Organoids comprising isolated kidney cells and uses thereof

Abstract

Described herein are organoids comprising a mixture of selected biologically active primary kidney cells and biologically active cell populations, such as endothelial cell populations, such as HUVEC cells, and methods of using the organoids to treat a subject in need. Further, the isolated kidney cells can include a population of renal tubular cells and a population of erythropoietin {EPO}-producing kidney cells, which can be autologous, syngeneic, allogeneic, or xenogeneic in origin, or any combination thereof. Further, methods of using the organoids to treat a subject in need are provided.

Description

[0001] This application is a divisional application of the invention patent application filed on May 8, 2014, with application number 201480025693.8 and invention title "Organoids comprising isolated kidney cells and their uses thereof". Invention Field

[0002] This invention relates to a mixture of selected bioactive primary kidney cells and other bioactive cell populations, and a method for treating a subject in need. The invention further relates to organoids comprising isolated kidney cells (including populations of renal tubular cells and populations of renal cells that produce erythropoietin (EPO), and a method for treating a subject in need using said organoids. Background of the Invention

[0003] In the United States, chronic kidney disease (CKD) affects more than 19 million people and is often a consequence of metabolic disorders including obesity, diabetes, and hypertension. Data analysis reveals an increasing rate of increase due to the development of hypertension-induced renal failure and non-insulin-dependent diabetes mellitus (NIDDM) (United States Renal Data System: Costs of CKD and ESRD. Bethesda, MD, ed., National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, 2007, pp. 223-238) – both of which are also increasing worldwide. Obesity, hypertension, and poor glycemic control have all been shown to be independent risk factors for kidney damage, causing glomerular and tubular lesions and leading to proteinuria and other systemic detectable changes in renal filtration function (Aboushwareb, et al.). , World Journal of Urol, 26: 295-300, 2008; Amann, K. et al. ,Nephrol DialTransplant, 13: 1958-66, 1998. Patients with CKD in stages 1-3 are managed through lifestyle modifications and pharmacological interventions aimed at controlling the underlying disease state, while those in stages 4-5 are managed through dialysis and medication regimens, which typically include antihypertensive agents, erythropoiesis-stimulating agents (ESAs), iron, and vitamin D supplements. According to the United States Renal Data Service (USRDS), patients with end-stage renal disease (ESRD) spend an average of >$600 per month on injectable erythropoiesis-stimulating agents (ESAs), vitamin D supplements, and iron supplements (United States Renal Data System: Costs of CKD and ESRD. Bethesda, MD, ed., National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, 2007, pp. 223-238). When combined with the average annual cost of dialysis ($65,405), the cost of maintaining health care for a single patient rises to >$72,000 per year (United States Renal Data System: Costs of CKD and ESRD. Bethesda, MD, ed., National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, 2007, pp. 223-238) – this figure reflects only the cost of standard procedures and does not include treatment for other complications, emergency procedures, or ancillary procedures such as placement of vascular grafts for dialysis access. In 2005, the combined medical costs for CKD and ESRD totaled $62 billion – representing 19% of all medical expenditures that year (United States Renal Data System: Costs of CKD and ESRD. Bethesda, MD, ed., National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, 2007, pp. 223-238).Kidney transplantation is an effective option for stage 4-5 patients, and it is the preferred measure to avoid dialysis or when dialysis is no longer sufficient to control the disease state. However, the number of stage 5 CKD patients who can benefit from whole kidney transplantation in the United States (>400,000) far exceeds the number of suitable donor kidneys available in any given year (approximately 16,000) (Powe, NR, etc.). , (Am J Kidney Dis, 53:S37-45, 2009). Therefore, new treatment modalities are needed to delay or reduce dependence on dialysis and fill the gap left by the shortage of donor kidneys.

[0004] Progressive CKD is caused by a combination of initial disease damage (e.g., hypertension) followed by a maladaptive renal response to that damage. This response includes the production of pro-inflammatory and pro-fibrotic cytokines and growth factors. Therefore, a strategy to slow CKD progression is to improve the inflammatory and fibrotic response, and to mitigate or reverse renal degeneration through repair and / or regeneration of renal tissue.

[0005] Chronic kidney failure is prevalent in humans and some domesticated animals. Patients with kidney failure not only suffer from the loss of kidney function (uremia) but also develop anemia because the bone marrow is unable to produce an adequate number of red blood cells (RBCs) through erythropoiesis. Erythroblast homeostasis depends on the production of erythropoietin (EPO) by specialized interstitial fibroblasts residing in the kidneys and the ability of target erythroblast-like progenitor cells in the bone marrow to respond to EPO and produce more RBCs. Anemia in kidney failure is due to reduced EPO production in the kidneys and the negative impact of uremic factors on the action of EPO in the bone marrow.

[0006] To date, clinical approaches to treating chronic renal failure have involved dialysis and kidney transplantation to restore renal filtration and urine production, as well as systemic delivery of recombinant EPO or EPO analogues to restore erythrocytocyte-like cell (RBC) quality. Dialysis provides survival benefits for patients in intermediate to advanced renal failure but results in significant quality-of-life problems. Kidney transplantation is a highly desirable (and often the only) option for patients with end-stage renal failure, but the supply of high-quality donor kidneys is insufficient to meet the needs of the renal failure population. Treatment of anemia with recombinant EPO bolus administration has now been associated with serious downstream health risks, prompting a black-box warning from the FDA and compelling further research into alternative treatments to restore RBC homeostasis in this population. Preclinical studies have examined the in vivo potency and safety of EPO-producing cells generated through gene therapy. These studies have shown that in vivo delivery of EPO-producing cells has the potential to transiently stimulate erythropoiesis and RBC count. However, to date, none of these approaches have provided regulated RBC homeostasis or long-term in vivo functionality. Consequently, HCT and RBC counts often increase beyond normal values, leading to polycythemia vera and other complications. Treatment-related delivery of EPO-producing cells, which offers advantages over recombinant EPO delivery, must not only increase hematocrit (HCT) but also restore erythrocyte-like cell homeostasis while maintaining the integrity of both positive and negative regulatory mechanisms. It is important to note that while EPO deficiency anemia is common in patients with kidney disease, it can also originate from other disease states, including heart failure, multiple organ failure, and other chronic diseases.

[0007] Regenerative medicine technologies offer next-generation treatment options for chronic kidney disease (CKD). Presnell et al. (WO / 2010 / 056328) and Ilagan et al. (PCT / US2011 / 036347) describe isolated bioactive kidney cells (including populations of renal tubular cells and populations of renal cells that produce erythropoietin (EPO), methods for isolating and culturing said bioactive kidney cells, and methods for treating subjects in need using said cell populations.

[0008] There is a need for an improved and more targeted regenerative medicine treatment option for subjects in need. Invention Overview

[0009] This article provides information on organoids, their preparation methods, and applications.

[0010] As described in this article, organoids provide therapeutic benefits to subjects in need without the use of scaffolds.

[0011] On one hand, a method for forming an organoid comprising a population of xenogeneic kidney cells and a population of bioactive cells is provided. In one embodiment, the method includes culturing the xenogeneic kidney cell population and the population of bioactive cells in a culture system selected from: i) 2D culture; ii) 3D culture: COL(I) gel; iii) 3D culture: Matrigel; iv) 3D culture: rotator followed by COL(I) / Matrigel; and v) 3D culture: COL(IV) gel. In some embodiments, the xenogeneic kidney cell population comprises a population of bioactive kidney cells. In some embodiments, the xenogeneic kidney cell population comprises a population of B2 cells containing an enriched population of renal tubular cells, wherein the xenogeneic kidney cell population discards a population of B1 cells and / or a population of B5 cells or a combination thereof. In some embodiments, the xenogeneic kidney cell population comprises a population of cells selected from B2, B2 / B3, B2 / B4, and B2 / B3 / B4. In embodiments, the xenogeneic kidney cell population comprises cells that produce erythropoietin (EPO).

[0012] In another embodiment, the bioactive cell population is an endothelial cell population. In some embodiments, the endothelial cell population is a cell line. In some embodiments, the endothelial cell population is derived from human umbilical cord. In some embodiments, the bioactive cell population includes endothelial progenitor cells. In some embodiments, the bioactive cell population includes mesenchymal stem cells. In some embodiments, the endothelial cell population is of adult origin. In some embodiments, the cell population is selected from xenogeneic, syngeneic, allogeneic, autologous, and combinations thereof.

[0013] In another embodiment, xenogeneic kidney cell populations and bioactive cell populations are cultured separately for a first time period, then combined and cultured for a second time period. In some embodiments, the kidney cell populations and bioactive cell populations are combined in a 1:1 ratio. In some embodiments, the kidney cell populations and bioactive cell populations are combined, for example, by suspending them in a growth medium. In some embodiments, the length of the second time period is between 24 hours and 72 hours, preferably 24 hours.

[0014] On the other hand, an organoid is provided. In some embodiments, the organoid is prepared according to the methods described herein. In all embodiments, the organoid comprises a xenogeneic kidney cell population and a bioactive cell population. In some embodiments, the bioactive cell population is an endothelial cell population. In some embodiments, the endothelial cell population is a cell line. In some embodiments, the endothelial cell population comprises HUVEC cells.

[0015] In some embodiments, the xenogeneic kidney cell population comprises a B2 cell population containing an enriched population of renal tubular cells, and wherein the B1 cell population is discarded. In some embodiments, the xenogeneic kidney cell population further discards the B5 cell population. In selected embodiments, the xenogeneic kidney cell population comprises a cell population selected from B2, B2 / B3, B2 / B4, and B2 / B3 / B4. In some embodiments, the xenogeneic kidney cell population comprises cells that produce erythropoietin (EPO).

[0016] On the other hand, an injectable formulation comprising at least one organoid and a liquid culture medium is provided. In one embodiment, the liquid culture medium is selected from cell growth media, DPBS, and combinations thereof. In another embodiment, the organoid is suspended in the liquid culture medium.

[0017] In a second embodiment, the injectable formulation comprises an organoid and a temperature-sensitive cell-stabilizing biomaterial, said biomaterial (i) remaining substantially solid at about 8°C or below, and (ii) remaining substantially liquid at about ambient temperature or above. In another embodiment, the bioactive cells comprise kidney cells as described herein. In another embodiment, the bioactive cells are substantially uniformly dispersed throughout the entire volume of the cell-stabilizing biomaterial. In other embodiments, the biomaterial has a solid-to-liquid transition state between about 8°C and about ambient temperature or above. In one embodiment, the substantially solid state is a gel state. In another embodiment, the cell-stabilizing biomaterial comprises a hydrogel. In another embodiment, the hydrogel comprises gelatin. In other embodiments, gelatin is present in the formulation at about 0.5% to about 1% (w / v). In one embodiment, gelatin is present in the formulation at about 0.75% (w / v).

[0018] On one hand, a method for treating kidney disease in a subject of need is provided, the method comprising administering at least one organoid comprising a population of xenogeneic kidney cells and a population of bioactive cells. In some embodiments, the bioactive cell population is a population of endothelial cells. In some embodiments, the endothelial cell population is a cell line. In one embodiment, the endothelial cell population comprises HUVEC cells.

[0019] In most embodiments, the xenogeneic kidney cell population comprises a B2 cell population containing an enriched population of renal tubular cells, and wherein the B1 cell population is discarded. In selected embodiments, the xenogeneic kidney cell population further discards the B5 cell population. In some embodiments, the xenogeneic kidney cell population comprises a cell population selected from B2, B2 / B3, B2 / B4, and B2 / B3 / B4. In most embodiments, the xenogeneic kidney cell population comprises cells that produce erythropoietin (EPO).

[0020] In one implementation, the method of treating a subject with kidney disease in need comprises administering an injectable formulation as described herein. In all implementations, the subject is a mammal selected from dogs, cats, horses, rabbits, zoo animals, cows, pigs, sheep, and primates. In a specific implementation, the mammal is a human. In all implementations, the subject suffers from kidney disease. In the implementations, improvement in any of the following measures of disease is observed: anemia (Hct, Hgb, RBC), inflammation (WBC), urine concentration (spGrav), and azotemia (BUN).

[0021] On the other hand, it provides the use of organoids in the preparation of drugs for treating kidney diseases. Brief description of the attached diagram

[0022] Figure 1 Figure 1A , Figure 1B , Figure 1C , Figure 1D , Figure 1E The image shows the morphology of primary ZSF1 cell cultures on a fibronectin-coated plate. A. In 5x B. Pre-sorting of p0 without grade separation observed at magnification; 5x p1 CD31 observed at magnification + C. Primary culture of ungraded kidney cells grown in fibronectin-treated flasks in KGM at 21% O2 at the end of generation 0; D. From Miltenyi Microbead-selected negative effluent (CD31) - ); and E. 90% CD31 selected at the end of the first generation in fibronectin-coated flasks with fully supplemented EMG2 medium. + cell.

[0023] Figure 2 Figure 2A , Figure 2B , Figure 2C , Figure 2D ) shows in 5 x The following are examples of morphological observations of human cell cultures: A. Ungraded kidney cells grown in KGM in TC-treated flasks at 21% O2 at the end of p0; B. Ungraded kidney cells grown in KGM in TC-treated flasks at the end of passage 0, exposed to 2% O2 O / N; C. Ungraded kidney cells in fully supplemented EMG2 medium in fibronectin-coated flasks at the end of passage 0; D. CD31 cells in fully supplemented EMG2 medium in fibronectin-coated flasks at the end of passage 1. + Positively selected cells.

[0024] Figure 3 ( Figure 3A , Figure 3B , Figure 3C , Figure 3D , Figure 3E , Figure 3F The FACS analysis shows the percentage of positive CD31 endothelial cells in the selected samples during culture. At p0, the ungraded (UNFX) endothelial composition was <3%, and at p1 it was enriched by approximately 15-fold when coated on fibronectin and cultured in EGM-2 medium.

[0025] Figure 3-1 Figure 3-1A , Figure 3-1B , Figure 3-1C The Bigeneic Cre / loxP reporter used for lineage tracing studies is shown. A. Six2-Cre x R26td tomato red cross tracer epithelial cells: apical, proximal, and distal epithelial cells, but not mesenchymal or collecting duct epithelial cells; B. Unlabeled control; C. Six-2 positive cells from p1 culture (3 days of normal oxygen culture followed by 1 day of hypoxic culture).

[0026] Figure 4 Figure 4A , Figure 4B The image shows a rotating flask (A) for forming SRC organoids and a low-binding plate (B) on the rotator.

[0027] Figure 5 ( Figure 5A , Figure 5B , Figure 5C This shows phase imaging of A. rats ( 10x B. shows phase imaging of a uniformly sized human SRC. 10x C. Phase imaging of organoids expressing the ubiquitin phenotype (green) and nucleus (blue). 20x ).

[0028] Figure 6 ( Figure 6A , Figure 6B , Figure 6C , Figure 6D The image shows human organs cultured in 3D collagen I / IV gels. A. Low-magnification phase image of organoid tube formation (circled in white); B. High-magnification phase image of the organoids along with the remaining organs (circled in white); C. GGT-1 phenotype expression (green), nucleus (blue); D. CK18 expression (green), nucleus (blue) at 20x magnification.

[0029] Figure 7 ( Figure 7A , Figure 7BThe following figures show organoids labeled with membrane dyes. A. SRC labeled with DiL (red) only at x100 magnification; B. Organoid+, SRC labeled with DiL (red), and HuVEC labeled with DiO (green) at x100 magnification.

[0030] Figure 8 ( Figure 8A , Figure 8B , Figure 8C Figures A, B, and C show 3D tubular development assays in Col I / IV gels containing autogenous organoids, illustrating the presence of both SRC groups (red) and endothelial cells (green). When combined, the groups appear yellow. Nuclear staining (blue) is shown at 20x magnification.

[0031] Figure 9 The organoids labeled with SPIO and rhodamine before injection are shown (in red).

[0032] Figure 10 ( Figure 10A , Figure 10B The images show magnetic resonance imaging (MRI) of organoid survival after transplantation, with green representing cells. A. 24 hours after injection; B. 48 hours after injection.

[0033] Figure 11 Prussian blue staining of implanted organoids is shown at low and high magnification, revealing cell survival and biodistribution. A. Left kidney implanted 24 hours post-implantation; B. Left kidney implanted 48 hours post-implantation.

[0034] Figure 12 ( Figure 12A , Figure 12B , Figure 12C , Figure 12D , Figure 12E , Figure 12F The following is a series of photographs showing HLA1 staining of human cells in rat kidneys that have been treated with the organoids described herein. A. Normal human kidney; B. Human kidney cells in rat kidney; C. Untreated nephrectomized rat kidney; D. NKO-treated (low magnification); E. NKO-treated (high magnification); F. Animal treated with a second NKO. Figures A and B show staining (brown) of normal human kidney tissue and human kidney cells (green arrows) acutely delivered to rodent kidneys. Background staining, as indicated by yellow arrows in Figures C and F, is present at the end of studies on untreated rat kidneys that may be diseased due to the “sticky” nature of protein casts and damaged renal tubules. This staining is usually light in color but sometimes dark and is smaller than the cells. Figures D, E, and F show lower magnification used for screening to identify darkly stained cells, followed by higher magnification to confirm HLA1-stained cells (green arrows).

[0035] The patent or application documents contain at least one color drawing. Upon request and payment of the necessary fees, the government agency will provide a copy of the patent or patent publication with the color drawing. Invention Details

[0036] Many cell types make up the nephron, the functional unit within the renal parenchyma. Recently, the ability to isolate therapeutically bioactive renal cells from normal and diseased animals and humans has been established. 1-3 The methods used in these studies relied on centrifugation using buoyancy density gradients to separate mixtures of kidney cells present in biopsies from diseased tissue.

[0037] On one hand, this invention relates to the isolation, identification, and expansion of individual cell populations selected from various nephron compartments or microenvironments, enabling the combination of novel, multi-enriched cell types as selective mixtures. These novel, selective mixtures provide targeting of specific structural and functional defects associated with the clinical and pathophysiological basis of kidney diseases. The isolation and enrichment of multiple individual cell types constituting nephrons and combined into selective mixtures can improve targeted therapy for specific groups of kidney diseases.

[0038] Cell types, such as vascular endothelial cells, renal tubular and collecting duct epithelial cells, interstitial cells, glomerular cells, and mesenchymal stem cells, can be isolated, identified, and expanded in vitro. While each cell type may require unique methods and culture medium formulations for subculture, they can be added back as organoid clusters in selective combinations or mixtures to provide enhanced delivery and improved therapy for underlying renal tissue / cell defects associated with specific acute and / or chronic kidney disease syndromes / groups. 8 .

[0039] This invention covers a method for treating kidney damage associated with vascular diseases (e.g., hypertension, microangiogenic anemia) using a defined ratio of renal tubular epithelial cells to endothelial cells. The ability to isolate, characterize, and expand resident renal endothelial cells using selective culture systems and magnetic sorting allows for the enrichment of previously characterized selectively regenerating renal epithelial cell (SRC) populations. 2，4 The invention comprises a specific percentage of purified renal epithelial cells (SRC+). The SRC+ cell population of the present invention comprises selected renal cells (SRC or BRC) as previously described and also described herein, as well as additional bioactive cell populations (including, but not limited to, endothelial cells, endothelial progenitor cells, mesenchymal stem cells, and / or adipose-derived progenitor cells). In one embodiment, the additional bioactive cell population is derived from a renal source. In another embodiment, the additional bioactive cell population is derived from a non-renal source.

[0040] On one hand, the present invention relates to organoids comprising and / or formed from a heterogeneous mixture or fraction of selected renal cells or bioactive renal cells (SRC or BRC) or SRC+ cell populations, methods for isolating and culturing said organoids, and methods for therapeutic use using the organoids described herein. Targeted delivery of organoid-formed SRC or SRC+ cell populations to the kidneys is expected to improve cell survival, thereby improving overall therapeutic outcomes. The invention also relates to methods for therapeutic use using SRC+ cell populations.

[0041] definition

[0042] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention pertains. Principles of Tissue Engineering The third edition (by R. Lanza, R. Langer, & J. Vacanti), 2007, provides general guidance to those skilled in the art on many of the terms used in this application. Those skilled in the art will recognize that many methods and materials similar to or equivalent to those described herein can be used to practice the invention. In fact, the invention is by no means limited to the methods and materials described.

[0043] As used herein, the term "cell population" refers to a large number of cells obtained by direct isolation from a suitable tissue source, typically from mammals. The isolated cell population can then be cultured in vitro. Those skilled in the art will appreciate the various methods for isolating and culturing cell populations used in conjunction with this invention, as well as the various cell numbers suitable for use in the cell populations of this invention. The cell population can be an ungraded xenogeneic cell population derived from the kidney. For example, the xenogeneic cell population can be isolated from a kidney biopsy or from whole kidney tissue. Alternatively, the xenogeneic cell population can be derived from an in vitro culture of mammalian cells established from a kidney biopsy or whole kidney tissue. An ungraded xenogeneic cell population can also be referred to as a non-enriched cell population.

[0044] The term "natural kidney" should refer to the kidney of a living subject. The subject can be healthy or unhealthy. An unhealthy subject may have kidney disease.

[0045] The term "regenerative effect" should refer to effects that provide benefits to the natural kidney. These effects may include, but are not limited to, reducing the degree of damage to the natural kidney or improving the recovery or stabilization of natural kidney function. Kidney injury can take the form of fibrosis, inflammation, glomerular hypertrophy, etc., and is associated with kidney disease in the subjects.

