Compositions comprising cell-derived vesicles and uses thereof

Abstract

In particular, extracellular products (e.g., vesicles, e.g., microvesicles, e.g., exosomes) produced by renal cells (e.g., biologically active renal cells, e.g., selected renal cells) are provided herein. Methods for altering the components (e.g., miRNA or proteins) of vesicles produced by cells, as well as methods for producing vesicles containing various compounds, are also included. Diagnostic and therapeutic methods are also provided.

Description

Background Technology

[0001] Research on extracellular vesicles, most notably exosomes, is rapidly increasing. Numerous papers have been published on exosome biogenesis (Kowal et al., 2014, Curr Opin Cell Biol. 29C:116-125), its diagnostic and prognostic potential (Revenfeld et al., 2014, Clin Ther. 36(6):830-846), and potential therapeutic uses in tissue engineering and regenerative medicine (Lamichhane et al., 2014, Tissue Eng Part B Rev.). These vectors of mRNA, miRNA, proteins, and lipid mediators can act on target cells to facilitate cell-to-cell communication and the exchange of functional genetic information (Simons and Raposo, 2009, Curr Opin Cell Biol. 21(4):575-581; Stoorvogel et al., 2002, Traffic 3(5):321-330; Nieuwland and Sturk, 2010, Thrombosis Research 125(Supplement 1):S49-S51).

[0002] In particular, extracellular products (e.g., vesicles, e.g., microvesicles, e.g., exosomes) produced by renal cells (e.g., bioactive renal cells, e.g., selected renal cells) are provided herein. In certain embodiments, such products are used to treat renal diseases, e.g., chronic renal disease. Methods for altering the components (e.g., miRNA or proteins) of vesicles produced by cells, as well as methods for producing vesicles containing various compounds, are also included. Diagnostic and therapeutic methods are also provided.

[0003] In one aspect, a method for treating renal disease in a subject is provided herein. In a specific embodiment, the method comprises administering an effective amount of isolated secreted renal cell vesicles to a subject, wherein the vesicles are administered by intravenous injection or by tube delivery.

[0004] In one aspect, a method for detecting at least one compound in a vesicle is provided herein. In certain embodiments, the method comprises the steps of 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 a miRNA, wherein the miRNA is at least two of miR-145, miR-22, miR-7, miR-10a, miR-143, and / or let7b; and (iii) at least one compound is not expressed or produced by renal cells in a natural kidney.

[0005] In one aspect, a method for monitoring treatment by a bioactive renal cell population in a subject to which a bioactive renal cell population has been administered is provided herein. In certain embodiments, the method comprises detecting, according to the method disclosed herein, whether at least one compound is present in a vesicle from the subject.

[0006] In one aspect, a method for determining whether a vesicle is regenerated is provided herein. In a specific embodiment, the method comprises: (i) detecting whether a protein and / or miRNA is present in the vesicle according to the method disclosed herein; and (ii) determining that the vesicle is regenerated if the protein and / or miRNA is detected in the vesicle.

[0007] In one aspect, a method for detecting the level of at least one miRNA in vesicles from a population of bioactive renal cells is provided herein. In a specific embodiment, the method comprises the step of (i) detecting whether one or more of the following miRNA molecules: 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 are increased in vesicles compared to a control group; and (ii) a step of 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 reduced in vesicles compared to a control. In certain embodiments, the miRNA is mammalian miRNA, e.g., human miRNA.

[0008] In one aspect, a method for treating renal disease in a subject is provided herein. In a specific embodiment, the method comprises administering an effective amount of vesicles from a vesicle preparation to a subject, wherein the vesicles from the vesicle preparation are identified as regenerative according to the method disclosed herein.

[0009] In a specific embodiment, a method for treating a renal disease in a subject is provided herein, 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.

[0010] In one aspect, a method for altering the level of at least one miRNA and / or protein in vesicles produced by a population of bioactive renal cells is provided herein, comprising culturing a population of bioactive renal cells under hypoxic conditions.

[0011] In one aspect, a vesicle containing a compound not produced by renal cells in a natural kidney is provided herein.

[0012] In one aspect, a composition comprising the vesicles disclosed herein and pharmaceutically acceptable carriers is provided herein.

[0013] In one aspect, a composition comprising renal cell vesicles and non-renal cell vesicles is provided herein.

[0014] In one aspect, a composition comprising vesicles produced by primary renal cells and vesicles produced by selected renal cells is provided herein.

[0015] In one aspect, a method for treating renal disease in a subject is provided herein. In certain embodiments, the method comprises administering an effective amount of the composition disclosed herein to the subject.

[0016] In one aspect, a method for producing vesicles (e.g., microvesicles, e.g., exosomes) from cells is provided herein, wherein the vesicles comprise a compound not produced by the cells. In certain embodiments, the method comprises isolating vesicles from a cell culture supernatant, wherein the cell culture supernatant is from a culture of cells that have come into contact with the compound (e.g., incubated in a medium containing the compound). In certain embodiments, the method comprises isolating vesicles (e.g., microvesicles, e.g., exosomes) from the cell culture supernatant and then incorporating the compound into the vesicles by permeating the exosome membrane (e.g., by sonication, lipofection, electroporation, etc.) to facilitate the entry of the compound.

[0017] In one aspect, a method for producing renal exosomes comprising a compound not produced by renal cells in a natural kidney is provided herein. In a specific embodiment, the method comprises isolating vesicles from a renal cell culture supernatant that is from a culture of renal cells comprising a population of bioactive renal cells in contact with the compound. Brief explanation of the drawing

[0018] Fig. 1: Flowchart of a non-limiting example of the overall NKA manufacturing process. FIGS. 2a-d: Flowcharts providing additional details of the non-limiting exemplary process illustrated in FIGS. 1. Fig. 3a-d: Flowchart of a non-limiting example of the production of NKA supplemented with exosomes from SRC. Figures 4a-d: Graphs showing the analysis of surface proteins in secreted exosomes isolated from both TCHK0012 and TCHK0013. The analysis suggested that the expression of CD133, CD326, and CD49e is upregulated in the case of SRC compared to BRC. Although the exact function of CD133 is still unknown, it has been suggested to act as a constructor of the cell membrane phase. Epithelial cell adhesion molecule (EpCAM) (also known as CD326) is a transmembrane glycoprotein that mediates Ca2+-independent homologous cell-cell adhesion in the epithelium. EpCAM is also involved in cell signaling, migration, proliferation, and differentiation. In addition to adhesion, integrins such as CD49e are known to participate in cell-surface-mediated signaling. Arrows were added to Figures 4b-d to highlight the comparison between exosomes from BRC-3A and SRC. Fig. 5: Graph from FACs analysis of exosome fusion into cells. Fluorescent labeling of cells, indicating the transfer of lipophilic dye from exosomes to the cell membrane, causes a shift in the histogram line from left to right. Exosomes will not attach to the cell membrane and will not integrate with it at 4°C. This is a negative control. Incubation at 37°C enables attachment and integration, thereby enabling fluorescent labeling of cells and shifting the histogram from left to right. Fig. 6: Graph showing cell proliferation as the mean cell count (y-axis) in response to a variable dose of exosomes supplied from a renal cell population (1X = dose / response by x ng / ml exosomes). Fig. 7A-C: Cell imaging. 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 10E10 exosomes (test sample). C. Serum-free medium supplemented with growth factors (positive control). Figure 8: Significantly different miRNA populations between pairs of experimental conditions E1 vs D1 are presented as a volcano plot. The volcano plot shows the distribution of differentially expressed miRNAs according to the fold change (x-axis) and significance (negative logarithm of the P-value on the y-axis). The horizontal dashed line is the P-value cut-off (0.05), and the vertical dashed line is the fold change cut-off (|Log2 fold change|≥1). Also refer to Tables 11-13. Figure 9: Significantly different miRNA populations between pairs of experimental conditions F1 vs A1 are presented as a volcano plot. Also refer to Tables 14-16. Fig. 10: Flowchart of a non-limiting example of an exosome manufacturing process. Specific details for implementing the invention

[0019] In particular, extracellular products (e.g., vesicles, e.g., microvesicles, e.g., exosomes) produced by renal cells (e.g., biologically active renal cells, e.g., selected renal cells) are provided herein.

[0020] All references cited throughout this disclosure are expressly incorporated herein by reference in their entirety. In the event that one or more of the included literature, patents, and similar materials, including but not limited to defined terms, usage of terms, and described techniques, differ from or contradict this application, this application shall prevail.

[0021] definition

[0022] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention pertains. The document [Principles of Tissue Engineering, 3rd Ed. (Edited by R Lanza, R Langer, & J Vacanti), 2007] provides general guidance to a person skilled in the art regarding many of the terms used in this application. A person skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of this invention. In fact, this invention is not limited in any way to the methods and materials described herein.

[0023] The singular form as used herein is intended to include the plural form unless the context otherwise clearly indicates. The term "and / or" as used herein includes any and all combinations of one or more associated listed items.

[0024] In this disclosure, terms such as “comprising,” “comprising,” “containing,” and “having,” etc., may have the meanings given to them by the U.S. Patent Act and may mean “comprising,” “comprising,” etc. “essentially made up” or “essentially made up” likewise have the meanings given to them by the U.S. Patent Act, and the terms are open-ended, allowing for the existence of anything beyond what is mentioned, provided that the basic features or novel features of what is mentioned are not altered by the existence of anything beyond what is mentioned, but excluding prior art embodiments.

[0025] As used herein, in the context of numbers or ranges, the term “about” means ±10% of the quoted or claimed number or range, 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, typically from mammals. In certain embodiments, the isolated cell population may subsequently be cultured in vitro. A person skilled in the art will recognize various methods for isolating and culturing cell populations for use with the present disclosure and for isolating and culturing varying numbers of cells within a cell population suitable for use in the present disclosure. In certain embodiments, the cell population may be an unfractionated heterogeneous cell population or an enriched homogeneous cell population derived from an organ or tissue, e.g., a kidney. In certain embodiments, the heterogeneous cell population may be isolated from a tissue biopsy or from whole organ tissue. In certain embodiments, the heterogeneous cell population may be derived from an in vitro culture of mammalian cells established from a tissue biopsy or whole organ tissue. An unfractionated heterogeneous cell population may also be referred to as a non-enriched cell population. In certain embodiments, the cell population contains bioactive cells. A homogeneous cell population contains a larger 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 homogeneous cell population may be isolated, extracted, or enriched from a heterogeneous kidney cell population. In certain embodiments, the enriched cell population is obtained as a cell fraction using separation by centrifugation across a density boundary, barrier, or interface of a heterogeneous cell suspension. In certain embodiments, the enriched cell population is obtained as a cell fraction using continuous or discontinuous (single-stage or multi-stage) density gradient separation of a heterogeneous cell suspension. In certain embodiments, the cell population may contain one, two, three, four, or more types of kidney cells.In certain embodiments, a homogeneous or heterogeneous cell population supplied from the kidney is combined with a homogeneous or heterogeneous cell population supplied from tissues or organs other than the kidney without further limitation.

[0027] As used herein, the term “biological activity” means “possessing biological activity,” such as pharmacological activity or therapeutic activity. In certain embodiments, biological activity is enhancement of renal function and / or enhancement of effects on renal homeostasis. In certain embodiments, biological activity is, without limitation, analgesic; antiviral; anti-inflammatory; antineoplastic; immune stimulation; immune modulation; enhancement of cell viability, antioxidant, oxygen transport, cell mobilization, cell adhesion, immunosuppression, angiogenesis, wound healing activity, mobilization of host stem or progenitor cells, cell proliferation, stimulation of cell migration to the site of injury, improvement of cell and tissue fibrosis, disruption of the epithelial-mesenchymal signaling cascade, and enhancement of the secretion of cytokines, growth factors, proteins, nucleic acids, exosomes, microvesicles, or any combination thereof.

[0028] As used herein, the terms “biologically active renal cell” or “BRC” refer to renal cells having one or more of the following characteristics when administered to the kidney of a subject: the ability to reduce (e.g., slow down or halt) the exacerbation or progression of chronic kidney disease or its symptoms; the ability to enhance renal function; the ability to influence (improve) renal homeostasis; and the ability to promote healing, repair, and / or regeneration of renal tissue or the kidney. In certain embodiments, microvesicles from BRC and / or BRC may be administered to a patient, wherein the haploid type of BRC differs from the haploid type of the patient. In certain embodiments, BRC is a cell capable of enhancing renal function and / or influencing (improving) renal homeostasis and / or promoting 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., based on improved creatinine excretion and protein retention), glomerular cells (e.g., based on improved protein retention), vascular cells, and / or other cells of the corticomedullary junction. In certain embodiments, the BRC has a regenerative effect on the kidney. In certain embodiments, the BRC comprises, is essentially composed of, or is composed of selected renal cells (SRC). In certain embodiments, the BRC is an SRC. In certain embodiments, the BRC is obtained from the isolation and expansion of renal cells from kidney tissue. In certain embodiments, the BRC is obtained from the isolation and expansion of renal cells from kidney tissue using a method for selecting bioactive cells (e.g., cells having regenerative capacity).

[0029] In certain embodiments, SRC is a cell obtained from the isolation and expansion of renal cells from a suitable kidney tissue source, wherein SRC contains one or more cell types in a greater percentage compared to the starting renal cell population and one or more other cell types are absent or have a lower percentage. In certain embodiments, SRC contains an increased proportion of BRC compared to the starting renal cell population. In certain embodiments, the SRC population is an isolated renal cell population in which specific bioactive components and / or cell types are enriched and / or specific inactive and / or undesirable components or cell types are depleted, for use in the treatment of renal disease, i.e., providing stabilization and / or improvement and / or regeneration of renal function. In certain embodiments, microvesicles from SRC and / or SRC may be administered to a patient, wherein the haploid type of SRC differs from the haploid type of the patient. In certain embodiments, SRC may provide stabilization and / or improvement and / or regeneration of renal function. SRC provides superior therapeutic and regenerative results compared to the starting population. In certain embodiments, SRCs are obtained from the patient's renal cortical tissue via a kidney biopsy. In certain embodiments, SRCs are selected based on the expression of one or more of their markers (e.g., by MACS or FACS). In certain embodiments, SRCs are depleted from one or more cell types (e.g., by MACS or FACS) based on the expression of one or more of their markers on the cell types. In certain embodiments, cell depletion or selection involves bead / antibody coupling to attract cells to specific proteins on their cell surfaces. 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, the SRC is selected by separation of expanded renal cells by centrifugation across a density boundary, barrier, or interface, or by single-stage discontinuous-stage gradient separation. In certain embodiments, the SRC is selected by continuous or discontinuous density gradient separation of expanded renal cells cultured under hypoxic conditions. In certain embodiments, the SRC is selected by density gradient separation of expanded renal cells cultured under hypoxic conditions for at least about 8, 12, 16, 20, or 24 hours. In certain embodiments, the SRC is selected by separation by centrifugation across a density boundary, barrier, or interface of proliferated renal cells cultured under hypoxic conditions. In certain embodiments, the SRC is selected by separation of expanded renal cells cultured under hypoxic conditions for at least about 8, 12, 16, 20, or 24 hours by centrifugation across a density boundary, barrier, or interface (e.g., single-stage discontinuous density gradient separation). In certain embodiments, the SRC consists mainly of renal tubular cells. In certain embodiments, other parenchymal (e.g., vascular) and stromal (e.g., collecting duct) cells may be present within the SRC. In certain embodiments, about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% of the cells in the SRC population are vascular cells. In certain embodiments, about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% of the cells in the SRC population are collecting duct cells. In certain embodiments, about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% of the cells in the SRC population are vascular or collecting duct cells. Methods for obtaining SRC are disclosed, for example, in Example 1 of the present invention, WO / 2010 / 056328 (Presnell et al.), PCT / US2011 / 036347 (Ilagan et al.), and PCT / US2016 / 044866 (Jain et al.).

[0030] The term "natural organ" refers to the organs of a living subject. The subject may be healthy or unhealthy. An unhealthy subject may have a disease associated with a specific organ.

[0031] The term "natural kidney" will refer to the kidney of a living subject. The subject may be healthy or unhealthy. An unhealthy subject may have kidney disease.

[0032] The term "regenerative effect" will refer to an effect that provides benefit to a natural organ, such as a kidney. The effect may, without limitation, include a reduction in the degree of damage to the natural organ or the improvement, repair, or stabilization of the function or structure of the natural organ. Kidney damage may take the form of fibrosis, inflammation, glomerular hypertrophy, atrophy, etc., and may be associated with diseases related to the subject's natural organ.

[0033] "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 a kidney) that contains a greater percentage of a specific cell type than the percentage of such cell type in the starting population. For example, a starting kidney cell population may be enriched for the first, second, third, fourth, fifth, etc. of cell populations 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 subjected to a reduced level of available oxygen in the culture system compared to standard culture conditions in which cells are cultured at atmospheric oxygen levels (about 21%). In certain embodiments, hypoxic culture conditions are in which the level of oxygen in the culture system is 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1%.

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

[0036] The term “hydrogel” is used herein to refer to a material formed when an organic polymer (natural or synthetic) is crosslinked via covalent, ionic, or hydrogen bonds to create a three-dimensional structure (e.g., an open-lattice structure) that captures water molecules to form a gel. Examples of materials that may be used to form a hydrogel include polysaccharides that are crosslinked by thermal conductivity, such as alginates, polyphosphazines, and polyacrylates, or block copolymers that are crosslinked by temperature or pH, such as Pluronics™ or Tetronics™, and polyethylene oxide-polypropylene glycol block copolymers. In certain embodiments, the hydrogel is a biodegradable gelatin-based hydrogel.

[0037] In certain embodiments, the biocompatible material comprises, for example, an extracellular matrix derived from an existing kidney of human or animal origin, wherein the natural cell population has been removed through the application of detergents and / or other chemical agents known to those skilled in the art. In certain embodiments, the biocompatible material is in the liquid form of a solution that may become a hydrogel and is layered in the presence or absence of a specific cell population by the application of a three-dimensional bioprinting methodology known to those skilled in the art. In certain embodiments, the biocompatible material 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 "slow release" refers to a formulation that releases an activator, such as a bioactive cell, over a period of time or at more than one point in time following administration to an individual. Modified release of the activator, which may occur over a desired time range depending on the formulation—e.g., minutes, hours, days, weeks, or longer—is in contrast to formulations where substantially the entire dose unit is available immediately after administration. In certain embodiments, for tissue engineering and regenerative medicine applications, modified-release formulations provide release of the activator at multiple points in time following local administration (e.g., direct administration of the activator to a parenchymal organ). For example, a modified-release formulation of a bioactive cell may provide an initial release of the cell immediately upon administration and a subsequent second release of the cell later. In certain embodiments, the time delay for the second release of the activator may be minutes, hours, or days after the initial administration. Generally, the release delay period corresponds to the time required for the biocompatible carrier of the activator to lose its structural integrity. The delayed release of the activator begins as soon as this integrity starts to decompose and is completed around the time the integrity is completely gone. A person skilled in the art will recognize other suitable release mechanisms.

[0039] The term “ambient temperature” refers to the temperature at which the formulation of the present disclosure is administered to a subject. Generally, the ambient temperature is the temperature of a temperature-controlled environment. The ambient temperature is in the range of about 18°C ​​to about 30°C. In certain embodiments, the 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 associated with acute or chronic renal failure of any stage or degree that cause a loss of the kidney's ability to perform the function of filtration of blood and removal of excess fluids, electrolytes, and waste products from the blood. In certain embodiments, kidney disease may also include endocrine dysfunction, such as anemia (erythropoietin deficiency) and mineral imbalance (vitamin D deficiency). Kidney disease may originate in the kidneys or may be secondary to various conditions including (but not limited to) heart failure, hypertension, diabetes, autoimmune diseases, or liver disease. Kidney disease may be a state of chronic renal failure that occurs after acute injury to the kidneys. For example, damage to the kidneys caused by ischemia and / or exposure to toxic substances can cause acute renal failure; and incomplete recovery after acute kidney injury can lead to the development of chronic renal failure.

[0041] The term “treatment” refers to both curative treatment and prophylactic or preventive measures for renal disease, anemia, tubular transport deficiency, or glomerular filtration deficiency, wherein the purpose is to reverse, prevent, or slow the targeted impairment (e.g., to reduce its deterioration). Those requiring treatment include those who already have renal disease, anemia, tubular transport deficiency, or glomerular filtration deficiency, as well as those who are prone to or at risk of having renal disease, anemia, tubular transport deficiency, or glomerular filtration deficiency, or those for whom renal disease, anemia, tubular transport deficiency, or glomerular filtration deficiency must be prevented. As used herein, the term “treatment” includes the stabilization and / or improvement of renal function.

[0042] In certain embodiments, "in vivo contact" of a natural organ and an activator (e.g., an enriched population of cells and / or its product) refers to direct in vivo contact between the activator and the natural organ. For example, a product secreted by an enriched population of renal cells may come into in vivo contact with a natural kidney (alone or, for example, together with cells within a structure). In certain embodiments, direct in vivo contact may be parasecretory, endocrine, or proximate in nature. In certain embodiments, the secreted product may be a heterogeneous population of the different products described herein.

[0043] In certain embodiments, a “construction” or “formulation” is provided herein, comprising one or more cell populations and / or one or more cell products (e.g., microvesicles, e.g., exosomes) deposited on or on the surface of a biocompatible material (e.g., a scaffold or matrix composed of one or more synthetic or naturally occurring biocompatible materials). In certain embodiments, one or more cell populations may be coated, deposited thereon, embedded thereon, attached thereon, seeded thereon, or captured thereon with a biocompatible material composed of one or more synthetic or naturally occurring biocompatible materials, polymers, proteins, or peptides. In certain embodiments, the naturally occurring biocompatible material is a decellularized kidney of human or animal origin. In certain embodiments, the biocompatible material is structurally engineered via three-dimensional bioprinting. In certain embodiments, one or more cell populations and / or cell products may be combined with the biocompatible material or scaffold or matrix in vitro or in vivo. In certain embodiments, one or more biocompatible materials used to create a construct or formulation may be selected to direct, facilitate, or allow the dispersion and / or integration of the cellular components of the construct and endogenous host tissue, or to direct, facilitate, or allow the viability, engraftment, tolerance, or functional performance of the cellular components of the construct or formulation. In certain embodiments, one or more biocompatible materials used to form a scaffold / biocompatible material are selected to direct, facilitate, or allow the formation of at least one multicellular, three-dimensional organization among the cell populations deposited thereon. In certain embodiments, the biocompatible material directs, promotes, or facilitates the assembly of a defined three-dimensional cell aggregate or organoid that reproduces aspects of natural kidney tissue, including but not limited to organizational polarity.In certain embodiments, the biocompatible material directs the assembly of a defined tubular structure that reproduces aspects of natural kidney tissue, including the lumen. In certain embodiments, the biocompatible material promotes or facilitates the secretion of proteins, nucleic acids, and microvesicles from a cell population. In certain embodiments, one or more biocompatible materials used to create the construct may also be selected to mimic or reproduce aspects of specific three-dimensional organization or environmental depressions within the natural kidney or renal parenchyma that represent the original biological environment from which the cell population originated. While not bound by any scientific theory, the regeneration of the original biological depressions from which these cell populations were supplied is believed to further promote or facilitate cell viability and efficacy.

[0044] The term “cell aggregate” or “ellipsoid” refers to an aggregate or assembly of cultured cells that enables 3D growth in contrast to growth as a monolayer. Note that the term “ellipsoid” does not imply that the aggregate is a geometric sphere. In certain embodiments, the aggregate may be highly organized with a well-defined shape and polarity, or it may be an unorganized mass; it may contain a single cell type or more than one cell type. In certain embodiments, the cells may be a primary isolate, a permanent cell line, or a combination of both. These definitions include organoids and organoid cultures. In certain embodiments, the ellipsoid (e.g., cell aggregate or organoid) is formed in a spinner flask. In certain embodiments, the ellipsoid (e.g., cell aggregate or organoid) is formed as a three-dimensional matrix.

[0045] The term "renal-renal enhancer (NKA)" refers to a bioactive cell preparation that is an injectable product comprising SRCs formulated in a biocompatible material consisting of a gelatin-based hydrogel. The term "advanced cell therapy (ACT)" is also used in relation to therapy using NKA. In certain embodiments, NKA is an injectable product comprising an immuno-compatible renal cell population (e.g., immuno-compatible SRCs) formulated in a biocompatible material consisting of a gelatin-based hydrogel. In certain embodiments, NKA is an injectable product comprising immune-rejection-incapable genome-modified immuno-privileged homologous SRCs formulated in a biocompatible material consisting of a gelatin-based hydrogel.

[0046] The term “subject” will mean any single human subject, including a patient eligible for treatment, who is experiencing or has experienced one or more signs, symptoms, or other indicators of kidney disease. Such subjects include, without limitation, subjects newly diagnosed with kidney disease regardless of cause, subjects previously diagnosed with kidney disease who are currently experiencing a recurrence or relapse, or subjects at risk of kidney disease. Subjects may or may not have previously been treated for kidney disease.

