CN113462636A - Improved method for differentiation of epidermal stem cells into liver cells - Google Patents
Improved method for differentiation of epidermal stem cells into liver cells Download PDFInfo
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- 210000002514 epidermal stem cell Anatomy 0.000 title claims abstract description 259
- 210000005229 liver cell Anatomy 0.000 title claims abstract description 141
- 230000004069 differentiation Effects 0.000 title claims abstract description 120
- 238000000034 method Methods 0.000 title claims abstract description 107
- 238000004113 cell culture Methods 0.000 claims abstract description 54
- 230000001939 inductive effect Effects 0.000 claims abstract description 36
- 230000000644 propagated effect Effects 0.000 claims abstract description 32
- 239000006285 cell suspension Substances 0.000 claims abstract description 31
- 239000007788 liquid Substances 0.000 claims abstract description 17
- 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
- 238000000926 separation method Methods 0.000 claims abstract description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 62
- 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 34
- 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 31
- 235000010413 sodium alginate Nutrition 0.000 claims description 31
- 239000000661 sodium alginate Substances 0.000 claims description 31
- 229940005550 sodium alginate Drugs 0.000 claims description 31
- 239000011780 sodium chloride Substances 0.000 claims description 31
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 30
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 17
- 239000001110 calcium chloride Substances 0.000 claims description 17
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 17
- 235000011056 potassium acetate Nutrition 0.000 claims description 17
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims description 16
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 claims description 15
- 229930003268 Vitamin C Natural products 0.000 claims description 15
- 235000019154 vitamin C Nutrition 0.000 claims description 15
- 239000011718 vitamin C Substances 0.000 claims description 15
- 229960002901 sodium glycerophosphate Drugs 0.000 claims description 13
- 239000001569 carbon dioxide Substances 0.000 claims description 8
- 229910002092 carbon dioxide Inorganic materials 0.000 claims description 8
- 210000004027 cell Anatomy 0.000 abstract description 8
- 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 6
- 239000002609 medium Substances 0.000 description 4
- 210000001671 embryonic stem cell Anatomy 0.000 description 3
- DHCLVCXQIBBOPH-UHFFFAOYSA-N Glycerol 2-phosphate Chemical compound OCC(CO)OP(O)(O)=O DHCLVCXQIBBOPH-UHFFFAOYSA-N 0.000 description 2
- 210000004504 adult stem cell Anatomy 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000024245 cell differentiation Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 230000001172 regenerating effect Effects 0.000 description 2
- -1 sodium chloride Chemical compound 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 210000003014 totipotent stem cell Anatomy 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 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
- 210000001185 bone marrow Anatomy 0.000 description 1
- 210000004271 bone marrow stromal cell Anatomy 0.000 description 1
- 210000004413 cardiac myocyte Anatomy 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 210000001612 chondrocyte Anatomy 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
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000004409 osteocyte Anatomy 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 210000002444 unipotent stem cell Anatomy 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) separation treatment of epidermal stem cells: and (2) carrying out secondary culture on the epidermal cells propagated in the step (1) until the epidermal stem cells grow to have the fusion degree of 70%, and adding 1: 3, passage, transferring to 3 generations, and freezing and storing; (3) differentiation treatment of epidermal stem cells: mixing the epidermal stem cells frozen in the step (2) with the liver cells, preparing into cell suspension, and then placing into an inducing solution until the differentiation process is completed. The invention is based on a co-culture system of epidermal stem cells and liver cells, skillfully leads the epidermal stem cells and the liver cells to work in a division way by utilizing the co-culture system, and adopts an inducing liquid to lead transforming growth factors IGF-1 and IGF-2 into the epidermal stem cells.
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 in history for decades, but have been considered as unipotent stem cells due to their difficulty in inducing differentiation, i.e., capable of differentiating only into relevant components of the epidermis. Regenerative medicine has been well known in the medical field, and research on stem cells has been an important place in the regenerative medicine field, and stem cells have been spotlighted by scientists for their ability to differentiate into various tissues and organs under specific conditions due to their multi-differentiation potential. An embryonic stem cell is a totipotent stem cell that can be differentiated into all tissues and organs except the placenta. However, since the research of embryonic stem cells involves ethical problems, those skilled in the art are always working on finding another totipotent stem cell to replace the embryonic stem cell.
