CN110628713A - Method for extracting and preparing adipose-derived stem cells - Google Patents
Method for extracting and preparing adipose-derived stem cells Download PDFInfo
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- 210000000130 stem cell Anatomy 0.000 title claims abstract description 31
- 238000000034 method Methods 0.000 title claims description 6
- 210000004027 cell Anatomy 0.000 claims abstract description 89
- 210000000577 adipose tissue Anatomy 0.000 claims abstract description 43
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims abstract description 26
- 238000005406 washing Methods 0.000 claims abstract description 26
- 102000029816 Collagenase Human genes 0.000 claims abstract description 18
- 108060005980 Collagenase Proteins 0.000 claims abstract description 18
- 229960002424 collagenase Drugs 0.000 claims abstract description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 16
- 238000012258 culturing Methods 0.000 claims abstract description 14
- 238000001514 detection method Methods 0.000 claims abstract description 14
- 238000011534 incubation Methods 0.000 claims abstract description 14
- 238000005070 sampling Methods 0.000 claims abstract description 14
- 238000000926 separation method Methods 0.000 claims abstract description 14
- 238000002360 preparation method Methods 0.000 claims abstract description 13
- 238000000605 extraction Methods 0.000 claims abstract description 8
- 230000000249 desinfective effect Effects 0.000 claims abstract description 4
- 239000012634 fragment Substances 0.000 claims description 38
- 210000001519 tissue Anatomy 0.000 claims description 27
- 229960001484 edetic acid Drugs 0.000 claims description 25
- 239000000243 solution Substances 0.000 claims description 24
- 239000011259 mixed solution Substances 0.000 claims description 14
- 238000004659 sterilization and disinfection Methods 0.000 claims description 12
- 229920000742 Cotton Polymers 0.000 claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- 238000001914 filtration Methods 0.000 claims description 10
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- 238000002156 mixing Methods 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 10
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- 239000000725 suspension Substances 0.000 claims description 10
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- 241000894006 Bacteria Species 0.000 abstract description 4
- 239000011780 sodium chloride Substances 0.000 abstract description 3
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
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- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
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Abstract
The invention discloses an adipose-derived stem cell extraction preparation method, which comprises the following steps: collagenase type I, EDTA, PBS and saline. The preparation process comprises the following steps: sampling, shearing, disinfecting and washing, enzymolysis, separation, incubation, detection and directional culture. The invention uses PBS to wash before extracting and culturing, and uses normal saline to soak, has eliminated the bacterium that the adipose tissue surface adheres to, has avoided the problem that the bacterium pollutes the adipose tissue and causes the wrong culture in the course of culturing; the cells are cultured in the environment with the temperature of 37 ℃ to simulate the temperature environment of a human body, so that the cultured cells can adapt to the environment of the human body.
Description
Technical Field
The invention belongs to the field of stem cell preparation, and particularly relates to an extraction and preparation method of an adipose-derived stem cell.
Background
The term "stem" means "origin and occurrence" of a stem cell. Stem cells, as the name implies, are cells that develop into other tissues and organs of the body, meaning cells that have not yet developed to maturity and that have a multi-differentiation potential and a self-replicating function. Under certain conditions, it can differentiate into different functional cells, forming various tissues and organs. The medical community expects that stem cells can be used to repair damaged tissues or organs, and are therefore referred to as "universal cells". The application of stem cells in the fields of treating chronic diseases, beautifying, resisting aging, skin care products and the like is very wide, and the promotion of stem cells in the journal of the United states 'science' in 1999 is one of the most important ten research fields of the 21 st century and is the first place. In 2000, stem cells were re-selected in the ten-major scientific achievement of "science" review. The science in the end of 2001 was put into the first of six popular science and technology fields worth paying attention in 2002. Stem cells have become the most valuable field of research in the 21 st century, with great prospects for development.
Adipose-derived stem cells are a kind of stem cells with multi-differentiation potential which have been isolated from adipose tissues in recent years. The cell has strong proliferation capacity, can maintain stable growth and proliferation activity in vitro culture, has multiple differentiation potentials, and has been applied to cosmetic treatment. The adipose-derived stem cells mainly restore the repair capability of tissue cells, promote the regeneration of the cells, restore the young face, simultaneously fully improve the physical function, effectively improve diseases such as sub-health, premature senility and the like, and really and effectively resist aging from inside to outside.
Disclosure of Invention
The invention aims to provide a method for extracting and preparing adipose-derived stem cells.
