Method for extracting and culturing urine-derived stem cells

Abstract

The invention relates to an extraction and culture method of urine-derived stem cells, and belongs to the field of cell biology and regenerative medicine. The method comprises the following steps: coating a culture plate with a gelatin solution; collecting clean midstream urine of healthy people and adding antibiotics; centrifugally separating the cell precipitate; washing with a phosphate buffer solution; re-suspending the precipitate by using a urine stem cell complete culture medium for primary culture; and after the cells grow to a certain fusion degree, carrying out passage amplification. The urine stem cell complete medium comprises a basic medium, fetal calf serum, epidermal growth factors, platelet-derived growth factors and basic fibroblast growth factors. By adopting the method disclosed by the invention, the urine-derived stem cells can be efficiently and stably separated from urine, and the obtained cells have high multiplication capacity, good stemness maintaining capacity, multidirectional differentiation potential and immunoregulation function, and can provide an ideal seed cell source for treating diseases such as diabetic erectile dysfunction.

Description

Technical Field

[0001] This invention relates to the fields of cell biology and regenerative medicine, and more specifically, to a method for extracting and culturing urinary stem cells from human urine. Background Technology

[0002] Erectile dysfunction (ED) is a common disease affecting men's health, severely impacting their quality of life, mental health, and interpersonal relationships. Diabetic erectile dysfunction (DMED), caused or exacerbated by diabetes, has an extremely high incidence in men with diabetes and is one of the most common and challenging complications of diabetes. Currently, conventional treatments for DMED include oral phosphodiesterase-5 inhibitors, intracavernosal vasoactive drug injections, and surgical procedures such as penile prosthesis implantation. However, these methods have limited efficacy, are ineffective for some patients, and may be accompanied by side effects. Therefore, there is an urgent need to develop safer and more effective new treatment strategies.

[0003] In recent years, stem cell-based regenerative medicine has opened up new avenues for the treatment of diabetic erectile dysfunction (DMED). For example, patent application CN116036132A discloses the application of cryopreserved allogeneic human adipose-derived mesenchymal stem cells in the preparation of drugs for treating diabetic erectile dysfunction, which can improve the ICP / MAP ratio and the number of erections to varying degrees, and has a restorative effect on the smooth muscle / collagen ratio of the corpus cavernosum. Patent application CN117982539A discloses an sPL compound preparation for treating ED, which contains sPL and umbilical cord mesenchymal stem cell exosomes, and can improve the repair effect of ED. These studies all indicate that mesenchymal stem cells and their derivatives have broad application prospects in the treatment of ED.

[0004] Urinary stem cells (USCs) are a novel type of adult stem cell isolated from human urine in recent years, possessing strong proliferative capacity and multi-lineage differentiation potential. Compared with bone marrow mesenchymal stem cells or adipose-derived stem cells, USCs have significant advantages: their acquisition method is completely non-invasive, avoiding ethical controversies, and their sources are widely available and inexpensive. More importantly, because they originate from the urinary system, they may be more suitable for the repair and reconstruction of urogenital tissues. Existing research shows that USCs can be induced to differentiate into multiple lineages in vitro, including osteoblasts, adipocytes, endothelial cells, and smooth muscle cells, and are considered ideal seed cells for urinary system tissue engineering. For example, US Patent Publication No. US11135248B2 was the first to disclose a method for isolating stem cells from human urine and demonstrated that they can differentiate into multiple lineages, including osteoblasts, adipocytes, chondrocytes, endothelial cells, neural cells, and myogenic cells. Patent application CN110051694A discloses a urinary stem cell preparation and its preparation method, and confirms that it can significantly prolong the survival of rats after kidney transplantation, reduce blood urea nitrogen and creatinine levels, and reduce T cell infiltration in kidney tissue.

[0005] However, urine contains complex cellular components, extremely low levels of mesenchymal stem cells (USCs), and requires a demanding culture environment. While existing research has explored methods for isolating and culturing urinary stem cells—for example, patent application CN120230708A discloses a method for extracting mesenchymal stem cells from urine—this method requires specialized stem cell culture medium for the first two weeks, and further optimization is needed to improve the adhesion efficiency and proliferation capacity of primary cells. Therefore, establishing a stable, efficient, and reproducible method for isolating and culturing USCs is crucial for both basic research and clinical applications. Summary of the Invention

[0006] The present invention aims to provide an improved method for the extraction and culture of urinary stem cells to solve the problems of low isolation efficiency, unstable culture success rate and insufficient stem cell quality of urinary stem cells in the prior art, and to provide high-quality seed cells for the treatment of diseases such as diabetic erectile dysfunction (DMED).