One gradually locks the target on adult stem cells. At present, those skilled in the art have succeeded in inducing adult stem cells such as MSC (mesenchymal stem cell), ADSC (adipose stem cell) and the like to differentiate into other types of cells such as chondrocyte, osteocyte, cardiomyocyte and the like. MSCs were originally found in bone marrow and have received increasing attention due to their multipotentiality, hematopoietic support, and stem cell engraftment, immune regulation, and self-replication. Epidermal stem cells, also known as specialized stem cells or unipotent cells, refer to cells that produce only one cell type, but have the property of self-renewal. The skilled person has classified epidermal stem cells as being unipotent. The range of applications for epidermal stem cells is also limited by the influence of unipotent cells.
Disclosure of Invention
Problems to be solved
Aiming at the problems in the prior art, the invention provides an improved method for differentiating epidermal stem cells into liver cells, which has simple process and can effectively realize the cell differentiation between two cells.
Technical scheme
In order to solve the above problems, the present invention adopts the following technical solutions.
An improved method for differentiation of 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) separation treatment of epidermal stem cells: and (2) carrying out secondary culture on the epidermal cells propagated in the step (1) until the epidermal stem cells grow to have the fusion degree of 70%, and adding 1: 3, passage, transferring to 3 generations, and freezing and storing;
(3) differentiation treatment of epidermal stem cells: mixing the epidermal stem cells frozen in the step (2) with the liver cells, preparing into cell suspension, and then placing into an inducing solution until the differentiation process is completed.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the density of the cultured epidermal stem cells in the step (1) is 1x105one/mL.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the components of the cell culture solution in the step (1) are as follows:
10-20 parts of Ham's F-10 culture medium,
1 to 3 parts of sodium chloride,
0.5-0.8 part of potassium acetate;
wherein the culture temperature is 37 deg.C, culture time is 7d, and carbon dioxide concentration is 5%.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the components of the cell culture solution in the step (1) are as follows:
12-16 parts of Ham's F-10 culture medium,
1 to 3 parts of sodium chloride,
0.5 to 0.8 portion of potassium acetate.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the components of the cell culture solution in the step (1) are as follows:
ham's F-10 culture medium 14 portions,
2 parts of sodium chloride, namely 2 parts of sodium chloride,
and 0.7 part of potassium acetate.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the components of the cell culture solution for secondary culture in the step (2) are as follows:
10-20 parts of Ham's F-10 culture medium,
1 to 3 parts of sodium alginate, namely,
0.5 to 0.8 portion of calcium chloride;
wherein the culture temperature is 37 deg.C, culture time is 7d, and carbon dioxide concentration is 5%.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the components of the cell culture solution for secondary culture in the step (2) are as follows:
12-18 parts of Ham's F-10 culture medium,
1 to 3 parts of sodium alginate, namely,
0.5 to 0.8 portion of calcium chloride.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the components of the cell culture solution for secondary culture in the step (2) are as follows:
15 parts of Ham's F-10 culture medium,
2 parts of sodium alginate, namely 2 parts of sodium alginate,
0.6 part of calcium chloride.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
in the step (3), the number ratio of the epidermal stem cells to the liver cells is 1: 0.5.
in the above-described method for improving differentiation of epidermal stem cells into liver cells,
the density of the cell suspension in the step (3) is 1x106Per mL;
the components of the inducing liquid in the step (3) are as follows:
the vitamin C accounts for 60 mu g/L,
15mol/L of beta-sodium glycerophosphate,
the volume fraction of the FBS is 10 percent,
IGF-1 25μg/L，
IGF-2 20μg/L。
has the beneficial effects of
Compared with the prior art, the invention has the beneficial effects that:
the invention is based on the co-culture system of the epidermal stem cells and the liver cells, the co-culture system is utilized to skillfully divide and cooperate the epidermal stem cells and the liver cells, the inducing liquid is adopted to introduce transforming growth factors IGF-1 and IGF-2 into the epidermal stem cells, the transfected epidermal stem cells provide continuous, local and over-expressed beta-glycerophosphate sodium for the liver cells, the epidermal stem cells are stimulated to direct the liver cell differentiation, the transforming growth factors are not required to be added externally 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 the better differentiation capability of the epidermal stem cells.
Detailed Description
The invention is further described with reference to specific examples.