[0046] As used herein, the terms “regenerative potential” or “potential regenerative bioactivity” refer to the potential of organoids containing the bioactive cell preparations and / or mixtures described herein to provide regenerative effects.

[0047] As used herein, the term "mixture" refers to a combination of two or more isolated, enriched cell populations derived from ungraded heterogeneous cell populations. According to some embodiments, the cell population of this invention is a kidney cell population.

[0048] "Enriched" cell populations or formulations refer to cell populations derived from a starting kidney cell population (e.g., an ungraded xenogeneic cell population) that contain a greater percentage of a specific cell type than the percentage of that cell type in the starting population. For example, a starting kidney cell population can be enriched with a first, second, third, fourth, fifth, etc., cell population of interest. As used herein, the terms "cell population," "cell formulation," and "cell prototype" are used interchangeably.

[0049] On the one hand, as used herein, the term "enriched" cell population refers to a cell population derived from a starting kidney cell population (e.g., a cell suspension from a kidney biopsy or cultured mammalian kidney cells) containing a greater percentage of EPO-producing cells than the starting population. For example, the term "B4" refers to a cell population derived from a starting kidney cell population containing a greater percentage of EPO-producing cells, glomerular cells, and vascular cells compared to the starting population. The cell populations of the present invention can be enriched with one or more cell types and discarded with one or more other cell types. For example, an enriched EPO-producing cell population can be enriched with mesenchymal fibroblasts and discarded with renal tubular cells and collecting duct epithelial cells relative to a non-enriched cell population (i.e., the starting cell population from which the enriched cell population originates). In all embodiments referencing EPO enrichment or the “B4” group, the enriched cell population is a heterogeneous cell population containing cells capable of producing EPO in an oxygen-regulated manner, as demonstrated by oxygen-regulated EPO expression from an endogenous natural EPO gene.

[0050] On the other hand, an enriched cell population containing a greater percentage of a specific cell type (e.g., vascular cells, glomerular cells, or endocrine cells) than the initial kidney cell population derived from a healthy individual or subject may also lack or be deficient in one or more specific cell types (e.g., vascular cells, glomerular cells, or endocrine cells). For example, the term "B4'" or "B4 apostrophe" refers to a cell population derived from an initial kidney cell population that, depending on the disease state of the initial sample, lacks or is deficient in one or more cell types, such as vascular cells, glomerular cells, or endocrine cells, compared to a healthy individual. In one embodiment, the B4' cell population is derived from a subject with chronic kidney disease. In one embodiment, the B4' cell population is derived from a subject with focal segmental glomerulosclerosis (FSGS). In another embodiment, the B4' cell population is derived from a subject with autoimmune glomerulonephritis. On the other hand, B4' is a cell population derived from an initial cell population including all cell types (e.g., vascular cells, glomerular cells, or endocrine cells), which is later discarded or lacks one or more cell types (e.g., vascular cells, glomerular cells, or endocrine cells). In yet another aspect, B4' is a cell population derived from an initial cell population including all cell types (e.g., vascular cells, glomerular cells, or endocrine cells), wherein one or more specific cell types (e.g., vascular cells, glomerular cells, or endocrine cells) are enriched. For example, in one embodiment, the B4' cell population may be enriched with vascular cells but discard glomerular cells and / or endocrine cells. In another embodiment, the B4' cell population may be enriched with glomerular cells but discard vascular cells and / or endocrine cells. In another embodiment, the B4' cell population may be enriched with endocrine cells but discard vascular cells and / or glomerular cells. In yet another embodiment, the B4' cell population may be enriched with both vascular cells and endocrine cells but discard glomerular cells. In a preferred embodiment, the therapeutic properties are retained either alone or in combination with another enriched cell population, such as B2 and / or B3. The B4' cell population is described herein, for example, in the embodiments of Examples 7-9.

[0051] On the other hand, the enriched cell population can also refer to a cell population derived from the initial kidney cell population as discussed above, wherein the percentage of cells expressing one or more vascular, glomerular, and proximal tubular markers, along with some EPO-producing cells, is greater than the percentage of cells expressing one or more vascular, glomerular, and proximal tubular markers, along with some EPO-producing cells, in the initial population. For example, the term "B3" refers to a cell population derived from the initial kidney cell population that contains a greater percentage of proximal tubular cells, as well as vascular and glomerular cells, compared to the initial population. In one embodiment, the B3 cell population contains a greater percentage of proximal tubular cells than the initial population, but a smaller percentage of proximal tubular cells compared to the B2 cell population. In another embodiment, the B3 cell population contains a greater percentage of vascular and glomerular cell markers, along with some EPO-producing cells, compared to the initial population, but a smaller percentage of vascular and glomerular cell markers, along with some EPO-producing cells, compared to the B4 cell population.

[0052] On the other hand, the enriched cell population can also refer to a cell population derived from the initial kidney cell population discussed above, wherein the percentage of cells expressing one or more renal tubular cell markers is greater than the percentage of cells expressing one or more renal tubular cell markers in the initial population. For example, the term "B2" refers to a cell population derived from the initial kidney cell population that contains a larger percentage of renal tubular cells compared to the initial population. Furthermore, the cell population enriched with cells expressing one or more renal tubular cell markers (or "B2") may contain some epithelial cells from the collecting duct system. Although the cell population enriched with cells expressing one or more renal tubular cell markers (or "B2") relatively discards EPO-producing cells, glomerular cells, and vascular cells, the enriched population may contain a smaller percentage of these cells (EPO-producing cells, glomerular cells, and vascular cells) compared to the initial population. Typically, one or more cell types in the xenogeneic cell population are discarded so that the discarded cell population contains a smaller proportion of cell types relative to the proportion of cell types originally included in the xenogeneic cell population. The cell types that can be discarded are any type of kidney cell. For example, in some embodiments, cell types that can be discarded include large-grained cells from the collecting ducts and renal tubules with a density < about 1.045 g / ml, referred to as "B1". In some other embodiments, cell types that can be discarded include low-grained and viable debris and small cells with a density > about 1.095 g / ml, referred to as "B5". In some embodiments, a cell population enriched with renal tubular cells relatively discards all of the following cells: "B1", "B5", oxygen-tunable EPO-expressing cells, glomerular cells, and vascular cells.

[0053] As used herein, “hypoxic” culture conditions refer to culture conditions in which cells are subjected to a reduced level of available oxygen in the culture system compared to standard culture conditions where cells are cultured at atmospheric oxygen levels (approximately 21%). Non-hypoxic conditions, as used herein, refer to normal or oxygen-rich culture conditions.

[0054] As used in this article, “oxygen-tunable” refers to the ability of a cell to regulate gene expression (up or down) based on the amount of oxygen available to the cell. “Hypoxia-inducible” refers to the upregulation of gene expression in response to a decrease in oxygen tension (whether pre-induced or initiated by oxygen tension).

[0055] The term "sphere" refers to an aggregate or ensemble of cells cultured to allow 3D growth, in contrast to monolayer growth. It should be noted that the term "sphere" does not imply that the aggregate is geometrically spherical. An aggregate can be highly organized with a well-defined morphology or it can be an unorganized mass; it can comprise a single cell type or more than one cell type. Cells can be primary isolates or permanent cell lines or a combination of both. Organoids and organoid cultures are included in this definition.

[0056] As used herein, the term "organoid" refers to xenogeneic 3D aggregations of cells, encompassing aspects of cellular self-organization, structural, and signaling interactions present in natural organs. The term "organoid" includes spheroids or clusters of cells formed from suspension cell cultures.

[0057] As used herein, the term "biomaterial" refers to a natural or synthetic biocompatible material suitable for introduction into living tissue. Natural biomaterials are materials made from living systems. Synthetic biomaterials are materials not made from living systems. The biomaterials disclosed herein may be combinations of natural and synthetic biocompatible materials. As used herein, biomaterials include, for example, polymer matrices and scaffolds. Those skilled in the art will appreciate that biomaterials can be configured in various forms, such as liquid hydrogel suspensions, porous foams, and may include one or more natural or synthetic biocompatible materials.

[0058] The term "construction" refers to one or more cell populations deposited on or within the surface of a scaffold or matrix composed of one or more synthetic or naturally occurring biocompatible materials. One or more cell populations may be coated with, deposited on, embedded in, attached to, seeded in, or captured in biomaterials composed of one or more synthetic or naturally occurring biocompatible polymers, proteins, or peptides. One or more cell populations may be combined with biomaterials, scaffolds, or matrices in vitro or in vivo. Typically, one or more biocompatible materials used to form the scaffold / biomaterial are selected to guide, promote, or permit the formation of a multicellular, three-dimensional tissue structure from at least one cell population deposited thereon. One or more biomaterials used to generate the construct may also be selected to guide, promote, or permit the dispersion and / or binding of the construct or its cellular components to endogenous host tissues, or to guide, promote, or permit the survival, transplantation, tolerance, or functional properties of the construct or its cellular components.

[0059] The term "marker" or "biomarker" generally refers to a molecular marker of DNA, RNA, protein, carbohydrate, or glycolipid-based molecules, the expression or presence of which in a cultured cell population can be detected by standard methods (or the methods disclosed herein) and is consistent with one or more cell types within the cultured cell population. A marker may be a polypeptide expressed by a cell or an identifiable physical location on a chromosome, such as a gene, a restriction endonuclease recognition site, or a nucleic acid (e.g., mRNA) encoding a polypeptide expressed by a native cell. A marker may be an expression region of a gene, referred to as a "gene expression marker," or segments of DNA that do not have a known coding function. A biomarker may be a cell-derived (e.g., secreted) product.

[0060] The terms “differentially expressed gene,” “differential gene expression,” and their synonyms, used interchangeably, refer to a gene whose expression in a first cell or cell population is activated to a higher or lower level relative to its expression in a second cell or cell population. The term also includes genes whose expression is activated to a higher or lower level over time at different stages during passage of the first or second cell in culture. It should also be understood that differentially expressed genes can be activated or repressed at the nucleic acid or protein level, or can be subject to alternative splicing to produce different polypeptide products. Such differences can be demonstrated by variations, for example, in mRNA levels, surface expression, secretion, or other aspects of polypeptide distribution. Differential gene expression can include comparisons of expression between two or more genes or their gene products, or comparisons of expression ratios between two or more genes or their gene products, or even comparisons of two different processed products of the same gene, the comparisons differing between the first and second cells. Differential expression includes, for example, quantitative and qualitative differences in the temporal or cellular expression patterns of genes or their expression products in the first and second cells. For the purposes of this invention, “differential gene expression” is considered to exist when there is a difference in the expression of a given gene between the first and second cells. Differential expression of the marker can exist in cells from the patient (first cells) prior to application of the cell population, mixture, or construct, relative to expression in cells from the patient after application (second cells).

[0061] The terms “inhibition,” “downregulation,” “low expression,” and “reduction” are used interchangeably and refer to a reduction in gene expression or the level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits, relative to one or more controls, such as one or more positive and / or negative controls. Low expression may be present in cells from the patient prior to administration of the cell population, mixture, or construct, relative to cells from the patient after administration.

[0062] The terms “upregulation” or “overexpression” are used to refer to an increase in gene expression or the level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits, relative to one or more controls, such as one or more positive and / or negative controls. Overexpression can be present in patient cells after administration of a cell population, mixture, or construct, relative to cells prior to administration.

[0063] As used herein, the term "anemia" refers to a deficiency in red blood cell count and / or hemoglobin level caused by improper production of functional EPO protein by EPO-producing cells in a subject, and / or improper release of EPO protein into the systemic circulation, and / or the inability of erythroblasts in the bone marrow to respond to EPO protein. Subjects with anemia are unable to maintain erythroblast-like cell homeostasis. Anemia can typically occur in cases of renal failure or loss of function (e.g., chronic renal failure), is associated with relative EPO deficiency, is associated with congestive heart failure, is associated with myelosuppressive therapy such as chemotherapy or antiviral therapy (e.g., AZT), is associated with non-myeloid cancer, is associated with viral infections such as HIV, and is associated with chronic diseases such as autoimmune diseases (e.g., rheumatoid arthritis), liver disease, and multiple organ failure.

[0064] The term “EPO deficiency” refers to any condition or symptom that can be treated with erythropoietin receptor agonists (e.g., recombinant EPO or EPO analogs), including anemia.

[0065] As used in this article, “organ-related disease” refers to any stage or degree of acute or chronic organ failure that results in an organ losing its ability to perform its functions.

[0066] As used herein, the term "kidney disease" refers to any stage or degree of acute or chronic renal failure that results in the kidneys losing their ability to perform their function of filtering blood and removing excess fluids, electrolytes, and waste products from the blood. Kidney disease also includes endocrine disorders such as anemia (erythropoietin deficiency) and mineral imbalances (vitamin D deficiency). Kidney disease may originate in the kidneys or can secondary to a variety of conditions, including (but not limited to) heart failure, hypertension, diabetes, autoimmune diseases, or liver disease. Kidney disease can be a symptom of chronic renal failure that develops following acute kidney injury. For example, damage to the kidneys through local ischemia and / or exposure to toxins can cause acute renal failure; incomplete recovery after acute kidney injury can lead to the development of chronic renal failure.

[0067] The term "treatment" refers to therapeutic and preventative or defensive measures for kidney disease, anemia, EPO deficiency, tubular transport defects, or glomerular filtration defects, with the goal of reversing, preventing, or alleviating the target condition. Those in need of treatment include those who already have kidney disease, anemia, EPO deficiency, tubular transport defects, or glomerular filtration defects, as well as those predisposed to developing these conditions or those in whom preventative measures against these conditions are desired. As used herein, the term "treatment" also includes stabilizing and / or improving kidney function.

[0068] The term "subject" should mean any single individual subject, including patients who are experiencing or have experienced one or more signs, symptoms, or other indicators of kidney disease, anemia, or EPO deficiency and are suitable for treatment. Subjects include, but are not limited to, subjects who have recently been diagnosed or previously diagnosed and are currently experiencing recurrence or relapse of kidney disease, anemia, or EPO deficiency, or who are at risk for any reason. Subjects may or may not have been previously treated for kidney disease, anemia, or EPO deficiency.

[0069] The term "patient" refers to any single animal, more preferably a mammal (including, for example, non-human animals such as dogs, cats, horses, rabbits, zoo animals, cows, pigs, sheep, and non-human primates). Most preferably, the patient in this document is a human.

[0070] The terms “sample,” “patient sample,” or “biological sample” should generally mean any biological sample obtained from a subject or patient, bodily fluids, body tissues, cell lines, tissue cultures, or other sources. The term includes tissue biopsies, such as kidney biopsies. The term includes cultured cells, such as cultured mammalian kidney cells. Methods for obtaining tissue biopsies and cultured cells from mammals are well known in the art. If the term “sample” is used alone, it should mean that “sample” is a “biological sample” or a “patient sample,” i.e., these terms are used interchangeably.

[0071] The term "test sample" refers to a sample from a subject who has been treated by the methods of this invention. Test samples can originate from various sources in mammalian subjects, including but not limited to blood, semen, serum, urine, bone marrow, mucous membranes, and tissues.

[0072] The term "control" or "control sample" refers to a negative or positive control in which a negative or positive result is expected to help correct for results in a test sample. Controls suitable for the present invention include, but are not limited to, samples known to exhibit indicator characteristics of normal erythrocyte-like cell homeostasis, samples known to exhibit indicator characteristics of anemia, samples obtained from subjects known not to be anemic, and samples obtained from subjects known to be anemic. Additional controls suitable for use in the methods of the present invention include, but are not limited to, samples derived from subjects who have been treated with pharmaceutical preparations known to regulate erythropoiesis (e.g., recombinant EPO or EPO analogs). Furthermore, controls may be samples obtained from subjects prior to treatment by the methods of the present invention. Additional suitable controls may be test samples obtained from subjects known to have any type or stage of kidney disease and samples from subjects known not to have any type or stage of kidney disease. Controls may be normally healthy matched controls. Other controls used in the present invention will be appreciated by those skilled in the art.

[0073] "Regenerative prognosis," "regenerative outcome," or "prognostic regeneration" generally refers to a prediction or forecast of the likely regenerative process or outcome of the application or implantation of the cell population, mixture, or construct described herein. For regenerative prognosis, a prediction or forecast may be provided by one or more of the following: improvement of a functional organ (e.g., kidney) after implantation or application; development of a functional kidney after implantation or application; development of improved kidney function or capacity after implantation or application; and expression of certain markers by the natural kidney after implantation or application.

[0074] “Regenerated organ” refers to a natural organ following the implantation or administration of a cell population, mixture, or construct as described herein. Regenerated organs are characterized by a variety of indicators, including but not limited to developing, improving, or expressing certain markers of a natural organ. Those skilled in the art will appreciate that other indicators may be suitable for characterizing regenerated organs.

[0075] "Regenerated kidney" refers to a natural kidney following the implantation or administration of cell populations, mixtures, or constructs as described herein. A regenerated kidney is characterized by a variety of indicators, including but not limited to developing, improving, or expressing certain markers found in a natural kidney. Those skilled in the art will appreciate that other indicators may be suitable for characterizing a regenerated kidney.

[0076] SRC+ cell population

[0077] This invention provides cell populations comprising: isolated xenogeneic kidney cell populations enriched with specific bioactive components or cell types and / or discarded with specific inactive or undesirable components or cell types; and additional bioactive cell populations for the treatment of acute or chronic kidney disease, including but not limited to endothelial cells, endothelial progenitor cells, mesenchymal stem cells, and adipose-derived progenitor cells. The isolated xenogeneic kidney cell populations enriched with specific bioactive components or cell types and / or discarded with specific inactive or undesirable components or cell types may include any cell populations described herein. In one embodiment, additional bioactive components, such as endothelial cells, endothelial progenitor cells, mesenchymal stem cells, and adipose-derived progenitor cells, are mixed with the isolated xenogeneic kidney cell populations enriched with specific bioactive components or cell types and / or discarded with specific inactive or undesirable components or cell types. Alternatively, this invention provides a method for preparing the SRC+ cell populations described herein. Other bioactive components of the cell population, such as endothelial cells, endothelial progenitor cells, mesenchymal stem cells, and adipose-derived progenitor cells, may be present in any percentage sufficient to improve cell or tissue defects.

[0078] The cell population of the present invention may comprise one or more additional bioactive components, such as, but not limited to, endothelial cells, endothelial progenitor cells, mesenchymal stem cells, and adipose-derived progenitor cells. In some embodiments, the cell population comprises two additional bioactive components. In some embodiments, the cell population comprises three additional bioactive components. In some embodiments, the cell population comprises four additional bioactive components. In some embodiments, the cell population comprises five additional bioactive components. In some embodiments, the cell population comprises six additional bioactive components. In some embodiments, the cell population comprises seven additional bioactive components. In some embodiments, the cell population comprises eight additional bioactive components. In some embodiments, the cell population comprises nine additional bioactive components. In some embodiments, the cell population comprises ten additional bioactive components.

[0079] In one embodiment, the additional bioactive component is epithelial cells. In one embodiment, the epithelial cells are proximal tubular epithelial cells. In another embodiment, the epithelial cells are distal tubular epithelial cells. In yet another embodiment, the epithelial cells are parietal epithelial cells.

[0080] In another embodiment, the additional bioactive component is epithelial cells. In one embodiment, the epithelial cells are venous endothelial cells. In one embodiment, the epithelial cells are arterial endothelial cells. In one embodiment, the epithelial cells are capillary endothelial cells. In one embodiment, the epithelial cells are lymphatic endothelial cells.

[0081] In another embodiment, the additional bioactive component is collecting duct cells. In yet another embodiment, the additional bioactive component is smooth muscle cells. In another embodiment, the additional bioactive component is mesenchymal stem cells. In another embodiment, the additional bioactive component is progenitor cells of endothelial, mesenchymal, epithelial, or hematopoietic lineages. In another embodiment, the additional bioactive component is progenitor cells derived from endoderm, ectoderm, or embryonic mesenchyme. In some embodiments, the additional bioactive component is stem cells of any source or origin, including but not limited to embryonic (ES) stem cells and induced pluripotent (iPS) stem cells. In some embodiments, the additional bioactive component is a derivative of stem cells of any source or origin, including but not limited to embryonic (ES) stem cells and induced pluripotent (iPS) stem cells, wherein the derivative can be produced by directed differentiation of stem cells by a defined combination or mixture of small molecules and / or proteins and / or nucleic acid molecules. In some embodiments, the additional bioactive component is a derivative of progenitor cells of endothelial, mesenchymal, epithelial, or hematopoietic lineages, wherein the derivative can be produced by directed differentiation of stem cells by a defined combination or mixture of small molecules and / or proteins and / or nucleic acid molecules. In some embodiments, the additional bioactive component is a derivative of progenitor cells derived from the endoderm, ectoderm, or embryonic mesenchyme, wherein said derivative can be produced by directed differentiation of stem cells through a defined combination or mixture of small molecules and / or proteins and / or nucleic acid molecules. In one embodiment, the additional bioactive component is a genetically modified cell of any lineage or origin.

[0082] In a further embodiment, the additional bioactive component is mesenchymal cells. In one embodiment, the mesenchymal cells are supporting fibroblasts. In another embodiment, the mesenchymal cells are specialized cortical fibroblasts that produce erythropoietin.

[0083] In one embodiment, the additional bioactive component is derived from the subject's own source. In another embodiment, the additional bioactive component is derived from an allogeneic source. In certain embodiments, the additional bioactive component is derived from the subject's own source, and said additional bioactive component is a mixture of another bioactive component derived from an allogeneic source.