[0007] To achieve the above objectives, the present invention provides a method for extracting and culturing urinary stem cells, comprising the following steps: S1. Coating culture dishes: Coat cell culture plates with gelatin solution and place them in a carbon dioxide incubator to complete the coating; S2. Urine collection and pretreatment: Under aseptic conditions, clean midstream urine from healthy individuals is collected and antibiotics are added. S3. Initial centrifugation: Centrifuge the urine in separate containers, carefully discarding the supernatant and retaining the cell precipitate; S4. Washing: Add phosphate-buffered saline (PBS) to the cell pellet to resuspend the cells, then centrifuge and wash again to remove impurities; S5. Primary culture: Resuspend the cell pellet in urine stem cell complete culture medium, seed it into a coated culture plate, place it in an incubator for primary culture, and change the medium regularly. S6. Subculture: After the primary cells have grown to a certain degree of confluence, they are digested with trypsin and then passaged for expansion.

[0008] Preferably, in step S1, the mass-volume concentration of the gelatin solution is 0.05%-0.2%, more preferably 0.1%. The coating conditions are standing at 37°C for at least 30 minutes, preferably at least 1 hour. This step, through gelatin coating, aims to improve the hydrophilicity of the culture surface, mimicking the extracellular matrix environment, thereby promoting the primary adhesion of urinary stem cells. Insufficient coating time will not significantly improve the adhesion effect; too low a concentration will result in incomplete coating, while too high a concentration may lead to an uneven coating.

[0009] Preferably, in step S2, the urine collection volume is 100-300 mL, more preferably 200-250 mL. The added antibiotic is a penicillin-streptomycin mixture, added at a volume of 1.5%-2.5% of the urine volume. This protocol specifies the urine collection volume and initial treatment method. Too low a collection volume will result in an insufficient total cell count, making it difficult to successfully isolate the extremely low concentration of urine-derived stem cells; too high a collection volume will be inconvenient to operate and increase the risk of contamination. Adding antibiotics can effectively inhibit bacterial contamination and ensure a sterile environment for subsequent culture.

[0010] Preferably, in steps S3 and S4, the centrifugal force is 200-600g, more preferably 400g; the centrifugation time is 5-15min. Step S3 is preferably centrifuged at 400g for 10min, and step S4 is preferably centrifuged at 400g for 6min. This setting of centrifugal force and time aims to effectively settle cells while avoiding irreversible mechanical damage to the cells due to excessive centrifugal force or prolonged centrifugation time.

[0011] Preferably, in step S4, the washing step is as follows: add 10-30 mL of phosphate buffer, gently mix by pipetting, centrifuge at 200-600 g for 5-10 min, and repeat the washing 1-3 times. More preferably, add 20 mL of phosphate buffer, centrifuge at 400 g for 6 min, and repeat this step once. This washing protocol aims to thoroughly remove metabolic waste, broken cells, and residual antibiotics from urine, avoiding these components from adversely affecting primary culture.

[0012] Preferably, in step S5, the complete urine stem cell culture medium comprises basal culture medium, fetal bovine serum, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF). The volume percentage of fetal bovine serum is 5%-20%, the mass-volume concentration of EGF is 5-50 ng / mL, the mass-volume concentration of PDGF is 5-50 ng / mL, and the mass-volume concentration of bFGF is 5-50 ng / mL.

[0013] More preferably, the formulation of the complete urine stem cell culture medium is: 450 mL of mesenchymal stem cell culture medium or DMEM / F12 culture medium, 50 mL of fetal bovine serum, 10 ng / mL of EGF, 10 ng / mL of PDGF, and 10 ng / mL of bFGF.

[0014] Resuspend 100 mL of urine sediment in 1-4 mL of complete culture medium, more preferably 2 mL. Inoculate into 6-well plates, adding culture medium to each well to a total volume of 1-3 mL, more preferably 2 mL. Incubate with medium every 2-4 days, more preferably every 3 days.

[0015] This preferred scheme defines the core nutritional system for primary cell culture. The basal culture medium and fetal bovine serum provide the essential nutrients required for cell growth. A specific combination of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF) is one of the key aspects of this invention: EGF strongly promotes mitosis and proliferation of adherent cells; PDGF can chemotactically attract and promote the growth of mesenchymal-derived cells; and bFGF plays an important role in maintaining the undifferentiated state of stem cells and promoting their long-term self-renewal. The synergistic effect of these three factors at specific concentrations can significantly improve the survival rate and colony-forming ability of the very small number of stem cells in urine. The absence of any one factor or a concentration deviating from the range defined in this invention may lead to slow proliferation of primary cells or even culture failure.

[0016] Preferably, in step S6, passage is performed when the primary cells reach 70%-90% confluence, more preferably 70%-80%. The digestion conditions are 0.05%-0.25% trypsin, more preferably 1 mL of 0.25% trypsin, at 37°C for 1-10 min (based on microscopic observation of cell rounding and increased intercellular spaces), and centrifugation conditions are 200-600g for 3-8 min, more preferably 400g for 5 min. This protocol sets the optimal timing and operating parameters for passage culture. Passaging during the logarithmic growth phase results in optimal cell condition, high survival rate, and strong proliferation capacity after passage. The concentration of trypsin and the digestion time need to be precisely controlled to fully dissociate the cells while avoiding over-digestion and cell damage.

