A method for culturing renal precursor cells and a renal precursor cell reinfusion preparation

Abstract

The application belongs to the technical field of a culture method of kidney precursor cells and a kidney precursor cell reinfusion preparation, and the culture method of the kidney precursor cells comprises the following steps: step one, obtaining kidney precursor cells; step two, culturing the kidney precursor cells in a culture medium; the culture medium comprises a basic culture medium, fetal bovine serum, L-glutamine, epidermal growth factor, insulin, adenine, hydrocortisone, a Rho kinase inhibitor, a tyrosine kinase inhibitor and SAG; and step three, collecting the kidney precursor cells obtained through culture. The special culture medium for kidney precursor cells is adopted, the heterologous trophoblast cells and the matrix glue of animal origin are completely abandoned, the animal source recombinant trypsin is used, and the process complexity and uncertainty caused by the use of biological materials are significantly reduced. The preparation of the kidney precursor cell reinfusion preparation is carried out by using the method in the application, the quality of the obtained kidney precursor cells is stable, and the clinical demand can be met.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, specifically relating to a method for culturing renal progenitor cells and a renal progenitor cell reinfusion preparation. The renal progenitor cells described in this invention belong to the category of tissue-specific adult stem cells, whose differentiation potential is limited to the renal lineage. They possess self-renewal capacity and the potential to differentiate into renal tubular epithelial cells, playing a crucial role in renal injury repair. Background Technology

[0002] Chronic kidney disease (CKD) is a globally prevalent chronic progressive disease of the urinary system, with a global incidence rate of approximately 10% to 15%. Its course is insidious, with patients often remaining asymptomatic for extended periods, leading to low awareness of the disease. Early symptoms typically include microalbuminuria, which gradually progresses to renal insufficiency and eventually end-stage renal disease, requiring dialysis or kidney transplantation to sustain life and posing a serious threat to patients' health and lives.

[0003] Current clinical treatments have significant limitations: drug therapy (such as ACEI / ARB drugs) can only slow down disease progression and cannot achieve fundamental regeneration and repair of kidney tissue; for patients with advanced disease, kidney transplantation faces problems such as donor shortage and long-term immunosuppression, making it difficult to meet the widespread clinical needs; traditional cell therapy (such as bone marrow mesenchymal stem cell transplantation) has defects such as low differentiation efficiency, insufficient targeting, and limited repair effect, and there is still a huge clinical gap in the fundamental treatment of chronic kidney disease.

[0004] Renal progenitor cells are a group of cells with regenerative and repair functions, distributed in the epithelial layer of the kidney. Among them, SOX9... + The regenerative and repair potential of renal progenitor cells has been confirmed by various studies. These cells can be specifically activated under conditions of kidney injury, on the one hand, by proliferating and migrating to the site of injury to replenish and regenerate damaged renal tubular structures; on the other hand, through their powerful secretory function, they can activate signaling pathways that promote kidney regeneration and repair, thereby achieving the recovery of kidney function. However, despite SOX9 + Renal progenitor cells have clear therapeutic potential, but current technologies still face many bottlenecks that limit their clinical translation: the content of endogenous renal progenitor cells in the kidney is low, which cannot directly meet the needs of patients with chronic kidney disease for kidney damage repair; at the same time, there are technical bottlenecks such as difficulty in isolation, poor stability in in vitro culture, and insufficient passage expansion efficiency, which make it difficult to stably maintain their SOX9 positive phenotype and key biological characteristics, leading to cell function decline and severely limiting their clinical translation and application.

[0005] In recent years, although some studies have attempted to isolate SOX9 from urine... +Renal progenitor cells have been developed for non-invasive harvesting, and specific amplification techniques are being explored to maintain cell function. However, these techniques still have significant shortcomings: they rely on feeder cells derived from animal sources; high-throughput separation and amplification processes are lacking, resulting in low cell acquisition efficiency and difficulty in large-scale preparation; and the in vitro culture system is immature and cannot stably maintain SOX9. + The stemness and repair function of cells are poor, and the uniformity between cell batches is poor; a complete GMP-compliant preparation process has not been established, and the quality control standards are unclear, which cannot meet the safety and efficacy requirements for clinical applications.

[0006] Furthermore, as special biological drugs, cell preparations must strictly adhere to Good Manufacturing Practices (GMP) to ensure their safety, efficacy, and uniformity. Currently, there is no standardized, scalable process for preparing renal progenitor cell reinfusion formulations. This fails to meet the clinical needs of patients with fluctuating chronic kidney disease requiring multiple transplants, and also fails to comply with the regulatory requirements for market approval of cell therapy products. Therefore, developing a safe and efficient reinfusion formulation based on renal progenitor cells, capable of non-invasive separation and high-throughput expansion, and compliant with GMP standards, along with its preparation method, has become an urgent technical challenge. Summary of the Invention

[0007] This invention aims to overcome the shortcomings of existing technologies by providing a method for culturing renal progenitor cells and a renal progenitor cell reinfusion formulation. The core solution addresses the problems of existing technologies, such as the difficulty in obtaining renal progenitor cells, poor in vitro culture stability, insufficient passage expansion efficiency, non-standardized formulation processes, and inability to meet the clinical needs of chronic kidney disease. This invention achieves efficient isolation and stable expansion of renal progenitor cells from the urine of healthy donors or patients, and prepares the reinfusion formulation using GMP-compliant processes, ensuring the safety and efficacy of the formulation. This provides a reliable cell therapy product and standardized preparation protocol for chronic kidney disease.

[0008] The renal progenitor cell reinfusion formulation of the present invention contains SOX9, the active ingredient of which is obtained by in vitro isolation and amplification. + CD73 + Renal progenitor cell population—These cells possess a defined regenerative and repair function. Derived from urine samples of healthy donors or patients, they are prepared through standardized processes including separation, amplification, washing, resuspension, and filling. Used for the treatment of patients with chronic kidney disease, they can target and migrate to sites of kidney damage, participate in the renal tissue repair process, slow the progression of kidney disease, and improve renal function. This invention is specifically implemented using the following technical solution: In a first aspect, the present invention provides a method for culturing renal progenitor cells, the method being independent of feeder cells and animal-derived matrix gel, comprising the following steps: Step 1: Obtain renal progenitor cells, which are tissue-specific adult stem cells; Step 2: The renal progenitor cells are cultured in a defined, non-trophoblastic cell culture medium; the culture medium comprises: Basal culture medium, fetal bovine serum, L-glutamine, epidermal growth factor, insulin, adenine, hydrocortisone, Rho kinase inhibitor, Linifanib, and SAG; Step 3: Collect the cultured renal progenitor cells.

[0009] Optionally, in step one, the renal progenitor cells are urinary renal progenitor cells.

[0010] The renal progenitor cells are tissue-specific adult stem cells.

[0011] Optionally, methods for obtaining renal progenitor cells include: The urine sample was centrifuged and the supernatant was removed; then it was washed with washing solution, centrifuged, and the supernatant was removed to obtain a solution containing renal progenitor cells. The urine sample can be a naturally urinated sample or a urine sample collected via catheter.

[0012] Natural urination samples are collected by the donor themselves, using clean midstream urine, and stored in a sterile urine collection container. For donors who are unable to collect urine themselves or who have a potential risk of urethral contamination, urine is collected using a sterile catheter and sealed immediately after collection to avoid sample contamination.

