Construction method of adipose-derived mesenchymal stem cell bank
阅读说明：本技术 一种脂肪间充质干细胞库的构建方法 (Construction method of adipose-derived mesenchymal stem cell bank ) 是由 刘建丽 李明 于 2021-08-24 设计创作，主要内容包括：本发明公开了一种脂肪间充质干细胞库的构建方法,包括以下步骤：(1)对选取的合格离体脂肪样本供体建立登记档案；(2)取离体的脂肪组织经消化、分离得到脂肪间充质干细胞；(3)将步骤(2)分离得到脂肪间充质干细胞接种在纯化培养基上,培养至细胞汇合度达到80-85%时消化,离心,向细胞中加入纯化培养基重悬,记为P0代细胞；(4)将P0代细胞采用上述纯化培养基进行传代培养,取传代培养得到的P3代细胞进行冻存,构建脂肪间充质干细胞库；本发明还具体限定了纯化培养基的组成。本发明提供的细胞库的构建方法可以充分满足脂肪间充质干细胞的科研和临床应用。(The invention discloses a method for constructing an adipose-derived mesenchymal stem cell bank, which comprises the following steps of: (1) establishing a registration file for the selected qualified in vitro fat sample donor; (2) taking isolated adipose tissues, digesting and separating to obtain adipose mesenchymal stem cells; (3) inoculating the adipose-derived mesenchymal stem cells separated in the step (2) on a purification culture medium, culturing until the cell confluence reaches 80-85%, digesting, centrifuging, adding the purification culture medium into the cells for heavy suspension, and marking as P0 generation cells; (4) subculturing the P0 generation cells by adopting the purified culture medium, freezing and storing the P3 generation cells obtained by subculturing to construct an adipose-derived mesenchymal stem cell bank; the invention also specifically defines the composition of the purification medium. The construction method of the cell bank provided by the invention can fully meet the scientific research and clinical application of the adipose-derived mesenchymal stem cells.)
1. A method for constructing an adipose-derived mesenchymal stem cell bank is characterized by comprising the following steps of:
(1) establishing a registration file for the selected qualified in vitro fat sample donor;
(2) taking isolated adipose tissues, digesting and separating to obtain adipose mesenchymal stem cells;
(3) inoculating the adipose-derived mesenchymal stem cells separated in the step (2) on a purification culture medium, sucking the culture medium when the cell confluence reaches 80-85%, adding 0.25% pancreatin for digestion, adding PBS for dilution, centrifuging, adding the purification culture medium into the cells for resuspension, and marking as P0 generation cells;
(4) p0 passage cells were cultured using the above purification medium according to 1: 2-3, performing subculture, and performing cryopreservation on the P3 generation cells obtained by subculture to construct an adipose-derived mesenchymal stem cell bank;
the composition of the purification medium is as follows: a basal medium and the following components added in the basal medium: glutathione, D-limonene, ranolazine, canavalin A, vitamin C, sodium selenite and L-arginine.
2. The method for constructing an adipose-derived mesenchymal stem cell bank according to claim 1, wherein the composition of the purification medium is as follows: basal medium DMEM/F12, each ingredient added to DMEM/F12 medium at final concentration: 20-25 mug/mL of glutathione, 8.5-11.5ng/mL of D-limonene, 5.8-7.2ng/mL of ranolazine, 6.5-8.9ng/mL of jack bean protein A, 1-5 mug/mL of vitamin C, 2.5-4.5ng/mL of sodium selenite and 5-10 mug/mL of L-arginine.
3. The method for constructing an adipose-derived mesenchymal stem cell bank according to claim 2, wherein the components added in the DMEM/F12 medium are added in final concentration: 23 mu g/mL of glutathione, 10ng/mL of D-limonene, 6.5ng/mL of ranolazine, 7.4ng/mL of jack bean protein A, 2.5 mu g/mL of vitamin C, 3ng/mL of sodium selenite and 8 mu g/mL of L-arginine.
