided herein comprise producing a blood circuit, wherein the blood circuit comprises blood from the subject in fluid communication with a kidney or kidney tissue provided herein. In some embodiments, the blood circuit comprises a bioreactor. A bioreactor can be utilized as part of a system or an ex-vivo blood circuit provided herein to supply a kidney or kidney tissue with physical stimulation, electrical stimulation, chemical stimulation, gas exchange, or a combination of these. In some embodiments, a bioreactor can comprise means for increasing the level of oxygen in a culture media. In some embodiments, disclosed herein can be a system comprising any of the compositions provided herein.

[0283] In some embodiments, a system can comprise at least one of a bioreactor, pump, housing, tubing, oxygen permeable tubing, incubator, motor, computer or controller, storage medium, biological safety cabinet, incubator, or any combination thereof. In some embodiments, cells areAttorney Docket No. 224638-702601 stored in an incubator. In some embodiments, an incubator can regulate temperature, gaseous concentration, humidity, and any combination thereof. Fluid pressure, flow characteristics and geometry of the bioreactor can be varied to apply a desired fluid shear stress to the in vitro kidney and kidney tissues provided herein.

[0284] In some embodiments, a pump can comprise a peristaltic pump, centrifugal, syringe, or a vacuum pump. In some cases, a system can further comprise a cannula, a perfusion apparatus, a holding container, a tubing, a pump, a sensor, a thermometer, an electrode, a valve, a balloon, a pacemaker, a thermostat, a user interface, or any combination thereof. In some embodiments, a sensor can comprise a glucose sensor, an ammonia sensor, an oxygen sensor, a fluid sensor, a temperature sensor, a pressure sensor, or any combination thereof.

[0285] In some embodiments, a biological safety cabinet can provide laminar airflow to prevent contamination of the biological materials. In some embodiments, sterile techniques are performed to prevent contamination. In some embodiments, sterile techniques can include sterilizing surfaces and equipment with about 70% isopropyl alcohol, use of UV radiation, use of personal protective equipment such as gloves, lab coats, or body suits, or any combination thereof.

[0286] In some embodiments, a bioreactor can be utilized as part of a system provided herein. A bioreactor may need to supply the biological materials or portions thereof with physical stimulation, electrical stimulation, chemical stimulation, or a combination thereof, depending on what is needed for growth, maintenance, or differentiation of the biological materials. In some embodiments, the kidney tissues provided herein can be cultured with growth factors described herein. For example, convert growth factor b 1 and trans-retinoic acid may allow for renal proximal tubule cells grow as a monolayer and produce lumens with polarized epithelial layers, microvilli, and tight junction complexes.

[0287] In some embodiments, the bioreactor is adapted for use with the bioprinting systems described herein. In some embodiments, the bioreactor can be upstream and / or downstream from the bioprinting systems.

[0288] In some embodiments, a bioreactor performs real time monitoring of certain parameters, such as pH, pCh, pCCh, temperature, electrolyte levels, glucose or lactate concentrations, and perfusion parameters, such as perfusion pressure and flow rates. The bioreactor may maintain conditions stable and adjustable, particularly during long-term culture. This monitoring further allows for calculation of other important parameters, such as vascular resistance. In some embodiments, various assays can be used to investigate cellular viability and proliferation during kidney tissue vascularization. Monitoring can be performed at any time, for example a measurement can be taken before perfusion, during perfusion, and after perfusion. In some cases,Attorney Docket No. 224638-702601 a measurement can be taken from about 1 hour, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 17 days, 20 days, 22 days, 24 days, 28 days, 30 days, 2 months, 5 months, 7 months, 8 months, 10 months, or up to 1 year after perfusion of a population of cells into the kidney tissues or portions thereof. In some embodiments, the temperature is maintained between about 4° C to 45° C.

[0289] In some embodiments, the bioreactor can be configured to allow for fluid perfusion at varying or constant flow rates. In some embodiments, fluid perfusion is applied in closed-loop or open-loop flow systems. In some embodiments, the flow rate comprises a range of about 0.01 mm per min to 50 mm per min. In some embodiments, the flow rate comprises a range of about 0.01 mL / min to 0.1 mL / min. In some embodiments, the flow rate comprises range of about 0.1 mL / min to 1.0 mL / min. In some embodiments, the flow rate comprises a range of about 1.0 mL / min to 2.0 mL / min. In some embodiments, the flow rate comprises a range of about 2.0 mL / min to 3.0 mL / min. In some embodiments, the flow rate comprises a range of about 3.0 mL / min to 4.0 mL / min. In some embodiments, the flow rate comprises a range of about 4.0 mL / min to 5.0 mL / min.some embodiments, the flow rate comprises a range of about 5.0 mL / min to 10.0 mL / min. In some embodiments, the flow rate comprises a range of about 10 mL / min to 15 mL / min. In some embodiments, the flow rate comprises a range of about 15 mL / min to 20 mL / min. In some embodiments, the flow rate comprises a range of about 20 mL / min to 25 mL / min. In some embodiments, the flow rate comprises a range of about 30 mL / min to 35 mL / min.In some embodiments, the flow rate comprises a range of about 35 mL / min to 40 mL / min. In some embodiments, the flow rate comprises a range of about 40 mL / min to 45 mL / min. In some embodiments, the flow rate comprises a range of about 45 mL / min to 50 mL / min.

[0290] In some embodiments, a bioreactor can comprise means for increasing the level of oxygen in a culture media. In some embodiments, oxygen levels range from about 2% to about22% oxygen to mimic hypoxic conditions. In some embodiments, the oxygen levels comprise from about 22% to about 25%, from about 25% to about 30%, from about 30% to about 35%, from about 35% to about 40%, from about 40% to about 45%, from about 45% to about 50%, from about 50% to about 55%, from about 55% to about 60%, from about 60% to about 65%, from about 65% to about 70%, from about 70% to about 75%, from about 75% to about 80%, from about 80% to about 85%, from about 85% to about 90%, from about 90% to about 95%, from about 95% to about 100% oxygen. In some embodiments a level of oxygen can vary over culture time.

[0291] In some embodiments, elevated oxygen levels can be produced by direct oxygenation, in-line oxygenation, gas permeable materials, or any combination thereof. In some embodiments,Attorney Docket No. 224638-702601 direct oxygenation can comprise using a membrane oxygenating chamber. In some embodiments, direct oxygenating can comprise using a bubbler. In some embodiments, in-line oxygenation can comprise use of in-line oxygenators. In some embodiments, a media can be pumped by a peristaltic pump. In some embodiments a media can be passed through gas permeable material allowing gaseous exchange through the material. In some embodiments, gaseous exchange may comprise oxygen exchange, nitrogen exchange, carbon dioxide exchange, or any combination thereof. In some embodiments, an at least partly permeable tubing may comprise silicone tubing. In some embodiments, silicone tubing may allow oxygen exchange, creating heightened oxygen levels in a media. In some embodiments oxygen levels in the media can be heightened by a direct injection of a mixture of oxygen and one or more other gases. In some embodiments one or more other gases can comprise nitrogen, carbon dioxide, or a combination of the two. In some embodiments oxygen levels in the media can be heightened by an injection of a gas comprising about 40% oxygen, about 45% oxygen, about 50% oxygen, about 55% oxygen, about 60% oxygen, about 65% oxygen, about 70% oxygen, about 75% oxygen, about 80% oxygen, about 85% oxygen, about 90% oxygen, about 95% oxygen, or about 100% oxygen. In some embodiments oxygen levels in the media can be heightened by an injection of about 100% pure oxygen. In some embodiments heightened oxygen levels in the media can be facilitated by oxygen carrying molecules to increase access to cells within the biological materials. In some embodiments, the biological materials comprise isolated cells, cellular aggregates, tissues, organoids or organs, or portion thereof. In some embodiments, cells may comprise seeded glomerular cells, glomerular progenitor cells, pluripotent stem cells, posterior intermediate mesoderm cells, or other cells provided herein. In some embodiments, an isolated organ, organoid, or portion thereof may comprise an extracellular matrix (ECM). In some embodiments, a media can be hyperoxygenated prior to seeding cells into an isolated organ, organoid, or portion thereof. In some embodiments, a media can be hyperoxygenated prior to seeding cells into an ECM graft. In some embodiments, oxygen levels can be adjusted based on metrics. In some embodiments, metrics can be evaluated or adjusted and can comprise media glucose levels, lactate levels, pCO2, pH, ammonia levels, pyruvate levels, other measurable parameters, and any combination thereof.

[0292] In some embodiments, a bioreactor comprises a vertical wheel bioreactor, a perfusion bioreactor, an air-lift bioreactor, a continuous stirred-tank bioreactor, a fluidized bed bioreactor, a packed bed bioreactor, or the like. In some embodiments, the cells, cellular aggregates, tissues and organoids provided herein are maintained in the bioreactor at different stages during development. In some embodiments, the biological materials described herein are cultured in the bioreactor prior to differentiation. In some embodiments, the biological materials described herein are cultured inAttorney Docket No. 224638-702601 the bioreactor during differentiation. In some embodiments, the biological materials described herein are cultured in the bioreactor after differentiation. In some embodiments, the biological materials are cultured in the bioreactor such that specific cellular markers are expressed, such as, for example, glomerular markers described herein. In some embodiments, the biological materials are cultured in the bioreactor such that specific structures are formed, such as, for example, penetration of kidney tissues from at least one location or repeating units at kidney tissue surfaces.

[0293] In some embodiments, the bioreactors can be used in a batch mode, fed batch mode, circulation and perfusion mode and can be fully controlled in a closed, aseptic environment and can be implemented for a single use (to be disposed after one culturing cycle) as well as for multiple cycle uses.

[0294] In some embodiments, the computer or controller of the systems comprise at least one processor and at least one non-transitory computer-readable medium. Data and / or instructions can be stored on the at least one non-transitory computer-readable medium. In some embodiments, the instructions comprise receiving one or more instructions for operating the systems herein. In some embodiments, the one or more instructions comprise a file or a set of files that can be loaded into the controller(s). The file or set of files may correspond to one or more protocols to be managed by the systems herein. The file or set of files may correspond to a plurality of conditions for each of the biological materials culture within the systems. In some embodiments, the instructions can be determined by one or more computer model, such as, for example, a machine learning model.(5) Kits and Reagents.

[0295] The compositions provided herein can be made using a reagent or series of reagents that promote the formation of vascularized kidney tissues. A reagent provided herein can comprise: a basal medium supplemented with one or more growth factors, metabolites, antioxidants, antigens, small molecules, and / or proteins that permit differentiation of a stem cell to a glomerular progenitor cell or a glomerular cell.

[0296] A reagent provided herein can comprise: a basal medium supplemented with one or more growth factors, metabolites, antioxidants, antigens, small molecules, and / or proteins that permit differentiation of a stem cell to glomerular cell. In some embodiments, the reagent further comprises serum.

