CN114686426A - Culture method and application of primary amniotic stem cells - Google Patents
Culture method and application of primary amniotic stem cells Download PDFInfo
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Abstract
The invention belongs to the technical field of stem cell culture, and particularly relates to a culture method of primary amniotic stem cells and application thereof. The invention provides a method for culturing primary amniotic stem cells, which mainly comprises the following steps: peeling placenta under aseptic condition, washing with normal saline, washing with mixed solution containing bactericide, treating with digestive enzyme, and culturing in culture medium; the mixed liquid containing the bactericide comprises the following components in percentage by weight: 1-2% of dandelion extract, 0.5-0.8% of rosemary hydrolat, 0.3-0.8% of vitamin C and 96.4-98.2% of purified water; the culture medium consists of LB liquid culture medium, Tween-20, IV type collagen and BI recombinant glass fibronectin. By adopting the culture method provided by the invention, the adherence of cells can be accelerated, tissues in the cells can be cleaned, and the purity of the cells is improved on the premise of shortening the culture time of the amniotic stem cells.
Description
Technical Field
The invention belongs to the technical field of stem cell culture, and particularly relates to a culture method of primary amniotic stem cells and application thereof.
Background
Human amniotic derived stem cells include human amniotic epithelial cells (hAECs) and human amniotic mesenchymal stem cells (hAMCs). The human amniotic epithelial cells have the characteristics of expressing various embryonic stem cell markers and comprehensive multidirectional differentiation potential, and the hMSCs have stronger proliferation capacity and stem cell characteristics than bone marrow mesenchymal stem cells. Therefore, because of its totipotency, amniotic stem cells are commonly used in various cytology and molecular biology research procedures.
However, in the conventional amnion stem cell culture method, the amnion tissue is thin, so that the cells are difficult to adhere to the wall, the tissue digestion process is slow, so that the removal is complex, the culture time of the amnion stem cells is about 20 days, and in the cell culture process, even if the amnion stem cells are cultured in an aseptic environment, the pollution of a small amount of bacteria cannot be avoided, so that the purity of the cultured cells is low.
Disclosure of Invention
Aiming at the defects generally existing in the prior art, the invention provides a culture method of primary amniotic stem cells and application thereof. By adopting the culture method provided by the invention, the adherence of cells can be accelerated, tissues in the cells can be cleaned, and the purity of the cells is improved on the premise of shortening the culture time of the amniotic stem cells.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for culturing primary amniotic membrane stem cells comprises the following steps:
s1, peeling the amnion from the placenta under the aseptic condition, washing with normal saline, removing blood filaments and sticky substances on the amnion, then cutting into small tissue blocks of 6cm multiplied by 6cm in a culture dish, and placing the smooth surface of the amnion downwards to obtain an initially processed amnion tissue block;
s2, placing the preliminarily processed amniotic tissue blocks obtained in the step S1 in culture dishes, placing a small tissue block in each culture dish, adding digestive enzymes into each culture dish according to the amount of 2.5g/L, digesting at 23-27 ℃ for 28-32min, and washing with physiological saline to obtain digested amniotic tissue;
s3, cutting the digested amniotic tissue obtained in the step S2 into 1-2 cm fragments, soaking the fragments in a mixed solution containing a bactericide for 10-20 min, washing the fragments with physiological saline, centrifuging after washing each time, and finally collecting precipitates to obtain cells to be cultured;
s4, resuspending the cells to be cultured obtained in the step S3 in 1.5-2 mL of culture medium, inoculating the cells into a T25 culture flask, inverting the culture flask, drying for 15-20 min, and placing the culture flask at 37 ℃ in 5% by volume of CO2Culturing for 24 hours in an incubator, adding 1.5-2 mL of culture medium, adding the culture medium every three days, observing the growth condition of the cells after 5-7 days, taking pictures, recording as P0 when the cells are fused to 80%, and harvesting the cells to obtain the cell.
Preferably, the size of the culture dish in step S2 is 90 mm; the digestive enzyme is prepared from trypsin, chymosin and xylanase according to a weight ratio of 8-15: 2-4: 1 to 3.
Preferably, the mixed solution containing the bactericide in step S3 includes the following components by weight percent: 1-2% of dandelion extract, 0.5-0.8% of rosemary hydrolat, 0.3-0.8% of vitamin C and 96.4-98.2% of purified water.