[0084] In some aspects, the present invention provides methods for achieving targeted regeneration of kidney quality and function by directed delivery of the cell populations and / or organoids and / or biomaterials described herein. In other aspects, the present invention further provides methods for salvaging and / or restoring kidney function in patients with acute or chronic kidney disease by administering the cell populations and / or organoids and / or biomaterials described herein.

[0085] Therapeutic organoids

[0086] The present invention also provides organoids comprising and / or formed therefrom bioactive components described herein (e.g., B2, B4, and B3), wherein inactive or unwanted components (e.g., B1 and B5) are discarded, alone or in combination, for the treatment of acute and / or chronic kidney disease. In one aspect, the present invention provides organoids containing, alone or when mixed with other bioactive subfractions (e.g., B2 and / or B3), a specific subfraction B4 (i.e., B4') that retains therapeutic properties (e.g., stabilization and / or improvement and / or regeneration of kidney function) that is discarded or lacks one or more cell types (e.g., vascular, endocrine, or endothelial cell types). In a preferred embodiment, the bioactive cell population is B2. In some embodiments, the B2 cell population is mixed with B4 or B4'. In other embodiments, the B2 cell population is mixed with B3. In other embodiments, the B2 cell population is mixed with B3 and B4, or specific cellular components of B3 and / or B4. In all embodiments, the organoids of the present invention are formed and cultured in vitro. In all embodiments, the organoid may also comprise or be formed from additional bioactive cell populations, including but not limited to endothelial cells, endothelial progenitor cells, mesenchymal stem cells, and adipose-derived progenitor cells. In one embodiment, additional bioactive cell populations (including but not limited to endothelial cells, endothelial progenitor cells, mesenchymal stem cells, and adipose-derived progenitor cells) are mixed with isolated xenogeneic kidney cell populations enriched with specific bioactive components or cell types, and / or discarded with specific inactive or undesirable components or cell types.

[0087] In one embodiment, the organoid of the present invention comprises or is formed from a population of B2 cells, wherein the B2 cell population comprises an enriched population of renal tubular cells. In another embodiment, the xenogeneic kidney cell population further comprises a population of B4 cells. In yet another embodiment, the xenogeneic kidney cell population further comprises a population of B3 cells. In still another embodiment, the xenogeneic kidney cell population further comprises a population of B5 cells.

[0088] In some embodiments, the cell population comprises a B2 cell population, wherein the B2 cell population contains an enriched population of renal tubular cells and the B1 and / or B5 cell populations are discarded.

[0089] On the other hand, the present invention provides a method for forming organoids comprising, and / or formed from, the bioactive components of the present invention (e.g., B2, B4, and B3), wherein the bioactive components discard inactive or undesirable components (e.g., B1 and B5), either alone or in mixture. In another aspect, the present invention provides organoids comprising, alone or when mixed with other bioactive subfractions (e.g., B2 and / or B3), retaining therapeutic properties (e.g., stabilization and / or improvement and / or regeneration of renal function), a specific subfraction B4 (i.e., B4') that discards or lacks one or more cell types (e.g., vascular, endocrine, or endothelial cell types), and / or formed therefrom. In a preferred embodiment, the bioactive cell population is B2. In some embodiments, the B2 cell population is mixed with B4 or B4'. In other embodiments, the B2 cell population is mixed with B3. In other embodiments, the B2 cell population is mixed with specific cell components of B3 and B4, or B3 and / or B4.

[0090] In one embodiment, the organoid of the present invention comprises or is formed from a population of B2 cells, wherein the B2 cell population comprises an enriched population of renal tubular cells. In another embodiment, the xenogeneic kidney cell population further comprises a population of B4 cells. In yet another embodiment, the xenogeneic kidney cell population further comprises a population of B3 cells. In still another embodiment, the xenogeneic kidney cell population further comprises a population of B5 cells.

[0091] In some embodiments, the cell population comprises a B2 cell population, wherein the B2 cell population contains an enriched population of renal tubular cells and the B1 and / or B5 cell populations are discarded.

[0092] On the other hand, the present invention provides a method for forming organoids using the bioactive cell preparations and / or mixtures described herein. General methods for culturing renal tubules from primary kidney cell populations using 3D COL(I) gels are known in the art, for example, as in Joraku et al., Methods. Feb. 2009;47(2):129-33. In all embodiments, the organoids of the present invention are formed and cultured in vitro.

[0093] In some embodiments, organoids and renal tubules may be induced, for example and not limited to, using the following culture methods or systems with the bioactive cell preparations and / or mixtures described herein: i) 2D culture; ii) 3D culture: COL(I) gel; iii) 3D culture: Matrigel; iv) 3D culture: rotator followed by COL(I) / Matrigel; and v) 3D culture: COL(IV) gel. Specific examples of organoid and renal tubule formation by NKA are provided in sections 2 and 4 below.

[0094] In one embodiment, organoids formed from the bioactive cell preparations and / or mixtures described herein can be induced in 2D culture. In one embodiment, the bioactive cell preparations and / or mixtures described herein are seeded onto standard 2D plastic dishes. In one embodiment, cells are seeded at a rate of approximately 5000 cells / cm². 2 Cells are seeded at a suitable density. They can be seeded in a suitable culture medium, such as Renal Cell Complete Growth Medium (RCGM). Generally, cell populations can grow to confluence over approximately 7, 8, 9, 10, 11, 12, 13, 14, 15, or more days, with the culture medium changed periodically approximately every 3-4 days. In one embodiment, cells exhibit spontaneous self-organization into spherical structures, i.e., organoids and renal tubules, between approximately 7 and approximately 15 days.

[0095] In another embodiment, organoids formed from the bioactive cell formulations and / or mixtures described herein can be induced in 3D culture. In one embodiment, organoids are generated using cell populations of the present invention along with a scaffold of biomaterials of natural or synthetic origin. In one embodiment, the formulated bioactive cell formulations and / or mixtures described herein can be incorporated into collagen (I) gel, collagen (IV) gel, Matrigel, or any mixture of these as previously described (see Guimaraees-Souza et al., 2012). In vitro reconstitution of human kidney structures for renal cell therapy Nephrol Dial Transplant 0: 1-9). The liquid gel can be adjusted to a neutral pH and the bioactive cell preparation and / or mixture described herein can be mixed at about 500-2500 cells / µl. In one embodiment, it can be mixed at about 1000 cells / µl. The cell / gel mixture is aliquoted into the wells of a 24-well plate, for example (about 200 to about 400 µl / well), and allowed to cure at 37°C for several hours. Cell culture medium is then added and the culture is allowed to mature for about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days, while the culture medium is changed periodically. In one embodiment, the renal tubular structural network is organized in lattice and ring form throughout the gel matrix by the bioactive cell preparation and / or mixture described herein.

[0096] In another embodiment, organoids can be formed by suspending and culturing the bioactive cell preparations and / or mixtures described herein in a rotary flask or a low-binding plastic dish. In one embodiment, cells can be cultured in a medium at approximately 80 rpm for up to 4 days in a rotary flask. For example, the spheres can then be cultured on Matrigel-coated plates for approximately 7, 8, 9, or 10 days. In one embodiment, spheres formed from the bioactive cell preparations and / or mixtures described herein show the potential to generate renal tubules, as evidenced by the rebudding of renal tubular structures from the cultured spheres.

[0097] cell population

[0098] The SRC+ cell populations and / or organoids of the present invention may contain isolated xenogeneic kidney cell populations and mixtures thereof and / or be formed therefrom, said isolated xenogeneic kidney cell populations and mixtures thereof being enriched with specific bioactive components or cell types, and / or having discarded specific inactive or undesirable components or cell types for the purpose of treating kidney diseases, i.e., providing stability and / or improvement and / or regeneration of kidney function, as previously described in Presnell et al. US 2011-0117162 and Ilagan et al. PCT / US2011 / 036347, the entire contents of which are incorporated herein by reference. Organoids may contain isolated fractions of kidney cells lacking cellular components compared to healthy individuals but retaining therapeutic properties (i.e., providing stability and / or improvement and / or regeneration of kidney function). The cell populations, cell fractions, and / or mixtures of cells described herein may be derived from healthy individuals, individuals with kidney disease, or subjects as described herein.

[0099] Bioactive cell population

[0100] This invention encompasses SRC+ cell populations of target organs or tissues to be administered to subjects in need, and therapeutic organoids containing bioactive cell populations. Bioactive cell populations generally refer to cell populations that may possess therapeutic properties when administered to a subject. For example, when administered to a subject in need, organoids containing bioactive kidney cell populations may provide stabilization and / or improvement and / or regeneration of kidney function in the subject. Therapeutic properties may include regenerative effects.

[0101] Bioactive cell populations include, but are not limited to, stem cells (e.g., pluripotent, multipotent, oligopotent, or unipotent) such as embryonic stem cells, amniotic stem cells, adult stem cells (e.g., hematopoietic, breast, intestinal, mesenchymal, placental, lung, bone marrow, blood, umbilical cord, endothelial, dental pulp, adipose, neural, olfactory, neurogenic, and testicular), induced pluripotent stem cells; genetically modified cells; and cell populations or tissue explants derived from any source in the body. Bioactive cell populations can be isolated, enriched, purified, allogeneic, or xenogeneic in nature. Those skilled in the art will recognize other bioactive cell populations suitable for generating the organoids of the present invention.

[0102] In one embodiment, the cells are derived from the same source as the intended target organ or tissue. For example, kidney cells may be derived from the kidney from which organoids to be administered to the kidney are produced. In another embodiment, the cells are derived from a different source than the intended target organ or tissue. For example, cells expressing erythropoietin may be derived from renal fat from which organoids to be administered to the kidney are produced.

[0103] On one hand, the present invention provides organoids comprising certain subfractions of xenogeneic kidney cell populations enriched with bioactive components and discarded inactive or unwanted components to provide superior therapeutic and regenerative outcomes compared to the starting population. For example, the bioactive kidney cells described herein (e.g., B2, B4, and B3, individually mixed, with discarded inactive or unwanted components (e.g., B1 and B5)) can be used to generate organoids for stabilizing and / or improving and / or regenerating kidney function.

[0104] On the other hand, organoids contain a specific subfraction B4 (i.e., B4') that retains therapeutic properties (e.g., stabilization and / or improvement and / or regeneration of renal function) alone or when mixed with other bioactive subfractions (e.g., B2 and / or B3), and is either discarded or lacking one or more cell types (e.g., vascular, endocrine, or endothelial cell types). In a preferred embodiment, the bioactive cell population is B2. In some embodiments, the B2 cell population is mixed with B4 or B4'. In other embodiments, the B2 cell population is mixed with B3. In other embodiments, the B2 cell population is mixed with B3 and B4, or specific cellular components of B3 and / or B4.

[0105] The B2 cell population is characterized by the expression of renal tubular cell markers selected from one or more of the following groups: megalin, cubilin, hyaluronic acid synthase 2 (HAS2), vitamin D3 25-hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin (Ecad), aquaporin-1 (Aqp1), aquaporin-2 (Aqp2), RAB17 (a member of the RAS oncogene family) (Rab17), GATA-binding protein 3 (Gata3), ion transport regulator 4 containing the FXYD domain (Fxyd4), solute carrier family 9 (sodium / hydrogen exchanger) member 4 (Slc9a4), aldehyde dehydrogenase 3 family member B1 (Aldh3b1), aldehyde dehydrogenase 1 family member A3 (Aldh1a3), calpain-8 (Capn8), and collecting duct marker aquaporin-4 (Aqp4). B2 is larger and more abundant in particles than B3 and / or B4, and therefore has a buoyant density between about 1.045 g / ml and about 1.063 g / ml (rodents), between about 1.045 g / ml and about 1.052 g / ml (humans), and between about 1.045 g / ml and about 1.058 g / ml (dogs).

[0106] The B3 cell population is characterized by expressing vascular, glomerular, and proximal tubular markers, as well as some EPO-producing cells. It has a moderate size and granularity compared to B2 and B4, and therefore a buoyant density between approximately 1.063 g / ml and approximately 1.073 g / ml (rodents), between approximately 1.052 g / ml and approximately 1.063 g / ml (humans), and between approximately 1.058 g / ml and approximately 1.063 g / ml (dogs). B3 is characterized by the expression of markers selected from one or more of the following groups: aquaporin 7 (Aqp7), ion transport regulator 2 containing the FXYD domain (Fxyd2), solute carrier family 17 (sodium phosphate) member 3 (Slc17a3), solute carrier family 3 member 1 (Slc3a1), tight junction protein 2 (claudin 2) (Cldn2), neoaspartic protease A aspartate peptidase (Napsa), solute carrier family 2 (promoted glucose transporter) member 2 (Slc2a2), alanyl (membrane) aminopeptidase (Anpep), transmembrane protein 27 (Tmem27), acyl-CoA synthase mid-chain family member 2 (Acsm2), glutathione peroxidase 3 (Gpx3), fructose-1,6-bisphosphatase 1 (Fbp1), and alanine-glyoxylate transaminase 2 (Agxt2). B3 is also characterized by the vascular expression marker platelet endothelial cell adhesion molecule (Pecam) and the glomerular expression marker glomerular podocyte slit membrane protein (Podn).

[0107] The B4 cell population is characterized by expressing one or more of the following vascular markers: PECAM, VEGF, KDR, HIF1a, CD31, CD146; one or more of the following glomerular markers: glomerular podocyte slit membrane protein (Podn) and glomerular podocyte-specific protein (Neph); and oxygen-tunable, EPO-enriched populations, B2 and B3, compared to the ungraded (UNFX) population. B4 is also characterized by expressing one or more of the following markers: chemokine (CXC motif) receptor 4 (Cxcr4), endothelin receptor type B (Ednrb), type V collagen α2 (Col5a2), cadherin 5 (Cdh5), plasminogen activator, tissue (Plat), angiopoietin 2 (Angpt2), kinase insertion domain protein receptor (Kdr), acidic cysteine-rich secretory protein (osteo-adhesion protein) (Sparc), seriganan (Srgn), TIMP metallopeptidase inhibitor 3 (Timp3), Wilms' tumor 1 (Wt1), wingless MMTV integration site family member 4 (Wnt4), G-protein signal transduction regulator 4 (Rgs4), platelet endothelial cell adhesion molecule (Pecam), and erythropoietin (Epo). B4 is also characterized by smaller and fewer granular cells compared to B2 or B3, and a buoyant density between approximately 1.073 g / ml and approximately 1.091 g / ml (rodents), and between approximately 1.063 g / ml and approximately 1.091 g / mL (humans and dogs).

[0108] The B4' cell population is defined as having a buoyant density between 1.063 g / mL and 1.091 g / mL and expressing one or more of the following markers: PECAM, vEGF, KDR, HIF1a, glomerular podocyte slit membrane protein, glomerular podocyte-specific protein, EPO, CK7, CK8 / 18 / 19. In one embodiment, the B4' cell population is characterized by expressing a set of vascular markers containing one or more of the following: PECAM, vEGF, KDR, HIF1a, CD31, CD146. In another embodiment, the B4' cell population is characterized by expressing the endocrine marker EPO. In one embodiment, the B4' cell population is characterized by expressing a set of glomerular markers containing one or more of the following: glomerular podocyte slit membrane protein (Podn) and glomerular podocyte-specific protein (Neph). In certain embodiments, the B4' cell population is characterized by expressing a set of vascular markers containing one or more of the following: PECAM, vEGF, KDR, HIF1a and is characterized by expressing the endocrine marker EPO. In another embodiment, B4' is also characterized by having smaller and fewer granular cells compared to B2 or B3, and having a buoyancy density between about 1.073 g / ml and about 1.091 g / ml (rodents), and between about 1.063 g / ml and about 1.091 g / mL (humans and dogs).

[0109] On one hand, the present invention provides organoids containing isolated, enriched populations of B4' human kidney cells, said B4' human kidney cells comprising at least one of erythropoietin-producing cells, vascular cells, and glomerular cells at densities between 1.063 g / mL and 1.091 g / mL. In one embodiment, the B4' cell population is characterized by expression of vascular markers. In some embodiments, the B4' cell population is not characterized by expression of glomerular markers. In some embodiments, the B4' cell population is capable of oxygen-tunable erythropoietin (EPO) expression.

[0110] In one embodiment, the organoids of the present invention contain a B4' cell population but exclude a B2 cell population, the B2 cell population comprising renal tubular cells with a density between 1.045 g / mL and 1.052 g / mL. In another embodiment, the organoids containing a B4' cell population exclude a B1 cell population, the B1 cell population comprising large granular cells of the collecting ducts and renal tubular system with a density <1.045 g / mL. In yet another embodiment, the B4' cell population organoids exclude a B5 cell population, the B5 cell population comprising fragments and small cells with low granularity and viability with a density >1.091 g / mL.

[0111] In one embodiment, the organoid containing the B4' cell population does not include the B2 cell population, which comprises renal tubular cells with a density between 1.045 g / mL and 1.052 g / mL; the B1 cell population, which comprises large granular cells of the collecting ducts and renal tubular system with a density <1.045 g / mL; and the B5 cell population, which comprises low-granularity and viable fragments and small cells with a density >1.091 g / mL. In some embodiments, the B4' cell population may be derived from a subject with kidney disease.

[0112] On one hand, the present invention provides an organoid comprising a mixture of human kidney cells comprising a first cell population B2 and a second cell population B4', wherein the first cell population B2 comprises isolated, enriched tubular cell populations at densities between 1.045 g / mL and 1.052 g / mL, and the second cell population B4' comprises erythropoietin-producing cells and vascular cells, but discarded glomerular cells, at densities between approximately 1.063 g / mL and 1.091 g / mL, wherein the mixture does not include a B1 cell population or a B5 cell population, wherein the B1 cell population comprises large granular cells of the collecting duct and tubular system at densities <1.045 g / mL, and the B5 cell population comprises fragments and small cells with low granularity and viability at densities >1.091 g / mL. In some embodiments, the B4' cell population is characterized by expression of vascular markers. In one embodiment, the B4' cell population is not characterized by expression of glomerular markers. In some embodiments, B2 further comprises collecting duct epithelial cells. In one embodiment, the organoid comprises or is formed from a mixture of cells capable of receptor-mediated albumin uptake. In another embodiment, the mixture of cells is capable of oxygen-tunable erythropoietin (EPO) expression. In one embodiment, the mixture comprises HAS-2-expressing cells capable of producing and / or stimulating the production of high molecular weight hyaluronic acid (HA) in vitro and in vivo. In all embodiments, the first and second cell populations may be derived from kidney tissue or cultured kidney cells (Basu et al., Lipids in Health and Disease, 2011, 10:171).

[0113] In one embodiment, the organoid contains a mixture capable of providing regenerative stimulation upon in vivo delivery. In other embodiments, the mixture is capable of reducing, stabilizing, or improving glomerular filtration, tubular reabsorption, urine production, and / or endocrine function upon in vivo delivery. In one embodiment, the B4' cell population is derived from a subject with kidney disease.

[0114] On one hand, the present invention provides organoids containing isolated, enriched populations of B4' human kidney cells, said B4' human kidney cells comprising at least one of erythropoietin-producing cells, vascular cells, and glomerular cells at densities between 1.063 g / mL and 1.091 g / mL. In one embodiment, the B4' cell population is characterized by expression of vascular markers. In some embodiments, the B4' cell population is not characterized by expression of glomerular markers. The unexpressed glomerular marker may be a glomerular podocyte slit membrane protein (see Example 10). In some embodiments, the B4' cell population is capable of oxygen-tunable erythropoietin (EPO) expression.

[0115] In one embodiment, the organoid containing the B4' cell population does not include the B2 cell population, which comprises renal tubular cells with a density between 1.045 g / mL and 1.052 g / mL. In another embodiment, the B4' cell population organoid does not include the B1 cell population, which comprises large granular cells of the collecting ducts and renal tubular system with a density <1.045 g / mL. In yet another embodiment, the B4' cell population organoid does not include the B5 cell population, which comprises low-granularity and viable fragments and small cells with a density >1.091 g / mL.

[0116] In one embodiment, the organoid containing the B4' cell population does not include the B2 cell population, which comprises renal tubular cells with a density between 1.045 g / mL and 1.052 g / mL; the B1 cell population, which comprises large granular cells of the collecting ducts and renal tubular system with a density <1.045 g / mL; and the B5 cell population, which comprises low-granularity and viable fragments and small cells with a density >1.091 g / mL. In some embodiments, the B4' cell population may be derived from a subject with kidney disease.

[0117] On one hand, the present invention provides an organoid comprising a mixture of human kidney cells comprising a first cell population B2 and a second cell population B4', wherein the first cell population B2 comprises isolated, enriched tubular cell populations at densities between 1.045 g / mL and 1.052 g / mL, and the second cell population B4' comprises erythropoietin-producing cells and vascular cells, but discarded glomerular cells, at densities between approximately 1.063 g / mL and 1.091 g / mL, wherein the mixture does not include a B1 cell population or a B5 cell population, wherein the B1 cell population comprises large granular cells of the collecting duct and tubular system at densities <1.045 g / mL, and the B5 cell population comprises fragments and small cells with low granularity and viability at densities >1.091 g / mL. In some embodiments, the B4' cell population is characterized by expression of vascular markers. In one embodiment, the B4' cell population is not characterized by expression of glomerular markers. In some embodiments, B2 further comprises collecting duct epithelial cells. In one embodiment, the cell mixture is capable of receptor-mediated albumin uptake. In another embodiment, the cell mixture is capable of oxygen-tuned erythropoietin (EPO) expression. In one embodiment, the mixture contains HAS-2-expressing cells capable of producing and / or stimulating the production of high molecular weight hyaluronic acid (HA) in vitro and in vivo. In all embodiments, the first and second cell populations can be derived from kidney tissue or cultured kidney cells.