[0297] Provided herein are reagents comprising one or more agent selected from the group consisting of: fetal bovine serum (FBS), Human leukocyte antigen B27 (B27), L-Glutamine, insulin-transferrin-selenium (ITS), one or more non-essential amino acids (NEAA), 2- mercaptoethanol, Y27632, Activin A, BMP4, CHIR99021, retinoic acid, FGF9, LDN193189,Atorney Docket No. 224638-702601SB431542, GDNF, FGF1, FGF7, RSP01, and a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma (MATRIGEL®). In some embodiments, the reagents provided herein comprise at least about 0.1%, at least about 0.25%, at least about 0.5%, at least about 0.75%, at least about 1%, at least about 1.25%, at least about 1.5%, at least about 1.75%, at least about 2%, at least about 3%, at least about 4%, up to 5% B27 percent volume of the total volume of the reagent. In some embodiments, the reagents provided herein comprise at least about 0.1%, at least about 0.25%, at least about 0.5%, at least about 0.75%, at least about 1%, at least about 1.25%, at least about 1.5%, at least about 1.75%, at least about 2%, at least about 3%, at least about 4%, up to 5% L-glutamine percent volume of the total volume of the reagent. In some embodiments, the reagents provided herein comprise at least about 0.1%, at least about 0.25%, at least about 0.5%, at least about 0.75%, at least about 1%, at least about 1.25%, at least about 1.5%, at least about 1.75%, at least about 2%, at least about 3%, at least about 4%, up to 5% ITS percent volume of the total volume of the reagent. In some embodiments, the reagents provided herein comprise at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, 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 65%, at least about 70%, up to 75% solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma (MATRIGEL®) percent volume of the total volume of the reagent (v / v). In some embodiments, the reagents provided herein comprise at least about 0.1%, at least about 0.25%, at least about 0.5%, at least about 0.75%, at least about 1%, at least about 1.25%, at least about 1.5%, at least about 1.75%, at least about 2%, at least about 3%, at least about 4%, up to 5% ITS percent volume of the total volume of the reagent. In some embodiments, the reagents provided herein comprise at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, 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%, up to 50% fetal bovine serum percent volume of the total volume of the reagent. In some embodiments, a reagent provided herein comprises at least about a 50 micromolar (pM), at least about a 60 micromolar (pM), at least about a 10 micromolar (pM), at least about a 20 pM, at least about a 30 pM, at least about a 40 pM, at least about a 50 pM, at least about a 60 pM, at least about a 70 pM, at least about a 80 pM, at least about a 90 pM, at least about a 100 pM up to a 500 pM concentration of 2-mercaptoethanol. In some embodiments, a reagent provided herein comprises at least about a 10 micromolar (pM), at least about a 20 pM, at least about a 30 pM, at least about a 40 pM, at least about a 50 pM, at least about a 60 pM, at least about a 70 pM, at least about a 80 pM, at least about a 90 pM, at leastAtorney Docket No. 224638-702601 about a 100 pM up to a 200 pM concentration of SB431542. In some embodiments, a reagent provided herein comprises at least about a 0.1 micromolar (pM), at least about a 1 pM, at least about a 2 pM, at least about a 3 pM, at least about a 4 pM, at least about a 5 pM, at least about a 6 pM, at least about a 7 pM, at least about a 8 pM, at least about a 9 pM, at least about a 10 pM, at least about a 50 pM, up to a 100 pM concentration of CHIR99021. In some embodiments, the reagent provided herein comprises about 1 pM up to a 10 pM concentration of CHIR99021. In some embodiments, a reagent provided herein comprises at least about a 0.1 micromolar (pM), at least about a 1 pM, at least about a 2 pM, at least about a 3 pM, at least about a 4 pM, at least about a 5 pM, at least about a 6 pM, at least about a 7 pM, at least about a 8 pM, at least about a 9 pM, at least about a 10 pM, at least about a 50 pM, up to a 100 pM concentration of Y27632. In some embodiments, a reagent provided herein comprises at least about a 0.1 nanogram / milliliter (ng / mL), at least about a 1 ng / mL, at least about a 2 ng / mL, at least about a 3 ng / mL, at least about a 4 ng / mL, at least about a 5 ng / mL, at least about a 6 ng / mL, at least about a 7 ng / mL, at least about a 8 ng / mL, at least about a 9 ng / mL, at least about a 10 ng / mL, at least about a 50 ng / mL, up to a 100 ng / mL concentration of Activin A. In some embodiments, a reagent provided herein comprises at least about a 0.01 nanogram / milliliter (ng / mL), at least about a 0.1 ng / mL, at least about a 0.5 ng / mL, at least about a 1 ng / mL, at least about a 1.5 ng / mL, at least about a 2 ng / mL, at least about a 2.5 ng / mL, at least about a 3 ng / mL, at least about a 3.5 ng / mL, at least about a 4 ng / mL, at least about a 4.5 ng / mL, at least about a 5 ng / mL, up to a 10 ng / mL concentration of bone morphogenetic protein 4 (BMP4). In some embodiments, a reagent provided herein comprises at least about a 0.01 nanogram / milliliter (ng / mL), at least about a 0.1 ng / mL, at least about a 0.5 ng / mL, at least about a 1 ng / mL, at least about a 1.5 ng / mL, at least about a 2 ng / mL, at least about a 2.5 ng / mL, at least about a 3 ng / mL, at least about a 3.5 ng / mL, at least about a 4 ng / mL, at least about a 4.5 ng / mL, at least about a 5 ng / mL, up to a 10 ng / mL concentration of glial cell line-derived neurotrophic factor (GDNF). In some embodiments, a reagent provided herein comprises at least about a 1 nanogram / milliliter (ng / mL), at least about a 10 ng / mL, at least about a 20 ng / mL, at least about a 30 ng / mL, at least about a 40 ng / mL, at least about a 50 ng / mL, at least about a 60 ng / mL, at least about a 70 ng / mL, at least about a 80 ng / mL, at least about a 90 ng / mL, at least about a 100 ng / mL, at least about a 500 ng / mL, up to a 1000 ng / mL concentration of fibroblast growth factor 9 (FGF9). In some embodiments, a reagent provided herein comprises at least about a 1 nanogram / milliliter (ng / mL), at least about a 10 ng / mL, at least about a 20 ng / mL, at least about a 30 ng / mL, at least about a 40 ng / mL, at least about a 50 ng / mL, at least about a 60 ng / mL, at least about a 70 ng / mL, at least about a 80 ng / mL, at least about a 90 ng / mL, at least about a 100 ng / mL, at least about a 500 ng / mL, up to a 1000 ng / mL concentration of FGF1. InAttorney Docket No. 224638-702601 some embodiments, a reagent provided herein comprises at least about a 1 nanogram / milliliter (ng / mL), at least about a 10 ng / mL, at least about a 20 ng / mL, at least about a 30 ng / mL, at least about a 40 ng / mL, at least about a 50 ng / mL, at least about a 60 ng / mL, at least about a 70 ng / mL, at least about a 80 ng / mL, at least about a 90 ng / mL, at least about a 100 ng / mL, at least about a 500 ng / mL, up to a 1000 ng / mL concentration of FGF7. In some embodiments, a reagent provided herein comprises at least about a 0.1 nanogram / milliliter (ng / mL), at least about a 1 ng / mL, at least about a 10 ng / mL, at least about a 20 ng / mL, at least about a 30 ng / mL, at least about a 40 ng / mL, at least about a 50 ng / mL, at least about a 60 ng / mL, at least about a 70 ng / mL, at least about a 80 ng / mL, at least about a 90 ng / mL, at least about a 100 ng / mL, at least about a 500 ng / mL, up to a 1000 ng / mL concentration of R-spondin 1 (RSPO1). In some embodiments, a reagent provided herein comprises at least about 1 nanoMolar (nM), at least about 5 nM, at least about 10 nM, at least about 15 nM, at least about 20 nM, at least about 25 nM, at least about 30 nM, at least about 35 nM, at least about 40 nM, at least about 45 nM, at least about 50 nM, at least about 75 nM, at least about 100 nM, up to a 500 nM concentration of LDN 193189. In some embodiments, a reagent provided herein comprises at least about 1 nanoMolar (nM), at least about 5 nM, at least about 10 nM, at least about 15 nM, at least about 20 nM, at least about 25 nM, at least about 30 nM, at least about 35 nM, at least about 40 nM, at least about 45 nM, at least about 50 nM, at least about 75 nM, at least about 100 nM, at least about 500 nM, up to a 1000 nM concentration of retinoic acid.

[0298] Provided herein are reagents comprising: fetal bovine serum (FBS), R-spondin 1 (RSPO1) protein, (GNDF), fibroblast growth factor 1 (FGF1), fibroblast growth factor 7 (FGF7), LDN193189, and solubilized basement membrane preparation extracted from Engelbreth-Holm- Swarm (EHS) mouse sarcoma. In some embodiments, the FBS is present in an amount that is at least about 1% up to 20% volume by total volume of the reagent (v / v). In some embodiments, the retinoic acid is present in an amount that is at least about 10 nanoMolar (nM) concentration up to 200 nM concentration. In some embodiments, the RSPO1 is present in an amount that is at least about 10 nanograms per milliliter (ng / mL) up to 200 ng / mL. In some embodiments, the GDNF is present in an amount that is at least about 0.1 nanograms per milliliter (ng / mL) up to 5 ng / mL. In some embodiments, the FGF1 is present in an amount that is at least about 10 nanograms per milliliter (ng / mL) up to 200 ng / mL. In some embodiments, the FGF7 is present in an amount that is at least about 3 nanograms per milliliter (ng / mL) up to 60 ng / mL. In some embodiments, the LDN193189 is present in an amount that is at least about 1 nanoMolar (nM) concentration up to 20 nM concentration. In some embodiments, the solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma is present in an amount of at least 5% up to 80% volume by total volume of the reagent (v / v). In some embodiments, the reagentsAttorney Docket No. 224638-702601 comprise: 10% FBS, 100 nM retinoic acid, 100 ng / mL RSPOl, 2 ng / mL GDNF, 100 ng / mL FGFl, 30 ng / mL FGF7, 10 nM LDN193189, and 50% solubilized basement membrane preparation extracted from Engelbreth-Holm- Swarm (EHS) mouse sarcoma (MATRIGEL®) (volume / total volume or v / v). In some embodiments, the reagents promote glomerulus formation and vascularization (e.g., a cell culture medium provided herein).

[0299] Provided herein are kits for making the in vitro kidneys, kidney tissues, and compositions provided herein. In some embodiments, a formulation of a composition described herein is prepared in a single container for administration. In some embodiments, a formulation of a composition described herein is prepared in multiple containers for administration. A container provided herein can include a vessel, a vial, an ampule, a tube, a cup, a box, a bottle, a flask, ajar, a dish, a well of a single-well or a multi-well apparatus, a reservoir, a tank, or the like, or other device in which the compositions and reagents provided herein can be placed, stored and / or transported, and accessed to remove the contents. Examples of such containers include glass and / or plastic sealed or re-sealable tubes and ampules, including those having a rubber septum or other sealing means that is compatible with withdrawal of the contents using a needle and syringe. In some implementations, the containers are RNase free.

[0300] Provided herein are kits comprising: a first container comprising: a population of glomerular cells and a population of MVFs; and a second container comprising media, growth factors, and agents for making an in vitro kidney tissue. Provided herein is a kit comprising: a first container comprising: (a) a population of glomerular kidney cells, (b) a population of MVFs, or (c) an in vitro kidney tissue provided herein; and a second container comprising: a reagent provided herein. In some embodiments, the kits provided herein comprise a reagent provided herein. In some embodiments, the kits provided herein comprise glomerular cells. In some embodiments, the kits provided herein comprise MVFs. In some embodiments, the kits provided herein comprise human induced pluripotent stem cells (iPSCs). In some embodiments, the kits provided herein comprise endothelial cells. In some embodiments, the kits provided herein comprise an extracellular matrix.

[0301] Provided herein are kits and reagents for screening for nephrotoxicity or screening for a therapeutic drug candidate. Provided herein is a kit comprising: a first container comprising: (a) a population of glomerular kidney cells, (b) a population of MVFs, or (c) an in vitro kidney tissue provided herein; and a second container comprising: reagents, a positive control for nephrotoxicity, and a test agent. The kits for screening for nephrotoxicity can be used to determine if a test agent is a viable candidate for clinical development or clinical trials in animals or humans. The test agent can be any therapeutic drug of interest for screening, for example, a lead candidateAttorney Docket No. 224638-702601 therapeutic agent. The test agent can be a small molecule, protein, cells, antibody, or chemical. In some embodiments, the kits provided herein comprise a reagent provided herein. In some embodiments, the kits provided herein comprise glomerular cells. In some embodiments, the kits provided herein comprise MVFs. In some embodiments, the kits provided herein comprise human induced pluripotent stem cells (iPSCs). In some embodiments, the kits provided herein comprise endothelial cells. In some embodiments, the kits provided herein comprise podocytes. In some embodiments, the kits provided herein comprise proximal tubule cells. In some embodiments, the kits provided herein comprise renal tubule cells. In some embodiments, the kits provided herein comprise endothelial cells. In some embodiments, the kits provided herein comprise an in vitro- vascularized kidney tissue. In some embodiments, the kits provided herein comprise an in vitro- kidney tissue provided herein. In some embodiments, the kits provided herein comprise an extracellular matrix. In some embodiments, the kits provided herein comprise a vessel, a vial, a multi-well plate, or a cell culture dish. In some embodiments, the kits provided herein comprise a therapeutic agent. In some embodiments, the therapeutic agent comprises a sodium-glucose cotransporter-2 (SGLT2) inhibitor, an angiotensin-converting enzyme (ACE) inhibitor, an angiotensin receptor blocker (ARB), a beta blocker, a diuretic, a potassium binder, a statin, an erythropoiesis-stimulating agent, an iron supplement, a phosphate binder, a calcium supplement, a vitamin D supplement, a calcimimetic, an aldosterone antagonist, a corticosteroid, metformin, a GLP-1 inhibitor, aspirin, a non-steroidal anti-inflammatory drug (NS AID), or any combination thereof. In some embodiments, the kits provided herein comprise an agent that causes kidney damage that can be used as a positive control in a screening assay. Non-limiting examples of agents that cause kidney damage include aminoglycosides, antifungals (amphotericin B), beta-lactams (e.g., cephalosporins, penicillins), quinolones (e.g., ciprofloxacin), rifampin (Rifadin), and vancomycin (Vancocin).

[0302] Provided herein is a method of screening a test agent for nephrotoxicity, the method comprising: contacting a first in vitro kidney tissue with a test agent; immunostaining the first in vitro kidney tissue and a second in vitro kidney tissue for biomarkers indicative of nephrotoxicity, wherein nephrotoxicity is characterized as the first in vitro kidney tissue having an increased level of the biomarker relative to the second in vitro kidney tissue that was not contacted with the test agent. Non-limiting examples of biomarkers indicative of nephrotoxicity can include: kidney injury molecule- 1 (KIM-1), cystatin C, neutrophil gelatinase-associated lipocalin (NGAL), clusterin, an interleukin 18.

[0303] Provided herein are kits and reagents for treating a subject with a kidney disease. Provided herein is a kit comprising: a first container comprising: (a) a population of glomerularAttorney Docket No. 224638-702601 kidney cells, (b) a population of MVFs, or (c) an in vitro kidney tissue provided herein; and a second container comprising: (a) a therapeutic agent for treating a kidney disease; (b) one or more immunosuppressants; or (c) a therapeutic agent for treating a kidney disease and an immunosuppressant. The one or more immunosuppressants can be administered before kidney tissue transplantation, simultaneously with kidney tissue transplantation, and / or after kidney tissue transplantation. The therapeutic agent for treating a kidney disease can be administered to a subject before, simultaneous, or after treatment with a kidney tissue provided herein or treating a subject with a kidney -tissue filtered blood circuit. Methods of treating a subject are described further below.(6) Methods of Treatment, Dosing, and Administration.

[0304] Provided herein are methods of treating a subject with a disease or a condition. In some embodiments, the methods comprise using a kidney tissue-filtered blood circuit. The kidney tissues provided here can be included as part of a blood circuit that can be used ex-vivo while a subject is waiting for an organ transplantation, to assist with organ transplantation, or to prolong survival of a subject with a lethal kidney disease. In some embodiments, the methods comprise producing a blood circuit, wherein the blood circuit comprises blood from the subject in fluid communication with a kidney or a kidney tissue(s) provided herein, wherein the kidney or the kidney tissue(s) filters blood from the subject, thereby treating a kidney disease in the subject. The kidney tissues can be maintained in a bioreactor receiving the patient’s blood for filtration. Following filtration of the patient’s blood by the kidney tissues, the filtered blood is returned back to the patient with excess waste chemicals and water removed from the blood.

[0305] Provided herein are methods of treating a disease in a subject, wherein the methods comprise: transplanting an in vitro kidney, an in vitro kidney tissue, a composition, or a system provided herein into a subject. In some embodiments, the transplanting comprises surgical removal of a diseased kidney and replacing the diseased kidney with the in vitro kidney, in vitro kidney tissue, composition, or system. In some embodiments, the transplanting comprises engrafting the in vitro kidney, in vitro kidney tissue, composition, or system into an existing kidney or kidney tissue in the subject.

[0306] The transplant composition can comprise a given number or dose of cells sufficient to perform blood filtration. In some embodiments, a dose of cells is administered to subjects in accord with the provided methods. In some embodiments, the size or timing of the doses is determined as a function of the particular disease or condition in the subject. It is within the level of a skilled artisan to empirically determine the size or timing of the doses for a particular disease in view ofAttorney Docket No. 224638-702601 the provided description. In certain embodiments, the cells, or individual populations of sub-types of cells, are administered to the subject at a range of about 0.1 million to about 100 billion cells and / or that amount of cells per kilogram of body weight of the subject, such as, e.g. , 0.1 million to about 50 billion cells (e.g. , about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), 1 million to about 50 billion cells (e.g. , about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g. , about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g. , about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges and / or per kilogram of body weight of the subject. Dosages may vary depending on attributes particular to the disease or disorder and / or patient and / or other treatments.