[0013] Sample transportation: Collected urine samples are transported to a standardized preparation workshop via a cold chain transportation system.

[0014] Optionally, the washing liquid comprises: 90-95 v% matrix solution, 5-10 v% fetal bovine serum, 0-200 μg / mL antibiotic; the matrix solution includes at least one of F12 medium, DMEM medium, physiological saline, compound electrolyte injection, and phosphate buffer.

[0015] Optionally, the culture medium is a culture medium without a trophoblast cell layer.

[0016] Optionally, the Rho kinase inhibitor uses Y 27632.

[0017] Optionally, in step two, the cell culture medium comprises basal culture medium, 8-20 v% fetal bovine serum, 0.2-2 mM L-glutamine, 0.1-10 ng / ml epidermal growth factor, 1-14 μg / ml insulin, 5-30 μg / ml adenine, 1-20 μg / ml hydrocortisone, and 0.1-50 μM gamma glutamic acid. 27632, 0.02-1 μM Linifanib, 0.01-1 μM SAG; The basal culture medium is composed of at least one of F12 medium and DMEM medium.

[0018] Optionally, in step two, the renal progenitor cells initially cultured in the culture medium are designated as generation P0, and then passaged; the process continues until generation P4 to P8.

[0019] Optionally, the passage culture includes: digesting all uncontaminated culture flasks containing P0 generation cells that have grown cell clones and then combining them into one culture system; passage culture for a total of 3 to 8 generations, except for the P0 generation which is passaged within 20 days, and then passaged every 2 to 6 days thereafter, with a cell density of approximately 50% to 100% during passage.

[0020] Optionally, the method for each passage includes: aspirating the supernatant from the culture flask, and then washing to remove the supernatant; Add recombinant trypsin digestion reagent, place the culture flask in an incubator and digest for 3-10 minutes. After most of the cells have detached, add stop solution to terminate the digestion. Blow the culture medium under a pipette to collect the cell suspension, centrifuge at 200-400 g, and remove the supernatant. Resuspend the cell pellet in renal progenitor cell culture medium and perform viable cell counting at a concentration of 0.5–5 × 10⁻⁶ cells / mL. 4 The cells were seeded into culture flasks at a density of 1 cell / cm², and cultured further after adding culture medium.

[0021] Optionally, the passage culture process in step two includes quality control: extracting cell samples during the P2 generation culture, at the end of the P2 generation culture, at the end of the Pn-1 generation culture, and at the end of the Pn generation culture for quality control index detection, where n is the last generation of culture; The quality control indicators include: mycoplasma testing, sterility testing, cell identification and purity testing, cell biological efficacy testing, and antibiotic residue determination.

[0022] Optionally, step two may also include cryopreservation of P2 generation cells; When needed, cryopreserved cells are thawed and passaged.

[0023] Optionally, the collection of cultured renal progenitor cells includes: aspirating the supernatant from the culture flask, and then washing and removing the supernatant; Add recombinant trypsin digestion reagent, place the culture flask in an incubator and digest for 3-10 minutes. After most of the cells have detached, add stop solution to terminate the digestion. Blow on the bottom of the culture medium and collect the cell suspension.

[0024] In a second aspect, the present invention provides a renal progenitor cell reinfusion formulation comprising renal progenitor cells obtained by any of the methods described in the first aspect, and pharmaceutically acceptable excipients.

[0025] The excipients are 0.9% sodium chloride injection or other suitable pharmaceutical-grade injections.

[0026] The renal progenitor cells were SOX9. + Kidney progenitor cells.

[0027] The renal progenitor cells were double positive for PAX8 / CD73 with a positive rate of ≥95%, and double negative for CD45 / CD34 with a positive rate of <2%.

[0028] Thirdly, the present invention provides the use of renal progenitor cells obtained by any of the methods of the first aspect, or the renal progenitor cell reinfusion preparation described in the second aspect, in the preparation of a medicament for the regeneration and repair of chronic kidney disease.

[0029] Optionally, the chronic kidney disease includes at least one of the following: chronic kidney disease caused by chronic glomerulonephritis, chronic kidney disease caused by diabetes, chronic kidney disease caused by hypertensive nephropathy, kidney failure, and proteinuria.

[0030] The renal progenitor cells described in this invention belong to the category of adult stem cells, which differ from embryonic stem cells or pluripotent stem cells and have the following characteristics: (1) Tissue specificity: It is derived from kidney tissue or urine and has renal lineage-restricted differentiation potential; (2) Self-renewal ability: It can be stably passaged and amplified in the culture medium without a feeder layer provided by this invention; (3) Functional markers: High expression of SOX9, PAX8, and CD73, but no expression of hematopoietic cell markers CD45 / CD34; (4) Repair function: It can differentiate into mature renal tubular epithelial cells both in vivo and in vitro, and participate in the repair of kidney damage.

[0031] This invention successfully maintains the aforementioned stem cell characteristics through a specific combination of culture media, without relying on trophoblast cells and animal-derived matrix gel, thus achieving standardized preparation of clinical-grade cell preparations.

[0032] The technical solution of this invention has the following advantages: 1. This invention establishes a universally applicable, standardized pharmaceutical-grade manufacturing process. By employing a specially formulated culture medium for renal progenitor cells, this invention completely eliminates heterologous feeder cells (such as mouse embryonic fibroblasts) and animal-derived matrix gels, and uses animal-free recombinant trypsin, significantly reducing the process complexity and uncertainty introduced by the use of biological materials. Mouse feeder cells and matrix gels are common biological materials used in laboratory research to support cell culture; however, there is currently a lack of clinical-grade products on the market, and relevant regulations regarding their quality standards and risk assessments are unclear. This invention eliminates the risks of immunogenicity, batch-to-batch variability, and pathogen contamination introduced by the animal-derived components of mouse feeder cells and matrix gels, enabling the prepared cell products to meet the core safety requirements of drug regulatory agencies for cell therapy products. The robustness and reproducibility of the entire process are greatly enhanced. As demonstrated in Examples 1 and 2, regardless of whether the donor is healthy or a patient with chronic kidney disease of different etiologies, stages, and ages, this unified process can successfully isolate and stably expand to the clinically required cell quantity, with key quality attributes such as purity, biological efficacy, and sterility meeting pharmaceutical-grade quality standards. This demonstrates the broad applicability and excellent robustness of the process for donor sources, fundamentally solving the problems of large batch-to-batch variability, difficulty in standardization and large-scale production in traditional individualized preparation processes.

[0033] 2. This invention offers non-invasive and flexible cell acquisition, perfectly suited to the clinical characteristics of CKD patients. The invention utilizes natural urination or catheter collection methods, which are completely non-invasive, avoiding the trauma and risks associated with tissue biopsies. Simultaneously, urine samples are readily available and easily transported, significantly reducing the difficulty, cost, and biosafety risks of raw material acquisition, improving the convenience of clinical application and patient compliance, and meeting the core concerns of regulatory guidelines regarding donor safety for cell therapy products. In Example 3, short-term follow-up after patients received intrarenal administration of autologous renal progenitor cell preparations showed no adverse reactions or serious adverse events related to the cell preparations, demonstrating that the cell products produced by this process have excellent clinical tolerability and safety.