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

[0048] The terms "sample," "patient sample," or "biological sample" generally refer to any biological sample obtained from a subject or patient, body fluid, body tissue, cell line, tissue culture, or other source. The term includes tissue biopsies, e.g., kidney biopsies. The term includes cultured cells, e.g., cultured mammalian kidney cells. Methods for obtaining tissue biopsies and cultured cells from mammals are widely known in the relevant art. Depending on the context, where the term "sample" is used alone, it will still mean "biological sample" or "patient sample"; that is, the terms are used interchangeably.

[0049] The term “test sample” refers to a sample from a subject treated by the method of the present disclosure. The test sample may originate from various sources of mammalian subjects, including, but not limited to, blood, semen, serum, urine, bone marrow, mucous membranes, tissues, etc.

[0050] The terms “control group” or “control sample” refer to a negative or positive control group in which a negative or positive result is expected to help correlate the result in the test sample. Control groups suitable for the present disclosure include, but not limited to, a sample known to exhibit characteristic indicators of normal kidney function, a sample obtained from a subject known not to have kidney disease, and a sample obtained from a subject known to have kidney disease. In certain embodiments, a control group may be a sample obtained from a subject prior to treatment by the method of the present disclosure. In certain embodiments, a suitable control group may be a test sample obtained from a subject known to have kidney disease of any type or stage, and a sample from a subject known not to have kidney disease of any type or stage. A control group may be a normal, healthy matching control group. A person skilled in the art will recognize other control groups suitable for use in the present disclosure.

[0051] "Regenerative prognosis," "regenerative prognosis," or "prognosis for regeneration" generally refers to the prospect or prediction of a possible regenerative process or outcome of the administration or transplantation of the cell population, cell product, or construct described herein. In the case of a regenerative prognosis, the prospect or prediction may be made 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 capacity after transplantation or administration; and expression of specific markers by the natural kidney after transplantation or administration.

[0052] “Regenerated organ” refers to a natural organ following the transplantation or administration of a cell population, cell product, or construct as described herein. In certain embodiments, the regenerated organ is characterized by various indicators, including, but not limited to, the development of function or capacity in the natural organ, improvement of function or capacity in the natural organ, improvement of specific markers and physiological indices associated with the disease, and / or expression of specific markers in the natural organ. A person skilled in the art will recognize that other indicators may be suitable for characterizing the regenerated organ.

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

[0054] "Small molecule" is a compound with a mass of less than 2,000 daltons. The molecular mass of the small molecule is preferably less than 1,000 daltons, more preferably less than 600 daltons, and, for example, the compound is less than 500 daltons, 400 daltons, 300 daltons, 200 daltons, or 100 daltons. In certain embodiments, the small molecule is an organic compound.

[0055] “Microves” are cell-derived membranous extracellular vesicles with a diameter of 30 to 1,000 nanometers (nm). “Exosomes” are cell-derived membranous microvesicles with a diameter of about 30 to 150 nm. In certain embodiments, exosomes are cell-derived membranous microvesicles with a diameter of about 50 to 100 nm. Additional features typically shared by exosomes are known in the art. A non-limiting description of microvesicles and exosomes is provided in the literature [Zhang et al. (2016) Am J Physiol Renal Physiol. 311(5):F844-F851], the entire contents of which are incorporated herein by reference.

[0056] When referring to the amount of therapeutic agent used herein (e.g., microvesicles alone or in combination with biologically active renal cells, e.g., exosomes), “effective” refers to an amount of agent sufficient to produce a desired therapeutic response without excessive adverse effects (e.g., toxicity, irritation, or allergic reaction) corresponding to a reasonable benefit / risk ratio when used in the manner of the present disclosure.

[0057] Secreted product

[0058] Products secreted by biologically active renal cells (e.g., SRC), such as vesicles, are provided herein. In certain embodiments, the vesicles comprise microvesicles.

[0059] In certain embodiments, the microvesicles have a diameter of about 30-150, 30-200, 30-500, 30-1000, 500-1000, 50-1000, 50-200, 50-150, 50-100, 100-150, 100-200, or 100-300 nm.

[0060] In certain embodiments, the vesicle contains exosomes, is essentially composed of exosomes, or is composed of exosomes. In certain embodiments, the exosomes have a diameter of about 50-100 nm. In certain embodiments, the exosomes have a diameter of 30-100, 50-150, 50-100, 100-150, or 30-150 nm. In certain embodiments, the exosomes have a diameter of about 30, 35, 40, 45, 50, 55, or 60 nm to about 100, 110, 120, 130, 140, or 150 nm.

[0061] In certain embodiments, the vesicle contains an activator (e.g., a compound) on its outer surface, within its lipid bilayer, and / or within its lumen. 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 present in the membrane of the vesicle. In certain embodiments, the compound is lipophilic and is present within the lipid bilayer of the exosome. In certain embodiments, the polynucleotide is a miRNA molecule.

[0062] In certain embodiments, the compound is expressed or produced by bioactive renal cells. In certain embodiments, the compound is not expressed or produced by bioactive renal cells. In certain embodiments, the compound is added to the medium of the cell that produces the vesicle (e.g., the cell is incubated in a medium containing the compound). In certain embodiments, the compound enters the cell and is contained within the vesicle produced by the cell. In certain embodiments, the vesicle is purified or isolated from the cell and then incubated in a solution (e.g., medium) containing the compound. In certain embodiments, the vesicle is isolated or purified from the cell and then the compound is incorporated into the vesicle by a technique that permeates the vesicle membrane to facilitate the entry of the compound (e.g., sonication, repopulation, electroporation, etc.).

[0063] In certain embodiments, the compound is not produced by naturally occurring renal 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 bioagent. In certain embodiments, the artificial compound is not expressed or produced by renal cells in the natural kidney. In certain embodiments, the compound is a cell viability agent. In certain embodiments, the compound is a compound used to treat a disease (e.g., kidney disease or some other disease). In certain embodiments, the compound is immunotolerant or anti-inflammatory.

[0064] In certain embodiments, the compound has been 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 for the healing, alleviation, treatment, or prevention of a disease. In certain embodiments, the compound is used to alter the structure or function of mammalian cells or organisms.

[0065] In certain embodiments, the vesicle comprises a compound that attenuates plasminogen activator-1 (PAI-1) signaling and / or transforming growth factor beta (TGFβ) signaling. In certain embodiments, the vesicle comprises a compound that attenuates normal Wnt signaling. In certain embodiments, the vesicle comprises a compound that attenuates non-normal Wnt signaling. In certain embodiments, the vesicle comprises a compound that attenuates CXCR4-mediated signaling. In certain embodiments, the vesicle comprises a compound that downregulates an inflammatory cytokine. In certain embodiments, the inflammatory cytokine is IL8. In certain embodiments, the vesicle comprises a compound that attenuates Notch signaling.

[0066] In certain embodiments, the compound is a cell surface molecule used to determine the immunophenotype of a cell. In certain embodiments, the compound is CD9, CD63, CD81, CD133, CD146, CD326, CD40, CD42a, CD44, or CD49e.

[0067] In certain embodiments, the compound is a protein receptor. In certain embodiments, the protein receptor is a retinoid-associated receptor (ROR4).

[0068] In certain embodiments, the compound is a developmental stage marker. In certain embodiments, the developmental stage marker is stage-specific embryonic antigen-4 (SSEA-4).

[0069] In certain embodiments, the compound is a stress-protective protein. In certain embodiments, the stress-protective protein is heat shock protein (HSP) 70, or HSP90.

[0070] In certain embodiments, the compound is a scaffolding protein. In certain embodiments, the scaffolding protein is TST101.

[0071] In certain embodiments, the compound is miRNA and is present within the lumen of a vesicle.

[0072] In certain embodiments, the miRNA is a cell cycle regulatory miRNA. In certain embodiments, the cell cycle regulatory miRNA is let7a, miR-143, or miR22.

[0073] In certain embodiments, the miRNA is a cellular senescence-regulating miRNA. In certain embodiments, the cellular senescence-regulating miRNA is miR-34.

[0074] In certain embodiments, the miRNA is a cell migration-regulating miRNA. In certain embodiments, the cell migration-regulating miRNA is miR30-C.

[0075] In a specific embodiment, the miRNA is a cell growth regulatory miRNA. In a specific embodiment, the cell growth regulatory miRNA is miR194-2.

[0076] In certain embodiments, the miRNA is a cell signaling pathway-regulating miRNA. In certain embodiments, the cell signaling pathway-regulating miRNA is miR-142.

[0077] In certain embodiments, the miRNA is an inflammation-regulating miRNA. In certain embodiments, the inflammation-regulating miRNA is miR-10a.

[0078] In certain embodiments, the miRNA is angiogenesis-coordinating miRNA. In certain embodiments, the angiogenesis-coordinating miRNA is miR-296 and / or miR-146a.

[0079] In certain embodiments, the miRNA is a kinase activity-coordinating miRNA. In certain embodiments, the kinase activity-coordinating miRNA is miR-83.

[0080] In certain embodiments, the compound is a miRNA that inhibits PAI-1, TGFβ, normal Wnt signaling, non-normal Wnt signaling, CXCR4-mediated signaling, and / or Notch signaling.

[0081] In certain embodiments, renal fibrosis is reduced or prevented by inhibition of epithelial-mesenchymal transition (EMT).

[0082] In certain embodiments, the parcels provided herein include miR-145, miR-22, miR-7, miR-10a, miR-143, and / or let7b.

[0083] In certain embodiments, the vesicles provided herein include 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.

[0084] In certain embodiments, the parcels provided herein include CD9, CD63, CD81, CD133, CD146, CD326, CD40, CD42a, CD44, CD49e, and / or SSEA-4.

[0085] In certain embodiments, the vesicle provided herein comprises CD63, CD9, and / or CD81, and CD63, CD9, and / or CD81 or a portion thereof on the outer surface of the vesicle. In certain embodiments, a portion of one or more of these proteins is present inside the vesicle.

[0086] In certain embodiments, the vesicle provided herein comprises CD133, CD326, and / or CD49e, and CD133, CD326, and / or CD49e are present on the outer surface of the vesicle.

[0087] In certain embodiments, proliferation of renal cells contacted by vesicles is increased compared to renal cells not contacted by vesicles. In certain embodiments, angiogenesis by endothelial cells contacted by vesicles is increased compared to endothelial cells not contacted by vesicles. In certain embodiments, nephron tubule formation of renal cells contacted by vesicles is increased compared to renal cells not contacted by vesicles.

[0088] In certain embodiments, the vesicles contain phospholipids, sphingolipids, cholesterol, cerimide, and / or phosphatidylcholine.

[0089] In certain embodiments, the vesicle is present 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.

[0090] In certain embodiments, the vesicles were produced by BRCs, e.g., primary renal cells. In certain embodiments, the vesicles were produced by SRCs. Compositions comprising vesicles from primary renal cells as well as vesicles from SRCs are included herein. Additionally, compositions are provided that further comprise vesicles secreted by endothelial cells or mesenchymal stem cells. In certain embodiments, the compositions provided herein comprise non-renal cell vesicles. In certain embodiments, non-renal cell vesicles were secreted by non-renal endothelial progenitor cells, non-renal mesenchymal stem cells, or non-renal adipose-derived progenitor cells.

[0091] In one aspect, a method for detecting at least one compound in a vesicle is provided herein. In certain embodiments, the method comprises the steps of 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 a miRNA, wherein the miRNA is at least two of miR-145, miR-22, miR-7, miR-10a, miR-143, and / or let7b; and (iii) at least one compound is not expressed or produced by renal cells in a natural kidney.

[0092] In certain embodiments, the vesicle is obtained from or from a biological sample from a subject. In certain embodiments, the biological sample is urine. In certain embodiments, the vesicle is obtained from or from the supernatant of a renal cell culture. In certain embodiments, the vesicle was secreted by a renal cell. In certain embodiments, the renal cell is a bioactive renal cell. In certain embodiments, the renal cell is a selected renal cell.

[0093] In certain embodiments, the step of detecting whether a protein is present within a vesicle includes an immunoassay. In certain embodiments, the step of detecting whether a protein is present within a vesicle includes an enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, immunoelectrophoresis, Western blot, or protein immunostaining. In certain embodiments, the step of detecting whether a protein is present within a vesicle includes a spectroscopic method. In certain embodiments, the step of detecting whether a protein is present within a vesicle includes high-performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS).

[0094] In certain embodiments, the step of detecting whether miRNA is present in a vesicle includes a polymerase chain reaction (PCR). In certain embodiments, the step of detecting whether miRNA is present in a vesicle includes a reverse-transscriptase PCR. In certain embodiments, the step of detecting whether miRNA is present in a vesicle includes microarray analysis. In certain embodiments, the step of detecting whether miRNA is present in a vesicle includes RNA sequencing. In certain embodiments, the step of detecting whether miRNA is present in a vesicle includes contacting a processed sample suspected of containing nucleic acids from a vesicle or a vesicle with a probe or primer complementary to the miRNA. In certain embodiments, the step of detecting whether miRNA is present in a vesicle does not include microarray analysis. In certain embodiments, the step of detecting whether miRNA is present in a vesicle includes microarray analysis using a microarray, wherein the microarray includes probes for 1,000, 500, or fewer than 100 different miRNAs.

[0095] In certain embodiments, the compound is a small molecule.

[0096] In certain embodiments, the compound is expressed or produced by a bioactive renal cell. In certain embodiments, the compound is not expressed or produced by a bioactive renal cell. In certain embodiments, the compound is expressed or produced by a bioactive renal cell. In certain embodiments, the compound is not expressed or produced by a bioactive renal cell. In certain embodiments, the compound is added to the medium of a cell that produces a vesicle (e.g., the cell is incubated in a medium containing the compound). In certain embodiments, the compound enters the cell and is contained within a vesicle produced by the cell. In certain embodiments, the vesicle is purified or isolated from the cell and then incubated in a solution (e.g., medium) containing the compound. In certain embodiments, the vesicle is isolated or purified from the cell and then the compound is incorporated into the vesicle by a technique that permeates the exosome membrane to facilitate the entry of the compound (e.g., sonication, lipofection, electroporation, etc.).

[0097] In certain embodiments, the compound is not produced by naturally occurring renal 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 renal cells in the natural kidney. In certain embodiments, the compound is a cell viability agent. In certain embodiments, the compound is a compound used to treat a disease (e.g., kidney disease or some other disease). In certain embodiments, the compound is immunotolerant 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 for the healing, alleviation, treatment, or prevention of a disease. In certain embodiments, the compound is used to alter the structure or function of mammalian cells or organisms.

[0098] In one aspect, a method for monitoring treatment by a bioactive renal cell population in a subject to which a bioactive renal cell population has been administered is provided herein. In certain embodiments, the method comprises detecting, according to the method disclosed herein, whether at least one compound is present in a vesicle from the subject.

[0099] In certain embodiments, the method comprises detecting, in accordance with the method disclosed herein, whether at least one compound is present in a vesicle from a subject at a first time point and a second time point. 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 confirming 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.

[0100] In certain embodiments, the method further comprises confirming a regenerative effect in a subject when the level of the compound is higher than that of the control. In certain embodiments, the method further comprises the control being the level in a corresponding subject to which a population of bioactive kidney cells has not been administered.

[0101] Additionally, a method for determining whether a vesicle is regenerated is included herein. In certain embodiments, the method comprises: (i) detecting whether a protein and / or miRNA is present in the vesicle according to the method disclosed herein; and (ii) determining that the vesicle is regenerated if the protein and / or miRNA is detected in the vesicle.

[0102] In one aspect, a method for detecting the level of at least one miRNA in vesicles from a population of bioactive renal cells is provided herein. In a specific embodiment, the method comprises the step of (i) detecting whether one or more of the following miRNA molecules: 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 are increased in vesicles compared to a control group; and / or (ii) the step of 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 reduced in vesicles compared to a 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 molecule vesicles from a primary renal cell population. In certain embodiments, the control is the level of one or more miRNA molecule vesicles from another bioactive renal cell population.

[0103] The subject of the present invention also provides a method for altering the level of at least one miRNA and / or protein in vesicles produced by a population of bioactive renal cells, comprising culturing a population of bioactive renal cells under hypoxic conditions. In certain embodiments, culturing the population under hypoxic conditions comprises culturing the population in the presence of about 5%, 4%, 3%, 2%, or less than 1% oxygen for, for example, 8 to 72 hours. In certain embodiments, culturing the population under hypoxic conditions comprises culturing the population in the presence of about 1-5%, 2-5%, 2-4%, 1-3%, or 1.5-2.5% oxygen for, for example, 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 for about 8, 12, 16, 20, 24, or 48 hours in the presence of about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5% oxygen.

[0104] In certain embodiments, the method further comprises subculturing the bioactive renal cells at least about 1, 2, or 3 times before culturing the population under hypoxic conditions.

[0105] In certain embodiments, (a) 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; (b) At least one protein is CD9, CD63, CD81, CD133, CD146, CD326, CD40, CD42a, CD44, CD49e, SSEA-4, TST101, HSP70, HSP90, and / or ROR4.

[0106] In one aspect, a method for producing exosomes from cells is provided herein, wherein the exosomes comprise a compound not produced by the cells. In certain embodiments, the method comprises isolating exosomes from the supernatant of a cell culture, wherein the supernatant is from a culture of cells in contact with the compound. In one aspect, a method for producing renal exosomes comprising a compound not produced by renal cells in a natural kidney is provided herein. In certain embodiments, the method comprises isolating vesicles from the supernatant of a renal cell culture, which is from a culture of renal cells comprising a population of bioactive renal cells in contact with the 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.

[0107] In one aspect, vesicles (e.g., microvesicles, e.g., exosomes) containing compounds not produced by renal cells in a natural kidney are included herein.

[0108] 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 neoplasm cells cultured in the absence of the compound. In certain embodiments, the compound is an artificial compound.

[0109] In certain embodiments, a compound (e.g., a protein or a small molecule drug) is “passively loaded” into a vesicle (e.g., a microvesicle, e.g., an exosome) by incubating a cell with a medium containing the compound, for example, or by incubating a purified vesicle (e.g., a microvesicle, e.g., an exosome) with a medium containing the compound. In certain embodiments, a compound (e.g., a protein or a small molecule drug) is “actively loaded” into a purified or isolated vesicle (e.g., a microvesicle, e.g., an exosome) by permeating the vesicle membrane (e.g., by sonication, lipofection, electroporation, etc.) to facilitate the entry of the compound.

[0110] In certain embodiments, the vesicle is present in a composition comprising a cell that produces the vesicle. In certain embodiments, the vesicle is isolated from the cell that produced it. In certain embodiments, the vesicle is a renal vesicle. In certain embodiments, the vesicle is produced by a bioactive renal cell.

[0111] In certain embodiments, the composition provided herein comprises renal cell vesicles and non-renal cell vesicles. In certain embodiments, the renal cell vesicles were secreted by bioactive renal cells. In certain embodiments, the non-renal cell vesicles were secreted by non-renal endothelial progenitor cells, non-renal mesenchymal stem cells, or non-renal adipose-derived progenitor cells.

[0112] In certain embodiments, the composition provided herein comprises vesicles produced by primary renal cells and vesicles produced by selected renal cells.

[0113] In certain embodiments, the vesicle further comprises a pharmaceutically acceptable carrier.

[0114] In certain embodiments, the regenerative effect may be provided by products (e.g., vesicles) secreted by cells and / or from bioactive renal cells. In certain embodiments, the regenerative effect may be characterized by one or more of the following: a reduction in epithelial-mesenchymal transition (which may be achieved through attenuation of TGF-β signaling); a reduction in renal fibrosis; a reduction in renal inflammation; differential expression of stem cell markers in the natural kidney; migration of transplanted cells and / or natural cells to renal damage, e.g., a site of tubular damage; engraftment of transplanted cells at renal damage, e.g., a site of tubular damage; stabilization of one or more indicators of renal function (as described herein); neoplastic formation of sigmoid / comma-like structures associated with renal regeneration, neoplastic formation of renal tubules or nephrons, and restoration of erythrocyte 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 full contents of each of these incorporated herein by reference).

[0115] In certain embodiments, as an alternative to tissue biopsy, the regenerative outcome in the subject being treated may be evaluated from the examination of body fluids, e.g., urine. It has been found that microvesicles (e.g., exosomes) obtained from a subject-derived urine source contain, to a non-limiting extent, specific components including specific proteins and miRNAs ultimately derived from the renal cell population affected by treatment. These components may, to a non-limiting extent, include factors associated with stem cell replication and differentiation, apoptosis, inflammation and immune modulation, fibrosis, epithelial-mesenchymal transition, TGF-β signaling, and / or PAI-1 signaling. In certain embodiments, temporal analysis of microvesicle (e.g., exosome)-associated miRNA / protein expression patterns enables continuous monitoring of the regenerative outcome within the kidney of a subject provided with the cell population, cell product, or construct of the present disclosure.

[0116] In certain embodiments, the present disclosure provides a method for evaluating whether a patient with kidney disease (KD) is responsive to treatment with a therapeutic agent. In certain embodiments, the method may include determining or detecting an amount of microvesicles (or their luminal contents), e.g., exosomes, in a test sample obtained from a KD patient treated with the therapeutic agent, in comparison to or in contrast to an amount of microvesicles (e.g., exosomes) in a control sample (e.g., a sample derived from the same patient prior to 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 indicates responsiveness of the patient to treatment with the therapeutic agent.

[0117] In certain embodiments, kidney-derived microvesicles (e.g., exosomes) and / or the lumen contents of kidney-derived microvesicles (e.g., exosomes) may be exuded into the subject's urine and analyzed for biomarkers indicating regenerative results or therapeutic efficacy. In certain embodiments, the non-invasive prognostic method provided herein may include obtaining a urine sample from the subject before and / or after administration or transplantation of the bioactive renal cell population, cell product, or construct described herein. Microvesicles and other secreted products may be isolated from urine samples using standard techniques, including, but not limited to, centrifugation to remove unwanted debris (Zhou et al. 2008. Kidney Int. 74(5):613-621; U.S. Patent Publication No. 20110053157 (Skog et al.), the full text of which is incorporated herein by reference), precipitation to separate microvesicles (e.g., exosomes) from urine, polymerase chain reaction and nucleic acid sequencing to identify specific nucleic acids, and mass spectrometry and / or 2D gel electrophoresis to identify specific proteins associated with the regeneration results.

[0118] cell population

[0119] Compositions and formulations comprising vesicles (e.g., microvesicles, e.g., exosomes) produced by a population of new cells (e.g., BRCs, e.g., primary cells and / or SRCs) are included herein. In certain embodiments, the vesicles may be isolated from cells, for example, or may be combined with cells that do not produce them. Non-limiting examples and characteristics of BRCs useful for the production of vesicles are provided herein.

[0120] In certain embodiments, the therapeutic composition or formulation provided herein contains microvesicles (e.g., exosomes) secreted by an isolated heterogeneous renal cell population enriched with a specific bioactive component or cell type and / or depleted of a specific inactive or undesirable component or cell type. In certain embodiments, the therapeutic composition or formulation provided herein contains or additionally contains an isolated heterogeneous renal cell population enriched with a specific bioactive component or cell type and / or depleted of a specific inactive or undesirable component or cell type. In certain embodiments, such compositions and formulations are used for the treatment of kidney disease, for example, to provide stabilization and / or improvement and / or regeneration of renal function and / or structure. In certain embodiments, the composition contains microvesicles (e.g., exosomes) secreted by an isolated renal cell fraction that lacks cellular components compared to a healthy individual but still retains therapeutic properties, for example, to provide stabilization and / or improvement and / or regeneration of renal function. In certain embodiments, the composition contains isolated renal cell fractions that lack cellular components compared to healthy individuals but still retain therapeutic properties, for example, providing stabilization and / or improvement and / or regeneration of kidney function. In certain embodiments, the cell population described herein may be derived from a healthy individual, an individual with kidney disease, or a subject as described herein.

[0121] Therapeutic compositions of microvesicles (e.g., exosomes) and / or selected renal cell populations to be administered to a target organ or tissue of a subject are included herein. In certain embodiments, a composition comprising microvesicles (e.g., exosomes) secreted by a BRC (e.g., SRC) is provided herein. In certain embodiments, the composition further comprises a BRC (e.g., SRC) that does not secrete microvesicles (e.g., exosomes). In certain embodiments, the composition comprises an NKA "spikeed" with microvesicles (e.g., microvesicles, such as exosomes, are added to the NKA to produce a vesicle-enhanced NKA). In certain embodiments, the BRC (e.g., SRC) from which microvesicles (e.g., exosomes) may be obtained comprises, 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 formulation provided herein is an NKA to which isolated vesicles (e.g., microvesicles, e.g., exosomes) have been added (the NKA is "spiked" or supplemented with vesicles).

[0122] In certain embodiments, a bioactive selected renal cell population generally refers to a cell population that has potentially therapeutic properties when administered to a subject. In certain embodiments, when administered to a subject in need, the bioactive renal cell population may provide stabilization and / or improvement and / or repair and / or regeneration of renal function in the subject. In certain embodiments, the therapeutic properties may include repair or regenerative effects.

