CN108315297B

CN108315297B - Method for separating and purifying adipose-derived stem cells from adipose tissues

Info

Publication number: CN108315297B
Application number: CN201810158962.8A
Authority: CN
Inventors: 苏爱盟; 肖桂清; 谢燕萍; 郑得胜; 李科
Original assignee: Fujian Yinfeng Stem Cell Engineering Co ltd
Current assignee: Fujian Yinfeng Stem Cell Engineering Co ltd
Priority date: 2018-02-26
Filing date: 2018-02-26
Publication date: 2022-04-05
Anticipated expiration: 2038-02-26
Also published as: CN108315297A

Abstract

The invention provides a method for separating and purifying adipose-derived stem cells from adipose tissues, which comprises the steps of adipose-derived stem cell preparation, adipose-derived mesenchymal stem cell subculture and cell harvesting. 5ng/ml EGF +5ng/ml FGF +5ng/ml IFN-gamma +5ng/ml TNF-alpha +1% L-GlutaMAX is added into the culture medium, and the factors are simultaneously added into the culture system to effectively promote the proliferation of MSC, thereby effectively shortening the culture time. The method can effectively reduce the cost, can quickly obtain high-quality adipose-derived stem cells, reduces the damage to the cells, shortens the culture period, reduces the pollution and can improve the purity of the cells.

Description

Method for separating and purifying adipose-derived stem cells from adipose tissues

Technical Field

The invention belongs to the technical field of biology, and particularly provides a method for separating and purifying adipose-derived stem cells from adipose tissues.

Background

Adipose-derived stem cells (ADSCs) are stem cells with the potential for multi-directional differentiation that are isolated from adipose tissue. Adipose stem cells can differentiate into multi-embryonic layer cells, including mesodermal, endodermal and ectodermal cells, and secrete a number of cytokines that are beneficial to human tissues. Adipose-derived stem cells are a group of multifunctional mesenchymal stem cells, can be differentiated into other cell lines, have a large number of adipose-derived stem cells in the non-adipose (mesenchymal) debris of adipose tissues, and are easy to separate and obtain. The adipose-derived stem cells can restore the repairing function of histiocytes, promote the regeneration of cells and restore the young face; meanwhile, the health care product can fully improve the body function, effectively improve diseases such as sub-health, premature senility and the like, and really and effectively resist aging from inside to outside.

A number of studies have demonstrated that ADSCs have some advantages over other types of mesenchymal stem cells, such as bone marrow mesenchymal stem cells: the adipose tissue has wide sources, convenient material taking, large acquisition amount of stem cells, easy separation and purification, small damage to adipose tissue supply areas and strong differentiation capability. Therefore, the adipose-derived stem cells are good seed cells for tissue engineering and regenerative medicine.

Adipose-derived stem cells have been clinically applied and have remarkable effects, are widely applied in medical cosmetology, immunity improvement and other aspects at present, and have been developed in clinical researches on major diseases which harm human life and health, such as cardiovascular diseases, diabetes, neurological diseases, digestive tract diseases, reproductive system diseases and the like.

In summary, adipose-derived stem cells have very outstanding advantages in terms of application advantages, practical application, potential development and the like, and are called as "star stem cells". At present, the preparation process of products of adipose-derived stem cells is complex, the biological activity is limited by various external conditions, and the products are inconvenient to store.

The most recent technical scheme currently available is as follows: chinese patent CN104560868A discloses a primary isolation culture method of adipose-derived stem cells, which involves digesting adipose tissue with a mixed liquid of type I collagenase and trypsin; the adipose stem cells were cultured with DMEM containing EGF and FBS.

The prior art has the defects that: in the prior art, the steps of removing blood vessels by a mechanical method, performing single enzyme digestion, cracking red blood cells by using a lysate and the like are mostly adopted, so that cell populations of ADSCs with high purity are obtained as much as possible; although the addition of antibiotics such as penicillin-streptomycin during culture can prevent pollution, the addition of antibiotics causes various degrees of damage to cells and is controversial in use. The separation culture process of the adipose-derived stem cells has the disadvantages of complex operation, long time consumption and high production cost. In addition, because more chemical reagents are used, the ADSCs have lower activity and lower proliferation speed after separation. The single-variety digestive enzyme is used for digesting the adipose tissues, and the incomplete digestion causes the low separation efficiency of the adipose-derived stem cells. In general, there is much room for improvement in the isolation and culture methods of ADSCs.

