CN113462636B - Improved method for differentiating epidermal stem cells into liver cells - Google Patents
Improved method for differentiating epidermal stem cells into liver cells Download PDFInfo
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- 210000002514 epidermal stem cell Anatomy 0.000 title claims abstract description 250
- 210000005229 liver cell Anatomy 0.000 title claims abstract description 132
- 238000000034 method Methods 0.000 title claims abstract description 98
- 230000004069 differentiation Effects 0.000 claims abstract description 37
- 238000004113 cell culture Methods 0.000 claims abstract description 33
- 230000006698 induction Effects 0.000 claims abstract description 33
- 239000006285 cell suspension Substances 0.000 claims abstract description 32
- 230000000644 propagated effect Effects 0.000 claims abstract description 32
- 238000012258 culturing Methods 0.000 claims abstract description 31
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 claims abstract description 16
- 102000004218 Insulin-Like Growth Factor I Human genes 0.000 claims abstract description 16
- 108090001117 Insulin-Like Growth Factor II Proteins 0.000 claims abstract description 16
- 102000048143 Insulin-Like Growth Factor II Human genes 0.000 claims abstract description 16
- 210000001339 epidermal cell Anatomy 0.000 claims abstract description 16
- 230000008014 freezing Effects 0.000 claims abstract description 16
- 238000007710 freezing Methods 0.000 claims abstract description 16
- 230000004927 fusion Effects 0.000 claims abstract description 16
- 238000002955 isolation Methods 0.000 claims abstract description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 36
- 239000001963 growth medium Substances 0.000 claims description 34
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 claims description 30
- 239000011780 sodium chloride Substances 0.000 claims description 18
- 239000012930 cell culture fluid Substances 0.000 claims description 17
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 16
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims description 16
- 235000010413 sodium alginate Nutrition 0.000 claims description 16
- 239000000661 sodium alginate Substances 0.000 claims description 16
- 229940005550 sodium alginate Drugs 0.000 claims description 16
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 15
- 239000001110 calcium chloride Substances 0.000 claims description 15
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 15
- 235000011056 potassium acetate Nutrition 0.000 claims description 15
- 229940088594 vitamin Drugs 0.000 claims description 15
- 229930003231 vitamin Natural products 0.000 claims description 15
- 235000013343 vitamin Nutrition 0.000 claims description 15
- 239000011782 vitamin Substances 0.000 claims description 15
- 229960002901 sodium glycerophosphate Drugs 0.000 claims description 14
- 239000001569 carbon dioxide Substances 0.000 claims description 8
- 229910002092 carbon dioxide Inorganic materials 0.000 claims description 8
- 239000000203 mixture Substances 0.000 abstract description 15
- 210000004027 cell Anatomy 0.000 abstract description 9
- 238000003501 co-culture Methods 0.000 abstract description 4
- 108010009583 Transforming Growth Factors Proteins 0.000 abstract description 3
- 102000009618 Transforming Growth Factors Human genes 0.000 abstract description 3
- 230000000052 comparative effect Effects 0.000 description 17
- 238000006243 chemical reaction Methods 0.000 description 13
- 210000000130 stem cell Anatomy 0.000 description 5
- 210000001671 embryonic stem cell Anatomy 0.000 description 3
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 3
- 210000004504 adult stem cell Anatomy 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 210000003014 totipotent stem cell Anatomy 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 210000002449 bone cell Anatomy 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 210000004413 cardiac myocyte Anatomy 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 210000001612 chondrocyte Anatomy 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 238000012637 gene transfection Methods 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 230000003394 haemopoietic effect Effects 0.000 description 1
- 230000003832 immune regulation Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 210000001778 pluripotent stem cell Anatomy 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
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Abstract
The invention discloses an improved method for differentiating epidermal stem cells into liver cells, and belongs to the technical field of cell engineering. It comprises the following steps: (1) pretreatment of epidermal stem cells: transferring the epidermal stem cells into a cell culture solution for culture to obtain propagated epidermal stem cells; (2) isolation treatment of epidermal stem cells: culturing the epidermal cells propagated in the step (1) for the second time until the epidermal stem cells grow to 70% of fusion degree, wherein the steps are as follows: 3, carrying out passage, transferring to 3 generations, and then carrying out freezing storage; (3) differentiation treatment of epidermal stem cells: mixing the frozen epidermal stem cells in the step (2) with liver cells, preparing a cell suspension, and then placing the mixture in an induction solution until the differentiation process is completed. The invention is based on a co-culture system of the epidermal stem cells and the liver cells, skillfully makes the epidermal stem cells and the liver cells work separately by using the co-culture system, and introduces the transforming growth factors IGF-1 and IGF-2 into the epidermal stem cells by using an induction solution.