Preferably, the mixed solution containing the bactericide in step S3 includes the following components by weight percent: 1.5 percent of dandelion extract, 0.7 percent of rosemary hydrosol, 0.6 percent of vitamin C and 97.2 percent of purified water.
Preferably, the centrifugation condition in step S3 is 1500-2000 r/min for 5-10 min.
Preferably, the culture medium of step S4 is composed of LB liquid medium, tween-20, type IV collagen, BI recombinant vitronectin, and is prepared as follows: adding Tween-20, IV type collagen and BI recombinant glass fibronectin into an LB liquid culture medium respectively, wherein the final concentration of the IV type collagen in the culture medium is 2-5 mu g/mL, the concentration of the BI recombinant glass fibronectin is 0.1-0.2 mg/mL, and the addition amount of the Tween-20 is 8-15 mu L/mL.
Preferably, the final concentration of type IV collagen in the culture medium in the step S4 is 4 μ g/mL, the concentration of BI recombinant vitronectin is 0.15mg/mL, and the addition amount of Tween-20 is 12 μ L/mL.
Preferably, the specific operation of observing the cell growth condition in step S4 is: observing under an inverted microscope, if cells climb out around the tissue block, changing the culture solution for half amount once, discarding half of the culture solution in the original culture bottle, adding an equal amount of fresh culture solution, sucking out the tissue block when the cell climbing-out area reaches 30% of the culture bottle area, supplementing the culture solution, changing the culture solution once every other day, and carrying out passage when the cells grow to 80% of fusion.
The invention also provides application of the culture method in culturing the primary amniotic stem cells.
Based on the fact that the amniotic membrane tissue is light and thin and is not easy to adhere to the wall, the wall adhering culture time is possibly too long, the primary amniotic membrane stem cells are mainly prepared by optimizing a wall adhering method, and in the research process, the inventor finds that a tissue block is placed in a culture medium prepared from trypsin, chymosin and xylanase according to the weight ratio of 8-15: 2-4: 1-3, and can help to remove redundant tissues on the basis of promoting cell adherence, the effect of the digestive enzyme is higher than that of common trypsin digestion, and in the digestion process, in order to improve the digestion effect and the cell purity, only one layer of amnion (namely, epithelial layer, and the rest 4 layers are not digested) is digested by adding the digestive enzyme once. And further soaking in the mixed solution containing the bactericide for a period of time can further remove dirt on the surface of the amniotic tissue and avoid the pollution of other tissues, and the addition of the bactericide can avoid the pollution of cells by other bacteria in the soaking process and improve the purity of the cultured cells.
Meanwhile, in the research of promoting the cell adherence process, the obtained cells to be cultured are re-suspended and inoculated in a T25 culture bottle by using a culture medium, the culture bottle is inverted and dried for 15-20 min, namely, the amnion in the culture bottle is dried temporarily, so that the amnion is easier to adhere to the wall, and when the cells are cultured, the culture medium consisting of LB liquid culture medium, IV type collagen and BI recombinant glass adhesion protein is adopted, so that the adherence of the cells can be accelerated, and the cell culture time can be shortened. The preparation time of the primary amniotic membrane stem cells in the existing preparation process is more than 20 days, and the preparation time is shortened to 12.5-14.5 days and is shortened to 12.5 days at least after the optimization.
In the screening process of various substances, the IV type collagen is mainly used for adherent culture of neuronal cells and glial cells, the adherent effect on endothelial cells, hepatic cells, epithelial cells and the like is poor, and the inventor finds that the addition amount of the BI recombinant glass adhesion protein can be reduced on the premise of adding a small amount of the IV type collagen in the culture medium adopted by the invention, through further deep research, when the final concentration of the IV type collagen is 2-5 mug/mL, the use concentration of the BI recombinant glass adhesion protein can be reduced to 0.1-0.2 mg/mL, and under the condition of low-concentration BI recombinant glass adhesion protein, the use concentration of the BI recombinant glass adhesion protein still has a good promoting effect on the adherent of the amniotic stem cells, the culture time of the cells is effectively reduced, and the optimal addition concentration ratio is 4 mug/mL: 0.15 mg/mL. Moreover, the whole culture process is carried out under the aseptic condition, and the soaking process of the mixed solution containing the bactericide avoids bacteria from polluting the amnion stem cells, and improves the purity of the cells.
