CN114686426B - 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 and application of primary amniotic stem cells. The culture method of the primary amniotic stem cells provided by the invention mainly comprises the following steps: peeling placenta under aseptic condition, washing with physiological saline, washing with mixed solution containing bactericide, treating with digestive enzyme, culturing with culture medium, etc.; the mixed solution containing the bactericide comprises the following components in percentage by weight: 1-2% of dandelion extract, 0.5-0.8% of rosemary hydrosol, 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, type IV collagen and BI recombinant vitronectin. The culture method provided by the invention can accelerate cell adhesion, clean tissues in cells, and improve the purity of cells on the premise of shortening the culture time of amniotic stem cells.
Description
Technical Field
The invention belongs to the technical field of stem cell culture, and particularly relates to a culture method and application of primary amniotic stem cells.
Background
Human amniotic membrane derived stem cells include human amniotic epithelial cells (hAEC), human amniotic mesenchymal stem cells (hAMSC). Wherein 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 the mesenchymal stem cells. Therefore, amniotic stem cells are often used in various cytology and molecular biology research processes due to their totipotency.
However, in the existing amniotic stem cell culture method, the amniotic tissue is thinner, so that the cell adhesion is difficult, the removal is complex due to the slower tissue digestion process, the culture time of the amniotic stem cells is about 20 days finally, and in the cell culture process, even if the amniotic stem cells are cultured in a sterile environment, the pollution of a small amount of bacteria cannot be avoided, so that the purity of the cultured cells is lower.
Disclosure of Invention
Aiming at the common defects in the prior art, the invention provides a culture method and application of primary amniotic stem cells. The culture method provided by the invention can accelerate cell adhesion, clean tissues in cells, and improve the purity of cells on the premise of shortening the culture time of amniotic stem cells.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a method for culturing primary amniotic stem cells, comprising the following steps:
s1, stripping the amniotic membrane from the placenta under the aseptic condition, washing with normal saline to remove blood filaments and sticky matters on the amniotic membrane, cutting into tissue small blocks of 6cm multiplied by 6cm in a culture dish, and placing the smooth surface of the amniotic membrane downwards to obtain a primarily treated amniotic membrane tissue block;
s2, placing the primarily treated amniotic membrane tissue blocks obtained in the step S1 in culture dishes, placing a tissue small block in each culture dish, adding digestive enzyme into each culture dish according to the amount of 2.5g/L, digesting for 28-32min at the temperature of 23-27 ℃, and washing with normal saline to obtain digested amniotic membrane tissue;
s3, cutting the digested amniotic membrane tissue obtained in the step S2 into fragments of 1-2 cm, soaking the fragments in a mixed solution containing a bactericide for 10-20 min, washing the fragments with normal saline, centrifuging the fragments after washing each time, and finally collecting the precipitate to obtain cells to be cultured;
s4, the cells to be cultured obtained in the step S3 are inoculated in a T25 culture bottle in a suspension manner by using 1.5-2 mL of culture medium, the culture bottle is inverted and dried for 15-20 min, and then the cells are placed at 37 ℃ and the volume fraction of CO is 5% 2 Culturing for 24h in an incubator, adding 1.5-2 mL of culture medium, adding the culture medium once every three days, observing the growth condition of cells after 5-7 days, photographing, recording as P0 when the cells reach 80% fusion, and harvesting the cells to obtain the culture medium.
Preferably, the culture dish in step S2 has a specification of 90mm; the digestive enzyme is prepared from trypsin, chymosin and xylanase in a weight ratio of 8-15: 2-4: 1-3 parts.
Preferably, the mixed solution containing the bactericide in the step S3 comprises the following components in percentage by weight: 1-2% of dandelion extract, 0.5-0.8% of rosemary hydrosol, 0.3-0.8% of vitamin C and 96.4-98.2% of purified water.
Preferably, the mixed solution containing the bactericide in the step S3 comprises 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.
Preferably, the centrifugation condition in the step S3 is centrifugation at 1500-2000 r/min for 5-10 min.