Description
Technical Field
The invention belongs to the technical field of cell engineering, and particularly relates to an improved method for differentiating epidermal stem cells into liver cells.
Background
Human studies on epidermal stem cells have been made for decades, but it has been considered as a pluripotent stem cell due to its difficulty in inducing differentiation, i.e., only differentiation into the relevant components of the epidermis. The research of stem cells has taken an important role in the field of regenerative medicine, because of their multipotency, the ability of stem cells to differentiate into various tissues and organs under specific conditions has been attracting attention of scientists. An embryonic stem cell is a totipotent stem cell that can differentiate into all tissues and organs except placenta. However, since research on embryonic stem cells involves many problems of ethics, those skilled in the art have been striving to find another totipotent stem cell to replace embryonic stem cells.
One gradually locks the target onto adult stem cells. At present, those skilled in the art have successfully induced differentiation of adult stem cells such as MSCs (mesenchymal stem cells), ADSCs (adipose stem cells), etc. into other types of cells such as chondrocytes, bone cells, cardiomyocytes, etc. MSCs were originally found in bone marrow and have been increasingly attracting attention due to their multipotent differentiation potential, hematopoietic support, and promotion of stem cell engraftment, immune regulation, and self-replication. Epidermal stem cells, also known as specific stem cells or unipotent cells, refer to cells that produce only one cell type, but have self-renewing properties. The skilled artisan always classifies epidermal stem cells as pluripotent cells. The range of applications for epidermal stem cells is also limited by the influence of pluripotent cells.
Disclosure of Invention
Problems to be solved
Aiming at the problems in the prior art, the invention provides an improved method for differentiating the epidermal stem cells into the liver cells, which has simple process and can effectively realize the cell differentiation between two cells.
Technical proposal
In order to solve the problems, the invention adopts the following technical scheme.
An improved method for differentiating epidermal stem cells into liver cells, comprising the steps of:
(1) Pretreatment of epidermal stem cells: transferring the epidermal stem cells into a cell culture solution for culture to obtain propagated epidermal stem cells;
(2) Isolation treatment of epidermal stem cells: culturing the epidermal cells propagated in the step (1) for the second time until the epidermal stem cells grow to 70% of fusion degree, wherein the steps are as follows: 3, carrying out passage, transferring to 3 generations, and then carrying out freezing storage;
(3) Differentiation treatment of epidermal stem cells: mixing the frozen epidermal stem cells in the step (2) with liver cells, preparing a cell suspension, and then placing the mixture in an induction solution until the differentiation process is completed.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the density of the epidermal stem cells after culturing in the step (1) is 1x10 5 And each mL.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the components of the cell culture fluid in step (1) are as follows:
10 to 20 parts of Ham's F to 10 culture medium,
1 to 3 parts of sodium chloride,
0.5-0.8 part of potassium acetate;
wherein the culture temperature is 37 ℃, the culture time is 7d, and the carbon dioxide concentration is 5%.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the components of the cell culture fluid in step (1) are as follows:
12 to 16 parts of Ham's F-10 culture medium,
1 to 3 parts of sodium chloride,
0.5-0.8 part of potassium acetate.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the components of the cell culture fluid in step (1) are as follows:
14 parts of Ham's F-10 culture medium,
2 parts of sodium chloride,
0.7 parts of potassium acetate.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the components of the cell culture solution for the secondary culture in the step (2) are as follows:
10 to 20 parts of Ham's F to 10 culture medium,
1 to 3 parts of sodium alginate,
0.5-0.8 part of calcium chloride;
wherein the culture temperature is 37 ℃, the culture time is 7d, and the carbon dioxide concentration is 5%.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the components of the cell culture solution for the secondary culture in the step (2) are as follows:
12 to 18 parts of Ham's F to 10 culture medium,
1 to 3 parts of sodium alginate,
0.5-0.8 part of calcium chloride.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the components of the cell culture solution for the secondary culture in the step (2) are as follows:
15 parts of Ham's F-10 culture medium,
2 parts of sodium alginate,
0.6 part of calcium chloride.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the quantitative ratio between the epidermal stem cells and the liver cells in the step (3) is 1:0.5.
