Methods and Uses of Differentiated Cells

Abstract

Methods for producing, culturing, and maintaining modified podocyte-like cells are disclosed herein. These methods include growing glomerular cells under in vitro conditions to differentiate them into podocyte-like cells. Kits comprising isolated and purified podocyte-like cells and culture media used for differentiating modified podocyte-like cells and / or podocyte-like cells are also disclosed herein. Modified podocyte-like cells can be used for recellularization of decellularized extracellular matrix. In some cases, recellularized isolated organs containing modified podocyte-like cells are disclosed herein.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit and priority of U.S. Provisional Patent Application Publication No. 63 / 470,248, filed on 1 June 2023, which are incorporated by reference and jointly owned in their entirety. [Overview of the project]

[0002] A method for producing manipulated podocyte-like cells is disclosed herein. In some embodiments, the method may include a) culturing glomerular cells for about 2 to 4 days in a first medium containing at least one of retinoic acid, corticosteroids, calcitriol, or a salt of any one thereof; b) removing the glomerular cells from the first medium; and c) culturing the glomerular cells for about 6 to 12 days in a second medium containing at least one of SB431542, a salt thereof, IWR-1-endo, or a salt thereof, wherein culturing the glomerular cells in the second medium for about 6 to 12 days results in differentiation of the glomerular cells into manipulated podocyte-like cells.

[0003] In some embodiments, after glomerular cells are cultured in a second medium, the glomerular cells can be differentiated into engineered podocyte-like cells having increased expression of one or more of podosin, nephrin, podocalyxin, or synaptopodin compared to pre-growth glomerular cells in the first medium. In some embodiments, the corticosteroid may include dexamethasone or a salt thereof. In some embodiments, the first medium may further include a basal medium, a nutrient mixture, an antibiotic, insulin-transferrin-selenium (ITS), fetal bovine serum, a salt of any of these, or any combination thereof. In some embodiments, the antibiotic may include penicillin, a salt thereof, streptomycin, a salt thereof, or any combination thereof. In some embodiments, the concentration of retinoic acid or a salt thereof in the first medium may be about 1 μM to about 1 mM. In some embodiments, the concentration of corticosteroid or a salt thereof in the first medium may be about 100 nM to about 10 mM. In some embodiments, the concentration of calcitriol or a salt thereof in the first medium may be about 1 nM to about 300 nM. In some embodiments, the second medium may further include a basal medium, a nutrient mixture, an antibiotic, insulin-transferrin-selenium (ITS), fetal bovine serum, a salt of any of these, or any combination thereof. In some embodiments, the antibiotic may include penicillin, a salt thereof, streptomycin, a salt thereof, or any combination thereof. In some embodiments, the concentration of SB431542 or a salt thereof in the second medium may be about 1 μM to about 10 μM. In some embodiments, the concentration of IWR-1-endo or a salt thereof in the second medium may be about 1 μM to about 10 μM. In some embodiments, the manipulated podocyte-like cells may have increased gene expression of NPHS1, NPHS2, SYNPO, or any combination thereof, compared to glomerular cells. In some embodiments, the increased gene expression may be determined by quantitative reverse transcriptase PCR. In some embodiments, glomerular cells may be glomerular elongation cells. In some embodiments, the manipulated podocyte-like cells may include cytoskeletal tissue having multiple elongations compared to the cytoskeletal tissue of glomerular cells.In some embodiments, growing glomerular cells in a first medium for about 3 days may further include replacing the first medium with fresh first medium approximately 48 hours after cell growth. In some embodiments, growing glomerular cells in a second medium for about 7 days may include replacing the second medium with fresh second medium every 48 hours after cell growth. In some embodiments, the manipulated podocyte-like cells may have reduced expression of one or more of podosins, nephrins, podocalyxins, or synaptopodins compared to primary podocytes. In some embodiments, the glomerular cells may be animal glomeruli. In some embodiments, the animal glomeruli may be human glomeruli. In some embodiments, the animal glomeruli may be porcine glomeruli, sheep glomeruli, goat glomeruli, monkey glomeruli, bovine glomeruli, canine glomeruli, or feline glomeruli. In some embodiments, the first medium may contain retinoic acid or a salt thereof. In some embodiments, the first medium may contain a corticosteroid or a salt thereof. In some embodiments, the first medium may contain calcitriol or a salt thereof. In some embodiments, the second medium may contain SB431542 or a salt thereof. In some embodiments, the second medium may contain IWR-1-endo or a salt thereof. In some embodiments, glomerular cells may be grown in the first medium for about 3 days. In some embodiments, glomerular cells may be grown in the second medium for about 7 days. In some embodiments, glomerular cells may be grown in at least partially decellularized kidney extracellular matrix. In some embodiments, increased expression of one or more of podosin, nephrin, podocalyxin, or synaptopodin may be determined by fluorescence microscopy, Western blotting, flow cytometry, or any combination thereof. Manipulated podocyte-like cells prepared by the methods described above are also disclosed herein.

[0004] Furthermore, engineered podocyte-like cells are disclosed herein. In some embodiments, engineered podocyte-like cells may include a) increased expression of one or more of podosins, nephrins, podocalyxins, or synaptopodins compared to glomerular cells, and b) decreased expression of one or more of podosins, nephrins, podocalyxins, or synaptopodins compared to primary podocytes. In some embodiments, engineered podocyte-like cells may include a cytoskeletal structure with multiple elongations compared to the cytoskeletal structure of glomerular cells. In some embodiments, engineered podocyte-like cells may have increased gene expression of NPHS1, NPHS2, SYNPO, or any combination thereof, compared to glomerular cells. In some embodiments, engineered podocyte-like cells may have decreased localization of one or more of podosins, nephrins, podocalyxins, or synaptopodins compared to primary podocytes.

[0005] Also disclosed herein is a method for engrafting cells onto at least partially decellularized kidney extracellular matrix, comprising contacting the at least partially decellularized kidney extracellular matrix with a plurality of engineered podocyte-like cells. In some embodiments, the engineered podocyte-like cells may include a) increased expression of one or more of podosin, nephrin, podocalyxin, or synaptopodin compared to glomerular cells, and b) decreased expression of one or more of podosin, nephrin, podocalyxin, or synaptopodin compared to primary podocyte cells. In some embodiments, the engineered podocyte-like cells may include cytoskeletal tissue with multiple elongations compared to the cytoskeletal tissue of glomerular cells. In some embodiments, the engineered podocyte-like cells may have increased gene expression of NPHS1, NPHS2, SYNPO, or any combination thereof, compared to glomerular cells. In some embodiments, the manipulated podocyte-like cells may have reduced localization of one or more of podocin, nephrin, podocalyxin, or synaptopodin compared to primary podocytes. In some embodiments, contact may be carried out in a bioreactor chamber. In some embodiments, contact includes depositing a plurality of manipulated podocyte-like cells in an aqueous composition onto glomeruli of at least partially decellularized kidney extracellular matrix through the ureters of at least partially decellularized kidney extracellular matrix, thereby engrafting the cells onto the at least partially decellularized kidney extracellular matrix. In some embodiments, the method may further include seeding a plurality of mesangial cells, a plurality of human umbilical vein endothelial cells (HUVECs), or both. In some embodiments, deposition through the ureters may include generating a vacuum in a bioreactor chamber. In some embodiments, the method may further include continuously perfusing a culture medium through at least partially decellularized kidney extracellular matrix after engraftment. In some embodiments, the culture medium may be changed approximately every 24 hours.

[0006] Also disclosed herein are at least partially recellularized isolated organs or parts thereof, including engineered podocyte-like cells. In some embodiments, at least partially recellularized isolated organs or parts thereof in a closed-loop ambient temperature perfusion system may a) maintain urine / serum protein levels in urine less than 30% after 1 hour of ambient temperature perfusion and less than 65% after 4 hours of transplantation, or b) maintain urine / serum hematocrit levels in urine less than 30% after 1 hour of ambient temperature perfusion and less than 1% after 4 hours of transplantation. In some embodiments, engineered podocyte-like cells may include a) increased expression of one or more of podosin, nephrin, podocalyxin, or synaptopodin compared to glomerular cells, and b) decreased expression of one or more of podosin, nephrin, podocalyxin, or synaptopodin compared to primary podocyte cells. In some embodiments, engineered podocyte-like cells may include cytoskeletal tissue with multiple elongations compared to the cytoskeletal tissue of glomerular cells. In some embodiments, the manipulated podocyte-like cells may have increased gene expression of NPHS1, NPHS2, SYNPO, or any combination thereof, compared to glomerular cells. In some embodiments, the manipulated podocyte-like cells may have decreased localization of one or more of podosins, nephrins, podocalixins, or synaptopodins, compared to primary podocytes. In some embodiments, at least a partially recellularized isolated organ or part thereof may include a kidney or part thereof. In some embodiments, levels of creatinine, urea, sodium, potassium, glucose, lactate, bicarbonate, or any combination thereof may be determined in a closed-loop ambient temperature perfusion system. In some embodiments, at least a partially recellularized isolated organ or part thereof may be homogeneous with respect to the manipulated podocyte-like cells. In some embodiments, at least a partially recellularized isolated organ or part thereof may be autologous with respect to the manipulated podocyte-like cells. In some embodiments, at least a partially recellularized isolated organ or part thereof may be heterogeneous with respect to the manipulated podocyte-like cells.

[0007] Also disclosed herein is a kit comprising a first medium for growing podocyte-like cells, containing in a container at least one of retinoic acid, its salts, corticosteroids, its salts, calcitriol, or its salts, and a second medium for growing podocyte-like cells, containing in a container at least one of SB431542, its salts, IWR-1-endo, or its salts. In some embodiments, the first medium, the second medium, or both may contain glomerular cells.

[0008] Also disclosed is a method for producing manipulated podocyte-like cells, comprising: a) culturing glomerular cells for about 4 to 8 days in a first medium containing at least one of a transforming growth factor beta pathway inhibitor and a Wnt pathway inhibitor, or a salt thereof; b) removing the glomerular cells from the first medium; and c) culturing the glomerular cells for about 2 to 6 days in a second medium containing at least one of retinoic acid, a Rho kinase (ROCK) inhibitor, or a salt thereof, wherein culturing the glomerular cells in the second medium for about 2 to 6 days results in differentiation of the glomerular cells into manipulated podocyte-like cells.

[0009] In some embodiments, the manipulated podocyte-like cells have an increase in finger-like embedding foot processes compared to glomerular cells before culture in the first medium. In some embodiments, the manipulated podocyte-like cells express at least one of F-actin and vimentin. In some embodiments, the manipulated podocyte-like cells express at least one of podosin, nephrin, podocalyxin, or synaptopodin.

[0010] In some embodiments, the first culture medium comprises a transforming growth factor beta pathway inhibitor, wherein the transforming growth factor beta pathway inhibitor comprises SB431542. In some cases, the concentration of SB431542 is about 2 μM to 10 μM. In some cases, the concentration of SB431542 is 4 μM. In some embodiments, the first culture medium comprises a Wnt pathway inhibitor, wherein the Wnt pathway inhibitor comprises IWR-1. The method according to claim 57, wherein the concentration of IWR-1 is about 2 μM to 20 μM. In some cases, the concentration of IWR-1 is 2 μM.

[0011] In some embodiments, the second culture medium contains retinoic acid. In some cases, the concentration of retinoic acid is 0.2 μM. In some embodiments, the second culture medium contains a ROCK inhibitor, which is Y-27632. In some cases, the concentration of Y-27632 is approximately 2 μM to approximately 15 μM. In some cases, the concentration of Y-27632 is 10 μM.

[0012] In some embodiments, at least one of the first and second media further comprises at least one of heparin, a hormone, and a glycoprotein. In some cases, at least one of the first and second media further comprises insulin-transferrin-selenium or a salt thereof. In some cases, at least one of the first and second media further comprises an antibiotic. In some cases, at least one of the first and second media further comprises at least one of penicillin and streptomycin.

[0013] Also disclosed is a method for producing manipulated podocyte-like cells, comprising culturing glomerular cells in a medium containing a histone deacetylase inhibitor for at least about 4 to 8 days, wherein the culturing results in differentiation of the glomerular cells into manipulated podocyte-like cells. In some cases, the histone deacetylase inhibitor includes hydroxamic acid or a salt thereof. In some cases, the histone deacetylase inhibitor includes panobinostat or a salt thereof. In some cases, the concentration of panobinostat is about 50 nM to about 200 nM. In some cases, the concentration of panobinostat is 50 nM.

[0014] In some embodiments, the culture medium further comprises at least one of hormones and glycoproteins. In some cases, the culture medium further comprises insulin-transferrin-selenium (ITS). In some cases, the culture medium further comprises an antibiotic. In some cases, the culture medium further comprises at least one of penicillin and streptomycin.

[0015] Also disclosed is a method for maintaining manipulated podocyte-like cells, comprising culturing the manipulated podocyte-like cells in a medium containing at least one of penicillin-streptomycin, fetal bovine serum, heparin, ascorbic acid, hydrocortisone, rh FGF, rh VEGF, rh EGF, Long R3 IGF, insulin, triiodothyronine, epinephrine, holotransferrin, and SB431542. In some cases, the concentration of penicillin-streptomycin is 1%. In some cases, the concentration of fetal bovine serum is 2%. In some cases, the concentration of heparin is 1.05 U / mL. In some cases, the concentration of ascorbic acid is 50 μg / mL. In some cases, the concentration of hydrocortisone is 1.15 μg / mL. In some cases, the concentration of rh FGF is 20 ng / mL. In some cases, the concentration of rh VEGF is 5 ng / mL. In some cases, the rh EGF concentration is 15 ng / mL. In some cases, the Long R3 IGF concentration is 15 ng / mL. In some cases, the insulin concentration is 0.125 U / mL. In some cases, the triiodothyronine (T3) concentration is 10 nM. In some cases, the epinephrine concentration is 1 μM. In some cases, the holotransferrin concentration is 5 μg / mL. In some cases, the SB431542 concentration is 4 μM.

[0016] Built-in by reference All publications, patents, and patent applications referenced herein are incorporated by reference to the same extent as any individual publication, patent, or patent application is specifically and individually indicated as being incorporated by reference.

[0017] Novel features of this disclosure are described in particular in the accompanying claims. A better understanding of the features and advantages of this disclosure can be obtained by referring to the detailed description below, which describes exemplary embodiments in which the principles of this disclosure are utilized, and to the accompanying drawings. [Brief explanation of the drawing]

[0018] [Figure 1] This diagram shows that glomerular elongating cells (small, elongated / round cells) differentiate into podocyte-like cells (large, dendrite-bearing cells) when exposed to the displayed culture medium scheme. Each square represents one day of culture, and the arrows indicate when a medium change is performed using a specific medium. The cells were cultured in medium 1 for 3 days and in medium 2 for 7 days. [Figure 2-1] This figure shows functional data of a bioengineered kidney and a 3D-manipulated podocyte-like cell culture. Figure 2A shows that a porcine kidney graft containing manipulated podocyte-like cells demonstrated sustained (over 3 hours) protein retention (indicated by protein levels less than 100%) and blood cell retention (indicated by low hematocrit %) in the blood. This data demonstrates the filtration function of a bioengineered kidney containing manipulated podocyte-like cells when ectopically transplanted in pigs. Figure 2B shows that filtration function is demonstrated by a bioengineered kidney with a dual culture (HUVEC and manipulated podocyte-like cells) ectopically transplanted. Protein levels (g / L) and hematocrit (HCT) (%) in serum and urine from a porcine ectopic kidney transplant model are shown. Urine protein levels started at 40 g / L and decreased over time to approximately 30 g / L. Urine HCT levels were less than approximately 2% throughout the entire 5-hour experiment. Figure 2C shows the regrowth of glomeruli by podocyte-like cells exhibiting immunofluorescence labeling and a functional podocyte phenotype in recellularized kidney cells (recellularized with manipulated podocyte-like cells), expressing podosin (a functional protein of podocytes) and exhibiting primary protrusion formation (a definitive characteristic of podocytes, indicated by the arrows). [Figure 2-2] (As stated above.) [Figure 2-3] (As stated above.) [Figure 3-1]Figures 3A-3F: Diagrams showing engineered podocyte-like cells in a two-dimensional cell culture. Figure 3A shows immunofluorescent labeling of a second media-treated glomerular extension cell (e.g., engineered podocyte-like cell) cultured on collagen I against F-actin. Figure 3B shows immunofluorescent labeling of a second media-treated glomerular extension cell (e.g., engineered podocyte-like cell) cultured on collagen I against vimentin. Figure 3C shows immunofluorescence of a second media-treated glomerular extension cell (e.g., engineered podocyte-like cell) showing prominent nephrin. Figure 3D shows changes in podocyte gene expression (nephrin (NPHS1), podocin (NPHS2), and synaptopodin (SYNPO)) in engineered podocyte-like cells after culture in a second media as compared to standard R-endothelial media. Figures 3E and 3F show immunofluorescent labeling of engineered podocyte-like cells against vimentin. Arrows indicate where cell secondary processes interact. [Figure 3-2] (As described above.) [Figure 3-3] (As described above.) [Figure 3-4] (As described above.) [Figure 3-5] (As described above.) [Figure 3-6] (As described above.) [Figure 4] A diagram showing a comparison of podocin expression in primary podocytes (left image) directly obtained from the kidney as compared to engineered podocyte-like cells (right image) created using the methods described herein. [Figure 5] A diagram showing immunofluorescent images of podocalyxin expression in glomerular extension cells before (prior to differentiation) and 6 days after culturing in a second media. Six days after differentiation, the glomerular extension cells are differentiated into engineered podocyte-like cells and have increased expression and proper localization of expression in podocalyxin. The spots in the image prior to differentiation indicate either very limited expression of podocalyxin or the stained nuclei of cells with no expression. In the image 6 days after differentiation, podocalyxin is scattered on the cell membrane of the engineered podocyte-like cells. [Figure 6-1]Figures 6A-B show the in vitro functional properties of bioengineered kidney constructs, including urine flow rate, normalized protein, and normalized erythrocyte volume ratio (PCV). Figure 6A shows ambient perfusion data from bioengineered kidney constructs of independent HUVEC alone (n=5) and dual culture (n=5). Dual culture bioengineered kidney constructs contained HUVEC and engineered podocyte-like cells. PCV (%) readings are expressed as normalized value ([urine value] / [serum value]) × 100. Error bars indicate the mean and standard deviation at each time point. * indicates p ≤ 0.005 when comparing the HUVEC-only graft to the dual culture graft at each time point, as determined by two-way ANOVA / Sidak multiple comparison test. Figure 6B shows that the filtration function of the dual culture (HUVEC and engineered podocyte-like cell) kidney graft is demonstrated by ambient perfusion experiments. Dual-culture urine HCT was less than approximately 15% at 30 and 60 minutes. Dual-culture urine protein was less than approximately 10 g / L at 30 and 60 minutes. The graph shows the mean and standard deviation of bioengineered kidney constructs for independent HUVEC alone (n=3) and dual culture (n=5). * indicates p ≤ 0.005 when dual-culture urine values ​​are compared to all other values ​​by two-way ANOVA / Sidak multiple comparison test. [Figure 6-2] (As stated above.) [Figure 7] This figure shows the results of ectopic transplantation of kidney constructs bioengineered from co-cultures (HUVEC and engineered podocyte-like cells) in a porcine ectopic kidney transplantation model. The graphs show urine flow rate, normalized protein, and normalized erythrocyte volume ratio (PCV). Measurements of renal filtration function from three independent acute porcine dual-culture implants (N=1) show improvement in function in each test. Protein (mg / dL) and PCV (%) readings are expressed as normalized values ​​([urine value] / [serum value]) × 100. [Figure 8] This figure outlines the culture strategy for developing a kidney by bioengineering using the modified podocyte-like cells described herein. [Figure 9]This diagram shows a schematic example of a PSM-YoDa differentiation culture schedule, where cells are cultured in PSM for 6 days, followed by 4 days in YoDa. The culture medium is changed every 48 hours (indicated by arrows). [Figure 10] This figure shows the morphology of glomerular elongating cells treated with the PSM-YoDa differentiation scheme. The left panel is a bright-field image showing finger-like indentation foot processes between adjacent differentiated podocytes (circular), a characteristic of mature podocytes that form a filtration barrier. The right panel is an immunofluorescence image of the cytoskeletal proteins F-actin (green) and vimentin (red), showing the formation of cytoskeletal elongation (arrows) that form foot processes in mature podocytes. The scale bar is 50 microns. [Figure 11] This figure shows podocyte protein expression after differentiation according to the PSM-YoDa differentiation schedule. (Panel A) Demonstrates the presence of cells with high levels of podocyte protein expression and primary, secondary, and tertiary foot processes (arrows). (Panel B) Immunofluorescence labeling of PSM-YoDa-treated glomerular elongating cells for podosin (green) and (Panel C) nephrin (red). (Panel D) Flow cytometry demonstrated that 75% of cells express the podocyte protein nephrin after differentiation. [Figure 12] This diagram shows a schematic example of a panobinostat differentiation culture schedule, where cells are cultured in panobinostat medium for 6 days. The medium is replaced every 48 hours (indicated by arrows). [Figure 13] This figure shows the morphology of glomerular elongating cells treated with panobinostat medium. Immunofluorescence staining with cytoskeletal markers vimentin (red) and F-actin (green) shows protrusion formation, a characteristic of mature podocytes, 2 days after panobinostat treatment (DMEM (A, D) compared to panobinostat conditions (B, C, E, F)). Morphological changes were widespread and observed at both 50 nM and 200 nM concentrations of panobinostat. [Figure 14]This figure shows podocyte gene expression after panobinostat differentiation. Podocyte marker gene expression was upregulated 2 days after treatment with 200 nM panobinostat compared to control medium (RM). Six days of treatment with 50 nM panobinostat resulted in higher synaptopodin and podocalixin gene expression (gene expression levels compared to RM control medium) than in PSM. These results were demonstrated using two different cell lots (GO-HK-220604 and GO-HK-220702). P = passage number. [Figure 15-1] Figures 15A-B: These figures show podocyte protein expression after maintenance culture using KCM+SB. (Figure 15A) Immunofluorescence labeling of nephrin (green), podosin (red) and (Figure 15B) synaptopodin (green), podocalyxin (red) demonstrates high podocyte protein expression in glomerular elongating cells differentiated using either PSM (top panel) or YoDa (bottom panel), followed by maintenance culture in KCM+SB. Cells were counterstained with DAPI. Scale bar = 100 μm. [Figure 15-2] (As stated above.) [Modes for carrying out the invention]

