Treatment of kidney disease in subjects with kidney and / or urinary tract anomalies

Abstract

To provide a method of treating a subject having kidney disease.SOLUTION: A method of treating kidney disease in a subject having chronic kidney disease, the method comprising administering to the subject an effective amount of a composition comprising (i) a bioactive renal cell population, (ii) vesicles secreted by the renal cell population, and / or (iii) spheroids comprising the renal cell population and at least one non-renal cell population, wherein the subject has an anomaly of a kidney and / or urinary tract.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates, inter alia, to methods, compositions, and cell populations for treating subjects with kidney disease. [Background technology]

[0002] Kidney abnormalities, such as congenital anomalies of the kidney and urinary tract (CAKUT) and / or acquired anomalies, can lead to kidney damage, including chronic kidney disease and end-stage renal disease. CAKUT constitute approximately 20% to 30% of all anomalies identified in the prenatal period. Queisser-Luft et al. (2002) Malformations in newborn: results based on 30,940 infants and fetuses from the Mainz congenital birth defect monitoring system (1990-1998). 2002;266(3):163 See, the entire contents of which are incorporated herein by reference. Summary of the Invention

[0003] Provided herein are, inter alia, methods, cell populations, and compositions for treating kidney disease in subjects with congenital anomalies of the kidney and / or urinary tract.

[0004] In one aspect, provided herein is a method of treating kidney disease in a subject suffering from chronic kidney disease (CKD), comprising administering to the subject an effective amount of (i) a bioactive renal cell population, (ii) vesicles secreted by the renal cell population, and / or (iii) spheroids comprising a renal cell population and at least one non-renal cell population, wherein the subject has a kidney and / or urinary tract abnormality.

[0005] In embodiments, the subject has a kidney defect. In embodiments, the subject has a urinary tract defect. In embodiments, the subject has a kidney and urinary tract defect. In embodiments, the defect is acquired prenatally. In embodiments, the defect is acquired postnatally. In embodiments, the defect is a congenital defect. In embodiments, the subject has a congenital kidney defect. In embodiments, the subject has a congenital urinary tract defect. In embodiments, the subject has a congenital kidney and urinary tract defect. As used herein, a "congenital" defect is one that is present at or before birth. In embodiments, the congenital defect worsens or causes additional defects after birth.

[0006] In one aspect, provided herein is a method of treating kidney disease in a subject having a kidney and / or urinary tract abnormality, comprising, consisting essentially of, or consisting of administering to the subject an effective amount of (i) a bioactive renal cell population, (ii) one or more products secreted by the renal cell population, and / or (iii) a spheroid comprising the renal cell population and at least one other cell population.

[0007] In one aspect, provided herein is a method for treating kidney disease in a subject having a kidney and / or urinary tract abnormality, comprising administering to the subject an effective amount of spheroids comprising (i) a bioactive renal cell population, (ii) one or more products (e.g., vesicles) secreted by the renal cell population, and / or (iii) the renal cell population and at least one other cell population, such as a non-renal cell population.

[0008] In one aspect, provided herein is a method of treating kidney disease in a subject having a kidney and / or urinary tract abnormality, comprising administering to the subject an effective amount of (i) a bioactive renal cell population, (ii) vesicles secreted by the renal cell population, or (iii) spheroids comprising the renal cell population and at least one non-renal cell population.

[0009] In one aspect, provided herein is a method of treating kidney disease in a subject having a kidney and / or urinary tract abnormality, the method comprising administering to the subject an effective amount of (i) a bioactive renal cell population, (ii) vesicles secreted by the renal cell population, and (iii) spheroids comprising the renal cell population and at least one non-renal cell population.

[0010] In one aspect, provided herein is a method for treating kidney disease in a subject having a kidney and / or urinary tract abnormality, comprising administering to the subject an effective amount of a composition comprising a bioactive renal cell population. In embodiments, the composition further comprises vesicles secreted by the renal cell population. In embodiments, the composition further comprises spheroids comprising the renal cell population and at least one non-renal cell population.

[0011] In one aspect, provided herein is a method of treating kidney disease in a subject having a kidney and / or urinary tract abnormality, the method comprising administering to the subject an effective amount of vesicles secreted by a population of kidney cells.

[0012] In one aspect, provided herein is a method for treating kidney disease in a subject having a kidney and / or urinary tract abnormality, the method comprising administering to the subject an effective amount of spheroids comprising a renal cell population and at least one non-renal cell population.

[0013] In one aspect, provided herein are bioactive renal cell populations and uses thereof for treating kidney disease in subjects with kidney and / or urinary tract abnormalities.

[0014] In one aspect, provided herein are products (e.g., vesicles) secreted by bioactive renal cell populations and their use to treat kidney disease in subjects with kidney and / or urinary tract abnormalities.

[0015] In one aspect, provided herein are spheroids comprising a population of bioactive renal cells and uses thereof for treating kidney disease in subjects with kidney and / or urinary tract abnormalities. [Brief explanation of the drawings]

[0016] [Figure 1] 1 is a graph showing estimated glomerular filtration rate (eGFR) before and after REACT treatment. [Figure 2] 1 is a graph showing serum creatinine before and after REACT treatment. [Figure 3] A photo of the REACT product delivery system. [Figure 4] This is a photo of the REACT shipping container. [Figure 5] Flow diagram of the study design. [Figure 6] 1 is a flow diagram of a non-limiting example of an overall NKA manufacturing method. [Figure 7A-D] 7 is a flow chart illustrating further details of the non-limiting exemplary method shown in FIG. 6. [Figure 8] 1 is a graph showing improvement in renal function, as measured by eGFR, in patients receiving REACT treatment for kidney disease caused by CAKUT. The asterisk indicates the patient's initial renal function before the effects of CAKUT. The solid gray line (plotted from -1 month to 0 months relative to injection) indicates the patient's decline in renal function, as measured by eGFR, before REACT injection. The dashed black line (plotted from 0 months to 3 months relative to injection) indicates the patient's eGFR after REACT injection. [Figure 9] 1 is a graph showing improvement in kidney function as measured by albumin to creatinine ratio in patients receiving REACT treatment for kidney disease caused by CAKUT. DETAILED DESCRIPTION OF THE INVENTION

[0017] Reference is made herein to certain specific embodiments and examples encompassed by the present invention. While the present invention will be described in conjunction with exemplary embodiments, it will be understood that the exemplary embodiments are not intended to limit the invention to those embodiments. On the contrary, the present invention is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0018] All references cited throughout this disclosure are expressly incorporated herein by reference in their entirety. In the event that one or more of the literature, patents, and similar materials incorporated herein by reference, including but not limited to defined terms, term usage, described techniques, etc., differs from or conflicts with this application, this application will control.

[0019] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.

[0020] As used herein, the term "about" in the context of a numerical value or range means ±10% of the numerical value or range recited or claimed, unless the context requires a more limited range.

[0021] In the detailed description and claims herein, a conjunctive list of elements or features may be preceded by a phrase such as "at least one of" or "one or more of." The term "and / or" may also be present within a list of two or more elements or features. Unless otherwise implicitly or explicitly stated by the context, such a phrase is intended to refer to any of the list of elements or features individually, or to any listed element or feature in combination with any other listed element or feature. For example, the phrases "at least one of A and B," "one or more of A and B," and "A and / or B" are intended to mean "A alone, B alone, or A and B together," respectively. Similar interpretation applies to lists containing more than two items. For example, the phrases "at least one of A, B, and C," "one or more of A, B, and C," and "A, B, and / or C" are intended to mean "A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B, and C together," respectively. Furthermore, use of the term "based on" above and in the claims is intended to mean "based at least in part on," so that unrecited features or elements may be allowed for.

[0022] When a range of a parameter is stated, it is understood that all integers and tenths thereof within that range are also encompassed by the present invention. For example, "0.2 mg to 5 mg" discloses 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, etc., including up to 5.0 mg.

[0023] The transitional term "comprising," which is synonymous with "including," "containing," or "characterized by," is used to describe a non-exclusive or exclusive In contrast, the transitional phrase "consisting of" is open-ended and does not exclude additional, unrecited elements or method steps. The transitional phrase "consisting essentially of" excludes any element, step, or ingredient not specified in the enclosed paragraph. Limited to the specified materials or steps of the described invention "and materials or steps that do not materially affect the basic and novel characteristic(s)."

[0024] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0025] As used herein, "treating" includes, for example, preventing, reversing, or halting the progression of a disorder. "Treating" also includes preventing or ameliorating any symptom or symptoms of a disorder. As used herein, "preventing" disease progression or disease complications in a subject means preventing or reducing disease progression and / or disease complications in a subject.

[0026] As used herein, a "symptom" associated with a disorder includes any clinical or laboratory sign associated with the disorder, including but not limited to what may be felt or noticed by a subject.

[0027] As used herein, "effective" when referring to an amount of a therapeutic agent refers to an amount of the therapeutic agent sufficient to produce a desired therapeutic response when used as disclosed herein without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit / risk ratio.

[0028] As used herein, the term "bioactive renal cells" or "BRCs" refers to renal cells that, when administered to a subject's kidney, have one or more of the following properties: the ability to reduce (e.g., slow or halt) the deterioration or progression of chronic kidney disease or its symptoms, enhance renal function, affect (improve) renal homeostasis, and promote healing, repair, and / or regeneration of renal tissue or the kidney. In 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 other cells of the corticomedullary junction. In embodiments, BRCs are obtained by the isolation and expansion of renal cells from kidney tissue. In embodiments, BRCs are obtained by the isolation and expansion of renal cells from kidney tissue using a method for selecting bioactive cells. In embodiments, BRCs have a regenerative effect on the kidney. In embodiments, BRCs comprise, consist essentially of, or consist of selected renal cells (SRCs). In embodiments, BRCs are SRCs.

[0029] In embodiments, SRCs are cells obtained by isolation and expansion of renal cells from a suitable renal tissue source, wherein the SRCs contain a higher percentage of one or more cell types and lack or have a lower percentage of one or more other cell types compared to the starting renal cell population. In embodiments, the SRCs contain an increased proportion of BRCs compared to the starting renal cell population. In embodiments, the SRC population is a population of isolated renal cells enriched for specific bioactive components and / or cell types and / or depleted of specific inactive and / or undesirable components or cell types used in the treatment of kidney disease, i.e., resulting in stabilization and / or improvement and / or regeneration of kidney function. SRCs result in superior therapeutic and regenerative outcomes compared to the starting population. In embodiments, SRCs are obtained from a patient's renal cortical tissue via kidney biopsy. In embodiments, the SRCs are selected based on their expression of one or more markers (e.g., by fluorescence-activated cell sorting, or "FACS"). In embodiments, SRCs are depleted of one or more cell types (e.g., by fluorescence activated cell sorting, or "FACS") based on the expression of one or more markers for the cell types. The SRCs are selected from a population of viable renal cells. In embodiments, the SRCs are selected by density gradient separation of expanded renal cells. In embodiments, the SRCs are selected by separation of expanded renal cells by centrifugation across a density boundary, density barrier, or density interface, or by single-step discontinuous density gradient separation. In embodiments, the SRCs are selected by continuous or discontinuous density gradient separation of expanded renal cells cultured under hypoxic conditions. In embodiments, the SRCs are selected by density gradient separation of expanded renal cells cultured under hypoxic conditions for at least about 8 hours, 12 hours, 16 hours, 20 hours, or 24 hours. In embodiments, the SRCs are selected by separation by centrifugation across a density boundary, density barrier, or density interface of expanded renal cells cultured under hypoxic conditions for at least about 8 hours, 12 hours, 16 hours, 20 hours, or 24 hours. In embodiments, the SRCs are selected by separation by centrifugation across a density boundary, density barrier, or density interface (e.g., single-step discontinuous density gradient separation) of expanded renal cells cultured under hypoxic conditions for at least about 8 hours, 12 hours, 16 hours, 20 hours, or 24 hours. In embodiments, the SRCs are primarily composed of renal tubular cells. In embodiments, other parenchymal (e.g., vascular) and stromal (e.g., collecting duct) cells may be present in SRCs. In embodiments, less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the cells in a population of SRCs are vascular cells. In embodiments, less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the cells in a population of SRCs are collecting duct cells. In embodiments, less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the cells in a population of SRCs are vascular cells or collecting duct cells.

[0030] The term "spheroid" refers to an aggregate or assembly of cells cultured to allow three-dimensional growth rather than growth as a monolayer. It is noted that the term "spheroid" does not imply that the aggregate is a geometric sphere. In embodiments, the aggregate may be highly organized with a well-defined morphology, or the aggregate may be an unorganized mass. In embodiments, the spheroid may comprise a single cell type or two or more cell types. In embodiments, the cells may be primary isolates or permanent cell lines, or a combination of the two. In embodiments, the spheroids (e.g., cell aggregates or organoids) are formed in a spinner flask. In embodiments, the spheroids (e.g., cell aggregates or organoids) are formed in a three-dimensional matrix.

[0031] The term "natural organ" is intended to mean an organ of a living subject. In embodiments, the subject may be healthy or unhealthy. In embodiments, an unhealthy subject may have a disease associated with that particular organ.

[0032] The term "native kidney" is intended to mean a kidney of a living subject. In embodiments, the subject may be healthy or unhealthy. In embodiments, an unhealthy subject may have kidney disease. In embodiments, an unhealthy subject may have a kidney and / or urinary tract abnormality.

[0033] Provided herein are cell types and populations of cells (e.g., SRCs) that provide a benefit to a native organ, such as the kidney. In embodiments, the benefit includes halting or slowing the progression of chronic kidney disease (e.g., worsening of one or more symptoms). In embodiments, the benefit includes a regenerative effect, e.g., reducing symptoms of chronic kidney disease and / or improving native kidney function. In embodiments, the benefit includes, but is not limited to, reducing the extent of damage to the native organ, or improving, restoring, or stabilizing the function or structure of the native organ. Renal damage can be in the form of fibrosis, inflammation, glomerular hypertrophy, atrophy, etc.

[0034] In embodiments, an enriched cell population or preparation is a starting organ cell population (e.g., a spheroid) that contains a higher percentage of a particular cell type than the percentage of that cell type in the starting population. For example, the starting kidney cell population can be enriched for a first cell type, a second cell type, a third cell type, a fourth cell type, a fifth cell type, etc., of interest.

[0035] As used herein, the term "hypoxic" culture conditions refers to culture conditions in which cells are exposed to reduced levels of available oxygen in a culture system compared to standard culture conditions in which cells are cultured at atmospheric oxygen levels (approximately 21%). Non-hypoxic conditions are referred to herein as normal or normoxic culture conditions.

[0036] Included herein are compositions comprising a biomaterial and one or more cell types. In embodiments, the biomaterial is a natural or synthetic biocompatible material suitable for introduction into living tissue that supports cells in a viable state. Natural biomaterials are materials produced by or derived from biological systems. Synthetic biomaterials are materials not produced by or derived from biological systems. In embodiments, the biomaterials disclosed herein can be a combination of natural and synthetic biocompatible materials. As used herein, biomaterials include, for example (but are not limited to), polymer matrices and scaffolds. Those skilled in the art will recognize that biomaterial(s) can be configured in various forms, such as porous foams, gels, liquids, beads, and solids, and can include one or more natural or synthetic biocompatible materials. In embodiments, the biomaterial is in the liquid form of a solution capable of becoming a hydrogel. In embodiments, the biomaterial is a hydrogel capable of becoming a liquid.

[0037] As used herein, the term "renal disease" includes disorders associated with any stage or degree of acute or chronic kidney disease (e.g., acute renal failure or chronic renal failure) that result in a decline or loss of the kidney's ability to perform the functions of blood filtration and removal of excess fluid, electrolytes, and waste products from the blood. In embodiments, kidney disease also includes endocrine dysfunction, such as anemia (erythropoietin deficiency) and mineral imbalance (vitamin D deficiency). In embodiments, kidney disease can originate in the kidney or can be secondary to various conditions, including (but not limited to) congenital anomalies of the renal urinary tract (CAKUT), vesicoureteral reflux, heart failure, hypertension, diabetes, autoimmune disease, or liver disease. In embodiments, kidney disease can be a state of chronic renal failure that develops after acute injury to the kidney. For example, kidney damage due to ischemia and / or exposure to toxic substances can cause acute renal failure. Incomplete recovery after acute kidney injury can lead to the development of chronic renal failure.

[0038] In embodiments, the term "treatment" may refer to therapeutic treatment and / or preventative or preventative measures for kidney disease, anemia, renal tubular transport disorder, or glomerular filtration disorder, the purpose of which is to reverse, prevent, or slow down (alleviate) the targeted disorder. Those in need of treatment include those who already have kidney disease, anemia, renal tubular transport disorder, or glomerular filtration disorder, as well as those who are susceptible to kidney disease, anemia, renal tubular transport disorder, or glomerular filtration disorder, or those in whom kidney disease, anemia, renal tubular transport disorder, or glomerular filtration disorder should be prevented. In embodiments, the subject in need of treatment includes those with congenital abnormalities of the kidney and / or urinary tract. As used herein, the term "treatment" includes stabilizing and / or improving kidney function.

[0039] Included herein are constructs or formulations comprising one or more cell types (e.g., cell populations such as SRCs) deposited on or within a scaffold or matrix made of one or more synthetic or naturally occurring biocompatible materials. In embodiments, the one or more cell populations are coated with a biomaterial made of one or more synthetic or naturally occurring biocompatible biomaterials, polymers, proteins, or peptides, and thereon The cell populations may be deposited, embedded in, attached to, seeded within, or entrapped within a biomaterial or scaffold or matrix, in embodiments, in vitro or in vivo. In embodiments, the one or more biomaterials used to fabricate the construct or formulation may be selected to induce, promote, or enable the dispersion and / or integration of the cellular components of the construct with endogenous host tissue, or to induce, promote, or enable the survival, engraftment, tolerance, or functional performance of the cellular components of the construct or formulation.

[0040] The term "Neo-Kidney Augment (NKA)" refers to a bioactive cell preparation that is an injectable product consisting of autologous and allogeneic SRCs formulated in a biomaterial composed of a gelatin-based hydrogel. The term "Advance Cell Therapy (ACT)" also refers to treatment with NKA.

[0041] In embodiments, the subject is a living animal. In embodiments, the subject is a mammal, such as a dog, cat, horse, rabbit, zoo animal, cow, pig, sheep, goat, camel, mouse, rat, or guinea pig. In embodiments, the subject is a primate, such as a human, chimpanzee, orangutan, monkey, or baboon. In embodiments, the subject is a human. In embodiments, the subject is a treatment-eligible patient who is experiencing or has experienced one or more signs, symptoms, or other indicators of kidney disease. Such subjects include, but are not limited to, newly diagnosed or previously diagnosed subjects currently experiencing or at risk for kidney disease, regardless of cause. In embodiments, the subject may or may not have been previously treated for kidney disease. In embodiments, the subject has a congenital anomaly of the kidney and / or urinary tract. In embodiments, the subject is a human with a congenital kidney and urinary tract anomaly. In embodiments, the subject has or has previously had one or more signs, symptoms, or other indicators of organ-related disease, such as kidney disease, anemia, or erythropoietin (EPO) deficiency. In embodiments, the subject does not have diabetes. In embodiments, the subject does not have type I diabetes. In embodiments, the subject does not have type II diabetes.

[0042] Congenital anomalies of the kidney and urinary tract (CAKUT) include a group of disorders of various anatomical spectrum, including anomalies of the kidney as well as anomalies of the bladder and urethra. In embodiments, the term "CAKUT" (e.g., when referring to a subject with CAKUT) refers to one congenital anomaly. In embodiments, the term CAKUT (e.g., when referring to a subject with CAKUT) refers to two or more congenital anomalies. In embodiments, a subject with CAKUT has one or more anomalies of the kidney, bladder, and / or urethra. In embodiments, a subject with CAKUT has an anomaly in one or two kidneys. In embodiments, a subject with CAKUT has an anomaly in the urethra. In embodiments, CAKUT is caused by a genetic mutation or genetic anomaly. In embodiments, CAKUT is caused by an environmental factor. In embodiments, a subject with CAKUT has an anomaly in the bladder. Non-limiting descriptions related to CAKUT include those described by Ristoska-Bojkovska et al. (2017) Pril (Makedon Akad Nauk Umet Odd Med Nauki) 38(1):59-62, and Rodriguez (2014) Fetal Pediatr Pathol. 33(5-6):293-320, the full contents of which are available at: The present disclosure is incorporated herein by reference. In an embodiment, the subject with CAKUT does not suffer from diabetes. In an embodiment, the subject with CAKUT does not suffer from type I diabetes. In an embodiment, the subject with CAKUT does not suffer from type II diabetes.

[0043] CAKUT constitutes approximately 20% to 30% of all abnormalities identified in the prenatal period. Queisser-Luft et al. (2002) Malformations in newborn: resu lts based on 30,940 infants and fetuses from the Mainz congenital birth defect monitoring system (1990-1998). See 2002;266(3):163 (the entire contents of which are hereby cited). In embodiments, the defects can be bilateral or unilateral, and different defects often coexist in an individual child.

[0044] In embodiments, CAKUT refers to a wide range of disorders resulting from abnormal embryonic kidney development due to malformations of the renal parenchyma, abnormalities in renal migration, or abnormalities in the developing urinary collecting system. In embodiments, CAKUT refers to a wide range of disorders resulting from abnormal kidney development processes. In embodiments, malformations of the renal parenchyma result in failure of normal nephron development, as seen in renal dysplasia, rheumatoid arthritis (RA), renal tubular dysplasia, and some types of nephronophthisis. Without being bound by any scientific theory, research using molecular genetics has shown that renal malformations result from defects in genes encoding signal transduction and transcription factors. In embodiments, environmental factors, such as prenatal exposure to teratogens, can disrupt kidney morphogenesis, resulting in CAKUT. In embodiments, abnormalities include abnormal embryonic kidney migration, as seen in renal ectopia (e.g., pelvic kidney), and fusion abnormalities such as horseshoe kidney. In embodiments, abnormalities in the developing urinary collecting system, such as those seen in duplicated collecting systems, posterior urethral valves, and ureteropelvic junction obstruction, can lead to CKD / ESRD. Renal dysplasia can be unilateral or bilateral and occurs in 2 to 4 per 1000 live births. The male-to-female ratio for bilateral renal dysplasia is 1.3:1, and the male-to-female ratio for unilateral dysplasia is 1.9:1.

[0045] Because CAKUTs are responsible for 30% to 50% of cases of end-stage renal disease (ESRD) in children (Seikaly et al. 2003 Chronic renal insufficiency in children: the 2001 Annual Report Pediatr Nephrol. 18(8):796), it is important to diagnose these abnormalities and initiate therapy to minimize renal damage, prevent or delay the onset of ESRD, and provide supportive care to avoid ESRD complications. Patients with malformations associated with a reduction in the number or size of kidneys are most likely to have a poor renal prognosis (Sanna-Cherchi et al. al. 2009 Renal outcome in patients with congenital anomalies of the kidney and urinary tract. Kidney Int. 76(5):528). In some embodiments, by age 30, most patients will be undergoing dialysis.

[0046] The risk of dialysis is increased by the presence of a solitary kidney or renal hypodysplasia associated with a posterior urethral valve. The risk of CKD is significantly higher for patients with renal hypoplasia (hypodysplasia) compared with patients with unilateral or bilateral renal hypodysplasia, or polycystic or horseshoe kidneys. In embodiments, the subclinical defect of a solitary kidney may contribute to a poorer prognosis compared with more benign forms of CAKUT. In embodiments, without being limited by any scientific theory, children with a solitary kidney are at risk for long-term CKD, thought to be due to glomerular hyperfiltration. In embodiments, approximately one-third of patients may have evidence of kidney damage, defined as proteinuria (e.g., a urinary protein-to-creatinine ratio greater than 0.2 mg / mg [e.g., greater than 22.6 mg / mmol for children over 2 years of age]), hypertension (e.g., blood pressure equal to or greater than the 95th percentile for age, sex, and height), elevated serum creatinine and estimated creatinine clearance based on the Schwartz formula, or use of renal-protective medications (e.g., angiotensin-converting enzyme inhibitors).

[0047] In embodiments, renal dysplasia may be discovered during routine prenatal screening or after birth when a renal ultrasound is performed on a dysmorphic infant. In embodiments, bilateral dysplasia is more likely to be diagnosed earlier than unilateral dysplasia, especially in the presence of oligohydramnios. In embodiments, renal ultrasound features include abnormal renal parenchyma, poor corticomedullary differentiation, and increased echogenicity as a result of parenchymal cysts.

[0048] In embodiments, infants with bilateral dysplasia may have impaired renal function at birth and may subsequently experience progressive renal failure. Clinical findings include abnormalities of the renal pelvis, calyces (e.g., congenital hydronephrosis), and ureters, such as megaureter with a double collecting system, ureteral strictures, and vesicoureteral reflux (VUR). In embodiments, symptomatic symptoms can result due to complications associated with these urological abnormalities, including urinary tract infection (UTI), hematuria, fever, and abdominal pain.

[0049] In embodiments, because renal dysplasia is frequently associated with urinary collecting system abnormalities, excretory cystourethrography may be considered in patients with renal dysplasia with or without UTI. In embodiments, children with unilateral renal dysplasia may be at increased risk for long-term sequelae of renal scarring due to recurrent UTI if the normal contralateral kidney has associated urological abnormalities such as VUR. In embodiments, DMSA radionuclide scans may provide further information about the various functions of each kidney. In embodiments, multicystic dysplastic kidney (MCDK) typically lacks viable functional renal tissue and therefore has no detectable renal blood flow or function. However, in embodiments, rare variants of segmental dysplasia may exist. In embodiments, imaging studies may be useful in defining baseline renal function and risk of future renal damage, as well as the ability to regenerate normally functioning renal parenchyma.

[0050] The terms "sample" or "patient sample" or "biological sample" are generally intended to include 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, such as, for example, a kidney biopsy. The term includes cultured cells, such as, for example, cultured mammalian kidney cells. Methods for obtaining tissue biopsies and cultured cells from mammals are known in the art. In embodiments, samples may be derived from a variety of sources in a mammalian subject, including, but not limited to, blood, semen, serum, urine, bone marrow, mucosa, tissue, etc.

[0051] The term "control sample" refers to a negative or positive control sample whose negative or positive results are expected to be useful for correlating with test sample results. In embodiments, suitable control samples include, but are not limited to, samples known to exhibit indicators characteristic of normal kidney function, samples obtained from subjects known to not have kidney disease, and samples obtained from subjects known to have kidney disease. In embodiments, the control sample may be a sample obtained from a subject prior to treatment with the methods provided herein. In embodiments, the control sample may be a test sample obtained from a subject known to have any type or stage of kidney disease, and a sample from a subject known not to have any type or stage of kidney disease. In embodiments, the control sample may be a normal, healthy, matched control. Those skilled in the art will recognize other control samples suitable for use.

[0052] Provided herein are methods and compositions for treating, inter alia, chronic kidney disease in subjects with kidney and / or urinary tract abnormalities (eg, subjects with CAKUT).

[0053] In one aspect, provided herein is a method of treating kidney disease in a subject suffering from chronic kidney disease, comprising, consisting essentially of, or consisting of administering to a subject an effective amount of (i) a bioactive renal cell population, (ii) vesicles secreted by the renal cell population, and / or (iii) spheroids comprising the renal cell population and at least one non-renal cell population, wherein the subject has a kidney and / or urinary tract abnormality. In embodiments, the subject has a kidney abnormality.

[0054] In one aspect, provided herein is a method for treating kidney disease in a subject having a kidney and / or urinary tract abnormality, comprising administering to the subject an effective amount of (i) a bioactive renal cell population, (ii) one or more products secreted by the renal cell population, and / or (iii) a spheroid comprising the renal cell population and at least one other cell population. or administering.

[0055] In one aspect, provided herein is a method for treating kidney disease in a subject having a kidney and / or urinary tract abnormality, comprising administering to the subject an effective amount of spheroids comprising (i) a bioactive renal cell population, (ii) one or more products (e.g., vesicles) secreted by the renal cell population, and / or (iii) the renal cell population and at least one other cell population, such as a non-renal cell population.

[0056] In one aspect, provided herein is a method of treating kidney disease in a subject having a kidney and / or urinary tract abnormality, comprising administering to the subject an effective amount of (i) a bioactive renal cell population, (ii) vesicles secreted by the renal cell population, or (iii) spheroids comprising the renal cell population and at least one non-renal cell population.

[0057] In one aspect, provided herein is a method of treating kidney disease in a subject having a kidney and / or urinary tract abnormality, the method comprising administering to the subject an effective amount of (i) a bioactive renal cell population, (ii) vesicles secreted by the renal cell population, and (iii) spheroids comprising the renal cell population and at least one non-renal cell population.

[0058] In one aspect, provided herein is a method for treating kidney disease in a subject with a kidney and / or urinary tract abnormality, comprising administering to the subject an effective amount of a composition comprising a bioactive renal cell population. In embodiments, the composition further comprises vesicles secreted by the renal cell population. In embodiments, the composition further comprises spheroids comprising the renal cell population and at least one non-renal cell population.

[0059] In one aspect, provided herein is a method of treating kidney disease in a subject having a kidney and / or urinary tract abnormality, the method comprising administering to the subject an effective amount of vesicles secreted by a population of kidney cells.

[0060] In one aspect, provided herein is a method for treating kidney disease in a subject having a kidney and / or urinary tract abnormality, the method comprising administering to the subject an effective amount of spheroids comprising a renal cell population and at least one non-renal cell population.

[0061] In one aspect, provided herein are bioactive renal cell populations and uses thereof for treating kidney disease in subjects with kidney and / or urinary tract abnormalities.

[0062] In one aspect, provided herein are products (e.g., vesicles) secreted by bioactive renal cell populations and their use to treat kidney disease in subjects with kidney and / or urinary tract abnormalities.

[0063] In one aspect, provided herein are spheroids comprising a population of bioactive renal cells and uses thereof for treating kidney disease in subjects with kidney and / or urinary tract abnormalities.

[0064] In embodiments, the subject has a urinary tract abnormality. In embodiments, the subject has a kidney and urinary tract abnormality. In embodiments, the abnormality is acquired prenatally. In embodiments, the abnormality is acquired postnatally. In embodiments, the abnormality is a congenital abnormality. In embodiments, the subject has a congenital kidney abnormality. In embodiments, the subject has a congenital urinary tract abnormality. In embodiments, the subject has a congenital renal and urinary tract abnormality. In embodiments, the congenital abnormality worsens or causes additional abnormalities after birth. In embodiments, the subject has an abnormality in one kidney. In embodiments, the subject has one or more abnormalities in each kidney. In embodiments, the subject has an abnormality of the urinary tract, wherein the abnormality is in the urethra. In embodiments, the subject has an abnormality of the urinary tract, wherein the abnormality is in the bladder. In embodiments, the subject has an abnormality of the urinary tract, wherein the abnormality is in the ureter. In embodiments, the abnormality is present at birth but does not develop or show symptoms until after birth.

[0065] In an embodiment, the kidney disease is CKD. In an embodiment, the subject has CKD from an abnormality (e.g., congenital) of the kidney and urinary tract.

[0066] In an embodiment, the abnormality comprises a congenital abnormality. In an embodiment, the subject has CAKUT.

[0067] In an embodiment, the abnormality is a morphological abnormality. In an embodiment, the subject has abnormally developed kidneys.

[0068] In embodiments, the subject has or has suffered from primary vesicoureteral reflux, reflux nephropathy, renal scarring, or renal hypodysplasia. In embodiments, the subject has or has suffered from reflux nephropathy. In embodiments, the subject has or has suffered from renal scarring. In embodiments, the subject has or has suffered from renal hypodysplasia.

[0069] In embodiments, the subject is susceptible to urinary tract infections, hi embodiments, the subject has had at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 urinary tract infections.

[0070] In an embodiment, the subject suffers from hypertension or proteinuria.

[0071] In an embodiment, the subject has undergone post-antireflux surgery.

