Isolated culture method for primary umbilical cord mesenchymal stem cells

Abstract

Provided is an isolated culture method for primary umbilical cord mesenchymal stem cells. The method comprises the following steps: S1, pretreating umbilical cord tissue; S2, adding the pretreated umbilical cord tissue to a digestive fluid containing type II collagenase, and performing digestion via shaking to obtain a digested mixed solution; S3, diluting the digested mixed solution with normal saline, performing centrifugation to collect crude precipitates, re-suspending same with normal saline, and performing centrifugation and washing to obtain precipitates; S4, transferring the precipitates into a culture solution, and performing suspension culture in a carbon dioxide incubator; and S5, transferring cells which have been subjected to the suspension culture to a sterile centrifuge tube, pipetting and mixing uniformly, performing centrifugation and washing to collect cell precipitates, and performing adherent culture on the cell precipitates to obtain primary umbilical cord mesenchymal stem cells. By means of the method, highly viable and highly proliferative primary UCMSCs can be obtained within 4–8 days from a serum-free and serum-substitute-free culture solution with clear components, thereby establishing a foundation for downstream development of new drugs and clinical research.

Description

A method for isolating and culturing primary umbilical cord mesenchymal stem cells Technical Field

[0001] This invention relates to the field of cell isolation and culture methods, particularly to the field of IPCC12N5, and more specifically, to a method for isolating and culturing primary umbilical cord mesenchymal stem cells. Background Technology

[0002] Mesenchymal stem cells (MSCs) are pluripotent stem cells found in various tissues, possessing self-renewal and multipotent differentiation potential. Due to their ease of isolation and expansion, multi-lineage differentiation, hematopoietic support, low immunogenicity, and immunomodulatory properties, they have become ideal seed cells in tissue engineering and regenerative medicine.

[0003] Umbilical cord mesenchymal stem cells (UCMSCs) are MSCs derived from discarded umbilical cords of newborn fetuses. As "zero-day cells," UCMSCs exhibit high cell purity and strong proliferative capacity, making them one of the most promising MSC types for future clinical research. Currently, primary UCMSCs can be extracted in vitro using tissue adhesion and enzymatic digestion methods. Tissue adhesion methods have a longer cycle for obtaining primary cells. Inconsistent cell migration times within the tissue lead to inconsistent cell expansion times, resulting in some cells becoming over-proliferated and senescent, increasing cell heterogeneity and affecting subsequent passage. Enzymatic digestion avoids the problems of over-proliferation, senescence, and increased heterogeneity associated with adhesion methods. However, currently, enzymatic digestion is only suitable for obtaining primary UCMSCs in serum-derived or serum-alternative-derived culture media, and cannot be used in serum-free or serum-alternative-derived culture media with clear composition. Furthermore, serum-derived or serum-alternative-derived culture media contain a large amount of heterologous or allogeneic proteins with unclear composition, which is detrimental to the use of cell-related new drug processes.

[0004] CN117603909A discloses a highly immunosuppressive UCMSCs, its culture method, reagents, and applications. The method involves obtaining P0 generation human umbilical cord mesenchymal stem cells from neonatal umbilical cord tissue; passage-culturing the obtained P0 generation human umbilical cord mesenchymal stem cells to obtain P1 generation human umbilical cord mesenchymal stem cells; and inducing the culture of P1 generation human umbilical cord mesenchymal stem cells using low concentrations of the two inflammatory factors IFN-γ and TNF-α to obtain human umbilical cord mesenchymal stem cells with highly immunosuppressive capacity. However, this invention uses enzymatic digestion for separation and extraction in serum, which limits its application scenarios. Summary of the Invention

[0005] This invention provides a method for isolating and culturing primary umbilical cord mesenchymal stem cells, comprising the following steps:

[0006] S1, Pretreatment of umbilical cord tissue;

[0007] S2, the pretreated umbilical cord tissue is added to a digestive solution containing type II collagenase and shaken to digest, resulting in a digested mixture;

[0008] S3, dilute the digested mixture with physiological saline, centrifuge to collect the coarse precipitate, then resuspend it with physiological saline, centrifuge and wash to obtain the precipitate;

[0009] S4. Transfer the precipitate into the culture medium and place it in a carbon dioxide incubator for suspension culture.