in the above-described improved method for differentiating epidermal stem cells into liver cells,
the density of the cell suspension in step (3) was 1X10 6 individual/mL;
the components of the induction liquid in the step (3) are as follows:
vitamin C60 mug/L,
beta-sodium glycerophosphate 15mol/L,
10% by volume of FBS,
IGF-1 25μg/L，
IGF-2 20μg/L。
beneficial effects of
Compared with the prior art, the invention has the beneficial effects that:
the invention is based on a co-culture system of the epidermal stem cells and the liver cells, skillfully makes the epidermal stem cells and the liver cells work separately by using the co-culture system, introduces the transforming growth factors IGF-1 and IGF-2 into the epidermal stem cells by adopting an induction solution, provides continuous, local and over-expressed beta-sodium glycerophosphate for the liver cells by the transfected epidermal stem cells, stimulates the epidermal stem cells to differentiate towards the liver cells, ensures that the transforming growth factors do not need to be added exogenously and are provided by the transfected liver cells, thereby preventing the growth factors from being decomposed by protease, avoiding the possible damage to the epidermal stem cells in the gene transfection process, and keeping the higher survival rate and better differentiating capability of the epidermal stem cells.
Detailed Description
The invention is further described below in connection with specific embodiments.
Example 1
The improved method for differentiating the epidermal stem cells into the liver cells comprises the following steps:
(1) Pretreatment of epidermal stem cells: transferring the epidermal stem cells into a cell culture solution for culture to obtain propagated epidermal stem cells;
(2) Isolation treatment of epidermal stem cells: culturing the epidermal cells propagated in the step (1) for the second time until the epidermal stem cells grow to 70% of fusion degree, wherein the steps are as follows: 3, carrying out passage, transferring to 3 generations, and then carrying out freezing storage;
(3) Differentiation treatment of epidermal stem cells: mixing the frozen epidermal stem cells in the step (2) with liver cells, preparing a cell suspension, and then placing the mixture in an induction solution until the differentiation process is completed.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the density of the epidermal stem cells after culturing in the step (1) is 1x10 5 And each mL.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the components of the cell culture fluid in step (1) are as follows:
10 parts of Ham's F-10 culture medium,
3 parts of sodium chloride,
0.5 parts of potassium acetate;
wherein the culture temperature is 37 ℃, the culture time is 7d, and the carbon dioxide concentration is 5%.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the components of the cell culture solution for the secondary culture in the step (2) are as follows:
10 parts of Ham's F-10 culture medium,
3 parts of sodium alginate,
0.5 parts of calcium chloride;
wherein the culture temperature is 37 ℃, the culture time is 7d, and the carbon dioxide concentration is 5%.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the quantitative ratio between the epidermal stem cells and the liver cells in the step (3) is 1:0.5.
in the above-described improved method for differentiating epidermal stem cells into liver cells,
the density of the cell suspension in step (3) was 1X10 6 individual/mL;
the components of the induction liquid in the step (3) are as follows:
vitamin C60 mug/L,
beta-sodium glycerophosphate 15mol/L,
10% by volume of FBS,
IGF-1 25μg/L，
IGF-2 20μg/L。
example 2
The improved method for differentiating the epidermal stem cells into the liver cells comprises the following steps:
(1) Pretreatment of epidermal stem cells: transferring the epidermal stem cells into a cell culture solution for culture to obtain propagated epidermal stem cells;
(2) Isolation treatment of epidermal stem cells: culturing the epidermal cells propagated in the step (1) for the second time until the epidermal stem cells grow to 70% of fusion degree, wherein the steps are as follows: 3, carrying out passage, transferring to 3 generations, and then carrying out freezing storage;
(3) Differentiation treatment of epidermal stem cells: mixing the frozen epidermal stem cells in the step (2) with liver cells, preparing a cell suspension, and then placing the mixture in an induction solution until the differentiation process is completed.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the density of the epidermal stem cells after culturing in the step (1) is 1x10 5 And each mL.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the components of the cell culture fluid in step (1) are as follows:
20 parts of Ham's F-10 culture medium,
1 part of sodium chloride, and the total weight of the sodium chloride,
0.8 parts of potassium acetate;
wherein the culture temperature is 37 ℃, the culture time is 7d, and the carbon dioxide concentration is 5%.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the components of the cell culture solution for the secondary culture in the step (2) are as follows:
20 parts of Ham's F-10 culture medium,
1 part of sodium alginate,
0.8 parts of calcium chloride;
wherein the culture temperature is 37 ℃, the culture time is 7d, and the carbon dioxide concentration is 5%.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the quantitative ratio between the epidermal stem cells and the liver cells in the step (3) is 1:0.5.