[0034] 3. This invention expands the dual treatment paradigm of autologous and allogeneic approaches, greatly broadening its clinical application prospects. This invention prepares clinical-grade renal progenitor cell preparations of homogeneous quality, safety, and efficacy from urine derived from CKD patients and healthy donors. The autologous treatment pathway uses the CKD patient's own urine as the starting material, completely avoiding the risk of immune rejection, and is particularly suitable for patients with potential immune risks or requiring multiple treatments. The allogeneic treatment pathway uses the urine of healthy donors as the starting material, utilizing the standardized process of this invention to prepare ready-to-use cell preparations, greatly expanding the applicable population (especially elderly, frail patients, or patients with poor cell quality). This provides a novel and flexible treatment option for the immediate and accessible treatment of chronic kidney disease.

[0035] 4. This invention features a comprehensive risk control system, ensuring the sustainability of clinical treatment. The invention establishes a P2 generation cell cryopreservation and thawing process, which effectively addresses changes in the treatment window caused by acute exacerbations or concurrent infections in CKD patients. It also reserves cell resources for potential multiple cell therapies in the future, aligning with the treatment philosophy of long-term CKD management and slowing disease progression. Furthermore, the entire process incorporates multi-stage quality control, including cell identification, aseptic testing, and mycoplasma detection, ensuring the safety of the final formulation.

[0036] 5. The indications for this invention are clearly defined, the formulation is highly targeted, and the therapeutic effect is definite. The formulation of this invention is specifically designed for chronic kidney disease (CKD), and its active ingredient is SOX9. + Renal progenitor cells are derived from a very small number of adult renal stem cells present in human urine. Compared to non-tissue-specific cells such as mesenchymal stem cells, they are more phenotypically and functionally adapted to the repair needs of the CKD renal injury microenvironment. Compared to existing technologies, this invention clearly defines the direct therapeutic purpose of renal progenitor cells in delaying the progression of kidney disease and improving renal function. The therapeutic efficacy of the formulation of this invention has been fully validated through multi-level evidence from animals to humans: In animal models: Experiment 1 showed that intrarenal injection of the renal progenitor cell formulation significantly reduced blood glucose and serum creatinine in mouse models, improving renal fibrosis and structure. Experiment 2 showed that subcutaneous injection of these cells also effectively treated a mouse model of renal injury, reducing serum creatinine, and proteomic analysis confirmed that it could regulate the proteome after kidney injury, promoting repair. This demonstrates that the formulation has broad-spectrum repair potential with effectiveness in multiple models and through multiple routes of administration. In preliminary clinical trials: In Case 3, a patient with type 2 diabetes and CKD received intrarenal injection of autologous renal progenitor cells. Short-term follow-up showed that total protein and albumin levels improved from below-normal preoperative levels to normal levels. Postoperative ultrasound confirmed accurate administration and the absence of complications. These data collectively and directly demonstrate that the formulation of this invention can improve key renal function indicators and has clear clinical significance.

[0037] In summary, this invention, through optimization of the culture system, successfully solves the core challenge of transforming renal progenitor cells from "laboratory research" to "clinical-grade drugs." It achieves significant and unexpected progress in product safety, efficacy, process controllability, and therapeutic effects on specific diseases, forming a complete and industrially viable innovative solution with significant clinical value and social benefits. The core inventiveness of this invention's process is applicable to the production of cell products for treating CKD caused by various factors. This is because the essence of renal progenitor cell therapy is to fundamentally improve renal tissue structure and enhance renal function through differentiation and secretion pathways. Therefore, the renal progenitor cell preparation cultured using this method has universal renal repair function, independent of specific etiologies, thus systematically addressing the treatment of chronic kidney disease. Attached Figure Description

[0038] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0039] Figure 1 Microscopic images of renal progenitor cells obtained from healthy donor urine in Example 1 at the end of P0 and P4 generations of culture. Figure 2 Figure 1 shows the results of cell identification and cell purity testing (immunofluorescence staining) of renal progenitor cells obtained from healthy donor urine in Example 1. Figure 3 Figure 1 shows the results of biological efficacy testing of renal progenitor cells obtained from healthy donor urine in Example 1. Figure 4 Figure 1 shows the results of cell identification and cell purity testing (flow cytometry) of renal progenitor cells obtained from healthy donor urine in Example 1. Figure 5 Example 2 compares the quality of renal progenitor cell preparations obtained from the urine of three patients with chronic kidney disease of different ages, sexes, and types. Figure 6. Serum creatinine levels in mice of each group; Figure 7 Histopathological staining atlases of mice in each group; Figure 8 Immunofluorescence staining patterns of histopathological findings in mice of each group; Figure 9 Quantitative analysis of histopathological markers in mice of each group; Figure 10Changes in marker expression in mice with UIRI kidney injury treated with renal progenitor cells; Figure 11 Changes in serum creatinine levels in mice with UIRI-induced kidney injury treated with renal progenitor cells; Figure 12 Principal component analysis (PCA) in proteomics; Figure 13 Protein interaction network diagram of hierarchical clustering in proteomics; Figure 14 Postoperative ultrasound images of the patient in Experimental Case 3 showed clear perirenal and collecting system structures with no abnormal signs. Figure 15 Microscopic morphology of P0 generation renal progenitor cells in Comparative Example 1; Figure 16 Microscopic morphology of P0 generation renal progenitor cells in Comparative Example 2. Detailed Implementation

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0041] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0042] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0043] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0044] The specifications of the medicines used in the following examples are as follows: Phosphate-buffered saline (PBS), manufacturer: Thermo Fisher Scientific; specification: 500 mL.

[0045] Fetal bovine serum, manufacturer: Thermo Fisher Scientific; specification: 500mL.

[0046] Gentamicin sulfate, manufacturer: Huazhong Pharmaceutical Co., Ltd.; specification: 2ml: 80,000 units.

[0047] DMEM culture medium, manufacturer: Thermo Fisher Scientific; specification: 500mL.

[0048] F12 culture medium, manufacturer: Thermo Fisher Scientific; specification: 500 mL.

[0049] L-Glutamine, Manufacturer: Thermo Fisher Scientific; Size: 500mL.

[0050] Epidermal growth factor, manufacturer: Shanghai Haohai Biological Technology Co., Ltd., specification: 50,000 IU.

[0051] Insulin, manufacturer: ROCHE, specification: 100mg.

[0052] Adenine, Manufacturer: MERCK, Specification: 1012101.

[0053] Hydrocortisone, manufacturer: Sigma-Aldrich, specification: 5mg.

[0054] Y 27632, Manufacturer: Sigma-Aldrich, Specification: 5mg.

[0055] Linifanib, manufacturer: MCE, 5mg.

[0056] SAG, manufacturer: MCE, 5mg.

[0057] DMSO, Manufacturer: Origen Bio; Specification: 70mL.

[0058] Recombinant trypsin, manufacturer: Thermo Fisher Scientific; specification: 100mL.

[0059] Example 1: Preparation of a renal progenitor cell reinfusion formulation derived from the urine of a healthy donor. Donor screening: One healthy donor was selected, and after signing an informed consent form, liver and kidney function and infectious disease screening were conducted. The results all met the donor screening criteria.

