CN115651899A - Method for separating and preparing adipose-derived stem cells - Google Patents
Method for separating and preparing adipose-derived stem cells Download PDFInfo
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- CN115651899A CN115651899A CN202211330756.3A CN202211330756A CN115651899A CN 115651899 A CN115651899 A CN 115651899A CN 202211330756 A CN202211330756 A CN 202211330756A CN 115651899 A CN115651899 A CN 115651899A
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Abstract
The invention discloses a method for separating and preparing adipose-derived stem cells, which comprises the following steps of firstly cleaning adipose, repeatedly cleaning adipose tissues until the adipose is completely cleaned, then carrying out centrifugal separation, and adding collagenase solution into a centrifugal machine; the invention uses the stimulating liquid to stimulate the cells after the cells are centrifuged, uses mitogen, ionomycin, lipopolysaccharide, interleukin and 13-acetate to stimulate the cells to generate reaction, thereby promoting the cells to release protein, enzyme and factor, uses the protein, enzyme and factor to repair the damaged tissues and new growing tissues, thereby realizing the purpose of repairing the damaged adipose-derived stem cells.
Description
Technical Field
The invention relates to the technical field of adipose-derived stem cell separation and preparation, in particular to a method for separating and preparing adipose-derived stem cells.
Background
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 is expected to become an important source for repairing damaged tissues and organs in the future;
in the existing technology for separating and preparing the adipose-derived stem cells, the adipose-derived stem cells are prepared by directly culturing the adipose-derived stem cells by using a nutrient solution, however, in the process of separating the adipose-derived stem cells, the adipose-derived stem cells rapidly rotate under the action of high-speed centrifugation of a centrifuge, so that the adipose-derived stem cells are damaged in the rapid centrifugation process, and the cells cannot be repaired by using the direct culture method, so that the cell death situation is easily caused, and further, the situations of low cell survival rate, low cell vitality and the like after the separation and preparation of the adipose-derived stem cells are completed are solved.
Disclosure of Invention
The invention aims to provide a method for separating and preparing an adipose-derived stem cell, which solves the problems of low survival rate, low vitality and the like of a prepared cell caused by the fact that a nutrient solution direct culture mode is adopted in the existing process of separating and preparing the adipose-derived stem cell and damaged cells are not repaired.
In order to achieve the purpose, the invention provides the following technical scheme: a method for isolating and preparing adipose-derived stem cells, comprising the steps of:
step 1: fat cleaning: repeatedly cleaning adipose tissues until the fat is completely cleaned;
step 2: centrifugal separation: adding the collagenase solution into a centrifugal machine, synchronizing the cleaned fat to the centrifugal machine, starting the centrifugal machine to carry out centrifugal separation, and obtaining a fat tissue layer after the centrifugal separation is finished;
and step 3: and (3) centrifugal precipitation: putting the obtained adipose tissue layer into a centrifuge, adding physiological saline for centrifugal separation again, precipitating after the centrifugal separation is finished, and filtering to obtain the adipose-derived stem cells;
and 4, step 4: preparation and culture: and (2) putting the obtained adipose-derived stem cells into a stimulating solution and a nutrient solution for stimulating culture, wherein the stimulating solution is prepared by mixing mitogen, ionomycin, lipopolysaccharide, interleukin and 13-acetate, observing whether the damaged condition of the adipose-derived stem cells is repaired or not after the adipose-derived stem cells are cultured in the stimulating solution and the nutrient solution for 2-3 days, and if the damaged adipose-derived stem cells are continuously cultured until the adipose-derived stem cells are mature, culturing for 24 hours until the damaged adipose-derived stem cells are not repaired until the damaged adipose-derived stem cells are repaired.
Preferably, in step 1, the fat is washed clean by the standard that blood does not exist on the fat and the washing is repeated until the blood disappears.
Preferably, in step 1, after the fat is cleaned and no blood exists, the fat is wiped dry by using gauze to ensure that the surface of the fat is clean and no cleaning solution remains.
Preferably, in step 1, adipose tissue is taken as fresh tissue and cells inside the adipose tissue are checked for survival before use.
Preferably, in step 2, the volume ratio of the adipose tissue layer to the collagenase solution is 1.5, and the concentration of the collagenase solution is 100Units/mL.
Preferably, in step 2, the centrifugation time of the centrifuge is 10min, and the centrifugation rotation speed of the centrifuge is 1000r/min.
Preferably, in step 3, the volume ratio of the physiological saline to the adipose tissue layer is 1-1.5, the centrifugation time is 10min, the centrifugation speed of the centrifuge is 1000r/min, and the adipose-derived stem cells are screened and filtered by a 100-mesh cell sieve.