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) separation treatment of epidermal stem cells: and (2) carrying out secondary culture on the epidermal cells propagated in the step (1) until the epidermal stem cells grow to have the fusion degree of 70%, and adding 1: 3, passage, transferring to 3 generations, and freezing and storing;
(3) differentiation treatment of epidermal stem cells: mixing the epidermal stem cells frozen in the step (2) with the liver cells, preparing into cell suspension, and then placing into an inducing solution until the differentiation process is completed.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the density of the cultured epidermal stem cells in the step (1) is 1x105one/mL.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the components of the cell culture solution in the step (1) are as follows:
10 parts of Ham's F-10 culture medium,
3 parts of sodium chloride, namely 3 parts of sodium chloride,
0.5 part of potassium acetate;
wherein the culture temperature is 37 deg.C, culture time is 7d, and carbon dioxide concentration is 5%.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the components of the cell culture solution for secondary culture in the step (2) are as follows:
10 parts of Ham's F-10 culture medium,
3 parts of sodium alginate, namely 3 parts of sodium alginate,
0.5 part of calcium chloride;
wherein the culture temperature is 37 deg.C, culture time is 7d, and carbon dioxide concentration is 5%.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
in the step (3), the number ratio of the epidermal stem cells to the liver cells is 1: 0.5.
in the above-described method for improving differentiation of epidermal stem cells into liver cells,
the density of the cell suspension in the step (3) is 1x106Per mL;
the components of the inducing liquid in the step (3) are as follows:
the vitamin C accounts for 60 mu g/L,
15mol/L of beta-sodium glycerophosphate,
the volume fraction of the FBS is 10 percent,
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) separation treatment of epidermal stem cells: and (2) carrying out secondary culture on the epidermal cells propagated in the step (1) until the epidermal stem cells grow to have the fusion degree of 70%, and adding 1: 3, passage, transferring to 3 generations, and freezing and storing;
(3) differentiation treatment of epidermal stem cells: mixing the epidermal stem cells frozen in the step (2) with the liver cells, preparing into cell suspension, and then placing into an inducing solution until the differentiation process is completed.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the density of the cultured epidermal stem cells in the step (1) is 1x105one/mL.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the components of the cell culture solution in the step (1) are as follows:
20 parts of Ham's F-10 culture medium,
1 part of sodium chloride, namely sodium chloride,
0.8 part of potassium acetate;
wherein the culture temperature is 37 deg.C, culture time is 7d, and carbon dioxide concentration is 5%.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the components of the cell culture solution for secondary culture in the step (2) are as follows:
20 parts of Ham's F-10 culture medium,
1 part of sodium alginate, namely 1 part of sodium alginate,
0.8 part of calcium chloride;
wherein the culture temperature is 37 deg.C, culture time is 7d, and carbon dioxide concentration is 5%.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
in the step (3), the number ratio of the epidermal stem cells to the liver cells is 1: 0.5.
in the above-described method for improving differentiation of epidermal stem cells into liver cells,
the density of the cell suspension in the step (3) is 1x106Per mL;
the components of the inducing liquid in the step (3) are as follows:
the vitamin C accounts for 60 mu g/L,
15mol/L of beta-sodium glycerophosphate,
the volume fraction of the FBS is 10 percent,
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) separation treatment of epidermal stem cells: and (2) carrying out secondary culture on the epidermal cells propagated in the step (1) until the epidermal stem cells grow to have the fusion degree of 70%, and adding 1: 3, passage, transferring to 3 generations, and freezing and storing;
(3) differentiation treatment of epidermal stem cells: mixing the epidermal stem cells frozen in the step (2) with the liver cells, preparing into cell suspension, and then placing into an inducing solution until the differentiation process is completed.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the density of the cultured epidermal stem cells in the step (1) is 1x105one/mL.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the components of the cell culture solution in the step (1) are as follows:
ham's F-10 medium 12 portions,
3 parts of sodium chloride, namely 3 parts of sodium chloride,
and 0.5 part of potassium acetate.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the components of the cell culture solution for secondary culture in the step (2) are as follows:
ham's F-10 medium 12 portions,
3 parts of sodium alginate, namely 3 parts of sodium alginate,
0.5 part of calcium chloride.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
in the step (3), the number ratio of the epidermal stem cells to the liver cells is 1: 0.5.
