CN110791477A - Culture method of mesenchymal stem cells after cryopreservation and recovery of adipocytes - Google Patents
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Abstract
The invention relates to the technical field of biology, in particular to a method for culturing adipose cell cryopreserved and resuscitated mesenchymal stem cells, which comprises the steps of treating extracted adipose tissues, cryopreserving at a deep low temperature, resuscitating the cryopreserved adipose tissues to culture adipose mesenchymal stem cells according to the usage amount in the later period, completing cell culture, transplanting the cryopreserved adipose tissues and the resuscitated adipose tissues together after qualification identification.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for culturing mesenchymal stem cells after cryopreservation and recovery of adipocytes.
Background
With the development of fat suction technology and the increasing acceptance of people on plastic cosmetology in recent years, autologous adipose tissue is taken as an ideal soft tissue filling material, and fat filling is one of the most important technical methods in the field of medical cosmetology.
However, the low survival rate and high absorption rate of transplanted adipose tissue are major problems that plague the wide clinical development and application of fat transplantation. At present, the ideal method for improving the fat survival rate is a small amount of repeated injections, and the repeated fat transplantation needs to be performed again with fat pumping from the body of a patient, but the repeated fat pumping and transplantation not only increase the pain of the patient and the risk and the operation cost, but also increase the workload of doctors, so that aiming at the current situation, the development of a culture method of mesenchymal stem cells after fat cell cryopreservation and resuscitation is urgently needed to overcome the defects in the current practical application.
Disclosure of Invention
The invention aims to provide a method for culturing mesenchymal stem cells after cryopreservation and recovery of adipocytes, so as to solve the problems in the background art.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for culturing mesenchymal stem cells after cryopreservation and recovery of adipocytes comprises the following steps:
the method comprises the following steps: after liposuction, placing the adipose tissues in a collection bottle containing a preservation solution, timely transporting the adipose tissues back to a laboratory at the temperature of 2-8 ℃, and numbering the adipose tissues;
step two: placing the collection bottle in a biological safety cabinet for aseptic operation and opening, transferring the upper layer adipose tissues into a 50ml centrifuge tube by using a 25ml pipette, wherein each tube contains 20ml of the upper layer adipose tissues;
step three: adding a proper amount of cleaning solution containing double antibodies into each centrifuge tube respectively, lightly blowing, repeatedly blowing, uniformly mixing, supplementing physiological saline to a scale of 45ml, centrifuging at 1300rpm for 5min, obviously layering after centrifuging, removing oil drops on the upper layer by using a 5ml pipette, slowly extending into the physiological saline on the lower layer by using a 25ml pipette, sucking away the physiological saline and the precipitate, and repeating the operation for a plurality of times, wherein the upper layer is oil drop, the middle layer is white adipose tissue, the lowest layer is precipitate such as physiological saline, a small amount of connective tissue and red blood cells, and the like;
step four: collecting adipose tissues, and cutting the tissues to 1 × 1mm with tissue scissors2Uniformly mixing the tissue blocks with the freezing solution according to a certain proportion, adding a 5ml freezing tube, placing the freezing tube into a programmed cooling box, standing at-80 ℃ for 8 hours, and transferring into a gas phase tank for freezing;
step five: taking out the cryopreservation tube from the gas phase tank, immediately placing the tube into a water bath at 37 ℃, continuously shaking to rapidly thaw the cryopreserved adipose tissues within 2 minutes, taking out the cryopreservation tube from the water bath after the cryopreservation tube is completely thawed, wiping off water outside the cryopreservation tube, wiping the outer wall of the cryopreservation tube with an alcohol cotton ball to sterilize, placing the cryopreservation tube into a biological safety cabinet, opening a cover of the cryopreservation tube in the biological safety cabinet in an aseptic operation, transferring the cryopreserved adipose tissues into a 50ml centrifuge tube, adding physiological saline, slightly mixing the materials, centrifuging at 2000rpm for 8min, discarding supernatant, re-suspending the tissues with the physiological saline, centrifuging at 2000rpm for 8min, and discarding supernatant;