[0017] A type of urinary stem cell prepared by any of the methods described above.

[0018] The theoretical and technical mechanism of this invention lies in constructing a highly efficient culture system encompassing cell adhesion, nutrient supply, and proliferation through multi-dimensional synergistic optimization. First, gelatin coating physically simulates the extracellular matrix in vivo, providing a suitable substrate for primary adhesion of urinary stem cells. Second, the carefully designed complete culture medium not only ensures the basic needs for cell survival through its fundamental components but also, through the synergistic effect of three growth factors—EGF, PDGF, and bFGF—simultaneously activates multiple key pathways at the cell signaling level, including promoting adherent cell proliferation, maintaining stem cell stemness, and chemotactic cell growth. Finally, throughout the entire separation and culture process, from centrifugation control and washing frequency to passage timing, every process parameter is precisely balanced around the two core objectives of "maximizing cell viability" and "optimal proliferation efficiency." In this entire technical path, the creation of the physical microenvironment, precise supply of chemical signals, and meticulous control of process operations are interconnected, systematically solving the problems of low separation efficiency, unstable culture, and difficulty in maintaining stemness caused by factors such as difficulty in adhesion, insufficient nutrition, and operational damage in traditional methods.

[0019] Compared with the prior art, the method provided by the present invention has the following beneficial effects: From a component perspective, the technical advantage of this scheme lies in the redesign and optimization of the culture medium composition. By employing a composite system consisting of basal culture medium, fetal bovine serum, and three growth factors—EGF, PDGF, and bFGF—a nutritional environment is constructed that promotes efficient adhesion and survival of primary cells while maintaining their long-term self-renewal capacity. EGF, PDGF, and bFGF function in three different ways: promoting mitosis, chemotaxis and migration, and maintaining stemness. Their synergistic effect far exceeds the effect of simply adding one or two factors, which is the material basis for the successful isolation and efficient expansion of a very small number of stem cells in urine.

[0020] From a process perspective, the technical advantages of this approach lie in the refined and standardized control of the entire isolation and culture process. From the temperature and time of gelatin coating, the magnitude of centrifugation force, and the number of washing cycles, to the assessment of fusion degree and digestion conditions during passage, the parameters of each step have been systematically optimized and coordinated. This standardized process design aims to minimize the risk of cell damage and loss during operation, while creating a continuous and stable physicochemical environment for cell growth, thereby significantly improving the stability and reproducibility of the method.

[0021] From a cellular performance perspective, the urinary stem cells obtained using this method exhibit excellent biological characteristics. Flow cytometry analysis revealed that the cells highly express mesenchymal stem cell surface markers (such as CD73, CD90, and CD105) but do not express hematopoietic markers (such as CD34 and CD45), consistent with the phenotypic characteristics of mesenchymal stem cells. Osteogenic and adipogenic differentiation induction experiments confirmed their multi-lineage differentiation potential, and immunofluorescence detection showed that they express kidney development-related transcription factors PAX2 and SOX9. More importantly, co-culture experiments with immune cells demonstrated that the urinary stem cells isolated in this invention can significantly downregulate the expression levels of multiple inflammatory factors such as IL-5, IL-6, and IFN-γ, exhibiting good immunomodulatory function. This provides an important theoretical basis for their application in the treatment of inflammation-related diseases (such as DMED). Attached Figure Description

[0022] Figure 1 This is a cell morphology diagram of primary (P0) urinary stem cells isolated and cultured in Example 1 of the present invention; Figure 2 This is a cell morphology diagram of the first generation (P1) of urine-derived stem cells isolated and cultured in Example 1 of the present invention; Figure 3 The cell morphology of the second generation (P2) of urine-derived stem cells isolated and cultured in Example 1 of this invention; Figure 4 This is a diagram illustrating the multi-directional differentiation capacity of urinary stem cells in Example 1 of the present invention, showing the results of alizarin red staining 21 days after osteogenic induction. Figure 5 This is a diagram illustrating the multi-directional differentiation capacity of urinary stem cells in Example 1 of the present invention, showing the Oil Red O staining results 11 days after adipogenic induction. Figure 6 This is a flow cytometry diagram of CD73, a surface marker of urinary stem cells, in Example 1 of the present invention. Figure 7 This is a flow cytometry diagram of CD90, a surface marker of urinary stem cells, in Example 1 of the present invention. Figure 8 This is a flow cytometry diagram of CD105, a surface marker of urinary stem cells, in Example 1 of the present invention. Figure 9 This is a flow cytometry diagram of CD45, a surface marker of urinary stem cells, in Example 1 of the present invention. Figure 10 This is a flow cytometry diagram of CD34, a surface marker of urinary stem cells, in Example 1 of the present invention. Figure 11 This is an immunofluorescence image of PAX2 expression displayed on urinary stem cells in Example 1 of the present invention; Figure 12This is an immunofluorescence image of urine-derived stem cells showing SOX9 expression in Example 1 of the present invention (cell nuclei counterstained with DAPI). Detailed Implementation

[0023] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings and specific examples. However, it should be understood that these embodiments are only for explaining the present invention and are not intended to limit the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments in this application without creative effort should fall within the scope of protection of this application.

