Compositions Comprising Cell-Derived Vesicles and Uses Thereof

Abstract

A method for treating kidney disease in a subject is provided. Provided herein, inter alia, are extracellular products (e.g., vesicles such as microvesicles, e.g., exosomes) produced by renal cells (e.g., bioactive renal cells, e.g., selected renal cells). Also included are methods for modifying components (e.g., miRNA or proteins) of vesicles produced by the cells, as well as methods for producing vesicles containing various compounds. Diagnostic and therapeutic methods are also provided.

Description

[Background technology]

[0001] Research into extracellular vesicles, particularly exosomes, is rapidly increasing. Numerous papers have been published on exosome biogenesis (Non-Patent Document 1), their diagnostic and prognostic potential (Non-Patent Document 2), and potential therapeutic applications in tissue engineering and regenerative medicine (Non-Patent Document 3). These carriers of mRNA, miRNA, proteins, and lipid mediators can act on target cells to facilitate intercellular communication and the exchange of functional genetic information (Non-Patent Document 4, Non-Patent Document 5, Non-Patent Document 6). [Prior art documents] [Non-patent literature]

[0002] [Non-Patent Document 1] Kowal et al., 2014, Curr Opin Cell Biol. 29C:116-125 [Non-patent document 2] Revenfeld et al., 2014, Clin Ther. 36(6):830-846 [Non-patent document 3] Lamichhane et al., 2014, Tissue Eng Part B Rev. [Non-patent document 4] Simons and Raposo, 2009, Curr Opin Cell Biol. 21(4):575-581 [Non-patent document 5] Stoorvogel et al., 2002, Traffic 3(5):321-330 [Non-patent document 6] Nieuwland and Sturk, 2010, Thrombosis Research 125(Supplement 1):S49-S51 Summary of the Invention

[0003] Provided herein, inter alia, are extracellular products (e.g., vesicles such as microvesicles, e.g., exosomes) produced by renal cells (e.g., bioactive renal cells, e.g., selected renal cells). In certain embodiments, such products are used to treat renal diseases, such as chronic kidney disease. Also included are methods for modifying components of vesicles produced by the cells (e.g., miRNA or proteins), and methods for producing vesicles containing various compounds. Diagnostic and therapeutic methods are also provided.

[0004] In one aspect, provided herein are methods of treating kidney disease in a subject, hi certain embodiments, the methods comprise administering to the subject an effective amount of isolated secreted kidney cell vesicles, wherein the vesicles are administered by intravenous injection or transcatheter delivery.

[0005] In one aspect, provided herein is a method for detecting at least one compound in a vesicle. In certain embodiments, the method includes obtaining the vesicle and detecting whether the at least one compound is present in the vesicle, wherein (i) the at least one compound is a protein, and the protein is CD9, CD81, CD146, CD326, CD40, CD42a, CD44, CD49e, and / or SSEA-4, (ii) the at least one compound is an miRNA, and the miRNA includes at least two of miR-145, miR-22, miR-7, miR-10a, miR-143, and / or let7b, and / or (iii) the at least one compound is not expressed or produced by renal cells in a native kidney.

[0006] In one aspect, provided herein are methods for monitoring treatment with a bioactive renal cell population in a subject to which the bioactive renal cell population has been administered, hi certain embodiments, the methods comprise detecting the presence of at least one compound in vesicles from the subject according to the methods disclosed herein.

[0007] In one aspect, provided herein is a method for determining whether a vesicle is regenerative. In certain embodiments, the method includes (i) detecting whether a protein and / or miRNA is present in the vesicle according to a method disclosed herein, and (ii) determining the vesicle as regenerative if the protein and / or the miRNA is detected in the vesicle.

[0008] In one aspect, provided herein are methods for detecting the level of at least one miRNA in vesicles from a population of bioactive kidney cells. In certain embodiments, the methods include (i) detecting the expression of one or more of the following miRNA molecules in the vesicles: miR-1248, miR-3168, miR-7113-5p, miR-758-3p, miR-937-3p, miR-4455, miR-4521, miR-203a-3p, miR-22-3p, miR-574-3p, miR-181b-5p, miR-1260b, and / or miR-181b-5p. and (ii) detecting whether one or more of the following miRNA molecules: miR-1-3p, miR-1-3p, miR-143-3p, miR-150-5p, miR-509-3p, miR-653-5p, miR-204-5p, miR-192-5p, and / or miR-363-3p, are decreased in the vesicles compared to a control. In certain embodiments, the miRNA is a mammalian miRNA, such as a human miRNA.

[0009] In one aspect, provided herein are methods of treating kidney disease in a subject, hi certain embodiments, the methods comprise administering to the subject an effective amount of vesicles from a vesicle preparation, wherein the vesicles from the vesicle preparation have been identified as regenerative according to the methods disclosed herein.

[0010] In certain embodiments, provided herein are methods for treating renal disease in a subject, comprising administering to the subject an effective amount of a composition comprising a bioactive renal cell population supplemented with renal cell vesicles not secreted by the bioactive renal cell population.

[0011] In one aspect, provided herein is a method of altering the level of at least one miRNA and / or protein in vesicles produced by a population of bioactive kidney cells, the method comprising culturing the population under hypoxic conditions.

[0012] In one aspect, provided herein are vesicles comprising a compound that is not produced by kidney cells in a native kidney.

[0013] In one aspect, provided herein is a composition comprising a vesicle disclosed herein and a pharmaceutically acceptable carrier.

[0014] In one aspect, provided herein is a composition comprising renal cell vesicles and non-renal cell vesicles.

[0015] In one aspect, provided herein is a composition comprising vesicles produced by primary kidney cells and vesicles produced by selected kidney cells.

[0016] In one aspect, provided herein are methods of treating kidney disease in a subject, hi certain embodiments, the methods comprise administering to the subject an effective amount of a composition disclosed herein.

[0017] In one aspect, provided herein are methods for producing vesicles (e.g., microvesicles such as exosomes) from cells that contain compounds not produced by the cells. In some embodiments, the method includes isolating vesicles from a cell culture supernatant, where the cell culture supernatant is from a culture of cells that have been contacted with a compound (e.g., incubated in a medium containing the compound). In certain embodiments, the method includes isolating vesicles (e.g., microvesicles such as exosomes) from the cell culture supernatant, and then incorporating the compound into the vesicles by permeabilizing the exosome membrane to facilitate compound entry (e.g., by sonication, lipofection, electroporation, etc.).

[0018] In one aspect, provided herein are methods for producing renal exosomes comprising a compound not produced by renal cells in a native kidney. In certain embodiments, the method comprises isolating vesicles from a renal cell culture supernatant, wherein the renal cell culture supernatant is from a culture of renal cells comprising a bioactive renal cell population that has been contacted with the compound. [Brief explanation of the drawings]

[0019] [Figure 1] 1 is a flow diagram of a non-limiting example of an overall NKA manufacturing method. [Figure 2A-D] 2 is a flow chart illustrating further details of the non-limiting exemplary method shown in FIG. 1. [Figure 3A-D] 1 is a flow diagram of a non-limiting example of the production of exosome-supplemented NKA from an SRC. [Figure 4A-D]Figure 4 shows a graph depicting surface protein analysis of secreted exosomes isolated from both TCHK0012 and TCHK0013 cells. This analysis revealed that CD133, CD326, and CD49e were upregulated in SRC cells compared with BRC cells. While the exact function of CD133 remains unknown, it has been proposed that CD133 acts as an organizer of cell membrane topology. Epithelial cell adhesion molecule (EpCAM) (also known as CD326) is a transmembrane glycoprotein that mediates Ca2+-independent homotypic cell-cell adhesion in epithelia. EpCAM is also involved in cell signaling, migration, proliferation, and differentiation. In addition to adhesion, integrins such as CD49e are known to be involved in cell surface-mediated signaling. Arrows have been added to Figures 4B-4D to highlight the comparison between exosomes from BRC-3A and SRC cells. [Figure 5] Figure 1 shows a graph from a FACS analysis of exosome fusion to cells. Fluorescent labeling of cells, an indicator of lipophilic dye transfer from exosomes to the cell membrane, results in a left-to-right shift in the histogram line. Exosomes do not attach to the cell membrane and integrate at 4°C; this is the negative control. Incubation at 37°C allows for attachment and integration, resulting in fluorescent labeling of cells, which shifts the histogram from left to right. [Figure 6] FIG. 1 shows a graph depicting cell proliferation (y-axis) as mean cell number in response to varying doses of exosomes originating from renal cell populations (1X=dose / response at x ng / ml exosomes). [Figure 7A-C] Figure 1 shows images of cells. Cultures were incubated for 9 hours after treatment. A. Serum-free, growth factor-free medium (negative control). B. Serum-free, growth factor-free medium supplemented with 10 exosomes (test article). C. Serum-free medium supplemented with growth factors (positive control). [Figure 8]Figure 11 shows a volcano plot of significantly different miRNAs between the pair of experimental conditions E1 vs. D1. The volcano plot shows the distribution of differentially expressed miRNAs according to fold change (x-axis) and significance (negative logarithm of the P-value on the y-axis). The horizontal dotted line is the P-value cutoff (0.05), and the vertical dotted line is the fold change cutoff (|Log2 fold change|≧1). See also Tables 11-13. [Figure 9] Figure 10 shows a volcano plot of miRNAs that are significantly different between the pair of experimental conditions F1 vs. A1. See also Tables 14-16. [Figure 10] 1 is a flow chart of a non-limiting example of a method for producing exosomes. DETAILED DESCRIPTION OF THE INVENTION

[0020] Provided herein, inter alia, are extracellular products (e.g., vesicles such as microvesicles, e.g., exosomes) produced by renal cells (e.g., bioactive renal cells, e.g., selected renal cells).

[0021] All references cited throughout this disclosure are expressly incorporated herein by reference in their entirety. In the event that one or more of the literature, patents, and similar materials incorporated herein by reference, including but not limited to defined terms, term usage, described techniques, etc., differs from or conflicts with this application, this application will control.

[0022] definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Principles of Tissue Engineering, 3rd Edition (R Lanza, R Langer and J Vacanti, eds.), 2007, provides a This application provides a general guide to many of the terms used. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.

[0023] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0024] In this disclosure, the words "comprises," "comprising," "containing," and "having" and the like have the meanings given to them in U.S. patent law. "includes," "including," etc. "Consisting essentially of" or "consisting essentially of" can be used. "Consists essentially of" similarly has the meaning given in U.S. patent law, and the term is open-ended, allowing for more than what is recited to be present, but excluding prior art embodiments, so long as the basic or novel characteristics of what is recited are not altered by the presence of more than what is recited.

[0025] As used herein, the term "about" in the context of a numerical value or range means ±10% of the numerical value or range recited or claimed, unless the context requires a more limited range.

[0026] As used herein, the term "cell population" refers to a number of cells obtained by direct isolation from a suitable tissue source, usually a mammal. In certain embodiments, the isolated cell population can be subsequently cultured in vitro. One of skill in the art will recognize various methods for isolating and culturing cell populations used in the present disclosure, as well as various numbers of cells in cell populations suitable for use in the present disclosure. In certain embodiments, the cell population can be an unfractionated heterogeneous cell population or an enriched homogeneous cell population derived from an organ or tissue, such as the kidney. In certain embodiments, the heterogeneous cell population can be isolated from a tissue biopsy or whole organ tissue. In certain embodiments, the heterogeneous cell population can be derived from an in vitro culture of mammalian cells established from a tissue biopsy or whole organ tissue. An unfractionated heterogeneous cell population can also be referred to as an unenriched cell population. In certain embodiments, the cell population comprises biologically active cells. A homogeneous cell population contains a higher proportion of cells of the same cell type that share a common phenotype or have similar physical properties compared to an unfractionated heterogeneous cell population. In certain embodiments, the homogenous cell population is a heterogenous kidney cell population. The cell population may be isolated, extracted, or enriched from a population of cells. In certain embodiments, the enriched cell population is obtained as a cell fraction using centrifugation separation across a density boundary, density barrier, or density interface of a heterogeneous cell suspension. In certain embodiments, the enriched cell population is obtained as a cell fraction using continuous or discontinuous (single-step or multi-step) density gradient separation of a heterogeneous cell suspension. In certain embodiments, the cell population may comprise one, two, three, four, or more kidney cell types. In certain embodiments, a homogeneous or heterogeneous cell population of kidney origin is combined with, without further limitation, a homogeneous or heterogeneous cell population of kidney origin from a tissue or organ other than the kidney.

[0027] As used herein, the term "biologically active" means "having biological activity," such as pharmacological activity or therapeutic activity. In certain embodiments, the biological activity is enhancement of renal function and / or an effect on renal homeostasis. In certain embodiments, the biological activity is, but is not limited to, analgesic, antiviral, anti-inflammatory, anti-tumor, immunostimulatory, immunomodulatory, enhancing cell viability, antioxidant, oxygen carrier, cell recruitment, cell adhesion, immunosuppressive, angiogenic, wound healing activity, recruitment of host stem or progenitor cells, cell proliferation, stimulation of cell migration to the site of injury, amelioration of cell and tissue fibrosis, inhibition of epithelial-mesenchymal signaling cascades, secretion of cytokines, growth factors, proteins, nucleic acids, exosomes, microvesicles, or any combination thereof.

[0028] As used herein, the term "bioactive renal cells" or "BRCs" refers to renal cells that, when administered to a subject's kidney, have one or more of the following properties: the ability to reduce (e.g., slow or stop) the deterioration or progression of chronic kidney disease or its symptoms, the ability to enhance renal function, the ability to affect (improve) renal homeostasis, and the ability to promote healing, repair, and / or regeneration of renal tissue or the kidney. In certain embodiments, microvesicles from BRCs and / or BRCs can be administered to a patient, wherein the BRC hyplotype ) differs from the patient's haplotype. In certain embodiments, BRCs are cells that can enhance renal function, affect (improve) renal homeostasis, and / or promote healing, repair, and / or regeneration of renal tissue or the kidney without immunological rejection. In certain embodiments, these cells may include functional tubular cells (e.g., due to improved creatinine excretion and protein retention), glomerular cells (e.g., due to improved protein retention), vascular cells, and / or other cells of the corticomedullary junction. In certain embodiments, BRCs have a regenerative effect on the kidney. In certain embodiments, BRCs comprise, consist essentially of, or consist of selected renal cells (SRCs). In certain embodiments, BRCs are SRCs. In certain embodiments, BRCs are obtained from the isolation and expansion of renal cells from renal tissue. In certain embodiments, BRCs are obtained from the isolation and expansion of renal cells from kidney tissue using methods that select for bioactive cells (eg, cells with regenerative potential).

[0029] In certain embodiments, SRCs are cells obtained by isolating and expanding renal cells from a suitable renal tissue source, wherein the SRCs contain a higher percentage of one or more cell types and lack or have a lower percentage of one or more other cell types compared to the starting renal cell population. In certain embodiments, the SRCs contain an increased proportion of BRCs compared to the starting renal cell population. In certain embodiments, the SRC population is a population of isolated renal cells enriched for certain bioactive components and / or cell types and / or depleted of certain inactive and / or undesirable components or cell types used in the treatment of kidney disease, i.e., resulting in stabilization and / or improvement and / or regeneration of kidney function. In certain embodiments, microvesicles and / or SRCs from SRCs can be administered to a patient, wherein the haplotype of the SRC is different from the patient's haplotype. In certain embodiments, the SRCs can result in stabilization and / or improvement and / or regeneration of kidney function. The SRCs can result in superior therapeutic outcomes and regeneration compared to the starting population. In certain embodiments, SRCs are obtained from a patient's renal cortical tissue via kidney biopsy. In certain embodiments, SRCs are selected (e.g., by MACS or FACS) based on their expression of one or more markers. In certain embodiments, SRCs are depleted of one or more cell types (e.g., by MACS or FACS) based on the expression of one or more markers for the cell type. In certain embodiments, cell depletion or selection involves bead / antibody coupling to remove cells that have certain proteins on their cell surface. In certain embodiments, SRCs are selected from a population of bioactive renal cells. In certain embodiments, SRCs are selected by density gradient separation of expanded renal cells. In certain embodiments, SRCs are selected by separation of expanded renal cells by centrifugation across a density boundary, density barrier, or density interface, or by single-step discontinuous density gradient separation. In certain embodiments, SRCs are selected by continuous or discontinuous density gradient separation of expanded renal cells cultured under hypoxic conditions. In certain embodiments, SRCs are selected by density gradient separation of expanded renal cells cultured under hypoxic conditions for at least about 8 hours, 12 hours, 16 hours, 20 hours, or 24 hours. In certain embodiments, SRCs are selected by centrifugation across a density boundary, density barrier, or density interface of expanded renal cells cultured under hypoxic conditions. In certain embodiments, SRCs are selected by centrifugation across a density boundary, density barrier, or density interface (e.g., single-stage discontinuous density gradient separation) of expanded renal cells cultured under hypoxic conditions for at least about 8 hours, 12 hours, 16 hours, 20 hours, or 24 hours. In certain embodiments, SRCs are primarily composed of renal tubular cells. In certain embodiments, other parenchymal (e.g., vascular) and interstitial (e.g., collecting duct) cells may be present among the SRCs. In certain embodiments, less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the cells in a population of SRCs are vascular cells. In certain embodiments, less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the cells in a population of SRCs are collecting duct cells.In certain embodiments, less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the cells in a population of SRCs are vascular cells or collecting duct cells. Methods for obtaining SRCs are disclosed, for example, in Example 1 herein, WO 2010 / 056328 by Presnell et al., International Application No. PCT / US2011 / 036347 by Ilagan et al., and International Application No. PCT / US2016 / 044866 by Jain et al.

[0030] The term "natural organ" is intended to mean an organ of a living subject. The subject may be healthy or unhealthy. An unhealthy subject may have a disease associated with that particular organ.

[0031] The term "native kidney" is intended to mean a kidney of a living subject. The subject may be healthy or unhealthy. An unhealthy subject may have kidney disease.

[0032] The term "regenerative effect" is intended to mean an effect that benefits a native organ, such as the kidney. This effect may include, but is not limited to, a reduction in the extent of damage to the native organ, or an improvement, restoration, or stabilization of the function or structure of the native organ. Renal damage may be in the form of fibrosis, inflammation, glomerular hypertrophy, atrophy, etc., and may be associated with a disease associated with the native organ in a subject.

[0033] An "enriched" cell population or preparation refers to a cell population derived from a starting cell population (e.g., an unfractionated heterogeneous cell population from an organ such as the kidney) that contains a higher percentage of a particular cell type than the percentage of that cell type in the starting population. For example, a starting kidney cell population can be enriched for a first cell population, a second cell population, a third cell population, a fourth cell population, a fifth cell population, etc., of interest. As used herein, the terms "cell population," "cell preparation," and "cell phenotype" are used interchangeably.

[0034] As used herein, the term "hypoxic" culture conditions refers to culture conditions in which cells are exposed to reduced levels of available oxygen in a culture system compared to standard culture conditions in which cells are cultured at atmospheric oxygen levels (approximately 21%), and in certain embodiments, hypoxic culture conditions in which the oxygen level in the culture system is less than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0035] The term "biomaterial," as used herein, refers to a natural or synthetic biocompatible material suitable for introduction into living tissue that supports selected bioactive cells in a viable state. Natural biomaterials are materials produced by or derived from biological systems. Synthetic biomaterials are materials that are not produced by or directly derived from biological systems, but instead are synthesized or constructed using specific chemical methods and protocols well known to those skilled in the art. The biomaterials disclosed herein can be a combination of natural and synthetic biocompatible materials. As used herein, biomaterials include, for example, polymer matrices and scaffolds. Those skilled in the art will recognize that biomaterial(s) can be configured in various forms, such as porous foams, gels, liquids, beads, or solids, and can include one or more natural or synthetic biocompatible materials. In certain embodiments, the biomaterial is in the liquid form of a solution, which can be a hydrogel.

[0036] The term "hydrogel" is used herein to refer to a substance formed when organic polymers (natural or synthetic) are crosslinked via covalent, ionic, or hydrogen bonds to produce a three-dimensional structure (e.g., an open lattice structure) that traps water molecules to form a gel. Examples of materials that can be used to form hydrogels include tonically crosslinked polysaccharides such as alginates, polyphosphazines, and polyacrylates, or block copolymers such as Pluronic™ or Tetronic™, polyethylene oxide-polypropylene glycol block copolymers, which are crosslinked by temperature or pH, respectively. In certain embodiments, the hydrogel is a biodegradable gelatin-based hydrogel.

[0037] In certain embodiments, the biomaterial includes an extracellular matrix derived from, for example, a pre-existing kidney of human or animal origin from which the natural cell population has been eliminated by application of surfactants and / or other chemical agents known to those skilled in the art. In certain embodiments, the biomaterial is in the liquid form of a solution capable of becoming a hydrogel, which is layered with or without certain cell populations by application of three-dimensional bioprinting techniques known to those skilled in the art. In certain embodiments, the biomaterial is configured to mimic the three-dimensional fractal tissue of a decellularized kidney.

[0038] The term "modified release" or equivalent terms "controlled release," "delayed release," or "sustained release" refers to a formulation that releases an active agent, such as bioactive cells, over an extended period of time or at two or more time points after administration to an individual. Modified release of an active agent, which can occur over a desired time range, e.g., minutes, hours, days, weeks, or longer, depending on the formulation, is in contrast to formulations in which substantially the entire dosage unit is available immediately after administration. In certain embodiments, for tissue engineering and regenerative medicine applications, modified release formulations provide for release of the active agent at multiple time points after local administration (e.g., administration of the active agent directly to a solid organ). For example, a modified release formulation of bioactive cells may provide an initial release of cells immediately upon administration, followed by a second release of cells at a later time point. In certain embodiments, the time delay for the second release of the active agent may be minutes, hours, or days after the initial administration. Generally, the period of delayed release corresponds to the period it takes for the biomaterial carrier of the active agent to lose its structural integrity. Delayed release of the active agent may be achieved by: The release mechanism begins as soon as the integrity of the saccharin begins to deteriorate and is complete by the time the integrity is completely lost. Those skilled in the art will recognize other suitable release mechanisms.

[0039] The term "ambient temperature" refers to the temperature at which a formulation of the present disclosure is administered to a subject. Generally, ambient temperature is the temperature of a temperature-controlled environment. Ambient temperature ranges from about 18°C ​​to about 30°C. In certain embodiments, ambient temperature is about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, or about 30°C.

[0040] Non-limiting examples of kidney disease include disorders related to any stage or degree of acute or chronic renal failure that result in the loss of the kidney's ability to perform the functions of blood filtration and removal of excess fluid, electrolytes, and waste products from the blood. In certain embodiments, kidney disease can also include endocrine dysfunction, such as anemia (erythropoietin deficiency) and mineral imbalance (vitamin D deficiency). Kidney disease can originate in the kidney or can be secondary to various conditions, including (but not limited to) heart failure, hypertension, diabetes, autoimmune disease, or liver disease. Kidney disease can be a state of chronic renal failure that develops after acute kidney injury. For example, kidney damage due to ischemia and / or exposure to toxic substances can cause acute kidney failure. Incomplete recovery after acute kidney injury can lead to the development of chronic kidney failure.

[0041] The term "treatment" refers to both therapeutic treatment and preventative or preventative measures for kidney disease, anemia, renal tubular transport disorder, or glomerular filtration disorder, the purpose of which is to reverse, prevent, or slow down (e.g., reduce deterioration) the targeted disorder. Those in need of treatment include those who already have kidney disease, anemia, renal tubular transport disorder, or glomerular filtration disorder, as well as those who are susceptible to or at risk of developing kidney disease, anemia, renal tubular transport disorder, or glomerular filtration disorder, or those in whom kidney disease, anemia, renal tubular transport disorder, or glomerular filtration disorder should be prevented. As used herein, the term "treatment" includes stabilizing and / or improving kidney function.

[0042] In certain embodiments, "contacting" a native organ with an active agent (such as an enriched cell population and / or its products) in vivo refers to direct in vivo contact between the active agent and the native organ. For example, a product secreted by an enriched renal cell population may contact a native kidney in vivo (alone or together with cells, e.g., in a construct). In certain embodiments, the direct in vivo contact may be paracrine, endocrine, or juxtocrine in nature. In certain embodiments, the secreted product may be a heterogeneous population of various products described herein.

[0043] In certain embodiments, provided herein are "constructs" or "formulations" that include one or more cell populations and / or one or more cell products (such as microvesicles, e.g., exosomes) deposited on or within a biomaterial (such as a scaffold or matrix made of one or more synthetic or naturally occurring biocompatible materials). In certain embodiments, one or more cell populations can be coated with, deposited on, embedded within, bound to, seeded within, or entrapped within a biomaterial made of one or more synthetic or naturally occurring biocompatible biomaterials, polymers, proteins, or peptides. In certain embodiments, the naturally occurring biomaterial is a decellularized kidney of human or animal origin. In certain embodiments, the biomaterial is structurally engineered by three-dimensional bioprinting. In certain embodiments, one or more cell populations and / or cell products can be combined with the biomaterial, scaffold, or matrix in vitro or in vivo. In certain embodiments, one or more biomaterials used to make the construct or formulation are capable of dispersing and transporting the cellular components of the construct along with endogenous host tissue. The biomaterial may be selected to induce, promote, or enable the formation of at least one multicellular, three-dimensional tissue from the cell population deposited thereon, or to induce, promote, or enable the survival, implantation, tolerance, or functional performance of the cellular components of the construct or formulation. In certain embodiments, one or more biocompatible materials used to form the scaffold / biomaterial are selected to induce, promote, or enable the formation of at least one multicellular, three-dimensional tissue from the cell population deposited thereon. In certain embodiments, the biomaterial induces, promotes, or facilitates the assembly of defined three-dimensional cellular aggregates or organoids that recapitulate aspects of native kidney tissue, including, but not limited to, tissue polarity. In certain embodiments, the biomaterial induces the assembly of defined tubular structures that recapitulate aspects of native kidney tissue, including the lumen. In certain embodiments, the biomaterial enhances or promotes the secretion of proteins, nucleic acids, and microvesicles from the cell population. In certain embodiments, one or more biomaterials used to fabricate the construct may also be selected to mimic or replicate aspects of a particular three-dimensional tissue or environmental niche within the native kidney or renal parenchyma that corresponds to the biological environment from which the cell populations originate. Without being bound by any particular scientific theory, it is believed that replicating the biological niche from which these cell populations originate further enhances or promotes cell viability and efficacy.

[0044] The term "cell aggregate" or "spheroid" refers to an aggregate or assembly of cells cultured to allow three-dimensional growth rather than growth as a monolayer. It is noted that the term "spheroid" does not imply that the aggregate is a geometric sphere. In certain embodiments, the aggregate may be highly organized with a well-defined morphology and polarity, or may be a disorganized mass. The aggregate may contain a single cell type or two or more cell types. In certain embodiments, the cells may be primary isolates or permanent cell lines, or a combination of the two. This definition includes organoids and organotypic cultures. In certain embodiments, spheroids (e.g., cell aggregates or organoids) are formed in spinner flasks. In certain embodiments, spheroids (e.g., cell aggregates or organoids) are formed in a three-dimensional matrix.

[0045] The term "Neo-Kidney Augment (NKA)" refers to a bioactive cell preparation that is an injectable product composed of SRCs formulated in a biomaterial composed of a gelatin-based hydrogel. The term "Advance Cell Therapy (ACT)" is also used in reference to treatment with an NKA. In certain embodiments, an NKA is an injectable product comprising an immunocompatible kidney cell population (e.g., immunocompatible SRCs) formulated in a biomaterial composed of a gelatin-based hydrogel. In certain embodiments, an NKA is an injectable product composed of genomically modified, immunoprivileged, homogeneous SRCs that are incapable of immune rejection formulated in a biomaterial composed of a gelatin-based hydrogel.

[0046] The term "subject" is intended to mean any single human subject, including a patient, experiencing or eligible for treatment after experiencing one or more signs, symptoms, or other indicators of kidney disease. Such subjects include, but are not limited to, newly diagnosed or previously diagnosed subjects currently experiencing a recurrence or flare-up or at risk for kidney disease, regardless of cause. The subject may or may not have been previously treated for kidney disease.

[0047] The term "patient" refers to any animal, more preferably a mammal (including, for example, dogs, cats, horses, rabbits, zoo animals, cows, pigs, sheep, and non-human animals such as non-human primates), for which treatment is desired. Most preferably, the patient is a human.

[0048] The terms "sample" or "patient sample" or "biological sample" are generally intended to mean any biological sample obtained from a subject or patient, body fluid, body tissue, cell line, tissue culture, or other source. This term includes tissue biopsies, such as, for example, a kidney biopsy. This term includes cultured cells, such as, for example, cultured mammalian kidney cells. Methods for obtaining tissue biopsies and cultured cells from mammals are well known in the art. Depending on the context, the term "sample" when used alone shall mean that the "sample" is a "biological sample" or a "patient sample," i.e., the terms are used interchangeably.

[0049] The term "test sample" refers to a sample from a subject treated by the methods of the present disclosure. The test sample may be derived from a variety of sources in a mammalian subject, including, but not limited to, blood, semen, serum, urine, bone marrow, mucosa, tissue, etc.

[0050] The term "control" or "control sample" refers to a negative or positive control whose negative or positive results are expected to be useful in correlating with results from a test sample. Controls suitable for the present disclosure include, but are not limited to, samples known to exhibit indicators characteristic of normal kidney function, samples obtained from subjects known to be free of kidney disease, and samples obtained from subjects known to have kidney disease. In certain embodiments, a control may be a sample obtained from a subject prior to treatment with the methods of the present disclosure. In certain embodiments, suitable controls may be test samples obtained from subjects known to have any type or stage of kidney disease and samples from subjects known to not have any type or stage of kidney disease. A control may be a normal, healthy, matched control. Those skilled in the art will recognize other controls suitable for use in the present disclosure.

[0051] "Regenerative prognosis," "regenerative prognosis," or "prognosis for regeneration" generally refers to a prediction or forecast of the likely regenerative course or outcome of administration or transplantation of a cell population, cell product, or construct described herein. For a regenerative prognosis, the prediction or forecast may be informed by one or more of the following: improvement of a functional organ (e.g., kidney) after transplantation or administration, development of a functional kidney after transplantation or administration, development of improved kidney function or kidney capacity after transplantation or administration, and expression of certain markers by the native kidney after transplantation or administration.

