A method for culturing human urine-derived stem cells

Abstract

This invention discloses a method for culturing human urine-derived stem cells, belonging to the field of biomedical technology. The key technical points are: by collecting urine samples from pregnant women, using optimized conditions such as gelatin-coated culture plates, induction of adhesion, passage expansion, immunofluorescence identification, and functional testing to isolate and culture stem cells. Compared with urine-derived stem cells from normal adults, stem cells derived from pregnant women exhibit stronger viability, higher stemness, and faster proliferation capacity. This invention provides a novel and superior cell source for tissue engineering and regenerative medicine.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for culturing human urine-derived stem cells. Background Technology

[0002] Urinary stem cells (USCs) are considered ideal seed cells for tissue engineering and regenerative medicine due to their non-invasive collection method, abundant source, and low immunogenicity. However, most current research uses urinary stem cells from normal adults, which have limited stemness and proliferative capacity. The inventors unexpectedly discovered that stem cells isolated from the urine of pregnant and lactating women exhibit stronger viability, stemness, and proliferative potential than adult-derived USCs.

[0003] Therefore, this invention aims to establish a standardized culture method for activating and amplifying pregnant women-derived USCs for clinical and research applications. Summary of the Invention

[0004] The purpose of this invention is to solve the above-mentioned problems and provide a method for culturing human urine stem cells. By optimizing urine collection, gelatin coating, induction of adhesion, passage expansion, immunofluorescence identification, and proliferation detection, highly active, highly stem, and rapidly proliferating UCs can be obtained.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] This invention provides a method for culturing human urine-derived stem cells, the method comprising the following steps:

[0007] S1: Gelatin-coated culture plates. Add 0.1% gelatin solution at 0.5 mL / well to a 12-well plate, incubate at 37°C for 30 min in a 5% CO2 incubator, discard the gelatin solution and set aside.

[0008] S2: Urine collection and centrifugation. Collect 200 mL of urine from pregnant women, centrifuge at room temperature (400 g, 10 min), discard the supernatant, retain 1 mL of urine precipitate, add 10 mL of DPBS, mix well by pipetting, then centrifuge at 1500 r / min for 10 min, discard the supernatant, and collect the cell pellet.

[0009] S3: Stem cell induction and adherent culture. Take approximately 100 μL of the precipitate and add 1 mL of USCs complete culture medium (Guangzhou Wanlei, 20210701), at a concentration of 2×10⁻⁶. 5 Cells were seeded at a density of 12-well gelatin-coated plates and cultured at 37°C and 5% CO2. The medium was changed for the first time after 48 h to remove non-adherent cells and retain adherent cells. The medium was then changed every 2-3 days thereafter.

[0010] S4: Stem cell passage and expansion. Passage was performed when the cell adhesion area reached 75%-80%. The culture medium was discarded, and 0.25% trypsin (containing EDTA) was added for digestion for 3 min. An equal volume of high-glucose DMEM containing 10% FBS was added to terminate the process. The cells were centrifuged (1000 rpm, 7 min) and transferred to a new culture flask at a 1:3 ratio for further expansion.

[0011] S5: Stem cell collection and pre-experimental treatment. Wash the digested and collected cells once with PBS containing antibiotics, centrifuge (1500 rpm, 7-10 min), discard the supernatant, and resuspend in high glucose medium at the required concentration for animal experiments or subsequent applications.

[0012] S6: Immunofluorescence identification of stem cells (CD90+, CD44+). Cells were stained with CD90 and CD44 antibodies using immunofluorescence staining, and the nuclei were counterstained with DAPI. Observation under an inverted microscope showed that the USCs derived from pregnant women had a CD90⁺ / CD44⁺ phenotype.

[0013] S7: CCK-8 assay was used to detect cell viability and plot growth curves. Cells were loaded at 2~5×10⁶ cells / year. 3 Cells were seeded at a density of 1 cell / well in 96-well plates and cultured continuously for 7 days. The OD value (490 nm) of the cells was detected using a CCK-8 assay kit, and growth curves were plotted. The results showed that the viability and proliferation capacity of USCs derived from pregnant women were significantly higher than those of normal adult USCs.