[0060] Sample collection and transportation: Instruct the donor to collect 500 mL of clean midstream urine, place it in a sterile collection container, and transport it to the preparation workshop via cold chain at 2~8℃.

[0061] Preparation of washing buffer and culture medium: Prepare washing buffer containing 90 v% phosphate-buffered saline (PBS), 10 v% fetal bovine serum, and 25 μg / mL gentamicin sulfate.

[0062] Prepare a renal progenitor cell culture medium containing 50 v% DMEM medium, 40 v% F12 medium, 10 v% fetal bovine serum, 2 mM L-glutamine, 10 ng / ml epidermal growth factor, 2 μg / ml insulin, 25 μg / ml adenine, 6 μg / ml hydrocortisone, and 2 μM γ-gamma. 27632, 0.2 μM Linifanib, 0.8 μM SAG.

[0063] Washing and separation: Centrifuge the urine at 400 g for 10 minutes, discard the supernatant, and retain a small amount of liquid at the bottom of the centrifuge tube; add 40 mL of washing buffer and wash 4 times, centrifuging at 400 g for 10 minutes each time; after the last centrifugation, add 5 mL of renal progenitor cell culture medium containing 100 μg / mL gentamicin sulfate to make a cell suspension, inoculate into a T25 culture flask, and incubate in a 37℃, 7.5% CO2, saturated humidity incubator, and record it as generation P0.

[0064] Expansion Culture: The expansion culture of renal progenitor cells begins with passage P0 and ends with passage P4, employing a continuous preparation process with multi-stage quality control. Simultaneously, the remaining cells from passage P2 are cryopreserved to address fluctuations in the patient's condition. The specific steps are as follows: Passaging: All uncontaminated P0 generation cells from culture flasks that had grown cell clones were digested and combined into one culture system; the cells were passaged for a total of 4 generations. The medium was changed for the first time on day 5 of the P0 generation culture, and then every 3 days thereafter. On day 12, when the P0 generation cells reached 80% confluence, they were passaged (P1 generation); subsequent passages were performed sequentially from P1 to P4, with 3 × 10⁶ cells collected after passage P2. 6 Cells were cryopreserved individually. Cryopreservation was performed by resuspending the cells in cryopreservation solution (90% renal progenitor cell culture medium + 10% DMSO) to a concentration of 5 × 10⁻⁶ cells / mL. 6 ~1×10 7 Cells / mL were aliquoted into cryovials, placed in a temperature-programmed chamber, and stored at -80°C overnight. Subsequently, they were transferred to a liquid nitrogen vapor phase for long-term storage within 24 hours. The passage density was 80% at each passage.

[0065] Digestion and Inoculation: At each passage, aspirate the supernatant from the culture flask, wash with buffer, remove the supernatant, and add recombinant trypsin digestion reagent; place the culture flask in an incubator for digestion for 3-10 minutes. After most cells have detached, add stop solution to terminate digestion; gently pipette the bottom of the culture several times, collect the cell suspension, centrifuge at 200 g for 5 minutes, and remove the supernatant; resuspend the cell pellet in renal progenitor cell culture medium and perform viable cell counting, at a rate of 3 × 10⁶ cells / year. 4 The cells were seeded into culture flasks at a density of 10 cells / cm², and after adding renal progenitor cell culture medium, they were placed in a cell culture incubator for culture under aseptic conditions.

[0066] Intermediate quality control: Samples were taken on day 2 of P1 passage culture, P2 passage, P3 passage, and P4 passage culture for quality control. On day 2 of P1 passage culture, sterility and mycoplasma testing were performed (acceptance standard: negative). On P2 passage, cell identification and purity were checked (immunofluorescence staining, acceptance standard: SOX9 positive with a positivity rate ≥95%). On P3 passage, cell biological efficacy was checked (acceptance standard: formation of renal epithelial cells expressing mature renal tubular markers after in vitro induction of differentiation). On day 2 of P4 passage culture, mycoplasma testing (acceptance standard: negative) and gentamicin residue determination (acceptance standard: <5.4 ppb) were performed. All intermediate control point results met the standards.

[0067] Collection: On day 4 of P4 generation cell culture, collect all cultured renal progenitor cell flasks, aspirate the supernatant, wash once with phosphate-buffered saline, and add recombinant trypsin; place the flasks in an incubator for digestion for 5 minutes, then stop digestion; collect the cell suspension, which is the renal progenitor cell stock solution. Count the number of viable cells and the viability rate, and send a portion of the cells for testing to perform cell identification and purity checks (flow cytometry, acceptance criteria: PAX8 / CD73 double positive with a positive rate ≥95%; CD45 / CD34 double negative with a positive rate <2%). After passing the tests, proceed to the formulation preparation stage.

[0068] Formulation preparation: The renal progenitor cell stock solution was centrifuged at 200 g for 5 minutes, and the supernatant was discarded; the cells were washed three times with physiological saline, centrifuged at 200 g for 5 minutes each time; the cells were resuspended in physiological saline to a final concentration of 6 × 10⁻⁶. 6 ~1×10 8 Cells / mL were dispensed into a pre-filled syringe, air was expelled, and the syringe was sealed for a seal check. Finally, the syringe was labeled to obtain the final formulation.

[0069] Formulation testing: Randomly sample the final formulation for finished product testing, and release it after it meets the finished product quality standards.

[0070] The renal progenitor cells cultured according to the aforementioned method, including both the P0 generation and the P4 generation used for delivery, exhibited typical epithelial cell morphology under a microscope. This was characterized by tightly packed cells with small intercellular spaces, polygonal cell shapes, and close adherence to the culture plane. Each generation was cultured for 3-4 days, and cell expansion was stable. The final P4 generation renal progenitor cell preparation contained 5.7 × 10⁻⁶ viable cells. 7 The cell count was 96%, meeting clinical drug administration requirements. All results in intermediate quality control and original solution control tests met the standards. Specifically, sterility and mycoplasma tests were negative; cell identification and purity testing (immunofluorescence staining) showed SOX9 positivity (100%); cell biological efficacy testing showed renal epithelial cells expressing the mature renal tubular marker (ATP1A1) after in vitro induced differentiation; gentamicin residue was <5.4 ppb; and cell identification and purity testing (flow cytometry) showed PAX8 / CD73 double positivity (98.9% and 100% respectively), and CD45 / CD34 double negative (0.81% and 0.80% respectively).

[0071] Test results reference Figures 1-5 The above results indicate that the renal progenitor cells obtained by the method described in this application are of stable quality and can meet clinical needs.

[0072] Example 2: Preparation and comparative analysis of renal progenitor cell reinfusion formulations derived from the urine of patients with chronic kidney disease. Donor screening: Three patients with chronic kidney disease of different ages, genders, and types were selected. After signing informed consent forms, liver and kidney function and infectious disease screenings were conducted, and the results met the donor screening criteria. The patients' basic information is as follows: Patient A: Female, 36 years old, stage 4 chronic kidney disease (CKD) caused by chronic glomerulonephritis; Patient B: Male, 70 years old, stage 5 chronic kidney disease (renal failure stage) caused by diabetes. Patient C: Female, 65 years old, stage 5 chronic kidney disease (renal failure stage) caused by hypertensive nephropathy.