[0019] definition Throughout this application, various embodiments may be presented in range form. A range description should be considered to specifically disclose all conceivable subranges and individual numerical values ​​within that range. For example, a range description such as 1 to 6 should be considered to specifically disclose subranges within that range such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and individual values ​​such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0020] Unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" are used herein as including the plural forms. Therefore, unless the reverse is indicated, the numerical parameters described herein are approximations that may vary depending on the desired characteristics to be obtained.

[0021] Unless otherwise specified, open terms such as "contain," "contain," "include," and "include" mean "to include."

[0022] The terms “determining,” “measuring,” “judging,” “evaluating,” “assaying,” and “analyzing” are often used interchangeably herein to refer to forms of measurement, including determining whether an element may or may not exist (e.g., detection), or determining the quantity of an element. These terms may include quantitative and qualitative determinations. Alternatively, evaluation may be relative or absolute. “Detecting the presence” includes determining the quantity of something that exists, and determining whether an element may or may not exist.

[0023] The terms “substantially” or “essentially” refer to a qualitative state indicating the overall or nearly overall range or degree of the characteristics or properties of interest. In some cases, “substantially” refers to at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 99.99% of the overall range or degree of the characteristics or properties of interest. In some cases, “substantially” or “essentially” refers to an amount that may be about 100% of the total amount.

[0024] The term "at least partially" refers to a qualitative state indicating a partial range or degree of the desired characteristic or trait. In some cases, "at least partially" refers to at least approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the overall range or degree of the desired characteristic or trait.

[0025] Where used herein, the terms “about” or “approximately” mean an acceptable margin of error for a particular value, as determined by those skilled in the art, and which depends somewhat on the method by which the value is measured or determined, for example, on the limits of the measuring system. For example, “about” means + or -10%, in accordance with the practice of the art; or “about” means a range of + or -10%, + or -5%, or + or -1% of a given value; or, particularly with respect to biological systems or processes, the term means within one order of magnitude, within five times, within four times, within three times, or within two times the value. Where a particular value is described in this application and claims, unless otherwise specified, the term “about” should be assumed to mean an acceptable margin of error for that particular value. Also, where a range and / or subrange of a value is provided, the range and / or subrange may include the endpoint of the range and / or subrange.

[0026] As used herein, the percentage of a composition's materials (e.g., biological materials, excipients, compounds, and / or components) is relative to the total weight of the composition. In some cases, the percentage of a composition's materials is relative to the total volume of the composition. In some cases, "weight percentage" or "w / w" means the ratio of the mass of a specified component to the mass of the entire composition (e.g., a dosage unit).

[0027] The terms “subject,” “host,” “individual,” and “patient” are used interchangeably herein to refer to animals, usually mammals. Any suitable mammal may be administered compositions as described herein or treated in any manner as described herein. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), domesticated animals (e.g., dogs and cats), livestock (e.g., horses, cattle, goats, sheep, pigs), and laboratory animals (e.g., mice, rats, rabbits, guinea pigs). Mammals may be of any age or at any developmental stage; for example, a mammal may be neonatal, infant, adolescent, adult, or in utero. In some embodiments, the subject is human. Humans may be over or equal to approximately 1, 2, 5, 10, 20, 30, 40, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 years of age. Humans may be under approximately 1, 2, 5, 10, 20, 30, 40, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 years of age. In some cases, humans may be under approximately 18 years of age. In some cases, humans may be approximately 1 week to approximately 5 weeks of age, 1 month to approximately 12 months of age, approximately 1 year to approximately 20 years of age, approximately 15 years to approximately 50 years of age, approximately 40 years to approximately 80 years of age, or approximately 60 years to approximately 110 years of age. In some cases, humans may be over approximately 18 years of age. Human subjects may be children. Human subjects may be adults. Human subjects may be children. Mammals such as humans may be born male or female. In some embodiments, subjects may have or be suspected of having a disease or condition, such as kidney disease. Subjects may be patients, such as patients receiving treatment for a condition or disease, such as kidney disease. In some cases, subjects may be responders to therapy. In some cases, subjects may be non-responders to therapy. Subjects may have a predisposition to developing a condition or disease. Subjects may be in remission from a condition or disease. In some cases, subjects may be healthy. Subjects may be subjects requiring treatment.

[0028] The term "recipient" and its grammatical equivalents, as used herein, refer to the subject. The recipient may also require treatment, such as treatment for a disease like kidney disease. In some embodiments, the recipient may require prophylactic treatment.

[0029] "Therapeutally effective amount" refers to the amount of a composition disclosed herein that may or may not have additional agents effective in achieving its intended purpose, for example, in treating a disease. Individual patient needs may vary. Generally, the dose required to provide an effective amount of a composition will vary depending on the recipient's age, health condition, physical condition, sex, weight, severity of the disease, frequency of treatment, and the nature and extent of the disease or condition.

[0030] As used herein, the terms “treatment” or “treatment” refer to a pharmaceutical or other intervention regimen for obtaining a beneficial or desired outcome in a recipient. Beneficial or desired outcomes include, but are not limited to, therapeutic benefits and / or prophylactic benefits. A therapeutic benefit refers to the elimination or recovery of one or more symptoms of the underlying disorder being treated. A therapeutic benefit may also be achieved by the elimination or recovery of one or more physiological symptoms associated with the underlying disorder, such that improvement may be observed in the subject even if the subject still suffers from the underlying disorder. Prophylactic benefits include delaying, preventing, or eliminating the onset of a disease or condition; delaying or eliminating the onset of symptoms of a disease or condition; slowing, stopping, or reversing the progression of a disease or symptom; or any combination thereof. With respect to prophylactic benefits, subjects at risk of developing a particular disease, or subjects reporting one or more physiological symptoms of a disease, may receive the treatments disclosed herein, even if they have not been diagnosed with the disease.

[0031] As used herein, “cell” refers to a biological cell. A cell can be the basic structural, functional, and / or biological unit of an organism. A cell may originate from any organism that has one or more cells. Some non-limiting examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotes, protist cells, plant-derived cells, animal cells, cells of invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), cells of vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), and cells of mammals (e.g., pigs, cattle, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.). In some cases, cells may not originate from natural organisms (e.g., cells can be made synthetically and may be referred to as artificial cells). Mammalian cells, for example, derived from mammals including experimental animals and humans, are of particular interest. In some cases, the cells are kidney cells, such as glomerular cells, podocytes, or podocyte-like cells. In some cases, the cells are engineered cells, such as cells that have been modified by humans to express certain proteins and / or functional properties. In some cases, engineered cells are cultured under artificial conditions to express certain proteins and / or functional properties.

[0032] As used herein, a substance is “pure” or “substantially pure” if it substantially contains no other components. The terms “purify,” “to purify,” and “purified,” when applied to cells, may refer to cells that have been separated from at least some of the associated components at the time of their initial production or generation (e.g., whether in nature or in a laboratory environment) or at any time after their initial production. Cells or cell populations may be considered purified if they are isolated at the time of production or after production, for example, from material or environment containing the cells or cell population, or by subculturing in culture, and purified cells or cell populations may contain at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more than about 90% of other material and are still considered “isolated.” Purified cells and cell populations may be pure to at least about 80 wt(w / w)%, about 85 wt(w / w)%, about 90 wt(w / w)%, about 91 wt(w / w)%, about 92 wt(w / w)%, about 93 wt(w / w)%, about 94 wt(w / w)%, about 95 wt(w / w)%, about 96 wt(w / w)%, about 97 wt(w / w)%, about 98 wt(w / w)%, about 99 wt(w / w)%, or at least more than about 99 wt(w / w)%. In the case of cell compositions provided herein, one or more cell types present in the composition may be produced in the material or environment containing the cell type and / or purified independently from one or more other cells present. Cell compositions and their cellular components are generally purified from animal or biological samples.

[0033] Isolated cells may (1) be separated from at least some of the components to which they were initially associated (whether naturally or in an experimental environment), and / or (2) be produced, prepared, purified, and / or manufactured artificially using artificial culture conditions, such as, but not limited to, culturing in one or more culture media. Isolated cells may contain cultured cells even if such culture is not a single culture. Isolated cells may be separated from other components to which they were initially associated by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more. Isolated cells may be pure by more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99%. The cell populations of the biological sample provided herein may consist of one or more cells which may subsequently be isolated from such sample. The isolated cells may be provided in a form that does not exist in nature.

[0034] The terms “decellularized” or “decellularized,” as used herein, refer to a biological structure (e.g., an isolated organ or part thereof, or tissue) in which the cellular and tissue content has been reduced or removed, leaving a cell-free base. Organs such as kidneys may be composed of various specialized tissues. The specialized tissue structure, or parenchyma, of an organ may provide a specific function associated with the organ. The supporting fibrous network of an isolated organ may be stroma. Most organs have a stromatous framework composed of non-specialized connective tissue supporting specialized tissues. The decellularization process may remove at least partially the cellular portion of a tissue, leaving a complex three-dimensional network of extracellular matrix (ECM). The ECM base may be composed primarily of collagen, but may also include cytokines, proteoglycans, laminins, fibrillins, and other proteins secreted by cells. At least partially decellularized structures may provide a biocompatible substrate into which various cell populations can be injected, or they may be used for implantation as cell-free medical devices that allow for cell invasion and remodeling after transplantation or application. Decellularized biological structures may be rigid or semi-rigid and possess the ability to change their shape. Examples of decellularized isolated organs include, but are not limited to, solid organs such as the heart, kidneys, liver, lungs, pancreas, brain, bones, spleen, gallbladder, bladder, uterus, ureters, and urethra.

[0035] When used herein, the terms “recellularize” or “re-cellatilize” may refer to the engraftment or distribution of cells, as described herein, onto a decellularized extracellular matrix. A recellularized organ may include the morphology or activity of a naturally occurring, non-decellularized organ.

[0036] The term “functional” and its grammatical equivalents, as used herein, may refer to the ability to operate, have, or perform an intended purpose. Functional can include any percentage from the baseline up to 100% of the intended purpose. For example, functional can include 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 the intended purpose. In some embodiments, the term functional can mean more than 100% or about 100% of normal function, for example, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, or up to about 1000% of the intended purpose.

[0037] The section headings used herein are for structural purposes only and should not be construed as limiting the subject matter described herein.

[0038] Overview This specification describes manipulated podocyte-like cells and methods for producing, maintaining, and culturing them. These cells can be used in the recellularization of decellularized organs. The recellularization process described herein achieves kidney function by targeting appropriate cells to specific parts of the decellularized extracellular matrix, which can mimic the physiological ultrastructure of the kidney. Kidney function is to filter blood, allowing certain blood components to pass from the blood into the urine, a process completed in kidney structures called nephrons. The filtration process can be mediated by specialized cells called podocytes located within structures called glomeruli. Primary podocytes are non-proliferative and terminally differentiated cells, and their availability is very limited. This scarcity hinders the ability to produce functional kidneys for transplantation, as it requires approximately 500 million podocytes per decellularized kidney to establish adequate ultrafiltration capacity. Therefore, it is necessary to develop alternatives to primary podocytes.

[0039] Methods for producing manipulated podocyte-like cells are disclosed herein. Methods for using manipulated podocyte-like cells and for recellularizing decellularized organs are also disclosed herein. Manipulated podocyte-like cells can be used in the treatment of diseases such as kidney disease. In some embodiments, the methods for differentiating manipulated podocyte-like cells described herein can leverage the capabilities of glomerular elongating cells and accordingly prepare them as podocyte precursors to become podocyte-like cells under appropriate conditions.

[0040] Podocyte-like cells Modified podocyte-like cells are disclosed herein. Where used herein, podocyte-like cells refer to modified podocyte-like cells. Methods herein can be used to produce modified podocyte-like cells. Podocyte-like cells can be differentiated from other cells, such as glomerular cells.

[0041] In some embodiments, podocyte-like cells are functionally similar to wild-type podocyte cells, such as primary podocyte cells, and may possess one or more of the physiological characteristics of wild-type cells. Podocytes are cells in Bowman's capsule in the kidney, which surround the glomerular capillaries. Podocytes constitute the inner epithelial layer of Bowman's capsule and contribute to blood filtration. In some cases, podocytes filter blood and retain large molecules such as proteins. Small molecules in the blood, such as water, salts, and sugars, are filtered as a step in urine formation. In some cases, podocytes are specialized epithelial cells present in the visceral layer of the capsule. In some cases, podocyte-like cells described herein filter blood and other fluids. In some cases, podocyte-like cells filter and retain large molecules such as proteins, but can remove small molecules such as water, salts, and sugars. Podocyte-like cells can be used as a substitute for primary podocytes to restore organ function. In some cases, podocyte-like cells can be used in the recellularization of decellularized organs or parts thereof. In some cases, podocyte-like cells can be used to replace primary podocytes in the kidney.

[0042] In some embodiments, podocyte-like cells may possess long, foot-like projections called pedicels. In some cases, pedicels may enclose capillaries, leaving slits between them. In some cases, blood or other fluids may be filtered through these slits, known as filtration slits, slit diaphragms, or slit pores, respectively. In some cases, certain proteins, such as nephrin, may be required for pedicels to enclose capillaries and function. In some cases, nephrin is a zipper-like protein that forms slit diaphragms in podocytes or podocyte-like cells, where the space between the teeth of the zipper is large enough to allow sugar and water to pass through, but too small to allow proteins to pass through. In some cases, nephrin deficiency may cause renal failure.

[0043] In some embodiments, the manipulated podocyte-like cells may exhibit increased expression of one or more of podosin, nephrin, podocalyxin, or synaptopodin compared to cells. In some cases, the cells may include glomerular cells. In some embodiments, the manipulated podocyte-like cells may exhibit decreased expression of one or more of podosin, nephrin, podocalyxin, or synaptopodin compared to cells such as primary podocytes. In some cases, the manipulated podocyte-like cells may exhibit increased expression of one or more of podosin, nephrin, podocalyxin, or synaptopodin compared to glomerular cells, and decreased expression of one or more of podosin, nephrin, podocalyxin, or synaptopodin compared to primary podocytes. In some cases, protein expression, such as the expression of podosin, nephrin, podocalyxin, or synaptopodin, can be measured by microscopic assays (e.g., fluorescence microscopy), Western blotting, dot blotting, functional assays, or any combination thereof. In some cases, podocyte-like cells may contain cytoskeletal tissue with multiple elongations compared to the cytoskeletal tissue of cells such as glomeruli. In some cases, podocyte-like cells may have more than, less than, or equal to approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 elongations. In some cases, podocyte-like cells may have approximately 1 to 30 elongations, 1 to 10 elongations, 5 to 15 elongations, 3 to 18 elongations, or 10 to 20 elongations. In some cases, podocyte-like cells may have increased gene expression of NPHS1, NPHS2, SYNPO, or any combination thereof, compared to cells such as glomeruli. In some cases, increased gene expression can be determined by quantitative reverse transcriptase PCR, Northern blotting, RNA sequencing, or any combination thereof. In some cases, manipulated podocyte-like cells exhibit reduced localization of one or more of the following compared to primary podocytes: podosin, nephrin, podocalyxin, or synaptopodin. In some cases, localization can be determined by fluorescence microscopy.