[0072] In an embodiment, the subject receives a blood flow of 90 mL / min / 1.73 m 2 In an embodiment, the subject has a glomerular filtration rate (GFR) of less than 80 mL / min / 1.73 m 2 In an embodiment, the subject has a GFR of less than 70 mL / min / 1.73 m 2 In an embodiment, the subject has a GFR of less than 60 mL / min / 1.73 m 2 In an embodiment, the subject has a GFR of less than 50 mL / min / 1.73 m 2 In an embodiment, the subject has a GFR of less than 40 mL / min / 1.73 m 2 In an embodiment, the subject has a GFR of less than 30 mL / min / 1.73 m 2 In embodiments, the subject has a GFR of at least 10 mL / min / 1.73 m 2 In embodiments, the subject has a GFR of at least 15 mL / min / 1.73 m 2 In embodiments, the subject has a GFR of at least 20 mL / min / 1.73 m 2In embodiments, the subject has a GFR of at least 30 mL / min / 1.73 m 2 In an embodiment, the subject has a GFR of 10 mL / min / 1.73 m 2 , 15mL / min / 1.73m 2 , 20mL / min / 1.73m 2 , 25mL / min / 1.73m 2 , or 30 mL / min / 1.73 m 2 to 50 mL / min / 1.73 m 2 , 60mL / min / 1.73m 2 ,70mL / min / 1.73m 2 , 80mL / min / 1.73m 2 , or 90 mL / min / 1.73 m 2 In an embodiment, the GFR is estimated GFR (eGFR). In an embodiment, the subject has microalbuminuria. In an embodiment, the subject has macroalbuminuria.

[0073] In embodiments, the subject is under 18 years old. In embodiments, the subject is under 60 years old. In embodiments, the subject is under 50 years old. In embodiments, the subject is under 40 years old. In embodiments, the subject is under 35 years old. In embodiments, the subject is under 30 years old. In embodiments, the subject is under 25 years old. In embodiments, the subject is The subject is under 20 years old. In embodiments, the subject is between 1 and 16 years old. In embodiments, the subject is between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years old and 20, 25, 30, 35, or 40 years old.

[0074] In embodiments, the subject is 20, 25, 30, 35, or 40 years old to 50, 55, 65, 70, 75, 80, 85, 90, 95, or 100 years old. In embodiments, the subject is at least 50 years old. In embodiments, the subject is at least 55 years old. In embodiments, the subject is at least 60 years old. In embodiments, the subject is at least 65 years old. In embodiments, the subject is at least 70 years old.

[0075] In an embodiment, the subject has a renal parenchymal malformation.

[0076] In embodiments, the subject has ureteral duplication, ureteropelvic junction obstruction, renal agenesis, vesicoureteral reflux, renal dysplasia, hypoplastic kidney, renal hypodysplasia, congenital hydronephrosis, horseshoe kidney, posterior urethral valve and prune belly syndrome, obstructive renal dysplasia, or nonmotile cilia.

[0077] In embodiments, the abnormality is caused by or correlated with genetic factors. In embodiments, CAKUT is caused by or correlated with genetic factors. In embodiments, the abnormality is caused by or correlated with non-genetic factors. In embodiments, CAKUT is caused by or correlated with non-genetic factors. In embodiments, the non-genetic factors are environmental factors.

[0078] In embodiments, the subject suffers from urethral duplication, ureteropelvic junction obstruction, renal agenesis, vesicoureteral reflux, renal hypodysplasia, congenital hydronephrosis, horseshoe kidney, posterior urethral valve and prune belly syndrome, obstructive renal dysplasia, or nonmotile cilia. In embodiments, the subject suffers from urethral duplication. In embodiments, the subject suffers from ureteropelvic junction obstruction. In embodiments, the subject suffers from renal agenesis. In embodiments, the subject suffers from vesicoureteral reflux. In embodiments, the subject suffers from renal hypodysplasia. In embodiments, the subject suffers from congenital hydronephrosis. In embodiments, the subject suffers from horseshoe kidney. In embodiments, the subject suffers from posterior urethral valve and prune belly syndrome. In embodiments, the subject suffers from obstructive renal dysplasia. In embodiments, the subject suffers from nonmotile cilia. In embodiments, CAKUT is caused by or correlated with genetic factors.

[0079] In embodiments, the abnormality is Alagille syndrome, Apert syndrome, Bardet-Biedl syndrome, Beckwith-Wiedemann syndrome, Branchio-oto-renal syndrome (BOR), Ankle dysplasia, Senani-Lenz syndrome, DiGeorge syndrome, Fraser syndrome, Hypoparathyroidism, Sensorineural hearing loss and kidney disease (HDR), Kallmann syndrome, Ulno-Mast syndrome, Meckel-Gruber syndrome, Nephronophthisis, Okihiro syndrome, Pallister-Hall syndrome, Renal coloboma syndrome, Hypothyroidism, Dysplasia, Renal dysplasia aplasia, cystic dysplasia, non-cystic dysplasia, VUR cystic dysplasia, renal hypodysplasia, isolated cystic renal hypodysplasia, isolated non-cystic renal hypodysplasia, isolated renal tubular dysplasia, Rubinstein-Taybi syndrome, Simpson-Golabi-Behmel syndrome, Townes-Brocks syndrome, Zellweger syndrome, Smith-Lemli-Opitz syndrome, hydronephrosis, medullary dysplasia, unilateral / bilateral aplasia / dysplasia, urinary collecting system abnormalities, aplasia, ureteropelvic junction obstruction (UPJO) aplasia, dysplasia agenesis, unilateral aplasia, VUR, malrotation, crossed fused kidney, VUR dysplasia, double serine / threonine and tyrosine protein kinase (DSTYK) mutation, DSTYK mutation associated with UPJO Includes dysplasia, tubular dysplasia, cysts, and / or hypoplasia.

[0080] In embodiments, the kidney disease is chronic kidney disease. In embodiments, the chronic kidney disease is stage I, stage II, stage III, stage IV, or stage V kidney disease. In embodiments, the chronic kidney disease is stage I kidney disease. In embodiments, the chronic kidney disease is stage II kidney disease. In embodiments, the chronic kidney disease is stage III kidney disease. In embodiments, the chronic kidney disease is stage IV kidney disease. In embodiments, the chronic kidney disease is stage V kidney disease. In embodiments, the subject undergoes dialysis at least once, twice, or three times per week.

[0081] In embodiments, a population of bioactive renal cells is administered to a native organ as part of a formulation described herein. In embodiments, a secreted product of a population of bioactive renal cells is administered to a native organ as part of a formulation described herein. In embodiments, the cells originate from a source other than the native organ or target native organ to which they are administered.

[0082] In embodiments, the cells of the renal cell population are present in the form of spheroids. In embodiments, spheroids comprising bioactive renal cells are administered to a subject. In embodiments, the spheroids comprise at least one non-renal cell type or cell population.

[0083] In embodiments, the subject has renal disease as assessed by microalbuminuria, which may be defined by a urinary albumin-to-creatinine ratio (UACR) of 30 mg / g or greater or a urinary albumin excretion of 30 mg / day or greater in a 24-hour urine collection.

[0084] In embodiments, the patient's renal function improves as a result of the treatment. The improvement in the patient's renal function can be a stabilization of the patient's renal function or a change in renal function that improves renal function. In embodiments, improved renal function is indicated by a decrease, stabilization, or increase in the rate of decline in estimated glomerular filtration rate (eGFR). In embodiments where improved renal function is indicated by an increase in eGFR, the increase in eGFR can be an increase of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, or at least 25% over the patient's baseline eGFR. In such embodiments, the patient's baseline eGFR can be the patient's eGFR prior to the first dose of the treatment, for example, the patient's eGFR determined up to 3 months, 2 months, 1 month, 3 weeks, 2 weeks, 10 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day prior to administering the first dose of the treatment. The increase in the patient's baseline eGFR may be achieved within 1 to 6 months, or within 2 to 6 months, or within 3 to 6 months, or within 4 to 6 months, or within 5 to 6 months, or within 1 to 5 months, or within 1 to 4 months, or within 1 to 3 months, or within 2 to 5 months, or within 2 to 4 months, or within 3 to 4 months, or within 2 to 3 months, or within 2 months, or within 3 months, or within 4 months, or within 5 months, or within 6 months after administration of the first dose of the treatment. The increase relative to the patient's baseline eGFR need not be at a constant level or degree, i.e., the patient need not maintain the same initial level of increase relative to baseline for the treatment to "improve" kidney function. The increase in the patient's baseline eGFR, once achieved, may decline, provided that the patient's eGFR continues to increase compared to the patient's baseline eGFR. The increase in the patient's baseline eGFR, once achieved, may also further increase or maintain the increase relative to baseline at the same level as that initial level of increase relative to baseline.The increase in eGFR relative to the patient's baseline is at least 12 months, 12 months, at least 18 months, 18 months, at least 24 months, 24 months, at least 30 months, 30 months, or at least. It can be for 36 months, 36 months, at least 42 months, 42 months, at least 48 months, 48 ​​months, at least 54 months, 54 months, at least 60 months, 60 months, at least 66 months, 66 months, at least 72 months, 72 months, at least 78 months, 78 months, or for the remaining life of the patient.

[0085] In embodiments, improved renal function is indicated by a decrease in the albumin-to-creatinine ratio (ACR) in the patient. In embodiments where improved renal function is indicated by a decrease in the ACR in the patient, the decrease may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80%, or at least 90% relative to the patient's baseline ACR. Alternatively, the decrease in ACR may be such that if the patient's baseline ACR increases moderately, e.g., between 30 mg / g and 300 mg / g, the decrease in ACR may reduce the patient's ACR to a level in the mild to normal range, e.g., below 30 mg / g. Alternatively, the decrease in ACR may be such that if the patient's baseline ACR is severely increased, e.g., greater than 300 mg / g, the decrease in ACR may be to a level that moderately increases the patient's ACR, e.g., between 30 mg / g and 300 mg / g, or to a level that is mildly increased to normal, e.g., less than 30 mg / g. In such embodiments, the patient's baseline ACR may be the patient's ACR prior to the first dose of therapy, e.g., the patient's ACR determined up to 3 months, 2 months, 1 month, 3 weeks, 2 weeks, 10 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day prior to administering the first dose of therapy. A reduction in a patient's baseline ACR may be achieved within 1 to 6 months, or within 2 to 6 months, or within 3 to 6 months, or within 4 to 6 months, or within 5 to 6 months, or within 1 to 5 months, or within 1 to 4 months, or within 1 to 3 months, or within 2 to 5 months, or within 2 to 4 months, or within 3 to 4 months, or within 2 to 3 months, or within 2 months, or within 3 months, or within 4 months, or within 5 months, or within 6 months after administration of the first dose of treatment. The reduction relative to a patient's baseline ACR does not need to be at a constant level or degree, i.e., the patient does not need to maintain the same initial level of reduction relative to baseline for the treatment to "improve" kidney function.The reduction in the patient's baseline ACR, once achieved, may increase, provided that the patient's ACR continues to decrease compared to the patient's baseline ACR. The reduction in the patient's baseline ACR, once achieved, may also decrease further or maintain the same level of reduction relative to baseline as its initial level of reduction relative to baseline. The reduction in the patient's ACR relative to baseline may be over a period of at least 12 months, 12 months, at least 18 months, 18 months, at least 24 months, 24 months, at least 30 months, 30 months, at least 36 months, 36 months, at least 42 months, 42 months, at least 48 months, 48 ​​months, at least 54 months, 54 months, at least 60 months, 60 months, at least 66 months, 66 months, at least 72 months, 72 months, at least 78 months, 78 months, or the remainder of the patient's life.

[0086] In embodiments, improved renal function is indicated by a decrease in total serum creatinine or a rate of increase in serum creatine (sCr), or an equivalent measure (e.g., cystatin C, inulin, or other measure of glomerular filtration). In embodiments, improved renal function is indicated by improved renal cortical thickness. In embodiments, improved renal function may be indicated by structural and functional changes. In embodiments, improved renal size and / or structure is determined by renal imaging. In embodiments, the method of renal imaging is ultrasound, MRI, or renal scintigraphy. In embodiments, improved renal function is superior to a previous state of renal structure or function.

[0087] In embodiments, the effective treatment of kidney disease in a subject provided herein comprises administering to the kidney Renal function may be monitored through various indicators of kidney function. In embodiments, indicators of kidney function include, but are not limited to, serum albumin levels, albumin to globulin ratio (A / G ratio), serum phosphorus levels, serum sodium levels, kidney size (measurable by ultrasound), serum calcium levels, phosphorus:calcium ratio, serum potassium levels, proteinuria, urinary creatinine levels, serum creatinine levels, blood urea nitrogen (BUN) levels, cholesterol levels, triglyceride levels, and glomerular filtration rate (GFR). In embodiments, some indicators of general health and well-being include, but are not limited to, weight gain or loss, survival rate, blood pressure (mean systemic blood pressure, diastolic blood pressure, or systolic blood pressure), and physical endurance capacity.

[0088] In embodiments, effective treatment with a bioactive renal cell preparation is evidenced by stabilization of one or more indicators of renal function. In embodiments, stabilization of renal function is demonstrated by observing a change in the same indicator in a subject treated with a method provided herein compared to the same indicator in a subject not treated with the method provided herein. In embodiments, stabilization of renal function can be demonstrated by observing a change in the same indicator in the same subject treated with a method provided herein compared to the same indicator in the subject before treatment. In embodiments, the change in one indicator can be an increase or decrease in value. In embodiments, the treatment provided herein can include stabilization of serum creatinine levels in a subject, where the BUN levels observed in the subject are lower compared to subjects with a similar condition not treated with the method provided herein. In embodiments, the treatment can include stabilization of serum creatinine levels in a subject, where the serum creatinine levels observed in the subject are lower compared to subjects with a similar condition not treated with the method provided herein. In embodiments, stabilization of one or more of the above indicators of renal function is a result of treatment with a selected renal cell preparation.

[0089] One of ordinary skill in the art will appreciate that one or more additional indicators described herein or known in the art can be measured to determine effective treatment of kidney disease in a subject.

[0090] In embodiments, effective treatment with a bioactive renal cell preparation is evidenced by an improvement in one or more indicators of kidney function. In embodiments, the bioactive renal cell population results in improved serum creatinine levels. In embodiments, the bioactive renal cell population results in improved serum protein maintenance. In embodiments, the bioactive renal cell population results in improved serum cholesterol and / or triglyceride levels. In embodiments, the bioactive renal cell population results in improved vitamin D levels. In embodiments, the bioactive renal cell population results in an improved phosphorus:calcium ratio compared to a non-enriched cell population. In embodiments, the bioactive renal cell population results in improved hemoglobin levels compared to a non-enriched cell population. In embodiments, the bioactive renal cell population results in improved serum creatinine levels compared to a non-enriched cell population. In embodiments, an improvement in one or more of the above indicators of kidney function is a result of treatment with a selected renal cell preparation.

[0091] Included herein are methods for regenerating a native kidney in a subject in need of regeneration. In embodiments, the method comprises administering or transplanting a bioactive cell population, formulation, or construct described herein into the subject. In embodiments, the regenerated native kidney may be characterized by a number of indicators, including, but not limited to, development of function or capacity in the native kidney, improvement of function or capacity in the native kidney, and expression of certain markers in the native kidney. In embodiments, development or improvement of function or capacity may be observed based on various indicators of kidney function described above. In embodiments, the regenerated kidney is characterized by differential expression of one or more stem cell markers. In embodiments, the stem cell marker may be one or more of the following: SRY (sex determining region Y)-box 2 (Sox 2), undifferentiated germ cell transcription factor (UTF1), mouse-derived Nodal homolog (NODAL), prominin 1 (PROM1) or CD133 (CD133), CD24, and any combination thereof (see International Application No. PCT / US2011 / 036347 by Ilagan et al., incorporated herein by reference in its entirety) (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, incorporated herein by reference in its entirety). (See also part of the appended claims.) In embodiments, expression of the stem cell marker(s) is upregulated compared to a control.

[0092] In embodiments, the effect may be brought about by the cells themselves and / or by a product secreted from the cells. In embodiments, a product secreted from the cells is administered to a subject. In embodiments, the product is isolated from the cells, e.g., the cells that produced it. In embodiments, the product is a vesicle as described herein. In embodiments, the vesicles (e.g., exosomes) are isolated from the renal cell population that produced them. In embodiments, the vesicles may include one or more of the following: paracrine factors, endocrine factors, junctional factors, microvesicles, exosomes, and RNA. Secreted products may also include products not present in microvesicles, including, but not limited to, paracrine factors, endocrine factors, junctional factors, and RNA. In embodiments, the secreted product may be part of a vesicle derived from a renal cell. In embodiments, the vesicles are secreted vesicles. In embodiments, the secreted vesicles are exosomes, microvesicles, ectosomes, membrane particles, exosome-like vesicles, or apoptotic vesicles. In embodiments, the secreted vesicles are exosomes. In embodiments, the secreted vesicles are microvesicles. In embodiments, the secreted vesicles contain or comprise one or more cellular components. In embodiments, the components may be one or more of the following: membrane lipids, RNA, proteins, metabolites, cytosolic components, and any combination thereof. In embodiments, the secreted vesicles comprise one or more microRNAs. In embodiments, the one or more miRNAs include one or any combination of RNA (e.g., miRNA) molecules disclosed herein. In embodiments, the vesicles comprise miRNAs that inhibit plasminogen activator inhibitor-1 (PAI-1) and / or TGFβ1.In embodiments, the secreted product comprises a paracrine and / or juxta-crine factor such as α1 microglobulin, β2 microglobulin, calbindin, clusterin, connective tissue growth factor, cystatin C, glutathione-S-transferase α, kidney injury molecule-1, neutrophil gelatinase-associated lipocalin, osteopontin, trefoil factor 3, Tamm-Horsfall urinary glycoprotein, tissue inhibitor of metalloproteinases 1, vascular endothelial growth factor, fibronectin, interleukin-6, or monocyte chemotactic protein-1.

[0093] In embodiments, the effect may be caused by the cells themselves and / or by products secreted by the cells. In embodiments, the regenerative effect may be characterized by one or more of the following: a decrease in epithelial-mesenchymal transition (which may be via attenuation of TGF-β signaling), a reduction in renal fibrosis, a reduction in nephritis, a differential expression of stem cell markers in the native kidney, migration of transplanted cells and / or native cells to sites of renal injury, e.g., tubular injury, engraftment of transplanted cells at sites of renal injury, e.g., tubular injury, a stabilization of one or more indicators of renal function (as described herein), de novo formation of S-body / C-body associated with nephrogenesis, de novo formation of renal tubules or nephrons, restoration of erythroid homeostasis (as described herein), and any combination thereof (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, respectively. (The entire contents of which are incorporated herein by reference).

[0094] In embodiments, in addition to or as an alternative to a tissue biopsy, regenerative outcomes in treated subjects can be assessed from examination of bodily fluids, such as urine. Microvesicles obtained from a subject's urine source have been found to contain certain components, including, but not limited to, specific proteins and miRNAs that ultimately derive from renal cell populations. In embodiments, these components may include factors involved in stem cell replication and differentiation, apoptosis, inflammation, and immune regulation. In embodiments, temporal analysis of microvesicle-associated miRNA / protein expression patterns allows for continuous monitoring of regenerative outcomes within the kidneys of subjects administered with the cell populations or constructs described herein.

[0095] Also provided are methods for assessing whether a patient with renal disease will respond to treatment with a therapeutic formulation. In embodiments, the methods may include measuring or detecting the amount of vesicles or one or more luminal contents thereof in a test sample obtained from a patient with renal disease treated with a therapeutic agent, compared to or relative to the amount of vesicles in a control sample, wherein a greater or lesser amount of vesicles or one or more luminal contents thereof in the test sample compared to the amount of vesicles or their luminal content(s) in the control sample is an indication of the treated patient's response to treatment with the therapeutic agent.

[0096] In embodiments, these kidney-derived vesicles and / or the luminal contents of the kidney-derived vesicles can be released into the urine of a subject and analyzed for biomarkers indicative of regenerative outcome or therapeutic efficacy. In embodiments, non-invasive prognostic methods can include obtaining a urine sample from a subject before and / or after administration or transplantation of a cell population, composition, formulation, cell product, or construct described herein. Vesicles and other secretory products can be separated by methods such as, but not limited to, centrifugation to remove unwanted debris (Zhou et al. 2008. Kidney Int. 74(5):613-621; Skog et al., U.S. Patent Application Publication No. 20110053157). , each of which is incorporated herein by reference in its entirety), can be isolated from urine samples using standard techniques.

[0097] In embodiments, the vesicles may include one or more of the following: paracrine factors, endocrine factors, junctional factors, microvesicles, exosomes, and RNA. Secreted products may also include products not present in microvesicles, including, but not limited to, paracrine factors, endocrine factors, junctional factors, and RNA.

[0098] In embodiments, the secreted product may be part of a vesicle derived from a kidney cell. In embodiments, the vesicle is a secreted vesicle. In embodiments, the secreted vesicle is an exosome, a microvesicle, an ectosome, a membrane particle, an exosome-like vesicle, or an apoptotic vesicle. In embodiments, the secreted vesicle is an exosome. In embodiments, the secreted vesicle is a microvesicle. In embodiments, the secreted vesicle contains or comprises one or more cellular components. In embodiments, the components may be one or more of the following: membrane lipids, RNA, proteins, metabolites, cytosolic components, and any combination thereof. In embodiments, the secreted vesicle comprises one or more microRNAs. In embodiments, the one or more miRNAs include one or any combination of miR-30b-5p, miR-449a, miR-146a, miR-130a, miR-23b, miR-21, miR-124, and miR-151. In embodiments, the one or more miRNAs include let-7a-1, let-7a-2, let-7a-3, let-7b, let-7c, let-7d, let-7e, let-7f-1, let-7f-2, let-7g, let-7i, mir-1-1, mir-1-2, mir-7-1, mir-7-2, mir-7-3, mir-9-1, mir-9-2, mir-9-3, mir-10a, mir-10b, mir-15a, mir-15b, mir-16-1, mir-17-2, mir-18-3, mir-19-4, mir-20-4, mir-21-4, mir-22-4, mir-23-4, mir-24-4, mir-25-4, mir-26-4, mir-27-4, mir-28-4, mir-29-5, mir-29-6, mir-29-7, mir-29-8, mir-29-9, mir-30-5, mir-31-5, mir-32-5, mir-33-5, mir-34-5, mir-35-5, mir-36-5, mir-37-5, mir-38-5, mir-39-6, mir-39-7, mir-39-8, mir-39-9, mir-30-10, mir-31-11, mir-32-12, mir-33-13, mir-34-14, mir-35-15, mir-36-16, mir-37-17, mir-38-18, mir-39-19, mir-39-20, mir-3 r-16-2, mir-17, mir-18a, mir-18b, mir-19a, mir-19b-1, mir-19b-2, mir-20a, mir-20b, mir-21, mir-22, mir-23a, mir-23b, mir-23c, mir-24-1, mir-24-2, mir-25, mir-26a-1, mir-26a-2, mir-26b, mir-27a, mir-27b, mir-28, m IR-29a, IR-29b-1, IR-29b-2, IR-29c, IR-30a, IR-30b, IR-30c-1, IR-30c-2, IR-30d, IR-30e, IR-31, IR-32, IR-33a, IR-33b, IR-34a, IR-34b, IR-34c, IR-92a-1, IR-92a-2, IR-92b, IR-93, IR-95, IR-96 mir-98, mir-99a, mir-99b, mir-100, mir-101-1, mir-101-2, mir-103-1, mir-103-1-as, mir-103-2, mir-103-2-as, mir-105-1, mir-105-2, mir-106a, mir-106b, mir-107, mir-122, mir-124-1, mir-124-2, mir-124-3, mir-125a mir-125b-1, mir-125b-2, mir-126, mir-127, mir-128-1, mir-128-2, mir-129-1, mir-129-2, mir-130a, mir-130b, mir-132, mir-132, mir-133a-1, mir-133a-2, mir-133b, mir-134, mir-135a-1, mir-135a-2, mir-135b, mir-136 MI101351120、mir-137、mir-138-1、mir-138-2、mir-139、mir-140、mir-141、mir-142、mir-143、mir-144、mir-145、mir-146a、mir-146b、mir-147、mir-147b、mir-148a、mir-148b、mir-149、mir-150、mir-151、mir-152、mir-153-1、mir-153-2、mir-154、mir-155、mir-181a-1、mir-181a-2、mir-181b-1, mir-181b-2, mir-181c, mir-181d, mir-182, mir-183, mir-184, mir-185, mir-186, mir-187, mir-188, mir-190, mir-190b, mir-191, mir -192、mir-193a、mir-193b、mir-194-1、mir-194-2、mir-195、mir-196a-1、mir-196a-2、mir-196b、mir-197、mir-198、mir-199a-1、mir-199a-2、mir- 199b、mir-200a、mir-200b、mir-200c、mir-202、mir-203、mir-204、mir-205、mir-206、mir-208a、mir-208b、mir-210、mir-211、mir-212、mir-214、mi r-215, mir-216a, mir-216b, mir-217, mir-218-1, mir-218-2, mir-219-1, mir-219-2, mir-221, mir-222, mir-223, mir-224, mir-296, mir-297, mir-2 98, mir-299, mir-300, mir-301a, mir-301b, mir-302a, mir-302b, mir-302c, mir-302d, mir-302e, mir-302f, mir-320a, mir-320b-1, mir-320b-2, mi r-320c-1, mir-320c-2, mir-320d-1, mir-320d-2, mir-320e, mir-323, mir-323b, mir-324, mir-325, mir-326, mir-328, mir-329-1, mir-329-2, mir-3 30, mir-331, mir-335, mir-337, mir-338, mir-339, mir-340, mir-342, mir-345, mir-346, mir-361, mir-362, mir-363, mir-365-1, mir-365-2, mir-3 67, mir-369, mir-370, mir-37, mir-372, mir-373, mir-374a, mir-374b, mir-374c, mir-375, mir-376a-1, mir-376a-2, mir-376b, mir-376c, mir-377mir-378、 Mir-378b, Mir-378c, Mir-379, Mir-380, Mir-381, Mir-382, Mir-383, Mir-384, Mir-409, Mir-410, Mir-411, Mir-412, Mir-421, Mir-422a, Mir-423 mir-424, mir-425, mir-429, mir-431, mir-432, mir-433, mir-448, mir-449a, mir-449b, mir-449c, mir-450a-1, mir-450a-2, mir-450b, mir-451, mi r-452、mir-454、mir-455、mir-466、mir-483、mir-484、mir-485、mir-486、mir-487a、mir-487b、mir-488、mir-489、mir-490、mir-491、mir-492、mir- 493, 494, 495, 496, 497, 498, 499, 500a, 500b, 501, 502, 503, 504, 505, 506, 507 mir-508, mir-509-1, mir-509-2, mir-509-3, mir-510, mir-511-1, mir-511-2, mir-512-1, mir-512-2, mir-513a-1, mir-513a-2, mir-513b, mir-51 3c、mir-514-1、mir-514-2、mir-514-3、mir-514b、mir-515-1、mir-515-2、mir-516a-1、mir-516a-2、mir-516b-1、mir-516b-2、mir-517a、mir-517b、m ir-517c、mir-518a-1、mir-518a-2、mir-518b、mir-518c、mir-518d、mir-518e、mir-518f、mir-519a-1、mir-519a-2、mir-519b、mir-519c、mir-519d、 mir-519e, mir-520a, mir-520b, mir-520c, mir-520d, mir-520e, mir-520f, mir-520g, mir-520h, mir-521-1, mir-521-2, mir-522, mir-523, mir-524mir-525, mir-526a-1, mir-526a-2, mir-526b, mir-527, mir-532, mir-539, mir-541, mir-542, mir-543, mir-544, mir-544b, mir-545, mir-548a-1, m ir-548a-2、mir-548a-3、mir-548ah-1、mir-548ah-2、mir-548b、mir-548c、mir-548d-1、mir-548d-2、mir-548e、mir-548f-1、mir-548f-2、mir-548f- 3、mir-548f-4、mir-548f-5、mir-548g、mir-548h-1、mir-548h-2、mir-548h-3、mir-548h-4、mir-548i-1、mir-548i-2、mir-548i-3、mir-548i-4、mir- 548j、mir-548k、mir-5481、mir-548m、mir-548n、mir-548o、mir-548p、mir-548s、mir-548t、mir-548u、mir-548v、mir-548w、mir-548x、mir-548y、mir -548z、mir-549、mir-550a-1、mir-550a-2、mir-550b-1、mir-550b-2、mir-551a、mir-551b、mir-552、mir-553、mir-554、mir-555、mir-556、mir-557、m ir-558, mir-559, mir-561, mir-562, mir-563, mir-564, mir-566, mir-567, mir-568, mir-569, mir-570, mir-571, mir-572, mir-573, mir-574, mir-57 5、mir-576、mir-577、mir-578、mir-579、mir-580、mir-581、mir-582、mir-583、mir-584、mir-585、mir-586、mir-587、mir-588、mir-589、mir-590、mir -591、mir-592、mir-593、mir-595、mir-596、mir-597、mir-598、mir-599、mir-600、mir-601、mir-602、mir-603、mir-604、mir-605、mir-606、mir-607、mir-60、 8, mir-609, mir-610, mir-611, mir-612, mir-613, mir-614, mir-615, mir-616, mir-617, mir-618, mir-619, mir-620, mir-621, mir-622, mir-623, mir-624, mir-625, mir-626, mir-627, mir-628, mir-629, mir-630, mir-631, mir-632, mir-633, mir-634, mir-635, mir-636, mir-637, mir-63 8、mir-639、mir-640、mir-641、mir-642a、mir-642b、mir-643、mir-644、mir-645、mir-646、mir-647、mir-648、mir-649、mir-650、mir-651、mir-652、mir-653、mir-654、mir-655、mir-656、mir-657、mir-658、mir-659、mir-660、mir-661、mir-662、mir-663、mir-663b、mir-664、mir-665、mir- 668, mir-670, mir-671, mir-675, mir-676, mir-708, mir-711, mir-718, mir-720, mir-744, mir-758, mir-759, mir-760, mir-761, mir-762, mir-764 mir-765, mir-766, mir-767, mir-769, mir-770, mir-802, mir-873, mir-874, mir-875, mir-876, mir-877, mir-885, mir-887, mir-888, mir-889, mir-8 90, mir-891a, mir-891b, mir-892a, mir-892b, mir-920, mir-921, mir-922, mir-924, mir-933, mir-934, mir-935, mir-936, mir-937, mir-938, mir-9 39, mir-940, mir-941-1, mir-941-2, mir-941-3, mir-941-4, mir-942, mir-942, mir-943, mir-944, mir-1178, mir-1179, mir-1180, mir-1181, mir-11 82、mir-1183、mir-1184-1、mir-1184-2、mir-1184-3、mir-1185-1、mir-1185-2、mir-1193、mir-1197、mir-1200、mir-1202、mir-1203、mir-1204、mir -1205、mir-1206、mir-1207、mir-1208、mir-1224、mir-1225、mir-1226、mir-1227、mir-1228、mir-1229、mir-1231、mir-1233-1、mir-1233-2、mir-123 4、mir-1236、mir-1237、mir-1238、mir-1243、mir-1244-1、mir-1244-2、mir-1244-3、mir-1245、mir-1246、mir-1247、mir-1248、mir-1249、mir-1250、 mir-1251, mir-1252, mir-1253, mir-1254, mir-1255a, mir-1255b-1, mir-1255b-2, mir-1256, mir-1257, mir-1258, mir-1260, mir-1260b, mir-1261mir-1262, mir-1263, mir-1264, mir-1265, mir-1266, mir-1267, mir-1268, mir-1269, mir-1270-1, mir-1270-2, mir-1271, mir-1272, mir-1273, mir-1273c, mir-1273d, mir-1273e, mir-1274a, mir-1274b, mir-1275, mir-1276, mir-1277, mir-1278, mir-1279, mir-12 80、mir-1281、mir-1282、mir-1283-1、mir-1283-2、mir-1284、mir-1285-1、mir-1285-2、mir-1286、mir-1287、mir-1288、mir-1289-1、mir-1289-2、mir-1290、mir-1291、mir-1292、mir-1293、mir-1294、mir-1295、mir-1296、mir-1297、mir-1298、mir-1299、mir-1301、 、mir-1302-1、mir-1302-10、mir-1302-11、mir-1302-2、mir-1302-3、mir-1302-4、mir-1302-5、mir-1302-6、mir-1302-7、mir-1302-8、mir-1302-9、 mir-1303, mir-1304, mir-1305, mir-1306, mir-1307, mir-1321, mir-1322, mir-1323, mir-1324, mir-1468, mir-1469, mir-1470, mir-1471, mir-1537 Mir-1538, Mir-1539, Mir-1825, Mir-1827, Mir-1908, Mir-1909, Mir-1910, Mir-1911, Mir-1912, Mir-1913, Mir-1914, Mir-1915, Mir-1972-1, Mir-1 972-2、mir-1973、mir-1976、mir-2052、mir-2053、mir-2054、mir-2110、mir-2113、mir-2114、mir-2115、mir-2116、mir-2117、mir-2276、mir-2277、mi r-2278, mir-2355, mir-2861, mir-2909, mir-3065, mir-3074, mir-3115, mir-3116-1, mir-3116-2, mir-3117, mir-3118-1, mir-3118-2, mir-3118-3 、mir-3118-4、mir-3118-5、mir-3118-6、mir-3119-1;mir-3119-2、mir-3120、mir-3121、mir-3122、mir-3123、mir-3124、mir-3125、mir-3126、mir-31 27, mir-3128, mir-3129, mir-3130-1, mir-3130-2, mir-3131, mir-3132, mir-3133, mir-3134, mir-3135, mir-3136, mir-3137, mir-3138, mir-3139, m ir-3140, mir-3141, mir-3142, mir-3143, mir-3144, mir-3145, mir-3146, mir-3147, mir-3148, mir-3149, mir-3150, mir-3151, mir-3152, mir-3153mir-3154, mir-3155, mir-3156-1, mir-3156-2, mir-3156-3, mir-3157, mir-3158-1, mir-3158-2, mir-3159, mir-3160-1, mir-3160-2, mir-3161, mi r-3162、mir-3163、mir-3164、mir-3165、mir-3166、mir-3167、mir-3168、mir-3169、mir-3170、mir-3171、mir-3173、mir-3174、mir-3175、mir-3176、 mir-3177, mir-3178, mir-3179-1, mir-3179-2, mir-3179-3, mir-3180-1, mir-3180-2, mir-3180-3, mir-3180-4, mir-3180-5, mir-3181, mir-3182, mir-3183, mir-3184, mir-3185, mir-3186, mir-3187, mir-3188, mir-3189, mir-3190, mir-3191, mir-3192, mir-3193, mir-3194, mir-3195, mir-319 6、mir-3197、mir-3198、mir-3199-1、mir-3199-2、mir-3200、mir-3201、mir-3202-1、mir-3202-2、mir-3605、mir-3606、mir-3607、mir-3609、mir-36 10、mir-3611、mir-3612、mir-3613、mir-3614、mir-3615、mir-3616、mir-3617、mir-3618、mir-3619、mir-3620、mir-3621、mir-3622a、mir-3622b、mi r-3646、mir-3647、mir-3648、mir-3649、mir-3650、mir-3651、mir-3652、mir-3653、mir-3654、mir-3655、mir-3656mir-3657、mir-3658、mir-3659、m ir-3660、mir-3661、mir-3662、mir-3663、mir-3664、mir-3665、mir-3666、mir-3667、mir-3668、mir-3669、mir-3670、mir-3670、mir-3671、mir-3671、mir-3673、 mir-3673, mir-3675, mir-3675, mir-3676, mir-3663, mir-3677, mir-3678, mir-3679, mir-3680, mir-3681, mir-3682, mir-3683, mir-3684, mir-368 5、mir-3686、mir-3687、mir-3688、mir-3689a、mir-3689b、mir-3690、mir-3691、mir-3692、mir-3713、mir-3714、mir-3907、mir-3908、mir-3909、mir -3910-1、mir-3910-2、mir-3911、mir-3912、mir-3913-1、mir-3913-2、mir-3914-1、mir-3914-2、mir-3915、mir-3916、mir-3917、mir-3918、mir-391 9、mir-3920、mir-3921、mir-3922、mir-3923、mir-3924、mir-3925、mir-3926-1、mir-3926-2、mir-3927、mir-3928、mir-3929、mir-3934、mir-3935、mi r-3936、mir-3937、mir-3938、mir-3939、mir-3940、mir-3941、mir-3942、mir-3943、mir-3944、mir-3945、mir-4251、mir-4252、mir-4253、mir-4254、 mir-4255, mir-4256, mir-4257, mir-4258, mir-4259, mir-4260, mir-4261, mir-4262, mir-4263, mir-4264, mir-4265, mir-4266, mir-4267, mir-426 8, mir-4269, mir-4270, mir-4271, mir-4272, mir-4273, mir-4274, mir-4275, mir-4276, mir-4277, mir-4278, mir-4279, mir-4280, mir-4281, mir-4 282、mir-4283-1、mir-4283-2、mir-4284、mir-4285、mir-4286、mir-4287、mir-4288、mir-4289、mir-4290、mir-4291、mir-4292、mir-4293、mir-4294、mir-4295, mir-4296, mir-4297, mir-4298, mir-4299, mir-4300, mir-4301, mir-4302, mir-4303, mir-4304, m ir-4305, mir-4306, mir-4307, mir-4308, mir-4309, mir-4310, mir-4311, mir-4312, mir-4313, mir-4314, mi The present invention also includes one or any combination of mir-4315-1, mir-4315-2, mir-4316, mir-4317, mir-4318, mir-4319, mir-4320, mir-4321, mir-4322, mir-4323, mir-4324, mir-4325, mir-4326, mir-4327, mir-4328; mir-4329, mir-4329, and mir-4330.