step six: putting adipose tissues into a new 50mL centrifuge tube, wherein each tube contains about 10mL of tissues, adding 2mL of collagenase and 2mL of DNAse, supplementing physiological saline to 20mL, uniformly blowing, sealing the centrifuge tube, putting the centrifuge tube into a constant-temperature shaking table at 37 ℃, shaking and digesting for 40min at 145rpm, after digestion, adding a proper amount of physiological saline into a biological safety cabinet, centrifuging at 1300rpm for 5min, sucking and discarding tissues and physiological saline which are not completely digested at the upper layer by a pipette, adding a proper amount of physiological saline to resuspend cell precipitates at the bottom of the centrifuge tube, centrifuging at 1300rpm for 5min, and discarding supernatant;
step seven: adding 10ml of mesenchymal stem cell culture medium into the digested cells for resuspension, inoculating the cells into a T175 culture flask according to the cell quantity, putting the culture flask into a 37 ℃ and 5% carbon dioxide incubator, culturing until the day 3, carrying out first half liquid change, carrying out half liquid change or full liquid change on the day 6 according to the actual condition, and then carrying out full liquid change every 3 days;
step eight: and (5) subculturing the adipose tissue-derived mesenchymal stem cells.
As a further scheme of the invention: in the eighth step, the fusion degree of the primary cells is more than 60%, and the P1 generations can be transmitted, and the inoculation density is 8000 cells/cm2And then the cell fusion degree reaches more than 80 percent, and the cell can be continuously passaged.
As a further scheme of the invention: in the third step, the preparation method of the double-antibody cleaning solution comprises the following steps: the penicillin and streptomycin sulfate injection is prepared into 1 ten thousand units of stock solution per milliliter by using normal saline for injection, namely 1 percent double-resistant stock solution, 500mL of normal saline for injection is added with 5mL of double-resistant stock solution, and then 2mL of gentamicin sulfate for injection is added to prepare tissue preservation solution or cleaning solution.
As a further scheme of the invention: in the sixth step, the preparation of the collagenase I stock solution comprises the following steps: 1g of collagenase I is weighed and dissolved in 100mL of physiological saline, and filtered by a 0.22 mu m filter membrane to obtain 1% (m/v) collagenase I stock solution.
As a further scheme of the invention: in the sixth step, the preparation of the DNase I stock solution comprises the following steps: 0.2g of DNase I was weighed, dissolved in 100mL of physiological saline, and filtered through a 0.22 μm filter to obtain a 0.2% (m/v) DNase I stock solution.
As a further scheme of the invention: in the third step, the operation is repeated for no less than three times.
As a further scheme of the invention: in the fourth step, the mixing ratio of the tissue blocks to the frozen stock solution is 3: 2.
Compared with the prior art, the invention has the beneficial effects that:
1. the method treats the extracted adipose tissues, then cryopreserves the adipose tissues at a deep low temperature, recovers the cryopreserved adipose tissues according to the usage amount at the later stage to culture adipose mesenchymal stem cells, finishes cell culture, and transplants the cryopreserved adipose tissues and the recovered adipose tissues together after qualification identification, and compared with the existing adipose tissue cryopreserving and mesenchymal stem cell culture method, the method solves the problems of multiple times of liposuction and adipose mesenchymal stem cell culture;
2. the method is simple to operate, tissue and cell filtration is not needed, the DNase I is used for solving the problem that the adipose mesenchymal stem cells cannot be separated when entering digestive mucus, and the obtained stem cells are more in number;
3. the whole separation process has no animal-derived components, and is good in safety and convenient for subsequent research and application.
Drawings
FIG. 1 is a diagram showing the result of flow detection of P2 in the culture method of mesenchymal stem cells after cryopreservation and recovery of adipocytes.
FIG. 2 is a diagram showing the result of flow detection of P3 in the culture method of mesenchymal stem cells after cryopreservation and recovery of adipocytes.