[0052] "Regenerated organ" refers to a native organ after transplantation or administration of a cell population, cell product, or construct described herein. In certain embodiments, the regenerated organ is characterized by various indicators, including, but not limited to, development of function or capacity in the native organ, improvement of function or capacity in the native organ, restoration of certain markers and physiological indicators associated with disease, and / or expression of certain markers in the native organ. Those skilled in the art will understand that other indicators may be suitable for characterizing a regenerated organ.

[0053] "Regenerated kidney" refers to a native kidney after transplantation or administration of a cell population, mixture, or construct described herein. In certain embodiments, the regenerated kidney is characterized by various indicators, including, but not limited to, development of function or capacity in the native kidney, improvement of function or capacity in the native kidney, restoration of certain markers and physiological indicators associated with renal disease, and expression of certain markers in the native kidney. Those skilled in the art will understand that other indicators may be suitable for characterizing a regenerated kidney.

[0054] A "small molecule" is a compound with a mass of less than 2000 daltons. The molecular mass of a small molecule is preferably less than 1000 daltons, more preferably less than 600 daltons, e.g., the compound may be 500 daltons, 400 daltons, 300 daltons, 200 daltons, or 10 0 Daltons. In certain embodiments, the small molecule is an organic compound.

[0055] "Microvesicles" are cell-derived membranous extracellular vesicles with a diameter between 30 nanometers (nm) and 1000 nanometers (nm). "Exosomes" are cell-derived membranous microvesicles with a diameter of approximately 30 nm to 150 nm. In certain embodiments, exosomes are cell-derived membranous microvesicles with a diameter of approximately 50 nm to 100 nm. Additional characteristics typically shared by exosomes are known in the art. A non-limiting description of microvesicles and exosomes is provided in Zhang et al. (2016) Am J Physiol Renal Physiol. 311(5):F844-F851, the entire contents of which are incorporated herein by reference.

[0056] As used herein, "effective" when referring to an amount of therapeutic agent (such as microvesicles, e.g., exosomes, alone or in combination with bioactive renal cells) refers to an amount of therapeutic agent sufficient to produce a desired therapeutic response when used as disclosed herein without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit / risk ratio.

[0057] Secreted products Provided herein are products secreted by bioactive kidney cells (e.g., SRCs), such as vesicles. In certain embodiments, the vesicles comprise microvesicles.

[0058] In certain embodiments, the microvesicles are about 30 nm to 150 nm, 30 nm to 200 nm, 30 nm to 500 nm, 30 nm to 1000 nm, 500 nm to 1000 nm, 50 nm to 1000 nm, 50 nm to 200 nm, 50 nm to 150 nm, 50 nm to 100 nm, 100 nm to 150 nm, 100 nm to 200 nm, or 100 nm to 300 nm in diameter.

[0059] In certain embodiments, the vesicles comprise, consist essentially of, or consist of exosomes. In certain embodiments, the exosomes are about 50 nm to 100 nm in diameter. In certain embodiments, the exosomes are 30 nm to 100 nm, 50 nm to 150 nm, 50 nm to 100 nm, 100 nm to 150 nm, or 30 nm to 150 nm in diameter. In certain embodiments, the exosomes are about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, or 60 nm to about 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, or 150 nm in diameter.

[0060] In certain embodiments, the vesicle comprises an active agent (such as a compound) on its outer surface, in its lipid bilayer, and / or in its interior cavity. In certain embodiments, the compound attenuates one or more cellular pathways. In certain embodiments, the compound is a protein, a small molecule, or a polynucleotide. In certain embodiments, the protein is a transmembrane protein located in the membrane of the vesicle. In certain embodiments, the compound is lipophilic and located in the lipid bilayer of the exosome. In certain embodiments, the polynucleotide is an miRNA molecule.

[0061] In certain embodiments, the compound is expressed or produced by bioactive kidney cells. In certain embodiments, the compound is not expressed or produced by bioactive kidney cells. In certain embodiments, the compound was added to the culture medium of the vesicle-producing cells (e.g., the cells were incubated in culture medium containing the compound). In certain embodiments, the compound entered the cells and was contained in vesicles produced by the cells. In certain embodiments, the vesicles are purified or isolated from the cells and then incubated in a solution (e.g., culture medium) containing the compound. In certain embodiments, the vesicles are isolated or purified from the cells and then permeabilized by techniques (e.g., sonication, lipofection) to permeabilize the vesicle membrane and facilitate compound entry. The compound is then loaded into the vesicles (by injection, electroporation, etc.).

[0062] In certain embodiments, the compound is not produced by naturally occurring kidney cells. In certain embodiments, the compound is a cytokine. In certain embodiments, the compound is an artificial compound. In certain embodiments, the artificial compound is a drug. In certain embodiments, the artificial compound is a small molecule. In certain embodiments, the artificial compound is a biologic. In certain embodiments, the artificial compound is not expressed or produced by kidney cells in a native kidney. In certain embodiments, the compound is a cell survival agent. In certain embodiments, the compound is a compound used to treat a disease (such as a kidney disease or some other disease). In certain embodiments, the compound is tolerogenic or anti-inflammatory.

[0063] In certain embodiments, the compound is approved by the U.S. Food and Drug Administration for administration to humans for the treatment of a disease. In certain embodiments, the compound is used to cure, mitigate, treat, or prevent a disease. In certain embodiments, the compound is used to alter the structure or function of a mammalian cell or organism.

[0064] In certain embodiments, the vesicles comprise a compound that attenuates plasminogen activator inhibitor-1 (PAI-1) signaling and / or transforming growth factor β (TGFβ) signaling. In certain embodiments, the vesicles comprise a compound that attenuates canonical Wnt signaling. In certain embodiments, the vesicles comprise a compound that attenuates non-canonical Wnt signaling. In certain embodiments, the vesicles comprise a compound that attenuates CXCR4-mediated signaling. In certain embodiments, the vesicles comprise a compound that downregulates a pro-inflammatory cytokine. In certain embodiments, the pro-inflammatory cytokine is IL8. In certain embodiments, the vesicles comprise a compound that attenuates Notch signaling.

[0065] In certain embodiments, the compound is a cell surface molecule used in immunophenotyping of cells, hi certain embodiments, the compound is CD9, CD63, CD81, CD133, CD146, CD326, CD40, CD42a, CD44, or CD49e.

[0066] In certain embodiments, the compound is a protein receptor, hi certain embodiments, the protein receptor is retinoid-related receptor 4 (ROR4).

[0067] In certain embodiments, the compound is a developmental marker, hi certain embodiments, the developmental marker is stage-specific embryonic antigen-4 (SSEA-4).

[0068] In certain embodiments, the compound is a stress-protective protein, hi certain embodiments, the stress-protective protein is heat shock protein (HSP) 70 or HSP90.

[0069] In certain embodiments, the compound is a scaffold protein. In certain embodiments, the scaffold protein is TST101.

[0070] In certain embodiments, the compound is an miRNA and is in the lumen of the vesicle.

[0071] In certain embodiments, the miRNA is a cell cycle-regulating miRNA. In certain embodiments, the cell cycle-regulating miRNA is let7a, miR-143, or miR22.

[0072] In certain embodiments, the miRNA is a cellular senescence-regulating miRNA. In certain embodiments, the cellular senescence-regulating miRNA is miR-34.

[0073] In certain embodiments, the miRNA is a cell migration-regulating miRNA. In certain embodiments, the cell migration-regulating miRNA is miR30-C.

[0074] In certain embodiments, the miRNA is a cell growth regulatory miRNA. In certain embodiments, the cell growth regulatory miRNA is miR194-2.

[0075] In certain embodiments, the miRNA is a cell signaling pathway-regulating miRNA. In certain embodiments, the cell signaling pathway-regulating miRNA is miR-142.

[0076] In certain embodiments, the miRNA is an inflammation-regulating miRNA. In certain embodiments, the inflammation-regulating miRNA is miR-10a.

[0077] In certain embodiments, the miRNA is an angiogenesis-regulating miRNA. In certain embodiments, the angiogenesis-regulating miRNA is miR-296 and / or miR-146a.

[0078] In certain embodiments, the miRNA is a kinase activity-regulating miRNA. In certain embodiments, the kinase activity-regulating miRNA is miR-83.

[0079] In certain embodiments, the compound is an miRNA that inhibits PAI-1, TGFβ, canonical Wnt signaling, non-canonical Wnt signaling, CXCR4-mediated signaling, and / or Notch signaling.

[0080] In certain embodiments, renal fibrosis is reduced or prevented by inhibiting epithelial-mesenchymal transition (EMT).

[0081] In certain embodiments, the vesicles provided herein comprise miR-145, miR-22, miR-7, miR-10a, miR-143, and / or let7b.

[0082] In certain embodiments, the vesicles provided herein comprise miR-1248, miR-3168, miR-7113-5p, miR-758-3p, miR-937-3p, miR-4455, miR-4521, miR-203a-3p, miR-22-3p, miR-574-3p, miR-181b-5p, miR-1260b, and / or miR-181b-5p.

[0083] In certain embodiments, the vesicles provided herein comprise CD9, CD63, CD81, CD133, CD146, CD326, CD40, CD42a, CD44, CD49e, and / or SSEA-4.

[0084] In certain embodiments, the vesicles provided herein comprise CD63, CD9, and / or CD81, wherein CD63, CD9, and / or CD81, or portions thereof, are on the exterior surface of the vesicle, while in certain embodiments, portions of one or more of these proteins are on the interior of the vesicle.

[0085] In certain embodiments, the vesicles provided herein comprise CD133, CD326, and / or CD49e, wherein the CD133, CD326, and / or CD49e are on the outer surface of the vesicle.

[0086] In certain embodiments, proliferation of renal cells contacted with vesicles is increased compared to renal cells not contacted with vesicles. In certain embodiments, angiogenesis by endothelial cells contacted with vesicles is increased compared to endothelial cells not contacted with vesicles. In certain embodiments, nephron tubule formation by renal cells contacted with vesicles is increased compared to renal cells not contacted with vesicles.

[0087] In certain embodiments, the vesicles comprise phospholipids, sphingolipids, cholesterol, ceramide, and / or phosphatidylcholine.

[0088] In certain embodiments, the vesicles are in a composition comprising a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutically acceptable carrier comprises an aqueous solution. In certain embodiments, the pharmaceutically acceptable carrier is temperature-sensitive. In certain embodiments, the pharmaceutically acceptable carrier is a hydrogel. In certain embodiments, the pharmaceutically acceptable carrier comprises gelatin.

[0089] In certain embodiments, the vesicles are produced by BRCs, such as primary renal cells. In certain embodiments, the vesicles are produced by SRCs. Included herein are compositions comprising vesicles from primary renal cells and vesicles from SRCs. Also provided are compositions further comprising vesicles secreted by endothelial cells or mesenchymal stem cells. In certain embodiments, the compositions provided herein comprise non-renal cell vesicles. In certain embodiments, the non-renal cell vesicles are secreted by non-renal endothelial progenitor cells, non-renal mesenchymal stem cells, or non-renal adipose-derived progenitor cells.

[0090] In one aspect, the present disclosure provides a method for detecting at least one compound in a vesicle. In certain embodiments, the method includes obtaining a vesicle and detecting whether at least one compound is present in the vesicle, wherein (i) at least one compound is a protein, and the protein is CD9, CD81, CD146, CD326, CD40, CD42a, CD44, CD49e, and / or SSEA-4; (ii) at least one compound is an miRNA, and the miRNA includes at least two of miR-145, miR-22, miR-7, miR-10a, miR-143, and / or let7b; and / or (iii) at least one compound is not expressed or produced by renal cells in a native kidney.

[0091] In certain embodiments, the vesicles are obtained in or from a biological sample from a subject. In certain embodiments, the biological sample is urine. In certain embodiments, the vesicles are obtained in or from the supernatant of a renal cell culture. In certain embodiments, the vesicles are secreted by renal cells. In certain embodiments, the renal cells are bioactive renal cells. In certain embodiments, the renal cells are selected renal cells.

[0092] In certain embodiments, detecting whether a protein is present in a vesicle comprises an immunoassay. In certain embodiments, detecting whether a protein is present in a vesicle comprises an enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, immunoelectrophoresis, Western blot, or protein immunostaining. In certain embodiments, detecting whether a protein is present in a vesicle comprises spectroscopy. In certain embodiments, detecting whether a protein is present in a vesicle comprises high-performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS).

[0093] In certain embodiments, detecting whether the miRNA is in the vesicles comprises polymerase chain reaction (PCR). In certain embodiments, detecting whether the miRNA is in the vesicles comprises reverse transcriptase PCR. In certain embodiments, detecting whether the miRNA is in the vesicles comprises reverse transcriptase PCR. Detecting whether miRNA is present in a vesicle comprises microarray analysis. In certain embodiments, detecting whether miRNA is present in a vesicle comprises RNA sequencing. In certain embodiments, detecting whether miRNA is present in a vesicle comprises contacting the vesicle or a processed sample suspected of containing nucleic acid from the vesicle with a probe or primer complementary to the miRNA. In certain embodiments, detecting whether miRNA is present in a vesicle does not comprise microarray analysis. In certain embodiments, detecting whether miRNA is present in a vesicle comprises microarray analysis using a microarray containing probes for less than 1000, 500, or 100 different miRNAs.

[0094] In certain embodiments, the compound is a small molecule.

[0095] In certain embodiments, the compound is expressed or produced by bioactive kidney cells. In certain embodiments, the compound is not expressed or produced by bioactive kidney cells. In certain embodiments, the compound is expressed or produced by bioactive kidney cells. In certain embodiments, the compound is not expressed or produced by bioactive kidney cells. In certain embodiments, the compound was added to the culture medium of vesicle-producing cells (e.g., cells were incubated in medium containing the compound). In certain embodiments, the compound entered the cells and was contained in vesicles produced by the cells. In certain embodiments, vesicles are purified or isolated from cells and then incubated in a solution (e.g., medium) containing the compound. In certain embodiments, vesicles are isolated or purified from cells, and then the compound is incorporated into the vesicles by a technique that permeabilizes the exosome membrane to facilitate compound entry (e.g., by sonication, lipofection, electroporation, etc.).

[0096] In certain embodiments, the compound is not produced by naturally occurring kidney cells. In certain embodiments, the compound is a cytokine. In certain embodiments, the compound is an artificial compound. In certain embodiments, the compound is a drug. In certain embodiments, the artificial compound is not expressed or produced by kidney cells in a native kidney. In certain embodiments, the compound is a cell survival agent. In certain embodiments, the compound is a compound used to treat a disease (such as a kidney disease or some other disease). In certain embodiments, the compound is tolerogenic or anti-inflammatory. In certain embodiments, the compound is approved by the U.S. Food and Drug Administration for administration to humans for the treatment of a disease. In certain embodiments, the compound is used to cure, mitigate, treat, or prevent a disease. In certain embodiments, the compound is used to alter the structure or function of a mammalian cell or organism.

[0097] In one aspect, provided herein are methods for monitoring treatment with a bioactive renal cell population in a subject to which the bioactive renal cell population has been administered. In certain embodiments, the method comprises detecting whether at least one compound is present in vesicles from the subject according to the methods disclosed herein.

[0098] In certain embodiments, the method comprises detecting whether at least one compound is present in vesicles from a subject at a first time point and a second time point according to the methods disclosed herein. In certain embodiments, the first time point is before the subject is administered a bioactive renal cell population, and the second time point is after the subject is administered a bioactive renal cell population. In certain embodiments, the first time point and the second time point are after the subject is administered a bioactive renal cell population. In certain embodiments, the method further comprises determining a regenerative effect in the subject if the level of the compound is higher at the first time point compared to the second time point.

[0099] In certain embodiments, the method further comprises determining if the level of the compound is higher than the control. and determining the regenerative effect in the subject. In certain embodiments, the control is a level in a matched subject who has not been administered the bioactive renal cell population.

[0100] Also provided herein are methods for determining whether a vesicle is regenerative. In certain embodiments, the method includes (i) detecting whether a protein and / or miRNA is present in the vesicle according to a method disclosed herein, and (ii) determining the vesicle as regenerative if the protein and / or miRNA is detected in the vesicle.

[0101] In one aspect, provided herein are methods for detecting the level of at least one miRNA in vesicles from a population of bioactive kidney cells. In certain embodiments, the method comprises: (i) detecting that one or more of the following miRNA molecules are expressed in the vesicles: miR-1248, miR-3168, miR-7113-5p, miR-758-3p, miR-937-3p, miR-4455, miR-4521, miR-203a-3p, miR-22-3p, miR-574-3p, miR-181b-5p, miR-1260b, and / or miR-181b-5p; and / or (ii) detecting whether one or more of the following miRNA molecules: miR-1-3p, miR-1-3p, miR-143-3p, miR-150-5p, miR-509-3p, miR-653-5p, miR-204-5p, miR-192-5p, and / or miR-363-3p, are decreased in the vesicles compared to the control. In certain embodiments, the bioactive renal cells are selected renal cells. In certain embodiments, the control is the level of one or more miRNA molecules in vesicles from a primary renal cell population. In certain embodiments, the control is the level of one or more miRNA molecules in vesicles from another bioactive renal cell population.

[0102] The present subject matter also provides methods for altering the level of at least one miRNA and / or protein in vesicles produced by a population of bioactive renal cells, the method comprising culturing the population under hypoxic conditions. In certain embodiments, culturing the population under hypoxic conditions comprises culturing the population in the presence of less than about 5%, 4%, 3%, 2%, or 1% oxygen, for example, for 8 to 72 hours. In certain embodiments, culturing the population under hypoxic conditions comprises culturing the population in the presence of about 1% to 5%, 2% to 5%, 2% to 4%, 1% to 3%, or 1.5% to 2.5% oxygen, for example, for 8 to 72 hours. In certain embodiments, culturing the population under hypoxic conditions comprises culturing the population in the presence of less than 5% oxygen for at least about 8, 12, 16, 20, 24, or 48 hours. In certain embodiments, culturing the population under hypoxic conditions comprises culturing the population in the presence of about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5% oxygen for about 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, or 48 hours.

[0103] In certain embodiments, the method further comprises culturing the population under hypoxic conditions after passage of the bioactive kidney cells at least about 1, 2, or 3 times.

[0104] In certain embodiments, (a) the at least one miRNA is selected from the group consisting of miR-145, miR-22, miR-7, miR-10a, miR-143, let7b, miR-1248, miR-3168, miR-7113-5p, miR-758-3p, miR-937-3p, miR-4455, miR-4521, miR-203a-3p, miR- and / or (b) at least one protein is selected from the group consisting of CD9, CD63, CD81, CD133, CD146, CD326, CD40, CD42a, CD44, CD49e, SSEA-4, TST101, HSP70, and HSP9. 0, and / or ROR4.

[0105] In one aspect, provided herein is a method for producing exosomes from cells containing a compound not produced by the cells. In certain embodiments, the method comprises isolating exosomes from a cell culture supernatant, wherein the cell culture supernatant is from a culture of cells contacted with a compound. In one aspect, provided herein is a method for producing renal exosomes, wherein the exosomes contain a compound not produced by renal cells in a native kidney. In certain embodiments, the method comprises isolating vesicles from a renal cell culture supernatant, wherein the renal cell culture supernatant is from a culture of renal cells containing a bioactive renal cell population contacted with a compound. In certain embodiments, the compound is an artificial compound. In certain embodiments, the compound is a small molecule. In certain embodiments, the compound is a cell viability agent. In certain embodiments, the compound is a drug.

[0106] In one aspect, included herein are vesicles (microvesicles, such as exosomes) that contain compounds that are not produced by renal cells in the native kidney.

[0107] In certain embodiments, the compound is a protein, a small molecule, or a polynucleotide. In certain embodiments, the compound is not expressed or produced by primary kidney cells cultured in the absence of the compound. In certain embodiments, the compound is an artificial compound.

[0108] In certain embodiments, a compound (such as a protein or small molecule drug) is "passively loaded" into a vesicle (e.g., a microvesicle such as an exosome), for example, by incubating cells with medium containing the compound, or by incubating purified vesicles (e.g., a microvesicle such as an exosome) with medium containing the compound. In certain embodiments, a compound (such as a protein or small molecule drug) is "actively loaded" into a purified or isolated vesicle (e.g., a microvesicle such as an exosome) by permeabilizing the vesicle membrane to facilitate entry of the compound (e.g., by sonication, lipofection, electroporation, etc.).

[0109] In certain embodiments, the vesicles are present in a composition comprising cells that produced the vesicles. In certain embodiments, the vesicles are isolated from the cells that produced them. In certain embodiments, the vesicles are renal vesicles. In certain embodiments, the vesicles are produced by bioactive renal cells.

[0110] In certain embodiments, the compositions provided herein comprise renal cell vesicles and non-renal cell vesicles. In certain embodiments, the renal cell vesicles are secreted by bioactive renal cells. In certain embodiments, the non-renal cell vesicles are secreted by non-renal endothelial progenitor cells, non-renal mesenchymal stem cells, or non-renal adipose-derived progenitor cells.

[0111] In certain embodiments, the compositions provided herein comprise vesicles produced by primary kidney cells and vesicles produced by selected kidney cells.

[0112] In certain embodiments, the vesicles further comprise a pharmaceutically acceptable carrier.

[0113] In certain embodiments, the regenerative effect may be provided by the cells and / or by products (such as vesicles) secreted by the bioactive kidney cells. In certain embodiments, the regenerative effect may be the following: reduction in epithelial-mesenchymal transition (which may be via attenuation of TGF-β signaling), reduction in renal fibrosis, reduction in nephritis, differential expression of stem cell markers in the native kidney, migration of transplanted cells and / or native cells to sites of renal injury, e.g., tubular injury, reduction in renal injury, e.g., urinary This may be characterized by one or more of the following: engraftment of transplanted cells at the site of tubular injury, stabilization of one or more indicators of kidney function (as described herein), de novo formation of S-shaped / C-shaped bodies associated with nephrogenesis, de novo formation of renal tubules or nephrons, restoration of erythroid homeostasis (as described herein), and any combination thereof (see also Basu et al., 2011. Functional evaluation of primary renal cell / biomaterial neo-kidney augment prototypes for renal tissue engineering. Cell Transplantation 20: 1771-90; Bruce et al., 2015. Selected renal cells modulate disease progression in rodent models of chronic kidney disease via NF-κB and TGF-β1 pathways. Regenerative Medicine 10: 815-839, the entire contents of each of which are incorporated herein by reference).

[0114] In certain embodiments, as an alternative to tissue biopsies, regenerative outcomes in treated subjects can be assessed from examination of bodily fluids, such as urine. Microvesicles (e.g., exosomes) obtained from a subject's urine source have been found to contain certain components, including, but not limited to, specific proteins and miRNAs that ultimately derive from renal cell populations affected by the treatment. These components may include, but are not limited to, factors involved in stem cell replication and differentiation, apoptosis, inflammation and immunoregulation, fibrosis, epithelial-mesenchymal transition, TGF-β signaling, and / or PAI-1 signaling. In certain embodiments, temporal analysis of miRNA / protein expression patterns associated with microvesicles (e.g., exosomes) allows for continuous monitoring of regenerative outcomes within the kidney of subjects administered with the cell populations, cell products, or constructs of the present disclosure.

[0115] In certain embodiments, the present disclosure provides a method for assessing whether a kidney disease (KD) patient will respond to treatment with a therapeutic formulation. In certain embodiments, the method may include measuring or detecting the amount of microvesicles (or their luminal contents), e.g., exosomes, in a test sample obtained from a KD patient treated with a therapeutic agent, compared to or relative to the amount of microvesicles (e.g., exosomes) in a control sample (e.g., a sample from the same patient before treatment with the therapeutic agent), wherein a greater or lesser amount of microvesicles (e.g., exosomes) or their luminal contents in the test sample compared to the amount of microvesicles (e.g., exosomes) or their luminal contents in the control sample is an indication of the patient's response to treatment with the therapeutic agent.

[0116] In certain embodiments, kidney-derived microvesicles (e.g., exosomes) and / or the luminal contents of kidney-derived microvesicles (e.g., exosomes) can be released into a subject's urine and analyzed for biomarkers indicative of regenerative outcome or therapeutic efficacy. In certain embodiments, the non-invasive prognostic methods provided herein can include obtaining a urine sample from a subject before and / or after administration or transplantation of a bioactive renal cell population, cell product, or construct described herein. Microvesicles and other secreted products can be purified by methods such as, but not limited to, centrifugation to remove unwanted debris (Zhou et al. 2008. Kidney Int. 74(5):613-621; Skog et al., U.S. Patent Application Publication No. 20110053157; and others). These can be isolated from urine samples using standard techniques, including precipitation to isolate microvesicles (e.g., exosomes) from urine, polymerase chain reaction and nucleic acid sequencing to identify specific nucleic acids, and mass spectroscopy and / or 2D gel electrophoresis to identify specific proteins associated with regenerative outcomes (all of which are incorporated herein by reference in their entireties).

[0117] cell population Included herein are compositions and formulations comprising vesicles (e.g., microvesicles such as exosomes) produced by renal cell populations (e.g., primary cells and / or BRCs such as SRCs). In certain embodiments, vesicles can be isolated from or combined with cells that do not produce them, for example. Non-limiting examples and characteristics of BRCs useful for producing vesicles include: As shown herein.

[0118] In certain embodiments, the therapeutic compositions or formulations provided herein contain microvesicles (e.g., exosomes) secreted by an isolated heterogeneous kidney cell population enriched for specific bioactive components or cell types and / or depleted for specific inactive or undesirable components or cell types. In certain embodiments, the therapeutic compositions or formulations provided herein contain or further contain an isolated heterogeneous kidney cell population enriched for specific bioactive components or cell types and / or depleted for specific inactive or undesirable components or cell types. In certain embodiments, such compositions and formulations are used in the treatment of kidney disease, e.g., resulting in stabilization and / or improvement and / or regeneration of kidney function and / or structure. In certain embodiments, the compositions contain microvesicles (e.g., exosomes) secreted by an isolated kidney cell fraction that lacks cellular components compared to healthy individuals but retains therapeutic properties, e.g., resulting in stabilization and / or improvement and / or regeneration of kidney function. In certain embodiments, the compositions contain isolated renal cell fractions that lack cellular components compared to healthy individuals but retain therapeutic properties, e.g., result in stabilization and / or improvement and / or regeneration of kidney function. In certain embodiments, the cell populations described herein can be derived from healthy individuals, individuals suffering from kidney disease, or subjects described herein.

[0119] Included herein are therapeutic compositions for administering microvesicles (e.g., exosomes) and / or selected renal cell populations to a target organ or tissue in a subject. In certain embodiments, provided herein are compositions comprising microvesicles (e.g., exosomes) secreted by a BRC (e.g., SRC). In certain embodiments, the composition further comprises a BRC (e.g., SRC) that did not secrete microvesicles (e.g., exosomes). In certain embodiments, the composition comprises an NKA "spiked" with microvesicles (e.g., microvesicles such as exosomes are added to an NKA to create a vesicle-enriched NKA). In certain embodiments, the BRC (e.g., SRC) from which the microvesicles (e.g., exosomes) can be obtained includes, for example, any BRC (e.g., SRC) disclosed herein. In certain embodiments, the vesicles are obtained from an SRC produced according to the method described in Example 1. In certain embodiments, the formulations provided herein are NKAs to which isolated vesicles (such as microvesicles, e.g., exosomes) have been added (the NKAs are "spiked" or supplemented with vesicles).

[0120] In certain embodiments, a selected bioactive renal cell population generally refers to a cell population that potentially has therapeutic properties upon administration to a subject. In certain embodiments, when administered to a subject in need, the bioactive renal cell population may result in stabilization and / or improvement and / or repair and / or regeneration of kidney function in the subject. In certain embodiments, the therapeutic properties may include a repair or regenerative effect.

[0121] In certain embodiments, the renal cell population is an unfractionated heterogeneous cell population or an enriched homogeneous cell population derived from the kidney. In certain embodiments, the heterogeneous cell population is isolated from a tissue biopsy or whole organ tissue. In certain embodiments, the renal cell population is derived from an in vitro culture of mammalian cells established from a tissue biopsy or whole organ tissue. In certain embodiments, the renal cell population comprises a subfraction or subpopulation of a heterogeneous renal cell population enriched for bioactive components (e.g., bioactive renal cells) and depleted of inactive or undesirable components or cells.

[0122] In certain embodiments, the renal cell population expresses GGT and cytokeratin. In certain embodiments, GGT is expressed at greater than about 10%, about 15%, about 18%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% expression. In certain embodiments, the GGT is GGT-1. In certain embodiments, the cells of the renal cell population express GGT-1, cytokeratin, VEGF, and KIM-1. In certain embodiments, more than 18% of the cells in the renal cell population express GGT-1. In certain embodiments, more than 80% of the cells in the renal cell population express cytokeratin. In certain embodiments, the cytokeratin is selected from CK8, CK18, CK19, and combinations thereof. In certain embodiments, the cytokeratin is CK8, CK18, CK19, CK8 / CK18, CK8 / CK19, CK18 / CK19, or CK8 / CK18 / CK19, where " / " refers to the combination of the cytokeratin adjacent to it. In certain embodiments, the cytokeratin has an expression level of greater than about 80%, about 85%, about 90%, or about 95%. In certain embodiments, more than 80% of the cells in the renal cell population express cytokeratin. In certain embodiments, the renal cell population expresses AQP2. In certain embodiments, less than 40% of the cells express AQP2. In certain embodiments, at least 3% of the cells in the renal cell population express AQP2.

[0123] In certain embodiments, more than 18% of the cells in the cell population express GGT-1, and more than 80% of the cells in the cell population express cytokeratin. In certain embodiments, the cytokeratin is CK18. In certain embodiments, 4.5% to 81.2% of the cells in the cell population express GGT-1, 3.0% to 53.7% of the cells in the cell population express AQP2, and 81.1% to 99.7% of the cells in the cell population express CK18.

[0124] In certain embodiments, the renal cell population comprises AQP1, AQP2, AQP4, calbindin, calponin, CD117, CD133, CD146, CD24, CD31 (PECAM-1), CD54 (ICAM-1), CD73, CK18, CK19, CK7, CK8, CK8, CK18, CK19, a combination of CK8, CK18, and CK19, connexin 43, cubilin, CXCR4 (fusin), DBA, E-cadherin (CD324), EPO (erythropoietin), GGT1, GLEPP1 (glomerular epithelial protein 1), haptoglobulin, Itgbl, KIM-1 (kidney injury detection). The present invention also includes cells expressing one or more of any combination of biomarkers selected from: T1M-1 (T-cell immunoglobulin and mucin-containing molecule), MAP-2 (microtubule-associated protein 2), megalin, N-cadherin, nephrin, NKCC (Na-K-Cl-cotransporter), OAT-1 (organic anion transporter 1), osteopontin, pan-cadherin, PCLP1 (podocalyxin-like 1 molecule), podocin, SMA (smooth muscle alpha-actin), synaptopodin, THP (Tamm-Horsfall protein), vimentin, and αGST-1 (alpha glutathione S-transferase).