[0099] In embodiments, the miRNAs include any one or more of the following: miR-21, miR-23a, miR-30c, miR-1224, miR-23b, miR-92a, miR-100, miR-125b-5p, miR-195, miR-10a-5p, and any combination thereof. In embodiments, the miRNAs include any one or more of the following: miR-30b-5p, miR-449a, miR-146a, miR-130a, miR-23b, miR-21, miR-124, miR-151, and any combination thereof. In embodiments, the miRNAs include any one or more of the following: miR-24, miR-195, miR-871, miR-30b-5p, miR-19b, miR-99a, miR-429, let-7f, miR-200a, miR-324-5p, miR-10a-5p, and any combination thereof. In embodiments, the combination of miRNAs can include two, three, four, five, six, seven, eight, nine, ten, or more individual miRNAs.

[0100] In embodiments, the secreted product comprises a compound that attenuates the NFκB signaling pathway.

[0101] In embodiments, the secreted product comprises a paracrine factor. In embodiments, a paracrine factor is a molecule synthesized by a cell that can diffuse over short distances to induce or cause changes in adjacent cells, i.e., a paracrine interaction. In embodiments, diffusible molecules are referred to as paracrine factors. In embodiments, juxtacrine factors are molecules that facilitate intercellular communication transmitted through oligosaccharide, lipid, or protein components of the cell membrane and can affect either the releasing cell or an immediately adjacent cell. In embodiments, juxtacrine signaling typically involves physical contact between the two cells involved.

[0102] In embodiments, the vesicles comprise miRNA that inhibits plasminogen activator inhibitor-1 (PAI-1) and / or TGFβ1.

[0103] In embodiments, the secreted product comprises a paracrine and / or juxta-crine factor such as α1 microglobulin, β2 microglobulin, calbindin, clusterin, connective tissue growth factor, cystatin C, glutathione-S-transferase α, kidney injury molecule-1, neutrophil gelatinase-associated lipocalin, osteopontin, trefoil factor 3, Tamm-Horsfall urinary glycoprotein, tissue inhibitor of metalloproteinases 1, vascular endothelial growth factor, fibronectin, interleukin-6, or monocyte chemotactic protein-1.

[0104] In embodiments, the effective treatment of kidney disease in a subject using the methods disclosed herein can be monitored through various indicators of erythropoiesis and / or kidney function. In embodiments, indicators of erythrocyte homeostasis include, but are not limited to, hematocrit (HCT), hemoglobin (HB), mean corpuscular hemoglobin (MCH), red blood cell count (RBC), reticulocyte count, % reticulocytes, mean corpuscular volume (MCV), and red blood cell distribution width (RDW). In embodiments, indicators of kidney function include, but are not limited to, serum albumin, albumin-to-globulin ratio (A / G ratio), serum phosphorus, serum sodium, kidney size (measurable by ultrasound), serum calcium, phosphorus:calcium ratio, serum potassium, proteinuria, urinary creatinine, serum creatinine, blood urea nitrogen (BUN), cholesterol level, triglyceride level, and glomerular filtration rate (GFR). Additionally, some indicators of general health and well-being include, but are not limited to, weight gain or loss, survival rate, blood pressure (mean systemic blood pressure, diastolic blood pressure, or systolic blood pressure), and physical endurance capacity.

[0105] In embodiments, effective treatment with a bioactive renal cell preparation is evidenced by stabilization of one or more indicators of renal function. In embodiments, stabilization of renal function is demonstrated by observing a change in an indicator in a subject treated by a method provided herein compared to the same indicator in a subject not treated by the method. In embodiments, stabilization of renal function can be demonstrated by observing a change in the same indicator in the same subject treated by a method provided herein compared to the same indicator in the subject before treatment. In embodiments, the change in one indicator can be an increase or decrease in value. In embodiments, treatments provided by the present disclosure can include stabilization of blood urea nitrogen (BUN) levels in a subject, where the BUN levels observed in the subject are lower compared to subjects with a similar condition not treated by a method of the present disclosure. In embodiments, the treatments can include stabilization of serum creatinine levels in a subject, where the serum creatinine levels observed in the subject are lower compared to subjects with a similar condition not treated by a method of the present disclosure. In embodiments, the treatment may include stabilizing hematocrit (HCT) levels in the subject, wherein the HCT levels observed in the subject are higher compared to subjects with similar conditions not treated by the methods of the present disclosure. In embodiments, the treatment may include stabilizing red blood cell (RBC) levels in the subject. wherein the RBC values ​​observed in the subject are higher compared to a subject with a similar condition who is not treated by the methods of the present disclosure. In embodiments, one or more additional indicators described herein or known in the art can be measured to determine effective treatment of kidney disease in a subject.

[0106] In embodiments, the regenerated native kidney may be characterized by a number of indicators, including, but not limited to, the development of function or capacity in the native kidney, the improvement of function or capacity in the native kidney, and the expression of certain markers in the native kidney. In embodiments, the development or improvement of function or capacity may be observed based on various indicators of erythroid homeostasis and kidney function described herein. In embodiments, the regenerated kidney is characterized by the differential expression of one or more stem cell markers. In embodiments, the stem cell marker may be one or more of the following: Sox2, UTF1, NODAL, PROM1, or CD133, CD24, and any combination thereof (see International Application PCT / US2011 / 036347 by Ilagan et al., incorporated herein by reference in its entirety). In embodiments, the expression of the stem cell marker(s) is upregulated compared to a control.

[0107] In embodiments, the cell populations described herein, including enriched cell populations and / or mixtures thereof, and constructs comprising the same, can be used to provide a regenerative effect to the native kidney. In embodiments, the effect can be provided by the cells themselves and / or by products secreted by the cells. In embodiments, the regenerative effect can be characterized by one or more of the following: reduced epithelial-mesenchymal transition (which may be via attenuation of TGF-β signaling), reduced renal fibrosis, reduced nephritis, differential expression of stem cell markers in the native kidney, migration of transplanted cells and / or native cells to sites of renal injury, e.g., tubular injury, engraftment of transplanted cells at sites of renal injury, e.g., tubular injury, stabilization of one or more indicators of renal function (as described herein), restoration of erythroid homeostasis (as described herein), and any combination thereof.

[0108] In embodiments, therapeutic compositions or formulations provided herein contain isolated heterogeneous kidney cell populations enriched for particular bioactive components or cell types and / or depleted of particular inactive or undesirable components or cell types. In embodiments, such compositions and formulations are used in the treatment of kidney disease, e.g., resulting in stabilization and / or improvement and / or regeneration of kidney function and / or structure. In embodiments, the compositions contain isolated kidney cell fractions that lack cellular components compared to healthy individuals but retain therapeutic properties, e.g., resulting in stabilization and / or improvement and / or regeneration of kidney function. In embodiments, the cell populations described herein can be derived from healthy individuals, individuals suffering from kidney disease, or subjects described herein.

[0109] Included herein are therapeutic compositions of selected renal cell populations administered to a target organ or tissue in a subject. In embodiments, a selected bioactive renal cell population generally refers to a cell population that potentially has therapeutic properties upon administration to a subject. In embodiments, when administered to a subject in need, the bioactive renal cell population can result in stabilization and / or improvement and / or repair and / or regeneration of kidney function in the subject. In embodiments, the therapeutic properties can include a repair or regenerative effect.

[0110] In embodiments, the renal cell population is an unfractionated heterogeneous cell population or an enriched homogeneous cell population derived from the kidney. In embodiments, the heterogeneous cell population is isolated from a tissue biopsy or whole organ tissue. In 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 embodiments, the renal cell population is enriched for bioactive components (e.g., bioactive renal cells) and is free of inactive or undesired components. It includes a subfraction or subpopulation of a heterogeneous kidney cell population that has been depleted of components or cells.

[0111] In embodiments, the renal cell population expresses GGT and cytokeratin. In embodiments, GGT has an expression level of greater than about 10%, about 15%, about 18%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60%. In embodiments, the GGT is GGT-1. In embodiments, cells of the renal cell population express GGT-1, cytokeratin, VEGF, and KIM-1. In embodiments, greater than 18% of cells in the renal cell population express GGT-1. In embodiments, greater than 80% of cells in the renal cell population express cytokeratin. In embodiments, the cytokeratin is selected from CK8, CK18, CK19, and combinations thereof. In embodiments, the cytokeratin is CK8, CK18, CK19, CK8 / CK18, CK8 / CK19, CK18 / CK19, or CK8 / CK18 / CK19, where " / " refers to the combination of the cytokeratins adjacent to it. In embodiments, the cytokeratin has an expression level of greater than about 80%, about 85%, about 90%, or about 95%. In embodiments, greater than 80% of the cells in the renal cell population express the cytokeratin. In embodiments, the renal cell population expresses AQP2. In embodiments, less than 40% of the cells express AQP2. In embodiments, at least 3% of the cells in the renal cell population express AQP2.

[0112] In an 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 an embodiment, the cytokeratin is CK18. In an 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.

[0113] In embodiments, the renal cell population is selected from the group consisting of AQP1, AQP2, AQP4, calbindin, calponin, CD117, CD133, CD146, CD24, CD31 (PECAM-1), CD54 (ICAM-1), CD73, CK18, CK19, CK7, CK8, CK8, CK18, CK19, a combination of CK8, CK18, and CK19, connexin 43, cubilin, CXCR4 (fusin), DBA, E-cadherin (CD324), EPO (erythropoietin), GGT1, GLEPP1 (glomerular epithelial protein 1), haptoglobulin, Itgbl (integrin O1), KIM-1 (renal The present invention also includes cells expressing one or more of any combination of biomarkers selected from injury molecule-1, T1M-1 (T cell immunoglobulin and mucin-containing molecule), MAP-2 (microtubule-associated protein 2), megalin, N-cadherin, nephrin, NKCC (Na-K-Cl- cotransporter), OAT-1 (organic anion transporter 1), osteopontin, pan-cadherin, PCLP1 (podocalyxin-like 1 molecule), podocin, SMA (smooth muscle alpha-actin), synaptopodin, THP (Tamm-Horsfall protein), vimentin, and alphaGST-1 (alpha glutathione S-transferase).

[0114] In embodiments, the renal cell population is enriched for epithelial cells compared to a starting population, such as a cell population in a kidney tissue biopsy or primary culture thereof (e.g., the renal cell population comprises at least about 5%, 10%, 15%, 20%, or 25% more epithelial cells than the starting population). In embodiments, the renal cell population is enriched for tubular cells compared to a starting population, such as a cell population in a kidney tissue biopsy or primary culture thereof (e.g., the renal cell population comprises at least about 5%, 10%, 15%, 20%, or 25% more tubular cells than the starting population). In embodiments, the tubular cells comprise proximal tubule cells. In embodiments, the renal cell population has a lower proportion of distal tubule cells, collecting duct cells, endocrine cells, vascular cells, or progenitor-like cells compared to the starting population. In embodiments, the renal cell population has a lower proportion of distal tubule cells compared to the starting population. In embodiments, the renal cells The renal cell population has a lower proportion of collecting duct cells compared to the starting population. In embodiments, the renal cell population has a lower proportion of endocrine cells compared to the starting population. In embodiments, the renal cell population has a lower proportion of vascular cells compared to the starting population. In embodiments, the renal cell population has a lower proportion of progenitor-like cells compared to the starting population. In embodiments, the renal cell population has a higher proportion of tubular cells and a lower proportion of EPO-producing cells, glomerular cells, and vascular cells compared to a non-enriched population (e.g., the starting renal cell population). In embodiments, the renal cell population has a higher proportion of tubular cells and a lower proportion of EPO-producing cells and vascular cells compared to a non-enriched population. In embodiments, the renal cell population has a higher proportion of tubular cells and a lower proportion of glomerular cells and vascular cells compared to a non-enriched population.

[0115] In embodiments, the cells of the renal cell population express hyaluronic acid (HA). In embodiments, the size range of HA is from about 5 kDa to about 20,000 kDa. In embodiments, the HA has a molecular weight of 5 kDa, 60 kDa, 800 kDa, and / or 3,000 kDa. In embodiments, the renal cell population synthesizes and / or stimulates the synthesis of high molecular weight HA through expression of hyaluronan synthase 2 (HAS-2), particularly after intrarenal transplantation. In embodiments, the cells of the renal cell population express higher molecular weight species of HA in vitro and / or in vivo through the action of HAS-2. In embodiments, the cells of the renal cell population express higher molecular weight species of HA both in vitro and in vivo through the action of HAS-2. In embodiments, the higher molecular weight species of HA is HA having a molecular weight of at least 100 kDa. In an embodiment, the higher molecular weight species HA is HA having a molecular weight of about 800 kDa to about 3500 kDa. In an embodiment, the higher molecular weight species HA is HA having a molecular weight of about 800 kDa to about 3000 kDa. In an embodiment, the higher molecular weight species HA is HA having a molecular weight of at least 800 kDa. In an embodiment, the higher molecular weight species HA is HA having a molecular weight of at least 3000 kDa. In an embodiment, the higher molecular weight species HA is HA having a molecular weight of about 800 kDa. In an embodiment, the higher molecular weight species HA is HA having a molecular weight of about 3000 kDa. In an embodiment, the HAS-2 is 2×10 5 Da~2×10 6In embodiments, the smaller species of HA is formed by the action of a degradative hyaluronidase. In embodiments, the higher molecular weight species of HA is HA having a molecular weight of about 200 kDa to about 2000 kDa. In embodiments, the higher molecular weight species of HA is HA having a molecular weight of about 200 kDa. In embodiments, the higher molecular weight species of HA is HA having a molecular weight of about 200 kDa. In embodiments, the higher molecular weight species of HA is HA having a molecular weight of about 2000 kDa. In embodiments, the higher molecular weight species of HA is HA having a molecular weight of at least 200 kDa. In embodiments, the higher molecular weight species of HA is HA having a molecular weight of at least 2000 kDa. In embodiments, the higher molecular weight species of HA is HA having a molecular weight of at least 5000 kDa. In embodiments, the higher molecular weight species of HA is HA having a molecular weight of at least 10000 kDa. In embodiments, the higher molecular weight species of HA is HA having a molecular weight of at least 15000 kDa. In an embodiment, the higher molecular weight species of HA is HA having a molecular weight of about 20,000 kDa.

[0116] In an embodiment, the population comprises cells capable of receptor-mediated albumin transport.

[0117] In embodiments, the cells of the renal cell population are hypoxia tolerant.

[0118] In embodiments, the renal cell population comprises one or more cell types that express one or more of any combination of megalin, cubilin, N-cadherin, E-cadherin, aquaporin-1, and aquaporin-2.

[0119] In embodiments, the renal cell population comprises one or more cell types that express any combination of one or more of megalin, cubilin, hyaluronate synthase 2 (HAS2), vitamin D3 25-hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin (Ecad), aquaporin-1 (Aqp1), aquaporin-2 (Aqp2), RAB17, RAS oncogene family member (Rab17), GATA binding protein 3 (Gata3), FXYD domain-containing ion transport regulator 4 (Fxyd4), solute carrier family 9 (sodium / hydrogen exchanger) member 4 (Slc9a4), aldehyde dehydrogenase 3 family member B1 (Aldh3b1), aldehyde dehydrogenase 1 family member A3 (Aldh1a3), and calpain-8 (Capn8).

[0120] In embodiments, the renal cell population comprises one or more cell types that express one or more of megalin, cubilin, hyaluronate synthase 2 (HAS2), vitamin D3 25-hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin (Ecad), aquaporin-1 (Aqp1), aquaporin-2 (Aqp2), RAB17, RAS oncogene family member (Rab17), GATA binding protein 3 (Gata3), FXYD domain-containing ion transport regulator 4 (Fxyd4), solute carrier family 9 (sodium / hydrogen exchanger) member 4 (Slc9a4), aldehyde dehydrogenase 3 family member 81 (Aldh3b1), aldehyde dehydrogenase 1 family member A3 (Aldh1a3), and any combination of calpain-8 (Capn8) and aquaporin-4 (Aqp4).

[0121] In embodiments, the renal cell population is a population of renal cells expressing aquaporin-7 (Aqp7), FXYD domain-containing ion transport regulator 2 (Fxyd2), solute carrier family 17 (sodium phosphate) member 3 (Slc17a3), solute carrier family 3 member 1 (Slc3a1), claudin 2 (Cldn2), napsin The present invention also includes one or more cell types expressing any combination of one or more of the following: A-aspartic peptidase (Napsa), solute carrier family 2 (facilitated glucose transporter) member 2 (Slc2a2), alanyl (membrane) aminopeptidase (Anpep), transmembrane protein 27 (Tmem27), acyl-CoA synthase medium chain family member 2 (Acsm2), glutathione peroxidase 3 (Gpx3), fructose-1,6-bisphosphatase 1 (Fbp1), alanine-glyoxylate aminotransferase 2 (Agxt2), platelet endothelial cell adhesion molecule (Pecam), and podocin (Podn).

[0122] In embodiments, the renal cell population expresses PECAM, VEGF, KDR, HIF1a, CD31, CD146, podocin (Podn), and nephrin (Neph), chemokine (CXC motif) receptor 4 (Cxcr4), endothelin receptor type B (Ednrb), type V collagen alpha 2 (Col5a2), cadherin 5 (Cdh5), tissue plasminogen activator (Plat), angiopoietin 2 (Angpt2), kinase insert domain protein (KIP). The present invention also includes one or more cell types that express any combination of one or more of the following: protein receptor (Kdr), secreted cysteine-rich acidic protein (osteonectin) (Sparc), serglycin (Srgn), TIMP metallopeptidase inhibitor 3 (Timp3), Wilms' tumor 1 (Wt1), wingless MMTV integration site family member 4 (Wnt4), regulator of G-protein signaling 4 (Rgs4), and erythropoietin (EPO).

[0123] In embodiments, the renal cell population comprises one or more cell types that express one or more of any combination of PECAM, vEGF, KDR, HIF1a, podocin, nephrin, EPO, CK7, CK8 / 18 / 19.

[0124] In embodiments, the renal cell population comprises one or more cell types that express one or more of any combination of PECAM, vEGF, KDR, HIF1a, CD31, CD146.

[0125] In embodiments, the renal cell population comprises one or more cell types that express one or more of any combination of podocin (Podn) and nephrin (Neph).

[0126] In embodiments, the renal cell population comprises one or more cell types that express one or more of any combination of PECAM, vEGF, KDR, HIF1a, and EPO.

[0127] In embodiments, the presence (e.g., expression) and / or level / amount of various biomarkers in a sample or cell population can be analyzed by a number of techniques, many of which are known in the art and understood by those of skill in the art, including, but not limited to, immunohistochemistry ("IHC"), Western blot analysis, immunoprecipitation, molecular binding assays, ELISA, ELIFA, fluorescence activated cell sorting ("FACS"), MassARRAY, proteomics, biochemical enzyme activity assays, in situ hybridization, Southern analysis, Northern analysis, whole genome sequencing, polymerase chain reaction ("PCR") including quantitative real-time PCR ("qRT-PCR") and other amplification-based detection methods (e.g., branched-DNA, SISBA, TMA, etc.), RNA-Seq, FISH, microarray analysis, gene expression profiling, and / or serial analysis of gene expression ("SAGE"), as well as any one of a wide variety of assays that may be performed by protein, gene, and / or tissue array analysis. Non-limiting examples of protocols for assessing the status of genes and gene products include Northern blotting, Southern blotting, immunoblotting, and PCR analysis. In embodiments, multiplex immunoassays, such as those available from Rules Based Medicine or Meso Scale Discovery, can also be used. In embodiments, the presence (e.g., expression) and / or level / amount of various biomarkers in a sample or cell population can be analyzed by a number of techniques, many of which are known in the art and understood by those skilled in the art, including, but not limited to, "-omics" platforms such as genome-wide transcriptomics, proteomics, secretomics, lipidomics, phosphatomics, and exomics, where high-throughput techniques, coupled with computational biology and bioinformatics techniques, reveal the complete biological signature of genes, miRNAs, proteins, secreted proteins, lipids, and the like, expressed and not expressed by the cell population under study.

[0128] In embodiments, a method for detecting the presence of two or more biomarkers in a renal cell population includes contacting a sample with an antibody to the biomarker under conditions that allow binding of the antibody to its cognate ligand (i.e., biomarker), and detecting the presence of the bound antibody, e.g., by detecting whether a complex forms between the antibody and the biomarker. In embodiments, the presence of one or more biomarkers is detected by immunohistochemistry. As used herein, the term "detecting" includes quantitative detection and / or qualitative detection.

[0129] In embodiments, the renal cell population expresses or expresses a biomarker disclosed herein, such as AQP1, AQP2, AQP4, calbindin, calponin, CD117, CD133, CD146, CD24, CD31 (PECAM-1), CD54 (ICAM-1), CD73, CK18, CK19, CK7, CK8, CK8 / 18, CK8 / 18 / 19, connexin 43, cubilin, CXCR4 (fusin), DBA, E-cadherin (CD324), EPO (erythropoietin), GGT1, GLEPP1 (glomerular epithelial protein 1), haptoglobulin, Itgbl (integrin p), KIM-1 (kidney injury molecule-1), or the like. ), T1M-1 (T-cell immunoglobulin and mucin-containing molecule), MAP-2 (microtubule-associated protein 2), megalin, N-cadherin, nephrin, NKCC (Na-K-Cl- cotransporter), OAT-1 (organic anion transporter 1), osteopontin, pan-cadherin, PCLP1 (podocalyxin-like 1 molecule), podocin, SMA (smooth muscle α-actin), synaptopodin, THP (Tamm-Horsfall protein), vimentin, and αGST-1 (alpha glutathione 5-transferase). In embodiments, the biomarkers are detected by one or more reagents that allow for the detection of:

[0130] In embodiments, the source of the cells is the same as the intended target organ or tissue. In embodiments, the BRCs or SRCs can be derived from the kidney for use in formulations administered to the kidney. In embodiments, the cell population is derived from a kidney biopsy. In embodiments, the cell population is derived from whole kidney tissue. In embodiments, the cell population is derived from an in vitro culture of mammalian kidney cells established from a kidney biopsy or whole kidney tissue.

[0131] In embodiments, BRCs or SRCs comprise a heterogeneous mixture or fraction of bioactive renal cells. In embodiments, BRCs or SRCs can be derived from a healthy individual, or are renal cell fractions themselves from a healthy individual. In embodiments, included herein are renal cell populations or fractions obtained from unhealthy individuals (e.g., in the kidney or a biopsy thereof) that may lack certain cell types compared to renal cell populations of healthy individuals. In embodiments, provided herein are therapeutically active cell populations that lack cell types compared to healthy individuals. In embodiments, the cell populations are isolated and expanded from an autologous cell population.

[0132] In embodiments, SRCs are obtained by isolating and expanding renal cells from a patient's renal cortical tissue via a kidney biopsy. In embodiments, renal cells are isolated from the kidney tissue by enzymatic digestion, expanded by standard cell culture techniques, and selected from the expanded renal cells by centrifugation across a density boundary, density barrier, or density interface. In embodiments, renal cells are isolated from the kidney tissue by enzymatic digestion, expanded by standard cell culture techniques, and selected from the expanded renal cells by continuous or discontinuous single-step or multi-step density gradient centrifugation. In embodiments, SRCs are primarily composed of renal epithelial cells, which are known for their regenerative capacity. In embodiments, other parenchymal (vascular) and stromal cells may be present in the autologous SRC population.

[0133] In embodiments, bioactive renal cells are obtained from renal cells isolated from kidney tissue by enzymatic digestion and expanded by standard cell culture techniques. In embodiments, the cell culture medium is designed to expand bioactive renal cells with regenerative potential. In embodiments, the cell culture medium does not contain any recombinant or purified differentiation factors. In embodiments, the expanded heterogeneous renal cell mixture is cultured under hypoxic conditions to further enrich for cells with regenerative potential. Without wishing to be bound by theory, this may be due to one or more of the following phenomena: 1) selective survival, death, or proliferation of specific cellular components during the hypoxic culture period; 2) changes in cellular granularity and / or size in response to hypoxic culture, resulting in changes in buoyant density and subsequent changes in localization during density gradient separation or centrifugation across a density boundary, density barrier, or density interface; and 3) changes in cellular gene / protein expression in response to the hypoxic culture period, resulting in different characteristics of cells within the isolated and expanded population.

[0134] In embodiments, the bioactive renal cell population is obtained from the isolation and expansion of renal cells from kidney tissue (such as tissue obtained from a biopsy) under culture conditions that enrich for cells that enable kidney regeneration.

[0135] In embodiments, renal cells from kidney tissue (such as tissue obtained from a biopsy) are passaged one, two, three, four, five or more times to produce expanded bioactive renal cells (such as a cell population enriched for cells that enable kidney regeneration). In embodiments, renal cells from kidney tissue (such as tissue obtained from a biopsy) are passaged once to produce expanded bioactive renal cells. In embodiments, renal cells from kidney tissue (such as tissue obtained from a biopsy) are passaged two times to produce expanded bioactive renal cells. In embodiments, renal cells from kidney tissue (such as tissue obtained from a biopsy) are passaged three times to produce expanded bioactive renal cells. In embodiments, renal cells from kidney tissue (such as tissue obtained from a biopsy) are passaged four times to produce expanded bioactive renal cells. In embodiments, renal cells from kidney tissue (such as tissue obtained from a biopsy) are passaged five times to produce expanded bioactive renal cells. In embodiments, passaging the cells depletes the cell population of non-bioactive renal cells. In embodiments, passaging the cells depletes the cell population of at least one cell type. In embodiments, passaging the cells depletes the cell population of cells having a density greater than 1.095 g / ml. In embodiments, passaging the cells depletes the cell population of small cells with low granularity. In embodiments, passaging the cells depletes the cell population of cells smaller than red blood cells. In embodiments, passaging the cells depletes the cell population of cells having a diameter less than 6 μm. In embodiments, passaging the cells depletes the cell population of cells having a diameter less than 2 μm. In embodiments, passaging the cells depletes the cell population of cells having a granularity less than red blood cells. In embodiments, the viability of the cell population increases after one or more passagings. In embodiments, the terms small cells and low granularity are used when analyzing cells by fluorescence-activated cell sorting (FACs), for example, using the XY axis of a scatter plot of cell occurrence.

[0136] In embodiments, expanded bioactive renal cells are grown under hypoxic conditions for at least about 6 hours, 9 hours, 10 hours, 12 hours, or 24 hours but less than 48 hours, or for 6 to 9 hours, or 6 to 48 hours, or for about 12 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 embodiments, cells grown under hypoxic conditions are selected based on density. In embodiments, the bioactive renal cell population is a selected wiser cell (SRC) population obtained after continuous or discontinuous (single-step or multi-step) density gradient separation of expanded renal cells (e.g., after passaging and / or culture under hypoxic conditions). In embodiments, the bioactive renal cell population is a selected wiser cell (SRC) population obtained after centrifugation separation of expanded renal cells across a density boundary, density barrier, or density interface (e.g., after passaging and / or culture under hypoxic conditions). In embodiments, hypoxic culture conditions are culture conditions in which cells are exposed to a reduced level of available oxygen in a culture system compared to standard culture conditions in which cells are cultured at atmospheric oxygen levels (about 21%). In embodiments, cells cultured under hypoxic culture conditions are cultured at an oxygen level of about 5% to about 15%, or about 5% to about 10%, or about 2% to about 5%, or about 2% to about 7%, or about 2%, or about 3%, or about 4%, or about 5%. In embodiments, the SRC exhibits a buoyant density greater than about 1.0419 g / mL. In embodiments, the SRC exhibits a buoyant density greater than about 1.04 g / mL. In embodiments, the BRC or SRC contains a higher percentage of one or more cell populations and is devoid of or reduced in one or more other cell populations compared to the starting kidney cell population.

