Umbilical cord mesenchymal stem cell generation determination and optimal generation evaluation method

Abstract

The invention relates to the fields of cytobiology and regenerative medicine, in particular to an umbilical cord mesenchymal stem cell generation judgment and optimal generation evaluation method which comprises the following steps: S1, acquiring hUC-MSCs of a generation to be judged, and performing in-vitro culture until the end of the logarithmic phase; s2, detecting the methylation level of the age-related genes in the hUC-MSCs by adopting whole genome bisulfite methylation sequencing; s3, the epigenetic age of the hUC-MSCs is obtained according to the methylation level; s4, the epigenetic age is substituted into a generation-physiological age linear formula y = 1.75 x + 13.467, y is the epigenetic age, x is the cell generation, and the generation of the hUC-MSCs is obtained through calculation. Compared with the prior art, the method has the beneficial effects that a'generation-physiological age 'linear formula is innovatively obtained through gene methylation detection, accurate judgment of hUC-MSCs generations is realized, and the problem that the traditional method cannot accurately evaluate the cell generations is solved.

Description

Technical Field

[0001] This invention relates to the fields of cell biology and regenerative medicine, and in particular to a method for determining the passage number and evaluating the optimal passage number of umbilical cord mesenchymal stem cells. Background Technology

[0002] Human umbilical cord mesenchymal stem cells (hUC-MSCs) have shown great promise for clinical applications in regenerative medicine, tissue engineering, and the treatment of immune diseases due to their multi-lineage differentiation potential, strong immunomodulatory function, and rich paracrine effects.

[0003] However, the clinical application of hUC-MSCs relies on in vitro expansion to obtain a sufficient number of cells. Multiple passages inevitably lead to "replicative senescence," accompanied by a significant decline in their biological functions; this phenomenon is known as "generation-dependent functional decay." Its main manifestations include: decreased proliferation capacity, weakened colony-forming ability, impaired immunomodulatory function, weakened secretome (the spectrum of secreted growth factors and cytokines), and increased genomic and epigenetic instability.

[0004] Currently, there is no unified and scientific "gold standard" for the appropriate passage range for the clinical application of hUC-MSCs. Various studies and clinical trials typically use cells within a relatively wide passage range (e.g., P3 to P10 passages). This ambiguity and arbitrariness in passage selection poses significant challenges to the efficacy, safety, and batch-to-batch consistency of hUC-MSCs therapeutic products. Cell products from different laboratories, different donor sources, and different batches may exhibit significant differences in efficacy due to differences in the passages used, severely hindering the standardization and industrialization of hUC-MSCs therapy.

[0005] Therefore, there is an urgent need in this field for a clear and scientific method that can accurately identify the "golden generation" of hUC-MSCs with the best function during in vitro expansion and provide a comprehensive, quantitative, and operable quality assessment standard to ensure the consistency and efficiency of cell therapy products. Summary of the Invention

[0006] To address the problems mentioned in the background section, this invention provides a method for determining the passage number and evaluating the optimal passage number of umbilical cord mesenchymal stem cells.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A method for determining the passage number of umbilical cord mesenchymal stem cells includes the following steps: S1. Obtain hUC-MSCs of the passage number to be determined and culture them in vitro to the end of the logarithmic growth phase; S2. The methylation levels of age-related genes in the hUC-MSCs were detected by whole-genome bisulfite methylation sequencing; S3. Obtain the epigenetic age of the hUC-MSCs based on the methylation level; S4. Substitute the epigenetic age into the linear formula y=1.75x+13.467 of “generation-physiological age”, where y is the epigenetic age and x is the cell generation, to calculate the generation of the hUC-MSCs.

[0008] Preferably, in step S1, the end of the logarithmic growth phase is 7 days after in vitro culture.

