Culture method of human urine-derived stem cells and application thereof

Abstract

The application belongs to the field of cell culture. The application provides a culture method of human urine-derived stem cells and application thereof. A culture medium of the human urine-derived stem cells comprises DMEM culture medium, F-12 culture medium, FBS, penicillin-streptomycin solution and L-glutamine. In the culture medium, the volume ratio of the DMEM culture medium to the F-12 culture medium is 1:0.3-2.5, the volume percentage of the FBS is 9-12%, the volume percentage of the penicillin-streptomycin solution is 0.5-1.5%, and the volume concentration of the L-glutamine is 0.5-1.5 mmol / L. The culture method of the application can obtain human urine-derived stem cells with a large number and good activity, and effectively improves the in-vitro proliferation rate of the human urine-derived stem cells.

Description

Technical Field

[0001] This invention belongs to the field of cell biology, specifically relating to a method for culturing human urine-derived stem cells and its application. Background Technology

[0002] Human urinary stem cells (hUSCs) are a type of stem cell extracted from urine. They possess multi-lineage differentiation and self-renewal capabilities, and can differentiate into various tissues such as smooth muscle, endothelium, epithelium, cartilage, and bone. Due to their numerous advantages, including non-invasive extraction, direct access to urine, and lack of ethical restrictions, they are frequently used in various tissue engineering and cell therapies, such as bladder, urethra, and ureter repair and reconstruction. Furthermore, injection therapy with urinary stem cells shows promising therapeutic effects for urinary system diseases such as erectile dysfunction (ED) in men and stress urinary incontinence in women.

[0003] Current cell culture media often employ a single, "universal formula," meaning they are based on the basic nutritional needs of most cell types, without addressing the specific requirement of "cell proliferation rate." A key drawback is the limited selection of components, relying heavily on traditional, classic formulas (such as DMEM, RPMI-1640, and F12) for cell culture. Existing methods for culturing human urine-derived stem cells require further improvement in terms of the number and viability of cultured stem cells. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for culturing human urine-derived stem cells and its application. The method of this invention yields a large number of human urine-derived stem cells with good activity, effectively improving the in vitro proliferation rate of human urine-derived stem cells.

[0005] The purpose of this invention is to provide a method for culturing human urine-derived stem cells, wherein the culture medium for human urine-derived stem cells includes DMEM medium, F-12 medium, FBS, penicillin-streptomycin solution and L-glutamine.

[0006] In the culture medium, the volume ratio of DMEM medium to F-12 medium is 1:0.3-2.5, the volume percentage of FBS is 9-12%, the volume percentage of penicillin-streptomycin solution is 0.5-1.5%, and the volume concentration of L-glutamine is 0.5-1.5 mmol / L. In the penicillin-streptomycin solution, the concentration of penicillin is 5000-20000 U / mL, and the concentration of streptomycin is 5-20 mg / mL.

[0007] Under the culture medium of the above-mentioned human urine-derived stem cells, the different components exerted a synergistic effect on proliferation, which improved the in vitro proliferation rate of human urine-derived stem cells, shortened the cell culture cycle, reduced the unit cell production cost, and has industrialization potential.

[0008] Preferably, the volume ratio of DMEM medium to F-12 medium is 1:1.

[0009] Furthermore, the above-mentioned culture medium also includes the following components in varying amounts: Recombinant human insulin 12-18 μg / mL; Transferrin 7.5-15 μg / mL; Basic fibroblast growth factor (bFGF) 1.6-2.5 ng / mL.

[0010] Preferably, in the above-mentioned culture media, the DMEM culture medium is from Yuanye Biotechnology Co., Ltd., catalog number R28175, the F-12 culture medium is from Gibco, catalog number 21127022, the recombinant human insulin is from SolarBio, catalog number I8830, and the basic fibroblast growth factor (bFGF) is from MedChemExpress, catalog number HY-P5321.

[0011] Furthermore, the above-mentioned method for culturing human urine-derived stem cells includes the following steps:

[0012] (1) Collect urine, centrifuge to remove supernatant, wash with washing buffer, centrifuge to remove supernatant, resuspend in culture and inoculate into culture dish;

[0013] (2) After culturing in a constant temperature incubator for 24-72 hours, change the medium to remove impurity cells. Then change the culture medium every 2-3 days and add the above-mentioned human urine-derived stem cell culture medium until the cell density reaches 80%-90% to complete the culture and obtain human urine-derived stem cells.