Detailed Description
The technical solution of the present patent will be described in further detail with reference to the following embodiments.
Example 1
In the embodiment of the invention, a method for culturing mesenchymal stem cells after cryopreservation and recovery of adipocytes comprises the following steps:
the method comprises the following steps: after liposuction, placing the adipose tissues in a collection bottle containing a preservation solution, timely transporting the adipose tissues back to a laboratory at the temperature of 2-8 ℃, and numbering the adipose tissues;
step two: placing the collection bottle in a biological safety cabinet for aseptic operation and opening, transferring the upper layer adipose tissues into a 50ml centrifuge tube by using a 25ml pipette, wherein each tube contains 20ml of the upper layer adipose tissues;
step three: adding a proper amount of cleaning solution containing double antibodies into each centrifuge tube respectively, lightly blowing, repeatedly blowing, uniformly mixing, supplementing physiological saline to a scale of 45ml, centrifuging at 1300rpm for 5min, obviously layering after centrifuging, removing oil drops on the upper layer by using a 5ml pipette, slowly extending into the physiological saline on the lower layer by using a 25ml pipette, sucking away the physiological saline and the precipitate, and repeating the operation for a plurality of times, wherein the upper layer is oil drop, the middle layer is white adipose tissue, the lowest layer is precipitate such as physiological saline, a small amount of connective tissue and red blood cells, and the like;
step four: collecting adipose tissues, and cutting the tissues to 1 × 1mm with tissue scissors2Uniformly mixing the tissue blocks with the freezing solution according to a certain proportion, adding a 5ml freezing tube, placing the freezing tube into a programmed cooling box, standing at-80 ℃ for 8 hours, and transferring into a gas phase tank for freezing;
step five: taking out the cryopreservation tube from the gas phase tank, immediately placing the tube into a water bath at 37 ℃, continuously shaking to rapidly thaw the cryopreserved adipose tissues within 2 minutes, taking out the cryopreservation tube from the water bath after the cryopreservation tube is completely thawed, wiping off water outside the cryopreservation tube, wiping the outer wall of the cryopreservation tube with an alcohol cotton ball to sterilize, placing the cryopreservation tube into a biological safety cabinet, opening a cover of the cryopreservation tube in the biological safety cabinet in an aseptic operation, transferring the cryopreserved adipose tissues into a 50ml centrifuge tube, adding physiological saline, slightly mixing the materials, centrifuging at 2000rpm for 8min, discarding supernatant, re-suspending the tissues with the physiological saline, centrifuging at 2000rpm for 8min, and discarding supernatant;
step six: putting adipose tissues into a new 50mL centrifuge tube, wherein each tube contains about 10mL of tissues, adding 2mL of collagenase and 2mL of DNAse, supplementing physiological saline to 20mL, uniformly blowing, sealing the centrifuge tube, putting the centrifuge tube into a constant-temperature shaking table at 37 ℃, shaking and digesting for 40min at 145rpm, after digestion, adding a proper amount of physiological saline into a biological safety cabinet, centrifuging at 1300rpm for 5min, sucking and discarding tissues and physiological saline which are not completely digested at the upper layer by a pipette, adding a proper amount of physiological saline to resuspend cell precipitates at the bottom of the centrifuge tube, centrifuging at 1300rpm for 5min, and discarding supernatant;
step seven: adding 10ml of mesenchymal stem cell culture medium into the digested cells for resuspension, inoculating the cells into a T175 culture flask according to the cell quantity, putting the culture flask into a 37 ℃ and 5% carbon dioxide incubator, culturing until the day 3, carrying out first half liquid change, carrying out half liquid change or full liquid change on the day 6 according to the actual condition, and then carrying out full liquid change every 3 days;
step eight: and (5) subculturing the adipose tissue-derived mesenchymal stem cells.
Example 2
In the eighth step, the fusion degree of the primary cells is more than 60%, i.e., the cells can be transferred to P1 generations, and the inoculation density is 8000 cells/cm2And then the cell fusion degree reaches more than 80 percent, and the cell can be continuously passaged.
