CN113322225A - Additive for human embryonic stem cell in vitro amplification and amplification method - Google Patents
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- IXVMHGVQKLDRKH-VRESXRICSA-N Brassinolide Natural products O=C1OC[C@@H]2[C@@H]3[C@@](C)([C@H]([C@@H]([C@@H](O)[C@H](O)[C@H](C(C)C)C)C)CC3)CC[C@@H]2[C@]2(C)[C@@H]1C[C@H](O)[C@H](O)C2 IXVMHGVQKLDRKH-VRESXRICSA-N 0.000 claims abstract description 16
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- IXVMHGVQKLDRKH-KNBKMWSGSA-N brassinolide Chemical compound C1OC(=O)[C@H]2C[C@H](O)[C@H](O)C[C@]2(C)[C@H]2CC[C@]3(C)[C@@H]([C@H](C)[C@@H](O)[C@H](O)[C@@H](C)C(C)C)CC[C@H]3[C@@H]21 IXVMHGVQKLDRKH-KNBKMWSGSA-N 0.000 claims abstract description 16
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- HCXVJBMSMIARIN-PHZDYDNGSA-N stigmasterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)/C=C/[C@@H](CC)C(C)C)[C@@]1(C)CC2 HCXVJBMSMIARIN-PHZDYDNGSA-N 0.000 claims abstract description 16
- 229940032091 stigmasterol Drugs 0.000 claims abstract description 16
- BFDNMXAIBMJLBB-UHFFFAOYSA-N stigmasterol Natural products CCC(C=CC(C)C1CCCC2C3CC=C4CC(O)CCC4(C)C3CCC12C)C(C)C BFDNMXAIBMJLBB-UHFFFAOYSA-N 0.000 claims abstract description 16
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
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- C12N2500/00—Specific components of cell culture medium
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
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Abstract
The invention discloses an additive for in vitro amplification of human embryonic stem cells, which is added into a basic culture medium for in vitro amplification of embryonic stem cells, and comprises the following components: brassinolide, sodium mercaptosuccinate, stigmasterol, gentiopicrin, kanamycin and L-glutamine. The brassinolide, the sodium mercaptosuccinate, the stigmasterol and the gentiopicrin in the additive have synergistic effect to obviously promote the proliferation of embryonic stem cells, shorten the proliferation period and improve the proliferation efficiency. The additive and the basic culture medium are adopted to carry out in-vitro amplification on the embryonic stem cells, so that a large number of high-quality human embryonic stem cells can be obtained in a short time, animal serum is not needed, the safety is high, and the requirements of scientific research and clinic are fully met. The invention also provides a method for in vitro amplification of human embryonic stem cells, wherein the additive is added into a basic culture medium for cell amplification, so that the cell amplification efficiency is improved, the operation is simple and convenient, and the mass production of the human embryonic stem cells is facilitated.
Description
Technical Field
The invention relates to the technical field of biological pharmacy, in particular to an additive for human embryonic stem cell in vitro amplification and an amplification method.
Background
Human embryonic stem cells (hESCs) are pluripotent cells derived from an in vitro fertilized blastocyst inner cell mass. hESCs have the ability to differentiate towards ectoderm, endoderm and mesoderm, such as cardiomyocytes, neurons, glial cells, hepatocytes, islet cells, chondrocytes, skeletal muscle cells, adipocytes, and endothelial cells, among others. Therefore, the embryonic stem cell can provide an ideal biological platform for scientific research and clinical application, such as drug screening, molecular regulation mechanism research of cell development and differentiation, establishment of tissue engineering seed cells and gene therapy vectors, cell therapy, regenerative medicine and the like.
With the increasing depth of research, the hESCs knowledge is leading to a new stage. The most attractive prospect and use of embryonic stem cells is to produce tissues and cells for "cell therapy" to provide non-immunogenic material for cell transplantation. However, in the field of culturing human embryonic stem cells, the problems of high cost, unstable in vitro culture, easy contamination by infectious microbes and the like, incapability of ensuring the quality and quantity of the embryonic stem cells and the like exist all the time, and the method causes obstacles for the clinical application of the embryonic stem cells.