The purpose of the invention can be realized by the following technical scheme:
an adipose-derived stem cell extraction preparation method comprises the following preparation processes: sampling, cutting, disinfecting and washing, enzymolysis, separation, incubation, detection and directional culture, and the specific extraction preparation method comprises the following steps:
s1, sampling: sucking deep adipose tissue from the upper part of the hip by using a sterile needle tube;
s2, cutting: cutting the sucked deep fat tissue into pieces;
s3, disinfection and washing: washing the deep fat tissue fragments with PBS, wiping with alcohol cotton, and soaking with normal saline;
s4, enzymolysis: adding mixed solution of collagenase type I and EDTA into the deep adipose tissue fragments after disinfection and washing, and fully oscillating in water bath at 37 ℃;
s5, separation: adding a proper amount of PBS into the deep adipose tissue fragments subjected to enzymolysis, mixing, filtering, and centrifuging at a low speed;
s6, incubation: containing CO at 37 deg.C2Culturing in the incubator, changing the culture solution for the first time after 48 hours, discarding the suspension cells, then changing the culture solution once every 3 days, and after passage, growing the cells from the tissue blocks to primary cells;
s7, detection: detecting the 3 rd generation cells after twice passage by using a flow cytometer, and observing whether the cells are concentrated to 128 cells/liter;
s8, directional culture: when the concentration of the cells is 128/L, the cells are transferred into a culture medium for directional culture for 2 weeks, and the adipose-derived stem cells are obtained.
S1, sampling: sucking 10ml of deep adipose tissue from the upper part of the hip by using a sterile needle tube;
s2, cutting: cutting the sucked deep adipose tissues into 2mm fragments;
s3, disinfection and washing: washing the deep fat tissue fragment with PBS for 5 times, cleaning with alcohol cotton, and soaking in normal saline for 2 min;
s4, enzymolysis: adding 10ml of mixed solution of collagenase type I and EDTA (ethylene diamine tetraacetic acid) with the volume ratio of 1:3 into the disinfected and washed deep adipose tissue fragments, and fully oscillating the mixture for 30 minutes in a water bath at 37 ℃;
s5, separation: adding a proper amount of PBS into the deep adipose tissue fragments subjected to enzymolysis, mixing, filtering, and then centrifuging at a low speed for 10 minutes, wherein the centrifugal speed is controlled to be 800 r/min;
s6, incubation: CO at a temperature of 37 ℃ and a volume concentration of 5%2Culturing in the incubator, changing the culture solution for the first time after 48 hours, discarding the suspension cells, then changing the culture solution once every 3 days, and carrying out passage after 7 days, wherein the cells grow into primary cells from the tissue blocks;
s7, detection: detecting the 3 rd generation cells after twice passage by using a flow cytometer, and observing whether the cells are concentrated to 128 cells/liter;
s8, directional culture: when the concentration of the cells is 128/L, the cells are transferred into a culture medium for directional culture for 2 weeks, and the adipose-derived stem cells are obtained.
S1, sampling: sucking 10ml of deep adipose tissue from the upper part of the hip by using a sterile needle tube;
s2, cutting: cutting the sucked deep adipose tissues into 2mm fragments;
s3, disinfection and washing: washing the deep fat tissue fragment with PBS for 5 times, cleaning with alcohol cotton, and soaking in normal saline for 2 min;
s4, enzymolysis: adding 20ml of mixed solution of collagenase type I and EDTA (ethylene diamine tetraacetic acid) with the volume ratio of 1:2 into the disinfected and washed deep adipose tissue fragments, and fully oscillating the mixture for 30 minutes in a water bath at 37 ℃;
s5, separation: adding a proper amount of PBS into the deep adipose tissue fragments subjected to enzymolysis, mixing, filtering, and then centrifuging at a low speed for 10 minutes, wherein the centrifugal speed is controlled to be 800 r/min;
s6, incubation: CO at a temperature of 37 ℃ and a volume concentration of 5%2Culturing in the incubator, changing the culture solution for the first time after 48 hours, discarding the suspension cells, then changing the culture solution once every 3 days, and carrying out passage after 8 days, wherein the cells grow into primary cells from the tissue blocks;
s7, detection: detecting the 3 rd generation cells after twice passage by using a flow cytometer, and observing whether the cells are concentrated to 128 cells/liter;
s8, directional culture: when the concentration of the cells is 128/L, the cells are transferred into a culture medium for directional culture for 2 weeks, and the adipose-derived stem cells are obtained.
And stirring the mixed solution of EDTA and type I collagenase for 5-10 minutes at the rotating speed of 500r/min, and storing the mixed solution in an environment at the temperature of 2-8 ℃ after stirring.