[0014] The culture method of this invention yields a large number of human urine-derived stem cells with good activity.

[0015] Furthermore, after culturing human urine-derived stem cells to a density of 80%–90% confluence, they are digested with trypsin and cryopreserved by adding cryopreservation solution.

[0016] Preferably, the cryopreservation solution comprises DMSO, proliferation medium and FBS, wherein the volume ratio of DMSO, proliferation medium and FBS is 1:4:5.

[0017] Furthermore, in step (1), the inoculation density is 1-3 × 10⁻³. 5 per ml.

[0018] Furthermore, regarding the source of the urine sample, the sampler should drink water 1-3 hours in advance, collect midstream urine, and the urine should be stored for no more than 1 hour.

[0019] The present invention also aims to provide an application of human urine-derived stem cells prepared by the above-mentioned culture method in drug screening, physiological and pathological research, cell therapy and kidney tissue engineering for non-diagnostic and therapeutic purposes.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The method for culturing human urine-derived stem cells of the present invention, as observed by light microscopy, shows that the morphology of human urine-derived stem cells cultured using the method of the present invention is basically consistent with the cell morphology of human urine-derived stem cells. (2) The immunofluorescence identification results of CD44, CD90 and DAPI showed that the human urine-derived stem cells cultured by the method of the present invention had a cell purity of ≥90%, which is excellent and has the characteristics of stem cells; (3) The present invention compared the growth efficiency of human urine-derived stem cells using other culture methods under the same culture environment (37°C, 5% CO2) with that of human urine-derived stem cells cultured using human urine-derived stem cell proliferation medium under the same culture environment. It can be concluded that under the same culture environment, human urine-derived stem cells cultured using the human urine-derived stem cell proliferation medium of the present invention have a faster growth rate and higher cell purity. (4) It achieves efficient expansion, effectively improves the in vitro proliferation rate of human urine-derived stem cells, and has a simple and low-cost culture medium and cryopreservation solution composition. It has ideal culture effect and broad application prospects. Attached Figure Description

[0021] Figure 1 Cell morphology diagram of P0 generation human urine-derived stem cells after Day 4 of culture;

[0022] Figure 2 Cell morphology diagram of P0 generation human urine-derived stem cells after Day 9 of culture;

[0023] Figure 3 Cell morphology diagram of P1 generation human urine-derived stem cells cultured for Day 11;

[0024] Figure 4 Cell morphology diagram of P1 generation human urine-derived stem cells after Day 14 of culture;

[0025] Figure 5 Cell morphology diagram of human urine-derived stem cells cultured to generation P3;

[0026] Figure 6 The image shows the cell morphology of human urine-derived stem cells cultured to passage P5.

[0027] Figure 7 Immunofluorescence identification results of CD44, CD90, and DAPI in P3 generation human urinary stem cells;

[0028] Figure 8 This figure shows a comparison of cell proliferation efficiency in several culture media. Detailed Implementation

[0029] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0030] This invention aims to establish a relatively ideal human urine-derived stem cell culture system by isolating, purifying, and expanding human urine-derived stem cells in vitro, and identifying them through morphological observation (light microscopy) and immunofluorescence, thereby providing a theoretical basis and technical method for the application of human urine-derived stem cells.

[0031] In order to overcome the shortcomings of existing human urine-derived stem cell culture technology, the present invention aims to provide a method for culturing, expanding and cryopreserving human urine-derived stem cells (hUSCs), which effectively improves the in vitro proliferation rate of human urine-derived stem cells.

[0032] By using the aforementioned human urine-derived stem cell proliferation culture medium, the aforementioned human urine-derived stem cell culture operation, and the aforementioned human urine-derived stem cell cryopreservation solution, human urine-derived stem cells can be cultured efficiently and at low cost, resulting in a large number of human urine-derived stem cells with good activity.