Preferably, the culture medium in the step S4 consists of LB liquid culture medium, tween-20, type IV collagen and BI recombinant vitronectin, and the preparation process is as follows: adding Tween-20, type IV collagen and BI recombinant glass fibronectin into an LB liquid culture medium respectively, wherein the final concentration of the type IV collagen in the culture medium is 2-5 mug/mL, the concentration of the BI recombinant glass fibronectin is 0.1-0.2 mug/mL, and the adding amount of the Tween-20 is 8-15 mug/mL.
Preferably, the final concentration of type IV collagen in the medium described in step S4 is 4. Mu.g/mL, the concentration of BI recombinant vitronectin is 0.15mg/mL, and the addition of Tween-20 is 12. Mu.L/mL.
Preferably, the specific operation of observing the growth of cells in step S4 is: observing under an inverted microscope, if the cells climb out around the tissue blocks, half of the culture solution is replaced once, namely, half of the culture solution in the original culture flask is discarded, then the same amount of fresh culture solution is added, the tissue blocks are sucked out when the cell climbing area reaches 30% of the area of the culture flask, the culture solution is supplemented, the solution is replaced once every other day, and the passage is carried out when the cells grow to 80% of fusion.
The invention also provides application of the culture method in culturing primary amniotic stem cells.
Based on the fact that the amniotic membrane tissue is light and thin and is not easy to adhere, the main reason is probably that the adhering culture time is too long, based on the fact that the primary amniotic membrane stem cells are prepared by the method of mainly optimizing the adhering method, in the research process, the inventor finds that tissue blocks are placed in a culture medium prepared from trypsin, chymosin and xylanase according to the weight ratio of 8-15: 2-4: 1-3, which can help to remove redundant tissues on the basis of promoting cell adhesion, and has an effect higher than that of common trypsin digestion, in the digestion process, only one layer of amniotic membrane (namely the epithelial layer and the other 4 layers are not digested) is digested by adding one-time digestive enzyme in order to improve the digestion effect and the cell purity. The amnion tissue surface dirt can be further removed by further soaking in the mixed solution containing the bactericide for a period of time, other tissue pollution is avoided, the addition of the bactericide can prevent the cells from being polluted by other bacteria in the soaking process, and the purity of the cultured cells is improved.
Meanwhile, in the research of promoting the cell adherence process, the obtained cells to be cultured are inoculated in a T25 culture bottle in a suspending way by using a culture medium, the culture bottle is inverted and dried for 15-20 min, namely, the amniotic membrane in the culture bottle is dried briefly, so that the amniotic membrane is easier to adhere, and when the cells are cultured, the culture medium consisting of LB liquid culture medium, IV type collagen and BI recombinant glass-adhesive protein is adopted, so that the cell adherence can be quickened, and the cell culture time is shortened. The preparation time of the primary amniotic 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 the minimum is shortened to 12.5 days after the optimization of the invention.
In the screening process of substances, the IV type collagen is mainly used for the adherent culture of neuron cells and glial cells, the adherent effect on endothelial cells, liver cells, epithelial cells and the like is poor, the inventor discovers that the culture medium adopted by the invention can reduce the addition amount of BI recombinant vitronectin on the premise of adding a small amount of IV type collagen in the research process of the adherent culture of amniotic stem cells, and further intensively researches that the use concentration of BI recombinant vitronectin can be reduced to 0.1-0.2 mg/mL when the final concentration of IV type collagen is 2-5 mu g/mL, and the adherent effect on the amniotic stem cells is still good under the condition that the low concentration of BI recombinant vitronectin, so that the culture time of the cells is effectively reduced, and the optimal addition concentration ratio is 4 mu g/mL:0.15mg/mL. In addition, the whole culture process is carried out under aseptic conditions, and the mixed solution containing the bactericide is soaked, so that bacteria are doubly prevented from polluting amniotic stem cells, and the cell purity is improved.
Compared with the prior art, the culture method of the primary amniotic stem cells provided by the invention has the following advantages:
the culture method provided by the invention can accelerate cell adhesion, clean tissues in cells, and improve the purity of cells to more than 95% on the premise of shortening the culture time of amniotic stem cells.