in the above-described improved method for differentiating epidermal stem cells into liver cells,
the density of the cell suspension in step (3) was 1X10 6 individual/mL;
the components of the induction liquid in the step (3) are as follows:
vitamin C60 mug/L,
beta-sodium glycerophosphate 15mol/L,
10% by volume of FBS,
IGF-1 25μg/L，
IGF-2 20μg/L。
example 3
The improved method for differentiating the epidermal stem cells into the liver cells comprises the following steps:
(1) Pretreatment of epidermal stem cells: transferring the epidermal stem cells into a cell culture solution for culture to obtain propagated epidermal stem cells;
(2) Isolation treatment of epidermal stem cells: culturing the epidermal cells propagated in the step (1) for the second time until the epidermal stem cells grow to 70% of fusion degree, wherein the steps are as follows: 3, carrying out passage, transferring to 3 generations, and then carrying out freezing storage;
(3) Differentiation treatment of epidermal stem cells: mixing the frozen epidermal stem cells in the step (2) with liver cells, preparing a cell suspension, and then placing the mixture in an induction solution until the differentiation process is completed.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the density of the epidermal stem cells after culturing in the step (1) is 1x10 5 And each mL.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the components of the cell culture fluid in step (1) are as follows:
12 parts of Ham's F-10 culture medium,
3 parts of sodium chloride,
0.5 parts of potassium acetate.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the components of the cell culture solution for the secondary culture in the step (2) are as follows:
12 parts of Ham's F-10 culture medium,
3 parts of sodium alginate,
0.5 parts of calcium chloride.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the quantitative ratio between the epidermal stem cells and the liver cells in the step (3) is 1:0.5.
in the above-described improved method for differentiating epidermal stem cells into liver cells,
the density of the cell suspension in step (3) was 1X10 6 individual/mL;
the components of the induction liquid in the step (3) are as follows:
vitamin C60 mug/L,
beta-sodium glycerophosphate 15mol/L,
10% by volume of FBS,
IGF-1 25μg/L，
IGF-2 20μg/L。
example 4
The improved method for differentiating the epidermal stem cells into the liver cells comprises the following steps:
(1) Pretreatment of epidermal stem cells: transferring the epidermal stem cells into a cell culture solution for culture to obtain propagated epidermal stem cells;
(2) Isolation treatment of epidermal stem cells: culturing the epidermal cells propagated in the step (1) for the second time until the epidermal stem cells grow to 70% of fusion degree, wherein the steps are as follows: 3, carrying out passage, transferring to 3 generations, and then carrying out freezing storage;
(3) Differentiation treatment of epidermal stem cells: mixing the frozen epidermal stem cells in the step (2) with liver cells, preparing a cell suspension, and then placing the mixture in an induction solution until the differentiation process is completed.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the density of the epidermal stem cells after culturing in the step (1) is 1x10 5 And each mL.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the components of the cell culture fluid in step (1) are as follows:
16 parts of Ham's F-10 culture medium,
1 part of sodium chloride, and the total weight of the sodium chloride,
0.8 parts of potassium acetate.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the components of the cell culture solution for the secondary culture in the step (2) are as follows:
18 parts of Ham's F-10 culture medium,
1 part of sodium alginate,
0.8 parts of calcium chloride.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the quantitative ratio between the epidermal stem cells and the liver cells in the step (3) is 1:0.5.