in the above-described method for improving differentiation of epidermal stem cells into liver cells,
the density of the cell suspension in the step (3) is 1x106Per mL;
the components of the inducing liquid in the step (3) are as follows:
the vitamin C accounts for 60 mu g/L,
15mol/L of beta-sodium glycerophosphate,
the volume fraction of the FBS is 10 percent,
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) separation treatment of epidermal stem cells: and (2) carrying out secondary culture on the epidermal cells propagated in the step (1) until the epidermal stem cells grow to have the fusion degree of 70%, and adding 1: 3, passage, transferring to 3 generations, and freezing and storing;
(3) differentiation treatment of epidermal stem cells: mixing the epidermal stem cells frozen in the step (2) with the liver cells, preparing into cell suspension, and then placing into an inducing solution until the differentiation process is completed.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the density of the cultured epidermal stem cells in the step (1) is 1x105one/mL.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the components of the cell culture solution in the step (1) are as follows:
16 parts of Ham's F-10 culture medium,
1 part of sodium chloride, namely sodium chloride,
and 0.8 part of potassium acetate.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the components of the cell culture solution for secondary culture in the step (2) are as follows:
18 parts of Ham's F-10 culture medium,
1 part of sodium alginate, namely 1 part of sodium alginate,
0.8 part of calcium chloride.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
in the step (3), the number ratio of the epidermal stem cells to the liver cells is 1: 0.5.
in the above-described method for improving differentiation of epidermal stem cells into liver cells,
the density of the cell suspension in the step (3) is 1x106Per mL;
the components of the inducing liquid in the step (3) are as follows:
the vitamin C accounts for 60 mu g/L,
15mol/L of beta-sodium glycerophosphate,
the volume fraction of the FBS is 10 percent,
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) separation treatment of epidermal stem cells: and (2) carrying out secondary culture on the epidermal cells propagated in the step (1) until the epidermal stem cells grow to have the fusion degree of 70%, and adding 1: 3, passage, transferring to 3 generations, and freezing and storing;
(3) differentiation treatment of epidermal stem cells: mixing the epidermal stem cells frozen in the step (2) with the liver cells, preparing into cell suspension, and then placing into an inducing solution until the differentiation process is completed.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the density of the cultured epidermal stem cells in the step (1) is 1x105one/mL.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the components of the cell culture solution in the step (1) are as follows:
ham's F-10 culture medium 14 portions,
2 parts of sodium chloride, namely 2 parts of sodium chloride,
and 0.7 part of potassium acetate.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the components of the cell culture solution for secondary culture in the step (2) are as follows:
15 parts of Ham's F-10 culture medium,
2 parts of sodium alginate, namely 2 parts of sodium alginate,
0.6 part of calcium chloride.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
in the step (3), the number ratio of the epidermal stem cells to the liver cells is 1: 0.5.
in the above-described method for improving differentiation of epidermal stem cells into liver cells,
the density of the cell suspension in the step (3) is 1x106Per mL;
the components of the inducing liquid in the step (3) are as follows:
the vitamin C accounts for 60 mu g/L,
15mol/L of beta-sodium glycerophosphate,
the volume fraction of the FBS is 10 percent,
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) separation treatment of epidermal stem cells: and (2) carrying out secondary culture on the epidermal cells propagated in the step (1) until the epidermal stem cells grow to have the fusion degree of 70%, and adding 1: 3, passage, transferring to 3 generations, and freezing and storing;
(3) differentiation treatment of epidermal stem cells: mixing the epidermal stem cells frozen in the step (2) with the liver cells, preparing into cell suspension, and then placing into an inducing solution until the differentiation process is completed.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the density of the cultured epidermal stem cells in the step (1) is 1x105one/mL.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the components of the cell culture solution in the step (1) are as follows:
ham's F-10 culture medium 14 portions,
2 parts of sodium chloride.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the components of the cell culture solution for secondary culture in the step (2) are as follows:
15 parts of Ham's F-10 culture medium,
2 parts of sodium alginate, namely 2 parts of sodium alginate,
0.6 part of calcium chloride.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
in the step (3), the number ratio of the epidermal stem cells to the liver cells is 1: 0.5.