Compared with the prior art, the culture method of the primary amniotic membrane stem cells provided by the invention has the following advantages:
the culture method provided by the invention can accelerate cell adherence, clean tissues in the cells and improve the purity of the cells to more than 95% on the premise of shortening the culture time of the amniotic stem cells.
Detailed Description
The present invention is further explained with reference to the following specific examples, but it should be noted that the following examples are only illustrative of the present invention and should not be construed as limiting the present invention, and all technical solutions similar or equivalent to the present invention are within the scope of the present invention. The method and the device are operated according to the conventional technical method and the content of the instrument instruction, wherein the specific technology or condition is not indicated in the embodiment; the reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
The herba Taraxaci extract is extracted from Taraxacum officinale of Compositae, and is available from Nanjing Zeylang Biotech limited; the rosemary hydrolat can be purchased from Huamao biological technology, Inc., and has the CAS number of 084604-14-8; the type IV collagen may be purchased from shanghai constant distance biotechnology limited; the BI recombinant glass fibronectin is BioLamina human recombinant laminin, which can be purchased from Powana Biotechnology Limited, Guangzhou and has the model of LN111-LN 521; the LB liquid medium is commercially available from Beijing Baiaosentai Biotechnology, Inc.
Example 1A method for culturing Primary amniotic Stem cells
The primary amnion stem cell culture method comprises the following preparation processes:
s1, peeling the amnion from the placenta under the aseptic condition, washing the amnion for 3 times by using normal saline, removing blood filaments and sticky substances on the amnion, then cutting the amnion into small tissue blocks of 6cm multiplied by 6cm in a culture dish, and placing the smooth surface of the amnion downwards to obtain an initially treated amnion tissue block;
s2, placing the preliminarily processed amniotic membrane tissue blocks obtained in the step S1 in 90mm culture dishes, placing a small tissue block in each culture dish, and then mixing trypsin, chymosin and xylanase according to a weight ratio of 8: 2: 1 to prepare digestive enzyme, respectively adding the digestive enzyme into each culture dish, digesting for 28min at 23 ℃ according to the addition of 2.5g/L, and then washing for 2 times by using normal saline to prepare digested amniotic tissues;
s3, preparing a mixed solution containing the bactericide according to the following components in percentage by weight: 1% of dandelion extract, 0.5% of rosemary hydrolat, 0.3% of vitamin C and 98.2% of purified water, then cutting the digested amniotic tissue obtained in the step S2 into 1-2 cm fragments, soaking the fragments in a prepared mixed solution containing a bactericide for 10min, then washing the fragments with physiological saline for 2 times, centrifuging the fragments after each washing, centrifuging the fragments under the condition of 1500r/min for 10min, and finally collecting the precipitate to obtain cells to be cultured;
s4, preparation of a culture medium: respectively adding Tween-20, IV collagen and BI recombinant glass fibronectin into an LB liquid culture medium, wherein the final concentration of the IV collagen in the culture medium is 2 mu g/mL, the final concentration of the BI recombinant glass fibronectin is 0.2mg/mL, the addition amount of the Tween-20 is 8 mu L/mL, re-suspending and inoculating the cells to be cultured obtained in the step S3 into a T25 culture bottle by using 1.5mL of the prepared culture medium, inverting the culture bottle, drying for 15min, and thenThen placing the mixture at 37 ℃ with the volume fraction of 5 percent CO2Culturing for 24h in an incubator, adding 1.5mL of culture medium, then adding the culture medium every three days, observing the growth condition of the cells after 5-7 days, taking pictures, recording as P0 when the cells are fused to 80%, and specifically observing the process as follows: observing under an inverted microscope, if cells climb out around the tissue block, changing the culture solution for half amount once, discarding half of the culture solution in the original culture bottle, adding an equal amount of fresh culture solution, sucking out the tissue block when the cell climbing-out area reaches 30% of the culture bottle area, supplementing the culture solution, changing the culture solution once every other day, marking as P0 when the cells grow to 80% and are fused, and harvesting the cells.