in the above-described improved method for differentiating epidermal stem cells into liver cells,
the density of the cell suspension in step (3) was 1X10 6 individual/mL;
the components of the induction liquid in the step (3) are as follows:
vitamin C60 mug/L,
beta-sodium glycerophosphate 15mol/L,
10% by volume of FBS,
IGF-1 25μg/L，
IGF-2 20μg/L。
example 5
The improved method for differentiating the epidermal stem cells into the liver cells comprises the following steps:
(1) Pretreatment of epidermal stem cells: transferring the epidermal stem cells into a cell culture solution for culture to obtain propagated epidermal stem cells;
(2) Isolation treatment of epidermal stem cells: culturing the epidermal cells propagated in the step (1) for the second time until the epidermal stem cells grow to 70% of fusion degree, wherein the steps are as follows: 3, carrying out passage, transferring to 3 generations, and then carrying out freezing storage;
(3) Differentiation treatment of epidermal stem cells: mixing the frozen epidermal stem cells in the step (2) with liver cells, preparing a cell suspension, and then placing the mixture in an induction solution until the differentiation process is completed.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the density of the epidermal stem cells after culturing in the step (1) is 1x10 5 And each mL.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the components of the cell culture fluid in step (1) are as follows:
14 parts of Ham's F-10 culture medium,
2 parts of sodium chloride,
0.7 parts of potassium acetate.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the components of the cell culture solution for the secondary culture in the step (2) are as follows:
15 parts of Ham's F-10 culture medium,
2 parts of sodium alginate,
0.6 part of calcium chloride.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the quantitative ratio between the epidermal stem cells and the liver cells in the step (3) is 1:0.5.
in the above-described improved method for differentiating epidermal stem cells into liver cells,
the density of the cell suspension in step (3) was 1X10 6 individual/mL;
the components of the induction liquid in the step (3) are as follows:
vitamin C60 mug/L,
beta-sodium glycerophosphate 15mol/L,
10% by volume of FBS,
IGF-1 25μg/L，
IGF-2 20μg/L。
comparative example 1
The improved method for differentiating the epidermal stem cells into the liver cells comprises the following steps:
(1) Pretreatment of epidermal stem cells: transferring the epidermal stem cells into a cell culture solution for culture to obtain propagated epidermal stem cells;
(2) Isolation treatment of epidermal stem cells: culturing the epidermal cells propagated in the step (1) for the second time until the epidermal stem cells grow to 70% of fusion degree, wherein the steps are as follows: 3, carrying out passage, transferring to 3 generations, and then carrying out freezing storage;
(3) Differentiation treatment of epidermal stem cells: mixing the frozen epidermal stem cells in the step (2) with liver cells, preparing a cell suspension, and then placing the mixture in an induction solution until the differentiation process is completed.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the density of the epidermal stem cells after culturing in the step (1) is 1x10 5 And each mL.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the components of the cell culture fluid in step (1) are as follows:
14 parts of Ham's F-10 culture medium,
sodium chloride 2 parts.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the components of the cell culture solution for the secondary culture in the step (2) are as follows:
15 parts of Ham's F-10 culture medium,
2 parts of sodium alginate,
0.6 part of calcium chloride.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the quantitative ratio between the epidermal stem cells and the liver cells in the step (3) is 1:0.5.
in the above-described improved method for differentiating epidermal stem cells into liver cells,
the density of the cell suspension in step (3) was 1X10 6 individual/mL;
the components of the induction liquid in the step (3) are as follows:
vitamin C60 mug/L,
beta-sodium glycerophosphate 15mol/L,
10% by volume of FBS,
IGF-1 25μg/L，
IGF-2 20μg/L。
comparative example 2
The improved method for differentiating the epidermal stem cells into the liver cells comprises the following steps:
(1) Pretreatment of epidermal stem cells: transferring the epidermal stem cells into a cell culture solution for culture to obtain propagated epidermal stem cells;
(2) Isolation treatment of epidermal stem cells: culturing the epidermal cells propagated in the step (1) for the second time until the epidermal stem cells grow to 70% of fusion degree, wherein the steps are as follows: 3, carrying out passage, transferring to 3 generations, and then carrying out freezing storage;
(3) Differentiation treatment of epidermal stem cells: mixing the frozen epidermal stem cells in the step (2) with liver cells, preparing a cell suspension, and then placing the mixture in an induction solution until the differentiation process is completed.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the density of the epidermal stem cells after culturing in the step (1) is 1x10 5 And each mL.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the components of the cell culture fluid in step (1) are as follows:
14 parts of Ham's F-10 culture medium,
2 parts of sodium chloride,
0.7 parts of potassium acetate.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the components of the cell culture solution for the secondary culture in the step (2) are as follows:
15 parts of Ham's F-10 culture medium,
sodium alginate 2 parts.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the quantitative ratio between the epidermal stem cells and the liver cells in the step (3) is 1:0.5.