[0024] General Implementation Examples A method for extracting and culturing urinary stem cells, comprising the following steps: S1. Coating culture plates: Coat cell culture plates with a gelatin solution with a mass-volume concentration of 0.05%-0.2%, adding 1-2 mL to each well, and place them in a carbon dioxide incubator at 37°C for at least 30 minutes to complete the coating.

[0025] S2. Urine collection and pretreatment: Under aseptic conditions, collect 100-300 mL of clean midstream urine from healthy individuals, add 1.5%-3% penicillin-streptomycin antibiotic solution, and mix gently.

[0026] S3. Initial centrifugation: Aliquot the urine into 50mL centrifuge tubes and centrifuge at 200-600g at room temperature for 5-15 minutes. After centrifugation, carefully transfer the sample to a biosafety cabinet, aspirate the supernatant with a pipette, retain the liquid at the bottom, gently resuspend the cell pellet by pipetting, and combine the pellets from the same urine sample.

[0027] S4. Washing: Add 10-30 mL of phosphate buffer to the cell pellet, gently pipette to mix, centrifuge at 200-600 g for 5-10 minutes, discard the supernatant, and repeat this washing step 1-3 times.

[0028] S5. Primary Culture: Resuspend the precipitate of each 100 mL initial urine volume in 1-4 mL of complete urine stem cell culture medium. The complete urine stem cell culture medium contains basal culture medium, fetal bovine serum at a volume percentage of 5%-20%, epidermal growth factor at a mass-volume concentration of 5-50 ng / mL, platelet-derived growth factor at a mass-volume concentration of 5-50 ng / mL, and basic fibroblast growth factor at a mass-volume concentration of 5-50 ng / mL. Seed the cell suspension into pre-coated culture plates, adding 1-3 mL to each well, and incubate statically in a 37℃, 5% CO2 incubator. Change the medium every 2-4 days.

[0029] S6. Subculture: When the primary cells reach 70%-90% confluence, subculture them. Discard the old culture medium, gently wash the cells once with phosphate buffer, add 0.05%-0.25% trypsin-EDTA solution, and digest at 37°C for 1-10 minutes. Observe under a microscope that the cells become rounded and the gaps increase. Then add complete culture medium to stop digestion, gently pipette to detach the cells, transfer the cell suspension to a centrifuge tube, centrifuge at 200-600g for 3-8 minutes, discard the supernatant, resuspend the cells in fresh complete culture medium, and seed them into new culture plates at an appropriate ratio for continued culture.

[0030] Example 1 A method for extracting and culturing urinary stem cells, comprising the following steps: S1. Coating culture dishes: Coat 6-well plates with 0.1% gelatin solution, adding 1.5 mL to each well, and place them in a carbon dioxide incubator at 37°C for 1 hour to complete the coating.

[0031] S2. Urine collection and pretreatment: Under aseptic conditions, collect 200 mL of clean midstream urine from healthy volunteers into a sterile container, add 5 mL of penicillin-streptomycin solution, and mix gently.

[0032] S3. Initial centrifugation: Aliquot the urine into 50mL sterile centrifuge tubes and centrifuge at 400g for 10 minutes at room temperature. After centrifugation, carefully transfer the centrifuge tubes to a biosafety cabinet. Use a pipette to aspirate the supernatant, leaving about 2mL of liquid at the bottom of each tube. Gently pipette to resuspend the cell pellet and combine the pellets from the same urine sample into one tube.

[0033] S4. Washing: Add 20 mL of sterile phosphate buffer to the combined centrifuge tubes, gently pipette to mix, centrifuge at 400 g for 6 minutes, discard the supernatant, and repeat the washing step once.

[0034] S5. Primary Culture: The precipitate of every 100 mL initial urine volume is resuspended in 2 mL of urine stem cell complete culture medium. The formula of the urine stem cell complete culture medium is 450 mL mesenchymal stem cell basal culture medium, 50 mL fetal bovine serum, epidermal growth factor at a concentration of 10 ng / mL, platelet-derived growth factor at a concentration of 10 ng / mL, and basic fibroblast growth factor at a concentration of 10 ng / mL. The cell suspension is seeded into pre-coated 6-well plates, and culture medium is added to each well to a total volume of 2 mL. The culture plates are placed in a 37°C, 5% CO2 saturated humidity incubator for static culture, and the medium is changed every 3 days.

