Culture method for amplifying and purifying umbilical cord multi-line differentiation stress tolerance cells

Abstract

The invention belongs to the technical field of regenerative medicine and biology, and particularly relates to an amplification and purification culture method of umbilical cord multi-line differentiation stress tolerance cells. The method comprises the steps of umbilical cord stem cell primary culture, umbilical cord stem cell subculture and umbilical cord Muse cell inoculated culture. Compared with the prior art, the method has the advantages that the totipotent type of Muse cells is well stabilized and maintained by creatively adding a certain amount of the totipotent stem cell culture medium through a Muse culture formula; meanwhile, substances such as NEAA are added, the Muse cells are efficiently and accurately subjected to in-vitro amplification, and the method is simple in operation step, high in cell acquisition rate, sufficient in number of extracted cells and high in cell viability. The experiment operation time is short, the obtained cell viability is high, and the cell number is greatly increased.

Description

Technical Field

[0001] The invention belongs to the technical field of regenerative medicine and biology, and specifically relates to a method for amplifying, purifying and culturing umbilical cord multilineage differentiated stress-tolerant cells. Background Art

[0002] In 2010, the research team of Japanese scientists KURODA discovered that there is a special type of pluripotent stem cells in the dermis of human skin and bone marrow mesenchymal stem cells. These cells can express anti-stagespecific embryonic antigen 3 (SSEA-3) and can withstand a certain degree of stress. They are named multilineage differentiating stress-enduring (Muse) cells. Compared with other stem cells used for cell therapy, hAECs have the following biological characteristics, making them one of the most promising seed cells for clinical application in the field of regenerative medicine: Muse cells continuously self-renew in vitro, can be cultured in suspension to form cell colonies; express marker genes of embryonic stem cells (Nanog, Oct4, Sox2, TRA-1-60, SSEA-3); can be induced to differentiate into three germ layer cell lines; can migrate to defective tissues in vivo and spontaneously differentiate into defective cells; low telomerase activity, no tumorigenicity, no teratoma formation in immunodeficient mice, and can be isolated from a variety of tissues by double positive labeling of CD105 and SSEA-3. The content of Muse cells in tissues is extremely low, with the proportion of Muse cells in bone marrow mononuclear cells being 0.03%, the proportion of SSEA-3 positive cells in peripheral blood mononuclear cells being 0.04%, and the proportion of Muse cells in mesenchymal stem cells being 1%-8.8%. Therefore, how to obtain sufficient Muse cells has become the key to conducting clinical research.

[0003] The discovery of Muse cells is the first clear report of adult stem cells with self-renewal ability and pluripotent stem cell characteristics at the natural single cell level. Muse cells overcome many shortcomings of ESCs, iPSCs, NSCs and other adult directed stem cells in in vivo transplantation therapy. It has the following characteristics:

[0004] (1) Have stress tolerance;

[0005] (2) Double positivity for SSEA-3 and CD105;

[0006] (3) Single cells can form cell spheres and express pluripotent stem cell markers;

[0007] (4) It can self-renew;

[0008] (5) Ability to differentiate into cells of the three germ layers;

[0009] (6) No teratoma is formed in nude mice.

[0010] The shortcomings of the prior art are as follows:

[0011] 1. Muse cells are cultured after magnetic bead sorting. Amplification process: cell proliferation to form cell clusters - cell clusters adhere to the wall - cells radially adhere to the wall - resuspend to form a single cell suspension - continue suspension amplification culture; the steps are cumbersome and not conducive to industrial production.

[0012] 2. When Muse cells are expanded in vitro, beyond P5, the cells show slow proliferation, large differences in cell morphology, and unstable cell quality.