in the above-described improved method for differentiating epidermal stem cells into liver cells,
the density of the cell suspension in step (3) was 1X10 6 individual/mL;
the components of the induction liquid in the step (3) are as follows:
vitamin C60 mug/L,
beta-sodium glycerophosphate 15mol/L,
10% by volume of FBS,
IGF-1 25μg/L，
IGF-2 20μg/L。
comparative example 3
The improved method for differentiating the epidermal stem cells into the liver cells comprises the following steps:
(1) Pretreatment of epidermal stem cells: transferring the epidermal stem cells into a cell culture solution for culture to obtain propagated epidermal stem cells;
(2) Isolation treatment of epidermal stem cells: culturing the epidermal cells propagated in the step (1) for the second time until the epidermal stem cells grow to 70% of fusion degree, wherein the steps are as follows: 3, carrying out passage, transferring to 3 generations, and then carrying out freezing storage;
(3) Differentiation treatment of epidermal stem cells: mixing the frozen epidermal stem cells in the step (2) with liver cells, preparing a cell suspension, and then placing the mixture in an induction solution until the differentiation process is completed.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the density of the epidermal stem cells after culturing in the step (1) is 1x10 5 And each mL.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the components of the cell culture fluid in step (1) are as follows:
14 parts of Ham's F-10 culture medium.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the components of the cell culture solution for the secondary culture in the step (2) are as follows:
15 parts of Ham's F-10 culture medium,
2 parts of sodium alginate,
0.6 part of calcium chloride.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the quantitative ratio between the epidermal stem cells and the liver cells in the step (3) is 1:0.5.
in the above-described improved method for differentiating epidermal stem cells into liver cells,
the density of the cell suspension in step (3) was 1X10 6 individual/mL;
the components of the induction liquid in the step (3) are as follows:
vitamin C60 mug/L,
beta-sodium glycerophosphate 15mol/L,
10% by volume of FBS,
IGF-1 25μg/L，
IGF-2 20μg/L。
comparative example 4
The improved method for differentiating the epidermal stem cells into the liver cells comprises the following steps:
(1) Pretreatment of epidermal stem cells: transferring the epidermal stem cells into a cell culture solution for culture to obtain propagated epidermal stem cells;
(2) Isolation treatment of epidermal stem cells: culturing the epidermal cells propagated in the step (1) for the second time until the epidermal stem cells grow to 70% of fusion degree, wherein the steps are as follows: 3, carrying out passage, transferring to 3 generations, and then carrying out freezing storage;
(3) Differentiation treatment of epidermal stem cells: mixing the frozen epidermal stem cells in the step (2) with liver cells, preparing a cell suspension, and then placing the mixture in an induction solution until the differentiation process is completed.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the density of the epidermal stem cells after culturing in the step (1) is 1x10 5 And each mL.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the components of the cell culture fluid in step (1) are as follows:
14 parts of Ham's F-10 culture medium,
2 parts of sodium chloride,
0.7 parts of potassium acetate.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the components of the cell culture solution for the secondary culture in the step (2) are as follows:
15 parts of Ham's F-10 culture medium.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the quantitative ratio between the epidermal stem cells and the liver cells in the step (3) is 1:0.5.
in the above-described improved method for differentiating epidermal stem cells into liver cells,
the density of the cell suspension in step (3) was 1X10 6 individual/mL;
the components of the induction liquid in the step (3) are as follows:
vitamin C60 mug/L,
beta-sodium glycerophosphate 15mol/L,
10% by volume of FBS,
IGF-1 25μg/L，
IGF-2 20μg/L。
comparative example 5
The improved method for differentiating the epidermal stem cells into the liver cells comprises the following steps:
(1) Pretreatment of epidermal stem cells: transferring the epidermal stem cells into a cell culture solution for culture to obtain propagated epidermal stem cells;
(2) Isolation treatment of epidermal stem cells: culturing the epidermal cells propagated in the step (1) for the second time until the epidermal stem cells grow to 70% of fusion degree, wherein the steps are as follows: 3, carrying out passage, transferring to 3 generations, and then carrying out freezing storage;
(3) Differentiation treatment of epidermal stem cells: mixing the frozen epidermal stem cells in the step (2) with liver cells, preparing a cell suspension, and then placing the mixture in an induction solution until the differentiation process is completed.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the density of the epidermal stem cells after culturing in the step (1) is 1x10 5 And each mL.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the components of the cell culture fluid in step (1) are as follows:
14 parts of Ham's F-10 culture medium,
2 parts of sodium chloride,
0.7 parts of potassium acetate.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the components of the cell culture solution for the secondary culture in the step (2) are as follows:
15 parts of Ham's F-10 culture medium,
2 parts of sodium alginate,
0.6 part of calcium chloride.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the quantitative ratio between the epidermal stem cells and the liver cells in the step (3) is 1:0.5.