[0125] In certain embodiments, the renal cell population is enriched for epithelial cells compared to a starting population, such as a cell population in a kidney tissue biopsy or primary culture thereof (e.g., the renal cell population comprises at least about 5%, 10%, 15%, 20%, or 25% more epithelial cells than the starting population). In certain embodiments, the renal cell population is enriched for tubular cells compared to a starting population, such as a cell population in a kidney tissue biopsy or primary culture thereof (e.g., the renal cell population comprises at least about 5%, 10%, 15%, 20%, or 25% more tubular cells than the starting population). In certain embodiments, the tubular cells comprise proximal tubule cells. In certain embodiments, the renal cell population has a lower proportion of distal tubule cells, collecting duct cells, endocrine cells, vascular cells, or progenitor-like cells compared to the starting population. In certain embodiments, the renal cell population has a lower proportion of distal tubule cells compared to the starting population. In certain embodiments, the renal cell population has a lower percentage of collecting duct cells compared to the starting population. In certain embodiments, the renal cell population has a lower percentage of endocrine cells compared to the starting population. In certain embodiments, the renal cell population has a lower percentage of vascular cells compared to the starting population. In certain embodiments, the renal cell population has a lower percentage of ductal cells compared to the starting population. The renal cell population has a lower proportion of progenitor-like cells compared to the starting population. In certain embodiments, the renal cell population has a higher proportion of tubular cells and a lower proportion of EPO-producing cells, glomerular cells, and vascular cells compared to a non-enriched population (e.g., the starting renal cell population). In certain embodiments, the renal cell population has a higher proportion of tubular cells and a lower proportion of EPO-producing cells and vascular cells compared to a non-enriched population. In certain embodiments, the renal cell population has a higher proportion of tubular cells and a lower proportion of glomerular cells and vascular cells compared to a non-enriched population.

[0126] In certain embodiments, cells of the renal cell population express hyaluronic acid (HA). In certain embodiments, the size range of HA is from about 5 kDa to about 20,000 kDa. In certain embodiments, the HA has a molecular weight of 5 kDa, 60 kDa, 800 kDa, and / or 3,000 kDa. In certain embodiments, the renal cell population synthesizes and / or stimulates the synthesis of high molecular weight HA through expression of hyaluronan synthase 2 (HAS-2), particularly after intrarenal transplantation. In certain embodiments, cells of the renal cell population express higher molecular weight species of HA in vitro and / or in vivo through the action of HAS-2. In certain embodiments, cells of the renal cell population express higher molecular weight species of HA both in vitro and in vivo through the action of HAS-2. In certain embodiments, the higher molecular weight species of HA is HA having a molecular weight of at least 100 kDa. In certain embodiments, the higher molecular weight species HA is HA having a molecular weight of about 800 kDa to about 3500 kDa. In certain embodiments, the higher molecular weight species HA is HA having a molecular weight of about 800 kDa to about 3000 kDa. In certain embodiments, the higher molecular weight species HA is HA having a molecular weight of at least 800 kDa. In certain embodiments, the higher molecular weight species HA is HA having a molecular weight of at least 3000 kDa. In certain embodiments, the higher molecular weight species HA is HA having a molecular weight of about 800 kDa. In certain embodiments, the higher molecular weight species HA is HA having a molecular weight of about 3000 kDa. In certain embodiments, the HAS-2 is 2×10 5 Da~2×10 6In certain embodiments, the smaller species of HA are synthesized with a molecular weight of about 200 kDa to about 2000 kDa. In certain embodiments, the smaller species of HA are formed by the action of a degradative hyaluronidase. In certain embodiments, the higher molecular weight species of HA are HA with a molecular weight of about 200 kDa to about 2000 kDa. In certain embodiments, the higher molecular weight species of HA are HA with a molecular weight of about 200 kDa. In certain embodiments, the higher molecular weight species of HA are HA with a molecular weight of about 2000 kDa. In certain embodiments, the higher molecular weight species of HA are HA with a molecular weight of at least 200 kDa. In certain embodiments, the higher molecular weight species of HA are HA with a molecular weight of at least 2000 kDa. In certain embodiments, the higher molecular weight species of HA are HA with a molecular weight of at least 5000 kDa. In certain embodiments, the higher molecular weight species of HA are HA with a molecular weight of at least 10,000 kDa. In certain embodiments, the higher molecular weight species of HA is HA having a molecular weight of at least 15,000 kDa, hi certain embodiments, the higher molecular weight species of HA is HA having a molecular weight of about 20,000 kDa.

[0127] In certain embodiments, the population comprises cells capable of receptor-mediated albumin transport.

[0128] In certain embodiments, the cells of the renal cell population are hypoxia resistant.

[0129] In certain embodiments, the renal cell population comprises one or more cell types that express one or more of any combination of megalin, cubilin, N-cadherin, E-cadherin, aquaporin-1, and aquaporin-2.

[0130] In certain embodiments, the renal cell population comprises one or more cell types that express any combination of one or more of megalin, cubilin, hyaluronate synthase 2 (HAS2), vitamin D3 25-hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin (Ecad), aquaporin-1 (Aqp1), aquaporin-2 (Aqp2), RAB17, RAS oncogene family member (Rab17), GATA binding protein 3 (Gata3), FXYD domain-containing ion transport regulator 4 (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).

[0131] In certain embodiments, the renal cell population is characterized by the expression of megalin, cubilin, hyaluronic acid synthase 2 (HAS2), vitamin D3 The present invention also includes one or more cell types that express one or more of the following: 25-hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin (Ecad), aquaporin-1 (Aqp1), aquaporin-2 (Aqp2), RAB17, RAS oncogene family member (Rab17), GATA binding protein 3 (Gata3), FXYD domain-containing ion transport regulator 4 (Fxyd4), solute carrier family 9 (sodium / hydrogen exchanger) member 4 (Slc9a4), aldehyde dehydrogenase 3 family member 81 (Aldh3b1), aldehyde dehydrogenase 1 family member A3 (Aldh1a3), and any combination of calpain-8 (Capn8) and aquaporin-4 (Aqp4).

[0132] In certain embodiments, the renal cell population is selected from the group consisting of aquaporin-7 (Aqp7), FXYD domain-containing ion transport regulator 2 (Fxyd2), solute carrier family 17 (sodium phosphate) member 3 (Slc17a3), solute carrier family 3 member 1 (Slc3a1), claudin 2 (Cldn2), napsin The present invention also includes one or more cell types expressing any combination of one or more of the following: A-aspartic peptidase (Napsa), solute carrier family 2 (facilitated glucose transporter) member 2 (Slc2a2), alanyl (membrane) aminopeptidase (Anpep), transmembrane protein 27 (Tmem27), acyl-CoA synthase medium chain family member 2 (Acsm2), glutathione peroxidase 3 (Gpx3), fructose-1,6-bisphosphatase 1 (Fbp1), alanine-glyoxylate aminotransferase 2 (Agxt2), platelet endothelial cell adhesion molecule (Pecam), and podocin (Podn).

[0133] In certain embodiments, the renal cell population expresses PECAM, VEGF, KDR, HIF1a, CD31, CD146, podocin (Podn), and nephrin (Neph), chemokine (C-X-C motif) receptor 4 (Cxcr4), endothelin receptor type B (Ednrb), type V collagen alpha 2 (Col5a2), cadherin 5 (Cdh5), tissue plasminogen activator (Plat), angiopoietin 2 (Angpt2), kinase insert domain protein (KIP). The present invention also includes one or more cell types that express any combination of one or more of the following: protein receptor (Kdr), secreted cysteine-rich acidic protein (osteonectin) (Sparc), serglycin (Srgn), TIMP metallopeptidase inhibitor 3 (Timp3), Wilms' tumor 1 (Wt1), wingless MMTV integration site family member 4 (Wnt4), regulator of G-protein signaling 4 (Rgs4), and erythropoietin (EPO).

[0134] In certain embodiments, the renal cell population comprises one or more cell types that express one or more of any combination of PECAM, vEGF, KDR, HIF1a, podocin, nephrin, EPO, CK7, CK8 / 18 / 19.

[0135] In certain embodiments, the renal cell population comprises one or more cell types that express one or more of any combination of PECAM, vEGF, KDR, HIF1a, CD31, and CD146.

[0136] In certain embodiments, the renal cell population comprises one or more cell types that express one or more of any combination of podocin (Podn) and nephrin (Neph).

[0137] In certain embodiments, the renal cell population comprises one or more cell types that express one or more of any combination of PECAM, vEGF, KDR, HIF1a, and EPO.

[0138] In certain embodiments, the presence (e.g., expression) and / or level / amount of various biomarkers in a sample or cell population can be analyzed by a number of techniques, many of which are known in the art and understood by those of skill in the art, including, but not limited to, immunohistochemistry ("IHC"), Western blot analysis, immunoprecipitation, molecular binding assays, ELISA, ELIFA, fluorescence activated cell sorting ("FACS"), MassARRAY, proteomics, biochemical enzyme activity assays, in situ hybridization, Southern analysis, Northern analysis, whole genome sequencing, polymerase chain reaction ("PCR") including quantitative real-time PCR ("qRT-PCR") and other amplification-based detection methods (e.g., branched-DNA, SISBA, TMA, etc.), RNA-Seq, FISH, microarray analysis, gene expression profiling, and / or serial analysis of gene expression ("SAGE"), as well as any one of a wide variety of assays that can be performed by protein, gene, and / or tissue array analysis. Non-limiting examples of protocols for assessing the status of genes and gene products include Northern blotting, Southern blotting, Western blotting, immunoblotting, and PCR analysis. In certain embodiments, multiplex immunoassays, such as those available from Rules Based Medicine or Meso Scale Discovery, can also be used. In certain embodiments, the presence (e.g., expression) and / or level / amount of various biomarkers in a sample or cell population can be analyzed by a number of techniques, many of which are known in the art and understood by those skilled in the art, including, but not limited to, "-omics" platforms such as genome-wide transcriptomics, proteomics, secretomics, lipidomics, phosphatomics, and exomics, where high-throughput techniques, coupled with computational biology and bioinformatics techniques, reveal the complete biological signature of genes, miRNAs, proteins, secreted proteins, lipids, microvesicles, etc., expressed and / or not expressed by the cell population under study.

[0139] In certain embodiments, a method for detecting the presence of two or more biomarkers in a renal cell population comprises contacting a sample containing the population with an antibody against the biomarker under conditions that allow binding of the antibody to its cognate ligand (i.e., biomarker), and detecting the presence of the bound antibody, e.g., by detecting whether a complex forms between the antibody and the biomarker. In certain embodiments, the presence of one or more biomarkers is detected by immunohistochemistry. In certain embodiments, a method for detecting the presence of a biomarker in or on a microvesicle (such as an exosome) comprises contacting a sample of the microvesicles (e.g., a sample suspected of or thought to contain microvesicles) with an antibody against the biomarker under conditions that allow binding of the antibody to its cognate ligand (i.e., biomarker), and detecting the presence of the bound antibody, e.g., by detecting whether a complex forms between the antibody and the biomarker.

[0140] As used herein, the term "detect" refers to quantitative and / or qualitative detection. include.

[0141] In certain embodiments, the biomarkers are detected by monoclonal or polyclonal antibodies.

[0142] In certain embodiments, the renal cell populations express or express a biomarker disclosed herein, e.g., AQP1, AQP2, AQP4, calbindin, calponin, CD117, CD133, CD146, CD24, CD31 (PECAM-1), CD54 (ICAM-1), CD73, CK18, CK19, CK7, CK8, CK8 / 18, CK8 / 18 / 19, connexin 43, cubilin, CXCR4 (fusin), DBA, E-cadherin (CD324), EPO (erythropoietin), GGT1, GLEPP1 (glomerular epithelial protein 1), haptoglobulin, Itgbl (integrin p), KIM-1 (kidney injury molecule-1), T1M-1 (T cell immunoglobulin and mucin-containing molecule), MAP-2 (microtubule-associated protein 2), megalin, N-cadherin, nephrin, NKCC (Na-K-Cl- cotransporter), OAT-1 (organic anion transporter 1), osteopontin, pan-cadherin, PCLP1 (podocalyxin-like 1 molecule), podocin, SMA (smooth muscle α-actin), synaptopodin, THP (Tamm-Horsfall protein), vimentin, and αGST-1 (alpha glutathione 5-transferase).

[0143] In certain embodiments, the source of the cells is the same as the intended target organ or tissue. In certain embodiments, the BRCs or SRCs can be derived from the kidney for use in formulations administered to the kidney. In certain embodiments, the cell population is derived from a kidney biopsy. In certain embodiments, the cell population is derived from whole kidney tissue. In certain embodiments, the cell population is derived from an in vitro culture of mammalian kidney cells established from a kidney biopsy or whole kidney tissue.

[0144] In certain embodiments, BRCs or SRCs comprise a heterogeneous mixture or fraction of bioactive renal cells. In certain embodiments, BRCs or SRCs can be derived from a healthy individual or are a renal cell fraction itself from a healthy individual. In certain embodiments, the term "renal cell populations" includes renal cell populations or fractions obtained from unhealthy individuals that may lack certain cell types compared to renal cell populations of healthy individuals (e.g., in the kidney or a biopsy thereof). In certain embodiments, the term "renal cell populations" includes therapeutically active cell populations that lack cell types compared to healthy individuals, as well as microvesicles (e.g., exosomes) secreted by the populations. Methods for detecting such cells and microvesicles (e.g., exosomes) are also provided. In certain embodiments, the cell populations are isolated and expanded from an autologous cell population.

[0145] In certain embodiments, SRCs are obtained by isolating and expanding renal cells from a patient's renal cortical tissue via kidney biopsy. In certain embodiments, renal cells are isolated from kidney tissue by enzymatic digestion, expanded by standard cell culture techniques, and selected from the expanded renal cells by centrifugation across a density boundary, density barrier, or density interface. In certain embodiments, renal cells are isolated from kidney tissue by enzymatic digestion, expanded by standard cell culture techniques, and selected from the expanded renal cells by continuous or discontinuous single-step or multi-step density gradient centrifugation. In certain embodiments, SRCs are primarily composed of renal epithelial cells, which are known for their regenerative capacity. In certain embodiments, other parenchymal (vascular) and stromal cells may be present in the autologous SRC population.

[0146] In certain embodiments, BRCs are an isolated population of regenerative kidney cells that are naturally involved in kidney repair and regeneration. In certain embodiments, BRCs are extracted from kidney tissue by enzymatic digestion. The renal cell population is obtained from renal cells isolated by hypoxia and expanded by standard cell culture techniques. In certain embodiments, the cell culture medium can be designed to expand bioactive renal cells with regenerative potential. In certain embodiments, the cell culture medium does not contain any differentiation factors. In certain embodiments, the expanded heterogeneous renal cell population is cultured under hypoxic conditions to further enrich for cells with regenerative potential. Without wishing to be bound by theory, this may be due to one or more of the following phenomena: 1) selective survival, death, or proliferation of specific cellular components during the hypoxic culture period; 2) changes in cellular granularity and / or size in response to hypoxic culture, which result in changes in buoyant density and subsequently in changes in localization during density gradient separation; and 3) changes in cellular gene / protein expression in response to the hypoxic culture period, which result in different characteristics of cells within the isolated and expanded population.

[0147] In certain embodiments, the bioactive renal cell population is obtained from the isolation and expansion of renal cells from kidney tissue (such as tissue obtained from a biopsy) under culture conditions that enrich for cells that enable kidney regeneration.

[0148] In certain embodiments, renal cells from kidney tissue (such as tissue obtained from a biopsy) are passaged one, two, three, four, five, or more times to produce expanded bioactive renal cells (such as a cell population enriched for cells that enable kidney regeneration). In certain embodiments, renal cells from kidney tissue (such as tissue obtained from a biopsy) are passaged once to produce expanded bioactive renal cells. In certain embodiments, renal cells from kidney tissue (such as tissue obtained from a biopsy) are passaged two times to produce expanded bioactive renal cells. In certain embodiments, renal cells from kidney tissue (such as tissue obtained from a biopsy) are passaged three times to produce expanded bioactive renal cells. In certain embodiments, renal cells from kidney tissue (such as tissue obtained from a biopsy) are passaged four times to produce expanded bioactive renal cells. In certain embodiments, renal cells from kidney tissue (such as tissue obtained from a biopsy) are passaged five times to produce expanded bioactive renal cells. In certain embodiments, passaging the cells depletes the cell population of non-bioactive kidney cells. In certain embodiments, passaging the cells depletes the cell population of at least one cell type. In certain embodiments, passaging the cells depletes the cell population of cells with a density greater than 1.095 g / ml. In certain embodiments, passaging the cells depletes the cell population of small cells with low granularity. In certain embodiments, passaging the cells depletes the cell population of cells smaller than red blood cells. In certain embodiments, passaging the cells depletes the cell population of cells with a diameter of less than 6 μm. In certain embodiments, passaging the cells depletes the cell population of cells with a diameter of less than 2 μm. In certain embodiments, passaging the cells depletes the cell population of cells with a granularity lower than red blood cells. In certain embodiments, the viability of the cell population increases after one or more passages. In certain embodiments, the descriptions small cells and low granularity are used when analyzing cells by fluorescence activated cell sorting (FACS), for example, using the XY axis of a scatter plot of cell occurrence.

[0149] In certain embodiments, expanded bioactive renal cells are grown under hypoxic conditions for at least about 6 hours, 9 hours, 10 hours, 12 hours, or 24 hours but less than 48 hours, or for 6 to 9 hours, or 6 to 48 hours, or for about 12 hours to about 15 hours, or about 8 hours, or about 12 hours, or about 24 hours, or about 36 hours, or about 48 hours. In certain embodiments, cells grown under hypoxic conditions are selected based on density. In certain embodiments, the bioactive renal cell population is an SRC population obtained after continuous or discontinuous (single-step or multi-step) density gradient separation of expanded renal cells (e.g., after passaging and / or culturing under hypoxic conditions). In certain embodiments, the bioactive renal cell population is an SRC population obtained after separation by centrifugation across a density boundary, density barrier, or density interface of expanded renal cells (e.g., after passaging and / or culturing under hypoxic conditions). (after ). In certain embodiments, hypoxic culture conditions are culture conditions in which cells are exposed to a reduced level of available oxygen in a culture system compared to standard culture conditions in which cells are cultured at atmospheric oxygen levels (about 21%). In certain embodiments, cells cultured under hypoxic culture conditions are cultured at an oxygen level of about 5% to about 15%, or about 5% to about 10%, or about 2% to about 5%, or about 2% to about 7%, or about 2%, or about 3%, or about 4%, or about 5%. In certain embodiments, the SRCs exhibit a buoyant density greater than about 1.0419 g / mL. In certain embodiments, the SRCs exhibit a buoyant density greater than about 1.04 g / mL. In certain embodiments, the SRCs exhibit a buoyant density greater than about 1.045 g / mL. In certain embodiments, the BRCs or SRCs contain a higher percentage of one or more cell populations and are devoid of or reduced in one or more other cell populations compared to the starting kidney cell population.

[0150] In certain embodiments, expanded bioactive renal cells can be subjected to density gradient separation to obtain SRCs. In certain embodiments, BRCs are subjected to both hypoxic culture conditions and density gradient separation to obtain SRCs. In certain embodiments, continuous or discontinuous single-step or multi-step density gradient centrifugation is used to separate the harvested renal cell population based on cell buoyant density. In certain embodiments, expanded bioactive renal cells can be separated by centrifugation across a density boundary, density barrier, or density interface to obtain SRCs. In certain embodiments, centrifugation across a density boundary or density interface is used to separate the harvested renal cell population based on cell buoyant density. In certain embodiments, SRCs are produced in part using OPTIPREP (Axis-Shield) medium containing a 60% (weight / volume) aqueous solution of the non-ionic iodine compound iodixanol. However, those skilled in the art will appreciate that other media, density gradients (continuous or discontinuous), density boundaries, density barriers, density interfaces, or other means, such as immunological separation using cell surface markers known in the art, having the characteristics necessary for isolating the cell populations described herein, can be used to obtain bioactive renal cells. In certain embodiments, a cell fraction exhibiting a buoyant density greater than about 1.04 g / mL is collected as a separate pellet after centrifugation. In certain embodiments, cells maintaining a buoyant density less than 1.04 g / mL are rejected and discarded. In certain embodiments, a cell fraction exhibiting a buoyant density greater than about 1.0419 g / mL is collected as a separate pellet after centrifugation. In certain embodiments, cells maintaining a buoyant density less than 1.0419 g / mL are rejected and discarded. In certain embodiments, a cell fraction exhibiting a buoyant density greater than about 1.045 g / mL is collected as a separate pellet after centrifugation. In certain embodiments, cells maintaining a buoyant density less than 1.045 g / mL are rejected and discarded.

[0151] In certain embodiments, cell buoyant density is used to obtain an SRC population and / or determine whether a renal cell population is a bioactive renal cell population. In certain embodiments, cell buoyant density is used to isolate bioactive renal cells. In certain embodiments, cell buoyant density is determined by centrifugation across a single-stage OptiPrep (7% iodixanol, 60% (wt / vol) in OptiMEM) density interface (single-stage discontinuous density gradient). Optiprep is a 60% (wt / vol) solution of iodixanol in water. When used in the exemplary density interface or single-stage discontinuous density gradient, Optiprep is diluted with OptiMEM (basal cell culture medium) to form a final solution of 7% iodixanol (in water and OptiMEM). The OptiMEM formulation is a modification of Eagle's minimum essential medium buffered with HEPES and sodium bicarbonate and supplemented with hypoxanthine, thymidine, sodium pyruvate, L-glutamine or GLUTAMAX, trace elements, and growth factors. Protein levels are minimal (15 μg / mL), with insulin and transferrin being the only protein supplements. A low concentration of phenol red is included as a pH indicator. In certain embodiments, OptiMEM can be supplemented with 2-mercaptoethanol prior to use.

[0152] In certain embodiments, an OptiPrep solution is prepared and the refractive index indicative of the desired density is measured prior to use (refractive index 1.3456±0.0004). In certain embodiments, kidney cells are layered onto the solution. In certain embodiments, the density interface or single-stage discontinuous density gradient is centrifuged at 800 g for 20 minutes at room temperature (no brake) in either a centrifuge tube (e.g., a 50 ml conical tube) or a cell processing device (e.g., a COBE 2991). In certain embodiments, a cell fraction exhibiting a buoyant density greater than about 1.04 g / mL is collected as a separate pellet after centrifugation. In certain embodiments, cells maintaining a buoyant density less than 1.04 g / mL are discarded and discarded. In certain embodiments, a cell fraction exhibiting a buoyant density greater than about 1.0419 g / mL is collected as a separate pellet after centrifugation. In certain embodiments, cells maintaining a buoyant density less than 1.0419 g / mL are discarded and discarded. In certain embodiments, the cell fraction exhibiting a buoyant density greater than about 1.045 g / mL is collected as a separate pellet after centrifugation. In certain embodiments, cells that maintain a buoyant density less than 1.045 g / mL are discarded and discarded. In certain embodiments, prior to cell density assessment or density-based selection, cells are cultured until they are at least 50% confluent and incubated overnight (e.g., at least about 8 or 12 hours) at 37°C in a 5% CO environment in a hypoxic incubator set at 2% oxygen.

[0153] In certain embodiments, cells obtained from a kidney sample are expanded and then processed (e.g., by hypoxia and centrifugation) to obtain an SRC population. In certain embodiments, the SRC population is produced using the reagents and techniques described herein. In certain embodiments, a sample of cells from the SRC population is tested for viability before the cells of the population are administered to a subject. In certain embodiments, a sample of cells from the SRC population is tested for expression of one or more of the markers disclosed herein before the cells of the population are administered to a subject.

[0154] In certain embodiments, SRCs are produced by a process comprising expanding primary renal cells (e.g., by one, two, three, four, five, or more passages), culturing the expanded renal cells under hypoxic conditions, and then contacting the cells with a nephrotoxin (such as iodixanol, e.g., 7% iodixanol). In certain embodiments, SRCs are produced by a process comprising expanding primary renal cells (e.g., by one, two, three, four, five, or more passages), culturing the expanded renal cells under hypoxic conditions, and then selecting the cells using a density gradient as disclosed herein. In certain embodiments, SRCs are produced by a process that includes expanding primary renal cells (e.g., by one, two, three, four, five, or more passages), culturing the expanded renal cells under hypoxic conditions, and then enriching the cells for tubular cells and / or depleting the expanded cells cultured under hypoxic conditions for vascular cells or collecting duct cells.

[0155] Non-limiting examples of compositions and methods for making SRCs are disclosed in U.S. Patent Application Publication No. 2017 / 0281684, the entire contents of which are incorporated herein by reference.

[0156] In certain embodiments, the BRCs or SRCs are derived from a natural autologous or allogeneic kidney sample. In certain embodiments, the BRCs or SRCs are derived from a non-autologous kidney sample. In certain embodiments, the sample can be obtained by kidney biopsy.

[0157] In certain embodiments, the isolation and expansion of renal cells produces renal cells, including renal epithelial and stromal cells. A mixture of renal cell types containing the expanded renal cells is obtained. In certain embodiments, SRCs are obtained by continuous or discontinuous density gradient separation of expanded renal cells. In certain embodiments, the primary cell type in the density gradient-separated SRC population is a tubular epithelial phenotype. In certain embodiments, SRCs are obtained by separation of expanded renal cells by centrifugation across a density boundary, density barrier, or density interface. In certain embodiments, the primary cell type in the SRC population separated across a density boundary / density barrier / density interface is a tubular epithelial phenotype. In certain embodiments, the characteristics of SRCs obtained from expanded renal cells are evaluated using a multi-pronged approach. In certain embodiments, cell morphology, growth kinetics, and cell viability are monitored during the renal cell expansion process. In certain embodiments, the buoyant density and viability of SRCs are characterized by centrifugation and trypan blue exclusion on or through a density gradient medium. In certain embodiments, the SRC phenotype is characterized by flow cytometry, and SRC function is indicated by expression of VEGF and KIM-1. In certain embodiments, preformulation of SRC cellular function can also be assessed by measuring the activity of two specific enzymes found in the kidney proximal tubules: GGT (γ-glutamyl transpeptidase) and LAP (leucine aminopeptidase).

[0158] In certain embodiments, cell subpopulations can be separated via flow cytometry using cellular characteristics (size and granularity) that contribute to the separation of cell subpopulations via density media (forward scatter = reflecting size by flow cytometry, and side scatter = reflecting granularity). In certain embodiments, the density gradient or separation media should have low toxicity to the specific cells of interest. While in certain embodiments, the density media should have low toxicity to the specific cells of interest, the present disclosure contemplates the use of media that play a role in the selection process for cells of interest. In certain embodiments, without wishing to be bound by theory, cell populations disclosed herein recovered with media containing iodixanol appear to be iodixanol-resistant, as there is a significant loss of cells between the loading and recovery steps, suggesting that exposure to iodixanol under conditions of a density gradient, or density boundary, density barrier, or density interface, results in the elimination of certain cells. In certain embodiments, cells emerging after iodixanol density gradient or density interface separation are resistant to any adverse effects of iodixanol and / or density gradient or interface exposure. In certain embodiments, imaging agents containing mild to moderate nephrotoxins are used to isolate and / or select cell populations, such as SRC populations. In certain embodiments, a "mild" nephrotoxin is one that kills 10% or less of primary renal cells when the cells are incubated for 12 hours in a standard media formulation supplemented with 7% (weight / volume) nephrotoxin, as assessed by a standard Live / Dead dye exclusion cell viability assay. In certain embodiments, SRCs are iodixanol-resistant. In certain embodiments, the density medium should not bind to proteins in human plasma or adversely affect the primary function of the cells of interest.

[0159] In certain embodiments, the cell population is enriched and / or depleted for one or more kidney cell types using fluorescence-activated cell sorting (FACS). In certain embodiments, kidney cell types can be enriched and / or depleted using a BD FACSAria™ or equivalent. In certain embodiments, kidney cell types can be enriched and / or depleted using a FACSAria III™ or equivalent.

[0160] In certain embodiments, the cell population is enriched and / or depleted for one or more kidney cell types using magnetic cell sorting, hi certain embodiments, the cell population can be enriched and / or depleted for one or more kidney cell types using a Miltenyi autoMACS™ system or equivalent.

[0161] In certain embodiments, the renal cell population is subjected to three-dimensional culture. In certain embodiments, the method of culturing the cell population is by continuous perfusion. In certain embodiments, cell populations cultured via three-dimensional culture and continuous perfusion exhibit higher cellularity and interconnectivity compared to statically cultured cell populations. In certain embodiments, cell populations cultured via three-dimensional culture and continuous perfusion exhibit enhanced expression of renal tubule-associated genes, such as E-cadherin, as well as higher EPO expression compared to static cultures of such cell populations. In certain embodiments, cell populations cultured via continuous perfusion exhibit higher levels of glucose and glutamine consumption compared to statically cultured cell populations.

[0162] In certain embodiments, low or hypoxic conditions can be used in the methods of producing cell populations provided herein. In certain embodiments, the methods of producing cell populations can be used without a step of low oxygen conditions. In certain embodiments, normoxic conditions can be used.

[0163] In certain embodiments, the renal cell population is isolated and / or cultured from kidney tissue. Non-limiting examples of methods for separating and isolating renal cell components, e.g., enriched cell populations, for use in therapeutic formulations, including those for the treatment of kidney disease, anemia, EPO deficiency, impaired tubular transport, and impaired glomerular filtration, are disclosed herein. In certain embodiments, the cell population is isolated from freshly digested, i.e., mechanically or enzymatically digested, kidney tissue, or from a heterogeneous in vitro culture of mammalian kidney cells.