Preferably, in step 4, the culture temperature of the adipose-derived stem cells is 37 ℃ and CO is added 2 The volume concentration is 5%, the stimulating solution and the nutrient solution are changed every 12h, a small part of adipose-derived stem cells are taken out in the process of changing the solution and observed, and the damaged repair condition of the adipose-derived stem cells is checked.
Preferably, in step 4, after observing the repair of the adipose-derived stem cells, the use of the stimulating solution is cancelled, and the culture is continued for 10 to 20 days using the nutrient solution to complete a cycle of culture preparation.
Preferably, in step 4, the damaged culture time of the adipose-derived stem cells is not limited to 2 to 3 days, and the completion of repair of the adipose-derived stem cells is specifically determined according to the damage of the adipose-derived stem cells.
Compared with the prior art, the invention has the beneficial effects that:
the invention uses the stimulating liquid to stimulate the cells after the cells are centrifuged, uses mitogen, ionomycin, lipopolysaccharide, interleukin and 13-acetate to stimulate the cells to generate reaction, thereby promoting the cells to release protein, enzyme and factor, uses the protein, enzyme and factor to repair the damaged tissues and new growing tissues, thereby realizing the purpose of repairing the damaged adipose-derived stem cells.
Detailed Description
The present invention will now be described in more detail by way of examples, which are given by way of illustration only and are not intended to limit the scope of the present invention in any way.
The invention provides a technical scheme that: a method for isolating and preparing adipose-derived stem cells, comprising the steps of:
step 1: fat cleaning: repeatedly cleaning adipose tissues until the fat is completely cleaned;
step 2: centrifugal separation: adding the collagenase solution into a centrifuge, synchronizing the cleaned fat to the centrifuge, starting the centrifuge for centrifugal separation, and obtaining a fat tissue layer after the centrifugal separation is finished;
and 3, step 3: centrifugal precipitation: putting the obtained adipose tissue layer into a centrifuge, adding physiological saline for centrifugal separation again, precipitating after the centrifugal separation is finished, and filtering to obtain the adipose-derived stem cells;
and 4, step 4: preparation and culture: and (2) putting the obtained adipose-derived stem cells into a stimulating solution and a nutrient solution for stimulating culture, wherein the stimulating solution is prepared by mixing mitogen, ionomycin, lipopolysaccharide, interleukin and 13-acetate, observing whether the damaged condition of the adipose-derived stem cells is repaired or not after the adipose-derived stem cells are cultured in the stimulating solution and the nutrient solution for 2-3 days, and culturing for 24 hours until the damaged adipose-derived stem cells are repaired if the repair is continued until the adipose-derived stem cells are mature and a plurality of cells are not repaired.
The first embodiment is as follows:
firstly, cleaning fat, repeatedly cleaning adipose tissues until the fat is completely cleaned, then performing centrifugal separation, adding a collagenase solution into a centrifugal machine, synchronizing the cleaned fat to the centrifugal machine, starting the centrifugal machine to perform centrifugal separation, obtaining a fat tissue layer after the centrifugal separation is completed, performing centrifugal precipitation, putting the obtained fat tissue layer into the centrifugal machine, adding physiological saline to perform centrifugal separation again, performing precipitation after the centrifugal separation is completed, filtering to obtain a fat-derived stem cell after the centrifugal separation is completed, finally performing preparation culture, putting the obtained fat-derived stem cell into a stimulus solution and a nutrient solution to perform stimulus culture, wherein the stimulus solution is prepared by mixing mitogen, ionomycin, lipopolysaccharide, interleukin and 13-acetate, observing whether the damaged condition of the fat-derived stem cell is repaired after the culture time of the fat-derived stem cell in the stimulus solution and the nutrient solution reaches 2-3 days, and culturing for 24 hours until the damaged fat-derived stem cell is repaired if the fat-derived stem cell is continuously cultured until the fat-derived stem cell is mature.
In the second embodiment, the first embodiment of the method,
in the first embodiment, the following steps are added,
in the step 1, the standard of fat cleaning is that blood does not exist on fat, the fat is repeatedly cleaned until the blood disappears, after the fat is cleaned and the blood does not exist, the fat is wiped by using gauze to ensure that the surface of the fat is clean, no cleaning solution remains, the fat tissue is fresh tissue, and whether cells in the fat tissue survive is checked before use.