[0009] Preferably, in step S2, the age-related genes include ELOVL2, FHL2, KLF14, OTUD7A, RNF32, IRS2, AMER3, CLYBL, CDK5R2, DBH, ZYG11A, GL3, DIO3, PCDHB1, BMI1, ABHD14A, SYNGR3, RPA2, ENO2, SLC1A6, NR2E3, TRIP6, SAMD10, RNF180, GFPT2, USB1, DDO, LGALS1, ACBD4, EDARADD, ACSS3, SYT6, WLS, and NWD1.

[0010] A method for evaluating the optimal passage number of umbilical cord mesenchymal stem cells includes the following steps: A1. Primary isolation of MSCs from umbilical cord MSCs yielded MSC cells; A2. Cell passage was performed to obtain P1, P2, P3, P4, and P5 generations of cells sequentially. A3. Detect growth curves and population doubling time, surface markers, growth factor secretion, cytokine secretion, apoptosis rate, cellular senescence level, and telomere length to determine the optimal passage of umbilical cord mesenchymal stem cells.

[0011] Preferably, the specific method of step A1 is as follows: Wash the umbilical cord with DPBS containing gentamicin and DPBS without gentamicin in sequence until there is no blood. Cut the umbilical cord into 2cm segments and soak them in centrifuge tubes containing complete stem cell culture medium. Cut the veins of the umbilical cord, tear off the inner lining of the veins and arteries on the inner wall, turn it to the front, and tear off the outer skin of the umbilical cord to fully expose Wharton's jelly. Cut the umbilical cord into 3-5mm square pieces and spread them evenly on a culture dish. Add the complete stem cell culture medium along the side wall of the culture dish. The medium was completely replaced on days 7 and 10, and MSC cells were obtained on days 12-14.

[0012] Preferably, the specific method of step A2 is as follows: After culturing MSC cells for 60-72 hours, observe the cultured MSC cells under a microscope. When the cell confluence reaches 85%-90%, they can be passaged. Collect the culture supernatant, wash the cells with DPBS and discard them to avoid rinsing and damaging the cells, add a mild stem cell digestive enzyme to digest the cells, centrifuge the cultured cell suspension, resuspend the cells in the culture medium and count them with a counting chamber. Add complete culture medium and seed cells at a density of 8000-10000 cells / cm². Incubate the culture flask at 37℃, 5% CO2, and saturated humidity. After 60-72 hours (85%-90% confluence), the cells can be passaged and designated as P1. Obtain P2, P3, P4, and P5 cells sequentially.

[0013] Preferably, in step A3, the growth curve and the population doubling time are set at 1.25 × 10⁻⁶. 5 Cells were seeded in T25 flasks and cultured continuously for 7 days. One flask was taken each day for cell counting, and the cell count and viability were recorded. Surface markers were measured by flow cytometry according to the standards of the International Society for Cell and Gene Therapy, including the expression levels of CD73, CD90, CD105, CD44 (positive markers) and CD34, CD45, HLA-DR, CD3, CD19, and CD14 (negative markers).

[0014] Preferably, in step A3, the secretion of growth factors is detected by flow cytometry or enzyme-linked immunosorbent assay (ELISA) to measure the secretion levels of VEGF, GM-CSF, ADM, HGF, and IGF-1, as well as telomerase activity. Cytokine secretion was assessed by flow cytometry or enzyme-linked immunosorbent assay (ELISA) to detect the secretion levels of IL-6, IL-1β, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p70, IL-17A, IFN-α2, IFN-γ, and TNF-α.

[0015] Preferably, in step A3, the apoptosis rate is determined by Hoechst 33258 staining and observation of cell nucleus morphology under a fluorescence microscope. Normal cell nuclei appear blue, while apoptotic cell nuclei appear as densely stained or fragmented densely stained areas. Cellular senescence levels were determined using β-galactosidase staining with X-Gal as a substrate, and the formation of dark blue products was observed under an optical microscope. Telomere length was estimated by calculating the ratio of telomere amplification products to control gene amplification products using qPCR.