[0033] To achieve the above objectives, the present invention provides the following technical solution: (1) Prepare human urine-derived stem cell proliferation medium, which includes DMEM medium, F-12 medium, FBS, penicillin-streptomycin solution, and L-glutamine; (2) Sterilize the collection vessel by autoclaving in advance, and add 5 ml of penicillin-streptomycin solution to the sterile collection bottle; (3) Instruct the sampler to drink water 2 hours in advance, keep the sampling environment clean and tidy, disinfect the urethral opening and the outside twice, and collect midstream urine; transport and store at 4℃, and the storage time shall not exceed 1 hour; (4) After spraying the collection bottle with 75% alcohol, place it in a biosafety cabinet, aliquot the urine, centrifuge at 2000 rpm for 10 min, and discard the supernatant; resuspend the cells in PBS, wash them by pipetting, centrifuge at 2000 rpm for 10 min, discard the supernatant, resuspend the cells in complete culture medium, and inoculate them into culture dishes at a density of 2 × 10⁻⁶. 5 / ml; (5) After culturing in a constant temperature incubator at 37℃ and 5% CO2 for 48 h, change the medium to remove impurity cells, add human urine-derived stem cell proliferation medium, and then change the medium every 3 days until the cell density reaches 80% to complete the culture. (6) After digesting the cells with 0.25% trypsin-EDTA solution, the digested cells are resuspended in cryopreservation solution and then stored in liquid nitrogen at -80°C. Cell identification can be performed after passage to P3. The culture method provided by this invention yields a large number of human urine-derived stem cells with good activity.

[0034] Preferably, the method for preparing the human urine-derived stem cell proliferation culture medium includes the following in step (1): DMEM culture medium, F-12 culture medium, FBS, penicillin-streptomycin solution, and L-glutamine. The DMEM culture medium and F-12 culture medium are mixed in proportion, 10% FBS is added to the final volume, 1% penicillin-streptomycin solution is added to the final volume (penicillin-streptomycin solution components: penicillin 10000 U / mL, streptomycin 10 mg / mL), and L-glutamine with a final concentration of 1 mmol / L is added and mixed thoroughly to obtain the human urine-derived stem cell proliferation culture medium.

[0035] The above-mentioned culture medium also includes the following components in appropriate amounts: Recombinant human insulin 12-18 μg / mL; Transferrin 7.5-15 μg / mL; Basic fibroblast growth factor (bFGF) 1.6-2.5 ng / mL.

[0036] Preferably, in the method for culturing human urine-derived stem cells, step (5) involves changing the medium every 3 days after the step of changing the medium to remove impurity cells.

[0037] Preferably, in the method for culturing human urine-derived stem cells, the cryopreservation solution in step (6) is a mixture of DMSO, proliferation culture medium, and FBS in a certain proportion.

[0038] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention provides an improved and efficient method for culturing, expanding, and cryopreserving human urine-derived stem cells (hUSCs): Adult male urine is aseptically collected and expanded using a proliferation medium containing DMEM, F-12, FBS, penicillin-streptomycin solution, and L-glutamine. Light microscopic observation results show that the morphology of the hUSCs cultured using the method and proliferation medium of this invention is basically consistent with that of human urine-derived stem cells. Immunofluorescence identification results using CD44, CD90, and DAPI show that the hUSCs cultured using the method and proliferation medium of this invention have a cell purity ≥90%, exhibiting excellent purity and stem cell characteristics. The hUSC culture method used in this invention provides highly efficient expansion, and the formulation of the proliferation medium and cryopreservation solution is simple, resulting in ideal culture effects and broad application prospects.

[0039] This invention compares the growth efficiency of human urine-derived stem cells cultured using other culture methods under the same culture environment (37°C, 5% CO2). Compared with human urine-derived stem cells cultured using human urine-derived stem cell proliferation medium under the same culture environment, it can be concluded that, under the same culture environment, human urine-derived stem cells cultured using human urine-derived stem cell proliferation medium have a faster growth rate and higher cell purity.

[0040] Material information used in this invention:

[0041] Example 1

[0042] An improved and efficient method for culturing, expanding, and cryopreserving human urine-derived stem cells (hUSCs) includes the following steps:

[0043] (1) Prepare a human urine-derived stem cell proliferation medium, which includes DMEM medium, F-12 medium, FBS, penicillin-streptomycin solution, and L-glutamine.