Detailed Description
The present invention will be further explained with reference to specific examples, but it should be noted that the following examples are only for explaining the present invention, and are not intended to limit the present invention, and all technical solutions identical or similar to the present invention are within the scope of the present invention. The specific techniques or conditions are not noted in this example and are practiced according to methods and apparatus conventional in the art; the reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The whole herb of dandelion extract extracted from dandelion belonging to the family Compositae is available from Nanjing ze Lang Biotechnology Co., ltd; the rosemary hydrosol can be purchased from Huamao biotechnology Limited liability company, and the CAS number is 084604-14-8; the type IV collagen is available from Shanghai Hengyuan biosciences limited; the BI recombinant glass nectin, namely BioLamina human recombinant laminin, is available from Guangzhou Baiwang biotechnology Co., ltd, model LN111-LN521; the LB liquid medium is commercially available from Beijing Bai Octa Biotechnology Co.
Example 1A method for culturing Primary amniotic Stem cells
The culture method of the primary amniotic stem cells comprises the following preparation processes:
s1, stripping the amniotic membrane from the placenta under the aseptic condition, washing the amniotic membrane for 3 times by using normal saline, removing blood filaments and sticky matters on the amniotic membrane, cutting the amniotic membrane into tissue small blocks of 6cm multiplied by 6cm in a culture dish, and placing the smooth surface of the amniotic membrane downwards to obtain primarily treated amniotic membrane tissue blocks;
s2, placing the preliminarily treated amniotic membrane tissue blocks obtained in the step S1 in culture dishes with 90mm, placing a small tissue block in each culture dish, and then placing trypsin, chymosin and xylanase according to a weight ratio of 8:2:1, respectively adding the digestive enzymes into each culture dish, putting the culture dishes at 23 ℃ for digestion for 28min according to the adding amount of 2.5g/L, and then washing the culture dishes with physiological saline for 2 times to obtain digested amniotic membrane tissues;
s3, preparing a mixed solution containing bactericide according to the following components in percentage by weight: 1% of dandelion extract, 0.5% of rosemary hydrosol, 0.3% of vitamin C and 98.2% of purified water, then cutting the digested amniotic membrane tissue obtained in the step S2 into fragments of 1-2 cm, soaking for 10min with a prepared mixed solution containing a bactericide, washing for 2 times with normal saline, centrifuging after each washing for 10min under the centrifugation condition of 1500r/min, and finally collecting precipitates to obtain cells to be cultured;
s4, preparing a culture medium: adding Tween-20, type IV collagen and BI recombinant glass fibronectin into LB liquid culture medium respectively, wherein the final concentration of type IV collagen in the culture medium is 2 μg/mL, the final concentration of BI recombinant glass fibronectin is 0.2mg/mL, the adding amount of Tween-20 is 8 μl/mL, inoculating the cells to be cultured obtained in step S3 into T25 culture flask by using 1.5mL of the prepared culture medium, inverting the culture flask, drying for 15min, and placing in CO with volume fraction of 5% at 37deg.C 2 Culturing in an incubator for 24 hours, adding 1.5mL of culture medium, adding the culture medium once every three days, observing the growth condition of cells after 5-7 days, photographing, and recording as P0 when the cells reach 80% fusion, wherein the specific observation process is as follows: observing under an inverted microscope, if the cells climb out around the tissue blocks, half of the culture solution is replaced once, namely, half of the culture solution in the original culture flask is discarded, then the fresh culture solution with the same amount is added, the tissue blocks can be sucked out when the cell climbing area reaches 30% of the area of the culture flask, the culture solution is supplemented, the solution is replaced once every other day, P0 is recorded when the cells grow to 80% of the fusion, and the cells are harvested, thus obtaining the culture medium.