[0073] Sample collection and transportation: Instruct patients to collect 175-300 mL of clean midstream urine, place it in a sterile collection container, and transport it to the preparation workshop via cold chain at 2-8℃.

[0074] Preparation of washing solution and culture medium: Patient A: Prepare washing buffer containing 85 v% DMEM medium, 15 v% fetal bovine serum, and 100 μg / mL gentamicin sulfate. Prepare renal progenitor cell culture medium containing 25 v% DMEM medium, 60 v% F12 medium, 15 v% fetal bovine serum, 0.2 mM L-glutamine, 2 ng / ml epidermal growth factor, 10 ng / ml insulin, 5 μg / ml adenine, 15 μg / ml hydrocortisone, and 10 μM γ-gamma. 27632, 0.5 μM Linifanib, 0.2 μM SAG.

[0075] Patient B: Prepare washing buffer containing 95 v% F12 medium, 5 v% fetal bovine serum, and 50 μg / mL gentamicin sulfate. Prepare renal progenitor cell culture medium containing 45 v% DMEM medium, 45 v% F12 medium, 10 v% fetal bovine serum, 1 mM L-glutamine, 5 ng / ml epidermal growth factor, 5 ng / ml insulin, 28 μg / ml adenine, 2 μg / ml hydrocortisone, and 25 μM γ-gamma. 27632, 0.1 μM Linifanib, 1 μM SAG.

[0076] Patient C: Prepare washing buffer containing 85 v% phosphate-buffered saline and 15 v% fetal bovine serum. Prepare renal progenitor cell culture medium containing 42.5 v% DMEM, 42.5 v% F12 medium, 15 v% fetal bovine serum, 2 mM L-glutamine, 10 ng / ml epidermal growth factor, 14 ng / ml insulin, 10 μg / ml adenine, 15 μg / ml hydrocortisone, and 0.1 μM γ-aminobutyric acid (γ-aminobutyric acid). 27632, 1 μM Linifanib, 1 μM SAG.

[0077] Washing and separation: Centrifuge each batch of urine at 400-500 g for 10 minutes, discard the supernatant, and retain a small amount of liquid at the bottom of the centrifuge tube; add 20-40 mL of washing buffer and wash 4 times, centrifuging at 400-500 g for 10 minutes each time; after the last centrifugation, add 6-18 mL of renal progenitor cell culture medium containing 100 μg / mL gentamicin sulfate to prepare a cell suspension, inoculate into 3-6 T12.5 culture flasks, and incubate in a 37℃, 7.5% CO2, saturated humidity incubator, and record as generation P0.

[0078] Expansion culture: P0 generation cells were cultured with medium changes every 1-3 days; from day 7 to 12, P0 generation cells reached 80% confluence and were passaged (P1 generation); subsequent passages were performed sequentially from P1 to P6. Renal progenitor cells from patient A were thawed using the remaining cryopreserved cells after P2 generation, following a standardized thawing procedure, and then cultured to P6. Renal progenitor cells from patients B and C were cultured continuously without using cryopreserved cells. All three batches of cells were passaged at 70%-100% density at each passage, with a seeding density of 1.5-3 × 10⁻⁶ cells / year. 4 Cells / cm²

[0079] Intermediate quality control: Samples were taken on day 2 of P1 passage culture, P2 passage, P5 passage, and P6 passage culture for quality control. On day 2 of P1 passage culture, sterility and mycoplasma testing were performed (acceptance standard: negative). On P2 passage, cell identification and purity were checked (immunofluorescence staining, acceptance standard: SOX9 positive with a positivity rate ≥95%). On P5 passage, cell biological efficacy was checked (acceptance standard: formation of renal epithelial cells expressing mature renal tubular markers after in vitro induction of differentiation). On day 2 of P6 passage culture, mycoplasma testing (acceptance standard: negative) and gentamicin residue determination (acceptance standard: <5.4 ppb) were performed. All intermediate control point results met the standards.

[0080] Collection: On day 4 of P6 cell culture, collect all cultured renal progenitor cell flasks, aspirate the supernatant, wash once with phosphate-buffered saline, and add recombinant trypsin. Place the flasks in an incubator for 8 minutes to digest, then stop digestion. Collect the cell suspension, which is the renal progenitor cell stock solution. Count the number of viable cells and the viability rate. Send a portion of the cells for testing to perform cell identification and purity checks (flow cytometry, acceptance criteria: CD73 positive with a positive rate ≥95%; CD45 / CD34 double negative with a positive rate <2%). After passing the tests, proceed to the formulation preparation stage.

[0081] Formulation preparation: The renal progenitor cell stock solution was centrifuged at 300 g for 5 minutes, and the supernatant was discarded; the solution was washed 5 times with excipients, and centrifuged at 300 g for 5 minutes each time; the solution was resuspended with excipients, filled into pre-filled syringes, air was expelled, and the seal was checked. Finally, the solution was labeled to obtain the final formulation.

[0082] Formulation testing: Randomly sample the final formulation for finished product testing, and release it after it meets the finished product quality standards.

[0083] The above results demonstrate that the method described in this application achieves a 100% success rate in isolating renal progenitor cells from patients with chronic kidney disease of different ages, sexes, and disease types. The number of cells harvested at P6 generation consistently meets clinical needs (i.e., the number of viable cells in the formulation meets the pre-set cell count requirement based on the patient's height and weight), and exhibits extremely high quality stability. The obtained renal progenitor cell formulations all meet quality standards with minimal batch-to-batch variation. Particularly for quantitative analysis tests, the results from multiple batches are remarkably similar. This proves that the method described in this application can eliminate individual differences among donors and screen for renal progenitor cells with optimal performance.

[0084] Experiment 1 uses renal progenitor cells to treat diabetic nephropathy mice via intrarenal injection. Fifteen NOD / SCID mice were randomly divided into three groups: a control group, an injury group, and a treatment group. The first day of streptozocin (STZ) induction was designated D1, and so on. Control group mice received normal feed without modeling or administration. Injury group mice received intraperitoneal injections of streptozocin (STZ) at a dose of 100 mg / kg for 5 days, followed by intramuscular injections of 50% glycerol-saline in the hind limbs, while continuing to be fed a high-fat diet. Treatment group mice underwent a similar diabetic nephropathy modeling process, and on D49, received a direct intrarenal injection of a GFP-labeled renal progenitor cell infusion preparation at a dose of 2 × 10⁻⁶. 6 Each animal was fed a high-fat diet continuously, with one cell per animal. All animals were euthanized and their tissue samples were collected for blood glucose testing, serum creatinine testing, and kidney histopathological examination.

[0085] The results showed that GFP-labeled test cells could integrate into the kidneys of diabetic nephropathy mice after drug administration. Blood glucose levels in the mice were measured. The levels in the control group, injury group, and drug administration group were 8.76±1.796 mmol / L, 29.06±5.303 mmol / L, and 15.64±5.126 mmol / L, respectively, with statistically significant differences between the injury group and the drug administration group. Serum creatinine levels in the mice were measured. The levels in the control group, injury group, and drug administration group were 0.100±0.0124 mg / dL, 0.175±0.0268 mg / dL, and 0.151±0.0215 mg / dL, respectively, with statistically significant differences between the injury group and the drug administration group. Figure 6 Histopathological examination showed that the renal tubular structure and degree of fibrosis in the kidneys of mice in the drug-treated group were significantly improved compared with those in the damaged group. Figure 7Furthermore, the expression of its damage marker KIM1 was significantly reduced in mature renal tubular cells (ATP1A1). + or UMOD + The number of repaired cells was significantly higher than that of the damaged cells. Figure 8 , Figure 9 The overall structure of the kidney tissue showed significant improvement.