in the above-described improved method for differentiating epidermal stem cells into liver cells,
the density of the cell suspension in step (3) was 1X10 6 individual/mL;
the components of the induction liquid in the step (3) are as follows:
vitamin C60 mug/L,
10% by volume of FBS,
IGF-1 25μg/L，
IGF-2 20μg/L。
comparative example 6
The improved method for differentiating the epidermal stem cells into the liver cells comprises the following steps:
(1) Pretreatment of epidermal stem cells: transferring the epidermal stem cells into a cell culture solution for culture to obtain propagated epidermal stem cells;
(2) Isolation treatment of epidermal stem cells: culturing the epidermal cells propagated in the step (1) for the second time until the epidermal stem cells grow to 70% of fusion degree, wherein the steps are as follows: 3, carrying out passage, transferring to 3 generations, and then carrying out freezing storage;
(3) Differentiation treatment of epidermal stem cells: mixing the frozen epidermal stem cells in the step (2) with liver cells, preparing a cell suspension, and then placing the mixture in an induction solution until the differentiation process is completed.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the density of the epidermal stem cells after culturing in the step (1) is 1x10 5 And each mL.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the components of the cell culture fluid in step (1) are as follows:
14 parts of Ham's F-10 culture medium,
2 parts of sodium chloride,
0.7 parts of potassium acetate.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the components of the cell culture solution for the secondary culture in the step (2) are as follows:
15 parts of Ham's F-10 culture medium,
2 parts of sodium alginate,
0.6 part of calcium chloride.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the quantitative ratio between the epidermal stem cells and the liver cells in the step (3) is 1:0.5.
in the above-described improved method for differentiating epidermal stem cells into liver cells,
the density of the cell suspension in step (3) was 1X10 6 individual/mL;
the components of the induction liquid in the step (3) are as follows:
vitamin C60 mug/L,
beta-sodium glycerophosphate 15mol/L,
IGF-1 25μg/L，
IGF-2 20μg/L。
comparative example 7
The improved method for differentiating the epidermal stem cells into the liver cells comprises the following steps:
(1) Pretreatment of epidermal stem cells: transferring the epidermal stem cells into a cell culture solution for culture to obtain propagated epidermal stem cells;
(2) Isolation treatment of epidermal stem cells: culturing the epidermal cells propagated in the step (1) for the second time until the epidermal stem cells grow to 70% of fusion degree, wherein the steps are as follows: 3, carrying out passage, transferring to 3 generations, and then carrying out freezing storage;
(3) Differentiation treatment of epidermal stem cells: mixing the frozen epidermal stem cells in the step (2) with liver cells, preparing a cell suspension, and then placing the mixture in an induction solution until the differentiation process is completed.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the density of the epidermal stem cells after culturing in the step (1) is 1x10 5 And each mL.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the components of the cell culture fluid in step (1) are as follows:
14 parts of Ham's F-10 culture medium,
2 parts of sodium chloride,
0.7 parts of potassium acetate.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the components of the cell culture solution for the secondary culture in the step (2) are as follows:
15 parts of Ham's F-10 culture medium,
2 parts of sodium alginate,
0.6 part of calcium chloride.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the quantitative ratio between the epidermal stem cells and the liver cells in the step (3) is 1:0.5.
in the above-described improved method for differentiating epidermal stem cells into liver cells,
step (a)(3) The density of the medium cell suspension was 1x10 6 individual/mL;
the components of the induction liquid in the step (3) are as follows:
vitamin C60 mug/L,
beta-sodium glycerophosphate 15mol/L,
IGF-2 20μg/L。
comparative example 8
The improved method for differentiating the epidermal stem cells into the liver cells comprises the following steps:
(1) Pretreatment of epidermal stem cells: transferring the epidermal stem cells into a cell culture solution for culture to obtain propagated epidermal stem cells;
(2) Isolation treatment of epidermal stem cells: culturing the epidermal cells propagated in the step (1) for the second time until the epidermal stem cells grow to 70% of fusion degree, wherein the steps are as follows: 3, carrying out passage, transferring to 3 generations, and then carrying out freezing storage;
(3) Differentiation treatment of epidermal stem cells: mixing the frozen epidermal stem cells in the step (2) with liver cells, preparing a cell suspension, and then placing the mixture in an induction solution until the differentiation process is completed.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the density of the epidermal stem cells after culturing in the step (1) is 1x10 5 And each mL.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the components of the cell culture fluid in step (1) are as follows:
14 parts of Ham's F-10 culture medium,
2 parts of sodium chloride,
0.7 parts of potassium acetate.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the components of the cell culture solution for the secondary culture in the step (2) are as follows:
15 parts of Ham's F-10 culture medium,
2 parts of sodium alginate,
0.6 part of calcium chloride.