[0123] In certain embodiments, the renal cell population is an unfractionated heterogeneous cell population or an enriched homogeneous cell population derived from a kidney. In certain embodiments, the heterogeneous cell population is isolated from a tissue biopsy or from 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 in which a bioactive component (e.g., bioactive renal cell) is enriched and an inactive or undesirable component or cell is depleted.

[0124] In certain embodiments, the renal cell population expresses GGT and cytokeratin. In certain embodiments, GGT has an expression level of about 10%, about 15%, about 18%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or more than about 60%. In certain embodiments, GGT is GGT-1. In certain embodiments, 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, 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 a combination of adjacent cytokeratins. In certain embodiments, the cytokeratin has an expression level of about 80%, about 85%, about 90%, or more than 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.

[0125] In a specific embodiment, 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 a specific embodiment, the cytokeratin is CK18. In a specific embodiment, 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.

[0126] In certain embodiments, the renal cell population comprises a combination of AQP1, AQP2, AQP4, calbindin, calponin, CD117, CD133, CD146, CD24, CD31 (PECAM-1), CD54 (ICAM-1), CD73, CK18, CK19, CK7, CK8, CK8, CK18, CK19, CK8, CK18, and CK19, connexin 43, curbilin, CXCR4 (fusin), DBA, E-cadherin (CD324), EPO (erythropoietin), GGT1, GLEPP1 (glomerular epithelial protein 1), haptoglobulin, Itgbl (integrin O1), KIM-1 (renal injury molecule-1), T1M-1 (T-cell immunoglobulin and mucin-containing molecule), and MAP-2 It includes cells expressing any combination of biomarkers selected from (microtubule-associated protein 2), megalin, N-cadherin, nephrine, NKCC (Na-K-Cl-cotransporter), OAT-1 (organic anion transporter 1), osteopontin, pan-cadherin, PCLP1 (podocalicin-like molecule 1), dodosin, SMA (smooth muscle alpha-actin), synaptopodin, THP (tam-horsefol protein), vinientin, and αGST-1 (alpha-glutathione S-transferase).

[0127] In certain embodiments, the renal cell population is rich in epithelial cells compared to the population of cells in the starting population, e.g., a renal tissue biopsy or its primary culture (e.g., the renal cell population contains at least about 5%, 10%, 15%, 20%, or 25% more epithelial cells than the starting population). In certain embodiments, the renal cell population is rich in tubular cells compared to the population of cells in the starting population, e.g., a renal tissue biopsy or its primary culture (e.g., the renal cell population contains at least about 5%, 10%, 15%, 20%, or 25% more tubular cells than the starting population). In certain embodiments, the tubular cells include proximal tubular cells. In certain embodiments, the renal cell population has a lower proportion of distal tubular cells, collecting duct cells, endocrine cells, vascular cells, or progenitor cell-like cells compared to the starting population. In certain embodiments, the renal cell population has a lower proportion of distal tubular cells compared to the starting population. In certain embodiments, the renal cell population has a lower proportion of collecting duct cells compared to the starting population. In certain embodiments, the renal cell population has a lower proportion of endocrine cells compared to the starting population. In certain embodiments, the renal cell population has a lower proportion of vascular cells compared to the starting population. In certain embodiments, the renal cell population has a lower proportion of progenitor cell-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 the 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 the 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 the non-enriched population.

[0128] In certain embodiments, cells of a renal cell population express hyaluronic acid (HA). In certain embodiments, the size range of HA is about 5 kDa to about 20,000 kDa. In certain embodiments, 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 high molecular weight HA and / or stimulates its synthesis through the expression of hyaluronic acid synthase-2 (HAS-2), particularly after renal transplantation. In certain embodiments, cells of the renal cell population express a 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 a higher molecular weight species of HA in 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 of HA is HA having a molecular weight of about 800 kDa to about 3500 kDa. In certain embodiments, the higher molecular weight species of HA is HA having a molecular weight of about 800 kDa to about 3000 kDa. In certain embodiments, the higher molecular weight species of HA is HA having a molecular weight of at least 800 kDa. In certain embodiments, the higher molecular weight species of HA is HA having a molecular weight of at least 3000 kDa. In certain embodiments, the higher molecular weight species of HA is HA having a molecular weight of about 800 kDa. In certain embodiments, the higher molecular weight species of HA is HA having a molecular weight of about 3000 kDa. In certain embodiments, HAS-2 is 2x10 5 Up to 2x10 6HA having a molecular weight of Da is synthesized. In certain embodiments, a smaller species of HA is formed through the action of a degradable hyaluronidase. In certain embodiments, a higher molecular weight species of HA is HA having a molecular weight of about 200 kDa to about 2000 kDa. In certain embodiments, a higher molecular weight species of HA is HA having a molecular weight of about 200 kDa. In certain embodiments, a higher molecular weight species of HA is HA having a molecular weight of about 2000 kDa. In certain embodiments, a higher molecular weight species of HA is HA having a molecular weight of at least 200 kDa. In certain embodiments, a higher molecular weight species of HA is HA having a molecular weight of at least 2000 kDa. In certain embodiments, a higher molecular weight species of HA is HA having a molecular weight of at least 5000 kDa. In certain embodiments, a higher molecular weight species of HA is HA having a molecular weight of at least 10000 kDa. In certain embodiments, the higher molecular weight species of HA is HA having a molecular weight of at least 15,000 kDa. In certain embodiments, the higher molecular weight species of HA is HA having a molecular weight of about 20,000 kDa.

[0129] In certain embodiments, the population includes cells capable of receptor-mediated albumin transport.

[0130] In certain embodiments, the cells of the renal cell population are hypoxic resistant.

[0131] In certain embodiments, the renal cell population comprises one or more cell types expressing one or more of the following combinations: megalin, curbilin, N-cadherin, E-cadherin, aquaporin-1, and aquaporin-2.

[0132] In certain embodiments, the renal cell population comprises one or more cell types expressing any combination of the following: megalin, curbilin, hyaluronic acid synthase 2 (HAS2), vitamin D3 25-hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin (Ecad), aquaporin-1 (Aqp1), aquaporin-2 (Aqp2), RAB17, member RAS oncogene family (Rab17), GATA binding protein 3 (Gata3), FXYD domain-containing ion transport regulator 4 (Fxyd4), solute carrier family 9 (sodium / hydrogen exchange factor), member 4 (Slc9a4), aldehyde dehydrogenase 3 family, member B1 (Aldh3b1), aldehyde dehydrogenase 1 family, member A3 (Aldh1a3), and calpain-8 (Capn8).

[0133] In certain embodiments, the renal cell population comprises one or more cell types expressing any combination of the following: megalin, curbilin, hyaluronic acid synthase 2 (HAS2), vitamin D3 25-hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin (Ecad), aquaporin-1 (Aqp1), aquaporin-2 (Aqp2), RAB17, member RAS oncogene family (Rab17), GATA binding protein 3 (Gata3), FXYD domain-containing ion transport regulator 4 (Fxyd4), solute carrier family 9 (sodium / hydrogen exchange factor), member 4 (Slc9a4), aldehyde dehydrogenase 3 family, member 81 (Aldh3b1), aldehyde dehydrogenase 1 family, member A3 (Aldh1a3), and calpain-8 (Capn8), and aquaporin-4 (Aqp4).

[0134] In certain embodiments, the renal cell population comprises one or more cell types expressing any combination of the following: 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 A aspartate peptidase (Napsa), solute carrier family 2 (promoted glucose transporter), member 2 (Slc2a2), alanyl (membrane) aminopeptidase (Anpep), transmembrane protein 27 (Tmem27), acyl-CoA synthetase heavy chain family member 2 (Acsm2), glutathione peroxidase 3 (Gpx3), fructose-1,6-biphosphatase 1 (Fbp1), Alanine-glyoxylate aminotransferase 2 (Agxt2), platelet endothelial cell adhesion molecule (Pecam), and dodocin (Podn).

[0135] In certain embodiments, the renal cell population comprises one or more cell types expressing any combination of the following: PECAM, VEGF, KDR, HIF1a, CD31, CD146, dodosin (Podn), and nephrine (Neph), chemokine (CXC motif) receptor 4 (Cxcr4), endothelin receptor type B (Ednrb), collagen, type V, alpha 2 (Col5a2), cadherin 5 (Cdh5), plasminogen activator, tissue (Plat), angiopoietin 2 (Angpt2), kinase insertion domain protein receptor (Kdr), secreted protein, acidic, cysteine-rich (osteonectin) (Sparc), serglycine (Srgn), TIMP metallopeptidase inhibitor 3 (Timp3), Wilms tumor 1 (Wt1), wingless-type MMTV integration site family, member 4 (Wnt4), G-protein signaling regulator 4 (Rgs4), erythropoietin (EPO).

[0136] In certain embodiments, the renal cell population comprises one or more cell types expressing one or more of the following combinations: PECAM, vEGF, KDR, HIF1a, podocin, nephrine, EPO, CK7, CK8 / 18 / 19.

[0137] In certain embodiments, the renal cell population comprises one or more cell types expressing one or more of the following combinations: PECAM, vEGF, KDR, HIF1a, CD31, and CD146.

[0138] In certain embodiments, the renal cell population comprises one or more cell types expressing one or more of the following combinations: podn and nephrin.

[0139] In certain embodiments, the renal cell population comprises one or more cell types expressing one or more of the following combinations: PECAM, vEGF, KDR, HIF1a, and EPO.

[0140] In certain embodiments, the presence (e.g., expression) and / or level / quantity of various biomarkers within a sample or cell population may be analyzed by a number of methodologies, many of which are known in the relevant art and understood by those skilled in the art, including but not limited to any one of a wide variety of assays that can be performed by protein, gene, and / or tissue array analysis, as well as by immunohistochemistry ("IHC"), Western blot analysis, immunoprecipitation, molecular binding assay, ELISA, ELIFA, fluorescence-activated cell sorting ("FACS"), mass array, proteomics, biochemical enzymatic activity assay, in situ hybridization, Southern analysis, Northern analysis, whole genome sequencing, polymerase chain reaction ("PCR") including quantitative real-time polymerase chain reaction ("qRT-PCR") and other amplification type 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 protein, gene, and / or tissue array analysis. Non-limiting examples of protocols for evaluating the status of genes and gene products include Northern blotting, Southern blotting, Western blotting, Immunoblotting, and PCR analysis. In certain embodiments, multiplexed immunoassays available from, for example, Rules Based Medicine or Meso Scale Discovery, may also be used.In certain embodiments, the presence (e.g., expression) and / or level / quantity of various biomarkers within a sample or cell population may be analyzed by a number of methodologies, many of which are known in the relevant art and understood by those skilled in the art, including but not limited to “-e-e-e-genome” platforms such as genome-wide transcriptomics, proteomics, secretomics, lipidomics, phosphatomics, exosomics, etc., where high-throughput methodologies are combined with computational biology and bioinformatics techniques to identify the complete biological signature of genes, miRNAs, proteins, secreted proteins, lipids, microvesicles, etc., that are expressed or / or are not expressed by the cell population under consideration.

[0141] In a specific embodiment, a method for detecting the presence of two or more biomarkers in a population of renal cells comprises the steps of contacting a sample containing the population with an antibody directed for the biomarker under conditions allowing the antibody to bind to its cognate ligand (i.e., the biomarker), and detecting the presence of the bound antibody by detecting, for example, whether a complex is formed between the antibody and the biomarker. In a specific embodiment, the detection of the presence of one or more biomarkers is by immunohistochemistry. In a specific embodiment, a method for detecting the presence of a biomarker in or on a microvesicle (e.g., an exosome) comprises the steps of contacting a sample containing the microvesicle (e.g., a sample suspected of containing or believed to contain a microvesicle) with an antibody directed for the biomarker under conditions allowing the antibody to bind to its cognate ligand (i.e., the biomarker), and detecting the presence of the bound antibody by detecting, for example, whether a complex is formed between the antibody and the biomarker.

[0142] As used herein, the term "detecting" encompasses quantitative and / or qualitative detection.

[0143] In certain embodiments, the biomarker is detected by a monoclonal or polyclonal antibody.

[0144] In certain embodiments, the renal cell population comprises biomarkers 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, curbilin, CXCR4 (fusin), DBA, E-cadherin (CD324), EPO (erythropoietin), GGT1, GLEPP1 (glomerular epithelial protein 1), haptoglobulin, Itgbl (integrin p), KIM-1 (renal injury molecule-1), T1M-1 (T-cell immunoglobulin and mucin-containing molecule), MAP-2 (microtubule-associated protein 2), It is identified by one or more reagents that enable the detection of megalin, N-cadherin, nephrine, NKCC (Na-K-Cl-cotransporter), OAT-1 (organic anion transporter 1), osteopontin, pan-cadherin, PCLP1 (podocalixin-like molecule 1), dodosin, SMA (smooth muscle alpha-actin), synaptopodin, THP (tam-horsefol protein), vimentin and / or αGST-1 (alpha-glutathione 5-transferase).

[0145] In certain embodiments, the source of the cells is the same as the intended target organ or tissue. In certain embodiments, the BRC or SRC may be sourced from the kidney to be used in the formulation to be 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.

[0146] In certain embodiments, the BRC or SRC comprises a heterogeneous mixture or fraction of bioactive renal cells. In certain embodiments, the BRC or SRC may be derived from a renal cell fraction from a healthy individual or is itself. In certain embodiments, a renal cell population or fraction obtained from an unhealthy individual may be deficient in a specific cell type compared to a renal cell population of a healthy individual (e.g., in a kidney or its biopsy). In certain embodiments, a therapeutically active cell population deficient in a cell type compared to a healthy individual, as well as microvesicles (e.g., exosomes) secreted by the population, are provided herein. A method for detecting such cells and microvesicles (e.g., exosomes) is also provided. In certain embodiments, the cell population is isolated from and expanded from an autologous cell population.

[0147] In certain embodiments, SRC is obtained from the isolation and expansion of renal cells from a patient's renal cortical tissue via a kidney biopsy. In certain embodiments, renal cells are isolated from renal tissue by enzymatic digestion, expanded using standard cell culture techniques, and selected from the expanded renal cells by centrifugation across density boundaries, barriers, or interfaces. In certain embodiments, renal cells are isolated from renal tissue by enzymatic digestion, expanded using standard cell culture techniques, and selected from the expanded renal cells by continuous or discontinuous single or multi-stage density gradient centrifugation. In certain embodiments, SRC is primarily composed of renal epithelial cells whose regenerative potential is known. In certain embodiments, other parenchymal (vascular) and stromal cells may be present within the autologous SRC population.

[0148] In certain embodiments, BRC is an isolated population of regenerating renal cells naturally associated with kidney repair and regeneration. In certain embodiments, BRC is obtained from renal cells isolated from kidney tissue by enzymatic digestion and expanded using standard cell culture techniques. In certain embodiments, the cell culture medium may be designed to expand bioactive renal cells with regenerative capacity. 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 the composition of regenerative cells. While not wishing to be confined to theory, this may be attributed to one or more of the following phenomena: 1) selective survival, death, or proliferation of specific cellular components during periods of hypoxic culture; 2) changes in cell particle size and / or size in response to hypoxic culture, and the resulting changes in buoyancy density and subsequent localization during density gradient separation; and 3) alteration of cell gene / protein expression in response to a hypoxic culture period, resulting in isolation and the occurrence of differential characteristics of cells within an expanded population.

[0149] In certain embodiments, a population of bioactive renal cells is obtained from the isolation and expansion of renal cells from renal tissue (e.g., tissue obtained from a biopsy) under culture conditions that enrich cells capable of regenerating kidney.

[0150] In certain embodiments, renal cells from kidney tissue (e.g., tissue obtained from a biopsy) are subcultured 1, 2, 3, 4, 5, or more times to produce expanded bioactive renal cells (e.g., a cell population enriched with cells capable of regeneration). In certain embodiments, renal cells from kidney tissue (e.g., tissue obtained from a biopsy) are subcultured once to produce expanded bioactive renal cells. In certain embodiments, renal cells from kidney tissue (e.g., tissue obtained from a biopsy) are subcultured twice to produce expanded bioactive renal cells. In certain embodiments, renal cells from kidney tissue (e.g., tissue obtained from a biopsy) are subcultured three times to produce expanded bioactive renal cells. In certain embodiments, renal cells from kidney tissue (e.g., tissue obtained from a biopsy) are subcultured four times to produce expanded bioactive renal cells. In certain embodiments, renal cells from kidney tissue (e.g., tissue obtained from a biopsy) are passaged five times to produce expanded bioactive renal cells. In certain embodiments, passaged cells deplete a population of non-biologically active renal cells. In certain embodiments, passaged cells deplete a population of at least one cell type. In certain embodiments, passaged cells deplete a population of cells having a density greater than 1.095 g / ml. In certain embodiments, passaged cells deplete a population of small cells with low particle size. In certain embodiments, passaged cells deplete a population of cells smaller than red blood cells. In certain embodiments, passaged cells deplete a population of cells having a diameter of less than 6 μm. In certain embodiments, passaged cells deplete a population of cells having a diameter of less than 2 μm. In certain embodiments, passaged cells deplete a population of cells having a particle size lower than red blood cells.In certain embodiments, the viability of the cell population increases after one or more passages. In certain embodiments, descriptions of small cells and low particle size are used when analyzing cells by fluorescence-activated cell sorting (FACs), for example, using the XY axes of a scatter plot where the cells are presented.

[0151] In certain embodiments, expanded bioactive renal cells are grown under hypoxic conditions for at least about 6, 9, 10, 12, or 24 hours, but less than 48 hours, or 6 to 9 hours, or 6 to 48 hours, or about 12 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 sequential or discontinuous (single-stage or multi-stage) density gradient separation of expanded renal cells (e.g., after subculture and / or culture under hypoxic conditions). In certain embodiments, the bioactive renal cell population is an SRC population obtained after separation by centrifugation across the density boundary, barrier, or interface of the expanded renal cells (e.g., after subculture and / or culture under hypoxic conditions). In certain embodiments, hypoxic culture conditions are culture conditions in which cells are applied to a reduced available oxygen level in the 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 oxygen levels 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 SRC exhibits a buoyancy density exceeding about 1.0419 g / mL. In certain embodiments, the SRC exhibits a buoyancy density exceeding about 1.04 g / mL. In certain embodiments, SRC exhibits a buoyancy density exceeding about 1.045 g / mL. In certain embodiments, BRC or SRC contains one or more cell populations in a larger percentage compared to the starting kidney cell population, and one or more other cell populations are absent or deficient.

[0152] In certain embodiments, expanded bioactive renal cells may be subjected to density gradient separation to obtain SRC. In certain embodiments, BRC is subjected to both hypoxic culture conditions and density gradient separation to obtain SRC. In certain embodiments, continuous or discontinuous single-stage or multi-stage density gradient centrifugation is used to separate the collected renal cell population based on cell buoyancy density. In certain embodiments, expanded bioactive renal cells may be separated by centrifugation across density boundaries, barriers, or interfaces to obtain SRC. In certain embodiments, centrifugation across density boundaries or interfaces is used to separate the collected renal cell population based on cell buoyancy density. In certain embodiments, SRC is produced, in part, by using OPTIPREP (Axis-Shield) medium containing a solution of the nonionic iodide compound iodixanol at 60% (w / v) of water. However, a person skilled in the art will recognize that other media known in the art, density gradients (continuous or discontinuous), density boundaries, barriers, interfaces, or other means, such as cell surface markers, comprising the features necessary to isolate the cell populations described herein, may be used to obtain bioactive neoplasms. In certain embodiments, cell fractions exhibiting a buoyancy density greater than about 1.04 g / mL are collected as separate pellets after centrifugation. In certain embodiments, cells maintaining a buoyancy density of less than 1.04 g / mL are excluded and discarded. In certain embodiments, cell fractions exhibiting a buoyancy density greater than about 1.0419 g / mL are collected as separate pellets after centrifugation. In certain embodiments, cells maintaining a buoyancy density of less than 1.0419 g / mL are excluded and discarded. In certain embodiments, about 1.Cell fractions exhibiting a buoyancy density exceeding 0.45 g / mL are collected as separate pellets after centrifugation. In certain embodiments, cells maintaining a buoyancy density of less than 1.045 g / mL are excluded and discarded.

[0153] In certain embodiments, cell buoyancy density is used to obtain an SRC population and / or to determine whether the renal cell population is a bioactive renal cell population. In certain embodiments, cell buoyancy density is used to isolate bioactive renal cells. In certain embodiments, cell buoyancy density is determined by centrifugation across a density interface (single-step discontinuous density gradient) of single-step Optiprep (7% iodixanol; 60% (w / v) in OptiMEM). Optiprep is a 60% w / v solution of iodixanol in water. When used in an exemplary density interface or single-step discontinuous density gradient, Optiprep is diluted with OptiMEM (cell culture base medium) to form a final solution of 7% iodixanol (in water and OptiMEM). The Optimem formulation is a modification of Eagle Minimal 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), and insulin and transferrin are the only protein supplements. Phenol red is included at a reduced concentration as a pH indicator. In certain embodiments, Optimem may be supplemented with 2-mercaptoethanol before use.

[0154] In certain embodiments, an Optiprep solution is prepared, and the refractive index indicating the desired density is measured before use (RI 1.3456 + / - 0.0004). In certain embodiments, the renal cells are stratified at the top of the solution. In certain embodiments, the density interface or single-step discontinuous density gradient is centrifuged at 800 g for 20 minutes (without interruption) at room temperature in a centrifuge tube (e.g., 50 ml conical tube) or cell processor (e.g., COBE 2991). In certain embodiments, the cell fraction exhibiting a buoyancy density exceeding about 1.04 g / mL is collected as a separate pellet after centrifugation. In certain embodiments, cells maintaining a buoyancy density of less than 1.04 g / mL are excluded and discarded. In certain embodiments, the cell fraction exhibiting a buoyancy density exceeding about 1.0419 g / mL is collected as a separate pellet after centrifugation. In certain embodiments, cells maintaining a buoyancy density of less than 1.0419 g / mL are excluded and discarded. In certain embodiments, cell fractions exhibiting a buoyancy density exceeding about 1.045 g / mL are collected as separate pellets after centrifugation. In certain embodiments, cells maintaining a buoyancy density of less than 1.045 g / mL are excluded and discarded. In certain embodiments, prior to evaluation of cell density or selection based on density, cells are cultured until at least 50% full growth is achieved and incubated overnight (e.g., for at least about 8 or 12 hours) in a hypoxic incubator set to 2% oxygen under a 5% CO2 environment at 37°C.

[0155] In certain embodiments, cells obtained from a kidney sample are proliferated and then processed (e.g., by hypoxic and centrifugal separation) to provide an SRC population. In certain embodiments, the SRC population is produced using the reagents and procedures 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 the expression of one or more of the markers disclosed herein before the cells of the population are administered to a subject.

[0156] In certain embodiments, SRC is produced by a process comprising expanding primary renal cells (e.g., 1, 2, 3, 4, 5, or more subcultures), culturing the expanded renal cells under hypoxic conditions, and then contacting the cells with a nephrotoxicant (e.g., iodixanol, e.g., 7% iodixanol). In certain embodiments, SRC is produced by a process comprising expanding primary renal cells (e.g., 1, 2, 3, 4, 5, or more subcultures), culturing the expanded renal cells under hypoxic conditions, and then selecting cells by a density gradient as disclosed herein. In certain embodiments, SRC is produced by a process comprising expanding primary renal cells (e.g., 1, 2, 3, 4, 5, or more passages), culturing the expanded renal cells under hypoxic conditions, and then enriching tubular cells from the cells, or depleting vascular cells or collecting tubular cells from the expanded cells cultured under hypoxic conditions.

[0157] Non-limiting examples of compositions and methods for producing SRC are disclosed in U.S. Patent Application Publication No. 2017 / 0281684 A1, the entire contents of which are incorporated herein by reference.

[0158] In certain embodiments, the BRC or SRC is derived from a natural autologous or allogeneic kidney sample. In certain embodiments, the BRC or SRC is derived from a non-autologous kidney sample. In certain embodiments, the sample may be obtained by kidney biopsy.

[0159] In certain embodiments, renal cell isolation and expansion provide a mixture of renal cell types including renal epithelial cells and stromal cells. 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 of the tubular epithelial phenotype. In certain embodiments, SRCs are obtained by separation of expanded renal cells by centrifugation across a density boundary, barrier, or interface. In certain embodiments, the primary cell type in the SRC population separated across a density boundary / barrier / interface is of the tubular epithelial phenotype. In certain embodiments, the characteristics of the SRCs obtained from expanded renal cells are evaluated using a multifaceted approach. In certain embodiments, cell morphology, growth kinetics, and cell viability are monitored during the renal cell expansion process. In certain embodiments, SRC buoyancy density and viability are characterized by centrifugation on or through a density gradient medium and trypan blue exclusion. In certain embodiments, the SRC phenotype is characterized by flow cytometry, and the SRC function is demonstrated by the expression of VEGF and KIM-1. In certain embodiments, the cellular function of the SRC pre-preparation can also be evaluated by measuring the activity of two specific enzymes found in the renal proximal tubules; GGT (γ-glutamyl transpeptidase) and LAP (leucine aminopeptidase).