[0137] In embodiments, the expanded bioactive renal cells can be subjected to density gradient separation to obtain SRCs. In embodiments, continuous or discontinuous single-step or multi-step density gradient centrifugation is used to separate the harvested renal cell population based on cell buoyant density. In embodiments, the expanded bioactive renal cells can be separated by centrifugation across a density boundary, density barrier, or density interface to obtain SRCs. In embodiments, a density boundary or Centrifugation across a density boundary is used to separate the harvested renal cell population based on cell buoyant density. In embodiments, the SRC is performed in part using OPTIPREP (Axis-Shield) medium containing a 60% (weight / volume) aqueous solution of iodixanol, a non-ionic iodine compound. However, one skilled in the art will appreciate that other media, density gradients (continuous or discontinuous), density boundaries, density barriers, density interfaces, or other means, such as immunological separation using cell surface markers known in the art, having the characteristics necessary for isolating the cell populations described herein, can be used to obtain bioactive kidney cells. In embodiments, a cell fraction exhibiting a buoyant density greater than about 1.04 g / mL is collected as a separate pellet after centrifugation. In embodiments, cells maintaining a buoyant density less than 1.04 g / mL are rejected and discarded. In embodiments, a cell fraction exhibiting a buoyant density greater than about 1.0419 g / mL is collected as a separate pellet after centrifugation. In embodiments, cells maintaining a buoyant density less than 1.0419 g / mL are rejected and discarded. In embodiments, a cell fraction exhibiting a buoyant density greater than about 1.045 g / mL is collected as a separate pellet after centrifugation. In embodiments, cells maintaining a buoyant density less than 1.045 g / mL are rejected and discarded.

[0138] In embodiments, cell buoyant density is used to obtain an SRC population and / or determine whether a renal cell population is a bioactive renal cell population. In embodiments, cell buoyant density is used to isolate bioactive renal cells. In embodiments, cell buoyant density is determined by centrifugation across a single-stage OptiPrep (7% iodixanol, 60% (wt / vol) in OptiMEM) density interface (single-stage discontinuous density gradient). Optiprep is a 60% (wt / vol) aqueous solution of iodixanol. When used in an exemplary density interface or single-stage discontinuous density gradient, Optiprep is diluted with OptiMEM (basal cell culture medium) to form a final 7% iodixanol solution (in water and OptiMEM). The OptiMEM formulation is a modification of Eagle's minimum essential medium buffered with HEPES and sodium bicarbonate and supplemented with hypoxanthine, thymidine, sodium pyruvate, L-glutamine or GLUTAMAX, trace elements, and growth factors. Protein levels are minimal (15 μg / mL), with insulin and transferrin being the only protein supplements. Phenol red is included at low concentrations as a pH indicator. In embodiments, OptiMEM can be supplemented with 2-mercaptoethanol prior to use.

[0139] In embodiments, an OptiPrep solution is prepared and a refractive index indicative of the desired density is measured prior to use (refractive index 1.3456±0.0004). In embodiments, kidney cells are layered on top of the solution. In embodiments, the density interface or single-step discontinuous density gradient is centrifuged at 800 g for 20 minutes at room temperature (no brake) in either a centrifuge tube (e.g., a 50 ml conical tube) or a cell processing device (e.g., a COBE 2991). In embodiments, a cell fraction exhibiting a buoyant density greater than about 1.04 g / mL is collected as a separate pellet after centrifugation. In embodiments, cells maintaining a buoyant density less than 1.04 g / mL are discarded and discarded. In embodiments, a cell fraction exhibiting a buoyant density greater than about 1.0419 g / mL is collected as a separate pellet after centrifugation. In embodiments, cells maintaining a buoyant density less than 1.0419 g / mL are discarded and discarded. In embodiments, the cell fraction exhibiting a buoyant density greater than about 1.045 g / mL is collected as a separate pellet after centrifugation. In embodiments, cells maintaining a buoyant density less than 1.045 g / mL are discarded and discarded. In embodiments, prior to cell density assessment or density-based selection, cells are cultured until they are at least 50% confluent and incubated overnight (e.g., at least about 8 or 12 hours) at 37°C in a 5% CO environment in a hypoxic incubator set at 2% oxygen.

[0140] In embodiments, cells obtained from a kidney sample are expanded and then processed (e.g., by hypoxia and centrifugation) to obtain an SRC population. In embodiments, the SRC population is produced using the reagents and techniques described herein. In 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 embodiments, a sample of cells from the SRC population is tested for expression of one or more of the markers disclosed herein before the cells of the population are administered to a subject.

[0141] Non-limiting examples of compositions and methods for making SRCs are disclosed in U.S. Patent Application Publication No. 2017 / 0281684, the entire contents of which are incorporated herein by reference.

[0142] In embodiments, the BRCs or SRCs are derived from a natural autologous or allogeneic kidney sample. In embodiments, the BRCs or SRCs are derived from a non-autologous kidney sample. In embodiments, the sample may be obtained by kidney biopsy.

[0143] In embodiments, isolation and expansion of renal cells results in a mixture of renal cell types, including renal epithelial cells and interstitial cells. In embodiments, SRCs are obtained by continuous or discontinuous density gradient separation of expanded renal cells. In embodiments, the primary cell type in the density gradient-separated SRC population is of a tubular epithelial phenotype. In embodiments, SRCs are obtained by separation of expanded renal cells by centrifugation across a density boundary, density barrier, or density interface. In embodiments, the primary cell type in the SRC population separated across a density boundary / density barrier / density interface is of a tubular epithelial phenotype. In embodiments, the characteristics of SRCs obtained from expanded renal cells are evaluated using a multi-pronged approach. In embodiments, cell morphology, growth kinetics, and cell viability are monitored during the renal cell expansion process. In embodiments, the buoyant density and viability of SRCs are characterized by centrifugation on or through a density gradient medium and trypan blue exclusion. In embodiments, the SRC phenotype is characterized by flow cytometry, and SRC function is indicated by expression of VEGF and KIM-1. In embodiments, preformulation of SRC cellular function can also be assessed by measuring the activity of two specific enzymes found in the kidney proximal tubules, GGT (γ-glutamyl transpeptidase) and LAP (leucine aminopeptidase).

[0144] In embodiments, cell subpopulations can be separated via flow cytometry using cellular characteristics (size and granularity) that contribute to the separation of cell subpopulations via density media (forward scatter = reflecting size by flow cytometry, and side scatter = reflecting granularity). In embodiments, the density gradient or separation media should have low toxicity to the specific cells of interest. In embodiments, while the density media should have low toxicity to the specific cells of interest, the present disclosure contemplates the use of media that play a role in the selection process for cells of interest. In embodiments, without wishing to be bound by theory, cell populations disclosed herein recovered with media containing iodixanol appear to be iodixanol-resistant because there is a significant loss of cells between the loading and recovery steps, suggesting that exposure to iodixanol under conditions of a density gradient, or density boundary, density barrier, or density interface, results in the elimination of certain cells. In embodiments, cells emerging after iodixanol density gradient or density interface separation are resistant to any adverse effects of iodixanol and / or density gradient or interface exposure. In embodiments, imaging agents containing mild to moderate nephrotoxins are used to isolate and / or select cell populations, such as SRC populations. In embodiments, the SRCs are iodixanol resistant. In embodiments, the density medium should not bind to proteins in human plasma or adversely affect the primary function of the cells of interest.

[0145] In embodiments, the cell population is enriched and / or depleted for one or more kidney cell types using fluorescence-activated cell sorting (FACS). In embodiments, kidney cell types can be enriched and / or depleted using a BD FACSAria™ or equivalent. In embodiments, kidney cell types can be enriched and / or depleted using a FACSAria III™ or equivalent.

[0146] In embodiments, the cell population is enriched and / or depleted for one or more kidney cell types using magnetic cell sorting. In embodiments, the cell population can be enriched and / or depleted for one or more kidney cell types using Miltenyi's autoMACS™ system or equivalent.

[0147] In embodiments, a renal cell population is subjected to three-dimensional culture. In embodiments, the method of culturing the cell population is by continuous perfusion. In embodiments, cell populations cultured via three-dimensional culture and continuous perfusion exhibit greater cellularity and interconnectivity compared to statically cultured cell populations. In embodiments, cell populations cultured via three-dimensional culture and continuous perfusion exhibit enhanced expression of renal tubule-associated genes, such as E-cadherin, as well as greater EPO expression compared to static cultures of such cell populations. In embodiments, cell populations cultured via continuous perfusion exhibit higher levels of glucose and glutamine consumption compared to statically cultured cell populations.

[0148] In embodiments, low or hypoxic conditions can be used in the methods of producing cell populations provided herein. In embodiments, the methods of producing cell populations can be used without a step of low oxygen conditions. In embodiments, normoxic conditions can be used.

[0149] In embodiments, the renal cell population is isolated and / or cultured from kidney tissue. Non-limiting examples of methods for separating and isolating renal cell components, e.g., enriched cell populations, for use in therapeutic formulations, including those for treating kidney disease, anemia, EPO deficiency, impaired renal tubular transport, and impaired glomerular filtration, are disclosed herein. In embodiments, the cell populations are isolated from freshly digested, i.e., mechanically or enzymatically digested, kidney tissue, or from heterogeneous in vitro cultures of mammalian kidney cells.

[0150] In embodiments, the renal cell population comprises EPO-producing renal cells. In embodiments, the subject has anemia and / or EPO deficiency. In embodiments, the EPO-producing renal cell population is characterized by expression of EPO and biological responsiveness to oxygen, such that reduced oxygen tension in the culture system results in induced EPO expression. In embodiments, the EPO-producing cell population is enriched for EPO-producing cells. In embodiments, EPO expression is induced when the cell population is cultured under conditions in which the cells are exposed to reduced levels of available oxygen in the culture system, compared to a cell population cultured at standard atmospheric (approximately 21%) levels of available oxygen. In embodiments, EPO-producing cells cultured under lower oxygen conditions express higher levels of EPO, compared to EPO-producing cells cultured under normoxic conditions. Generally, culturing cells at reduced levels of available oxygen (also referred to as hypoxic culture conditions) means that the level of reduced oxygen is reduced compared to cell culture at standard atmospheric levels of available oxygen (also referred to as standard culture conditions or normoxic culture conditions). In embodiments, hypoxic cell culture conditions include culturing cells at less than about 1% oxygen, less than about 2% oxygen, less than about 3% oxygen, less than about 4% oxygen, or less than about 5% oxygen. In embodiments, normal or normoxic 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.

[0151] In embodiments, EPO induction or increased expression can be obtained and observed by culturing cells in less than about 5% available oxygen and comparing EPO expression levels with cells cultured in atmospheric oxygen (about 21%). In embodiments, EPO induction is obtained in a culture of cells capable of expressing EPO by a method comprising a first culture phase in which the cell culture is cultured in atmospheric oxygen (about 21%) for some period of time, and a second culture phase in which the same cells are cultured in less than about 5% available oxygen with reduced available oxygen levels. In embodiments, EPO expression in response to hypoxic conditions is regulated by HIF1α. In embodiments, other oxygen-manipulated culture conditions known in the art can be used for the cells described herein.

[0152] In embodiments, the formulation contains an enriched population of EPO-producing mammalian cells characterized by bioresponsiveness (e.g., EPO expression) to perfusion conditions. In embodiments, perfusion conditions include transient, intermittent, or continuous fluid flow (perfusion). In embodiments, EPO expression is mechanically induced when the medium in which the cells are cultured is circulated or agitated intermittently or continuously, such that dynamic forces are imparted to the cells via the flow. In embodiments, by culturing cells exposed to transient, intermittent, or continuous fluid flow, the cells exist as three-dimensional structures in or on a material that provides a scaffold and / or voids for the formation of the three-dimensional structure. In embodiments, the cells are cultured on porous beads and exposed to intermittent or continuous fluid flow via a rocking platform, an orbital platform, or a spinner flask. In embodiments, the cells are cultured on a three-dimensional scaffold and placed in a device that immobilizes the scaffold and allows fluid to flow directionally through or across the scaffold. Those skilled in the art will understand that other perfusion culture conditions known in the art can be used for the cells described herein.

[0153] In embodiments, the cell population is derived from a kidney biopsy. In embodiments, the cell population is derived from whole kidney tissue. In 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 embodiments, the kidney cell population is an SRC population. In embodiments, the cell population is an unfractionated cell population, also referred to herein as a non-enriched cell population.

[0154] Compositions containing various active agents (e.g., other than kidney cells) are included herein. Non-limiting examples of suitable active agents include, but are not limited to, cell aggregates, acellular biomaterials, secretory products from bioactive cells, macromolecular and small molecule therapeutics, and combinations thereof. For example, one bioactive cell type can be combined with a biomaterial-based microcarrier, with or without a therapeutic molecule or another bioactive cell type. In embodiments, non-adherent cells can be combined with acellular particles.

[0155] In embodiments, the cells of the renal cell population are present within spheroids. In embodiments, the renal cell population is present in the form of spheroids. In embodiments, spheroids comprising bioactive renal cells are administered to a subject. In embodiments, the spheroids comprise at least one non-renal cell type or cell population. In embodiments, the spheroids are produced by a method comprising: (i) combining a bioactive renal cell population and a non-renal cell population; and (ii) culturing the bioactive renal cell population and the non-renal cell population in a three-dimensional culture system comprising a spinner flask until spheroids form.

[0156] In embodiments, the non-renal cell population comprises an endothelial cell population or an endothelial progenitor cell population. In embodiments, the bioactive cell population is an endothelial cell population. In embodiments, the endothelial cell population The cell population is a cell lineage. In embodiments, the endothelial cell population comprises human umbilical vein endothelial cells (HUVECs). In embodiments, the non-renal cell population is a mesenchymal stem cell population. In embodiments, the non-renal cell population is a stem cell population of hematopoietic origin, breast origin, intestinal origin, placental origin, lung origin, bone marrow origin, blood origin, umbilical cord origin, endothelial origin, dental pulp origin, adipose origin, neural origin, olfactory organ origin, neural crest origin, or testicular origin. In embodiments, the non-renal cell population is an adipose-derived progenitor cell population. In embodiments, the cell population is a xenogeneic, syngeneic, allogeneic, autologous cell population, or combinations thereof. In embodiments, the bioactive renal cell population and the non-renal cell population are cultured at a ratio of 0.1:9.9 to 9.9:0.1. In embodiments, the bioactive renal cell population and the non-renal cell population are cultured at a ratio of about 1:1. In embodiments, the renal cell population and the bioactive cell population are suspended in growth medium.

[0157] The expanded bioactive renal cells can be further subjected to continuous or discontinuous density media separation to obtain SRCs. In particular, continuous or discontinuous single-step or multi-step density gradient centrifugation is used to separate the harvested renal cell population based on cell buoyant density. In embodiments, the expanded bioactive renal cells can be further subjected to separation by centrifugation across a density boundary, density barrier, or density interface to obtain SRCs. In particular, centrifugation across a density boundary, density barrier, or density interface is used to separate the harvested renal cell population based on cell buoyant density. In embodiments, SRCs can be obtained in part using OPTIPREP (Axis-Shield) medium containing a 60% aqueous solution of the non-ionic iodine compound iodixanol. However, one skilled in the art will recognize that any density gradient medium can be used, including but not limited to a particular medium or other means, such as immunological separation using cell surface markers known in the art, that have the characteristics necessary for isolating a cell population encompassed by the present invention. For example, Percoll or sucrose can be used to form a density gradient or density boundary. In embodiments, a cell fraction exhibiting a buoyant density greater than about 1.04 g / mL is collected as a separate pellet after centrifugation. In embodiments, cells that maintain a buoyant density less than 1.04 g / mL are rejected and discarded. In embodiments, a cell fraction exhibiting a buoyant density greater than about 1.0419 g / mL is collected as a separate pellet after centrifugation. In embodiments, cells that maintain a buoyant density less than 1.0419 g / mL are rejected and discarded. In embodiments, a cell fraction exhibiting a buoyant density greater than about 1.045 g / mL is collected as a separate pellet after centrifugation. In embodiments, cells that maintain a buoyant density less than 1.045 g / mL are rejected and discarded.

[0158] Therapeutic compositions and formulations thereof may contain isolated heterogeneous kidney cell populations and / or mixtures thereof enriched for specific bioactive components or cell types and / or depleted of specific inactive or undesirable components or cell types used to treat kidney disease, i.e., to stabilize and / or improve and / or regenerate kidney function and / or structure, e.g., as previously described in U.S. Pat. No. 8,318,484 to Presnell et al. and International Application PCT / US2011 / 036347 to Ilagan et al. (the entire contents of each are incorporated herein by reference). The compositions may contain isolated kidney cell fractions that lack cellular components compared to healthy individuals but retain therapeutic properties, i.e., to stabilize and / or improve and / or regenerate kidney function. The cell populations, cell fractions, and / or mixtures of cells described herein may be derived from healthy individuals, individuals suffering from kidney disease, or subjects described herein.

[0159] The present disclosure contemplates therapeutic compositions of selected renal cell populations administered to a target organ or tissue of a subject in need thereof. A selected bioactive renal cell population generally refers to a cell population that potentially has therapeutic properties upon administration to a subject. For example, when administered to a subject in need thereof, the bioactive renal cell population may result in stabilization and / or improvement and / or repair and / or regeneration of kidney function in the subject. Therapeutic properties may include regenerative effects.

[0160] In embodiments, the source of the cells is the same as the intended target organ or tissue. For example, BRCs and / or SRCs can be derived from the kidney for use in formulations administered to the kidney. In embodiments, the cell population is derived from a kidney biopsy. In embodiments, the cell population is derived from whole kidney tissue. In another embodiment, the cell population is derived from an in vitro culture of mammalian kidney cells established from a kidney biopsy or whole kidney tissue. In embodiments, the BRCs and / or SRCs comprise a heterogeneous mixture or fraction of bioactive kidney cells. The BRCs and / or SRCs can be derived from a healthy individual or are kidney cell fractions themselves from a healthy individual. Furthermore, the present disclosure provides kidney cell fractions obtained from unhealthy individuals that may lack certain cellular components compared to corresponding kidney cell fractions from healthy individuals, yet still maintain therapeutic properties. The present disclosure also provides therapeutically active cell populations that lack cellular components compared to healthy individuals, which, in embodiments, can be isolated and expanded from autologous sources for various disease states.

[0161] In one embodiment, SRCs are 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 the kidney tissue by enzymatic digestion, expanded using standard cell culture techniques, and selected by centrifugation of expanded renal cells across a density boundary, density barrier, or density interface. In this embodiment, SRCs are primarily composed of renal tubular epithelial cells, which are known for their regenerative capacity (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 in the autologous SRC population. In embodiments, kidney cells are selected by centrifugation through continuous or discontinuous single- or multi-step gradients.

[0162] As described herein, the present invention is based, in part, on the surprising discovery that certain subfractions of a heterogeneous population of renal cells, enriched for bioactive components and depleted of inactive or undesirable components, result in superior therapeutic and regenerative outcomes than the starting population.

[0163] Renal cell isolation and expansion yields a mixture of renal cell types, including renal tubular epithelial cells and interstitial cells. As described above, SRCs are obtained by separating expanded renal cells by centrifugation across a density boundary, density barrier, or density interface. The primary cell type in the isolated SRC population is of the tubular epithelial phenotype. The characteristics of SRCs obtained from expanded renal cells are evaluated using a multi-pronged approach. Cell morphology, growth kinetics, and cell viability are monitored during the renal cell expansion process. SRC buoyant density and viability are characterized by density interface and trypan blue exclusion. SRC phenotype is characterized by flow cytometry, and SRC function is indicated by expression of VEGF and KIM-1.

[0164] Those skilled in the art will understand that other isolation and culture methods known in the art can be used for the cells described herein. Those skilled in the art will also understand that bioactive cell populations can be derived from sources other than those specifically listed above, including, but not limited to, tissues and organs other than kidney, body fluids, and fat.

[0165] In embodiments, one or more of a variety of biomaterials can be combined with an active agent (e.g., a renal cell population, a product thereof, or a spheroid comprising a renal cell population and one or more non-renal cell types or populations) to provide a therapeutic formulation. In embodiments, the biomaterial can be in any suitable shape (e.g., beads) or form (e.g., liquid, gel, etc.). Non-limiting examples of suitable biomaterials in the form of polymer matrices include those described in U.S. Patent Application Publication No. 20070276507 to Bertram et al. (incorporated herein by reference). and is incorporated herein in its entirety. In embodiments, the polymer matrix can be a biocompatible material formed from a variety of synthetic or naturally occurring materials, including, but not limited to, open-cell polylactic acid (OPLA™), cellulose ethers, cellulose, cellulose esters, and the like. Examples of biomaterials include ethers, fluorinated polyethylene, phenols, poly-4-methylpentene, polyacrylonitrile, polyamides, polyamideimides, polyacrylates, polybenzoxazoles, polycarbonates, polycyanoaryl ethers, polyesters, polyestercarbonates, polyethers, polyetheretherketones, polyetherimides, polyetherketones, polyethersulfones, polyethylenes, polyfluoroolefins, polyimides, polyolefins, polyoxadiazoles, polyphenylene oxides, polyphenylene sulfides, polypropylenes, polystyrenes, polysulfides, polysulfones, polytetrafluoroethylenes, polythioethers, polytriazoles, polyurethanes, polyvinyls, polyvinylidene fluoride, regenerated cellulose, silicones, urea formaldehyde, collagen, gelatin, alginates, laminins, fibronectin, silk, elastin, alginates, hyaluronic acid, agarose, or copolymers or physical blends thereof. In embodiments, the biomaterial is a hydrogel. Scaffold compositions can range from soft porous scaffolds to rigid, shape-retaining porous scaffolds. In embodiments, the scaffold is configured as a liquid solution capable of becoming a hydrogel, e.g., a hydrogel above its melting temperature.

[0166] In embodiments, the scaffold is derived from a pre-existing kidney or other organ of human or animal origin from which the natural cell population has been eliminated by application of surfactants and / or other chemical agents and / or other enzymatic and / or physical techniques known to those skilled in the art. In this embodiment, the native three-dimensional structure of the organ of origin is maintained in their native bioactive state along with all associated extracellular matrix components. In embodiments, the scaffold is an extracellular matrix derived from a human or animal kidney or other organ. In embodiments, the construct is assembled into a tissue-like structure by application of three-dimensional bioprinting techniques. In embodiments, the construct is in the liquid form of a solution that can become a hydrogel.

[0167] Hydrogels can be formed from a variety of polymeric materials and are useful for a variety of biomedical applications. Hydrogels can be physically described as three-dimensional networks of hydrophilic polymers. Depending on the type of hydrogel, hydrogels contain varying percentages of water, but are generally insoluble in water. Despite their high water content, hydrogels can still bind large volumes of liquid due to the presence of hydrophilic residues. Hydrogels swell to a wide range without changing their gelatinous structure. Hydrogels swell to a wide range without changing their gelatinous structure. The basic physical characteristics of hydrogels can be specifically modified according to the properties of the polymers used and the device used to administer the hydrogel.

[0168] In embodiments, hydrogels are formed when organic polymers (e.g., natural or synthetic) are crosslinked via covalent, ionic, or hydrogen bonds to create a three-dimensional open lattice structure that traps water molecules to form a gel. In embodiments, materials used to form hydrogels include polymers such as alginate that are tonically crosslinked. Polysaccharides, polyphosphazines, and polyacrylates, or block copolymers such as Pluronic™ or Tetronic™, polyethylene oxide-polypropylene glycol block copolymers, which are crosslinked by temperature or pH, respectively, are also suitable. In embodiments, the hydrogel comprises gelatin (e.g., the hydrogel is a biodegradable gelatin-based hydrogel).

[0169] In embodiments, the hydrogel material does not induce an inflammatory response. Non-limiting examples of other materials that can be used to form hydrogels include (a) modified alginates, (b) polysaccharides (e.g., gellan gum and carrageenan) that gel upon exposure to monovalent cations, (c) polysaccharides (e.g., hyaluronic acid) that are highly viscous liquids or thixotropic and form gels over time by slow development of structure, (d) gelatin or collagen, and (e) hydrogel polymer precursors (e.g., polyethylene oxide-polypropylene glycol block copolymers and proteins). U.S. Patent No. 6,224,893 provides a detailed description of various polymers suitable for making hydrogels according to certain embodiments described herein and the chemical properties of such polymers.

[0170] In embodiments, the hydrogel used to formulate the biomaterial is gelatin-based. Gelatin is a non-toxic, biodegradable, water-soluble protein derived from collagen and is a major component of mesenchymal extracellular matrix (ECM). Gelatin possesses signaling signals containing arginine-glycine-aspartic acid (RGD) sequences, which promote cell adhesion, proliferation, and stem cell differentiation. A distinctive property of gelatin is that it exhibits upper critical solution temperature (UCST). In embodiments, above a certain temperature threshold of 40°C, gelatin can dissolve in water by forming flexible, random single helices. Upon cooling, hydrogen bonding and van der Waals interactions occur, resulting in the formation of triple helices. In embodiments, these collagen-like triple helices act as junction regions, thereby inducing a sol-gel transition. Gelatin is widely used in pharmaceutical and medical applications.

[0171] Collagen is the major structural protein in the extracellular space within various connective tissues in animal bodies. As the main component of connective tissue, collagen is the most abundant protein in mammals, comprising 25%–35% of the total body protein content. Depending on the degree of mineralization, collagen tissue can be rigid (bone), flexible (tendon), or have a gradient from rigid to flexible (cartilage). Collagen in the form of elongated fibrils is found primarily in fibrous tissues such as tendons, ligaments, and skin. Collagen is also abundant in the cornea, cartilage, bone, blood vessels, the digestive tract, intervertebral discs, and the dentin of teeth. In muscle tissue, collagen serves as the main component of the endomysium. Collagen comprises 1%–2% of muscle tissue and accounts for 6% of the weight of strong tendons. Collagen is present in many locations throughout the body. However, more than 90% of the collagen in the human body is type I.

[0172] To date, 28 types of collagen have been identified and described. Collagens can be divided into several groups according to the structure they form: fibrillar (types I, II, III, V, and XI), non-fibrillar FACIT (Fibril Associated Collagens with Interrupted Triple Helices) ( Types IX, XII, XIV, XVI, and XIX), short chain (Types VIII and X), basement membrane (Type IV), multiplexin (Multiple Triple Helix domains with Interruptions) (Types XV and XVIII), MACIT (Membrane Associated Collagens with Interrupted Triple Helices) (Types XIII and XVII), and others (Types VI The five most common types are: Type I: skin, tendons, blood vessels, ligaments, organs, and bone (the main component of the organic part of bone); Type II: cartilage (the main collagen component of cartilage); Type III: reticular (the main component of reticular fibers) (usually coexisting with Type I); Type IV: forming the basal layer (the layer of basement membrane secreted by epithelia); Type V: cell surfaces, hair, and placenta.

[0173] Gelatin contains information signals containing the arginine-glycine-aspartic acid (RGD) sequence, which promotes cell adhesion, proliferation, and stem cell differentiation. A distinctive property of gelatin is that it exhibits upper critical solution temperature behavior (UCST). Above a certain temperature threshold of 40°C, gelatin can dissolve in water by forming flexible, random single helices. Upon cooling, hydrogen bonding and van der Waals interactions result in the formation of triple helices. These collagen-like triple helices act as junction regions, thereby inducing the sol-gel transition. Gelatin is widely used in pharmaceutical and medical applications.

[0174] In embodiments, the hydrogels used to formulate the injectable cell compositions herein are based on porcine gelatin, which may be derived from pig skin and are available from, for example, Nitta Gelatin NA Inc. (North Carolina, USA) or Gelita USA Inc. (USA). Gelatin is commercially available from Biosciences, Inc. (Iowa, USA). Gelatin can be dissolved, for example, in Dulbecco's phosphate buffered saline (DPBS) to form a thermoresponsive hydrogel that can gel and liquefy at various temperatures. In embodiments, the hydrogels used to formulate the injectable cell compositions herein are based on recombinant human or animal gelatin expressed and purified by methods known to those skilled in the art. In embodiments, an expression vector containing all or part of the cDNA for type I alpha I human collagen is expressed in the yeast Pichia pastoris. Other expression vector systems and organisms are Known to those skilled in the art, in certain embodiments, gelatin-based hydrogels can be liquid at room temperature (22°C to 28°C) or above and gel when cooled to refrigerated temperatures (2°C to 8°C).

[0175] In embodiments, the gelatin-based hydrogel biomaterial used to formulate the SRC into the NKA is porcine gelatin, which forms a thermoresponsive hydrogel when dissolved in a buffer solution. In embodiments, this hydrogel is fluid at room temperature but gels when cooled to refrigerated temperatures (2°C to 8°C). The SRC is formulated with the hydrogel to obtain the NKA. In embodiments, the NKA is gelled by cooling and shipped to the clinic at refrigerated temperatures (2°C to 8°C). In embodiments, the NKA has a shelf life of 3 days. In embodiments, in the clinical setting, the product is warmed to room temperature before being injected into the patient's kidney. In embodiments, the NKA is implanted into the kidney cortex using a needle and syringe suitable for percutaneous or laparoscopic delivery of the NKA. In embodiments, the hydrogel is derived from gelatin or another extracellular matrix protein of recombinant origin. In embodiments, the hydrogel is derived from an extracellular matrix originating from the kidney or another tissue or organ. In embodiments, the hydrogel is derived from a recombinant extracellular matrix protein. In embodiments, the hydrogel comprises gelatin derived from recombinant collagen (ie, recombinant gelatin).

[0176] In embodiments, the properties of the scaffold or biomaterial may allow cells to attach to and interact with the scaffold or biomaterial material and / or may provide porous spaces in which cells may be entrapped. In embodiments, a porous scaffold or biomaterial allows for the addition or deposition of one or more cell populations onto the biomaterial configured as a porous scaffold (e.g., by cell attachment) and / or within the pores of the scaffold (e.g., by cell entrapment). In embodiments, the scaffold or biomaterial allows or promotes cell:cell and / or cell:biomaterial interactions within the scaffold to form the constructs described herein.

[0177] In embodiments, the biomaterial is from 5.1 kDa to 2 x 10 6 Up to over kDa The biomaterial is composed of hyaluronic acid (HA) in the form of a hydrogel containing HA molecules ranging in size from 5.1 kDa to 2 x 10 6 The biomaterial is comprised of hyaluronic acid in the form of a porous foam containing HA molecules in the size range from 0.1 to over 100 kDa. In embodiments, the biomaterial is comprised of a polylactic acid (PLA)-based foam having an open-cell structure and pore sizes of about 50 microns to about 300 microns. In embodiments, the renal cell population directly induces and / or stimulates the synthesis of high molecular weight hyaluronic acid via hyaluronan synthase 2 (HAS-2), particularly after intrarenal transplantation.

[0178] In embodiments, the biomaterials described herein respond to certain external conditions, e.g., in vitro or in vivo. In embodiments, the biomaterials are temperature-sensitive (e.g., either in vitro or in vivo). In embodiments, the biomaterials respond to exposure to enzymatic degradation (e.g., either in vitro or in vivo). In embodiments, the response of the biomaterials to external conditions can be fine-tuned as described herein. In embodiments, the temperature sensitivity of the described formulations can be varied by adjusting the percentage of biomaterial in the formulation. For example, the percentage of gelatin in solution can be adjusted to modulate the temperature sensitivity of the gelatin in the final formulation (e.g., liquid, gel, beads, etc.). In embodiments, the gelatin solution can be provided in PBS, DMEM, or another suitable solvent. In embodiments, the biomaterials can be chemically crosslinked to provide greater resistance to enzymatic degradation. For example, chemically crosslinking gelatin beads using a carbodiimide crosslinker can result in reduced susceptibility to endogenous enzymes.