The traditional hESCs culture method needs to place hESCs on mitomycin-treated feeder cells (such as embryonic fibroblasts, MEFs), and although the method can maintain the growth and proliferation of hESCs, the method faces the problems of complicated feeder cell preparation process, difficulty in separating feeder cells and the like. In addition, feeder layer cell quality can also affect hESCs growth, leading to inconsistent hESCs quality and large batch-to-batch variation, and feeder layer cells can also be a source of animal pathogen and mycoplasma contamination. In order to further improve the quality of hESCs in vitro culture, a culture system completely free of heterologous sources needs to be developed on the premise of not affecting the quality and quantity of hESCs.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide the additive for the in vitro expansion of the human embryonic stem cells, which has clear components and safe use and effectively improves the proliferation efficiency of the embryonic stem cells.
The second purpose of the invention is to provide a method for expanding human embryonic stem cells in vitro.
One of the purposes of the invention is realized by adopting the following technical scheme:
an additive for in vitro amplification of human embryonic stem cells, which is added to a basic culture medium for in vitro amplification of embryonic stem cells, and comprises the following components: brassinolide, sodium mercaptosuccinate, stigmasterol, gentiopicrin, kanamycin and L-glutamine.
Further, the concentration of each component in the additive is as follows according to the final concentration of the culture medium: brassinolide 1.5-4.5ng/mL, sodium mercaptosuccinate 2.8-5.0ng/mL, stigmasterol 13.3-17.5ng/mL, gentiopicrin 10.0-14.5ng/mL, kanamycin 5-10ng/mL, and L-glutamine 15-20 ng/mL.
Further, the concentration of each component in the additive is as follows according to the final concentration of the culture medium: 2.0ng/mL of brassinolide, 3.5ng/mL of sodium mercaptosuccinate, 15.8ng/mL of stigmasterol, 13.0 ng/mL of gentiopicrin, 8ng/mL of kanamycin and 16ng/mL of L-glutamine.
The second purpose of the invention is realized by adopting the following technical scheme:
an in vitro amplification method of human embryonic stem cells comprises the following steps:
(1) inoculating the human embryonic stem cells separated in vitro into a basic culture medium, and pre-culturing for 24 hours in a culture dish coated with fibronectin;
(2) adding the additive according to any one of claims 1 to 3 to a basal medium to obtain an amplification medium, inoculating the embryonic stem cells of step (1) in the amplification medium, and culturing the cells at 37 ℃ in 5% CO2And culturing for 7 days under the environment of partial pressure and saturated humidity.
Further, the medium for amplification was replaced every 1 day in step (2).
Further, the basic medium is a high-glucose DMEM medium.
Further, the seeding density of the cells in the step (2) is 5-8X 104one/mL.
Compared with the prior art, the invention has the beneficial effects that: the invention provides an additive for in vitro amplification of human embryonic stem cells, wherein brassinolide, sodium mercaptosuccinate, stigmasterol and gentiopicrin in the additive have synergistic effect to remarkably promote the proliferation of the embryonic stem cells, shorten the proliferation period and improve the proliferation efficiency. Kanamycin in the additive effectively avoids the pollution of embryonic stem cells by mixed bacteria. The additive and the basic culture medium are adopted to carry out in-vitro amplification on the embryonic stem cells, so that a large number of high-quality human embryonic stem cells can be obtained in a short time, animal serum is not needed, the safety is high, and the requirements of scientific research and clinic are fully met.
The invention also provides a method for in vitro amplification of human embryonic stem cells, wherein the additive is added into a basic culture medium for cell amplification, so that the cell amplification efficiency is improved, the operation is simple and convenient, and the mass production of the human embryonic stem cells is facilitated.
Detailed Description
The present invention is further described below with reference to specific embodiments, and it should be noted that, without conflict, any combination between the embodiments or technical features described below may form a new embodiment.
The following examples, in which the source of human embryonic stem cells is human embryonic stem cells isolated in vitro according to methods known to those skilled in the art, are mainly used to demonstrate the beneficial effects on the proliferation culture of human embryonic stem cells, regardless of the source of human embryonic stem cells and the in vitro isolation method, and human embryonic stem cells isolated in vitro by any method known to those skilled in the art can be used in the following examples to produce almost equivalent proliferation effects.
Example 1
An additive for in vitro amplification of human embryonic stem cells is added into a high-sugar DMEM culture medium for in vitro amplification of embryonic stem cells, and the concentration of each component in the additive is as follows according to the final concentration of the culture medium: 2.0ng/mL of brassinolide, 3.5ng/mL of sodium mercaptosuccinate, 15.8ng/mL of stigmasterol, 13.0 ng/mL of gentiopicrin, 8ng/mL of kanamycin and 16ng/mL of L-glutamine.