Example 2A method of culturing Primary amniotic Stem cells
The culture method of the primary amniotic stem cells comprises the following preparation processes:
s1, stripping the amniotic membrane from the placenta under the aseptic condition, washing the amniotic membrane for 5 times by using normal saline, removing blood filaments and sticky matters on the amniotic membrane, cutting the amniotic membrane into tissue small blocks of 6cm multiplied by 6cm in a culture dish, and placing the smooth surface of the amniotic membrane downwards to obtain primarily treated amniotic membrane tissue blocks;
s2, placing the preliminarily treated amniotic membrane tissue blocks obtained in the step S1 in culture dishes with 90mm, placing a small tissue block in each culture dish, and then placing trypsin, chymosin and xylanase according to a weight ratio of 15:4:3, respectively adding the digestive enzymes into each culture dish, putting the culture dishes at the temperature of 27 ℃ for digestion for 32min according to the amount of 2.5g/L, and then washing the culture dishes with physiological saline for 4 times to obtain digested amniotic membrane tissues;
s3, preparing a mixed solution containing bactericide according to the following components in percentage by weight: 2% of dandelion extract, 0.8% of rosemary hydrosol, 0.8% of vitamin C and 96.4% of purified water, then shearing the digested amniotic membrane tissue obtained in the step S2 into fragments of 1-2 cm by using an ophthalmic lens, soaking for 20min by using a prepared mixed solution containing a bactericide, washing for 3 times by using normal saline, centrifuging after washing for 5min at the centrifugation condition of 2000r/min, and finally collecting precipitates to obtain cells to be cultured;
s4, preparing a culture medium: respectively adding Tween-20, type IV collagen and BI recombinant glass fibronectin into LB liquid culture medium, wherein the final concentration of type IV collagen in the culture medium is 5 mug/mL, the final concentration of BI recombinant glass fibronectin is 0.1mg/mL, and the adding amount of Tween-20 is 15 mug/mL; then the cells to be cultured obtained in the step S3 are inoculated in a T25 culture flask in a suspension manner by using 1.8mL of the prepared culture medium, the culture flask is inverted and dried for 20min, and then the cells are placed at 37 ℃ and the volume fraction of CO is 5% 2 Culturing for 24 hours in an incubator, adding 1.8mL of culture medium, adding the culture medium once every three days, observing the growth condition of cells after 5-7 days, photographing, recording as P0 when the cells reach 80% fusion, and harvesting the cells.
Example 3A method of culturing Primary amniotic Stem cells
The culture method of the primary amniotic stem cells comprises the following preparation processes:
s1, stripping the amniotic membrane from the placenta under the aseptic condition, washing for 4 times by using normal saline, removing blood filaments and sticky matters on the amniotic membrane, cutting into tissue small blocks of 6cm multiplied by 6cm in a culture dish, and placing the smooth surface of the amniotic membrane downwards to obtain a primarily treated amniotic membrane tissue block;
s2, placing the preliminarily treated amniotic membrane tissue blocks obtained in the step S1 in culture dishes with 90mm, placing a small tissue block in each culture dish, and then placing trypsin, chymosin and xylanase according to a weight ratio of 12:3:2, respectively adding the digestive enzymes into each culture dish, putting the culture dishes at the temperature of 25 ℃ for digestion for 30min according to the amount of 2.5g/L, and then washing the culture dishes with physiological saline for 3 times to obtain digested amniotic membrane tissues;
s3, preparing a mixed solution containing 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 shearing the digested amniotic membrane tissue obtained in the step S2 into fragments of 1-2 cm by using an ophthalmic lens, soaking the fragments for 15min by using a mixed solution containing a bactericide, washing for 3 times by using normal saline, centrifuging after each washing, treating for 17min under the centrifugation condition of 1800rpm, and finally collecting precipitates to obtain cells to be cultured;
s4, preparing a culture medium: adding Tween-20, type IV collagen and BI recombinant glass fibronectin into LB liquid culture medium, wherein the final concentration of type IV collagen in the culture medium is 4 μg/mL, the final concentration of BI recombinant glass fibronectin is 0.15mg/mL, the adding amount of Tween-20 is 12 μl/mL, then re-suspending the cells to be cultured obtained in step S3 in a T25 culture flask with 2mL of the prepared culture medium, inverting the culture flask, drying for 20min, and placing in CO with volume fraction of 5% at 37deg.C 2 Culturing in an incubator for 24 hours, adding 2mL of culture medium, adding the culture medium once every three days, observing the growth condition of cells after 5-7 days, photographing, recording as P0 when the cells reach 80% fusion, and harvesting the cells.