[0179] In embodiments, the response by a biomaterial to external conditions relates to the loss of structural integrity of the biomaterial. While temperature sensitivity and resistance to enzymatic degradation are demonstrated herein, there are other mechanisms by which loss of material integrity can occur in various biomaterials. These mechanisms can include, but are not limited to, thermodynamic mechanisms (e.g., phase transitions such as melting, diffusion (e.g., diffusion of ionic crosslinkers from the biomaterial into surrounding tissue)), chemical mechanisms, enzymatic mechanisms, pH (e.g., pH-sensitive liposomes), ultrasound, and photoinstability mechanisms (light penetration). In embodiments, the exact mechanism by which a biomaterial loses structural integrity varies, but typically, this mechanism is triggered either at the time of implantation or after implantation.

[0180] In embodiments, the formulations described herein include a biomaterial with properties that create a favorable environment for an active agent (e.g., a renal cell population, a product thereof, or a spheroid comprising a renal cell population and one or more non-renal cell types or populations) administered to a subject. In embodiments, the formulation contains a first biomaterial that provides a favorable environment from the time the active agent is formulated with the biomaterial to the time it is administered to a subject. In embodiments, the favorable environment relates to the advantage of having one or more active agents (e.g., a renal cell population, a product thereof, or a spheroid comprising a renal cell population and one or more non-renal cell types or populations) suspended in a substantially solid state in a fluid (described herein) prior to administration to a subject. In embodiments, the first biomaterial is a temperature-sensitive biomaterial. In embodiments, the temperature-sensitive biomaterial can have (i) a substantially solid state at about 8°C or below and (ii) a substantially liquid state at ambient temperature or above. In embodiments, ambient temperature refers to the temperature at which the composition is administered. In embodiments, ambient temperature is the temperature of a temperature-controlled environment. In embodiments, ambient temperature is about room temperature. In an embodiment, the ambient temperature is in the range of about 18° C. to about 30° C. In an embodiment, 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 is about 30° C. In embodiments, one or more active agents described herein (e.g., a renal cell population, products thereof, or spheroids comprising a renal cell population and one or more non-renal cell types or populations) can be coated with, deposited on, embedded within, bound to, seeded within, suspended within, or entrapped within a temperature-sensitive biomaterial.

[0181] In embodiments, one or more active agents (e.g., a renal cell population, a product thereof, or a spheroid comprising a renal cell population and one or more non-renal cell types or populations) are uniformly dispersed throughout the volume of the cell-stabilizing biomaterial.

[0182] In embodiments, the formulation is an injectable formulation comprising one or more active agents (e.g., a renal cell population, products thereof, or spheroids comprising a renal cell population and one or more non-renal cell types or populations) and a temperature-sensitive cell-stabilizing biomaterial that (i) maintains a substantially solid state at or below 8°C and (ii) maintains a substantially liquid state at or above ambient temperature, wherein the biomaterial comprises a hydrogel, the biomaterial is in a solid-liquid transition phase between 8°C and above ambient temperature, and the one or more active agents are suspended in and dispersed throughout the cell-stabilizing biomaterial. In embodiments, the ambient temperature is in the range of 18°C ​​to 30°C. In embodiments, the biomaterial exists in a liquid state at 37°C. In embodiments, the substantially solid state is a gel state. In embodiments, the hydrogel comprises gelatin. In embodiments, the gelatin is present in the formulation at 0.5% (wt / vol) to 1% (wt / vol). In embodiments, the gelatin is present in the formulation at 0.75% (wt / vol).

[0183] In embodiments, the formulation further comprises an antioxidant, an oxygen carrier, an immunomodulatory factor, a cell recruitment factor, a cell adhesion factor, an anti-inflammatory agent, an immunosuppressant, an angiogenic factor, or a wound healing factor.

[0184] In embodiments, the formulation further comprises an antioxidant. In embodiments, the antioxidant is 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid. In embodiments, the 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid is present at 50 μM to 150 μM. In embodiments, the 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid is present at 100 μM.

[0185] In embodiments, the formulation further comprises an oxygen carrier, hi embodiments, the oxygen carrier is a perfluorocarbon.

[0186] In an embodiment, the formulation further comprises an immunomodulatory factor.

[0187] In an embodiment, the formulation further comprises an immunosuppressant.

[0188] In an embodiment, the formulation comprises 0.75% (wt / vol) gelatin and 100 μM 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid.

[0189] In embodiments, the formulation further comprises biocompatible beads comprising a biomaterial. In embodiments, the beads are crosslinked. In embodiments, the crosslinked beads have reduced susceptibility to enzymatic degradation compared to non-crosslinked biocompatible beads. In embodiments, the crosslinked beads are carbodiimide crosslinked beads. In embodiments, the carbodiimide is selected from the group consisting of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), DCC (N,N'-dicyclohexylcarbodiimide (DCC)), and N,N'-diisopropylcarbodiimide (DIPC). In embodiments, the carbodiimide is 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC).

[0023] propyl] carbodiimide hydrochloride (EDC). In embodiments, the crosslinked beads have a reduced number of free primary amines compared to non-crosslinked beads. In embodiments, the number of free primary amines is detectable spectrophotometrically at 355 nm. In embodiments, the beads are seeded with an active agent (e.g., a renal cell population, a product thereof, or a spheroid comprising a renal cell population and one or more non-renal cell types or populations). In embodiments, the formulation further comprises additional biocompatible beads comprising a temperature-sensitive biomaterial that (i) maintains a substantially solid state at or below ambient temperature and (ii) maintains a substantially liquid state at or above 37°C. In embodiments, the biomaterial has a solid-liquid transition state between ambient temperature and 37°C. In embodiments, the substantially solid state is a gel state. In embodiments, the biomaterial comprises a hydrogel. In embodiments, the hydrogel comprises gelatin. In embodiments, the beads comprise 5% (wt / vol) to 10% (wt / vol) gelatin. In embodiments, the additional biocompatible beads are spacer beads. In embodiments, the spacer beads are not seeded with an active agent (eg, a renal cell population, a product thereof, or a spheroid comprising a renal cell population and one or more non-renal cell types or populations).

[0190] In embodiments, the formulation comprises or further comprises a product secreted by a renal cell population. In embodiments, the product comprises a paracrine factor. In embodiments, the product comprises an endocrine factor. In embodiments, the product comprises a juxtocrine factor. In embodiments, the product comprises a vesicle. In embodiments, the vesicle comprises a microvesicle. In embodiments, the vesicle comprises an exosome.

[0191] In embodiments, the vesicles comprise a secreted product selected from the group consisting of a paracrine factor, an endocrine factor, a junctional factor, and RNA. In embodiments, the RNA is miRNA. In embodiments, the vesicles comprise miRNA that inhibits plasminogen activator inhibitor-1 (PAI-1) and / or TGFβ1.

[0192] In embodiments, the secreted product comprises a paracrine and / or juxta-crine factor such as α1 microglobulin, β2 microglobulin, calbindin, clusterin, connective tissue growth factor, cystatin C, glutathione-S-transferase α, kidney injury molecule-1, neutrophil gelatinase-associated lipocalin, osteopontin, trefoil factor 3, Tamm-Horsfall urinary glycoprotein, tissue inhibitor of metalloproteinases 1, vascular endothelial growth factor, fibronectin, interleukin-6, or monocyte chemotactic protein-1.

[0193] Further included by the present disclosure are formulations containing biomaterials that degrade over periods on the scale of seconds, minutes, hours, or days. This contrasts with numerous studies that have focused on implanting solid materials that slowly degrade over days, weeks, or months. In embodiments, the biomaterial possesses one or more of the following attributes: biocompatibility, biodegradability / bioabsorbability, a substantially solid state before and during implantation into a subject, loss of structural integrity (substantially solid state) after implantation, and a cytocompatible environment that supports cell viability. In embodiments, the biomaterial's ability to maintain space between implanted particles during implantation promotes natural tissue ingrowth. In embodiments, the biomaterial also facilitates implantation of solid formulations. In embodiments, the biomaterial provides localization of the formulations described herein, as the insertion of a solid unit helps prevent the delivered material from dispersing within the tissue during implantation. In embodiments, in the case of cell-based formulations, the solid biomaterial also improves the stability and viability of anchorage-dependent cells compared to cells suspended in a fluid. In embodiments, a short duration of structural integrity means that immediately after implantation, the biomaterial does not pose a significant impediment to tissue ingrowth or integration of the delivered cells / materials with the host tissue.

[0194] In embodiments, the construct includes a biomaterial configured as a three-dimensional (3D) porous biomaterial suitable for entrapment and / or attachment of compounds. In embodiments, the construct includes a biomaterial configured as a liquid or semi-liquid gel suitable for embedding, attaching, suspending, or coating mammalian cells. In embodiments, the construct includes a biomaterial composed primarily of high molecular weight species of hyaluronic acid (HA) in the form of a hydrogel. In embodiments, the construct includes a biomaterial composed primarily of high molecular weight species of hyaluronic acid in the form of a porous foam. In embodiments, the construct includes a biomaterial composed of a polylactic acid-based foam having pores between about 50 microns and about 300 microns. In embodiments, the construct includes one or more cell populations that may be derived from a kidney sample autologous to a subject in need of improved kidney function. In embodiments, the sample is a kidney biopsy. In embodiments, the subject is afflicted with kidney disease. In embodiments, the cell population is derived from a non-autologous kidney sample. In embodiments, the construct results in increased kidney function. In embodiments, the construct results in kidney regeneration. In an embodiment, the construct effects red blood cell homeostasis.

[0195] In embodiments, the formulation contains bioactive cells combined with a second biomaterial that provides a favorable environment for the cell combination from the time of formulation until after administration to a subject. In embodiments, the favorable environment provided by the second biomaterial relates to the advantage of administering cells in a biomaterial that retains its structural integrity up until and after administration to a subject. In embodiments, the structural integrity of the second biomaterial after implantation is minutes, hours, days, or weeks. In embodiments, the structural integrity is less than one month, less than one week, less than one day, or less than one hour. In embodiments, the relatively short-term structural integrity results in a formulation that allows the active agent and biomaterial to be delivered to a target site within a tissue or organ by controlled handling, placement, or dispersion without interfering with or interfering with the interaction of the incorporated elements with the tissue or organ in which they are placed.

[0196] In embodiments, the second biomaterial is a temperature-sensitive biomaterial having a different sensitivity than the first biomaterial. The second biomaterial can (i) have a substantially solid state at about ambient temperature or below, and (ii) have a substantially liquid state at about 37° C. or above. In embodiments, ambient temperature is about room temperature.

[0197] In embodiments, the second biomaterial is a crosslinked bead. In embodiments, the crosslinked bead can have a finely adjustable in vivo residence time depending on the degree of crosslinking, as described herein. In embodiments, the crosslinked bead contains bioactive cells and is resistant to enzymatic degradation, as described herein.

[0198] In embodiments, the formulations of the present disclosure may include a first biomaterial combined with an active agent, e.g., bioactive cells, with or without a second biomaterial combined with the active agent, e.g., bioactive cells. In embodiments, when the formulation includes a second biomaterial, the second biomaterial may be temperature-sensitive beads and / or crosslinked beads.

[0199] In embodiments, the bioactive cell preparations and / or constructs described herein may be administered as a bioactive cell formulation. In embodiments, the formulation comprises cells and one or more biomaterials that provide stability to the bioactive cell preparations and / or constructs described herein. In embodiments, the biomaterial is a temperature-sensitive biomaterial that can maintain at least two different phases or states depending on the temperature. In embodiments, the biomaterial maintains a first state at a first temperature. The biomaterial may be in a first state at a first temperature, may maintain the second state at a second temperature, and / or may maintain the third state at a third temperature. In embodiments, the first state, second state, or third state may be a substantially solid state, a substantially liquid state, or a substantially semi-solid or semi-liquid state. In embodiments, the biomaterial has a first state at a first temperature and a second state at a second temperature, where the first temperature is lower than the second temperature.

[0200] In embodiments, the temperature-sensitive biomaterial is in a substantially solid state at a temperature of about 8°C or below. In 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 embodiments, the substantially solid state has the form of a gel. In embodiments, the temperature-sensitive biomaterial is in a substantially liquid state at ambient temperature or above. In 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 embodiments, the ambient temperature is about room temperature.

[0201] In embodiments, the temperature-sensitive biomaterial is in a substantially solid state at temperatures below about ambient temperature. In embodiments, ambient temperature is about room temperature. In embodiments, the substantially solid state is maintained at about 17°C, about 16°C, about 15°C, about 14°C, about 13°C, about 12°C, about 11°C, about 10°C, about 9°C, about 8°C, about 7°C, about 6°C, about 5°C, about 4°C, about 3°C, about 2°C, or about 1°C. In embodiments, the substantially solid state has the form of beads. In embodiments, the temperature-sensitive biomaterial is in a substantially liquid state at temperatures above about 37°C. In embodiments, the substantially solid state is maintained at about 37°C, about 38°C, about 39°C, or about 40°C.

[0202] In embodiments, the temperature-sensitive biomaterial may be provided in the form of a solution, in the form of beads, or in other suitable forms described herein and / or known to those of skill in the art. In embodiments, the cell populations and preparations described herein may be coated with, deposited on, embedded in, bound to, seeded in, suspended in, or entrapped in the temperature-sensitive biomaterial. In embodiments, the temperature-sensitive biomaterial may be provided without any cells, for example, in the form of spacer beads.

[0203] In embodiments, the temperature-sensitive biomaterial has a transition state between a first state and a second state. In embodiments, the transition state is a solid-liquid transition state between a temperature of about 8°C and about ambient temperature. In embodiments, ambient temperature is about room temperature. In embodiments, the solid-liquid transition state occurs at one or more temperatures of about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, and about 18°C.

[0204] In embodiments, the temperature-sensitive biomaterial has a certain viscosity at a given temperature, measured in centipoise (cP). In embodiments, the biomaterial has a viscosity at 25°C of about 1 cP to about 5 cP, about 1.1 cP to about 4.5 cP, about 1.2 cP to about 4 cP, about 1.3 cP to about 3.5 cP, about 1.4 cP to about 3.5 cP, about 1.5 cP to about 3 cP, about 1.55 cP to about 2.5 cP, or about 1.6 cP to about 2 cP. In embodiments, the biomaterial has a viscosity at 37°C of about 1.0 cP to about 1.15 cP. The viscosity at 37°C can be about 1.0 cP, about 1.01 cP, about 1.02 cP, about 1.03 cP, about 1.04 cP, about 1.05 cP, about 1.06 cP, about 1.07 cP, about 1.08 cP, about 1.09 cP, about 1.10 cP, about 1.11 cP, about 1.12 cP, about 1.13 cP, about 1.14 cP, or about 1.15 cP. In embodiments, the biomaterial is a gelatin solution. In embodiments, the gelatin is present in the solution at about 0.5%, about 0.5%, about 1.15%, or about 1.15%. The biomaterial may be present at about 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, or about 1% (weight / volume). In embodiments, the biomaterial is a 0.75% (weight / volume) gelatin solution in PBS. In embodiments, a 0.75% (weight / volume) solution has a viscosity of about 1.6 cP to about 2 cP at 25°C. In embodiments, a 0.75% (weight / volume) solution has a viscosity of about 1.07 cP to about 1.08 cP at 37°C. In embodiments, the gelatin solution may be provided in PBS, DMEM, or another suitable solvent.

[0205] In embodiments, the bioactive cell formulation also includes a cell viability agent, which in embodiments is selected from the group consisting of antioxidants, oxygen carriers, immunomodulators, cell recruitment factors, cell adhesion factors, anti-inflammatory agents, angiogenic factors, matrix metalloproteinases, wound healing factors, and products secreted by bioactive cells.

[0206] In embodiments, antioxidants are characterized by their ability to inhibit the oxidation of other molecules. Antioxidants include, but are not limited to, one or more of 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox™), carotenoids, flavonoids, isoflavones, ubiquinone, glutathione, lipoic acid, superoxide dismutase, ascorbic acid, vitamin E, vitamin A, mixed carotenoids (e.g., beta-carotene, alpha-carotene, gamma-carotene, lutein, lycopene, phytopene, phytofluene, and astaxanthin), selenium, coenzyme Q10, indole-3-carbinol, proanthocyanidins, resveratrol, quercetin, catechins, salicylic acid, curcumin, bilirubin, oxalic acid, phytic acid, lipoic acid, vanillic acid, polyphenols, ferulic acid, theaflavins, and derivatives thereof. Those of ordinary skill in the art will recognize other antioxidants suitable for use in the present disclosure.

[0207] In embodiments, an oxygen carrier is an agent characterized by its ability to carry and release oxygen. Oxygen carriers include, but are not limited to, perfluorocarbons and perfluorocarbon-containing pharmaceuticals. Suitable perfluorocarbon-based oxygen carriers include, but are not limited to, perfluorooctyl bromide (CF). 17 Br), perfluorodichlorooctane (CF 16 C l2), perfluorodecyl bromide, perflubron, perfluorodecalin, perfluorotripropylamine, perfluoromethylcyclopiperidine, Fluosol™ (perfluorodecalin and perfluorotripropylamine), Perftoran™ (perfluorodecalin and perfluoromethylcyclopiperidine), Oxygent™ (perfluorodecyl bromide and perflubron), Ocycyte™ (perfluoro(tert-butylcyclohexane)). Those skilled in the art will recognize other perfluorocarbon-based oxygen carriers suitable for use in the present disclosure.

[0208] Immunomodulatory factors include, but are not limited to, osteopontin, FAS ligand factor, interleukins, transforming growth factor beta, platelet-derived growth factor, clusterin, transferrin, regulated upon action, normal T-cell expressed, secreted protein (RANTES), plasminogen activator inhibitor-1 (Pai-1), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), alpha 1 microglobulin, and beta 2 microglobulin. Those of skill in the art will recognize other immunomodulatory factors suitable for use in the present disclosure.

[0209] In embodiments, anti-inflammatory or immunosuppressant agents may also be part of the formulation. Those skilled in the art will recognize other antioxidants suitable for use in the present formulations and / or treatments.

[0210] Cell recruitment factors include, but are not limited to, monocyte chemoattractant protein 1 (MCP-1) and CXCL-1. Those skilled in the art will recognize other cell recruitment factors suitable for use in the present formulations and / or treatments.

[0211] Cell adhesion factors include, but are not limited to, fibronectin, procollagen, collagen, ICAM-1, connective tissue growth factor, laminin, proteoglycans, and certain cell adhesion peptides such as RGD and YSIGR. Those of skill in the art will recognize other cell adhesion factors suitable for use in the present formulations and / or treatments.

[0212] Angiogenic factors include, but are not limited to, vascular endothelial growth factor F (VEGF) and angiopoietin-2 (ANG-2). Those skilled in the art will recognize other angiogenic factors suitable for use in certain embodiments of the present disclosure.

[0213] Matrix metalloproteinases include, but are not limited to, matrix metalloproteinase 1 (MMP1), matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP-9), and tissue inhibitor of metalloproteinase-1 (TIMP-1).

[0214] 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. Those skilled in the art will recognize other wound healing factors suitable for use in the present formulations and / or treatments.

[0215] The present disclosure also provides a bioactive cell preparation containing a biomaterial and an implantable construct comprising bioactive renal cells for the treatment of kidney disease. In embodiments, the construct is made from a biocompatible material or biomaterial, a scaffold or matrix composed of one or more synthetic or naturally occurring biocompatible materials, and one or more cell populations described herein deposited on or embedded in the surface of the scaffold by attachment and / or entrapment. In embodiments, the construct is made from a biomaterial and one or more cell populations described herein coated with, deposited on, deposited within, attached to, entrapped within, embedded in, seeded within, or combined with the biomaterial component(s). Any of the cell populations described herein, including enriched cell populations (e.g., SRCs), can be used in combination with a matrix to form the construct. In embodiments, the bioactive cell preparation is made from a biocompatible material or biomaterial and an SRC population described herein.

[0216] In an embodiment, the bioactive cell preparation is Neo-Kidney Augment (NKA), an injectable product composed of autologous and allogeneic selected renal cells (SRCs) formulated in a biomaterial (gelatin-based hydrogel). In an embodiment, the autologous and allogeneic SRCs are obtained by isolating and expanding renal cells from a patient's renal cortical tissue via a kidney biopsy, and selecting the expanded renal cells by separation across a density boundary, density barrier, or density interface (e.g., single-step discontinuous density gradient separation). In an embodiment, the autologous SRCs are obtained by isolating and expanding renal cells from a patient's renal cortical tissue via a kidney biopsy, and selecting the expanded renal cells against a continuous or discontinuous single-step or multi-step density gradient. In an embodiment, the SRCs are primarily composed of renal epithelial cells, which are known for their regenerative capacity (Humphreys et al. (2008) Intrinsic epithelial cells repair the kidney after injury. Cell Stem Cell. 2(3):284-91). In embodiments, injection of SRC into the recipient kidney results in significant improvements in animal survival, urinary concentration, and filtration function. In embodiments, SRC has a limited shelf life and stability. In embodiments, formulating SRC in a gelatin-based hydrogel biomaterial results in enhanced cell stability, thereby extending the shelf life of the product and improving the stability of the NKA during transport and delivery to the kidney cortex for clinical utility.

[0217] In embodiments, NKAs are produced by first obtaining renal cortical tissue from a donor using standard-of-care kidney biopsy procedures. In embodiments, the donor is the subject being treated. In embodiments, renal cells are isolated from the kidney tissue by enzymatic digestion and expanded using standard cell culture techniques. In embodiments, the cell culture medium used to expand the primary renal cells does not contain any differentiation factors. In embodiments, the harvested renal cells are subjected to separation across a density boundary or interface or density gradient separation to obtain SRCs.

[0218] In embodiments, the formulation includes a biomaterial designed or adapted to respond to external conditions as described herein. As a result, the nature of the association of the bioactive cell population with the biomaterial in the construct changes depending on the external conditions. In embodiments, the association of the cell population with the temperature-sensitive biomaterial changes with temperature. In embodiments, the construct contains a bioactive renal cell population and a biomaterial having a substantially solid state at about 8°C or below and a substantially liquid state at about ambient temperature or above, wherein the cell population is suspended in the biomaterial at about 8°C or below. In embodiments, the cell population is substantially free to move throughout the volume of the biomaterial at about ambient temperature or above. In embodiments, the suspension of the cell population in a substantially solid phase at lower temperatures provides a stability advantage to cells, such as anchorage-dependent cells, compared to cells in a fluid. In embodiments, the cells are suspended in a substantially solid state, providing one or more of the following advantages: i) preventing settling of the cells, ii) keeping the cells fixed to the biomaterial in a suspended state, iii) keeping the cells more uniformly dispersed throughout the volume of the biomaterial, iv) preventing the formation of cell aggregates, and v) providing better protection of the cells during storage and transport of the formulation. Formulations that can retain such characteristics up until administration to a subject are advantageous, at least because of better overall health of the cells in the formulation and a more uniform and consistent dose of cells administered.

[0219] In embodiments, the manufacturing process for the bioactive cell preparation is designed to deliver the product in approximately four weeks from patient biopsy to product implantation. In embodiments, tissue variability between patients poses challenges to delivering the product on a fixed implantation schedule. In embodiments, the expanded renal cells are cryopreserved during cell expansion to accommodate this patient-specific variability in cell growth. In embodiments, the cryopreserved renal cells provide a continuous source of cells to manufacture multiple doses for re-implantation as needed in the event that another treatment is required (e.g., delay due to patient illness, unanticipated course of events, etc.).

[0220] In embodiments, the bioactive cell composition is comprised of autologous and allogeneic cells formulated in a biomaterial (gelatin-based hydrogel). In embodiments, the composition is comprised of approximately 20 x 10 cells per mL in a gelatin solution with Dulbecco's phosphate buffered saline (DPBS). 6 ~ Approximately 200 x 10 cells per mL 6 In embodiments, the number of cells per mL of product is about 20 x 10 cells per mL. 6 Approximately 40 x 10 cells per mL 6 Approximately 60 x 10 cells per mL 6 cells, approximately 100 x 10 per mL 6 cells, approximately 120 x 10 per mL 6 cells, approximately 140 x 10 per mL 6 Approximately 160 x 10 cells per mL 6 Approximately 180 x 10 cells per mL 6 cells, or approximately 200 x 10 per mL 6 In embodiments, the gelatin is present in solution at about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7% %, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, or about 1% (weight / volume). In embodiments, the biomaterial is a 0.88% (weight / volume) gelatin solution in DPBS. In embodiments, the injectable formulation comprises a biomaterial comprising about 0.88% (weight / volume) gelatin and a composition comprising a bioactive renal cell population (BRC), wherein the BRC comprises an enriched population of tubular renal cells having a density greater than about 1.04 g / mL. In embodiments, the injectable formulation comprises a biomaterial comprising about 0.88% (weight / volume) gelatin and a composition comprising a bioactive renal cell population (BRC), wherein the BRC comprises an enriched population of tubular renal cells having a density greater than about 1.0419 g / mL or about 1.045 g / mL.

[0221] In an embodiment, the NKA is provided in a sterile, disposable 10 mL syringe. In an embodiment, the final volume is 100 x 10 per mL of NKA. 6 The concentration of SRC was 3.0 x 10 per gram of kidney weight. 6 The target dose of SRC is calculated from the kidney weight. In an embodiment, the kidney weight is estimated by MRI. In an embodiment, the therapeutic dose is determined (e.g., by a medical professional such as a surgeon) based on the patient's kidney weight at the time of injection. In an embodiment, the dose is about 2.5 x 10 per gram of kidney weight. 6 Approximately 3.5 x 10 SRC per gram of kidney weight 6 This is the SRC.

[0222] In embodiments, the total number of cells for the formulation can be selected, and the volume of the formulation can be adjusted to achieve an appropriate therapeutic dose. In embodiments, the formulation can include a dose of cells to the subject that is a single dose or a single dose plus additional doses. In embodiments, the dose can be provided by a construct described herein. In embodiments, a therapeutically effective amount of the bioactive renal cell populations described herein can range from the maximum number of cells safely tolerated by a subject to the minimum number of cells required to treat kidney disease, e.g., stabilize, reduce the rate of decline, or improve one or more kidney functions.

[0223] In embodiments, a therapeutically effective amount of the bioactive renal cell population described herein can be suspended in a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, basal culture medium plus 1% serum albumin, saline, buffered saline, dextrose, water, collagen, alginate, hyaluronic acid, fibrin glue, polyethylene glycol, polyvinyl alcohol, carboxymethylcellulose, and combinations thereof. The formulation should be compatible with the mode of administration.

[0224] In embodiments, the bioactive kidney cell preparation or composition is formulated according to routine procedures as a pharmaceutical composition adapted for administration to humans. In embodiments, for example, compositions for intravenous, intra-arterial, or intrarenal capsule administration are solutions in sterile isotonic aqueous buffer. In embodiments, the composition may also include a local anesthetic to alleviate any pain at the injection site. In embodiments, these ingredients are supplied separately or mixed together in unit dosage form, for example, as a lyophilized concentrate in a hermetically sealed container such as an ampule indicating the quantity of active agent. In embodiments, when the composition is administered by infusion, the composition can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline. In embodiments, when the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0225] In embodiments, pharmaceutically acceptable carriers can be determined, in part, by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of pharmaceutical compositions (see, e.g., Alfonso R Gennaro (ed), Remington: The Science and Practice of Pharmacy, formerly Remington's See Pharmaceutical Sciences 20th ed., Lippincott, Williams & Wilkins, 2003, incorporated herein by reference in its entirety. In embodiments, pharmaceutical compositions are generally formulated to be sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0226] In embodiments, the bioactive cell formulation comprises a cell survival agent selected from the group consisting of antioxidants, oxygen carriers, immunomodulatory factors, cell recruitment factors, cell adhesion factors, anti-inflammatory agents, angiogenic factors, wound healing factors, and products secreted from bioactive cells.

[0227] In embodiments, products secreted from the bioactive cells described herein can also be added to bioactive cell formulations as cell viability agents.

[0228] In embodiments, the formulation comprises a temperature-sensitive biomaterial described herein and a population of biocompatible beads containing the biomaterial. In embodiments, the beads are crosslinked. Crosslinking can be performed using any suitable crosslinking agent known to those skilled in the art, such as carbodiimides; aldehydes (e.g., furfural, acrolein, formaldehyde, glutaraldehyde, glyceraldehyde); succinimide-based crosslinkers (bis(sulfosuccinimidyl) suberate (BS3), disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS), dithiobis(succinimidyl propionate), ethylene glycol bis(sulfosuccinimidyl succinate), ethylene glycol bis(succinimidyl succinate) (E Crosslinking can be achieved using bis(sulfosuccinimidyl)glutarate (BS2G), disuccinimidyl tartrate (DST); epoxides (ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl abis); sugars (glucose and aldose sugars); sulfonic and p-toluenesulfonic acids; carbonyldiimidazole; genipin; imines; ketones; diphenylphosphoryl azide (DDPA); terephthaloyl chloride; cesium(III) nitrate hexahydrate; microbial transglutaminase; and hydrogen peroxide. Those skilled in the art will recognize other crosslinking agents and methods suitable for use in the present methods, formulations, and / or treatments.

[0229] In embodiments, the beads are carbodiimide-crosslinked beads. In 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).

[0230] In embodiments, crosslinked beads have reduced susceptibility to enzymatic degradation compared to non-crosslinked biocompatible beads, resulting in beads with finely tunable in vivo residence times. In embodiments, crosslinked beads are resistant to endogenous enzymes such as collagenase. In embodiments, the provision of crosslinked beads is part of a delivery system that facilitates one or more of the following: (a) delivery of attached cells to a desired site, creating space for regeneration and ingrowth of native tissue and a vascular supply; (b) the ability to persist at the site long enough to allow the cells to establish, function, remodel, and secrete their own extracellular matrix (ECM); (c) promotion of integration of the implanted cells with the surrounding tissue; (d) the ability to implant cells in a substantially solid form; (e) short-term structural integrity that does not pose a significant impediment to tissue ingrowth or integration of the delivered cells / material with the host tissue; (f) localized in vivo delivery in a substantially solid form, preventing dispersion of cells within the tissue during implantation; (g) improved stability and viability of anchorage-dependent cells compared to cells suspended in a fluid; and (h) the ability of cells to: i) remain in a substantially solid form (e.g., attached to beads); and ii) a biphasic release profile when delivered in a substantially liquid form (e.g., suspended in a fluid).

[0231] In embodiments, the present disclosure provides cross-linked beads containing gelatin. In embodiments, non-cross-linked gelatin beads are not suitable for bioactive cell formulations because they rapidly lose integrity and cells dissipate from the injection site. In embodiments, highly cross-linked gelatin beads may persist at the injection site for an excessively long time, potentially interfering with de novo ECM secretion, cell integration, and tissue regeneration. In embodiments, the present disclosure allows for fine-tuning of the in vivo residence time of cross-linked beads. In embodiments, various cross-linker concentrations of carbodiimide are used to tailor the biodegradability of the biomaterial, while keeping the overall reaction conditions constant for all samples. In embodiments, the enzyme sensitivity of carbodiimide-cross-linked beads can be fine-tuned by varying the cross-linker concentration from about 0 M to about 1 M. In embodiments, the concentration is about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM, about 20mM, about 21mM, about 22mM, about 23mM, about 24mM, about 25mM, about 26mM, about 27mM, about 28mM, about 29mM, about 30mM, about 31mM, about 32mM , about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM. The concentration of the cross-linking agent can also be about 0.15M, about 0.2M, about 0.25M, about 0.3M, about 0.35M, about 0.4M, about 0.45M, about 0.5M, about 0.55M, about 0.6M, about 0.65M, about 0.7M, about 0.75M, about 0.8M, about 0.85M, about 0.9M, about 0.95M, or about 1M. In another embodiment, the cross-linking agent is 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC). In an embodiment, the EDC-cross-linked beads are gelatin beads.