[0118] On the other hand, the present invention provides organoids containing xenogeneic kidney cell populations comprising a combination of cell fractions or enriched cell populations (e.g., B1, B2, B3, B4 (or B4'), and B5). In one embodiment, the combination has a buoyant density between about 1.045 g / ml and about 1.091 g / ml. In another embodiment, the combination has a buoyant density less than about 1.045 g / ml and between about 1.099 g / ml or about 1.100 g / ml. In yet another embodiment, the combination has a buoyant density as determined by separation over a density gradient (e.g., by centrifugation). In yet another embodiment, the cell fraction combination contains B2, B3, and B4 (or B4') with B1 and / or B5 discarded. In some embodiments, the cell fraction combination contains B2, B3, B4 (or B4'), and B5 but discards B1. Once B1 and / or B5 are discarded, the combination can then be cultured in vitro prior to preparing organoids containing B2, B3, and B4 (or B4') cell fractions.

[0119] The inventors of this invention have unexpectedly discovered that the combination of B2, B3, B4, and B5 in vitro culture of discarded B1 results in the discarding of B5. In one embodiment, B5 is discarded after at least one, two, three, four, or five passages. In another embodiment, the combination of B2, B3, B4, and B5 cell fractions passaged under the conditions described herein provides a passaged cell population with B5 percentages of less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, or less than about 0.5% of the passaged cell population.

[0120] In another embodiment, B4' is part of a combination of cell fractions. In yet another embodiment, in vitro culture of discarded B5 occurs under hypoxic conditions.

[0121] In one embodiment, the organoid contains a mixture capable of providing regenerative stimulation upon in vivo delivery. In other embodiments, the mixture is capable of reducing, stabilizing, or improving glomerular filtration, tubular reabsorption, urine production, and / or endocrine function upon in vivo delivery. In one embodiment, the B4' cell population is derived from a subject with kidney disease.

[0122] In one preferred embodiment, the organoid comprises, and / or is formed from, a mixture of B2 in combination with B3 and / or B4. In another preferred embodiment, the mixture comprises B2 in combination with B3 and / or B4'. In other preferred embodiments, the mixture consists of, or is primarily composed of, (i) B2 in combination with B3 and / or B4; or (ii) B2 in combination with B3 and / or B4'.

[0123] The mixture containing the B4' cell population may also contain B2 and / or B3 cell populations obtained from non-healthy subjects. Non-healthy subjects may be the same subjects from whom the B4' fraction was obtained. In contrast to the B4' cell population, the B2 and B3 cell populations obtained from non-healthy subjects typically do not lack one or more specific cell types compared to the initial kidney cell population derived from healthy individuals.

[0124] As described in Presnell et al., WO / 2010 / 056328, B2 and B4 cell populations have been found to express higher molecular weight hyaluronic acid (HA) in vitro and in vivo through the action of hyaluronic acid synthase-2 (HAS-2) (more precisely, a marker enriched in the B2 cell population). Treatment with B2 in the 5 / 6 Nx model showed reduced fibrosis, accompanied by strong in vivo expression of HAS-2 and the expected production of high molecular weight HA in the treated tissues. Significantly, the untreated 5 / 6 Nx model resulted in fibrosis with limited HAS-2 detection and little production of high molecular weight HA. Not wishing to be bound by theory, it is hypothesized that this anti-inflammatory high molecular weight HA, primarily produced by B2 (and to some extent by B4), synergistically works with cellular agents to reduce renal fibrosis and aid renal regeneration. Therefore, this invention includes organoids containing the bioactive renal cells described herein along with biomaterials containing hyaluronic acid. This invention also covers biomaterial components that provide regenerative stimulation through direct or stimulated production by implanted cells.

[0125] On one hand, the present invention provides organoids containing and / or derived from isolated, xenogeneic, EPO-producing kidney cell populations for treating kidney disease, anemia, and / or EPO deficiency in subjects in need. In one embodiment, the cell population is derived from a kidney biopsy. In another embodiment, the cell population is derived from whole kidney tissue. In yet another embodiment, the cell population is derived from an in vitro culture of mammalian kidney cells derived from a kidney biopsy or whole kidney tissue. In all embodiments, these populations are unsegregated and ungraded cell populations, also referred to herein as non-enriched cell populations.

[0126] On the other hand, the present invention provides organoids containing and / or derived from isolated erythropoietin-producing (EPO)-generating kidney cell populations, wherein the isolated EPO-producing kidney cell populations are further enriched such that the proportion of EPO-producing cells in the enriched subpopulation is greater than the proportion of EPO-producing cells in the initial or unenriched cell population. In one embodiment, the enriched EPO-producing cell fraction contains a larger proportion of interstitial fibroblasts and a smaller proportion of renal tubular cells compared to the interstitial fibroblasts and tubular cells contained in the unenriched initial population. In some embodiments, the enriched EPO-producing cell fraction contains a larger proportion of glomerular cells and vascular cells and a smaller proportion of collecting duct cells compared to the glomerular cells, vascular cells, and collecting duct cells contained in the unenriched initial population. In the embodiments described herein, these populations are referred to herein as the "B4" cell population.

[0127] On the other hand, the present invention provides organoids containing and / or derived from EPO-producing kidney cell populations mixed with one or more additional kidney cell populations. In one embodiment, the EPO-producing cell population is a first cell population enriched with EPO-producing cells, such as B4. In another embodiment, the EPO-producing cell population is a first cell population without enrichment of EPO-producing cells, such as B2. In another embodiment, the first cell population is mixed with a second kidney cell population. In some embodiments, the second cell population is enriched with renal tubular cells, which can be indicated by the presence of a renal tubular cell phenotype. In another embodiment, the renal tubular cell phenotype can be indicated by the presence of a renal tubular cell marker. In yet another embodiment, the renal tubular cell phenotype can be indicated by the presence of one or more renal tubular cell markers. Renal tubular cell markers include, but are not limited to, macroprotein, cubilin, hyaluronic acid synthase 2 (HAS2), vitamin D325-hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin (Ecad), aquaporin-1 (Aqp1), aquaporin-2 (Aqp2), RAB17 (a member of the RAS oncogene family) (Rab17), GATA-binding protein 3 (Gata3), ion transport regulator 4 containing the FXYD domain (Fxyd4), solute carrier family 9 (sodium / hydrogen exchanger) member 4 (Slc9a4), aldehyde dehydrogenase 3 family member B1 (Aldh3b1), aldehyde dehydrogenase 1 family member A3 (Aldh1a3), and calpain-8 (Capn8). In another embodiment, the first cell group is mixed with at least one of several types of kidney cells, including but not limited to interstitial cells, tubular cells, collecting duct cells, glomerular cells, and / or cells derived from blood or the vascular system.

[0128] The organoids of the present invention may include or be formed of EPO-producing kidney cell populations, said EPO-producing kidney cell populations containing B4 or B4' in the form of a mixture with B2 and / or B3 or in the form of an enriched cell population (e.g., B2+B3+B4 / B4').

[0129] On one hand, the organoid contains and / or produces EPO-producing kidney cell populations characterized by EPO expression and a biological responsiveness to oxygen such that a reduction in oxygen stress in the culture system leads to the induction of EPO expression. In one embodiment, the EPO-producing cell population is enriched with EPO-producing cells. In one embodiment, EPO expression is induced when the cell population is cultured under conditions in which the cells are subjected to a reduced level of available oxygen in the culture system compared to a cell population cultured at normal atmospheric (approximately 21%) oxygen levels. In one embodiment, EPO-producing cells cultured under lower oxygen conditions express higher levels of EPO than EPO-producing cells cultured under normal oxygen conditions. Generally, culturing cells at reduced levels of available oxygen (also known as hypoxic culture conditions) means a reduced oxygen level compared to culturing cells at normal atmospheric (also known as normal or oxygen-rich) oxygen levels. In one embodiment, hypoxic cell culture conditions include culturing cells at approximately less than 1% oxygen, approximately less than 2% oxygen, approximately less than 3% oxygen, approximately less than 4% oxygen, or approximately less than 5% oxygen. In another embodiment, normal or oxygen-rich culture conditions include culturing cells at approximately 10% oxygen, approximately 12% oxygen, approximately 13% oxygen, approximately 14% oxygen, approximately 15% oxygen, approximately 16% oxygen, approximately 17% oxygen, approximately 18% oxygen, approximately 19% oxygen, approximately 20% oxygen, or approximately 21% oxygen.

[0130] In another embodiment, the induction or increase of EPO expression is obtained and can be observed by culturing cells at about less than 5% available oxygen and comparing the EPO expression level of cells cultured at atmospheric (about 21%) oxygen. In another embodiment, EPO induction is obtained in a cell culture capable of expressing EPO by a method comprising a first culture phase in which the cell culture is cultured at atmospheric oxygen (about 21%) for a certain period of time and a second culture phase in which the available oxygen level is reduced and the cells are cultured at about less than 5% available oxygen. In another embodiment, EPO expression in response to hypoxic conditions is regulated by HIF1α. Those skilled in the art will recognize that other oxygen-manipulated culture conditions known in the art can be used for the cells described herein.

[0131] On the one hand, organoids contain and / or are formed from an enriched population of EPO-producing mammalian cells characterized by a biological response (e.g., EPO expression) to perfusion conditions. In one embodiment, the perfusion conditions include transient, intermittent, or continuous fluid flow (perfusion). In one embodiment, EPO expression is mechanically induced when the culture medium in which cells are cultured is intermittently or continuously circulated or agitated in such a manner that power is transferred to the cells by flow. In one embodiment, cells subjected to transient, intermittent, or continuous fluid flow are cultured in such a manner that they exist in a three-dimensional structure within or on a material that provides a framework and / or gaps for forming the three-dimensional structure. In one embodiment, cells are cultured on porous beads and subjected to intermittent or continuous fluid flow by means of a shaker, track platform, or rotating flask. In another embodiment, cells are cultured on a three-dimensional scaffold and placed in a device whereby the scaffold is fixed and fluid flows directionally through or across the scaffold. Those skilled in the art will appreciate that other perfusion culture conditions known in the art can be used for the cells described herein.

[0132] Inactive cell population

[0133] As described herein, the present invention is based in part on the surprising discovery that organoids comprising certain subfractions and / or composed of xenogeneic kidney cell populations, enriched with bioactive components and discarding inactive or unwanted components, provide superior therapeutic and regenerative outcomes compared to the starting population. In a preferred embodiment, the organoids provided by the present invention contain cell populations that have discarded B1 and / or B5 cell populations. For example, the following substances may be discarded B1 and / or B5: mixtures of two or more of B2, B3, and B4 (or B4'); enriched cell populations of B2, B3, and B4 (or B4').

[0134] The B1 cell population contains large granular cells of the collecting ducts and renal tubules, with a buoyant density of less than approximately 1.045 g / ml. The B5 cell population consists of low-granularity and viable fragments and small cells, with a buoyant density greater than approximately 1.091 g / ml.

[0135] Methods for isolating and culturing cell populations

[0136] On one hand, the SRC+ cell populations and / or organoids of the present invention contain and / or are formed from cell populations isolated and / or cultured from kidney tissue. Methods for separating and isolating kidney cell components, such as enriched cell populations contained in organoids for therapeutic purposes (including treatment of kidney disease, anemia, EPO deficiency, tubular transport defects, and glomerular filtration defects), are provided herein. In one embodiment, the cell populations are isolated from freshly digested (i.e., mechanically or enzymatically digested) kidney tissue or from xenogeneic in vitro cultures of mammalian kidney cells. Methods for isolating additional bioactive cell populations constituting the SRC+ cell populations of the present invention are further described in the embodiments.

[0137] Organoids may contain, and / or be formed from, xenogeneic mixtures of renal cells, wherein the xenogeneic mixture of renal cells has been cultured under hypoxic conditions prior to separation on a density gradient in fractions B4 (including B4') and B2 and / or B3, providing an enhanced distribution and composition of cells. For renal cells isolated from diseased and disease-free kidneys, enrichment of oxygen-dependent cells from B2 in B4 has been observed. It is not intended to be theoretically constrained, but this may be due to one or more of the following phenomena: 1) selective survival, death, or proliferation of specific cell components during a hypoxic culture cycle; 2) changes in cell granularity and / or size in response to hypoxic culture, thus affecting buoyancy density and subsequent localization during density gradient separation; and 3) changes in cell gene / protein expression in response to a hypoxic culture cycle, thus leading to differential cell characteristics within any given gradient fraction. Therefore, in one embodiment, the organoid contains, and / or is formed from, a hypoxia-resistant cell population enriched with renal tubular cells, such as B2.

[0138] Exemplary techniques for separating and isolating cell populations of the present invention include separation on a density gradient based on different proportions of different cell types contained within the population of interest. The proportion of any given cell type can be affected by intracellular granularity, intracellular water volume, and other factors. In one aspect, the present invention provides optimal gradient conditions for isolating cell preparations of the present invention (e.g., B2 and B4, including B4') in multiple species, including but not limited to humans, dogs, and rodents. In a preferred embodiment, the density gradient is used to obtain a novel enriched tubular cell-level subset derived from xenogeneic kidney cell populations, namely the B2 cell population. In one embodiment, the density gradient is used to obtain a novel enriched EPO-producing cell-level subset derived from xenogeneic kidney cell populations, namely the B4 cell population. In other embodiments, the density gradient is used to obtain enriched subpopulations of renal tubular cells, glomerular cells, and endothelial cells of the kidney. In one embodiment, the EPO-producing cells and tubular cells are isolated from erythrocytes and cell debris. In one embodiment, EPO-producing cells, glomerular cells, and vascular cells are isolated from other cell types and from erythrocytes and cell debris, while subsets of renal tubular cells and collecting duct cells are also isolated from other cell types and from erythrocytes and cell debris. In another embodiment, endocrine cells, glomerular cells, and / or vascular cells are isolated from other cell types and from erythrocytes and cell debris, while subsets of renal tubular cells and collecting duct cells are also isolated from other cell types and from erythrocytes and cell debris.

[0139] On one hand, the organoids of the present invention contain OPTIPREP by partially using an aqueous solution of 60% nonionic iodide compound iodixanol based on certain key features described below. ®Cell populations prepared and / or formed from (Axis-Shield) density gradient media. However, those skilled in the art will recognize that any density gradient or other means, including the necessary characteristics for separating the cell populations of the invention, can be used according to the invention, such as immunoisolation using cell surface markers known in the art. Those skilled in the art should also recognize that the same cellular characteristics that facilitate the separation of cell subpopulations by density gradient (size and particle size) can be utilized by flow cytometry (forward scattering = size reflection by flow cytometry, and side scattering = particle size reflection). Importantly, the density gradient media should be low in toxicity to the specific cells of interest. While the density gradient media should be low in toxicity to the specific cells of interest, the invention covers the use of gradient media that function in the selection process for the cells of interest. It is not intended to be theoretically restrictive, but it can be seen that the cell populations of the invention recovered by a gradient containing iodixanol are iodixanol resistant, as there is significant cell loss between the loading and recovery steps, indicating that exposure to iodixanol under gradient conditions leads to the elimination of some cells. Cells appearing in specific bands after the iodixanol gradient are resistant to any adverse effects of iodixanol and / or density gradient exposure. Therefore, the use of additional contrast agents for the isolation and / or selection of cell populations used in the organoids described herein is also covered, wherein the additional contrast agents are mild to moderate nephrotoxic. Furthermore, the density gradient culture medium should not bind to proteins in human plasma or adversely affect the important functions of the cells of interest.

[0140] On the other hand, the present invention provides cell populations containing kidney cell types that have been enriched and / or discarded using fluorescence-activated cell sorting (FACS), and / or organoids formed therefrom. In one embodiment, the kidney cell types can be enriched and / or discarded using BD FACSAria™ or equivalents.

[0141] On the other hand, organoids contain cell populations that have been enriched and / or discarded kidney cell types using magnetic cell sorting, and / or formed therefrom. In one embodiment, the kidney cell types can be obtained using Miltenyi autoMACS. ® The system or equivalent is used to enrich and / or discard.

[0142] On the other hand, organoids may include, and / or be formed from, a population of kidney cells that has undergone three-dimensional culture. One method of culturing the cell population is by continuous perfusion. In one embodiment, cell populations cultured via three-dimensional culture and continuous perfusion exhibit greater cellularity and interconnectivity compared to statically cultured cell populations. In another embodiment, cell populations cultured via three-dimensional culture and continuous perfusion exhibit greater EPO expression and enhanced expression of tubular-related genes such as epithelial cadherin compared to statically cultured cell populations. In yet another embodiment, cell populations cultured via continuous perfusion exhibit greater glucose and glutamine consumption levels compared to statically cultured cell populations.

[0143] As described herein, hypoxic or hypoxic conditions can be used in methods for preparing cell populations for the organoids of the present invention. However, methods for preparing cell populations can be used without requiring a hypoxia regulation step. In one embodiment, conditions with normal oxygen levels can be used.

[0144] Those skilled in the art will recognize that other methods of isolation and culture known in the art can be used for the cells described herein.

[0145] biomaterials

[0146] As described in U.S. Application 20070276507 published by Bertram et al., the polymer matrix or scaffold can be shaped into any number of desired configurations to meet any number of overall system, geometric, or spatial constraints. In one embodiment, the matrix or scaffold of the present invention may be three-dimensional and shaped to conform to the size and shape of an organ or tissue structure. For example, a three-dimensional (3-D) matrix may be used in the use of polymer scaffolds to treat kidney disease, anemia, EPO deficiency, tubular transport defects, or glomerular filtration defects. A variety of different shapes of 3-D scaffolds may be used. Naturally, the polymer matrix may be shaped to different sizes and shapes to suit patients of different sizes. The polymer matrix may also be shaped in other ways to suit the specific needs of a patient. In another embodiment, the polymer matrix or scaffold may be a biocompatible porous polymer scaffold. The scaffold may be formed from a variety of synthetic or naturally occurring materials, including but not limited to open-cell polylactic acid (OPLA). ®Cellulose ethers, cellulose, cellulose esters, fluorinated polyethylene, phenolic resins, poly-4-methylpentene, polyacrylonitrile, polyamide, polyamide-imide, polyacrylate, polybenzoxazole, polycarbonate, polycyanoaryl ether, polyester, polyester carbonate, polyetheretherketone, polyetherimide, polyetherketone, polyethersulfone, polyethylene, polyfluoroolefin, polyimide, polyolefin, polyoxadiazole, polyphenylene ether, polyphenylene sulfide, polypropylene, polystyrene, polysulfide, polysulfone, polytetrafluoroethylene, polysulfide, polytriazole, polyurethane, polyethylene, polyvinylidene fluoride, regenerated cellulose, silicone, urea-formaldehyde, collagen, laminin, fibronectin, glycosaminoglycans, silk, elastin, alginate, hyaluronic acid, agarose, or copolymers or physical blends thereof. The range of scaffold configurations can be from liquid hydrogel suspensions to flexible porous scaffolds to rigid, fixed-shape porous scaffolds.

[0147] The scaffold can be made of any form of biomaterial, including but not limited to diluents, cell carriers, microbeads, material fragments, scaffolds of synthetic compositions (including but not limited to PGA, PLGA, PLLA, OPLA, and electrospun nanofibers), and foams of synthetic compositions (including but not limited to PGA, PLGA, PLLA, OPLA, and electrospun nanofibers).

[0148] Hydrogels can be formed from a variety of polymer materials and are used in a wide range of biomedical applications. Hydrogels can be physically described as a three-dimensional network of hydrophilic polymers. Depending on the type of hydrogel, they contain varying percentages of water, but are generally insoluble in water. Despite their high water content, hydrogels are able to bind a significant amount of liquid due to the presence of hydrophilic residues. Hydrogels swell considerably without altering their gel-like structure. The fundamental physical characteristics of hydrogels can be precisely modified depending on the properties of the polymers used and the additional specialized instruments used in the product.

[0149] Preferably, the hydrogel is made of a bioinert and physiologically compatible polymer, a biologically derived material, a synthetically derived material, or a combination thereof. The hydrogel material preferably does not induce an inflammatory response. Examples of other materials that can be used to form hydrogels include (a) modified alginates, (b) polysaccharides that gel by exposure to monovalent cations (e.g., glucan gum and carrageenan), (c) polysaccharides that are very viscous liquids or thixotropic and gel over time through slow structural evolution (e.g., hyaluronic acid), and (d) polymeric hydrogel precursors (e.g., polyethylene oxide-polypropylene glycol block copolymers and proteins). U.S. Patent No. 6,224,893 B1 provides a detailed description of various polymers suitable for preparing hydrogels according to the invention and the chemical properties of said polymers.

[0150] The scaffold or biomaterial features enable cells to attach to and interact with the scaffold or biomaterial, and / or provide porous spaces in which cells can be captured. In one embodiment, the porous scaffold or biomaterial of the present invention allows one or more cell populations or mixtures of cells to be added to or deposited on a biomaterial configured as a porous scaffold (e.g., by cell attachment) and / or within the pores of the scaffold (e.g., by cell capture). In another embodiment, the scaffold or biomaterial allows or facilitates cell-to-cell and / or cell-to-biomaterial interactions within the scaffold to form a construct as described herein.