An in vitro amplification method of human embryonic stem cells comprises the following steps:
(1) inoculating the human embryonic stem cells separated in vitro into a high-glucose DMEM culture medium, and pre-culturing for 24 hours in a culture dish coated with fibronectin;
(2) adding the additives into a high-glucose DMEM culture medium to obtain a culture medium for amplification, and inoculating the human embryonic stem cells separated in vitro in the step (1) into the culture medium for amplification at a density of 7 x 104one/mL, 5% CO at 37 ℃2Culturing for 7 days under partial pressure and saturated humidity environment, and replacing the culture medium for amplification every 1 day during the culture processAnd then the process is finished.
Example 2
An additive for in vitro amplification of human embryonic stem cells is added into a high-sugar DMEM culture medium for in vitro amplification of embryonic stem cells, and the concentration of each component in the additive is as follows according to the final concentration of the culture medium: brassinolide 1.5ng/mL, sodium mercaptosuccinate 2.8ng/mL, stigmasterol 13.3ng/mL, gentiopicrin 10.0ng/mL, kanamycin 5ng/mL, L-glutamine 15 ng/mL.
An in vitro amplification method of human embryonic stem cells comprises the following steps:
(1) inoculating the human embryonic stem cells separated in vitro into a high-glucose DMEM culture medium, and pre-culturing for 24 hours in a culture dish coated with fibronectin;
(2) adding the additives into a high-glucose DMEM culture medium to obtain a culture medium for amplification, and inoculating the human embryonic stem cells separated in vitro in the step (1) into the culture medium for amplification at a density of 5 x 104one/mL, 5% CO at 37 ℃2Culturing for 7 days under the environment of partial pressure and saturated humidity, and replacing the culture medium for amplification every 1 day in the culture process to finish the process.
Example 3
An additive for in vitro amplification of human embryonic stem cells is added into a high-sugar DMEM culture medium for in vitro amplification of embryonic stem cells, and the concentration of each component in the additive is as follows according to the final concentration of the culture medium: brassinolide 4.5ng/mL, sodium mercaptosuccinate 5.0ng/mL, stigmasterol 17.5ng/mL, gentiopicrin 14.5ng/mL, kanamycin 10ng/mL, L-glutamine 20 ng/mL.
An in vitro amplification method of human embryonic stem cells comprises the following steps:
(1) inoculating the human embryonic stem cells separated in vitro into a high-glucose DMEM culture medium, and pre-culturing for 24 hours in a culture dish coated with fibronectin;
(2) adding the additives into a high-glucose DMEM culture medium to obtain a culture medium for amplification, and inoculating the human embryonic stem cells separated in vitro in the step (1) into the culture medium for amplification at the inoculation density of 8 multiplied by 104one/mL, 5% CO at 37 ℃2Culturing for 7 days under partial pressure and saturated humidity environmentReplacing the culture medium for amplification every 1 day in the culture process.
Comparative example 1
Comparative example 1 provides an additive for in vitro amplification of human embryonic stem cells, which is different from example 1 in that: brassinolide was omitted and the rest was the same as in example 1.
Comparative example 2
Comparative example 2 provides an additive for in vitro amplification of human embryonic stem cells, which is different from example 1 in that: sodium mercaptosuccinate was omitted and the procedure was as in example 1.
Comparative example 3
Comparative example 3 provides an additive for in vitro amplification of human embryonic stem cells, which is different from example 1 in that: sodium mercaptosuccinate was omitted and the amount of brassinolide was adjusted to 5.5ng/mL, the remainder being the same as in example 1.
Comparative example 4
Comparative example 4 provides an additive for in vitro amplification of human embryonic stem cells, which is different from example 1 in that: brassinolide was omitted, and the amount of sodium mercaptosuccinate was adjusted to 5.5ng/mL, the remainder being the same as in example 1.
Comparative example 5
Comparative example 5 provides an additive for in vitro amplification of human embryonic stem cells, which is different from example 1 in that: stigmasterol was omitted and the procedure was as in example 1.
Comparative example 6
Comparative example 6 provides an additive for in vitro amplification of human embryonic stem cells, which is different from example 1 in that: gentiopicroside was omitted and the procedure was as in example 1.
Comparative example 7
Comparative example 7 provides an additive for in vitro amplification of human embryonic stem cells, which is different from example 1 in that: gentiopicroside was omitted and the amount of stigmasterol was adjusted to 28.8 ng/mL, the remainder being the same as in example 1.
Comparative example 8
Comparative example 8 provides an additive for in vitro amplification of human embryonic stem cells, which is different from example 1 in that: stigmasterol was omitted and the amount of gentiopicroside used was adjusted to 28.8 ng/mL, the remainder being the same as in example 1.