Example 2 method for culturing Primary amniotic Stem cells
The primary amnion stem cell culture method comprises the following preparation processes:
s1, peeling the amnion from the placenta under the aseptic condition, washing the amnion for 5 times by using normal saline, removing blood filaments and sticky substances on the amnion, then cutting the amnion into small tissue blocks of 6cm multiplied by 6cm in a culture dish, and placing the smooth surface of the amnion downwards to obtain an initially treated amnion tissue block;
s2, placing the preliminarily processed amniotic membrane tissue blocks obtained in the step S1 in 90mm culture dishes, placing a small tissue block in each culture dish, and then mixing trypsin, chymosin and xylanase according to a weight ratio of 15: 4: 3 to prepare digestive enzyme, adding the digestive enzyme into each culture dish respectively, digesting for 32min at 27 ℃ according to the amount of 2.5g/L, and then washing for 4 times by using normal saline to prepare digested amniotic tissues;
s3, preparing a mixed solution containing the bactericide according to the following components in percentage by weight: 2% of dandelion extract, 0.8% of rosemary hydrolat, 0.8% of vitamin C and 96.4% of purified water, then cutting the digested amniotic tissue obtained in the step S2 into 1-2 cm fragments by using an ophthalmic lens, soaking the fragments in a prepared mixed solution containing a bactericide for 20min, washing the fragments for 3 times by using physiological saline, centrifuging the washed fragments under the centrifugal condition of 2000r/min for 5min, and finally collecting precipitates to obtain cells to be cultured;
s4, preparation of a culture medium: respectively mixing Tween-20 and type IVAdding collagen and BI recombinant glass adhesion protein into an LB liquid culture medium, wherein the final concentration of IV type collagen in the culture medium is 5 mu g/mL, the final concentration of the BI recombinant glass adhesion protein is 0.1mg/mL, and the addition amount of Tween-20 is 15 mu L/mL; then, the cells to be cultured obtained in step S3 were re-suspended in 1.8mL of the prepared medium and inoculated into a T25 flask, the flask was inverted, dried for 20min, and then placed at 37 ℃ in a 5% volume fraction of CO2Culturing for 24h in an incubator, adding 1.8mL of culture medium, adding the culture medium every three days, observing the growth condition of the cells after 5-7 days, taking pictures, recording as P0 when the cells are fused to 80%, and then harvesting the cells.
Example 3 method for culturing Primary amniotic Stem cells
The primary amnion stem cell culture method comprises the following preparation processes:
s1, peeling the amnion from the placenta under the aseptic condition, washing the amnion for 4 times by using normal saline, removing blood filaments and sticky substances on the amnion, then cutting the amnion into small tissue blocks of 6cm multiplied by 6cm in a culture dish, and placing the smooth surface of the amnion downwards to obtain an initially treated amnion tissue block;
s2, placing the preliminarily processed amniotic membrane tissue blocks obtained in the step S1 in 90mm culture dishes, placing a small tissue block in each culture dish, and then mixing trypsin, chymosin and xylanase according to a weight ratio of 12: 3: 2 to prepare digestive enzyme, adding the digestive enzyme into each culture dish respectively, digesting for 30min at 25 ℃ according to the amount of 2.5g/L, and then washing for 3 times by using normal saline to prepare digested amniotic tissues;
s3, preparing a mixed solution containing the bactericide according to the following components in percentage by weight: 1.5% of dandelion extract, 0.7% of rosemary hydrosol, 0.6% of vitamin C and 97.2% of purified water, then cutting the digested amniotic tissue obtained in the step S2 into 1-2 cm fragments by using an ophthalmoscope, soaking the fragments in a mixed solution containing a bactericide for 15min, washing the fragments with physiological saline for 3 times, centrifuging after each washing, treating the fragments for 17min under the condition of a rotation speed of 1800rpm, and finally collecting precipitates to obtain cells to be cultured;
s4, preparation of a culture medium: respectively mixing Tween-20, IV type collagen,Adding the BI recombinant glass fibronectin into an LB liquid culture medium, wherein the final concentration of the IV type collagen in the culture medium is 4 mu g/mL, the final concentration of the BI recombinant glass fibronectin is 0.15mg/mL, the adding amount of the Tween-20 is 12 mu L/mL, then re-suspending the cells to be cultured obtained in the step S3 by using 2mL of the prepared culture medium, inoculating the cells to be cultured into a T25 culture bottle, inverting the culture bottle, drying for 20min, then placing the culture bottle at 37 ℃, and placing CO with the volume fraction of 5%2Culturing for 24h in an incubator, adding 2mL of culture medium, adding the culture medium every three days, observing the growth condition of the cells after 5-7 days, taking pictures, recording as P0 when the cells are fused to 80%, and then harvesting the cells.