[0160] In certain embodiments, cell subpopulations may be separated via flow cytometry using cell characteristics that contribute to the separation of cell subpopulations through density media (size and particle size) (forward scattering = reflection of size via flow cytometry, and lateral scattering = reflection of particle size). In certain embodiments, the density gradient or separation medium must have low toxicity to specific cells of interest. In certain embodiments, the density medium must have low toxicity to specific cells of interest, but the present disclosure considers the use of a medium that plays a specific role in the selection process of the cells of interest. In certain embodiments, although not wishing to be bound by theory, there is a recognizable loss of cells between the loading and recovery steps, and since this implies that exposure to iodixanol under conditions of a density gradient or density boundary, density, barrier, or density interface leads to the removal of specific cells, the cell population disclosed herein recovered by a medium containing iodixanol appears to be iodixanol-resistant. In certain embodiments, cells appearing 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, a contrast medium containing a weak to moderate nephrotoxicity is used for the isolation and / or selection of cell populations, e.g., SRC populations. In certain embodiments, a "weak" nephrotoxicity is a nephrotoxicity that kills 10% or less of primary neocellular cells, as evaluated by a standard live / death dye exclusion cell viability assay, when cells are incubated for 12 hours in a standard medium formulation supplemented with 7% w / v nephrotoxicity. In certain embodiments, SRC is iodixanol-resistant. In certain embodiments, the density medium must not bind to proteins in human plasma or adversely affect the primary function of the cells of interest.

[0161] In certain embodiments, the cell population is enriched with and / or depleted of one or more kidney cell types using fluorescence-activated cell sorting (FACS). In certain embodiments, the kidney cell types may be enriched with and / or depleted using BD FACSAria™ or an equivalent. In certain embodiments, the kidney cell types may be enriched with and / or depleted using FACSAria III™ or an equivalent.

[0162] In certain embodiments, the cell population is enriched with and / or depleted of one or more kidney cell types using self-cell sorting. In certain embodiments, one or more kidney cell types may be enriched with and / or depleted using a Miltenyi autoMACS® system or an equivalent.

[0163] In certain embodiments, a new cell population was applied to three-dimensional culture. In certain embodiments, the method of culturing the cell population is through continuous perfusion. In certain embodiments, a cell population cultured through three-dimensional culture and continuous perfusion demonstrates greater cellularity and interconnectivity compared to a cell population cultured in static culture. In certain embodiments, a cell population cultured through three-dimensional culture and continuous perfusion demonstrates greater EPO expression, as well as enhanced expression of renal tubule-associated genes such as E-cadherin, compared to a static culture of these cell populations. In certain embodiments, a cell population cultured through continuous perfusion demonstrates higher levels of glucose and glutamine consumption compared to a cell population cultured in static culture.

[0164] In certain embodiments, low or low oxygen conditions may be used in the method for producing the cell population provided herein. In certain embodiments, the method for producing the cell population may be used without a low oxygen conditioning step. In certain embodiments, normal oxygen conditions may be used.

[0165] In certain embodiments, a renal cell population was isolated and / or cultured from renal tissue. Non-limiting examples of methods for isolating and isolating an enriched cell population to be used in formulations for therapeutic uses, including the treatment of renal cell components, e.g., renal disease, anemia, EPO deficiency, tubular transport deficiency, and glomerular filtration deficiency, are disclosed herein. In certain embodiments, the cell population is isolated from freshly digested, i.e., mechanically or enzymatically digested renal tissue, or from a heterogeneous in vitro culture of mammalian renal cells.

[0166] 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 EPO expression and bioresponsiveness to oxygen, such that a decrease in the partial pressure of oxygen in the culture system results in the induction of EPO expression. In certain embodiments, the EPO-producing cell population is enriched with EPO-producing cells. In certain embodiments, EPO expression is induced when the cell population is cultured under conditions where the cells are subjected to a reduced level of available oxygen in the culture system, compared to a cell population cultured at a normal atmospheric level of available oxygen (about 21%). 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, culture of cells at a reduced level of available oxygen (also referred to as hypoxic culture conditions) means that the reduced oxygen level is lower compared to the culture of cells at a normal atmospheric level of available oxygen (also referred to as normal or normal oxygen culture conditions). In certain embodiments, hypoxic cell culture conditions include culturing cells at about 1% oxygen, about 2% oxygen, about 3% oxygen, about 4% oxygen, or about 5% oxygen. In certain embodiments, 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.

[0167] In certain embodiments, the induction or increased expression of EPO can be obtained and observed by culturing cells at less than about 5% available oxygen and comparing the EPO expression level with that of cells cultured at atmospheric oxygen (about 21%). In certain embodiments, the induction of EPO is obtained in a culture of cells capable of expressing EPO by a method comprising a first culture vessel in which the cells are cultured at atmospheric oxygen (about 21%) for a certain period and a second culture vessel in which the available oxygen level is reduced and the same cells are cultured at less than about 5% available oxygen. In certain embodiments, EPO expression responsive to hypoxic conditions is regulated by HIF1α. In certain embodiments, other oxygen manipulation culture conditions known in the art may be used for the cells described herein.

[0168] In certain embodiments, the formulation contains an enriched population of EPO-producing mammalian cells characterized by bio-responsiveness to perfusion conditions (e.g., EPO expression). 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 stirred intermittently or continuously in such a manner that dynamic forces are transmitted to the cells through the flow. In certain embodiments, cells subjected to transient, intermittent, or continuous fluid flow are cultured in such a manner that they exist as such a three-dimensional structure within or on a material that provides a framework and / or space for the formation of a three-dimensional structure. In certain embodiments, cells are cultured on porous beads and subjected to intermittent or continuous fluid flow by a oscillating platform, an orbital platform, or a spinner flask. In certain embodiments, cells are cultured on a three-dimensional scaffold and placed within a device in which the scaffold is fixed and fluid flows directionally through or across the scaffold. A person skilled in the art will recognize that other perfusion culture conditions known in the art may be used for the cells described herein.

[0169] 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 renal cell population is an SRC population. In certain embodiments, the cell population is an unfractionated cell population, also referred herein to as a non-enriched cell population.

[0170] Compositions containing various active agents (e.g., other than renal cells or microvesicles) are included herein. In certain embodiments, the microvesicles (e.g., exosomes) provided herein comprise a compound present in the culture medium of a population of renal cells secreting microvesicles (e.g., exosomes). In certain embodiments, the microvesicles (e.g., exosomes) provided herein comprise a compound present in a population of renal cells secreting microvesicles (e.g., exosomes).

[0171] Non-limiting examples of suitable active agents include, without limitation, cell aggregates, cell-free biocompatible materials, products secreted from bioactive cells, macromolecule and small molecule therapeutic agents, as well as combinations thereof. For example, one type of bioactive cell may be combined with a biocompatible material-based microcarrier or another type of bioactive cell in the presence or absence of a therapeutic molecule. In certain embodiments, non-attached cells may be combined with cell-free particles.

[0172] In certain embodiments, the cells of the renal cell population exist within an ellipsoid. In certain embodiments, the renal cell population is in the form of an ellipsoid. In certain embodiments, an ellipsoid containing bioactive renal cells is administered to a subject. In certain embodiments, the ellipsoid comprises at least one non-renal cell type or cell population. In certain embodiments, the ellipsoid is produced by a method comprising (i) combining the bioactive renal cell population and the 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 the ellipsoid is formed.

[0173] In certain embodiments, the non-renal cell population comprises an endothelial cell population or 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 line. In certain embodiments, the endothelial cell population comprises human umbilical vein endothelial cells (HUVEC). In certain embodiments, the non-renal cell population is a mesenchymal stem cell population. In certain embodiments, the non-renal cell population is a stem cell population of hematopoietic, breast, intestinal, placental, lung, bone marrow, blood, umbilical, endothelium, pulp, fat, nerve, 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 xenogeneic, syngeneic, allogeneic, autologous, or a combination thereof. In certain embodiments, the bioactive renal cell population and the non-renal cell population are cultured in 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 in a ratio of about 1:1. In certain embodiments, the new cell population and the bioactive cell population are suspended in a growth medium.

[0174] Expanded bioactive renal cells may be further subjected to continuous or discontinuous density medium separation to obtain SRC. Specifically, continuous or discontinuous single-stage or multi-stage density gradient centrifugation is used to separate the collected renal cell population based on cell buoyancy density. In certain embodiments, expanded bioactive renal cells may be further subjected to separation by centrifugation across density boundaries, barriers, or interfaces to obtain SRC. Specifically, centrifugation across density boundaries, barriers, or interfaces is used to separate the collected renal cell population based on cell buoyancy density. In certain embodiments, SRC is produced, in part, by using Optiprep (Axis-Shield) medium containing a 60% solution of the nonionic iodide compound iodixanol in water. However, a person skilled in the art will recognize that any density gradient medium without limitation of specific medium, or other means, such as immunological separation using cell surface markers known in the art, which possess the characteristics necessary to isolate the cell population of this disclosure, may be used in accordance with this disclosure. For example, a density gradient or density boundary may be formed using Percoll® [colloidal silica particles with a diameter of 15–30 nm (23% w / w in water) coated with polyvinylpyrrolidone (PVP)] or sucrose. In certain embodiments, cell fractions exhibiting a buoyancy density greater than about 1.04 g / mL are collected as separate pellets after centrifugation. In certain embodiments, cells maintaining a buoyancy density of less than 1.04 g / mL are excluded and discarded. In certain embodiments, cell fractions exhibiting a buoyancy density greater than about 1.0419 g / mL are collected as separate pellets after centrifugation. In certain embodiments, cells maintaining a buoyancy density of less than 1.0419 g / mL are excluded and discarded. In certain embodiments, about 1.Cell fractions exhibiting a buoyancy density exceeding 0.45 g / mL are collected as separate pellets after centrifugation. In certain embodiments, cells maintaining a buoyancy density of less than 1.045 g / mL are excluded and discarded.

[0175] In certain embodiments, the therapeutic compositions and formulations thereof of the present disclosure may contain (i) a population of isolated heterogeneous renal cells enriched with a specific bioactive component or cell type and / or depleted of a specific inactive or undesirable component or cell type, and / or (ii) microvesicles (e.g., exosomes) secreted by such cells for use in the treatment of renal disease, i.e., for providing stabilization and / or improvement and / or regeneration of renal function and / or structure. Non-limiting examples of cells providing such stabilization and / or improvement have been previously described in U.S. 8,318,484 (Presnell et al.) and PCT / US2011 / 036347 (Ilagan et al.) and PCT / US2016 / 044866 (Jain et al.), the full contents of each of which are incorporated herein by reference. In certain embodiments, the composition provided herein may contain isolated renal cell fractions that lack cellular components compared to healthy individuals but still retain therapeutic properties, namely, providing stabilization and / or improvement and / or regeneration of renal function. In certain embodiments, the cell population, cell fraction, and / or secreted products of the cells described herein may be derived from healthy individuals, individuals with renal disease, or subjects as described herein.

[0176] In certain embodiments, the source of the cells is the same as the intended target organ or tissue. For example, BRC and / or SRC may be sourced from a kidney to be used in a formulation to be 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, BRC and / or SRC comprise a heterogeneous mixture or fraction of bioactive renal cells. BRC and / or SRC may be derived from a renal cell fraction from a healthy individual or are themselves. Additionally, the present disclosure provides a renal cell fraction obtained from an unhealthy individual that may lack certain cellular components compared to the corresponding renal cell fraction of a healthy individual but may still possess therapeutic properties. The present disclosure also provides a therapeutically active cell population lacking cellular components compared to a healthy individual, and the cell population may be isolated and expanded from an autogenous source of various disease states in certain embodiments.

[0177] In certain embodiments, SRC is obtained from the isolation and expansion of renal cells from a patient's renal cortical tissue via a kidney biopsy. Renal cells are isolated from kidney tissue by enzymatic digestion, expanded using standard cell culture techniques, and selected by centrifugation of the expanded renal cells across density boundaries, barriers, or interfaces. In this embodiment, the SRC is mainly composed of renal tubular epithelial cells whose regenerative potential is known (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 stromal cells may be present within the autologous SRC population. In certain embodiments, new cells are selected by centrifugation through a continuous or discontinuous single-stage or multi-stage gradient.

[0178] Therapeutic compositions comprising both vesicles (e.g., microvesicles, e.g., exosomes) and selected renal cells are included herein. In certain embodiments, the combination of vesicles and cells provides stabilization and / or improvement and / or recovery and / or regeneration of renal function in a subject. The therapeutic properties may include recovery or regenerative effects.

[0179] In certain embodiments, the cell is an immune-privileged BRC (e.g., SRC) that is genetically modified (e.g., genome modified and / or modified via RNAi).

[0180] In certain embodiments, the vesicle is obtained from a genetically modified (e.g., genome modified and / or modified via RNAi) immune-privileged BRC (e.g., SRC).

[0181] In certain embodiments, the genetically modified BRC is a genomically modified BRC (i.e., a BRC having genetic modification in its genome). In certain embodiments, the genetically modified BRC comprises an exogenous polynucleotide (e.g., a plasmid or a viral vector) expressing 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 short interference or short hairpin RNA molecule. In certain embodiments, the method comprises genetically modifying a genomic immunogenic gene in the BRC.

[0182] In certain embodiments, the gene codes for a protein within a major histocompatibility complex (MHC) class I molecule or an MHC class II molecule. In certain embodiments, the gene is the beta-2 microglobulin (B2M, also known as β2M), human leukocyte antigen (HLA)-A, HLA-B, HLA-C, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DPA1, HLA-DPA2, HLA-DQA1, or HLA-DQB1 gene.

[0183] In certain embodiments, the gene codes for a secondary histocompatibility antigen (MiHA or mHA). In certain embodiments, the gene is the HA-1, HA-2, HA-8, HB-1, HY-A1, HY-A2, HY-B7, HY-B8, HY-B60, or HY-DQ5 gene.

[0184] In certain embodiments, any allelic variant of the HLA gene, B2M, or mHA gene mentioned herein may be modified (e.g., deleted) or targeted by RNA interference.

[0185] In certain embodiments, genetically modifying a gene includes mutating the gene. In certain embodiments, mutating the gene includes deleting the gene or a part thereof.

[0186] In certain embodiments, genetically modifying the cell comprises mutating any combination of two or more of the B2M, HLA-A, HLA-B, HLA-C, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DPA1, HLA-DPA2, HLA-DQA1, and / or HLA-DQB1 genes.

[0187] In certain embodiments, the genetically modified BRC is a genetically modified primary renal cell. In certain embodiments, the genetically modified primary renal cell was subcultured at least about 1, 2, 3, 4, 5 times or more before or after genetic modification. In certain embodiments, the method further comprises obtaining an SRC from the genetically modified BRC. In certain embodiments, the SRC is obtained and then genetically modified.

[0188] In certain embodiments, BRC is SRC. Various non-limiting examples of SRC are disclosed herein.

[0189] In certain embodiments, BRCs are genetically modified while within a BRC population, wherein fewer cells than all cells within the BRC population are genetically modified. In certain embodiments, the method further comprises isolating or enriching genetically modified BRCs from a population of BRCs. In certain embodiments, the method further comprises isolating or enriching genetically modified SRCs from a population of SRCs. In certain embodiments, a population of BRCs (e.g., SRCs) is subjected to genetic modification to produce a BRC population 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 the expression of a protein encoded by a gene. In certain embodiments, the mutation reduces the level of protein on the surface of the modified cell. In certain embodiments, cells expressing the protein are depleted or excluded. In certain embodiments, the mutation reduces the level of MHC Class I molecules and / or MHC Class II molecules on the surface of the cell. In certain embodiments, the cell having the mutation does not have MHC Class I molecules and / or MHC Class II molecules on its surface. In certain embodiments, cells expressing MHC Class I molecules on its surface are depleted or excluded. In certain embodiments, cells expressing MHC Class II molecules on its surface are depleted or excluded. In certain embodiments, a cell sorting method is used to remove cells expressing protein, MHC Class I molecules, and / or MHC Class II molecules from the population.In certain embodiments, the cell sorting method comprises an agent (e.g., an antibody) that binds to a protein, an MHC class I molecule, and / or an MHC class II molecule. In certain embodiments, cell depletion or selection involves bead / antibody coupling to attract the cell to a specific protein on its cell surface. In certain embodiments, the cell sorting method is self-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, and accordingly, FACS and / or MACS technology can distinguish living cells (e.g., based on antibody binding to the surface). In certain embodiments, MACs are used to separate cells expressing MHC molecules (e.g., MHC class I molecules or MHC class II molecules) from cells that do not. In certain embodiments, an integrated or non-integrated vector may be used to express another HLA component polypeptide to further modify or modulate the adaptive or innate immune system to prevent targeting and lysis by natural killer (NK) cells, for example.

[0190] In certain embodiments, genetically modifying a gene (e.g., mutating it) 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, genetically modifying the gene further comprises (i) expressing a guide single-guide RNA (gRNA) in the BRC; or (ii) delivering a guide single-guide RNA (gRNA) across the cell membrane of the BRC. In certain embodiments, the Cas9 protein and gRNA are part of a ribonucleoprotein complex.

[0191] In certain embodiments, genetically modifying a gene reduces the amount of MHC class I on the surface of a cell. In certain embodiments, genetically modifying a gene reduces the amount of MHC class II on the surface of a cell. In certain embodiments, the method comprises genetically modifying two or more genes, wherein at least one of the genes codes for a protein in an MHC class I molecule and at least one of the genes codes for a protein in an MHC class II molecule. In certain embodiments, at least one of the genes is an HLA gene.

[0192] A non-limiting description of genetically modified BRCs, including methods of production, is provided in PCT application number PCT / US18 / 38801 filed on June 21, 2018.

[0193] In certain embodiments, the source of the cells is the same as the intended target organ or tissue from the same or different sources. For example, BRC and / or SRC may be sourced from a kidney to be used in a preparation to be administered to the kidney (along with or separately from the vesicles). In certain embodiments, BRC and / or SRC may be sourced from a kidney to be used to produce vesicles to be 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, BRC and / or SRC comprise a heterogeneous mixture or fraction of genetically modified (e.g., genomically modified or modified via RNAi) immune-privileged bioactive renal cells. BRC and / or SRC may be derived from a renal cell fraction from a healthy individual or are the renal cell fraction itself. Furthermore, the present invention provides a renal cell fraction obtained from an unhealthy individual that may lack certain cellular components compared to the corresponding renal cell fraction of a healthy individual but may still possess therapeutic properties. The present invention also provides a therapeutically active cell population lacking cellular components compared to a healthy individual, wherein, in one embodiment, the cell population may be isolated and expanded from kidneys supplied from various mammals.

[0194] In certain embodiments, SRC is obtained from the isolation and expansion of renal cells from renal cortical tissue of different patients via renal biopsy. In certain embodiments, renal cells are isolated from renal tissue by enzymatic digestion, expanded using standard cell culture techniques, and selected from the expanded renal cells by density gradient centrifugation. In certain embodiments, SRC is primarily composed of renal epithelial cells whose immune privilege and regenerative potential are known. Other parenchymal (vascular) and stromal cells may be sparsely present within the SRC population.

[0195] As described herein, the present invention is based, in part, on the surprising discovery that a specific subfraction of a heterogeneous population of novel cells, enriched with bioactive components and depleted of inactive or undesirable components, provides superior therapeutic and regenerative results compared to the starting population.

[0196] In certain embodiments, renal cell isolation and expansion provide a mixture of renal cell types including renal epithelial cells and stromal cells. In certain embodiments, SRCs are obtained by density gradient separation of 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 demonstrated by the expression of VEGF and KIM-1.

[0197] A person skilled in the art will recognize that other isolation and culture methods known in the art may be used for the cells described herein. A person skilled in the art will also recognize that biologically active cell populations may be derived from sources other than those specifically listed above, including, but not limited to, kidneys, body fluids, and tissues and organs other than fat.

[0198] SRC phenotype

[0199] In certain embodiments, microvesicles (e.g., exosomes) secreted by SRC and / or SRC are administered to subjects who have kidney disease or are at risk of kidney disease.

[0200] In certain embodiments, cell phenotypes are monitored by analyzing the expression of novel cell markers using flow cytometry. The analysis of cell phenotypes is based on the use of antigen markers specific to the cell type to be analyzed. Flow cytometry analysis provides a quantitative measurement of cells within a sample population expressing the antigen marker to be analyzed.

[0201] Various markers have been reported in the literature as useful for characterizing the phenotype of renal tubular epithelial cells: (i) cytokeratin; (ii) transport membrane proteins (aquaporins and curbilins); (iii) cell binding molecules (cadherins, differentiation clusters, and lectins); and (iv) metabolic enzymes (glutathione and gamma-glutamyl transpeptidase (GGT)). (Table 1) Since most cells found in cultures derived from whole renal digests are epithelial and endothelial cells, the markers examined focused on the expression of proteins specific to these two groups.

[0202] Table 1. Phenotypic markers for SRC characterization

[0203]

[0204] Table 2 provides the selected markers, ranges, and mean percentage values ​​of phenotypes in the SRC population and the rationale for their selection.

[0205] Table 2. Markers selected for phenotypic analysis of SRC

[0206]

[0207] Cell function

[0208] SRC actively secretes proteins, which can be detected through the analysis of the conditioned medium. Cell function is evaluated by the ability of cells to metabolize PrestoBlue and secrete VEGF (vascular endothelial growth factor) and KIM-1 (kidney injury molecule-1).

[0209] Table 3 shows the amounts of VEGF and KIM-1 present in the conditioning medium from renal cell and SRC cultures. Renal cells were cultured at nearly full growth rates. The conditioning medium from overnight exposure of renal cell cultures was tested for VEGF and KIM-1.

[0210] Table 3. Production of VEGF and KIM-1 by human renal cells and SRC

[0211]

[0212] SRC enzymatic activity

[0213] The cellular function of SRC pre-preparation can also be evaluated by measuring the activity of two specific enzymes found in the proximal tubules of the kidney; GGT (γ-glutamyl transpeptidase) and LAP (leucine aminopeptidase).

[0214] Although microvesicles (e.g., exosomes) and selected novel cell compositions are described herein, the present invention considers compositions containing various other active agents. Other suitable active agents include, but are not limited to, cell aggregates, cell-free biocompatible materials, large and small molecule therapeutic agents, as well as combinations thereof. For example, one type of bioactive cell may be combined with a biocompatible material-based microcarrier or another type of bioactive cell in the presence or absence of a therapeutic molecule, and non-adherent cells may be combined with cell-free particles.

[0215] cell aggregates

[0216] In one aspect, the formulation of the present disclosure contains cell aggregates or ellipsoids and / or microvesicles (e.g., exosomes) secreted by such aggregates or ellipsoids and / or microvesicles (e.g., exosomes) secreted by bioactive cells that are not aggregates or ellipsoids.

[0217] In certain embodiments, the cell aggregate comprises the bioactive cell populations described herein. In certain embodiments, the cell aggregate comprises bioactive renal cells, e.g., combinations of renal cell mixtures, enriched renal cell populations, and renal cell fractions, and mixtures of renal cells and mesenchymal stem cells, endothelial progenitor cells, cells derived from the stromal vascular fraction of adipose tissue, or, without limitation, any other non-renal cell populations.

[0218] In certain embodiments, the bioactive renal cells of the present disclosure may be cultured in a 3D format as further described herein. In certain embodiments, the term “organoid” refers to an accumulation of cells having a phenotype and / or function that reproduces a side of a natural kidney. In certain embodiments, the organoid comprises a mixed population of cells of various lineages typically found in vivo in a given tissue. In certain embodiments, the organoid of the present disclosure is formed in vitro by any means, thereby causing the cells of the present disclosure to form aggregates, which in turn may form ellipsoids, organoids, or a combination thereof. In certain embodiments, such aggregates, ellipsoids, or organoids assume a structure consistent with a specific organ. In certain embodiments, such aggregates, ellipsoids, or organoids express surface markers typically expressed by cells of a specific organ. In certain embodiments, such aggregates, ellipsoids, or organoids produce compounds or substances typically expressed by cells of a specific organ. In certain embodiments, the cells of the present disclosure may be cultured on a natural substrate, for example, gelatin. In certain embodiments, the cells of the present disclosure may be cultured on a synthetic substrate, for example, PLGA.