[0044] Methods and compositions for producing and maintaining manipulated podocyte-like cells. Methods and compositions for producing manipulated podocyte-like cells are disclosed herein. In some embodiments, a method for producing manipulated podocyte-like cells may include culturing cells in one or more culture medium compositions. In some embodiments, a method for producing manipulated podocyte-like cells may include culturing manipulated podocyte-like cells in a first medium and a second medium. In some embodiments, the method may include culturing manipulated podocyte-like cells in a third medium. In some cases, the third medium is a maintenance medium. In some cases, the compositions herein may include a first medium, a second medium, or any combination thereof, having or not having cells such as manipulated podocyte-like cells or glomerular cells. In some embodiments, cells are cultured in the first medium and then transferred to the second medium. In some cases, cells may be cultured in the second medium and then transferred to the first medium. In some cases, cells may be cultured in the first medium only or the second medium only. In some cases, the first medium or the second medium may be mixed with another medium. In some cases, the first and second culture media can be mixed (for example, in a 1:1 ratio).

[0045] In some cases, glomerular cells can be seeded into a decellularized organ, such as a decellularized kidney, cultured in a culture medium, and differentiated into engineered podocyte-like cells. A decellularized kidney may consist of, or be essentially composed of, the extracellular matrix of a native kidney. For example, glomerular cells can be transplanted into the glomeruli of a decellularized kidney (e.g., a pig kidney) and differentiated into engineered podocyte-like cells using the method described herein. In some cases, glomerular cells can be transplanted into the glomeruli of a decellularized kidney and differentiated into engineered podocyte-like cells by culturing in a first culture medium and / or a second culture medium. In some cases, glomerular cells can be differentiated in situ. In some cases, further cell populations, such as human umbilical vein endothelial cells (HUVECs), can be co-cultured with engineered podocyte-like cells.

[0046] In some embodiments, the method for producing manipulated podocyte-like cells includes culturing glomerular cells in a first medium for about 2 to 4 days. In some embodiments, the method for producing manipulated podocyte-like cells includes culturing glomerular cells in a first medium for about 4 to 8 days. In some cases, the method includes culturing glomerular cells in a first medium for about 3, 4, 5, or 6 days. In some cases, the method includes culturing glomerular cells in a first medium for about 6 days. In some cases, the first medium includes at least one of retinoic acid, its salts, corticosteroids, its salts, calcitriol, or its salts. In some embodiments, the method includes transferring glomerular cells from a first medium to a second medium and culturing the glomerular cells in a second medium for about 6 to 12 days. In some cases, the method includes culturing glomerular cells in a second medium for about 7 to 10 days or about 7 to 9 days. In some cases, the method involves culturing glomerular cells in a second medium for approximately 7 days. In some cases, the second medium contains at least one of SB431542, its salt, IWR-1-endo, or its salt. In some embodiments, after the glomerular cells have been cultured in the second medium, they are differentiated into engineered podocyte-like cells. In some cases, the engineered podocyte-like cells may have increased expression of one or more of podosin, nephrin, podocalyxin, or synaptopodin compared to glomerular cells before culturing in the first medium, the second medium, or both. In some cases, the expression of podosin, nephrin, podocalyxin, or synaptopodin can be determined by fluorescence microscopy, Western blotting, flow cytometry, or any combination thereof. In some cases, the first medium is used before the second medium. In some cases, the second medium is used before the first medium. In some cases, glomerular cells may include glomerular elongating cells, primary glomerular cells, or established glomerular cell lines. Glomerular cells can be obtained from any animal, such as humans. In some cases, glomerular cells may be porcine glomerulosides, sheep glomerulosides, goat glomerulosides, monkey glomerulosides, bovine glomerulosides, canine glomerulosides, feline glomerulosides, or mixtures thereof.

[0047] In some embodiments, the method may include culturing glomerular cells in a first medium for about 1 to 12 days, 2 to 8 days, 2 to 4 days, 3 to 7 days, 3 to 4 days, 4 to 8 days, or 5 to 6 days. In some embodiments, the method may include culturing glomerular cells in a first medium for a period of time longer than, less than, or equal to, about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days. In some embodiments, the method may include culturing glomerular cells in a second medium for about 1 to 24 days, 4 to 18 days, 1 to 8 days, 6 to 12 days, 5 to 15 days, 8 to 16 days, or 9 to 20 days. In some embodiments, the method may include culturing glomerular cells in a second medium for a period of time longer than, less than, or equal to, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 days.

[0048] In some embodiments, the first medium, the second medium, or both can be replaced with fresh medium approximately 48 hours after cell culture. In some cases, the first medium, the second medium, or both can be replaced with fresh medium approximately 1 to 96 hours after cell culture. In some cases, the first medium, the second medium, or both can be replaced with fresh medium after approximately 6, 12, 24, 48, 72, or 96 hours of cell culture, or after a time longer than, less than, or equal to those times. In some cases, the fresh medium may consist of fresh first medium or fresh second medium. In some cases, cells can be cultured at any temperature suitable for growth, for example, approximately 34°C, 35°C, 36°C, 37°C, or 38°C. In some cases, cells can be cultured at a CO2 concentration of approximately 2% to approximately 15% CO2 (v / v).

[0049] In some cases, the first medium includes a basal medium, a nutrient mixture, antibiotics, insulin-transferrin-selenium (ITS), serum, retinoic acid, corticosteroids, calcitriol, salts of any of these, or any combination thereof, or salts thereof. In some cases, the basal medium may include Dulbecco's Modified Eagle Medium, Basic Medium Eagle, Glasgow Minimum Essential Medium, Iskov Modified Dulbecco's Medium, Grace Insect Medium, Minimum Essential Medium, RPMI Medium, McCoy 5A, or any combination thereof. In some cases, the basal medium may include a compound medium. In some cases, the nutrient mixture may include F-12, F-10, a non-essential amino acid solution, or any combination thereof. In some embodiments, the serum may include fetal bovine serum, horse serum, calf serum, rabbit serum, pig serum, goat serum, human serum, or any combination thereof. In some cases, the medium may be serum-free or low-serum medium. In some cases, the first medium includes ITS. In some cases, ITS may be used to replace serum in the culture medium. In some cases, the culture medium may contain serum substitutes (e.g., serum replacements) or modified serum. In some cases, the antibiotic may include penicillin, streptomycin, beta-lactam, tetracycline, trimethoprim-sulfamethoxazole, lincosamide, fluoroquinolone, cephalosporin, macrolide, aminoglycoside, amphotericin, chloramphenicol, ampicillin, vancomycin, lincomycin, carbenicillin, gentamicin, neomycin, benzylpenicillin, rifampicin, mitomycin C, kanamycin, erythromycin, fosmidomycin, salts of any of these, or any combination thereof. In some cases, the antibiotic may include penicillin or a salt thereof and streptomycin or a salt thereof. In some cases, the first medium may include equilibrium salt solutions such as phosphate-buffered saline, Dulbecco's phosphate-buffered saline, Hanks equilibrium salt solution, Earl equilibrium salt, or any combination thereof. In some cases, the first medium may include endothelial growth medium.In some cases, the endothelial growth medium may consist of an endothelial cell growth base medium supplemented with one or more of the following: fetal bovine serum, ascorbic acid, hydrocortisone, fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), R3 IGF, heparin, acetate, and / or antibiotics.

[0050] In some embodiments, the culture media described herein may contain antibiotics in amounts (by weight / weight or volume / volume) greater than, less than, or equal to about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In some cases, the culture media described herein may contain antibiotics in amounts (by weight / weight or volume / volume) of about 0.1% to 1%, 0.1% to 10%, 1% to 10%, or 3% to 8%. In some embodiments, the culture media described herein may be approximately 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35% The culture media may contain serum in amounts (weight / weight or volume / volume) greater than, less than, or equal to, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%. In some cases, the culture media described herein may contain serum in amounts (weight / weight or volume / volume) of about 0.1% to 60%, 1% to 10%, 5% to 25%, 10% to 20%, 15% to 40%, or 25% to 50%, or 30% to 60%. In some embodiments, the culture media described herein may be approximately 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35% It may contain ITS in amounts (by weight / weight or volume / volume) greater than, less than, or equal to, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%.In some cases, the culture media described herein may contain ITS in amounts (weight / weight or volume / volume) of approximately 0.1% to 60%, 1% to 10%, 5% to 25%, 10% to 20%, 15% to 40%, or 25% to 50%, or 30% to 60%. In some cases, the culture media described herein may contain ITS in amounts (weight / weight or volume / volume) of approximately 0.5 times, 1 time, 2 times, 3 times, 4 times, or 5 times.

[0051] In some embodiments, the first medium comprises at least one of retinoic acid, its salts, corticosteroids, its salts, calcitriol, or its salts. In some cases, the first medium comprises at least one of a transforming growth factor beta pathway inhibitor or its salt, and a Wnt pathway inhibitor or its salt. In some cases, the medium comprises panobinostat.

[0052] In some cases, the first culture medium may contain retinoic acid. In some cases, retinoic acid can exert multifaceted cellular effects, such as inhibiting proliferation and inflammation while simultaneously inducing cell differentiation. In some cases, retinoic acid can be used to protect and / or differentiate podocytes. In some cases, retinoic acid may contain vitamin A, its derivatives, or salts of either of these. In some cases, retinoic acid may contain all-trans retinoic acid. In some cases, retinoic acid may contain isomers of retinoic acid, such as 12-cis or 9-cis retinoic acid. In some cases, retinoic acid may contain precursors of retinoic acid, such as retinol, its derivatives, or salts thereof. In some cases, the culture medium may contain retinoic acid or salts thereof at concentrations ranging from approximately 0.1 μM to approximately 1 mM. In some cases, the culture medium may contain retinoic acid or a salt thereof at concentrations of approximately 0.1 μM to 10 μM, 1 μM to 10 μM, 0.1 μM to 1 μM, 1 μM to 5 μM, 5 μM to 25 μM, 10 μM to 100 μM, 50 μM to 500 μM, 250 μM to 1000 μM, 0.1 mM to 1 mM, or 1 mM to approximately 10 mM.In some cases, the culture medium contains approximately 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 21 μM, and 22 μM. , 23μM, 24μM, 25μM, 26μM, 27μM, 28μM, 29μM, 30μM, 31μM, 32μM, 33μM, 34μM, 35μM, 36μM, 37μM, 38μM, 39μM M, 40μM, 41μM, 42μM, 43μM, 44μM, 45μM, 46μM, 47μM, 48μM, 49μM, 50μM, 60μM, 70μM, 80μM, 90μM, 100μM, 1 10μM, 120μM, 130μM, 140μM, 150μM, 160μM, 170μM, 180μM, 190μM, 200μM, 210μM, 220μM, 230μM, 240μM, 2 50μM, 260μM, 270μM, 280μM, 290μM, 300μM, 310μM, 320μM, 330μM, 340μM, 350μM, 360μM, 370μM, 380μM, 3 Contains retinoic acid or a salt thereof in concentrations higher than, lower than, or equal to, 90 μM, 400 μM, 410 μM, 420 μM, 430 μM, 440 μM, 450 μM, 460 μM, 470 μM, 480 μM, 490 μM, 500 μM, 1000 μM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM.

[0053] In some cases, the first culture medium may contain a corticosteroid or a salt thereof. In some cases, glomerular podocytes may contain functional receptors for corticosteroids. In some cases, corticosteroids may exhibit clinical effects and rescue podocyte function in cells. Corticosteroids such as dexamethasone may increase the stability of the cytoskeletal protein F-actin in podocytes and / or at least partially inhibit podocyte apoptosis. In some cases, the corticosteroid may contain dexamethasone or a salt thereof. In some cases, the corticosteroid may contain cortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, hydrocortisone, bethamethasone, triamcinolone, any salt thereof, or any combination thereof. In some cases, the corticosteroid may contain a synthetic corticosteroid. In some cases, the culture medium may contain corticosteroids or their salts at concentrations of approximately 100 nM to approximately 10 mM. In some cases, the culture medium may contain corticosteroids or their salts at concentrations of approximately 10 nM to 1000 nM, 50 nM to 250 nM, 75 nM to 500 nM, 100 nM to 1000 nM, 0.1 μM to 1 μM, 1 μM to 5 μM, 5 μM to 25 μM, 10 μM to 100 μM, 50 μM to 500 μM, 250 μM to 1000 μM, 0.1 mM to 1 mM, or 1 mM to approximately 10 mM. In some cases, the culture medium contains corticosteroids or their salts at concentrations higher than, lower than, or equal to, approximately 5 nM, 10 nM, 20 nM, 50 nM, 70 nM, 80 nM, 90 nM, 100 nM, 150 nM, 250 nM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 10 μM, 50 μM, 100 μM, 500 μM, 1 mM, or 10 mM.

[0054] In some cases, the first culture medium may contain calcitriol or a salt thereof. Calcitriol, a biologically active form of vitamin D, can exert its biological effects by activating the vitamin D receptor. Calcitriol may be effective in reducing podocyte damage and / or promoting podocyte gene expression. In some cases, calcitriol may contain vitamin D, its derivatives, or salts thereof. In some cases, calcitriol may contain 1,25-dihydroxycholecalciferol. In some cases, calcitriol may contain vitamin D3 (e.g., cholecalciferol) and vitamin D2 (e.g., ergocalciferol), vitamin D4 (e.g., 22-dihydroergocalciferol), vitamin D5 (e.g., citocalciferol), derivatives of any of these, salts of any of these, or any combination thereof. In some cases, calcitriol may contain precursors of calcitriol. In some cases, the culture medium may contain calcitriol or its salts at concentrations of approximately 1 nM to approximately 300 nM. In some cases, the culture medium may contain calcitriol or its salts at concentrations of approximately 1 nM to 1000 nM, 10 nM to 300 nM, 50 nM to 250 nM, 75 nM to 500 nM, 30 nM to 200 nM, 50 nM to 275 nM, 100 nM to 400 nM, 100 nM to 1000 nM, or 0.1 μM to 1 μM. In some cases, the culture medium was approximately 1nM, 5nM, 10nM, 20nM, 30nM, 40nM, 50nM, 60nM, 70nM, 80nM, 90nM, 100nM, 110nM, 120nM, 130nM, 140nM, 150nM, 160nM, 170nM, 180nM, 190nM, 200nM, 210nM, 220nM, 230nM, 240nM, 250nM, 260nM, 2 Contains calcitriol or its salts at concentrations of 70 nM, 280 nM, 290 nM, 300 nM, 500 nM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 10 μM, 50 μM, 100 μM, 500 μM, 1 mM, or higher than, lower than, or equal to, 10 mM.

[0055] In some cases, the second medium includes a basal medium, a nutrient mixture, antibiotics, insulin-transferrin-selenium (ITS), fetal bovine serum, SB431542, IWR-1-endo (also referred to herein as IWR-1), salts of any of these, or any combination thereof, or salts thereof. In some cases, the second medium includes at least one of retinoic acid or a salt thereof, and a ROCK inhibitor or a salt thereof. In some cases, the basal medium may include Dulbecco's Modified Eagle Medium, Basic Medium Eagle, Glasgow Minimum Essential Medium, Iskov Modified Dulbecco's Medium, Grace Insect Medium, Minimum Essential Medium, RPMI Medium, McCoy 5A, or any combination thereof. In some cases, the basal medium may include a compound medium. In some cases, the nutrient mixture may include F-12, F-10, a non-essential amino acid solution, or any combination thereof. In some embodiments, the serum may include fetal bovine serum, horse serum, calf serum, rabbit serum, pig serum, goat serum, human serum, or any combination thereof. In some cases, the culture medium may be serum-free or low-serum medium. In some cases, the second medium may include ITS. In some cases, ITS can be used to replace serum in the culture medium. In some cases, the culture medium may include serum substitutes (e.g., serum replacements) or modified serum. In some cases, antibiotics may include penicillin, streptomycin, beta-lactam, tetracycline, trimethoprim-sulfamethoxazole, lincosamide, fluoroquinolone, cephalosporin, macrolide, aminoglycoside, amphotericin, chloramphenicol, ampicillin, vancomycin, lincomycin, carbenicillin, gentamicin, neomycin, benzylpenicillin, rifampicin, mitomycin C, kanamycin, erythromycin, fosmidomycin, salts of any of these, or any combination thereof. In some cases, antibiotics may include penicillin or a salt thereof and streptomycin or a salt thereof. In some cases, the culture medium, such as the second medium, may include equilibrium salt solutions such as phosphate-buffered saline, Dulbecco's phosphate-buffered saline, Hanks equilibrium salt solution, Earl equilibrium salt solution, or any combination thereof.

[0056] In some embodiments, the second culture medium may comprise at least one of SB431542, its salts, IWR-1-endo, or its salts.

[0057] In some cases, the first medium and / or the second medium may contain SB431542 or a salt thereof. SB431542 may be a potent transforming growth factor beta pathway inhibitor. In podocytes, SB431542 has been shown to promote podocyte function and / or protect podocytes from injury. In some cases, SB431542 has been shown to have the formula C 22 H 16It may contain N4O3. In some cases, SB431542 may contain CAS number 301836-41-9. In some cases, SB431542 may contain derivatives of SB431542 or salts thereof. In some cases, the second medium may contain growth factor beta pathway inhibitors. In some cases, the medium may contain SB431542 or salts thereof at concentrations of approximately 0.1 μM to approximately 100 μM. In some cases, the medium may contain SB431542 or salts thereof at concentrations of approximately 0.1 μM to 100 μM, 1 μM to 10 μM, 1 μM to 15 μM, 5 μM to 15 μM, 0.1 μM to 1 μM, 1 μM to 5 μM, 5 μM to 25 μM, 10 μM to 100 μM, 50 μM to 75 μM, or 80 μM to 100 μM. In some cases, the culture medium contains approximately 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 21 μM, 22 μM, 23 μM, 24 μM, 25 μM, 26 μM, 27 μM, 28 μM, 29 μM, 30 μM, 31 μM, 32 μM, and 33 μM. , containing SB431542 or a salt thereof in concentrations higher than, lower than, or equal to, 34 μM, 35 μM, 36 μM, 37 μM, 38 μM, 39 μM, 40 μM, 41 μM, 42 μM, 43 μM, 44 μM, 45 μM, 46 μM, 47 μM, 48 μM, 49 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 110 μM, 120 μM, 130 μM, 140 μM, 150 μM, 160 μM, 170 μM, 180 μM, 190 μM, or 200 μM.