[0164] In certain embodiments, the renal cell population comprises EPO-producing renal cells. In certain embodiments, the subject has anemia and / or EPO deficiency. In certain embodiments, the EPO-producing renal cell population is characterized by expression of EPO and biological responsiveness to oxygen, such that induction of EPO expression occurs as a result of reducing the oxygen tension in the culture system. In certain embodiments, the EPO-producing cell population is enriched for EPO-producing cells. In certain embodiments, EPO expression is induced when the cell population is cultured under conditions in which the cells are exposed to a reduced level of available oxygen in the culture system, compared to a cell population cultured at standard atmospheric (approximately 21%) levels of available oxygen. In certain embodiments, EPO-producing cells cultured under lower oxygen conditions express higher levels of EPO, compared to EPO-producing cells cultured under normal oxygen conditions. Generally, culturing cells at reduced levels of available oxygen (also referred to as hypoxic culture conditions) means that the level of oxygen is reduced compared to culturing cells at standard atmospheric levels of available oxygen (also referred to as standard or normoxic culture conditions). In certain embodiments, hypoxic cell culture conditions include culturing cells at less than about 1% oxygen, less than about 2% oxygen, less than about 3% oxygen, less than about 4% oxygen, or less than about 5% oxygen. In certain embodiments, the culture conditions include culturing cells at about 10% oxygen, about 12% oxygen, about 13% oxygen, about 14% oxygen, about 15% oxygen, about 16% oxygen, about 17% oxygen, about 18% oxygen, about 19% oxygen, about 20% oxygen, or about 21% oxygen.

[0165] In certain embodiments, EPO induction or increased expression can be obtained and observed by culturing cells in less than about 5% available oxygen and comparing EPO expression levels with cells cultured in atmospheric oxygen (about 21%). In certain embodiments, EPO induction is obtained in a culture of cells capable of expressing EPO by a method comprising a first culture phase in which the cell culture is cultured in atmospheric oxygen (about 21%) for some period of time, and a second culture phase in which the effective oxygen level is reduced and the same cells are cultured in less than about 5% available oxygen. In certain embodiments, EPO expression in response to hypoxic conditions is regulated by HIF1α. In certain embodiments, other oxygen-manipulated culture conditions known in the art can be used for the cells described herein.

[0166] In certain embodiments, the formulation contains an enriched population of EPO-producing mammalian cells characterized by bioresponsiveness (e.g., EPO expression) to perfusion conditions. In certain embodiments, perfusion conditions include transient, intermittent, or continuous fluid flow (perfusion). In certain embodiments, EPO expression is mechanically induced when the medium in which the cells are cultured is circulated or agitated intermittently or continuously, such that dynamic forces are imparted to the cells via the flow. In certain embodiments, by culturing cells exposed to transient, intermittent, or continuous fluid flow, the cells exist as three-dimensional structures in or on a material that provides a scaffold and / or void space for the formation of the three-dimensional structure. In certain embodiments, the cells are cultured on porous beads and exposed to intermittent or continuous fluid flow via a rocking platform, an orbital platform, or a spinner flask. In certain embodiments, the cells are cultured on a three-dimensional scaffold and placed in a device that immobilizes the scaffold and allows fluid to flow directionally through or across the scaffold. Those skilled in the art will understand that other perfusion culture conditions known in the art can be used for the cells described herein.

[0167] In certain embodiments, the cell population is derived from a kidney biopsy. In certain embodiments, the cell population is derived from whole kidney tissue. In certain embodiments, the cell population is derived from an in vitro culture of mammalian kidney cells established from a kidney biopsy or whole kidney tissue. In certain embodiments, the kidney cell population is an SRC population. In certain embodiments, the cell population is an unfractionated cell population, also referred to herein as a non-enriched cell population.

[0168] Compositions containing various active agents (e.g., other than kidney cells or microvesicles) are included herein. In certain embodiments, the microvesicles (e.g., exosomes) provided herein comprise compounds that were present in the culture medium of the kidney cell population that secreted the microvesicles (e.g., exosomes). In certain embodiments, the microvesicles (e.g., exosomes) provided herein comprise compounds that were present in the kidney cell population that secreted the microvesicles (e.g., exosomes).

[0169] Non-limiting examples of suitable active agents include, but are not limited to, cell aggregates, acellular biomaterials, secretory products from bioactive cells, macromolecular and small molecule therapeutics, and combinations thereof. For example, one bioactive cell type can be combined with a biomaterial-based microcarrier, with or without a therapeutic molecule or another bioactive cell type. In certain embodiments, unattached cells can be combined with acellular particles.

[0170] In certain embodiments, the cells of the renal cell population are present within spheroids. In certain embodiments, the renal cell population is present in the form of spheroids. In certain embodiments, spheroids comprising bioactive renal cells are administered to a subject. In certain embodiments, the spheroids comprise at least one non-renal cell type or cell population. In certain embodiments, the spheroids are produced by a method comprising: (i) combining a bioactive renal cell population and a non-renal cell population; and (ii) culturing the bioactive renal cell population and the non-renal cell population in a three-dimensional culture system comprising a spinner flask until spheroids form.

[0171] In certain embodiments, the non-renal cell population comprises an endothelial cell population and an endothelial progenitor cell population. In certain embodiments, the bioactive cell population is an endothelial cell population. In certain embodiments, the endothelial cell population is a cell lineage. In certain embodiments, the endothelial cell population comprises human umbilical vein endothelial cells (HUVECs). In certain embodiments, the non-renal cell population is a mesenchymal stem cell population. In certain embodiments, the non-renal cell population is of hematopoietic origin, breast origin, intestinal origin, placental origin, lung origin, bone marrow origin, blood origin, umbilical cord origin, endothelial origin, or dental pulp origin. The non-renal cell population is a stem cell population of adipose, neural, olfactory, neural crest, or testicular origin. In certain embodiments, the non-renal cell population is an adipose-derived progenitor cell population. In certain embodiments, the cell population is a xenogeneic, syngeneic, allogeneic, autologous cell population, or a combination thereof. In certain embodiments, the bioactive renal cell population and the non-renal cell population are cultured at a ratio of 0.1:9.9 to 9.9:0.1. In certain embodiments, the bioactive renal cell population and the non-renal cell population are cultured at a ratio of about 1:1. In certain embodiments, the renal cell population and the bioactive cell population are suspended in growth medium.

[0172] The expanded bioactive renal cells can be further subjected to continuous or discontinuous density media separation to obtain SRCs. In particular, continuous or discontinuous single-step or multi-step density gradient centrifugation is used to separate the harvested renal cell population based on cell buoyant density. In certain embodiments, the expanded bioactive renal cells can be further subjected to separation by centrifugation across a density boundary, density barrier, or density interface to obtain SRCs. In particular, centrifugation across a density boundary, density barrier, or density interface is used to separate the harvested renal cell population based on cell buoyant density. In certain embodiments, SRCs are obtained using, in part, OPTIPREP (Axis-Shield) medium containing a 60% aqueous solution of the non-ionic iodine compound iodixanol. The density gradient medium is produced by using a centrifugation system. However, one skilled in the art will recognize that any density gradient medium can be used in accordance with the present disclosure, without limitation to a particular medium or other means, such as immunological separation using cell surface markers known in the art, that possess the characteristics required for isolating the cell populations of the present disclosure. For example, Percoll™ (15-30 nm diameter colloidal silica particles (23% w / w in water) coated with polyvinylpyrrolidone (PVP)) or sucrose can be used to form a density gradient or density boundary. In certain embodiments, a cell fraction exhibiting a buoyant density greater than about 1.04 g / mL is collected as a separate pellet after centrifugation. In certain embodiments, cells that maintain a buoyant density less than 1.04 g / mL are rejected and discarded. In certain embodiments, a cell fraction exhibiting a buoyant density greater than about 1.0419 g / mL is collected as a separate pellet after centrifugation. In certain embodiments, cells that maintain a buoyant density less than 1.0419 g / mL are rejected and discarded. In certain embodiments, the cell fraction exhibiting a buoyant density greater than about 1.045 g / mL is collected as a separate pellet after centrifugation, hi certain embodiments, cells that maintain a buoyant density less than 1.045 g / mL are discarded and discarded.

[0173] In certain embodiments, therapeutic compositions and formulations thereof disclosed herein may contain (i) isolated heterogeneous kidney cell populations enriched for particular bioactive components or cell types and / or depleted of particular inactive or undesirable components or cell types, and / or (ii) microvesicles (e.g., exosomes) secreted by such cells, used to treat kidney disease, i.e., to stabilize and / or improve and / or regenerate kidney function and / or structure. Non-limiting examples of cells for effecting such stabilization and / or improvement have previously been described in U.S. Patent No. 8,318,484 to Presnell et al., International Application No. PCT / US2011 / 036347 to Ilagan et al., and International Application No. PCT / US2016 / 044866 to Jain et al., the entire contents of each of which are incorporated herein by reference. In certain embodiments, the compositions provided herein may contain isolated renal cell fractions that lack cellular components compared to healthy individuals but retain therapeutic properties, i.e., result in stabilization and / or improvement and / or regeneration of kidney function. In certain embodiments, the cell populations, cell fractions, and / or secretory products of the cells described herein may be derived from healthy individuals, individuals suffering from kidney disease, or subjects described herein.

[0174] In certain embodiments, the source of the cells is the same as the intended target organ or tissue. For example, BRCs and / or SRCs may be derived from the kidney for use in formulations administered to the kidney. The cell population can be derived from a kidney biopsy. In certain embodiments, the cell population is derived from whole kidney tissue. In certain embodiments, the cell population is derived from an in vitro culture of mammalian kidney cells established from a kidney biopsy or whole kidney tissue. In certain embodiments, the BRCs and / or SRCs comprise a heterogeneous mixture or fraction of bioactive kidney cells. The BRCs and / or SRCs can be derived from a healthy individual or are kidney cell fractions themselves from a healthy individual. Furthermore, the present disclosure provides kidney cell fractions obtained from unhealthy individuals that may lack certain cellular components compared to corresponding kidney cell fractions from healthy individuals, yet still maintain therapeutic properties. The present disclosure also provides therapeutically active cell populations that lack cellular components compared to healthy individuals, which, in certain embodiments, can be isolated and expanded from autologous sources for various disease states.

[0175] In certain embodiments, SRCs are obtained from the isolation and expansion of renal cells from a patient's renal cortical tissue via a kidney biopsy. The renal cells are isolated from the kidney tissue by enzymatic digestion, expanded by standard cell culture techniques, and selected by centrifugation of expanded renal cells across a density boundary, density barrier, or density interface. In this embodiment, the SRCs are primarily composed of renal tubular epithelial cells, which are known for their regenerative capabilities (Bonventre JV. Dedifferentiation and proliferation of surviving epithelial cells in acute renal failure. J Am Soc Nephrol. 2003;14(Suppl. 1):S55-61; Humphreys BD, Czerniak S, DiRocco DP, et al. Repair of injured proximal tubule does not involve specialized progenitors. PNAS. 2011;108:9226-31; Humphreys BD, Valerius MT, Kobayashi A, et al. Intrinsic epithelial cells repair the kidney after injury. Cell Stem Cell. 2008;2:284-91). Other parenchymal (vascular) and interstitial cells may also be present in the autologous SRC population. In certain embodiments, kidney cells are selected by centrifugation through continuous or discontinuous single-step or multi-step gradients.

[0176] Included herein are therapeutic compositions comprising both vesicles (e.g., microvesicles such as exosomes) and selected kidney cells. In certain embodiments, the combination of vesicles and cells results in stabilization and / or improvement and / or repair and / or regeneration of kidney function in a subject. Therapeutic properties can include a repair or regenerative effect.

[0177] In certain embodiments, the cells are genetically modified (eg, modified via genomic modification and / or RNAi) immune-privileged BRCs (such as SRCs).

[0178] In certain embodiments, the vesicles are obtained from genetically modified (e.g., genomically modified and / or modified via RNAi) immune-privileged BRCs (such as SRCs).

[0179] In certain embodiments, the genetically modified BRC is a genomically modified BRC (i.e., a BRC with a genetic modification in its genome). In certain embodiments, the genetically modified BRC comprises an exogenous polynucleotide (such as a plasmid or viral vector) that expresses an RNA interference (RNAi) molecule that reduces the expression of a genomic immunogenic gene in the BRC. In certain embodiments, the RNAi molecule is a small interfering RNA molecule or a small hairpin RNA molecule. In certain embodiments, the method comprises genetically modifying a genomic immunogenic gene in the BRC.

[0180] In certain embodiments, the gene encodes a protein within a major histocompatibility complex (MHC) class I molecule or an MHC class II molecule. In certain embodiments, the gene encodes a beta-2 microglobulin (B2M, also known as β2M) gene, a human leukocyte antigen (HLA)-A gene, an HLA-B gene, an HLA-C gene, an HLA-DRA gene, an HLA-DRB1 gene, an HLA-DRB3 gene, an HLA-DRB4 gene, an HLA-DRB5 gene, an HLA-DRB6 gene, an HLA-DRB7 gene, an HLA-DRB8 gene, an HLA-DRB9 gene, an HLA-DRB10 gene, an HLA-DRB11 gene, an HLA-DRB12 gene, an HLA-DRB13 gene, an HLA-DRB14 gene, an HLA-DRB15 gene, an HLA-DRB16 gene, an HLA-DRB17 gene, an HLA-DRB18 gene, an HLA-DRB19 gene, an HLA-DRB20 gene, an HLA-DRB210 gene, an HLA-DRB22 gene, an HLA-DRB23 gene, an HLA-DRB24 gene, an HLA-DRB25 gene, an HLA-DRB26 gene, an HLA-DRB27 gene, an HLA-DRB28 gene, an HLA-DRB29 ... 4 gene, HLA-DRB5 gene, HLA-DPA1 gene, HLA-DPA2 gene, HLA-DQA1 gene, or HLA-DQB1 gene.

[0181] In certain embodiments, the gene encodes a minor histocompatibility antigen (MiHA or mHA). In certain embodiments, the gene is the HA-1 gene, the HA-2 gene, the HA-8 gene, the HB-1 gene, the HY-A1 gene, the HY-A2 gene, the HY-B7 gene, the HY-B8 gene, the HY-B60 gene, or the HY-DQ5 gene.

[0182] In certain embodiments, any allelic variant of an HLA gene, B2M gene, or mHA gene described herein may be modified (e.g., deleted) or targeted with RNA interference.

[0183] In certain embodiments, the genetic modification of the gene comprises a mutation of the gene, hi certain embodiments, the mutation of the gene comprises a deletion of the gene or a portion thereof.

[0184] In certain embodiments, the genetic modification of the cell includes mutations in any combination of two or more of the B2M gene, HLA-A gene, HLA-B gene, HLA-C gene, HLA-DRA gene, HLA-DRB1 gene, HLA-DRB3 gene, HLA-DRB4 gene, HLA-DRB5 gene, HLA-DPA1 gene, HLA-DPA2 gene, HLA-DQA1 gene, and / or HLA-DQB1 gene.

[0185] In certain embodiments, the genetically modified BRCs are genetically modified primary kidney cells. In certain embodiments, the genetically modified primary kidney cells have been passaged at least about 1, 2, 3, 4, 5, or more times before or after genetic modification. In certain embodiments, the method further includes obtaining SRCs from the genetically modified BRCs. In certain embodiments, the SRCs are obtained and then genetically modified.

[0186] In certain embodiments, the BRC is an SRC. Various non-limiting examples of SRCs are disclosed herein.

[0187] In certain embodiments, BRCs are genetically modified while within a population of BRCs, resulting in fewer than all cells in the population of BRCs being genetically modified. In certain embodiments, the method further comprises isolating or enriching genetically modified BRCs from the population of BRCs. In certain embodiments, the method further comprises isolating or enriching genetically modified SRCs from the population of SRCs. In certain embodiments, a population of BRCs (e.g., SRCs) is subjected to genetic modification to obtain a population of BRCs in which some cells are genetically modified and others are not. In certain embodiments, some of the genetically modified cells are homozygous for the modification. In certain embodiments, some of the genetically modified cells are heterozygous for the modification. In certain embodiments, cells that are homozygous for the modification are enriched or selected. In certain embodiments, cells that are heterozygous for the modification are enriched or selected. In certain embodiments, cells that are homozygous or heterozygous for the modification are enriched or selected. In certain embodiments, the modification is a mutation that reduces expression of a protein encoded by the gene. In certain embodiments, the mutation reduces the level of the protein on the surface of the modified cell. In certain embodiments, cells that express the protein are depleted or eliminated. In certain embodiments, the mutation reduces the level of MHC class I and / or MHC class II molecules on the surface of the cell. In certain embodiments, cells with the mutation do not have MHC class I and / or MHC class II molecules on their surface. In certain embodiments, cells that express MHC class I molecules on their surface Cells are depleted or eliminated. In certain embodiments, cells expressing MHC class II molecules on their surface are depleted or eliminated. In certain embodiments, cell sorting methods are used to remove cells expressing proteins, MHC class I molecules, and / or MHC class II molecules from a population. In certain embodiments, the cell sorting method includes an agent (such as an antibody) that binds to proteins, MHC class I molecules, and / or MHC class II molecules. In certain embodiments, cell depletion or selection includes bead / antibody coupling to remove cells that have a specific protein on their cell surface. In certain embodiments, the cell sorting method is magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS). In certain embodiments, the gene selected for genetic modification is one whose protein is expressed on the cell surface; thus, live cells can be sorted (e.g., based on antibodies binding to the surface) using FACS and / or MACS techniques. In certain embodiments, MACS is used to remove cells that express MHC molecules (such as MHC class I or MHC class II molecules) from cells that do not. In certain embodiments, integrating or non-integrating vectors can be used to express additional HLA component polypeptides to further modify or regulate the adaptive or innate immune system, for example, to prevent targeting and lysis by natural killer (NK) cells.

[0188] In certain embodiments, genetic modification (e.g., mutation) of the gene comprises (i) expressing a gene-editing protein in the BRC, or (ii) delivering the gene-editing protein across the cell membrane of the BRC. In certain embodiments, the gene-editing protein is a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a megaTAL, or an RNA-guided endonuclease. In certain embodiments, the RNA-guided endonuclease is a Cas protein. In certain embodiments, the Cas protein is a Cas9 protein. In certain embodiments, genetic modification of the gene further comprises (i) expressing a single-guide guide RNA (gRNA) in the BRC, or (ii) delivering a single-guide guide RNA (gRNA) across the cell membrane of the BRC. In certain embodiments, the Cas9 protein and gRNA are part of a ribonucleoprotein complex.

[0189] In certain embodiments, the genetic modification of the gene reduces the amount of MHC class I on the surface of the cell. In certain embodiments, the genetic modification of the gene reduces the amount of MHC class II on the surface of the cell. In certain embodiments, the method comprises genetically modifying two or more genes, wherein at least one of the genes encodes a protein in an MHC class I molecule, and at least one of the genes encodes a protein in an MHC class II molecule. In certain embodiments, at least one of the genes is an HLA gene.

[0190] A non-limiting description of genetically modified BRCs, including methods of production, is provided in International Application No. PCT / US18 / 38801, filed June 21, 2018.

[0191] In certain embodiments, the source of the cells is the same as the intended target organ or tissue, from the same or a different source. For example, BRCs and / or SRCs can originate from the kidney for use in a formulation administered to the kidney (together with or separately from the vesicles). In certain embodiments, BRCs and / or SRCs can originate from the kidney for use in the production of vesicles administered to the kidney. In certain embodiments, the cell population is derived from a kidney biopsy. In certain embodiments, the cell population is derived from whole kidney tissue. In certain embodiments, the cell population is derived from an in vitro culture of mammalian kidney cells established from a kidney biopsy or whole kidney tissue. In certain embodiments, the BRCs and / or SRCs are genetically modified (e.g., modified via genomic modification and / or RNAi). ) comprise a heterogeneous mixture or fraction of immune-privileged, bioactive renal cells. The BRCs and / or SRCs may be derived from healthy individuals or may be renal cell fractions themselves from healthy individuals. Additionally, the present invention provides renal cell fractions obtained from unhealthy individuals that may lack certain cellular components compared to corresponding renal cell fractions from healthy individuals, yet still maintain therapeutic properties. The present invention also provides therapeutically active cell populations that lack cellular components compared to healthy individuals, which, in one embodiment, may be isolated and expanded from kidneys of various mammalian origins.

[0192] In certain embodiments, SRCs are obtained by isolating and expanding renal cells from renal cortical tissue of various patients via renal biopsy. In certain embodiments, renal cells are isolated from kidney tissue by enzymatic digestion, expanded by standard cell culture techniques, and selected from the expanded renal cells by density gradient centrifugation. In certain embodiments, SRCs are primarily composed of renal epithelial cells, which are known for their immune privilege and regenerative capabilities. Other parenchymal (vascular) and interstitial cells may be sparsely present in the SRC population.

[0193] As described herein, the present invention is based, in part, on the surprising discovery that certain subfractions of a heterogeneous population of renal cells, enriched for bioactive components and depleted of inactive or undesirable components, result in superior therapeutic and regenerative outcomes than the starting population.

[0194] In certain embodiments, isolation and expansion of renal cells results in a mixture of renal cell types, including renal epithelial cells and stromal cells. In certain embodiments, SRCs are obtained by density gradient separation of the expanded renal cells. In certain embodiments, the primary cell type in the density gradient-separated SRC population is of the tubular epithelial phenotype. In certain embodiments, the SRC phenotype is characterized by flow cytometry, and SRC function is indicated by expression of VEGF and KIM-1.

[0195] Those skilled in the art will understand that other isolation and culture methods known in the art can be used for the cells described herein. Those skilled in the art will also understand that bioactive cell populations can be derived from sources other than those specifically listed above, including, but not limited to, tissues and organs other than kidney, body fluids, and fat.

[0196] SRC phenotype In certain embodiments, microvesicles (eg, exosomes) secreted by SRCs and / or SRCs are administered to a subject suffering from or at risk for kidney disease.

[0197] In certain embodiments, cell phenotype is monitored by analysis of the expression of renal cell markers using flow cytometry. Cell phenotype analysis is based on the use of antigen markers specific to the cell type being analyzed. Flow cytometry analysis provides a quantitative indication of cells in a sample population that express the antigen marker being analyzed.

[0198] Various markers useful for phenotypic characterization of renal tubular epithelial cells have been reported in the literature: (i) cytokeratins, (ii) transport membrane proteins (aquaporins and cubilins), (iii) cell-binding molecules (adherins and cluster of differentiation antigens and lectins), and (iv) metabolic enzymes (glutathione and γ-glutamyl transpeptidase (GGT)) (Table 1). Because the majority of cells found in cultures obtained from whole kidney digests are epithelial and endothelial cells, the markers examined focus on the expression of proteins specific to these two groups.

[0199] TIFF2026035692000002.tif133170

[0200] Table 2 shows the selected markers, ranges, and mean percentage values ​​of the phenotypes in the SRC population, as well as the rationale for their selection.

[0201] TIFF2026035692000003.tif43170

[0202] cell function SRCs actively secrete proteins that can be detected by analysis of conditioned medium. Cell function is assessed by the ability of cells to metabolize PrestoBlue and secrete VEGF (vascular endothelial growth factor) and KIM-1 (kidney injury molecule 1).

[0203] Table 3 shows the amount of VEGF and KIM-1 present in conditioned medium from renal cell and SRC cultures. Renal cells were cultured until near confluence. Conditioned medium from overnight exposure to renal cell cultures was tested for VEGF and KIM-1.

[0204] TIFF2026035692000004.tif40170

[0205] SRC enzyme activity Preformulation of SRC cellular function can also be assessed by measuring the activity of two specific enzymes found in the kidney proximal tubules: GGT (γ-glutamyl transpeptidase) and LAP (leucine aminopeptidase).

[0206] While microvesicles (e.g., exosomes) and selected renal cell compositions are described herein, the present invention contemplates compositions containing a variety of other active agents. Other suitable active agents include, but are not limited to, cell aggregates, acellular biomaterials, macromolecular and small molecule therapeutics, and combinations thereof. For example, a bioactive cell type can be combined with a biomaterial-based microcarrier, with or without a therapeutic molecule or another bioactive cell type, and unattached cells can be combined with acellular particles.

[0207] cell aggregates In one aspect, the formulations of the present disclosure contain cellular aggregates or spheroids and / or microvesicles (e.g., exosomes) secreted by such aggregates or spheroids and / or microvesicles (e.g., exosomes) secreted by bioactive cells not present in the aggregates or spheroids.

[0208] In certain embodiments, the cell aggregates comprise bioactive cell populations described herein, including, but not limited to, bioactive renal cells such as renal cell mixtures, enriched renal cell populations, and renal cell fractions and mixtures of renal cells in combination with mesenchymal stem cells, endothelial progenitor cells, cells derived from the stromal vascular fraction of adipose, or any other non-renal cell population.

[0209] In certain embodiments, the bioactive kidney cells of the present disclosure can be cultured in a 3D format as further described herein. In certain embodiments, the term "organoid" refers to an accumulation of cells with a phenotype and / or function that recapitulates the appearance of a native kidney. In certain embodiments, organoids typically comprise a mixed population of cells of various lineages found in a given tissue in vivo. In certain embodiments, organoids of the present disclosure are formed in vitro by any means, whereby cells of the present disclosure form aggregates, which may also form spheroids, organoids, or combinations thereof. Such aggregates, spheroids, or organoids, in certain embodiments, adopt a structure consistent with a specific organ. In certain embodiments, such aggregates, spheroids, or organoids express surface markers typically expressed by cells of a specific organ. In certain embodiments, such aggregates, spheroids, or organoids produce compounds or substances typically expressed by cells of a specific organ. In certain embodiments, the cells of the present disclosure can be cultured on a natural substrate, such as gelatin, hi certain embodiments, the cells of the present disclosure can be cultured on a synthetic substrate, such as PLGA.

[0210] Biomaterials Various biomaterials can be combined with active agents to provide the therapeutic formulations of the present disclosure. In certain embodiments, the biomaterial can take any suitable shape (e.g., beads) or form (e.g., liquid, gel, etc.). Suitable biomaterials in the form of polymer matrices are described in U.S. Patent Application Publication No. 2007020200702 by Bertram et al. No. 76507, incorporated herein by reference in its entirety. In certain embodiments, the polymeric matrix or scaffold can be shaped into many desired configurations to meet many overall system, geometric, or spatial constraints. In certain embodiments, the biomaterial is present in the form of a liquid suspension. In certain embodiments, the matrix or scaffold of the present disclosure is three-dimensional and can be shaped to match the dimensions and shape of an organ or tissue structure. For example, in using polymeric scaffolds to treat kidney disease, tubular transport disorders, or glomerular filtration disorders, three-dimensional (3-D) matrices can be used that replicate aspects or all of the native kidney tissue structure and organization, as well as aspects or all of the renal parenchyma.

[0211] A variety of different shapes of 3-D scaffolds can be used. Of course, the polymer matrix can be shaped into various sizes and shapes to accommodate patients of different sizes. The polymer matrix can also be shaped in other ways to accommodate the special needs of the patient. In certain embodiments, the polymer matrix or scaffold can be a biocompatible material (such as a porous polymeric scaffold). The scaffold can be formed from a variety of synthetic or naturally occurring materials, including, but not limited to, open-cell polylactic acid (OPLA™), cell Examples of suitable polymeric materials include cellulose ethers, cellulose, cellulose esters, fluorinated polyethylene, phenols, poly-4-methylpentene, polyacrylonitrile, polyamides, polyamideimides, polyacrylates, polybenzoxazoles, polycarbonates, polycyanoaryl ethers, polyesters, polyestercarbonates, polyethers, polyetheretherketones, polyetherimides, polyetherketones, polyethersulfones, polyethylenes, polyfluoroolefins, polyimides, polyolefins, polyoxadiazoles, polyphenylene oxides, polyphenylene sulfides, polypropylenes, polystyrenes, polysulfides, polysulfones, polytetrafluoroethylenes, polythioethers, polytriazoles, polyurethanes, polyvinyls, polyvinylidene fluoride, regenerated cellulose, silicones, urea formaldehyde, collagen, gelatin, alginates, laminins, fibronectin, silk, elastin, alginates, hyaluronic acid, agarose, or copolymers or physical blends thereof. Scaffold compositions can range from soft porous scaffolds to rigid, shape-retaining porous scaffolds, hi certain embodiments, the scaffold is configured as a liquid solution capable of becoming a hydrogel, e.g., a hydrogel above its melting point.

[0212] In certain embodiments, the scaffold is derived from a pre-existing kidney or other organ of human or animal origin from which the natural cell population has been eliminated by application of surfactants and / or other chemical agents and / or other enzymatic and / or physical techniques known to those skilled in the art. In this embodiment, the native three-dimensional structure of the organ of origin is preserved in their native bioactive state along with all associated extracellular matrix components. In certain embodiments, the scaffold is an extracellular matrix derived from a human or animal kidney or other organ. In certain embodiments, the construct is assembled into a tissue-like structure by application of three-dimensional bioprinting techniques. In certain embodiments, the construct is in the liquid form of a solution that can become a hydrogel.

[0213] In certain embodiments, the biomaterial is a hydrogel. Hydrogels can be formed from a variety of polymeric materials and are useful in a variety of biomedical applications. Hydrogels include: Physically, hydrogels can be described as a three-dimensional network of hydrophilic polymers. Depending on the type of hydrogel, hydrogels contain various percentages of water, but are generally insoluble in water. Despite their high water content, hydrogels can still bind large volumes of liquid due to the presence of hydrophilic residues. Hydrogels swell to a wide range without changing their gelatinous structure. The basic physical characteristics of hydrogels can be specifically modified according to the properties of the polymers used and the device used to administer the hydrogel.

[0214] 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 (e.g., gellan gum and carrageenan) that gel upon exposure to monovalent cations, (c) polysaccharides (e.g., hyaluronic acid) that are highly viscous liquids or thixotropic and form gels over time by slow development of structure, (d) gelatin or collagen, and (e) hydrogel polymer precursors (e.g., polyethylene oxide-polypropylene glycol block copolymers and proteins). U.S. Patent No. 6,224,893 provides a detailed description of various polymers suitable for making hydrogels according to the present disclosure and the chemical properties of such polymers.

[0215] In certain embodiments, the hydrogels used to formulate the biomaterials of the present disclosure are gelatin-based. Gelatin is a non-toxic, biodegradable, water-soluble protein derived from collagen and is a major component of the mesenchymal extracellular matrix (ECM). Collagen is the major structural protein in the extracellular space within various connective tissues in animal bodies. As the main component of connective tissue, collagen is the most abundant protein in mammals, constituting 25% to 35% of the total body protein content. Depending on the degree of mineralization, collagen tissues can be rigid (bone), flexible (tendon), or have a gradient from rigid to flexible (cartilage). Collagen in the form of elongated fibrils is found primarily in fibrous tissues such as tendons, ligaments, and skin. Collagen is also abundant in the cornea, cartilage, bone, blood vessels, the digestive tract, intervertebral discs, and the dentin of teeth. In muscle tissue, collagen serves as the main component of the endomysium. Collagen makes up 1% to 2% of muscle tissue and accounts for 6% of the weight of muscle in strong tendons. Collagen is found in many places throughout the body. However, more than 90% of the collagen in the human body is type I.