The invention has the beneficial effects that:
1. the invention uses PBS to wash before extracting and culturing, and uses normal saline to soak, has eliminated the bacterium that the adipose tissue surface adheres to, has avoided the problem that the bacterium pollutes the adipose tissue and causes the wrong culture in the course of culturing;
2. the invention cultures cells under the environment with the temperature of 37 ℃ and simulates the temperature environment of human body, so that the cultured cells can adapt to the environment of human body.
Drawings
In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.
FIG. 1 is a flow chart of the extraction preparation according to an embodiment of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The preparation process comprises the following steps: sampling, shearing, disinfecting and washing, enzymolysis, separation, incubation, detection and directional culture.
The extraction preparation process of the invention is shown in figure 1.
Example 1
S1, sampling: sucking 10ml of deep adipose tissue from the upper part of the hip by using a sterile needle tube;
s2, cutting: cutting the sucked deep adipose tissues into 2mm fragments;
s3, disinfection and washing: washing the deep fat tissue fragment with PBS for 5 times, cleaning with alcohol cotton, and soaking in normal saline for 2 min;
s4, enzymolysis: adding 10ml of mixed solution of collagenase type I and EDTA (ethylene diamine tetraacetic acid) with the volume ratio of 1:3 into the disinfected and washed deep adipose tissue fragments, and fully oscillating the mixture for 30 minutes in a water bath at 37 ℃;
s5, separation: adding a proper amount of PBS into the deep adipose tissue fragments subjected to enzymolysis, mixing, filtering, and then centrifuging at a low speed for 10 minutes, wherein the centrifugal speed is controlled to be 800 r/min;
s6, incubation: CO at a temperature of 37 ℃ and a volume concentration of 5%2Culturing in the incubator, changing the culture solution for the first time after 48 hours, discarding the suspension cells, then changing the culture solution once every 3 days, and carrying out passage after 7 days, wherein the cells grow into primary cells from the tissue blocks;
s7, detection: detecting the 3 rd generation cells after twice passage by using a flow cytometer, and observing whether the cells are concentrated to 128 cells/liter;
s8, directional culture: when the concentration of the cells is 128/L, the cells are transferred into a culture medium for directional culture for 2 weeks, and the adipose-derived stem cells are obtained.
Example 2
S1, sampling: sucking 10ml of deep adipose tissue from the upper part of the hip by using a sterile needle tube;
s2, cutting: cutting the sucked deep adipose tissues into 2mm fragments;
s3, disinfection and washing: washing the deep fat tissue fragment with PBS for 5 times, cleaning with alcohol cotton, and soaking in normal saline for 2 min;
s4, enzymolysis: adding 20ml of mixed solution of collagenase type I and EDTA (ethylene diamine tetraacetic acid) with the volume ratio of 1:2 into the disinfected and washed deep adipose tissue fragments, and fully oscillating the mixture for 30 minutes in a water bath at 37 ℃;
s5, separation: adding a proper amount of PBS into the deep adipose tissue fragments subjected to enzymolysis, mixing, filtering, and then centrifuging at a low speed for 10 minutes, wherein the centrifugal speed is controlled to be 800 r/min;
s6, incubation: CO at a temperature of 37 ℃ and a volume concentration of 5%2Culturing in the incubator, changing the culture solution for the first time after 48 hours, discarding the suspension cells, then changing the culture solution once every 3 days, and carrying out passage after 8 days, wherein the cells grow into primary cells from the tissue blocks;
s7, detection: detecting the 3 rd generation cells after twice passage by using a flow cytometer, and observing whether the cells are concentrated to 128 cells/liter;
s8, directional culture: when the concentration of the cells is 128/L, the cells are transferred into a culture medium for directional culture for 2 weeks, and the adipose-derived stem cells are obtained.
Example 3
S1, sampling: sucking 10ml of deep adipose tissue from the upper part of the hip by using a sterile needle tube;
s2, cutting: cutting the sucked deep adipose tissues into 2mm fragments;
s3, disinfection and washing: washing the deep fat tissue fragment with PBS for 5 times, cleaning with alcohol cotton, and soaking in normal saline for 2 min;
s4, enzymolysis: adding 20ml of mixed solution of collagenase type I and EDTA (ethylene diamine tetraacetic acid) with the volume ratio of 1:3 into the disinfected and washed deep adipose tissue fragments, and fully oscillating the mixture for 30 minutes in a water bath at 37 ℃;
s5, separation: adding a proper amount of PBS into the deep adipose tissue fragments subjected to enzymolysis, mixing, filtering, and then centrifuging at a low speed for 10 minutes, wherein the centrifugal speed is controlled to be 800 r/min;
s6, incubation: CO at a temperature of 37 ℃ and a volume concentration of 6%2Culturing in the incubator, changing the culture solution for the first time after 48 hours, discarding the suspension cells, then changing the culture solution once every 3 days, and carrying out passage after 5 days, wherein the cells grow into primary cells from the tissue blocks;
s7, detection: detecting the 3 rd generation cells after twice passage by using a flow cytometer, and observing whether the cells are concentrated to 128 cells/liter;
s8, directional culture: when the concentration of the cells is 128/L, the cells are transferred into a culture medium for directional culture for 2 weeks, and the adipose-derived stem cells are obtained.