[0014] The human urinary stem cells are derived from urine samples from pregnant women. Activated urinary stem cells obtained through optimized culture conditions exhibit stronger viability, higher stemness, and faster proliferation capacity, making them ideal seed cells for tissue engineering and regenerative medicine.

[0015] The activated human urine-derived stem cells exhibited high adhesion and survival rates in in vitro culture, quickly forming typical spindle-shaped cell morphology and maintaining stable expression of stem cell markers (CD90 positive, CD44 positive). CCK-8 assays revealed that the growth curve of the activated urine-derived stem cells was significantly superior to that of stem cells derived from normal adult urine, demonstrating their advantage in cell proliferation.

[0016] Compared with existing technologies, the beneficial effects of this solution are:

[0017] This invention innovatively utilizes stem cells derived from the urine of pregnant and postpartum women, significantly improving the viability and expansion capacity of urine-derived stem cells. By optimizing urine collection, gelatin coating, induction of adhesion, and passage culture, the culture process is made more efficient, yielding a larger number of higher-quality cells. This technology reduces culture risks while ensuring cell stability and biosafety; the stem cells obtained from this culture exhibit greater plasticity and application potential, providing a new high-quality cell source and culture technology for tissue repair, regenerative medicine, and related cell research. Attached Figure Description

[0018] Figure 1 Bright-field images showing cell morphology at 7, 9, and 11 days of primary culture for activating human urine-derived stem cells; A: USC primary culture at 7, 9, and 11 days; B: Cell number quantification histogram; C: Cell viability curve; D: Red fluorescence indicates CD90-positive cells, green fluorescence indicates CD44-positive cells, and blue indicates DAPI-stained nuclei; Merge fluorescence merged image; Scale bar = 100 μm (bright-field image), scale bar = 50 μm (fluorescence image); E: Immunofluorescence identification quantification histogram; F: Flow cytometry identification image.

[0019] Figure 2 A comparison of the proliferation of human urine-derived stem cells at different culture time points; the horizontal axis represents different culture time points of urine-derived stem cells, and the vertical axis represents the number of cells; the blue curve represents normal human urine-derived stem cells, and the red curve represents activated human urine-derived stem cells from pregnant women; P0 represents primary tissue culture; P1 represents primary passage to the first generation; P2 represents primary passage to the second generation; and P3 represents primary passage to the third generation.

[0020] Figure 3 This is a comparison of the viability of human urinary stem cells at different culture time points; the horizontal axis represents different culture time points of urinary stem cells, and the vertical axis represents the cell viability OD value; the blue curve represents normal human urinary stem cells, and the red curve represents activated human urinary stem cells after childbirth in pregnant women; P0 represents primary culture; P1 represents primary culture to the first generation; P2 represents primary culture to the second generation; and P3 represents primary culture to the third generation. Detailed Implementation

[0021] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be described in further detail below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0022] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0023] Example 1:

[0024] Isolation and culture of human urine-derived stem cells from pregnant women

[0025] 1. Gelatin-coated culture plates

[0026] Add 0.1% gelatin solution to 0.5 mL / well of a 12-well plate and incubate at 37°C with 5% CO2 for 30 min to complete the coating. Discard the gelatin solution before cell seeding and air dry for later use.

[0027] 2. Sample Source

[0028] The samples were obtained from postpartum women in their recovery period. Urine was collected within 24–72 hours post-surgery, after the subjects resumed spontaneous urination. All subjects had stable vital signs post-surgery and no history of urinary tract infection or antibiotic use. The clean-catch midstream urine method was used, collecting approximately 200 mL of urine in a sterile container. Samples were immediately sent to the laboratory and processed within 4 hours; if delayed, they were briefly stored at 4°C and separated within 24 hours. All postpartum samples were reviewed and approved by the Ethics Committee of Kunming Medical University, and written informed consent was obtained from the subjects.

[0029] 3. Urine collection and treatment

[0030] Collect approximately 200 mL of midstream urine from pregnant or postpartum women, centrifuge at 400 g for 10 min at room temperature. Discard the supernatant, retain 1 mL of urine precipitate at the bottom, add 10 mL of DPBS, mix well by pipetting, and then centrifuge at 1500 rpm for 10 min. Discard the supernatant and collect the cell pellet for later use.