[0010] S5. After suspension culture, the cells are transferred to a sterile centrifuge tube, mixed by pipetting, centrifuged and washed, and the cell pellet is collected. The cell pellet is then cultured in an adherent environment to obtain primary umbilical cord mesenchymal stem cells.

[0011] This study found that primary UCMSCs with high viability and high proliferation capacity can be obtained from serum-free and serum-free substitute culture media with clear composition in 4-8 days through suspension culture, which may be due to the uniformity of cell growth promoted during cell suspension culture. Compared with the traditional tissue adhesion method, not only is the culture time significantly shortened, but cell proliferation is also more uniform. Compared with the traditional enzyme digestion method, primary culture can be carried out in serum-free and serum-free substitute culture media with clear composition.

[0012] The umbilical cord tissue pretreatment includes the following steps: washing the obtained umbilical cord tissue with physiological saline, removing the umbilical vein and umbilical artery, and cutting the umbilical cord tissue into small pieces.

[0013] The type II collagenase content in the digestion solution is 0.05-0.1 wt%.

[0014] The suspension culture temperature is 36-37℃.

[0015] The suspension culture time is 1-4 days.

[0016] Preferably, the suspension culture time can be one of 1 day, 2 days, 3 days, or 4 days.

[0017] The concentration of carbon dioxide is 4-6%.

[0018] Preferably, the concentration of the carbon dioxide is 4.8-5.2%.

[0019] The culture medium is a complete culture medium without serum or serum-free substitutes.

[0020] The time for the oscillation digestion is 0.5-2 hours.

[0021] Preferably, the time for the oscillation digestion can be one of 0.5h, 0.8h, 1h, 1.2h, 1.4h, 1.6h, 1.8h, and 2h.

[0022] The volume ratio of the umbilical cord tissue to the digestive fluid is 1:(0.5-2).

[0023] Preferably, the volume ratio of the umbilical cord tissue to the digestive fluid can be one of 1:0.5, 1:1, 1:0.8, 1:1.2, 1:1.5, and 1:2.

[0024] The adherent culture time is 2-3 days. Beneficial effects

[0025] 1. This invention addresses the current status of primary isolation, extraction, and culture of umbilical cord mesenchymal stem cells (UCSMCs) both domestically and internationally. After digesting umbilical cord tissue, the tissue is cultured in its original niche in a culture medium with clear components, free of serum and serum-free substitutes, ultimately yielding high-purity primary umbilical cord mesenchymal stem cells (UCSMCs-PO) with high proliferative performance.

[0026] 2. Compared with the traditional tissue adhesion method, the original ecological suspension culture method provided in this application not only significantly shortens the culture time, but also results in uniform cell proliferation.

[0027] 3. Compared with the enzyme digestion method, the original suspension culture method of this application can be used for primary culture in a serum-free or serum-free culture medium with clear composition.

[0028] 4. The original suspension culture method provided in this application can obtain primary UCMSCs with high viability and high proliferation capacity from serum-free and serum-free substitute culture media with clear composition in 4-8 days, laying the foundation for downstream new drug development and clinical research. Attached Figure Description

[0029] Figure 1 shows micrographs of primary cells cultured in Example 1 and Comparative Examples 1-2. The left, middle, and right sides of the figure correspond to Comparative Example 1, Comparative Example 2, and Example 1, respectively.

[0030] Figure 2 shows the average time for different primary culture processes. The left bar chart corresponds to the enzyme digestion method, and the right bar chart corresponds to the original suspension culture method. Detailed Implementation Example 1

[0031] A method for isolating and culturing primary umbilical cord mesenchymal stem cells (original suspension culture method) comprises the following steps:

[0032] S1, Pretreatment of umbilical cord tissue (Yuaneng Cell Bank Co., Ltd.): The obtained umbilical cord tissue was washed with physiological saline (Dongguan Puji Pharmaceutical Co., Ltd., H20065567), the umbilical vein and umbilical artery were removed, and the umbilical cord tissue was cut into pieces;

[0033] S2, take 10 mL of pretreated umbilical cord tissue, add an equal volume of 0.1 wt% type II collagenase solution (Worthington, LS004176), and digest at 37°C with shaking for 60 min to obtain the digested mixture;

[0034] S3, dilute the digested mixture 15 times with physiological saline, centrifuge to collect the coarse precipitate, then resuspend it with physiological saline and wash it twice by centrifugation to obtain the precipitate;