[0016] Compared with the prior art, the beneficial effects of the present invention are: 1. P3 and P4 generations have been identified as the "golden generations" for the treatment of hUC-MSCs. Cells of these generations are superior to P5 generation in key indicators such as proliferation capacity, secretory function, and apoptosis rate, providing a clear basis for generation selection in clinical applications. 2. Innovatively, a linear formula of "generation-physiological age" was obtained through gene methylation detection, which enabled accurate determination of the generation of hUC-MSCs and solved the problem that traditional methods could not accurately assess cell generation; 3. A multi-parameter quality assessment system covering proliferation, stemness, secretion, viability and epigenetic levels has been established, which can comprehensively and quantitatively evaluate the quality of hUC-MSCs and ensure the efficacy, safety and batch-to-batch consistency of cell therapy products; 4. It provides key technical support for the standardization and industrialization of hUC-MSCs therapy, and helps promote the clinical application of hUC-MSCs in regenerative medicine, tissue engineering and the treatment of immune diseases. Attached Figure Description

[0017] Figure 1 The growth curves and viability of P3-P5 generation hUC-MSCs show that P3 and P4 generations are significantly superior to P5 generation in terms of viable cell count, viability, and total cell count.

[0018] Figure 2 The expression levels of cell surface markers showed that the expression of positive markers was more stable in generations P3 and P4. Figure 3 The levels of growth factor expression were shown, indicating that P3 and P4 generations had higher levels of VEGF and GM-CSF secretion. Figure 4 The expression levels of 12 common cytokines are shown, illustrating their secretion at different generations. Figure 5 The expression levels of cytokines related to aging were measured, showing that the secretion of IL-6, IL-1β, and GM-CSF did not decrease in P3 and P4 generations. Figure 6 This represents the cellular senescence level, indicating that P3 and P4 generation cells have not entered a significant senescence phase; Figure 7 The apoptosis level was shown to be lower in P3 and P4 generations than in P5 generation. Figure 8 shows the age-related telomere length levels, indicating that there is no significant change in telomere length between generations P3 and P5; Figure 9 shows the age levels associated with gene methylation, indicating that P3 represents the youngest observable age, while P5 represents the highest. Figure 10 shows the linear formula of generation-physiological age, illustrating the linear relationship between generation and epigenetic age (y=1.75x+13.467, R²=0.9681). Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Unless otherwise stated, all raw materials and reagents used in this invention are commercially available or can be prepared by known methods.

[0021] Example 1: Primary cell culture and passage preparation Primary isolation of MSCs from umbilical cord (175 vials): (1) Scissors, scalpels, and forceps should be sterilized at high temperature in advance; (2) Clean the umbilical cord three times with DPBS containing gentamicin (25 mg / L), each time for 5-10 minutes, until there is no blood, and change to a new tube each time; (3) Clean the umbilical cord twice with DPBS without gentamicin until there is no blood. Cut off the small sections at both ends of the umbilical cord. If there is blood seepage, continue cleaning. (4) Cut the umbilical cord into small segments of about 2 cm. If there is blood seepage, wash it with DPBS and soak it in a centrifuge tube containing complete culture medium. (5) In a 150 mm culture dish, cut open the umbilical vein and tear off the venous intima and the two arteries; (6) Turn the umbilical cord over, peel off the outer skin to expose Wharton's jelly, and keep it moist throughout the process; (7) Cut the umbilical cord into 3-5mm square pieces. Inoculate a 2cm section of umbilical cord into a 150mm culture dish, spread it evenly, and add 10-15ml of culture medium along the side wall to prevent the tissue pieces from floating. (8) After 24 hours, replenish the fluid to 30 ml; (9) Replace the entire solution (30ml) on the 7th day and replace the entire solution (30ml) again on the 10th day; (10) Harvest MSC cells in 14 days.