[0219] biocompatible materials

[0220] Various biocompatible materials may be combined with activators to provide the therapeutic formulations of the present disclosure. In certain embodiments, the biocompatible material may be any suitable shape (e.g., beads) or form (e.g., liquid, gel, etc.). Biocompatible materials suitable for forming a polymer matrix are described in U.S. Publication 20070276507 (Bertram et al.) (the full text of which is incorporated herein by reference). In certain embodiments, the polymer matrix or scaffold may be shaped into any number of preferred configurations to satisfy any number of overall systems, geometry, or spatial constraints. In certain embodiments, the biocompatible material is in the form of a liquid suspension. In certain embodiments, the matrix or scaffold of the present disclosure may be three-dimensional and may be shaped to correspond to the dimensions and shape of an organ or tissue structure. For example, in the use of polymer scaffolds to treat kidney disease, tubular transport deficiency, or glomerular filtration deficiency, a three-dimensional (3-D) matrix can be used to reproduce aspects or the whole of the natural kidney tissue structure and organization, as well as aspects or the whole of the kidney parenchyma.

[0221] Various differently shaped 3-D scaffolds may be used. Naturally, the polymer matrix may be shaped into different sizes and shapes to accommodate patients of different sizes. The polymer matrix may also be shaped in other ways to accommodate the patient's specific needs. In certain embodiments, the polymer matrix or scaffold may be a biocompatible material (e.g., a porous polymer scaffold). The scaffold is continuous-cell polylactic acid (OPLA®), cellulose ether, cellulose, cellulose ester, fluorinated polyethylene, phenolic, poly-4-methylpentene, polyacrylonitrile, polyamide, polyamideimide, polyacrylate, polybenzoxazole, polycarbonate, polycyanoaryl ether, polyester, polyester carbonate, polyether, polyetheretherketone, polyetherimide, polyetherketone, polyethersulfone, polyethylene, polyfluoroolefin, polyimide, polyolefin, polyoxadiazole, polyphenylene oxide, polyphenylene sulfide, polypropylene, polystyrene, polysulfide, polysulfone, polytetrafluoroethylene, polythioether, polytriazole, polyurethane, polyvinyl, polyvinylidene fluoride, regenerated cellulose, silicone, urea-formaldehyde, collagen, It may be formed from various synthetic or naturally occurring materials, including but not limited to gelatin, alginate, laminin, fibronectin, silk, elastin, alginate, hyaluronic acid, agarose, or copolymers or physical blends thereof. The scaffolding configuration may range from a soft porous scaffold to a hard shape-retaining porous scaffold. In certain embodiments, the scaffold is composed of a liquid solution that may be a hydrogel, for example, a hydrogel exceeding its melting temperature.

[0222] In certain embodiments, the scaffold is derived from an existing kidney or other organ of human or animal origin, wherein the natural cell population has been removed through the application of detergents and / or other chemical agents and / or other enzymatic and / or physical methodologies known to those skilled in the art. In these embodiments, the natural three-dimensional structure of the source organ is retained along with all associated extracellular matrix components in their natural, biologically active state. In certain embodiments, the scaffold is an extracellular matrix derived from a human or animal kidney or other organ. In certain embodiments, the composition is assembled into a tissue-like structure through the application of three-dimensional bioprinting methodologies. In certain embodiments, the composition is in the liquid form of a solution that can become a hydrogel.

[0223] In certain embodiments, the biocompatible material is a hydrogel. Hydrogels can be formed from various polymeric materials and are useful for various biomedical applications. Hydrogels can be described as a three-dimensional network of physically hydrophilic polymers. Depending on the type of hydrogel, they contain varying percentages of water but are not entirely soluble in water. Despite their high water content, hydrogels can further bind with large volumes of liquid due to the presence of hydrophilic residues. Hydrogels expand extensively without altering their gelatinous structure. The basic physical characteristics of the hydrogel can be specifically modified depending on the polymer used and the characteristics of the device used to administer the hydrogel.

[0224] Hydrogel materials preferably do not induce an inflammatory response. Examples of other materials that may be used to form a hydrogel include (a) modified alginates, (b) polysaccharides that gel upon exposure to monovalent cations (e.g., gellan gum and carrageenan), (c) polysaccharides that are highly viscous liquids or thixotropic and form a gel over time through slow structural evolution (e.g., hyaluronic acid), (d) gelatin or collagen, and (e) polymeric hydrogel precursors (e.g., polyethylene oxide-polypropylene glycol block copolymers and proteins). U.S. Patent No. 6,224,893 B1 provides a detailed description of various polymers suitable for producing hydrogels according to the present disclosure and the chemical properties of such polymers.

[0225] In certain embodiments, the hydrogel used to formulate the biocompatible material of the present disclosure is a gelatin-based hydrogel. Gelatin is a non-toxic, biodegradable, and water-soluble protein derived from collagen, which is a major component of the extracellular matrix (ECM) of mesenchymal tissues. Collagen is the major structural protein in the extracellular space of various connective tissues within the animal body. As a major component of connective tissue, it is the most abundant protein in mammals, accounting for 25% to 35% of total protein content. Depending on the degree of mineralization, collagen tissue can be hard (bone), soft (tendon), or have a gradient from hard to soft (cartilage). Collagen in the form of elongated fibrils is found mostly in fibrous tissues, such as tendons, ligaments, and skin. It is also abundant in the cornea, cartilage, bone, blood vessels, intestines, intervertebral discs, and the dentin of teeth. In muscle tissue, it serves as a major component of the endomysium. Collagen makes up 1 to 2% of muscle tissue and accounts for 6% of strong dry muscle weight. Collagen occurs in many locations throughout the body. However, more than 90% of collagen in the human body is Type I.

[0226] To date, 28 types of collagen have been identified and described. These can be divided into several groups based on the structures they form: fibrinous (Types I, II, III, V, XI); non-fibrinous; FACIT (fibrin-associated collagen with interrupted triple helix) (Types IX, XII, XIV, XVI, XIX); short-chain (Types VIII, X); basement membrane (Type IV); multiplexin (multiple triple helix domains with interruptions) (Types XV, XVIII); MACIT (membrane-associated collagen with interrupted triple helix) (Types XIII, XVII); and others (Types VI, VII). The five most common types are as follows: Type I: Skin, tendons, vascular ligations, organs, bone (major component of the organic part of bone); Type II: Cartilage (major collagen component of cartilage); Type III: Reticulo (major component of reticular fibers), typically found together with Type I; Type IV: Forms the basement membrane, which is the epithelial-secretory layer of the basement membrane. Type V: Cell surface, hair, and placenta.

[0227] Gelatin possesses informational signals containing arginine-glycine-aspartic acid (RGD) sequences that promote cell adhesion, proliferation, and stem cell differentiation. A characteristic property of gelatin is that it exhibits upper critical solution temperature behavior (UCST). In certain embodiments, when a specific temperature threshold of 40°C is exceeded, gelatin can dissolve in water through the formation of flexible, random single coils. Upon cooling, hydrogen bonding and van der Waals interactions occur, forming triple helices. These collagen-like triple helices act as junction zones and thus trigger the sol-gel transition. Gelatin is widely used in pharmaceutical and medical applications.

[0228] In certain embodiments, the hydrogel used to formulate the injectable cell composition of the present invention is based on porcine gelatin that may be supplied from porcine skin, which is commercially available, for example, from Nitta Gelatin NA Inc. (North Carolina, USA) or Gelita USA Inc. (Iowa, USA). The gelatin may be dissolved, for example, in Dulbecco phosphate-buffered saline (DPBS) to form a heat-responsive hydrogel that may gel and liquefy at different temperatures. In certain embodiments, the hydrogel used to formulate the injectable cell composition of the present invention is based on recombinant human or animal gelatin that has been expressed and purified using methodologies known to those skilled in the art. In certain embodiments, an expression vector containing all or part of cDNA for type I alpha I human collagen is expressed in the yeast Pichia pastoris. Other expression vector systems and organisms will be known to those skilled in the art. In certain embodiments, the gelatin-based hydrogel of the present disclosure is liquid at room temperature (22-28°C) or higher and is a gel when cooled to a refrigeration temperature (2-8°C).

[0229] A person skilled in the art will recognize that other types of synthetic or naturally occurring materials known in the art may be used to form scaffolds as described herein.

[0230] In certain embodiments, the biocompatible material used according to the present disclosure is 5.1 kDA to > 2x10 5It consists of hyaluronic acid (HA) in the form of a hydrogel containing HA molecules in the kDa size range. HA can promote branched morphogenesis and three-dimensional self-organization of associated bioactive cell populations. In certain embodiments, the biocompatible material used according to the present disclosure is 5.1 kDa to >2 x 10 5 It consists of hyaluronic acid in the form of a porous foam that also contains HA molecules in the kDa size range. In certain embodiments, the hydrogel is derived from, or contains, an extracellular matrix supplied from, but not limited to, a kidney or any other tissue or organ. In another embodiment, the biocompatible material used according to the present disclosure consists of a poly-lactic acid (PLA)-based foam having a continuous-bubble structure and a pore size of about 50 micrometers to about 300 micrometers.

[0231] Temperature-sensitive biocompatible materials

[0232] The biocompatible materials described herein may also be designed or adapted to respond to specific external conditions, for example, in vitro or in vivo. In certain embodiments, the biocompatible materials are temperature-sensitive (e.g., in vitro or in vivo). In certain embodiments, the biocompatible materials are adapted to respond to exposure to enzymatic degradation (e.g., in vitro or in vivo). The response of the biocompatible materials to external conditions may be fine-tuned as described herein. The temperature sensitivity of the described formulations may be varied by adjusting the percentage of the biocompatible material in the formulation. For example, the temperature sensitivity of the gelatin in the final formulation (e.g., liquid, gel, beads, etc.) may be modulated by adjusting the percentage of gelatin in the solution. Alternatively, the biocompatible materials may be chemically crosslinked to provide greater resistance to enzymatic degradation. For example, a carbodiimide crosslinking agent may be used to chemically crosslink gelatin beads to thereby provide reduced sensitivity to endogenous enzymes.

[0233] In one aspect, the formulation described herein comprises a biocompatible material having properties that create a favorable environment for an activator to be administered to a subject, such as microvesicles (e.g., exosomes) and / or bioactive renal cells. In certain embodiments, the formulation contains a first biocompatible material that provides a favorable environment from the time the activator is formulated with the biocompatible material until the time of administration to the subject. In certain embodiments, the favorable environment relates to the advantage of having bioactive cells suspended in a substantially solid state compared to cells in a fluid (as described herein) prior to administration to the subject. In certain embodiments, the first biocompatible material is a temperature-sensitive biocompatible material. A temperature-sensitive biocompatible material may have (i) a substantially solid state at about 8°C or lower, and (ii) a substantially liquid state at ambient temperature or higher. In certain embodiments, the ambient temperature is approximately room temperature.

[0234] In certain embodiments, the biocompatible material is a temperature-sensitive biocompatible material capable of maintaining at least two different phases or states depending on temperature. The biocompatible material may 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 states may be substantially solid, substantially liquid, or substantially semi-solid or semi-liquid states. In certain embodiments, the biocompatible material has a first state at a first temperature and a second state at a second temperature, wherein the first temperature is lower than the second temperature.

[0235] In certain embodiments, the state of the temperature-sensitive biocompatible material is substantially solid at a temperature of about 8°C or lower. 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 a gel form. In certain embodiments, the state of the temperature-sensitive biocompatible material is substantially liquid at ambient temperature or higher. 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 a specific embodiment, the ambient temperature is approximately room temperature.

[0236] In certain embodiments, the state of the temperature-sensitive biocompatible material is substantially solid at temperatures below approximately ambient temperature. In certain embodiments, the ambient temperature is approximately room temperature. In certain embodiments, the substantially solid state is maintained at approximately 17°C, approximately 16°C, approximately 15°C, approximately 14°C, approximately 13°C, approximately 12°C, approximately 11°C, approximately 10°C, approximately 9°C, approximately 8°C, approximately 7°C, approximately 6°C, approximately 5°C, approximately 4°C, approximately 3°C, approximately 2°C, or approximately 1°C. In certain embodiments, the substantially solid state has a bead form. In certain embodiments, the state of the temperature-sensitive biocompatible material is substantially liquid at temperatures above approximately 37°C. In certain embodiments, the substantially solid state is maintained at approximately 37°C, approximately 38°C, approximately 39°C, or approximately 40°C.

[0237] Temperature-sensitive biocompatible materials may be provided in solution form, solid form, bead form, or other suitable forms described herein or known to those skilled in the art. Microvesicles (e.g., exosomes) and / or cell populations and formulations described herein may be coated with, deposited upon, embedded therein, attached thereto, seeded therein, suspended therein, or captured therein with the temperature-sensitive biocompatible material. In certain embodiments, the cell populations described herein may be assembled as three-dimensional cell aggregates, organelles, or three-dimensional tubular structures before complexing with the temperature-sensitive biocompatible material, or may be assembled as is at the time of complexing with the temperature-sensitive biocompatible material. In certain embodiments, the temperature-sensitive biocompatible material may be provided without any cells, for example, in the form of spacer beads. In these embodiments, the temperature-sensitive biocompatible material functions in a purely passive role to create space within a target organ for regenerative biological activity, e.g., angiogenesis or the infiltration and migration of host cell populations.

[0238] In certain embodiments, the temperature-sensitive biocompatible material has a transition state between a first state and a second state. In certain embodiments, the transition state is a solid-to-liquid transition state between a temperature of about 8°C and an ambient temperature. In certain embodiments, the ambient temperature is approximately room temperature. In certain embodiments, the solid-to-liquid transition state occurs at one or more of the following temperatures: 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.

[0239] Temperature-sensitive biocompatible materials have a specific viscosity, measured in centipoise (cP), at a given temperature. In certain embodiments, the biocompatible material has a viscosity 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 at 25°C. In certain embodiments, the biocompatible material has a viscosity of about 1.0 cP to about 1.15 cP at 37°C. The viscosity at 37°C may 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 biocompatible material is a gelatin solution. 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% (w / v). In one example, the biocompatible material is a 0.75% (w / v) gelatin solution in PBS. In certain embodiments, the 0.75% (w / v) solution has a viscosity of about 1.6 cP to about 2 cP at 25°C. In certain embodiments, the 0.75% (w / v) solution has a viscosity of about 1.07 cP to about 1.08 cP at 37°C. The gelatin solution may be provided in PBS, DMEM, or another suitable solvent.

[0240] In one aspect, the formulation contains microvesicles (e.g., exosomes) and / or bioactive cells combined with a second biocompatible material that provides a favorable environment for the combined microvesicles (e.g., exosomes) and / or cells from the time of formulation until the time after administration to the subject. In certain embodiments, the favorable environment provided by the second biocompatible material relates to the advantage of administering microvesicles (e.g., exosomes) and / or cells within the biocompatible material that maintain structural integrity until the time of administration to the subject and for a certain period after administration. In certain embodiments, the structural integrity of the second biocompatible material 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. Relatively short-term structural integrity provides a formulation capable of delivering the activator and biocompatible material to a target site within the tissue or organ with controlled handling, placement, or dispersion without interfering with or acting as a barrier to the interaction between the incorporated element and the tissue or organ to which it is placed.

[0241] In certain embodiments, the second biocompatible material is a temperature-sensitive biocompatible material having a sensitivity different from that of the first biocompatible material. The second biocompatible material may have (i) a substantially solid state at or below about ambient temperature, and (ii) a substantially liquid state at or above about 37°C. In certain embodiments, the ambient temperature is approximately room temperature.

[0242] In certain embodiments, the second biocompatible material is a cross-linked bead. The cross-linked bead may have an in vivo residence time that is finely adjustable depending on the degree of crosslinking as described herein. In certain embodiments, the cross-linked bead comprises a microvesicle (e.g., exosome) and / or a bioactive cell and is resistant to enzymatic degradation as described herein. In certain embodiments, the formulation of the present disclosure may comprise a first biocompatible material combined with an activator, e.g., a microvesicle (e.g., exosome) and / or a bioactive cell, in the presence or absence of a second biocompatible material combined with an activator, e.g., a microvesicle (e.g., exosome) and / or a bioactive cell. In certain embodiments, where the formulation comprises the second biocompatible material, it may be a temperature-sensitive bead and / or a cross-linked bead.

[0243] In one aspect, the present disclosure provides a formulation containing a biocompatible material that degrades over a period of approximately minutes, hours, or days. This contrasts with most research that focuses on the implantation of solid materials that degrade slowly over days, weeks, or months. In certain embodiments, the biocompatible material has one or more of the following characteristics: biocompatibility, biodegradability / bioreabsorbability, 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 survival and proliferation. The ability of the biocompatible material to keep the implanted particles separated during implantation promotes natural tissue growth. In certain embodiments, the biocompatible material also facilitates the implantation of the solid formulation. In certain embodiments, because the insertion of the solid unit helps prevent the delivered material from dispersing within the tissue during implantation, the biocompatible material provides localization of the formulation described herein. In the case of cell-based formulations, solid biocompatible materials also improve the stability and viability of fixation-dependent cells compared to cells suspended in fluid. However, the short duration of structural integrity implies that the biocompatible material does not provide a significant barrier to tissue growth or the integration of delivered cells / materials into the host tissue immediately after transplantation.

[0244] In one aspect, the present disclosure provides a formulation containing a biocompatible material that is implanted in a substantially solid form and subsequently liquefies / melts or otherwise loses structural integrity after implantation into the body. This contrasts with significant research focusing on the use of materials that can be injected as liquids and subsequently solidify within the body.

[0245] Biocompatible beads

[0246] In one aspect, the formulation comprises a group of biocompatible beads containing the temperature-sensitive biocompatible material and the biocompatible material described herein. In certain embodiments, the beads are crosslinked. The crosslinking is performed using any suitable crosslinking agent known to a person skilled in the art, e.g., carbodiimide; Aldehydes (e.g., furfural, acrolein, formaldehyde, glutaraldehyde, glycerylaldehyde), succinimide-based crosslinking agents {bis(sulfosuccinimidyl) suberate (BS3), disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS), dithiobis(succinimidyl propionate), ethylene glycol bis(sulfosuccinimidyl succinate), ethylene glycol bis(succinimidyl succinate) (EGS), bis(sulfosuccinimidyl) glutarate (BS2G), disuccinimidyl tartrate (DST); epoxides (ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether); saccharides (glucose and aldose sugars); sulfonic acids and p-toluenesulfonic acids; carbonyldiimidazole; This can be achieved using genipin; imine; ketone; diphenylphosphorylazide (DDPA); terephthaloyl chloride; cerium (III) nitrate hexahydrate; microbial transglutaminase; and hydrogen peroxide. A person skilled in the art will recognize other crosslinking agents and crosslinking methods suitable for use in accordance with the present disclosure.

[0247] In certain embodiments, the beads are carbodiimide-crosslinked beads. In certain embodiments, the carbodiimide-crosslinked beads may be crosslinked with a carbodiimide selected from the group consisting of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), DCC-N,N'-dicyclohexylcarbodiimide (DCC), and N,N'-diisopropylcarbodiimide (DIPC). Beads treated with a lower concentration of EDC are expected to have a greater number of free primary amines, whereas samples treated with a high concentration of crosslinking agent will have most of the primary amines involved in amide bonding. The intensity of the orange color developed by the covalent bonding between the primary amine and picrylsulfonic acid, detectable by a spectrophotometer at 335 nm, is proportional to the number of primary amines present in the sample. When normalized per milligram of protein present in the sample, an inverse correlation may be observed between the number of free amines present and the initial concentration of EDC used for crosslinking. These results indicate differential bead crosslinking, which is governed by the amount of carbodiimide used in the reaction. Generally, crosslinked beads exhibit a reduced number of free primary amines compared to non-crosslinked beads.

[0248] In certain embodiments, the cross-linked beads provide a finely tuned in vivo residence time by having reduced susceptibility to enzymatic degradation compared to non-cross-linked biocompatible beads. In certain embodiments, the cross-linked beads are resistant to endogenous enzymes such as collagenase. In certain embodiments, the provision of the cross-linked beads provides (a) delivery of attached cells to a desired site and creation of space for the regeneration and endogeneity of natural tissue and vascular supplies; (b) the ability to persist at that site long enough to enable the cells to establish, function, remodel, and secrete their own extracellular matrix (ECM); (c) promotion of integration of the transplanted cells with surrounding tissues; (d) the ability to transplant the cells in a substantially solid form; (e) short-term structural integrity that does not provide significant barriers to tissue endogeneity, neovascularization, or integration of the delivered cells / materials with the host tissue; and (f) localized in vivo delivery in a substantially solid form, thereby preventing the dispersion of cells within the tissue during transplantation. (g) improved stability and viability of fixation-dependent cells compared to cells suspended in fluid; and (h) a two-phase release profile when cells are delivered in 1) a substantially solid form (e.g., attached to beads) and 2) a substantially liquid form (e.g., suspended in fluid); i) part of a delivery system that facilitates one or more of the reproduction and mimicry of the three-dimensional biological depression or elongated parenchyma from which the bioactive cell population is derived.

[0249] 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 preparations because they rapidly lose integrity and cells disappear from the injection site. In certain embodiments, highly cross-linked gelatin beads may persist at the injection site for too long and may interfere with neo-ECM secretion, cell integration, angiogenesis, and tissue regeneration. The present disclosure enables fine-tuning of the in vivo residence time of the cross-linked beads. To tune the biodegradability of the biocompatible material, different concentrations of carbodiimide crosslinker are used while keeping the overall reaction conditions constant for all samples. For example, the enzymatic susceptibility of the carbodiimide-cross-linked beads can be fine-tuned by varying the concentration of the crosslinker from about 0 to about 1 M. In certain embodiments, the concentration is 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, The values ​​are approximately 46 mM, approximately 47 mM, approximately 48 mM, approximately 49 mM, approximately 50 mM, approximately 55 mM, approximately 60 mM, approximately 65 mM, approximately 70 mM, approximately 75 mM, approximately 80 mM, approximately 85 mM, approximately 90 mM, approximately 95 mM, or approximately 100 mM. The crosslinker concentrations are also approximately 0.15 M, approximately 0.2 M, approximately 0.25 M, approximately 0.3 M, and approximately 0.It may be 35 M, about 0.4 M, about 0.45 M, about 0.5 M, about 0.55 M, about 0.6 M, about 0.65 M, about 0.7 M, about 0.75 M, about 0.8 M, about 0.85 M, about 0.9 M, about 0.95 M, or about 1 M. In certain embodiments, the crosslinking agent is 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC). In certain embodiments, the EDC-crosslinked beads are gelatin beads. The % degradation of the beads can be finely adjusted depending on the concentration of the crosslinking agent. In certain embodiments, the gelatin beads may be mixed with other beads or microparticles other than gelatin (e.g., alginate or HA, to a non-limiting extent) to further facilitate the effect of the bioactive cell population being delivered.

[0250] Cross-linked beads may possess specific features advantageous for the 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 be substantially hollow. In certain embodiments, the presence of pores provides an increased cell attachment surface that allows a greater number of cells to attach compared to a non-porous or smooth surface. Additionally, the porous structure may support the formation of new tissue by supporting host tissue integration with the porous beads. In certain embodiments, the beads have a size distribution that can be fitted to a Weibull plot corresponding to a typical particle distribution pattern. In certain embodiments, the cross-linked beads have an average diameter of 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 less than about 90 μm. In certain embodiments, the characteristics of the cross-linked beads vary depending on the casting process. In certain embodiments, a process of aerosolizing a liquid gelatin solution using a stream of air and spraying it into liquid nitrogen using a thin-layer chromatography reagent sprayer (ACE Glassware) is used to provide beads having the aforementioned features. A person skilled in the art will recognize that modulating the parameters of the casting process provides an opportunity to adjust different features of the beads, for example, different size distributions. In certain embodiments, the microtopography, surface, and internal features of the beads may be further modified to facilitate cell attachment.

[0251] In certain embodiments, the cytocompatibility of the cross-linked beads is evaluated in vitro using cell culture techniques in which the beads are cultured with cells corresponding to the final bioactive cell preparation prior to formulation. In certain embodiments, the beads are cultured with primary renal cells prior to the preparation of the bioactive renal cell preparation, and a viability / death cell assay is used to confirm cytocompatibility. In addition to cell viability, specific functional tests for measuring cell metabolic activity, the secretion of specific key cytokines, growth factors, and exosomes, and the expression of nucleic acid markers, including specific key proteins and miRNAs associated with functional bioactive renal cell populations, are widely known to those skilled in the art and are additionally used to confirm cellular efficacy upon formulation with the cross-linked beads.

[0252] In certain formulations, biocompatible cross-linked beads are combined with a temperature-sensitive biocompatible material in solution at about 5% (w / w) to about 15% (w / w) of the solution volume. The cross-linked beads may be present at about 5% (w / w), about 5.5% (w / w), about 6% (w / w), about 6.5% (w / w), about 7% (w / w), about 7.5% (w / w), about 8% (w / w), about 8.5% (w / w), about 9% (w / w), about 9.5% (w / w), about 10% (w / w), about 10.5% (w / w), about 11% (w / w), about 11.5% (w / w), about 12% (w / w), about 12.5% ​​(w / w), about 13% (w / w), about 13.5% (w / w), about 14% (w / w), about 14.5% (w / w), or about 15% (w / w) of the solution volume.