4. The method for constructing the adipose-derived mesenchymal stem cell bank according to claim 1, wherein the adipose tissue at the upper layer is taken after the adipose sample is centrifuged in the step (2), the adipose tissue is washed by PBS and then centrifuged again, 1% type II collagenase is added into the centrifuged adipose tissue to carry out shock digestion for 30min, PBS is added after digestion is finished, the adipose tissue is diluted and then centrifuged, and the precipitate is the adipose-derived mesenchymal stem cells.
5. The method for constructing an adipose-derived mesenchymal stem cell bank according to claim 1, wherein the adipose-derived mesenchymal stem cells isolated in the step (3) have a density of 1-5 x 10 in a purified culture medium6Cells at 37 ℃ in 5% CO/mL2The culture box is used for culturing, and the culture medium is replaced every 2 days in the culture process.
6. The method for constructing adipose-derived mesenchymal stem cell bank according to claim 1, wherein the steps ofThe density of the P0 generation cells in the purification medium after resuspension in the (3) step is 1-5X 105one/mL.
7. The method for constructing the adipose-derived mesenchymal stem cell bank according to claim 1, wherein the P3 generation cells cultured to have a confluency of 70-80% in step (4) are digested with 0.25% pancreatin, diluted with PBS, a part of the cells are counted, the cell viability is measured, the cell proliferation activity, purity and multi-directional differentiation capacity are detected, the remaining cells are centrifuged, the supernatant is separated from the cells, the centrifuged cells are resuspended in a cryopreservation solution with a cell density of 1-8 x 107And (2) each/mL, the freezing medium is respectively filled in 2mL freezing tubes, each tube is 1mL, the freezing date and the cell information file are marked, and then the freezing storage is carried out in liquid nitrogen.
8. The method for constructing the adipose-derived mesenchymal stem cell bank according to claim 1, wherein the frozen stock solution is prepared by adding 1-5% of DMSO into a purification medium.
Technical Field
The invention relates to a method for constructing a cell bank, in particular to a method for constructing a human adipose tissue-derived mesenchymal stem cell bank.
Background
Stem cells are a class of cells with self-renewal and differentiation potential that, under certain conditions, can differentiate into a variety of functional cells. Stem cells can be classified into embryonic stem cells and adult stem cells according to developmental stages. Embryonic stem cells, although capable of differentiating into a variety of different tissues, have limited their clinical utility due to regulatory and ethical limitations on cell differentiation. Mesenchymal stem cells are currently of great interest as a class of adult stem cells with multipotentiality.
Adipose-derived mesenchymal stem cells are a class of mesenchymal stem cells with multipotential differentiation potential obtained from adipose tissue, which can differentiate into adipocytes, osteoblasts, chondrocytes, cardiomyocytes, and even neural cells under specific induction conditions. Compared with mesenchymal stem cells from bone marrow, the adipose-derived stem cells have more stability and difficult aging property, and are rare high-quality seed cells in tissue engineering; adipose-derived mesenchymal stem cells (ADSCs) are used as mesenchymal stem cells derived from fat, the material is easy to obtain, a large amount of stem cells can be obtained from a small amount of tissues, the mass culture is suitable, and the damage to organisms caused by the material obtaining process is small. Compared with the bone marrow mesenchymal stem cells, the adipose mesenchymal stem cells have lower immunogenicity, can not be rejected by organisms as foreign matters during transplantation, are suitable for autologous transplantation, have high safety performance, and gradually become one of new research hotspots in recent years.
The existing adipose-derived mesenchymal stem cell bank is complicated in establishing process, and exogenous serum such as fetal calf serum and the like added in the cell obtaining process influences the quality of adipose-derived mesenchymal stem cells in the cell bank, so that the clinical application of the adipose-derived mesenchymal stem cells is greatly limited. With the increase of the demand of stem cells in various fields, it is necessary to improve the establishment process of the adipose-derived mesenchymal stem cell bank, improve the establishment efficiency of the adipose-derived mesenchymal stem cell bank, and promote the wide application of adipose-derived mesenchymal stem cells in various fields.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for constructing an adipose-derived mesenchymal stem cell bank, which effectively improves the purity and activity of cells in the cell bank and provides guarantee for clinical application of adipose-derived mesenchymal stem cells.