[0151] In one embodiment, the biomaterial used according to the invention comprises hyaluronic acid (HA) in the form of a hydrogel, said hydrogel containing a size of 5.1 kDa to >2 x 10⁻⁶ kDa. 6 HA molecules in the kDa range. In another embodiment, the biomaterial used according to the invention comprises hyaluronic acid in the form of a porous foam, said porous foam also containing HA molecules with sizes ranging from 5.1 kDa to >2 x 10⁻⁶ kDa. 6 HA molecules in the kDa range. In yet another embodiment, the biomaterial used according to the invention is composed of polylactic acid (PLA)-based foam having an open-cell structure and a pore size of about 50 micrometers to about 300 micrometers. In yet another embodiment, specific cell populations (preferably B2 and B4) directly provide and / or stimulate the synthesis of high molecular weight hyaluronic acid via hyaluronic acid synthase-2 (HAS-2), especially after intrarenal implantation.

[0152] The biomaterials described herein can also be designed or adapted to respond to certain external conditions, such as in vitro or in vivo. In one embodiment, the biomaterial is temperature-sensitive (e.g., in vitro or in vivo). In another embodiment, the biomaterial is adapted to respond to exposure to enzymatic degradation (e.g., in vitro or in vivo). As described herein, the response of the biomaterial to external conditions can be fine-tuned. The temperature sensitivity of the described organoids can be altered by adjusting the percentage of biomaterial in the organoid. Alternatively, the biomaterial can be chemically crosslinked to provide greater resistance to enzymatic degradation. For example, carbodiimide crosslinking agents can be used to chemically crosslink gelatin beads to provide reduced sensitivity to endogenous enzymes.

[0153] On the one hand, the response of biomaterials to external conditions involves the loss of their structural integrity. While the above provides for temperature sensitivity and resistance to enzymatic degradation, other mechanisms exist in different biomaterials through which the loss of material integrity can occur. These mechanisms can include, but are not limited to, thermodynamic (e.g., phase transitions such as dissolution, diffusion (e.g., allowing ionic crosslinking agents to diffuse from the biomaterial into surrounding tissues)), chemical, enzymatic, pH (e.g., pH-sensitive liposomes), ultrasound, and photoinstability (light penetration). The exact mechanism by which biomaterials lose their structural integrity will vary, but it is typically triggered during or after implantation.

[0154] Those skilled in the art will understand that other types of synthetic or naturally occurring materials known in the art can be used to form the scaffolds described herein.

[0155] On the one hand, the present invention provides the constructs described herein made of the aforementioned scaffolds or biomaterials.

[0156] Buildings

[0157] On one hand, the present invention provides organoids containing implantable constructs having one or more cell populations as described herein for treating kidney disease, anemia, or EPO deficiency in subjects in need. In one embodiment, the construct is composed of a biocompatible material or biomaterial, a scaffold, or a matrix, the scaffold or matrix consisting primarily of one or more synthetic or naturally occurring biocompatible materials and a mixture of one or more cell populations or cells as described herein, deposited on or embedded in the scaffold surface by attachment and / or capture. In some embodiments, the construct is composed of a biomaterial and a mixture of one or more cell populations or cells as described herein, the mixture of which is coated with, deposited on, deposited in, attached to, captured in, embedded in, seeded in, or combined with a biomaterial component. Any cell population described herein (including enriched cell populations or mixtures thereof) can be used in combination with a matrix to form the construct.

[0158] In another embodiment, the deposited cell population or cell component of the construct is a first kidney cell population enriched with oxygen-tunable EPO-producing cells. In another embodiment, the first kidney cell population contains glomerular cells and vascular cells in addition to oxygen-tunable EPO-producing cells. In one embodiment, the first kidney cell population is a B4' cell population. In another embodiment, the deposited cell population or cell component of the construct comprises a first enriched kidney cell population and a second kidney cell population. In some embodiments, the second cell population is not enriched with oxygen-tunable EPO-producing cells. In another embodiment, the second cell population is enriched with renal tubular cells. In another embodiment, the second cell population is enriched with renal tubular cells and contains collecting duct epithelial cells. In other embodiments, the renal tubular cells are characterized by expressing one or more renal tubular cell markers, which may include, but are not limited to, macroprotein, cubilin, hyaluronic acid synthase 2 (HAS2), vitamin D3 25-hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin (Ecad), aquaporin-1 (Aqp1), aquaporin-2 (Aqp2), RAB17 (a member of the RAS oncogene family) (Rab17), GATA-binding protein 3 (Gata3), ion transport regulator 4 containing the FXYD domain (Fxyd4), solute carrier family 9 (sodium / hydrogen exchanger) member 4 (Slc9a4), aldehyde dehydrogenase 3 family member B1 (Aldh3b1), aldehyde dehydrogenase 1 family member A3 (Aldh1a3), and calpain-8 (Capn8).

[0159] In one embodiment, the cell population deposited on or combined with biomaterials or scaffolds to form the constructs of the present invention is derived from a variety of sources, such as autologous sources. Non-autologous sources are also suitable for use, including but not limited to allogeneic or syngeneic (autogenous or syngeneic) sources.

[0160] Those skilled in the art will recognize that there are several suitable methods for depositing or otherwise combining cell populations with biological materials to form constructs.

[0161] On one hand, the constructs of the present invention are suitable for use in the methods of use described herein. In one embodiment, the construct is suitable for administration to a subject who requires treatment for kidney disease of any cause, anemia, or EPO deficiency of any cause. In other embodiments, the construct is suitable for administration to a subject who requires improvement or restoration of erythrocyte-like cell homeostasis. In yet another embodiment, the construct is suitable for administration to a subject who requires improvement in kidney function.

[0162] In yet another aspect, the present invention provides a construct for implantation into a subject requiring improved kidney function, the construct comprising: a) a biomaterial comprising one or more biocompatible synthetic polymers or naturally occurring proteins or peptides; and b) a mixture of mammalian kidney cells derived from a subject with kidney disease comprising a first cell population B2 and a second cell population B4', the first cell population B2 comprising isolated, enriched populations of renal tubular cells at densities between 1.045 g / mL and 1.052 g / mL, and the second cell population B4' comprising erythropoietin-producing cells and vascular cells, but discarded glomerular cells, at densities between 1.063 g / mL and 1.091 g / mL, coated with the biomaterial, deposited on or within the biomaterial, captured in the biomaterial, suspended in the biomaterial, embedded in the biomaterial, and / or otherwise combined with the biomaterial. In some embodiments, the mixture does not include B1 or B5 cell populations, wherein the B1 cell population comprises large granular cells of the collecting duct and renal tubular system with a density <1.045 g / ml, and the B5 cell population comprises fragments and small cells with low particle size and viability with a density >1.091 g / ml.

[0163] In one embodiment, the construct includes a B4' cell population characterized by expressing vascular markers. In some embodiments, the B4' cell population is not characterized by expressing glomerular markers. In some embodiments, the mixture is capable of oxygen-tuned erythropoietin (EPO) expression. In all embodiments, the mixture may be derived from mammalian kidney tissue or cultured kidney cells.

[0164] In one embodiment, the construct comprises a biomaterial configured to be suitable for capturing and / or attaching a mixture of three-dimensional (3-D) porous biomaterials. In another embodiment, the construct comprises a biomaterial configured to be suitable for embedding, attaching, suspending, or coating mammalian cells as a liquid or semi-liquid gel. In yet another embodiment, the construct comprises a biomaterial configured to consist primarily of high molecular weight hyaluronic acid (HA) in the form of a hydrogel. In another embodiment, the construct comprises a biomaterial consisting primarily of high molecular weight hyaluronic acid in the form of a porous foam. In yet another embodiment, the construct comprises a biomaterial consisting of polylactic acid-based foam with pore sizes between about 50 micrometers and about 300 micrometers. In yet another embodiment, the construct comprises one or more cell populations, which may be derived from an autologous kidney sample for a subject requiring improved kidney function. In some embodiments, the sample is a kidney biopsy. In some embodiments, the subject has kidney disease. In yet another embodiment, the cell population is derived from a non-autologous kidney sample. In one embodiment, the construct provides erythrocyte-like cell homeostasis.

[0165] How to use

[0166] On one hand, the present invention provides a method for treating kidney disease, anemia, or EPO deficiency in a subject in need by using SRC+ cell populations and / or organoids formed therefrom containing the kidney cell populations and mixtures of kidney cells described herein, and / or formed therefrom. In one embodiment, the method includes administering to a subject a first kidney cell population comprising EPO-enriched cells and / or organoids formed therefrom. In another embodiment, the first cell population is enriched with EPO-enriched cells, glomerular cells, and vascular cells. In another embodiment, the organoid may further comprise one or more additional kidney cell populations, and / or may be formed from one or more additional kidney cell populations. In one embodiment, the additional cell population is a second cell population that does not contain EPO-enriched cells. In another embodiment, the additional cell population is a second cell population that does not contain EPO-enriched cells, glomerular cells, or vascular cells. In another embodiment, the organoid also includes a population or mixture of kidney cells deposited in, on, embedded in, coated with, or captured in a biomaterial to form an implantable construct for treating the disease or condition described herein, and / or formed from said population or mixture of kidney cells. In one embodiment, the organoid is used alone or in combination with other cells or biomaterials (e.g., hydrogels, porous scaffolds, or natural or synthetic peptides or proteins) to stimulate regeneration in acute or chronic disease states.

[0167] On the other hand, effective treatment of kidney disease, anemia, or EPO deficiency in subjects using the methods of the present invention can be observed through various indicators of erythropoiesis and / or kidney function. In one embodiment, indicators of erythrocyte-like cell homeostasis include, but are not limited to, hematocrit (HCT), hemoglobin (HB), mean corpuscular hemoglobin (MCH), red blood cell count (RBC), reticulocyte count, reticulocyte percentage, mean corpuscular volume (MCV), and red blood cell distribution width (RDW). In another embodiment, indicators of kidney function include, but are not limited to, serum albumin, albumin / globulin ratio (A / G ratio), serum phosphorus, serum sodium, kidney size (measurable by ultrasound), serum calcium, phosphorus / calcium ratio, serum potassium, proteinuria, urinary creatinine, serum creatinine, blood urea nitrogen (BUN), cholesterol levels, triglyceride levels, and glomerular filtration rate (GFR). Furthermore, several indicators of general health and well-being include, but are not limited to, weight gain or loss, survival, blood pressure (mean general blood pressure, diastolic or systolic), and physical endurance performance.

[0168] In another embodiment, the effectiveness of treatment using SRC+ cell populations or bioactive renal cell organoids is demonstrated by stabilizing one or more indicators of renal function. Stabilization of renal function is demonstrated by observing changes in indicators in subjects treated with the methods of the present invention, compared to the same indicators in subjects not treated with the methods of the present invention. Alternatively, stabilization of renal function can be demonstrated by observing changes in indicators in subjects treated with the methods of the present invention, compared to the same indicators in the same subjects before treatment. Changes in the first indicator may be an increase or decrease in value. In one embodiment, the treatment provided by the present invention may include stabilizing blood urea nitrogen (BUN) levels in the subject, wherein the BUN levels observed in the subject are lower than those observed in subjects with a similar disease state who have not been treated with the methods of the present invention. In another embodiment, the treatment may include stabilizing serum creatinine levels in the subject, wherein the serum creatinine levels observed in the subject are lower than those observed in subjects with a similar disease state who have not been treated with the methods of the present invention. In another embodiment, the treatment may include stabilizing hematocrit (HCT) levels in the subject, wherein the HCT levels observed in the subject are higher than those observed in subjects with a similar disease state who have not been treated with the methods of the present invention. In another embodiment, treatment may include stabilizing red blood cell (RBC) levels in the subject, wherein the RBC levels observed in the subject are higher than those in subjects with a similar disease state who have not been treated with the methods of the present invention. Those skilled in the art will appreciate that one or more additional indicators described herein or known in the art may be measured to determine an effective treatment for kidney disease in the subject.

[0169] Application methods and routes

[0170] The SRC+ cell populations and / or bioactive organoids of the present invention can be administered alone or in combination with other bioactive components. The SRC+ cell populations and / or organoids are suitable for injecting or implanting combined tissue-engineered elements into the interior of solid organs to regenerate tissue.

[0171] On one hand, the present invention provides a method for providing bioactive cell organoids as described herein to subjects in need. In one embodiment, the bioactive cells may be derived from autologous or allogeneic, syngeneic (autogenous or syngeneic), and any combination thereof. Where the source is not autologous, the method may include administration of an immunosuppressant. Suitable immunosuppressant drugs include, but are not limited to, azathioprine, cyclophosphamide, imidazolidin, cyclosporine, tacrolimus hydrate, chlorambucil, chlorbenzalofin, auranofen, alprostadil, guanitolimus hydrochloride, biosynsorb, muromonab, alefacept, pentostatin, daclizumab, sirolimus, mycophenolate mofetil, leflunomide. lonomide, basiliximab, streptokinase alpha, bindarid, cladribine, pimecrolimus, ilodecakin, cedelizumab, efalizumab, everolimus, anisperimus, gavilimomab, faralimomab, chloramphenicol Clofarabine, Rapamycin, Siplizumab, Saireito, LDP-03, CD4, SR-43551, SK&F-106615, IDEC-114, IDEC-131, FTY-720, TSK-204, LF-080299, A-86281, A-802715, GVH-313, HMR-1279, ZD-7349, IPL-423323, CBP-1011, MT- 1345, CNI-1493, CBP-2011, J-695, LJP-920, L-732531, ABX-RB2, AP-1903, IDPS, BMS-205820, BMS-224818, CTLA4-1g, ER-49890, ER-38925, ISAtx-247, RDP-58, PNU-156804, LJP-1082, TMC-95A, TV-4710, PTR-262-MG, and AGI-1096 (see U.S. Patent No. 7,563,822). Other suitable immunosuppressant drugs will be apparent to those skilled in the art.

[0172] The therapeutic methods of the present invention involve delivering the SRC+ cell populations and / or organoids described herein. In one embodiment, direct application of the cells to the site of intended benefit is preferred.

[0173] In view of this specification, various methods of administering organoids to subjects will be apparent to those skilled in the art. These methods include, but are not limited to, intraparenchymal injection, subcapsular placement, transurethral catheter insertion, and intrarenal artery catheter insertion.

[0174] Cells can be inserted into a delivery device or medium that facilitates introduction by injection or implantation into a subject. In some embodiments, the delivery medium may comprise a natural material. In some embodiments, the delivery medium may comprise a synthetic material. In one embodiment, the delivery medium provides a structure that mimics or precisely conforms to organ structure. In other embodiments, the delivery medium is essentially fluid-like. Such delivery devices may include tubes, such as catheters, for injecting cells and fluids into the body of a recipient subject. In a preferred embodiment, the tube additionally has a needle (e.g., a syringe) through which the cells of the present invention can be introduced into the subject at a desired location. In some embodiments, a cell population derived from mammalian kidneys is formulated for administration into a blood vessel via a catheter (wherein the term "catheter" is intended to include any of the various tubular systems used for delivering substances into a blood vessel). Alternatively, cells can be inserted into or onto biomaterials or scaffolds, including but not limited to textiles such as woven fabrics, knitted fabrics, braided fabrics, and mesh fabrics; nonwovens, perforated membranes, sponges, and foams; and beads such as solid or porous beads, microparticles, nanoparticles, etc. (e.g., Cultispher-S gelatin beads - Sigma). Cells can be prepared for delivery in a variety of different forms. For example, cells can be suspended in solutions or gels. Cells can be mixed with pharmaceutically acceptable carriers or diluents in which the cells of the present invention remain viable. Pharmaceutically acceptable carriers and diluents include saline, buffered aqueous solutions, solvents, and / or dispersion media. The use of such carriers and diluents is well known in the art. The solution is preferably sterile and fluid, and will generally be isotonic. Preferably, the solution is stable under the conditions of manufacture and storage and is preserved against contamination by microorganisms such as bacteria and fungi using, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. Those skilled in the art will understand that the delivery medium used in delivering the cell populations and mixtures thereof of the present invention may include a combination of the features mentioned above.

[0175] Administration modalities for organoids containing isolated renal cell populations (e.g., isolated B2 cell populations or a mixture with B4' and / or B3), and / or formed therefrom, include, but are not limited to, intraparenchymal injection, subcapsular placement, or renal artery delivery. Additional administration modalities to be used according to the invention include single or multiple injections via direct laparotomy, direct laparoscopy, or transabdominal or percutaneous delivery. Further additional administration modalities to be used according to the invention include, for example, retrograde infusion and ureteropelvic infusion. Surgical means of administration include one-step procedures such as, but not limited to, partial nephrectomy and construct implantation, partial nephrectomy, partial pyelectomy, omental ± peritoneal vascularization, multifocal biopsy needle tracing of a cone or pyramid to a cylinder, and renal columnar replacement; and two-step procedures including, for example, an organoid internal bioreactor for re-transplantation. In one embodiment, organoids containing a cell mixture and / or composed of a cell mixture are delivered simultaneously via the same route. In another embodiment, each organoid is delivered individually to a specific location or simultaneously or in a temporarily controlled manner via one or more methods described herein.

[0176] The appropriate cell or organoid implantation dose in humans can be determined from existing information on organoid activity (e.g., EPO production) or inferred from dosing studies conducted in preclinical research. The amount of organoids can be quantified from in vitro cultures and in vivo animal experiments, and this can be used to calculate the appropriate dose of implanted material. Furthermore, patients can be monitored to determine whether additional implantations can be performed or, accordingly, whether the amount of implanted material can be reduced.

[0177] One or more other components may be added to the cell populations and mixtures thereof comprising the present invention and / or organoids composed thereof, including selected extracellular matrix components such as one or more types of collagen or hyaluronic acid, growth factors, and / or cytokines (including but not limited to VEGF, PDGF, TGFβ, FGF, IGF, platelet-rich plasma, and drugs).

[0178] Those skilled in the art will understand the various administration methods suitable for the organoids described herein.

[0179] Products and reagent kits

[0180] The present invention also includes a kit comprising the polymer matrix and scaffold of the present invention and related materials and / or cell culture medium and instructions for use. Instructions for use may include, for example, instructions for culturing cells during the formation of SRC+ cell populations or organoids of the present invention and / or instructions for administering SRC+ cell populations or organoids. In one embodiment, the present invention provides a kit comprising the scaffold described herein and instructions for use. In yet another embodiment, the kit comprises reagents for detecting marker expression, reagents for using said reagents, and instructions for use. This kit can be used for the purpose of determining the regenerative prognosis of natural kidneys in subjects after implantation or administration of the organoids described herein. The kit can also be used to determine the biotherapeutic efficacy of the organoids described herein.

[0181] Another embodiment of the invention is an article containing organoids, said organoids containing and / or formed from bioactive cells that can be used to treat a subject in need. The article includes a container and a label or instruction manual on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. Containers can be formed from various materials such as glass or plastic. The container contains a composition effective for treating a condition and may have a sterile access port (e.g., the container may be a solution bag or a vial with a stop that can be punctured by an injection needle). The label or instruction manual indicates that the organoid is used to treat a specific condition. The label or instruction manual will also include instructions for administering the organoid to a patient. Articles and kits containing combination therapies described herein are also covered. Instructions for use refer to instructions customarily included in the commercial packaging of a therapeutic product, containing information about indications, usage, dosage, administration, contraindications, and / or warnings regarding the use of said therapeutic product. In one embodiment, the instruction manual indicates that the organoid is used to treat a disease or condition, such as kidney disease or condition. It may also include other materials desirable from a commercial and user perspective, including additional buffers, diluents, filters, needles, and syringes. Kits useful for various purposes, such as evaluating regeneration results, are also provided. Kits containing detection agents for urinary vesicles and / or their contents, such as nucleic acids (e.g., miRNAs), vesicles, exosomes, etc., as described herein, may be provided. Detection agents include, but are not limited to, nucleic acid primers and probes, and antibodies for in vitro detection of desired targets. Like the product, the kit includes a container and a label or package insert on or associated with the container. The container holds a composition containing at least one detection agent. Additional containers may be included, containing, for example, diluents and buffers or control detection agents. The label or package insert provides a description of the composition and instructions for intended in vitro, prognostic, or diagnostic use.

[0182] The present invention also relates to the following embodiments: 1. A method for forming an organoid comprising a population of xenogeneic kidney cells and a population of bioactive cells, the method comprising culturing the xenogeneic kidney cells and the population of bioactive cells in a culture system selected from: i) 2D culture; ii) 3D culture: COL(I) gel; iii) 3D culture: Matrigel; iv) 3D culture: rotator followed by COL(I) / Matrigel; and v) 3D culture: COL(IV) gel.

[0183] 2. The method according to embodiment 1, wherein the xenogeneic kidney cell population comprises a bioactive kidney cell population.

[0184] 3. The method according to embodiment 1, wherein the xenogeneic kidney cell population comprises a B2 cell population containing an enriched population of renal tubular cells, and wherein the xenogeneic kidney cell population discards the B1 cell population.

[0185] 4. The method according to embodiment 3, wherein the xenogeneic kidney cell population further discards the B5 cell population.

[0186] 5. The method according to embodiment 1, wherein the xenogeneic kidney cell population comprises a cell population selected from B2, B2 / B3, B2 / B4 and B2 / B3 / B4.

[0187] 6. The method according to embodiment 1, wherein the xenogeneic kidney cell population comprises cells that produce erythropoietin (EPO).

[0188] 7. The method according to embodiment 1, wherein the bioactive cell population is an endothelial cell population.

[0189] 8. The method according to embodiment 7, wherein the endothelial cell population is a cell line.