In this embodiment, in the third step, the preparation of the double-antibody cleaning solution includes: the penicillin and streptomycin sulfate injection is prepared into 1 ten thousand units of stock solution per milliliter by using normal saline for injection, namely 1 percent double-resistant stock solution, 500mL of normal saline for injection is added with 5mL of double-resistant stock solution, and then 2mL of gentamicin sulfate for injection is added to prepare tissue preservation solution or cleaning solution.
In this embodiment, in step six, the preparation of the collagenase i stock solution includes: 1g of collagenase I is weighed and dissolved in 100mL of physiological saline, and filtered by a 0.22 mu m filter membrane to obtain 1% (m/v) collagenase I stock solution.
In this embodiment, in step six, the preparation of the DNase i stock solution includes: 0.2g of DNase I was weighed, dissolved in 100mL of physiological saline, and filtered through a 0.22 μm filter to obtain a 0.2% (m/v) DNase I stock solution.
In the third step, the number of times of the repeated operation is not less than three.
In the fourth step, the mixing ratio of the tissue block and the frozen stock solution is 3: 2.
In this embodiment, the preparation of the frozen stock solution includes: human serum albumin, trehalose, medium, DMSO in 4: 3: 2: 1, and placing the mixture into a refrigerator for precooling and balancing at 4-8 ℃.
Example 3
In order to verify the rationality of the method, the flow detection is respectively carried out on the P2 and P3 generation adipose-derived mesenchymal stem cells, and the operation steps are as follows:
1) taking the single cell suspension, centrifuging at 1200rpm for 5 min;
2) resuspending the cell pellet with PBS to adjust the cell concentration to 1.0X 107cell/ml；
3) Adding antibodies CD90, CD105 and CD166, gently blowing, beating and mixing uniformly, incubating at 4 ℃ in dark for 30min, and setting isotype control;
4) adding 1ml PBS into a flow detection tube, 1200rpm for 5min, and removing the supernatant;
5) adding 100ul PBS, gently blowing, mixing, and detecting on a machine.
As a result: the positive rates of all three surface markers were 90% or more, and the results are shown in fig. 1 and 2.
According to the method for culturing the adipose cell cryopreserved and the resuscitated mesenchymal stem cells, the extracted adipose tissues are treated and then cryopreserved at a deep low temperature, the adipose tissue is resuscitated according to the using amount in the later period to culture the adipose mesenchymal stem cells, the cell culture is completed, and the qualified adipose tissues and the resuscitated adipose tissues are transplanted together.
The above is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, it is possible to make several variations and modifications without departing from the concept of the present invention, and these should be considered as the protection scope of the present invention, which will not affect the effect of the implementation of the present invention and the utility of the patent.