[0232] In embodiments, crosslinked beads may have certain properties that are advantageous for seeding, attachment, or encapsulation. In embodiments, the beads may have a porous surface and / or may be substantially hollow. In embodiments, the presence of pores provides an increased cell attachment surface, allowing for the attachment of a greater number of cells compared to a non-porous, i.e., smooth, surface. In embodiments, the pore structure may support host tissue integration with the porous beads, supporting de novo tissue formation. In embodiments, the beads have a size distribution that can be fitted to a Weibull plot corresponding to a general particle distribution pattern. In embodiments, the crosslinked beads have an average diameter of less than about 120 μm, about 115 μm, about 110 μm, about 109 μm, about 108 μm, about 107 μm, about 106 μm, about 105 μm, about 104 μm, about 103 μm, about 102 μm, about 101 μm, about 100 μm, about 99 μm, about 98 μm, about 97 μm, about 96 μm, about 95 μm, about 94 μm, about 93 μm, about 92 μm, about 91 μm, or about 90 μm. In embodiments, the characteristics of the crosslinked beads vary depending on the casting method. In embodiments, an air stream is used to aerosolize a liquid gelatin solution, which is then sprayed onto a thin layer chromatography reagent sprayer (ACE Glassware) using liquid nitrogen. Using the method of spraying into a powder, beads having the above-mentioned properties can be obtained. Those skilled in the art will appreciate that adjustment of the parameters of the casting method provides the opportunity to tailor different properties of the beads, such as different size distributions.

[0233] In embodiments, the cytocompatibility of the crosslinked beads is evaluated in vitro prior to formulation using cell culture techniques in which the beads are cultured with cells corresponding to the final bioactive cell preparation. In embodiments, the beads are cultured with primary renal cells prior to production of the bioactive renal cell preparation. and confirm cytocompatibility using a live / dead cell assay. In embodiments, the biocompatible crosslinked beads are combined with the temperature-sensitive biomaterial in solution at about 5% (wt / wt) to about 15% (wt / wt) of the volume of the solution. In embodiments, the crosslinked beads may be present at about 5% (wt / wt), about 5.5% (wt / wt), about 6% (wt / wt), about 6.5% (wt / wt), about 7% (wt / wt), about 7.5% (wt / wt), about 8% (wt / wt), about 8.5% (wt / wt), about 9% (wt / wt), about 9.5% (wt / wt), about 10% (wt / wt), about 10.5% (wt / wt), about 11% (wt / wt), about 11.5% (wt / wt), about 12% (wt / wt), about 12.5% ​​(wt / wt), about 13% (wt / wt), about 13.5% (wt / wt), about 14% (wt / wt), about 14.5% (wt / wt), or about 15% (wt / wt) of the volume of the solution.

[0234] In embodiments, the present disclosure provides formulations containing biomaterials that degrade over periods of time on the scale of minutes, hours, or days. This contrasts with numerous studies that focus on implanting solid materials that slowly degrade over days, weeks, or months. In embodiments, the biomaterials possess one or more of the following attributes: biocompatibility, biodegradability / bioabsorbability, a substantially solid state before and during implantation into a subject, loss of structural integrity (substantially solid state) after implantation, and a cytocompatible environment that supports cell viability and proliferation. The biomaterial's ability to maintain space between implanted particles during implantation promotes natural tissue ingrowth. The biomaterial also facilitates implantation of solid formulations. The insertion of solid units helps prevent the delivered material from dispersing within the tissue during implantation, so the biomaterial provides localization of the formulations described herein. In the case of cell-based formulations, solid biomaterials also improve the stability and viability of anchorage-dependent cells compared to cells suspended in a fluid. However, the short duration of structural integrity means that immediately after implantation the biomaterial does not pose a significant impediment to tissue ingrowth or integration of the delivered cells / materials with the host tissue.

[0235] In one aspect, the present disclosure provides formulations containing biomaterials that are implanted in a substantially solid form and then liquefy / melt or otherwise lose structural integrity after implantation in the body, in contrast to many studies that have focused on the use of materials that can be injected as a liquid and then solidify in the body.

[0236] In embodiments, the present disclosure provides a formulation having a delivery matrix with biocompatible crosslinked beads seeded with bioactive cells. In embodiments, the delivery matrix has one or more of the following attributes: biocompatibility, biodegradability / bioabsorbability, a substantially solid state before and during implantation into a subject, loss of structural integrity (substantially solid state) after implantation, and a cytocompatible environment that supports cell viability. In embodiments, the ability of the delivery matrix to maintain space between implanted particles (e.g., crosslinked beads) during implantation promotes natural tissue ingrowth. In embodiments, without the delivery matrix, compression of cellularized beads during implantation can result in spaces unsuitable for sufficient tissue ingrowth. In embodiments, the delivery matrix also facilitates implantation of solid formulations. Furthermore, in embodiments, the short duration of structural integrity means that the matrix does not pose a significant obstacle to tissue ingrowth or integration of delivered cells / materials with host tissue immediately after implantation. In embodiments, the delivery matrix provides localization of the formulations described herein, as the insertion of a solid unit helps prevent the delivered material from dispersing within the tissue during implantation. In embodiments, in the case of cell-based formulations, a solid delivery matrix improves the stability and viability of anchorage-dependent cells compared to cells suspended in a fluid.

[0237] In embodiments, the delivery matrix is ​​a biocompatible bead matrix that is not seeded with cells. In embodiments, unseeded beads are interspersed throughout the individual cell-seeded beads. In embodiments, the unseeded beads serve as "spacer beads" between the cell-seeded beads before and immediately after implantation. In embodiments, the spacer beads contain a temperature-sensitive biomaterial that has a substantially solid state at a first temperature and a substantially liquid state at a second temperature, the first temperature being lower than the second temperature. In embodiments, the spacer beads contain a biomaterial, such as a biomaterial described herein, that has a substantially solid state at about ambient temperature or below and a substantially liquid state at about 37°C. In embodiments, the ambient temperature is about room temperature. In embodiments, the biomaterial is a gelatin solution. In embodiments, the gelatin solution is present at about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 10.5%, or about 11% (weight / volume). In embodiments, the gelatin solution may be provided in PBS, cell culture medium (e.g., DMEM), or another suitable solvent.

[0238] In embodiments, the present disclosure provides formulations containing biomaterials that are implanted in a substantially solid form (e.g., spacer beads) and then liquefy / melt or otherwise lose structural integrity after implantation in the body.

[0239] In embodiments, the temperature sensitivity of spacer beads can be evaluated in vitro prior to formulation. In embodiments, spacer beads can be labeled and mixed with unlabeled, temperature-insensitive beads. In embodiments, the mixture is then incubated at 37°C and changes in physical transition are observed. In embodiments, the shape change of labeled temperature-sensitive beads at higher temperatures is observed over time. In embodiments, temperature-sensitive gelatin beads can be made using Alcian blue dye to serve as a marker for the physical transition. In embodiments, blue gelatin beads are mixed with Cultispher S beads (white) and loaded into a catheter, then extruded and incubated in 1x PBS (pH 7.4) at 37°C. In embodiments, the shape change of the blue gelatin beads is tracked by microscopy at various time points. In embodiments, changes in the physical state of the blue gelatin beads are visible after 30 minutes and become more pronounced with extended incubation times. In embodiments, due to the viscosity of the material, the beads do not completely dissipate.

[0240] In embodiments, the bioactive cell formulations described herein can be used to prepare renal cell-based formulations for injection into the kidney. However, those skilled in the art will understand that the formulations are suitable for many other types of bioactive cell populations. For example, the present disclosure contemplates formulations of bioactive cells for injection into any solid organ or tissue.

[0241] In embodiments, the bioactive cell formulations described herein will contain a defined number of cells. In embodiments, the total number of cells for a formulation is about 10 4 pieces, about 10 5 pieces, about 10 6 pieces, about 10 7 pieces, about 10 8 pieces, or about 10 9In embodiments, the cell dosage for the formulations described herein may be calculated based on the estimated mass or functional mass of the target organ or tissue. In embodiments, the bioactive cell formulation comprises a dosage corresponding to the number of cells based on the weight of the host organ to be treated with the formulation. In embodiments, the bioactive renal cell formulation is based on an average weight of about 150 grams for a human kidney. In embodiments, the number of cells per gram (g) of kidney is about 600 cells / g to about 7.0 x 10 7 In embodiments, the number of cells per gram of kidney is about 600 cells / g, about 1000 cells / g, about 1500 cells / g, about 2000 cells / g, about 2500 cells / g, about 3000 cells / g, about 3500 cells / g, about 4000 cells / g, about 4500 cells / g, about 500 0 cells / g, about 5500 cells / g, about 6000 cells / g, about 6500 cells / g, about 7000 cells / g, about 7500 cells / g, about 8000 cells / g, about 8500 cells / g, about 9000 cells / g, about 9500 cells / g, or about 10000 cells / g.

[0242] In embodiments, the number of cells per gram of kidney is about 1.5 x 10 4 cells / g, approximately 2.0 x 10 4 cells / g, approximately 2.5 x 10 4 cells / g, approximately 3.0 x 10 4 cells / g, approximately 3.5 x 10 4 cells / g, approximately 4.0 x 10 4 cells / g, approximately 4.5 x 10 4 cells / g, approximately 5.0 x 10 4 cells / g, approximately 5.5 x 10 4 cells / g, approximately 6.0 x 10 4 cells / g, approximately 6.5 x 10 4 cells / g, approximately 7.0 x 10 4 cells / g, approximately 7.5 x 10 4 cells / g, approximately 8.0 x 10 4 cells / g, approximately 9.5 x 10 4cells / g.

[0243] In embodiments, the number of cells per gram of kidney is about 1.0 x 10 5 cells / g, approximately 1.5 x 10 5 cells / g, approximately 2.0 x 10 5 cells / g, approximately 2.5 x 10 5 cells / g, approximately 3.0 x 10 5 cells / g, approximately 3.5 x 10 5 cells / g, approximately 4.0 x 10 5 cells / g, approximately 4.5 x 10 5 cells / g, approximately 5.0 x 10 5 cells / g, approximately 5.5 x 10 5 cells / g, approximately 6.0 x 10 5 cells / g, approximately 6.5 x 10 5 cells / g, approximately 7.0 x 10 5 cells / g, approximately 7.5 x 10 5 cells / g, approximately 8.0 x 10 5 cells / g, approximately 8.5 x 10 5 cells / g, approximately 9.0 x 10 5 cells / g, or approximately 9.5 x 10 5 cells / g.

[0244] In embodiments, the number of cells per gram of kidney is about 1.0 x 10 6 cells / g, approximately 1.5 x 10 6 cells / g, approximately 2.0 x 10 6 cells / g, approximately 2.5 x 10 6 cells / g, approximately 3.0 x 10 6 cells / g, approximately 3.5 x 10 6 cells / g, approximately 4.0 x 10 6 cells / g, approximately 4.5 x 10 6 cells / g, approximately 5.0 x 10 6 cells / g, approximately 5.5 x 10 6 cells / g, approximately 6.0 x 10 6 cells / g, approximately 6.5 x 10 6 cells / g, approximately 7.0 x 10 6 cells / g, approximately 7.5 x 10 6cells / g, approximately 8.0 x 10 6 cells / g, approximately 8.5 x 10 6 cells / g, approximately 9.0 x 10 6 cells / g, approximately 9.5 x 10 6 cells / g, 1.0 x 10 7 cells / g, or approximately 1.5 x 10 7 cells / g.

[0245] In embodiments, the total number of cells for the formulation can be selected and the volume of the formulation can be adjusted to achieve an appropriate dosage.

[0246] In embodiments, the formulation may include an administration amount of cells to a subject that is a single dose or a single dose plus additional doses. In embodiments, the administration amount may be provided by a construct described herein. In embodiments, a therapeutically effective amount of a renal cell population described herein may range from the maximum number of cells safely tolerated by a subject to the minimum number of cells required to treat a kidney disease, e.g., stabilize, reduce the rate of decline, or improve one or more kidney functions.

[0247] In embodiments, a therapeutically effective amount of the renal cell population described herein can be suspended in a pharmaceutically acceptable carrier or additive. Such carriers include, but are not limited to, basal culture medium plus 1% serum albumin, saline, buffered saline, dextrose, water, collagen, alginate, hyaluronic acid, fibrin glue, polyethylene glycol, polyvinyl alcohol, carboxymethylcellulose, and combinations thereof. The formulation should be compatible with the mode of administration.

[0248] In embodiments, the present disclosure provides a method for manufacturing a medicament for treating kidney disease in a subject. Use of a preparation containing a renal cell population is provided. In embodiments, the medicament further comprises a recombinant polypeptide such as a growth factor, a chemokine, or a cytokine. In embodiments, the medicament comprises a cell population derived from a human kidney. In embodiments, the cells used to manufacture the medicament can be isolated, derived, or enriched using any of the variations indicated for the methods described herein.

[0249] In embodiments, the kidney cell preparation(s) or compositions disclosed herein are formulated according to routine procedures as pharmaceutical compositions adapted for administration to humans. In embodiments, for example, compositions for intravenous, intra-arterial, or intrarenal capsule administration are solutions in sterile isotonic aqueous buffer. In embodiments, the compositions may also include a local anesthetic to alleviate any pain at the injection site. In embodiments, these ingredients are supplied separately or mixed together in unit dosage form, for example, as a lyophilized concentrate in a hermetically sealed container, such as an ampule, indicating the quantity of active agent. In embodiments, when the composition is administered by infusion, the composition can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline. In embodiments, when the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0250] In embodiments, pharmaceutically acceptable carriers are determined, in part, by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions (see, e.g., Alfonso R Gennaro (ed), Remington: The Science and Practice of Pharmacy, formerly Remington's Pharmaceutical Sciences 20th ed., Uppincott, Williams & Wilkins, 2003 (cited herein). (See, e.g., U.S. Pat. No. 6,223,999, filed Dec. 1, 2002, which is hereby incorporated by reference in its entirety.) In embodiments, pharmaceutical compositions are generally formulated to be sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0251] In embodiments, the formulations of the present disclosure are provided as modified-release formulations. In embodiments, the modified release is characterized by an initial release of a first active agent upon administration, followed by at least one additional subsequent release of a second active agent. In embodiments, the first active agent and the second active agent may be the same or different. In embodiments, the formulation provides modified release through multiple components in the same formulation. In embodiments, the modified-release formulation contains the active agent as part of a first component that allows the active agent to move freely throughout the volume of the formulation, thereby enabling immediate release at the target site upon administration. In embodiments, the first component may be a temperature-sensitive biomaterial having a substantially liquid phase and a substantially solid phase, where the first component is in a substantially liquid phase at the time of administration. In embodiments, the active agent is present in a substantially liquid phase such that it is substantially free to move throughout the volume of the formulation, thereby providing immediate release at the target site upon administration.

[0252] In embodiments, the modified release formulation has an active agent attached to, deposited on, coated with, embedded in, seeded on, or entrapped within the second component as part of the second component, which persists before and after administration to the target site. In embodiments, the second component contains structural elements to which the active agent can associate, thereby preventing immediate release of the active agent from the second component upon administration. In embodiments, the second component is provided in a substantially solid form, e.g., biocompatible beads that can be crosslinked to prevent or delay enzymatic degradation in vivo. In embodiments, the substantially solid active agent retains its structural integrity within the formulation before and after administration, and therefore does not immediately release the active agent to the target site upon administration. Suitable carriers for modified release formulations are described herein, and those of skill in the art will readily understand the nature of the modified release formulations described herein. Other carriers suitable for use herein will be understood.

[0253] In embodiments, the formulations provide for an initial rapid delivery / release of the delivered element, including cells, nanoparticles, therapeutic molecules, etc., followed by a delayed release of subsequent elements. In embodiments, the formulations of the present disclosure may be designed for a biphasic release profile such that the delivered agent is provided in both an unattached form (e.g., cells in solution) and an attached form (e.g., cells together with beads or another suitable carrier). In embodiments, upon initial administration, the unhindered agent is immediately delivered to the delivery site, while release of the hindered agent is delayed until the structural integrity of the carrier (e.g., beads) is compromised, at which point the pre-attached agent is released. As discussed herein, other suitable release mechanisms will be recognized by those of skill in the art.

[0254] In embodiments, the release delay time can be adjusted based on the properties of the active agent. In embodiments, the release delay time for bioactive cell formulations can be on the order of seconds, minutes, hours, or days. In embodiments, a delay on the order of weeks can be appropriate. In embodiments, for other active agents, such as small molecules or large molecules, the release delay time for the formulation can be on the order of seconds, minutes, hours, days, weeks, or months. In embodiments, the formulation can also contain different biomaterials that provide different time-delay release profiles. In embodiments, a first biomaterial having a first active agent can exhibit a first release time, and a second biomaterial having a second active agent can exhibit a second release time. In embodiments, the first active agent and the second active agent can be the same or different.

[0255] In embodiments, the period of delayed release may generally correspond to the time it takes for the biomaterial to lose structural integrity. However, those skilled in the art will recognize other delayed-release mechanisms. In embodiments, the active agent may be released continuously over an extended period of time, independent of the degradation time of any particular biomaterial, for example, by diffusion of the drug from the polymer matrix. In embodiments, bioactive cells may migrate out of a formulation containing the biomaterial and the bioactive cells and into native tissue. In embodiments, bioactive cells migrate from a biomaterial, e.g., beads, into native tissue.

[0256] In embodiments, biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. In embodiments, extended absorption of injectable formulations can be achieved by including in the formulation an agent that delays absorption, such as monostearate salts and gelatin. Many non-limiting, exemplary methods for making such formulations are patented or generally known to those skilled in the art. See, for example, *Sustained and Controlled Release Drug Delivery Systems*, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. Further non-limiting, exemplary methods applicable to the controlled or sustained release of polypeptide agents are described, for example, in U.S. Patent Nos. 6,306,406 and 6,346,274, as well as U.S. Patent Application Publication Nos. 20020182254 and 20020051808, all of which are incorporated herein by reference.

[0257] In embodiments, the formulations provided herein are administered alone. In embodiments, the formulations provided herein are administered in combination with one or more other active compositions. In embodiments, the formulations are suitable for injecting or implanting incorporated tissue engineering components into the interior of solid organs to regenerate tissue. In embodiments, the formulations are used for injecting or implanting tissue engineering components into the wall of hollow organs to regenerate tissue.

[0258] The present disclosure also provides a method for delivering a bioactive cell preparation to a subject. In embodiments, the source of the bioactive cells can be autologous, allogeneic, syngeneic (autologous or allogeneic), and any combination thereof. In embodiments where the source is not autologous, the method can include administering an immunosuppressant (see, e.g., U.S. Patent No. 7,563,822). Examples of immunosuppressants include, but are not limited to, azathioprine, cyclophosphamide, mizoribine, cyclosporine, tacrolimus hydrate, chlorambucil, lobenzarit disodium, auranofin, alprostadil, gusperimus hydrochloride, biosynsorb, muromonab, alefacept, pentostatin, daclizumab, sirolimus, mycophenolate mofetil, leflunomide, basiliximab, dornase alfa, bindaride, cladribine, pimecrolimus, ilodecaquine, cedelizumab, efalizumab, everolimus, anisperimus, gavilimomab, faralimomab, clofarabine, rapamycin, siplizumab, and shiitake mushroom. Hot water, LDP-03, CD4, SR-43551, SK&F-106615, IDEC-114, IDEC-131, FTY-720, TSK-204, LF-080299, A-86281, A-8 02715, GVH-313, HMR-1279, ZD-7349, IPL-423323, CBP-1011, MT-1345, CNI-1493, CBP-2011, J-695, UP-920 , L-732531, ABX-RB2, AP-1903, IDPS, BMS-205820, BMS-224818, CTLA4-1g, ER-49890, ER-38925, ISAtx-247, RDP-58, PNU-156804, UP-1082, TMC-95A, TV-4710, PTR-262-MG, and AGI-1096 (see U.S. Patent No. 7,563,822). Those of skill in the art will recognize other suitable immunosuppressive drugs.

[0259] In embodiments, at least one active agent (e.g., a renal cell population, a product thereof, or a spheroid comprising a renal cell population and one or more non-renal cell types or populations) is administered directly to the site of intended benefit, e.g., by injection. In embodiments, a subject may be treated by contacting their native kidney in vivo with a bioactive cell preparation described herein, together with a product secreted from one or more enriched renal cell populations and / or a mixture or construct containing said product. In embodiments, the in vivo contacting step produces a regenerative effect on the native kidney.

[0260] Various means of administering compositions of active agents, such as selected kidney cells, to a subject will be apparent to those skilled in the art in light of this specification, including injection of cells into a target site in a subject.

[0261] The administration method of the formulation includes, but is not limited to, systemic injection, intrarenal (e.g., parenchymal) injection, intravenous injection or intraarterial injection, and direct injection into the tissue at the intended active site. Additional administration methods used in certain embodiments herein include single or multiple injections via direct laparotomy, direct laparoscopy, transperitoneal, or percutaneous injection. Still further administration methods used in embodiments include, for example, retrograde injection and ureteral injection into the renal pelvis. Surgical administration methods include, but are not limited to, one-stage procedures such as partial nephrectomy and construct transplantation, partial nephrectomy, partial pyelectomy, vascularization using the retinoperitoneum, multifocal biopsy needle tracking, and renal pole replacement from a conical or pyramidal shape to a cylindrical shape, as well as two-stage procedures, for example, including organoid-in-vivo bioreactors for reimplantation. In embodiments, formulations containing various active agents are delivered simultaneously via the same route. In embodiments, the active agents are delivered to a specific location or separately via a specific technique, either simultaneously or in a time-controlled manner, by one or more of the methods described herein. In embodiments, at least one active agent (e.g., a renal cell population, a product thereof, or a spheroid comprising a renal cell population and one or more non-renal cell types or populations) is injected percutaneously into the renal cortex of the kidney. In this embodiment, a guide cannula is inserted percutaneously and used to puncture the kidney capsule before injecting the composition into the kidney.

[0262] In embodiments, laparoscopic or percutaneous techniques can be used to access the kidney for injection of the formulated BRC or SRC population. In embodiments, laparoscopic techniques allow direct visualization of the kidney, allowing any bleeding or other adverse events during injection to be identified and immediately addressed. In embodiments, the use of percutaneous approaches to the kidney has been used for over a decade, primarily to remove intrarenal masses. In embodiments, these procedures involve inserting an electrode or cryogenic needle into a defined mass within the kidney and maintaining contact for (typically) 10 to 20 minutes while the lesion is removed. In embodiments, for injection of therapeutic formulations, percutaneous devices are not particularly large or complex, and this approach offers the safety benefits of being non-surgical (avoiding abdominal puncture wounds and gas distension) and minimizing occlusion time. In embodiments, a biodegradable hemostatic material may be placed in the access tract to further reduce the possibility of significant bleeding.

[0263] In embodiments, the therapeutic formulation is injected into the renal cortex. In embodiments, it is important to distribute the therapeutic formulation as widely as possible within the renal cortex. In embodiments, distribution of the therapeutic formulation within the renal cortex is achieved by puncturing the renal cortex at an angle that allows for as much distribution of the therapeutic formulation within the renal cortex as possible. In embodiments, the kidney is imaged using a longitudinal or transverse approach using ultrasound guidance, or by axial computed tomography (CT) imaging, depending on the characteristics of the individual patient. In embodiments, the injection involves multiple placements as the injection needle / cannula is gradually withdrawn. In embodiments, the entire volume of the therapeutic formulation can be placed at a single or multiple puncture points. In embodiments, up to two puncture points can be used to place the entire volume of the therapeutic formulation within the kidney. In embodiments, one or more puncture points, for example, one or two puncture points, can be used to perform injections into a single kidney. In embodiments, both kidneys are injected using one or more puncture points, for example, one or two puncture points, in each kidney. In embodiments, the compositions provided herein are administered to a subject multiple times, for example, two or more times, over a given period of time, wherein each administration is at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 12 months after the previous administration. In embodiments, the SRC is administered into one kidney as a single treatment. In embodiments, the BRC (e.g., SRC) is administered into both kidneys as a single treatment by injection. In embodiments, the BRC (e.g., SRC) is administered into one or both kidneys as repeated or multiple injections. In embodiments, the first and second injections are administered at least three months apart, at least six months apart, or at least one year apart. In embodiments, the BRC (e.g., SRC) is administered over three or more injections. In embodiments, the composition is administered as a single injection or multiple injections over a specified period of time. In embodiments, the composition is administered as at least one injection into one kidney. In embodiments, the composition is administered as two or more injections.In embodiments, the first and second injections may be administered at any time up to 3 months apart, any time up to 6 months apart, or at annual intervals. In embodiments, the second injection is administered at any time up to 3 years after the first injection. In embodiments, the composition may also be administered as one, two, or more injections into one or both kidneys. In embodiments, the composition is administered to a subject who is concurrently receiving standard treatment for CKD before receiving an injection of NKA. In embodiments, more than one injection does not cause adverse immunogenic effects. In embodiments, the composition is injected into one kidney of a patient. In embodiments, the composition is injected into both kidneys of a patient. In embodiments, the composition can be injected into a patient's kidney using single or multiple puncture points. In embodiments, the injection is into the renal parenchyma. In embodiments, the patient is administered a therapeutic dose at any given injection site. In an embodiment, the patient receives 1 x 10 per gram of kidney. 6 SRC~9×10 6 A dose of SRC is administered at any given injection site.

[0264] In embodiments, contacting the native kidney in vivo with the secreted product may be accomplished by use / administration of a formulation comprising the secreted product population from cell culture medium, e.g., conditioned medium, and / or by implantation of an enriched cell population and / or construct capable of secreting the product in vivo. In embodiments, the in vivo contacting produces a regenerative effect on the native kidney.

[0265] Various means of administering the cells and / or secreted products to a subject will be apparent to those skilled in the art in light of this specification. In embodiments, such methods include injection of the cells into a target site in a subject.

[0266] In embodiments, the cells and / or secretory products may be inserted into a delivery device or vehicle that facilitates introduction by injection or implantation into a subject. In embodiments, the delivery vehicle may comprise natural materials. In embodiments, the delivery vehicle may comprise synthetic materials. In embodiments, the delivery vehicle results in a structure that mimics or appropriately matches an organ structure. In embodiments, the delivery vehicle is fluidic in nature. In embodiments, such a delivery device may include a tube, e.g., a catheter, for injecting the cells and fluid into the body of the recipient subject. In embodiments, the tube further has a needle, e.g., a syringe, that can introduce the cells into the subject at a desired location. In embodiments, the mammalian kidney-derived cell population is formulated for intravascular administration via a catheter (wherein the term "catheter" is intended to include any of a variety of tube-like systems that deliver substances to a blood vessel). In embodiments, the cells are supported on biomaterials including, but not limited to, textiles such as woven, knitted, braided, mesh, and nonwoven fabrics, perforated films, sponges and foams, and beads such as solid or porous beads, microparticles, nanoparticles (e.g., Cultispher-S gelatin beads, Sigma). The cells may be inserted into or onto a biomaterial or scaffold. In embodiments, the cells may be prepared in a variety of different forms for delivery. In embodiments, the cells may be suspended in a solution or gel. In embodiments, the cells may be mixed with a pharmaceutically acceptable carrier or diluent that maintains the cells in a viable state. In embodiments, pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents, and / or dispersion media. The use of such carriers and diluents is known in the art. In some embodiments, the solution is sterile, fluid, and often isotonic. In embodiments, the solution is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms, such as bacteria and fungi, by the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. One of skill in the art will understand that the delivery vehicle used in delivering the cell population and / or mixture thereof may include a combination of the above-mentioned characteristics.

[0267] In one aspect, provided herein is a method of treating kidney disease in a subject, the method comprising injecting into the subject a formulation, composition, or cell population disclosed herein. In embodiments, the formulation, composition, or cell population is injected through an 18- to 30-gauge needle. In embodiments, the formulation, composition, or cell population is injected through a needle smaller than 20-gauge. In embodiments, the formulation, composition, or cell population is injected through a needle smaller than 21-gauge. In embodiments, the formulation, composition, or cell population is injected through a needle smaller than 22-gauge. In embodiments, the formulation, composition, or cell population is injected through a needle smaller than 23-gauge. In embodiments, the formulation, composition, or cell population is injected through a needle smaller than 24-gauge. In embodiments, the formulation, composition, or cell population is injected through a needle smaller than 25-gauge. In embodiments, the formulation, composition, or cell population is injected through a needle smaller than 26-gauge. In embodiments, the formulation, composition, or cell population is injected through a needle smaller than 27 gauge. In embodiments, the formulation, composition, or cell population is injected through a needle smaller than 28 gauge. In embodiments, the formulation, composition, or cell population is injected through a needle smaller than 29 gauge. In embodiments, the formulation, composition, or cell population is injected through a needle of about 20 gauge. In embodiments, the formulation, composition, or cell population is injected through a needle of about 21 gauge.

[0268] In embodiments, the formulation, composition, or cell population is injected through an approximately 22-gauge needle. In embodiments, the formulation, composition, or cell population is injected through an approximately 23-gauge needle. In embodiments, the formulation, composition, or cell population is injected through an approximately 24-gauge needle. In embodiments, the formulation, composition, or cell population is injected through an approximately 25-gauge needle. In embodiments, the formulation, composition, or cell population is injected through an approximately 26-gauge needle. In embodiments, the formulation, composition, or cell population is injected through an approximately 27-gauge needle. In embodiments, the formulation, composition, or cell population is injected through an approximately 28-gauge needle. In embodiments, the formulation, composition, or cell population is injected through an approximately 29-gauge needle.

[0269] In embodiments, the inner diameter of the needle is less than 0.84 mm. In embodiments, the inner diameter of the needle is less than 0.61 mm. In embodiments, the inner diameter of the needle is less than 0.51 mm. In embodiments, the inner diameter of the needle is less than 0.41 mm. In embodiments, the inner diameter of the needle is less than 0.33 mm. In embodiments, the inner diameter of the needle is less than 0.25 mm. In embodiments, the inner diameter of the needle is less than 0.20 mm. In embodiments, the inner diameter of the needle is less than 0.15 mm. In embodiments, the outer diameter of the needle is less than 1.27 mm. In embodiments, the outer diameter of the needle is less than 0.91 mm. In embodiments, the outer diameter of the needle is less than 0.81 mm. In embodiments, the outer diameter of the needle is less than 0.71 mm. In embodiments, the outer diameter of the needle is less than 0.64 mm. In embodiments, the outer diameter of the needle is less than 0.51 mm. In embodiments, the outer diameter of the needle is less than 0.41 mm. In 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 following table:

[0270] TIFF2025160401000002.tif50170

[0271] A non-limiting example of a cell-containing therapeutic product is Neo Kidney Augment (NKA), which contains SRCs (i.e., allogeneic, autologous, selected kidney cells) as the biologically active component. Without being bound by any scientific theory, this cell population is inherently involved in kidney repair and regeneration (Bruce et al. Regen Med. 2015;10:815-39; Bruce et al. Experimental Biology Meeting, Washington, DC, 2011; Genheimer et al. Cells Tissues Organs. 2012;196:374-84; Ilagan et al. TERMIS Conference, Orlando, FL, 2010; Ilagan et al. TERMIS Conference, Orlando, FL, 2010; Ilagan et al. KIDSTEM Conference, Liverpool, UK, 2009; Kelley et al. Cell Transplant. 2013;22:1023-39; Kelley et al. ADA Conference, San Diego, CA, 2011; Kelley et al. ISC T Conference, Philadelphia, PA, 2010, Kelley et al. KIDSTEM Conference, Liverpool, UK, 2008, Kelley et al. TERMIS Conference, Orlando, FL, 2010, Presnell et al. Tissue Engineering Part C Methods. 2010;17:261-73, Presnell et al. Experimental Biology Meeting, New Orleans, LA, 2009, Wallace et al. ISCT Conference, Philadelphia, PA, 2010, Yamaleyeva et al. TERMIS Conference, Orlando, FL, 2010). implementation In this manner, therapeutic intervention with NKA improves renal function in subjects with CKD and CAKUT and delays the need for renal dialysis or kidney transplantation, which, based on the current standard of care, is inevitable for ESRD.

[0272] NKAs are made from expanded autologous selected renal cells (SRCs) obtained from each individual subject's kidney biopsy. In embodiments, to produce NKAs, kidney biopsy tissue from a subject is processed to expand kidney cells and select SRCs.