[0058] In some cases, the first medium and / or the second medium may contain a Wnt pathway inhibitor such as IWR-1-endo or a salt thereof. In some cases, IWR-1 is a Wnt pathway inhibitor that can promote podocyte differentiation. In some cases, the second medium may contain a Wnt pathway inhibitor. In some cases, IWR-1-endo may contain IWR-1. In some cases, IWR-1 may contain IWR-1-endo. In some cases, IWR-1-endo may contain formula C 25 H 19 It may contain N3O3. In some cases, IWR-1-endo may contain CAS number 1127442-82-3. In some cases, IWR-1-endo may contain derivatives of IWR-1-endo or salts thereof. In some cases, the culture medium may contain IWR-1-endo or salts thereof at concentrations of approximately 0.1 μM to approximately 100 μM. In some cases, the culture medium may contain IWR-1-endo or salts thereof at concentrations of approximately 0.1 μM to 100 μM, 1 μM to 10 μM, 1 μM to 15 μM, 5 μM to 15 μM, 0.1 μM to 1 μM, 1 μM to 5 μM, 5 μM to 25 μM, 10 μM to 100 μM, 50 μM to 75 μM, or 80 μM to 100 μM. In some cases, the culture medium contains approximately 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 21 μM, 22 μM, 23 μM, 24 μM, 25 μM, 26 μM, 27 μM, 28 μM, 29 μM, 30 μM, 31 μM, 32 μM, 33 μM. Contains IWR-1-endo or a salt thereof in concentrations higher than, lower than, or equal to, 34 μM, 35 μM, 36 μM, 37 μM, 38 μM, 39 μM, 40 μM, 41 μM, 42 μM, 43 μM, 44 μM, 45 μM, 46 μM, 47 μM, 48 μM, 49 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 110 μM, 120 μM, 130 μM, 140 μM, 150 μM, 160 μM, 170 μM, 180 μM, 190 μM, or 200 μM.

[0059] In some cases, the first culture medium and / or the second culture medium may contain a ROCK inhibitor or a salt thereof. In some cases, the ROCK inhibitor contains Y-27632. In some cases, Y-27632 is of formula C 14 H 21 Contains N3O. In some cases, Y-27632 may contain CAS number 129830-38-2. In some cases, Y-27632 may contain derivatives of Y-27632 or salts thereof. In some cases, the culture medium may contain Y-27632 or salts thereof at concentrations of approximately 0.1 μM to approximately 100 μM. In some cases, the culture medium may contain Y-27632 or salts thereof at concentrations of approximately 0.1 μM to 100 μM, 1 μM to 10 μM, 1 μM to 15 μM, 5 μM to 15 μM, 0.1 μM to 1 μM, 1 μM to 5 μM, 5 μM to 25 μM, 10 μM to 100 μM, 50 μM to 75 μM, or 80 μM to 100 μM. In some cases, the culture medium contains approximately 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 21 μM, 22 μM, 23 μM, 24 μM, 25 μM, 26 μM, 27 μM, 28 μM, 29 μM, 30 μM, 31 μM, 32 μM, and 33 μM. , containing Y-27632 or a salt thereof in concentrations higher than, lower than, or equal to, 34 μM, 35 μM, 36 μM, 37 μM, 38 μM, 39 μM, 40 μM, 41 μM, 42 μM, 43 μM, 44 μM, 45 μM, 46 μM, 47 μM, 48 μM, 49 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 110 μM, 120 μM, 130 μM, 140 μM, 150 μM, 160 μM, 170 μM, 180 μM, 190 μM, or 200 μM.

[0060] In some cases, the culture medium contains panobinostat or a salt thereof. In some cases, panobinostat is of formula C 21 H 23It contains N3O2. In some cases, panobinostat may contain the CAS number 404950-80-7. In some cases, panobinostat may contain a derivative of panobinostat or a salt thereof. In some cases, the medium may contain panobinostat or its salt at a concentration of about 0.1 nM to about 200 nM. In some cases, the medium may contain panobinostat or its salt at a concentration of about 0.1 nM to 100 nM, 1 nM to 10 nM, 1 nM to 15 nM, 5 nM to 15 nM, 0.1 nM to 1 nM, 1 nM to 5 nM, 5 nM to 25 nM, 10 nM to 100 nM, 50 nM to 75 nM, or 80 nM to 100 nM. In some cases, the medium may contain panobinostat or its salt at a concentration of about 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 21 nM, 22 nM, 23 nM, 24 nM, 25 nM, 26 nM, 27 nM, 28 nM, 29 nM, 30 nM, 31 nM, 32 nM, 33 nM, 34 nM, 35 nM, 36 nM, 37 nM, 38 nM, 39 nM, 40 nM, 41 nM, 42 nM, 43 nM, 44 nM, 45 nM, 46 nM, 47 nM, 48 nM, 49 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, or higher than, less than, or equal to 200 nM.

[0061] In some cases, the medium contains hydrocortisone. In some cases, hydrocortisone has the formula C 21 H 30Contains O5. In some cases, hydrocortisone may contain CAS number 50-23-7. In some cases, hydrocortisone may contain derivatives of hydrocortisone or salts thereof. In some cases, the culture medium may contain hydrocortisone or salts thereof at concentrations of approximately 0.5 μg / mL to 5 μg / mL. In some cases, the culture medium may contain hydrocortisone or salts thereof at concentrations of approximately 0.5 μg / mL, 0.6 μg / mL, 0.7 μg / mL, 0.8 μg / mL, 0.9 μg / mL, 1 μg / mL, 1.5 μg / mL, 2 μg / mL, 2.5 μg / mL, 3 μg / mL, 3.5 μg / mL, 4 μg / mL, 4.5 μg / mL, or 5 μg / mL. In some cases, the culture medium may contain hydrocortisone at approximately 1.15 μg / mL.

[0062] In some cases, the culture medium contains recombinant human fibroblast growth factor (rh FGF) or a variant thereof. In some cases, the rh FGF contains the amino acid sequence or a variant thereof described in Sequence ID No. 1. In some cases, the rh FGF contains human FGF basic, Pro143-Ser288, with N-terminal Ala, accession number P09038, derived from Escherichia coli (E. coli). In some cases, the rh FGF may contain derivatives of rh FGF or salts thereof. In some cases, the culture medium may contain rh FGF or salts thereof at concentrations of approximately 1 ng / mL to 100 ng / mL. In some cases, the culture medium may contain rh FGF or its variants or salts thereof at concentrations of approximately 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, or 100 ng / mL.

[0063] In some cases, the culture medium contains recombinant human vascular endothelial growth factor (rh VEGF) or a variant thereof. In some cases, the rh VEGF contains the amino acid sequence or a variant thereof described in Sequence ID No. 2. In some cases, the rh VEGF contains human VEGF 165, Ala27-Arg191, derived from Sf21 (baculovirus), with accession number NP_001165097. In some cases, the rh VEGF may contain derivatives of rh VEGF or a salt thereof. In some cases, the culture medium may contain rh VEGF or a salt thereof at a concentration of approximately 1 ng / mL to 100 ng / mL. In some cases, the culture medium may contain rh VEGF or its variants or salts thereof at concentrations of approximately 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, or 100 ng / mL.

[0064] In some cases, the culture medium contains recombinant human epidermal growth factor (rh EGF) or a variant thereof. In some cases, the rh EGF contains the amino acid sequence or a variant thereof described in Sequence ID No. 3. In some cases, the rh EGF contains the human EGF protein derived from Escherichia coli (E. coli), Asn971-Arg1023, with N-terminal Met, and accession number P01133. In some cases, the rh EGF may contain derivatives of rh EGF or salts thereof. In some cases, the culture medium may contain rh EGF or salts thereof at a concentration of approximately 1 ng / mL to 100 ng / mL. In some cases, the culture medium may contain rh EGF or its variants or salts thereof at concentrations of approximately 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, or 100 ng / mL.

[0065] In some cases, the culture medium contains recombinant human long R3 insulin-like growth factor (Long R3 IGF) or a variant thereof. In some cases, the Long R3 IGF contains the amino acid sequence or a variant thereof described in SEQ ID NO: 4. In some cases, the Long R3 IGF contains human IGF-I derived from Escherichia coli (E. coli), Gly49-Ala118 (Glu51Arg), N-terminal MFPAMPLSSLFVN (SEQ ID NO: 5), accession number P05019.1. In some cases, the Long R3 IGF may contain derivatives of Long R3 IGF or a salt thereof. In some cases, the culture medium may contain Long R3 IGF or a salt thereof at a concentration of approximately 1 ng / mL to 100 ng / mL. In some cases, the culture medium may contain Long R3 IGF or its variants or salts thereof at concentrations of approximately 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, or 100 ng / mL.

[0066] In some cases, the culture medium contains insulin derived from bovine pancreas. In some cases, the insulin is of formula C 254 H 377 N 65 O 75Contains S6. In some cases, insulin may contain CAS number 11070-73-8. In some cases, insulin may contain insulin derivatives or salts thereof. In some cases, the culture medium may contain insulin or salts thereof at concentrations of approximately 0.01 U / mL to 10 U / mL. In some cases, the culture medium may contain 0.01 U / mL, 0.02 U / mL, 0.03 U / mL, 0.04 U / mL, 0.05 U / mL, 0.06 U / mL, 0.07 U / mL, 0.08 U / mL, 0.09 U / mL, 0.1 U / mL, 0.125 U / mL, 0.150 U / mL, 0.175 U / mL, 0.2 U / mL, 0.225 U / mL L, 0.250U / mL, 0.275U / mL, 0.3U / mL, 0.325U / mL, 0.350U / mL, 0.375U / mL, 0.4U / mL, 0.425U / mL, 0.450U / mL, 0.475U / mL, 0.5U / mL, 0.525U / mL, 0.550U / mL, 0.575U / mL, 0.6U / mL, 0.62 5U / mL, 0.650U / mL, 0.675U / mL, 0.7U / mL, 0.725U / mL, 0.75U / mL, 0.775U / mL, 0.8U / mL, 0.8 25U / mL, 0.850U / mL, 0.875U / mL, 0.9U / mL, 0.925U / mL, 0.950U / mL, 0.975U / mL, 1U / mL, 1.5 It may contain insulin or a salt thereof at concentrations of U / mL, 2U / mL, 2.5U / mL, 3U / mL, 3.5U / mL, 4U / mL, 4.5U / mL, 5U / mL, 5.5U / mL, 6U / mL, 6.5U / mL, 7U / mL, 7.5U / mL, 8U / mL, 8.5U / mL, 9U / mL, 9.5U / mL, or 10U / mL.

[0067] In some cases, the culture medium contains triiodothyronine. In some cases, triiodothyronine is of formula C 15 H 12Contains I3NO4. In some cases, triiodothyronine may contain CAS number 6893-02-3. In some cases, triiodothyronine may contain derivatives of triiodothyronine or salts thereof. In some cases, the culture medium may contain triiodothyronine or salts thereof at concentrations of approximately 0.1 nM to 100 nM. In some cases, the culture medium contains approximately 0.1nM, 0.2nM, 0.3nM, 0.4nM, 0.5nM, 0.6nM, 0.7nM, 0.8nM, 0.9nM, 1nM, 2nM, 3nM, 4nM, 5nM, 6nM, 7nM, 8nM, 9nM, 10nM, 11nM, 12nM, 13nM, 14nM, 15nM, 16nM, 17nM, 18nM, 19nM, 20nM, 21nM, 22nM, 23nM, 24nM, and 25nM. It may contain triiodothyronine or a salt thereof at concentrations of M, 26nM, 27nM, 28nM, 29nM, 30nM, 31nM, 32nM, 33nM, 34nM, 35nM, 36nM, 37nM, 38nM, 39nM, 40nM, 41nM, 42nM, 43nM, 44nM, 45nM, 46nM, 47nM, 48nM, 49nM, 50nM, 60nM, 70nM, 80nM, 90nM, or 100nM.

[0068] In some cases, the culture medium contains epinephrine or a salt thereof. In some cases, epinephrine is of formula C9H 13 Contains NO3. In some cases, epinephrine may contain CAS number 329-63-5. In some cases, epinephrine may contain derivatives of epinephrine or salts thereof. In some cases, the culture medium may contain epinephrine or salts thereof at a concentration of approximately 0.1 μM to 10 μM. In some cases, the culture medium may contain epinephrine or a salt thereof at concentrations of approximately 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.5 μM, 9 μM, 9.5 μM, or 10 μM.

[0069] In some cases, the culture medium contains holotransferrin or a variant thereof. In some cases, the holotransferrin contains CAS number 11096-37-0. In some cases, the culture medium may contain holotransferrin or a variant thereof or a salt thereof at a concentration of approximately 0.1 μg / mL to 100 μg / mL. In some cases, the medium is approximately 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.4 μg / mL, 0.5 μg / mL, 0.6 μg / mL, 0.7 μg / mL, 0.8μg / mL, 0.9μg / mL, 1μg / mL, 2μg / mL, 3μg / mL, 4μg / mL, 5μg / mL, 6μg / mL, 7μg / m L, 8μg / mL, 9μg / mL, 10μg / mL, 11μg / mL, 12μg / mL, 13μg / mL, 14μg / mL, 15μg / mL, 16μg / mL , 17μg / mL, 18μg / mL, 19μg / mL, 20μg / mL, 21μg / mL, 22μg / mL, 23μg / mL, 24μg / mL, 25μg / m It may contain holotransferrin or its variants or salts in concentrations of L, 26 μg / mL, 27 μg / mL, 28 μg / mL, 29 μg / mL, 30 μg / mL, 31 μg / mL, 32 μg / mL, 33 μg / mL, 34 μg / mL, 35 μg / mL, 36 μg / mL, 37 μg / mL, 38 μg / mL, 39 μg / mL, 40 μg / mL, 41 μg / mL, 42 μg / mL, 43 μg / mL, 44 μg / mL, 45 μg / mL, 46 μg / mL, 47 μg / mL, 48 μg / mL, 49 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, or 100 μg / mL.

[0070] In some embodiments, the first, second, or third culture medium, such as maintenance medium, comprises at least one of penicillin-streptomycin, fetal bovine serum, heparin, ascorbic acid, hydrocortisone, rh FGF, rh VEGF, rh EGF, Long R3 IGF, insulin, triiodothyronine, epinephrine, holotransferrin, and SB431542.

[0071] Decellularized organs A kidney or a portion thereof, at least partially recellularized, prepared from a decellularized extracellular matrix, is disclosed herein. Methods relating to decellularization and recellularization are disclosed in U.S. patents, including U.S. Patent No. 8,470,520, No. 10,233,420, and No. 10,220,056, which are incorporated herein by reference in their entirety.

[0072] In some cases, the initial step in decellularizing an organ or tissue, such as the kidney, is to insert a cannula into the organ or tissue, if possible. Blood vessels, tubes, and / or lumens of the organ or tissue can be used to insert the cannula using common methods and materials. The next step in decellularizing an organ or tissue may be to perfuse the cannulated organ or tissue with cell disruption medium. Perfusion through the organ may be multidirectional (e.g., anterograde and retrograde). The cell disruption medium can be delivered by injection, a roller pump, or by constant hydrostatic pressure.

[0073] One or more cell disruption media may be used to decellularize an organ or tissue. Cell disruption media generally contain at least one washing agent, such as SDS, PEG, or Triton X. Cell disruption media may contain water so that the medium is osmotically incompatible with the cells. Alternatively, cell disruption media may contain a buffer (e.g., PBS) for osmotic compatibility with the cells. Cell disruption media may also contain enzymes, but are not limited to, one or more collagenases, one or more dispases, one or more DNases, or proteases such as trypsin. In some cases, cell disruption media may further or instead contain one or more enzyme inhibitors (e.g., protease inhibitors, nuclease inhibitors, and / or collagenase inhibitors).

[0074] In certain embodiments, the organ or tissue into which the cannula has been inserted may be sequentially perfused with two different cell disruption media. For example, the first cell disruption media may contain an anionic washing agent such as SDS, and the second cell disruption media may contain an ionic washing agent such as Triton X. After perfusion with at least one cell disruption media, the organ or tissue into which the cannula has been inserted may be perfused with a solution containing one or more enzymes, such as a washing solution and / or those disclosed herein. Alternating the direction of perfusion (e.g., antegrade and retrograde) may help to effectively decellularize the entire organ or tissue. Decellularization as described herein essentially decellularizes the organ from the inside out, resulting in very little damage to the ECM. The organ or tissue may be decellularized at a suitable temperature of 4–40°C. Depending on the size and weight of the organ or tissue, and the concentration of the specific washing agent(s) in the cell disruption medium, the organ or tissue is generally perfused in the cell disruption medium for about 0.1 to about 12 hours per gram of solid organ or tissue. Including washing, the organ may be perfused for up to about 12 to about 72 hours per gram of tissue. Perfusion is generally adjusted to physiological conditions, including pulsating flow, pulsation rate, and pulsating pressure. In some embodiments, the cell disruption solution is a solution that may contain at least one washing agent. The washing agent may be an amphiphilic molecule that may contain both a nonpolar "tail" and a polar "head" having aliphatic or aromatic properties. The ionic properties of the polar head group may form the basis of a broad classification of washing agents, which may be ionic (charged, anionic, or cationic), nonionic (uncharged), or zwitterionic (having both positively and negatively charged groups but with a net charge of zero). In some embodiments, the detergent may be denaturing or non-denaturing to the protein structure. Denaturing detergents may be anionic, such as sodium dodecyl sulfate (SDS), or cationic, such as ethyltrimethylammonium bromide (ETMAB). These detergents denature proteins by disrupting membranes and breaking protein-protein interactions.Non-denatured detergents can be classified into nonionic detergents such as Triton X-100, NP40, and Tween, bile salts such as cholate, and zwitterionic detergents such as CHAPS.

[0075] In some cases, decellularized organs or tissues are essentially composed of extracellular matrix (ECM) components in all or most areas of the organ or tissue, including ECM components of the vascular tree. ECM components may include any or all of the following: fibronectin, fibrillin, laminin, elastin, members of the collagen family (e.g., collagen I, III, and IV), glycosaminoglycans, basal layer, reticular fibers, and thrombospongin, which may remain organized as distinct structures such as the basal layer. Successful decellularization is defined as the absence of detectable myofilaments, endothelial cells, smooth muscle cells, and nuclei in tissue sections using standard tissue staining procedures. Preferably, but not necessarily, residual cellular debris is also removed from the decellularized organ or tissue. The morphology and structure of the ECM can be examined visually and / or histologically.

[0076] One or more compounds may be applied in or on a decellularized organ or tissue, for example, to preserve the decellularized organ or to prepare the decellularized organ or tissue for recellularization, and / or to assist or stimulate cells during the recellularization process. Such compounds include, but are not limited to, one or more growth factors (e.g., VEGF, DKK-1, FGF, bFGF, PDGF, HGF, BMP-1, BMP-4, SDF-1, IGF, and HGF), immunomodulators (e.g., cytokines, glucocorticoids, IL2R antagonists, leukotriene antagonists, antibody therapies, the use of stem cells to modulate immune responses), and / or factors that modify the coagulation cascade (e.g., aspirin, heparin-binding proteins, and heparin). Furthermore, the decellularized organ or tissue may be further treated, for example, with irradiation (e.g., UV, gamma), to reduce or eliminate the presence of any type of microorganism remaining on or within the decellularized organ or tissue.