in the above-described method for improving differentiation of epidermal stem cells into liver cells,
the density of the cell suspension in the step (3) is 1x106Per mL;
the components of the inducing liquid in the step (3) are as follows:
the vitamin C accounts for 60 mu g/L,
15mol/L of beta-sodium glycerophosphate,
the volume fraction of the FBS is 10 percent,
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) separation treatment of epidermal stem cells: and (2) carrying out secondary culture on the epidermal cells propagated in the step (1) until the epidermal stem cells grow to have the fusion degree of 70%, and adding 1: 3, passage, transferring to 3 generations, and freezing and storing;
(3) differentiation treatment of epidermal stem cells: mixing the epidermal stem cells frozen in the step (2) with the liver cells, preparing into cell suspension, and then placing into an inducing solution until the differentiation process is completed.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the density of the cultured epidermal stem cells in the step (1) is 1x105one/mL.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the components of the cell culture solution in the step (1) are as follows:
ham's F-10 culture medium 14 portions,
2 parts of sodium chloride, namely 2 parts of sodium chloride,
and 0.7 part of potassium acetate.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the components of the cell culture solution for secondary culture in the step (2) are as follows:
15 parts of Ham's F-10 culture medium,
and 2 parts of sodium alginate.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
in the step (3), the number ratio of the epidermal stem cells to the liver cells is 1: 0.5.
in the above-described method for improving differentiation of epidermal stem cells into liver cells,
the density of the cell suspension in the step (3) is 1x106Per mL;
the components of the inducing liquid in the step (3) are as follows:
the vitamin C accounts for 60 mu g/L,
15mol/L of beta-sodium glycerophosphate,
the volume fraction of the FBS is 10 percent,
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) separation treatment of epidermal stem cells: and (2) carrying out secondary culture on the epidermal cells propagated in the step (1) until the epidermal stem cells grow to have the fusion degree of 70%, and adding 1: 3, passage, transferring to 3 generations, and freezing and storing;
(3) differentiation treatment of epidermal stem cells: mixing the epidermal stem cells frozen in the step (2) with the liver cells, preparing into cell suspension, and then placing into an inducing solution until the differentiation process is completed.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the density of the cultured epidermal stem cells in the step (1) is1x105one/mL.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the components of the cell culture solution in the step (1) are as follows:
ham's F-10 medium 14 parts.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the components of the cell culture solution for secondary culture in the step (2) are as follows:
15 parts of Ham's F-10 culture medium,
2 parts of sodium alginate, namely 2 parts of sodium alginate,
0.6 part of calcium chloride.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
in the step (3), the number ratio of the epidermal stem cells to the liver cells is 1: 0.5.
in the above-described method for improving differentiation of epidermal stem cells into liver cells,
the density of the cell suspension in the step (3) is 1x106Per mL;
the components of the inducing liquid in the step (3) are as follows:
the vitamin C accounts for 60 mu g/L,
15mol/L of beta-sodium glycerophosphate,
the volume fraction of the FBS is 10 percent,
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) separation treatment of epidermal stem cells: and (2) carrying out secondary culture on the epidermal cells propagated in the step (1) until the epidermal stem cells grow to have the fusion degree of 70%, and adding 1: 3, passage, transferring to 3 generations, and freezing and storing;
(3) differentiation treatment of epidermal stem cells: mixing the epidermal stem cells frozen in the step (2) with the liver cells, preparing into cell suspension, and then placing into an inducing solution until the differentiation process is completed.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the density of the cultured epidermal stem cells in the step (1) is 1x105one/mL.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the components of the cell culture solution in the step (1) are as follows:
ham's F-10 culture medium 14 portions,
2 parts of sodium chloride, namely 2 parts of sodium chloride,
and 0.7 part of potassium acetate.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the components of the cell culture solution for secondary culture in the step (2) are as follows:
ham's F-10 medium 15 parts.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
in the step (3), the number ratio of the epidermal stem cells to the liver cells is 1: 0.5.
in the above-described method for improving differentiation of epidermal stem cells into liver cells,
the density of the cell suspension in the step (3) is 1x106Per mL;
the components of the inducing liquid in the step (3) are as follows:
the vitamin C accounts for 60 mu g/L,
15mol/L of beta-sodium glycerophosphate,
the volume fraction of the FBS is 10 percent,
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) separation treatment of epidermal stem cells: and (2) carrying out secondary culture on the epidermal cells propagated in the step (1) until the epidermal stem cells grow to have the fusion degree of 70%, and adding 1: 3, passage, transferring to 3 generations, and freezing and storing;
(3) differentiation treatment of epidermal stem cells: mixing the epidermal stem cells frozen in the step (2) with the liver cells, preparing into cell suspension, and then placing into an inducing solution until the differentiation process is completed.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the density of the cultured epidermal stem cells in the step (1) is 1x105one/mL.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the components of the cell culture solution in the step (1) are as follows:
ham's F-10 culture medium 14 portions,
2 parts of sodium chloride, namely 2 parts of sodium chloride,
and 0.7 part of potassium acetate.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the components of the cell culture solution for secondary culture in the step (2) are as follows:
15 parts of Ham's F-10 culture medium,
2 parts of sodium alginate, namely 2 parts of sodium alginate,
0.6 part of calcium chloride.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
in the step (3), the number ratio of the epidermal stem cells to the liver cells is 1: 0.5.