The method can effectively reduce the cost, can quickly obtain high-quality adipose-derived stem cells, reduces the damage to the cells, shortens the culture period, reduces the pollution and can improve the purity of the cells.

Disclosure of Invention

The invention provides a method for separating adipose-derived stem cells from adipose tissues and carrying out subculture to finally obtain a cell product, and aims to solve the problems of low separation efficiency, long period, insufficient safety and the like which possibly occur in the processes of separating and culturing the adipose-derived stem cells.

The technical scheme adopted by the invention for solving the technical problems is as follows:

the method for obtaining the adipose tissue, transporting the adipose tissue, preparing the adipose tissue and culturing the cells adopts methods of detection and investigation, adding antibiotics and the like to ensure that the pollution-free adipose-derived stem cells are obtained, thereby ensuring the safety, and adopts an improved digestion method, a culture system and a culture method to improve the separation efficiency, shorten the culture period and obtain the cells with high purity and excellent representation in the separation and culture processes.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for separating and purifying adipose-derived stem cells from adipose tissues comprises the steps of adipose-derived stem cell preparation, adipose-derived mesenchymal stem cell subculture and cell harvesting.

The adipose-derived stem cells are prepared by the following steps:

(1) transferring the adipose tissues to a 50ml centrifuge tube, wherein each tube contains 40ml of adipose tissues and 800g of adipose tissues for 8 minutes;

(2) transferring the centrifuged adipose tissues to a new centrifugal tube, adding 0.9% physiological saline with the same volume, and 800g for 8 minutes;

(3) discarding the supernatant, and repeating the step 2 to wash the adipose tissues twice;

(4) adding digestive juice into the fat precipitate with equal volume, mixing, sealing, and placing into a shaker at 37 deg.C, 150rpm for 30-50 min;

(5) taking out the digested adipose tissues, centrifuging, and performing centrifugation at 800g for 10 minutes;

(6) discarding the upper fat layer, resuspending the centrifuged cell pellet with DMEM/F12 culture medium, centrifuging, and centrifuging for 10 minutes at 800 g; repeating step 6 to wash the cell pellet;

(7) discarding the centrifuged supernatant, resuspending the cell precipitate in DMEM/F12 culture medium, mixing, and filtering the cells with a 40um filter screen; centrifuging the filtered cells for 10 minutes at 800g, and discarding the supernatant;

(8) 1.0ml of DMEM/F12 culture medium is used for resuspending cell precipitates, 4.0ml of precooled 0.3% NaCl solution is added after uniform mixing, the mixture is placed for 10 minutes at room temperature, and erythrocytes are lysed;

(9) centrifuging at 400g for 5 min, and discarding the supernatant;

(10) resuspending the cell pellet with MSC serum-free medium, counting trypan blue stained cells, adjusting the cell density to 2.0 x 10^{^6}Adding 14ml of culture medium into each culture bottle, adding 1.0ml of cell suspension, and uniformly mixing; after 24 hours, the cells are changed, the cells which are not attached to the wall are removed, and the cells are changed every three days; after culturing to the fifth day, the cell growth density reaches 90%, and the adipose tissue-derived mesenchymal stem cells are subjected to subculture.

The digestive juice contains 0.075-0.5 wt.% of collagenase I +0.1wt.% of collagenase IV +0.1wt.% of trypsin.

The MSC serum-free culture medium is MSC serum-free culture medium +5ng/ml EGF +5ng/ml FGF +5ng/ml IFN-gamma +5ng/ml TNF-alpha +1wt.% L-GlutaMAX.