[0190] 9. The method according to embodiment 7, wherein the endothelial cell population comprises HUVEC cells.

[0191] 10. The method according to embodiment 1, wherein the cell population is selected from heterogeneous, homogeneous, allogeneic, autologous, and combinations thereof.

[0192] 11. The method according to embodiment 1, wherein the xenogeneic kidney cell population and the bioactive cell population are cultured separately for a first time period, and then combined and cultured for a second time period.

[0193] 12. The method according to implementation scheme 11, wherein the second time period is at least 24 hours.

[0194] 13. The method according to implementation scheme 12, wherein the second time period is 24 hours to 72 hours.

[0195] 14. The method according to embodiment 1, wherein the ratio of the renal cell population to the bioactive cell population is 1:1.

[0196] 15. The method according to embodiment 1, wherein the kidney cell population and the bioactive cell population are suspended in a growth medium.

[0197] 16. An organoid prepared according to any one of embodiments 1 to 15.

[0198] 17. An organoid comprising a population of xenogeneic kidney cells and a population of biologically active cells.

[0199] 18. The organoid according to embodiment 17, wherein the bioactive cell population is an endothelial cell population.

[0200] 19. The organoid according to embodiment 18, wherein the xenogeneic kidney cell population comprises a B2 cell population containing an enriched population of renal tubular cells, and wherein the xenogeneic kidney cell population discards the B1 cell population.

[0201] 20. The organoid according to embodiment 19, wherein the xenogeneic kidney cell population further discards the B5 cell population.

[0202] 21. The organoid according to embodiment 17, wherein the xenogeneic kidney cell population comprises cell populations selected from B2 / B3, B2 / B4, and B2 / B3 / B4.

[0203] 22. The organoid according to embodiment 17, wherein the xenogeneic kidney cell population comprises cells that produce erythropoietin (EPO).

[0204] 23. The organoid according to embodiment 18, wherein the endothelial cell population is a cell line.

[0205] 24. The organoid according to embodiment 18, wherein the endothelial cell population comprises HUVEC cells.

[0206] 25. An injectable formulation comprising at least one organoid and a liquid culture medium.

[0207] 26. The formulation according to embodiment 25, wherein the liquid culture medium is selected from cell growth media, DPBS, and combinations thereof.

[0208] 27. The formulation according to embodiment 26, wherein the liquid culture medium is DPBS.

[0209] 28. The formulation according to embodiment 25, wherein the organoid is suspended in the liquid culture medium.

[0210] 29. An injectable formulation comprising an organoid and a temperature-sensitive cell-stabilizing biomaterial, said cell-stabilizing biomaterial

[0211] (i) Remains substantially solid at or below about 8°C, and

[0212] (ii) Remains substantially liquid at or above ambient temperature.

[0213] 30. The formulation according to embodiment 29, wherein the organoid comprises bioactive kidney cells.

[0214] 31. The formulation according to embodiment 30, wherein the organoid further comprises HUVEC.

[0215] 32. The formulation according to embodiment 29, wherein the bioactive cells are substantially uniformly dispersed throughout the entire volume of the cell-stabilizing biomaterial.

[0216] 33. The formulation according to embodiment 29, wherein the biomaterial comprises a solid-to-liquid transition state between about 8°C and about ambient temperature or above.

[0217] 34. The formulation according to embodiment 29, wherein the substantially solid state is a gel state.

[0218] 35. The formulation according to embodiment 29, wherein the cell-stabilizing biomaterial comprises a hydrogel.

[0219] 36. The formulation according to embodiment 35, wherein the hydrogel comprises gelatin.

[0220] 37. The formulation according to embodiment 36, wherein the gelatin is present in the formulation at about 0.5% to about 1% (w / v).

[0221] 38. The formulation according to embodiment 36, wherein the gelatin is present in the formulation at about 0.75% (w / v).

[0222] 39. A method for treating a subject with kidney disease, comprising administering at least one organoid comprising a population of xenogeneic kidney cells and a population of bioactive cells.

[0223] 40. The method according to embodiment 39, wherein the bioactive cell population is an endothelial cell population.

[0224] 41. The method according to embodiment 39, wherein the xenogeneic kidney cell population comprises a B2 cell population containing an enriched population of renal tubular cells, and wherein the xenogeneic kidney cell population discards the B1 cell population.

[0225] 42. The method according to embodiment 41, wherein the xenogeneic kidney cell population further discards the B5 cell population.

[0226] 43. The method according to embodiment 39, wherein the xenogeneic kidney cell population comprises a cell population selected from B2, B2 / B3, B2 / B4 and B2 / B3 / B4.

[0227] 44. The method according to embodiment 39, wherein the xenogeneic kidney cell population comprises cells that produce erythropoietin (EPO).

[0228] 45. The method according to embodiment 40, wherein the endothelial cell population is a cell line.

[0229] 46. ​​The method according to embodiment 40, wherein the endothelial cell population comprises HUVEC cells.

[0230] 47. A method of treating a subject with kidney disease, comprising administering an injectable formulation according to any one of embodiments 25-38.

[0231] 48. The method according to any one of embodiments 39-46, wherein the subject is a mammal.

[0232] 49. The method according to embodiment 48, wherein the mammal is a human.

[0233] 50. The method according to any one of embodiments 39-46, wherein the subject suffers from kidney disease.

[0234] 51. The method according to any one of embodiments 39-46, wherein improvement in any of the following measures of disease is observed: anemia (Hct, Hgb, RBC), inflammation (WBC), urine concentration (spGrav), and azotemia (BUN).

[0235] 52. Use of organoids in the preparation of pharmaceuticals for the treatment of kidney diseases.

[0236] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0237] Example

[0238] Example 1 – Isolation of Selected Renal Cell+ (SRC+) Cell Population: Isolation of Endothelial Cells from Renal Biopsy

[0239] The following method provides a digestion protocol using a previously adapted collagenase / dispersant enzyme. 2，3，5 An example of isolating endothelial cells after enzymatic digestion. This method has been applied to kidneys from diseased ZSF1 rats and kidney biopsies obtained from dialysis-induced hypertension patients with ESRD.

[0240] Endothelial cells (ECs), blood vessels, and lymphatic vessels are sources of this material. Through 100 µm Steriflip Filter ( Millipore Kidneys were digested using the standard operating procedure for kidney cell isolation as described below. The remaining cell suspension was neutralized with DMEM containing 5% FBS, followed by washing by centrifugation at 300 x g for 5 minutes. The cell pellet recovered through a 100 µm filter was resuspended in fully replenished EGM-2 growth medium. Lonza In ), and at 25K / cm 2 The cells were seeded at a density suitable for fibronectin coating onto culture dishes. The culture was fed every 3 days. When the EC cultures reached 80-90% confluence, they were trypsinized and counted. Trypsinized cells were labeled with CD31 (PECAM) primary antibody and then... Miltenyi Positive selection was performed using anti-CD31 microbeads. CD31... + The sorted cells were washed, counted, plated, and sprayed at 10 K / cm². 2 The cells were seeded at a density in fully supplemented EGM-2 medium. After 24 hours, the morphology of the endothelial cells should be clearly visible, and these cells can be expanded through multiple passages. Vascular and / or lymphatic vessel compositions can be analyzed using lineage-specific antibodies (e.g., VEGF3 – vascular capillaries; LYVE1 – lymphatic vessels); the same cell surface phenotypic markers can be used. Miltenyi Microbead selection methods can be used to further select / sort specific endothelial subgroups.

[0241] Following amplification and purification, the endothelial cell components of NKA SRCs can be enriched to a greater percentage (>30%) than the intrinsic frequency observed by buoyancy density gradient separation (<2%). In one embodiment, for the control of the active biological components or compositions of NKA, purified ECs can be combined with the previously described B2 and / or B2-B3-B4 fractions (e.g., 60% B2-B4 + 40% EC) at a selected frequency prior to transplantation.

[0242] Endothelial cell sorting using magnetic microbeads: Cells harvested from fibronectin-coated flasks in EGM2 medium were stained with mouse anti-human CD31 primary antibody at a concentration of 0.4 µg / million cells / 100 µl in supplement-free EGM2 medium at 4°C for 20 min. After 20 min, the cells were washed and resuspended in 12 ml of supplement-free EGM2 medium and 200 µl of goat anti-mouse IgG1. Miltenyi microbeads were added and the cells were incubated at 4°C without light for another 20 min. After 20 min, the cells were washed twice by centrifugation (5 min, 300 x g) and resuspended in 12 ml [10 million cells / ml] and purified using a Miltenyi auto-MACS instrument (double-positive selection in sensitive mode).

[0243] In the embodiment described in Figure 1, endothelial cells were selected (CD31+ selected) from primary ZSF1 cells. 3.6 million cells recovered from this process were counted and passaged in two T175 fibronectin-coated flasks at a concentration of 10 K / cm². The cells were cultured at 21% O² for 4 days and harvested at 85% confluence. After this selection and culture period, 9.5 million cells were counted and recovered. Samples collected for FACS phenotypic analysis were approximately 90% CD31 positive (CD31+). Figure 1E (FACS right figure).

[0244] Isolation, characterization and expansion of human CKD endothelial cells

[0245] Human kidney cell culture: Human kidney cells were isolated using the standard operating procedure for enzyme isolation and culture of primary human kidney cells as described below (donor information is shown in Table 1 below). In short, the cells were isolated and cultured at 25 K / cm². 2 Cells were cultured in T / C-treated flasks containing standard KGM or in fibronectin-coated flasks containing EGM2-completely replenished medium. Cells were grown at 21% oxygen for 3 days, then the medium was changed and the cultures were transferred to 2% oxygen for O / N exposure. Cells were imaged after 4 days for culture morphology analysis. Figure 2A Then harvest them and count them using standard methods (see HK027 BatchRecord). Collect samples and stain them to determine CD31. + Percentage of endothelial cells (Figure 2). Endothelial cells were sorted using mouse anti-human CD31 primary antibody (BD biosciences) and magnetic beads (Miltenyi). Then CD31... + Cells at 10K / cm 2The cells were re-seeded at a density similar to fibronectin-coated flasks and cultured for another 4 days (Figure 2, cell culture morphology). Cells were then counted and samples were collected to determine the percentage of positive endothelial cells using mouse anti-human CD31 primary and FACS analysis (Figure 3).

[0246] Table 1. Human kidney donor information:

[0247] In summary, primary cultures of diseased ZSF1 cells on fibronectin produced 9% CD31. + Culture (TC-treated <2%). 9% CD31 subcultured on p1 fibronectin. + The fractionation produced approximately 90% of CD31. + Cells expanded nearly 3-fold from p0 to p1. Primary cultures of human CKD cells in T / C-treated or fibronectin-coated flasks produced approximately the same percentage of CD31-positive cells (2.1% vs. 2.7%). As shown in the figure, subculturing positively selected CD31-positive cells in fully supplemented EGM2 medium on fibronectin-coated flasks was possible, and the percentage of CD31-positive cells increased 13-fold after one generation (from 2.7% to 35.5%).

[0248] Isolation of SRC+ cell population: Examples of isolation of other bioactive cell types: A. Kidney epithelial cells Ongoing assessments utilize representations of various epithelial cell compartments (parietal, proximal tubule, loop of Henle, distal tubular cells) in fractions separated by buoyancy density gradients. More specifically, lineage tracing studies provide Six2 + Direct and selective confirmation of epithelial cell isolation; cell-specific detection was confirmed by co-labeling Six2 with markers specific to nephron epithelial compartments (see Tables 2-3) (see Table 4). Table 4 provides a limited set of epithelial markers characterizing a mixture of p0 ZSF1 cultures (combined fractions B2-B3-B4) representing a mixture of proximal tube, distal tube, and collecting duct cell types. Enrichment of these specialized epithelial cell compartments is expected through culture selection (culture conditions and magnetic sorting) as previously described, using the same cell surface detection antibodies listed in Tables 2-4.

[0249] Table 2. Marker set for tissue-specific nephron staining

[0250] Pos = Positive, Pos-ind = Positive cells in the selected cell population, Med-ind = Positive staining of the collecting duct of the medulla.

[0251] Table 3. Antibody Sources

[0252]

[0253] The secondary antibodies consisted of the Alexa 647 molecular probe (goat anti-mouse IgG, goat anti-rabbit IgG, and donkey anti-goat IgG) and streptavidin A647. The dosage was 1 μg / ml / 1x10⁻¹. 6 Staining cells according to the following SOP

[0254] Table 4. Preliminary ZSF1 p0 cell-specific detection

[0255] B. Cells derived from the glomerulus (parietal epithelial cells and podocytes) Anatomical obstruction of the pelvis improved the separation of glomerular fractions. Cells were separated using the standard operating procedure for renal digestion described below and passed through 100µm... Steri- flip Filter ( Millipore Filter and re-digest cell particles / clumps larger than 100µm (containing glomerular fractions) for an additional 20 minutes. Neutralize the digested fractions with DMEM medium containing 10% FBS and wash by centrifugation (300xg for 5 minutes). Resuspend the cell pellet in VRADD medium. 6 (DMEM / F12, 1uM all-trans retinoic acid) Genzyme Cambridge, MA), 0.1µm dexamethasone Sigma-Aldrich Cells were cultured in 10-100 nM vitamin D (1,25(OH)2D3) to promote podocyte culture, or in a parietal epithelium-friendly serum-free medium, such as (50:50 DMEM / KSFM) completely supplemented without FBS, on collagen type 1 coated T / C plates. Cells were cultured at 25 K / cm². 2 The cells were seeded at a high density and subcultured until cell growth occurred. This was used in the first generation and... Miltenyi The microbead-bound primary antibody is used to expand and sort the grown cells using PEC-specific markers (such as, but not limited to, tight junction protein-1), progenitor-specific markers (e.g., CD146, CD117, SOX2, Oct 4A, CD24, CD133), mesangial-specific markers (such as, but not limited to, smooth muscle actin, vimentin, cardiomyin, calcium-binding protein), or glomerular capillary endothelial cells (e.g., VEFG3, CD31, or LIVE1). A higher proportion of glomerular-derived cells can be used in patients with glomerular disease when harvested at optimal cell yield and combined with the B2 component at a higher than intrinsic frequency.

[0256] C. Collecting duct epithelial cells Anatomical exclusion of the cortical and medullary regions of the kidney improves the isolation of cells located at or near the pelvis. Isolation and expansion of collecting duct epithelial cells followed the standard operating procedures described below for the isolation and expansion of primary kidney cells from rodents, dogs, and humans. The standard operating procedure for grading the separation of primary cultured kidney cell subpopulations enriched with collecting duct cells in cell fraction B1 is described below. These cells contain the highest percentage of collecting duct epithelial cells based on markers such as aquaporin 2 and digoxin (DBA). Alternatively, we adapted the papillary / inner medullary collecting duct (IMCD) culture protocol from Dr. Ben Humphreys (REGM medium supplemented with EGF; unpublished data). In any of these cases, the cell fraction enriched from collecting duct epithelial cells can be used in combination with the B2 fraction at a higher than intrinsic frequency, or amplified using standard KGM or equivalents and used to target disease etiologies associated with urine concentration, and can better demonstrate the bioactive components applicable to renal pelvis abnormalities and / or diseases (e.g., hydronephrosis, ureteral obstruction).

[0257] Example 2 – Generation and characterization of organoids from SRC and SRC+ groups

[0258] Self-generated organoid / spheroid formation

[0259] Organoids were generated from primary kidney culture, expansion, and buoyancy density gradient centrifugation to isolate SRCs (as described below in the standard operating procedure for NKA generation). In short, SRCs were centrifuged at [1 x 10⁻⁶]⁻¹. 6 The cells were resuspended at a concentration of [cells / ml] in 100 ml of kidney cell growth medium in a rotary flask (cornering) set to 80 rpm for 24–48 hours (Figure 4). Cellular organoids / spheres consisted of cell clusters ranging in size from 50–125 µM. The number of cells per organoid could vary based on the cell type and size prior to rotary flask culture. Organoids / spheres were derived from rat and human SRCs and expressed a renal tubular epithelial phenotype (Figure 5).

[0260] Organoid function and renal tubule development

[0261] The ability of SRCs to form renal tubules can be used to assess the potency of NKA. This assay can be applied to SRC organoids cultured for 3D in a 50:50 collagen 1 / collagen IV gelatin mixture. When the cultured organoids are stained with immunofluorescence, the resulting renal tubules continue to express the epithelial phenotype (Figure 6).

[0262] organoids+

[0263] When combined with other cell types, the ability of SRCs to form self-generating spheroids has proven advantageous. Adding vascular or stem cell components can enhance renal tubular development. By adding selective cell populations to cultures containing SRC populations during organoid formation, functional units can be formed, reproducing key cell signaling pathways activated during regeneration. Examples of such cell-cell interactions include, but are not limited to, epithelial-mesenchymal signaling events known to play a crucial role during organogenesis (see...). Basu and Ludlow 2012, Developmental engineering the kidney-leveraging morphogenic principles for renal regeneration. Birth Defects Research Part C 96:30-38 Although SRCs were generated using standard procedures, the epithelial cell line (HuVEC) was used as an example of organoid (+) consortia. Eighty million SRCs were labeled with a membrane dye (Invitrogen DiL red label) and added to 20 million HuVECs labeled with a different color membrane dye (Invitrogen DiO green label) in 100 ml of RCGM medium in a 125 ml rotary flask at 80 rpm for 48 hours (Figure 7). Individual SRC organoid populations were also started as controls. When organoids formed, in vitro tubulogenesis assays were performed in Col I / IV 3D gels to ensure the ability to form tubules with and without potential concomitant vascularization (Figure 8).

[0264] Example 3 Characterization / Biodistribution of SRC organoids in rodent kidney disease models

[0265] An acute study of ZSF1 evaluated the biodistribution and cell survival of SPIO-labeled organoids over a 48-hour period. (2.5 x 10⁻⁶ cells were used.) 6 One SPIO rhodamine-labeled cell (representing approximately 25,000 spontaneously generated organoids in PBS) at 50x10 6 A concentration of [value] ml was injected into the parenchyma at the left tail pole of six 40+ week old ZSF1 rats. Figure 9 Three animals were harvested at 24-hour and 48-hour intervals post-transplantation. Cell survival and biodistribution in the kidneys were assessed by MRI and histologically using Prussian blue and H&E staining methods (Figs. 10 and 11).

[0266] result

[0267] Organoids delivered along the target were easily labeled and traced. Organoid therapy was well tolerated, and no morphological changes were observed in the renal tubules or glomerular compartments. Frequent clusters of epithelioid cells (by Prussian blue positive staining) were observed in the renal cortex of the left kidney (intratubular / intertubular) 24 hours after injection, and their intensity decreased after 48 hours.

[0268] Example 4– In vivo studies to demonstrate the therapeutic efficacy of SRC+ and SRC / SRC+ derived organoids: CKD (5 / 6 nephrectomy) immunocompromised rodent studies.

[0269] The therapeutic efficacy of human-derived SRC+ and SRC / SRC+ organoids was evaluated using a 5 / 6-nephrectomy chronic kidney disease model in immunocompromised rodents (NIHRNU (naked) rats; athymic rats). Disease state establishment, treatment interventions, and clinical assessment of treatment response were as previously described (…). Genheimer et al., 2012. Molecular characterization of the regenerative response induced by intrarenal transplantation of selected renal cells in rodent model of chronic kidney disease. Cells Tissues Organs 196: 374-84 ).

[0270] Cell isolation and selection: Human kidney cells were isolated and expanded from the biopsy as described in Presnell (2011). Cells were subcultured and, after the second passage, selected at a density of (20 x 10⁻⁶) cells. 6 Cells were stored at a concentration of 1000 cells / ml / flask in cryogenic buffer (80% HTS / 10% DMSO / 10% FBS) and cooled to -80°C at a freezing rate of -1°C / min, then transferred to a liquid nitrogen freezer for long-term storage. After cryogenic storage, the cells were rapidly thawed at 37°C to assess recovery and viability using the standard trypan blue exclusion method. The cells were then stored at 3000 cells / ml / flask. 2 Cells were seeded onto tissue culture vessels and cultured in renal cell growth medium under normal oxygen conditions (21% O2 / 5% CO2 / 37°C) for 4 days. After 4 days, the medium was changed, and the cultures were incubated overnight under hypoxic conditions (2% O2 / 5% CO2 / 37°C). Cells were then harvested and SRCs were selected by density gradient separation. After cryopreservation, human umbilical vein endothelial cell (HUVEC) cultures (Lonza 2389, catalog # CC2517) were expanded from early passages to passage 3 in EGM2 fully supplemented medium. Cells were harvested, and viability was assessed using the standard trypan blue exclusion method.

[0271] Organoid formation: Organoid cultures were established by: 1) incorporating human SRC and HUVEC at a ratio of 2 x 10⁻⁶. 6 2) Resuspend the cells at a concentration of 10 cells / ml in their respective culture media. 6 Each cell preparation (cells / ml) is combined into a 125ml rotary flask (Corning). For combined cultures, 25ml of each culture medium is added to the flask to achieve 1x10 cells / ml. 6 The final cell concentration was [number of cells / ml]. The flask was placed in an incubator with a magnetic stirrer at 80 RPM and incubated for 24 hours.

[0272] Test items: The dosage of organoids was adjusted so that approximately (2-5 x 10) was administered to each rat kidney. 6 (1 cell / 50µl). Adjust the number of organoids to the above concentration in a larger volume of 0.5mL DPBS to compensate for any loss during shipment. Transfer an additional tube as a replacement. At 4–8°C, use a CREDO tube delivery system to deliver these doses via FEDEX to two 0.5mL microcentrifuge tubes for the implantation device.