[0013] Therefore, based on this, the technical solution of the present invention is proposed. Summary of the invention

[0014] In order to solve the problems existing in the prior art, the present invention provides a method for culturing and amplifying umbilical cord multilineage differentiated stress-tolerant cells, the culturing method comprising the following steps:

[0015] (I) Primary Culture of Umbilical Cord Stem Cells

[0016] (1) The umbilical cord is cleaned, the amniotic membrane and arteries and veins are peeled off, the Wharton's jelly is removed and cut into pieces, and then cultured to obtain cells that can be passed on;

[0017] (II) Subculture of umbilical cord stem cells

[0018] (2) Take the P0 cells that can be passaged, wash them first, then add trypsin, and finally terminate the digestion;

[0019] (3) After terminating the digestion, the cells are detached and a single cell suspension is formed, which is then centrifuged;

[0020] (4) After centrifugation, discard the supernatant, add basal culture medium to resuspend the cells, and perform cell subculture;

[0021] (5) When the cells grow to fusion, repeat the “subculture” operation to amplify them to obtain P5 to P10 cells;

[0022] (III) Umbilical cord Muse cell inoculation and culture

[0023] (6) P5 to P10 cells are first inoculated into a culture plate and then cultured to adhere to the wall. After the cell colonies are spread into a single cell layer, the cells are digested and collected with trypsin, and the obtained cell pellets are resuspended and cultured to expand the umbilical cord Muse cells.

[0024] To facilitate understanding of the present invention, some terms in the present invention are explained:

[0025] Muse cells: Multilineage-differentiating stress-enduring (Muse) is a type of cell with self-renewal and multidirectional differentiation potential, which can differentiate into three germ layers. It can be obtained from the skin, umbilical cord, and bone marrow, and can also be isolated from mesenchymal stem cells. It has the ability of continuous self-renewal, low telomerase activity and non-tumorigenicity, and can be integrated into the damaged site in the body. It has strong tissue repair ability and is an ideal seed cell in the field of tissue engineering, cell transplantation and gene therapy.

[0026] Preferably, in step (1), the culture is carried out using DMEM+10% FBS complete medium.

[0027] Preferably, in step (1), the culture is carried out under the conditions of 5% CO2, 37°C and 95% humidity.

[0028] Preferably, in step (2), PBS buffer is used for washing first, then 0.25% trypsin is added, and finally complete culture medium is added to terminate digestion.

[0029] Preferably, in step (3), the centrifugal speed is 1500 r / min, the centrifugal time is 5 min, and the centrifugal temperature is room temperature.

[0030] Preferably, in step (4), α-MEM basal medium + 10% FBS + 1% penicillin-streptomycin is added to resuspend the cells.

[0031] Preferably, in step (6), the adherent culture system is: GMP-grade mesenchymal stem cell serum-free complete medium + E8 complete medium + 100 μmol / mL NEAA.

[0032] Preferably, in step (6), the suspension culture system is: GMP-grade mesenchymal stem cell serum-free complete medium + E8 complete medium + 100 μmol / mL NEAA + 1% methylcellulose basal medium.

[0033] The beneficial effects of the present invention are:

[0034] Compared with the prior art, the present invention pioneered the use of Muse culture formula, by adding a certain amount of totipotent stem cell culture medium, to stabilize and maintain the totipotency of Muse cells; at the same time, by adding substances such as NEAA, Muse cells are efficiently and accurately expanded in vitro. The method has simple operation steps, high cell acquisition rate, sufficient number of extracted cells, and high cell viability. The experimental operation time is short, the cell viability is high, and the number of cells is greatly increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0036] Figure 1 This is the morphological picture of Muse cells on the 7th day in the comparative example (10×).

[0037] Figure 2 This is the morphological picture of Muse cells on the 7th day in the comparative example (40×).

[0038] Figure 3 This is a morphological picture of Muse cells adherent culture in the comparative example (40×).

[0039] Figure 4 This is a morphological picture of Muse cells on day 7 in the Example (10×).

[0040] Figure 5 This is a morphological picture of Muse cells on day 7 in the Example (40×).

[0041] Figure 6 This is a morphological picture of Muse cells adherent culture in the example (40×). DETAILED DESCRIPTION

[0042] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0043] Example

[0044] This embodiment provides a method for culturing and amplifying umbilical cord multilineage differentiated stress-tolerant cells, the method comprising the following steps:

[0045] (I) Primary Culture of Umbilical Cord Stem Cells

[0046] (1) The umbilical cord of a healthy full-term caesarean section baby was taken and aseptically cleaned in a clean bench. The amniotic membrane and arteries and veins of the umbilical cord were peeled off, and the Wharton's jelly was removed and cut into pieces with surgical scissors. After being resuspended in DMEM + 10% FBS complete culture medium, the pieces were inoculated into a 15 cm plate and transferred to an environment of 5% CO2, 37°C, and 95% CO2 for culture.