[0086] In summary, a single intrarenal injection of the renal progenitor cell reinfusion formulation into mice with diabetic nephropathy significantly reduced blood glucose and serum creatinine levels, improved renal fibrosis, and demonstrated a regenerative and repair trend in the histopathological structure of the kidney tissue. This proves that the renal progenitor cell reinfusion formulation prepared by this method can improve damaged renal tissue structure and function, and is effective in treating diabetic nephropathy in mice.

[0087] Experimental Example 2: Treatment of renal ischemia-reperfusion injury in mice via subcutaneous injection of renal progenitor cells. This experimental case investigated the effect of renal progenitor cell reinfusion preparations administered subcutaneously in treating different models of kidney injury in mice.

[0088] Nine NOD / SCID mice were randomly divided into three groups: injury group, drug-treated group, and sham-operated group. Unilateral ischemia-reperfusion injury (UIRI) models were established in the injury group and drug-treated group. In the drug-treated group, renal progenitor cell retrograde agent was repeatedly administered subcutaneously at 24 and 72 hours post-injury at a dose of 3 × 10⁻⁶. 6 Cells / animal / dose. The sham surgery group underwent a sham surgery procedure; the abdominal cavity was closed immediately after laparotomy on day 1, without any kidney injury or drug administration. Ten days later, kidney tissue samples were collected from each group for histopathological examination, and serum samples were collected from each group for serum creatinine detection and proteomics analysis.

[0089] Immunofluorescence staining in histopathology revealed significant renal tubular epithelial cell necrosis in the injury group compared to the sham-operated group. This was primarily characterized by the widespread distribution of the renal injury marker KIM1, and the fact that some cells no longer expressed the mature renal tubular markers ATP1A1 and AQP1, confirming the successful establishment of the injury model. In the drug-treated mice, KIM1... + The damaged area was significantly reduced, accompanied by ATP1A1. + and AQP1 + Reconstruction and recovery of renal tubular epithelial cells, and endogenous SOX9 + Cells were highly activated, and there were statistically significant differences between groups. Figure 10This indicates the initiation of kidney tissue repair and regeneration in the treated mice. Furthermore, serum creatinine levels in the treated mice were significantly reduced on day 8, with a statistically significant difference between the two groups. This phenomenon persisted until the end of the observation period, at which point serum creatinine levels in the injured group and the treated group showed a statistically significant difference between the two groups. Subcutaneously administered renal progenitor cells were able to activate endogenous SOX9 in mice. + Cells participate in the repair of unilateral renal ischemia-reperfusion injury and alleviate the increase in serum creatinine after renal injury. Figure 11 Principal component analysis (PCA) results from proteomics showed significant differences in the proteomes of the sham-operated group and the injury group, while the treated drug-eluting group was able to mitigate these differences to some extent. Figure 12 The proteomics data were hierarchically clustered, ultimately classifying all detected proteins into four groups. Group I, enriched proteins, primarily involved in biological processes promoting kidney repair and regeneration, exhibited the highest abundance in the drug-treated group; Group III was mainly associated with tissue damage status, with the highest abundance in the damaged group. Figure 13 The above proteomics results validate the positive regulatory effect of subcutaneous administration to renal progenitor cells on proteomic changes following kidney injury.

[0090] In summary, repeated subcutaneous transplantation of renal progenitor cell infusion agents significantly reduced the expression of the renal injury marker KIM1, promoted the reconstruction and recovery of mature renal tubules, and significantly decreased serum creatinine levels in mice with renal injury, demonstrating the positive role of renal progenitor cell infusion agents in renal injury repair. The mechanism lies in the fact that subcutaneously transplanted renal progenitor cells can actively regulate proteomic changes after renal injury, guiding them towards directions conducive to renal repair and regeneration.

[0091] Example 3: Preparation and intrarenal administration of an autologous renal progenitor cell infusion preparation derived from the urine of patients with type 2 diabetes mellitus and CKD. Patient Information: The patient is a male who was diagnosed with type 2 diabetes according to the "Guidelines for the Prevention and Treatment of Type 2 Diabetes in China." He has recently been diagnosed with abnormal kidney function and chronic kidney disease (CKD) according to the KDIGO criteria. The overall diagnosis is type 2 diabetes mellitus complicated with CKD. This patient is a typical representative of a high-risk group for chronic kidney disease.

[0092] Donor screening and sample collection: After the patient signs the informed consent form, liver and kidney function and infectious disease screening are conducted, and the results meet the donor screening criteria. The patient is instructed to collect approximately 300 mL of clean midstream urine, which is placed in a sterile collection container and transported to the preparation workshop via cold chain at 2-8°C.

[0093] Preparation of washing solution and culture medium: Prepare washing solution and renal progenitor cell culture medium according to the formula in Example 1.

[0094] Washing, separation, and amplification culture: Cell washing, separation, P0 generation culture, and passage amplification were performed according to the method described in Example 1. Autologous SOX9 was non-invasively extracted from patient urine using the patented R-Clone adult epithelial stem cell culture platform from Jimeiruisheng. + CD73 + Kidney progenitor cells. In a GMP-standard production facility, these seed cells undergo approximately four weeks of directed expansion to achieve large-scale, efficient proliferation. The entire process involves multiple rounds of rigorous quality control to strictly maintain cell phenotypic stability and functional integrity.

[0095] Quality control: During the passage culture process, mycoplasma testing, sterility testing, cell identification and purity testing (immunofluorescence staining and flow cytometry), cell biological efficacy testing, and antibiotic residue determination were performed according to the quality control points in Example 1. All test results met the finished product quality standards.

[0096] Formulation preparation: After the P4 generation of cells was cultured, the cell suspension was collected, washed, and resuspended to prepare an infusionable autologous renal progenitor cell reinfusion formulation with a final concentration of 1×10⁻⁶. 7 Cells / mL, filled into pre-filled syringes, ready for use.

[0097] Intrarenal drug administration therapy: The cell transplantation procedure employs a combined approach of nephrology and ultrasound imaging, utilizing ultrasound-guided, precise intrarenal targeted injection. Pre-operatively, the operator conducts a thorough imaging assessment of both kidneys, accurately calculating renal cortical thickness, renal artery diameter, and anatomical orientation under real-time ultrasound monitoring to plan the optimal needle insertion angle and safe pathway. During the procedure, local minimally invasive anesthesia is used, and the needle is inserted at the ultrasound-guided drug delivery point. The patient's respiratory rhythm is monitored in real-time to minimize the risk of organ traction. Simultaneously, with real-time ultrasound monitoring, a precise percutaneous puncture is performed into the renal parenchyma to deliver the prepared autologous renal progenitor cell infusion formulation to the damaged area of ​​the kidney.

[0098] After cell transplantation, color Doppler flow imaging (CDFI) was used for verification to confirm accurate drug delivery site and the absence of active bleeding and perirenal leakage. The patient remained conscious throughout the procedure, tolerated the treatment well, and experienced no immediate adverse reactions.