The subculture of the adipose tissue-derived mesenchymal stem cells comprises the following steps:

(1) taking out adipose mesenchymal stem cell cells to be passaged, and sucking out the culture medium;

(2) adding 1.0ml of 0.25% pancreatin into each culture bottle to digest cells, immediately adding 5.0ml of stop solution to stop digestion when cells are found to be round under a microscope, gently blowing and sucking the cells, transferring the cells into a 50ml centrifuge tube, washing the bottom of each culture bottle once by using 20.0ml of normal saline, transferring the washed cells into the centrifuge tube, and washing the cells for 800g for 6 minutes;

(3) the supernatant was aspirated off, the cell pellet was resuspended in serum-free medium, trypan blue stained cells were counted and cell density was adjusted to 4.0 x 10^{^5}And/ml, adding 23.0ml of serum-free culture medium into each culture bottle, adding 2.0ml of cell suspension, uniformly mixing, and placing in an incubator for static culture.

The cell harvest was:

1) observing the cells, and harvesting and freezing or supplying for use when the cells grow to 80-90%;

2) taking out the cell culture bottle, sterilizing the bottom of the bottle by using 75% alcohol, and putting the bottle into a biological safety cabinet;

3) the old medium was discarded by pipette;

4) adding 3ml of 0.25% pancreatin into each bottle, digesting at 37 ℃ for 1min, adding 10ml of stop solution into each bottle when the cells are obviously retracted, stopping digestion, collecting all the liquid into a 50ml centrifuge tube, adding 10ml of normal saline into each bottle, gently blowing and beating the solution, and merging the solution into the 50ml centrifuge tube;

5) centrifuging at 1200rpm for 6min, removing supernatant, suspending cell precipitate with 16ml physiological saline, mixing well, and taking 1ml for counting and flow detection;

6) adding physiological saline to 40ml, collecting 500 μ l supernatant for endotoxin detection, centrifuging at 1200rpm for 6 min;

7) pouring the supernatant into a clean centrifugal tube for bacteria detection and mycoplasma detection;

8) frozen cells: suspending the centrifugal precipitate with 2.5ml FBS, slowly adding 2.5ml of the frozen mother liquor, mixing uniformly, and subpackaging into 1ml of frozen tubes;

the cells used were: the centrifugal precipitation is resuspended by 0.9 percent of physiological saline solution, and the number and the volume of the cells are determined according to the use condition; transferring the resuspended cell suspension into a cell feedback bag or a feedback tube;

9) marking the cell algebra, density, bar code and operation time outside the tube wall;

10) and (4) carrying out cryopreservation on the cryopreserved cells according to a programmed cooling box method, and transporting the applied cells to an application site through a cold chain.

The invention has the advantages that:

1. the digestive juice is a mixed enzyme solution of 0.075-0.5% of collagenase I, 0.1% of collagenase IV and 0.1% of trypsin, so that the enzymolysis efficiency can be effectively improved, and the preparation time can be shortened.

2. The fat stem cells with higher purity can be obtained by using 0.3 percent NaCl solution to crack the red blood cells.

3. 5ng/ml EGF +5ng/ml FGF +5ng/ml IFN-gamma +5ng/ml TNF-alpha +1% L-GlutaMAX is added into the culture medium, and the factors are simultaneously added into the culture system to effectively promote the proliferation of MSC, thereby effectively shortening the culture time.

4. Counting, flow detection, endotoxin detection, mycoplasma detection, bacteria culture detection and the like are carried out during cell harvesting, only if all the detections are qualified, the cells are allowed to be put in storage or supplied to customers for use, and the effectiveness and the safety of cell products are ensured from the aspects of cell quantity, cell characteristics, microbial pollution and the like.

The method adopts detection and investigation and other methods in the processes of fat obtaining, fat preparation and cell culture to ensure that pollution-free adipose-derived stem cells are obtained, thereby ensuring the safety, adopts an improved separation method and a culture system in the processes of separation and culture to improve the separation efficiency, shortens the culture period and can obtain cells with high purity and excellent characteristics.

Drawings

FIG. 1 is a diagram of the state of cells after 2 days of plating adipose stem cells: most cells are round, a few cells are fusiform, and the number of cells is small.

FIG. 2 is a diagram of the state of the cells after 5 days of plating the adipose-derived stem cells: the number of cells is obviously increased, and the cells present a classical mesenchymal stem cell shape.