[0077] In some embodiments, perfusion decellularization involves inserting a cannula into an organ or part thereof. In some embodiments, at least one cannula insertion is introduced into an organ or part thereof. In some embodiments, at least two cannula insertions are introduced into an organ or part thereof. In some embodiments, approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 cannula insertions are introduced into an organ or part thereof. In some cases, a cannula may be part of a cannula insertion system. A cannula insertion system may include a hollow tube of a suitable size for introduction into a blood vessel, tube, lumen, or any combination thereof in an organ or tissue. Typically, a cannula is inserted into at least one blood vessel, tube, and / or lumen in the organ. The perfusion device or cannula insertion system may include a holding container for a solution (e.g., cell disruption medium) and a mechanism (e.g., a pump, pneumatics, gravity) for moving the liquid through one or more cannulas into the organ. The sterility of organs or tissues during decellularization and / or recellularization can be maintained using a variety of techniques known in the art, such as airflow control and filtration, and / or perfusion with antibiotics, antifungals, or other antimicrobial agents, to prevent the growth of undesirable microorganisms. In some embodiments, the systems described herein may have the ability to monitor certain perfusion characteristics (e.g., pressure, volume, flow pattern, temperature, gas, pH), mechanical forces (e.g., ventricular wall motion and stress), and electrical stimulation (e.g., pacing). In some embodiments, the vascular bed may change during the decellularization and recellularization process (e.g., vascular resistance, volume), and a pressure-regulated perfusion device or cannula insertion system may be suitable for avoiding or reducing fluctuations. The effectiveness of perfusion can be evaluated in the effluent and in tissue sections. Perfusion volume, flow pattern, temperature, O2 and CO2 partial pressures, and pH can be monitored using standard methods. In some embodiments, sensors may be used to monitor the system (e.g., bioreactor) and / or organs or tissues.Sonomicrometry, micromanometry, and / or conductance measurements can be used to obtain pressure-volume or preload mobilization work-per-stroke information regarding myocardial wall motion and performance. For example, sensors can be used to monitor the pressure of the fluid moving through the organ or tissue into which the cannula is inserted; the ambient temperature in the system; and / or the temperature of the organ or tissue; the pH and / or flow rate of the fluid moving through the organ or tissue into which the cannula is inserted; and / or the biological activity of the organ or tissue during recellularization. In addition to having sensors for monitoring such features, a system for decellularizing and / or recellularizing an organ or tissue may also include means for maintaining or regulating such features. Means for maintaining or regulating such features may include components such as thermometers, thermostats, electrodes, pressure sensors, overflow valves, valves for changing the fluid flow rate, valves for opening and closing fluid connections to a solution used to change the pH of the solution, balloons, external pacemakers, and / or compliance chambers. To help ensure stable conditions (e.g., temperature), the chamber, reservoir, and tubing may be covered with water. In some embodiments, cannula insertion is performed in a lumen, blood vessel, tube, or a combination thereof. In some embodiments, about 1–3, 1–5, 2–3, 2–5, or 1–8 solutions may be used for organ perfusion. In some embodiments, the solution is perfused at least twice. In some embodiments, the solution is perfused at least 3, 4, 5, 6, 7, 8, 9, or up to 10 times through the organ or a portion thereof. Various solutions and culture media may be used during recellularization. In some embodiments, the solutions may be selected from a group including cell disruption solutions, washing solutions, disinfection solutions, or a combination thereof.

[0078] In some embodiments, a washing solution may be used during decellularization. The washing solution may be used to remove residual solutions, such as cell disruption solutions, from organs or parts thereof, as well as residual cellular components, enzymes, or combinations thereof. Suitable washing solutions may include water, filtered water, phosphate-buffered saline (PBS), and combinations thereof. PBS can maintain a constant pH and molar osmotic concentration for cells. In some cases, the pH of most biological materials falls between approximately 6.8 and 7.6.

[0079] In some embodiments, a disinfectant solution may be used during decellularization. The disinfectant solution may contain any number of agents, such as antibiotics, disinfectants, or combinations thereof. In some embodiments, the antibiotics that can be used in the decellularization solution may be selected from the group including actinomycin, ampicillin, carbenicillin, cefotaxime, fosmidomycin, gentamicin, kanamycin, neomycin, amphotericin, penicillin, polymyxin, streptomycin, broad-spectrum selective antibiotics, and combinations thereof. Any concentration of antibiotic may be introduced into the disinfectant solution.

[0080] In some embodiments, a system, such as a system for generating an organ or part thereof or tissue, may be controlled by a computer-readable storage medium in combination with a programmable processor (for example, the computer-readable storage medium, as used herein, stores instructions therefor for the programmable processor to perform specific steps). For example, such a storage medium may, in combination with a programmable processor, receive and process information from one or more sensors. Furthermore, such a storage medium may, in conjunction with the programmable processor, transmit information and instructions back to the bioreactor and / or organ or tissue. In some embodiments, the organ or tissue undergoing recellularization may be monitored for its biological activity. Biological activity may include the biological activity of the organ or part thereof or the tissue itself, such as kidney tissue; electrical activity; mechanical activity; mechanical pressure; contractile force; and / or wall stress. Additionally, the biological activity of cells bound to or engrafted on the organ or part thereof or tissue, such as ion transport / exchange activity, cell division, and / or cell viability, may be monitored. In some embodiments, it may be useful to simulate the activity load on the organ or part thereof during recellularization. In some embodiments, a computer-readable storage medium may be used in combination with a programmable processor to harmonize components used to monitor and maintain the active load on an organ or tissue. In some cases, the weight of an organ or part thereof or tissue is entered into a computer-readable storage medium, as described herein, which, in combination with a programmable processor, can calculate the exposure time and perfusion pressure for that particular organ or tissue. Such a storage medium may record preload and afterload (pressure before and after perfusion, respectively) and flow rate. In this embodiment, for example, the computer-readable storage medium may be used in combination with a programmable processor to adjust the perfusion pressure, direction of perfusion, and / or type of perfusion solution via one or more pump and / or valve controls.

[0081] In some embodiments, immersion-based decellularization of an organ or part thereof may be carried out. In some embodiments, an entire organ or part thereof may be decellularized by removing the entire cellular and tissue contents from the organ. In some embodiments, decellularization may involve a series of sequential extractions. In some embodiments, the first step may include removal of cellular debris and solubilization of the cell membrane. This may be followed by solubilization of the nucleocytoplasmic and nuclear components. In some embodiments, an organ may be decellularized by removing the cell membrane and cellular debris surrounding the organ using a gentle mechanical destruction method. A gentle mechanical destruction method may destroy the cell membrane. However, the decellularization process should avoid damaging or disturbing the complex basis of the biological structure. A gentle mechanical destruction method may include scraping the organ surface, stirring the organ, or agitating the organ in an appropriate volume of fluid, such as distilled water. In some embodiments, a gentle mechanical destruction method may include magnetically agitating the organ or part thereof in an appropriate amount of distilled water (e.g., using magnetic stirring rods and magnetic plates) until the cell membranes are destroyed and cellular debris is removed from the organ or part thereof. After the cell membranes have been removed, the nuclear and cytoplasmic components of the biological structure are removed. This may be accomplished by solubilizing the cellular and nuclear components without destroying the substrate. Nonionic detergents or surfactants may be used to solubilize the nuclear components.Examples of nonionic detergents or surfactants include the Triton series available from Rohm and Haas in Philadelphia, Pennsylvania, including Triton X-100, Triton N-101, Triton X-114, Triton X-405, Triton X-705, and Triton DF-16, which are commercially available from many suppliers; the Tween series, e.g., monolaurate (Tween 20), monopalmitate (Tween 40), monooleate (Tween 80), and polyoxyethylene-23-lauryl ether (Brij. 35), polyoxyethylene ether W-1 (Polyox), as well as sodium cholate, deoxycholate, CHAPS, saponins, n-decyl β-D-glucopyranoside, n-heptyl β-D-glucopyranoside, n-octyl α-D-glucopyranoside, and nonidet. The P-40 is one example, but it is not limited to these.

[0082] In some cases, physical treatment of an organ or part thereof may be performed to achieve decellularization. Physical treatment may be used to lyse, kill, and remove cells from the ECM or part thereof. Physical treatment may utilize temperature, force, pressure, and electrical disruption. In some cases, the thermal method may be used in a rapid freeze-thaw mechanism. For example, by freezing the tissue, microscopic ice crystals can form around the plasma membrane, and the cells can be lysed. After the cells have been lysed, the tissue is further exposed to liquefied chemicals, which can break down and wash away any residual or undesirable components. In some cases, the thermal method can preserve the physical structure of the ECM scaffold. Organs or parts thereof, and tissues, can be decellularized at appropriate temperatures. Appropriate temperatures may be around 4°C, 8°C, 10°C, 12°C, 14°C, 16°C, 18°C, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, 45°C, 50°C, 55°C, 60°C, or up to approximately 70°C. Physical procedures may also involve the use of pressure. Pressure decellularization involves the controlled use of hydrostatic pressure applied to tissues, organs, or parts thereof. Pressure decellularization may be performed at high temperatures in some cases to avoid the formation of unmonitored ice crystals. In some cases, electrical disruption of organs or parts thereof may be performed. Electrical disruption may be performed to dissolve cells contained within the tissue or organ. Micropores may be formed in the plasma membrane by exposing tissues, organs, or parts thereof to electrical pulses. Cells may die after their homeostatic electrical equilibrium is disrupted by the applied stimulus. This electrical process has been documented as non-thermal irreversible electroporation (NTIRE).

[0083] In some cases, chemical treatment of an organ or part thereof may be carried out to achieve decellularization. Chemicals and / or salts thereof for use in chemical treatment may be selected with respect to decellularization depending on the thickness of the tissue or organ, the composition of the extracellular matrix, and the intended use. For example, enzymes may not be usable in collagenous tissue because they disrupt connective tissue fibers. However, enzymes may be a viable option for decellularization when collagen is not present in high concentrations or is not required in the tissue. Chemicals and / or salts thereof, which may include but are not limited to acids, alkali treatments, ionic detergents, nonionic detergents, and zwitterionic detergents, may be used to kill and remove cells. In some cases, one or more chemicals may constitute a cell disruption medium. A cell disruption medium may contain at least one detergent, such as sodium dodecyl sulfate (SDS), polyethylene glycol (PEG), or Triton X. The detergent may act to effectively dissolve the cell membrane and expose the contents to further degradation. For example, after SDS lyses the cell membrane, endonucleases and / or exonucleases can degrade the genetic contents, while other cellular components can be solubilized and washed away from the matrix. In some cases, washing agents may be mixed with alkaline and / or acidic treatments due to their ability to degrade nucleic acids and solubilize cytoplasmic inclusions.

[0084] One or more cell disruption media may be used to decellularize organs or tissues. The cell disruption media may contain at least one washing agent, such as SDS, PEG, or Triton X.

[0085] The cleaning agent is used for approximately 10 minutes, 30 minutes, 60 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, and 36 hours. The treatment may be administered for a period of time equal to or longer than, less than, or equal to, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 70, 80, 90 hours, or up to approximately 100 hours. In some cases, the treatment agent may be allowed to come into contact with an organ or part thereof with a solid organ or tissue containing cell disruption medium for a period of time equal to or longer than, less than, or equal to, approximately 20 hours per gram of organ or tissue.

[0086] Including lavage, organs may be perfused for up to approximately 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 70, 80, 90 hours, or up to approximately 100 hours. In some cases, organs or parts thereof can be perfused for approximately 12 to 72 hours per gram of tissue. In some embodiments, perfusion can be adjusted to physiological conditions including pulsating flow, pulsation rate, pulsation pressure, and any combination thereof.

[0087] In some cases, a sequential method of decellularization may include contacting an organ or part thereof with a cell disruption medium such as an SDS washing agent, a subsequent washing step, the addition of one or more chemicals, contact with a subsequent washing agent, and ending with at least one washing step. A sequential method of decellularization may include at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelfth, thirteen, fourteen, or up to fifteen contact steps with any medium or solution provided herein.

[0088] The buffers provided herein may exist at concentrations (volume / volume or weight-to-weight) higher than, lower than, or equal to approximately 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some cases, the buffers provided herein may exist at a concentration of approximately 100%.

[0089] Methods for engrafting cells Methods for engrafting cells, such as manipulated podocyte-like cells, onto a decellularized organ or a portion thereof are disclosed herein. In some cases, HUVEC cells can be engrafted onto a decellularized organ or a portion thereof, with or without the use of manipulated podocyte-like cells.

[0090] In some cases, decellularization removes cellular material from a kidney, such as a pig kidney, generating a three-dimensional, human-scale scaffold composed of an extracellular matrix that maintains the complex structure of a natural kidney. This decellularized extracellular matrix can then be regenerated in human cells, such as engineered podocyte-like cells derived from a suitable human kidney donor, to produce a functionally bioengineered kidney graft. In some cases, the decellularized extracellular matrix is ​​regenerated in HUVEC cells. The methods described herein describe the recellularization of the extracellular matrix.

[0091] Such methods include methods for engrafting cells onto at least partially decellularized renal extracellular matrix, comprising contacting the at least partially decellularized renal extracellular matrix with a plurality of manipulated podocyte-like cells and / or a plurality of HUVEC cells. In some cases, the contact is performed in a bioreactor chamber. In some cases, the contact involves depositing a plurality of manipulated podocyte-like cells in aqueous solution into the glomeruli of the at least partially decellularized renal extracellular matrix through the ureters of the at least partially decellularized renal extracellular matrix, thereby engrafting the cells onto the at least partially decellularized renal extracellular matrix. In some cases, the deposition through the ureters involves creating a vacuum in a bioreactor chamber.

[0092] In some cases, the method may further include seeding one or more additional cell types. In some cases, the method may include seeding multiple manipulated podocyte-like cells. In some cases, the method may further include seeding multiple mesangial cells, multiple human umbilical vein endothelial cells (HUVECs), or both. In some cases, the method may further include seeding multiple tubular epithelial cells, macula densa cells, glomerular endothelial cells, tubular cells, podocytes, smooth muscle cells, pericytes, juxtaglomerular cells, collecting duct cells (e.g., CD-PC, CD-Trans, or CD-IC), distal curved tubular cells (e.g., DCT1, DCT2), loop of Henle cells, proximal tubular cells (e.g., curved or straight), afferent arteriole (vas afferens) cells, efferent arteriole (vas efferens) cells, peritubular capillary cells, ascending rectal cells, descending rectal cells, immune cells, mesangial cells, parietal epithelial cells, or any combination thereof. In some cases, the method may include seeding endothelial cells, human umbilical vein endothelial cells (HUVEC), or both. In some cases, the cells may be cryopreserved before seeding. The cells may be any animal cells, such as human cells, pig cells, sheep cells, goat cells, monkey cells, bovine cells, dog cells, cat cells, or mixtures thereof. In some cases, the cells may be autologous, heterologous, or allologous cells for the decellularized organ.

[0093] In some embodiments, after engraftment, the cells grow in the extracellular matrix. In some cases, the culture medium is continuously perfused through a recellularized kidney after transplantation to supply nutrients to the engrafted cells. In some cases, the culture medium is replaced with fresh medium after approximately 1, 6, 12, 24, 48, 72, or 96 hours of cell growth, or after less than or equal to those hours.

[0094] Compositions and methods for generating a manipulated organ or a portion thereof containing a population of cells are also provided herein. In some embodiments, at least two populations of cells may be introduced into a decellularized organ or a portion thereof. In some cases, the isolated organ or a portion thereof, which has been at least partially recellularized, includes a kidney or a portion thereof.

[0095] The decellularized organs and parts thereof provided herein can be recellularized. Organs or tissues can be generated by bringing a decellularized organ or tissue, as described herein, into contact with a population of cells. In some embodiments, the population of cells may include manipulated podocyte-like cells. In some cases, the population of cells may be undifferentiated cells, partially differentiated cells, or fully differentiated cells. In some cases, the number of cells that can be introduced into a decellularized organ or part thereof to generate an organ or tissue may depend on both the organ (e.g., what organ it is, its size and weight) or tissue, as well as the cell type and developmental stage. Different cell types may have different tendencies with respect to the population density to which cells reach. Similarly, different organs or tissues can be recellularized at different densities. For example, a decellularized organ or tissue may be “seeded” with more than or equal to about 100, 1,000, 10,000, 100,000, 1,000,000, 10,000,000, or 100,000,000 cells (e.g., manipulated podocyte-like cells), or may have about 1,000 cells / tissue 1 mg (wet weight) to about 10,000,000 cells / tissue 1 mg (wet weight) bound to them. In some embodiments, cells may be introduced into a decellularized organ or tissue (“seeded”) by injection, physical placement, and / or deposition at one or more sites.

[0096] The cells described herein may be further cultured under conditions that result in fully differentiated cells. Furthermore, or alternatively, cells may be obtained from any number of sources, such as blood, kidneys, or any other tissue or organ that harbors cells. Representative cells, for example, may include tubular epithelial cells, macula densa cells, glomerular endothelial cells, podocytes, smooth muscle cells, pericytes, juxtaglomerular cells, collecting duct cells (e.g., CD-PC, CD-Trans, or CD-IC), distal curved tubular cells (e.g., DCT1, DCT2), loop of Henle cells, proximal tubular cells (e.g., curved or straight), afferent arterioles, efferent arterioles, peritubular capillary cells, ascending rectus cells, descending rectus cells, immune cells, mesangial cells, parietal epithelial cells, or any combination thereof. The cells may be any animal cells, such as human cells, pig cells, sheep cells, goat cells, monkey cells, bovine cells, canine cells, cat cells, or mixtures thereof. In some cases, the cells may be autologous, xenogeneic, or allogeneic. In some cases, the cells may be embryonic stem cells, umbilical cord blood cells, tissue-derived stem cells or progenitor cells, bone marrow-derived stem cells or progenitor cells, blood-derived stem cells or progenitor cells, adipose tissue-derived stem cells or progenitor cells, mesenchymal stem cells (MSCs), skeletal muscle-derived cells, induced pluripotent stem cells (iPSCs), genetically modified cells with immunogenic factors, including but not limited to HLA, removed, or pluripotent adult progenitor cells.

[0097] Compositions containing cells as described herein can be delivered to tissue or organ matrices in cell-compatible solutions (e.g., physiological compositions) under physiological conditions (e.g., 37°C) and non-physiological conditions (e.g., 4–35°C). Physiological compositions as described herein may include, but are not limited to, buffers, nutrients (e.g., sugars, carbohydrates), enzymes, growth and / or differentiation media, cytokines, antibodies, repressors, growth factors, salt solutions, or serum-derived proteins.

[0098] In some cases, cells can be introduced into an organ or tissue matrix by perfusion. Perfusion may occur via the vascular system or vascular-type structures of the organ or tissue matrix. Perfusion for recellularizing the organ or tissue matrix may have a flow rate sufficient to circulate the physiological composition of cells through the vascular system. Perfusion by cells may be multidirectional (e.g., anterograde and retrograde). After cell perfusion, a resting period may exist to enhance engraftment before reperfusion of the organ or tissue matrix.