Claims (7)
1. A method for culturing mesenchymal stem cells after cryopreservation and recovery of adipocytes is characterized by comprising the following steps:
the method comprises the following steps: after liposuction, placing the adipose tissues in a collection bottle containing a preservation solution, timely transporting the adipose tissues back to a laboratory at the temperature of 2-8 ℃, and numbering the adipose tissues;
step two: placing the collection bottle in a biological safety cabinet for aseptic operation and opening, transferring the upper layer adipose tissues into a 50ml centrifuge tube by using a 25ml pipette, wherein each tube contains 20ml of the upper layer adipose tissues;
step three: adding a proper amount of cleaning solution containing double antibodies into each centrifuge tube respectively, lightly blowing, repeatedly blowing, uniformly mixing, supplementing physiological saline to a scale of 45ml, centrifuging at 1300rpm for 5min, obviously layering after centrifuging, removing oil drops on the upper layer by using a 5ml pipette, slowly extending into the physiological saline on the lower layer by using a 25ml pipette, sucking away the physiological saline and the precipitate, and repeating the operation for a plurality of times, wherein the upper layer is oil drop, the middle layer is white adipose tissue, the lowest layer is precipitate such as physiological saline, a small amount of connective tissue and red blood cells, and the like;
step four: collecting adipose tissues, and cutting the tissues to 1 × 1mm with tissue scissors2Uniformly mixing the tissue blocks with the freezing solution according to a certain proportion, adding a 5ml freezing tube, placing the freezing tube into a programmed cooling box, standing at-80 ℃ for 8 hours, and transferring into a gas phase tank for freezing;
step five: taking out the cryopreservation tube from the gas phase tank, immediately placing the tube into a water bath at 37 ℃, continuously shaking to rapidly thaw the cryopreserved adipose tissues within 2 minutes, taking out the cryopreservation tube from the water bath after the cryopreservation tube is completely thawed, wiping off water outside the cryopreservation tube, wiping the outer wall of the cryopreservation tube with an alcohol cotton ball to sterilize, placing the cryopreservation tube into a biological safety cabinet, opening a cover of the cryopreservation tube in the biological safety cabinet in an aseptic operation, transferring the cryopreserved adipose tissues into a 50ml centrifuge tube, adding physiological saline, slightly mixing the materials, centrifuging at 2000rpm for 8min, discarding supernatant, re-suspending the tissues with the physiological saline, centrifuging at 2000rpm for 8min, and discarding supernatant;
step six: putting adipose tissues into a new 50mL centrifuge tube, wherein each tube contains about 10mL of tissues, adding 2mL of collagenase and 2mL of DNAse, supplementing physiological saline to 20mL, uniformly blowing, sealing the centrifuge tube, putting the centrifuge tube into a constant-temperature shaking table at 37 ℃, shaking and digesting for 40min at 145rpm, after digestion, adding a proper amount of physiological saline into a biological safety cabinet, centrifuging at 1300rpm for 5min, sucking and discarding tissues and physiological saline which are not completely digested at the upper layer by a pipette, adding a proper amount of physiological saline to resuspend cell precipitates at the bottom of the centrifuge tube, centrifuging at 1300rpm for 5min, and discarding supernatant;
step seven: adding 10ml of mesenchymal stem cell culture medium into the digested cells for resuspension, inoculating the cells into a T175 culture flask according to the cell quantity, putting the culture flask into a 37 ℃ and 5% carbon dioxide incubator, culturing until the day 3, carrying out first half liquid change, carrying out half liquid change or full liquid change on the day 6 according to the actual condition, and then carrying out full liquid change every 3 days;
step eight: and (5) subculturing the adipose tissue-derived mesenchymal stem cells.
2. The method for culturing mesenchymal stem cells after cryopreservation and recovery of adipocytes of claim 1,it is characterized in that in the eighth step, the fusion degree of primary cells is more than 60 percent, the cells can be transmitted for P1 generations, and the inoculation density is 8000 cells/cm2And the cell fusion degree reaches more than 80 percent, and the cell can be continuously passaged.
3. The method for culturing mesenchymal stem cells after cryopreservation and recovery of adipocytes according to claim 2, wherein in step three, the preparation of the double antibody cleaning solution comprises: the penicillin and streptomycin sulfate injection is prepared into 1 ten thousand units of stock solution per milliliter by using normal saline for injection, namely 1 percent double-resistant stock solution, 500mL of normal saline for injection is added with 5mL of double-resistant stock solution, and then 2mL of gentamicin sulfate for injection is added to prepare tissue preservation solution or cleaning solution.
4. The method for culturing mesenchymal stem cells after cryopreservation and recovery of adipocytes of claim 3, wherein in the sixth step, the preparation of the collagenase I stock solution comprises: 1g of collagenase I is weighed and dissolved in 100mL of physiological saline, and filtered by a 0.22 mu m filter membrane to obtain 1% (m/v) collagenase I stock solution.