The purpose of the invention is realized by adopting the following technical scheme:
a method for constructing an adipose-derived mesenchymal stem cell bank comprises the following steps:
(1) establishing a registration file for the selected qualified in vitro fat sample donor;
(2) taking isolated adipose tissues, digesting and separating to obtain adipose mesenchymal stem cells;
(3) inoculating the adipose-derived mesenchymal stem cells separated in the step (2) on a purification culture medium, sucking the culture medium when the cell confluence reaches 80-85%, adding 0.25% pancreatin for digestion, adding PBS for dilution, centrifuging, adding the purification culture medium into the cells for resuspension, and marking as P0 generation cells;
(4) p0 passage cells were cultured using the above purification medium according to 1: 2-3, performing subculture, and performing cryopreservation on the P3 generation cells obtained by subculture to construct an adipose-derived mesenchymal stem cell bank;
the composition of the purification medium is as follows: a basal medium and the following components added in the basal medium: glutathione, D-limonene, ranolazine, canavalin A, vitamin C, sodium selenite and L-arginine.
Further, the composition of the purification medium is: basal medium DMEM/F12, each ingredient added to DMEM/F12 medium at final concentration: 20-25 mug/mL of glutathione, 8.5-11.5ng/mL of D-limonene, 5.8-7.2ng/mL of ranolazine, 6.5-8.9ng/mL of jack bean protein A, 1-5 mug/mL of vitamin C, 2.5-4.5ng/mL of sodium selenite and 5-10 mug/mL of L-arginine.
Further, the components added in DMEM/F12 medium were measured in final concentration: 23 mu g/mL of glutathione, 10ng/mL of D-limonene, 6.5ng/mL of ranolazine, 7.4ng/mL of jack bean protein A, 2.5 mu g/mL of vitamin C, 3ng/mL of sodium selenite and 8 mu g/mL of L-arginine.
Further, centrifuging the fat sample in the step (2), taking the upper layer of fat tissue, washing with PBS, centrifuging again, adding 1% type II collagenase into the centrifuged fat tissue, oscillating and digesting for 30min, adding PBS for dilution after digestion, centrifuging, and precipitating to obtain the adipose mesenchymal stem cells.
Further, the density of the adipose-derived mesenchymal stem cells separated in the step (3) in a purification medium is 1-5 x 106Cells at 37 ℃ in 5% CO/mL2The culture box is used for culturing, and the culture medium is replaced every 2 days in the culture process.
Further, the density of the P0 generation cells in the step (3) after being resuspended in the purification medium is 1-5X 105one/mL.
Further, in the step (4), P3 generation cells cultured to 70-80% of fusion degree are digested by 0.25% pancreatin and then diluted by adding PBS, part of the cells are counted, the cell viability is measured, the cell proliferation activity, purity and multi-directional differentiation capacity are detected, the rest of the cells are centrifuged, then supernatant and cells are separated, frozen stock solution is added into the centrifuged cells for re-suspension, and the cell density is 1-8 × 107And (2) each/mL, the freezing medium is respectively filled in 2mL freezing tubes, each tube is 1mL, the freezing date and the cell information file are marked, and then the freezing storage is carried out in liquid nitrogen.
Further, the frozen stock solution is prepared by adding 1-5% DMSO to a purification medium.
Compared with the prior art, the invention has the beneficial effects that: the invention provides a method for constructing an adipose-derived mesenchymal stem cell bank, wherein in the construction process of the cell bank, in order to ensure the quality of cells in the cell bank, components such as ranolazine, D-limonene, canavalin A and the like are added into a purification culture medium, on one hand, the ranolazine can effectively screen out adipose-derived mesenchymal stem cells, the purity of the adipose-derived mesenchymal stem cells is improved, and a large amount of adipose-derived mesenchymal stem cells with higher purity can be obtained without multiple passage amplification; on the other hand, the added D-limonene and the canavalin A have a synergistic effect to improve the activity of the adipose-derived mesenchymal stem cells, a large number of adipose-derived mesenchymal stem cells with high proliferation activity and differentiation potential can be obtained through culture, animal serum does not need to be added in the whole process, and the cell quality in the adipose-derived mesenchymal stem cell bank is fully guaranteed.