FIG. 3 shows the state diagram of the cells of the adipose-derived stem cell P0 at day 3 of P1: the cells grow uniformly, have standard shapes and are more in number.

FIG. 4 is a diagram showing the results of flow assay of adipose-derived stem cells. From the flow detection results, the phenotype of the isolated cells in the scheme can be seen to be consistent with the characteristics of the adipose-derived stem cells (CD 44/CD105 is positive, and CD34/CD45 is negative).

Detailed Description

Example 1

1. Adipose-derived stem cell preparation

(1) And (4) reserving a fat specimen by a client qualified in infectious disease detection through a liposuction operation, storing at a low temperature and transporting to a laboratory as soon as possible.

(2) Adipose tissue was transferred to 50ml centrifuge tubes, 40ml per tube, 800g, 8 min.

(3) The centrifuged adipose tissues were transferred to a new centrifuge tube, and 0.9% physiological saline, 800g, was added in an equal volume for 8 minutes.

(4) The supernatant was discarded and step 3 was repeated to wash the adipose tissue twice.

(5) Adding digestive juice (0.5% collagenase type I +0.1% collagenase type IV +0.1% trypsin) into the fat precipitate at the same volume, mixing well, sealing, and placing in a shaker at 37 deg.C, 150rpm, 30 min.

(6) The digested adipose tissue was removed and centrifuged at 800g for 10 min.

(7) The upper fat was discarded, and the centrifuged cell pellet was resuspended in DMEM/F12 basal medium and centrifuged at 800g for 10 minutes.

(8) Step 7 was repeated to wash the cell pellet.

(9) The centrifuged supernatant was discarded, DMEM/F12 resuspended and mixed with the cell pellet, and the cells were first filtered through a 40um filter.

(10) The filtered cells were centrifuged at 800g for 10 min. The supernatant was discarded.

(11) 1.0ml DMEM/F12 medium heavy suspension cell precipitation, after mixing, adding 4.0ml precooled 0.3% NaCl solution, room temperature 10 minutes, lysis of red blood cells.

(12) Centrifuge, 400g for 5 min. The supernatant was discarded.

(13) Resuspending the cell pellet with MSC serum-free medium (MSC serum-free basal medium +5ng/ml EGF +5ng/ml FGF +5ng/ml IFN-. gamma. +5ng/ml TNF-. alpha. +1% L-GlutaMAX), counting trypan blue stained cells, adjusting the cell density to 2.0 x 10^{^6}And/ml. 14ml of medium and 1.0ml of cell suspension were added to each T75 flask and mixed well.

(14) After 24 hours, the cells were changed and the nonadherent cells were removed. The solution was changed every three days later.

(15) Culturing until about the fifth day, wherein the cell growth density reaches about 90%, and subculturing the adipose tissue-derived mesenchymal stem cells.

And subculturing adipose tissue-derived stem cells

(16) And taking out the adipose mesenchymal stem cells to be passaged, and sucking out the culture medium.

(17) 1.0ml of digested cells were added to each T75 flask with 0.25% trypsin, and when cells were found to be round under the microscope, 5.0ml of stop buffer was added immediately to stop the digestion, the cells were gently pipetted and transferred to a 50ml centrifuge tube, and the flasks were washed once with 20.0ml of physiological saline, and the washed cells were transferred to a centrifuge tube at 800g for 6 minutes.

(18) The supernatant was aspirated off, the cell pellet was resuspended in serum-free medium, trypan blue stained cells were counted and cell density was adjusted to 4.0 x 10^{^5}And/ml, adding 23.0ml of culture medium into each T175 culture flask, adding 2.0ml of cell suspension, mixing uniformly, and placing in an incubator for static culture.

And (3) cell harvesting:

1) cells were observed and harvested at 90% growth for cryopreservation or for use by the customer.

2) Taking out the cell culture bottle, sterilizing the bottom of the bottle by using 75% alcohol, and putting the bottle into a biological safety cabinet.

3) The old medium was pipetted off.