In the above-described improved method for differentiating epidermal stem cells into liver cells,
the quantitative ratio between the epidermal stem cells and the liver cells in the step (3) is 1:0.5.
in the above-described improved method for differentiating epidermal stem cells into liver cells,
the density of the cell suspension in step (3) was 1X10 6 individual/mL;
the components of the induction liquid in the step (3) are as follows:
vitamin C60 mug/L,
10% by volume of FBS,
IGF-1 25μg/L。
example 6
The products prepared in examples 1-5 and comparative examples 1-8 were selected for the following tests:
reference is made to the Chinese patent invention, application number: CN201910859880.0, publication No.: CN110684725a discloses a method for inducing stem cell-directed cartilage differentiation.
Wherein the conversion of the product prepared in example 1 was 65.4%;
wherein the conversion of the product prepared in example 2 was 66.7%;
wherein the conversion of the product prepared in example 3 was 66.1%;
wherein the product prepared in example 4 had a conversion of 68.4%;
wherein the conversion of the product prepared in example 5 was 70.2%;
wherein the conversion of the product prepared in comparative example 1 was 63.0%;
wherein the conversion of the product prepared in comparative example 2 was 60.5%;
wherein the conversion of the product prepared in comparative example 3 was 52.4%;
wherein the conversion of the product prepared in comparative example 4 was 58.2%;
wherein the conversion of the product prepared in comparative example 5 was 36.1%;
wherein the conversion of the product prepared in comparative example 6 was 20.5%;
wherein the conversion of the product prepared in comparative example 7 was 24.2%;
wherein the conversion of the product prepared in comparative example 8 was 11.8%.
The foregoing is a further elaboration of the present invention in connection with the detailed description, and it is not intended that the invention be limited to the specific embodiments shown, but rather that a number of simple deductions or substitutions be made by one of ordinary skill in the art without departing from the spirit of the invention, should be considered as falling within the scope of the invention as defined in the appended claims.
Claims (1)
1. An improved method for differentiating epidermal stem cells into liver cells, which is characterized in that:
the method comprises the following steps:
(1) Pretreatment of epidermal stem cells: transferring the epidermal stem cells into a cell culture solution for culture to obtain propagated epidermal stem cells;
(2) Isolation treatment of epidermal stem cells: culturing the epidermal cells propagated in the step (1) for the second time until the epidermal stem cells grow to 70% of fusion degree, wherein the steps are as follows: 3, carrying out passage, transferring to 3 generations, and then carrying out freezing storage;
(3) Differentiation treatment of epidermal stem cells: mixing the frozen epidermal stem cells in the step (2) with liver cells, preparing cell suspension, and then placing the cell suspension in an induction solution until the differentiation process is completed;
the density of the epidermal stem cells after culturing in the step (1) is 1x10 5 individual/mL;
the components of the cell culture fluid in step (1) are as follows:
14 parts of Ham's F-10 culture medium,
2 parts of sodium chloride,
0.7 parts of potassium acetate;
wherein the culture temperature is 37 ℃, the culture time is 7d, and the carbon dioxide concentration is 5%;
the components of the cell culture solution for the secondary culture in the step (2) are as follows:
15 parts of Ham's F-10 culture medium,
2 parts of sodium alginate,
0.6 parts of calcium chloride;
wherein the culture temperature is 37 ℃, the culture time is 7d, and the carbon dioxide concentration is 5%;
the quantitative ratio between the epidermal stem cells and the liver cells in the step (3) is 1:0.5;
the density of the cell suspension in step (3) was 1X10 6 individual/mL;
the components of the induction liquid in the step (3) are as follows:
vitamin C60 mug/L,
beta-sodium glycerophosphate 15mol/L,
10% by volume of FBS,
IGF-1 25μg/L，
IGF-2 20μg/L。
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