[0253] In one aspect, the present disclosure provides a formulation containing a biocompatible material that degrades over a period of approximately minutes, hours, or days. This contrasts with most research that focuses on the implantation of solid materials that degrade slowly over days, weeks, or months.

[0254] In one aspect, the present disclosure provides a formulation having biocompatible cross-linked beads seeded with bioactive cells together with a delivery matrix. In certain embodiments, the delivery matrix has one or more of the following features: biocompatibility, biodegradability / bioreabsorbability, a substantially solid state before and during implantation into a subject, loss of structural integrity (substantially solid state) after implantation, and a cellularly compatible environment that supports cell viability. In certain embodiments, the ability of the delivery matrix to keep the implanted particles (e.g., cross-linked beads) spaced apart during implantation promotes natural tissue incorporation. In certain embodiments, in the absence of a delivery matrix, compression of the cellularized beads during implantation may cause inadequate space for sufficient tissue incorporation. In certain embodiments, the delivery matrix facilitates the implantation of the solid formulation. In certain embodiments, the short duration of structural integrity means that immediately after implantation, the matrix does not provide a significant barrier to tissue incorporation, neovascularization, or integration of the delivered cells / materials into the host tissue. In certain embodiments, the delivery matrix provides localization of the formulation described herein because the insertion of solid units helps prevent the delivered material from dispersing within the tissue during transplantation. In certain embodiments, the application of a delivery matrix as described herein helps prevent rapid loss of transplanted cells through urination upon delivery to the renal parenchyma. In certain embodiments, for cell-based formulations, the solid delivery matrix improves the stability and viability of the fixation-dependent cells compared to cells suspended in fluid.

[0255] In certain embodiments, the delivery matrix is ​​a collection of biocompatible beads that are not seeded into cells. In certain embodiments, the seeded beads are dispersed among the individual cell-seeded beads. In certain embodiments, the seeded beads act as "spacer beads" between the cell-seeded beads before and immediately after transplantation. In certain embodiments, the spacer beads contain a temperature-sensitive biocompatible material having a substantially solid state at a first temperature and a substantially liquid state at a second temperature, wherein the first temperature is lower than the second temperature. For example, the spacer beads contain a biocompatible material having a substantially solid state at or below ambient temperature and a substantially liquid state at or below 37°C, as described herein. In certain embodiments, the ambient temperature is approximately room temperature. In certain embodiments, the biocompatible material 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% (w / v). In certain embodiments, the gelatin solution may be provided in PBS, cell culture medium (e.g., DMEM), or another suitable solvent. In certain embodiments, the biocompatible material is hyaluronic acid. In certain embodiments, the biocompatible material is a decellularized extracellular matrix supplied from a human or animal kidney that may be further reconstituted as a hydrogel.

[0256] In one aspect, the present disclosure provides a formulation containing a biocompatible material that is implanted in a substantially solid form (e.g., a spacer bead) and subsequently liquefies / melts or otherwise loses structural integrity after implantation into the body. This contrasts with significant research focusing on the use of materials that can be injected as liquids and subsequently solidify within the body.

[0257] The temperature sensitivity of the spacer beads can be evaluated in vitro prior to formulation. For example, in certain embodiments, the spacer beads may be labeled and mixed with unlabeled, non-temperature-sensitive beads. The mixture is then incubated at 37°C to observe changes in physical transition. Loss of shape of the labeled temperature-sensitive beads at higher temperatures is observed over time. For example, the temperature-sensitive gelatin beads may be prepared using an Alcian blue dye that acts as a marker for physical transition. The blue gelatin beads are mixed with cross-linked beads (white), loaded into a catheter, extruded, and incubated in 1X PBS, pH 7.4 at 37°C. The loss of shape of the blue gelatin beads is tracked under a microscope at different time points. Changes in the physical state of the blue gelatin beads become visible after 30 minutes and become more pronounced as the incubation time is extended. Due to the viscosity of the material, the beads do not completely disappear.

[0258] Modified-release formulations

[0259] In one aspect, the formulation of the present disclosure is provided as a modified-release formulation. Generally, modified release is characterized by an initial release of a first activator followed by at least one additional subsequent release of a second activator upon administration. The first and second activators may be the same or different. In certain embodiments, the formulation provides modified release through multiple components of the same formulation. In certain embodiments, the modified-release formulation contains an activator as part of the first component, which allows the activator to move freely throughout the volume of the formulation, thereby allowing immediate release at the target site upon administration. The first component may be a temperature-sensitive biocompatible material having substantially a liquid phase and substantially a solid phase, wherein the first component is substantially in the liquid phase upon administration. In certain embodiments, the activator exists substantially in the liquid phase so as to move substantially freely throughout the volume of the formulation and thus be immediately released to the target site upon administration.

[0260] In certain embodiments, the modified-release formulation has an activator as part of a second component, wherein the activator is attached to, deposited upon, coated upon, embedded upon, seeded upon, or captured upon the second component, and this persists before and after administration to a target site. The second component contains a structural element to which the activator can associate, thereby preventing the immediate release of the activator from the second component upon administration. For example, the second component is provided in a substantially solid form, e.g., biocompatible beads, which can be cross-linked to prevent or delay enzymatic degradation in vivo. In certain embodiments, the substantially solid activator maintains its structural integrity within the formulation before and after administration, and thus does not immediately release the activator to a target site upon administration. While carriers suitable for the modified-release formulation are described herein, those skilled in the art will recognize other carriers suitable for use in the present disclosure.

[0261] In certain embodiments, the formulation provides an initial rapid delivery / release of a delivered element, including cells, microvesicles (e.g., exosomes), nanoparticles, therapeutic molecules, etc., followed by a subsequent delayed release of the element. In certain embodiments, the formulation provides an initial rapid delivery / release of microvesicles (e.g., exosomes), miRNAs, and other bioactive nucleic acid or protein molecules that are soluble and secreted bioactive products supplied from kidneys or other cell populations. Other molecules or therapeutic agents associated with regenerative bioactivity will be recognized by those skilled in the art. The formulations of the present disclosure may be designed for such a two-phase release profile in which the agent to be delivered 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 with beads or another suitable carrier). At the initial administration, the unattached agent is delivered immediately to the delivery site, whereas the release of the unattached agent is delayed until the structural integrity of the carrier (e.g., beads) is lost at the point where the previously attached agent is released. As discussed below, other suitable emission mechanisms will be recognized by those skilled in the art in the relevant technical field.

[0262] In certain embodiments, the time delay for release may be adjusted based on the properties of the activator. For example, the time delay for release in microvesicles (e.g., exosomes) and / or bioactive cell formulations may be approximately seconds, minutes, hours, or days. In certain embodiments, a delay of approximately weeks may be appropriate. In certain embodiments, for other activators, e.g., small molecules or large molecules, the time delay for release in the formulation may be approximately seconds, minutes, hours, days, weeks, or months. It is also possible for the formulation to contain different biocompatible materials that provide different time-delayed release profiles. For example, a first biocompatible material having a first activator may have a first release time, and a second biocompatible material having a second activator may have a second release time. The first and second activators may be the same or different.

[0263] In certain embodiments, the delayed release period may generally correspond to the period during the loss of structural integrity of the biocompatible material. However, a person skilled in the art will recognize other mechanisms of delayed release. For example, the activator may be released continuously over time independently of the degradation time of any particular biocompatible material, e.g., the diffusion of the drug from the polymer matrix. Additionally, microvesicles (e.g., exosomes) and / or bioactive cells may migrate from a formulation containing biocompatible material and bioactive cells to natural tissue. In certain embodiments, bioactive cells migrate from the biocompatible material, e.g., beads, to natural tissue. In certain embodiments, bioactive cells migrate from the biocompatible material to natural tissue and induce the secretion of growth factors, cytokines, exosomes, miRNAs, and other nucleic acids and proteins associated with regenerative bioactivity. In certain embodiments, exosomes and other extracellular microvesicles, as well as miRNAs, other bioactive nucleic acids, and proteins, migrate from the biocompatible material. In certain embodiments, the bioactive cell moves away from the biocompatible material to the natural tissue, mediates the mobilization of host stem and progenitor cells, and then moves toward or returns to the site of injury or disease.

[0264] In certain embodiments, biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Sustained absorption of the injectable formulation may be achieved by including an absorption-delaying agent, such as a monostearate salt and gelatin, in the formulation. Many methods for manufacturing such formulations have been patented or are generally known to those skilled in the art. For example, see the document [Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978]. Additional methods applicable to the controlled or extended release of polypeptide agents are described, for example, in U.S. Patent Nos. 6,306,406 and 6,346,274, as well as, for example, in U.S. Patent Application Nos. US20020182254 and US20020051808, all of which are incorporated herein by reference.

[0265] Exemplary bioactive cell preparation

[0266] In certain embodiments, the vesicles provided herein (e.g., microvesicles, e.g., exosomes) are included in the bioactive cell preparation. Alternatively or additionally, the vesicles may be administered before, simultaneously with, or after the bioactive cell preparation.

[0267] In certain embodiments, the formulation described herein contains an implantable construct made of the above-mentioned biocompatible material having the bioactive renal cells described herein for the treatment of renal disease in subjects requiring treatment of renal disease. In certain embodiments, the bioactive cell formulation provided herein further comprises vesicles (e.g., microvesicles such as exosomes secreted by bioactive renal cells).

[0268] In certain embodiments, the construct is made of a biocompatible material or biocompatible material composed of one or more synthetic or naturally occurring biocompatible materials, a scaffold or matrix, and one or more cell populations or microvesicles (e.g., exosomes) described herein that are deposited on or embedded therein on the surface of the scaffold by attachment and / or capture. In certain embodiments, the construct is composed of a biocompatible material and one or more cell populations or products thereof (e.g., microvesicles, e.g., exosomes) described herein that are coated with, deposited upon, deposited within, attached thereto, captured therein, embedded therein, seeded therein, or combined therewith by biocompatible material component(s). Any of the microvesicles (e.g., exosomes) and / or cell populations described herein may be used in combination with the matrix to form the construct.

[0269] In certain embodiments, the bioactive cell preparation is an injectable product consisting of SRCs that have been genetically modified to reduce immunogenicity and formulated in a biocompatible material (e.g., gelatin-based hydrogel). In one aspect, allogeneic SRCs are obtained from the isolation and expansion of renal cells from the renal cortical tissue of a donor patient via kidney biopsy, the genetic modification of SRCs using gene editing technology, and selection by density gradient centrifugation from the expanded renal cells. In certain embodiments, SRCs are composed primarily of renal epithelial cells, which are widely known for their regenerative potential (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 stromal (collecting duct) cells may be sparsely present within the SRC population. Injection of SRCs into the recipient kidney resulted in significant improvements in animal survival, urine concentration, and filtration function in nonclinical studies. However, SRC has a limited shelf life and stability. Among gelatin-based hydrogel biocompatible materials, SRC formulations extend the product shelf life by providing enhanced cellular stability and offer improved stability during transport and delivery to the renal cortex for clinical utility.

[0270] In one aspect, the bioactive cell preparation is first prepared by obtaining renal cortical tissue from a donor using standard clinically managed kidney biopsy procedures. Renal cells are isolated from the kidney tissue by enzymatic digestion and expanded using standard cell culture techniques. The cell culture medium is designed to expand primary renal cells and does not contain any differentiation factors. The collected renal cells are subjected to density gradient separation to obtain SRCs. The use of gene editing technology to modify the immunogenicity of SRCs can be performed before or after density gradient separation.

[0271] In certain embodiments, the formulated cell population and / or microvesicles (e.g., exosomes) move substantially freely throughout the volume of the biocompatible material at temperatures above ambient temperature. Suspending the cell population substantially in a solid phase at lower temperatures provides stability advantages for the cells, e.g., for fixation-dependent cells, compared to cells in a fluid. Additionally, suspending the microvesicles (e.g., exosomes) and / or cells substantially in a solid state provides one or more of the following benefits: i) preventing sedimentation of the microvesicles and / or cells; ii) enabling the cells to be fixed and maintained in the biocompatible material in a suspended state; iii) enabling the microvesicles and / or cells to be maintained more uniformly dispersed throughout the volume of the biocompatible material; iv) preventing the formation of microvesicle and / or cell aggregates; and v) providing superior protection for the microvesicles and / or cells during storage and transport of the formulation. A formulation capable of possessing these characteristics leading to administration to a subject is advantageous because, at least, the overall health of the cells within the formulation will be superior, and a more uniform and consistent dosage of cells will be administered.

[0272] In certain embodiments, the gelatin-based hydrogel biocompatible material used to formulate the SRC is porcine gelatin that dissolves in a buffer to form a heat-reactive hydrogel. In certain embodiments, this hydrogel is fluid at room temperature but gels when cooled to a refrigerated temperature (2–8°C). In certain embodiments, the SRC is formulated with the hydrogel, gels upon cooling, and transported to a clinic under a refrigerated temperature (2–8°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 renal cortex using a needle and syringe suitable for delivery via a percutaneous or laparoscopic procedure.

[0273] Description and composition of an exemplary renal-kidney enhancing composition

[0274] In certain embodiments, the bioactive cell formulation is a renal-renal enhancer (NKA) which is an injectable product composed of autologous, selected renal cells (SRCs) formulated in a biocompatible material (gelatin-based hydrogel). In certain embodiments, the NKA is enhanced or supplemented by vesicles (e.g., microvesicles such as exosomes secreted by bioactive renal cells).

[0275] In one aspect, autologous SRC is obtained from the isolation and expansion of renal cells from a patient's renal cortical tissue via renal biopsy, and selection by centrifugation of the expanded renal cells across density boundaries, barriers, or interfaces. In certain embodiments, autologous SRC is obtained from the isolation and expansion of renal cells from a patient's renal cortical tissue via renal biopsy, and selection of the expanded renal cells across a continuous or discontinuous single-stage or multi-stage density gradient. SRC is primarily composed of renal tubular epithelial cells, which are widely known for their regenerative potential (Humphreys et al. (2008) Intrinsic epithelial cells repair the kidney after injury. Cell Stem Cell. 2(3):284-91). Other parenchymal (vascular) and stromal (collecting duct) cells may be sparsely present within the autologous SRC population.

[0276] In certain embodiments, NKA is supplemented with microvesicles (e.g., exosomes) produced and isolated from an SRC population.

[0277] Injection of SRC into recipient kidneys resulted in significant improvements in animal survival, urine concentration, and filtration function in preclinical studies. However, SRC has a limited shelf life and stability. Among gelatin-based hydrogel biocompatible materials, SRC formulations extend product shelf life by providing enhanced cellular stability and offer improved stability of NKA during transport and delivery to the renal cortex for clinical utility.

[0278] In one aspect, NKA is prepared by first obtaining renal cortical tissue from a donor / recipient using a standard clinically managed kidney biopsy procedure. In certain embodiments, renal cells are isolated from the renal 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 collected renal cells are subjected to separation across a density boundary or interface or density gradient to obtain SRC. In certain embodiments, SRC is genetically modified in accordance with the present disclosure.

[0279] Formulations composed of biocompatible materials designed or adapted to respond to external conditions as described herein are included herein. As a result, the nature of the association between the biocompatible material and the bioactive cell population and other active agents, such as microvesicles (e.g., exosomes), in the construct will vary depending on external conditions. For example, the association between the cell population and the temperature-sensitive biocompatible material depends on temperature. In certain embodiments, the construct contains a bioactive neocellular population and a biocompatible material that is substantially solid at about 8°C or lower and substantially liquid at about ambient temperature or higher, wherein the cell population is suspended in the biocompatible material at about 8°C or lower. However, the cell population moves substantially freely throughout the volume of the biocompatible material at about ambient temperature or higher. Suspending the cell population in a substantially solid phase at lower temperatures provides a stability advantage for the cells, e.g., for fixed-dependent cells, compared to cells in a fluid. Additionally, the suspension of microvesicles (e.g., exosomes) and cells in a substantially solid state provides one or more of the following benefits: i) preventing the sedimentation of microvesicles and cells; ii) enabling cells to be immobilized and maintained in a suspended state within a biocompatible material; iii) enabling microvesicles and cells to be maintained more uniformly dispersed throughout the volume of the biocompatible material; iv) preventing the formation of microvesicle and cell aggregates; and v) providing superior protection of microvesicles and cells during the storage and transport of the formulation. In certain embodiments, a formulation capable of possessing these features leading to administration to a subject is advantageous because, at least, the overall health of the cells within the formulation will be superior, and a more uniform and consistent dose of cells will be administered.

[0280] In certain embodiments, the gelatin-based hydrogel biocompatible material used to formulate SRC into NKA is porcine gelatin that dissolves in a buffer to form a heat-reactive hydrogel. This hydrogel is fluid at room temperature but gels when cooled to a refrigerated temperature (2–8°C). SRC is formulated with the hydrogel to obtain NKA. The NKA gels upon cooling and is transported to a clinic under refrigerated temperatures (2–8°C). The NKA has a shelf life of 3 days. In the clinical setting, the product is warmed to room temperature before being injected into the patient's kidney. The NKA is implanted into the renal cortex using a needle and syringe suitable for the delivery of NKA via percutaneous or laparoscopic procedures. 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 supplied 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 (i.e., recombinant gelatin).

[0281] Cell survival agents

[0282] In one aspect, the bioactive cell preparation also comprises a cell viability agent. In certain embodiments, the vesicle provided herein comprises a cell viability agent. In certain embodiments, the cell viability agent is selected from the group consisting of antioxidants, oxygen carriers, immunomodulators, cell mobilization factors, cell adhesion factors, anti-inflammatory agents, angiogenesis factors, matrix metalloproteases, wound healing factors, and products secreted from bioactive cells.

[0283] In one aspect, microvesicles (e.g., exosomes) contain a cell survival agent (e.g., within their lumen, in their lipid bilayer, or on their surface). In certain embodiments, microvesicles were secreted by cells cultured in the presence of a cell survival agent.

[0284] In certain embodiments, the cell viability agent is selected from the group consisting of antioxidants, oxygen carriers, immunomodulators, cell mobilization factors, cell adhesion factors, anti-inflammatory agents, angiogenesis factors, matrix metalloproteases, wound healing factors, and products secreted from bioactive cells.

[0285] Antioxidants are characterized by the 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, ubiquinones, glutathione, lipoic acid, superoxide dismutase, ascorbic acid, vitamin E, vitamin A, mixed carotenoids (e.g., beta-carotene, alpha-carotene, gamma-carotene, lutein, lycopene, phytophen, phytofluene, and astaxanthin), selenium, coenzyme Q10, indole-3-carbinol, proanthocyanidins, resveratrol, quercetin, catechin, salicylic acid, curcumin, bilirubin, oxalic acid, phytic acid, lipoic acid, vanillic acid, polyphenols, ferulic acid, theaflavin, and one or more derivatives thereof. A person skilled in the art will recognize that other suitable antioxidants may be used in specific embodiments of this disclosure.

[0286] Oxygen carriers are agents characterized by the ability to transport and release oxygen. These include, but are not limited to, perfluorocarbons and pharmaceuticals containing perfluorocarbons. Suitable perfluorocarbon-based oxygen carriers include, but are not limited to, perfluorooctyl bromide (C8F17Br); perfluorodicolotan (C8F16C12); perfluorodecyl bromide; perfluoroobron; perfluorodecalin; perfluorotriphophylamine; perfluoromethylcyclopiperidine; Fluosol® (perfluorodecalin & perfluorotriphophylamine); Perftoran® (perfluorodecalin & perfluoromethylcyclopiperidine); Oxygent® (perfluorodecyl bromide & perfluoroobron); Ocycyte™ (perfluoro(tert-butylcyclohexane)). A person skilled in the art will recognize that other suitable perfluorocarbon oxygen carriers may be used in specific embodiments of this disclosure.

[0287] Immunomodulatory factors include, but are not limited to, osteopontin, FAS ligand factor, interleukin, transforming growth factor beta, platelet-derived growth factor, clusterin, transferrin, normal T-cell expression secreted protein regulated upon action (RANTES), plasminogen activator inhibitor-1 (Pai-1), tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), alpha-1 microglobulin, and beta-2-microglobulin. A person skilled in the art will recognize that other suitable immunomodulatory factors may be used in specific embodiments of this disclosure.

[0288] Anti-inflammatory agents or immunosuppressants (described below) may also be part of the formulation. A person skilled in the art will recognize that other suitable antioxidants may be used in certain embodiments of this disclosure.

[0289] Cell recruitment factors include, but are not limited to, monocyte chemotactic protein 1 (MCP-1) and CXCL-1. A person skilled in the art will recognize that other suitable cell recruitment factors may be used in specific embodiments of this disclosure.

[0290] Cell adhesion factors include, but are not limited to, fibronectin, procollagen, collagen, ICAM-1, connective tissue growth factor, laminin, proteoglycans, and specific cell adhesion peptides such as RGD and YSIGR. A person skilled in the art will recognize that other suitable cell adhesion factors may be used in specific embodiments of this disclosure.

[0291] Angiogenic factors include, but are not limited to, vascular endothelial growth factor F (VEGF) and angiopoietin-2 (ANG-2). A person skilled in the art will recognize that other suitable angiogenic factors may be used in certain embodiments of this disclosure.

[0292] Matrix metalloproteases include, but are not limited to, matrix metalloprotease 1 (MMP1), matrix metalloprotease 2 (MMP2), matrix metalloprotease 9 (MMP-9), and tissue inhibitor and metalloprotease-1 (TIMP-1).

[0293] Wound healing factors include, but are not limited to, keratinocyte growth factor 1 (KGF-1), tissue plasminogen activator (tPA), calbindin, clusterin, cystatin C, and trefoil factor 3. A person skilled in the art will recognize that other suitable wound healing factors may be used in specific embodiments of this disclosure.

[0294] The product secreted from the bioactive cell described herein may also be added to the bioactive cell preparation as a cell viability agent.

[0295] Compositions supplied from body fluids, tissues, or organs from human or animal sources, including human plasma, human platelet lysate, bovine fetal plasma, or bovine pituitary gland extract, may also be added to bioactive cell preparations as cell viability agents.

[0296] A person skilled in the art will recognize that there are various suitable methods for forming structures by depositing cell populations with biocompatible materials or otherwise combining them.

[0297] In a specific embodiment, a BRC (e.g., SRC) cultured in a medium containing a cell viability agent produces a vesicle containing a cell viability agent.

[0298] How to use

[0299] In one aspect, a method for treating renal disease in a subject is provided herein. In certain embodiments, the method comprises administering an effective amount of isolated secreted renal cell vesicles (e.g., microvesicles, e.g., exosomes) to the subject. In certain embodiments, the vesicles comprise a compound that is not produced by the cell generating the vesicles. In certain embodiments, the vesicles are present in the composition or formulation disclosed herein.

[0300] In one aspect, a method for treating renal disease in a subject is provided herein. In a specific embodiment, the method comprises administering an effective amount of vesicles from a vesicle preparation to a subject, wherein the vesicles from the vesicle preparation are identified as regenerative according to the method disclosed herein.

[0301] In one aspect, a method for treating renal disease in a subject is provided herein. In a specific embodiment, the method comprises administering to the subject an effective amount of a composition comprising a population of bioactive renal cells supplemented with renal cell vesicles not secreted by the bioactive renal cell population.

[0302] In certain embodiments, the composition is administered by intravenous injection. In certain embodiments, the composition is administered by tube delivery. In certain embodiments, the vesicle is injected intravenously into a peripheral blood vessel. In certain embodiments, tube delivery is performed into the subject's left renal artery or right renal artery.

[0303] In certain embodiments, the subject has chronic kidney disease. In certain embodiments, the chronic kidney disease is stage I, stage II, stage III, stage IV, or stage V kidney disease.

[0304] In certain embodiments, treating kidney disease includes reducing or preventing renal fibrosis in the subject.

[0305] In certain embodiments, the subject received dialysis at least 1, 2, or 3 times per week for at least 1 or 2 weeks.

[0306] In certain embodiments, the subject has type II diabetes.

[0307] In certain embodiments, the subject has a congenital malformation (CAKUT) of the kidney and urinary tract.

[0308] In a specific embodiment, the object is 90 mL / min / 1.73 m 2 He has a glomerular filtration rate (GFR) of less than 1, microalbuminuria, or macroalbuminuria.

[0309] In certain embodiments, cells are not administered to the subject. In certain embodiments, cells (e.g., BRC, e.g., SRC) are administered to the subject. In certain embodiments, vesicles are administered separately from the cells. In certain embodiments, vesicles are administered before, together with, or after the cells. In certain embodiments, isolated vesicles are administered into 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 were secreted by a population of bioactive renal cells having the same origin as and / or containing the same cell type as the bioactive renal cell population in the composition.

[0310] In a specific embodiment, the vesicle was produced by a BRC group. In a specific embodiment, the BRC group is an SRC group.

[0311] In a specific embodiment, a vesicle secreted by a primary renal cell is administered to a subject. In a specific embodiment, a vesicle secreted by a primary renal cell and a vesicle secreted by an SRC are administered to a subject.