Example 4
S1, sampling: sucking 10ml of deep adipose tissue from the upper part of the hip by using a sterile needle tube;
s2, cutting: cutting the sucked deep adipose tissues into 2mm fragments;
s3, disinfection and washing: washing the deep fat tissue fragment with PBS for 5 times, cleaning with alcohol cotton, and soaking in normal saline for 2 min;
s4, enzymolysis: adding 20ml of mixed solution of collagenase type I and EDTA (ethylene diamine tetraacetic acid) with the volume ratio of 1:3 into the disinfected and washed deep adipose tissue fragments, and fully oscillating the mixture for 30 minutes in a water bath at 37 ℃;
s5, separation: adding a proper amount of PBS into the deep adipose tissue fragments subjected to enzymolysis, mixing, filtering, and then centrifuging at a low speed for 10 minutes, wherein the centrifugal speed is controlled to be 800 r/min;
s6, incubation: CO at a temperature of 37 ℃ and a volume concentration of 5%2Culturing in the incubator, changing the culture solution for the first time after 48 hours, discarding the suspension cells, then changing the culture solution once every 3 days, and subculturing after 6 days, wherein the cells grow into primary cells from the tissue blocks;
s7, detection: detecting the 3 rd generation cells after twice passage by using a flow cytometer, and observing whether the cells are concentrated to 128 cells/liter;
s8, directional culture: when the concentration of the cells is 128/L, the cells are transferred into a culture medium for directional culture for 2 weeks, and the adipose-derived stem cells are obtained.
It is noted that the mixed solution of EDTA and collagenase type I needs to be stirred for 5-10 minutes at a rotation speed of 500r/min, and is stored in an environment of 2-8 ℃ after stirring.
Several culture solutions of the present invention are briefly described below:
1. PBS: PBS is a phosphate buffered saline solution, generally acting as a solvent, which acts to dissolve the protective agent. It is a buffer solution which is most widely used in biochemical research, and the main component of the buffer solution is Na2HPO4、KH2PO4NaCl and KCl due to Na2HPO4And KH2PO4They have secondary dissociation, and the buffered pH value range is wide; while NaCl and KCl mainly act to increase the salt ion concentration.
2. EDTA: EDTA is EDTA, an organic compound of formula C10H16N2O8White powder at normal temperature and pressure. It is a compound capable of reacting with Mg2+、Ca2+、Mn2+、Fe2+And divalent metal ion-binding chelating agents. Mg is required for the action of most nucleases and some proteases2+Therefore, it is often used as an inhibitor of nuclease and protease; can also be used for removing the inhibition effect of heavy metal ions on enzyme.
3. Collagenase: collagenase, chemically known as a collagen hydrolase, specifically hydrolyzes the three-dimensional helical structure of native collagen under physiological PH and temperature conditions without damaging other proteins and tissues. Collagenase is a protein in its chemical nature, and therefore, it is very sensitive to temperature, PH and various factors that cause protein denaturation, and is very susceptible to external conditions to change its conformation and properties.
The following table shows the results of four comparative examples:
from the above table, it can be seen that:
1. according to examples 1 and 4, it can be seen that the more the amount of the mixed solution is, the faster the passage speed is when the other conditions are the same;
2. from examples 1 and 3, it can be seen that when other conditions are the same, CO is present2The larger the volume concentration is, the faster the passage speed is;
3. according to examples 2 and 4, it is found that the smaller the proportion of the mixture (collagenase type I and EDTA) is, the higher the passaging speed is when the other conditions are the same.
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed.