The proliferation rate and the cell viability of the embryonic stem cells of examples 1 to 3 and comparative examples 1 to 8 were counted, wherein the proliferation rate = total number of cells counted for 7 days in culture/initial inoculation number, and the cell viability rate = number of viable cells/total number of cells × 100%, and the number of cells was counted by trypan blue staining method, and the results are shown in table 1.
TABLE 1
It can be seen from table 1 that the cell proliferation fold was much higher in examples 1 to 3 than in comparative examples 1 to 8, and the cell survival rate was slightly higher than in comparative examples 1 to 8. The composition of the additives was adjusted in comparative examples 1 to 8, as can be seen from table 1: the brassinolide, the sodium mercaptosuccinate, the stigmasterol and the gentiopicroside in the additive have synergistic effect to remarkably promote the proliferation of embryonic stem cells, shorten the proliferation period, greatly improve the proliferation multiple of the cells, and finally obtain a large number of cells through amplification and have high cell survival rate.
The additive and the basic culture medium are adopted to carry out in-vitro amplification on the embryonic stem cells, so that a large number of high-quality human embryonic stem cells can be obtained in a short time, animal serum is not needed, the safety is high, and the requirements of scientific research and clinic are fully met.
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.
Claims (7)
1. An additive for in vitro amplification of human embryonic stem cells, which is added to a basic culture medium for in vitro amplification of embryonic stem cells, wherein the additive comprises the following components: brassinolide, sodium mercaptosuccinate, stigmasterol, gentiopicrin, kanamycin and L-glutamine.
2. The additive for in vitro expansion of human embryonic stem cells according to claim 1, wherein the concentration of each component in the additive is, based on the final concentration of the culture medium: brassinolide 1.5-4.5ng/mL, sodium mercaptosuccinate 2.8-5.0ng/mL, stigmasterol 13.3-17.5ng/mL, gentiopicrin 10.0-14.5ng/mL, kanamycin 5-10ng/mL, and L-glutamine 15-20 ng/mL.
3. The additive for in vitro expansion of human embryonic stem cells according to claim 2, wherein the concentration of each component in the additive is, based on the final concentration of the culture medium: 2.0ng/mL of brassinolide, 3.5ng/mL of sodium mercaptosuccinate, 15.8ng/mL of stigmasterol, 13.0 ng/mL of gentiopicrin, 8ng/mL of kanamycin and 16ng/mL of L-glutamine.
4. An in vitro amplification method of human embryonic stem cells, which is characterized by comprising the following steps:
(1) inoculating the human embryonic stem cells separated in vitro into a basic culture medium, and pre-culturing for 24 hours in a culture dish coated with fibronectin;
(2) adding the additive according to any one of claims 1 to 3 to a basal medium to obtain an amplification medium, inoculating the embryonic stem cells of step (1) in the amplification medium, and culturing the cells at 37 ℃ in 5% CO2And culturing for 7 days under the environment of partial pressure and saturated humidity.
5. The method for in vitro expansion of human embryonic stem cells according to claim 4, wherein the medium for expansion is replaced every 1 day in step (2).
6. The in vitro human embryonic stem cell expansion method of claim 4, wherein the basic medium is a high-glucose DMEM medium.
7. The method for in vitro expansion of human embryonic stem cells according to claim 4, wherein the seeded cell density in step (2) is 5-8X 104one/mL.
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Citations (3)
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| WO2006029198A2 (en) * | 2004-09-08 | 2006-03-16 | Wisconsin Alumni Research Foundation | Culturing human embryonic stem cells |
| CN1827766A (en) * | 2001-06-28 | 2006-09-06 | 徐荣祥 | In vitro cell cultivation method |
| CN1844374A (en) * | 2006-05-17 | 2006-10-11 | 北京大学 | Culture method for human embryonic stem cell and special culture medium thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1827766A (en) * | 2001-06-28 | 2006-09-06 | 徐荣祥 | In vitro cell cultivation method |
| WO2006029198A2 (en) * | 2004-09-08 | 2006-03-16 | Wisconsin Alumni Research Foundation | Culturing human embryonic stem cells |
| CN1844374A (en) * | 2006-05-17 | 2006-10-11 | 北京大学 | Culture method for human embryonic stem cell and special culture medium thereof |
Non-Patent Citations (1)
| Title |
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| 庾石山: "《中华医学百科全书 天然药物化学》", vol. 1, 31 March 2021, 中国协和医科大学出版社, pages: 137 * |
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