[0307] In some embodiments, the methods provided herein comprise administering to a subject a kidney tissue provided herein that comprises at least or at least about 0.1 x 106cells / kg body weight of the subject, 0.2 x 106cells / kg, 0.3 x 106cells / kg, 0.4 x 106cells / kg, 0.5 x 106cells / kg, 1 x 106cell / kg, 2.0 x 106cells / kg, 3 x 106cells / kg or 5 x 106cells / kg. In some embodiments, the methods provided herein comprise administering to a subject a kidney tissue provided herein that comprises a number of cells that is between or between about 0.1 x 106cells / kg body weight of the subject and 1.0 x 107cells / kg, between or between about 0.5 x 106cells / kg and 5 x 106cells / kg, between or between about 0.5 x 106cells / kg and 3 x 106cells / kg, between or between about 0.5 x 106cells / kg and 2 x 106cells / kg, between or between about 0.5 x 106cells / kg and 1 x 106cell / kg, between or between about 1.0 x 106cells / kg body weight of the subject and 5 x 106cells / kg, between or between about 1.0 x 106cells / kg and 3 x 106cells / kg, between or between about 1.0 x 106cells / kg and 2 x 106cells / kg, between or between about 2.0 x 106cells / kg body weight of the subject and 5 x 106cells / kg, between or between about 2.0 x 106cells / kg and 3 x 106cells / kg, or between or between about 3.0 x 106cells / kg body weight of the subject and 5 x 106cells / kg, each inclusive.Attorney Docket No. 224638-702601

[0308] In some embodiments, the subj ect that is to be treated with a kidney tissue, a composition, a blood circuit, or a system provided herein has, is diagnosed with, or is suspected of having a disease or condition. Relevant diseases that may require organ transplantation include but are not limited to: organ failure, kidney failure, diabetes, polycystic kidney disease, cardiovascular disease, edema, birth defects, genetic diseases, autoimmune disease, and any combinations thereof. In some embodiments, the subject to be treated with a kidney tissue, a composition, a blood circuit, or a system provided herein has, is diagnosed with, or is suspected of having a kidney disease or a kidney condition. Non-limiting examples of kidney diseases and conditions include: atypical hemolytic uremic syndrome (aHUS), Alport syndrome, amyloidosis, POLl-mediated kidney disease, cancer, cardiovascular kidney metabolic (CKM) syndrome, complement 3 glomerulopathy (C3G), cystinosis, diabetic kidney disease, end-stage renal failure, Fabry disease, focal segmental glomerulosclerosis (FSGS). Glomerulonephritis (Glomerular Disease) Goodpasture syndrome, granulomatosis with polyangiitis (GPA), hemolytic uremic syndrome (HUS), Henoch-Schbnlein purpura (HSP), IgA nephropathy, interstitial nephritis, kidney failure, Lupus nephritis, minimal change disease, polycystic kidney disease, chronic kidney disease (CKD), primary hyperoxaluria and oxalate, thrombotic thrombocytopenic purpura (TTP), and vasculitis of the kidney. In some embodiments, the subject to be treated with a kidney tissue, a composition, a blood circuit, or a system provided herein has, is diagnosed with, or is suspected of having kidney failure. In some embodiments, the kidney failure is caused by one or more conditions selected from the group consisting of: diabetes, high blood pressure, glomerulonephritis, polycystic kidney disease, lupus nephritis, IgA nephropathy, alcoholism, and nephrotoxicity. In some embodiments, the subject to be treated with a kidney tissue, a composition, a blood circuit, or a system provided herein has, or is diagnosed with a kidney injury. In some embodiments, the kidney injury is caused by an infection (e.g., acute pyelonephritis or septicemia), a pregnancy complication (e.g., placental abruption or placenta previa), a urinary tract obstruction, kidney stones, or a physical injury (e.g., an automobile accident). In some embodiments, the subject to be treated with a kidney tissue, a composition, a blood circuit, or a system provided herein has, or is diagnosed with a congenital abnormality. In some embodiments, the subject has or is diagnosed with renal agenesis, renal dysplasia, renal hypoplasia. Renal agenesis is the absence of one or both kidneys at birth. In some embodiments, the subject has or is diagnosed with polycystic kidney disease (PKD). PKD is a genetic condition in which multiple cysts (abnormal sacs containing fluid) grow in the kidneys. If not properly treated and managed, PKD can lead to kidney failure. There are two types of PKD which include Autosomal dominant polycystic kidney disease (ADPKD) and Autosomal recessive polycystic kidney disease (ARPKD). AutosomalAttorney Docket No. 224638-702601 dominant PKD is represents about 90 percent of all PKD cases. Symptoms of ADPKD typically present between the ages of 30 and 40. However, some patients do develop symptoms as children. Autosomal recessive polycystic kidney disease (ARPKD) is a rare form of PKD and symptoms of this condition begin very early in life, even while still in the womb.

[0309] The methods of treating a subject with a disease or condition provided herein can ameliorate at least one symptom of a disease. In some embodiments, the methods can ameliorate at least one symptom of a disease by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, or at least 20% relative to the symptom prior to treatment. Non-limiting examples of symptoms associated with a disease, such as a kidney disease include: formation of renal cysts, renal insufficiency, urinary tract infections, hematuria (blood in the urine), high blood pressure, kidney stones, aneurysms (bulges in the walls of blood vessels), burning or difficulty during urination, an increase in the frequency of urination, volume retention (e.g. puffiness around the eyes, swelling of the hands and feet), pain in the small of the back just below the ribs, fatigue, pale skin, joint pain, fingernail and toenail abnormalities, bruising, foamy urine, loss of appetite, nausea, vomiting, poor growth, fevers, swollen stomach, a reduction in the glomerular filtration rate (GFR), albuminuria, and proteinuria.

[0310] In some embodiments, the methods provided herein increase glomerular filtration rate (GFR) in a subject relative to the GFR of the subject prior to treatment with a composition or system provided herein as determined by a glomerular filtration rate blood test. GFR is a blood test that measures how well kidneys remove waste, toxins, and extra fluid from the blood. GFR is usually measured in milliliters per minute per 1.73 square meters of body surface area (mL / min / 1.73 m2). Serum creatinine level, age, and sex are used to calculate GFR. Generally, in humans, a GFR of 60 or higher indicates that the subject has functioning kidneys and is generally healthy. A GFR less than 60 can indicate that the human subject has a kidney disease. A GFR that is less than 15 indicates that a patient has kidney failure. A GFR level of less than 20 over 6 to 12 months indicates that a subject is in need of a kidney transplantation or dialysis to survive. In some embodiments, the methods provided herein increase glomerular filtration rate (GFR) in a subject by at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, or at least 60 mL / min / 1.73 m2.

[0311] In some embodiments, the methods provided herein decrease the level of urea in the blood relative to the level of urea in the blood of a subject prior to treatment with a composition or system provided herein as determined by a creatinine test. In some embodiments, the methods provided herein decrease the level of creatinine in the blood relative to the level of creatinine inAttorney Docket No. 224638-702601 the subject prior to treatment with a composition or system provided herein as determined by a blood urea nitrogen test.

[0312] In some embodiments, the methods provided herein decrease blood pressure in a subject relative to the blood pressure in the subject prior to treatment with a composition or system provided herein.

[0313] In some embodiments, the methods provided herein reduce the level of a biomarker indicative of nephrotoxicity in a kidney of a subject relative to the level of the biomarker indicative of nephrotoxicity prior to treatment. The biomarker can be measured by taking a kidney biopsy or a blood sample from a subject prior to treatment and after treatment with a transplant composition or an ex-vivo blood circuit; and performing an assay that measures the level of the biomarker. For example, the assay can include immunohistochemistry and microscopy techniques, RT-PCR, or sequencing.

[0314] The methods provided herein can further comprise administering to the subject a therapeutically effective amount of an additional therapeutic agent. The additional therapeutic agent can include, for example, an immunosuppressant, an anti-inflammatory, a therapeutic for treating a kidney disease, an immunotherapy, or an adoptive cell therapy. In some embodiments, the subject is administered a therapeutic for treating a kidney disease before, after, or during transplantation of a transplant composition provided herein. In some embodiments, the subject is administered a therapeutic for treating a kidney disease before, after, or during treatment with an ex-vivo blood circuit provided herein. In some embodiments, the subject is administered an immunosuppressant before, after, or during transplantation of a transplant composition provided herein.

[0315] In some embodiments, the therapeutic for treating a kidney disease comprises a sodiumglucose cotransporter-2 (SGLT2) inhibitor, an angiotensin-converting enzyme (ACE) inhibitor, an angiotensin receptor blocker (ARB), a beta blocker, a diuretic, a potassium binder, a statin, an erythropoiesis-stimulating agent, an iron supplement, a phosphate binder, a calcium supplement, a vitamin D supplement, a calcimimetic, an aldosterone antagonist, a corticosteroid, metformin, a GLP-1 inhibitor, aspirin, a non-steroidal anti-inflammatory drug (NS AID), or any combination thereof. In some embodiments, the SGLT2 inhibitor comprises canagliflozin (Invokana), dapagliflozin (Forxiga), empagliflozin (Jardiance), ertugliflozin (Steglatro), analogues, derivatives, salts, or a combination thereof. In some embodiments, the ACE inhibitor comprises: benazepril (Lotensin), captopril (Capoten), enalapril (Vasotec), fosinopril (Monopril), lisinopril (Zestril and Prinivil), moexipril (Univasc), perindopril (Aceon), quinapril (Accupril), ramipril: (Altace), trandolapril (Mavik), analogues, derivatives, salts, or a combination thereof. In someAttorney Docket No. 224638-702601 embodiments, the diuretic comprises indapamide, bumetanide, chlorothiazide, furosemide, hydrochlorothiazide, metolazone, spironolactone, thiazide, amiloride, chlorthalidone, eplerenone, ethacrynic acid, torsemide, bendroflumethiazide, dyrenium, edecrin, triamterene analogues, derivatives, salts, or a combination thereof. In some embodiments, the statin comprises atorvastatin (Lipitor), fluvastatin (Lescol XL), lovastatin (Altoprev), pitavastatin (Livalo), pravastatin (Pravachol), rosuvastatin (Crestor), simvastatin (Zocor), analogues, derivatives, salts, or a combination thereof. Acebutolol. In some embodiments, the beta blocker comprises atenolol (Tenormin), bisoprolol, metoprolol (Lopressor, Toprol XL), nadolol (Corgard), nebivolol (Bystolic), propranolol (Inderal LA, InnoPran XL), analogues, derivatives, salts, or a combination thereof.Exemplary Embodiments

[0316] Embodiment 1. An in vitro vascularized tissue comprising:

[0317] i. a plurality of stem cell-derived glomeruli or precursors thereof; and

[0318] ii.a plurality of adipose-derived microvessel fragments (MVFs), wherein the plurality of MVFs are sufficient to form a network of microvessels (MVs) that penetrate or are penetrated by the plurality of stem cell-derived glomeruli at a density that is at least 10% greater than stem cell- derived glomeruli that have not been cultivated with the plurality of MVFs, wherein the density of penetrated stem cell-derived glomeruli or precursors thereof is measured based on an amount of penetrated stem cell-derived glomeruli or precursors thereof in a cross-sectional area of the in vitro vascularized tissue.

[0319] Embodiment 2. The in vitro vascularized tissue of Embodiment 1, wherein the in vitro vascularized tissue comprises at least one developing stem cell-derived glomerulus; and the plurality of MVFs or the network of MVs within the developing stem cell-derived glomerulus.

[0320] Embodiment 3. The in vitro vascularized tissue of Embodiment 1, wherein the in vitro vascularized tissue comprises at least one formed stem cell-derived glomeruli; and the plurality of MVFs or the network of MVs within the formed stem cell-derived glomeruli.

[0321] Embodiment 4. The in vitro vascularized tissue of Embodiment 1, further comprising at least one kidney tissue component selected from a proximal tubule, a loop of Henle, a distal tubule, or a combination thereof.

[0322] Embodiment 5. The in vitro vascularized tissue of Embodiment 1, wherein an expression profile of endothelial cells in the stem cell-derived glomeruli or precursors thereof comprises at least one of UEA1+, CD31+ or VEGFR2+.Attorney Docket No. 224638-702601

[0323] Embodiment 6. The in vitro vascularized tissue of Embodiment 1, wherein an expression profile of podocytes in the stem cell-derived glomeruli or precursors thereof comprises PODXL+.

[0324] Embodiment 7. The in vitro vascularized tissue of Embodiment 1, wherein an expression profile of renal tubule cells in the stem cell-derived glomeruli or precursors thereof comprises EPCAM+.

[0325] Embodiment 8. The in vitro vascularized tissue of Embodiment 1, wherein an expression profile of proximal tubule cells in the stem cell-derived glomeruli or precursors thereof comprises LTL+.

[0326] Embodiment 9. The in vitro vascularized tissue of Embodiment 1, wherein an expression profile of cells in the stem cell-derived glomeruli or precursors thereof vary along a z-direction.

[0327] Embodiment 10. The in vitro vascularized tissue of Embodiment 1, further comprising an extracellular matrix (ECM).

[0328] Embodiment 11. The in vitro vascularized tissue of Embodiment 10, wherein the ECM comprises at least one of solubilized basement membrane preparation extracted from Engelbreth- Holm-Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibrin, fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, decellularized kidney ECM, or a combination thereof.

[0329] Embodiment 12. The in vitro vascularized tissue of Embodiment 1, wherein the network of adipose-derived MVs or plurality of MVFs comprise at least a portion or segment of at least one artery, arteriole, capillary, venule, vein, or a combination thereof.

[0330] Embodiment 13. The in vitro vascularized tissue of Embodiment 1, wherein the network of adipose-derived MVs or plurality of MVFs are obtained from subcutaneous fat, perirenal fat, pericardial fat, omental fat, breast fat, epididymal fat, properitoneal fat, or a combination thereof.

[0331] Embodiment 14. The in vitro vascularized tissue of Embodiment 1, further comprising allogeneic components and autologous components.

[0332] Embodiment 15. The in vitro vascularized tissue of Embodiment 1, further comprising synthetic or semi -synthetic materials.

[0333] Embodiment 16. The in vitro vascularized tissue of Embodiment 1, further comprising a three-dimensional framework selected from at least one of a membrane, a mesh, a grid, a sponge, a foam, or a combination thereof.