[0022] Cell passage: (1) After culturing for 72 hours, the cell confluence was observed to be 88% under a microscope, and the cells were then passaged. (2) Collect the culture supernatant in the clean bench for later use. Add 10 mL of DPBS to wash the cells and then discard the supernatant. (3) Add 2 mL of stem cell mild digestive enzyme and digest in an incubator for 3 minutes; (4) Under the microscope, the cells were observed to shrink and become round and flow slightly. 4 mL of culture medium supernatant was added to dilute the cells and the mixture was gently pipetted to mix. (5) Transfer to a 15 mL centrifuge tube, centrifuge at 1300 rpm for 5 min, resuspend in culture medium and count; (6) Add 15 mL of complete culture medium to the petri dish and inoculate at a density of 9000 cells / cm²; (7) After culturing at 37℃, 5% CO2 and saturated humidity for 72 hours, the cell confluence reached 89%, and the passage was recorded as P1. P2, P3, P4 and P5 cells were obtained in sequence.

[0023] Example 2: Generation determination Cell culture: P3, P4 and P5 generation hUC-MSCs were cultured in vitro for 7 days until the end of the logarithmic growth phase; (1) The methylation levels of age-related genes (ELOVL2, FHL2, KLF14, OTUD7A, RNF32, IRS2, AMER3, CLYBL, CDK5R2, DBH, ZYG11A, GL3, DIO3, PCDHB1, BMI1, ABHD14A, SYNGR3, RPA2, ENO2, SLC1A6, NR2E3, TRIP6, SAMD10, RNF180, GFPT2, USB1, DDO, LGALS, ACBD4, EDARADD, ACSS3, SYT6, WLS, NWD1) were detected by WGBS. (2) Calculate the epigenetic age: P3 generation is 18.9 years, P4 generation is 20.1 years, and P5 generation is 22.4 years; (3) Substituting into the linear formula y=1.75x+13.467, it was verified that the calculated generations of P3, P4 and P5 cells were consistent with the actual generations.

[0024] Optimal generation determination: (1) Growth curve: The proliferation of P3 generation is better than that of P4 and P5 generation. The early stage of P4 generation (before d4) is better than that of P5 generation. The difference is small between d4 and d5 days. (2) Surface markers: The expression of positive markers CD73, CD90, CD105 and CD44 in P3 and P4 generations is higher than that in P5 generation, and the expression of negative markers meets the ISCT criteria; (3) Growth factor secretion: The secretion levels of VEGF and GM-CSF in P3 and P4 generations were significantly higher than those in P5 generation; (4) Cytokine secretion: The secretion of IL-6, IL-1β, and GM-CSF did not decrease, and the cells did not enter the senescent phase; (5) Apoptosis rate: P3 and P4 generations were lower than P5 generation; (6) Cellular senescence level: P3, P4, and P5 generations did not enter a significant senescence phase; (7) Telomere length: There was no significant change in the three generations of cells, and the physiological age level was basically the same.

[0025] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for determining the passage of umbilical cord mesenchymal stem cells, characterized by, Comprising the following steps: S1. Obtain hUC-MSCs of the generation to be determined, and culture in vitro to the end of the logarithmic growth phase; S2. Detect the methylation level of age-related genes in the hUC-MSCs by whole genome bisulfite sequencing; S3. Obtain the epigenetic age of the hUC-MSCs according to the methylation level; S4. Substitute the epigenetic age into the linear formula "generation-physiological age" y = 1.75x + 13.467, where y is the epigenetic age and x is the cell generation, to calculate the generation of the hUC-MSCs.

2. The method of claim 1, wherein the method is characterized by, In step S1, the end of the logarithmic growth phase is after 7 days of in vitro culture.

3. The method of claim 1, wherein the method is characterized by, In step S2, the age-related genes include ELOVL2, FHL2, KLF14, OTUD7A, RNF32, IRS2, AMER3, CLYBL, CDK5R2, DBH, ZYG11A, GL3, DIO3, PCDHB1, BMI1, ABHD14A, SYNGR3, RPA2, ENO2, SLC1A6, NR2E3, TRIP6, SAMD10, RNF180, GFPT2, USB1, DDO, LGALSL, ACBD4, EDARADD, ACSS3, SYT6, WLS, NWD1.