[0035] S6. Subculture: When the primary cells reach 70%-80% confluence, subculture them. Discard the old culture medium, gently wash the cells once with 2 mL of phosphate buffer, add 1 mL of 0.25% trypsin-EDTA solution, and digest at 37°C for 2-3 minutes. Observe under a microscope that the cells become rounded and the gaps between them increase. Then add 2 mL of complete culture medium to stop the digestion. Gently pipette to detach the cells, transfer the cell suspension to a centrifuge tube, centrifuge at 400g for 5 minutes, discard the supernatant, resuspend the cells in fresh complete culture medium, and seed them into a new culture plate at a ratio of 1:2 for continued culture.

[0036] Example 2 A method for extracting and culturing urine-derived stem cells, comprising the following steps: S1. Coating culture dishes: Coat 6-well plates with 0.05% gelatin solution, adding 1 mL to each well, and place them in a carbon dioxide incubator at 37°C for 30 minutes to complete the coating.

[0037] S2. Urine collection and pretreatment: Under aseptic conditions, collect 100 mL of clean midstream urine from healthy volunteers into a sterile container, add 1.5 mL of penicillin-streptomycin solution, and mix gently.

[0038] S3. Initial centrifugation: Aliquot the urine into 50mL sterile centrifuge tubes and centrifuge at 200g for 15 minutes at room temperature. After centrifugation, carefully transfer the centrifuge tubes to a biosafety cabinet. Use a pipette to aspirate the supernatant, leaving about 2mL of liquid at the bottom of each tube. Gently pipette to resuspend the cell pellet and combine the pellets from the same urine sample into one tube.

[0039] S4. Washing: Add 10 mL of sterile phosphate buffer to the combined centrifuge tubes, gently mix by pipetting, centrifuge at 200 g for 10 minutes, discard the supernatant, and repeat this washing step once.

[0040] S5. Primary Culture: Resuspend the precipitate of every 100 mL initial urine volume in 1 mL of complete urine stem cell culture medium. The complete urine stem cell culture medium is formulated as follows: DMEM / F12 basal medium, 5% fetal bovine serum (FBS), 5 ng / mL epidermal growth factor (EGFR), 5 ng / mL platelet-derived growth factor (PDGF), and 5 ng / mL basic fibroblast growth factor (BGF). Seed the cell suspension into pre-coated 6-well plates, add culture medium to each well to a total volume of 1 mL, and place the culture plates in a 37°C, 5% CO2 saturated humidity incubator for static culture. Change the medium every 4 days.

[0041] S6. Subculture: When the primary cells reach 90% confluence, subculture them. Discard the old culture medium, gently wash the cells once with 2 mL of phosphate buffer, add 1 mL of 0.05% trypsin-EDTA solution, and digest at 37°C for 5 minutes. Observe under a microscope that the cells become rounded and the gaps between them increase. Then add 2 mL of complete culture medium to stop the digestion. Gently pipette to detach the cells, transfer the cell suspension to a centrifuge tube, centrifuge at 200g for 8 minutes, discard the supernatant, resuspend the cells in fresh complete culture medium, and seed them into a new culture plate at a ratio of 1:2 for continued culture.

[0042] Example 3 A method for extracting and culturing urine-derived stem cells, comprising the following steps: S1. Coating culture dishes: Coat 6-well plates with 0.2% gelatin solution, adding 2 mL to each well, and place them in a carbon dioxide incubator at 37°C for 2 hours to complete the coating.

[0043] S2. Urine collection and pretreatment: Under aseptic conditions, collect 300 mL of clean midstream urine from healthy volunteers into a sterile container, add 6 mL of penicillin-streptomycin solution, and mix gently.

[0044] S3. Initial centrifugation: Aliquot the urine into 50mL sterile centrifuge tubes and centrifuge at 600g for 5 minutes at room temperature. After centrifugation, carefully transfer the centrifuge tubes to a biosafety cabinet. Use a pipette to aspirate the supernatant, leaving about 2mL of liquid at the bottom of each tube. Gently pipette to resuspend the cell pellet and combine the pellets from the same urine sample into one tube.

[0045] S4. Washing: Add 30 mL of sterile phosphate buffer to the combined centrifuge tubes, gently pipette to mix, centrifuge at 600 g for 5 minutes, discard the supernatant, and repeat this washing step three times.

[0046] S5. Primary Culture: The precipitate of every 100 mL initial urine volume is resuspended in 4 mL of urine stem cell complete culture medium. The formula of the urine stem cell complete culture medium is mesenchymal stem cell basal culture medium, fetal bovine serum at a volume percentage of 20%, epidermal growth factor at a mass / volume concentration of 50 ng / mL, platelet-derived growth factor at a mass / volume concentration of 50 ng / mL, and basic fibroblast growth factor at a mass / volume concentration of 50 ng / mL. The cell suspension is seeded into pre-coated 6-well plates, and culture medium is added to each well to a total volume of 3 mL. The culture plates are placed in a 37°C, 5% CO2 saturated humidity incubator for static culture, and the medium is changed every 2 days.