The purification culture medium can be used as the frozen stock solution only by adding 1-5% of DMSO, so that the using amount of DMSO in the frozen stock solution is reduced, and the freezing efficiency is effectively ensured.
Detailed Description
The present invention is further described below with reference to specific embodiments, and it should be noted that, without conflict, any combination between the embodiments or technical features described below may form a new embodiment.
Example 1
A method for constructing an adipose-derived mesenchymal stem cell bank comprises the following steps:
(1) establishing a registration file for the selected qualified ex vivo fat sample donor, wherein the age is between 20 and 35 years, the registration file comprises the elimination of the existing medical history, family genetic history, infectious disease history and other health abnormal conditions, and the following items are subjected to physical examination: syphilis spiral antibody, hepatitis B surface antigen, hepatitis C antibody, transaminase, and AIDS antibody, and qualified patients can be used as donor of adipose tissue;
(2) taking isolated adipose tissues, respectively packaging in a 50mL centrifuge tube, centrifuging at 1500r/min for 10min, taking the upper layer adipose tissues, adding PBS with the same volume, cleaning, centrifuging at 1000r/min for 10min, adding 1% II-type collagenase into the centrifuged adipose tissues, oscillating and digesting at 37 ℃ for 30min, adding PBS for dilution after digestion, centrifuging at 1000r/min again for 10min, and obtaining adipose mesenchymal stem cells as precipitates;
(3) separating the adipose-derived mesenchymal stem cells obtained in the step (1) to obtain 3 x 10 adipose-derived mesenchymal stem cells6one/mL of the culture was inoculated on purified medium in a T75 flask at 37 ℃ with 5% CO2Culturing in the culture box, replacing culture medium every 2 days, removing culture medium when cell confluence reaches 80%, adding 0.25% pancreatin for digestion, adding PBS for dilution, centrifuging, adding purified culture medium into cells, and resuspending at density of 2 × 105one/mL, designated as P0 generation cells,
(4) the above P0 generation cells were cultured in the above purification medium according to the following ratio of 1: 2, digesting P3 generation cells cultured to the fusion degree of 75% by using 0.25% pancreatin, adding PBS for dilution, taking part of the cells for counting, measuring the cell viability, detecting the proliferation capacity and the multidirectional differentiation capacity of the cells, centrifuging the rest cells to separate supernatant and the cells, adding freezing medium into the centrifuged cells for resuspension, wherein the freezing medium is prepared by adding 3% DMSO into a purification culture medium, and the cell density in the freezing medium is 5 multiplied by 107each/mL, the frozen stock solution is respectively filled in 2mL frozen stock tubes, each tube is 1mL, frozen and preserved in liquid nitrogen to construct an adipose mesenchymal stem cell bank, and the frozen stock date and the fine stem cell are markedThe cell information file is convenient for retrieval and query;
the composition of the purification medium is as follows: basal medium DMEM/F12, each ingredient added to DMEM/F12 medium at final concentration: 23 mu g/mL of glutathione, 10ng/mL of D-limonene, 6.5ng/mL of ranolazine, 7.4ng/mL of jack bean protein A, 2.5 mu g/mL of vitamin C, 3ng/mL of sodium selenite and 8 mu g/mL of L-arginine.