[0047] After 3 days, replenish the fluid until the cells crawl out, discard the tissue pieces, add new complete medium and continue culturing. When the cells form larger clones, they can be passaged.

[0048] (II) Subculture of umbilical cord stem cells

[0049] (2) Take the P0 cells that can be subcultured, discard the original culture medium in the dish, add 10 mL of PBS buffer to gently rinse the cell growth surface, and discard the washing solution; then add 2 mL of 0.25% trypsin, shake the dish left and right to make the trypsin evenly cover the bottom of the dish; under the microscope, it can be seen that the cell gap is enlarged and the cytoplasm shrinks; when the long spindle-shaped cells become round and bright, immediately add 10 times the volume of complete culture medium to terminate the digestion.

[0050] (3) Repeatedly blow and beat until all cells are detached into a single-cell suspension. Transfer the suspension to a centrifuge tube and centrifuge at 1500 rpm for 5 min at room temperature.

[0051] (4) Discard the supernatant and add α-MEM basal medium + 10% FBS + 1% penicillin-streptomycin to resuspend the cells at a density of 1×10 5 For subculture of cells, the culture flasks were placed in a cell culture incubator at 37°C, 5% CO2, and saturated humidity.

[0052] (5) When the cells grow to confluence, repeat the "subculture" operation to expand. In addition, unless otherwise specified, the human umbilical cord stem cells used in this study were all P5 to P10.

[0053] (III) Umbilical cord Muse cell inoculation and culture

[0054] The umbilical cord mesenchymal stem cells treated with the above enzymes for a long time were resuspended and cultured in the following systems, at 5×10 3 The cells were inoculated into ultra-low adsorption culture plates at a concentration of 0.5 mL per well, and the solution was replenished every other day. After 5-8 days of culture, the cell colonies with a diameter greater than 25 μm were picked out with a glass pipette and inoculated into ordinary culture plates for adherent culture. After the cell colonies spread into a single cell layer, they were digested and collected with 0.25% trypsin containing EDTA, and the obtained cell pellets were resuspended and cultured in ultra-low adsorption culture dishes to expand the umbilical cord Muse cells.

[0055] The adherent culture system is: GMP-grade mesenchymal stem cell serum-free complete medium + E8 complete medium + 100 μmol / mL NEAA; wherein: GMP-grade mesenchymal stem cell serum-free complete medium is prepared by GMP-grade mesenchymal stem cell serum-free complete medium (brand: Youkang, product number: NC0106) and GMP-grade mesenchymal stem cell serum-free medium additive (brand: Youkang, product number: NC0106.S).

[0056] The brand of NEAA is Gibco, the volume is 100mL, and the product number is 2093372.

[0057] The suspension culture system is: 80% to 90% by volume of GMP-grade mesenchymal stem cell serum-free complete medium + E8 complete medium + 100 μmol / mL NEAA + 1% methylcellulose basal medium. The cell spheres formed by inoculation in the PolyHEMA-coated culture plate are more uniform and are cultured in suspension; the brand of the methylcellulose basal medium is STEMCELL Technologies, the product number is 04100, and the name is MethoCult TM H4100 / basic methylcellulose culture medium, product specification is 40mL.

[0058] E8 complete medium is composed of Essential 8 TM Culture medium and Essential 8 TM Supplement (brand: Gibco, item number: A1517001).

[0059] Comparative Example

[0060] The difference between the comparative example and the embodiment is that "(III) umbilical cord Muse cell inoculation culture". In this comparative example, the method described in the existing literature is used for adherent culture and suspension culture. Among them, the existing literature is: "Isolation and identification of stress-tolerant multi-lineage differentiated cells in human umbilical cord and human adipose mesenchymal stem cells and comparison of differentiation ability", Chinese Journal of Tissue Engineering Research [J]. 2022.26(24):3802-3807, Zhang Kun, Li Fang, Xiao Dongjie.

[0061] The adherent culture system (used in the literature) is: α-MEM medium + 10% fetal bovine serum + 1% penicillin-streptomycin + 10 μg / L recombinant basic fibroblast growth factor + 10 μg / L epidermal growth factor in volume fraction.