[0216] To date, 28 types of collagen have been identified and described. Collagens can be divided into several groups according to the structure they form: fibrillar (types I, II, III, V, and XI), non-fibrillar FACIT (Fibril Associated Collagens with Interrupted Triple Helices) ( Types IX, XII, XIV, XVI, and XIX), short chain (Types VIII and X), basement membrane (Type IV), multiplexin (Multiple Triple Helix domains with Interruptions) (Types XV and XVIII), MACIT (Membrane Associated Collagens with Interrupted Triple Helices) (Types XIII and XVII), and others (Types VI The five most common types are: Type I: skin, tendons, blood vessels, ligaments, organs, and bone (the main component of the organic part of bone); Type II: cartilage (the main collagen component of cartilage); Type III: reticular (the main component of reticular fibers) (usually coexisting with Type I); Type IV: forming the basal layer (the layer of basement membrane secreted by epithelia); Type V: cell surfaces, hair, and placenta.

[0217] Gelatin carries information signals containing the arginine-glycine-aspartic acid (RGD) sequence, which promotes cell adhesion, proliferation, and stem cell differentiation. A distinctive property of gelatin is that it exhibits upper critical solution temperature behavior (UCST). In certain embodiments, above a certain temperature threshold of 40°C, gelatin can dissolve in water by forming a flexible, random single helix. Upon cooling, hydrogen bonds and phagocytosis occur. Gelatin is widely used in pharmaceutical and medical applications.

[0218] In certain embodiments, the hydrogels used to formulate the injectable cell compositions herein are based on porcine gelatin, which can be derived from pig skin and is commercially available, for example, from Nitta Gelatin NA Inc. (North Carolina, USA) or Gelita USA Inc. (Iowa, USA). Gelatin can be, for example, dulbeccoline. It can be dissolved in dilute acid-buffered saline (DPBS) to form a thermoresponsive hydrogel that can gel and liquefy at various temperatures. In certain embodiments, the hydrogels used to formulate the injectable cell compositions herein are based on recombinant human or animal gelatin expressed and purified by techniques known to those skilled in the art. In certain embodiments, an expression vector containing all or part of the cDNA for type I alpha I human collagen is expressed in the yeast Pichia pastoris. Other developments include the use of αI human collagen. Current vector systems and organisms are known to those skilled in the art. In certain embodiments, the gelatin-based hydrogels of the present disclosure are liquid at or above room temperature (22°C-28°C) and gel upon cooling to refrigerated temperatures (2°C-8°C).

[0219] Those skilled in the art will appreciate that other types of synthetic or naturally occurring materials known in the art can be used to form the scaffolds described herein.

[0220] In certain embodiments, biomaterials used in accordance with the present disclosure have a molecular weight of 5.1 kDa to 2×10 5 It is composed of hyaluronic acid (HA) in the form of a hydrogel containing HA molecules ranging in size from 5.1 kDa to over 2 x 10. HA can promote branching morphogenesis and three-dimensional self-organization of associated bioactive cell populations. In certain embodiments, biomaterials used in accordance with the present disclosure also range in size from 5.1 kDa to 2 x 10. 5 The biomaterials used in accordance with the present disclosure are comprised of hyaluronic acid in the form of a porous foam containing HA molecules ranging in size from 0.1 kDa to over 100 kDa. In certain embodiments, the hydrogel is derived from or contains, but is not limited to, extracellular matrix derived from the kidney or any other tissue or organ. In yet another embodiment, the biomaterial used in accordance with the present disclosure is comprised of a polylactic acid (PLA)-based foam having an open-cell structure and a pore size of about 50 microns to about 300 microns.

[0221] Temperature-Sensitive Biomaterials The biomaterials described herein can also be designed or adapted to respond to certain external conditions, e.g., in vitro or in vivo. In certain embodiments, the biomaterials are temperature-sensitive (e.g., both in vitro and in vivo). In certain embodiments, the biomaterials are adapted to respond to exposure to enzymatic degradation (e.g., both in vitro and in vivo). The response of the biomaterials to external conditions can be fine-tuned as described herein. The temperature sensitivity of the described formulations can be varied by adjusting the proportion of biomaterial in the formulation. For example, the percentage of gelatin in solution can be adjusted to modulate the temperature sensitivity of the gelatin in the final formulation (e.g., liquid, gel, beads, etc.). Alternatively, the biomaterials can be chemically crosslinked to provide greater resistance to enzymatic degradation. For example, chemically crosslinking gelatin beads using a carbodiimide crosslinker can result in reduced susceptibility to endogenous enzymes.

[0222] In one aspect, the formulations described herein are administered to a subject using microvesicles (e.g., exosporins). The formulation comprises a biomaterial with properties that create a favorable environment for an active agent, such as a soluble ...

[0223] In certain embodiments, the biomaterial is a temperature-sensitive biomaterial that can maintain at least two different phases or states depending on the temperature. The biomaterial can maintain a first state at a first temperature, a second state at a second temperature, and / or a third state at a third temperature. The first, second, or third state can be a substantially solid state, a substantially liquid state, or a substantially semi-solid or semi-liquid state. In certain embodiments, the biomaterial has a first state at a first temperature and a second state at a second temperature, where the first temperature is lower than the second temperature.

[0224] In certain embodiments, the temperature-sensitive biomaterial is in a substantially solid state at a temperature of about 8°C or less. In certain embodiments, the substantially solid state is maintained at about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, or about 8°C. In certain embodiments, the substantially solid state has the form of a gel. In certain embodiments, the temperature-sensitive biomaterial is in a substantially liquid state at ambient temperature or above. In certain embodiments, the substantially liquid state is maintained at about 25°C, about 25.5°C, about 26°C, about 26.5°C, about 27°C, about 27.5°C, about 28°C, about 28.5°C, about 29°C, about 29.5°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, or about 37°C. In certain embodiments, the ambient temperature is about room temperature.

[0225] In certain embodiments, the temperature-sensitive biomaterial is in a substantially solid state at temperatures below about ambient temperature. In certain embodiments, ambient temperature is about room temperature. In certain embodiments, the substantially solid state is maintained at about 17°C, about 16°C, about 15°C, about 14°C, about 13°C, about 12°C, about 11°C, about 10°C, about 9°C, about 8°C, about 7°C, about 6°C, about 5°C, about 4°C, about 3°C, about 2°C, or about 1°C. In certain embodiments, the substantially solid state has the form of beads. In certain embodiments, the temperature-sensitive biomaterial is in a substantially liquid state at temperatures above about 37°C. In certain embodiments, the substantially solid state is maintained at about 37°C, about 38°C, about 39°C, or about 40°C.

[0226] The temperature-sensitive biomaterial may be provided in solution, solid, bead, or other suitable form described herein and / or known to those skilled in the art. The microvesicles (e.g., exosomes) and / or cell populations and preparations described herein may be coated with, deposited on, embedded in, bound to, seeded in, suspended in, or entrapped in the temperature-sensitive biomaterial. In certain embodiments, the cell populations described herein may be assembled as three-dimensional cell aggregates or organoids or three-dimensional tubular structures prior to forming a complex with the temperature-sensitive biomaterial, or may be so assembled upon forming a complex with the temperature-sensitive biomaterial. In certain embodiments, the temperature-sensitive biomaterial may be provided without any cells, for example, in the form of spacer beads. In this embodiment, the temperature-sensitive biomaterial functions in a purely passive role, thereby promoting regenerative biological activity, such as angiogenesis or host cell aggregation. Space is created within the target organ for the infiltration and migration of the cells.

[0227] In certain embodiments, the temperature-sensitive biomaterial has a transition state between a first state and a second state. In certain embodiments, the transition state is a solid-liquid transition state between a temperature of about 8°C and about ambient temperature. In certain embodiments, ambient temperature is about room temperature. In certain embodiments, the solid-liquid transition state occurs at one or more temperatures of about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, and about 18°C.

[0228] A temperature-sensitive biomaterial has a certain viscosity at a given temperature, measured in centipoise (cP). In certain embodiments, the biomaterial has a viscosity at 25°C of about 1 cP to about 5 cP, about 1.1 cP to about 4.5 cP, about 1.2 cP to about 4 cP, about 1.3 cP to about 3.5 cP, about 1.4 cP to about 3.5 cP, about 1.5 cP to about 3 cP, about 1.55 cP to about 2.5 cP, or about 1.6 cP to about 2 cP. In certain embodiments, the biomaterial has a viscosity at 37°C of about 1.0 cP to about 1.15 cP. The viscosity at 37°C can be about 1.0 cP, about 1.01 cP, about 1.02 cP, about 1.03 cP, about 1.04 cP, about 1.05 cP, about 1.06 cP, about 1.07 cP, about 1.08 cP, about 1.09 cP, about 1.10 cP, about 1.11 cP, about 1.12 cP, about 1.13 cP, about 1.14 cP, or about 1.15 cP. In certain embodiments, the biomaterial is a gelatin solution. The gelatin is present in the solution at about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, or about 1% (weight / volume). In one example, the biomaterial is a 0.75% (wt / vol) gelatin solution in PBS. In certain embodiments, the 0.75% (wt / vol) solution has a viscosity of about 1.6 cP to about 2 cP at 25° C. In certain embodiments, the 0.75% (wt / vol) solution has a viscosity of about 1.07 cP to about 1.08 cP at 37° C. The gelatin solution can be provided in PBS, DMEM, or another suitable solvent.

[0229] In one aspect, the formulation contains microvesicles (e.g., exosomes) and / or bioactive cells combined with a second biomaterial that provides a favorable environment for the combination of microvesicles (e.g., exosomes) and / or cells from the time of formulation until after administration to a subject. In certain embodiments, the favorable environment provided by the second biomaterial relates to the advantage of administering microvesicles (e.g., exosomes) and / or cells in a biomaterial that retains its structural integrity up until and for a period of time after administration to a subject. In certain embodiments, the structural integrity of the second biomaterial after implantation is minutes, hours, days, or weeks. In certain embodiments, the structural integrity is less than one month, less than one week, less than one day, or less than one hour. The relatively short-term structural integrity results in a formulation that allows the active agent and biomaterial to be delivered to a target site within a tissue or organ by controlled handling, placement, or dispersion without interfering with or interfering with the interaction of the incorporated elements with the tissue or organ in which they are located.

[0230] In certain embodiments, the second biomaterial is a temperature-sensitive biomaterial that has a different sensitivity than the first biomaterial. The second biomaterial can have (i) a substantially solid state at about ambient temperature or below, and (ii) a substantially liquid state at about 37° C. or above. In certain embodiments, ambient temperature is about room temperature.

[0231] In certain embodiments, the second biomaterial is a crosslinked bead. The crosslinked bead can have a tunable in vivo residence time depending on the degree of crosslinking, as described herein. In certain embodiments, the crosslinked bead can have a microscopically small diameter. The formulations may comprise microvesicles (e.g., exosomes) and / or bioactive cells, and are resistant to enzymatic degradation as described herein. In certain embodiments, the formulations of the present disclosure may comprise a first biomaterial combined with an active agent, e.g., microvesicles (e.g., exosomes) and / or bioactive cells, with or without a second biomaterial combined with the active agent, e.g., microvesicles (e.g., exosomes) and / or bioactive cells. In certain embodiments, when the formulation comprises a second biomaterial, the second biomaterial may be temperature-sensitive beads and / or crosslinked beads.

[0232] In one aspect, the present disclosure provides formulations containing biomaterials that degrade over a period of time on the scale of minutes, hours, or days. This contrasts with numerous studies that focus on implanting solid materials that slowly degrade over days, weeks, or months. In certain embodiments, the biomaterials possess one or more of the following attributes: biocompatibility, biodegradability / bioabsorbability, a substantially solid state before and during implantation into a subject, loss of structural integrity (substantially solid state) after implantation, and a cytocompatible environment that supports cell viability and proliferation. The biomaterial's ability to maintain space between implanted particles during implantation promotes natural tissue ingrowth. In certain embodiments, the biomaterial also facilitates implantation of solid formulations. In certain embodiments, the biomaterial provides localization of the formulations described herein, as the insertion of a solid unit helps prevent the delivered material from dispersing within the tissue during implantation. In the case of cell-based formulations, solid biomaterials also improve the stability and viability of anchorage-dependent cells compared to cells suspended in a fluid. However, the short duration of structural integrity means that immediately after implantation the biomaterial does not pose a significant impediment to tissue ingrowth or integration of the delivered cells / materials with the host tissue.

[0233] In one aspect, the present disclosure provides formulations containing biomaterials that are implanted in a substantially solid form and then liquefy / melt or otherwise lose structural integrity after implantation in the body, in contrast to many studies that have focused on the use of materials that can be injected as a liquid and then solidify in the body.

[0234] Biocompatible beads In one aspect, the formulation comprises a temperature-sensitive biomaterial described herein and a population of biocompatible beads containing the biomaterial. In certain embodiments, the beads are crosslinked. Crosslinking can be achieved using any suitable crosslinking agent known to those skilled in the art, such as carbodiimides; aldehydes (e.g., furfural, acrolein, formaldehyde, glutaraldehyde, glyceraldehyde); succinimide-based crosslinkers (bis(sulfosuccinimidyl) suberate (BS3), disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS), dithiobis(succinimidyl propionate), ethylene glycol bis(sulfosuccinimidyl succinate), ethylene glycol bis(succinimidyl succinate) (E Cross-linking can be achieved using bis(sulfosuccinimidyl)glutarate (BS2G), disuccinimidyl tartrate (DST); epoxides (ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether); sugars (glucose and aldose sugars); sulfonic and p-toluenesulfonic acids; carbonyldiimidazole; genipin; imines; ketones; diphenylphosphoryl azide (DDPA); terephthaloyl chloride; cesium(III) nitrate hexahydrate; microbial transglutaminase; and hydrogen peroxide. Those skilled in the art will recognize other cross-linking agents and methods suitable for use in accordance with the present disclosure.

[0235] In certain embodiments, the beads are carbodiimide cross-linked beads. In certain embodiments, the carbodiimide cross-linked beads are 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), DCC (N,N'-dicyclohexyl The beads can be crosslinked with a carbodiimide selected from the group consisting of diisopropylcarbodiimide (DCC) and N,N'-diisopropylcarbodiimide (DIPC). Beads treated with lower concentrations of EDC are expected to have a higher number of free primary amines, while samples treated with higher concentrations of crosslinker will have most of the primary amines engaged in amide bonds. The intensity of the orange color, detectable spectrophotometrically at 335 nm due to the covalent bond between primary amines and picrylsulfonic acid, is proportional to the number of primary amines present in the sample. When normalized to the number of milligrams of protein present in the sample, an inverse correlation can be observed between the number of free amines present and the initial concentration of EDC used for crosslinking. This result is an indication of the difference in bead crosslinking, which is determined by the amount of carbodiimide used in the reaction. Generally, crosslinked beads show a reduced number of free primary amines compared to non-crosslinked beads.

[0236] In certain embodiments, crosslinked beads have reduced susceptibility to enzymatic degradation compared to non-crosslinked biocompatible beads, resulting in beads whose in vivo residence time can be finely tuned. In certain embodiments, crosslinked beads are resistant to endogenous enzymes such as collagenase. In certain embodiments, the provision of crosslinked beads is part of a delivery system that facilitates one or more of the following: (a) delivery of attached cells to a desired site, creating space for natural tissue regeneration and ingrowth and vascular supply; (b) the ability to persist at the site long enough to allow the cells to establish, function, remodel, and secrete their own extracellular matrix (ECM); (c) promotion of integration of implanted cells with surrounding tissue; (d) the ability to implant cells in a substantially solid form; (e) short-term structural integrity that does not pose significant obstacles to tissue ingrowth, de novo vascularization, or integration of delivered cells / materials with host tissue; and (f) localized in a substantially solid form. (g) improved stability and viability of anchorage-dependent cells compared to cells suspended in fluid; and (h) a two-stage release profile when cells are delivered 1) in a substantially solid form (e.g., attached to beads) and 2) in a substantially liquid form (e.g., suspended in a liquid); (i) recapitulation and mimicry of the three-dimensional biological niche or renal parenchyma from which the bioactive cell population is derived.

[0237] In certain embodiments, the present disclosure provides cross-linked beads containing gelatin. In certain embodiments, non-cross-linked gelatin beads are not suitable for bioactive cell formulations because they rapidly lose integrity and cells dissipate from the injection site. In certain embodiments, highly cross-linked gelatin beads may persist at the injection site for an excessively long time, potentially interfering with de novo ECM secretion, cellular integration, angiogenesis, and tissue regeneration. The present disclosure allows for fine-tuning of the in vivo residence time of cross-linked beads. To tailor the biodegradability of the biomaterial, various cross-linker concentrations of carbodiimide are used, while the overall reaction conditions are kept constant for all samples. For example, the enzyme sensitivity of carbodiimide-cross-linked beads can be fine-tuned by varying the cross-linker concentration from about 0 M to about 1 M. In certain embodiments, the concentrations are about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, about 50 mM, about 51 mM, about 52 mM, about 53 mM, about 54 mM, about 55 mM, about 56 mM, about 57 mM, about 58 mM, about 59 mM, about 60 mM, about 61 mM, about 62 mM, about 63 mM, about 64 mM, about 65 mM, about 66 mM, about 67 mM, about 68 mM, about 69 mM, about 70 mM, about 71 mM, about 72 mM, about 73 mM, about 74 mM, about 75 mM, about 76 mM, about 77 mM, about 78 mM, about 79 mM, about 80 mM, about 81 mM, about 82 mM, about 83 mM, about 84 mM, about 85 mM, about 86 mM, about 87 mM, about 88 mM, about 89 mM The cross-linking agent concentration may also be about 0.15M, about 0.2M, about 0.25M, about 0.3M, about 0.35M, about 0.4M, about 0.45M, about 0.5M, about 0.55M, about 0.60M, about 0.65M, about 0.70M, about 0.75M, about 0.80M, about 0.85M, about 0.90M, about 0.95M, or about 100mM. The cross-linking agent may be about 0.6M, about 0.65M, about 0.7M, about 0.75M, about 0.8M, about 0.85M, about 0.9M, about 0.95M, or about 1M. In certain embodiments, the cross-linking agent is 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC). In certain embodiments, the EDC-crosslinked beads are gelatin beads. The percentage of bead degradation can be fine-tuned depending on the concentration of the cross-linking agent. In certain embodiments, the gelatin beads can be mixed with non-gelatin (e.g., but not limited to, alginate or HA) beads or microparticles to further enhance the efficacy of the delivered bioactive cell population.

[0238] Crosslinked beads may have certain properties that are advantageous for seeding, attachment, or encapsulation of bioactive cell populations and / or microvesicles (e.g., exosomes). For example, the beads may have a porous surface and / or may be substantially hollow. In certain embodiments, the presence of pores provides an increased cell attachment surface, allowing for the attachment of a greater number of cells compared to non-porous, i.e., smooth, surfaces. Furthermore, the pore structure may support host tissue integration with the porous beads, supporting de novo tissue formation. In certain embodiments, the beads have a size distribution that can be fitted to a Weibull plot, corresponding to a general particle distribution pattern. In certain embodiments, the crosslinked beads have an average diameter of less than about 120 μm, about 115 μm, about 110 μm, about 109 μm, about 108 μm, about 107 μm, about 106 μm, about 105 μm, about 104 μm, about 103 μm, about 102 μm, about 101 μm, about 100 μm, about 99 μm, about 98 μm, about 97 μm, about 96 μm, about 95 μm, about 94 μm, about 93 μm, about 92 μm, about 91 μm, or about 90 μm. In certain embodiments, the characteristics of the crosslinked beads vary depending on the casting method. In certain embodiments, crosslinked beads are cast using a method in which a liquid gelatin solution is aerosolized using an air stream and sprayed into liquid nitrogen using a thin layer chromatography reagent sprayer (ACE Glassware). , resulting in beads with the above-mentioned properties. Those skilled in the art will appreciate that adjusting the parameters of the casting method provides the opportunity to tailor different properties of the beads, such as different size distributions. In certain embodiments, the microtopography, surface and internal properties of the beads can be further modified to promote cell attachment.

[0239] In certain embodiments, the cytocompatibility of the crosslinked beads is assessed in vitro prior to formulation using cell culture techniques in which beads are cultured with cells corresponding to the final bioactive cell preparation. In certain embodiments, the beads are cultured with primary renal cells and live / dead cell assays are used to confirm cytocompatibility prior to production of the bioactive renal cell preparation. In addition to cell viability, specific functional tests that measure cellular metabolic activity, secretion of certain key cytokines and growth factors and exosomes, and expression of certain key protein and nucleic acid markers, including miRNAs associated with functional bioactive renal cell populations, are well known to those skilled in the art and are further used to confirm cell performance when formulated with crosslinked beads.

[0240] In certain formulations, the biocompatible crosslinked beads are combined with the temperature-sensitive biomaterial in a solution at about 5% (wt / wt) to about 15% (wt / wt) of the volume of the solution. The crosslinked beads may be present at about 5% (wt / wt), about 5.5% (wt / wt), about 6% (wt / wt), about 6.5% (wt / wt), about 7% (wt / wt), about 7.5% (wt / wt), about 8% (wt / wt), about 8.5% (wt / wt), about 9% (wt / wt), about 9.5% (wt / wt), about 10% (wt / wt), about 10.5% (wt / wt), about 11% (wt / wt), about 11.5% (wt / wt), about 12% (wt / wt), about 12.5% ​​(wt / wt), about 13% (wt / wt), about 13.5% (wt / wt), about 14% (wt / wt), about 14.5% (wt / wt), or about 15% (wt / wt) of the volume of the solution.

[0241] In one aspect, the present disclosure provides formulations containing biomaterials that degrade over periods on the scale of minutes, hours, or days, in contrast to many studies that have focused on implanting solid materials that slowly degrade over days, weeks, or months.

[0242] In one aspect, the present disclosure provides a formulation having a delivery matrix with biocompatible crosslinked beads seeded with bioactive cells. In certain embodiments, the delivery matrix has one or more of the following attributes: biocompatibility, biodegradability / bioabsorbability, a substantially solid state before and during implantation into a subject, loss of structural integrity (substantially solid state) after implantation, and a cytocompatible environment that supports cell viability. In certain embodiments, the ability of the delivery matrix to maintain space between implanted particles (e.g., crosslinked beads) during implantation promotes natural tissue ingrowth. In certain embodiments, without the delivery matrix, compression of cellularized beads during implantation can result in spaces unsuitable for sufficient tissue ingrowth. In certain embodiments, the delivery matrix also facilitates implantation of solid formulations. In certain embodiments, the short duration of structural integrity means that the matrix does not pose a significant obstacle to tissue ingrowth, de novo vascularization, or integration of delivered cells / materials with host tissue immediately after implantation. In certain embodiments, the delivery matrix provides localization of the formulations described herein, as the insertion of a solid unit helps prevent the delivered material from dispersing within the tissue during implantation. In certain embodiments, application of the delivery matrix described herein helps prevent rapid loss of transplanted cells through urination when delivered to the renal parenchyma. In certain embodiments, in the case of cell-based formulations, a solid delivery matrix improves the stability and viability of anchorage-dependent cells compared to cells suspended in a fluid.

[0243] In certain embodiments, the delivery matrix is ​​a collection of biocompatible beads that are not seeded with cells. In certain embodiments, unseeded beads are interspersed throughout the individual cell-seeded beads. In certain embodiments, the unseeded beads serve as "spacer beads" between the cell-seeded beads before and immediately after implantation. In certain embodiments, the spacer beads contain a temperature-sensitive biomaterial that has a substantially solid state at a first temperature and a substantially liquid state at a second temperature, the first temperature being lower than the second temperature. For example, the spacer beads contain a biomaterial, such as a biomaterial described herein, that has a substantially solid state at about ambient temperature or below and a substantially liquid state at about 37°C. In certain embodiments, the ambient temperature is about room temperature. In certain embodiments, the biomaterial is a gelatin solution. In certain embodiments, the gelatin solution is present at about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 10.5%, or about 11% (weight / volume). In certain embodiments, the gelatin solution can be provided in PBS, cell culture medium (e.g., DMEM), or another suitable solvent. In certain embodiments, the biomaterial is hyaluronic acid. In certain embodiments, the biomaterial is a decellularized extracellular matrix of human or animal kidney origin that can be further reconstituted as a hydrogel.

[0244] In one aspect, the present disclosure provides formulations containing biomaterials that are implanted in a substantially solid form (e.g., spacer beads) and then liquefy / melt or otherwise lose structural integrity after implantation in the body, in contrast to many studies that have focused on the use of materials that can be injected as a liquid and then solidify in the body.

[0245] The temperature sensitivity of spacer beads can be evaluated in vitro prior to formulation. For example, in certain embodiments, spacer beads can be labeled and unlabeled, non-temperature sensitive beads can be used. The mixture can be mixed with the cross-linked beads (white) and then incubated at 37°C to observe changes in the physical transition. The deformation of the labeled temperature-sensitive beads at higher temperatures is observed over time. For example, temperature-sensitive gelatin beads can be made using Alcian blue dye to act as a marker for the physical transition. Blue gelatin beads can be mixed with cross-linked beads (white) and loaded into a catheter, then extruded and incubated in 1x PBS (pH 7.4) at 37°C. The deformation of the blue gelatin beads is tracked by microscopy at various time points. The change in the physical state of the blue gelatin beads becomes visible after 30 minutes and becomes more pronounced with extended incubation times. Due to the viscosity of the material, the beads do not completely dissipate.

[0246] Modified-release formulations In one aspect, the formulations of the present disclosure are provided as modified-release formulations. Generally, the modified release is characterized by an initial release of a first active agent upon administration, followed by at least one additional subsequent release of a second active agent. The first active agent and the second active agent may be the same or different. In certain embodiments, the formulation provides modified release through multiple components in the same formulation. In certain embodiments, the modified-release formulation contains the active agent as part of a first component that allows the active agent to move freely throughout the volume of the formulation, thereby enabling immediate release at the target site upon administration. The first component may be a temperature-sensitive biomaterial having a substantially liquid phase and a substantially solid phase, where the first component is in a substantially liquid phase at the time of administration. In certain embodiments, the active agent is present in a substantially liquid phase such that it is substantially free to move throughout the volume of the formulation, thereby resulting in immediate release at the target site upon administration.

[0247] In certain embodiments, the modified-release formulation has an active agent attached to, deposited on, coated with, embedded in, seeded on, or entrapped within the second component as part of the second component, which persists before and after administration to a target site. The second component contains a structural element with which the active agent can associate, thereby preventing immediate release of the active agent from the second component upon administration. For example, the second component is provided in a substantially solid form, e.g., biocompatible beads that can be crosslinked to prevent or delay enzymatic degradation in vivo. In certain embodiments, the substantially solid-phase active agent retains its structural integrity within the formulation before and after administration, and therefore does not immediately release the active agent to a target site upon administration. Suitable carriers for modified-release formulations are described herein; however, those skilled in the art will recognize other carriers suitable for use in the present disclosure.

[0248] In certain embodiments, the formulations provide for an initial rapid delivery / release of delivered elements, including cells, microvesicles (e.g., exosomes), nanoparticles, therapeutic molecules, etc., followed by a delayed release of subsequent elements. In certain embodiments, the formulations provide for an initial rapid delivery / release of microvesicles (e.g., exosomes), miRNA, and other bioactive nucleic acid or protein molecules that are soluble and secreted bioactive products originating from renal cell populations or other cell populations. Other molecules or therapeutic agents associated with regenerative bioactivity will be understood by those skilled in the art. The formulations of the present disclosure can be designed for a two-stage release profile such that the delivered agent is provided in both an unattached form (e.g., microvesicles and / or cells in solution) and an attached form (e.g., microvesicles and / or cells together with beads or another suitable carrier). Upon initial administration, unhindered agents are immediately delivered to the delivery site, while release of hindered agents is delayed until the structural integrity of the carrier (e.g., beads) is compromised, at which point the pre-attached agent is released. Other suitable release mechanisms will be appreciated by those skilled in the art, as discussed below.

[0249] In certain embodiments, the release delay time can be adjusted based on the properties of the active agent. For example, the release delay time for microvesicles (e.g., exosomes) and / or bioactive cell formulations can be on the order of seconds, minutes, hours, or days. In certain embodiments, a delay of several weeks may be appropriate. In certain embodiments, for other active agents, such as small or large molecules, the release delay time for the formulation can be on the order of seconds, minutes, hours, days, weeks, or months. The formulation can also contain different biomaterials that provide different time-delay release profiles. For example, a first biomaterial with a first active agent can exhibit a first release time, and a second biomaterial with a second active agent can exhibit a second release time. The first and second active agents can be the same or different.

[0250] In certain embodiments, the period of delayed release may generally correspond to the time it takes for the biomaterial to lose its structural integrity. However, those skilled in the art will recognize other delayed-release mechanisms. For example, the active agent may be released continuously over an extended period of time, independent of the degradation time of any particular biomaterial, e.g., by diffusion of the drug from the polymer matrix. Furthermore, microvesicles (e.g., exosomes) and / or bioactive cells may migrate out of the formulation containing the biomaterial and bioactive cells into the native tissue. In certain embodiments, the bioactive cells migrate out of the biomaterial, e.g., beads, into the native tissue. In certain embodiments, the bioactive cells migrate out of the biomaterial into the native tissue, inducing the secretion of growth factors, cytokines, exosomes, miRNA, and other nucleic acids and proteins associated with regenerative bioactivity. In certain embodiments, exosomes and other extracellular microvesicles, as well as miRNA, other bioactive nucleic acids, and proteins, migrate out of the biomaterial. In certain embodiments, the bioactive cells migrate from the biomaterial into the native tissue and mediate the recruitment of host stem and progenitor cells, which then migrate or home towards the site of injury or disease.