[0334] Embodiment 17. The in vitro vascularized tissue of Embodiment 16, wherein the three- dimensional framework is selected from at least one of solubilized basement membrane preparation extracted from Engelbreth-Holm- Swarm (EHS) mouse sarcoma, laminin, collagen IV,Attorney Docket No. 224638-702601 heparan sulfate proteoglycans, entactin, fibrin, fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, or a combination thereof.

[0335] Embodiment 18. An in vitro vascularized tissue comprising:

[0336] i. a plurality of stem cell-derived glomeruli or precursors thereof; and

[0337] ii. a network of adipose-derived microvessels (MVs) or a plurality MVFs, wherein the network of adipose-derived MVs or plurality of MVFs are sufficient to obtain a branching index that is at least 10% greater than a plurality of stem cell-derived glomeruli that have not been cultivated with the network of adipose-derived MVs or the plurality MVFs, wherein the branching index is measured based on an amount of branch points in a cross-sectional area of the stem cell- derived glomeruli.

[0338] Embodiment 19. The in vitro vascularized tissue of Embodiment 18, wherein the in vitro vascularized tissue comprises at least one developing stem cell-derived glomerulus; and the plurality of MVFs or the network of MVs within the developing stem cell-derived glomerulus.

[0339] Embodiment 20. The in vitro vascularized tissue of Embodiment 18, wherein the in vitro vascularized tissue comprises at least one formed stem cell-derived glomeruli; and the plurality of MVFs or the network of MVs within the formed stem cell-derived glomeruli.

[0340] Embodiment 21. An in vitro vascularized tissue comprising:

[0341] i.a plurality of stem cell-derived glomeruli or precursors thereof; and

[0342] ii. a network of adipose-derived MVs or plurality of MVFs, wherein the network of adipose-derived MVs or plurality of MVFs are sufficient to obtain a total vessel length that is at least 10% greater than stem cell-derived glomeruli that have not been cultivated with the network of adipose-derived MVs or plurality of MVFs, the total vessel length is measured based on a linear extent of identified vessels in a cross-sectional area of the stem cell-derived glomeruli.

[0343] Embodiment 22. The in vitro vascularized tissue of Embodiment 21, wherein the in vitro vascularized tissue comprises at least one developing stem cell-derived glomerulus; and the plurality of MVFs or the network of MVs within the developing stem cell-derived glomerulus.

[0344] Embodiment 23. The in vitro vascularized tissue of Embodiment 21, wherein the in vitro vascularized tissue comprises at least one formed stem cell-derived glomeruli; and the plurality of MVFs or the network of MVs within the formed stem cell-derived glomeruli.

[0345] Embodiment 24. A method for vascularizing glomeruli in vitro, the method comprising:

[0346] Embodiment 25. contacting a plurality of stem cell-derived glomeruli or precursors thereof with a network of adipose-derived microvessels (MVs) or a plurality of MVFs; and

[0347] Embodiment 26. cultivating the contacted plurality of stem cell-derived glomeruli or precursors thereof in a cell culture medium, thereby allowing the network of adipose-derived MVsAttorney Docket No. 224638-702601 or plurality of MVFs to penetrate or be penetrated by the plurality of stem cell-derived glomeruli or precursors thereof at a density that is at least 10% greater than stem cell-derived glomeruli or precursors thereof that have not been cultivated with the network of adipose-derived MVs or plurality of MVFs, the density of penetrated stem cell-derived glomeruli or precursors thereof is measured based on an amount of penetrated stem cell-derived glomeruli or precursors thereof in a cross-sectional area of an in vitro vascularized tissue comprising the stem cell-derived glomeruli or precursors thereof.

[0348] Embodiment 27. The method of Embodiment 24, wherein the in vitro vascularized tissue comprises at least one developing stem cell-derived glomerulus; and the plurality of MVFs or the network of MVs within the developing stem cell-derived glomerulus.

[0349] Embodiment 28. The method of Embodiment 24, wherein the in vitro vascularized tissue comprises at least one formed stem cell-derived glomeruli; and the plurality of MVFs or the network of MVs within the formed stem cell-derived glomeruli.

[0350] Embodiment 29. The method of Embodiment 24, wherein the contacting comprises incorporating the stem cell-derived glomeruli or precursors thereof and the network of adipose- derived MVs or plurality of MVFs into a bioink, wherein the bioink comprises a granular tissue, where the granular tissue comprises stem cell-derived spherical aggregates, spheroids, organoids, embryoid bodies, or a combination thereof.

[0351] Embodiment 30. The method of Embodiment 24, wherein the contacting comprises incorporating the stem cell-derived glomeruli or precursors thereof and the network of adipose- derived MVs or plurality of MVFs into a bioink, wherein the bioink comprises a suspension culture.

[0352] Embodiment 31. The method of Embodiment 30, further comprising extruding the bioink at least one spatially defined location(s) on or within a three-dimensional framework.

[0353] Embodiment 32. The method of Embodiment 31, wherein the three-dimensional framework is selected from at least one of a membrane, mesh, a grid, a sponge, a foam, or a combination thereof.

[0354] Embodiment 33. The method of Embodiment 31, wherein the three-dimensional framework is selected from at least one of solubilized basement membrane preparation extracted from Engelbreth-Holm- Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibrin, fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, decellularized kidney ECM, or a combination thereof.Attorney Docket No. 224638-702601

[0355] Embodiment 34. The method of Embodiment 24, wherein an expression profile of cells in the stem cell-derived glomeruli or precursors thereof comprises at least one of UEA1+, CD31+ or VEGFR2+.

[0356] Embodiment 35. The method of Embodiment 24, wherein an expression profile of podocytes in the stem cell-derived glomeruli or precursors thereof comprises PODXL+.

[0357] Embodiment 36. The method of Embodiment 24, wherein an expression profile of renal tubule cells in the stem cell-derived glomeruli or precursors thereof comprises EPCAM+.

[0358] Embodiment 37. The method of Embodiment 24, wherein an expression profile of proximal tubule cells in the stem cell-derived glomeruli or precursors thereof comprises LTL+.

[0359] Embodiment 38. The method of Embodiment 24, wherein an expression profile of cells in the stem cell-derived glomeruli or precursors thereof varies along a z-direction.

[0360] Embodiment 39. The method of Embodiment 24, wherein the cultivating comprises exposure of the network of adipose-derived MVs or plurality of MVFs to flow and / or shear.

[0361] Embodiment 40. The method of Embodiment 24, wherein the cell culture medium comprises growth factors.

[0362] Embodiment 41. The method of Embodiment 40, wherein the growth factors comprise at least one of an agent selected from the group consisting of: CHIR99021, Noggin, Activin A, FGF1, FGF7, FGF9, BMP7, GDNF BMP4, Retinoic Acid, GDNF1, RSPO1, or a combination thereof.

[0363] Embodiment 42. The method of Embodiment 24, wherein the cultivating is performed on an extracellular matrix (ECM).

[0364] Embodiment 43. The method of Embodiment 42, wherein the ECM comprises at least one of solubilized basement membrane preparation extracted from Engelbreth-Holm- Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibrin, fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, decellularized kidney ECM, or a combination thereof.

[0365] Embodiment 44. The method of Embodiment 24, wherein the network of adipose-derived MVs or plurality of MVFs comprise at least a portion or segment of at least one artery, arteriole, capillary, venule, vein, or a combination thereof.

[0366] Embodiment 45. The method of Embodiment 24, wherein the network of adipose-derived MVs or plurality of MVFs are obtained from subcutaneous fat, perirenal fat, pericardial fat, omental fat, breast fat, epididymal fat, properitoneal fat, or a combination thereof.

[0367] Embodiment 46. The method of Embodiment 24, further comprising allogeneic components and autologous components.Attorney Docket No. 224638-702601

[0368] Embodiment 47. The method of Embodiment 24, further comprising synthetic or semisynthetic materials.

[0369] Embodiment 48. The method of Embodiment 24, further comprising a three-dimensional framework selected from at least one of a membrane, a mesh, a grid, a sponge, a foam, or a combination thereof.

[0370] Embodiment 49. The method of Embodiment 48, wherein the three-dimensional framework is selected from at least one of solubilized basement membrane preparation extracted from Engelbreth-Holm- Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibrin, fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, decellularized kidney ECM, or a combination thereof.

[0371] Embodiment 50. The method of Embodiment 24, wherein the network of adipose-derived MVs or plurality of MVFs and cells in the plurality of stem cell-derived glomeruli or precursors thereof are in a ratio of 1 :500 or 1 : 1650.

[0372] Embodiment 51. An in vitro vascularized kidney tissue comprising:

[0373] i.a plurality of stem cell-derived kidney tissue components comprising: a plurality of glomeruli or precursors thereof, a plurality of podocytes, a plurality of proximal tubule cells, a plurality of renal tubule cells, a plurality of distal tubule cells, a loop of Henle, a plurality of endothelial cells, or a combination thereof; and

[0374] ii. a network of adipose-derived microvessels (MVs) or plurality of MVFs, in an amount sufficient to form a network of MVs that penetrate or are penetrated by the plurality of stem cell- derived kidney tissue components at a density that is at least 10% greater than a plurality of stem cell-derived kidney tissue components that have not been cultivated with the network of adipose- derived MVs or plurality of MVFs, the density of penetrated kidney tissue components is measured based on an amount of penetrated kidney tissue components in a cross-sectional area of the in vitro vascularized kidney tissue comprising the stem cell-derived kidney tissue components.

[0375] Embodiment 52. The in vitro vascularized kidney tissue of Embodiment 51, wherein an expression profile of endothelial cells in the plurality of glomeruli or precursors thereof comprises at least one of UEA1+, CD31+ or VEGFR2+.

[0376] Embodiment 53. The in vitro vascularized kidney tissue of Embodiment 51, wherein an expression profile of podocytes in the stem cell-derived kidney tissue components comprises PODXL+.

[0377] Embodiment 54. The in vitro vascularized kidney tissue of Embodiment 51, wherein an expression profile of renal tubule cells in the stem cell-derived kidney tissue components comprises EPCAM+.Attorney Docket No. 224638-702601

[0378] Embodiment 55. The in vitro vascularized kidney tissue of Embodiment 51, wherein an expression profile of proximal tubule cells in the stem cell-derived kidney tissue components comprises LTL+.

[0379] Embodiment 56. The in vitro vascularized kidney tissue of Embodiment 51, wherein an expression profile of cells in the stem cell-derived kidney tissue components vary along a z- direction.

[0380] Embodiment 57. The in vitro vascularized kidney tissue of Embodiment 51, further comprising an extracellular matrix (ECM).

[0381] Embodiment 58. The in vitro vascularized kidney tissue of Embodiment 57, wherein the ECM comprises at least one of solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibrin, fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, decellularized kidney ECM, or a combination thereof.

[0382] Embodiment 59. The in vitro vascularized kidney tissue of Embodiment 51, wherein the network of adipose-derived MVs or plurality of MVFs comprise at least a portion or segment of at least one artery, arteriole, capillary, venule, vein, or a combination thereof.

[0383] Embodiment 60. The in vitro vascularized kidney tissue of Embodiment 51, wherein the network of adipose-derived MVs or plurality of MVFs are obtained from subcutaneous fat, perirenal fat, pericardial fat, omental fat, breast fat, epididymal fat, properitoneal fat, or a combination thereof.

[0384] Embodiment 61. The in vitro vascularized kidney tissue of Embodiment 51, further comprising allogeneic components and autologous components.

[0385] Embodiment 62. The in vitro vascularized kidney tissue of Embodiment 51, further comprising synthetic or semi-synthetic materials.

[0386] Embodiment 63. The in vitro vascularized kidney tissue of Embodiment 51, further comprising a three-dimensional framework selected from at least one of a membrane, a mesh, a grid, a sponge, a foam, or a combination thereof.

[0387] Embodiment 64. The in vitro vascularized kidney tissue of Embodiment 63, wherein the three-dimensional framework is selected from at least one of solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibrin, fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, decellularized kidney ECM, or a combination thereof.

[0388] Embodiment 65. An in vitro vascularized tissue comprising:Attorney Docket No. 224638-702601

[0389] i.a plurality of stem cell-derived kidney tissue components comprising: a plurality of glomeruli or precursors thereof, a plurality of podocytes, a plurality of proximal tubule cells, a plurality of renal tubule cells, a plurality of distal tubule cells, a loop of Henle, a plurality of endothelial cells, or a combination thereof; and

[0390] ii. a network of adipose-derived microvessels (MVs) or plurality of MVFs, wherein the network of adipose-derived MVs or plurality of MVFs are in an amount sufficient to obtain a branching index that is at least 10% greater than stem cell-derived kidney tissue components that have not been cultivated with the network of adipose-derived MVs or plurality of MVFs, the branching index is measured based on an amount of branch points in a cross-sectional area of the stem cell-derived kidney tissue components.

[0391] Embodiment 66. An in vitro vascularized tissue comprising:

[0392] i.a plurality of stem cell-derived kidney tissue components comprising: a plurality of glomeruli or precursors thereof, a plurality of podocytes, a plurality of proximal tubule cells, a plurality of renal tubule cells, a plurality of distal tubule cells, a loop of Henle, a plurality of endothelial cells, or a combination thereof; and

[0393] ii. a network of adipose-derived microvessels (MVs) or plurality of MVFs, wherein the network of adipose-derived MVs or plurality of MVFs are in an amount sufficient to obtain a total vessel length that is at least 10% greater than stem cell-derived kidney tissue components that have not been cultivated with the network of adipose-derived MVs or plurality of MVFs, the total vessel length is measured based on a linear extent of identified vessels in a cross-sectional area of the stem cell-derived kidney tissue components.

[0394] Embodiment 67. A method for making an in vitro vascularized kidney tissue comprising:

[0395] Embodiment 68. combining a plurality of stem cell-derived glomeruli or precursors thereof with a plurality of stem cell-derived kidney tissue components selected from: a plurality of podocytes, a plurality of proximal tubule cells, a plurality of renal tubule cells, a plurality of distal tubule cells, a loop of Henle, a plurality of endothelial cells, or a combination thereof;

[0396] Embodiment 69. contacting the combination with a network of adipose-derived microvessels (MVs) or plurality of MVFs; and

[0397] Embodiment 70. cultivating the contacted combination in a cell culture medium such that the combination comprises a network of adipose-derived MVs at a density that is at least 10% greater than a plurality of stem cell-derived kidney tissue components that have not been cultivated with the network of adipose-derived MVs or plurality of MVFs, the density of penetrated kidney tissue components is measured based on an amount of penetrated kidney tissue components in aAttorney Docket No. 224638-702601 cross-sectional area of the in vitro vascularized kidney tissue comprising the stem cell-derived kidney tissue components.