in the above-described method for improving differentiation of epidermal stem cells into liver cells,
the density of the cell suspension in the step (3) is 1x106Per mL;
the components of the inducing liquid in the step (3) are as follows:
the vitamin C accounts for 60 mu g/L,
the volume fraction of the FBS is 10 percent,
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) separation treatment of epidermal stem cells: and (2) carrying out secondary culture on the epidermal cells propagated in the step (1) until the epidermal stem cells grow to have the fusion degree of 70%, and adding 1: 3, passage, transferring to 3 generations, and freezing and storing;
(3) differentiation treatment of epidermal stem cells: mixing the epidermal stem cells frozen in the step (2) with the liver cells, preparing into cell suspension, and then placing into an inducing solution until the differentiation process is completed.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the density of the cultured epidermal stem cells in the step (1) is 1x105one/mL.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the components of the cell culture solution in the step (1) are as follows:
ham's F-10 culture medium 14 portions,
2 parts of sodium chloride, namely 2 parts of sodium chloride,
and 0.7 part of potassium acetate.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the components of the cell culture solution for secondary culture in the step (2) are as follows:
15 parts of Ham's F-10 culture medium,
2 parts of sodium alginate, namely 2 parts of sodium alginate,
0.6 part of calcium chloride.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
in the step (3), the number ratio of the epidermal stem cells to the liver cells is 1: 0.5.
in the above-described method for improving differentiation of epidermal stem cells into liver cells,
the density of the cell suspension in the step (3) is 1x106Per mL;
the components of the inducing liquid in the step (3) are as follows:
the vitamin C accounts for 60 mu g/L,
15mol/L of beta-sodium glycerophosphate,
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) separation treatment of epidermal stem cells: and (2) carrying out secondary culture on the epidermal cells propagated in the step (1) until the epidermal stem cells grow to have the fusion degree of 70%, and adding 1: 3, passage, transferring to 3 generations, and freezing and storing;
(3) differentiation treatment of epidermal stem cells: mixing the epidermal stem cells frozen in the step (2) with the liver cells, preparing into cell suspension, and then placing into an inducing solution until the differentiation process is completed.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the density of the cultured epidermal stem cells in the step (1) is 1x105one/mL.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the components of the cell culture solution in the step (1) are as follows:
ham's F-10 culture medium 14 portions,
2 parts of sodium chloride, namely 2 parts of sodium chloride,
and 0.7 part of potassium acetate.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the components of the cell culture solution for secondary culture in the step (2) are as follows:
15 parts of Ham's F-10 culture medium,
2 parts of sodium alginate, namely 2 parts of sodium alginate,
0.6 part of calcium chloride.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
in the step (3), the number ratio of the epidermal stem cells to the liver cells is 1: 0.5.
in the above-described method for improving differentiation of epidermal stem cells into liver cells,
the density of the cell suspension in the step (3) is 1x106Per mL;
the components of the inducing liquid in the step (3) are as follows:
the vitamin C accounts for 60 mu g/L,
15mol/L of beta-sodium glycerophosphate,
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) separation treatment of epidermal stem cells: and (2) carrying out secondary culture on the epidermal cells propagated in the step (1) until the epidermal stem cells grow to have the fusion degree of 70%, and adding 1: 3, passage, transferring to 3 generations, and freezing and storing;
(3) differentiation treatment of epidermal stem cells: mixing the epidermal stem cells frozen in the step (2) with the liver cells, preparing into cell suspension, and then placing into an inducing solution until the differentiation process is completed.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the density of the cultured epidermal stem cells in the step (1) is 1x105one/mL.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the components of the cell culture solution in the step (1) are as follows:
ham's F-10 culture medium 14 portions,
2 parts of sodium chloride, namely 2 parts of sodium chloride,
and 0.7 part of potassium acetate.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
the components of the cell culture solution for secondary culture in the step (2) are as follows:
15 parts of Ham's F-10 culture medium,
2 parts of sodium alginate, namely 2 parts of sodium alginate,
0.6 part of calcium chloride.