[0047] S6. Subculture: When the primary cells reach 70% confluence, subculture them. Discard the old culture medium, gently wash the cells once with 2 mL of phosphate buffer, add 1 mL of 0.25% trypsin-EDTA solution, and digest at 37°C for 1 minute. Observe under a microscope that the cells become rounded and the gaps increase. Then add 2 mL of complete culture medium to stop the digestion. Gently pipette to detach the cells, transfer the cell suspension to a centrifuge tube, centrifuge at 600g for 3 minutes, discard the supernatant, resuspend the cells in fresh complete culture medium, and seed them into a new culture plate at a ratio of 1:2 for continued culture.

[0048] Comparative Example 1 The difference from Example 1 is that in step S1, gelatin coating is not performed, and the cell suspension is directly seeded into an untreated 6-well plate.

[0049] Comparative Example 2 The difference from Example 1 is that the centrifugal force in all centrifugation steps was reduced to 100g and the centrifugation time was adjusted to 20 minutes.

[0050] Comparative Example 3 The difference from Example 1 is that the centrifugal force in all centrifugation steps is increased to 800g and the centrifugation time is adjusted to 3 minutes.

[0051] Comparative Example 4 The difference from Example 1 is that washing is not performed in step S4, and primary inoculation is performed directly after centrifugation.

[0052] Comparative Example 5 The difference from Example 1 is that the washing is performed 5 times in step S4.

[0053] Comparative Example 6 The difference from Example 1 is that fetal bovine serum is not added in step S5.

[0054] Comparative Example 7 The difference from Example 1 is that the complete culture medium in step S5 does not contain epidermal growth factor (EGF), but only PDGF and bFGF.

[0055] Comparative Example 8 The difference from Example 1 is that platelet-derived growth factor (PDGF) is not added to the complete culture medium in step S5, but only EGF and bFGF are added.

[0056] Comparative Example 9 The difference from Example 1 is that basic fibroblast growth factor (bFGF) is not added to the complete culture medium in step S5, only EGF and PDGF are added.

[0057] Comparative Example 10 The difference from Example 1 is that in the complete culture medium of step S5, the concentrations of EGF, PDGF and bFGF are all 1 ng / mL.

[0058] Comparative Example 11 The difference from Example 1 is that in the complete culture medium of step S5, the concentrations of EGF, PDGF and bFGF are all 100 ng / mL.

[0059] Comparative Example 12 The difference from Example 1 is that in step S5, conventional DMEM medium is used instead of the mesenchymal stem cell-specific medium, while the amount of fetal bovine serum and growth factors added remains unchanged.

[0060] Comparative Example 13 The difference from Example 1 is that in step S5, 0.5 mL of complete culture medium was used to resuspend every 100 mL of urine sediment, resulting in an excessively high inoculation density.

[0061] Comparative Example 14 The difference from Example 1 is that in step S5, 8 mL of complete culture medium was used to resuspend every 100 mL of urine sediment, resulting in an excessively low inoculation density.

[0062] Comparative Example 15 The difference from Example 1 is that the cells were passaged when they grew to be completely covered and reached 100% confluence.

[0063] Comparative Example 16 The difference from Example 1 is that the cells were passaged when they reached 30% confluence.

[0064] Comparative Example 17 The difference from Example 1 is that 0.25% trypsin was used for digestion for 10 minutes during passaging.

[0065] Comparative Example 18 The difference from Example 1 is that trypsin was not used during passage; cells were directly scraped off with a cell scraper.

[0066] The detection methods are shown in Table 1: Table 1 Detection Methods ; Table 2 Performance data for examples and comparative examples ; Note: In Comparative Example 6, all cells died within 3 days due to serum-free culture, making subsequent testing impossible; +++ indicates strong differentiation ability, ++ indicates moderate differentiation ability, and + indicates weak differentiation ability. The isolated and cultured urinary stem cells were identified and their performance was tested. The results shown in the attached figures are typical representatives of Example 1.

[0067] Cell morphology observation: The growth and cell morphology of primary and passaged cells were observed under an inverted microscope; the cell morphology of primary (P0) urinary stem cells isolated and cultured in Example 1 of this invention is shown in the figure below. Figure 1 As shown, the cell morphology diagram of the first generation (P1) is as follows. Figure 2 As shown, the cell morphology diagram of the second generation (P2) is as follows. Figure 3 As shown in Table 2, the time of the first appearance of adherent clones in primary cells was recorded.