[0398] Embodiment 71. The method of Embodiment 67, wherein an expression profile of endothelial cells in the network of MVs comprise at least one markers selected from UEA1+, CD31+ or VEGFR2+.

[0399] Embodiment 72. The method of Embodiment 67, wherein an expression profile of podocytes in the network of MVs comprise PODXL+.

[0400] Embodiment 73. The method of Embodiment 67, wherein an expression profile of the renal tubule cells in the network of MVs comprise EPCAM+.

[0401] Embodiment 74. The method of Embodiment 67, wherein an expression profile of the proximal tubule cells in the network of MVs comprise LTL+.

[0402] Embodiment 75. The method of Embodiment 67, wherein the contacting comprises incorporating the stem cell-derived kidney tissue components and the network of adipose-derived MVs or plurality of MVFs into a bioink, wherein the bioink comprises a granular tissue, where the granular tissue comprises stem cell-derived spherical aggregates, spheroids, organoids, embryoid bodies, or a combination thereof.

[0403] Embodiment 76. The method of Embodiment 67, wherein the contacting comprises incorporating the stem cell-derived kidney tissue components and the network of adipose-derived MVs or plurality of MVFs into a bioink, wherein the bioink comprises a suspension culture.

[0404] Embodiment 77. The method of Embodiment 76, further comprising extruding the bioink at least one spatially defined location(s) on or within a three-dimensional framework.

[0405] Embodiment 78. The method of Embodiment 77, wherein the three-dimensional framework is selected from at least one of a membrane, mesh, a grid, a sponge, a foam, or a combination thereof.

[0406] Embodiment 79. The method of Embodiment 77, wherein the three-dimensional framework is selected from at least one of solubilized basement membrane preparation extracted from Engelbreth-Holm- Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibrin, fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, decellularized kidney ECM, or a combination thereof.

[0407] Embodiment 80. The method of Embodiment 67, wherein an expression profile of cells in the stem cell-derived glomeruli or precursors thereof varies along a z-direction.

[0408] Embodiment 81. The method of Embodiment 67, wherein the cultivating comprises exposure of the network of MVs to flow and / or shear.Attorney Docket No. 224638-702601

[0409] Embodiment 82. The method of Embodiment 67, wherein the cell culture medium comprises growth factors.

[0410] Embodiment 83. The method of Embodiment 82, wherein the growth factors comprise at least one of an agent selected from the group consisting of: CHIR99021, Noggin, Activin A, FGF1, FGF7, FGF9, BMP7, GDNF BMP4, Retinoic Acid, GDNF1, RSPO1, or a combination thereof.

[0411] Embodiment 84. The method of Embodiment 67, wherein the cultivating is performed on an extracellular matrix (ECM).

[0412] Embodiment 85. The method of Embodiment 84, wherein the ECM comprises at least one of solubilized basement membrane preparation extracted from Engelbreth-Holm- Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibrin, fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, decellularized kidney ECM, or a combination thereof.

[0413] Embodiment 86. The method of Embodiment 67, wherein the network of adipose-derived MVs or plurality of MVFs comprise at least a portion or segment of at least one artery, arteriole, capillary, venule, vein, or a combination thereof.

[0414] Embodiment 87. The method of Embodiment 67, wherein the network of adipose-derived MVs or plurality of MVFs are obtained from subcutaneous fat, perirenal fat, pericardial fat, omental fat, breast fat, epididymal fat, properitoneal fat, or a combination thereof.

[0415] Embodiment 88. The method of Embodiment 67, wherein the cell culture medium comprises allogeneic components and autologous components.

[0416] Embodiment 89. The method of Embodiment 67 wherein the cell culture medium comprises synthetic or semi-synthetic materials.

[0417] Embodiment 90. An in vitro composition comprising the in vitro vascularized tissue of any one of claims 1 to Embodiment 16 or the in vitro vascularized kidney tissue of any one of claims Embodiment 51 to Embodiment 63.

[0418] Embodiment 91. The composition of Embodiment 90, further comprising a cell culture medium.

[0419] Embodiment 92. The composition of Embodiment 90, further comprising growth factors.

[0420] Embodiment 93. The composition of Embodiment 92, wherein the growth factors comprise CHIR99021, Noggin, Activin A, FGF1, FGF7, FGF9, BMP7, GDNF BMP4, Retinoic Acid, GDNF1, RSPO1, or a combination thereof.

[0421] Embodiment 94. The composition of Embodiment 90, further comprising at least one metabolite.Attorney Docket No. 224638-702601

[0422] Embodiment 95. The composition of Embodiment 94, wherein the at least one metabolite comprise: urea, creatinine, uric acid, ammonium phosphate, or a combination thereof.

[0423] Embodiment 96. The composition of Embodiment 90, further comprising an extracellular matrix.

[0424] Embodiment 97. The composition of Embodiment 96, wherein the extracellular matrix comprises: a solubilized basement membrane preparation extracted from Engelbreth-Holm- Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibronectin, vitronectin, retronectin, elastin, hyaluronic acid, methylcellulose, a gelatin, solubilized ECM from decellularized kidney?, or any combination thereof.

[0425] Embodiment 98. The composition of Embodiment 90, further comprising a diluent.

[0426] Embodiment 99. The composition of Embodiment 98, wherein the diluent comprises a cryopreservation agent, a serum, or a suspension.

[0427] Embodiment 100. The composition of Embodiment 90, further comprising a population of glomerular progenitor cells or a differentiated progeny thereof.

[0428] Embodiment 101. The composition of Embodiment 90, further comprising a population of human blood cells.

[0429] Embodiment 102. An in vitro composition comprising:

[0430] i .an in vitro-differentiated kidney tissue comprising a population of glomerular tissues with a network of microvessels of a population of microvascular fragments (MVFs),

[0431] ii .wherein:

[0432] iii.the in vitro-differentiated kidney tissue that comprises spatially-organized progenitors of a collecting duct or a differentiated population of cells thereof,

[0433] iv.and the in vitro-differentiated kidney tissue comprises two or more markers selected from: LRP2, GAT A3, MAFB, and CK8.

[0434] Embodiment 103. The in vitro composition of Embodiment 102, wherein the in vitro- differentiated kidney tissue comprises two or more markers, wherein the two or more markers comprise:

[0435] (i)MAFB and LRP2;

[0436] (ii)MAFB and CK8;

[0437] (iii)MAFB and GAT A3;

[0438] (iv)LRP2 and CK8;

[0439] (v)LRP2 and GAT A3; or

[0440] (vi)CK8 and GAT A3.Attorney Docket No. 224638-702601

[0441] Embodiment 104. The in vitro composition of Embodiment 102, wherein the in vitro- differentiated kidney tissue comprises three or more markers, wherein the three or more markers comprise:

[0442] (i)MAFB, LRP2, and CK8;

[0443] (ii)MAFB, LRP2, and GAT A3;

[0444] (iii)MAFB, CK8, and GAT A3; or

[0445] (iv)LRP2, CK8, and GAT A3.

[0446] Embodiment 105. The in vitro composition of Embodiment 102, wherein the in vitro- differentiated kidney tissue comprises four or more markers, wherein the four or more markers comprise: MAFB, LRP2, CK8, and GAT A3.

[0447] Embodiment 106. The in vitro composition of Embodiment 102, wherein the population of glomerular tissues are derived from human stem cells.

[0448] Embodiment 107. The in vitro composition of Embodiment 102, wherein the population of MVFs are derived from human adipose tissue.

[0449] Embodiment 108. The in vitro composition of Embodiment 106, wherein the human stem cells are embryonic stem cells, induced pluripotent stem cells (iPSCs), or adult stem cells.

[0450] Embodiment 109. The in vitro composition of Embodiment 102, wherein a portion of a core of the glomerular tissues comprise epithelial cells.

[0451] Embodiment 110. The in vitro composition of Embodiment 102, wherein a portion of a core of the glomerular tissues comprise renal stromal cells.

[0452] Embodiment 111. The in vitro composition of Embodiment 102, wherein a portion of a core of the glomerular tissues comprise epithelial cells and renal stromal cells.

[0453] Embodiment 112. The in vitro composition of Embodiment 102, further comprising a cell culture medium.

[0454] Embodiment 113. The in vitro composition of Embodiment 102, further comprising growth factors.

[0455] Embodiment 114. The in vitro composition of Embodiment 113, wherein the growth factors comprise CHIR99021, Noggin, Activin A, FGF1, FGF7, FGF9, BMP7, GDNF BMP4, Retinoic Acid, GDNF1, RSPO1, or a combination thereof.

[0456] Embodiment 115. The in vitro composition of Embodiment 102, further comprising at least one metabolite.

[0457] Embodiment 116. The in vitro composition of Embodiment 115, wherein the at least one metabolite comprise urea, creatinine, uric acid, ammonium phosphate, or a combination thereof.Attorney Docket No. 224638-702601

[0458] Embodiment 117. The in vitro composition of Embodiment 102, further comprising an extracellular matrix.

[0459] Embodiment 118. The in vitro composition of Embodiment 117, wherein the extracellular matrix comprises: a solubilized basement membrane preparation extracted from Engelbreth-Holm- Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibronectin, vitronectin, retronectin, elastin, hyaluronic acid, methylcellulose, a gelatin, or any combination thereof.

[0460] Embodiment 119. The in vitro composition of Embodiment 102, further comprising a diluent.

[0461] Embodiment 120. The in vitro composition of Embodiment 119, wherein the diluent comprises a cryopreservation agent, a serum, or a suspension.

[0462] Embodiment 121. A method of treating a kidney disease in a subject, the method comprising:

[0463] (a) culturing a plurality of the in vitro composition of any one of claims Embodiment 90 to Embodiment 120 in a bioreactor; and

[0464] (b)forming a blood circuit between the subject’s blood and the in vitro composition to remove excess waste and fluid, thereby treating the kidney disease.

[0465] Embodiment 122. A method of treating a disease in a subject, the method comprising:

[0466] i. administering to the subject the in vitro composition of any one of claims Embodiment 90 to Embodiment 120, thereby treating the disease in the subject.

[0467] Embodiment 123. The method of Embodiment 122, wherein the disease is a kidney disease.

[0468] Embodiment 124. The method of Embodiment 123, wherein the kidney disease is a chronic kidney disease.

[0469] Embodiment 125. The method of Embodiment 123, wherein the kidney disease is selected from the group consisting of: atypical hemolytic uremic syndrome (aHUS), Alport syndrome, amyloidosis, POLI -mediated kidney disease, cancer, cardiovascular kidney metabolic (CKM) syndrome, complement 3 glomerulopathy (C3G), cystinosis, diabetic kidney disease, end-stage renal failure, Fabry disease, focal segmental glomerulosclerosis (FSGS), glomerulonephritis (Glomerular Disease) Goodpasture syndrome, granulomatosis with polyangiitis (GPA), hemolytic uremic syndrome (HUS), Henoch-Schbnlein purpura (HSP), IgA nephropathy, interstitial nephritis, kidney failure, Lupus nephritis, minimal change disease, polycystic kidney disease, primary hyperoxaluria and oxalate, thrombotic thrombocytopenic purpura (TTP), and vasculitis of the kidney.Attorney Docket No. 224638-702601

[0470] Embodiment 126. The method of Embodiment 122, wherein the subject has, is diagnosed with, or is suspected of having kidney failure.

[0471] Embodiment 127. The method of Embodiment 126, wherein the kidney failure is caused by at least one condition selected from the group consisting of: diabetes, high blood pressure, glomerulonephritis, polycystic kidney disease, lupus nephritis, IgA nephropathy, alcoholism, and nephrotoxicity.

[0472] Embodiment 128. The method of Embodiment 122, wherein the subject has, or is diagnosed with a kidney injury.

[0473] Embodiment 129. The method of Embodiment 128, wherein the kidney injury is caused by an infection, a pregnancy complication, a urinary tract obstruction, a kidney stone, or a physical injury.

[0474] Embodiment 130. The method of Embodiment 122, wherein the subject has, or is diagnosed with a congenital abnormality.

[0475] Embodiment 131. The method of Embodiment 122, wherein the subject has, or is diagnosed with a congenital abnormality, wherein the congenital abnormality comprises renal agenesis, renal dysplasia, or renal hypoplasia.

[0476] Embodiment 132. The method of Embodiment 122, wherein the administering comprises surgical transplantation of the in vitro composition in the subject.

[0477] Embodiment 133. A reagent comprising: fetal bovine serum (FBS), retinoic acid, R- spondin 1 (RSPO1) protein, glial-derived neurotrophic factor (GDNF), fibroblast growth factor 1 (FGF1), fibroblast growth factor 7 (FGF7), LDN193189, and solubilized basement membrane preparation extracted from Engelbreth -Holm- Swarm (EHS) mouse sarcoma.

[0478] Embodiment 134. The reagent of Embodiment 133, wherein the FBS is present in an amount that is at least about 1% up to 20% volume by total volume of the reagent (v / v).

[0479] Embodiment 135. The reagent of Embodiment 133, wherein the retinoic acid is present in an amount that is at least about 10 nanoMolar (nM) concentration up to 200 nM concentration.

[0480] Embodiment 136. The reagent of Embodiment 133, wherein the RSPO1 is present in an amount that is at least about 10 nanograms per milliliter (ng / mL) up to 200 ng / mL.

[0481] Embodiment 137. The reagent of Embodiment 133, wherein the GDNF is present in an amount that is at least about 0.1 nanograms per milliliter (ng / mL) up to 5 ng / mL.

[0482] Embodiment 138. The reagent of Embodiment 133, wherein the FGF1 is present in an amount that is at least about 10 nanograms per milliliter (ng / mL) up to 200 ng / mL.

[0483] Embodiment 139. The reagent of Embodiment 133, wherein the FGF7 is present in an amount that is at least about 3 nanograms per milliliter (ng / mL) up to 60 ng / mL.Attorney Docket No. 224638-702601

[0484] Embodiment 140. The reagent of Embodiment 133, wherein the LDN193189 is present in an amount that is at least about 1 nanoMolar (nM) concentration up to 20 nM concentration.

[0485] Embodiment 141. The reagent of Embodiment 133, wherein the solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma is present in an amount of at least 5% up to 80% volume by total volume of the reagent (v / v).