Firstly, fat is cleaned, fat tissue is taken and repeatedly cleaned until the fat is completely cleaned, the standard of the clean fat cleaning is that no blood exists on the fat, the blood is required to be repeatedly cleaned until the blood disappears, after the fat is cleaned and no blood exists, the fat is wiped by using gauze to ensure that the fat surface is clean, no cleaning liquid is left, the fat tissue is taken as a fresh tissue, whether cells inside the fat tissue survive or not is checked before use, then centrifugal separation is carried out, collagenase solution is added into a centrifugal machine, the cleaned fat is synchronously added into the centrifugal machine, centrifugal separation is carried out by starting the centrifugal machine, a fat tissue layer is obtained after the centrifugal separation is completed, then centrifugal precipitation is carried out, the obtained fat tissue layer is put into the centrifugal machine, physiological saline is added for centrifugal separation again, precipitation is carried out after the centrifugal separation is completed, after the fat source stem cell is obtained by filtration, finally, preparation culture is carried out, the obtained fat source is put into stimulation culture liquid and nutrient solution for stimulation culture, wherein the stimulation liquid is prepared by mixing mitogen, ionomycin, lipopolysaccharide, interleukin and 13-acetate, and the stem cell culture is continuously repaired when the fat source stem cell is obtained after the stem cell is cultured for 24-after the stem cell is mature, and the fat source is continuously repaired, and the condition is obtained.
In the third embodiment of the present invention, the following steps are carried out,
in the second embodiment, the following steps are added,
in step 2, the volume ratio of the adipose tissue layer to the collagenase solution is 1.5, the concentration of the collagenase solution is 100Units/mL, the centrifugation time of the centrifuge is 10min, and the centrifugation rotating speed of the centrifuge is 1000r/min.
Firstly, fat is cleaned, adipose tissues are taken and repeatedly cleaned until the adipose tissues are completely cleaned, the standard of the clean adipose tissues is that no blood exists on the adipose tissues, the blood is required to be repeatedly cleaned until the blood disappears, after the adipose tissues are cleaned and no blood exists, the adipose tissues are wiped by using gauze to ensure that the adipose surfaces are clean, no cleaning solution is left, the adipose tissues are taken as fresh tissues, whether cells inside the adipose tissues survive or not is checked before use, then centrifugal separation is carried out, collagenase solution is added into a centrifuge, the cleaned adipose tissues are synchronously transferred to the centrifuge, centrifugal separation is carried out by starting the centrifuge, an adipose tissue layer is obtained after the centrifugal separation is finished, the volume ratio of the adipose tissue layer to the collagenase solution is 1.
In the fourth embodiment, the first step is that,
in the third example, in addition to the following steps,
in step 3, the volume ratio of the physiological saline to the adipose tissue layer is 1-1.5, the centrifugation time is 10min, the centrifugation speed of the centrifuge is 1000r/min, and the adipose-derived stem cells are screened and filtered by a 100-mesh cell sieve.
Firstly, adipose tissues are taken and repeatedly cleaned until the adipose tissues are completely cleaned, the standard of the clean adipose tissues is that no blood exists on the adipose tissues and blood medicine disappears, after the adipose tissues are cleaned and no blood exists, the adipose tissues are wiped by using gauze to ensure that the adipose surfaces are clean without residual cleaning liquid, the adipose tissues are taken as fresh tissues, before the use, whether cells inside the adipose tissues survive or not is checked, then centrifugal separation is carried out, collagenase solution is added into a centrifuge, the cleaned adipose tissues are synchronously transferred to the centrifuge, the centrifugal separation is carried out by starting the centrifuge, an adipose tissue layer is obtained after the centrifugal separation is finished, the volume ratio of the adipose tissue layer to the collagenase solution is 1, and if the repair is continued to be cultured until the adipose-derived stem cells are mature and a plurality of cells are not repaired, culturing for 24h until the damaged adipose-derived stem cells are repaired.
In the fifth embodiment, the first step is,
in the fourth example, the following steps were added,
in step 4, the culture temperature of the adipose-derived stem cells was 37 ℃ and CO was added 2 The volume concentration is 5%, the stimulation solution and the nutrient solution are changed once every 12h, a small part of adipose-derived stem cells are taken in the solution changing process for observation, the damaged repair condition of the adipose-derived stem cells is checked, the use of the stimulation solution is cancelled after the adipose-derived stem cells are observed to be repaired, the nutrient solution is used for continuously culturing for 10-20 days to finish the culture preparation of a period, the damaged culture time of the adipose-derived stem cells is not limited to 2-3 days, and the completion of the repair of the adipose-derived stem cells is specifically taken as the standard according to the damaged condition of the adipose-derived stem cells.