[0068] Multipotent differentiation potential identification: P2 generation cells were used for osteogenic and adipogenic induction differentiation. After 21 days of osteogenic induction, Alizarin Red staining was performed, revealing numerous mineralized nodules in the extracellular matrix. The identification results, represented by Example 1, are shown in the figure below. Figure 4 As shown in the figure. Oil Red O staining was performed 11 days after adipogenic induction, and the results showed that a large number of red lipid droplets were formed in the cells. The identification results, represented by Example 1, are shown in the figure below. Figure 5 As shown in Table 2, the osteogenic / adipogenic differentiation capacity ratings for each embodiment and comparative example are presented.

[0069] Flow cytometry identification of cell surface markers: P2 generation cells were subjected to flow cytometry analysis. The results showed that the cells highly expressed mesenchymal stem cell markers CD73, CD90, and CD105, with positive rates all above 95%; while the expression of hematopoietic markers CD34 and CD45 was negative. Example 1 is a representative example; the flow cytometry identification chromatogram of the urinary stem cell surface marker CD73 is shown below. Figure 6 As shown, the flow cytometry identification diagram of CD90 is as follows. Figure 7 As shown, the flow cytometry identification diagram of CD105 is as follows. Figure 8 As shown in Figure 9, the flow cytometry identification pattern of CD45 is shown in Figure 9, and the flow cytometry identification pattern of CD34 is shown in Figure 9. Figure 10 As shown in Table 2, the positive rate data for flow cytometry identification of each embodiment and comparative example are presented in Table 2.

[0070] Immunofluorescence identification: P2 generation cells were subjected to immunofluorescence staining. The results showed that PAX2 and SOX9 were expressed in the cell nuclei, suggesting that they may have characteristics of the renal lineage. As exemplified by Example 1, the immunofluorescence detection image of PAX2 expression in urinary stem cells is shown below. Figure 11 As shown, the immunofluorescence assay of SOX9 expression in urine-derived stem cells (cell nuclei counterstained with DAPI) is shown below. Figure 12 As shown.

[0071] The background of this invention reveals a key challenge commonly encountered in existing methods for isolating and culturing urinary stem cells: urine contains complex cellular components, the target stem cell content is extremely low, and the culture environment is demanding. Conventional methods struggle to simultaneously achieve efficient primary adhesion, stable in vitro expansion, and maintenance of stem cell characteristics, resulting in low isolation efficiency and unstable culture success rates, thus limiting their widespread application in regenerative medicine. The embodiments of this invention, through the construction of a complete culture medium comprising gelatin coating pretreatment, a specific combination of growth factors, and a fully integrated synergistic culture system involving stepwise centrifugation washing and precise passage timing, successfully achieve efficient isolation, rapid expansion, and maintenance of stem cell characteristics in urinary stem cells. Its theoretical advantages lie in the fact that gelatin coating treatment physically simulates the extracellular matrix in vivo, creating a suitable substrate for the initial adhesion of rare stem cells; the precise combination of three growth factors, EGF, PDGF, and bFGF, exerts a synergistic effect in promoting mitosis, chemotaxis and migration, and maintaining stemness, respectively, simultaneously activating multiple key pathways that promote cell survival and proliferation at the level of cell signal transduction; and the fine optimization of process parameters such as centrifugation force control, number of washes, and passage timing minimizes the risk of cell damage and loss during operation, providing a continuous and stable physicochemical environment for cell growth, thus systematically solving the problem of low isolation and culture efficiency caused by factors such as difficulty in adhesion, insufficient nutrition, and operational damage in traditional methods.

[0072] Compared to the examples, none of the comparative examples achieved the aforementioned synergistic effect due to the absence or deviation from any key technical feature. Comparative Example 1 omitted the gelatin coating treatment, resulting in a lack of a suitable extracellular matrix environment on the culture surface. This made it difficult for the already low-content urine-derived stem cells to adhere, significantly reducing colony formation efficiency and consequently affecting the total number of cells obtained from subsequent expansion and the maintenance of stemness. Comparative Examples 2 and 3 used excessively low or high centrifugation forces, respectively. Insufficient centrifugation forces led to incomplete cell sedimentation and cell loss, while excessive forces caused irreversible mechanical damage to the cells. Both compromised the foundation for effectively recovering live cells from urine samples. Comparative Examples 4 and 5 used insufficient or excessive washing, respectively. Insufficient washing resulted in metabolic waste and residual antibiotics in the urine, continuously inhibiting primary culture; excessive washing caused unnecessary cell loss. Neither approach achieved a balance between purification level and cell retention. Comparative Example 6 omitted fetal bovine serum, leaving the culture medium lacking the essential nutrients necessary for cell growth, leading to the death of all cells within three days, confirming the fundamental role of serum in supporting cell survival. Comparative Examples 7, 8, and 9 lacked one of EGF, PDGF, or bFGF, respectively, disrupting the synergistic effect of the three growth factors. Regardless of the specific factor missing, all three resulted in slowed cell proliferation, decreased colony formation efficiency, and poor stemness maintenance, confirming that their synergistic effect is indispensable. Comparative Examples 10 and 11 used excessively low or high concentrations of growth factors, respectively. Too low a concentration failed to fully activate the proliferative signaling pathway, while too high a concentration may have triggered receptor saturation or non-specific activation, neither achieving optimal culture results. Comparative Example 12 used conventional DMEM basal medium instead of the mesenchymal stem cell-specific medium. Although the growth factor combination was retained, the difference in the basal nutritional environment still resulted in culture effects inferior to the present invention. Comparative Examples 13 and 14 used excessively high or low seeding densities, respectively. Excessively high densities led to cell-cell contact inhibition and nutrient competition, while excessively low densities resulted in a lack of necessary paracrine support between cells, both detrimental to primary cell survival and colony formation. Comparative Examples 15 and 16, using excessively late or early passaging, resulted in contact inhibition and senescence after excessive cell fusion, while premature passaging led to insufficient cell numbers and suboptimal cell condition, both affecting subsequent expansion capacity. Comparative Example 17, with its excessively long digestion time, caused excessive degradation of cell membrane surface proteins, impairing cell viability; Comparative Example 18, using physical scraping instead of enzyme digestion, caused severe mechanical damage to the cells, both disrupting cell integrity during passaging. These comparative examples collectively demonstrate that the technical solution of this invention is an interconnected and precisely coordinated organic whole. Each of its technical features is indispensable for simultaneously achieving efficient isolation, stable expansion, and maintenance of stem cell characteristics from urinary stem cells, thus systematically resolving long-standing contradictions in the prior art.