Comparative example 1 culture method of primary amniotic Stem cells
The culture method of the primary amniotic membrane stem cells is similar to that of the example 3;
the difference from example 2 is that the digestive enzyme in comparative example 1 is trypsin.
Comparative example 2 culture method of Primary amniotic Stem cells
The culture method of the primary amniotic membrane stem cells is similar to that of the example 3;
the difference from example 2 is that in comparative example 2, the dandelion extract in the mixed solution containing the bactericide was replaced with the licorice extract.
Comparative example 3 culture method of Primary amniotic Stem cells
The culture method of the primary amniotic membrane stem cells is similar to that of the example 3;
the difference from example 2 is that in the culture medium prepared in comparative example 3, type IV collagen was replaced with type I collagen in a constant amount.
Comparative example 4 culture method of Primary amniotic Stem cells
The culture method of the primary amniotic membrane stem cells is similar to that of the example 3;
the difference from example 2 is that the culture medium of comparative example 4 was prepared with a concentration of 0.5mg/mL of BI recombinant vitronectin and no type IV collagen.
Comparative example 5 culture method of Primary amniotic Stem cells
The existing culture method is adopted.
Slowly tearing amnion at the outer layer of the fetal membrane by using surgical forceps, placing the amnion in a culture dish of 150mm, repeatedly cleaning surface contaminated blood by using basic balanced salt solution, and shearing the amnion into amnion tissue small blocks with the diameter of 5 mm; filtering with 300 mesh filter screen, washing residual blood with normal saline, placing tissue block at 75cm2In the culture flask, the tissue mass can cover half of the culture area in the flask, adding complete culture medium to submerge the tissue, shaking uniformly, and adding 5% CO2Culturing in an incubator at 37 ℃; after 2d, supplementing 10mL of DMEM complete culture medium, and continuing to culture; removing tissues after more mesenchymal stem cells climb out, periodically changing the liquid (every 2 days), and carrying out subculture when the cells reach 80% fusion.
Test example 1 comparison of incubation time
1. The test method comprises the following steps: the culture methods of examples 1 to 3 and comparative examples 1 to 5;
2. and (3) test results: counting the time required for culturing each culture method to 80% fusion; specific test results are shown in table 1.
TABLE 1 time required for different test methods to incubate to 80% confluence
Test example 2 comparison of cell purity
1. Test samples: p0 generation cells obtained by the culture methods of examples 1 to 3 and comparative example 2;
2. the detection method comprises the following steps: the phenotype and the cell purity of the P0 generation cells are detected by flow cytometry, and the surface markers comprise CD73, CD90, CD44, CD105, CD166, CD11b, CD19, CD34, CD45, HLA-DR and the like. The specific detection process is as follows: collecting cell suspension, centrifuging, removing supernatant at 1000rpm for 10min, washing cell precipitate with 4 deg.C pre-cooled 0.01 mol/L PBS for 2 times at 1000rpm for 10min, re-suspending the cell precipitate with 200 μ L PBS buffer solution, and collecting 1 × 106The individual cells were resuspended in 0.1mL of precooled 0.01 mol/L PBS, 2uL of the corresponding CD73, CD90, CD105, CD44, CD166, negative marker (CD 34, CD45, CD19, CD11b, HLA-DR) fluorescent antibody was added in the dark, incubated in the dark at 4 ℃ for 30min, 2mL of PBS was added, L000 rpm, L0min, washedThe cells were washed to remove unbound antibody, the supernatant removed, the cell pellet resuspended in PBS, submitted to 4 ℃ and examined by flow cytometry.
3. And (3) test results: cryopreserved cells of greater than or equal to 2 × 107(obtained by automatic counting by a cell counter), the phenotype of P0 is qualified, and the expression results of the surface markers are shown in tables 2 and 3 respectively.
TABLE 2 results of expression of various factors
TABLE 3 comparison of expression results of different factors
As is clear from tables 2 and 3 above, in examples 1 to 3, the expression rates of CD73, CD90, CD44, CD105 and CD166 were 95% or more, and the expression rates of CD11b, CD19, CD34, CD45 and HLA-DR molecules were all less than 1%, with the highest cell purity in example 2. Compared with the comparative example 2, the expression rate of CD73, CD90, CD44, CD105 and CD166 is not up to 95%, the expression rate of CD11b, CD19, CD34, CD45 and HLA-DR is between 1% and 2%, and the purity of the group of the comparative example 2 is obviously reduced compared with the group of the examples 1 to 3.