[0044] DMEM and F-12 media were mixed at volume ratios (DMEM and F-12 media were divided into three groups with volume ratios of 7:3, 5:5, and 3:7). 10% FBS was added to the final volume, followed by 1% penicillin-streptomycin solution, then L-glutamine at a final concentration of 1 mmol / L. 12-18 μg / mL of recombinant human insulin, 7.5-15 μg / mL of transferrin, and 1.6-2.5 ng / mL of basic fibroblast growth factor (bFGF) were added. The mixture was thoroughly mixed to obtain the human urine-derived stem cell proliferation medium.

[0045] (2) Sterilize the collection vessel by autoclaving in advance, and add 5 ml of penicillin-streptomycin solution to the sterile collection bottle;

[0046] (3) Instruct the sampler to drink water 2 hours in advance, keep the sampling environment clean and tidy, disinfect the urethral opening and the outside twice, and collect midstream urine; transport and store at 4℃, and the storage time shall not exceed 1 hour;

[0047] (4) Spray the collection bottle with 75% alcohol and place it in a biosafety cabinet. Aliquot the urine and centrifuge at 2000 rpm for 10 min. Remove the supernatant. Resuspend the cells in PBS, wash them by pipetting, centrifuge at 2000 rpm for 10 min, remove the supernatant, and resuspend them in complete culture medium. Inoculate the cells into culture dishes at a density of 2 × 10⁻⁶ cells / mL. 5 / ml;

[0048] (5) After culturing in a constant temperature incubator at 37℃ and 5% CO2 for 48 hours, change the medium to remove the impurity cells, add human urine-derived stem cell proliferation medium, and then change the medium every 3 days until the cell density reaches 80% to complete the culture.

[0049] (6) After digesting the cells with 0.25% trypsin-EDTA solution, the digested cells were resuspended in cryopreservation solution and then frozen in liquid nitrogen at -80°C and passaged to P3 (see Figure 5 Cell identification can be performed using a cryopreservation solution, which is a mixture of DMSO, proliferation medium, and FBS in a volume ratio of 1:4:5.

[0050] The proliferation efficiency of human urine-derived stem cells was compared among three groups (DMEM medium to F-12 medium volume ratios of 7:3, 5:5, and 3:7, respectively). The results are as follows: Figure 8 As shown, the highest proliferation efficiency was achieved when the volume ratio of DMEM medium to F-12 medium was 5:5.

[0051] Example 2: Light microscopic observation and immunofluorescence identification of human urinary stem cells

[0052] 1. Observation under a light microscope

[0053] Cell morphology of P0 generation human urine-derived stem cells cultured for Day 4 is shown in the figure below. Figure 1 As shown;

[0054] Cell morphology of P0 generation human urine-derived stem cells cultured for Day 9 is shown in the figure below. Figure 2 As shown;

[0055] Cell morphology of P1 generation human urine-derived stem cells cultured for Day 11 is shown in the figure. Figure 3 As shown;

[0056] Cell morphology of P1 generation human urine-derived stem cells cultured for 14 days is shown in the figure below. Figure 4As shown;

[0057] Cell morphology diagram of human urine-derived stem cells cultured to passage P3 is shown below. Figure 5 As shown;

[0058] Cell morphology diagram of human urine-derived stem cells cultured to passage P5 is shown below. Figure 6 As shown.

[0059] Immunofluorescence identification results of CD44 and CD90 in human urinary stem cells cultured to passage P3 are as follows: Figure 7 As shown.

[0060] The comparison results of cell proliferation efficiency of several culture media are shown in the figure below. Figure 8 As shown.

[0061] The results of light microscopy observation show that the morphology of human urine-derived stem cells cultured using the culture method and proliferation culture medium used in this invention is basically consistent with the cell morphology of human urine-derived stem cells.