4. A method for evaluating the optimal passage of umbilical cord mesenchymal stem cells, characterized by, Comprising the following steps: A1. Primary separation of umbilical cord MSCs to obtain MSC cells; A2. Cell passage to obtain P1, P2, P3, P4, and P5 cells in turn; A3. Detect the growth curve, population doubling time, surface markers, growth factor secretion, cytokine secretion, apoptosis rate, cell aging level, and telomere length to determine the optimal generation of umbilical cord mesenchymal stem cells.

5. The method of claim 4, wherein the umbilical cord mesenchymal stem cells are selected from the group consisting of: Wharton's jelly mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, and combinations thereof. The specific method of step A1 is: Wash the umbilical cord with DPBS containing gentamicin and DPBS not containing gentamicin in turn until no blood color is observed; Cut the umbilical cord into 2 cm small pieces and soak them in a centrifuge tube containing stem cell complete medium; Cut the umbilical cord into 3-5 mm square small pieces and evenly spread them on the culture dish, and add stem cell complete medium along the side wall of the culture dish; Change the medium in full amount on the 7th and 10th days, and obtain MSC cells on the 12th to 14th days. The specific method of step A2 is:

6. The method of claim 4, wherein the umbilical cord mesenchymal stem cells are evaluated for the optimal passage, and After culturing the MSC cells for 60-72 h, observe the cultured MSC cells under a microscope, and when the cell confluence reaches 85%-90%, passaging can be performed; Collect the culture supernatant, discard it after washing the cells with DPBS to avoid damaging the cells, add stem cell gentle digestion enzyme to digest the cells, centrifuge the suspended cells, and count the cells after resuspending the culture medium with a counting plate; ​ Add complete medium, inoculate cells at a density of 8000-10000 cells / cm², and place the culture bottle in a 37℃, 5% CO, saturated humidity environment. After 60-72 hours (85%-90% confluence), the cells can be subcultured, recorded as P1, and the P2, P3, P4, and P5 generations of cells are obtained in turn.

7. The method of claim 4, wherein the umbilical cord mesenchymal stem cells are selected from the group consisting of: Wharton's jelly mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, and combinations thereof. The growth curve and population doubling time in step A3 are 1.25 x 10 5 Cells were inoculated in T25 flasks and cultured continuously for 7 days, and one flask was counted every day to count the number of cells and the viability. The expression levels of CD73, CD90, CD105, CD44 (positive markers) and CD34, CD45, HLA-DR, CD3, CD19, and CD14 (negative markers) are detected by flow cytometry according to the International Society for Cellular and Gene Therapy standards.

8. The method of claim 4, wherein the umbilical cord mesenchymal stem cells are evaluated for the optimal passage, and In step A3, the secretion of growth factors is detected by flow cytometry or enzyme-linked immunosorbent assay to detect the secretion levels of VEGF, GM-CSF, ADM, HGF, and IGF-1, and telomerase activity. The secretion of cytokines is detected by flow cytometry or enzyme-linked immunosorbent assay to detect the secretion levels of IL-6, IL-1β, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p70, IL-17A, IFN-α2, IFN-γ, and TNF-α.

9. The method of claim 4, wherein the umbilical cord mesenchymal stem cells are evaluated for the optimal passage, and In step A3, the apoptosis rate is determined by Hoechst 33258 staining, and the nuclear morphology is observed under a fluorescence microscope. Normal cell nuclei appear blue, and apoptotic cell nuclei appear dense and chunky. The level of cell senescence is determined by β-galactosidase staining, using X-Gal as a substrate, and the formation of dark blue products is observed under a light microscope. The length of the telomere is calculated by qPCR to estimate the average length of the telomere.

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