[0099] In some embodiments, at least one cell type can be introduced into the decellularized organ or a portion thereof. For example, a cocktail or population of cells can be injected and / or deposited at multiple locations in the decellularized organ or tissue, or different cell types can be injected and / or deposited in different parts of the decellularized organ or a portion thereof. Alternatively, or in addition to injection, cells or a cocktail of cells can be introduced into the decellularized organ or a portion thereof by perfusion through cannula insertion. For example, cells may be perfused into the decellularized organ using perfusion medium, which can then be replaced with growth and / or differentiation medium to induce cell growth and / or differentiation. During recellularization, the organ or tissue can be maintained under conditions that allow at least a portion of the cells to proliferate, reproduce, differentiate, and any combination thereof in the decellularized organ or a portion thereof. In some embodiments, such conditions may include, but are not limited to, appropriate temperature, pressure, electrical activity, mechanical activity, force, appropriate amounts of O2 and / or CO2, appropriate amounts of humidity, sterile or semi-sterile conditions, and any combination thereof. During recellularization, the decellularized organ or tissue and the cells attached thereto can be maintained in a suitable environment. For example, the manipulated podocyte-like cells may require nutritional supplements (e.g., nutrients and / or carbon sources such as glucose), exogenous hormones or growth factors, and / or a specific pH. In some embodiments, the cells provided herein may be allogeneic, heterogeneic, or autologous to the decellularized organ or part thereof.

[0100] In some embodiments, the manipulated podocyte-like cells can be deposited in a decellularized kidney. The cell population can engraft on the decellularized kidney matrix. In some cases, upon engraftment on the kidney matrix, the manipulated podocyte-like cells may have increased gene expression of NPHS1, NPHS2, and / or SYNPO. In some cases, upon engraftment on the kidney matrix, the manipulated podocyte-like cells may have increased protein expression of podosin, nephrin, podocalyxin, and / or synaptopodin.

[0101] Recellularized organs Recellularized organs or parts thereof, such as recellularized kidneys, are disclosed herein. In some cases, the recellularized organs are recellularized with manipulated podocyte-like cells. The recellularized organs herein may have functions similar to wild-type or primary organs so that the organs can sustain life in animals. In some cases, the scaffold of the recellularized organ (e.g., a decellularized organ or part thereof) is heterogeneous, homogeneous, or autologous to one or more cell populations used to recellularize the decellularized organ or part thereof. In some cases, one or more cell populations may be heterogeneous, homogeneous, or autologous to the decellularized organ or part thereof.

[0102] For example, at least partially recellularized organs or parts thereof may contain the manipulated podocyte-like cells described herein. In some cases, at least partially recellularized isolated organs or parts thereof in a closed-loop ambient perfusion system maintain urine / serum protein levels in urine of less than or equal to 30% about 1 hour after ambient perfusion and / or less than or equal to 65% about 4 hours after transplantation. In some cases, the percentage value indicates the urine / serum normalized value. The normalized value can be calculated as ([total urine protein (g / L)] / [total serum protein (g / L)]) × 100. The percentage of urine / serum protein levels in urine is used to indicate the urine protein concentration compared to the protein concentration in the perfusing blood. For example, a urine / serum normalized protein of less than 1% indicates that the urine protein concentration is less than 1% of the protein concentration perfusing the serum. In some cases, urine protein levels are sustained in liquid. In some cases, isolated organs or portions thereof that have been at least partially re-celled in a closed-loop ambient perfusion system maintain urine / serum protein levels in urine of approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% or more, or equal to, those levels in urine. In some cases, isolated organs or parts thereof that have been at least partially recellularized in a closed-loop ambient perfusion system maintain urine / serum protein levels of approximately 1%–40%, 1%–30%, 10%–35%, 15%–30%, 15%–25%, 20%–30%, or 25%–35% after approximately 1, 2, 3, 4, or 5 hours (or more) of ambient perfusion.In some cases, the isolated organ or part thereof, which was at least partially recellularized after transplantation, was approximately 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52% after approximately 1, 2, 3, 4, or 5 hours (multiple) of transplantation. Sustained urine / serum protein levels in urine that are less than, more than, or equal to 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%. In some cases, the isolated organ or part thereof, which has been at least partially recellularized after transplantation, maintains urine / serum protein levels of approximately 20%–80%, 30%–70%, 35%–65%, 45%–70%, 50%–60%, 30%–55%, or 55%–65% approximately 1, 2, 3, 4, or 5 hours (or more) after transplantation.

[0103] In some cases, the isolated organ or part thereof, which was at least partially recellularized after transplantation, showed a concentration of approximately 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, 21 g / L, 22 g / L after transplantation. Sustained urinary protein levels of 23g / L, 24g / L, 25g / L, 26g / L, 27g / L, 28g / L, 29g / L, 30g / L, 31g / L, 32g / L, 33g / L, 34g / L, 35g / L, 36g / L, 37g / L, 38g / L, 39g / L, 40g / L, 41g / L, 42g / L, 43g / L, 44g / L, or less than, or greater than or equal to, 45g / L. In some cases, the isolated organ or part thereof, which has been at least partially recellularized after transplantation, maintains urinary protein levels of approximately 5 g / L to 45 g / L, 10 g / L to 40 g / L, 20 g / L to 35 g / L, 25 g / L to 35 g / L, 20 g / L to 30 g / L, 24 g / L to 32 g / L, or 30 g / L to 40 g / L approximately 1, 2, 3, 4, or 5 hours (or more) after transplantation. In some cases, isolated organs or parts thereof that were at least partially recellularized in a closed-loop ambient temperature perfusion system showed levels of approximately 0.01 g / L, 0.05 g / L, 0.1 g / L, 0.15 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, approximately 30 minutes or 1 hour after ambient temperature perfusion. Sustained urinary protein levels of 0.9 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, or less than, or greater than or equal to, 20 g / L.In some cases, at least partially recellularized isolated organs or parts thereof in a closed-loop ambient perfusion system maintain urinary protein levels of approximately 0.1 g / L to 20 g / L, 0.1 g / L to 1 g / L, 1 g / L to 10 g / L, 5 g / L to 15 g / L, 8 g / L to 12 g / L, 5 g / L to 10 g / L, or 10 g / L to 17 g / L about 30 minutes or 1 hour after ambient perfusion. In some cases, normal urinary protein is about 0.15 g / L when using a primary kidney. In some cases, normal urinary protein is about 10 g / L when using at least partially recellularized isolated organs or parts thereof. In some cases, urinary protein levels can be determined from the amount of protein in the fluid before and after circulation through the recellularized organ or parts thereof.

[0104] In some cases, isolated organs or parts thereof that were at least partially recellularized in a closed-loop ambient temperature perfusion system showed a concentration of approximately 40 g / L, 41 g / L, 42 g / L, 43 g / L, 44 g / L, 45 g / L, 46 g / L, 47 g / L, 48 g / L, 49 g / L, 50 g / L, 51 g / L, 52 g / L, 53 g / L, and 54 g / L after about 30 minutes or 1 hour of ambient temperature perfusion. Sustained serum protein levels of 55g / L, 56g / L, 57g / L, 58g / L, 59g / L, 60g / L, 61g / L, 62g / L, 63g / L, 64g / L, 65g / L, 66g / L, 67g / L, 68g / L, 69g / L, 70g / L, 71g / L, 72g / L, 73g / L, 74g / L, or less than, or greater than or equal to, 75g / L. In some cases, isolated organs or parts thereof that have been at least partially recellularized in a closed-loop ambient perfusion system maintain serum protein levels of approximately 40 g / L to 75 g / L, 50 g / L to 70 g / L, 55 g / L to 65 g / L, 60 g / L to 70 g / L, 65 g / L to 75 g / L, 58 g / L to 65 g / L, or 63 g / L to 68 g / L approximately 30 minutes or 1 hour after ambient perfusion.

[0105] In some embodiments, at least partially recellularized isolated organs or portions thereof in a closed-loop ambient temperature perfusion system maintain urinary hematocrit levels of less than or equal to 30% after 1 hour of ambient temperature perfusion and / or less than or equal to 1% after 4 hours of transplantation. In some cases, these percentage values ​​represent urine / serum normalized values. Normalized values ​​can be calculated as ([urinary hematocrit%] / [serum hematocrit%]) × 100. The percentage of urine / serum hematocrit levels is used to indicate the concentration of red blood cells in the urine compared to the concentration of red blood cells in the perfusing blood. In some cases, urine / serum hematocrit levels are maintained in liquid. In some cases, isolated organs or portions thereof that were at least partially recellularized in a closed-loop ambient temperature perfusion system showed regeneration rates of approximately 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 0.5%, and 1% respectively after approximately 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, or 5 hours of ambient temperature perfusion. Sustained urine / serum hematocrit levels in urine that are less than, more than, or equal to, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or less than, more than, or equal to, 40%. In some cases, isolated organs or parts thereof that have been at least partially recellularized in a closed-loop ambient temperature perfusion system maintain urine / serum hematocrit levels of approximately 1%–40%, 1%–30%, 10%–35%, 15%–30%, 15%–25%, 20%–30%, or 25%–35% in urine after approximately 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, or 5 hours of ambient temperature perfusion. In some cases, the urine hematocrit level can be determined from the amount of hematocrit in the fluid before and after circulation through the recellularized organ or parts thereof.

[0106] In some cases, isolated organs or portions thereof that have been at least partially re-celled in a closed-loop ambient perfusion system maintain serum urine / serum hematocrit levels of less than, greater than, or equal to, approximately 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, or 55% of these values, or less, greater than, or equal to these values. In some cases, isolated organs or portions thereof that have been at least partially recellularized in a closed-loop ambient perfusion system maintain serum urine / serum hematocrit levels of approximately 25%–55%, 25%–45%, 30%–45%, 35%–45%, 40%–45%, 40%–50%, or 45%–55% after approximately 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, or 5 hours of ambient perfusion.

[0107] In some cases, at least partially recellularized isolated organs or portions thereof after transplantation maintain urine / serum hematocrit levels in urine that are less than, greater than, or equal to, or equal to, those of approximately 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the transplanted isolated organs or portions thereof. In some cases, at least partially recellularized isolated organs or parts thereof after transplantation maintain urine / serum hematocrit levels of approximately 0.01%–20%, 0.1%–3%, 0.1%–1%, 0.5%–1.5%, 0.5%–4%, 1%–5%, 3%–10%, or 8%–18% in the urine approximately 1, 2, 3, 4, or 5 hours after transplantation. For example, the percentage of urine / serum hematocrit levels is used to indicate the concentration of red blood cells in the urine compared to the concentration of red blood cells in the perfuming blood. Therefore, a urine / serum hematocrit value of less than 1% indicates that the concentration of red blood cells in the urine is less than 1% of the concentration of red blood cells in the perfuming blood.

[0108] In some embodiments, the isolated organ or any part thereof, which has been at least partially re-celled after transplantation, can sustain a urine flow rate of less than, faster than, or equal to, approximately 1 mL / h (hour), 2 mL / h, 3 mL / h, 4 mL / h, 5 mL / h, 6 mL / h, 7 mL / h, 8 mL / h, 9 mL / h, 10 mL / h, 11 mL / h, 12 mL / h, 13 mL / h, 14 mL / h, 15 mL / h, 16 mL / h, 17 mL / h, 18 mL / h, 19 mL / h, 20 mL / h, 21 mL / h, 22 mL / h, 23 mL / h, 24 mL / h, 25 mL / h, 26 mL / h, 27 mL / h, 28 mL / h, 29 mL / h, 30 mL / h, 31 mL / h, 32 mL / h, 33 mL / h, 34 mL / h, or 35 mL / h. In some embodiments, the isolated organ or part thereof, at least partially recellularized after transplantation, can maintain urine flow rates of approximately 1 mL / h to approximately 35 mL / h, 10 mL / h to approximately 25 mL / h, 5 mL / h to approximately 15 mL / h, 10 mL / h to approximately 20 mL / h, 15 mL / h to approximately 25 mL / h, 18 mL / h to approximately 24 mL / h, or 20 mL / h to approximately 30 mL / h. In some cases, the urine flow rate can be determined at 10, 20, 30, 40, 50, 60 minutes, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, or more than 5 hours after transplantation.

[0109] In some embodiments, at least partially recellularized isolated organs or portions thereof in a closed-loop ambient temperature perfusion system can sustain urine flow rates of less than, faster than, or equal to, approximately 1 mL / h (hour), 2 mL / h, 3 mL / h, 4 mL / h, 5 mL / h, 6 mL / h, 7 mL / h, 8 mL / h, 9 mL / h, 10 mL / h, 11 mL / h, 12 mL / h, 13 mL / h, 14 mL / h, 15 mL / h, 16 mL / h, 17 mL / h, 18 mL / h, 19 mL / h, 20 mL / h, 21 mL / h, 22 mL / h, 23 mL / h, 24 mL / h, 25 mL / h, 26 mL / h, 27 mL / h, 28 mL / h, 29 mL / h, 30 mL / h, 31 mL / h, 32 mL / h, 33 mL / h, 34 mL / h, or 35 mL / h. In some embodiments, at least partially recellularized isolated organs or portions thereof in a closed-loop ambient perfusion system can maintain urine flow rates of approximately 1 mL / h to approximately 35 mL / h, 10 mL / h to approximately 25 mL / h, 5 mL / h to approximately 15 mL / h, 10 mL / h to approximately 20 mL / h, 15 mL / h to approximately 25 mL / h, 18 mL / h to approximately 24 mL / h, or 20 mL / h to approximately 30 mL / h. In some cases, the urine flow rate can be determined in a closed-loop ambient perfusion system after 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, or more than 5 hours.

[0110] In some embodiments, in a closed-loop ambient perfusion system, or at least a portion of an isolated organ or a portion of an isolated organ that has been partially re-celled after transplantation, can sustain a flow rate of about 0.1 L / min, 0.2 L / min, 0.3 L / min, 0.4 L / min, 0.5 L / min, 0.6 L / min, 0.7 L / min, 0.8 L / min, 0.9 L / min, 1 L / min, 2 L / min, 3 L / min, 4 L / min, or less than, faster than, or equal to, 5 L / min. In some embodiments, in a closed-loop ambient perfusion system, or at least a portion of an isolated organ or a portion of an isolated organ that has been partially re-celled after transplantation, can sustain a flow rate of about 0.1 L / min to about 5 L / min, 0.1 L / min to about 1.5 L / min, 0.5 L / min to about 2 L / min, or 1 L / min to about 3 L / min.

[0111] In some embodiments, isolated organs or portions thereof in a closed-loop ambient temperature perfusion system, or at least partially recellularized after transplantation, can maintain a erythrocyte volume ratio (PCV) greater than or equal to about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35%, as determined by normalized values ​​in urine (e.g., ([urine value] / [serum value]) × 100). In some embodiments, isolated organs or portions thereof in a closed-loop ambient perfusion system, or at least partially recellularized after transplantation, can maintain erythrocyte volume ratio (PCV) (%) as determined by normalized values ​​in urine (e.g., ([urine value] / [serum value]) × 100) in the ranges of approximately 0.1% to approximately 35%, 1% to approximately 10%, 0.5% to approximately 5%, 5% to approximately 20%, 10% to approximately 15%, 25% to approximately 35%, 20% to approximately 30%, 10% to approximately 35%, or 30% to approximately 40%.

[0112] In some embodiments, a closed-loop ambient perfusion system can determine the levels of biological compounds, such as metabolites, in the circulating fluid. In some cases, a closed-loop ambient perfusion system can determine the levels of creatinine, urea, sodium, potassium, glucose, lactate, bicarbonate, salt, blood components, proteins, or any combination thereof. In some cases, samples can be taken from the closed-loop ambient perfusion system before and after circulation in a recellularized organ or a portion thereof to determine the levels of compounds.

[0113] Use of recellularized organs Methods of using recellularized organs or parts thereof are disclosed herein. In some cases, the methods herein may include transplanting a recellularized organ or part thereof into a subject in need of transplantation, for example, a subject with kidney disease. In some cases, a method of treating a disease may include transplanting a recellularized organ or part thereof. The recellularized organs or parts thereof provided herein can be used in a variety of applications. For example, an organ or part thereof can be transplanted into a subject. In some embodiments, compositions herein, such as an organ or part thereof, can be transplanted into a subject with a disease. Related diseases that may require organ transplantation include, but are not limited to, organ failure, cardiomyopathy, cirrhosis, chronic obstructive pulmonary disease, pulmonary edema, biliary atresia, emphysema and pulmonary hypertension, coronary heart disease, valvular heart disease, congenital heart disease, coronary artery disease, pancreatitis, cystic fibrosis, diabetes mellitus, hepatitis, hypertension, idiopathic pulmonary fibrosis, polycystic kidney disease, short bowel syndrome, injury, congenital anomalies, genetic diseases, autoimmune diseases, and any combination thereof. In some cases, the disease may include kidney disease. In some cases, the disease may include end-stage renal disease. In some cases, kidney disease may include Fabry disease, cystinosis, glomerulonephritis, IgA nephropathy, lupus nephritis, atypical hemolytic uremic syndrome, and polycystic kidney disease. In some cases, kidney disease may include chronic kidney disease or acute kidney disease. In some cases, the disease may include acute kidney injury. In some cases, the disease may include Alport syndrome, amyloidosis, Goodpasture disease, glomerular disease, infectious diseases, interstitial nephritis, lupus nephritis, nephrotic syndrome, renal tubular acidosis, unilateral kidney, or any combination thereof. In some cases, the implant may be used to replace or enhance existing tissue by replacing the dysfunctional kidney of the subject with an exogenous or manipulated kidney, such as a recellularized kidney as described herein, for example, to treat a subject with kidney impairment. The subjects may be monitored for recovery from kidney damage after transplantation of an exogenous kidney. Any decellularized organ or part thereof provided herein may be used for transplantation to the subjects.

[0114] In some cases, compositions provided herein, such as solid organs or parts thereof, may have about 1% to about 100% of their natural function after decellularization. In some cases, compositions provided herein, such as solid organs or parts thereof, may have about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or up to about 100% of their natural function after decellularization. In some cases, compositions provided herein, such as recellularized organs or parts thereof, may have about 1% to about 100% of their natural function after recellularization. In some cases, compositions provided herein, such as recellularized organs or parts thereof, may have more, less than, or equal to about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or up to about 100% of their natural function after recellularization.

[0115] In some embodiments, certain organs or parts thereof may be suitable for transplantation if they function below the level of their natural counterparts. For example, a kidney may require approximately 20% of its total organ function to provide the organ function necessary to save a person from kidney failure. In some embodiments, a kidney may require approximately 20-30%, 30-40%, 20-50%, 20-60%, or 40-60% of its total organ function to be suitable for transplantation. In some embodiments, an organ may function equally well as its natural counterpart.

