A method for promoting the survival of pluripotent stem cells and its application

Abstract

The present invention discloses a method for promoting the survival of pluripotent stem cells and its application, the method comprising the following steps: using a culture medium containing an anti-apoptosis inhibitor and albumin to culture monoclonal cells. The method of the present invention promotes the survival of monoclonal cells by combining an anti-apoptosis inhibitor and albumin to prepare a culture medium; the prepared culture medium has clear components and is simple to prepare, and can effectively increase the survival success rate of monoclonal cells and reduce the experimental cost in scale-up production.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a method for promoting the survival of pluripotent stem cells and an application thereof. Background Art

[0002] Human pluripotent stem cells (hESC) and induced pluripotent stem cells (iPSC) can directly differentiate into tissues and organs and replace diseased parts, which has a very broad application prospect. In the process of clinical application, cell tracing generally requires iPSC monocloning, but iPSC cannot survive as a single cell under normal circumstances, which greatly limits its industrial production. Therefore, it is of great significance to develop a culture scheme that promotes the survival of single cells of pluripotent stem cells. Summary of the invention

[0003] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a method for promoting the survival of pluripotent stem cells, which can effectively promote the survival of single pluripotent stem cells.

[0004] The present invention also proposes the application of the above method.

[0005] The invention also provides a method for culturing monoclonal cells.

[0006] According to one aspect of the present invention, a method for promoting the survival of pluripotent stem cells is provided, comprising the following steps: culturing monoclonal cells using a culture medium containing an anti-apoptosis inhibitor and albumin.

[0007] In some embodiments of the present invention, the anti-apoptosis inhibitor includes but is not limited to one of a ROCK inhibitor, a Caspase inhibitor and a Blebbistatin inhibitor.

[0008] In some embodiments of the present invention, the ROCK inhibitor includes but is not limited to one of Y27632 and Thiazovivin.

[0009] In some embodiments of the present invention, the Caspase inhibitor includes but is not limited to z-VAD-fmk.

[0010] In some embodiments of the present invention, the concentration of the ROCK inhibitor is 0.1-15 μM.

[0011] In some embodiments of the present invention, when the ROCK inhibitor is Y27632, the concentration of the ROCK inhibitor in the culture medium is 5-15 μM.

[0012] In some embodiments of the present invention, when the ROCK inhibitor is Y27632, the concentration of the ROCK inhibitor in the culture medium is 8-12 μM.

[0013] In some embodiments of the present invention, when the ROCK inhibitor is Y27632, the concentration of the ROCK inhibitor in the culture medium is 10 μM.

[0014] In some embodiments of the present invention, when the ROCK inhibitor is Thiazovivin, the concentration of the ROCK inhibitor in the culture medium is 0.1-10 μM.

[0015] In some embodiments of the present invention, when the ROCK inhibitor is Thiazovivin, the concentration of the ROCK inhibitor in the culture medium is 0.5-1 μM.

[0016] In some embodiments of the invention, the albumin comprises human serum albumin or bovine serum albumin.

[0017] In some embodiments of the invention, the albumin is human serum albumin.

[0018] In some embodiments of the present invention, the concentration of albumin in the culture medium is 1-10 mg / mL.

[0019] In some embodiments of the present invention, the concentration of albumin in the culture medium is 4-7 mg / mL.

[0020] In some embodiments of the present invention, the concentration of albumin in the culture medium is 5 mg / mL.

[0021] In some embodiments of the present invention, the culture medium further comprises a basal culture medium.

[0022] In some embodiments of the present invention, the basal culture medium comprises E8 medium.

[0023] According to a second aspect of the present invention, an application of the above method is provided, wherein the application is application in culturing pluripotent stem cells.

[0024] In some embodiments of the present invention, the use is for preparing a product for promoting the survival of pluripotent stem cells.

[0025] In some embodiments of the present invention, the pluripotent stem cells are monoclonal cells.

[0026] In some embodiments of the invention, the cells are embryonic stem cells or induced pluripotent stem cells.

[0027] According to a third aspect of the present invention, a method for culturing pluripotent stem cells is provided, the method comprising the following steps: culturing pluripotent stem cells using a culture medium comprising an anti-apoptosis inhibitor and albumin.