[0273] Delivery of the test item: The remaining left kidney was assessed via a left ventral incision. Prior to delivery, the organoid was gently resuspended by tapping or flicking the bottom of the tube (without vortexing). The organoid was aspirated into a syringe using an 18G blunt-tipped needle. The needle was then replaced with a 23G cutting needle for delivery. Targeted delivery was performed by injecting 50 µL into the cortical-medullary region of the kidney. Untreated control animals remained untreated and did not undergo the procedure.

[0274] RKM model procedure: Male NIHRNU rats aged 8-12 weeks (average weight approximately 200 g) underwent the following two-step nephrectomy procedure: For each individual animal, their right kidney was removed and weighed, allowing for a one-week recovery period. Then, the tail and head poles of the left kidney were removed, and the left kidney was also weighed. A 2-3 week recovery period followed by an acclimatization period, after which the study began. After the acclimatization period, blood and urine were collected and analyzed for two consecutive weeks. Subsequent sampling was performed every two weeks thereafter. All rodents were fed commercially available feed and water was provided freely.

[0275] Animals were observed to have some signs of increased urinary protein / urinary protein creatinine (UPC) after nephrectomy, and human organs were used to treat the animals 12 weeks after nephrectomy. Table 5 below provides an overview of the study.

[0276] Table 5: Research Design

[0277] Measurements: Body weight (g) was collected every two weeks. Serum biochemical and hematological assessments were performed every two weeks during the study period. Urinalysis was performed every two weeks. At the scheduled necropsy, the study animals were observed, the remaining kidneys were removed and fixed, and the animals were kept in formalin. All animal and tissue samples were collected, weighed, measured, and prepared for histological processing as needed. The methods used in the histological assessment of the kidneys were performed. KMR was defined as kidney mass reduction.

[0278] result: The efficacy of human NKO was confirmed in a rat model of renal insufficiency with athymia.

[0279] RKM Model Overview

[0280] In the athymic KMR model, disease progression is consistent with previously described nephrectomy models and CKD-related sequelae, including impaired renal protein processing, azotemia, hypercholesterolemia, anemia, and progressive uremia. The NIHRNU nephrectomy model's outcomes regarding disease status are sensitive to the amount of renal tissue removed during the period of quality reduction, and the animals used here, based on clinicopathological monitoring and final histological assessment, demonstrate a slowly developing early disease state.

[0281] The impact of NKO phenotype on in-life clinicopathology

[0282] Clinicopathological measures of renal function were examined prior to treatment and compared in pairs with those obtained before the end of the study. Based on this comparison, as summarized in the table below, changes consistent with disease progression were observed in the untreated control animals over a 4-month study. Significant differences were observed in paired BUN, Hct, Hgb, RBC, WBC, sChol, sProt, sAlb, uPro, UPC, and spGrav. Changes in these markers are characteristic of early CKD and indicate that the decline in renal function has not yet led to acute azotemia, late-stage loss of tubular filtration / concentration, and phosphate hyperphosphatemia. Changes in BUN, Hct, Hgb, RBC, WBC, and spGrav in animals treated with NKO No As expected based on results from untreated control animals (see Table 6 below).

[0283] Table 6. Clinical measurements of the treatment group before and after treatment

[0284]

[0285]

[0286] WBCs here will not respond to T cell-mediated responses because these animals do not have a thymus.

[0287] Histological overview of the kidney from the model

[0288] Pathological histology showed that the control animals had progressed to mild progressive kidney disease and were euthanized at the end stage.

[0289] The damage-related outcomes are essentially mainly tubular atrophy and dilation, and proteinaceous casts, which are usually mild in severity. Tubular basophilia and fibrosis are rarely observed, and interstitial inflammation is usually rare or absent. Glomerular severity changes are minimal.

[0290] The severity of cystic fibrosis / inflammation was generally very low and characterized by minimal inflammatory cellular contents; the response may have been weakened by the incomplete immune system in athymic nude rats. In the first batch of animals, the incidence and severity of mineralization and lymphocytic infiltration were low. Focal mineralization, common in laboratory rats, was occasionally observed.

[0291] No significant differences were observed between the untreated group and the NKO-treated group.

[0292] Table 7

[0293] The impact of NKO on survival rate

[0294] All animals (59% KMR) survived to the end of the study (119 days after treatment and 204 days after nephrectomy), therefore no identifiable survival benefit was detected from NKO treatment.

[0295] Transplantation of human NKO using staining methods in EOS

[0296] Because this model allows for xenogeneic application of human products, it is possible to trace human cells in rat kidneys using human cell recognition antibodies. Four months post-treatment, human HLA1 staining was performed against a background of cells identified in RKM rat kidneys (see Figure 12). Two independent reviewers confirmed the identification of staining considered consistent with human cell transplantation. Although a cluster was identified within the interstitial tissue, typically one or two cells were identified within one of four stained sections for each animal and bound to the renal tubules.

[0297] in conclusion: In control animals, delivery of organoids (NKOs) mitigated significant changes associated with kidney disease progression and included measures achieved by delivering homologous / autologous NKAs to the disease model.

[0298] The achieved measures are consistent with those observed when homologous / autologous NKA is delivered to disease models – measures such as anemia (Hct, Hgb, RBC), inflammation (WBC), urine concentration (spGrav), and possibly azotemia (BUN).

[0299] Approximately four months after treatment with the NKO prototype, a very low number of human cells were detected.

[0300] Nephrectomy in nude rats (mean 59% KMR) resulted in an early stage of renal insufficiency and chronic progressive renal failure, characterized by possible indications of developmental anemia, proteinuria, dyslipidemia, and early azotemia.

[0301] All animals survived until the end of the study.

[0302] The procedure used to deliver human organs to the RKM model is well tolerated.

[0303] Example 5 – Isolation and Characterization of Bioresponsive Kidney Cells

[0304] Adult male wild boars suffering from idiopathic progressive chronic kidney disease (CKD) and anemia. Sus scrofa In this case, fresh diseased kidney tissue was provided and directly compared with age-matched normal pig kidney tissue for assessment of cellular composition and characterization. Histological examination of the kidney tissue at harvest confirmed kidney disease characterized by severe diffuse chronic interstitial fibrosis and crescentic glomerulonephritis with multifocal fibrosis. Clinical chemistry confirmed azotemia (elevated serum urinary nitrogen and serum creatinine) and mild anemia (mildly decreased hematocrit and decreased hemoglobin levels). Cells were isolated, expanded, and characterized from both diseased and normal kidney tissues. As shown in Figure 1 of Presnell et al. WO / 2010 / 056328 (incorporated hereinously by reference), Gomori's trichrome staining highlights fibrosis in the diseased kidney tissue compared to normal kidney tissue (blue staining indicated by arrows). Functional renal tubular cells expressing cupulin:macroprotein and capable of receptor-mediated albumin transport proliferated from both normal and diseased kidney tissues. Cells expressing erythropoietin (EPO) were also present in the culture and were preserved through multiple passages and freeze / thaw cycles. Furthermore, molecular analysis confirmed that EPO-expressing cells from both normal and diseased tissues were present in HIF1 cells. In vitro responses to hypoxia were investigated to drive the induction of EPO and other hypoxia-regulated target genes (including vEGF). Cells were isolated from porcine kidney tissue by enzymatic digestion with collagenase and dispersase, and also by simple mechanical digestion and explant culture in separate experiments. In a second generation, explant-derived cell cultures containing cells expressing EPO were subjected to atmospheric pressure (21%) and varying hypoxia (<5%) to determine whether exposure to hypoxia would peak the upregulation of EPO gene expression. As noted in rodent cultures (see Example 3), normal pigs exhibited oxygen-dependent expression and regulation of the EPO gene. Surprisingly, despite uremic / anemic conditions in CKD pigs (hematocrit <34, creatinine >9.0), EPO-expressing cells remained readily isolated from tissue and proliferated, and EPO gene expression remained hypoxia-regulated, as shown in Figure 2 of Presnell et al. WO / 2010 / 056328 (incorporated herein by reference in its entirety). As shown in Figure 3 of Presnell et al. (WO / 2010 / 056328, incorporated herein by reference in its entirety), cells in proliferating cultures exhibit the ability to self-organize into renal tubule-like structures. As shown in Figure 4 of Presnell et al. (WO / 2010 / 056328, incorporated herein by reference in its entirety), the presence of functional renal tubular cells in the culture (at passage 3) was confirmed by the observation of receptor-mediated uptake of FITC-coupled albumin in cultured cells. Green dots (indicated by thin white arrows) represent endocytosis of fluorescein-coupled albumin, mediated by renal tubule-specific receptors macroprotein and Cubilin, indicating protein reabsorption by functional renal tubular cells. Blue staining (indicated by thick white arrows) represents Hoescht-stained cell nuclei. In summary, these data demonstrate that functional renal tubular and endocrine cells can be isolated and proliferated from porcine kidney tissue, even from kidney tissue already severely damaged by CKD. Furthermore, these findings support the development of autologous cell-based therapeutic products for the treatment of CKD.

[0305] Furthermore, EPO-producing cells were enzymatically isolated from normal adult kidneys (as described in Example 1 above). As shown in Figure 5 of Presnell et al. WO / 2010 / 056328 (incorporated herein by reference in its entirety), the isolation procedure resulted in relatively higher EPO expression after isolation compared to the initial tissue. As shown in Figure 6 of Presnell et al. WO / 2010 / 056328 (incorporated herein by reference in its entirety), it is possible to maintain human EPO-producing cells in culture while preserving EPO gene expression. Human cells were cultured / proliferated on plastic treated with standard tissue culture methods or on plastic coated with some extracellular matrix (such as fibronectin or collagen), and all were found to support EPO expression over time.

[0306] Example 6 – Isolation and enrichment of specific bioactive kidney cells

[0307] Kidney cell isolation In summary, ten batches of kidneys from two-week-old male Lewis rats were obtained from a commercial supplier (Hilltop Lab Animals Inc.) and transported overnight in Viaspan preservation medium at approximately 4°C. All steps described herein were performed in a biosafety cabinet (BSC) to maintain sterility. The kidneys were washed three times with Hank's Balanced Salt Solution (HBSS) to flush out the Viaspan preservation medium. After the third wash, any remaining renal cysts and any remaining matrix tissue were removed. The major calyces of the kidneys were also removed using a microdissection technique. The kidneys were then finely minced into a paste using a sterile scalpel. The paste was then transferred to 50 ml conical centrifuge tubes and weighed. A small sample was collected for RNA and placed in sterile RNase-free 1.5 ml microcentrifuge tubes and rapidly frozen in liquid nitrogen. Once frozen, it was then transferred to a freezer at -80°C until analysis. The tissue weight of 10 juvenile kidneys was approximately 1 gram. Based on the batch weight, the digestion medium was adjusted to deliver 20 ml of digestion medium per 1 gram of tissue. The digestion buffer used in this procedure contains 4 units of dispersive enzyme 1 (Stem Cell Tech) in HBSS solution, 300 units / ml of collagenase type IV (Worthington) and 5 mM CaCl2 (Sigma).

[0308] Add an appropriate volume of preheated digestion buffer to the tube, then seal the tube and place it on a shaker in an incubator at 37°C for 20 minutes. This first digestion step removes many red blood cells and enhances the digestion of the remaining tissue. After 20 minutes, remove the tube and place it in BSC. Allow the tissue to settle to the bottom of the tube and then remove the supernatant. Then, add fresh digestion buffer equal to the initial volume to the remaining tissue. Place the tube back on a shaker in an incubator at 37°C for an additional 30 minutes.

[0309] After 30 minutes, the digestion mixture was aspirated through a 70µm cell filter (BD Falcon) into an equal volume of neutralization buffer (DMEM containing 10% FBS) to terminate the digestion reaction. The cell suspension was then washed by centrifugation at 300 x g for 5 minutes. After centrifugation, the pellet was resuspended in 20 ml of KSFM medium, and samples were obtained for cell counting and viability assessment using the trypan blue exclusion method. Once the cell count was calculated, 1 million cells were collected for RNA analysis, washed in PBS, and rapidly frozen in liquid nitrogen. The remaining cell suspension was brought to 50 ml using KSFM medium and washed again by centrifugation at 300 x g for 5 minutes. After washing, the cell pellet was resuspended at a concentration of 15 million cells / ml KSFM.

[0310] Add 5 ml of 30% (w / v) Optiprep to a 15 ml conical centrifuge tube (BD Falcon). ® Add 5 ml of kidney cell suspension and mix by inverting 6 times. This forms 15% (w / v) Optiprep. ® The final mixture was obtained. After inversion, the tube was carefully separated into layers using 1 mL PBS. The tube was centrifuged continuously at 800 xg for 15 minutes. After centrifugation, the tube was removed and a cell band formed at the top of the mixing gradient. A precipitate was also formed, containing red blood cells, dead cells, and a small population of viable cells, including some small granular cells, some EPO-producing cells, some renal tubular cells, and some endothelial cells. The band was carefully removed using a pipette and transferred to another 15 mL conical tube. The gradient medium was removed by aspiration and the precipitate was collected by resuspending it in 1 mL KSFM. The cells in the band and the precipitate were then reconstituted and resuspended in the collected band volume diluted at least 3 times with KSFM and washed by centrifugation at 300 xg for 5 minutes. After washing, the cells were resuspended in 20 mL KSFM and the sample was collected for cell counting. Once the cell count is calculated using the trypan blue exclusion method, one million cells are collected for RNA sampling, washed in PBS, and rapidly frozen in liquid nitrogen.

[0311] Density gradient separation was used to "clean" precultures to improve the viability and culture of specific bioactive kidney cells. performance To obtain a clean, viable cell population for culture, a cell suspension is first generated in the “Kidney Cell Isolation” process as described above. As an optional step and method for cleaning up the initial formulation, up to 100 million total cells suspended in sterile isotonic buffer are mixed with an equal volume of 30% Optiprep prepared at room temperature from 60% (w / v) stock iodine. ®(Thus obtaining the final 15% w / v Optiprep solution) Mix thoroughly at a 1:1 ratio and invert 6 times. After mixing, carefully layer 1 ml of PBS buffer onto the top layer of the mixed cell suspension. Then carefully load the gradient tube into the centrifuge, ensuring proper equilibration. Place the gradient tube in... Centrifuge continuously at 800 xg for 15 minutes at C. The cleaned cell population (containing viable and functional collecting duct, renal tubule, endocrine, glomerular, and vascular cells) is then centrifuged at 6% and 8% (w / v) with Optiprep. ® The tubes are divided into segments corresponding to densities between 1.025 and 1.045 g / mL. Other cells and debris precipitate to the bottom of the tube.

[0312] Kidney cell culture The combined cell strips and precipitate were then seeded at a concentration of 30,000 cells / cm² in 150 ml of a DMEM (high glucose) / KSFM50:50 mixture in a tissue culture-treated triple flask (Nunc T500) or equivalent, containing 5% (v / v) FBS, 2.5 µg EGF, 25 mg BPE, 1X ITS (insulin / transferrin / sodium selenite medium supplement) with antibiotics / antifungal agents. Cells were cultured in a humidified 5% CO2 incubator for 2–3 days, providing 21% atmospheric oxygen. After two days, the medium was changed and the culture was placed in a CO2 / nitrogen multi-gas humidified incubator (Sanyo) at 2% oxygen for 24 hours. After 24 hours of incubation, cells were washed with 60 ml of 1X PBS, followed by cell removal using 40 ml of 0.25% (w / v) trypsin / EDTA (Gibco). After removal, the cell suspension was neutralized with an equal volume of KSFM containing 10% FBS. Cells were then washed by centrifugation at 300 x g for 10 minutes. After washing, cells were resuspended in 20 ml of KSFM and transferred to 50 ml conical tubes, and samples were collected for cell counting. Once the viable cell count was determined using the trypan blue exclusion method, 1 million cells were collected for RNA sampling, washed in PBS, and rapidly frozen in liquid nitrogen. Cells were washed again in PBS and collected by centrifugation at 300 x g for 5 minutes. The washed cell pellet was resuspended in KSFM at a concentration of 37.5 million cells / ml.

[0313] Density gradient separation and enrichment of specific bioactive kidney cellsCultured kidney cells, primarily composed of tubular cells but containing other cell types (collecting ducts, glomeruli, blood vessels, and endocrine cells), were separated into their constituent subpopulations using a density gradient prepared from multiple w / v concentrations of iodixanol (Optiprep). The cultures were placed under hypoxic conditions for up to 24 hours before harvesting and application to the gradient. The density gradient was created by layering four different density culture media onto each other in 15 mL sterile conical tubes, with the highest density solution at the bottom and the lowest density solution layer on top. Cells were applied to the top of the density gradient and centrifuged, resulting in the separation of the populations into multiple bands based on size and particle size.

[0314] In short, Optiprep at densities of 7%, 11%, 13%, and 16% were prepared using KFSM medium as a diluent. ® (60% w / v iodixanol). For example: for 50 ml 7% (w / v) Optiprep ® Add 5.83 ml of 60% (w / v) stock iodixanol to 44.17 ml of KSFM medium and mix thoroughly by inversion. Set the peristaltic pump (Master Flex L / S) loaded with an L / S 16 Tygon tube connected to a sterile capillary to a flow rate of 2 ml / min, and load 2 mL of each of the four solutions into a 15 mL sterile conical tube, starting with the 16% solution, followed by the 13%, 11%, and 7% solutions. Finally, load 2 mL of a cell suspension containing 75 million cultured rodent kidney cells onto the top of the graded gradient (the suspension has been prepared as described above in "Kidney Cell Culture"). Importantly, when the pump begins delivering the gradient solutions to the tubes, carefully maintain a flow rate of 45°C. The gradient was flowed slowly along the side of the tube to ensure proper interfaces were formed between the layers. The cell-loaded gradient was then centrifuged continuously at 800 xg for 20 minutes. After centrifugation, the tube was carefully removed to avoid disturbing the interfaces. Five distinct cell fractions were produced (four bands and one precipitate) (B1–B4, + precipitate) (left conical tube). Each fraction was collected using sterile disposable bulb pipettes or 5 ml pipettes and characterized phenotypically and functionally (see Example 10, Presnell et al., WO / 2010 / 056328). When rodent kidney cell suspensions were subjected to gradient gradation immediately after separation, the fractions enriched with renal tubular cells (and containing some cells from collecting ducts) had a fractionation density between 1.062 and 1.088 g / mL. Conversely, when density gradient separation was performed after in vitro culture, the fraction enriched with renal tubular cells (and containing some cells from collecting ducts) was separated at densities between 1.051 and 1.062 g / mL. Similarly, when rodent kidney cell suspensions were subjected to graded gradient separation immediately after separation, the fraction enriched with epo-producing cells, glomerular podocytes, and vascular cells (“B4”) was separated at densities of 1.025–1.035 g / mL. Conversely, when density gradient separation was performed after in vitro culture, the fraction enriched with epo-producing cells, glomerular podocytes, and vascular cells (“B4”) was separated at densities of 1.073–1.091 g / mL. Importantly, by exposing the cultures to a hypoxic culture environment (defined as <21% (atmospheric) oxygen level) prior to harvesting and grading gradient procedures (for a period of approximately 1 hour to approximately 24 hours), the distribution of cells in the “B2” and “B4” fractions after culture was enhanced (further details regarding the effect of hypoxia on the band distribution are provided in Example 7).

[0315] Each band was washed with a 3-fold dilution of KSFM, thoroughly mixed, and centrifuged at 300 xg for 5 minutes. The precipitate was resuspended in 2 ml of KSFM, and viable cells were counted using the trypan blue exclusion method and a hemocytometer. One million cells were collected for RNA sampling, washed in PBS, and rapidly frozen in liquid nitrogen. Cells from B2 and B4 were used for transplantation into uremic and anemic female rats induced by a two-step 5 / 6 nephrectomy procedure at the Charles River Laboratory. Characterization of B4 cells, including the expression of erythropoietin and vEGF for oxygen regulation, the expression of glomerular markers (glomerular podocyte-specific proteins, glomerular podocyte slit membrane proteins), and the expression of vascular markers (PECAM), were confirmed by quantitative real-time PCR. The phenotype of the 'B2' fraction was confirmed by the expression of E-cadherin, N-cadherin, and aquaporin-2. See Figures 49a and 49b in Presnell et al., WO / 2010 / 056328.

[0316] Therefore, the use of a graded gradient strategy not only allows for the enrichment of rare EPO-producing cell populations (B4), but also provides a method for generating a relatively enriched fraction of functional renal tubular cells (B2) (see Figures 50 and 51 of Presnell et al., WO / 2010 / 056328). The graded gradient strategy also allows for the separation of EPO-producing cells and renal tubular cells from erythrocytes, cell debris, and other potentially undesirable cell types, such as large cell aggregates and certain immune cell types.