[0312] In certain embodiments, vesicles secreted by endothelial cells or mesenchymal stem cells are also administered to the subject.

[0313] In certain embodiments, non-renal cell vesicles are also administered to the subject. In certain embodiments, the non-renal cell vesicles were secreted by non-renal endothelial progenitor cells, non-renal mesenchymal stem cells, or non-renal adipose-derived progenitor cells.

[0314] In certain embodiments, the effective amount of the vesicle is an effective amount 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 the bioactive renal cells is an effective amount in the absence of administration of the vesicle (e.g., an amount sufficient for treatment without administration of the vesicle). In certain embodiments, the effective amount of the vesicle is a smaller amount than the amount effective without co-administration of bioactive renal cells. In certain embodiments, the effective amount of the bioactive renal cells is a smaller amount than the amount effective without co-administration of the vesicle.

[0315] 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 may be administered for the treatment of kidney disease. In certain embodiments, microvesicles (e.g., exosomes) and / or bioactive cells may be administered to a natural organ as part of the formulations described herein. In certain embodiments, microvesicles (e.g., exosomes) and / or bioactive cells may be supplied from the natural organ to be administered or from a source other than the target natural organ.

[0316] In certain embodiments, the present disclosure provides a method for treating renal disease in a subject requiring treatment of renal disease by means of a formulation containing microvesicles (e.g., exosomes) and / or a population of bioactive renal cells as described herein. In certain embodiments, the formulation is suitable for administration to a subject requiring improved renal function.

[0317] In one aspect, effective treatment of renal disease in a subject by the method of the present disclosure may be observed through various indicators of renal function. In certain embodiments, indicators of renal function include, but are not limited to, serum albumin, albumin to globulin ratio (A / G ratio), serum phosphorus, serum sodium, renal size (measurable by ultrasound), serum calcium, phosphorus:calcium ratio, serum potassium, proteinuria, urinary creatinine, serum creatinine, blood nitrous urea (BUN), cholesterol levels, triglyceride levels, and glomerular filtration rate (GFR). Additionally, various indicators of general health and well-being include, but are not limited to, weight gain or loss, survival, blood pressure (mean body blood pressure, diastolic blood pressure, or systolic blood pressure), and physical endurance performance.

[0318] In one aspect, effective treatment by the formulation is demonstrated by the stabilization of one or more indicators of renal function. In certain embodiments, stabilization of renal function is demonstrated by observing a change in the indicator in a subject treated by the method of the present disclosure compared to the same indicator in a subject not treated by the method of the present disclosure. In certain embodiments, stabilization of renal function may be demonstrated by observing a change in the indicator in a subject treated by the method of the present disclosure compared to the same indicator in the same subject prior to treatment. The change in the first indicator may be an increase or a decrease in value. In certain embodiments, the treatment provided by the present disclosure may include stabilization of serum creatinine and / or blood urea nitrogen (BUN) levels in a subject such that the BUN level observed in the subject is lower than that observed in a subject having a similar disease state not treated by the method of the present disclosure. In certain embodiments, the treatment may include stabilization of serum creatinine levels in a subject such that the serum creatinine level observed in the subject is lower than that observed in a subject having a similar disease state not treated by the method of the present disclosure.

[0319] A person skilled in the art will recognize that effective treatment for kidney disease in a subject can be determined by measuring one or more additional indicators described herein or known in the art.

[0320] In certain embodiments, effective treatment by the formulation is demonstrated by the improvement of one or more indicators of renal structure and / or function. In certain embodiments, microvesicles (e.g., exosomes) and / or bioactive renal cells provide improved levels of serum creatinine and / or blood urea nitrogen (BUN). In certain embodiments, microvesicles (e.g., exosomes) and / or bioactive renal cells provide improved protein retention in serum. In certain embodiments, microvesicles (e.g., exosomes) and enriched bioactive renal cells provide improved levels of serum albumin compared to a non-enriched cell population or cells without added microvesicles (e.g., exosomes). In certain embodiments, microvesicles (e.g., exosomes) provide improved levels of serum albumin compared to bioactive renal cells. In certain embodiments, a population of microvesicles (e.g., exosomes) and / or enriched bioactive renal cells provides an improved A:G ratio compared to a non-enriched cell population. In certain embodiments, a bioactive renal cell population provides improved levels of serum cholesterol and / or triglycerides. In certain embodiments, microvesicles (e.g., exosomes) and / or a bioactive renal cell population provide improved levels of vitamin D. In certain embodiments, microvesicles (e.g., exosomes) and / or an enriched bioactive renal cell population provide an improved phosphorus-to-calcium ratio compared to a non-enriched cell population. In certain embodiments, microvesicles (e.g., exosomes) and / or a bioactive renal cell population provide improved levels of hemoglobin compared to a non-enriched cell population. In certain embodiments, microvesicles (e.g., exosomes) and / or a bioactive renal cell population provide improved levels of serum creatinine compared to a non-enriched cell population. In certain embodiments, microvesicles (e.g., exosomes) and / or an enriched bioactive renal cell population provide improved levels of hematocrit compared to a non-enriched cell population.In certain embodiments, improvement in one or more of the indicators of renal function is the result of treatment with microvesicles (e.g., exosomes) and / or selected renal cell preparations. In one embodiment, improvement in one or more of the indicators of renal function is the result of treatment with microvesicles (e.g., exosomes) and / or selected renal cell preparations.

[0321] In one aspect, the present disclosure provides a formulation for use in a method for regenerating a natural kidney in a subject requiring regeneration of a natural kidney. In certain embodiments, the method comprises the step of administering or implanting the bioactive cell population, cell product, or construct described herein to the subject. The regenerated natural kidney may be characterized by a number of indicators, including, but not limited to, the development of natural kidney function or capacity, improvement of natural kidney function or capacity, and the expression of specific markers in the natural kidney. In certain embodiments, the developed or improved function or capacity may be observed based on various indicators of kidney function described above. In certain embodiments, the regenerated kidney is characterized by differential expression of one or more stem cell markers. The stem cell markers may be one or more of the following: SRY (sex-determining region Y)-box 2 (Sox2); undifferentiated embryonic cell transcription factor (UTF1); nodular homolog from mouse (NODAL); prominin 1 (PROM1) or CD133 (CD133); CD24; and any combination thereof (see PCT / US2011 / 036347 (Ilagan et al.), the full text of which is incorporated herein by reference). Also see the literature [Genheimer et 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], the full text of which is incorporated herein. In certain embodiments, the expression of stem cell marker(s) is upregulated compared to the control group.

[0322] In one aspect, a method for treating kidney disease in a subject is provided herein, comprising injecting the preparation, composition, cell population, or microvesicle (e.g., exosome) disclosed herein into the subject. In certain embodiments, the preparation, composition, cell population, or cell product (e.g., microvesicle, e.g., exosome) is injected through a needle of 18 to 30 gauge. In certain embodiments, the preparation, 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 preparation, 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 preparation, composition, cell population, or cell product (e.g., microvesicle, e.g., exosome) is injected through a needle smaller than 22 gauge. In certain embodiments, a formulation, composition, cell population, or cell product (e.g., microvesicles, e.g., exosomes) is injected through a needle smaller than 23 gauge. In certain embodiments, a formulation, composition, cell population, or cell product (e.g., microvesicles, e.g., exosomes) is injected through a needle smaller than 24 gauge. In certain embodiments, a formulation, composition, cell population, or cell product (e.g., microvesicles, e.g., exosomes) is injected through a needle smaller than 25 gauge. In certain embodiments, a formulation, composition, cell population, or cell product (e.g., microvesicles, e.g., exosomes) is injected through a needle smaller than 26 gauge. In certain embodiments, a formulation, composition, cell population, or cell product (e.g., microvesicles, e.g., exosomes) is injected through a needle smaller than 27 gauge. In certain embodiments, a formulation, composition, cell population, or cell product (e.g., microvesicles, e.g., exosomes) is injected through a needle smaller than 28 gauge.In certain embodiments, a formulation, composition, cell population, or cell product (e.g., microvesicles, e.g., exosomes) is injected through a needle smaller than 29 gauge. In certain embodiments, a formulation, composition, cell population, or cell product (e.g., microvesicles, e.g., exosomes) is injected through a needle of about 20 gauge. In certain embodiments, a formulation, composition, cell population, or cell product (e.g., microvesicles, e.g., exosomes) is injected through a needle of about 21 gauge.

[0323] In certain embodiments, a formulation, composition, cell population, or cell product (e.g., microvesicles, e.g., exosomes) is injected through a needle approximately 22 gauge. In certain embodiments, a formulation, composition, cell population, or cell product (e.g., microvesicles, e.g., exosomes) is injected through a needle approximately 23 gauge. In certain embodiments, a formulation, composition, cell population, or cell product (e.g., microvesicles, e.g., exosomes) is injected through a needle approximately 24 gauge. In certain embodiments, a formulation, composition, cell population, or cell product (e.g., microvesicles, e.g., exosomes) is injected through a needle approximately 25 gauge. In certain embodiments, a formulation, composition, cell population, or cell product (e.g., microvesicles, e.g., exosomes) is injected through a needle approximately 26 gauge. In certain embodiments, a formulation, composition, cell population, or cell product (e.g., microvesicles, e.g., exosomes) is injected through a needle approximately 27 gauge. In certain embodiments, a formulation, composition, cell population, or cell product (e.g., microvesicles, e.g., exosomes) is injected through a needle approximately 28 gauge. In certain embodiments, a formulation, composition, cell population, or cell product (e.g., microvesicles, e.g., exosomes) is injected through a needle approximately 29 gauge.

[0324] In a specific embodiment, the inner diameter of the needle is less than 0.84 mm. In a specific embodiment, the inner diameter of the needle is less than 0.61 mm. In a specific embodiment, the inner diameter of the needle is less than 0.51 mm. In a specific embodiment, the inner diameter of the needle is less than 0.41 mm. In a specific embodiment, the inner diameter of the needle is less than 0.33 mm. In a specific embodiment, the inner diameter of the needle is less than 0.25 mm. In a specific embodiment, the inner diameter of the needle is less than 0.20 mm. In a specific embodiment, the inner diameter of the needle is less than 0.15 mm. In a specific embodiment, the outer diameter of the needle is less than 1.27 mm. In a specific embodiment, the outer diameter of the needle is less than 0.91 mm. In a specific embodiment, the outer diameter of the needle is less than 0.81 mm. In a specific embodiment, the outer diameter of the needle is less than 0.71 mm. In a specific embodiment, the outer diameter of the needle is less than 0.64 mm. In a specific embodiment, the outer diameter of the needle is less than 0.51 mm. In certain embodiments, the outer diameter of the needle is less than 0.41 mm. In certain embodiments, the outer diameter of the needle is less than 0.30 mm. In certain embodiments, the needle has one of the sizes in the table below:

[0325]

[0326] Method and route of administration

[0327] In certain embodiments, vesicles (e.g., microvesicles, e.g., renal exosomes) are administered in the absence of cells (e.g., BRCs, e.g., SRCs). In certain embodiments, vesicles are administered together with cells. In certain embodiments, vesicles are administered with the same composition as cells, as well as individually with cells. In certain embodiments, vesicles and cells are administered by different routes of administration. In certain embodiments, vesicles and cells are administered by one route of administration, and vesicles are also administered individually by another route of administration. In certain embodiments, vesicles are administered more frequently than cells.

[0328] In certain embodiments, the vesicle is administered intravenously. In certain embodiments, the vesicle is administered intravenously into a peripheral blood vessel. In certain embodiments, the vesicle is administered by tube delivery. In certain embodiments, tube delivery is performed into the subject's left renal artery or right renal artery.

[0329] The formulations of the present invention may be administered alone or in combination with other bioactive ingredients. In certain embodiments, the formulations are suitable for regenerating tissue by injecting or implanting the incorporated tissue engineering elements into the interior of a parenchymal organ. In certain embodiments, the formulations are used to regenerate tissue by injecting or implanting the tissue engineering elements into the walls of a hollow organ. In certain embodiments, these formulations are administered in combination with additional formulations suitable for systemic or tube delivery.

[0330] In one aspect, the present invention provides a method for providing the bioactive cell preparation described herein to a subject who requires the bioactive cell preparation described herein. In certain embodiments, vesicles are supplemented to the bioactive cell preparation. In certain embodiments, vesicles are not supplemented to the bioactive cell preparation, but the subject is additionally provided with an individual preparation containing vesicles. In certain embodiments, the source of the bioactive cells and / or vesicles may be homologous or homologous, and any combination thereof. In certain embodiments, the method may include the administration of an immunosuppressant (see, for example, U.S. Patent No. 7,563,822).

[0331] In certain embodiments, the therapeutic method of the present invention involves the delivery of the bioactive cell preparation described herein. In certain embodiments, direct administration of cells and / or vesicles to the intended benefit site is performed. Additionally, a subject requiring treatment may be treated by bringing the natural kidney and the bioactive cell preparation described herein into in vivo contact with a product secreted from one or more enriched renal cell populations, and / or a mixture or construct containing such product. The in vivo contact step provides a regenerative effect to the natural kidney. In certain embodiments, isolated vesicles (e.g., vesicles in the absence of cells) are administered before, together with, or after the bioactive cell preparation (e.g., via the same or different route of administration).

[0332] In light of this specification, various means for administering a composition of selected kidney cells to a subject will be apparent to a person skilled in the art. Such methods include injecting cells into a target site within the subject.

[0333] delivery vehicle

[0334] In certain embodiments, cells and / or secreted products may be inserted into a delivery device or vehicle that facilitates introduction into a subject by injection or implantation. In certain embodiments, the delivery vehicle may contain natural materials. In certain embodiments, the delivery vehicle may contain synthetic materials. In certain embodiments, the delivery vehicle provides a structure that mimics the architecture of an organ or fits it appropriately. In certain embodiments, the delivery vehicle is virtually fluid-like. Such a delivery device may include a tube, e.g., a catheter, for injecting cells and fluid into the body of a recipient subject. In certain embodiments, the tube additionally has a needle, e.g., a syringe, through which the cells of the present invention can be introduced to a desired location within the subject. In certain embodiments, a mammalian kidney-derived cell population is formulated for administration into a blood vessel through a catheter (wherein the term “catheter” is intended to include any of various tube-like systems for the delivery of a substance into a blood vessel). In certain embodiments, cells may be inserted into or onto a biocompatible material or scaffold, including but not limited to textiles, such as woven, knitted, woven, mesh, 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). Cells may be prepared for delivery in various different forms. In certain embodiments, cells may be suspended in a solution or gel. In certain embodiments, cells may be mixed with a pharmaceutically acceptable carrier or diluent that keeps the cells of the present invention viable.

[0335] Pharmaceutically acceptable carriers and diluents include brine, aqueous buffered solutions, solvents, and / or dispersion media. The use of such carriers and diluents is widely known in the relevant art. The solution is preferably sterile and fluid, and often isotonic. Preferably, the solution is stable under manufacturing and storage conditions and is preserved against the action of microorganisms, such as bacteria and fungi, through the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. A person skilled in the art will recognize that the delivery vehicle used for the delivery of the cell population and mixtures thereof of the present invention may include a combination of the aforementioned features.

[0336] In certain embodiments, a subject is administered (i) a preparation containing vesicles, (ii) a preparation containing bioactive renal cells, and / or (iii) a preparation containing both bioactive renal cells and vesicles.

[0337] In certain embodiments, biologically active renal cells (e.g., selected renal cells) are administered to the subject 1 to 3 times. In certain embodiments, biologically active renal cells (e.g., selected renal cells) are administered to the subject 1 time. In certain embodiments, biologically active renal cells (e.g., selected renal cells) are administered to the subject 2 times. In certain embodiments, biologically active renal cells (e.g., selected renal cells) are administered to the subject 3 times.

[0338] In certain embodiments, kidney vesicles (e.g., microvesicles, e.g., exosomes) are administered to the subject 1 to 10 times. In certain embodiments, kidney vesicles (e.g., microvesicles, e.g., exosomes) are administered to the subject 1 time. In certain embodiments, kidney vesicles (e.g., microvesicles, e.g., exosomes) are administered to the subject 2 times. In certain embodiments, kidney vesicles (e.g., microvesicles, e.g., exosomes) are administered to the subject 3 times. In certain embodiments, kidney vesicles (e.g., microvesicles, e.g., exosomes) are administered to the subject 4 times. In certain embodiments, kidney vesicles (e.g., microvesicles, e.g., exosomes) are administered to the subject 5 times. In certain embodiments, kidney vesicles (e.g., microvesicles, e.g., exosomes) are administered to the subject 6 times. In certain embodiments, kidney vesicles (e.g., microvesicles, e.g., exosomes) are administered to the subject 7 times. In a specific embodiment, renal vesicles (e.g., microvesicles, e.g., exosomes) are administered to the subject 8 times. In a specific embodiment, renal vesicles (e.g., microvesicles, e.g., exosomes) are administered to the subject 9 times. In a specific embodiment, renal vesicles (e.g., microvesicles, e.g., exosomes) are administered to the subject 10 times.

[0339] Administration method

[0340] The method of administration of the formulation includes, but is not limited to, systemic, intrarenal (e.g., parenchyma), intravenous or intra-arterial injection, tube delivery, and direct injection into the tissue of the intended active site. In certain embodiments, the method of administration used according to the present invention includes direct laparotomy, direct laparoscopy, and single or multiple injection(s) via the transabdominal or percutaneous. In certain embodiments, the method of administration used according to the present invention includes, for example, retrograde and ureteropelvic infusion. Surgical means of administration include a 1-step procedure, for example, but not limited to, partial nephrectomy and retractor implantation, partial nephrectomy, partial pyelonephrectomy, vascularization using a retina ± peritoneum, multifocal biopsy needle track, conical or pyramidal to cylindrical, and renal pole-like replacement, as well as a 2-step procedure including, for example, an intra-organ bioreactor for re-implantation. In certain embodiments, the formulation containing a mixture of cells and vesicles is delivered simultaneously through the same route. In certain embodiments, the cell composition and the vesicle composition are delivered to a specific location individually or via a specific methodology, either simultaneously or in a transient-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 renal capsule before injecting the composition into the kidney. In certain embodiments, the vesicles are administered by intravenous injection or tube delivery.

[0341] Laparoscopic or percutaneous techniques may be used to access the kidney for injecting formulated BRC or SRC populations (e.g., together with or individually with vesicles). The use of laparoscopic surgical techniques enables direct visualization of the kidney, allowing for the detection and immediate resolution of any bleeding or other adverse events during injection. Percutaneous approaches to the kidney have been used for over a decade, primarily to remove intrarenal masses. These procedures involve inserting an electrode or cryo-needle into a defined mass within the kidney and maintaining contact (typically) for 10 to 20 minutes while the lesion is removed. For the injection of therapeutic agents, percutaneous devices are not larger or more complex, and this approach offers safety benefits and minimal immobilization time without surgery (avoiding abdominal perforation wounds and gas distension). Additionally, the access track can leave hemostatic biodegradable material at the site, further reducing the potential for significant bleeding.

[0342] In a specific embodiment of delivery by injection, a therapeutic bioactive cell preparation (which may or may not be supplemented with vesicles) is injected into the renal cortex. In a specific embodiment, it is important to distribute the therapeutic preparation as widely as possible within the renal cortex, which can be achieved, for example, by entering the renal cortex at an angle that enables the deposition of the therapeutic preparation within the renal cortex to be distributed as widely as feasible. This may require imaging the kidney using ultrasound guidance or a longitudinal or transverse approach via axial computed tomography (CT) imaging, depending on individual patient characteristics. Ideally, the injection will entail multiple depositions as the injection needle / cannula is gradually withdrawn. The entire volume of the therapeutic preparation may be deposited at a single or multiple entry points. In a specific embodiment, up to two entry points may be used to deposit the entire volume of the therapeutic preparation into the kidney. In a specific embodiment, the injection may be administered to a single kidney using one or more entry points, for example, one or two entry points. In certain embodiments, the injection is performed into both kidneys using one or more entry points in each kidney, for example, one or two entry points.

[0343] The description provided above is deemed sufficient to enable a person skilled in the art to practice the present invention. Although the present invention has been specifically disclosed by preferred embodiments and optional features, it should be understood that variations and modifications of the concept disclosed herein may be reclassified by a person skilled in the art and that such variations and modifications 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. In fact, in addition to what is presented and described herein, various variations of the present invention will become apparent from the description above to a person skilled in the art and will fall within the scope of the appended claims.

[0344] All patents, patent applications, and references cited in this specification are incorporated herein by reference in their entirety.

[0345] Examples

[0346] Example 1: Non-limiting example of a method and composition for producing SRC

[0347] Example 1.1 - Preparation of Solution

[0348] This section of the embodiments provides compositions of various culture medium formulations and solutions used in the isolation and characterization of heterogeneous renal cell populations and the preparation of regenerative therapy products in this embodiment.

[0349] Table 6: Culture media and solutions

[0350]

[0351] Dulbecco phosphate buffered saline (DPBS) was used for washing all cells.

[0352] Example 1.2 - Isolation of a heterogeneous, unfractionated renal cell population

[0353] This section of the examples illustrates the isolation of an unfractionated (UNFX) heterogeneous renal cell population from humans. An initial tissue dissociation was performed to produce a heterogeneous cell suspension from human kidney tissue.

[0354] Renal tissue obtained through renal biopsy provided source material for a heterogeneous renal cell population. Renal tissue containing one or more of cortical, corticomedullary junction, or medullary tissue may be used. It is preferable to use corticomedullary junction tissue. Multiple biopsy cores (at least two) avoiding scar tissue were required from CKD kidneys. Renal tissue was obtained from the patient at the clinical site by a clinical investigator 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.

[0355] Next, the tissue was washed with the tissue washing solution of Example 1.1 to reduce bioburden inflow before processing the tissue for cell extraction.

[0356] Kidney tissue was minced, weighed, and dissociated in the digestion solution of Example 1.1. The resulting cell suspension was neutralized in Dulbecco Modified Eagle Medium (D-MEM) + 10% Fetal Bovine Serum (FBS) (Invitrogen, Carlsbad, California), washed, and resuspended in serum-free, supplement-free Keratinocyte Medium (KSFM) (Invitrogen). Subsequently, the cell suspension was centrifuged at the density boundary of 15% (w / v) iodixanol (OptiPrep™, Sigma) to remove red blood cells and debris, and then resuspended in the renal cell growth medium of Example 1.1 cm 2 Culture was initiated on polystyrene flasks or dishes treated with tissue culture medium at a density of 25,000 cells per cell. For example, 25 x 10 cells in 150 ml of 50:50 medium 6 Can be plated on a T500 Nunk flask with individual cells / flasks.

[0357] Example 1.3 - Cell expansion of isolated renal cell population

[0358] Renal cell expansion depends on the amount of donated tissue and the success of renal cell isolation from the source tissue. Isolated cells may be cryopreserved if necessary (see below). Renal cell growth kinetics may vary from sample to sample due to the inherent variability of cells isolated from individual patients.

[0359] A defined cell expansion process has been developed to accommodate the range of cell recovery resulting from the variability of the incoming tissue (Table 7). The expansion of neoplasms involves continuous subculture in closed culture vessels (e.g., T-flasks, Cell Factories, HyperStacks®) in neoplasm growth medium (Table 6) using a defined cell culture procedure.

[0360] To eliminate the inherent risks associated with the use of BPE, a BPE-free medium was developed for human clinical trials. Cell growth, phenotype (CK18), and cellular function (GGT and LAP enzymatic activity) were evaluated in the BPE-free medium and compared to the BPE-containing medium used in animal studies. Renal cell growth, phenotype, and function were equivalent in the two media. (Data not presented)

[0361] Table 7 Cell recovery from human kidney biopsy

[0362]

[0363] Once cell growth was observed in the initial T-flask (subculture 0) and no signs of visual contamination were present, the culture medium was replaced every 2–4 days thereafter (Fig. 2b). New cell morphology was confirmed by evaluating the cells through visual observation of the culture under a microscope. Due to the clustering of cells, the culture exhibited a characteristically dense pavement or cobblestone appearance. These morphological features change during expansion and may not be present in all subcultures. The cell culture overall growth rate was estimated at varying levels of overall growth rate in the culture vessels used throughout the cell expansion.

[0364] Renal cells were subcultured by trypsin treatment when they had grown to at least 50% full coverage in the culture vessel (Fig. 2b). Desorbed cells were collected into a vessel containing renal cell growth medium, counted, and cell viability was calculated. In each cell subculture, 500–4000 cells / cm² were placed in a sufficient number of culture vessels to expand the cell count to the level required for NKA formulation. 2 Seeding was performed (Fig. 2b). The culture vessels were placed in a 37°C incubator under a 5% CO2 environment. As described above, cell morphology and overall growth rate were monitored, and the tissue culture medium was replaced every 2–4 days. Table 8 lists the viability of human renal cells observed during cell isolation and expansion of six kidney biopsies from human donors.