[0486] Embodiment 142. A kit comprising: a first container comprising: a population of glomerular cells and a network of adipose-derived MVs or plurality of MVFs; and a second container comprising media, growth factors, and agents for making an in vitro kidney tissue.

[0487] Embodiment 143. A transplant composition comprising: a first container comprising: the in vitro vascularized tissue of any one of claims 1 to Embodiment 16 or the in vitro vascularized kidney tissue of any one of claims Embodiment 51 to Embodiment 63 or a plurality thereof ; and a second container comprising: (a) an additional therapeutic agent; (b) at least one immunosuppressant; or (c) a combination thereof.

[0488] Embodiment 144. A method of treating a subject with a kidney disease, the method comprising: administering to the subject a transplant composition of Embodiment 143, thereby treating the kidney disease.

[0489] Embodiment 145. The method of Embodiment 144, wherein the second container of the transplant composition is administered to the subject prior to the first container.

[0490] Embodiment 146. The method of Embodiment 144, wherein the second container of the transplant composition is administered to the subject at a same time as the first container.

[0491] Embodiment 147. The method of Embodiment 144, wherein the second container of the transplant composition is administered to the subject after the first container.

[0492] Embodiment 148. A method of transplanting a kidney in a subject, the method comprising: engrafting a plurality of the in vitro vascularized tissue of any one of claims 1 to Embodiment 16 or the in vitro vascularized kidney tissue of any one of claims Embodiment 51 to Embodiment 63 into a kidney of the subject.

[0493] Embodiment 149. A method comprising:

[0494] (a)transplanting the in vitro vascularized tissue of any one of claims 1 to Embodiment 16 or the in vitro vascularized kidney tissue of any one of claims Embodiment 51 to Embodiment 63 into a subject having a kidney disease; and

[0495] (b)administering to the subject at least one immunosuppressive agent,

[0496] ii .wherein the method increases a glomerular filtration rate (GFR) of the subject relative to the GFR of the subject prior to transplantation.

[0497] Embodiment 150. A method comprising:Attorney Docket No. 224638-702601

[0498] (a)transplanting the in vitro vascularized tissue of any one of claims 1 to Embodiment 16 or the in vitro vascularized kidney tissue of any one of claims Embodiment 51 to Embodiment 63 into a subject having a kidney disease; and

[0499] (b)administering to the subject at least one immunosuppressive agent,

[0500] ii .wherein the method increases reduces a level of a biomarker indicative of nephrotoxicity in the subject relative to the level of the biomarker indicative of nephrotoxicity in the subject prior to transplantation.

[0501] Embodiment 151. The method of any one of claims Embodiment 148 to Embodiment 150, wherein the method further comprises surgically removing a diseased kidney from the subject.

[0502] Embodiment 152. The method of any one of claims Embodiment 148 to Embodiment 150, wherein the subject has a kidney disease.

[0503] Embodiment 153. The method of any one of claims Embodiment 148 to Embodiment 150, wherein the subject has a kidney injury.EXAMPLESExample 1. Exemplary Method of preparing adipose-derived microvessel fragments.

[0504] Microvessel fragments were isolated from human adipose tissue isolated from discarded abdominoplasty or lipoaspirate tissue. Under aseptic conditions, adipose tissue was finely minced with scissors, digested in approximately 2-4 mg / mL collagenase plus 2 mg / mL EFAF-BSA in phosphate-buffered saline (PBS) for 8 minutes at 37°C with vigorous shaking and washed in 0.1% BSA-PBS. Tissue debris and large vessel pieces were removed by filtering the suspension through a sterile 500-pm-pore nylon screen. Microvessel fragments were captured by filtration of the remaining suspension on a 30-pm-pore nylon screen and recovered by vigorous flushing of the screen surface with 0.1% BSA-PBS. The type and lot number of collagenase used were predetermined to optimize fragment yield while maintaining microvessel integrity. For 3- dimensional culture and endothelial cell expansion, microvessel fragments (MF) were suspended (12,000 to 15,000 MF / mL) in ice-cold type I collagen (BD BioSciences, Bedford, Mass) prepared with cell culture medium. The vessel / collagen suspensions were plated into individual wells (0.25 mL / well) of a 48-well plate and placed in a 37°C incubator for 20 minutes to polymerize the collagen. Human fat microvessel fragments were prepared for bioprinting and co-culture with the in vitro-differentiated human kidney tissues prior to aforementioned suspension in 3D type I collagen gel preparations.Example 2. Bioprinting with Microvascular Fragments (MVF)Atorney Docket No. 224638-702601

[0505] Vascularized kidney tissues were produced. Pluripotent stem cells were exposed to a guided 7-day differentiation protocol as described in Lawlor, Vanslambrouck, Higgins et al Nature Materials, 2021 with minor adaptations. Prior to harvesting day 7 cultures, 2D monolayers of posterior intermediate mesoderm cells were exposed to a 1 hour, 5 pM CHIR99021 in E6 media pulse before preparation for bioprinting. At the time of print, adipose-derived microvascular fragments (MVFs) (Advanced Solutions Inc. Catalog # ASLS-0000171) were used directly from cryopreservation following vendor protocols. In short, a vial containing 20k adipose-derived microvascular fragments was quick thawed in a 37°C water bath, then transferred into lOmL of RPMI supplemented with 10% FBS (v / v). Thawed MVFs were centrifuged for 4 minutes at 400g. The resulting supernatant was aspirated, and the pellet was resuspended in 2mL E6 supplemented with 2% FBS (v / v). MVFs were incorporated into bioink at 100,000 MVFs / mL (10,000 MVFs and 10,000,000 posterior intermediate mesoderm cells per 100 pL bioink). The combined cell populations were pelleted for 3 minutes at 200g. Following centrifugation, the supernatant was aspirated, and the pellet was resuspended in ImL of E6 media supplemented with 2% FBS (v / v). The freshly resuspend material was transferred to a 1.5 mL centrifuge tube and spun a 2ndtime for 3 minutes at 200g with the resulting supernatant being aspirated manually with a pl 000 pipette. 50 pL E6 media supplemented with 2% FBS (v / v) was added back to the cell pellet to facilitate loosening of the cell pellet prior to direct transfer into a lOOpL Gastight syringe (Hamilton catalog # 7656-05) with a 21-gauge needle (Hamilton catalog # 7804-12). Loaded syringe was placed within the mechanical dispense tool (ASLS catalog # ASLS-0000227) prior to bioprint execution. Bioink extrusion was performed in a spatially defined location on 0.4 pM polyester membranes of 6-well Transwell permeable supports (Coming Costar catalog # 3450). Following print, all bioprinted tissues were maintained at the air-liquid interface with media refreshed every other day. For the day of print (PPD0), PPD2 and PPD4, tissues were cultured in E6 media supplemented with 2% FBS (v / v), 200ng / mL FGF9, 1 pg / mL heparin, and 25 ng / mL VEGF. For PPD5 on, tissues were cultured in E6 media supplemented with 2% FBS (v / v) and 25 ng / mL VEGF. For tissues incorporated into microfluidic chambers, media was additionally supplemented with 500 units / mL of penicillin and streptomycin.Example 3. Endothelial Progenitors in Bioprinted Kidney

[0506] Endothelial progenitor cells exist within nephron tissues, but there is minimal network formation, and glomeruli remain largely avascular. Nephron-rich kidney tissues were made by the methods described in Example 1 to form vascularized kidney tissues. FIG. 2A illustrates bioprinted nephron-rich kidney tissues, including podocytes (PODXL), renal tubule (EPCAM),Attorney Docket No. 224638-702601 proximal tubule (LTL), and endothelial cells (CD31). FIG. 2B illustrates bioprinted nephron-rich kidney tissue, with inclusion of human adipose-derived microvascular fragments (MVFs). As shown, the inclusion of MVFs and associated media cues result in increased arborization and density of endothelia within nephron tissues. FIG. 2C illustrates a comparison of developing endothelial network in bioprinted nephron-rich kidney tissues with / without MVFs. All cultures under static conditions.

[0507] FIG. 2D provides a quantitative analysis of vascular networks of the results from FIGS. 2A-2C via AngioTool software. As shown, inclusion of MVFs in bioink results in increased vessel length, vessel density, and branching index, with reduction in lacunarity. Findings indicate improved tissue vascularization upon inclusion of MVFs, even under static culture conditions.Example 4. Bioprinted Kidney Tissues + MVFs and Flow

[0508] The purpose of this example is to illustrate the impact of shear / flow conditions on bioprinted kidney tissues and MVFs.

[0509] FIG. 3 illustrates bioprinted nephron-rich kidney tissues containing MVF s that have been cultured under perfusion and exposed to flow / shear stress. Podocytes (POD XL); renal tubule (EPCAM), proximal tubule (LTL), endothelial cells (CD31). As shown, the addition of flow exhibit increased vascular density.Example 5. Morphology and Complexity of Vascular Network Varies in Z-D

[0510] The purpose of this example is to illustrate the varying morphology of a vascular network in bioprinted kidney tissues in Z-dimension.

[0511] FIG. 4 illustrates reference raw data of vascularized kidney tissues, z.e., an initial evaluation by fluorescence microscopy, which suggested increased complexity of microvascular network in tissues containing MVFs and exposed to flow / shear cues. Left to right illustrates different staining, z.e., podocytes (PODXL), human endothelial cells (UEA1), and DNA (DAPI; bottom). Standard microscopy suggests differences in morphology of microvascular network depending on tissue region and position in Z-plane along the Z-dimension.

[0512] FIG. 5 illustrates a high resolution 3D confocal microscopy image of a vascularized kidney tissue. FIG. 5 reveals evidence of glomerular wrapping by endothelial cells, capillary invasion, and vessels with patent lumens. Triple axial sections shown: XY section (center,), xz section (bottom), yz section (right). Podocytes (PODXL), human endothelial cells (UEA1).

[0513] FIG. 6 illustrates bioprinted kidney tissues with segregation of vascular morphology in Z-dimension. Endothelial cell expansion is shown at a top of the bioprinted kidney tissue, capillaryAttomey Docket No. 224638-702601 invasion of developing glomeruli is shown at a middle of the bioprinted kidney tissue, and a network of mature vessels with patent lumens is shown at a bottom of the bioprinted kidney tissue. Podocytes (PODXL), human endothelial cells (UEA1).Example 6. Wolffian duct progenitors form an epithelial tube.

[0514] Wolffian duct progenitors were enriched for and used to generate recombined kidney tissues comprising stem cell-derived nephron progenitor cells (NPCs). Wolffian duct progenitors form an epithelial tube with repeated points of connections with the stem cell-derived NPCs along its length. Kidney tissues were co-cultured in vitro with adipose-derived microvesicle fragments.

[0515] The vascularized kidney tissues were surgically implanted onto the surface of a recipient mouse kidney. For longitudinal, intravital imaging and analysis, a circular titanium ring enclosing a thin glass window was then placed directly over the implanted kidney tissue and the underlying host tissue (FIG. 7). Mice were then recovered and returned to their cages. The mouse kidney and implanted kidney tissue were visible via the overlying glass window for monitoring of the engraftment over time The implanted human kidney tissues developed an extensive vascular network of human and mouse blood vessels within the kidney graft. (FIG. 8A and FIG. 10).Example 7. Vascularization of Kidney Tissues in vivo.

[0516] Vascularized kidney tissues were prepared and engrafted onto the surface the kidney of a mouse model described in Examples 1-6. For intravital imaging experiments, mice were anesthetized, a left lateral incision made, the left kidney externalized, the renal capsule incised and removed on the exposed surface, the vascularized kidney tissue implanted on the exposed surface of the kidney, in some cases, following a superficial corticotomy. For serial imaging, a titanium ring encircling a thin, glass window was placed directly over the implanted kidney tissue and the underlying mouse kidney, then sutured into place. To assay the functionality of the blood vessel network after 7, 12, 14, 21 or 28 days, mice bearing implanted kidney tissues were given a retroorbital injection of fluorescently labeled albumin and small molecular weight dextran. After labeled compound administration, implanted kidney tissues were imaged by multiphoton imaging and the developing nephrons and associated vasculature were identified and characterized. Robust microvascular network formation was observed, complete with hierarchical, arborized vasculature noted throughout the implanted tissues. Human glomeruli within the implanted kidney tissue were found to be well vascularized, with mature capillary loops and functional afferent and efferent arterioles developing as a function of implant duration, such that by post-implantation Day 12, the majority of identified glomeruli were well perfused. The implanted kidney tissue exhibited size-Attorney Docket No. 224638-702601 selective sieving of small (10 kDa Dextran) compounds and retention of large compounds (67 kDa Albumin) by the glomerulus of implanted bioengineered kidney tissue (FIG. 8A-FIG. 8C). The implanted bioengineered kidney tissue formed a tubular network with connected glomeruli.

[0517] Injection of Angiotensin II induced vasoconstriction of afferent / efferent arterioles and glomerular mesangium, suggesting physiological activity and functional maturation of glomerular vasculature (FIG. 9A). Vessel diameter was measured before administration (FIG. 9A, brackets) and after administration (FIG. 9B, brackets) and quantified (FIG. 9C). Vessel diameter was reduced by approximately 25% after Angiotensin II infusion.

[0518] Upon implantation on or in a host kidney, the bioengineered kidney tissues containing microvascular fragments developed into an arborized, dense vascular network, which includes direct perfusion of nephron progenitor cell-derived glomeruli (FIG. 10). Implanted microvessels within the bioengineered kidney tissues were perfused, such that circulating CD71+ reticulocytes were observed within human vessel. Moreover the human vessel tissues formed connections to the mouse circulation (FIG. 11).

Claims

Attorney Docket No. 224638-702601CLAIMSWHAT IS CLAIMED IS:

1. An in vitro vascularized tissue comprising: a plurality of stem cell-derived glomeruli or precursors thereof; and a plurality of adipose-derived microvessel fragments (MVFs), wherein the plurality of MVFs are sufficient to form a network of microvessels (MVs) that penetrate or are penetrated by the plurality of stem cell-derived glomeruli at a density that is at least 10% greater than stem cell-derived glomeruli that have not been cultivated with the plurality of MVFs, wherein the density of penetrated stem cell-derived glomeruli or precursors thereof is measured based on an amount of penetrated stem cell-derived glomeruli or precursors thereof in a cross-sectional area of the in vitro vascularized tissue.

2. The in vitro vascularized tissue of claim 1, wherein the in vitro vascularized tissue comprises at least one developing stem cell-derived glomerulus; and the plurality of MVFs or the network of MVs within the developing stem cell-derived glomerulus.