In the above-described method for improving differentiation of epidermal stem cells into liver cells,
in the step (3), the number ratio of the epidermal stem cells to the liver cells is 1: 0.5.
in the above-described method for improving differentiation of epidermal stem cells into liver cells,
the density of the cell suspension in the step (3) is 1x106Per mL;
the components of the inducing liquid in the step (3) are as follows:
the vitamin C accounts for 60 mu g/L,
the volume fraction of the FBS is 10 percent,
IGF-1 25μg/L。
example 6
The products prepared in examples 1 to 5 and the products prepared in comparative examples 1 to 8 were selected and subjected to the following tests:
referring to the Chinese invention patent, the application number: CN201910859880.0, publication No.: CN110684725A, discloses a method for inducing stem cells to differentiate into directional cartilage.
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 conversion of the product prepared in example 4 was 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%.
While the invention has been described in further detail in connection with specific embodiments thereof, it will be understood that the invention is not limited thereto, and that various other modifications and substitutions may be made by those skilled in the art without departing from the spirit of the invention, which should be considered to be within the scope of the invention as defined by the appended claims.
Claims (10)
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) separation treatment of epidermal stem cells: and (2) carrying out secondary culture on the epidermal cells propagated in the step (1) until the epidermal stem cells grow to have the fusion degree of 70%, and adding 1: 3, passage, transferring to 3 generations, and freezing and storing;
(3) differentiation treatment of epidermal stem cells: mixing the epidermal stem cells frozen in the step (2) with the liver cells, preparing into cell suspension, and then placing into an inducing solution until the differentiation process is completed.
2. The method of improving differentiation of epidermal stem cells into liver cells of claim 1, wherein:
the density of the cultured epidermal stem cells in the step (1) is 1x105one/mL.
3. The method of improving differentiation of epidermal stem cells into liver cells of claim 1, wherein:
the components of the cell culture solution in the step (1) are as follows:
10-20 parts of Ham's F-10 culture medium,
1 to 3 parts of sodium chloride,
0.5-0.8 part of potassium acetate;
wherein the culture temperature is 37 deg.C, culture time is 7d, and carbon dioxide concentration is 5%.
4. The method of improving differentiation of epidermal stem cells into liver cells according to claim 3, wherein:
the components of the cell culture solution in the step (1) are as follows:
12-16 parts of Ham's F-10 culture medium,
1 to 3 parts of sodium chloride,
0.5 to 0.8 portion of potassium acetate.
5. The method of improving differentiation of epidermal stem cells into liver cells according to claim 4, wherein:
the components of the cell culture solution in the step (1) are as follows:
ham's F-10 culture medium 14 portions,
2 parts of sodium chloride, namely 2 parts of sodium chloride,
and 0.7 part of potassium acetate.
6. The method of improving differentiation of epidermal stem cells into liver cells of claim 1, wherein:
the components of the cell culture solution for secondary culture in the step (2) are as follows:
10-20 parts of Ham's F-10 culture medium,
1 to 3 parts of sodium alginate, namely,
0.5 to 0.8 portion of calcium chloride;
wherein the culture temperature is 37 deg.C, culture time is 7d, and carbon dioxide concentration is 5%.
7. The method of improving differentiation of epidermal stem cells into liver cells according to claim 6, wherein:
the components of the cell culture solution for secondary culture in the step (2) are as follows:
12-18 parts of Ham's F-10 culture medium,
1 to 3 parts of sodium alginate, namely,
0.5 to 0.8 portion of calcium chloride.
8. The method of improving differentiation of epidermal stem cells into liver cells of claim 7, wherein:
the components of the cell culture solution for secondary culture in the step (2) are as follows:
15 parts of Ham's F-10 culture medium,
2 parts of sodium alginate, namely 2 parts of sodium alginate,
0.6 part of calcium chloride.
9. The method of improving differentiation of epidermal stem cell 3 into liver cells of claim 1, wherein:
in the step (3), the number ratio of the epidermal stem cells to the liver cells is 1: 0.5.
10. the method of improving differentiation of epidermal stem cells into liver cells of claim 1, wherein:
the density of the cell suspension in the step (3) is 1x106Per mL;
the components of the inducing liquid in the step (3) are as follows:
the vitamin C accounts for 60 mu g/L,
15mol/L of beta-sodium glycerophosphate,
the volume fraction of the FBS is 10 percent,
IGF-1 25μg/L，
IGF-2 20μg/L。
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