[0116] In some cases, the lifespan of a subject may be extended after transplantation of a composition such as an organ or a part thereof provided herein. For example, the lifespan of a subject may be extended to about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, 20 years, 30 years, 40 years, 50 years, 60 years, 70 years, 80 years, 90 years, or up to about 100 years after transplantation. In some embodiments, transplantation of a composition such as a recellularized organ or a part thereof provided herein may reduce the need for secondary treatment in the subject. Secondary treatment may refer to dialysis (e.g., hemodialysis and / or peritoneal dialysis), pacemakers, ventilators, and combinations thereof. In some cases, secondary treatment may be drug therapy such as renal drug therapy. In some cases, the methods herein may include further secondary treatment of the transplanted subject.

[0117] The decellularized and recellularized organs or parts thereof provided herein can also be used in vitro to screen a wide variety of compounds for the efficacy and cytotoxicity of pharmaceuticals, chemical agents, and growth / regulatory factors. Cultures can be maintained in vitro and exposed to the compounds to be tested. The activity of cytotoxic compounds can be measured by their ability to damage or kill cells in the culture. This can be readily assessed by staining techniques. The effects of growth / regulatory factors can be assessed by analyzing the cellular contents of the matrix, for example, by total cell number and differential cell number. This can be achieved using standard cytological and / or histological techniques, including the use of immunocytochemical techniques with antibodies defining type-specific cell antigens. The effects of various drugs on normal cells cultured in reconstructed artificial organs can be evaluated.

[0118] The decellularized and recellularized organs or parts thereof provided in the present invention can be used in vitro to filter aqueous solutions; for example, a modified artificial kidney can be used to filter blood. Using a modified kidney provides a system having morphological features similar to in vivo kidney products. This system may be suitable for hemodialysis. In some embodiments, the system may also be useful for hemofiltration to remove water and low molecular weight solutes from blood. The artificial kidney can be maintained in vitro and exposed to blood that can be injected into the luminal side of the artificial kidney. The treated aqueous solution can be collected from the non-luminal side of the modified kidney. The efficiency of filtration can be evaluated by measuring the ion or metabolic waste content of the filtered and unfiltered blood.

[0119] The decellularized and recellularized organs or parts thereof provided herein can be used as vehicles for introducing genes and gene products in vivo to assist or improve transplant outcomes and / or for use in gene therapy. For example, cultured cells such as kidney cells can be engineered to express gene products. Cells can be engineered to express gene products transiently and / or under inducible control, or as chimeric fusion proteins immobilized on the cells. In another embodiment, cells can be genetically engineered to express genes that are deficient in a patient or that would exert a therapeutic effect. The target gene engineered into the cells may be related to the disease being treated. For example, with respect to kidney injury, endothelial or cultured kidney cells can be engineered to express gene products that restore kidney injury.

[0120] Furthermore, compositions and methods for generating an artificial organ or a portion thereof, including populations of cells such as artificial podocyte-like cells, are provided herein. In some embodiments, at least two populations of cells may be introduced into a decellularized organ or a portion thereof. Organs that can be manipulated include, but are not limited to, the heart, kidney, liver, pancreas, spleen, bladder, ureter, urethra, skeletal muscle, small and large intestines, esophagus, stomach, brain, spinal cord, and bone. In some cases, the kidney is an example of an organ that can be manipulated.

[0121] In some cases, recellularized kidneys can be transplanted into recipients. Recellularized kidneys, as described herein, can be transplanted as functional organs. In some cases, function can be determined through fluid filtration by the recellularized organ. In some embodiments, functionality can be assessed by determining the consumption of certain metabolites (i.e., glucose, lactate, glutamine, glutamate, and ammonia). Such consumption can be determined by perfusing through a continuous line of metabolites and by measuring the rate of metabolite consumption over time, for example, using changes in electrochemical potential. In some cases, the rate of metabolite consumption can be used to determine the successful engraftment of endothelial cells onto the decellularized matrix.

[0122] In some cases, recellularized kidneys can be transplanted with systemic administration of immunosuppressants. Immunosuppressant administration can extend the patency of the transplanted organ. In some cases, the immunosuppressants may be corticosteroids, Janus kinase inhibitors, calcineurin inhibitors, mTOR inhibitors, IMDH inhibitors, biologics, monoclonal antibodies, or any combination thereof. Examples of corticosteroids include prednisone, budesonide, prednisolone, and methylprednisolone. An example of a Janus kinase inhibitor is tofacitinib. Examples of calcineurin inhibitors include cyclosporine and tacrolimus. Examples of mTOR inhibitors include sirolimus and everolimus. Examples of IMDH inhibitors include azathioprine, leflunomide, and mycophenolate. Examples of immunosuppressive biological agents include abatacept, adalimumab, anakinra, certolizumab, etanercept, golimumab, infliximab, ixekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinumab, and vedolizumab. Examples of immunosuppressive monoclonal antibodies include basiliximab and daclizumab. Such immunosuppressants may be administered to recipients of recellularized kidneys via enteral routes (including oral, gastric or duodenal feeding tubes, rectal suppositories and rectal enemas), parenteral routes (injection or infusion, intra-arterial, intracardiac, intraventricular, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreous, epidural and subcutaneous), inhalation, percutaneous, transmucosal, sublingual, buccal or topical (including supercutaneous, dermal, enema, eye drops, ear drops, intranasal, and vaginal) administration.Immunosuppressants are administered to the recipient in doses of approximately 0.001mg to 1mg, 0.01mg to 1mg, 0.1mg to 10mg, 1mg to 1000mg, 5mg to 1000mg, 10mg to 1000mg, 15mg to 1000mg, 20mg to 1000mg, 25mg to 1000mg, 30mg to 1000mg, and 35mg or more. About 1000mg, about 40mg to about 1000mg, about 45mg to about 1000mg, about 50mg to about 1000mg, about 55mg to about 1000mg, about 60mg to about 1000m g, about 65mg to about 1000mg, about 70mg to about 1000mg, about 75mg to about 1000mg, about 80mg to about 1000mg, about 85mg to about 1000mg, about 90mg ~1000mg, approx. 95mg~1000mg, approx. 100mg~1000mg, approx. 150mg~1000mg, approx. 200mg~1000mg, approx. 250mg~approx. 1000mg, about 300mg to about 1000mg, about 350mg to about 1000mg, about 400mg to about 1000mg, about 450mg to about 1000mg, about 500mg to about 10 It can be administered in doses of 00 mg, approximately 550 mg to 1000 mg, approximately 600 mg to 1000 mg, approximately 650 mg to 1000 mg, approximately 700 mg to 1000 mg, approximately 750 mg to 1000 mg, approximately 800 mg to 1000 mg, approximately 850 mg to 1000 mg, approximately 900 mg to 1000 mg, or approximately 950 mg to 1000 mg.

[0123] kit Kits are also disclosed herein. In some cases, a kit may include compositions described herein. In some embodiments, a kit may include one or more culture media disclosed herein, for example, a first culture medium and / or a second culture medium in a container. In some cases, a kit may include cells such as glomerular cells or manipulated podocyte-like cells. In some cases, a kit may include at least a partially recellularized organ or part thereof and a container. In some cases, a kit may include at least a partially decellularized organ or part thereof and a container. Kits herein may include containers. In some cases, the container may include glass, metal, plastic, and / or a material suitable for a container.

[0124] In some embodiments, the kit may include a first medium for culturing podocyte-like cells containing at least one of retinoic acid, its salts, corticosteroids, their salts, calcitriol, or their salts in a container, and / or a second medium for culturing podocyte-like cells containing at least one of SB431542, its salts, IWR-1-endo, or its salts in a container. In some cases, the first medium, the second medium, or both may contain glomerular cells. [Examples]

[0125] [Example 1]: Preparation of manipulated podocyte-like cells To initiate the podocyte differentiation protocol, glomerular elongating cells are cultured in medium 1 for a total of 3 days, with the first medium change occurring 48 hours after the initial culture. Three days after exposure to medium 1, glomerular elongating cells are cultured in medium 2 for a further 7 days, with fresh medium change of medium 2 every 48 hours thereafter. After a 10-day differentiation protocol, the manipulated podocyte-like cells are widely found in the bioengineered kidney, as shown in Figure 1, and confer filtration function. Table 1 shows the components of medium 1 and medium 2.

[0126] [Table 1]

[0127] [Example 2]: Manipulated podocyte-like cells are functional in the manipulated kidney. Perfusion at room temperature and with porcine cells was performed using bioengineered kidneys containing manipulated podocyte-like cells. The kidneys exhibited sustained filtration function, indicated by significantly lower urinary protein levels than serum levels, e.g., protein retention for longer than 4 hours, as well as sustained hematocrit, indicated by low (less than 15%) hematocrit in the urine, as is evident from Figures 2B and 6B. Data from Figure 2C show that bioengineered kidneys recellularized with manipulated podocyte-like cells exhibit glomerular regrowth by cells expressing podosin, a protein used in podocyte function. Furthermore, the bioengineered kidneys exhibit an assembly of cells showing primary processes, as indicated by the arrows. Primary processes are unique functionally relevant structural features possessed by podocytes, maintaining the complex cellular buildup that enables them to perform glomerular filtration.

[0128] [Example 3]: Manipulated podocyte-like cells exhibit podocyte characteristics. The manipulated podocyte-like cells were differentiated as described in Example 1. The expression of podocyte proteins was determined by fluorescence microscopy of the manipulated podocyte-like cells. Figure 3A shows immunofluorescence labeling for F-actin in second medium-treated glomerular elongation cells (e.g., manipulated podocyte-like cells) cultured on collagen I, and Figure 3B shows immunofluorescence labeling for vimentin, a type III intermediate filament protein. These images show characteristic podocyte cytoskeleton tissue with multiple elongations, indicated by circles. Figure 3C shows that second medium-treated glomerular elongation cells (e.g., manipulated podocyte-like cells) exhibit protruding nephrin, a protein involved in filtration. Nephrin expression was localized to the cell membrane, as indicated by arrows. Figure 3D shows that glomerular elongating cells treated with a second medium (e.g., manipulated podocyte-like cells) evoke upregulation of several podocyte markers (NPHS1, NPHS2, and SYNPO) as indicated by quantitative reverse transcriptase polymerase chain reaction (PCR). NPHS1 encodes nephrin, NPHS2 encodes podosin, and SYNPO encodes synaptopodin. NPHS1 showed an approximately 8-fold increase in expression after growth in the second medium compared to growth in RM medium. NPHS2 showed an approximately 4-fold increase in expression after growth in the second medium compared to growth in RM medium. SYNPO showed an approximately 2.5-fold increase in expression after growth in the second medium compared to growth in RM medium. Glomerular elongating cells treated with the second medium were compared to glomerular elongating cells grown in standard R-endothelial medium after a 6-day culture period. Figures 3E to 3F show high-magnification images of immunofluorescence labeling for vimentin, indicating the region where characteristic secondary processes of podocytes interact (as shown by arrowheads).

[0129] [Example 4]: Differences between manipulated podocyte-like cells and primary podocytes Using fluorescence microscopy, we determined the differences between engineered podocyte-like cells developed using the methods disclosed herein and primary podocytes obtained directly from kidney. Podosin expression was compared between the two cell types. As shown in Figure 4, primary podocytes (left image) had higher and more appropriately localized podosin expression than differentiated engineered podocyte-like cells (right image). Other podocyte proteins, such as nephrin and synaptopodin, showed similar trends (not shown). Images were taken from cells grown in culture. Therefore, the images may suggest that differentiated engineered podocyte-like cells may lack biological artifacts, such as cellular factors and intercellular interactions, present in native kidney cells that drive their distinctive phenotype.

[0130] [Example 5]: Seeding and culturing of renal vesicles and evaluation of filtration function To initiate kidney cell seeding, the culture medium was removed from the bioreactor chamber and replaced with fresh preheated medium, and the perfusion was switched from arterial to ureteral.

[0131] Podocytes, including manipulated podocyte-like cells and mesangial cells, were seeded into decellularized porcine kidney extracellular matrix via ureteral perfusion. A small vacuum (20–40 mmHg) was applied in the bioreactor chamber while the kidney was positioned above the culture medium volume to allow the seeded cells to reach the glomeruli of the decellularized porcine kidney extracellular matrix. Perfusion was stopped, and the cell suspension was drawn from a sterile bottle into the renal ureter through a tube secured to the bioreactor chamber using the vacuum. These cells were drawn up to the glomeruli, at which point they reached a physical barrier (i.e., the glomerular basement membrane) and were retained within the glomeruli.

[0132] Next, the recellularized kidneys were continuously perfused with culture medium. In some cases, procedures requiring pauses in perfusion should be performed. Once the first seeding was initiated, the culture medium in the bioreactor was replaced with fresh medium every 24 hours. During medium changes, perfusion was paused and the medium was replaced with fresh preheated medium. Similarly, if perfusion was switched to a different conduit, perfusion was paused while moving the connection. To ensure sufficient nutrients throughout the culture period, samples were collected daily before medium changes to monitor glucose and increase the medium volume as needed (up to the maximum volume allowed by the bioreactor assembly).

[0133] To evaluate the filtration function of bioengineered kidneys, our proprietary closed-loop ambient perfusion system facilitated the collection of both blood and urine samples, and functionally relevant analytes, including creatinine, urea, sodium, potassium, total protein, hematocrit, glucose, lactate, and bicarbonate, were measured. Protein and hematocrit (HCT) readings were specifically used to assess the degree of filtration performed by the engineered podocyte-like cells.

[0134] [Example 6]: Kidney transplantation by bioengineering The subjects are diagnosed with renal failure due to kidney disease. To treat the disease, the subjects receive a transplant of a recellularized kidney containing the modified podocyte-like cells described herein. After transplantation, the recellularized kidney will have at least partial renal function, and the subjects will not have renal failure.

[0135] [Example 7]: Seed sowing method for obtaining filtration function To prepare the vascular pathways, decellularized porcine kidney grafts were perfused over the renal artery with epithelial growth medium (EGM) for 30 minutes, followed by seeding of HUVECs. Initial seeding of 150 million HUVECs at a concentration of 2 million cells / mL was performed using manual syringe seeding at a rate of 50 mL / min. This seeding helped to re-endothelialize the glomerular capillaries. The renal artery was perfused for a further 30 minutes after HUVEC seeding. To prepare the urinary pathways, the grafts were perfused over the ureter with endothelial growth medium (EGM) for 30 minutes, followed by seeding of glomerular elongating cells (GOCs). 500 million GOCs were seeded through the ureter for 30 minutes while maintaining the bioreactor under a vacuum pressure of -40 mmHg. This seeding helped to regrow the urinary side of the glomerular basement membrane. Following the seeding described above, the grafts were cultured in EGM on days 0-2 of the bioengineered kidney culture to promote the growth of HUVECs and GOCs. On day 3 of the bioengineered kidney culture, the culture medium was changed from EGM to a second medium (e.g., a medium containing SB431542 and IWR-1-endo) to promote podocyte differentiation. The second medium culture was continued until day 8. On day 9 of the bioengineered kidney culture, the culture medium was changed to a transition medium composed of equal parts of the second medium and EGM. To re-endothelialize the renal vascular system, a series of HUVEC seedings were performed on days 10-12 of the bioengineered kidney culture: on day 10, 150 million HUVECs were syringe-seeded through the renal vein; on day 11, 150 million HUVECs were syringe-seeded through the renal artery; and on day 12, 150 million HUVECs were syringe-seeded through the renal artery. Following a series of HUVEC seedings, the bioengineered kidneys were perfused arterially with EGM, starting at a flow rate of 50 mL / min and gradually increasing by 50 mL / min daily until either the flow rate or arterial pressure reached a maximum of 500 mL / min or 80 mmHg, respectively. Perfusion was maintained at these maximum values ​​for the remainder of the bioengineered kidney culture period (days 24-25). Throughout the entire bioengineered kidney culture period, the second culture medium was freshened every other day, and the EGM was freshened with daily medium changes.Figure 8 shows an overview of the development of kidney grafts by bioengineering using the methods described herein.

[0136] [Example 8] Functional test by room temperature mechanical perfusion Pig blood was circulated by a digitally controlled pump and maintained at a temperature of 37°C by a water bath circulator (aerated through an oxygen supply with a gas mixture of 20% O2, 5% CO2, and 75% N2). The flow rate of the gas mixture was adjusted to 150 mL / min using a steel ball rotometer. From the blood reservoir, blood was pumped through the oxygen supply into the renal artery, drained from the kidney through the renal vein, and circulated back to the blood reservoir. A target arterial pressure of 100 mmHg was maintained by a CompactRIO-based PID system. The resulting perfusion flow rate ranged from 300 mL / min to 800 mL / min. Pressure and flow rate data were continuously acquired using custom software. Urine and perfusion fluid samples were collected every 15 minutes for 1 hour. Samples were processed to obtain plasma, which was stored at -80°C for subsequent analysis.

[0137] [Example 9]: Transplantation Method The peritoneum of the pigs was opened via a midline incision, and the kidneys were ectopically transplanted into the inferior vena cava (IVC) and abdominal aorta (AA). The ureters were led into 0.25-inch diameter silicone tubes connected to a 50 mL conical tube for effluent collection. After graft reperfusion, samples of the perfusing pig blood and collected urine were taken to assess renal function.

[0138] [Example 10]: Further exemplary podocyte differentiation protocol To initiate the podocyte differentiation protocol, glomerular elongating cells were cultured in podocyte-designated medium (PSM) for 4–6 days, with PSM medium changes performed every 48 hours after the initial culture. After 4–6 days of PSM medium exposure, glomerular elongating cells were cultured in YoDa medium for a further 2–4 days, with YoDa medium changes every 48 hours. Figure 9 shows the medium scheme used to induce the conversion of glomerular elongating cells to podocytes, where each square represents one day of culture and the arrows indicate when medium changes should be performed using a specific medium. After a culture period of 6–10 days, podocytes were widely observed as shown in Figures 10 and 11. Table 2 shows the components of the PSM and YoDa mediums used in this example and exemplary amounts / concentrations of those components.

[0139] [Table 2]

[0140] [Example 11]: Further exemplary podocyte differentiation protocol To initiate the podocyte differentiation protocol, glomerular elongating cells were cultured in panobinostat medium for 2–6 days, with medium changes using panobinostat medium performed every 48 hours after the initial culture. Figure 12 shows the medium scheme for inducing the conversion of glomerular elongating cells into podocytes, where each square represents one day of culture and the arrows indicate when medium changes should be performed using a specific medium. After the culture period, podocytes were present as shown in Figures 13 and 14. Table 3 shows the components of the panobinostat medium.