Comparative example 1 culture method of Primary amniotic Stem cells
The culture method of the primary amniotic stem cells is similar to that of example 3;
the difference from example 2 is that the digestive enzyme in comparative example 1 is trypsin.
Comparative example 2A method of culturing Primary amniotic Stem cells
The culture method of the primary amniotic stem cells is similar to that of example 3;
the difference from example 2 is that dandelion extract in the mixed solution containing bactericide is replaced with licorice extract in comparative example 2.
Comparative example 3 culture method of Primary amniotic Stem cells
The culture method of the primary amniotic stem cells is similar to that of example 3;
the difference from example 2 is that the type IV collagen was replaced with type I collagen in the preparation of the medium of comparative example 3, in a constant amount.
Comparative example 4A method of culturing Primary amniotic Stem cells
The culture method of the primary amniotic stem cells is similar to that of example 3;
the difference from example 2 is that the concentration of BI recombinant vitronectin was 0.5mg/mL when the medium was prepared in comparative example 4, and no type IV collagen was contained.
Comparative example 5A method of culturing Primary amniotic Stem cells
The existing culture method is adopted.
Slowly tearing the outer amniotic membrane of the fetal membrane by using surgical forceps, placing the amniotic membrane in a 150mm culture dish, repeatedly cleaning the surface dirty blood by using basic balanced salt solution, and shearing into amniotic membrane tissue blocks with the diameter of 5 mm; filtering with 300 mesh sieve, washing residual blood with normal saline, and placing tissue block at 75cm 2 In the culture flask, the tissue block can cover half of the culture area in the culture flask, the complete culture medium is added without the tissue, and the culture flask is evenly shaken and then subjected to 5% CO 2 Culturing in a 37 ℃ incubator; supplementing 10mL of DMEM complete medium after 2d, and continuing to culture; after more mesenchymal stem cells climb out, tissues are removed, liquid is periodically changed (every 2 days), and subculture is carried out when the cells reach 80% fusion.
Test example 1 incubation time comparison
1. The test method comprises the following steps: the culture methods of examples 1-3 and comparative examples 1-5;
2. test results: counting the time required for each culture method to culture to 80% fusion; the specific test results are shown in Table 1.
TABLE 1 time required for different test methods to culture to 80% confluence
Test example 2 cell purity comparison
1. Test sample: 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: detection of P0 generation using flow cytometryCell phenotype and cell purity, including surface markers such as CD73, CD90, CD44, CD105, CD166, CD11b, CD19, CD34, CD45, HLA-DR, and the like. The specific detection process comprises the following steps: collecting cell suspension, centrifuging at 1000rpm for 10min, removing supernatant, washing cell precipitate with 0.01 mol/L PBS (4 deg.C precooled for 2 times at 1000rpm for 10 min), re-suspending cell precipitate with 200 μl PBS buffer, collecting 1×10 6 Individual cells were resuspended in 0.1ml of pre-chilled 0.01 mol/L PBS, 2uL of the corresponding CD73, CD90, CD105, CD44, CD166, negative markers (CD 34, CD45, CD19, CD11b, HLA-DR) fluorescent antibodies were added in the dark, incubated at 4 ℃ for 30min in the dark, 2mL PBS,l000 rpm,l0min was added, the cells were washed to remove unbound antibodies, the supernatant removed, the cell pellet resuspended in PBS, and examined by flow cytometry at 4 ℃.
3. Test results: cryopreserved cells of 2X 10 or more 7 The P0 phenotype was detected as acceptable (by automatic counting with a cytometer), and the surface marker expression results are shown in tables 2 and 3, respectively.
TABLE 2 results of different factor expression
TABLE 3 comparison of the expression results of different factors
As is clear from tables 2 and 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 were all lower than 1%, among which the cell purity of example 2 was the highest. In comparative example 2, the expression rates of CD73, CD90, CD44, CD105 and CD166 were not 95%, and the expression rates of CD11b, CD19, CD34, CD45 and HLA-DR were 1% -2%, and the purity of comparative example 2 was significantly reduced as compared with examples 1-3.