[0365] Table 8 Cell viability of human kidney cells in culture

[0366]

[0367] The inherent variability of tissues from different patients resulted in different cell yields in culture. Therefore, it is not practical to strictly define the timing of cell passages or the number and type of culture vessels required for each passage to achieve a target number of cells. Typically, new cells undergo two or three passages; however, the duration of culture and cell yield may vary depending on the cell growth rate.

[0368] Cells were detached for collection or subculture using 0.25% trypsin containing EDTA (Invitrogen). Viability was evaluated by trypan blue exclusion, and counting was performed manually using a hemocytometer or using an automated cell counting system (Nexcelom Bioscience, Lawrence, Massachusetts).

[0369] Example 1.4 Cryopreservation of cultured cells

[0370] Expanded neoplasms were commercially cryopreserved to accommodate the unique variability of cell growth from individual patients and to deliver the product according to a predetermined clinical schedule. The cryopreserved cells also provide a backup source of cells in cases where another NKA is required (e.g., patient disease, delays due to unexpected process events, etc.). Conditions used to cryopreserve cells and restore them to viable, functional cells upon thawing were established.

[0371] For cryopreservation, place approximately 50 x 10 cells in the cryopreservation solution 6 The cells were suspended to a final concentration of 10 cells / mL (see Example 1.1) and dispensed into vials. Approximately 50 x 10 6 A 1 ml vial containing cells / mL was placed in the freezing chamber 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.

[0372] Example 1.5 Preparation of SRC cell population

[0373] Selected neoplasm cells (SRCs) can be prepared from cryopreserved cells or from end culture vessels grown directly from an extended culture according to a schedule (Fig. 2b).

[0374] When using cryopreserved cells, the cells were thawed and plated on tissue culture vessels for a single final expansion step. When the cells had grown to approximately 50–100% full growth in the final culture vessel, they were prepared for processing for SRC isolation. Medium exchange and a final wash with NKA diluted any residual cryopreservation solution in the final product.

[0375] Once the final cell culture vessel reached at least 50% full growth rate, the vessel was transferred to a hypoxic incubator set to 2% oxygen under a 5% CO2 environment at 37°C (Fig. 2c) and cultured overnight. Cells can be maintained for as long as 48 hours in an oxygen-controlled incubator set to 2% oxygen. Exposure to a more physiologically relevant hypoxic (2%) environment improved cell separation efficiency and enabled the detection of more hypoxia-induced markers such as VEGF.

[0376] After exposing cells to hypoxic conditions for a sufficient period (e.g., overnight to 48 hours), cells were desorbed with 0.25% trypsin containing EDTA (Invitrogen). Viability was assessed by trypan blue exclusion, and counting was performed manually using a hemocytometer or using an automated Cellometer® counting system (Nexelom Biosciences, Lawrence, Massachusetts). Cells were washed once with DPBS, and approximately 850 x 10⁶ cells were placed in DPBS. 6 It was resuspended in cells / mL.

[0377] The collected renal cell population was separated based on cell buoyancy density using centrifugation across density boundaries / interfaces. The renal cell suspension was separated by centrifugation in a 7% iodixanol solution (Optifrep; 60% (w / v) in Optimem; see Example 1.1).

[0378] A 7% Optiprep density interface solution was prepared, and the refractive index indicating the target density was measured prior to use (RI 1.3456 + / - 0.0004). Collected renal cells were layered on top of the solution. The density interface was centrifuged at 800 g for 20 minutes at room temperature (without interruption) in a centrifuge tube or cell processor (e.g., Cobb 2991). Cell fractions exhibiting a buoyancy density greater than approximately 1.045 g / mL were collected as separate pellets after centrifugation. Cells maintaining a buoyancy density less than 1.045 g / mL were excluded and discarded.

[0379] The SRC pellet was resuspended in DPBS (Fig. 2c). Carryover of residual Optiprep, FBS, culture medium, and auxiliary materials in the final product is minimized by four DPBS washes and one gelatin solution step.

[0380] Example 2: Exosome composition for the treatment of kidney disease and function and its use

[0381] Example 2.1 - Technical Field

[0382] The present embodiment relates to a kidney cell exosome composition and a formulation thereof for uses including tissue engineering and regenerative medicine applications for kidney repair and function.

[0383] Example 2.2 - General Description

[0384] Significant research continues to focus on utilizing exosomes in biological fluids for disease biomarkers. The therapeutic potential of exosomes has been addressed more recently, with most of these focused on cancer immunotherapy, vaccine development, treatment of autoimmune diseases, and the delivery of therapeutic agents (compounds, siRNA). Exosomes have garnered attention over the past seven years as potential therapeutic agents for modulating angiogenesis, a key element in tissue regeneration (7, 8).

[0385] Secreted extracellular vesicles (EVs), such as exosomes, are packed with potent repair-promoting proteins and RNA cargoes that are not only cell type-specific but also produced and secreted differentially depending on the cellular environment. An excellent review of these targets, as they are directly related to kidney disease, was published in the literature [Zhang at 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.)].

[0386] Chronic kidney disease (CKD) is a global health issue; the growing gap between the number of patients awaiting transplants and the number of organs actually transplanted highlights the need for new therapies to restore kidney function. Regenerative medicine is a promising approach that has emerged as a treatment for organ-level dysfunction and has been translated into clinical practice. In vitro extended regenerative templates composed of biodegradable materials and autologous cells stimulate natural-like organ tissue regeneration after transplantation.

[0387] Recent studies have demonstrated the emerging role of EVs in mediating cell-to-cell or intercellular communication (9, 10). The unique biological activity of EVs has indicated potential benefits for the correction of cellular dysfunction, and in turn, for the treatment of diseases (11). EVs have also been considered ideal nanovectors for biotransportation, specifically for drug delivery in clinical applications (12). In the kidney, renal EVs are produced and secreted by renal cells and have been implicated in renal function and diseases (10).

[0388] Mechanistically, several studies attributed the protective effect of EV against kidney disease mostly to its RNA content, particularly microRNA (13, 14).

[0389] There is intensive research on the potential of EVs as biomarkers for CKD. In contrast, the therapeutic effects of EVs in CKD are very limited and well-known. Although not bound by any scientific theory, the inventors reasonably explain that the success of their CKD treatment using SRC is, at least in part, due to the hypoxic treatment of SRC, which causes 'tuned' EVs to be secreted from transplanted cells, and in turn 'saves' the affected cells, thereby improving their function.

[0390] In a specific embodiment, hypoxic conditioning of selected neoplasm cells after gradient banding prior to exosome isolation provides an EV having enhanced regenerative properties.

[0391] Example 2.3 - Exosome Isolation - Quantification and Size Determination

[0392] Based on the purification method (15) used, exosomes are described as being in the range of 30–150 nm (16, 17) with a density of approximately 1.10–1.20 g / mL (18, 19), depending on the density gradient material (sucrose or optiprep) used for analysis. Microvesicles are described as being larger than exosomes and are often described as having a diameter of 100–300 nm. The degree of overlap in size for these classes of EVs depends on the publications and techniques used for the measurements. As used herein, the term “microvesicle” refers to cell-derived membranous extracellular vesicles with a diameter of 30 to 1,000 nanometers (nm). As used herein, the term “exosome” refers to cell-derived membranous microvesicles with a diameter of approximately 30–150 nm. Thus, as used herein, the term “microvesicle” encompasses exosomes as well as larger vesicles. Although a position statement (20) was released by leading opinion leaders of the International Society for Extracellular Vesicles (ISEV) in late 2014 and 2015, there is no unified consensus on the best methods for isolating, sizing, and characterizing exosomes. As the field advances, particularly in the area of ​​biological function, techniques for isolating, sizing, and characterizing particles are expected to be driven by methods that provide the desired biological effects. In certain embodiments, EVs containing exosomes are obtained by a process of pelleting cell debris by centrifuging EV-containing culture medium at 3000xg for 20 minutes, and then pelleting the EVs by ultracentrifuging the purified supernatant at 100,000xg. These preparations currently demonstrate biological activity (see proliferation and tubular regeneration assays below).

[0393] Cells were grown in serum-free culture medium for 24 hours. The medium was collected. To isolate exosomes, the collected serum-free conditioned medium was subjected to centrifugation in two stages: 1) 3000 xg for 20 minutes to remove cell debris; and 2) 100,000 xg for 2 hours to pellet the exosomes. The exosomes were resuspended in DPBS and stored at -80°C until use.

[0394] To determine the size distribution and concentration of exosomes, the inventors analyzed samples using variable resistance pulse sensing (TRPS; qNano, Izon Science Ltd) with an NP150 nanopore membrane at a 47 mm stretch. The particle concentration was 1.0 x 10⁶ 13 Standardization was performed using 114 nm carboxylated polystyrene beads at a concentration of 1 particle / mL using multiple pressure correction. Samples were diluted 1:100 in DPBS immediately prior to analysis. Results and yields for four different lots are presented in the table below (Table 9). Although size and concentration variability existed across the lots, the particle sizes for all lots were sufficiently within the operational definition of exosomes (30–150 nm).

[0395] Table 9: Results and Yields of Exosome Isolation, Quantification, and Size Determination

[0396]

[0397] In this embodiment and other embodiments, "BRC-0" is a bioactive renal cell that is a primary cell that has not been passaged. "BRC-1" is a bioactive renal cell that has been passaged once. "BRC-2" is a bioactive renal cell that has been passaged twice. "BRC-3" is a bioactive renal cell that has been passaged three times. "BRC-3A" is a bioactive renal cell that has been passaged three times and then cultured under hypoxic conditions (representing the cell after the hypoxic culture is completed). "SRC" represents a selected renal cell.

[0398] Example 2.4 - Characterization of SRC Exosomal MicroRNA (miRNA)

[0399] Because exosomal cargo contents can include various analytes, including proteins, metabolites, and RNA, the inventors have characterized microRNAs found associated with SRC-derived exosomes. MicroRNAs (miRNAs) are small non-coding RNAs containing approximately 18–23 nucleotides that bind to the 3'-untranslating region of messenger RNA to inhibit translation or promote degradation. miRNA profiles reflect various physiological and pathological conditions. They are expressed in tissue- or cell-specific ways. The expression levels of miRNAs vary depending on various physiological processes, and most human protein-coding genes are thought to be targeted by miRNAs.

[0400] When its function is essentially negative—that is, by inhibiting RNA translation, perhaps through competitive binding or promoting degradation—the miRNA possesses an overarching function of regulating cell growth and proliferation. For example, cell proliferation promotion may involve miRNAs that function to inhibit the translation and, consequently, the expression of growth-inhibiting proteins. Alternatively, inhibiting the translation or expression of growth-inhibiting proteins can inhibit cell proliferation.

[0401] It should be recognized that the functions of these miRNAs were largely determined by studying abnormal cell growth, as observed in cancer cells and other disease states. Therefore, the extrapolation of their functions to normal cells needs to be carefully evaluated. For example, just because miRNAs were found to be upregulated or repressed in cancer cells does not necessarily imply that they function to promote cell transformation; the balance between cell proliferation and growth inhibition has shifted toward cell proliferation in cancer cells. With this in mind, it is not surprising that most of the aforementioned miRNAs were identified in these kidney cells.

[0402] Experiment Overview:

[0403] miRNA was isolated from each sample using the miRNeasy Mini Kit, which can purify total RNA containing more than approximately 18 nucleotides (nt) of RNA. miRNA was quantified using a nanodrop spectrophotometer.

[0404] Sequencing service provided: Small RNA-Seq. Sequencing platform: Illumina NextSeq 500. Sequencing platform reagent: NextSeq MID Output Kit v2. Product used for library preparation: Norgen Biotek Small RNA Library Preparation Kit. Small RNA-Seq data analysis workflow used: exceRpt Small RNA-seq Pipeline (v4.6.2). (Weblink: genboree.org / theCommons / projects / exrnatools-may2014 / wiki / Small_RNA-seq_Pipeline). Sources for small RNA reference sequences: miRNAs miRBase version 21; tRNAs gtRNAdb, piRNAs, RNAdb, Genome GeneCode version 21 (hg38).

[0405] result:

[0406] Computer analysis of the generated sequences shows the following miRNAs differentially expressed in exosomes secreted by SRC:

[0407] miR-145 - Hypothesized to be a tumor suppressor

[0408] mir-22- can function as a tumor suppressor

[0409] miR-7 - A highly conserved miRNA exhibiting restricted spatiotemporal expression during development and maturation. It can also function as a cell growth / tumor suppressor.

[0410] miR-10a regulates the inflammatory phenotype, a marker for kidney injury. It has been experimentally demonstrated that miR-10a downregulates human HOXA1 and HOXA3 genes. The control of Hox genes by miR-10 suggests that these microRNAs may play a significant role in development.

[0411] miR-143 - Tumor suppressor, growth inhibitor

[0412] Considering that the expression levels of let7b—a member of let-7—are significantly low in human cancers and cancer stem cells, the primary function of the let-7 gene may be to promote telomere differentiation and tumor suppression during development.

[0413] Example 2.5 - Exosome MACS-flex Surface Labeling

[0414] Exosome surface marker characterization was performed using a multiplex assay consisting of FACS analysis of 39 surface markers reported to be present on EVs.

[0415] Experiment Overview:

[0416] Exosomes were isolated from cultured cells (BRC-0, BRC-3, BRC-3A, SRC) conditioned medium by ultracentrifugation.

[0417] Particle diameter and concentration were evaluated by variable resistance pulse sensing (TRPS; Q-Nano, Ezone Science Limited) using an NP150 nanopore membrane at a 47 mm stretch. Particle concentration was standardized using multi-pressure correction with 110 nm carboxylated polystyrene beads at a concentration of 1.1 x 10E13 particles / mL.

[0418] 1 x 10⁸ particles were used for each sample.

[0419] Exosomes are immunoisolated using a cocktail of CD63, CD9, and CD81.

[0420] After that, these groups are screened for the expression of 37 different markers.

[0421] Result: See Fig. 4

[0422] In exosomes isolated from both TCHK0012 and TCHK0013, expression of SRC is upregulated compared to BRC.

[0423] CD133 - Although its exact function is still unknown, it has been suggested to act as a component of the cell membrane phase.

[0424] CD326—the epithelial cell adhesion molecule (EpCAM)—is a transmembrane glycoprotein that mediates Ca2+-independent homologous cell-cell adhesion in the epithelium. EpCAM is also involved in cell signaling, migration, proliferation, and differentiation.

[0425] In addition to CD49e attachment, integrins are known to participate in cell-surface mediated signaling.

[0426] Example 2.6 - Exosome-mediated delivery of lipophilic dye to kidney cells

[0427] It is hypothesized that for an exosome to deliver its protein or nucleic acid cargo, the exosome must attach to and fuse with the recipient cell membrane for this purpose. Exosome-mediated delivery of lipophilic dyes to the cell membrane is one way to demonstrate exosome fusion to the recipient cell (21).

[0428] Experiment Overview:

[0429] Dye delivery. To evaluate the ability of exosomes to deliver their cargo, the inventors monitored the ability of exosomes to deliver lipophilic dyes from a culture to kidney cells by flow cytometry.

[0430] Exosomes (BRC-0, BRC-3, SRC) fractions were labeled with Vybrant DiI cell labeling solution at 37°C for 20 minutes.

[0431] After removing excess dye by ultracentrifugation, 5 x 09 labeled exosomes were added to each well of a 6-well dish containing approximately 250,000 cells / well.

[0432] After incubating at 4°C or 37°C for 4 hours, the culture was washed to remove any unmixed, labeled exosomes.

[0433] Next, the recovered cells were retrieved and analyzed by FACs.

[0434] Fluorescently labeled cells showing the transfer of lipophilic dyes from exosomes to the cell membrane cause a shift from left to right on the histogram line.

[0435] Result: See Fig. 5

[0436] As expected, exosome binding was not observed at 4°C, as indicated by the lack of delivery of lipophilic dyes. This supports the concept that cells need to be biologically active for exosomes to be absorbed.

[0437] Also, as expected, in the histogram of the exosome-free control group, there is no shift to the right compared to the 4°C histogram.

[0438] The area between the histograms, as indicated by the arrows, represented the population of cells that absorbed the dye.

[0439] Based on this experiment, it appears that there is a difference in exosome binding between BRC-0 (maximum) and BRC-3 and SRC (approximately equal degree of binding).

[0440] Example 2.7 - Cell proliferation assay

[0441] In the case of exosomes that play a specific role in kidney tissue repair and regeneration, it can be discussed that having the ability to stimulate kidney cell proliferation is a desirable function.

[0442] Experiment Overview:

[0443] Human kidney cells were plated on a 12-well plate at a density of 50,000 cells / well.

[0444] Exosomes were isolated from the presented cell supernatant.

[0445] Exosome volumes of 25, 50, and 100 uL (1X, 2X, 4X) were added to each well.

[0446] The samples were tested in duplicate.

[0447] The plates were enumerated 3 days after processing using ArrayScan.

[0448] SF = Serum-free medium (negative control).

[0449] Growth medium = serum-containing medium (positive control).

[0450] Result: See Fig. 6

[0451] All treatment dilutions of TCHK006 BRC-3 and SRC increased cell proliferation to nearly the same level, exceeding that of the negative control.

[0452] Treatment dilutions of TCHK004 BRC-3 and SRC demonstrated a dose-like effect on proliferation; only 4X volume of SRC increased proliferation beyond that of the negative control.

[0453] Example 2.7 - Tubular Formation / Angiogenesis Test

[0454] The ability to stimulate angiogenesis is also assumed to be a desirable feature for tissue engineering / regenerative medicine products. One of the most widely used in vitro assays to model the reorganization phase of angiogenesis is the tube formation assay. The assay measures the ability of endothelial cells plated at sub-frontal growth densities with an appropriate extracellular matrix support to form capillary-like structures (also known as tubes). Scientists typically use this assay to determine the ability of various compounds to promote or inhibit tube formation. Upon plating, endothelial cells attach to the surrounding extracellular matrix support and generate mechanical forces to create tracks or guide pathways that facilitate cell movement. The resulting cellular cords will eventually form a hollow lumen.

[0455] Compounds capable of inhibiting tube formation may be useful for various diseases, such as cancer, where tumors stimulate the formation of new blood vessels to receive oxygen and nutrients in order to grow beyond a relatively small size. In contrast, compounds or bioagents capable of stimulating tube formation (i.e., exosomes) may be useful for tissue engineering / regenerative medicine applications.

[0456] Experiment Overview:

[0457] 50,000 vascular endothelial cells were plated on an extracellular matrix (GelTrex) in a 48-well plate.

[0458] Cells were incubated in serum-free growth medium supplemented with growth factors as a positive control.

[0459] Cells were incubated in serum-free growth factor medium as a negative control.

[0460] Cells incubated in serum-free and growth factor-free medium supplemented with exosomes (TCHK004, SRC, 10E10 particles) are the test items.

[0461] Result: See Fig. 7

[0462] Tubule formation occurred more rapidly at T=9 hours in the positive control (tubule became visible at T=2 hours), and robust tubule formation was observed in exosome-treated cells.

[0463] Exosomes successfully replaced a prescribed growth factor cocktail containing hEGF, bFGF, IGF-1, and VEGF in supporting tubule formation.

[0464] The 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 10E10 exosomes (test sample). C. Serum-free medium supplemented with growth factors (positive control).

[0465] Example 2.8 - References

[0466]

[0467]

[0468]

[0469] Example 3: Profiling of miRNAs present in secreted exosomes and intracellular vesicles of human SRC, BRC3 / 3A, and BRC0

[0470] Bioinformatics analysis of SRC secreted proteins and exosomal miRNAs identifies specific signaling pathways capable of modulating the progression of CKD upon activation or deactivation by SRC-derived factors. These signaling pathways can be used in vitro as quantitative efficacy assays directly linked to putative SRC MOAs, and / or the identified proteins / miRNAs can be applied as proxies for efficacy and evaluated by ELISA / PCR-based methodologies from conditioned media. This approach leads to the identification of signaling pathways associated with renal disease progression and SRC MOA function via paracrine mechanisms utilizing the activity of secreted biologically active ligands and miRNAs delivered from the donor to the host cell through the activation of exosomes and other secreted microvesicular elements.

[0471] miRNAs were isolated from exosomes purified from secreted media or intracellular pits. Using n=6 independent donors, statistically rigorous comparisons were performed for BRC3 / 3A and BRC0 / SRC. The miRNAs distinguishing these intermediates were identified.

[0472] approach

[0473] 1. Application of Computer / Bioinformatics Approaches to Cell Secretomes

[0474] The inventors characterized secretome and miRNA profiles (intracellular and exosomal) from SRC and SRC production intermediates (n=6). The dataset is being sequentially analyzed by bioinformatics methodologies to computer-identify disease-related and regeneration-associated signaling networks directly affected by secreted proteins or miRNAs generated by SRC.

[0475] To date, these data demonstrate that the BRC3-BRC3A transition (hypoxic phase) induces major changes in the biological signature of the BRC cell population, thereby clearly distinguishing the final product (SRC) from the starting material (BRC0).

[0476] 2. Profiling of miRNAs present in secreted exosomes and intracellular vesicles of human SRC, BRC3 / 3A, and BRC0

[0477] miRNAs were isolated from exosomes purified from secreted media or intracellular pits. Using n=6 independent donors, statistically rigorous comparisons were performed sequentially for BRC3 / 3A and BRC0 / SRC.

[0478] We identified miRNAs that distinguish these manufacturing intermediates. Unlike standard phenotypic descriptions and functional analyses, the approach of characterizing SRC and BRC-0 cell secretomes and miRNAs provides an increased depth of understanding regarding not only how SRC differs from BRC-0 but also how it offers a unique and useful approach for efficacy testing. The data collected by the inventors clearly distinguishes SRC from BRC-0. Although not bound by any specific scientific theory, since differences also exist between BRC-3, BRC-3A, and SRC, many of these differences are most likely due to the hypoxic phase.

[0479] Example 3.1: Experimental Design

[0480]

[0481] n=6, TCHK007-TCHK012

[0482] Exosomes produced by A1 BRC0 cells.

[0483] Exosomes produced by D1 BRC3 cells.

[0484] Exosomes produced by E1 BRC3A cells.

[0485] Exosomes produced by F1 SRC.

[0486] Example 3.2 - Differentially expressed miRNA

[0487] Based on the experimental design, the number of differentially expressed miRNAs for each comparison is presented in the table below. The standard selection criteria for identifying differentially expressed miRNAs are as follows: | Loge-fold change | ≥ 1 and P-value < 0.05.

[0488] Table 10: Number of differentially expressed miRNAs

[0489]

[0490] In the table below, "hsa-" represents human miRNA.

[0491] Table 11: Top 10 differentially expressed miRNAs in E1 vs. D1 (in order of increasing p-value)

[0492]

[0493] Table 12: Top 10 upregulated miRNAs in E1 vs. D1 (in order of decreasing fold change)

[0494]

[0495] Table 13: Top 10 down-regulated miRNAs in E1 vs. D1 (in order of increasing fold change)

[0496]

[0497] Table 14: Top 10 differentially expressed miRNAs in F1 vs. A1 (in order of increasing p-value)

[0498]

[0499] Table 15: Top 10 upregulated miRNAs in F1 vs. A1 (in order of decreasing fold change)

[0500]

[0501] Table 16: Top 10 down-regulated miRNAs in F1 vs. A1 (in order of increasing fold change)

[0502]

[0503] Significantly different miRNA groups between pairs of experimental conditions E1 vs D1 are presented as a volcano plot in Fig. 8. Significantly different miRNA groups between pairs of experimental conditions F1 vs A1 are presented as a volcano plot in Fig. 9.

Claims

Claim 1 A method for determining whether a vesicle secreted by a renal cell is regenerative, comprising: (i) a step of detecting whether at least one protein is upregulated or downregulated in the vesicle compared with a vesicle secreted by an unpassed primary renal cell, wherein the at least one protein is CD9, CD326, CD40, CD42a, CD44, CD49e and / or SSEA-4; and (ii) a step of confirming the vesicle as regenerative when (a) the at least one protein is CD9, CD326, CD40, CD44 and / or CD49e and the at least one protein is upregulated in the vesicle and / or, or (b) the at least one protein is CD42a and / or SSEA-4 and the at least one protein is downregulated in the vesicle. Claim 2 A method according to claim 1, wherein the vesicle is obtained from or within a biological sample from a subject. Claim 3 In paragraph 2, the method in which the biological sample is urine. Claim 4 A method according to claim 1, wherein the vesicle is obtained in or from the supernatant of a new cell culture. Claim 5 A method according to any one of claims 1 to 4, wherein the renal cell is a biologically active renal cell. Claim 6 In paragraph 5, the method wherein the biologically active kidney cell is a selected kidney cell. Claim 7 A method according to any one of claims 1 to 4, wherein at least one of the proteins is CD326 and / or CD49e. Claim 8 In claim 5, the method wherein at least one of the proteins is CD326 and / or CD49e. Claim 9 In claim 6, the method wherein at least one of the proteins is CD326 and / or CD49e. Claim 10 A method according to any one of claims 1 to 4, wherein the vesicle is an exosome or a microvesicle.

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