It should be noted that although the above embodiments have been described, once the basic inventive concept is obtained, other variations and modifications can be made to these embodiments by those skilled in the art, so that the above embodiments are only examples of the present invention, and not to limit the scope of the present invention, and all the modifications made by the equivalent structures or equivalent processes in the present specification, or directly or indirectly applied to other related technical fields are included in the scope of the present invention.
Claims (9)
1. A method for culturing primary amniotic stem cells is characterized by comprising the following steps:
s1, peeling the amnion from the placenta under the aseptic condition, washing with normal saline, removing blood filaments and sticky substances on the amnion, then cutting into small tissue blocks of 6cm multiplied by 6cm in a culture dish, and placing the smooth surface of the amnion downwards to obtain an initially processed amnion tissue block;
s2, placing the preliminarily processed amniotic tissue blocks obtained in the step S1 in culture dishes, placing a small tissue block in each culture dish, adding digestive enzymes into each culture dish according to the amount of 2.5g/L, digesting at 23-27 ℃ for 28-32min, and washing with physiological saline to obtain digested amniotic tissue;
s3, cutting the digested amniotic tissue obtained in the step S2 into 1-2 cm fragments, soaking the fragments in a mixed solution containing a bactericide for 10-20 min, washing the fragments with physiological saline, centrifuging after washing each time, and finally collecting precipitates to obtain cells to be cultured;
s4, resuspending the cells to be cultured obtained in the step S3 in 1.5-2 mL of culture medium, inoculating the cells into a T25 culture flask, inverting the culture flask, drying for 15-20 min, and placing the culture flask at 37 ℃ in 5% by volume of CO2Culturing for 24 hours in an incubator, adding 1.5-2 mL of culture medium, adding the culture medium every three days, observing the growth condition of the cells after 5-7 days, taking pictures, recording as P0 when the cells are fused to 80%, and harvesting the cells to obtain the cell.
2. The culture method according to claim 1, wherein the size of the dish in step S2 is 90 mm; the digestive enzyme is prepared from trypsin, chymosin and xylanase according to a weight ratio of 8-15: 2-4: 1 to 3.
3. The culture method according to claim 2, wherein the mixed solution containing the bactericide in step S3 comprises the following components in percentage by weight: 1-2% of dandelion extract, 0.5-0.8% of rosemary hydrolat, 0.3-0.8% of vitamin C and 96.4-98.2% of purified water.
4. The culture method according to claim 3, wherein the mixed solution containing the bactericide in step S3 comprises the following components in percentage by weight: 1.5 percent of dandelion extract, 0.7 percent of rosemary hydrosol, 0.6 percent of vitamin C and 97.2 percent of purified water.
5. The culture method according to claim 1, wherein the centrifugation in step S3 is performed at 1500 to 2000r/min for 5 to 10 min.
6. The culture method of claim 1, wherein the culture medium of step S4 comprises LB broth, tween-20, type IV collagen, BI recombinant vitronectin, and is prepared as follows: adding Tween-20, IV type collagen and BI recombinant glass adhesion protein into an LB liquid culture medium respectively, wherein the final concentration of the IV type collagen in the culture medium is 2-5 mu g/mL, the final concentration of the BI recombinant glass adhesion protein is 0.1-0.2 mg/mL, and the addition amount of the Tween-20 is 8-15 mu L/mL.
7. The culture method of claim 6, wherein the final concentration of type IV collagen in the culture medium of step S4 is 4 μ g/mL, the final concentration of BI recombinant vitronectin is 0.15mg/mL, and the amount of Tween-20 added is 12 μ L/mL.
8. The culture method according to claim 1, wherein the observation of the growth of the cells in step S4 is performed by: observing under an inverted microscope, if cells climb out around the tissue block, changing the culture solution for half amount once, discarding half of the culture solution in the original culture bottle, adding an equal amount of fresh culture solution, sucking out the tissue block when the cell climbing-out area reaches 30% of the culture bottle area, supplementing the culture solution, changing the culture solution once every other day, and carrying out passage when the cells grow to 80% of fusion.
9. Use of a culture method according to any one of claims 1 to 8 in primary amniotic stem cells.
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