[0273] In an embodiment, the NKA is given in a sterile, disposable syringe. In an embodiment, the SRC is 100 x 10 6 The cells are formulated in a gelatin-based hydrogel at a concentration of 100 x 10 cells / mL, packaged in 10 mL syringes, and shipped to the clinical site for use. In an embodiment, the final volume is 100 x 10 cells / mL of NKA. 6 The concentration of SRC was 3.0 x 10 per gram of kidney weight. 6 The volume of the kidney is calculated from the target dose of SRC (e.g., estimated by MRI). In embodiments, kidney volume measurements in mL obtained by various methods, as described in the literature, are approximately 92% to 97% of the dry weight measurement in grams obtained by measuring the excised organ trimmed of perinephric fat. In embodiments, the dose of NKA is calculated using the conversion 1 g equals 1 mL. In embodiments, the dosage is determined based on the patient's kidney weight at the time of injection. In embodiments, the maximum volume for any patient is 8.0 mL. That is, if the calculated weight of any subject's left kidney is 259 g or greater, the subject will receive 8 mL of NKA.

[0274] The expanded renal cells may be cryopreserved during cell expansion to accommodate patient-specific variability in cell growth. Cryopreserved renal cells provide a continuous source of cells for producing multiple doses of bioactive cell preparations for reinjection in the event that another treatment is needed (e.g., delay due to patient illness, unanticipated course of events, etc.).

[0275] In order to facilitate a more complete understanding of the present invention, the following examples are provided. The following examples illustrate exemplary ways of making and practicing the present invention. However, the scope of the present invention is not limited to the specific embodiments disclosed in these examples, which are for illustrative purposes only, as alternative methods may be utilized to achieve similar results. [Example]

[0276] Example 1 - A Phase I, Open-Label Safety, Tolerability, and Early Efficacy Study of Renal Autologous Cell Therapy (REACT) in Patients with Chronic Kidney Disease from Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) (REGEN-044) Protocol Synopsis therapeutic products REACT is made from expanded autologous selected kidney cells (SRCs) obtained from an individual subject's kidney biopsy. To manufacture REACT, kidney biopsy tissue from each enrolled subject is sent to Twin City Bio LLC, where the kidney cells are expanded and SRCs are selected. SR C is 100 x 10 6 It is formulated in a gelatin-based hydrogel at a concentration of 10 cells / mL, packaged in 10 mL syringes, and shipped to the clinical site for use.

[0277] Research purpose Primary Objective: The primary objective of this study is to evaluate the safety of REACT injected into one recipient kidney.

[0278] Primary endpoint: Changes in eGFR over 6 months after two REACT injections Incidence of renal-specific procedural and / or product-related adverse events (AEs) through 6 months post-injection

[0279] Secondary Objectives: The secondary objective of this study is to evaluate the safety and tolerability of REACT administration by assessing renal-specific adverse events over a 24-month period following injection.

[0280] Secondary endpoints: Renal-specific laboratory evaluations through 24 months post-injection

[0281] Exploratory Objectives: The exploratory objectives of this study are designed to evaluate the impact of REACT on renal function over a 24-month period following injection.

[0282] Exploratory endpoints: Clinical diagnostic and laboratory evaluation of renal structure and function (including eGFR, serum creatinine, and proteinuria) to assess changes in the rate of progression of renal disease Vitamin D levels Iohexol imaging Blood pressure control MRI assessment of kidney volume

[0283] Study design Multicenter, prospective, open-label, single-arm study. All subjects will be treated with two REACT injections 3 months (+12 weeks) apart after biopsy.

[0284] Randomization Open-label, non-randomized

[0285] Control group Each subject will serve as his or her own control. The patient's previous medical history, which must include observation of renal function for a minimum period of 6 months, will serve as a control for the rate of progression of renal failure.

[0286] Sample Size A maximum of 15 patients will be treated with REACT. Because this is a Phase I safety trial, robust statistical analysis is not required. Therefore, the proposed sample size for this study is typical of a Phase I study, allowing for confirmation of safety outcomes in a limited population.

[0287] Study population CAKUT results in 14 mL / min / 1.73 m 2 ~50mL / min / 1.73m 2 Male or female patients aged 18-65 years with CKD, defined as eGFR of 100 mg / kg / day. Patients should have sufficient historical clinical data (three or more eGFR measurements) to determine their individual rate of progression of CKD.

[0288] Inclusion Criteria: Unless otherwise stated, subjects must meet each inclusion criterion to participate in the study. Inclusion criteria should be assessed at the screening visit, before kidney biopsy, and before each REACT injection unless otherwise specified. 1. Male or female patients aged 18-65 years on the date of informed consent. 2. Patients with a documented history of renal and / or urinary tract abnormalities in addition to a documented history of CAKUT. 3. 14 mL / min / 1.73 m including the border at the screening visit before REACT injection 2 to 50 mL / min / 1.73 m 2 Patients with a confirmed diagnosis of stage III / IV CKD who do not require renal dialysis and have an eGFR between 0.1 and 0.2. 4. Subjects with blood pressure less than 140 / 90 at the screening visit, before kidney biopsy, and before REACT injection(s). Note that blood pressure should not fall significantly below 115 / 70. 5. A minimum of three measurements of eGFR or sCr at least 3 months apart should be obtained prior to the screening visit and within the past 24 months to define the rate of CKD progression. 6. Patients who are willing and able to refrain from taking NSAIDs (including aspirin), as well as clopidogrel, prasugrel, or other platelet inhibitors, for the period beginning 7 days before and ending 7 days after both the renal biopsy and REACT injection(s). 7. Patients willing and able to refrain from taking platelet aggregation inhibitors, such as fish oil and dipyridamole (i.e., Persantine™), starting 7 days prior to and ending 7 days after both the renal biopsy and REACT injection(s). 8. Patients willing and able to cooperate with all aspects of the protocol. 9. Patients willing and able to provide signed informed consent.

[0289] Exclusion Criteria: Subjects who meet the exclusion criteria listed below are not eligible to participate in the study. Exclusion criteria will be assessed at the screening visit, prior to kidney biopsy, and prior to each REACT injection unless otherwise noted. 1.Patients with a history of kidney transplantation. 2.Patients diagnosed with SFU grade 4 or SFU grade 5 hydronephrosis. 3. Patients with uncorrected VUR grade 5. 4. Patients with cortical thickness less than 5mm on MRI. 5. Patients with known allergies or contraindications, or history of severe systemic reaction(s) to kanamycin or structurally related aminoglycoside antibiotic(s). 6. Patients with a history of anaphylactic or severe systemic reaction(s) or contraindication(s) to human blood products or materials of animal origin (e.g., bovine, porcine). 7. Patients with a history of severe systemic reaction(s) to local anesthetics or sedatives or any contraindication. 8. Patients with a clinically significant infection requiring parenteral antibiotics within 6 weeks of REACT injection. 9. Patients who have suffered from acute kidney injury or rapid decline in renal function within the past 3 months prior to REACT injection. 10. Patients with any of the following conditions prior to REACT injection: renal tumor, polycystic kidney disease, anatomic abnormalities that would interfere with the REACT injection procedure, or evidence of urinary tract infection. NOTE: Anatomic abnormalities are not excluded as long as the kidney is accessible and meets the criteria to receive a REACT injection. 11. Patients with class III or class IV heart failure (NYHA functional class). 12. Patients with FEV1 / FVC ≥ 70%. 13. Patients with a history of cancer within the past 3 years (non-melanoma skin cancer and cervical intraepithelial cancer) (Excluding cancer). 14. Patients with clinically significant liver disease (ALT or AST >3x upper limit of normal) when assessed at the screening visit. 15. Patients who are positive for active infection with Hepatitis B virus (HBV) or Hepatitis C virus (HCV), and / or Human Immunodeficiency Virus (HIV) when evaluated at the screening visit. 16. Patients with a history of active tuberculosis (TB) requiring treatment within the past 3 years. 17. Patients who are immunocompromised or receiving immunosuppressive medications, including individuals who have been treated for chronic glomerulonephritis within 3 months of REACT injection. NOTE: Inhaled corticosteroids and chronic low-dose corticosteroids (7.5 mg per day or less) are allowed, as are short-course pulse corticosteroids for intermittent symptoms (e.g., asthma). Patients with a life expectancy of less than 18.2 years. 19. Female patients who are pregnant, lactating (breastfeeding), or planning to become pregnant during the course of the study, or who are of childbearing potential and are not using highly effective contraceptive methods(s) that include sexual abstinence, or who are unwilling to continue using highly effective contraceptive methods throughout the study period. 20. Patients with an active alcohol and / or substance abuse history that, in the investigator's judgment, would impair their ability to comply with the protocol. 21. Patients whose health status, in the opinion of the investigator, would be at risk from participating in the study. 22. Patients who have used any investigational drug within 3 months prior to REACT injection without written consent from the medical monitor.

[0290] Research period Treatment will begin as soon as the REACT product becomes available, with a 1-month interval before receiving the first REACT injection, a 3-month interval before receiving the second injection, and a 24-month follow-up period after the final injection. 28 months for a series of two REACT injections This becomes:

[0291] Study registration A maximum of 15 subjects will be enrolled in the study. Patients who complete the screening procedure and meet all I / E criteria will be enrolled in the study immediately prior to biopsy. Patients who do not meet all criteria before biopsy is taken will be considered screen-ineligible. Patients who undergo biopsy but are not injected for any reason will be discontinued from the study and may be replaced. Once patients are injected, they will complete treatment, and every effort will be made to ensure that patients complete all follow-up visits.

[0292] Clinical trial planning Screening: Subjects who meet the eligibility criteria and provide written informed consent may participate in the study. Subjects should have adequate historical clinical data that will provide a reasonable estimate of the rate of CKD progression after consultation with a medical monitor. Screening procedures include a complete physical examination, ECG, and clinical laboratory evaluation (hematology, clinical chemistry, and urinalysis). An ultrasound is performed to confirm the anatomical features of the kidney that will receive the biopsy and injection. An MRI or ultrasound is completed to measure kidney size and volume and determine the administration volume.

[0293] Renal Biopsy: Within 3 days prior to undergoing a renal biopsy, enrolled subjects will present to the clinic for a preliminary biopsy along with an ECG and renal MRI (if not completed during or after the screening visit). Subjects undergo a physical examination. Laboratory tests, including renal function tests, hemoglobin tests, and, for women, a pregnancy test, are also performed. Eligible subjects who meet all inclusion and exclusion criteria are admitted to a hospital / clinical research center for kidney biopsy. To provide sufficient material for the production of REACT, a minimum of two tissue cores measuring 1.5 cm each must be collected using a 16-gauge biopsy needle. Subjects who do not experience complications from the biopsy can be discharged the same day, according to local standard practice. The kidney biopsy tissue of each individual subject is then transferred to a Twin It will be sent to City Bio LLC.

[0294] REACT Injection: 10-14 days prior to the scheduled injection date, subjects will undergo an interim physical examination for ongoing verification of inclusion and exclusion criteria. Subjects will also undergo a renal scintigraphy (i.e., a split renal function scan) to determine the percentage contribution of each kidney to overall baseline renal function. On the scheduled REACT injection date, eligible subjects will be admitted to the hospital / clinical research unit. After warming and liquefying the hydrogel, REACT will be injected into the same kidney previously biopsied using a percutaneous approach. This procedure follows standard techniques, such as those used for radiofrequency or cryogenic resection of renal masses. Subjects without complications may be discharged the same day, in accordance with local standard practice. An ultrasound will be performed the day after injection to detect any potential subclinical AEs. Subjects will receive two REACT injections, 3 months (+12 weeks) apart. The first and second injections will be administered into the same kidney from which the biopsy was taken. Therefore, only one kidney will be used for the duration of the study.

[0295] Follow-up: Subjects complete follow-up assessments at days 1, 7, 14, and 28 (± 3 days) and 2 months (± 7 days) after the first and second REACT injections. Depending on when the second injection is administered (i.e., 3 months [+ 12 weeks]), subjects may be assessed 3 and 6 months after the first REACT injection. Following the final REACT injection, subjects complete long-term follow-up assessments of safety and efficacy through 6, 9, 12, 15, 18, 21, and 24 months after treatment.

[0296] Safety Monitoring: Bleeding after REACT injection is a known and predictable risk for subjects participating in this study. Therefore, hemoglobin will be measured by the local laboratory at the following times: a) pre-procedure, b) post-procedure, according to local standard practice.

[0297] Investigational drug, dosage, and administration route Investigational Product: REACT is made from expanded autologous selected kidney cells obtained from each individual subject's kidney biopsy. To manufacture REACT, biopsy tissue from each enrolled subject will be sent to Twin City Bio LLC, where the kidney cells will be expanded and SRCs selected. SRC is 100 x 10 per mL 6 The cells are formulated in a gelatin-based hydrogel at a concentration of 1000 cells per 1000 cells, packaged in 10 mL syringes, and shipped to the clinical site.

[0298] Dosage: The volume of REACT administered is determined by pre-procedure MRI volumetric 3D assessment or by the ellipsoid formula (length x width AP plane x width transverse plane x 0.62). Based on preclinical data, the dose of REACT is determined per gram of estimated kidney weight (gKW). est ) 3 x 10 6 The concentration of SRC per mL of reactant is 100 x 10 per mL. 6Because there are 100 cells, the dose volume is 3.0 mL per 100 g kidney weight. Using this dosing paradigm, the table below shows the dose volume and number of SRCs delivered for each estimated kidney weight. The maximum volume of REACT injected into a biopsied kidney would be 8.0 mL.

[0299] TIFF2025160401000003.tif53170

[0300] Subjects will receive two planned injections of REACT to allow for titration and to assess duration of effect. The first and second injections will be given in the same kidney from which the biopsy was taken. In some cases, the subject or investigator may decide to postpone or withhold the second REACT injection. For example, the second REACT injection should not be administered if there appears to be any undue safety risk, or rapid deterioration of renal function, or the development of uncontrolled diabetes or uncontrolled hypertension, or the development of malignancy or intercurrent infection.

[0301] Administration: REACT is injected into the biopsied kidney using a percutaneous approach using standard techniques (such as those utilized for radiofrequency or cryogenic ablation of renal masses).

[0302] statistical analysis method Statistical analyses were primarily descriptive in nature, and no statistical hypothesis testing was planned for this study. Unless otherwise specified, continuous variables will be summarized by presenting the number of nonmissing observations (n), mean, standard deviation, median, minimum, and maximum. Categorical variables will be summarized by presenting frequency counts and percentages for each category.

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[0306] 1. CAKUT and chronic kidney disease A common component of CAKUT is vesicoureteral reflux, defined as the reflux of urine from the bladder into one or both ureters, the renal pelvis, or both. Primary vesicoureteral reflux (VUR) is the most common congenital urinary tract abnormality in childhood, and it is usually diagnosed after an episode of urinary tract infection (UTI). VUR is thought to predispose to UTIs and renal scarring. Renal scarring associated with VUR is also known as reflux nephropathy (RN). Potential long-term complications of RN include hypertension, proteinuria, and progression to end-stage renal disease (ESRD). [5] Patients with abnormally developed kidneys are most vulnerable to the development of ESRD because their kidneys continue to deteriorate even after VUR is corrected (Brakeman). Ardissino et al. reported that nearly 26% of end-stage renal disease cases in patients with low dysplasia were due to urinary bladder obstruction. The exact incidence of RN in children or adults is unknown. RN accounts for 12% to 21% of all children with chronic renal failure. [1、2] According to a 2008 report from the North American Pediatric Renal Trials and Collaborative Studies, RNs reported that 8.4% of children had chronic kidney disease. It is the fourth most common cause of glaucoma and the primary medical condition in 5.2% of transplant patients and 3.5% of dialysis patients. [3] In the CKID study involving a cohort of 586 children aged 1 to 16 years, 30 mL / min / 1.73 m 2 ~90mL / min / 1.73m 2RN was the underlying cause of CKD in 87 (14.8%) patients. In adults, obstructive uropathy was the point prevalent case of ESRD in 2005. CKD accounts for 0.3% of cases, some of which may be attributable to RN. Few cohort studies have performed long-term follow-up of patients after antireflux surgery. In one cohort from Israel, only 1 in 100 patients developed CKD after 20 years. In another cohort of patients identified as having renal scarring by IVP, 18% developed CKD after 20 years. Regardless, the incidence of ESRD in adults due to RN is low.

[0307] Chronic kidney disease (CKD) is characterized by progressive nephropathy that will worsen without therapeutic intervention until patients reach ESRD. CKD is defined as a decline in kidney function supported by evidence of kidney damage, such as a decreased glomerular filtration rate (GFR) or increased urinary albumin excretion. The global prevalence of CKD is estimated to be 8%–16%. To survive, patients with ESRD require renal replacement therapy (dialysis or kidney transplantation). Preventing or delaying adverse outcomes of CKD through early intervention is the primary strategy for CKD management. Nevertheless, early treatment is suboptimal, resulting in a significant unmet medical need for improved intervention strategies to manage CKD and delay progression to ESRD.

[0308] Treatment for patients with CKD focuses on slowing progression and preparing for kidney failure / replacement. In many patients, CKD occurs as part of a complex comorbidity cluster. Once patients reach ESRD, renal replacement therapy (i.e., dialysis or transplant) is indicated. The majority of patients in stage 5 undergo hemodialysis. [4] Dialysis replaces approximately 5% to 15% of kidney function, depending on the intensity and frequency of use. Dialysis also helps restore fluid and electrolyte balance when the kidneys fail. However, ESRD patients who begin hemodialysis have an average life expectancy of only 4 to 5 years. [5]Furthermore, hemodialysis is associated with many serious complications, such as the need to undergo dialysis up to three times a week, as well as an interference with quality of life. Currently, kidney transplantation remains the most effective form of treatment. Organs are in chronic shortage. If a patient is able to secure a kidney for transplant, long-term immunosuppressive therapy is required to prevent rejection. The use of these regimens leads to a higher incidence of infections and, in the long term, some types of cancer. [6] In summary, there is a significant medical need for improved therapies for CKD that can dramatically slow disease progression and significantly delay or reduce the need for kidney transplantation. Table 4 defines the stages of CKD according to GFR measurements. The early stage of nephropathy (Stage 1) occurs over a period of several years and is characterized by microalbuminuria (30 mg / 240 ml). It is characterized by a rise in serum creatinine (up to 300 mg / 24 hours) followed by overt albuminuria (greater than 300 mg / 24 hours). As the kidneys' ability to filter blood waste declines, serum creatinine rises. As kidney damage increases (Stages 2-4), elevated blood pressure further worsens kidney disease. When the kidneys completely stop functioning (Stage 5 [ESRD]), kidney replacement therapy (dialysis or transplantation) is required.

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[0310] 1.1 Nonclinical pharmacology studies In a series of preclinical studies, Tengion (a former regenerative medicine company) has characterized the pharmacological properties of SRC. Defined and slowed progression of CKD in experimental models by enhancing kidney structure and function [7~12] Subsequently, Tengion conducted safety pharmacology and GLP toxicology studies. A summary of these non-clinical studies is shown in Table 5.

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[0312] Pharmacodynamics Proof-of-principle for SRC as the biologically active component of REACT has been established in multiple animal models of CKD. For example, the 5 / 6 nephrectomy (Nx) rodent mass reduction model of CKD allowed for optimized selection of therapeutically relevant SRC cell populations. The 70% Nx canine model of CKD confirmed SRC activity in a large mammal, while the ZSF-1 rat served as proof-of-principle to demonstrate the efficacy of SRC in a model relevant to T2DM.

[13] In multiple experimental models of CKD, SRC delivered directly to the renal cortex induced a regenerative response via direct engraftment or tissue replacement, as well as secreted factors via a putative paracrine mechanism. [9、14~17] .

[0313] This intervention strategy significantly improved survival, stabilized disease progression, and extended lifespan in both the 5 / 6Nx and ZSF-1 rodent models of CKD. Morphological normalization of multiple nephron structures was accompanied by functional improvements, including glomerular filtration, tubular protein transport, electrolyte balance, and the ability to concentrate urine. Lower blood pressure and reduced circulating renin levels were also observed in the ZSF-1 rat model. The functional improvements observed after SRC treatment were accompanied by significant reductions in glomerular sclerosis, tubular degeneration, and interstitial inflammation and fibrosis. No toxicologically significant in-life clinical pathology or histological changes were observed in target organs or other tissues. Based on the results of numerous preclinical studies conducted in various CKD animal models, SRC (i.e., the active component of REACT) was effective in significantly slowing CKD progression when injected into affected organs before irreversible nephropathy. These results provide a rationale for investigating the efficacy of this cell-based intervention in patients with pre-ESRD.

[0314] 1.1.2. Safety Pharmacology 1.1.2.1. Extrarenal activity REACT (i.e., SRC formulated in a gelatin-based hydrogel) was administered to various rat and dog models to evaluate its immediate cardiovascular and respiratory pharmacological effects. The acute effects of lower and higher SRC concentrations formulated in various percentages of gelatin (0.75% to 1.0%) were evaluated in a rodent 5 / 6Nx model. Potential changes in blood pressure were assessed immediately before, during, and after REACT delivery in a normal dog model. Studies of REACT's effects on the central nervous system were not performed because 1) animals exhibited normal behavior before, during, and after REACT injection, 2) no central nervous system effects were expected from an investigational drug containing intact renal cells, and 3) REACT was delivered to the kidney.

[0315] Hemodynamic effects Rats in the 5 / 6Nx study (Study No. 4) were administered REACT or vehicle control and monitored for potential hemodynamic effects over a 4-day period. Of the 77 animals treated in this REACT formulation study, 16 animals experienced apnea during or immediately after REACT delivery. A total of 9 animals died. Causes of death were classified as apnea (n=3), renal hemorrhage (n=2), and CKD-related death (n=4). Six of the 16 apneic animals were not pretreated with atropine. Of these apneic animals, two died under the effects of anesthesia prior to the use of atropine. Ten of the 16 apneic animals were treated with atropine, and all recovered from the surgical procedure and REACT injection.

[0316] In contrast, the apnea, renal hemorrhage, and death that occurred in the 5 / 6Nx rat study were not observed in the ZSF-1 rat study, or in the dog pharmacology study, or in two (intact) dog pilot studies evaluating the short-term effects of high-dose administration on blood pressure. Without being bound by any theory, in summary, the hemodynamic responses unique to this model may be due to: 1) altered hemodynamics of the remnant kidney in severely mass-depleted rodents;

[18] , 2) the kidney, which triggers a central autonomic response Transient changes in hepatic interstitial pressure control [19、20] 3) Hemorrhage after delivery to the kidney may have resulted in tissue insufficiency or acute hypoxia. Pretreatment with atropine, a competitive antagonist of the parasympathetic nervous system, helped to mitigate the hemodynamic changes specific to this model. The effect of atropine suggested a possible autonomic response to REACT delivery specific to the severely mass-depleted 5 / 6Nx rodent model of CKD.

[0317] 1.1.2.3. Dosage Using various doses, volumes, and concentrations of REACT (Study No. 5), normal dogs were selected to assess blood pressure immediately before, during, and after delivery of REACT to the kidney. In this study, 2.5 mL of REACT was administered to each pole of each kidney, thus delivering a total of 10 mL / 120 g, or 0.083 mL / g, or 12.5 x 10 per gram of kidney mass. 6 REACT treatment was well tolerated, with no systemic side effects (physical or serological) and no significant toxicological or histomorphological changes indicative of kidney or other tissue damage as a result of REACT delivery. In contrast to rodent models of severely mass-depleted CKD, no apnea, renal hemorrhage, or death was observed.

[0318] 1.1.3. Dynamics, migration, and persistence As with other cell-based therapies targeting soft organs, data on the biodistribution of the investigational drug are limited. Therefore, three additional studies will provide evidence regarding the potential translocation and persistence of REACT within the kidney at selected sampling times after delivery. [16、17、21] The results of these studies are summarized in this section; further information is provided in the Investigator Brochure.

[0319] ZSF-1 rats SRCs were labeled with rhodamine B superparamagnetic iron oxide (SPIO) particles. This imaging agent is specifically formulated for cell labeling and is readily taken up by non-phagocytic cells. SPIO-labeled cells were administered to the kidneys of ZSF-1 rats. 24 hours after delivery, SPIO-labeled cells were detected by MRI and whole-organ optical imaging. Furthermore, ZSF-1 rats were administered CelSense-19F-labeled SRCs, which were quantified by nuclear magnetic resonance imaging 3 hours, 24 hours, and 7 days after injection. [16、22] .

[0320] Both acute ZSF-1 detection and long-term donor cell detection using a 5 / 6Nx model of CKD demonstrated significant retention of SPIO-labeled cells. Clinically relevant MRI detection 24 hours after cell delivery revealed the area of ​​the anterior pole of the kidney where the SPIO-labeled cells were injected. Whole kidney sectioning and Prussian blue staining showed a bolus of iron-labeled SPIO cells migrating and distributing from the cortical injection site, confirming their presence in the tubules and peritubular spaces of the renal cortex and medulla.

[0321] Similarly, whole-organ fluorescence imaging highlighted cellular detection at and around the injection site, located in the superior cortex of the anterior pole of the ZSF-1 rat kidney. Detection of 19F-labeled SRC 3 and 24 hours after delivery confirmed nearly 100% retention in the kidney. After 7 days, detection of 19F-labeled SRC decreased by orders of magnitude, consistent with continued urinary excretion.

[0322] 1.1.3.2. Non-GLP Analysis of Non-Kidney Tissues in Pig In a preclinical, non-GLP study, SPIO-labeled SRC was delivered to the kidneys of live pigs (n=11). Cell distribution was monitored over the 30-day study period using MRI. Labeled SRC was distributed to two major compartments: the bladder and the renal parenchyma. The primary route of excretion was via urine. Notably, no evidence of ectopic SRC migration or site-specific engraftment in non-target organ sites was observed.

[23] .

[0323] 1.1.3.3. Dog Non-GLP Analysis of Non-renal Tissues In a preclinical, non-GLP study, SPIO-labeled SRC was delivered to the kidneys of living canine hosts. Cellular distribution was monitored via MRI 30 minutes after injection. Consistent with observations from a living pig model showing that injected SPIO-labeled SRC is retained in the renal parenchyma or excreted in the urine, SPIO-labeled SRC was also retained within the renal parenchyma at the injection site 30 minutes later.

[0324] 1.1.3.4. Conclusion SRC was distributed to the injection site (renal parenchyma) and excreted via urine based on SRC labeling studies using SPIO and CelSense-19F. SRC delivered to the kidneys of rats, pigs, and dogs was not detected in non-target organs (other than the excretory urinary tract) based on extensive histological evaluation. Based on reports on allogeneic mesenchymal stem cells and published data on the safety of autologous mesenchymal stem cells in clinical trials, autologous REACT-related materials should also not cause ectopic tissue growth, organ dysfunction, or tumorigenesis. [24~28] .

[0325] 1.2. Toxicological research Three GLP safety studies were conducted to evaluate the safety of REACT: one study was conducted in the rat ZSF-1 disease model of CKD, and two other studies were conducted in normal dogs.

[0326] ZSF-1 Rat Single-Dose Study The purpose of this study was to evaluate the safety of a single dose of REACT in ZSF-1 rats, a model of uncontrolled metabolic syndrome including T2DM, hypertension, and severe obesity. Rats received 1) a high dose of REACT, 2) a low dose of REACT, 3) a sham, or 4) biomaterial alone. Each animal received four injections of the test article, one into each pole of each kidney. Results were evaluated after 3 and 6 months of treatment.

[0327] 1.2.1.1. Renal findings After evaluation of eight regions of each kidney (three stains per region), including evaluation and scoring of 150 glomeruli per kidney, no treatment-related renal findings were observed. With the exception of changes related to injection and / or linear scarring at the injection site, all kidneys were considered normal within the context of the disease model. No test article-related renal findings were observed after 3 or 6 months of treatment. All macroscopic and microscopic renal changes were considered to be related to the natural progression of renal disease in ZSF-1 obese rats or to the injection treatment.

[0328] Renal changes in all groups were more severe in men, consistent with differences in disease stage between the sexes. Overall, there was a clear trend toward lower renal histological severity scores (i.e., lower glomerular injury score, tubulointerstitial injury score, and global nephron score) consistently observed in the low-concentration REACT-treated group compared with the sham control group after 6 months of treatment.

[0329] Based on the lack of differences between study groups, the no observed adverse effect level (NOAEL) was the high dose of 6.25 × 10 6 cells / gKW est It was.

[0330] 1.2.1.2. Non-renal findings No findings related to REACT safety were observed in non-target tissues. No REACT-related changes were observed in the ureter or bladder (the major pathways for REACT excretion). No REACT-related effects were seen, and no observable REACT cellular material was found in any of the draining (lymph nodes) or filtering (liver, lung, spleen) tissues examined.

[0331] 1.2.1.3.Clinical pathology Results of clinical laboratory tests (including hematology, clinical chemistry, and special urinalysis panels) were evaluated for differences between baseline and end of study (after 3 or 6 months of treatment) and between treatment and control groups. No REACT-related clinically significant laboratory abnormalities were identified.

[0332] 1.2.1.4. Conclusion All animals survived to the end of the study (after 3 or 6 months of treatment). There were no treatment-related significant safety-related clinical pathology or toxicologically significant safety-related findings. No REACT-related clinically significant laboratory abnormalities were identified. The observed NOAEL is 6.25 x 10 6 cells / gKW est It was.

[0333] 1.2.2. Initial single-dose dog study The initial canine toxicology study evaluated the safety of a single dose of two different doses of REACT compared with sham treatment or biomaterial treatment. The test article was delivered to one pole of each kidney. A total of 32 mixed-breed dogs were enrolled in the study. 16 were evaluated at 1 month and 16 at 3 months.

[0334] 1.2.2.1. General Results All 32 animals survived to the designated endpoints after 1 or 3 months of treatment. Animals appeared healthy throughout the study. No significant clinical pathology findings were noted. There were no signs of renal failure (azotemia) or indications of reduced GFR.

[0335] 1.2.2.2. Kidney-related outcomes No macroscopic or microscopic findings related to REACT delivery were observed at the 1-month or 3-month endpoints. After evaluation of eight regions of each kidney (three stains per region), including evaluation and scoring of 150 glomeruli per kidney, no treatment-related renal findings were noted. All kidneys were normal, except for changes related to scarring at the injection site. All macroscopic and microscopic renal changes were considered background findings or related to the injection treatment.

[0336] 1.2.2.3. Non-renal findings No test article-related findings were identified in other (non-renal) tissues. All macroscopic and microscopic changes were considered background changes and within normal limits.

[0337] 1.2.2.4. Procedure-related findings The most common abnormalities included swelling at the incision site (seroma formation) and weight loss at the end of the study. Regarding incision site swelling, 10 of 16 (10 / 16) animals developed sterile seromas after injection and 9 of 16 (9 / 16) developed them after treatment.

[0338] Animals had varying degrees of swelling at the time of retroperitoneal incision and were treated as deemed necessary by a veterinarian.

[0339] Many animals showed mild anorexia after REACT administration (29 of 32) and after treatment (18 of 32). Most animals (28 of 32) experienced weight loss from baseline (pre-injection) to termination. Notably, greater weight loss occurred between baseline (2 weeks prior to treatment) and treatment (day 0; kidney injection) than between treatment and termination. Weight loss occurred across all treatment groups, suggesting a more stressful period in the study. It was determined that the nature of the

[0340] 1.2.2.5. Conclusion All animals survived to the designated endpoints and were in good general health throughout the study based on clinical pathology, urinalysis, and veterinary evaluation. Neither low dose REACT nor high dose REACT caused any macroscopic or microscopic adverse effects at 1 or 3 months after treatment, similar to those observed after sham treatment or biomaterial treatment. Pathological evaluation revealed no findings (macroscopic or microscopic) related to the safety of REACT in the target organs (kidneys) or non-target organs examined. Based on anatomic pathology, the observed NOAEL was at the higher tested concentration, i.e., 11.7 × 10 6cells / gKW est It was.

[0341] 1.2.3. Repeated-dose dog studies A second dog toxicology study evaluated the safety of two repeat doses of REACT. Each dose was delivered to both kidneys at baseline (time zero) and at 3 months. All animals underwent two renal biopsies per kidney 4-6 weeks before the baseline injection treatment. Control animals were injected with PBS. Animals were monitored for 6 months after the baseline injection.

[0342] 1.2.3.1.Research results All eight animals remained in good clinical health throughout the study and survived to their designated endpoint of 6 months. Five of the eight animals experienced mild or slight weight loss, with two animals losing more than 3% of their body weight over the study period. Greater weight loss occurred between renal injection and initial treatment than between initial treatment and termination. Clinical pathology and urinalysis data showed no abnormal trends. There were no signs of renal failure or indications of reduced GFR.