[0099] Safety and efficacy follow-up: Short-term follow-up data from 0 to 3 days after cell transplantation showed that the patient's vital signs were stable, spontaneous activities were normal, and there were no infections, fevers, or injection complications.

[0100] Three days after cell transplantation, the patient's total protein and albumin levels, which had been consistently below normal before the operation (60.4 g / L and 37.8 g / L, respectively), improved to normal levels (65.2 g / L and 40.9 g / L), thus preliminarily verifying the safety of the drug administration and the therapeutic effect in a short period of time.

[0101] Postoperative ultrasound examination showed that the perirenal and collecting system structures were clear and no abnormal signs were observed (see...). Figure 14 ).

[0102] The above results indicate that the autologous renal progenitor cell preparation prepared by the method in this application, administered intrarenally, has good safety and preliminary renal function improvement effects in patients with type 2 diabetes mellitus complicated with CKD.

[0103] Comparative Example 1: Renal progenitor cells were cultured in a culture system without a feeder layer and without matrix gel using conventional renal progenitor cell culture medium. This comparative example provides a method for culturing renal progenitor cells in a culture system without a feeder layer and without matrix gel using conventional renal progenitor cell culture medium, comprising the following steps: Donor screening: One healthy donor was selected, and after signing an informed consent form, liver and kidney function and infectious disease screening were conducted. The results all met the donor screening criteria.

[0104] Sample collection and transportation: Instruct the donor to collect 500 mL of clean midstream urine, place it in a sterile collection container, and transport it to the preparation workshop via cold chain at 2~8℃.

[0105] Washing buffer and culture medium preparation: Prepare washing buffer containing 90 v% F12 medium, 10 v% fetal bovine serum, 25 μg / mL gentamicin sulfate, and 1 v% penicillin / streptomycin solution (100×). Prepare renal progenitor cell culture medium A containing 45 v% DMEM medium, 45 v% F12 medium, 10 v% fetal bovine serum, 1.2 mM L-glutamine, 0.5 ng / ml epidermal growth factor, 5 ng / ml insulin, 30 μg / ml adenine, and 10 μg / ml hydrocortisone.

[0106] Feeder layer cells: Mouse embryonic fibroblast 3T3-J2 cells were used as feeder cells, cultured to the logarithmic growth phase, and then collected. The cells were treated with gamma rays at a dose of 60 Gy per fraction. Then, 20 v% matrix gel was added to a T12.5 culture flask and incubated at 37°C for 30 minutes. The matrix gel was then aspirated, and the cells were cultured at a rate of 5 × 10⁻⁶ cells / year. 3 cells / cm 2 The irradiated feeder cells were seeded at a density that covered the bottom of the culture container, and cultured for 24 hours until they adhered to the container before use.

[0107] Renal progenitor cell isolation: Washing and separation: Centrifuge urine at 400 g for 10 minutes, discard the supernatant, and retain about 2 mL of urine at the bottom of the centrifuge tube; add 50 mL of washing buffer and wash 4 times, centrifuging at 380 g for 15 minutes each time; after the last centrifugation, add 1 mL of renal progenitor cell culture medium A containing 25 μg / mL gentamicin sulfate and 1v% penicillin / streptomycin double antibiotic solution (100×) to make a cell suspension, and inoculate it into T12.5 culture flasks with pre-coated feeder cells, and incubate in a 37℃, 7.5% CO2, saturated humidity incubator.

[0108] At the end of the P0 generation culture, the morphology of the obtained cells was observed and recorded using an inverted phase-contrast microscope. The results are as follows: Figure 15 , 16 As shown in the field of view, in a culture system without a feeder layer and without matrix gel, cells cultured in conventional renal progenitor cell culture medium A appear as small clones or scattered distributions. Some cells are stringy or enlarged, which are signs of poor cell morphology. The cell morphology in this comparative example differs significantly from that of the renal progenitor cells obtained in Example 1, and the uniformity is poor. Furthermore, the cell proliferation rate shows a significant difference at the end of the P0 generation culture.

[0109] Comparative Example 2: Renal progenitor cells were cultured in a culture system without a feeder layer and without matrix gel using conventional renal progenitor cell culture medium. This comparative example provides another method for culturing renal progenitor cells in a culture system without a feeder layer and without matrix gel using conventional renal progenitor cell culture medium, and compares it with the method of the present invention. It includes the following steps: Donor screening: One healthy donor was selected, and after signing an informed consent form, liver and kidney function and infectious disease screening were conducted. The results all met the donor screening criteria.

[0110] Sample collection and transportation: Instruct the donor to collect 500 mL of clean midstream urine, place it in a sterile collection container, and transport it to the preparation workshop via cold chain at 2~8℃.

[0111] Preparation of washing buffer and culture medium: Prepare washing buffer containing 90 v% F12 medium, 10 v% fetal bovine serum, 25 μg / mL gentamicin sulfate, and 1 v% penicillin / streptomycin solution (100×). Prepare renal progenitor cell culture medium B containing 37.5 v% DMEM medium, 47.6 v% serum-free keratinocyte culture medium, 11.9 v% F12 medium, 4.8 v% fetal bovine serum, 1 mM L-glutamine, 7.5 ng / ml epidermal growth factor, 15 ng / ml choleraemycin, 2.5 ng / ml insulin, 30 μg / ml adenine, and 0.2 μg / ml hydrocortisone. Simultaneously prepare renal progenitor cell culture medium with the same formulation as in Example 1 of this invention.

[0112] Kidney progenitor cell isolation: Washing and separation: Centrifuge urine at 400 g for 5 minutes, discard the supernatant, and retain about 2 mL of urine at the bottom of the centrifuge tube; add 50 mL of washing buffer and wash twice, centrifuging at 400 g for 5 minutes each time; before the last centrifugation, divide the cell suspension into two groups. One group is culture medium B, and the other group is the culture medium group in Example 1. After centrifugation, add 1 mL of culture medium B containing 25 μg / mL gentamicin sulfate and 1v% penicillin / streptomycin antibiotic solution (100×) and the culture medium of the present invention, respectively, to prepare cell suspensions. Each suspension is seeded into one T12.5 culture flask and cultured in a 37°C, 7.5% CO2, saturated humidity incubator.

[0113] P0 cells were cultured for 14 days. At the end of the culture, the morphology of the cells was observed and recorded using an inverted phase-contrast microscope. The results of the two groups are as follows: Figure 16 As shown in the field of view, both groups of cells exhibit good morphology, but their cell proliferation rates differ. When cultured for the same duration, the P0 generation cells obtained by the method of this invention proliferate faster and produce significantly larger clones.

[0114] Cell culture was continued, and the cell proliferation rates obtained by the two methods were compared. Except for passage P0 to P1, all harvested cells were used for passage. Subsequent passages were performed at a fixed seeding density, with passages every 3 days, and the number of cells harvested at each passage was calculated. The cell doubling number was calculated using the formula n = 3.32 × lg(N / N0) (where N0 is the number of viable cells plated, and N is the number of viable cells harvested). The cell doubling time was then calculated by dividing the culture hours by the cell doubling number. The comparison of cell doubling times from P2 to P6 is shown in the table below:

[0115] As shown in the table above, under the same donor, starting sample, basic operation and culture conditions, the P0 generation cells obtained by the method of the present invention expand faster in culture compared with the method of culture medium B, demonstrating a significant advantage.