3. The in vitro vascularized tissue of claim 1, wherein the in vitro vascularized tissue comprises at least one formed stem cell-derived glomeruli; and the plurality of MVFs or the network of MVs within the formed stem cell-derived glomeruli.

4. The in vitro vascularized tissue of claim 1, further comprising at least one kidney tissue component selected from a proximal tubule, a loop of Henle, a distal tubule, or a combination thereof.

5. The in vitro vascularized tissue of claim 1, wherein an expression profile of endothelial cells in the stem cell-derived glomeruli or precursors thereof comprises at least one of UEA1+, CD31+ or VEGFR2+.

6. The in vitro vascularized tissue of claim 1, wherein an expression profile of podocytes in the stem cell-derived glomeruli or precursors thereof comprises PODXL+.

7. The in vitro vascularized tissue of claim 1, wherein an expression profile of renal tubule cells in the stem cell-derived glomeruli or precursors thereof comprises EPCAM+.

8. The in vitro vascularized tissue of claim 1, wherein an expression profile of proximal tubule cells in the stem cell-derived glomeruli or precursors thereof comprises LTL+.

9. The in vitro vascularized tissue of claim 1, wherein an expression profile of cells in the stem cell-derived glomeruli or precursors thereof vary along a z-direction.

10. The in vitro vascularized tissue of claim 1, further comprising an extracellular matrix (ECM).Attorney Docket No. 224638-70260111. The in vitro vascularized tissue of claim 10, wherein the ECM comprises at least one of solubilized basement membrane preparation extracted from Engelbreth-Holm- Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibrin, fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, decellularized kidney ECM, or a combination thereof.

12. The in vitro vascularized tissue of claim 1, wherein the network of adipose-derived MVs or plurality of MVFs comprise at least a portion or segment of at least one artery, arteriole, capillary, venule, vein, or a combination thereof.

13. The in vitro vascularized tissue of claim 1, wherein the network of adipose-derived MVs or plurality of MVFs are obtained from subcutaneous fat, perirenal fat, pericardial fat, omental fat, breast fat, epididymal fat, properitoneal fat, or a combination thereof.

14. The in vitro vascularized tissue of claim 1, further comprising allogeneic components and autologous components.

15. The in vitro vascularized tissue of claim 1, further comprising synthetic or semisynthetic materials.

16. The in vitro vascularized tissue of claim 1, further comprising a three-dimensional framework selected from at least one of a membrane, a mesh, a grid, a sponge, a foam, or a combination thereof.

17. The in vitro vascularized tissue of claim 16, wherein the three-dimensional framework is selected from at least one of solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibrin, fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, or a combination thereof.

18. An in vitro vascularized tissue comprising: a plurality of stem cell-derived glomeruli or precursors thereof; and a network of adipose-derived microvessels (MVs) or a plurality MVFs, wherein the network of adipose-derived MVs or plurality of MVFs are sufficient to obtain a branching index that is at least 10% greater than a plurality of stem cell-derived glomeruli that have not been cultivated with the network of adipose-derived MVs or the plurality MVFs, wherein the branching index is measured based on an amount of branch points in a cross-sectional area of the stem cell- derived glomeruli.

19. The in vitro vascularized tissue of claim 18, wherein the in vitro vascularized tissue comprises at least one developing stem cell-derived glomerulus; and the plurality of MVFs or the network of MVs within the developing stem cell-derived glomerulus.Attorney Docket No. 224638-70260120. The in vitro vascularized tissue of claim 18, wherein the in vitro vascularized tissue comprises at least one formed stem cell-derived glomeruli; and the plurality of MVFs or the network of MVs within the formed stem cell-derived glomeruli.

21. An in vitro vascularized tissue comprising: a plurality of stem cell-derived glomeruli or precursors thereof; and a network of adipose-derived MVs or plurality of MVFs, wherein the network of adipose-derived MVs or plurality of MVFs are sufficient to obtain a total vessel length that is at least 10% greater than stem cell-derived glomeruli that have not been cultivated with the network of adipose-derived MVs or plurality of MVFs, the total vessel length is measured based on a linear extent of identified vessels in a cross-sectional area of the stem cell-derived glomeruli.

22. The in vitro vascularized tissue of claim 21, wherein the in vitro vascularized tissue comprises at least one developing stem cell-derived glomerulus; and the plurality of MVFs or the network of MVs within the developing stem cell-derived glomerulus.

23. The in vitro vascularized tissue of claim 21, wherein the in vitro vascularized tissue comprises at least one formed stem cell-derived glomeruli; and the plurality of MVFs or the network of MVs within the formed stem cell-derived glomeruli.

24. A method for vascularizing glomeruli in vitro, the method comprising:(a) contacting a plurality of stem cell-derived glomeruli or precursors thereof with a network of adipose-derived microvessels (MVs) or a plurality of MVFs; and(b) cultivating the contacted plurality of stem cell-derived glomeruli or precursors thereof in a cell culture medium, thereby allowing the network of adipose-derived MVs or plurality of MVFs to penetrate or be penetrated by the plurality of stem cell-derived glomeruli or precursors thereof at a density that is at least 10% greater than stem cell-derived glomeruli or precursors thereof that have not been cultivated with the network of adipose-derived MVs or plurality of MVFs, the density of penetrated stem cell-derived glomeruli or precursors thereof is measured based on an amount of penetrated stem cell-derived glomeruli or precursors thereof in a cross- sectional area of an in vitro vascularized tissue comprising the stem cell-derived glomeruli or precursors thereof.

25. The method of claim 24, wherein the in vitro vascularized tissue comprises at least one developing stem cell-derived glomerulus; and the plurality of MVFs or the network of MVs within the developing stem cell-derived glomerulus.

26. The method of claim 24, wherein the in vitro vascularized tissue comprises at least one formed stem cell-derived glomeruli; and the plurality of MVFs or the network of MVs within the formed stem cell-derived glomeruli.Attorney Docket No. 224638-70260127. The method of claim 24, wherein the contacting comprises incorporating the stem cell- derived glomeruli or precursors thereof and the network of adipose-derived MVs or plurality of MVFs into a bioink, wherein the bioink comprises a granular tissue, where the granular tissue comprises stem cell-derived spherical aggregates, spheroids, organoids, embryoid bodies, or a combination thereof.

28. The method of claim 24, wherein the contacting comprises incorporating the stem cell- derived glomeruli or precursors thereof and the network of adipose-derived MVs or plurality of MVFs into a bioink, wherein the bioink comprises a suspension culture.

29. The method of claim 28, further comprising extruding the bioink at least one spatially defined location(s) on or within a three-dimensional framework.

30. The method of claim 29, wherein the three-dimensional framework is selected from at least one of a membrane, mesh, a grid, a sponge, a foam, or a combination thereof.

31. The method of claim 29, wherein the three-dimensional framework is selected from at least one of solubilized basement membrane preparation extracted from Engelbreth-Holm- Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibrin, fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, decellularized kidney ECM, or a combination thereof.

32. The method of claim 24, wherein an expression profile of cells in the stem cell-derived glomeruli or precursors thereof comprises at least one of UEA1+, CD31+ or VEGFR2+.

33. The method of claim 24, wherein an expression profile of podocytes in the stem cell- derived glomeruli or precursors thereof comprises PODXL+.

34. The method of claim 24, wherein an expression profile of renal tubule cells in the stem cell-derived glomeruli or precursors thereof comprises EPCAM+.

35. The method of claim 24, wherein an expression profile of proximal tubule cells in the stem cell-derived glomeruli or precursors thereof comprises LTL+.

36. The method of claim 24, wherein an expression profile of cells in the stem cell-derived glomeruli or precursors thereof varies along a z-direction.

37. The method of claim 24, wherein the cultivating comprises exposure of the network of adipose-derived MVs or plurality of MVFs to flow and / or shear.

38. The method of claim 24, wherein the cell culture medium comprises growth factors.

39. The method of claim 38, wherein the growth factors comprise at least one of an agent selected from the group consisting of: CHIR99021, Noggin, Activin A, FGF1, FGF7, FGF9, BMP7, GDNF BMP4, Retinoic Acid, GDNF1, RSPO1, or a combination thereof.Attorney Docket No. 224638-70260140. The method of claim 24, wherein the cultivating is performed on an extracellular matrix (ECM).

41. The method of claim 40, wherein the ECM comprises at least one of solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibrin, fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, decellularized kidney ECM, or a combination thereof.

42. The method of claim 24, wherein the network of adipose-derived MVs or plurality of MVFs comprise at least a portion or segment of at least one artery, arteriole, capillary, venule, vein, or a combination thereof.

43. The method of claim 24, wherein the network of adipose-derived MVs or plurality of MVFs are obtained from subcutaneous fat, perirenal fat, pericardial fat, omental fat, breast fat, epididymal fat, properitoneal fat, or a combination thereof.

44. The method of claim 24, further comprising allogeneic components and autologous components.

45. The method of claim 24, further comprising synthetic or semi -synthetic materials.

46. The method of claim 24, further comprising a three-dimensional framework selected from at least one of a membrane, a mesh, a grid, a sponge, a foam, or a combination thereof.

47. The method of claim 46, wherein the three-dimensional framework is selected from at least one of solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibrin, fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, decellularized kidney ECM, or a combination thereof.

48. The method of claim 24, wherein the network of adipose-derived MVs or plurality of MVFs and cells in the plurality of stem cell-derived glomeruli or precursors thereof are in a ratio of 1 :500 or 1 : 1650.

49. An in vitro vascularized kidney tissue comprising: a plurality of stem cell-derived kidney tissue components comprising: a plurality of glomeruli or precursors thereof, a plurality of podocytes, a plurality of proximal tubule cells, a plurality of renal tubule cells, a plurality of distal tubule cells, a loop of Henle, a plurality of endothelial cells, or a combination thereof; and a network of adipose-derived microvessels (MVs) or plurality of MVFs, in an amount sufficient to form a network of MVs that penetrate or are penetrated by the plurality of stem cell-derived kidney tissue components at a density that is at least 10% greater than a pluralityAttorney Docket No. 224638-702601 of stem cell-derived kidney tissue components that have not been cultivated with the network of adipose-derived MVs or plurality of MVFs, the density of penetrated kidney tissue components is measured based on an amount of penetrated kidney tissue components in a cross-sectional area of the in vitro vascularized kidney tissue comprising the stem cell-derived kidney tissue components.

50. The in vitro vascularized kidney tissue of claim 49, wherein an expression profile of endothelial cells in the plurality of glomeruli or precursors thereof comprises at least one of UEA1+, CD31+ or VEGFR2+.

51. The in vitro vascularized kidney tissue of claim 49, wherein an expression profile of podocytes in the stem cell-derived kidney tissue components comprises PODXL+.

52. The in vitro vascularized kidney tissue of claim 49, wherein an expression profile of renal tubule cells in the stem cell-derived kidney tissue components comprises EPCAM+.

53. The in vitro vascularized kidney tissue of claim 49, wherein an expression profile of proximal tubule cells in the stem cell-derived kidney tissue components comprises LTL+.

54. The in vitro vascularized kidney tissue of claim 49, wherein an expression profile of cells in the stem cell-derived kidney tissue components vary along a z-direction.

55. The in vitro vascularized kidney tissue of claim 49, further comprising an extracellular matrix (ECM).

56. The in vitro vascularized kidney tissue of claim 55, wherein the ECM comprises at least one of solubilized basement membrane preparation extracted from Engelbreth-Holm- Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibrin, fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, decellularized kidney ECM, or a combination thereof.

57. The in vitro vascularized kidney tissue of claim 49, wherein the network of adipose- derived MVs or plurality of MVFs comprise at least a portion or segment of at least one artery, arteriole, capillary, venule, vein, or a combination thereof.

58. The in vitro vascularized kidney tissue of claim 49, wherein the network of adipose- derived MVs or plurality of MVFs are obtained from subcutaneous fat, perirenal fat, pericardial fat, omental fat, breast fat, epididymal fat, properitoneal fat, or a combination thereof.

59. The in vitro vascularized kidney tissue of claim 49, further comprising allogeneic components and autologous components.

60. The in vitro vascularized kidney tissue of claim 49, further comprising synthetic or semi -synthetic materials.Attorney Docket No. 224638-70260161. The in vitro vascularized kidney tissue of claim 49, further comprising a three- dimensional framework selected from at least one of a membrane, a mesh, a grid, a sponge, a foam, or a combination thereof.

62. The in vitro vascularized kidney tissue of claim 61, wherein the three-dimensional framework is selected from at least one of solubilized basement membrane preparation extracted from Engelbreth-Holm- Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibrin, fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, decellularized kidney ECM, or a combination thereof.

63. An in vitro vascularized tissue comprising: a plurality of stem cell-derived kidney tissue components comprising: a plurality of glomeruli or precursors thereof, a plurality of podocytes, a plurality of proximal tubule cells, a plurality of renal tubule cells, a plurality of distal tubule cells, a loop of Henle, a plurality of endothelial cells, or a combination thereof; and a network of adipose-derived microvessels (MVs) or plurality of MVFs, wherein the network of adipose-derived MVs or plurality of MVFs are in an amount sufficient to obtain a branching index that is at least 10% greater than stem cell-derived kidney tissue components that have not been cultivated with the network of adipose-derived MVs or plurality of MVFs, the branching index is measured based on an amount of branch points in a cross-sectional area of the stem cell-derived kidney tissue components.

64. An in vitro vascularized tissue comprising: a plurality of stem cell-derived kidney tissue components comprising: a plurality of glomeruli or precursors thereof, a plurality of podocytes, a plurality of proximal tubule cells, a plurality of renal tubule cells, a plurality of distal tubule cells, a loop of Henle, a plurality of endothelial cells, or a combination thereof; and a network of adipose-derived microvessels (MVs) or plurality of MVFs, wherein the network of adipose-derived MVs or plurality of MVFs are in an amount sufficient to obtain a total vessel length that is at least 10% greater than stem cell-derived kidney tissue components that have not been cultivated with the network of adipose-derived MVs or plurality of MVFs, the total vessel length is measured based on a linear extent of identified vessels in a cross-sectional area of the stem cell-derived kidney tissue components.