[0343] 1.2.3.2. Renal findings At 6 months, no macroscopic or microscopic findings related to the safety of REACT injections were observed. After improved evaluation of eight regions of each kidney (three stains per region), including evaluation and scoring of 150 glomeruli per kidney, no treatment-related renal findings were noted. All kidneys appeared normal, except for scar-related changes at the injection site (fibrosis / chronic inflammation within the capsule; linear fibrosis / chronic inflammation; and inflammatory cells in the cortex / medulla).

[0344] 1.2.3.3. Non-renal findings No findings related to the safety of the test article were identified in non-target tissues. All macroscopic and microscopic changes were considered background changes and thus within normal limits.

[0345] 1.2.3.4. Conclusion All animals survived to the designated termination time points and appeared to be in good health based on clinical pathology, urinalysis, and veterinary evaluation data. Pathological evaluation revealed no findings (macroscopic or microscopic) related to the safety of REACT in any of the target organs (kidneys) or non-target organs examined. At 6 months, no adverse effects of two repeated doses of REACT were observed compared to PBS-injected control animals.

[0346] 1.3. Non-clinical conclusions Multiple animal studies over a wide range of doses (3 to 15 million SRCs per gram of injected kidney tissue) and long periods (up to 1 year) after REACT treatment, including three GLP studies Evidence from the study showed that the potential risk of complications from delivering REACT to the kidney was similar to the potential risk of complications associated with performing a standard kidney biopsy. [1~3] .

[0347] With the exception of changes related to the injection procedure and cardiovascular findings specific to the 5 / 6 nephrectomized rodent mass reduction model of CKD, no unexpected in-life hematologic, urological, serological, or histological changes were observed in target organs or non-target tissues following delivery of REACT.

[0348] 1.4. Phase 1 Clinical Trial: Preliminary Results 1.4.1. In April 2013, a first-in-human clinical trial was initiated at Karolinska University Hospital in Huddinge, Stockholm, Sweden: A Phase 1, Open-Label, Safety, and Delivery Optimization Study (RMTX-CL001) of Autologous Kidney Augmentation Agent (REACT) in Patients with Chronic Kidney Disease. This is a Phase 1, Open-Label, Safety, and Delivery Optimization Study of REACT injected into subjects with CKD. REACT is manufactured from SRC obtained from a subject's kidney biopsy, formulated with gelatin biomaterial, and injected back into the subject's left kidney. The primary objective is to evaluate the safety and optimal delivery of REACT injected into a single site in the recipient's kidney, as measured by treatment-related and / or product-related adverse events (AEs) through 12 months post-treatment. A secondary objective is to evaluate renal function by comparing the results of clinical tests from baseline to 12 months post-REACT injection, followed by an additional 18-month observation period. Each subject's baseline CKD disease progression rate served as their own "control" to monitor changes in kidney failure over time. Six subjects recruited from Karolinska University Hospital were enrolled in the study. Additionally, one subject was enrolled in the study at the University of North Carolina.

[0349] Adverse events In a cohort of seven male subjects aged 53 to 70 years with predialysis diabetic nephropathy (CKD stage 3b / 4), all subjects recovered from the laparoscopic REACT delivery procedure without immediate perioperative complications. Notably, no subjects experienced hematuria, which was prospectively considered the most likely adverse event. One subject developed intestinal volvulus two days after REACT injection, necessitating a partial colectomy and concomitant anastomotic bleeding. In the investigator's judgment, this event was unrelated to the study drug or treatment. One subject developed a skin infection associated with the laparoscopic injection procedure. Another subject recovered from the surgical procedure with airway inflammation. All serious adverse events (SAEs) associated with the clinical trial are presented in Table 6.

[0350] Eight of the nine SAEs were considered possibly related to the injection procedure. No AEs or SAEs were considered related to the biopsy procedure. To date, no delayed or late adverse reactions associated with REACT or other study treatments have been identified (e.g., negative immune-mediated reactions). Based on current data, the highest risks associated with REACT treatment appear to be attributable to the injection procedure. As a result, measures are being taken to shorten the duration of surgical procedures and improve surgical outcomes.

[0351] TIFF2025160401000013.tif41170

[0352] 1.4.3. Estimated glomerular filtration rate (eGFR) Seven male patients with T2DM and stage 3b / 4 CKD were injected with REACT into the left kidney. Pre-injection information from these subjects indicated a mean decline in eGFR of 6.1 ml / min / year. After REACT treatment, the decline in eGFR for the combined group (all subjects) was -3.1 ml / min / year (gray line in Figure 1).

[0353] After approximately 1 year of monitoring the potential impact of REACT treatment on CKD progression in this cohort, the expected decline in kidney function appears to have been altered by a single injection of REACT into a single kidney. In Figure 1, comparing eGFR after REACT treatment (gray line) with eGFR before REACT treatment (black line), 6 of 7 subjects showed a reduction in the rate of decline in eGFR after treatment. The annual rate of change for eGFR before and after REACT treatment is shown for each subject in Table 7.

[0354] TIFF2025160401000014.tif54170

[0355] Serum creatinine Pre-treatment serum creatinine (sCR) values ​​were generally elevated in this cohort, as would be expected for subjects with T2DM and moderate to severe renal failure (CKD stage 3b / 4). The annualized rate of change for sCR before and after REACT treatment is shown for each subject in Table 8. All subjects experienced a decrease in their individual rate of increase in sCr after REACT treatment compared to the rate of increase in sCr observed before REACT treatment.

[0356] TIFF2025160401000015.tif56170

[0357] Pretreatment total sCR values ​​for this cohort were greater than 100 μmol / L / year. After REACT treatment, sCR decreased to less than 50 μmol / L / year. As shown in Figure 2, comparing sCR after REACT treatment (gray line) with sCr before REACT treatment (black line), this cohort showed a decrease in the rate of increase in sCr after REACT treatment. This change was consistent for each subject.

[0358] 1.4.5. Renal cortical thickness Patients with chronic kidney disease suffer from thinning of the functional portion of the kidney, the cortex. Renal cortical thickness decreases in CKD as the disease progresses as a result of fibrosis and scarring. Increased cortical thickness has been associated with kidney regeneration in preclinical studies of REACT and was confirmed histologically in all four animal species studied. In clinical trials TNG-CL010 and TNG-CL011, cortical thickness was assessed using imaging techniques. No biopsies were taken to confirm the basis of the increased thickness. Cortical thickness was measured in both the right and left kidneys to determine whether the injected left kidney showed any changes in cortical thickness that could be attributed to REACT injection. The right kidney served as an uninjected control.

[0359] On average, cortical thickness increased from 14 mm at baseline to approximately 16 mm after 1 year of REACT treatment in the left kidney. This change in cortical thickness was not sufficient to cause an increase in total kidney volume (data not shown). No change in cortical thickness was observed in the cortex of the right kidney.

[0360] Hemoglobin CKD may be associated with metabolic abnormalities resulting from chronic uremia as well as anemia due to altered renal erythropoietin production.

[29] In clinical trial RMTX-CL001, three of seven subjects showed improvement in hemoglobin levels after REACT treatment, while the remaining four subjects maintained normal levels throughout the study.

[0361] Blood Pressure Blood pressure was monitored during clinical trials TNG-CL010 and TNG-CL011. Subjects were taking medications to control their blood pressure. Specifically, antihypertensive medication intake decreased in 3 of 6 subjects during the first 6 months of REACT treatment.

[0362] 1.5. Potential Risks In general, the potential risks associated with the clinical use of REACT can be broadly divided into three categories: renal biopsy, REACT product, and delivery to the recipient's kidney. An assessment of the potential risks associated with each of these steps is presented in this section.

[0363] At this time, there are no specific warnings or precautions associated with the use of REACT. However, warnings and precautions for renal biopsy and percutaneous injection procedures should be considered when using this product. The risks of renal biopsy have been well characterized over the 100 years that this procedure has been used and developed. Renal percutaneous needle devices have a shorter history.

[0364] Risks of kidney biopsy and percutaneous needle kidney injection include: 1. Pain in the flank / injection site / biopsy site, 2. Bleeding at the time of injection / biopsy, which may occur anywhere around the kidney or along the needle track and may be sufficient to cause clinically significant anemia, acute kidney injury (AKI), hematoma, and, in the case of subcapsular hemorrhage, "page kidney" and acute hypertension. 3. Surgical injury to the kidney due to needle injury and damage to other structures including connective tissue, bone, and intra-abdominal organs.

[0365] 1.5.1. Potential Risks Associated with Renal Biopsy Autologous kidney cells are a standard medical procedure [1~3] The renal biopsy will be obtained from each individual subject via a renal biopsy performed in accordance with the REA and consistent with standard operating procedures at the participating hospital / institution. A minimum of two tissue cores from a single kidney biopsy is required to obtain enough renal cortical tissue for CT imaging. A 16-gauge biopsy needle approximately 10 mm long removes 0.01%–0.02% of the average total volume of the affected kidney. Approximately 0.001% of the total number of glomeruli is collected. [3] , biopsy is not expected to adversely affect kidney function.

[0366] Kidney biopsies as diagnostic procedures are low risk and are often performed under sedation on an outpatient basis in the United States. [30、31] When performed by a qualified interventionalist, a properly cortically directed renal biopsy causes limited renal damage.

[27] On the other hand, reports of kidney damage at the biopsy site describe vascular injury and varying degrees of ischemia and infarction. The severity of the injury depends on the size and number of blood vessels damaged during the biopsy procedure.

[32] .

[0367] Bleeding is the most common adverse event associated with routine renal biopsy. Nearly all patients develop microscopic hematuria as a result of the biopsy, but this is not clinically significant. [2、31] On the other hand, gross hematuria occurs in 3% to 9% of patients. [30、31], which generally resolves by 24 hours after biopsy. The most serious complication is severe bleeding, which may require a blood transfusion and / or result in patient death. Blood transfusion is required in less than 1% of renal biopsies, and death occurs in less than 0.01% of cases. [33~35] .

[0368] 1.5.2. Potential Risks Associated with REACT Products The investigational drug, REACT, is composed of autologous selected kidney cells obtained via kidney biopsy from the same subject. Based on experience with autologous stem cell transplantation, the risk of an immune response (e.g., graft rejection) caused by injection of REACT into the kidney is unlikely.

[0369] Because the kidney is a highly perfused organ, it is doubtful that injected SRC will remain localized at the injection site. The three most likely destinations for translocated SRC are 1) the subcapsular space, 2) the systemic circulation, and 3) the urinary tract. Leakage of SRC into the subcapsular space is not expected to pose a risk to the subject. For example, the subcapsular space is commonly used for injection of endocrine tissues such as pancreatic islet cells.

[36] The renal capsule also serves as a niche for innate stem cells that can migrate into the renal parenchyma.

[37] Furthermore, direct injection into the kidney reduces the possibility of SRCs entering the systemic circulation by providing a natural route of excretion via the urinary tract. Furthermore, intravenous administration of heterologous, allogeneic stem cells (i.e., mesenchymal stem cells) from different species has been evaluated in clinical trials and posed no significant risks to subjects. [24~28] .

[0370] The pigskin type B gelatin used in the formulation of REACT conforms to the Pharmaceutical and Edible Gelatin Monograph (European Pharmacopoeia 7.0, Meets the requirements of the United States Pharmacopeia - National Formulary (USP35 NF30). Gelatin is widely used in pharmaceutical and medical applications, including cell transplantation for regenerative products. Gelatin is not expected to cause adverse effects in study subjects based on its biocompatibility, widespread use, and the results of GLP toxicity studies with porcine gelatin, including REACT.

[0371] 1.5.3. Potential Risks Associated with REACT Treatment Percutaneous techniques are used to access the kidney for REACT delivery. Percutaneous approaches have been used for resection of renal masses for over a decade. A concise review of this method is provided by Salagierski and Salagierski (2010).

[38] Safety precautions are implemented during REACT treatment and postoperative follow-up to reduce the possibility of excessive bleeding and other adverse events. Patients are closely monitored, as discussed in Section 6.

[0372] Cain and colleagues (1976)

[39] reported that renal cell homogenates injected into the kidneys of rodents did not cause any significant adverse events. Similarly, the morphological effects observed after REACT delivery to the kidney were consistent with those reported for repeated kidney biopsies taken from dogs. That is, the presence of mature connective tissue tracts without functional impairment leads to minimal structural changes.

[32] In dogs, increased intracapsular renal water not only increases intrarenal pressure but also may enhance the transient increase in kidney weight and systemic blood pressure. [19、20] However, in a pilot study in dogs evaluating the short-term effects of large doses on blood pressure, there were no adverse effects on blood pressure after increasing the volume of REACT to 6 mL per kidney.

[0373] 1.6. Potential Benefits The potential for achieving clinically significant improvements in CKD is supported by studies that tested REACT in preclinical animal models of renal failure, i.e., the ZSF-1 rat model of T2DM, in addition to surgical models of declining kidney function in otherwise healthy rats and dogs. The primary finding was that REACT significantly reduced the rate of structural and functional deterioration in already damaged kidneys to a clinically relevant extent in the animal model. Thus, there is potential for subjects participating in this clinical trial to realize therapeutic benefits from REACT treatment, such as a potential reduction in the rate of CKD progression.

[0374] 2. Purpose and Objectives of Phase I Trials This clinical trial involves Kidney Augment (REACT), a regenerative cell-based product aimed at improving renal function in subjects with CKD and T2DM. Therapeutic intervention with REACT is intended to delay the need for renal replacement therapy (dialysis or transplantation), which is inevitable for patients with end-stage CKD based on current standard of care. The purpose of this study is to compare the safety and efficacy of up to two injections of REACT given 3 months (+12 weeks) apart (maximum) in subjects randomized to receive the first injection as soon as the REACT product becomes available, to subjects randomized to receive concurrent standard of care for CKD during the first 12 to 18 months before receiving up to two injections of REACT. Additionally, each subject's annual rate of renal function decline, based on appropriate historical clinical data from 18 months prior to the screening visit, serves as a comparator to monitor the rate of progression of renal failure before and after REACT injection.

[0375] REACT treatment reduces the rate (slope) of decline in eGFR and improves renal function over a period of 24 months after the last REACT injection.

[0376] 2.1. Main purpose To evaluate the safety of REACT injected into one recipient's kidney.

[0377] Primary endpoint: Changes in eGFR over 6 months after two REACT injections Incidence of renal-specific procedural and / or product-related adverse events (AEs) through 6 months post-injection

[0378] 2.2. Secondary Objectives To evaluate the safety and tolerability of REACT administration by assessing kidney-specific adverse events for 24 months after injection.

[0379] Secondary endpoints: Renal-specific clinical laboratory assessments through 24 months post-injection

[0380] 2.3. Exploratory purpose To evaluate the effect of REACT on renal function over 24 months after injection.

[0381] Exploratory endpoints: Clinical diagnostic and laboratory evaluation of renal structure and function (including eGFR, serum creatinine, and proteinuria) to assess changes in the rate of progression of renal disease Vitamin D levels Iohexol imaging Blood pressure control MRI assessment of kidney volume

[0382] 3. Investigational drugs 3.1. Study Drug Description REACT is an injectable product composed of SRC formulated in a biomaterial (gelatin-based hydrogel). Table 9 provides an overview of the investigational product. For a detailed description of SRC and REACT, as well as manufacturing methods, please refer to the Investigational Product Brochure.

[0383] TIFF2025160401000016.tif57170

[0384] 3.2. Procurement and Manufacturing of REACT REACT is manufactured at Twin City Bio LLC's GMP facility in Winston-Salem, North Carolina, USA.

[0385] Biopsy Biopsies are collected using standard surgical techniques to evaluate either the left or right kidney. To provide sufficient material for the production of autologous REACT, a minimum of two tissue cores measuring 1.5 cm each must be collected using a 16-gauge biopsy needle. Upon receipt of the biopsy at Twin City Bio LLC, the sample is labeled and documented for product traceability. Strict documentation measures are in place to ensure that the product is maintained. The site will be notified regarding the adequacy and quality of the biopsy specimen for the production of CT and the scheduled date for REACT injection. If the biopsy material is not available, the subject should be withdrawn from the study.

[0386] 3.2.2.SRC Selection Approximately four weeks prior to the subject's planned REACT treatment, autologous kidney cells are removed from the vapor phase of a liquid nitrogen freezer, thawed, and separated from the kidney tissue by enzymatic digestion. The cells are cultured and expanded using standard techniques.

[0387] The cell culture medium is designed to grow primary kidney cells and does not contain any differentiation factors. The harvested kidney cells are subjected to density gradient separation to obtain SRCs, which are primarily composed of kidney epithelial cells known to have regenerative potential.

[40] In the autologous SRC population, other parenchymal (vascular) and interstitial (collecting duct) cells may be sparsely present.

[0388] If enough cells are available, the same biopsy material will be used to generate additional REACT preparations for research studies and stored in the vapor phase of a liquid nitrogen freezer under GMP conditions.

[0389] All subjects will receive a series of two REACT injections. The time and event table indicates that the series of two REACT injections will be administered three months apart over a 12-week study visit period. Regardless, every attempt will be made to ensure that the second REACT injection is administered three months after the first injection. Twin City Bio LLC will notify the patient of the second injection. Notify the field to obtain information on the planned date of

[0390] Formulations SRC is formulated in a gelatin-based hydrogel to improve stability during transport and delivery upon injection into the renal cortex. Porcine gelatin is dissolved in a buffer solution to form a thermoresponsive hydrogel. This biomaterial is fluid at room temperature but gels upon cooling to refrigerated temperatures (2°C to 8°C). Prior to injection, the REACT investigational drug must be warmed to above 20°C to 26°C to liquefy the hydrogel.

[0391] 3.2.4. REACT products for injection Ten to fourteen days before the scheduled REACT injection, subjects will appear in the clinic for an evaluation to confirm continued eligibility. If the subject is not eligible for a REACT injection, the investigator and sponsor will discuss possible options, such as whether the subject is stable enough to attempt a future REACT injection. If the subject is still eligible, the REACT product will be manufactured and shipped to the clinical center. It is the site's responsibility to ensure that shipments of REACT are delivered directly to site personnel. REACT will be injected into the biopsied kidney of eligible subjects using a percutaneous approach. The percutaneous method uses standard techniques (such as those utilized for radiofrequency or cryogenic resection of renal masses).

[38] .

[0392] Two injections of REACT are planned for each subject. However, if there appears to be any undue safety risk, or rapid deterioration of renal function, or development of uncontrolled diabetes or uncontrolled hypertension, or development or occurrence of malignancy, the second injection of REACT should not be administered.

[0393] Kidney cells that may be frozen but are not used to manufacture REACT remain in the vapor phase of Twin City Bio LLC's liquid nitrogen freezer until your EOS visit. At that time, these When the kidney cells are no longer needed, all personal information is anonymized and they are stored in the vapor phase of a liquid nitrogen freezer for up to five years. The purpose is to test these kidney cells in laboratory research studies. During the informed consent process, each subject provides written consent for the storage and future use of autologous cells not used for REACT injections. Subjects have the option to discard these cells upon completion of the study.

[0394] 3.2.5.REACT Dose The dose of REACT for subjects in the Phase 1 clinical trials (TNG-CL010 and TNG-CL011) was 1 g of estimated kidney weight (gKW) est ) 3 x 10 6 Similarly, in this study, each REACT injection contained 3 x 10 SRCs. 6 cells / gKW est The concentration of SRC is 100 x 10 per mL of REACT. 6 Because the total number of cells is 1, the administration volume is 3.0 mL per 100 g of kidney weight. The volume of REACT administered is determined by pre-treatment MRI volumetric 3D assessment or the ellipsoid formula (length x width AP plane x width transverse plane x 0.62). An example of the administration volume based on estimated kidney weight is shown in Table 10.

[0395] TIFF2025160401000017.tif89170

[0396] The REACT dose is based on kidney volume calculated via MRI. In contrast to other methods, measuring kidney volume using MRI is more accurate, obtaining true tomographic data along any direction without the risk of ionizing radiation or nephrotoxic contrast agents. MRI-estimated kidney volume measurements (mL) are approximately 92%–97% of the dry weight measurement in grams for excised organs trimmed of perinephric fat. Traditionally, the REACT dose is calculated using the 1 g to 1 mL conversion. The volume of REACT administered is determined by pre-treatment 3D MRI volumetric assessment or the ellipsoid formula (length × width AP plane × width transverse plane × 0.62). This ensures that subjects are not administered a higher dose of REACT than previously tested in animal studies.

[0397] 3.2.5.1. Rationale for Two REACT Injections All subjects are intended to receive two scheduled injections of REACT to allow for dose titration and to assess duration of effect. Scientific evidence, based on nonclinical studies, suggests that the biologically active component of REACT (allogeneic autologous SRC) slows progression in experimental models of CKD by enhancing renal structure and function. [7~12] As a result, the more cells that can be injected, the greater the potential improvement in kidney function. The total number of cells that can be delivered to the kidney at one time is limited not only by the size of the kidney but also by the inelasticity of the renal capsule. Consequently, it may be possible to improve therapeutic efficacy by administering a larger number of SRCs via a second injection, administered after the cells from the first injection have been incorporated into the kidney.

[0398] Apart from increasing the number of SRCs by administering two REACT injections to the same kidney, we can evaluate the duration of effect. The process by which functional nephrons fail in CKD kidneys may, over time, adversely affect the "new" cells delivered via REACT injection. As a result, REACT may not provide a long-term therapeutic effect. Examining the effect of a second REACT injection administered at an appropriate interval after the first injection will address this issue.

[0399] In this study, subjects will receive a second REACT injection three months after the first injection and will have a 12-week study visit period. Regardless, every attempt should be made to ensure that the second REACT injection is administered three months after the first injection. However, if there appears to be any undue safety risk, or rapid deterioration of renal function, or the development of uncontrolled diabetes or uncontrolled hypertension, or the development of malignancy or intercurrent infection, a second REACT injection will not be administered.

[0400] 3.2.5.2.Safety of two REACT injections A canine GLP toxicity study was conducted to evaluate the safety of administering two doses of REACT to biopsied kidneys (see Section 1.2.2). Similar to the clinical study design, study animals (n=8) underwent renal biopsies 4-6 weeks prior to baseline. Each dose was delivered to both kidneys at baseline and 3 months, and animals were observed for 6 months after the baseline injection. Control animals received PBS, while REACT-treated animals received a dose twice as high as that used in this clinical study.

[0401] Briefly, no adverse effects of the two doses of REACT on biopsied kidneys were observed compared to control animals after 6 months of baseline treatment. Pathological evaluation revealed no findings (macroscopic or microscopic) related to the safety of REACT in any of the target organs (kidneys) or non-target organs examined. After improved evaluation of eight regions of each kidney (three stains per region), including evaluation and scoring of 150 glomeruli per kidney, no treatment-related renal findings were noted. With the exception of changes related to scarring at the injection sites, all kidneys appeared normal. No signs of renal failure or indications of reduced GFR were observed. Further information is provided in the Investigator's Brochure.

[0402] 3.3. Study Drug Packaging The product delivery system has three components: 1) a 10 mL standard Luer-Lok™ syringe; 2) packaging containing the syringe; 3) REACT shipping containers that transport the packages to the clinical site; It consists of:

[0403] Syringes containing REACT are shipped to the clinical site in packaging designed to maintain product integrity and sterility of the product and syringe. A representative image of the product delivery system is shown in Figure 3.

[0404] The product delivery system is made from the components listed in Table 11. Materials in contact with the REACT product are USP Class VI or equivalent. Syringes, tubing, and accessories are obtained from vendors listed in Table 11 or other vendors that meet the requirements for biocompatibili...

Claims

1. 1. A method of treating kidney disease in a subject suffering from chronic kidney disease (CKD), comprising: (i) a bioactive renal cell population; (ii) vesicles secreted by said renal cell population, and / or (iii) a spheroid comprising said renal cell population and at least one non-renal cell population; administering to the subject an effective amount of a composition comprising wherein the subject has a kidney and / or urinary tract abnormality. method.

2. 10. The method of claim 1, wherein the subject suffers from CKD from congenital anomalies of the kidney and urinary tract (CAKUT).

3. 3. The method of claim 1 or 2, wherein the subject has an abnormality in kidney development.

4. 4. The method of any one of claims 1 to 3, wherein the subject has or has previously suffered from primary or secondary vesicoureteral reflux, reflux nephropathy, renal scarring, or renal hypodysplasia, with or without infection and / or inflammation.

5. The method of any one of claims 1 to 4, wherein the subject is susceptible to a urinary tract infection.

6. The method of any one of claims 1 to 5, wherein the subject suffers from hypertension or proteinuria.

7. The method of any one of claims 1 to 6, wherein the subject has undergone post-antireflux surgery.

8. The target is 90 mL / min / 1.73 m ２ The method of any one of claims 1 to 7, wherein the patient has a glomerular filtration rate (GFR) of less than 100 mg / kg, microalbuminuria, or macroalbuminuria.

9. The method of any one of claims 1 to 8, wherein the subject is under 18 years of age.

10. The method according to any one of claims 1 to 9, wherein the subject has a renal parenchymal malformation.

11. 11. The method of any one of claims 1 to 10, wherein the subject is suffering from urethral duplication, ureteropelvic junction obstruction, renal agenesis, vesicoureteral reflux, renal dysplasia, hypoplastic kidney, renal hypodysplasia, congenital hydronephrosis, horseshoe kidney, posterior urethral valve and prune belly syndrome, obstructive renal dysplasia, or nonmotile cilia.

12. The method according to any one of claims 2 to 11, wherein the CAKUT is caused by or correlated with a genetic factor.

13. The method according to any one of claims 2 to 11, wherein the CAKUT is caused by or correlated with a non-genetic factor.

14. The method of claim 13 , wherein the non-genetic factor is an environmental factor.

15. The disorders include Alagille syndrome, Apert syndrome, Bardet-Biedl syndrome, Beckwith-Wiedemann syndrome, Branchio-oto-renal syndrome (BOR), flexor limb dysplasia, Senani-Lenz syndrome, DiGeorge syndrome, Fraser syndrome, hypoparathyroidism, sensorineural hearing loss, and HDR, Kallmann syndrome, Ulnar-Mammary syndrome, Meckel-Gruber syndrome, Nephronophthisis, Okihiro syndrome, Pallister-Hall syndrome, Renal coloboma syndrome, Hypoplasia, Dysplasia, Renal dysplasia, Cystic dysplasia, Non-cystic dysplasia, VUR cystic dysplasia, Renal hypodysplasia, Isolated cystic renal hypodysplasia, Isolated non-cystic renal hypodysplasia, Isolated renal tubular dysplasia, Rubinstein-Taybi syndrome, Simpson-Golabi-Behmel syndrome, Townes-Brocks syndrome, Zerbe 15. The method of any one of claims 1 to 14, comprising Garr syndrome, Smith-Lemli-Opitz syndrome, hydronephrosis, medullary dysplasia, unilateral / bilateral aplasia / dysplasia, urinary collecting system abnormalities, aplasia, ureteropelvic junction obstruction (UPJO) aplasia, dysplasia aplasia, unilateral aplasia, VUR, malrotation, crossed fused kidney, VUR dysplasia, double serine / threonine and tyrosine protein kinase (DSTYK) mutation, DSTYK mutation associated with UPJO, tubular dysplasia, cysts, and / or hypoplasia.

16. The method of any one of claims 1 to 15, wherein the subject is suffering from end-stage renal disease.

17. 17. The method of any one of claims 1 to 16, wherein the chronic kidney disease is stage I, stage II, stage III, stage IV, or stage V kidney disease.

18. 18. The method of any one of claims 1 to 17, wherein the subject undergoes dialysis at least once, twice, or three times per week.

19. 19. The method of any one of claims 1 to 18, wherein at least 80% of the cells in said bioactive renal cell population express GGT-1.

20. 20. The method of any one of claims 1-19, wherein between 4.5% and 81.2% of cells in said bioactive renal cell population express GGT-1, between 3.0% and 53.7% of cells in said bioactive renal cell population express AQP2, and between 81.1% and 99.7% of cells in said bioactive renal cell population express CK18.

21. 21. The method of any one of claims 1 to 20, wherein the bioactive renal cell population is enriched for renal tubular cells compared to primary cultures of renal cells from kidney biopsies, and the tubular cells express higher molecular weight species of hyaluronic acid (HA) both in vitro and in vivo through the action of hyaluronan synthase 2 (HAS-2).

22. 22. The method of any one of claims 1-21, wherein the bioactive renal cell population has a lower proportion of distal tubule cells, collecting duct cells, endocrine cells, vascular cells, and / or progenitor-like cells compared to a primary culture of renal cells from a kidney biopsy.

23. The method of any one of claims 1 to 22, wherein the vesicles comprise a paracrine factor.

24. The method of any one of claims 1 to 23, wherein the vesicles comprise miRNA that inhibits plasminogen activator inhibitor-1 (PAI-1) and / or TGFβ1.

25. The method of any one of claims 1 to 24, wherein the at least one non-renal cell population is an endothelial cell population or an endothelial progenitor cell population.

26. The method of any one of claims 1 to 24, wherein the at least one non-renal cell population is a mesenchymal stem cell population.

27. 10. The method of claim 9, wherein the administration is by injection into one or both kidneys of the subject.

27. The method of any one of 1 to 26.

28. 28. The method of any one of claims 1 to 27, wherein the composition further comprises a temperature-sensitive cell-stabilizing biomaterial that (i) maintains a substantially solid state at or below 8°C and (ii) maintains a substantially liquid state at or above ambient temperature, wherein the biomaterial comprises a hydrogel, and the biomaterial is in a solid-liquid transition stage between 8°C and above ambient temperature.

29. 29. The method of claim 28, wherein the bioactive renal cell population, the vesicles, and / or the spheroids are suspended in and dispersed throughout a biomaterial that stabilizes the cells.

30. 30. The method of claim 28 or 29, wherein the hydrogel comprises gelatin.

31. 31. The method of any one of claims 1 to 30, wherein the bioactive renal cell population, the vesicles, and / or the spheroids are administered by injection through an 18-gauge to 30-gauge needle.

32. 32. The method of claim 31, wherein the needle has a diameter of about 27 gauge, about 26 gauge, about 25 gauge, about 24 gauge, about 23 gauge, about 22 gauge, about 21 gauge, or about 20 gauge.

33. 18. The method of any one of claims 1 to 17, wherein the treatment of kidney disease comprises improving kidney function in the subject.

34. 34. The method of claim 33, wherein the improved renal function comprises a decrease in albumin to creatinine ratio (ACR) in the subject.

35. 35. The method of claim 34, wherein the reduction in ACR is at least a 50% reduction relative to the subject's baseline ACR.

36. 36. The method of claim 35, wherein the reduction in ACR is at least a 60% reduction relative to the subject's baseline ACR.

37. 35. The method of claim 34, wherein the reduction in ACR is a reduction in ACR to between 30 mg / g and 300 mg / g, wherein the subject has an ACR greater than 300 mg / g prior to administering the first dose of the composition.

38. 35. The method of claim 34, wherein the reduction in ACR is a reduction in ACR to less than 30 mg / g, wherein the subject has an ACR of between 30 mg / g and 300 mg / g prior to administering the first dose of the composition.

39. 37. The method of any one of claims 34-36, wherein the reduction in ACR is achieved within 3 to 6 months after administering the first dose of the composition.

40. 37. The method of any one of claims 34-36, wherein the reduction in ACR is achieved within 2 to 3 months after administering the first dose of the composition.

41. 34. The method of claim 33, wherein the improved renal function comprises an increase in eGFR in the subject.

42. 42. The method of claim 41, wherein the increase in eGFR is achieved within 2 to 4 months after administering the first dose of the composition.

43. 42. The method of claim 41, wherein the increase in eGFR is achieved within two months after administering the first dose of the composition.

44. 44. The method of any one of claims 41 to 43, wherein the increase in eGFR is at least 5% relative to the subject's baseline eGFR.

45. 45. The method of claim 44, wherein the increase in eGFR is at least 10% relative to the subject's baseline eGFR.

46. 46. ​​The method of any one of claims 33 to 45, wherein the kidney and / or urinary tract abnormality comprises a posterior urethral valve.

47. The method of any one of claims 1 to 46, wherein the composition comprises (i) a population of bioactive kidney cells.

48. The effective amount of the bioactive renal cell population is 3×10 per gram of estimated kidney weight of the subject. ６ 48. The method of claim 47, comprising cells.

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