It should be noted that, although the foregoing embodiments have been described in some detail, those skilled in the art, once having the basic inventive concept, may make other changes and modifications to the embodiments, so that the foregoing description is only an embodiment of the present invention, and therefore, the scope of the present invention is not limited thereto, and all equivalent structures or equivalent flow modifications made by the present invention or directly or indirectly applied to other related technical fields are included in the scope of the present invention.
Claims (6)
1. A method for culturing primary amniotic stem cells, comprising the following steps:
s1, stripping the amniotic membrane from the placenta under the aseptic condition, washing with normal saline to remove blood filaments and sticky matters on the amniotic membrane, cutting into tissue small blocks of 6cm multiplied by 6cm in a culture dish, and placing the smooth surface of the amniotic membrane downwards to obtain a primarily treated amniotic membrane tissue block;
s2, placing the primarily treated amniotic membrane tissue blocks obtained in the step S1 in culture dishes, placing a tissue small block in each culture dish, adding digestive enzyme into each culture dish according to the amount of 2.5g/L, digesting for 28-32min at the temperature of 23-27 ℃, and washing with normal saline to obtain digested amniotic membrane tissue;
s3, cutting the digested amniotic membrane tissue obtained in the step S2 into fragments of 1-2 cm, soaking the fragments in a mixed solution containing a bactericide for 10-20 min, washing the fragments with normal saline, centrifuging the washed fragments after washing each time, and finally collecting the precipitate to obtain cells to be cultured;
s4, the cells to be cultured obtained in the step S3 are inoculated in a T25 culture flask in a suspension manner by using 1.5-2 mL of culture medium, the culture flask is inverted and dried for 15-20 min, and then the cells are placed at 37 ℃ and the volume fraction of CO is 5% 2 Culturing in an incubator for 24 hours, adding 1.5-2 mL of culture medium, adding the culture medium once every three days, observing the growth condition of cells after 5-7 days, photographing, recording as P0 when the cells reach 80% fusion, and harvesting the cells to obtain the culture medium;
the specification of the culture dish in the step S2 is 90mm; the digestive enzyme is prepared from trypsin, chymosin and xylanase according to the weight ratio of 8-15: 2 to 4:1 to 3;
the mixed solution containing the bactericide in the step S3 comprises the following components in percentage by weight: 1 to 2 percent of dandelion extract, 0.5 to 0.8 percent of rosemary hydrosol, 0.3 to 0.8 percent of vitamin C and 96.4 to 98.2 percent of purified water;
the culture medium in the step S4 consists of LB liquid culture medium, tween-20, IV type collagen and BI recombinant vitronectin, and the preparation process is as follows: adding Tween-20, IV type collagen and BI recombinant glass fibronectin into LB liquid culture medium respectively, wherein the final concentration of the IV type collagen in the culture medium is 2-5 mug/mL, the final concentration of the BI recombinant glass fibronectin is 0.1-0.2 mg/mL, and the adding amount of the Tween-20 is 8-15 mug/mL.
2. The culture method according to claim 1, wherein the mixed solution containing the bactericide in the step S3 comprises 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.
3. The culture method according to claim 1, wherein the centrifugation conditions in the step S3 are centrifugation at 1500 to 2000r/min for 5 to 10min.
4. The method of claim 1, wherein the culture medium of step S4 has a final concentration of type IV collagen of 4 μg/mL, a final concentration of BI recombinant vitronectin of 0.15mg/mL, and tween-20 is added in an amount of 12 μl/mL.
5. The culture method according to claim 1, wherein the specific operation of observing the growth of cells in step S4 is as follows: observing under an inverted microscope, if the cells climb out around the tissue blocks, half of the culture solution is replaced once, namely, half of the culture solution in the original culture flask is discarded, then the same amount of fresh culture solution is added, the tissue blocks are sucked out when the cell climbing area reaches 30% of the area of the culture flask, the culture solution is supplemented, the solution is replaced once every other day, and the passage is carried out when the cells grow to 80% of fusion.
6. Use of the culture method of any one of claims 1-5 in primary amniotic stem cells.
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