4) Adding 3ml of 0.25% pancreatin into each bottle, digesting at 37 ℃ for 1min, adding 10ml of stop solution into each bottle when the cells are obviously retracted, stopping digestion, collecting all the liquid into a 50ml centrifuge tube, adding 10ml of normal saline into each bottle, gently blowing and beating, and merging into the 50ml centrifuge tube.

5) Centrifuging at 1200rpm for 6min, discarding supernatant, suspending cell precipitate with 16ml physiological saline, mixing well, and taking 1ml for counting and flow detection.

6) Adding physiological saline to 40ml, collecting 500 μ l supernatant for endotoxin detection, centrifuging at 1200rpm for 6 min.

7) And pouring the supernatant into a clean centrifugal tube for bacteria detection and mycoplasma detection.

8) Frozen cells: suspending the centrifugal precipitate with 2.5ml FBS, slowly adding 2.5ml of the frozen mother liquor, mixing uniformly, and subpackaging into 1ml of frozen tubes.

The cells used were: the pellet was resuspended in 0.9% saline solution, and the number and volume of cells were determined according to the customer's usage. The resuspended cell suspension is transferred to a cell return bag or return tube.

9) The number of cell generations, density, barcode and time of operation were marked outside the vessel wall.

Example 2

1. Adipose-derived stem cell preparation

(19) And (4) reserving a fat specimen by a client qualified in infectious disease detection through a liposuction operation, storing at a low temperature and transporting to a laboratory as soon as possible.

(20) Adipose tissue was transferred to 50ml centrifuge tubes, 40ml per tube, 800g, 8 min.

(21) The centrifuged adipose tissues were transferred to a new centrifuge tube, and 0.9% physiological saline, 800g, was added in an equal volume for 8 minutes.

(22) The supernatant was discarded and step 3 was repeated to wash the adipose tissue twice.

(23) Adding digestive juice (0.1% collagenase type I +0.1% collagenase type IV +0.1% trypsin) into the fat precipitate at the same volume, mixing well, sealing, and placing in a shaker at 37 deg.C, 150rpm, 40 min.

(24) The digested adipose tissue was removed and centrifuged at 800g for 10 min.

(25) The upper fat was discarded, and the centrifuged cell pellet was resuspended in DMEM/F12 basal medium and centrifuged at 800g for 10 minutes.

(26) Step 7 was repeated to wash the cell pellet.

(27) The centrifuged supernatant was discarded, DMEM/F12 resuspended and mixed with the cell pellet, and the cells were first filtered through a 40um filter.

(28) The filtered cells were centrifuged at 800g for 10 min. The supernatant was discarded.

(29) 1.0ml DMEM/F12 medium heavy suspension cell precipitation, after mixing, adding 4.0ml precooled 0.3% NaCl solution, room temperature 10 minutes, lysis of red blood cells.

(30) Centrifuge, 400g for 5 min. The supernatant was discarded.

(31) Resuspend the cell pellet with MSC serum free medium (MSC serum free basal medium +5ng/ml EGF +5ng/ml FGF +5ng/ml IFN-. gamma. +5ng/ml TNF-. alpha. +1% L-GlutaMAX), count trypan blue stained cells, adjust cell density to 2.0 x 10^ 6/ml. 14ml of medium and 1.0ml of cell suspension were added to each T75 flask and mixed well.

(32) After 24 hours, the cells were changed and the nonadherent cells were removed. The solution was changed every three days later.

(33) Culturing until about the fifth day, wherein the cell growth density reaches about 90%, and subculturing the adipose tissue-derived mesenchymal stem cells.

And subculturing adipose tissue-derived stem cells

(34) And taking out the adipose mesenchymal stem cells to be passaged, and sucking out the culture medium.

(35) 1.0ml of digested cells were added to each T75 flask with 0.25% trypsin, and when cells were found to be round under the microscope, 5.0ml of stop buffer was added immediately to stop the digestion, the cells were gently pipetted and transferred to a 50ml centrifuge tube, and the flasks were washed once with 20.0ml of physiological saline, and the washed cells were transferred to a centrifuge tube at 800g for 6 minutes.