[0251] In certain embodiments, biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Prolonged absorption of injectable formulations can be brought about by including in the formulation an agent that delays absorption, such as monostearate salts and gelatin. Many methods for making such formulations are patented or generally known to those skilled in the art. See, for example, *Sustained and Controlled Release Drug Delivery Systems*, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. Additional methods applicable to controlled or sustained release of polypeptide agents are described, for example, in U.S. Patent Nos. 6,306,406 and 6,346,274, as well as U.S. Patent Application Publication Nos. 20020182254 and 20020051808, all of which are incorporated herein by reference.

[0252] Exemplary Bioactive Cell Formulations In certain embodiments, the vesicles (microvesicles, e.g., exosomes, etc.) provided herein are included in a bioactive cell formulation. Alternatively, or additionally, the vesicles can be administered before, simultaneously with, or after the bioactive cell formulation.

[0253] In certain embodiments, the formulations described herein contain implantable constructs made from the above-referenced biomaterials with bioactive renal cells described herein for the treatment of kidney disease in a subject in need thereof. In certain embodiments, the bioactive cell formulations provided herein further comprise vesicles (e.g., microvesicles such as exosomes secreted by bioactive renal cells).

[0254] In certain embodiments, constructs are made from a scaffold or matrix composed of a biocompatible material or biomaterial, one or more synthetic or naturally occurring biocompatible materials, and one or more cell populations or microvesicles (e.g., exosomes) described herein deposited on or embedded in the surface of the scaffold by attachment and / or entrapment. In certain embodiments, constructs are made from a biomaterial and one or more cell populations or products thereof (such as microvesicles, e.g., exosomes) described herein coated with, deposited on, deposited within, attached to, entrapped within, embedded in, seeded with, or combined with the biomaterial component(s). Any of the microvesicles (e.g., exosomes) and / or cell populations described herein can be used in combination with a matrix to form a construct.

[0255] In certain embodiments, the bioactive cell preparation is an injectable product composed of SRCs genetically modified to reduce immunogenicity and formulated in a biomaterial (e.g., a gelatin-based hydrogel). In one aspect, allogeneic SRCs are obtained by isolating and expanding kidney cells from a donor patient's renal cortical tissue via kidney biopsy, genetically modifying the SRCs using gene editing technology, and selecting the expanded kidney cells by density gradient centrifugation. In certain embodiments, SRCs are primarily composed of renal epithelial cells, which are well known for their regenerative capabilities (Humphreys et al. (2008) Intrinsic epithelial cells repair the kidney after injury. Cell Stem Cell. 2(3):284-91). In certain embodiments, other parenchymal (vascular) and interstitial (collecting duct) cells may be sparsely present in the SRC population. Injection of SRCs into recipient kidneys has resulted in significant improvements in animal survival, urinary concentration, and filtration function in non-clinical studies. However, SRCs have a limited shelf life and stability. Formulating SRC in a gelatin-based hydrogel biomaterial results in enhanced cell stability, thus extending the product's shelf life and improving stability during transport and delivery to the kidney cortex for clinical utility.

[0256] In one embodiment, bioactive cell preparations are produced by first obtaining renal cortical tissue from a donor using standard clinical care kidney biopsy procedures. Renal cells are isolated from the kidney tissue by enzymatic digestion and grown using standard cell culture techniques. The cell culture medium is designed to grow primary kidney cells and does not contain any differentiation factors. The harvested kidney cells are subjected to density gradient separation to obtain SRCs. Gene editing techniques can be used to alter the immunogenicity of SRCs either before or after density gradient separation.

[0257] In certain embodiments, the formulated cell population and / or microvesicles (e.g., exosomes) are substantially free to move throughout the volume of the biomaterial at about ambient temperature or above. Having the cell population suspended in a substantially solid phase at lower temperatures provides a stability advantage for cells, such as anchorage-dependent cells, compared to cells in a fluid. Furthermore, having the microvesicles (e.g., exosomes) and / or cells suspended in a substantially solid state provides one or more of the following advantages: i) preventing settling of the microvesicles and / or cells; ii) keeping the cells fixed to the biomaterial in a suspended state; iii) keeping the microvesicles and / or cells more uniformly dispersed throughout the volume of the biomaterial; iv) preventing the formation of microvesicle and / or cell aggregates; and v) providing better protection of the microvesicles and / or cells during storage and transport of the formulation. Formulations that can retain such characteristics up until administration to a subject are advantageous, at least due to better overall health of the cells in the formulation and a more uniform and consistent dose of cells administered.

[0258] In certain embodiments, the gelatin-based hydrogel biomaterial used to formulate the SRC is porcine gelatin, which forms a thermoresponsive hydrogel when dissolved in a buffer solution. In certain embodiments, the hydrogel is fluid at room temperature but gels when cooled to refrigerated temperatures (2°C-8°C). In certain embodiments, SRC is formulated with the hydrogel, gelled by cooling, and shipped to the clinic at refrigerated temperatures (2°C-8°C). In certain embodiments, at the clinical site, the product is warmed to room temperature before being injected into the patient's kidney. In certain embodiments, the bioactive cell preparation (e.g., supplemented with microvesicles such as exosomes from bioactive renal cells) is implanted into the kidney cortex using a needle and syringe suitable for percutaneous or laparoscopic delivery.

[0259] Description and Composition of Exemplary Neo-Kidney Augment Compositions In certain embodiments, the bioactive cell preparation is Neo-Kidney Augment (NKA), which is an injectable product composed of autologous selected kidney cells (SRCs) formulated in a biomaterial (gelatin-based hydrogel). In certain embodiments, the NKA is augmented or supplemented with vesicles (e.g., microvesicles such as exosomes secreted by bioactive kidney cells).

[0260] In one aspect, autologous SRCs are obtained by isolating and expanding renal cells from a patient's renal cortical tissue via a kidney biopsy, and selecting the expanded renal cells by centrifugation across a density boundary, density barrier, or density interface. In certain embodiments, autologous SRCs are obtained by isolating and expanding renal cells from a patient's renal cortical tissue via a kidney biopsy, and selecting the expanded renal cells against a continuous or discontinuous single-step or multi-step density gradient. SRCs are primarily composed of renal tubular epithelial cells, which are well known for their regenerative capacity (Humphreys et al. (2008) Intrinsic epithelial cells repair the kidney after injury. Cell Stem Cell. 2(3):284-91). Other parenchymal (vascular) and interstitial (collecting duct) cells may be present sparsely in the autologous SRC population.

[0261] In certain embodiments, the NKA is supplemented with microvesicles (e.g., exosomes) produced and isolated from a population of SRCs.

[0262] Infusion of SRC into recipient kidneys has significantly improved animal survival, urinary concentration, and filtration function in preclinical studies. However, SRC has a limited shelf life and stability. Formulation of SRC in a gelatin-based hydrogel biomaterial results in enhanced cell stability, thus extending the shelf life of the product and improving the stability of NKA during transport and delivery to the kidney cortex for clinical utility.

[0263] In one aspect, NKAs are produced by first obtaining renal cortical tissue from a donor / recipient using standard clinical care kidney biopsy procedures. In certain embodiments, renal cells are isolated from the kidney tissue by enzymatic digestion and expanded using standard cell culture techniques. In certain embodiments, the cell culture medium is designed to expand primary renal cells and does not contain any differentiation factors. In certain embodiments, the harvested renal cells are subjected to separation across a density boundary or density interface or density gradient to obtain SRCs. In certain embodiments, the SRCs are genetically modified according to the present disclosure.

[0264] Included herein are formulations made from biomaterials designed or adapted to respond to external conditions as described herein. As a result, the nature of the association of bioactive cell populations and other active agents, such as microvesicles (e.g., exosomes), with the biomaterial in the construct will change depending on the external conditions. For example, the association of a cell population with a temperature-sensitive biomaterial will change with temperature. In certain embodiments, the construct contains a bioactive renal cell population and a biomaterial that has a substantially solid state at about 8°C or below and a substantially liquid state at about ambient temperature or above, wherein the cell population The cell population is suspended in the biomaterial at about 8°C or below. However, the cell population is substantially free to move throughout the volume of the biomaterial at about ambient temperature or above. Having the cell population suspended in a substantially solid phase at lower temperatures provides a stability advantage for cells, such as anchorage-dependent cells, compared to cells in a fluid. Furthermore, having the microvesicles (e.g., exosomes) and cells suspended in a substantially solid state provides one or more of the following advantages: i) preventing settling of the microvesicles and cells; ii) keeping the cells fixed to the biomaterial in a suspended state; iii) keeping the microvesicles and cells more uniformly dispersed throughout the volume of the biomaterial; iv) preventing the formation of microvesicle or cell aggregates; and v) providing better protection of the microvesicles and cells during storage and transport of the formulation. In certain embodiments, formulations that can retain such characteristics until administration to a subject are advantageous, at least due to better overall health of the cells in the formulation and a more uniform and consistent dose of cells administered.

[0265] In certain embodiments, the gelatin-based hydrogel biomaterial used to formulate the SRC into the NKA is porcine gelatin, which forms a thermoresponsive hydrogel when dissolved in a buffer solution. This hydrogel is fluid at room temperature but gels when cooled to refrigerated temperatures (2°C-8°C). The SRC is formulated with the hydrogel to obtain the NKA. The NKA is gelled by cooling and transported to the clinic at refrigerated temperatures (2°C-8°C). The NKA has a shelf life of 3 days. In clinical settings, the product is warmed to room temperature before injection into the patient's kidney. The NKA is implanted into the kidney cortex using a needle and syringe suitable for percutaneous or laparoscopic delivery of the NKA. In certain embodiments, the hydrogel is derived from gelatin or another extracellular matrix protein of recombinant origin. In certain embodiments, the hydrogel is derived from an extracellular matrix originating from the kidney or another tissue or organ. In certain embodiments, the hydrogel is derived from a recombinant extracellular matrix protein. In certain embodiments, the hydrogel comprises gelatin derived from recombinant collagen (ie, recombinant gelatin).

[0266] cell viability agents In one aspect, the bioactive cell formulation also includes a cell viability agent. In certain embodiments, the vesicles provided herein include a cell viability agent. In certain embodiments, the cell viability agent is selected from the group consisting of antioxidants, oxygen carriers, immunomodulatory factors, cell recruitment factors, cell adhesion factors, anti-inflammatory agents, angiogenic factors, matrix metalloproteinases, wound healing factors, and products secreted from bioactive cells.

[0267] In one aspect, the microvesicles (e.g., exosomes) comprise a cell viability agent (e.g., within their lumen, within their lipid bilayer, or on their surface). In certain embodiments, the microvesicles are secreted by cells cultured in the presence of the cell viability agent.

[0268] In certain embodiments, the cell viability agent is selected from the group consisting of antioxidants, oxygen carriers, immunomodulatory factors, cell recruitment factors, cell adhesion factors, anti-inflammatory agents, angiogenic factors, matrix metalloproteinases, wound healing factors, and bioactive cell-secreted products.

[0269] Antioxidants are characterized by their ability to inhibit the oxidation of other molecules. Antioxidants include, but are not limited to, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox™), carotenoids, flavonoids, isoflavones, ubiquinone, glutathione, lipoic acid, superoxide dismutase, ascorbic acid, vitamin E, vitamin A, mixed carotenoids (e.g., beta-carotene, alpha-carotene, gamma-carotene, lutein, lycopene, phytopene, phytofluene, and astaxanthin), selenium, coenzyme Q10, indole-3-carbinol, proanthocyanidins, resveratrol, quercetin, catechins, salicylic acid, curcumin, bilirubin, Antioxidants include one or more of oxalic acid, phytic acid, lipoic acid, vanillic acid, polyphenols, ferulic acid, theaflavins, and derivatives thereof. Those skilled in the art will appreciate that other suitable antioxidants may be used in certain embodiments of the present disclosure.

[0270] Oxygen carriers are agents characterized by their ability to carry and release oxygen. Oxygen carriers include, but are not limited to, perfluorocarbons and perfluorocarbon-containing pharmaceuticals. Suitable perfluorocarbon-based oxygen carriers include, but are not limited to, perfluorooctyl bromide (CF 17 Br), perfluorodichlorooctane (CF 16 C l2), perfluorodecyl bromide, perflubron, perfluorodecalin, perfluorotripropylamine, perfluoromethylcyclopiperidine, Fluosol™ (perfluorodecalin and perfluorotripropylamine), Perftoran™ (perfluorodecalin and perfluoromethylcyclopiperidine), Oxygent™ (perfluorodecyl bromide and perflubron), Ocycyte™ (perfluoro(tert-butylcyclohexane)). Those skilled in the art will appreciate that other suitable perfluorocarbon oxygen carriers may be used in certain embodiments of the present disclosure.

[0271] Immunomodulatory factors include, but are not limited to, osteopontin, FAS ligand factor, interleukins, transforming growth factor beta, platelet-derived growth factor, clusterin, transferrin, regulated upon action, normal T-cell expressed, secreted protein (RANTES), plasminogen activator inhibitor-1 (Pai-1), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), alpha-1-microglobulin, and beta-2-microglobulin. One skilled in the art will appreciate that other suitable immunomodulatory factors can be used in certain embodiments of the present disclosure.

[0272] Anti-inflammatory or immunosuppressant agents (described below) may also be part of the formulation. Those skilled in the art will appreciate that other suitable antioxidants may be used in certain embodiments of the present disclosure.

[0273] Cell recruitment factors include, but are not limited to, monocyte chemoattractant protein 1 (MCP-1) and CXCL-1. Those skilled in the art will appreciate that other suitable cell recruitment factors may be used in certain embodiments of the present disclosure.

[0274] Cell adhesion factors include, but are not limited to, fibronectin, procollagen, collagen, ICAM-1, connective tissue growth factor, laminin, proteoglycans, and certain cell adhesion peptides such as RGD and YSIGR. Those skilled in the art will appreciate that other suitable cell adhesion factors may be used in certain embodiments of the present disclosure.

[0275] Angiogenic factors include, but are not limited to, vascular endothelial growth factor F (VEGF) and angiopoietin-2 (ANG-2). Those skilled in the art will appreciate that other suitable angiogenic factors may be used in certain embodiments of the present disclosure.

[0276] Matrix metalloproteinases include, but are not limited to, matrix metalloproteinase 1 (MMP1), matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP-9), and tissue inhibitor of metalloproteinase-1 (TIMP-1).

[0277] Wound healing factors include, but are not limited to, keratinocyte growth factor 1 (KGF-1), tissue plasminogen activator (tPA), calbindin, clusterin, cysteine, and cysteine. These include tatin C, trefoil factor 3. Those skilled in the art will appreciate that other suitable wound healing factors may be used in certain embodiments of the present disclosure.

[0278] Products secreted from the bioactive cells described herein can also be added to bioactive cell formulations as cell viability agents.

[0279] Compositions derived from body fluids, tissues, or organs from human or animal sources, including, but not limited to, human plasma, human platelet lysate, fetal bovine plasma, or bovine pituitary extract, can also be added to the bioactive cell preparations as cell viability agents.

[0280] Those skilled in the art will appreciate that there are several suitable methods for depositing or otherwise combining cell populations with biomaterials to form constructs.

[0281] In certain embodiments, BRCs (such as SRCs) cultured in medium containing a cell viability agent produce vesicles containing the cell viability agent.

[0282] How to use In one aspect, provided herein are methods for treating kidney disease in a subject. In certain embodiments, the methods include administering to a subject an effective amount of isolated secreted kidney cell vesicles (e.g., microvesicles such as exosomes). In certain embodiments, the vesicles include a compound not produced by the cells that produce the vesicles. In certain embodiments, the vesicles are present in a composition or formulation disclosed herein.

[0283] In one aspect, provided herein are methods for treating kidney disease in a subject. In certain embodiments, the methods comprise administering to the subject an effective amount of vesicles from a vesicle preparation, wherein the vesicles from the vesicle preparation have been identified as regenerative according to the methods disclosed herein.

[0284] In one aspect, provided herein are methods of treating a renal disease in a subject, hi certain embodiments, the method comprises administering to the subject an effective amount of a composition comprising a bioactive renal cell population supplemented with renal cell vesicles not secreted by the bioactive renal cell population.

[0285] In certain embodiments, the composition is administered by intravenous injection. In certain embodiments, the composition is administered by transcatheter delivery. In certain embodiments, the vesicles are injected intravenously into a peripheral blood vessel. In certain embodiments, the transcatheter delivery is delivery to the left or right renal artery of the subject.

[0286] In certain embodiments, the subject has chronic kidney disease, hi certain embodiments, the chronic kidney disease is stage I, stage II, stage III, stage IV, or stage V kidney disease.

[0287] In certain embodiments, treating kidney disease comprises reducing or preventing kidney fibrosis in a subject.

[0288] In certain embodiments, the subject is undergoing dialysis at least once, twice, or three times per week for at least one or two weeks.

[0289] In certain embodiments, the subject has type II diabetes.

[0290] In certain embodiments, the subject has a congenital anomaly of the kidney and urinary tract (CAKUT).

[0291] In certain embodiments, the subject receives a blood flow of 90 mL / min / 1.73 m 2 have a glomerular filtration rate (GFR) of less than 0.05, microalbuminuria, or macroalbuminuria.

[0292] In certain embodiments, no cells are administered to the subject. In certain embodiments, cells (e.g., BRCs, such as SRCs) are administered to the subject. In certain embodiments, the vesicles are administered separately from the cells. In certain embodiments, the vesicles are administered before, simultaneously with, or after the cells. In certain embodiments, the isolated vesicles are administered in a composition further comprising cells (e.g., cells other than the cells from which the vesicles were isolated). In certain embodiments, the renal cell vesicles are secreted by a bioactive renal cell population that has the same origin and / or comprises the same cell type as the bioactive renal cell population in the composition.

[0293] In certain embodiments, the vesicles are produced by a BRC population. In certain embodiments, the BRC population is an SRC population.

[0294] In certain embodiments, vesicles secreted by primary renal cells are administered to a subject. In certain embodiments, vesicles secreted by primary renal cells and vesicles secreted by SRCs are administered to a subject.

[0295] In certain embodiments, vesicles secreted by endothelial cells or mesenchymal stem cells are also administered to the subject.

[0296] In certain embodiments, non-renal cell vesicles are also administered to the subject, hi certain embodiments, the non-renal cell vesicles are secreted by non-renal endothelial progenitor cells, non-renal mesenchymal stem cells, or non-renal adipose-derived progenitor cells.

[0297] In certain embodiments, the effective amount of vesicles is an amount that would be effective in the absence of administration of bioactive renal cells (e.g., an amount sufficient for treatment without administration of bioactive renal cells). In certain embodiments, the effective amount of bioactive renal cells is an amount that would be effective in the absence of administration of vesicles (e.g., an amount sufficient for treatment without administration of vesicles). In certain embodiments, the effective amount of vesicles is an amount that is less than the effective amount without co-administration of bioactive renal cells. In certain embodiments, the effective amount of bioactive renal cells is an amount that is less than the effective amount without co-administration of vesicles.

[0298] The microvesicles (e.g., exosomes), cells, and formulations of the present invention are suitable for use in the methods of use described herein. In certain embodiments, the formulations of the present invention can be administered for the treatment of kidney disease. In certain embodiments, the microvesicles (e.g., exosomes) and / or bioactive cells can be administered to a native organ as part of the formulations described herein. In certain embodiments, the microvesicles (e.g., exosomes) and / or bioactive cells can originate from a source other than the native organ or target native organ to which they are administered.

[0299] In certain embodiments, the present disclosure provides methods of treating kidney disease in a subject in need thereof using a formulation comprising the microvesicles (e.g., exosomes) and / or bioactive kidney cell populations described herein. In certain embodiments, the formulation is suitable for administration to a subject in need of improved kidney function.

[0300] In one aspect, effective treatment of kidney disease in a subject by the methods of the present disclosure can be monitored through various indicators of kidney function. In certain embodiments, indicators of kidney function include, but are not limited to: These include, but are not limited to, serum albumin, albumin to globulin ratio (A / G ratio), serum phosphorus, serum sodium, kidney size (which can be measured 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). Additionally, some indicators of general health and well-being include, but are not limited to, weight gain or loss, survival rate, blood pressure (mean systemic, diastolic, or systolic), and physical endurance capacity.

[0301] In one aspect, effective treatment with the formulation is evidenced by stabilization of one or more indicators of renal function. In certain embodiments, stabilization of renal function is demonstrated by observing a change in one or more indicators in a subject treated with the disclosed method compared to the same indicator in a subject not treated with the disclosed method. In certain embodiments, stabilization of renal function can be demonstrated by observing a change in the same indicator in the same subject treated with the disclosed method compared to the same indicator in the subject before treatment. The change in one indicator can be an increase or decrease in value. In certain embodiments, treatment provided by the present disclosure can include stabilization of serum creatinine and / or blood urea nitrogen (BUN) levels in a subject, where the BUN levels observed in the subject are lower compared to subjects with a similar condition not treated with the disclosed method. In certain embodiments, the treatment can include stabilization of serum creatinine levels in a subject, where the serum creatinine levels observed in the subject are lower compared to subjects with a similar condition not treated with the disclosed method.

[0302] One of ordinary skill in the art will appreciate that one or more additional indicators described herein or known in the art can be measured to determine effective treatment of kidney disease in a subject.

[0303] In certain embodiments, effective treatment with the formulation is evidenced by an improvement in one or more indicators of kidney structure and / or kidney function. In certain embodiments, the microvesicles (e.g., exosomes) and / or bioactive renal cells result in improved serum creatinine and / or blood urea nitrogen (BUN) levels. In certain embodiments, the microvesicles (e.g., exosomes) and / or bioactive renal cells result in improved serum protein retention. In certain embodiments, the microvesicles (e.g., exosomes) and enriched bioactive renal cells result in improved serum albumin levels compared to a non-enriched cell population or cells to which the microvesicles (e.g., exosomes) have not been added. In certain embodiments, the microvesicles (e.g., exosomes) result in improved serum albumin levels compared to bioactive renal cells. In certain embodiments, the microvesicles (e.g., exosomes) and / or enriched bioactive renal cell population result in an improved A:G ratio compared to a non-enriched cell population. In certain embodiments, the bioactive renal cell population results in improved serum cholesterol and / or triglyceride levels. In certain embodiments, the microvesicles (e.g., exosomes) and / or bioactive renal cell population result in improved vitamin D levels. In certain embodiments, the microvesicles (e.g., exosomes) and / or enriched bioactive renal cell population result in improved phosphorus:calcium ratios compared to non-enriched cell populations. In certain embodiments, the microvesicles (e.g., exosomes) and / or bioactive renal cell population result in improved hemoglobin levels compared to non-enriched cell populations. In certain embodiments, the microvesicles (e.g., exosomes) and / or bioactive renal cell population result in improved serum creatinine levels compared to non-enriched cell populations. In certain embodiments, the microvesicles (e.g., exosomes) and / or enriched bioactive renal cell population result in improved hematocrit levels compared to non-enriched cell populations. In certain embodiments, improvement in one or more of the above indicators of kidney function is a result of treatment with microvesicles (e.g., exosomes) and / or selected kidney cell preparations. In one embodiment, improvement in kidney function is a result of treatment with microvesicles (e.g., exosomes) and / or selected kidney cell preparations. Improvement in one or more of the above indicators is a result of treatment with the microvesicles (e.g., exosomes) and / or selected renal cell preparations.

[0304] In one aspect, the present disclosure provides a formulation for use in a method for regenerating a native kidney in a subject in need of regeneration. In certain embodiments, the method comprises administering or transplanting a bioactive cell population, cell product, or construct described herein into a subject. The regenerated native kidney can be characterized by a number of indicators, including, but not limited to, the development of function or capacity in the native kidney, the improvement of function or capacity in the native kidney, and the expression of certain markers in the native kidney. In certain embodiments, the development or improvement of function or capacity can be observed based on various indicators of kidney function described above. In certain embodiments, the regenerated kidney is characterized by the differential expression of one or more stem cell markers. Stem cell markers can be one or more of the following: SRY (Sex Determining Region Y)-box 2 (Sox2), undifferentiated germline transcription factor (UTF1), Nodal homolog from mouse (NODAL), prominin 1 (PROM1) or CD133 (CD133), CD24, and any combination thereof (see International Application No. PCT / US2011 / 036347 by Ilagan et al., which is incorporated herein by reference in its entirety) (Genheimer et al. al., 2012. Molecular characterization of the regenerative response induced by intrarenal transplantation of selected renal cells in a rodent model of chronic kidney disease. Cells Tissue Organs 196: 374-384 (incorporated by citation in its entirety) See also (part of the specification). In certain embodiments, expression of the stem cell marker(s) is upregulated compared to a control.

[0305] In one aspect, provided herein is a method of treating kidney disease in a subject, the method comprising injecting a formulation, composition, cell population, or microvesicle (e.g., exosome) disclosed herein into the subject. In certain embodiments, the formulation, composition, cell population, or cell product (e.g., microvesicle, e.g., exosome) is injected through an 18- to 30-gauge needle. In certain embodiments, the formulation, composition, cell population, or cell product (e.g., microvesicle, e.g., exosome) is injected through a needle smaller than 20-gauge. In certain embodiments, the formulation, composition, cell population, or cell product (e.g., microvesicle, e.g., exosome) is injected through a needle smaller than 21-gauge. In certain embodiments, the formulation, composition, cell population, or cell product (e.g., microvesicle, e.g., exosome) is injected through a needle smaller than 22-gauge. In certain embodiments, the formulation, composition, cell population, or cell product (e.g., microvesicle, e.g., exosome) is injected through a needle smaller than 23-gauge. In certain embodiments, the formulation, composition, cell population, or cell product (such as a microvesicle, e.g., an exosome) is injected through a needle smaller than 24 gauge. In certain embodiments, the formulation, composition, cell population, or cell product (such as a microvesicle, e.g., an exosome) is injected through a needle smaller than 25 gauge. In certain embodiments, the formulation, composition, cell population, or cell product (such as a microvesicle, e.g., an exosome) is injected through a needle smaller than 26 gauge. In certain embodiments, the formulation, composition, cell population, or cell product (such as a microvesicle, e.g., an exosome) is injected through a needle smaller than 27 gauge. In certain embodiments, the formulation, composition, cell population, or cell product (such as a microvesicle, e.g., an exosome) is injected through a needle smaller than 28 gauge. In certain embodiments, the formulation, composition, cell population, or cell product (such as a microvesicle, e.g., an exosome) is injected through a needle smaller than 29 gauge. In certain embodiments, the formulation, composition, cell population, or cell product (such as a microvesicle, e.g., an exosome) is injected through an approximately 20-gauge needle. In certain embodiments, the formulation, composition, cell population, or cell product (such as a microvesicle, e.g., an exosome) is injected through an approximately 21-gauge needle.

[0306] In certain embodiments, the formulation, composition, cell population, or cell product (such as a microvesicle, e.g., an exosome) is injected through an approximately 22 gauge needle. The agent, composition, cell population, or cell product (such as a microvesicle, e.g., an exosome) is injected through an approximately 23-gauge needle. In certain embodiments, the agent, composition, cell population, or cell product (such as a microvesicle, e.g., an exosome) is injected through an approximately 24-gauge needle. In certain embodiments, the agent, composition, cell population, or cell product (such as a microvesicle, e.g., an exosome) is injected through an approximately 25-gauge needle. In certain embodiments, the agent, composition, cell population, or cell product (such as a microvesicle, e.g., an exosome) is injected through an approximately 26-gauge needle. In certain embodiments, the agent, composition, cell population, or cell product (such as a microvesicle, e.g., an exosome) is injected through an approximately 27-gauge needle. In certain embodiments, the agent, composition, cell population, or cell product (such as a microvesicle, e.g., an exosome) is injected through an approximately 28-gauge needle. In certain embodiments, the formulation, composition, cell population, or cell product (such as a microvesicle, e.g., an exosome) is injected through an approximately 29 gauge needle.

[0307] In certain embodiments, the inner diameter of the needle is less than 0.84 mm. In certain embodiments, the inner diameter of the needle is less than 0.61 mm. In certain embodiments, the inner diameter of the needle is less than 0.51 mm. In certain embodiments, the inner diameter of the needle is less than 0.41 mm. In certain embodiments, the inner diameter of the needle is less than 0.33 mm. In certain embodiments, the inner diameter of the needle is less than 0.25 mm. In certain embodiments, the inner diameter of the needle is less than 0.20 mm. In certain embodiments, the inner diameter of the needle is less than 0.15 mm. In certain embodiments, the outer diameter of the needle is less than 1.27 mm. In certain embodiments, the outer diameter of the needle is less than 0.91 mm. In certain embodiments, the outer diameter of the needle is less than 0.81 mm. In certain embodiments, the outer diameter of the needle is less than 0.71 mm. In certain embodiments, the outer diameter of the needle is less than 0.64 mm. In certain embodiments, the needle has an outer diameter of less than 0.51 mm. In certain embodiments, the needle has an outer diameter of less than 0.41 mm. In certain embodiments, the needle has an outer diameter of less than 0.30 mm. In certain embodiments, the needle has one of the sizes in the following table:

[0308] TIFF2026035692000005.tif99170

[0309] Administration method and route In certain embodiments, the vesicles (e.g., microvesicles such as renal exosomes) are administered in the absence of cells (e.g., BRCs such as SRCs). In certain embodiments, the vesicles are administered together with the cells. In certain embodiments, the vesicles are administered in the same composition as the cells, as well as separately from the cells. In certain embodiments, the vesicles and the cells are administered by different routes of administration. In certain embodiments, the vesicles and the cells are administered by one route of administration and the vesicles are administered separately by another route of administration. In certain embodiments, the vesicles are administered more frequently than the cells.

[0310] In certain embodiments, the vesicles are administered intravenously. In certain embodiments, the vesicles are administered intravenously into a peripheral blood vessel. In certain embodiments, the vesicles are administered by transcatheter delivery. In certain embodiments, the transcatheter delivery is delivery into the left or right renal artery of a subject.

[0311] The formulations of the present invention can be administered alone or in combination with other bioactive components. In certain embodiments, the formulations are suitable for injecting or implanting incorporated tissue engineering elements into the interior of solid organs to regenerate tissue. In certain embodiments, the formulations are used for injecting or implanting tissue engineering elements into the wall of hollow organs to regenerate tissue. In certain embodiments, such formulations are administered in combination with additional formulations suitable for systemic or transcatheter delivery.

[0312] In one aspect, the present invention provides a method of delivering a bioactive cell preparation described herein to a subject in need thereof. In certain embodiments, the bioactive cell preparation is supplemented with vesicles. In certain embodiments, the bioactive cell preparation is not supplemented with vesicles, and a separate preparation containing vesicles is administered to the subject. In certain embodiments, the source of the bioactive cells and / or vesicles can be allogeneic or syngeneic, and any combination thereof. In certain embodiments, the method can include administration of an immunosuppressant (see, e.g., U.S. Patent No. 7,563,822).