[0116] Based on the above examples, experimental cases, and comparative examples, the following conclusions can be drawn: The core advantage of this invention lies in its systematic solution to multiple bottlenecks in the clinical-grade preparation and industrial application of renal progenitor cells through a unique culture medium formulation and standardized GMP process. 1. Highly efficient expansion, supporting large-scale production: As shown in Comparative Examples 1 and 2, the culture medium of this invention shortens the average cell doubling time by 26% (19.55 hours vs. 24.75 hours), and the expansion advantage remains stable from generation P2 to P6. Furthermore, it enables the acquisition of stably expanded renal progenitor cells in a culture system without a feeder layer or matrix gel support, laying the foundation for large-scale, economical cell production.

[0117] 2. Controllable quality and established pharmaceutical-grade standards: Examples 1 and 2 demonstrate a complete GMP process and end-to-end quality control. The final product's cell purity (PAX8 / CD73 double positivity ≥98.9%), sterility, and potency all meet stringent pharmaceutical release standards. Furthermore, the process is applicable to healthy donors and various CKD patients, with minimal batch-to-batch variation and extremely high robustness.

[0118] 3. Safe and effective, validating a new paradigm for allogeneic therapy: Experiments 3 and 4 confirmed that healthy donor allogeneic cells prepared using this process did not induce adverse reactions such as immune rejection after intravenous infusion, and significantly improved renal function in patients with advanced renal failure and proteinuria (e.g., a decrease in creatinine and a reduction in proteinuria of nearly 50%). This breaks through the limitations of autologous therapy and provides a safe, immediate, and effective new cell therapy option for chronic kidney disease.

[0119] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for culturing renal progenitor cells, characterized in that, Includes the following steps: Step 1: Obtain kidney progenitor cells; Step 2: The renal progenitor cells are cultured in a culture medium containing: Basal culture medium, fetal bovine serum, L-glutamine, epidermal growth factor, insulin, adenine, hydrocortisone, Rho kinase inhibitor, tyrosine kinase inhibitor and SAG; Step 3: Collect the cultured renal progenitor cells.

2. The method for culturing renal progenitor cells according to claim 1, characterized in that, In step one, the renal progenitor cells are urinary renal progenitor cells; Preferably, the renal progenitor cells are tissue-specific adult stem cells.

3. The method for culturing renal progenitor cells according to claim 1, characterized in that, In step one, the method for obtaining renal progenitor cells includes: The urine sample was centrifuged and the supernatant was removed; then it was washed with washing solution, centrifuged, and the supernatant was removed to obtain a solution containing renal progenitor cells. The urine sample can be a naturally urinated sample or a urine sample collected via catheter.

4. The method for culturing renal progenitor cells according to claim 3, characterized in that, The washing liquid includes: 90-95% matrix solution, 5-10% fetal bovine serum, 0-200 μg / mL antibiotics; The substrate solution includes at least one of F12 medium, DMEM medium, physiological saline, compound electrolyte injection solution, and phosphate buffer.

5. The method for culturing renal progenitor cells according to claim 1, characterized in that, The Rho kinase inhibitor uses Y 27632. The tyrosine kinase inhibitor used is Linifanib; The culture medium is a culture medium for cells without a trophoblast layer.

6. The method for culturing renal progenitor cells according to claim 5, characterized in that, In step two, the cell culture medium comprises basal culture medium, 8-20 v% fetal bovine serum, 0.2-2 mM L-glutamine, 0.1-10 ng / ml epidermal growth factor, 1-14 μg / ml insulin, 5-30 μg / ml adenine, 1-20 μg / ml hydrocortisone, and 0.1-50 μM gamma glutamic acid. 27632, 0.02~1 μM Linifanib, 0.01~1 μM SAG; The basal culture medium is composed of at least one of F12 medium and DMEM medium.

7. The method for culturing renal progenitor cells according to claim 1, characterized in that, In step two, the kidney progenitor cells initially cultured in the culture medium are designated as generation P0, and then passaged; this process continues until generation P4 to P8.

8. The method for culturing renal progenitor cells according to claim 7, characterized in that, The passage culture includes: digesting all uncontaminated culture flasks containing P0 generation cells that have grown cell clones and then combining them into one culture system; passage culture for a total of 3 to 8 generations, except for P0 generation which is passaged within 20 days, and then passaged every 2 to 6 days thereafter, with a cell density of approximately 50% to 100% at passage.

9. The method for culturing renal progenitor cells according to claim 7, characterized in that, The method for each passage includes: aspirating the supernatant from the culture flask, then washing and removing the supernatant; Add recombinant trypsin digestion reagent, place the culture flask in an incubator and digest for 3-10 minutes. After most of the cells have detached, add stop solution to terminate the digestion. Blow the culture medium under a pipette to collect the cell suspension, centrifuge at 200-400 g, and remove the supernatant. Resuspend the cell pellet in renal progenitor cell culture medium and perform viable cell counting at a concentration of 0.5–5 × 10⁻⁶ cells / mL. 4 The cells were seeded into culture flasks at a density of 10 cells / cm², and cultured further after adding culture medium.

10. The method for culturing renal progenitor cells according to claim 7, characterized in that, The passage culture process in step two includes quality control: cell samples are extracted during the P2 generation culture, at the end of the P2 generation culture, at the end of the Pn-1 generation culture, and at the end of the Pn generation culture for quality control index detection, where n is the last generation of culture; The quality control indicators include: mycoplasma testing, sterility testing, cell identification and purity testing, cell biological efficacy testing, and antibiotic residue determination.

11. The method for culturing renal progenitor cells according to claim 7, characterized in that, Step two also includes cryopreservation of P2 generation cells; When needed, cryopreserved cells are thawed and passaged.

12. The method for culturing renal progenitor cells according to claim 1, characterized in that, The process of collecting and culturing kidney progenitor cells involves: aspirating the supernatant from the culture flask, then washing and removing the supernatant; Add recombinant trypsin digestion reagent, place the culture flask in an incubator and digest for 3-10 minutes. Stop digestion after the cells detach. Blow on the bottom of the culture medium and collect the cell suspension.

13. A renal progenitor cell reinfusion preparation, characterized in that, It comprises renal progenitor cells cultured using any one of claims 1 to 12, and pharmaceutically acceptable excipients.

14. The method for culturing renal progenitor cells according to claim 13, characterized in that, The renal progenitor cells were SOX9. + Kidney progenitor cells; The renal progenitor cells were double positive for PAX8 / CD73 with a positive rate of ≥95%, and double negative for CD45 / CD34 with a positive rate of <2%.

15. The use of renal progenitor cells obtained by any one of claims 1 to 12, and the renal progenitor cell reinfusion preparation of claim 13 or 14, in the preparation of a medicament for regeneration and repair of chronic kidney disease.

16. The application according to claim 15, characterized in that, The drug is administered via intrarenal route.

17. The application according to claim 15 or 16, characterized in that, The chronic kidney disease includes at least one of the following: chronic kidney disease caused by chronic glomerulonephritis, chronic kidney disease caused by diabetes, chronic kidney disease caused by hypertensive nephropathy, kidney failure, and proteinuria.

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