Claims (4)
1. An adipose-derived stem cell extraction preparation method comprises the following preparation processes: sampling, cutting, disinfecting and washing, enzymolysis, separation, incubation, detection and directional culture, and is characterized in that the specific extraction and preparation method comprises the following steps:
s1, sampling: sucking deep adipose tissue from the upper part of the hip by using a sterile needle tube;
s2, cutting: cutting the sucked deep fat tissue into pieces;
s3, disinfection and washing: washing the deep fat tissue fragments with PBS, wiping with alcohol cotton, and soaking with normal saline;
s4, enzymolysis: adding mixed solution of collagenase type I and EDTA into the deep adipose tissue fragments after disinfection and washing, and fully oscillating in water bath at 37 ℃;
s5, separation: adding a proper amount of PBS into the deep adipose tissue fragments subjected to enzymolysis, mixing, filtering, and centrifuging at a low speed;
s6, incubation: containing CO at 37 deg.C2Culturing in the incubator, changing the culture solution for the first time after 48 hours, discarding the suspension cells, then changing the culture solution once every 3 days, and after passage, growing the cells from the tissue blocks to primary cells;
s7, detection: detecting the 3 rd generation cells after twice passage by using a flow cytometer, and observing whether the cells are concentrated to 128 cells/liter;
s8, directional culture: when the concentration of the cells is 128/L, the cells are transferred into a culture medium for directional culture for 2 weeks, and the adipose-derived stem cells are obtained.
2. The method for extracting and producing adipose-derived stem cells according to claim 1, wherein,
s1, sampling: sucking 10ml of deep adipose tissue from the upper part of the hip by using a sterile needle tube;
s2, cutting: cutting the sucked deep adipose tissues into 2mm fragments;
s3, disinfection and washing: washing the deep fat tissue fragment with PBS for 5 times, cleaning with alcohol cotton, and soaking in normal saline for 2 min;
s4, enzymolysis: adding 10ml of mixed solution of collagenase type I and EDTA (ethylene diamine tetraacetic acid) with the volume ratio of 1:3 into the disinfected and washed deep adipose tissue fragments, and fully oscillating the mixture for 30 minutes in a water bath at 37 ℃;
s5, separation: adding a proper amount of PBS into the deep adipose tissue fragments subjected to enzymolysis, mixing, filtering, and then centrifuging at a low speed for 10 minutes, wherein the centrifugal speed is controlled to be 800 r/min;
s6, incubation: at a temperature of 37 ℃ and a volume concentration of 5%CO of2Culturing in the incubator, changing the culture solution for the first time after 48 hours, discarding the suspension cells, then changing the culture solution once every 3 days, and carrying out passage after 7 days, wherein the cells grow into primary cells from the tissue blocks;
s7, detection: detecting the 3 rd generation cells after twice passage by using a flow cytometer, and observing whether the cells are concentrated to 128 cells/liter;
s8, directional culture: when the concentration of the cells is 128/L, the cells are transferred into a culture medium for directional culture for 2 weeks, and the adipose-derived stem cells are obtained.
3. The method for extracting and producing adipose-derived stem cells according to claim 1, wherein,
s1, sampling: sucking 10ml of deep adipose tissue from the upper part of the hip by using a sterile needle tube;
s2, cutting: cutting the sucked deep adipose tissues into 2mm fragments;
s3, disinfection and washing: washing the deep fat tissue fragment with PBS for 5 times, cleaning with alcohol cotton, and soaking in normal saline for 2 min;
s4, enzymolysis: adding 20ml of mixed solution of collagenase type I and EDTA (ethylene diamine tetraacetic acid) with the volume ratio of 1:2 into the disinfected and washed deep adipose tissue fragments, and fully oscillating the mixture for 30 minutes in a water bath at 37 ℃;
s5, separation: adding a proper amount of PBS into the deep adipose tissue fragments subjected to enzymolysis, mixing, filtering, and then centrifuging at a low speed for 10 minutes, wherein the centrifugal speed is controlled to be 800 r/min;
s6, incubation: CO at a temperature of 37 ℃ and a volume concentration of 5%2Culturing in the incubator, changing the culture solution for the first time after 48 hours, discarding the suspension cells, then changing the culture solution once every 3 days, and carrying out passage after 8 days, wherein the cells grow into primary cells from the tissue blocks;
s7, detection: detecting the 3 rd generation cells after twice passage by using a flow cytometer, and observing whether the cells are concentrated to 128 cells/liter;
s8, directional culture: when the concentration of the cells is 128/L, the cells are transferred into a culture medium for directional culture for 2 weeks, and the adipose-derived stem cells are obtained.
4. The method for extracting and preparing adipose-derived stem cells according to claim 1, wherein the mixture of EDTA and collagenase type I is stirred at a rotation speed of 500r/min for 5-10 minutes, and then stored at 2-8 ℃.
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