Claims

1. A method for extracting and culturing urinary stem cells, characterized in that, Includes the following steps: S1. Coating culture dishes: Coat cell culture plates with gelatin solution and place them in a carbon dioxide incubator to complete the coating; S2. Urine collection and pretreatment: Under aseptic conditions, clean midstream urine from healthy individuals is collected and antibiotics are added. S3. Initial centrifugation: Centrifuge the urine in separate containers, discard the supernatant, and retain the cell precipitate; S4. Washing: Add phosphate buffer to the cell pellet to resuspend the cells, then centrifuge and wash again to remove impurities; S5. Primary culture: The cell pellet is resuspended in urine stem cell complete culture medium, seeded into a coated culture plate, placed in an incubator for primary culture, and the medium is changed regularly; the urine stem cell complete culture medium contains basal culture medium, fetal bovine serum, epidermal growth factor, platelet-derived growth factor and basic fibroblast growth factor. S6. Subculture: After the primary cells have grown to 70%-90% confluence, they are digested with trypsin and then passaged for expansion.

2. The extraction and cultivation method according to claim 1, characterized in that, The mass-volume concentration of the gelatin solution in step S1 is 0.05%-0.2%, and the coating condition is standing at 37°C for more than 30 minutes.

3. The extraction and cultivation method according to claim 1, characterized in that, In step S2, the urine collection volume is 100-300 mL, and the antibiotic is a penicillin-streptomycin mixture, with an addition volume of 1.5%-2.5% of the urine volume.

4. The extraction and cultivation method according to claim 1, characterized in that, The centrifugal force in steps S3 and S4 is 200-600g, and the centrifugation time is 5-15min.

5. The extraction and cultivation method according to claim 1, characterized in that, The washing process in step S4 involves adding 10-30 mL of phosphate buffer, centrifuging at 200-600 g for 5-10 min, and repeating the washing process 1-3 times.

6. The extraction and cultivation method according to claim 1, characterized in that, In step S5, the volume percentage of fetal bovine serum in the complete urine stem cell culture medium is 5%-20%, the mass-volume concentration of epidermal growth factor is 5-50 ng / mL, the mass-volume concentration of platelet-derived growth factor is 5-50 ng / mL, and the mass-volume concentration of basic fibroblast growth factor is 5-50 ng / mL.

7. The extraction and cultivation method according to claim 6, characterized in that, The formula for the complete urine stem cell culture medium is as follows: 450 mL of mesenchymal stem cell culture medium or DMEM / F12 culture medium, 50 mL of fetal bovine serum, 10 ng / mL of epidermal growth factor, 10 ng / mL of platelet-derived growth factor, and 10 ng / mL of basic fibroblast growth factor.

8. The extraction and cultivation method according to claim 1, characterized in that, In step S5, the precipitate of every 100 mL initial urine volume is resuspended in 1-4 mL of urine stem cell complete culture medium and seeded into a culture plate with 1-3 mL added to each well. The medium is changed every 2-4 days.

9. The extraction and cultivation method according to claim 1, characterized in that, The conditions for passage culture described in step S6 are as follows: when the primary cells grow to 70%-90% confluence, digest them with trypsin at a concentration of 0.05%-0.25% at 37°C for 1-10 minutes, and centrifuge them at 200-600g for 3-8 minutes.

10. A type of urine-derived stem cell, characterized in that, Obtained by the extraction and cultivation method according to any one of claims 1-9.

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