[0031] 4. Stem cell induction and adhesion

[0032] Take approximately 100 μL of the precipitate, resuspend it in 1 mL of complete culture medium for human urine-derived stem cells (USCs) (Guangzhou Wanlei, 20210701), and adjust the concentration by 2 × 10⁻⁶. 5 10 cells / well were seeded into gelatin-coated 12-well plates. The culture plates were incubated at 37°C in a 5% CO2 incubator. The medium was changed for the first time after 48 h, and the non-adherent cells were discarded. The adherent cells were retained and cultured for a further period of time. The medium was then changed every 2–3 days.

[0033] 5. Cell morphology observation and passage

[0034] Under a microscope, when the adherent cells reached 75%–80% confluence, the culture medium was discarded under aseptic conditions. 0.25% trypsin-EDTA (250 μL) was added for digestion for 3 min, followed by the addition of an equal volume of high-glucose DMEM containing 10% FBS and 1% antibiotics to terminate the digestion. The cells were centrifuged at 1000 rpm for 7 min, and the precipitate was collected and passaged at a ratio of 1:2–3.

[0035] like Figure 1 As shown, USCs derived from pregnant women exhibited typical spindle-shaped or rice-grain-like arrangements on days 7, 9, and 11 of primary culture. Figure 1 A) The number of cells increased significantly over time ( ). Figure 1 B), cell viability increases with culture time ( Figure 1 C). Immunofluorescence results showed that CD90 was red fluorescently positive, CD44 was green fluorescently positive, and DAPI showed a clear blue signal on the nucleus. The merged image showed high expression of mesenchymal stem cell markers on the cell surface. Figure 1 D). No significant difference was found in the number of DAPI, CD90, and CD44 positive cells in the quantitative immunofluorescence histogram. This indicates that almost all cells in the detected cell population expressed CD90 and CD44, demonstrating high positivity, good uniformity, and high purity and stability. Figure 1 E). Flow cytometry further confirmed that it was positive for CD90, CD73, CD44, and CD105, but negative for CD34, CD45, CD11B, and HLA-DR. Figure 1 F) indicates that the isolated USCs have a typical mesenchymal stem cell phenotype.

[0036] Example 2:

[0037] Identification and viability testing of human urinary stem cells

[0038] 1. Cell collection

[0039] USCs passaged to generation 3 (P3) were removed and digested aseptically with 0.25% trypsin (T25 flask: 1–2 mL, 37°C for 3–5 min). The reaction was terminated by adding an equal volume of serum-containing culture medium. After centrifugation (1500 rpm, 7–10 min), the cells were washed once with PBS (containing 1% penicillin and antibiotics), centrifuged again to collect the cells, and a cell suspension was prepared for subsequent detection.

[0040] 2. Identification by immunofluorescence staining

[0041] Cells were seeded in 96-well plates and, after adhesion, were washed with PBS, fixed with 4% paraformaldehyde for 15 min, and blocked with 0.3% Triton + 5% sheep serum for 30 min. CD90 (1:100) and CD44 (1:100) antibodies were added, and the plates were incubated overnight at 4°C. The next day, after washing with PBS, secondary antibodies (488 green rabbit antibody and 594 red mouse antibody, diluted 1:200) were added and incubated at 37°C for 1 h. Cells were counterstained with DAPI and observed under a fluorescence microscope. The results showed positive results for CD90 and CD44, confirming the mesenchymal stem cell characteristics.

[0042] 3. Flow cytometry identification

[0043] After digestion and collection of P4 generation cells, they were resuspended in PBS and the concentration was adjusted to 1×10⁻⁶. 6 / 5 μL. Flow cytometry antibodies CD90, CD73, CD44, CD105, CD34, CD45, CD11b, and HLA-DR were added, and the mixture was incubated on ice in the dark for 20 min. After washing with PBS, the samples were analyzed using Cytoflex (LX). The results showed that USCs primarily expressed mesenchymal stem cell-specific markers.

[0044] 4. CCK-8 cell viability assay

[0045] USCs are priced at 2–5 × 10 3 Cells were seeded into 96-well plates, with 100 μL of culture medium added to each well. CCK-8 assays were performed at different culture time points. After incubation at 37°C for 2 h, the OD value was measured at 490 nm using a microplate reader.