[0035] S4, the precipitate was transferred into 8 times the volume of culture medium (YT-SFM complete medium, Shanghai Haitai Pharmaceutical Co., Ltd., M02-CD-g-500), and suspended in culture at 37℃ in a 5% carbon dioxide incubator for 1 day;

[0036] S5, on day 2, centrifuge to collect cells, resuspend the cells in a cell culture flask pretreated with 0.1% gelatin to promote adhesion (stemcell, #07903) and contain YT-SFM complete medium, and culture in a 37℃, 5% CO2 incubator (Thermo, Steri-Cyclei 160); as shown in Figure 1, on day 4, when cell confluence is >90%, discard the supernatant, wash once with PBS buffer (Shanghai Yuanpei Biotechnology, B310KJ), add recombinase (Trypleexpress, Gibco, 12604021) to digest until the cells become round and detach, then add 3 times the volume of PBS to dilute, centrifuge at 500×g for 5 min (low-temperature refrigerated centrifuge, Thermo, ST16R), collect the cell pellet, and obtain primary umbilical cord mesenchymal stem cells.

[0037] Example 2-11

[0038] The specific implementation method is the same as in Example 1, with the differences shown in Table 1: the donor source of umbilical cord tissue is different, the volume of umbilical cord tissue is different, and the concentration and digestion time of collagenase (type II) are different.

[0039] Table 1

[0040] Umbilical cord tissue volume (mL) Collagenase (Type II) concentration, digestion time Example 2 13.5 0.1%, 75 min Example 3 20 0.1%, 60 min Example 4 10 0.05%, 60 min Example 5 7.5 0.05%, 60 min Example 6 50 0.075%, 50 min Example 7 20 0.1%, 40 min Example 8 15 0.08%, 90 min Example 9 40 0.075%, 50 min Example 10 30 0.075%, 50 min Example 11 21 0.075%, 60 min

[0041] Comparative Example 1

[0042] Primary umbilical cord mesenchymal stem cell isolation and culture method using tissue adherence: 10 mL of pretreated umbilical cord tissue from Example 1 was adhered to four 15 cm culture dishes pretreated with 0.1% gelatin adherent agent (stemcell, #07903). 12 mL of YT-SFM complete culture medium (not enough to cause tissue to float) was added, and the cells were cultured at 37°C in a 5% CO2 incubator. 3 mL of medium was added every 3 days, as shown in Figure 1. After 11 days, when cell confluence was >90%, the supernatant was discarded, and the cells were washed once with PBS buffer (Shanghai Yuanpei Biotechnology, B310KJ). Recombinant enzyme (Trypleexpress, Gibco, 12604021) was added to digest the cells until they became rounded and detached. The cells were then diluted with 3 times the volume of PBS buffer, centrifuged at 500×g for 5 min, and the cell pellet was collected to obtain primary umbilical cord mesenchymal stem cells.

[0043] Comparative Example 2

[0044] Enzymatic digestion method for isolating and culturing primary umbilical cord mesenchymal stem cells: 10 mL of pretreated umbilical cord tissue from Example 1 was taken and an equal volume of 0.1% type II collagenase solution (Worthington, LS004176) was added. The mixture was digested at 37°C with shaking for 60 min. The digested mixture was diluted with physiological saline, and the precipitate was collected by centrifugation. The precipitate was then resuspended with physiological saline and washed twice by centrifugation. The precipitate was transferred to a cell culture flask containing MSCBM (Shenzhen Dakwei Biotechnology Co., Ltd., catalog number 6114021) + 5% Elitegro-Adv serum substitute (EliteCell Biomedicalcorp, catalog number: EPA-500) complete medium and cultured at 37°C in a 5% CO2 incubator. The medium was changed every 3 days. After 11 days, the cell confluence was still low, as shown in Figure 1. After 11 days, the cell confluence was still <90%, which was insufficient for passage culture.

[0045] Comparative Example 3-12

[0046] The specific implementation method is the same as in Example 1, with the differences shown in Table 2: the donor sources of umbilical cord tissue are different, and the volume of umbilical cord tissue is different.

[0047] Table 2

[0048] Umbilical cord tissue volume (mL) Comparative example 3 20 Comparative example 4 17.5 Comparative example 5 20 Comparative example 6 25 Comparative example 7 20 Comparative example 8 17.5 Comparative example 9 20 Comparative example 10 50 Comparative example 11 30 Comparative example 12 20

[0049] Performance testing methods

[0050] 1. Cells of Example 1 and Comparative Examples 1-2 were observed using an inverted biological microscope (Leica, DMI1). The micrographs are shown in Figure 1.