[0062] The suspension culture system (used in the literature) is: α-MEM medium + 10% fetal bovine serum + 10 ng / mL recombinant basic fibroblast growth factor + 10 ng / mL epidermal growth factor + 1% basic methylcellulose medium H4100 + 200 μmol / mL glutamine.

[0063] This comparative example was cultured according to the procedure of the existing literature.

[0064] Verification Example

[0065] (A) Flow cytometry detection of umbilical cord mesenchymal stem cells, as shown in Table 1.

[0066] Table 1

[0067] P5 P6 P7 P8 P9 P10 CD73 99.12％ 99.08％ 99.55％ 99.26％ 98.92％ 98.76％ CD90 99.34％ 99.65％ 99.17％ 99.33％ 99.80％ 99.86％ CD105 99.04％ 99.58％ 99.43％ 99.37％ 99.23％ 99.12％ CD11b 0.02％ 0.04％ 0.05％ 0.03％ 0.02％ 0.04％ CD19 0.01％ 0.03％ 0.04％ 0.02％ 0.01％ 0.04％ CD34 0.00％ 0.00％ 0.00％ 0.00％ 0.00％ 0.00％ CD45 0.03％ 0.02％ 0.01％ 0.03％ 0.02％ 0.01％ HLA-DR 0.05％ 0.02％ 0.03％ 0.02％ 0.03％ 0.04％ SSEA-3 1.28％ 1.35％ 1.42％ 1.39％ 1.47％ 1.45％

[0068] As can be seen from Table 1, the flow cytometry detection of P5 to P10 of umbilical cord mesenchymal stem cells all meet the minimum identification standards of mesenchymal stem cells issued by the International Society for Cellular Therapy (ISCT): CD73 ≥ 95%, CD90 ≥ 95%, CD105 ≥ 95%; CD11b, CD19, CD34, CD45, HLA-DR ≤ 2.0%.

[0069] In addition, it can be seen from the table that each generation of umbilical cord mesenchymal stem cells P5-P10 contains less than 2.0% of Muse cells (SSEA3 + ), the content is low and difficult to purify, and the number of Muse cells does not meet the requirements for clinical application.

[0070] (II) Muse cell identification (cell morphology, cell number, SSEA3 + CD105 + Cell content)

[0071] (1) Morphology of Muse cells

[0072] Comparative Example:

[0073] The subculture cycle of Muse cells is about 7 days. Usually, a cell cluster consisting of several cells can be formed on the second day, and a cell sphere consisting of dozens of cells to hundreds of round cells can be formed on the seventh day. The volume of the cell sphere varies greatly, the size is uneven, and it is easy to fuse (such as Figure 1 , Figure 2 shown).

[0074] Muse cells were inoculated into the adherent culture system. After the cell clumps adhered to the wall, they grew radially in an "island-like" manner, with a spindle-shaped morphology as the main feature. The cell proliferation rate was 4 days (e.g. Figure 3 shown).

[0075] Example:

[0076] The subculture cycle of Muse cells is about 5 days. Usually, a cell cluster consisting of several cells can be formed on the first day, and a cell sphere consisting of dozens of cells to hundreds of round cells can be formed on the 5th to 7th day. The cell sphere has a uniform volume and is not easy to fuse (such as Figure 4 , Figure 5 shown).

[0077] Muse cells were inoculated into the adherent culture system. The cells grew evenly and were mainly spindle-shaped. The cells adhered to the wall and proliferated quickly. Fusion was completed in 3 days (e.g. Figure 6 shown).

[0078] (2) Number of Muse cells

[0079] Muse cell spheres from P5 to P10 (10 cm dishes, 12 mL / dish) were collected and digested with 0.25% trypsin for 10 to 15 min to disperse the Muse cell spheres into a single cell suspension. 2 mL of fetal bovine serum was added to terminate the enzymatic hydrolysis, and the mixture was centrifuged at 1500 rpm for 5 min. The supernatant was discarded.

[0080] The cells were resuspended in DMEM high-glucose basal medium, and the volume ratio of 1:1 = 0.4% trypan blue: cell suspension was used for quantity and viability detection. This was repeated 3 times. The results are shown in Table 2.

[0081] Table 2

[0082]

[0083] *Indicates significant difference compared with cells from the same batch, P<0.05.