5. The method for culturing mesenchymal stem cells after cryopreservation and recovery of adipocytes of claim 1, wherein in step six, the preparation of the DNase I stock solution comprises: 0.2g of DNase I was weighed, dissolved in 100mL of physiological saline, and filtered through a 0.22 μm filter to obtain a 0.2% (m/v) DNase I stock solution.
6. The method for culturing mesenchymal stem cells after cryopreservation and recovery of adipocytes of claim 5, wherein in step three, the operation is repeated no less than three times.
7. The method for culturing mesenchymal stem cells after cryopreservation and recovery of adipocytes of claim 6, wherein in step four, the mixing ratio of the tissue mass to the cryopreservation solution is 3: 2.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112155008A (en) * | 2020-09-30 | 2021-01-01 | 北京银丰鼎诚生物工程技术有限公司 | Cryopreservation and recovery method of adipose tissues and adipose tissue cryopreservation recovery kit |
| CN113201489A (en) * | 2021-05-14 | 2021-08-03 | 达瑟儿(上海)生命科技有限公司 | Preparation method of adipose-derived mesenchymal stem cell working cell bank |
| CN113577106A (en) * | 2020-04-30 | 2021-11-02 | 中国医学科学院北京协和医院 | Adipose-derived stem cell preparation and preparation method thereof |
| CN114832016A (en) * | 2022-04-19 | 2022-08-02 | 芙普瑞生物细胞科学(苏州)有限公司 | Adipose SVF cell preparation and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008049281A1 (en) * | 2006-10-27 | 2008-05-02 | Beijing Institute Of Transfusion Medicine | Construction method of hepatic tissue engineering construct and the hepatic tissue engineering construct |
| CN103849598A (en) * | 2014-03-05 | 2014-06-11 | 青岛中天干细胞工程有限公司 | Method for preparing human adipose tissue-derived stromal cells |
| CN105219707A (en) * | 2015-11-17 | 2016-01-06 | 广州赛莱拉干细胞科技股份有限公司 | A kind of method of recovery fat mesenchymal stem cell |
| CN109321517A (en) * | 2018-10-26 | 2019-02-12 | 陕西九州细胞基因工程有限公司 | The efficiently separating of a kind of human amnion mesenchymal stem cell, amplification method |
-
2019
- 2019-11-21 CN CN201911149709.7A patent/CN110791477A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008049281A1 (en) * | 2006-10-27 | 2008-05-02 | Beijing Institute Of Transfusion Medicine | Construction method of hepatic tissue engineering construct and the hepatic tissue engineering construct |
| CN103849598A (en) * | 2014-03-05 | 2014-06-11 | 青岛中天干细胞工程有限公司 | Method for preparing human adipose tissue-derived stromal cells |
| CN105219707A (en) * | 2015-11-17 | 2016-01-06 | 广州赛莱拉干细胞科技股份有限公司 | A kind of method of recovery fat mesenchymal stem cell |
| CN109321517A (en) * | 2018-10-26 | 2019-02-12 | 陕西九州细胞基因工程有限公司 | The efficiently separating of a kind of human amnion mesenchymal stem cell, amplification method |
Non-Patent Citations (1)
| Title |
|---|
| 谢甬淋;戚飞腾;童洋萍;毕涌;张旭;: "人脂肪间充质干细胞的分离培养及冻存前后生物学变化" * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113577106A (en) * | 2020-04-30 | 2021-11-02 | 中国医学科学院北京协和医院 | Adipose-derived stem cell preparation and preparation method thereof |
| CN112155008A (en) * | 2020-09-30 | 2021-01-01 | 北京银丰鼎诚生物工程技术有限公司 | Cryopreservation and recovery method of adipose tissues and adipose tissue cryopreservation recovery kit |
| CN113201489A (en) * | 2021-05-14 | 2021-08-03 | 达瑟儿(上海)生命科技有限公司 | Preparation method of adipose-derived mesenchymal stem cell working cell bank |
| CN114832016A (en) * | 2022-04-19 | 2022-08-02 | 芙普瑞生物细胞科学(苏州)有限公司 | Adipose SVF cell preparation and application thereof |
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