65. A method for making an in vitro vascularized kidney tissue comprising:(a) combining a plurality of stem cell-derived glomeruli or precursors thereof with a plurality of stem cell-derived kidney tissue components selected from: a plurality of podocytes,Attorney Docket No. 224638-702601 a plurality of proximal tubule cells, a plurality of renal tubule cells, a plurality of distal tubule cells, a loop of Henle, a plurality of endothelial cells, or a combination thereof;(b) contacting the combination with a network of adipose-derived microvessels (MVs) or plurality of MVFs; and(c) cultivating the contacted combination in a cell culture medium such that the combination comprises a network of adipose-derived MVs at a density that is at least 10% greater than a plurality of stem cell-derived kidney tissue components that have not been cultivated with the network of adipose-derived MVs or plurality of MVFs, the density of penetrated kidney tissue components is measured based on an amount of penetrated kidney tissue components in a cross- sectional area of the in vitro vascularized kidney tissue comprising the stem cell-derived kidney tissue components.

66. The method of claim 65, wherein an expression profile of endothelial cells in the network of MVs comprise at least one markers selected from UEA1+, CD31+ or VEGFR2+.

67. The method of claim 65, wherein an expression profile of podocytes in the network of MVs comprise PODXL+.

68. The method of claim 65, wherein an expression profile of the renal tubule cells in the network of MVs comprise EPCAM+.

69. The method of claim 65, wherein an expression profile of the proximal tubule cells in the network of MVs comprise LTL+.

70. The method of claim 65, wherein the contacting comprises incorporating the stem cell- derived kidney tissue components and the network of adipose-derived MVs or plurality of MVFs into a bioink, wherein the bioink comprises a granular tissue, where the granular tissue comprises stem cell-derived spherical aggregates, spheroids, organoids, embryoid bodies, or a combination thereof.

71. The method of claim 65, wherein the contacting comprises incorporating the stem cell- derived kidney tissue components and the network of adipose-derived MVs or plurality of MVFs into a bioink, wherein the bioink comprises a suspension culture.

72. The method of claim 71, further comprising extruding the bioink at least one spatially defined location(s) on or within a three-dimensional framework.

73. The method of claim 72, wherein the three-dimensional framework is selected from at least one of a membrane, mesh, a grid, a sponge, a foam, or a combination thereof.

74. The method of claim 72, wherein the three-dimensional framework is selected from at least one of solubilized basement membrane preparation extracted from Engelbreth-Holm- Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibrin,Attorney Docket No. 224638-702601 fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, decellularized kidney ECM, or a combination thereof.

75. The method of claim 65, wherein an expression profile of cells in the stem cell-derived glomeruli or precursors thereof varies along a z-direction.

76. The method of claim 65, wherein the cultivating comprises exposure of the network of MVs to flow and / or shear.

77. The method of claim 65, wherein the cell culture medium comprises growth factors.

78. The method of claim 77, wherein the growth factors comprise at least one of an agent selected from the group consisting of CHIR99021, Noggin, Activin A, FGF1, FGF7, FGF9, BMP7, GDNF BMP4, Retinoic Acid, GDNF1, RSPO1, or a combination thereof.

79. The method of claim 65, wherein the cultivating is performed on an extracellular matrix (ECM).

80. The method of claim 79, wherein the ECM comprises at least one of solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibrin, fibronectin, gelatin, chitosan, polyethylene glycol, dextran, hyaluronic acid, decellularized kidney ECM, or a combination thereof.

81. The method of claim 65, wherein the network of adipose-derived MVs or plurality of MVFs comprise at least a portion or segment of at least one artery, arteriole, capillary, venule, vein, or a combination thereof.

82. The method of claim 65, wherein the network of adipose-derived MVs or plurality of MVFs are obtained from subcutaneous fat, perirenal fat, pericardial fat, omental fat, breast fat, epididymal fat, properitoneal fat, or a combination thereof.

83. The method of claim 65, wherein the cell culture medium comprises allogeneic components and autologous components.

84. The method of claim 65, wherein the cell culture medium comprises synthetic or semisynthetic materials.

85. An in vitro composition comprising the in vitro vascularized tissue of any one of claims 1 to Error! Reference source not found, or the in vitro vascularized kidney tissue of any one of claims 49 to 62.

86. The composition of claim 85, further comprising a cell culture medium.

87. The composition of claim 85, further comprising growth factors.Attorney Docket No. 224638-70260188. The composition of claim 87, wherein the growth factors comprise CHIR99021, Noggin, Activin A, FGF1, FGF7, FGF9, BMP7, GDNF BMP4, Retinoic Acid, GDNF1, RSPO1, or a combination thereof.

89. The composition of claim 85, further comprising at least one metabolite.

90. The composition of claim 89, wherein the at least one metabolite comprise: urea, creatinine, uric acid, ammonium phosphate, or a combination thereof.

91. The composition of claim 85, further comprising an extracellular matrix.

92. The composition of claim 91, wherein the extracellular matrix comprises: a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibronectin, vitronectin, retronectin, elastin, hyaluronic acid, methylcellulose, a gelatin, solubilized ECM from decellularized kidney?, or any combination thereof.

93. The composition of claim 85, further comprising a diluent.

94. The composition of claim 93, wherein the diluent comprises a cryopreservation agent, a serum, or a suspension.

95. The composition of claim 85, further comprising a population of glomerular progenitor cells or a differentiated progeny thereof.

96. The composition of claim 85, further comprising a population of human blood cells.

97. An in vitro composition comprising: an in iv / ra-differentiated kidney tissue comprising a population of glomerular tissues with a network of microvessels of a population of microvascular fragments (MVFs), wherein: the in vv / ra-differentiated kidney tissue that comprises spatially-organized progenitors of a collecting duct or a differentiated population of cells thereof, and the in vztro-differentiated kidney tissue comprises two or more markers selected from: LRP2, GATA3, MAFB, and CK8.

98. The in vitro composition of claim 97, wherein the in vztro-differentiated kidney tissue comprises two or more markers, wherein the two or more markers comprise:(i) MAFB and LRP2;(ii) MAFB and CK8;(iii) MAFB and GAT A3;(iv) LRP2 and CK8;(v) LRP2 and GAT A3; or(vi) CK8 and GAT A3.Attorney Docket No. 224638-70260199. The in vitro composition of claim 97, wherein the in vztro-differentiated kidney tissue comprises three or more markers, wherein the three or more markers comprise:(i) MAFB, LRP2, and CK8;(ii) MAFB, LRP2, and GAT A3;(iii) MAFB, CK8, and GAT A3; or(iv) LRP2, CK8, and GAT A3.

100. The in vitro composition of claim 97, wherein the in vztro-differentiated kidney tissue comprises four or more markers, wherein the four or more markers comprise: MAFB, LRP2, CK8, and GAT A3.

101. The in vitro composition of claim 97, wherein the population of glomerular tissues are derived from human stem cells.

102. The in vitro composition of claim 97, wherein the population of MVFs are derived from human adipose tissue.

103. The in vitro composition of claim 101, wherein the human stem cells are embryonic stem cells, induced pluripotent stem cells (iPSCs), or adult stem cells.

104. The in vitro composition of claim 97, wherein a portion of a core of the glomerular tissues comprise epithelial cells.

105. The in vitro composition of claim 97, wherein a portion of a core of the glomerular tissues comprise renal stromal cells.

106. The in vitro composition of claim 97, wherein a portion of a core of the glomerular tissues comprise epithelial cells and renal stromal cells.

107. The in vitro composition of claim 97, further comprising a cell culture medium.

108. The in vitro composition of claim 97, further comprising growth factors.

109. The in vitro composition of claim 108, wherein the growth factors comprise CHIR99021, Noggin, Activin A, FGF1, FGF7, FGF9, BMP7, GDNF BMP4, Retinoic Acid, GDNF1, RSPO1, or a combination thereof.

110. The in vitro composition of claim 97, further comprising at least one metabolite.

111. The in vitro composition of claim 110, wherein the at least one metabolite comprise urea, creatinine, uric acid, ammonium phosphate, or a combination thereof.

112. The in vitro composition of claim 97, further comprising an extracellular matrix.

113. The in vitro composition of claim 112, wherein the extracellular matrix comprises: a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, laminin, collagen IV, heparan sulfate proteoglycans, entactin, fibronectin,Attorney Docket No. 224638-702601 vitronectin, retronectin, elastin, hyaluronic acid, methylcellulose, a gelatin, or any combination thereof.

114. The in vitro composition of claim 97, further comprising a diluent.

115. The in vitro composition of claim 114, wherein the diluent comprises a cryopreservation agent, a serum, or a suspension.

116. A method of treating a kidney disease in a subject, the method comprising:(a) culturing a plurality of the in vitro composition of any one of claims 85 to 115 in a bioreactor; and(b) forming a blood circuit between the subj ecf s blood and the in vitro composition to remove excess waste and fluid, thereby treating the kidney disease.

117. A method of treating a disease in a subject, the method comprising: administering to the subj ect the in vitro composition of any one of claims 85 to 115, thereby treating the disease in the subject.

118. The method of claim 117, wherein the disease is a kidney disease.

119. The method of claim 118, wherein the kidney disease is a chronic kidney disease.

120. The method of claim 118, wherein the kidney disease is selected from the group consisting of: atypical hemolytic uremic syndrome (aHUS), Alport syndrome, amyloidosis, POL1- mediated kidney disease, cancer, cardiovascular kidney metabolic (CKM) syndrome, complement 3 glomerulopathy (C3G), cystinosis, diabetic kidney disease, end-stage renal failure, Fabry disease, focal segmental glomerulosclerosis (FSGS), glomerulonephritis (Glomerular Disease) Goodpasture syndrome, granulomatosis with polyangiitis (GPA), hemolytic uremic syndrome (HUS), Henoch-Schbnlein purpura (HSP), IgA nephropathy, interstitial nephritis, kidney failure, Lupus nephritis, minimal change disease, polycystic kidney disease, primary hyperoxaluria and oxalate, thrombotic thrombocytopenic purpura (TTP), and vasculitis of the kidney.

121. The method of claim 117, wherein the subject has, is diagnosed with, or is suspected of having kidney failure.

122. The method of claim 121, wherein the kidney failure is caused by at least one condition selected from the group consisting of: diabetes, high blood pressure, glomerulonephritis, polycystic kidney disease, lupus nephritis, IgA nephropathy, alcoholism, and nephrotoxicity.

123. The method of claim 117, wherein the subject has, or is diagnosed with a kidney injury.

124. The method of claim 123, wherein the kidney injury is caused by an infection, a pregnancy complication, a urinary tract obstruction, a kidney stone, or a physical injury.Attorney Docket No. 224638-702601125. The method of claim 117, wherein the subject has, or is diagnosed with a congenital abnormality.

126. The method of claim 117, wherein the subject has, or is diagnosed with a congenital abnormality, wherein the congenital abnormality comprises renal agenesis, renal dysplasia, or renal hypoplasia.

127. The method of claim 117, wherein the administering comprises surgical transplantation of the in vitro composition in the subject.

128. A reagent comprising: fetal bovine serum (FBS), retinoic acid, R-spondin 1 (RSPO1) protein, glial-derived neurotrophic factor (GDNF), fibroblast growth factor 1 (FGF1), fibroblast growth factor 7 (FGF7), LDN193189, and solubilized basement membrane preparation extracted from Engelbreth-Holm- Swarm (EHS) mouse sarcoma.

129. The reagent of claim 128, wherein the FBS is present in an amount that is at least about 1% up to 20% volume by total volume of the reagent (v / v).

130. The reagent of claim 128, wherein the retinoic acid is present in an amount that is at least about 10 nanoMolar (nM) concentration up to 200 nM concentration.

131. The reagent of claim 128, wherein the RSPO1 is present in an amount that is at least about 10 nanograms per milliliter (ng / mL) up to 200 ng / mL.

132. The reagent of claim 128, wherein the GDNF is present in an amount that is at least about 0.1 nanograms per milliliter (ng / mL) up to 5 ng / mL.

133. The reagent of claim 128, wherein the FGF1 is present in an amount that is at least about 10 nanograms per milliliter (ng / mL) up to 200 ng / mL.

134. The reagent of claim 128, wherein the FGF7 is present in an amount that is at least about 3 nanograms per milliliter (ng / mL) up to 60 ng / mL.

135. The reagent of claim 128, wherein the LDN193189 is present in an amount that is at least about 1 nanoMolar (nM) concentration up to 20 nM concentration.

136. The reagent of claim 128, wherein the solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma is present in an amount of at least 5% up to 80% volume by total volume of the reagent (v / v).

137. A kit comprising: a first container comprising: a population of glomerular cells and a network of adipose-derived MVs or plurality of MVFs; and a second container comprising media, growth factors, and agents for making an in vitro kidney tissue.

138. A transplant composition comprising: a first container comprising: the in vitro vascularized tissue of any one of claims 1 to 23 or the in vitro vascularized kidney tissue of anyAttorney Docket No. 224638-702601 one of claims 49 to 62 or a plurality thereof ; and a second container comprising: (a) an additional therapeutic agent; (b) at least one immunosuppressant; or (c) a combination thereof.

139. A method of treating a subject with a kidney disease, the method comprising: administering to the subject a transplant composition of claim 138, thereby treating the kidney disease.

140. The method of claim 139, wherein the second container of the transplant composition is administered to the subject prior to the first container.

141. The method of claim 139, wherein the second container of the transplant composition is administered to the subject at a same time as the first container.

142. The method of claim 139, wherein the second container of the transplant composition is administered to the subject after the first container.

143. A method of transplanting a kidney in a subject, the method comprising: engrafting a plurality of the in vitro vascularized tissue of any one of claims 1 to 23 or the in vitro vascularized kidney tissue of any one of claims 49 to 62 into a kidney of the subject.

144. A method comprising:(a) transplanting the in vitro vascularized tissue of any one of claims 1 to 23 or the in vitro vascularized kidney tissue of any one of claims 49 to 62 into a subject having a kidney disease; and(b) administering to the subject at least one immunosuppressive agent, wherein the method increases a glomerular filtration rate (GFR) of the subject relative to the GFR of the subject prior to transplantation.

145. A method comprising:(a) transplanting the in vitro vascularized tissue of any one of claims 1 to 23 or the in vitro vascularized kidney tissue of any one of claims 49 to 62 into a subject having a kidney disease; and(b) administering to the subject at least one immunosuppressive agent, wherein the method increases reduces a level of a biomarker indicative of nephrotoxicity in the subject relative to the level of the biomarker indicative of nephrotoxicity in the subject prior to transplantation.

146. The method of any one of claims 143 to 145, wherein the method further comprises surgically removing a diseased kidney from the subject.

147. The method of any one of claims 143 to 145, wherein the subject has a kidney disease.

148. The method of any one of claims 143 to 145, wherein the subject has a kidney injury.

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