[0051] 2. In Examples 2-11, the time when cell confluence was >90% was recorded as the P0 generation harvest time. Each group of cells was resuspended and diluted with YT-SFM complete medium, and the number of P0 generation harvested cells was counted using a cell counter (Nexcelom, cellometermini). The results are shown in Table 3.

[0052] 3. For Comparative Example 3-12, the time when cell confluence was >90% was recorded as the harvest time of generation P0. The results are shown in Table 4.

[0053] Performance test data

[0054] Table 3

[0055] P0 generation harvest time (days) P0 generation harvest cell quantity (×10⁶) Example 2 41.58 Example 3 82.98 Example 4 83.32 Example 5 83.08 Example 6 79.2 Example 7 82.68 Example 8 87.42 Example 9 613.8 Example 10 710.5 Example 11 83.98

[0056] Table 4

[0057] P0 generation harvest time (days) Comparison 3 1 2 Comparison 4 1 2 Comparison 5 1 0 Comparison 6 1 0 Comparison 7 1 1 Comparison 8 1 1 Comparison 9 1 1 Comparison 10 1 3 Comparison 1 1 1 1 Comparison 1 2 1 1

[0058] As shown in Figure 2, the average culture time of primary cells using donor umbilical cords in nutrient-rich serum substitute culture medium via enzymatic digestion in Comparative Examples 3-12 was 11.2 days (maximum culture time 13 days, minimum 10 days), which is much longer than the 7.2 days (maximum culture time 8 days, minimum 4 days) of donor umbilical cords in pristine suspension culture medium with clearly defined components used in Examples 2-11.

Claims

1. A method for isolating and culturing primary umbilical cord mesenchymal stem cells, characterized in that, Includes the following steps: S1, Pretreatment of umbilical cord tissue; S2, the pretreated umbilical cord tissue is added to a digestive solution containing type II collagenase and shaken to digest, resulting in a digested mixture; S3, dilute the digested mixture with physiological saline, centrifuge to collect the coarse precipitate, then resuspend it with physiological saline, centrifuge and wash to obtain the precipitate; S4. Transfer the precipitate into the culture medium and place it in a carbon dioxide incubator for suspension culture. S5. After suspension culture, the cells are transferred to a sterile centrifuge tube, mixed by pipetting, centrifuged and washed, and the cell pellet is collected. The cell pellet is then cultured in an adherent environment to obtain primary umbilical cord mesenchymal stem cells.

2. The method for isolating and culturing primary umbilical cord mesenchymal stem cells according to claim 1, characterized in that, The umbilical cord tissue pretreatment includes the following steps: washing the obtained umbilical cord tissue with physiological saline, removing the umbilical vein and umbilical artery, and cutting the umbilical cord tissue into small pieces.

3. The method for isolating and culturing primary umbilical cord mesenchymal stem cells according to claim 2, characterized in that, The type II collagenase content in the digestion solution is 0.05-0.1 wt%.

4. The method for isolating and culturing primary umbilical cord mesenchymal stem cells according to claim 1 or 3, characterized in that, The suspension culture temperature is 36-37℃.

5. The method for isolating and culturing primary umbilical cord mesenchymal stem cells according to claim 1, characterized in that, The suspension culture time is 1-4 days.

6. The method for isolating and culturing primary umbilical cord mesenchymal stem cells according to claim 5, characterized in that, The volume concentration of the carbon dioxide is 4-6%.

7. The method for isolating and culturing primary umbilical cord mesenchymal stem cells according to claim 6, characterized in that, The culture medium is a complete culture medium without serum or serum-free substitutes.

8. The method for isolating and culturing primary umbilical cord mesenchymal stem cells according to claim 7, characterized in that, The time for the oscillation digestion is 0.5-2 hours.

9. The method for isolating and culturing primary umbilical cord mesenchymal stem cells according to claim 1, characterized in that, The volume ratio of the umbilical cord tissue to the digestive fluid is 1:(0.5-2).

10. The method for isolating and culturing primary umbilical cord mesenchymal stem cells according to claim 8, characterized in that, The adherent culture time is 2-3 days.

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