[0317] Hierarchical gradient procedures may require adjustments regarding the specific density used to provide good separation of cell components. Preferred methods for adjusting the gradient include 1) running a continuous density gradient from a high density at the bottom of the gradient (e.g., 16-21% Optiprep) to a relatively low density at the top of the gradient (e.g., 5-10%). A continuous gradient can be prepared using any standard density gradient solution (Ficoll, Percoll, sucrose, iodixanol) according to standard methods (AxisShield). The cells of interest are loaded onto the continuous gradient and centrifuged continuously at 800xG for 20 minutes. Cells of similar size and particle size tend to separate together in the gradient, allowing for the measurement of their relative positions within the gradient, and also the measurement of the specific gravity of the solution at that position. Thus, a defined hierarchical gradient can then be derived, focusing on separating a specific cell population based on its ability to traverse the density gradient under specific conditions. Such optimization may be necessary when separating cells from unhealthy tissue versus healthy tissue, or when separating specific cells from different species. For example, canine and human kidney cell cultures were optimized to ensure that specific B2 and B4 subpopulations identified in rats could be isolated from other species. The optimal gradients for isolating rodent B2 and B4 subpopulations consisted of (w / v) 7%, 11%, 13%, and 16% Optiprep. The optimal gradients for isolating canine B2 and B4 subpopulations consisted of (w / v) 7%, 10%, 11%, and 16% Optiprep. The optimal gradients for isolating human B2 and B4 subpopulations consisted of (w / v) 7%, 9%, 11%, and 16%. Therefore, Table 8 provides the density ranges for locating B2 and B4 from cultured rodent, canine, and human kidney cells.

[0318] Table 8. Species Density Range

[0319] Example 7 - Hypoxia culture prior to gradient influencing band distribution, composition, and gene expression

[0320] To determine the effect of oxygen conditions on the distribution and composition of phenotypes B2 and B4, novel kidney cell formulations from different species were exposed to different oxygen conditions prior to the gradient step. A rodent neonatal kidney enlargement (NKA) cell formulation (RK069) was established using the standard procedure described above for rat cell isolation and culture initiation. All flasks were cultured in 21% (atmospheric) oxygen for 2–3 days. The culture medium was replaced, and half of the flasks were then repositioned in an oxygen-controlled incubator set to 2% oxygen, while the remaining flasks were kept at 21% oxygen for an additional 24 hours. Cells were then harvested from each condition using the standard enzymatic harvesting procedure described above. A graded gradient was prepared according to the standard procedure, and “normal oxygen” (21% oxygen) and “hypoxia” (2% oxygen) cultures were harvested separately and applied side-by-side to the same graded gradient. Although four bands and one precipitate were produced in both conditions, the cell distribution across the gradient differed between the batches cultured in 21% and 2% oxygen (Table 3). Specifically, under hypoxic conditions, the yield of B2 increased, accompanied by a decrease in B3. Furthermore, in the gradient obtained from cells produced in hypoxic cultures (Figure 73 of Presnell et al. WO / 2010 / 056328), the expression of B4-specific genes (such as erythropoietin) was enhanced.

[0321] A canine NKA cell formulation (DK008) was established using the standard procedure for canine cell isolation and culture described above (similar to the procedure for rodent isolation and culture). All flasks were cultured in 21% (atmospheric) oxygen for 4 days, then subgroup flasks were transferred to hypoxia (2%) for 24 hours while maintaining the subgroup flasks at 21%. Subsequently, each group of flasks was harvested and subjected to the same grading gradient. Similar to the rat results (Example 6), canine cells cultured under hypoxia differed from canine cells cultured under atmospheric oxygen in distribution across the entire gradient (Table 9). Again, prior to the gradient, the yield of B2 increased under hypoxia exposure, accompanied by a decrease in the distribution of B3.

[0322] Table 9.

[0323] The data above show that pre-gradient exposure to hypoxia enhances the composition of B2 cells and the distribution of specific specialized cells (erythropoietin-producing cells, vascular cells, and glomerular cells) into B4 cells. Therefore, hypoxic culture followed by density-gradient segregation as described above is an effective method for generating 'B2' and 'B4' cell populations in different species.

[0324] Example 8 - Isolation of renal tubular / glomerular cells from human kidney

[0325] Renal tubular and glomerular cells were isolated from normal human kidney tissue and proliferated using the enzymatic isolation method described throughout. In vitro enrichment fractions of renal tubular cells were then cultured using the gradient method described above. Phenotypic characteristics were maintained during isolation and proliferation, as shown in Figure 68 of Presnell et al. (WO / 2010 / 056328, incorporated herein by reference in its entirety). Renal tubular cell function, assessed by labeled albumin uptake, was also preserved after repeated passages and cryopreservation. Figure 69 of Presnell et al. (WO / 2010 / 056328, incorporated herein by reference in its entirety) shows a significant increase in the expression of the renal tubular marker (cadherin) in the enriched population when both enriched and discarded populations were cultured in a 3D dynamic environment. This confirms that renal tubular cell enrichment can be maintained beyond the initial enrichment when cells are cultured in a 3D dynamic environment. The same cultured kidney cell populations described above in Example 7 were analyzed by flow cytometry to examine forward and lateral scattering. Small, granular EPO-producing cell populations were identifiable (8.15%) and were separated by positive selection of small, granular populations using the sorting capability of the flow cytometer (see Figure 70 of Presnell et al., WO / 2010 / 056328 (incorporated herein by reference in its entirety)).

[0326] Example 9 - Ungraded renal cells isolated from samples of patients with autoimmune glomerulonephritis Characterization of cell mixtures

[0327] As described above, an ungraded mixture of renal cells was isolated from samples from patients with autoimmune glomerulonephritis. To determine the unbiased genotypic composition of specific renal cell subsets isolated and expanded from kidney tissue, quantitative real-time PCR (qRTPCR) analysis (Brunskill et al., ibid., 2008) was used to identify differential cell type-specific and pathway-specific gene expression patterns within the cell subfractions. As shown in Table 6.1 of Ilagan et al., PCT / US2011 / 036347, HK20 represents a sample from a patient with autoimmune glomerulonephritis. As shown in Table 6.2 of Ilagan et al., PCT / US2011 / 036347, cells generated from HK20 lacked glomerular cells, as determined by qRTPCR.

[0328] Example 11 - Enrichment / Disposal of Viable Kidney Cell Types Using Fluorescence Activated Cell Sorting (FACS)

[0329] Fluorescence-activated cell sorting (FACS) can enrich one or more isolated kidney cells and / or discard one or more specific kidney cell types from isolated primary kidney tissue.

[0330] Reagents:70% ethanol; wash buffer (PBS); 50:50 kidney cell culture medium (50% DMEM high glucose): 50% keratinocyte-SFM; trypan blue 0.4%; primary antibodies targeting kidney cell populations such as CD31 for renal endothelial cells and targeting glomerular podocyte-specific proteins for renal glomerular cells. Matched allotype-specific fluorescent secondary antibody; staining buffer (PBS solution of 0.05% BSA).

[0331] program: Following standard procedures for cleaning a biosafety cabinet (BSC), a single-cell suspension of kidney cells from primary isolated or cultured cells is obtained from a T500 T / C treated flask and resuspended in kidney cell culture medium and placed on ice. Cell counts and viability are then determined using the trypan blue exclusion method. For kidney cells enriched / discarded from heterologous populations, such as glomerular cells or endothelial cells, 10⁻⁵ and 50x10⁻⁵ cells with at least 70% viability are obtained. 6 Live cells between. Then use staining buffer at an initial concentration of 1 µg / 0.1 ml / 1 x 10 6 Primary antibodies specific to the target cell type (at a titer if necessary) stain xenogeneic kidney cell populations. Target antibodies can be conjugated, such as CD31 PE (specific to renal endothelial cells), or unconjugated, such as glomerular podocyte-specific proteins (specific to renal glomerular cells).

[0332] Cells were then protected from light and stained on ice or at 4°C for 30 minutes. After 30 minutes of incubation, the cells were washed by centrifugation at 300 x g for 5 minutes. The precipitate was then resuspended in PBS or staining buffer, depending on whether a type-specific secondary antibody was required. If cells were labeled with a fluorescently conjugated primary antibody, the cells were resuspended in 2 ml PBS / 10 x 10 7 Cells are processed using FACS aria or an equivalent cell sorting system. If the cells are not labeled with fluorescent dye-conjugated antibodies, then the cells are started at a concentration of 1 μg / 0.1 ml / 1 x 10⁻⁶. 6 Secondary antibody labeling with a type-specific fluorescent dye conjugated to each cell.

[0333] Cells were then protected from light and stained on ice or at 4°C for 30 minutes. After 30 minutes of incubation, the cells were washed by centrifugation at 300 x g for 5 minutes. After centrifugation, the precipitate was resuspended in a solution of 5 x 10⁻⁶ cells / mL. 6 Add 4 ml of PBS solution to a sterile tube and then transfer it to a 12x75 mm tube.

[0334] Prepare the FACs Aria for aseptic live cell sorting according to the manufacturer's instructions (BD FACs Aria User Manual). Load the sample tubes into the FACs Aria and adjust the PMT voltage after acquisition begins. Use a specific wavelength to draw gates using fluorescence intensity to select specific kidney cell types. Draw another gate to select the negative population. Once the desired gates have been drawn to encapsulate the positive and negative populations, sort the cells using the manufacturer's instructions.

[0335] Positive target populations were collected in a 15 ml conical tube and negative populations were collected in another 15 ml conical tube filled with 1 ml of kidney cell culture medium. After collection, samples from each tube were analyzed by flow cytometry to determine purity.

[0336] The collected cells were washed continuously by centrifugation at 300 x g for 5 minutes, and the precipitate was resuspended in kidney cell culture medium for further analysis and experiments.

[0337] Example 12 - Enrichment / Disposal of Kidney Cell Types Using Magnetic Cell Sorting

[0338] One or more isolated kidney cells can be enriched and / or one or more specific kidney cell types can be discarded from isolated primary kidney tissue.

[0339] Reagents: 70% ethanol, wash buffer (PBS), 50:50 kidney cell culture medium (50% DMEM high glucose): 50% keratinocytes-SFM, trypan blue 0.4%, running buffer (PBS, 2 mM EDTA, 0.5% BSA), wash buffer (PBS, 2 mM EDTA), washing solution (70% v / v ethanol), Miltenyi FCR blocking reagent, Miltenyi microbeads specific to IgG isotypes, target antibodies such as CD31 (PECAM) or glomerular podocyte-specific proteins or secondary antibodies.

[0340] program: Following standard procedures for cleaning a biosafety cabinet (BSC), a single-cell suspension of kidney cells from primary isolation or culture was obtained and resuspended in kidney cell culture medium. Cell counts and viability were determined using the trypan blue exclusion method. For kidney cells enriched / discarded from heterologous populations, such as glomerular cells or endothelial cells, at least 10 x 10⁻⁶ cells with at least 70% viability were obtained. 6 Up to 4x10 9 One living cell.

[0341] The optimal separation for enrichment / discarding methods is determined based on the target cells of interest. For enrichment of less than 10% of the target frequency, such as glomerular cells, using glomerular podocyte-specific protein antibodies, Miltenyi autoMACS or the equivalent instrument program POSSELDS (double-positive selection in sensitive mode) is used. For discarding of more than 10% of the target frequency, Miltenyi autoMACS or the equivalent instrument program DEPLETES (discarding in sensitive mode) is used.

[0342] By 1 µg / 10 x 10 6 One cell / 0.1 ml of PBS along with 0.05% BSA was added to a 15 ml conical centrifuge tube, and then incubated at 4°C for 15 minutes to label live cells with a target-specific primary antibody, such as a recombinant polyclonal antibody against glomerular podocyte-specific proteins for glomerular cells.

[0343] After labeling, add 1-2 ml of buffer / 10 x 10 7 Cells were collected and then washed by centrifugation at 300xg for 5 minutes to remove unbound primary antibody. After washing, 1 μg / 10x10 cells were added. 6 0.1 ml of PBS along with 0.05% BSA of a type-specific secondary antibody, such as chicken anti-rabbit PE, was incubated at 4°C for 15 minutes.

[0344] After incubation, add 1-2 ml of buffer / 10 x 10 7 Cells were collected and then washed by centrifugation at 300 x g for 5 minutes to remove unbound secondary antibody. The supernatant was removed, and the cell pellet was diluted with 60 µl buffer / 10 x 10 cells / ml. 7 Resuspend the total cells, then add 20 µl of FCR blocking reagent / 10 x 10 7 Take a total of 100 cells and then mix them thoroughly.

[0345] Add 20 µl of direct MACS beads (such as anti-PE beads) and mix, then incubate at 4 °C for 15 minutes.

[0346] After incubation, the cell suspension was centrifuged at 300xg for 5 minutes by adding 10-20 times the labeled volume of buffer and the cell pellet was then separated into 500 µl - 2 ml buffer / 10 x 10 8 Cells are resuspended to wash them.

[0347] Clean the autoMACS system according to the manufacturer's instructions and prepare it for magnetic cell separation using autoMACS. Place a new sterile collection tube under the outlet port. Select the autoMACS cell separation program. For optional, select the POSSELDS program. For optional, select the DEPLETES program.

[0348] Insert the tagged cells into the uptake port and then start the procedure.

[0349] After cell selection or disposal, samples are collected and kept on ice until use. The purity of discarded or selected samples is confirmed by flow cytometry.

[0350] Example 13 - Cells with therapeutic potential can be derived from normal kidney tissue and kidney tissue with chronic disease. Separate and multiply

[0351] The aim of this study was to determine the functional characterization of human NKA cells using high-content analysis (HCA). High-content imaging (HCI) allows for the simultaneous imaging of multiple subcellular events across numerous samples using two or more fluorescent probes (multiplexed). High-content analysis (HCA) provides for the simultaneous quantitative measurement of multiple cellular parameters captured in high-content images. In short, ungraded (UNFX) cultures were independently generated from core biopsies of five kidneys from individuals with advanced chronic kidney disease (CKD) and three kidneys from individuals without CKD using standard biopsy procedures (Aboushwareb et al.). Same as above (2008) and maintained. After (2) generations of ex vivo UNFX, cells were harvested and subjected to a density gradient method (as in Example 2) to produce subfractions, including subfractions B2, B3 and / or B4.

[0352] Human kidney tissue was obtained from non-CKD and CKD human donors as summarized in Table 10.1 of Ilagan et al., PCT / US2011 / 036347. Figure 4 of Ilagan et al., PCT / US2011 / 036347 shows the histopathological features of HK17 and HK19 samples. Ex vivo cultures were established from all non-CKD (3 / 3) and CKD (5 / 5) kidneys. High-content analysis (HCA) of albumin transport in human NKA cell-defined regions of interest (ROI) is shown in Figure 5 of Ilagan et al., PCT / US2011 / 036347 (HCA of albumin transport in human NKA cells). A quantitative comparison of albumin transport in NKA cells derived from non-CKD and CKD kidneys is shown in Figure 6 of Ilagan et al., PCT / US2011 / 036347.

[0353] As shown in Figure 6 of Ilagan et al., PCT / US2011 / 036347, albumin transport was not impaired in CKD-derived NKA cultures. A comparative analysis of marker expression between the tubular-enriched B2 subfraction and the tubular-abandoned B4 subfraction is shown in Figure 7 (CK8 / 18 / 19) of Ilagan et al., PCT / US2011 / 036347.

[0354] A comparative functional analysis of albumin transport between the tubular-enriched B2 subfraction and the tubular-abandoned B4 subfraction is shown in Figure 8 of Ilagan et al., PCT / US2011 / 036347. Subfraction B2 is enriched in proximal tubular cells and therefore exhibits increased albumin transport function.

[0355] albumin intake Replace the culture medium for confluenced cells grown in 24-well collagen IV plates (BD Biocoat™) with phenol red-free, serum-free, low-glucose DMEM (pr- / s- / lg DMEM) containing 1X antifungal agent / antibiotic and 2 mM glutamine for 18–24 hours. Prior to assay, wash cells with pr- / s- / lg DMEM + 10 mM HEPES, 2 mM glutamine, 1.8 mM CaCl2, and 1 mM MgCl2 and incubate for 30 minutes. Expose cells to 25 μg / mL rhodamine-conjugated bovine albumin (Invitrogen) for 30 minutes, wash with ice-cold PBS to stop endocytosis, and immediately fix with 2% paraformaldehyde containing 25 μg / mL Hoechst nuclear dye. For inhibition assays, add 1 μM receptor-associated protein (RAP) (Ray Biotech, Inc., Norcross GA) 10 minutes before albumin addition. Microscopic imaging and analysis were performed using a BD Pathway™ 855 high-content biological imager (Becton Dickinson) (see Kelley et al. Am JPhysiol Renal Physiol. November 2010; 299(5):F1026-39. Electronic publication September 8, 2010).

[0356] Finally, HCA yields cellular-level data and can reveal population dynamics that are undetectable by other assays (i.e., gene or protein expression). A quantifiable ex vivo HCA assay for measuring albumin transport (HCA-AT) function can be used with human renal tubular cells characterizing components that are the prototypes of human NKA. HCA-AT enables a comparative assessment of cellular function, showing albumin-transporting competent cells preserved in NKA cultures derived from human CKD kidneys. Specific subfractions B2 and B4 of the NKA cultures are also shown to be phenotypic and functionally distinct, with B2 representing a fraction enriched in renal tubular cells with enhanced albumin transport activity. The B2 cell subpopulation from human CKD is phenotypic and functionally similar to rodent B2 cells that demonstrate efficacy in vivo (as shown above).

[0357] References

[0358] 1. Kelley, R. et al. Intra-renal Transplantation of Bioactive RenalCells Preserves Renal Functions and Extends Survival in the ZSFI model of Progressive Diabetic Nephropathy. 2011 American Diabetes Association (ADA)Conference. (2011).

[0359] 2. Kelley, R. et al. Tubular cell-enriched subpopulation of primaryrenal cells improves survival and augments kidney function in rodent model of chronic kidney disease. Am J Physiol Renal Physiol 299, F1026-1039.

[0360] 3. Presnell, S.C. et al. (2011) Isolation, Characterization, andExpansion Methods for Defined Primary Renal Cell Populations from Rodent,Canine, and Human Normal and Diseased Kidneys. Tissue Eng Part C Methods 17:261-273.

[0361] 4. Humphreys, B.D. et al. Repair of injured proximal tubule does notinvolve specialized progenitors. Proc Natl Acad Sci U S A 108, 9226-9231.

[0362] 5. Aboushwareb, T. et al. Erythropoietin producing cells forpotential cell therapy. World J Urol 26, 295-300 (2008).

[0363] 6. Takano, Y. et al. Recovery and maintenance of nephrin expressionin cultured podocytes and identification of HGF as a repressor of nephrin. AmJ Physiol Renal Physiol 292, F1573-1582 (2007).

[0364] 7. Basu, J. et al. Expansion of the human adipose-derived stromalvascular cell fraction yields a population of smooth muscle-like cells withmarkedly distinct phenotypic and functional properties relative tomesenchymal stem cells. Tissue Eng Part C Methods 17, 843-860.

[0365] 8. Basu, J. et al. Organ specific regenerative markers in peri-organadipose: kidney. Lipids Health Dis 10, 171.

[0366] 9. Xinaris C, et al. In vivo maturation of functional renal organoidsformed from embryonic cell suspensions. J Am Soc Nephrol 23,1-12. (2012).

[0367] 10. Buzhor et al. Kidney Spheroids Recapitulate Tubular OrganoidsLeading to Enhanced Tubulogenic Potency of Human Kideny-Derived Cells TissueEngineering Part A, (2011).

[0368] 11. Brown, SA et al., Impaired renal autoregulatory ability in dogswith reduced renal mass. J Am Soc Nephrol. 5:1768-74 (1995).

[0369] 12. Robertson, J.L. et al., Long-term renal responses to high dietaryprotein in dogs with 75% nephrectomy. Kidney Int. 29:511-9 (1986).

[0370] 13. Urie, B.K. et al., Evaluation of clinical status, renal function,and hematopoietic variables after unilateral nephrectomy in canine kidneydonors. J Am Vet Med Assoc. 230:1653-6 (2007).

Claims

1. A cell cluster for treating chronic kidney disease, wherein the cell cluster comprises: Heterogeneous renal cell population and non-renal bioactive cell population The heterologous kidney cell population contained enriched renal tubular cells, and The non-renal bioactive cell populations mentioned herein are non-renal endothelial cell populations, non-renal endothelial progenitor cell populations, non-renal mesenchymal stem cell populations, or progenitor cell populations not derived from renal adipose tissue. The cell clusters are cultured in suspension in the culture medium without attaching to the scaffold.

2. The cell cluster according to claim 1, wherein the non-renal bioactive cell population is the non-renal endothelial cell population.

3. The cell cluster of claim 2, wherein the non-renal endothelial cell population comprises human venous endothelial cells.

4. Articles, which include: Cell clusters according to any one of claims 1-3; and Containers containing labels or packaging instructions.

5. The article of claim 4, wherein the label or packaging instructions further include instructions for administering the cell clusters to a patient.

6. Use of the cell clusters according to any one of claims 1-3 in the preparation of a medicament for treating a subject's kidney disease.

7. A cell cluster for treating chronic kidney disease, wherein the cell cluster comprises: Heterogeneous kidney cell populations and non-renal bioactive cell populations The xenogeneic kidney cell population described herein is enriched with renal erythropoietin (EPO)-producing cells, and The non-renal bioactive cell populations mentioned herein are non-renal endothelial cell populations, non-renal endothelial progenitor cell populations, non-renal mesenchymal stem cell populations, or progenitor cell populations not derived from renal adipose tissue. The cell clusters are cultured in suspension in the culture medium without attaching to the scaffold.

8. The cell cluster according to claim 7, wherein the non-renal bioactive cell population is the non-renal endothelial cell population.

9. Articles, which include: The cell cluster as described in claim 7 or 8; and Containers containing labels or packaging instructions.

10. Use of the cell cluster of claim 7 or 8 in the preparation of a medicament for treating a subject with kidney disease.

Images