[0028] In some embodiments of the present invention, the anti-apoptosis inhibitor includes but is not limited to one of a ROCK inhibitor, a Caspase inhibitor and a Blebbistatin inhibitor.

[0029] In some embodiments of the present invention, the ROCK inhibitor includes but is not limited to one of Y27632 and Thiazovivin.

[0030] In some embodiments of the present invention, the Caspase inhibitor includes but is not limited to z-VAD-fmk.

[0031] In some embodiments of the present invention, before using the above-mentioned culture medium to culture the pluripotent stem cells, the method further comprises the step of using recombinant trypsin to dissociate the human pluripotent stem cells into monoclonal cells.

[0032] In some embodiments of the present invention, the pluripotent stem cells are cultured using a cell culture plate coated with recombinant laminin.

[0033] In some embodiments of the present invention, the recombinant laminin comprises LN511.

[0034] According to some embodiments of the present invention, at least the following beneficial effects are achieved: the present invention promotes the survival of pluripotent stem cells by preparing a culture medium by combining an anti-apoptosis inhibitor, a ROCK inhibitor, and albumin; the prepared culture medium has clear components and is easy to prepare, and can effectively increase the survival success rate of pluripotent stem cell monoclones and reduce experimental costs in scale-up production. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0036] Figure 1 The figure is a test result diagram of the survival rate of monoclonal cells in different culture media in the test examples of the present invention;

[0037] Figure 2 This is a graph showing the test results of the effects of different concentrations of HSA in the culture medium on the survival rate of monoclonal cells in the test example of the present invention. DETAILED DESCRIPTION

[0038] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0039] Example 1

[0040] In this example, a culture medium was prepared, which was an E8 basal medium (purchased from thermo) containing a ROCK inhibitor Y27632 (purchased from selleck, S1049) with a final concentration of 10 μM and human serum albumin (HSA) (purchased from Heyuan Biotechnology, HYC002M01) with a final concentration of 5 mg / mL.

[0041] The culture medium was prepared as follows:

[0042] 1) Preparation of Y27632 stock solution: 10 mg Y27632 + 3.1225 mL DMSO was dissolved at room temperature to prepare a 10 mM stock solution;

[0043] 2) Preparation of human serum albumin stock solution: 5 g HSA powder was added to 20 mL DMEM / F12, and dissolved by shaking at 37°C or at room temperature. After filtering with a 0.22 μm needle filter, a 250 mg / mL HSA stock solution was prepared.

[0044] 3) The culture medium was prepared by containing 50 μL of 10 mM Y27632 stock solution and 1 mL of 250 mg / mL HSA stock solution in 50 mL of E8 culture medium.

[0045] Example 2

[0046] In this example, a culture medium was prepared. The culture medium was an E8 basal culture medium containing a ROCK inhibitor Y27632 at a final concentration of 10 μM and human serum albumin (HSA) at a final concentration of 1 mg / mL.

[0047] The preparation method of the culture medium is the same as that in Example 1.

[0048] Example 3

[0049] In this example, a culture medium was prepared. The culture medium was an E8 basal culture medium containing a ROCK inhibitor Y27632 at a final concentration of 10 μM and human serum albumin (HSA) at a final concentration of 2 mg / mL.

[0050] The preparation method of the culture medium is the same as that in Example 1.

[0051] Example 4

[0052] In this example, a culture medium was prepared. The culture medium was an E8 basal culture medium containing a ROCK inhibitor Y27632 at a final concentration of 10 μM and human serum albumin (HSA) at a final concentration of 10 mg / mL.

[0053] The preparation method of the culture medium is the same as that in Example 1.

[0054] Comparative Example 1

[0055] This comparative example prepares a culture medium, which is different from Example 1 in that ROCK inhibitor Y27632 and human serum albumin are omitted.

[0056] The preparation method of the culture medium is the same as that in Example 1.

[0057] Comparative Example 2

[0058] This comparative example prepares a culture medium, which differs from Example 1 in that human serum albumin is omitted.

[0059] The preparation method of the culture medium is the same as that in Example 1.