[0313] In certain embodiments, the therapeutic methods of the subject invention involve delivery of a bioactive cell preparation described herein. In certain embodiments, cells and / or vesicles are administered directly to the intended site of benefit. A subject in need may also be treated by contacting the native kidney in vivo with a bioactive cell preparation described herein, together with products secreted from one or more enriched renal cell populations and / or mixtures or constructs containing such products. The in vivo contacting step produces a regenerative effect on the native kidney. In certain embodiments, isolated vesicles (e.g., vesicles in the absence of cells) are administered before, simultaneously with, or after (e.g., via the same or a different route of administration) the bioactive cell preparation.

[0314] Various means of administering compositions of selected kidney cells to a subject will be apparent to those of skill in the art in light of this specification, including injection of the cells into a target site in the subject.

[0315] delivery vehicle In certain embodiments, the cells and / or secretory products may be inserted into a delivery device or vehicle that facilitates introduction by injection or implantation into a subject. In certain embodiments, the delivery vehicle may comprise natural materials. In certain embodiments, the delivery vehicle may comprise synthetic materials. In certain embodiments, the delivery vehicle results in a structure that mimics or appropriately matches an organ structure. In certain embodiments, the delivery vehicle is fluidic in nature. Such a delivery device may include a tube, e.g., a catheter, that injects the cells and fluid into the body of the recipient subject. In certain embodiments, the tube further has a needle, e.g., a syringe, that can introduce the cells of the present invention into a subject at a desired location. In certain embodiments, a mammalian kidney-derived cell population is delivered via a catheter. (Here, the term "catheter" is intended to include any of a variety of tube-like systems that deliver substances to blood vessels.) In certain embodiments, the cells are placed in or on biomaterials or scaffolds, including, but not limited to, textiles such as woven, knitted, braided, meshed, and nonwoven fabrics, perforated films, sponges and foams, and beads, such as solid or porous beads, microparticles, nanoparticles, etc. (e.g., Cultispher-S gelatin beads, Sigma). The cells may be inserted into a variety of different forms for delivery. In certain embodiments, the cells may be suspended in a solution or gel. In certain embodiments, the cells may be mixed with a pharmaceutically acceptable carrier or diluent that maintains the viability of the cells of the invention.

[0316] Pharmaceutically acceptable carriers and diluents include physiological saline, aqueous buffer solutions, solvents, and / or dispersion media. The use of such carriers and diluents is well known in the art. The solutions are preferably sterile and fluid, and often isotonic. Preferably, the solutions are stable under the conditions of manufacture and storage and are preserved against the contaminating action of microorganisms, such as bacteria and fungi, through the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. Those skilled in the art will understand that the delivery vehicles used in delivering the cell populations and mixtures thereof of the present invention can contain combinations of the above-mentioned characteristics.

[0317] In certain embodiments, the subject is administered (i) a formulation comprising vesicles, (ii) a formulation comprising bioactive renal cells, and / or (iii) a formulation comprising both bioactive renal cells and vesicles.

[0318] In certain embodiments, the subject receives one to three doses of bioactive kidney cells (e.g., selected kidney cells). In certain embodiments, the subject receives one dose of bioactive kidney cells (e.g., selected kidney cells). In certain embodiments, the subject receives two doses of bioactive kidney cells (e.g., selected kidney cells). In certain embodiments, the subject receives three doses of bioactive kidney cells (e.g., selected kidney cells).

[0319] In certain embodiments, a subject receives between 1 and 10 doses of renal vesicles (e.g., microvesicles such as exosomes). In certain embodiments, a subject receives a single dose of renal vesicles (e.g., microvesicles such as exosomes). In certain embodiments, a subject receives two doses of renal vesicles (e.g., microvesicles such as exosomes). In certain embodiments, a subject receives three doses of renal vesicles (e.g., microvesicles such as exosomes). In certain embodiments, a subject receives four doses of renal vesicles (e.g., microvesicles such as exosomes). In certain embodiments, a subject receives five doses of renal vesicles (e.g., microvesicles such as exosomes). In certain embodiments, a subject receives six doses of renal vesicles (e.g., microvesicles such as exosomes). In certain embodiments, a subject receives seven doses of renal vesicles (e.g., microvesicles such as exosomes). In certain embodiments, a subject receives 8 doses of renal vesicles (e.g., microvesicles such as exosomes). In certain embodiments, a subject receives 9 doses of renal vesicles (e.g., microvesicles such as exosomes). In certain embodiments, a subject receives 10 doses of renal vesicles (e.g., microvesicles such as exosomes).

[0320] Administration method Modes of administration of the formulations include, but are not limited to, systemic injection, intrarenal (e.g., parenchymal) injection, intravenous or intra-arterial injection, transcatheter delivery, and direct injection into tissue at the intended site of activity. In certain embodiments, modes of administration used in accordance with the present invention include single or multiple injections via direct laparotomy, direct laparoscopy, transperitoneally, or percutaneously. In certain embodiments, modes of administration used in accordance with the present invention include: These include, for example, retrograde injection and ureteral injection. Surgical administration methods include, but are not limited to, one-stage procedures such as partial nephrectomy and construct transplantation, partial nephrectomy, partial pyelectomy, vascularization using the retinoperitoneum, multifocal biopsy needle tracking, and polar replacement of the kidney from a conical or pyramidal shape to a cylindrical shape, as well as two-stage procedures, for example, organoid-in-vivo bioreactors for reimplantation. In certain embodiments, a formulation containing a mixture of cells and vesicles is delivered simultaneously via the same route. In certain embodiments, the cell composition and vesicle composition are delivered separately to a specific location or via a specific technique, either simultaneously or in a temporally controlled manner, by one or more of the methods described herein. In certain embodiments, selected renal cells are injected percutaneously into the renal cortex of the kidney. In certain embodiments, a guide cannula is inserted percutaneously and used to puncture the kidney capsule before injecting the composition into the kidney. In certain embodiments, the vesicles are administered by intravenous injection or transcatheter delivery.

[0321] Laparoscopic or percutaneous techniques can be used to access the kidney for injection of formulated BRC or SRC populations (e.g., with or without vesicles). Laparoscopic techniques allow direct visualization of the kidney, allowing any bleeding or other adverse events during injection to be identified and addressed immediately. Percutaneous approaches to the kidney have been used for over a decade, primarily for the removal of intrarenal masses. These techniques involve inserting an electrode or cryogenic needle into a defined mass within the kidney and maintaining contact for (typically) 10 to 20 minutes while the lesion is removed. For the injection of therapeutic formulations, percutaneous devices are not particularly large or complex, and this approach offers the safety advantages of being non-surgical (avoiding abdominal puncture wounds and gas distension) and minimizing occlusion. Furthermore, biodegradable hemostatic materials may be placed in the access tract to further reduce the potential for significant bleeding.

[0322] In certain embodiments of delivery by injection, the therapeutic bioactive cell formulation (which may or may not be supplemented with vesicles) is injected into the renal cortex. In certain embodiments, it is important to distribute the therapeutic formulation as widely as possible within the renal cortex, which can be achieved, for example, by puncturing the renal cortex at an angle that allows for placement of the therapeutic formulation within the renal cortex and distributing it as widely as possible. This requires imaging of the kidney using a longitudinal or transverse approach with ultrasound guidance or axial computed tomography (CT) imaging, depending on individual patient characteristics. Ideally, the injection involves multiple placements as the needle / cannula is gradually withdrawn. The entire volume of the therapeutic formulation can be placed at a single or multiple puncture points. In certain embodiments, up to two puncture points can be used to place the entire volume of the therapeutic formulation within the kidney. In certain embodiments, one or more puncture points, for example, one or two puncture points, can be used to administer an injection into a single kidney. In certain embodiments, both kidneys are injected using more than one puncture point in each kidney, for example, one or two puncture points.

[0323] The foregoing description is deemed sufficient to enable one skilled in the art to practice the present invention. While the present invention has been specifically disclosed by preferred embodiments and optional features, it should be understood that modifications and variations of the concepts disclosed herein may be made by those skilled in the art, and that such modifications and variations are deemed to be within the scope of the present invention as defined by the appended claims. The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Indeed, various modifications of the present invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description, which are encompassed within the scope of the appended claims.

[0324] All patents, patent applications, and literature references cited herein are incorporated by reference in their entirety. [Example]

[0325] Example 1: Non-limiting examples of methods and compositions for producing SRC Example 1.1 - Solution Preparation This Examples section presents the compositions of the various media formulations and solutions used in this Example for the isolation and characterization of heterogeneous renal cell populations and the manufacture of regenerative therapy products.

[0326] TIFF2026035692000006.tif117170

[0327] Dulbecco's phosphate buffered saline (DPBS) was used for all cell washes.

[0328] Example 1.2 - Isolation of a heterogeneous unfractionated kidney cell population This Examples section demonstrates the isolation of an unfractionated (UNFX) heterogeneous renal cell population from humans. Tissue dissociation was first performed to generate a heterogeneous cell suspension from human kidney tissue.

[0329] Renal tissue via kidney biopsy provided the source material for the heterogeneous renal cell population. Renal tissue containing one or more of the cortical, corticomedullary junction, or medullary tissue can be used. It is preferable to use corticomedullary junction tissue. Multiple biopsy cores (minimum two) from CKD kidneys were required to avoid scar tissue. Renal tissue was obtained from patients by clinical researchers at clinical sites approximately four weeks prior to the planned transplantation of the final NKA. The tissue was transported in the tissue transport medium of Example 1.1.

[0330] The tissue was then washed with the tissue wash solution of Example 1.1 to reduce incoming bioburden, after which the tissue was processed to remove cells.

[0331] The kidney tissue was minced, weighed, and dissociated in the digestion solution of Example 1.1. The resulting cell suspension was resuspended in Dulbecco's Modified Eagle Medium (D-MEM) + 10% fetal bovine serum (FBS). The cells were neutralized in serum-free supplement-free keratinocyte medium (KSFM) (Invitrogen, Carlsbad, CA), washed, and resuspended in serum-free supplement-free keratinocyte medium (KSFM) (Invitrogen). The cell suspension was then centrifuged across a 15% (wt / vol) iodixanol (OptiPrep™, Sigma) density boundary to remove red blood cells and debris, followed by tissue reconstitution. 25,000 cells / cm in kidney cell growth medium of Example 1.1 on culture-treated polystyrene flasks or dishes 2 Cultures were started at a density of 25 × 10 cells in 150 ml of 50:50 medium on the surface of a T500 Nunc flask. 6 Cells can be seeded in cells / flask.

[0332] Example 1.3 - Cellular expansion of isolated renal cell populations Renal cell expansion depends on the amount of tissue received and the successful isolation of renal cells from the delivered tissue. Isolated cells can be cryopreserved if desired (see above). Renal cell growth kinetics can vary from sample to sample due to inherent variability in cells isolated from individual patients.

[0333] Defined cell expansion methods were developed to accommodate the range of cell recovery due to variability in delivered tissue in Table 7. Renal cell expansion requires serial passaging in Renal Cell Growth Medium in Table 6 in closed culture vessels (e.g., T-flasks, Cell Factories, HyperStacks™) using defined cell culture techniques.

[0334] A BPE-free medium was developed for human clinical trials to eliminate the inherent risks associated with the use of BPE. Cell growth, phenotype (CK18), and cell function (enzymatic activity of GGT and LAP) were evaluated in BPE-free medium and compared with the BPE-containing medium used in animal studies. Renal cell growth, phenotype, and function were comparable in the two media (data not shown).

[0335] TIFF2026035692000007.tif34170

[0336] Once cell growth was observed in the first T-flask (passage 0) and there were no visible signs of contamination, the culture medium was replaced, and then every 2–4 days thereafter (Figure 2B). Cells were evaluated to verify kidney cell morphology by visually observing the cultures under a microscope. Cultures characteristically exhibited a tightly packed paving or cobblestone appearance due to cell confluence. These morphological traits change during expansion and may not be present at all passages. Cell culture confluence was estimated at various confluence levels in the culture vessels used throughout cell expansion.

[0337] When the culture vessels reached at least 50% confluence, the kidney cells were passaged by trypsinization (Figure 2B). Detached cells were collected in a vessel containing kidney cell growth medium, counted, and cell viability was calculated. At each cell passage, 500 cells / cm were cultured in a sufficient number of culture vessels to expand the cell number to the cell number required for NKA formulation. 2 ~4000 cells / cm 2 Cells were seeded at 100°C (Figure 2B). Culture vessels were placed in a 37°C incubator with 5% CO2. Cell morphology and confluence were monitored as described above, and tissue culture medium was replaced every 2–4 days. Table 8 lists the viability of human kidney cells observed during cell isolation and expansion of six kidney biopsies from human donors.

[0338] TIFF2026035692000008.tif41170

[0339] The inherent variability of tissues from different patients resulted in differences in cell yields during culture. Therefore, it is not practical to strictly prescribe the timing of cell passage or the number and type of culture vessels required for each passage to achieve the target cell number. Typically, kidney cells undergo two or three passages, but the culture period and cell yield may vary depending on the cell growth rate.

[0340] Cells were detached with 0.25% trypsin containing EDTA (Invitrogen) for harvesting or passaging. Viability was assessed by trypan blue exclusion and counted manually using a hemocytometer or using the automated Cellometer™ counting system (Nexcelom Bioscience, Massachusetts). Counts were performed using a Quantitative Counting System (Quantitative Counting System, Lawrence, SS).

[0341] Example 1.4 Cryopreservation of cultured cells The expanded kidney cells were routinely cryopreserved to accommodate the inherent variability of cell growth from individual patients, and the product was delivered on a predetermined clinical schedule. The cryopreserved cells also provide a backup cell source in the event another NKA is needed (e.g., delay due to patient illness, unanticipated course events, etc.). The conditions used to cryopreserve the cells and recover viable, functional cells upon thawing were established.

[0342] For cryopreservation, cells were suspended in cryopreservation solution (see Example 1.1) to approximately 50 × 10 6 The final concentration of cells / mL was adjusted to approximately 50 x 10 cells / mL and dispensed into vials. 6 1 ml vials containing cells / ml were placed in the freezer compartment of a controlled rate freezer and frozen at a pre-programmed rate. After freezing, the cells were transferred to a liquid nitrogen freezer for in-process storage.

[0343] Example 1.5 Generation of SRC cell populations Selected renal cells (SRCs) can be generated from final culture vessels grown from cryopreserved cells or directly from expansion cultures, depending on scheduling (Figure 2B).

[0344] If using cryopreserved cells, the cells were thawed and seeded onto tissue culture vessels for one final expansion step. When the final culture vessels were approximately 50%-100% confluent, the cells were ready to be processed for SRC isolation. The NKA medium change and final wash dilute any remaining cryopreservation solution in the final product.

[0345] Once the final cell culture vessel reached at least 50% confluence, it was transferred to a hypoxic incubator set at 2% oxygen in a 5% CO2 environment at 37°C (Figure 2C) and cultured overnight. Cells can be maintained in the oxygen-controlled incubator set at 2% oxygen for as long as 48 hours. Exposure to a more physiologically relevant hypoxic (2%) environment improved cell isolation efficiency and allowed for greater detection of hypoxia-inducible markers, such as VEGF.

[0346] After the cells were exposed to hypoxic conditions for a sufficient period of time (e.g., overnight to 48 hours), they were detached using 0.25% trypsin containing EDTA (Invitrogen). Viability was assessed by trypan blue exclusion and counted manually using a hemocytometer or using the automated Cellometer™ counting system (Nexcelom Bioscience, Lauren, MA). The cells were counted using a microcentrifuge. The cells were washed once with DPBS and then counted at approximately 850 × 10 6 The cells were resuspended to 1000 cells / mL.

[0347] Centrifugation across a density boundary / interface was used to separate the harvested renal cell population based on cell buoyant density. Renal cell suspensions were separated by centrifugation across a 7% iodixanol solution (OptiPrep, 60% (wt / vol) in OptiMEM, see Example 1.1).

[0348] A 7% OptiPrep density interface solution was prepared, and the refractive index, which indicates the desired density, was measured before use (refractive index 1.3456 ± 0.0004). Harvested kidney cells were layered on the solution. The density interface was centrifuged at 800 g for 20 minutes at room temperature (no brake) in either a centrifuge tube or a cell processing device (e.g., COBE 2991). Cell fractions exhibiting a buoyant density greater than approximately 1.045 g / mL were collected as a separate pellet after centrifugation. Cells maintaining a buoyant density less than 1.045 g / mL were discarded.

[0349] The SRC pellet was resuspended in DPBS (Figure 2C). Four DPBS washes and one gelatin solution step minimize carryover of residual OptiPrep, FBS, culture medium, and supplementary materials into the final product.

[0350] Example 2: Exosome compositions and uses thereof for the treatment of kidney disease and function Example 2.1 - Technical Field This example relates to kidney cell exosome compositions and their production for uses including tissue engineering and regenerative medicine applications for kidney repair and function.

[0351] Example 2.2 - General Comment Considerable research continues to focus on utilizing exosomes in biological fluids for biomarkers of disease. The therapeutic potential of exosomes has been addressed more recently, with most of the research focused on cancer immunotherapy, vaccine development, autoimmune disease treatment, and delivery of therapeutic agents (chemical compounds, siRNA). Exosomes as potential therapeutic agents for modulating angiogenesis, a key component in tissue regeneration, have attracted attention over the past seven years (7, 8).

[0352] Secreted extracellular vesicles (EVs) such as exosomes are loaded with potent pro-repair proteins and RNA cargo that are not only cell-type specific but also differentially produced and secreted depending on the cellular environment. A similar review has been published by Zhang et al. (Am J Physiol Renal Physiol. 2016 Nov 1;311 (5):F844-F851. doi: 10.1152 / ajprenal.00429.2016. Epub 2016 Aug 31. Extracellular vesicles in diagnosis and therapy of kidney diseases. Zhang W, Zhou X, Zhang H, Yao Q, Liu Y, Dong Z.).

[0353] Chronic kidney disease (CKD) is a global health problem, and the widening gap between the number of patients awaiting transplantation and the number of organs actually transplanted highlights the need for new therapies to restore kidney function. Regenerative medicine is a promising approach from which treatments for organ-level disorders are emerging and being translated into the clinic. Regenerative templates, composed of biodegradable materials and autologous cells isolated and expanded ex vivo, can stimulate native-like organ tissue regeneration after transplantation. Become excited.

[0354] Recent studies have demonstrated novel roles for EVs in mediating cell-cell and cell-cell communication. (9, 10) The unique biological activities of EVs have demonstrated potential benefits for correcting cellular dysfunction and, ultimately, for disease therapy. (11) EVs are also considered ideal nanovectors for biodelivery, particularly drug delivery in clinical applications. (12) In the kidney, renal EVs are produced and secreted by kidney cells and are involved in renal function and disease. (10)

[0355] Mechanistically, several studies have attributed the protective effect of EVs against kidney disease primarily to their RNA content, particularly microRNAs ( 13 , 14 ).

[0356] There has been intensive research into the potential of EVs as biomarkers for CKD. In contrast, very little is known about the therapeutic effects of EVs in CKD. Without being limited by any scientific theory, we rationalize that our success in treating CKD with SRCs is due, at least in part, to the hypoxic treatment of SRCs resulting in the secretion of "conditioned" EVs from the transplanted cells, which then "rescue" the diseased cells, thereby improving their function.

[0357] In certain embodiments, hypoxic conditioning of selected kidney cells by gradient banding prior to exosome isolation confers enhanced regenerative properties to EVs.

[0358] Example 2.3 - Exosome isolation - quantification and sizing Based on the purification method used (15), exosomes have been described to range in size from 30 nm to 150 nm (16, 17) with an approximate density of 1.10 g / mL to 1.20 g / mL (18, 19) depending on the density gradient material (sucrose or OptiPrep) used for analysis. Microvesicles have been described as larger than exosomes, often ranging in diameter from 100 nm to 300 nm. The degree of size overlap between these classes of EVs varies depending on the publication and the technique used for measurement. As used herein, the term "microvesicles" refers to cell-derived membranous extracellular vesicles with diameters between 30 nanometers (nm) and 1,000 nanometers (nm). As used herein, the term "exosomes" refers to cell-derived membranous microvesicles with diameters of approximately 30 nm to 150 nm. Therefore, as used herein, the term "microvesicles" encompasses not only exosomes but also larger vesicles. Despite position statements (20) published by key opinion leaders within the International Society for Extracellular Vesicles (ISEV) in late 2014 and 2015, there is still no consensus on the best methods for isolating, sizing, and characterizing exosomes. As the field evolves, particularly in the area of ​​biological function, advances in particle isolation, sizing, and characterization techniques are expected, depending on which method provides the desired biological effect. In certain embodiments, exosome-containing EVs are obtained by centrifuging the EV-containing culture medium at 3,000 × g for 20 minutes to pellet cellular debris, followed by ultracentrifugation of the clarified supernatant at 100,000 × g to pellet the EVs. These preparations currently demonstrate biological activity (see proliferation and tubule formation assays below).

[0359] Cells are grown in serum-free culture medium for 24 hours. The medium is collected. To isolate exosomes, the collected serum-free conditioned medium is centrifuged in two steps: 1) at 3,000 x g for 20 minutes to remove cell debris, and 2) at 100,000 x g for 2 hours to pellet the exosomes. The exosomes are resuspended in DPBS and stored at -80°C until use.

[0360] To determine the size distribution and concentration of exosomes, samples were analyzed by tunable resistive pulse sensing (TRPS; qNano, Izon Science Ltd.) using a 47 mm wide NP150 nanopore membrane. 13 Particle concentrations were standardized using multi-pressure calibration with 114 nm carboxylated polystyrene beads at a concentration of particles / mL. Samples were diluted 1:100 in DPBS immediately prior to analysis. Results and yields for four different lots are shown in the table below (Table 9). While there is variability in size and concentration between lots, particle sizes for all lots are well within the operational definition of exosomes (30 nm to 150 nm).

[0361] TIFF2026035692000009.tif126170

[0362] In this and other examples, "BRC-0" refers to bioactive renal cells that are non-passaged primary cells. "BRC-1" refers to bioactive renal cells that have been passaged once. "BRC-2" refers to bioactive renal cells that have been passaged twice. "BRC-3" refers to bioactive renal cells that have been passaged three times. "BRC-3A" refers to bioactive renal cells that have been passaged three times and then cultured under hypoxic conditions (representing cells after completion of hypoxic culture). "SRC" refers to selected renal cells.

[0363] Example 2.4 - Characterization of microRNAs (miRNAs) from SRC exosomes Because the contents of exosomal cargo can include several analytes, including proteins, metabolites, and RNA, we specifically characterized the microRNAs found in association with SRC-derived exosomes. MicroRNAs (miRNAs) are small non-coding RNAs containing approximately 18-23 nucleotides that bind to the 3' untranslated region of messenger RNAs to repress translation or promote their degradation. miRNA profiles reflect various physiological and pathological states. miRNAs are expressed in a tissue- or cell-specific manner. The expression levels of miRNAs change in response to various physiological processes and are involved in histological processes. Most of the genes encoding proteins are thought to be targets of miRNAs.

[0364] Because miRNA functions are essentially negative, i.e., they inhibit RNA translation, possibly by competitive binding or promoting degradation, miRNAs have global functions that regulate cell growth and proliferation. For example, promoting cell proliferation may involve miRNAs that function to inhibit the translation, and therefore expression, of growth-inhibitory proteins. On the other hand, inhibiting the translation / expression of growth-promoting proteins can inhibit cell proliferation.

[0365] It should be recognized that the functions of these miRNAs have been primarily ascertained by studying abnormal cell growth, such as that seen in cancer cells and other pathologies. Therefore, their putative functions in normal cells must be carefully evaluated. For example, simply finding that a miRNA is elevated or suppressed in cancer cells does not necessarily mean that it functions solely to promote cellular transformation. In cancer cells, the balance between cell proliferation and growth suppression is shifted toward cell proliferation. With this in mind, it is not surprising that the majority of the miRNAs listed above were identified in these kidney cells.

[0366] Test Summary: miRNA from each sample was isolated using the miRNeasy Mini Kit, which allows for the purification of total RNA containing RNA of approximately 18 nucleotides (nt) or larger, and miRNA was quantified using a NanoDrop spectrophotometer. Sequencing service provided: Small RNA-Seq. Sequencing platform: Illumina NextSeq 500. Sequencing platform reagents: NextSeq Mid Output Kit v2. Library preparation products: Norgen Biotek Small RNA Library Prep Kit. t. Small RNA-Seq data analysis workflow used: exceRpt small RNA-seq pipeline (v4.6.2). (Web link: genboree.org / theCommons / projects / exrnatools-may2014 / wiki / Small_RNA-seq_Pipeline) Sources of RNA reference sequences: miRNAs miRBase version 21; tRNAs gtRNAdb, piRNAs RNAdb, Genome Gencode version 21(hg38).

[0367] result: Computational analysis of the resulting sequences reveals the following miRNAs that are differentially expressed in exosomes secreted by SRC: miR-145 (hypothesized tumor suppressor) mir-22 (may function as a tumor suppressor) miR-7, a highly conserved miRNA that shows restricted spatiotemporal expression during development and maturation and may also function as a cell growth / tumor suppressor miR-10a (regulates the pro-inflammatory phenotype, a marker of renal injury. It has been experimentally validated that miR-10a downregulates the human HOXA1 and HOXA3 genes. The regulation of Hox genes by miR-10 suggests that this microRNA may play an important role in development.) miR-143 (tumor suppressor, growth inhibitor) let7b (given that expression levels of let-7 members are significantly lower in human cancers and cancer stem cells, the primary function of the let-7 gene may be to promote terminal differentiation and tumor suppression during development).

[0368] Example 2.5 - MACSplex surface labeling of exosomes Characterization of exosomal surface markers was performed using a multiplex assay consisting of FACS analysis of 39 surface markers reported to be present on EVs.

[0369] Test Summary: Exosomes were isolated from the conditioned medium of cultured cells (BRC-0, BRC-3, BRC-3A, SRC) by ultracentrifugation. Particle size and concentration were assessed by tunable resistive pulse sensing (TRPS; qNano, Izon Science Ltd.) using a 47 mm wide NP150 nanopore membrane. 13 Particle concentrations were standardized using a multi-pressure calibration with 110 nm carboxylated polystyrene beads at a concentration of particles / mL. 1 x 10 for each sample 10 particles were used. A cocktail of CD63, CD9, and CD81 is used to immunoisolate exosomes. This population is then screened for the expression of 37 different markers.

[0370] Results: See Figure 4 In exosomes isolated from both TCHK0012 and TCHK0013, the following are upregulated in SRC compared to BRC: CD133 (although its exact function remains unknown, it has been proposed that CD133 acts as an organizer of cell membrane topology) CD326 (epithelial cell adhesion molecule (EpCAM)) is a Ca2+ receptor that mediates epithelial adhesion.2+ EpCAM is a transmembrane glycoprotein that mediates chromatin-independent homotypic cell-cell adhesion. EpCAM is also involved in cell signaling, migration, proliferation, and differentiation. CD49e (In addition to adhesion, integrins are known to be involved in signal transduction via the cell surface).

[0371] Example 2.6 - Exosome-mediated lipophilic dye transfer to kidney cells It is assumed that exosomes must attach to and fuse with the recipient cell membrane in order to deliver their protein or nucleic acid cargo. Exosome-mediated delivery of lipophilic dyes to the cell membrane is one way to demonstrate exosome fusion with recipient cells (21).

[0372] Test Summary: Dye Transfer. To assess the ability of exosomes to deliver their cargo, we monitored the ability of exosomes to transfer a lipophilic dye to kidney cells in culture by flow cytometry. Aliquots of exosomes (BRC-0, BRC-3, SRC) were labeled with Vybrant DiI cell labeling solution at 37°C for 20 minutes. Following removal of excess pigment by ultracentrifugation, 5 × 10 9 Labeled exosomes were added to each well of a 6-well dish, each containing approximately 250,000 cells / well. After 4 hours of incubation at either 4°C or 37°C, the cultures were washed to remove any uninternalized labeled exosomes. Recovered cells were then collected and analyzed by FACS. Fluorescently labeled cells, indicative of transfer of lipophilic dye from exosomes to the cell membrane, result in a shift of the histogram line from left to right.

[0373] Results: See Figure 5 As expected, at 4°C, exosomal activity was not observed, as indicated by the lack of lipophilic dye transfer. No binding of exosomes was observed, supporting the notion that cells must be biologically active to take up exosomes. Also as expected, the histogram of the control without exosomes does not show a shift to the right compared to the histogram at 4°C. The areas indicated by the arrows between the histograms indicate the population of cells that had taken up the dye. Based on this experiment, there appears to be a difference in exosome binding between BRC-0 (maximum) and BRC-3 and SRC (approximately equal binding).

[0374] Example 2.7 - Cell proliferation assay For exosomes to play a role in the repair and regeneration of kidney tissue, it may be desirable for them to have the ability to stimulate kidney cell proliferation.

[0375] Test Summary: Human kidney cells were seeded onto 12-well plates at 50,000 cells / well. Exosomes were isolated from the supernatants of the indicated cells. Exosome volumes of 25 μL, 50 μL, and 100 μL (1×, 2×, and 4×) were added to each well. Samples were tested in duplicate. Plates were counted using an ArrayScan 3 days after treatment. SF = serum-free medium (negative control). Growth medium = serum-containing medium (positive control).

[0376] Results: See Figure 6 All treatment dilutions of TCHK006 for BRC-3 and SRC increased cell proliferation to approximately the same level, higher than the negative control. Treatment dilutions of TCHK004 with BRC-3 and SRC showed a dose effect on proliferation, with only the 4x dose of SRC increasing proliferation above the negative control.

[0377] Example 2.7 - Tubulogenesis / Angiogenesis Assay The ability to stimulate angiogenesis is also assumed to be a desirable feature for tissue-engineered / regenerative medicine products. One of the most widely used in vitro assays to model the remodeling phase of angiogenesis is the tube formation assay. This assay measures the ability of endothelial cells seeded at subconfluent density with an appropriate extracellular matrix support to form capillary-like structures (also known as lumens). Scientists typically use this assay to determine the ability of various compounds to promote or inhibit tube formation. Once seeded, endothelial cells attach to the surrounding extracellular support matrix, generating mechanical forces and creating channels or guidance pathways that facilitate cell migration. The resulting cell cords ultimately form hollow lumens.