[0046] The results show that: Figure 2 As shown, the number of activated USCs from pregnant women (red curve) was significantly higher than that of USCs from normal individuals (blue curve) in each passage (P0–P3), indicating that their proliferation rate was faster.

[0047] like Figure 3 As shown, in the viability curves of the two groups of USCs at different time points, the OD value of the cells derived from pregnant and lactating women was consistently higher, indicating stronger metabolic activity and growth potential. The results collectively demonstrate that the activated USCs prepared in this embodiment possess excellent growth and viability characteristics. The growth curve of USCs derived from pregnant and lactating women was significantly higher than that of USCs derived from normal adults, suggesting that they have stronger viability and proliferative capacity, and can provide a high-quality cell source for subsequent functional studies or regenerative medicine applications.

[0048] The above specific embodiments are merely explanations of the present invention and are not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A method for culturing human urine-derived stem cells, characterized by: The method comprises the following steps: S1, gelatin-coated culture plate: 0.1% gelatin solution is added to a 12-well plate at 0.5 mL / well, incubated in a 5% CO2 incubator at 37°C for 30 min, and discarded after the gelatin solution is discarded; S2, urine collection and centrifugal precipitation: collect 200 mL of midstream urine of pregnant women, centrifuge at 400 g at room temperature for 10 min; discard the supernatant, retain 1 mL of urine sediment at the bottom of the tube, and add 10 mL of DPBS to mix evenly; Then centrifuge at 1500 r / min for 10 min, discard the supernatant, and collect the cell precipitate; the midstream urine of the pregnant woman is collected from the woman in the recovery period after delivery within 24-72 hours after surgery after the subject recovers from independent urination; S3, Stem cell induction and formation with adherent culture: Take 100 μL of the precipitate, add 1 mL of USCs complete medium, inoculate at 2x10 5 Cell density seeding to gelatin-coated 12-well plates; placed in a 37°C, 5% CO2 incubator, and after 48 h, the liquid was changed, the suspended cells were discarded, and the adherent cells were retained; every 2-3 d, the liquid was changed; S4, stem cell subculture and expansion: when the cell adhesion area reaches 75%-80%, subculture, discard the culture medium, place it in a biological safety cabinet for subculture, add 0.25% trypsin-EDTA and digest for 3 min; add an equal volume of high-sugar DMEM containing 10% FBS and 1% double antibody to terminate the reaction; centrifuge at 1000 rpm for 7 min in a low-speed centrifuge, collect the precipitate; then transfer it to a new culture bottle at a ratio of 1:3 for expansion; S5: stem cell collection and pre-treatment: the collected cells are washed once with double-antibody-containing PBS, centrifuged at 1500 rpm for 7-10 min in a low-speed centrifuge, the supernatant is discarded and the precipitate is retained, and then resuspended in high-sugar medium according to the required concentration for animal experiments or subsequent application; S6: identification of stem cells by immunofluorescence: CD90 and CD44 antibodies are used for immunofluorescence staining of the cells, DAPI is used for nuclear staining, and the results are observed under an inverted microscope, showing that the USCs from pregnant women are CD90⁺ / CD44⁺; S7: CCK-8 detection of cell activity and draw growth curve: cells were seeded in 96-well plates at a density of 2-5 x 10 3 cells / well, cultured for 7 consecutive days, and the OD value of the cells was detected using a CCK-8 kit to draw a growth curve; the results showed that the viability and proliferation ability of the pregnant woman-derived USCs were significantly higher than those of the normal adult USCs.

2. The method for culturing human urine-derived stem cells as described in claim 1, characterized in that: The human urine-derived stem cells are derived from urine samples of pregnant women, have stronger vitality, stemness and proliferation capacity than normal adult urine-derived stem cells, and can be used as cell therapy materials in tissue engineering and regenerative medicine; The pregnant woman-derived urine-derived stem cells can significantly improve cell activity and show higher growth curves in CCK-8 detection, which is helpful for cell proliferation; The pregnant woman-derived urine-derived stem cells show CD90 positivity and CD44 positivity in immunofluorescence identification, and have good biological safety and transplantation adaptability.

Images