(36) And (3) sucking and removing the supernatant, re-suspending the cell sediment by using a serum-free culture medium, counting trypan blue stained cells, adjusting the cell density to 4.0 x 10^5/ml, adding 23.0ml of culture medium into each T175 culture bottle, adding 2.0ml of cell suspension, uniformly mixing, and placing in an incubator for static culture.

And (3) cell harvesting:

1) cells were observed and harvested at 80% growth for cryopreservation or for use by the customer.

3) The old medium was pipetted off.

Comparative example 1

The same conditions were followed, using 0.05% collagenase type I +0.1% trypsin instead of the digestates of examples 1 and 2, for a digestion time of 40 min; the medium was high-glucose DMEM medium containing 10% FBS.

Example 1, example 2 and comparative example 1 three different samples were separately cultured, and the results of the present invention were examined and compared by the following 2 aspects.

1. And (3) comparing the enzymolysis efficiency of different digestive juices:

the results of digesting human adipose-derived stem cells with digestive juices of different formulations are as follows: the average number of viable cells harvested per 100ml of adipose tissue in example 1, example 2 and control example 1 were as follows: 3.25*10⁷3.20 x 10 pieces⁷2.50 x 10 pieces⁷And (4) respectively. As shown in Table 1, there was a very significant difference in the number of live cells obtained by digestion in examples 1 and 2 from that in comparative example 1. The data show that compared with other digestive juice formulas, the digestive juice formula provided by the invention can obviously improve the enzymolysis efficiency and has higher cell yield.

The enzymolysis condition explored by the invention is stable and efficient, the adipose tissues are completely digested, and cells in the tissues are released as much as possible; when the collagenase type I solubility is low (example 2), the digestion time needs to be prolonged properly, but the digestion effect is not affected.

2. Comparing the number of adherent cells cultured in different culture media and culture periods:

human adipose-derived stem cells were cultured in media of different formulations, and P0 generation cells were counted, with the following results: the average cell numbers of the cultures of example 1, example 2 and comparative example 1 were: 2.50*10⁶2.45 x 10 pieces⁶1.58 x 10⁶And (4) respectively. Culturing adipose-derived stem cells by adopting culture media with different formulas, and finally harvesting 2.50 x 10⁷The total average culture time of each cell in example 1, example 2 and comparative example 1 was calculated as follows: 14 days, 20 days. As shown in table 1, the number of cells harvested and the number of days of culture were significantly different between P0 in example 1 and example 2 from those in comparative example 1.

TABLE 1

The results show that the improved culture system and the improved culture method adopted in the separation and culture processes can improve the separation efficiency, shorten the culture period and obtain cells with high purity and excellent characteristics.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

Claims

1. A method for separating and purifying adipose-derived stem cells from adipose tissues is characterized by comprising the following steps: the method comprises the steps of adipose-derived stem cell preparation, adipose-derived mesenchymal stem cell subculture and cell harvesting;

the adipose-derived stem cells are prepared by the following steps:

(9) centrifuging at 400g for 5 min, and discarding the supernatant;

(10) resuspending the cell pellet with MSC serum-free medium, counting trypan blue stained cells, adjusting the cell density to 2.0 x 10^{^6}Adding 14ml of culture medium into each culture bottle, adding 1.0ml of cell suspension, and uniformly mixing; after 24 hours, the cells are changed, the cells which are not attached to the wall are removed, and the cells are changed every three days; after culturing to the fifth day, the cell growth density reaches 90%, and the adipose-derived mesenchymal stem cells are subjected to subculture;

the digestive juice contains 0.075-0.5 wt.% of collagenase I +0.1wt.% of collagenase IV +0.1wt.% of trypsin;

the MSC serum-free culture medium is MSC serum-free culture medium +5ng/ml EGF +5ng/ml FGF +5ng/ml IFN-gamma +5ng/ml TNF-alpha +1wt.% L-GlutaMAX;

(3) the supernatant was aspirated off, the cell pellet was resuspended in serum-free medium, trypan blue stained cells were counted and cell density was adjusted to 4.0 x 10^{^5}Adding 23.0ml of serum-free culture medium into each culture bottle, adding 2.0ml of cell suspension, uniformly mixing, and placing in an incubator for static culture;

the cell harvest was:

3) the old medium was discarded by pipette;