[0378] Compounds that can inhibit tube formation may be useful in various diseases, such as cancer, where tumors grow beyond their relatively small size by stimulating neovascularization to receive oxygen and nutrients. In contrast, compounds or biologics (i.e., exosomes) that can stimulate tube formation may be useful in tissue engineering / regenerative medicine applications.

[0379] Test Summary: 50,000 vascular endothelial cells were seeded on an extracellular matrix (GelTrex) in a 48-well plate. As a positive control, cells were incubated in serum-free growth medium supplemented with growth factors. As a negative control, cells were incubated in serum-free, growth factor-free medium. I did. Exosomes (TCHK004, SRC, 10 10 The test article is cells incubated in serum-free and growth factor-free medium supplemented with 1000 ng / ml of erythrocytes (1000 ng / ml particles).

[0380] Results: See Figure 7 Although tubule formation occurred more rapidly in the positive control (tubules were visible by T = 2 h), robust tubule formation was observed for exosome-treated cells by T = 9 h. Exosomes successfully replaced a defined growth factor cocktail containing hEGF, bFGF, IGF-1, and VEGF in supporting tubule formation.

[0381] Cultures were incubated for 9 hours after treatment. A. Serum-free growth factor-free medium (negative control). B.10 10 C. Serum-free, growth factor-free medium supplemented with exosomes (test article). D. Serum-free medium supplemented with growth factors (positive control).

[0382] Example 2.8 - References 1. Kowal J, Tkach M, Thery C. Biogenesis and secretion of exosomes. Curr Opin Cell Biol. 2014 Jun 21;29C:116-125. doi: 10.1016 / j.ceb.2014.05.004. [Epub ahead of Print Review. PMID: 24959705 2. Revenfeld AL, Baek R, Nielsen MH, Stensballe A, Varming K, Jorgensen M. Diagnostic and Prognostic Potential of Extracellular Vesicles in Peripheral Blood. Clin Ther. 2014 Jun 1;36(6):830-846. doi: 10.1016 / j.clinthera.2014.05.008. Review. PMID: 24952934 3. Lamichhane TN, Sokic S, Schardt JS, Raiker RS, Lin JW, Jay SM. Emerging roles for extracellular vesicles in tissue engineering and regenerative medicine. Tissue Eng Part B Rev. 2014 Jun 23. [Epub ahead of print] PMID: 24957510 4. Simons M, Raposo G. Exosomes-vesicular carriers for intercellular communication. Curr Opin Cell Biol 2009, Aug;21(4):575-581. doi: 10.1016 / j.ceb.2009.03.007.Epub 2009 May 11. Review. PMID: 19442504 5. Stoorvogel W, Kleijmeer MJ, Geuze HJ, Raposo G. The biogenesis and functions of exosomes. Traffic 2002, May;3(5):321-330. PMID: 11967126 6. Nieuwland R, Sturk A. Why do cells release vesicles? Thrombosis Research 2010, 125(Supplement 1):S49-S51. doi: 10.1016 / j.thromres.2010.01.037. Epub 2010 Feb 11. Review. PMID: 20149923 7. Martinez MC, Andriantsitohaina R: Microparticles in angiogenesis: therapeutic potential. Circ Res 2011, Jun 24, 109(1):110-119. doi: 10.1161 / CIRCRESAHA.110.233049. Review. PMID: 21700952 8. Sahoo S, Klychko E, Thorne T, Misener S, Schultz KM, Millay M, Ito A, Liu T, Kamide C, Agrawal H et al: Exosomes from human CD34(+) stem cells mediate their proangiogenic paracrine activity. Circ Res 2011, Sept 16, 109(7):724-728. doi: 10.1161 / CIRCRESAHA.111.253286. Epub 2011 Aug 11. PMID: 21835908 9. Camussi G, Deregibus MC, Bruno S, Cantaluppi V, Biancone L. Exosomes / microvesicles as a mechanism of cell-to-cell communication. Kidney Int 78: 838-848, 2010. 10. Krause M, Samoylenko A, Vainio SJ. Exosomes as renal inductive signals in health and disease, and their application as diagnostic markers and therapeutic agents. Front Cell Dev Biol 3: 65, 2015. 11. Erdbrugger U, Le TH. Extracellular vesicles in renal diseases: More than novel biomarkers? J Am Soc Nephrol 27: 12-26, 2016. 12. Vader P, Mol EA, Pasterkamp G, Schiffelers RM. Extracellular vesicles for drug delivery. Adv Drug Deliv Rev (Epub ahead of print). 13. Chen TS, Lai RC, Lee MM, Choo AB, Lee CN, Lim SK. Mesenchymal stem cell secretes microparticles enriched in pre-microRNAs. Nucleic Acids Res 38: 215-224, 2010. 14. Wang B, Yao K, Huuskes BM, Shen HH, Zhuang J, Godson C, Brennan EP, Wilkinson-Berka JL, Wise AF, Ricardo SD. Mesenchymal stem cells deliver exogenous microRNA-let7c via exosomes to attenuate renal fibrosis. Mol Ther 24: 1290-1301, 2016.15. Lane RE, Korbie D, Anderson W, Vaidyanathan R, Trau M. Analysis of exosome purification methods using a model liposome system and tunable-resistive pulse sensing. Sci Rep. 2015 Jan 6;5:7639. doi: 10.1038 / srep07639 16. Dragovic RA, Gardiner C, Brooks AS, Tannetta DS, Ferguson DJ, Hole P, Carr B, Redman CW, Harris AL, Dobson PJ, Harrison P, Sargent IL. Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis. Nanomedicine. 2011 Dec;7(6):780-8. doi: 10.1016 / j.nano.2011.04.003. Epub 2011 May 4 17. van der Pol E, Hoekstra AG, Sturk A, Otto C, van Leeuwen TG, Nieuwland R. Optical and non-optical methods for detection and characterization of microparticles and exosomes. J Thromb Haemost. 2010 Dec;8(12):2596-607. doi: 10.1111 / j.1538-7836.2010.04074.x. 18. Tauro BJ1, Greening DW, Mathias RA, Ji H, Mathivanan S, Scott AM, Simpson RJ. Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes. Methods. 2012 Feb;56(2):293-304. doi: 10.1016 / j.ymeth.2012.01.002. Epub 2012 Jan 21 19. Kalra H, Adda CG, Liem M, Ang CS, Mechler A, Simpson RJ, Hulett MD, Mathivanan S. Comparative proteomics evaluation of plasma exosome isolation techniques and assessment of the stability of exosomes in normal human blood plasma. Proteomics. 2013 Nov;13(22):3354-64. doi: 10.1002 / pmic.201300282. Epub 2013 Oct 18 20. Loetvall J, Hill AF, Hochberg F, Buzas EI, Di Vizio D, Gardiner C, Gho YS, Kurochkin IV, Mathivanan S, Quesenberry P, Sahoo S, Tahara H, Wauben MH, Witwer KW, Thery C. Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. J Extracell Vesicles. 2014 Dec 22;3:26913. doi: 10.3402 / jev.v3.26913. eCollection 2014 21. Deregibus, M.C., et al., Endothelial progenitor cell derived microvesicles activate an angiogenic program in endothelial cells by a horizontal transfer of mRNA. Blood, 2007. 110(7): p. 2440-8. 22. Dursun I, Poyrazoglu HM, Gunduz Z, Ulger H, Yykylmaz A, Dusunsel R, Patyroglu T, Gurgoze M. The relationship between circulating endothelial microparticles and arterial stiffness and atherosclerosis in children with chronic kidney disease. Nephrol Dial Transplant 24: 2511-2518, 2009.

[0383] Example 3: Profiling of miRNAs present in secreted exosomes and intracellular vesicles of human SRC, BRC3 / 3A, and BRC0 Bioinformatics analysis of SRC-secreted proteins and exosomal miRNAs identifies specific signaling pathways that may regulate CKD progression upon activation or inactivation by SRC-derived factors. These signaling pathways can be exploited in vitro by applying quantitative potency assays directly linked to the putative SRC MOA and / or identified proteins / miRNAs as surrogates for potency, which can be assessed by ELISA / PCR-based techniques from conditioned media. This approach will lead to the identification of signaling pathways involved in renal disease progression and SRC MOA function via a paracrine mechanism that utilizes the activity of secreted biologically active ligands and miRNAs transferred from donor to host cells by the activity of exosomes and other secreted microvesicle components.

[0384] miRNAs were isolated from exosomes purified from secreted media or intracellular niches. Using n=6 independent donors, statistically rigorous comparisons were performed for BRC3 / 3A and BRC0 / SRC. miRNAs that distinguish these manufacturing intermediates were identified.

[0385] approach 1. Application of computational / bioinformatics approaches to cellular secretome We characterized the secretome and miRNA profiles (intracellular and exosomal) from SRC and SRC manufacturing intermediates (n=6). Data sets were subsequently analyzed by bioinformatics techniques to computationally determine disease- and regeneration-related signaling networks directly affected by secreted proteins or miRNAs produced by SRC.

[0386] To date, these data indicate that the BRC3-BRC3A transition (hypoxia step) leads to significant changes in the biosignature of the BRC cell population, such that the manufactured end product (SRC) is clearly distinguishable from the starting material (BRC0).

[0387] 2. Profiling of miRNAs present in secreted exosomes and intracellular vesicles of human SRC, BRC3 / 3A, and BRC0 miRNAs were isolated from exosomes purified from secreted media or intracellular niches. Statistically rigorous comparisons were performed sequentially for BRC3 / 3A and BRC0 / SRC using n=6 independent donors.

[0388] miRNAs that distinguish these production intermediates were identified. Unlike standard phenotypic descriptions and functional analyses, characterizing the secretomes and miRNAs of SRC and BRC-0 cells not only provides a better understanding of how SRC differs from BRC-0, but also provides a useful approach specific to potency assays. The data we collected clearly distinguished SRC from BRC-0. Without wishing to be bound by any scientific theory, many of the differences are most likely due to the hypoxic step, as there are also differences between BRC-3, BRC-3A, and SRC.

[0389] Example 3.1: Experimental Design

[0390] TIFF2026035692000010.tif47170

[0391] Example 3.2 - Differentially expressed miRNAs Based on the experimental design, the number of differentially expressed miRNAs for each comparison is shown in the table below. The standard selection criteria for determining differentially expressed miRNAs are as follows: |Loge fold change|≧1 and P value<0.05.

[0392] TIFF2026035692000011.tif34170

[0393] In the table below, "hsa-" indicates human miRNA.

[0394] TIFF2026035692000012.tif89170

[0395] TIFF2026035692000013.tif89170

[0396] TIFF2026035692000014.tif89170

[0397] TIFF2026035692000015.tif89170

[0398] TIFF2026035692000016.tif89170

[0399] TIFF2026035692000017.tif89170

[0400] In Figure 8, the miRNAs that are significantly different between the pair of experimental conditions E1 vs. D1 are shown in a volcano plot. In Figure 9, the miRNAs that are significantly different between the pair of experimental conditions F1 vs. A1 are shown in a volcano plot.

Claims

1. 1. A method for treating a kidney disease in a subject, comprising administering to the subject an effective amount of isolated secreted kidney cell vesicles, wherein the vesicles are administered by intravenous injection or transcatheter delivery.

2. 10. The method of claim 1, wherein the vesicles are injected intravenously into a peripheral blood vessel.

3. 10. The method of claim 1, wherein the transcatheter delivery is to the left or right renal artery of the subject.

4. The method of any one of claims 1 to 3, wherein the subject suffers from chronic kidney disease.

5. 5. The method of claim 4, wherein the chronic kidney disease is stage I, stage II, stage III, stage IV, or stage V kidney disease.

6. The method of any one of claims 1 to 5, wherein the treating of the kidney disease comprises reducing or preventing kidney fibrosis in the subject.

7. 7. The method of any one of claims 1 to 6, wherein the subject undergoes dialysis at least once, twice, or three times per week for at least one or two weeks.

8. The method of any one of claims 1 to 7, wherein the subject suffers from type II diabetes.

9. The method of any one of claims 1 to 8, wherein the subject has a congenital anomaly of the kidney and urinary tract (CAKUT).

10. The target is 90 mL / min / 1.73 m ２ 10. The method of any one of claims 1 to 9, wherein the patient has a glomerular filtration rate (GFR), microalbuminuria, or macroalbuminuria of less than 100 mg / kg.

11. The method of any one of claims 1 to 10, wherein no cells are administered to the subject.

12. The method of any one of claims 1 to 12, wherein the vesicles comprise microvesicles.

13. 14. The method of any one of claims 1 to 13, wherein the vesicles comprise exosomes having a diameter of about 30 nm to 150 nm.

14. The method of any one of claims 1 to 14, wherein the vesicle comprises a compound on its outer surface, in its lipid bilayer, and / or in its lumen.

15. 15. The method of claim 14, wherein the compound attenuates one or more cellular pathways.

16. 16. The method of claim 14 or 15, wherein the compound is a protein, a small molecule, or a polynucleotide.

17. 17. The method of claim 16, wherein the polynucleotide is a miRNA molecule.

18. The method of any one of claims 14 to 17, wherein the compound is not produced by naturally occurring kidney cells.

19. The method of any one of claims 14 to 16, wherein the compound is a cytokine.

20. The method according to any one of claims 14 to 16, wherein the compound is an artificial compound.

21. 21. The method of claim 20, wherein the compound is a compound used in the treatment of kidney disease.

22. 22. The method of any one of claims 15 to 21, wherein the vesicles comprise a compound that attenuates plasminogen activator inhibitor-1 (PAI-1) signaling and / or transforming growth factor β (TGFβ) signaling.

23. 22. The method of any one of claims 15 to 21, wherein the vesicle comprises a compound that attenuates canonical Wnt signaling.

24. The method of any one of claims 15 to 21, wherein the vesicle comprises a compound that attenuates non-canonical Wnt signaling.

25. The method of any one of claims 15 to 21, wherein the vesicle comprises a compound that attenuates CXCR4-mediated signaling.

26. The method of any one of claims 15 to 21, wherein the vesicles comprise a compound that downregulates a pro-inflammatory cytokine.

27. 27. The method of claim 26, wherein the inflammatory cytokine is IL8.

28. 22. The method of any one of claims 15 to 21, wherein the vesicle comprises a compound that attenuates Notch signaling.

29. The method of any one of claims 14 to 16, wherein the compound is a cell surface molecule used in immunophenotyping of cells.

30. 30. The method of claim 29, wherein the compound is CD9, CD63, CD81, CD133, CD146, CD326, CD40, CD42a, CD44, or CD49e.

31. The method of claim 14 , wherein the compound is a protein receptor.

32. 32. The method of claim 31, wherein the protein receptor is a retinoid-related receptor (ROR4).

33. The method of claim 14, wherein the compound is a developmental marker.

34. 34. The method of claim 33, wherein the developmental stage marker is stage-specific embryonic antigen-4 (SSEA-4).

35. 15. The method of claim 14, wherein the compound is a stress-protective protein.

36. 36. The method of claim 35, wherein the stress-protective protein is heat shock protein (HSP) 70 or HSP90.

37. 15. The method of claim 14, wherein the compound is a scaffold protein.

38. 38. The method of claim 37, wherein the scaffold protein is TST101.

39. The method of any one of claims 14 to 17, wherein the compound is a miRNA and is in the lumen of the vesicle.

40. 40. The method of any one of claims 14 to 17 or claim 39, wherein the miRNA is a cell cycle regulatory miRNA.

41. 41. The method of claim 40, wherein the cell cycle-regulating miRNA is let7a, miR-143, or miR22.

42. 40. The method of any one of claims 14 to 17 or 39, wherein the miRNA is a cellular senescence-regulating miRNA.

43. The method of claim 42, wherein the cellular senescence-regulating miRNA is miR-34.

44. 40. The method of any one of claims 14 to 17 or 39, wherein the miRNA is a cell migration-regulating miRNA.

45. The method of claim 44, wherein the cell migration-regulating miRNA is miR30-C.

46. 40. The method of any one of claims 14 to 17 or claim 39, wherein the miRNA is a cell growth regulatory miRNA.

47. 47. The method of claim 46, wherein the cell growth-regulatory miRNA is miR194-2.

48. 40. The method of any one of claims 14 to 17 or claim 39, wherein the miRNA is a cell signaling pathway-regulating miRNA.

49. 49. The method of claim 48, wherein the cell signaling pathway-regulating miRNA is miR-142.

50. 40. The method of any one of claims 14 to 17 or 39, wherein the miRNA is an inflammation-regulating miRNA.

51. 51. The method of claim 50, wherein the inflammation-regulating miRNA is miR-10a.

52. 40. The method of any one of claims 14 to 17 or 39, wherein the miRNA is an angiogenesis-regulating miRNA.

53. The method of claim 52, wherein the angiogenesis-regulating miRNA is miR-296 and / or miR-146a.

54. The method of any one of claims 14 to 17 or 39, wherein the miRNA is a kinase activity-regulating miRNA.

55. The method of claim 54, wherein the kinase activity-regulating miRNA is miR-83.

56. 40. The method of any one of claims 14 to 17 or claim 39, wherein the compound is a miRNA that inhibits PAI-1, TGFβ, canonical Wnt signaling, non-canonical Wnt signaling, CXCR4-mediated signaling, and / or Notch signaling.

57. 57. The method of any one of claims 6 to 56, wherein renal fibrosis is reduced or prevented by inhibiting epithelial-mesenchymal transition (EMT).

58. 58. The method of any one of claims 1 to 57, wherein the vesicles comprise miR-145, miR-22, miR-7, miR-10a, miR-143, and / or let7b.

59. 59. The method of any one of claims 1 to 58, wherein the vesicles comprise miR-1248, miR-3168, miR-7113-5p, miR-758-3p, miR-937-3p, miR-4455, miR-4521, miR-203a-3p, miR-22-3p, miR-574-3p, miR-181b-5p, miR-1260b, and / or miR-181b-5p.

60. 60. The method of any one of claims 1 to 59, wherein the vesicles comprise CD9, CD63, CD81, CD133, CD146, CD326, CD40, CD42a, CD44, CD49e, and / or SSEA-4.

61. 61. The method of claim 60, wherein the vesicles comprise CD63, CD9, and / or CD81, and the CD63, CD9, and / or CD81 are on the outer surface of the vesicles.

62. 61. The method of claim 60, wherein the vesicles comprise CD133, CD326, and / or CD49e, and the CD133, CD326, and / or CD49e are on the outer surface of the vesicles.

63. 63. The method of any one of claims 1 to 62, wherein proliferation of renal cells contacted with the vesicles is increased compared to renal cells not contacted with the vesicles.

64. 64. The method of any one of claims 1 to 63, wherein angiogenesis by endothelial cells contacted with said vesicles is increased compared to endothelial cells not contacted with said vesicles.

65. 65. The method of any one of claims 1 to 64, wherein nephron tubule formation in renal cells contacted with the vesicles is increased compared to renal cells not contacted with the vesicles.

66. 66. The method of any one of claims 1 to 65, wherein the vesicles comprise phospholipids, sphingolipids, cholesterol, ceramide, and / or phosphatidylcholine.

67. 68. The method of any one of claims 1 to 67, wherein the vesicles are in a composition comprising a pharmaceutically acceptable carrier.

68. 68. The method of claim 67, wherein the pharmaceutically acceptable carrier comprises an aqueous solution.

69. 69. The method of any one of claims 1 to 68, wherein the vesicles are secreted by a primary renal cell population.

70. 70. The method of claim 69, wherein the cells have been passaged 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

71. 71. The method of any one of claims 1 to 70, wherein the vesicles are secreted by an enriched renal cell population, wherein the enriched renal cell population comprises bioactive renal cells.

72. The population is (a) an enriched population of renal tubular cells, or (b) an enriched population of tubular cells and one or more glomerular and vascular cells; 72. The method of claim 71, comprising:

73. 73. The method of claim 71 or 72, wherein the population is derived from a starting kidney cell population and comprises a higher percentage of tubular cells compared to the starting population.

74. 74. The method of claim 73, wherein the population comprises glomerular cells.

75. 75. The method of claim 74, wherein the population comprises vascular cells.

76. 76. The method of any one of claims 69 to 75, wherein the population is non-autologous to the subject.

77. 76. The method of any one of claims 69 to 75, wherein the population is autologous to the subject.

78. 76. The method of any one of claims 69 to 75, wherein the population is allogeneic.

79. 79. The method of any one of claims 70 to 78, wherein the cells of the population are hypoxia-resistant and / or iodixanol-resistant.

80. 80. The method of any one of claims 70 to 79, wherein the cells in the population express CK18 and / or GGT1.

81. The method of any one of claims 70 to 80, wherein the population has LAP enzyme activity and / or GGT enzyme activity.

82. 82. The method of any one of claims 70 to 81, wherein at least 80% of the cells in said population express GGT-1.

83. 83. The method of any one of claims 70 to 82, wherein the population expresses VEGF and / or KIM-1.

84. 84. The method of any one of claims 70 to 83, wherein 4.5% to 81.2% of the cells in the population express GGT-1, 3.0% to 53.7% of the cells in the population express AQP2, and 81.1% to 99.7% of the cells in the population express CK18.

85. 85. The method of any one of claims 70-84, wherein the population is enriched for renal tubular cells compared to primary cultures of renal cells from kidney biopsies, and wherein the tubular cells express higher molecular weight species of hyaluronic acid (HA) through the action of hyaluronan synthase-2 (HAS-2) both in vitro and in vivo.

86. The bioactive renal cell population is less potent than primary cultures of renal cells from kidney biopsies.

86. The method of any one of claims 70 to 85, comprising a proportion of distal tubule cells, collecting duct cells, endocrine cells, vascular cells, and / or progenitor-like cells.

87. 87. The method of any one of claims 1-68 or claims 70-86, further comprising administering to the subject vesicles secreted by primary renal cells.

88. 88. The method of any one of claims 1 to 87, further comprising administering to the subject vesicles secreted by endothelial cells or mesenchymal stem cells.

89. 89. The method of any one of claims 1 to 88, further comprising administering to the subject non-renal cell vesicles.

90. 90. The method of claim 89, wherein the non-renal cell vesicles are secreted by non-renal endothelial progenitor cells, non-renal mesenchymal stem cells, or non-renal adipose-derived progenitor cells.

91. 1. A method for detecting at least one compound in a vesicle, comprising obtaining the vesicle and detecting whether the at least one compound is present in the vesicle; (i) the at least one compound is a protein, and the protein is CD9, CD81, CD146, CD326, CD40, CD42a, CD44, CD49e, and / or SSEA-4; (ii) the at least one compound comprises a miRNA, wherein the miRNA comprises at least two of miR-145, miR-22, miR-7, miR-10a, miR-143, and / or let7b; and / or (iii) The method, wherein said at least one compound is not expressed or produced by kidney cells in a native kidney.

92. 92. The method of claim 91, wherein the vesicles are obtained in or from a biological sample from a subject.

93. 93. The method of claim 92, wherein the biological sample is urine.

94. 92. The method of claim 91, wherein the vesicles are obtained in or from the supernatant of a culture of kidney cells.

95. 95. The method of any one of claims 91 to 94, wherein the vesicles are secreted by kidney cells.

96. 96. The method of any one of claims 91 to 95, wherein detecting whether the protein is in the vesicle comprises an immunoassay.

97. 97. The method of any one of claims 91 to 96, wherein detecting whether the miRNA is present in the vesicles comprises contacting the vesicles or a processed sample suspected of containing nucleic acids from the vesicles with a probe or primer complementary to the miRNA.

98. 98. The method of any one of claims 91 to 97, wherein detecting whether the miRNA is in the vesicles does not involve microarray analysis.

99. 98. The method of any one of claims 91 to 97, wherein detecting whether the miRNA is in the vesicles comprises microarray analysis using a microarray comprising probes for less than 1000, 500, or 100 different miRNAs.

100. 100. The method of any one of claims 91 to 99, wherein detecting whether the miRNA is in the vesicles comprises polymerase chain reaction.

101. The method of any one of claims 91 to 100, wherein the compound is a small molecule.

102. The method of any one of claims 91 to 101, wherein the compound is a compound used in the treatment of kidney disease.

103. 103. A method for monitoring treatment with a bioactive renal cell population in a subject to whom said bioactive renal cell population has been administered, comprising detecting whether at least one compound is present in vesicles from said subject according to the method of any one of claims 91-102.

104. 104. The method of claim 103, comprising detecting whether at least one compound is present in vesicles from the subject at a first time point and a second time point according to the method of any one of claims 91 to 102.

105. 105. The method of claim 104, wherein the first time point is before the bioactive renal cell population is administered to the subject and the second time point is after the bioactive renal cell population is administered to the subject.

106. 105. The method of claim 104, wherein the first time point and the second time point are after the bioactive renal cell population has been administered to the subject.

107. 107. The method of any one of claims 104-106, further comprising determining a regenerative effect in the subject if the level of the compound is higher at the first time point compared to the second time point.

108. 107. The method of any one of claims 104 to 106, further comprising determining a regenerative effect in the subject if the level of the compound is higher than a control.

109. The method of claim 108, wherein the control is the level in a corresponding subject to which the bioactive renal cell population has not been administered.

110. 1. A method for determining whether a vesicle is regenerative, comprising: (i) detecting whether proteins and / or miRNAs are present in the vesicles according to the method of any one of claims 49 to 58; (ii) determining the vesicle as regenerative if the protein and / or the miRNA is detected in the vesicle; A method comprising:

111. 1. A method for detecting the level of at least one miRNA in vesicles from a population of bioactive kidney cells, comprising: (i) detecting whether one or more of the following miRNA molecules: miR-1248, miR-3168, miR-362-5p, miR-7113-5p, miR-758-3p, miR-937-3p, miR-4455, miR-4521, miR-203a-3p, miR-22-3p, miR-574-3p, miR-181b-5p, miR-1260b, and / or miR-181b-5p, are increased in the vesicles compared to a control; (ii) detecting whether one or more of the following miRNA molecules: miR-1-3p, miR-1-3p, miR-143-3p, miR-150-5p, miR-509-3p, miR-653-5p, miR-204-5p, miR-192-5p, and / or miR-363-3p, are decreased in the vesicles compared to a control; A method comprising:

112. 112. The method of claim 111, wherein the bioactive kidney cells are selected kidney cells.

113. 113. The method of claim 111 or 112, wherein the control is the level of the one or more miRNA molecules in vesicles from a primary renal cell population.

114. 113. The method of claim 111 or 112, wherein the control is the level of the one or more miRNA molecules in vesicles from another bioactive renal cell population.

115. A method for treating renal disease in a subject, comprising administering to the subject an effective amount of vesicles from a vesicle preparation, wherein the vesicles from the vesicle preparation have been identified as regenerative according to the method of claim 110.

116. A method for treating a renal disease in a subject, comprising administering to the subject an effective amount of a composition comprising a bioactive renal cell population supplemented with renal cell vesicles not secreted by the bioactive renal cell population.

117. 117. The method of claim 116, wherein the bioactive renal cell population is a selected renal cell population.

118. 118. The method of claim 116 or 117, wherein the renal cell vesicles are secreted by a bioactive renal cell population that has the same origin and / or comprises the same cell type as the bioactive renal cell population in the composition.

119. A method for altering the level of at least one miRNA and / or protein in vesicles produced by a population of bioactive kidney cells, the method comprising culturing the population under hypoxic conditions.

120. 120. The method of claim 119, wherein culturing the population under hypoxic conditions comprises culturing the population in the presence of less than 5% oxygen for at least about 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, or 48 hours.

121. 121. The method of claim 119 or 120, further comprising culturing the population under hypoxic conditions after passage of the bioactive renal cells at least about 1, 2, or 3 times.

122. (a) the at least one miRNA is miR-145, miR-22, miR-7, miR-10a, miR-143, let7b, miR-1248, miR-3168, miR-7113-5p, miR-758-3p, miR-937-3p, miR-4455, miR-4521, miR-203a-3p, miR-22-3p, miR-574-3p, miR-181b-5p, miR-1260b, and / or miR-181b-5p; and / or (b) the at least one protein is CD9, CD63, CD81, CD133, CD146, CD326, CD40, CD42a, CD44, CD49e, SSEA-4, TST101, HSP70, HSP90, and / or ROR4.

123. Vesicles containing compounds not produced by renal cells in the native kidney.

124. 124. The vesicle of claim 123, wherein the compound is a protein, a small molecule, or a polynucleotide.

125. 125. The vesicle of claim 123 or 124, wherein the compound is not expressed or produced by primary kidney cells cultured in the absence of the compound.

126. 126. The vesicle of claim 125, wherein the compound is an artificial compound.

127. The vesicle of any one of claims 123 to 126, wherein the compound is a compound used in the treatment of kidney disease.

128. A vesicle according to any one of claims 123 to 127, which is a microvesicle.

129. The vesicle of claim 128, which is an exosome.

130. 130. A vesicle according to any one of claims 123 to 129 in a composition comprising the cells that produced the vesicle.

131. 130. A vesicle according to any one of claims 123 to 129, isolated from cells that produced the vesicle.

132. 132. The vesicle of any one of claims 123 to 131, which is a renal vesicle.

133. 133. The vesicle of any one of claims 123 to 132, produced by bioactive kidney cells.

134. 134. A composition comprising a vesicle according to any one of claims 123 to 133 and a pharmaceutically acceptable carrier.

135. A composition comprising renal cell vesicles and non-renal cell vesicles.

136. 136. The composition of claim 135, wherein the renal cell vesicles are secreted by bioactive renal cells.

137. 137. The composition of claim 135 or 136, wherein the non-renal cell vesicles are secreted by non-renal endothelial progenitor cells, non-renal mesenchymal stem cells, or non-renal adipose-derived progenitor cells.

138. A composition comprising vesicles produced by primary kidney cells and vesicles produced by selected kidney cells.

139. 139. A method of treating kidney disease in a subject, comprising administering to said subject an effective amount of a composition according to any one of claims 134 to 138.

140. 1. A method for producing exosomes from cells comprising a compound not produced by the cells, the method comprising isolating the exosomes from a cell culture supernatant, wherein the cell culture supernatant is from a culture of cells contacted with the compound.

141. A method for producing renal exosomes containing compounds not produced by renal cells in the native kidney. and isolating vesicles from a renal cell culture supernatant, wherein the renal cell culture supernatant is a cell culture supernatant from a culture of renal cells comprising a bioactive renal cell population that has been contacted with the compound.

142. 142. The method of claim 140 or 141, wherein the compound is an artificial compound.

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