Firstly, cleaning fat, taking adipose tissue for repeated cleaning until the fat is completely cleaned, wherein the standard of the clean fat cleaning is that blood does not exist on the fat, the fat is required to be repeatedly cleaned until the blood disappears, after the fat is cleaned and the blood does not exist, the fat is wiped by using gauze to ensure that the surface of the fat is clean, no cleaning solution is remained, the adipose tissue is taken as fresh tissue, whether cells in the adipose tissue survive is checked before use, then centrifugal separation is carried out, collagenase solution is added into a centrifugal machine, and the cleaning is carried outSynchronously feeding the cleaned fat to a centrifugal machine, starting the centrifugal machine for centrifugal separation, obtaining a fat tissue layer after centrifugal separation is finished, wherein the volume ratio of the fat tissue layer to a collagenase solution is 1 2 The volume concentration is 5%, the stimulation solution and the nutrient solution are changed once every 12h, a small part of adipose-derived stem cells are taken in the solution changing process for observation, the damaged repair condition of the adipose-derived stem cells is checked, the use of the stimulation solution is cancelled after the adipose-derived stem cells are observed to be repaired, the nutrient solution is used for continuously culturing for 10-20 days to finish the culture preparation of a period, the damaged culture time of the adipose-derived stem cells is not limited to 2-3 days, and the completion of the repair of the adipose-derived stem cells is specifically taken as the standard according to the damaged condition of the adipose-derived stem cells.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. A method for separating and preparing adipose-derived stem cells, which is characterized by comprising the following steps: the method comprises the following steps:
step 1: fat cleaning: repeatedly cleaning adipose tissues until the fat is completely cleaned;
step 2: centrifugal separation: adding the collagenase solution into a centrifuge, synchronizing the cleaned fat to the centrifuge, starting the centrifuge for centrifugal separation, and obtaining a fat tissue layer after the centrifugal separation is finished;
and step 3: and (3) centrifugal precipitation: putting the obtained adipose tissue layer into a centrifuge, adding physiological saline for centrifugal separation again, precipitating after the centrifugal separation is finished, and filtering to obtain the adipose-derived stem cells;
and 4, step 4: preparation and culture: and (2) putting the obtained adipose-derived stem cells into a stimulating solution and a nutrient solution for stimulating culture, wherein the stimulating solution is prepared by mixing mitogen, ionomycin, lipopolysaccharide, interleukin and 13-acetate, observing whether the damaged condition of the adipose-derived stem cells is repaired or not after the adipose-derived stem cells are cultured in the stimulating solution and the nutrient solution for 2-3 days, and if the damaged adipose-derived stem cells are continuously cultured until the adipose-derived stem cells are mature, culturing for 24 hours until the damaged adipose-derived stem cells are not repaired until the damaged adipose-derived stem cells are repaired.
2. The method for separating adipose-derived stem cells according to claim 1, wherein: in step 1, the standard of fat cleaning is that blood does not exist on fat, and the fat cleaning needs to be repeated until blood disappears.
3. The method for separating adipose-derived stem cells according to claim 1, wherein: in the step 1, after the fat is cleaned and blood does not exist, the fat is wiped dry by using gauze, so that the surface of the fat is ensured to be clean, and no cleaning solution is left.
4. The method for separating and preparing adipose-derived stem cells according to claim 1, wherein: in step 1, adipose tissue is taken as fresh tissue and examined for survival of cells inside the adipose tissue before use.
5. The method for separating and preparing adipose-derived stem cells according to claim 1, wherein: in step 2, the volume ratio of the adipose tissue layer to the collagenase solution was 1.5, and the concentration of collagenase solution was 100Units/mL.
6. The method for separating and preparing adipose-derived stem cells according to claim 1, wherein: in the step 2, the centrifugal time of the centrifugal machine is 10min, and the centrifugal rotating speed of the centrifugal machine is 1000r/min.
7. The method for separating and preparing adipose-derived stem cells according to claim 1, wherein: in step 3, the volume ratio of the physiological saline to the adipose tissue layer is 1-1.5, the centrifugation time is 10min, the centrifugation speed of the centrifuge is 1000r/min, and the adipose-derived stem cells are screened and filtered by a 100-mesh cell sieve.
8. The method for separating and preparing adipose-derived stem cells according to claim 1, wherein: in step 4, the culture temperature of the adipose-derived stem cells was 37 ℃ and CO was used 2 The volume concentration is 5%, the stimulating solution and the nutrient solution are changed every 12h, a small part of adipose-derived stem cells are taken out in the process of changing the solution and observed, and the damaged repair condition of the adipose-derived stem cells is checked.
9. The method for separating and preparing adipose-derived stem cells according to claim 1, wherein: in step 4, after observing the repair of the adipose-derived stem cells, the use of the stimulating solution is cancelled, and the culture is continued for 10 to 20 days by using the nutrient solution to complete a period of culture preparation.
10. The method for separating and preparing adipose-derived stem cells according to claim 1, wherein: in step 4, the repair culture time of the adipose-derived stem cells is not limited to 2 to 3 days, and the repair of the adipose-derived stem cells is specifically determined according to the damage condition of the adipose-derived stem cells.
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| CN114288276A (en) * | 2021-12-29 | 2022-04-08 | 成都清科生物科技有限公司 | Anti-inflammatory amnion patch and preparation method thereof |
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