[0062] 2. Immunofluorescence identification

[0063] The identification method includes the following steps: human urine-derived stem cells are divided into 2×10 groups. 4 The cells were seeded at a density of / ml in 6-well plates pre-placed with coverslips. After the cells reached 80%–90% confluence, 4% paraformaldehyde was added for fixation in the dark for 5 minutes. The cells were washed three times with PBS, and CD90 and CD44 fluorescent primary antibodies were added. The cells were incubated overnight at 4°C. The cells were washed three times with PBS, and DAPI was added for incubation in the dark for 10 minutes. After washing three times with PBS, one drop of anti-fluorescence quencher was added to a glass slide. The slide was removed, and the cell-containing side was placed face down on the glass slide. The slide was then observed under a fluorescence microscope and stored at 4°C in the dark.

[0064] Immunofluorescence identification results of P3 generation human urinary stem cells for CD44, CD90, and DAPI are as follows: Figure 7 As shown, the results indicate that the human urine-derived stem cells cultured using the culture method and proliferation medium of this invention have a cell purity of ≥90%, which is excellent and possesses the characteristics of stem cells.

[0065] The human urine-derived stem cell culture method used in this invention can achieve efficient expansion. The formulation of the proliferation medium and cryopreservation solution used is simple, the culture effect is ideal, and it has broad application prospects.

[0066] The above embodiments are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can make various changes or modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions should also fall within the scope of the invention and should be defined by the claims.

Claims

1. A method for culturing human urine-derived stem cells, characterized in that, The culture medium for the human urinary stem cells includes DMEM medium, F-12 medium, FBS, penicillin-streptomycin solution, and L-glutamine. In the culture medium, the volume ratio of DMEM medium to F-12 medium is 1:0.3-2.5, the volume percentage of FBS is 9-12%, the volume percentage of penicillin-streptomycin solution is 0.5-1.5%, and the volume concentration of L-glutamine is 0.5-1.5 mmol / L. In the penicillin-streptomycin solution, the concentration of penicillin is 5000-20000 U / mL, and the concentration of streptomycin is 5-20 mg / mL.

2. The cultivation method according to claim 1, characterized in that, In the culture medium, the volume ratio of DMEM medium to F-12 medium is 1:

1.

3. The cultivation method according to claim 1, characterized in that, The culture medium also includes the following components in appropriate amounts: Recombinant human insulin 12-18 μg / mL; Transferrin 7.5-15 μg / mL; Basic fibroblast growth factor (bFGF) 1.6-2.5 ng / mL.

4. The cultivation method according to claim 3, characterized in that, In the culture medium, The DMEM culture medium was sourced from Yuanye Biotechnology Co., Ltd., catalog number R28175. F-12 medium is sourced from Gibco, catalog number 21127022; The recombinant human insulin is sourced from SolarBio, catalog number I8830; Basic fibroblast growth factor (bFGF) is derived from medchemexpress, catalog number HY-P5321.

5. The method for culturing human urine-derived stem cells according to claim 1, characterized in that, Includes the following steps, (1) Collect urine, centrifuge to remove supernatant, wash with washing buffer, centrifuge to remove supernatant, resuspend in culture and inoculate into culture dish; (2) After culturing in a constant temperature incubator for 24-72 hours, change the medium to remove impurity cells. Then change the culture medium every 2-3 days and add the culture medium of the human urine-derived stem cells until the cell density reaches 80%-90% to complete the culture and obtain human urine-derived stem cells.

6. The cultivation method according to claim 5, characterized in that, After culturing human urine-derived stem cells to a density of 80%–90% confluence, they are digested with trypsin and then cryopreserved with added cryopreservation solution.

7. The cultivation method according to claim 6, characterized in that, The cryopreservation solution comprises DMSO, growth medium, and FBS, wherein the volume ratio of DMSO, growth medium, and FBS is 1:4:

5.

8. The cultivation method according to claim 5, characterized in that, In step (1), the inoculation density is 1-3 × 10⁻³. 5 per ml.

9. The cultivation method according to claim 5, characterized in that, The urine sample was collected by having the sampler drink water 1-3 hours in advance, collecting midstream urine, and storing the urine for no more than 1 hour.

10. The use of human urine-derived stem cells prepared by the culture method according to any one of claims 1-9 in drug screening, physiological and pathological studies, cell therapy and kidney tissue engineering for non-diagnostic and therapeutic purposes.

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