[0084] As can be seen from Table 2, the number of Muse cells obtained in the examples is higher than that in the comparative examples, and the number of cells in Examples P5 to P10 is significantly different from that in the comparative examples, P<0.05. This indicates that the amount of Muse cells obtained in the examples is more than that in the comparative examples. From the viability data, there is no significant difference. The viability of the Muse cells in the comparative examples is about 90%, but the viability of the Muse cells obtained in the examples is above 94%, which is higher than that in the comparative examples.

[0085] (3) Purity of Muse cells

[0086] Muse cell spheres from P5 to P10 (10 cm flat dishes, 12 mL / dish) were collected and digested with 0.25% trypsin for 10 to 15 min to disperse the Muse cell spheres into a single cell suspension, and 2 mL of fetal bovine serum was added to terminate the enzymatic hydrolysis, and the mixture was centrifuged at 1500 rpm for 5 min; the supernatant was discarded. The results are shown in Table 3.

[0087] Table 3

[0088]

[0089] *Indicates significant difference compared with cells from the same batch, P<0.05.

[0090] As can be seen from the above table, the purity of Muse cells obtained in Example (CD105 + SSEA-3 + ) were expressed at 95% to 97%, while the purity of the Muse cells in the control group (CD105 + SSEA-3 + ) were both between 89% and 92%, and P<0.05, showing a significant difference. This indicates that the Muse cells obtained by the culture scheme provided in the embodiment have a higher purity.

[0091] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for amplifying and purifying umbilical cord multilineage differentiated stress-tolerant cells, characterized in that: The culture method comprises the following steps: (I) Primary Culture of Umbilical Cord Stem Cells (1) The umbilical cord is cleaned, the amniotic membrane and arteries and veins are peeled off, the Wharton's jelly is removed and cut into pieces, and then cultured to obtain cells that can be passed on; (II) Subculture of umbilical cord stem cells (2) Take the P0 cells that can be passaged, wash them first, then add trypsin, and finally terminate the digestion; (3) After terminating the digestion, the cells are detached and a single cell suspension is formed, which is then centrifuged; (4) After centrifugation, discard the supernatant, add basal culture medium to resuspend the cells, and perform cell subculture; (5) When the cells grow to fusion, repeat the "subculture" operation to amplify them to obtain P5 to P10 cells; (III) Umbilical cord Muse cell inoculation and culture (6) P5 to P10 cells are first inoculated into a culture plate and then cultured to adhere to the wall. After the cell colonies are spread into a single cell layer, the cells are digested and collected with trypsin, and the obtained cell pellets are resuspended and cultured to expand the umbilical cord Muse cells.

2. The method for amplifying and purifying umbilical cord multilineage differentiated stress-tolerant cells according to claim 1, characterized in that: In step (1), the culture is carried out using DMEM+10% FBS complete medium.

3. The method for amplifying and purifying umbilical cord multilineage differentiated stress-tolerant cells according to claim 1, characterized in that: In step (1), the culture is carried out under the conditions of 5% CO2, 37°C and 95% humidity.

4. The method for amplifying and purifying umbilical cord multilineage differentiated stress-tolerant cells according to claim 1, characterized in that: In step (2), PBS buffer is used for washing first, then 0.25% trypsin is added, and finally complete culture medium is added to terminate digestion.

5. The method for amplifying and purifying umbilical cord multilineage differentiated stress-tolerant cells according to claim 1, characterized in that: In step (3), the centrifugal speed is 1500 r / min, the centrifugal time is 5 min, and the centrifugal temperature is room temperature.

6. The method for amplifying and purifying umbilical cord multilineage differentiated stress-tolerant cells according to claim 1, characterized in that: In step (4), add α-MEM basal medium + 10% FBS + 1% penicillin-streptomycin to resuspend the cells.

7. The method for amplifying and purifying umbilical cord multilineage differentiated stress-tolerant cells according to claim 1, characterized in that: In step (6), the adherent culture system is: GMP-grade mesenchymal stem cell serum-free complete medium + E8 complete medium + 100 μmol / mL NEAA.

8. The method for amplifying and purifying umbilical cord multilineage differentiated stress-tolerant cells according to claim 1, characterized in that: In step (6), the suspension culture system is: GMP-grade mesenchymal stem cell serum-free complete medium + E8 complete medium + 100 μmol / mL NEAA + 1% methylcellulose basal medium.

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