[0141] [Table 3]

[0142] [Example 12]: Maintenance of podocytes The podocyte maintenance medium described below provides an excellent and innovative solution for maintaining the phenotype of differentiated podocytes in co-culture with other kidney cells for our kidney grafts. The following protocol specifies how the cell culture medium is formulated and used to maintain differentiated podocytes. After podocyte differentiation was complete, the podocytes were cultured in kidney co-culture medium (KCM) + 4 μM SB431542 for up to 14 days, with medium changes every other day. See Figure 15. Table 4 shows the components of the maintenance medium.

[0143] [Table 4]

[0144] amino acid sequence Sequence ID 1

[0145] [ka]

[0146] Sequence ID 2

[0147] [ka]

[0148] Sequence ID 3 NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR

[0149] Sequence ID 4 GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSA

[0150] Preferred embodiments of the Disclosure have been shown and described herein, but such embodiments are provided merely as examples. Numerous modifications, changes, and substitutions can be made without departing from the Disclosure. Various alternatives to the embodiments of the Disclosure described herein may be used to carry out the Disclosure. The accompanying claims define the scope of the Disclosure and are intended to encompass the methods and structures of these claims and their equivalents.

Claims

1. A method for producing manipulated podocyte-like cells, a) Culturing glomerular cells for about 2 to 4 days in a first medium containing at least one of retinoic acid, corticosteroids, calcitriol, or a salt of any one thereof, b) Removing the glomerular cells from the first culture medium, c) The glomerular cells are cultured for about 6 to 12 days in a second medium containing at least one of SB431542, its salt, IWR-1-endo, or its salt. Includes, Culturing the glomerular cells in the second culture medium for approximately 6 to 12 days leads to the differentiation of the glomerular cells into the manipulated podocyte-like cells. The method wherein the manipulated podocyte-like cells have increased expression of one or more of podosins, nephrins, podocalyxins, or synaptopodins when compared to the glomerular cells before culturing in the first medium.

2. The method according to claim 1, wherein the first culture medium comprises dexamethasone or a salt thereof.

3. The method according to claim 1 or 2, wherein the first culture medium further comprises a basal medium, a nutrient mixture, an antibiotic, insulin-transferrin-selenium (ITS), fetal bovine serum, a salt of any of these, or any combination thereof.

4. The method according to claim 3, wherein the first culture medium further comprises penicillin, streptomycin, a salt of either of these, or any combination thereof.

5. The method according to any one of claims 1 to 4, wherein the first culture medium comprises the retinoic acid or a salt thereof, and the concentration of the retinoic acid or salt thereof in the first culture medium is about 1 μM to about 1 mM.

6. The method according to any one of claims 1 to 5, wherein the first culture medium comprises the corticosteroid or a salt thereof, and the concentration of the corticosteroid or salt thereof in the first culture medium is about 100 nM to about 10 mM.

7. The method according to any one of claims 1 to 6, wherein the first culture medium comprises the calcitriol or a salt thereof, and the concentration of the calcitriol or salt thereof in the first culture medium is about 1 nM to about 300 nM.

8. The method according to any one of claims 1 to 7, wherein the second culture medium further comprises a basal medium, a nutrient mixture, an antibiotic, insulin-transferrin-selenium (ITS), fetal bovine serum, a salt of any of these, or any combination thereof.

9. The method according to claim 8, wherein the second culture medium comprises penicillin, streptomycin, a salt of either of these, or any combination thereof.

10. The method according to any one of claims 1 to 9, wherein the second culture medium comprises SB431542 or a salt thereof, and the concentration of SB431542 or a salt thereof in the second culture medium is about 1 μM to about 10 μM.

11. The method according to any one of claims 1 to 10, wherein the second culture medium comprises IWR-1-endo or a salt thereof, and the concentration of IWR-1-endo or the salt thereof in the second culture medium is about 1 μM to about 10 μM.

12. The method according to any one of claims 1 to 11, wherein the manipulated podocyte-like cells have increased gene expression of NPHS1, NPHS2, SYNPO, or any combination thereof, compared to the glomerular cells.

13. The method according to claim 12, wherein the increase in gene expression is determined by quantitative reverse transcriptase PCR.

14. The method according to any one of claims 1 to 13, wherein the glomerular cell is a glomerular elongation cell.

15. The method according to any one of claims 1 to 14, wherein the manipulated podocyte-like cells include cytoskeletal tissue having multiple elongations when compared to the cytoskeletal tissue of glomeruli.

16. The method according to any one of claims 1 to 15, wherein the first culture medium is replaced with fresh first culture medium during the culture of glomerular cells in the first culture medium approximately 48 hours after cell culture.

17. The method according to any one of claims 1 to 16, wherein the second medium is replaced with fresh second medium during the culture of glomerular cells in the second medium approximately 48 hours after cell culture.

18. The method according to any one of claims 1 to 17, wherein the manipulated podocyte-like cells have a reduced expression of one or more of podosin, nephrin, podocalixin, or synaptopodin compared to primary podocytes.

19. The method according to any one of claims 1 to 18, wherein the glomerular cells are animal glomerular cells.

20. The method according to claim 19, wherein the animal glomerular cells are human glomerular cells.

21. The method according to claim 19, wherein the animal glomeruli are pig glomeruli, sheep glomeruli, goat glomeruli, monkey glomeruli, bovine glomeruli, dog glomeruli, or cat glomeruli.

22. The method according to claim 1, wherein the first culture medium comprises the retinoic acid or a salt thereof.

23. The method according to claim 1, wherein the first culture medium comprises the corticosteroid or a salt thereof.

24. The method according to claim 1, wherein the first culture medium comprises the calcitriol or a salt thereof.

25. The method according to claim 1, wherein the second culture medium comprises SB431542 or a salt thereof.

26. The method according to claim 1, wherein the second culture medium comprises IWR-1-endo or a salt thereof.

27. The method according to any one of claims 1 to 26, wherein the glomerular cells are cultured in the first culture medium for about three days.

28. The method according to any one of claims 1 to 27, wherein the glomerular cells are cultured in the second culture medium for about seven days.

29. The method according to any one of claims 1 to 28, wherein the glomerular cells are engrafted on at least partially decellularized kidney extracellular matrix.

30. The method according to any one of claims 1 to 29, wherein the increase in the expression of one or more of podosin, nephrin, podocalyxin, or synaptopodin is determined by fluorescence microscopy, Western blotting, flow cytometry, or any combination thereof.

31. a) Increased expression of one or more of podosin, nephrin, podocalyxin, or synaptopodin compared to glomerular cells, b) Reduced expression of one or more of podosin, nephrin, podocalyxin, or synaptopodin compared to primary podocyte cells. Manipulated podocyte-like cells, including those mentioned above.

32. The manipulated podocyte-like cell according to claim 31, comprising a cytoskeletal tissue having multiple elongations when compared to the cytoskeletal tissue of the glomerular cell.

33. The manipulated podocyte-like cell according to claim 31 or 32, having increased gene expression of NPHS1, NPHS2, SYNPO, or any combination thereof, compared to the glomerular cell.

34. The manipulated podocyte-like cell according to any one of claims 31 to 33, wherein the manipulated podocyte-like cell has a reduced localization of one or more of podosins, nephrins, podocalixins, or synaptopodins compared to the primary podocyte.

35. A modified podocyte-like cell prepared by the method described in any one of claims 1 to 30.

36. A method for engrafting cells onto the extracellular matrix of kidney cells that has been at least partially decellularized, The at least partially decellularized kidney extracellular matrix is ​​brought into contact with the manipulated podocyte-like cells according to any one of the claims 31 to 35. The method, including the method described above.

37. The method according to claim 36, wherein the contact is performed in a bioreactor chamber, and the contact includes depositing the plurality of manipulated podocyte-like cells suspended in an aqueous composition onto the glomeruli of the at least partially decellularized renal extracellular matrix through the ureter, thereby engrafting the cells onto the at least partially decellularized renal extracellular matrix.

38. The method according to claim 36 or 37, further comprising seeding a plurality of mesangial cells, a plurality of human umbilical vein endothelial cells (HUVECs), or both.

39. The method according to claim 37 or 38, wherein the deposition through the ureter is carried out under reduced pressure in the bioreactor chamber.

40. The method according to any one of claims 37 to 39, further comprising continuously perfusing a culture medium through the at least partially decellularized kidney extracellular matrix after engraftment.

41. The method according to claim 40, wherein the culture medium is replaced approximately every 24 hours.

42. An isolated organ or part thereof that is at least partially recellularized, comprising the manipulated podocyte-like cells described in any one of claims 31 to 35, wherein the isolated organ or part thereof is at least partially recellularized in a closed-loop ambient temperature perfusion system. a) Persistent urine / serum protein levels of less than 30% one hour after normal temperature perfusion and less than 65% four hours after transplantation, or b) Maintain a urine / serum hematocrit level of less than 30% one hour after normal temperature perfusion and less than 1% four hours after transplantation. The aforementioned isolated organ or a portion thereof that has been at least partially recellularized.

43. An isolated organ or part thereof that has been at least partially recellularized according to claim 42, comprising a kidney or a part thereof.

44. The at least partially recellularized isolated organ or part thereof according to claim 42 or 43, wherein the levels of creatinine, urea, sodium, potassium, glucose, lactate, bicarbonate, or any combination thereof are determined in the closed-loop ambient temperature perfusion system.

45. A portion of an isolated organ or any part thereof that is at least partially recellularized according to any one of claims 42 to 44, which is homogeneous with respect to the aforementioned manipulated podocyte-like cells.

46. An isolated organ or part thereof that is at least partially recellularized according to any one of claims 42 to 44, which is autologous to the aforementioned manipulated podocyte-like cells.

47. A part of an isolated organ or a portion thereof that is heterogeneous to the aforementioned manipulated podocyte-like cells, according to any one of claims 42 to 44, which is at least partially recellularized.

48. A kit comprising: a first culture medium for culturing podocyte-like cells, containing at least one of retinoic acid, corticosteroid, calcitriol, or a salt thereof in a container; and a second culture medium for culturing podocyte-like cells, containing at least one of SB431542, IWR-1-endo, or a salt thereof in a container.

49. The kit according to claim 48, wherein the first culture medium, the second culture medium, or both contain glomerular cells.

50. A method for producing manipulated podocyte-like cells, a) Glomerular cells are cultured for about 4 to 8 days in a first medium containing at least one of a transforming growth factor beta pathway inhibitor and a Wnt pathway inhibitor, or a salt thereof. b) Removing the glomerular cells from the first culture medium, c) The glomerular cells are cultured for about 2 to 6 days in a second medium containing at least one of retinoic acid, a Rho kinase (ROCK) inhibitor, or a salt thereof. The method comprising culturing the glomerular cells in the second culture medium for about 2 to 6 days, thereby causing the glomerular cells to differentiate into the manipulated podocyte-like cells.

51. The method according to claim 50, wherein the manipulated podocyte-like cells have an increase in finger-like indentation foot processes compared to the glomerular cells before being cultured in the first culture medium.

52. The method according to claim 50, wherein the manipulated podocyte-like cells express at least one of F-actin and vimentin.

53. The method according to claim 50, wherein the manipulated podocyte-like cells express at least one of podosin, nephrin, podocalyxin, or synaptopodin.

54. The method according to claim 50, wherein the first culture medium comprises the transforming growth factor beta pathway inhibitor, and the transforming growth factor beta pathway inhibitor comprises SB431542.

55. The method according to claim 54, wherein the concentration of SB431542 is approximately 2 μM to 10 μM.

56. The method according to claim 54, wherein the concentration of SB431542 is 4 μM.

57. The method according to claim 50, wherein the first culture medium contains the Wnt pathway inhibitor, and the Wnt pathway inhibitor contains IWR-1.

58. The method according to claim 57, wherein the concentration of IWR-1 is approximately 2 μM to 20 μM.

59. The method according to claim 57, wherein the concentration of IWR-1 is 2 μM.

60. The method according to claim 50, wherein the second culture medium contains the retinoic acid.

61. The method according to claim 60, wherein the concentration of retinoic acid is 0.2 μM.

62. The method according to claim 50, wherein the second culture medium contains the ROCK inhibitor, and the ROCK inhibitor contains Y-27632.

63. The method according to claim 62, wherein the concentration of Y-27632 is approximately 2 μM to approximately 15 μM.

64. The method according to claim 62, wherein the concentration of Y-27632 is 10 μM.

65. The method according to claim 50, wherein at least one of the first culture medium and the second culture medium further comprises at least one of heparin, a hormone, and a glycoprotein.

66. The method according to claim 50, wherein at least one of the first culture medium and the second culture medium further comprises insulin-transferrin-selenium or a salt thereof.

67. The method according to claim 50, wherein at least one of the first culture medium and the second culture medium further comprises an antibiotic.

68. The method according to claim 50, wherein at least one of the first culture medium and the second culture medium contains at least one of penicillin and streptomycin.

69. A method for producing manipulated podocyte-like cells, Glomerular cells should be cultured in a culture medium containing a histone deacetylase inhibitor for at least approximately 4 to 8 days. The method comprising, wherein culturing the glomerular cells results in differentiation of the glomerular cells into the manipulated podocyte-like cells.

70. The method according to claim 69, wherein the histone deacetylase inhibitor comprises hydroxamic acid or a salt thereof.

71. The method according to claim 69, wherein the histone deacetylase inhibitor comprises panobinostat or a salt thereof.

72. The method according to claim 71, wherein the concentration of panobinostat is approximately 50 nM to approximately 200 nM.

73. The method according to claim 71, wherein the concentration of panobinostat is 50 nM.

74. The method according to claim 69, wherein the culture medium further comprises at least one of a hormone and a glycoprotein.

75. The method according to claim 69, wherein the culture medium further comprises insulin-transferrin-selenium (ITS).

76. The method according to claim 69, wherein the culture medium further comprises an antibiotic.

77. The method according to claim 69, wherein the culture medium further comprises at least one of penicillin and streptomycin.

78. A method for maintaining manipulated podocyte-like cells, comprising culturing the manipulated podocyte-like cells in a medium containing at least one of penicillin-streptomycin, fetal bovine serum, heparin, ascorbic acid, hydrocortisone, rh FGF, rh VEGF, rh EGF, Long R3 IGF, insulin, triiodothyronine, epinephrine, holotransferrin, and SB431542.

79. The method according to claim 78, wherein the culture medium comprises penicillin-streptomycin.

80. The method according to claim 79, wherein the concentration of penicillin-streptomycin is 1%.

81. The method according to claim 78, wherein the culture medium contains fetal bovine serum.

82. The method according to claim 81, wherein the concentration of fetal bovine serum is 2%.

83. The method according to claim 78, wherein the culture medium contains heparin.

84. The method according to claim 83, wherein the concentration of heparin is 1.05 U / mL.

85. The method according to claim 78, wherein the culture medium contains ascorbic acid.

86. The method according to claim 85, wherein the concentration of ascorbic acid is 50 μg / mL.

87. The method according to claim 78, wherein the culture medium contains hydrocortisone.

88. The method according to claim 87, wherein the concentration of hydrocortisone is 1.15 μg / mL.

89. The method according to claim 78, wherein the culture medium contains rh FGF.

90. The method according to claim 89, wherein the concentration of rh FGF is 20 ng / mL.

91. The method according to claim 78, wherein the culture medium contains rh VEGF.

92. The method according to claim 91, wherein the concentration of rh VEGF is 5 ng / mL.

93. The method according to claim 78, wherein the culture medium contains rh EGF.

94. The method according to claim 93, wherein the concentration of rh EGF is 15 ng / mL.

95. The method according to claim 78, wherein the culture medium contains Long R3 IGF.

96. The method according to claim 95, wherein the concentration of Long R3 IGF is 15 ng / mL.

97. The method according to claim 78, wherein the culture medium contains insulin.

98. The method according to claim 97, wherein the insulin concentration is 0.125 U / mL.

99. The method according to claim 78, wherein the culture medium contains triiodothyronine (T3).

100. The method according to claim 99, wherein the concentration of triiodothyronine (T3) is 10 nM.

101. The method according to claim 78, wherein the culture medium contains epinephrine.

102. The method according to claim 101, wherein the concentration of epinephrine is 1 μM.

103. The method according to claim 78, wherein the culture medium comprises holotransferrin.

104. The method according to claim 103, wherein the concentration of holotransferrin is 5 μg / mL.

105. The method according to claim 78, wherein the culture medium contains SB431542.

106. The method according to claim 105, wherein the concentration of SB431542 is 4 μM.

107. A modified podocyte-like cell prepared by the method described in any one of claims 50 to 106.

108. A method for engrafting cells onto the extracellular matrix of kidney cells that has been at least partially decellularized, The at least partially decellularized kidney extracellular matrix is ​​brought into contact with the manipulated podocyte-like cells according to any one of the claims 50 to 106. The method, including the method described above.

109. The method according to claim 108, wherein the contact is performed in a bioreactor chamber, and the contact includes depositing the plurality of manipulated podocyte-like cells suspended in an aqueous composition onto the glomeruli of the at least partially decellularized renal extracellular matrix through the ureter, thereby engrafting the cells onto the at least partially decellularized renal extracellular matrix.

110. The method according to claim 108 or 109, further comprising seeding a plurality of mesangial cells, a plurality of human umbilical vein endothelial cells (HUVECs), or both.

111. The method according to any one of claims 108 to 110, wherein the deposition through the ureter is carried out under reduced pressure in the bioreactor chamber.

112. The method according to any one of claims 108 to 110, further comprising continuously perfusing a culture medium through the at least partially decellularized kidney extracellular matrix after engraftment.

113. The method according to claim 112, wherein the culture medium is replaced approximately every 24 hours.

114. An isolated organ or part thereof that is at least partially recellularized, comprising manipulated podocyte-like cells according to any one of claims 50 to 106, wherein the isolated organ or part thereof is at least partially recellularized in a closed-loop ambient temperature perfusion system. a) Persistent urine / serum protein levels of less than 30% one hour after normal temperature perfusion and less than 65% four hours after transplantation, or b) Maintain a urine / serum hematocrit level of less than 30% one hour after normal temperature perfusion and less than 1% four hours after transplantation. The aforementioned isolated organ or a portion thereof that has been at least partially recellularized.

115. An isolated organ or part thereof that has been at least partially recellularized according to claim 114, comprising a kidney or a part thereof.

116. The at least partially recellularized isolated organ or part thereof according to claim 114 or 115, wherein the levels of creatinine, urea, sodium, potassium, glucose, lactate, bicarbonate, or any combination thereof are determined in the closed-loop ambient temperature perfusion system.

117. A portion of an isolated organ or any part thereof that is of the same type as the manipulated podocyte-like cells, according to any one of claims 114 to 116.

118. An isolated organ or part thereof that is at least partially recellularized according to any one of claims 114 to 116, which is autologous to the aforementioned manipulated podocyte-like cells.

119. A part of an isolated organ or a portion thereof that is heterogeneous with respect to the aforementioned manipulated podocyte-like cells, according to any one of claims 114 to 116, at least partially recellularized.

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