[0060] Comparative Example 3

[0061] This comparative example prepares a culture medium, which differs from Example 1 in that the ROCK inhibitor Y27632 is omitted.

[0062] The preparation method of the culture medium is the same as that in Example 1.

[0063] Test example

[0064] This test example tests the effects of the culture media prepared in Examples 1-4 and Comparative Examples 1-3 on the survival rate of a single clone of pluripotent stem cells.

[0065] 1. Determination of the survival rate of monoclonal stem cells

[0066] 1) Preparation of coating solution: 20 mL DPBS plus 200 μL 0.5 ug / μL LN511 (biogems, RL511S-350) to make 5 ug / mL coating solution;

[0067] 2) Coating: Add 1 mL of 5ug / mL LN511 to each well of a 6-well plate and incubate at 37°C for more than 2 hours;

[0068] 3) Inoculation: When human induced pluripotent stem cells grow to 80%-90% confluence, 600 μL tryple (gibco / 12605-028) is used in each well of a 6-well plate to dissociate human induced pluripotent stem cells (induced pluripotent stem cells derived from human urine cells) into single cells, and after counting, inoculate a 6-well plate (previously coated with LN511), inoculate 500 cells per well, and add 2 mL of the culture medium prepared in Example 1 and Comparative Examples 1-3 respectively. On the second day, change to normal stem cell culture medium E8, 2 mL per well, and change the medium every day;

[0069] 4) Detection of clone formation efficiency: After 10 days of culture, the cells were stained with alkaline phosphatase (Biyuntian / C3206, refer to the product manual for the specific protocol).

[0070] Test results such as Figure 1 As shown in the figure, it can be seen that only by adding human serum albumin and Y27632 at the same time can the survival rate of monoclonal cells be effectively promoted.

[0071] 2. Screening of HSA concentration

[0072] Human pluripotent stem cells were dissociated into single cells using tryple (recombinant pancreatic enzyme), and inoculated into 6-well plates (previously coated with LN511) after counting, with 500 cells inoculated into each well. The culture media prepared in Examples 1-4 and Comparative Example 2 were used for culture, and screening tests of different concentrations of HSA were performed.

[0073] Test results such as Figure 2 As shown in the figure, it can be seen that 1-10 mg / mL HSA has a more obvious effect of promoting survival, but 5 mg / mL HSA has the best effect.

[0074] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for promoting the survival of a pluripotent stem cell monoclone, characterized in that: The following steps are involved: Monoclonal cells were cultured using medium consisting of anti-apoptotic inhibitors and human serum albumin; The anti-apoptosis inhibitor includes one of a ROCK inhibitor, a Caspase inhibitor and a Blebbistatin inhibitor; ROCK inhibitors included one of Y27632 and Thiazovivin; The Caspase inhibitor includes z-VAD-fmk; The concentration of human serum albumin in the culture medium is 1-10 mg / mL; The concentration of the anti-apoptosis inhibitor in the culture medium is 0.1-15 μM.

2. The method according to claim 1, characterized in that The concentration of human serum albumin is 4-7 mg / mL.

3. The method according to claim 1, characterized in that The culture medium also includes a basal culture medium.

4. The method according to claim 3, characterized in that: The basic culture medium includes E8 culture medium.

5. Use of the method according to any one of claims 1 to 4 in culturing pluripotent stem cells.

6. Use of the method according to any one of claims 1 to 4 in the preparation of a product that promotes the survival of pluripotent stem cells.

7. The use according to claim 5 or 6, characterized in that: The pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

8. A method for culturing a monoclone of pluripotent stem cells, characterized in that: The method comprises the following steps: culturing monoclonal cells of pluripotent stem cells using a culture medium consisting of an anti-apoptosis inhibitor and human serum albumin; The anti-apoptosis inhibitor includes one of a ROCK inhibitor, a Caspase inhibitor and a Blebbistatin inhibitor; ROCK inhibitors included one of Y27632 and Thiazovivin; The Caspase inhibitor includes z-VAD-fmk; The concentration of the anti-apoptosis inhibitor in the culture medium is 0.1-15 μM; The concentration of human serum albumin in the culture medium is 1-10 mg / mL.

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