Example 2
A method for constructing an adipose-derived mesenchymal stem cell bank comprises the following steps:
(1) establishing a registration file for the selected qualified ex vivo fat sample donor, wherein the age is between 20 and 35 years, the registration file comprises the elimination of the existing medical history, family genetic history, infectious disease history and other health abnormal conditions, and the following items are subjected to physical examination: syphilis spiral antibody, hepatitis B surface antigen, hepatitis C antibody, transaminase, and AIDS antibody, and qualified patients can be used as donor of adipose tissue;
(2) taking isolated adipose tissues, respectively packaging in a 50mL centrifuge tube, centrifuging at 1500r/min for 10min, taking the upper layer adipose tissues, adding PBS with the same volume, cleaning, centrifuging at 1000r/min for 10min, adding 1% II-type collagenase into the centrifuged adipose tissues, oscillating and digesting at 37 ℃ for 30min, adding PBS for dilution after digestion, centrifuging at 1000r/min again for 10min, and obtaining adipose mesenchymal stem cells as precipitates;
(3) separating the adipose-derived mesenchymal stem cells obtained in the step (1) to obtain 1 × 10 adipose-derived mesenchymal stem cells6one/mL of the culture was inoculated on purified medium in a T75 flask at 37 ℃ with 5% CO2Culturing in the culture box, replacing culture medium every 2 days, removing culture medium when cell confluence reaches 85%, adding 0.25% pancreatin for digestion, adding PBS for dilution, centrifuging, adding purified culture medium into cells, and resuspending at density of 1 × 105one/mL, designated as P0 generation cells,
(4) the above P0 generation cells were cultured in the above purification medium according to the following ratio of 1: 3, digesting the P3 generation cells cultured to 70% of fusion degree with 0.25% of pancreatin, adding PBS for dilution, taking part of cells for counting, measuring cell viability, and detecting cell proliferationSeparating supernatant and cells by centrifuging the rest cells, adding freezing medium into centrifuged cells, resuspending, wherein the freezing medium is prepared by adding 3% DMSO into purified culture medium, and the cell density in the freezing medium is 1 × 107each/mL, respectively filling the cryopreservation liquid into 2mL of cryopreservation tubes, wherein each tube is 1mL, performing cryopreservation in liquid nitrogen to construct an adipose mesenchymal stem cell bank, and marking the cryopreservation date and a cell information file so as to facilitate retrieval and query;
the composition of the purification medium is as follows: basal medium DMEM/F12, each ingredient added to DMEM/F12 medium at final concentration: 20 mu g/mL of glutathione, 8.5ng/mL of D-limonene, 5.8ng/mL of ranolazine, 6.5ng/mL of concanavalin A, 1 mu g/mL of vitamin C, 2.5ng/mL of sodium selenite and 5 mu g/mL of L-arginine.
Example 3
A method for constructing an adipose-derived mesenchymal stem cell bank comprises the following steps:
(1) establishing a registration file for the selected qualified ex vivo fat sample donor, wherein the age is between 20 and 35 years, the registration file comprises the elimination of the existing medical history, family genetic history, infectious disease history and other health abnormal conditions, and the following items are subjected to physical examination: syphilis spiral antibody, hepatitis B surface antigen, hepatitis C antibody, transaminase, and AIDS antibody, and qualified patients can be used as donor of adipose tissue;
(2) taking isolated adipose tissues, respectively packaging in a 50mL centrifuge tube, centrifuging at 1500r/min for 10min, taking the upper layer adipose tissues, adding PBS with the same volume, cleaning, centrifuging at 1000r/min for 10min, adding 1% II-type collagenase into the centrifuged adipose tissues, oscillating and digesting at 37 ℃ for 30min, adding PBS for dilution after digestion, centrifuging at 1000r/min again for 10min, and obtaining adipose mesenchymal stem cells as precipitates;
(3) separating the adipose-derived mesenchymal stem cells obtained in the step (1) to obtain 5 x 10 adipose-derived mesenchymal stem cells6one/mL of the culture was inoculated on purified medium in a T75 flask at 37 ℃ with 5% CO2The culture box is used for culturing, the culture medium is replaced every 2 days in the culture process, the culture medium is removed when the cell confluence reaches 80 percent, 0.25 percent pancreatin is added for digestion, PBS is added for dilution, and then the cell is separatedHeart, adding purified culture medium to the cells, and resuspending at a density of 5 × 105one/mL, designated as P0 generation cells,
(4) the above P0 generation cells were cultured in the above purification medium according to the following ratio of 1: 3, digesting P3 generation cells cultured to the fusion degree of 80% by using 0.25% pancreatin, adding PBS for dilution, taking part of the cells for counting, measuring the cell viability, detecting the proliferation capacity and the multidirectional differentiation capacity of the cells, centrifuging the rest cells to separate supernatant and the cells, adding freezing medium into the centrifuged cells for resuspension, wherein the freezing medium is prepared by adding 5% DMSO into a purification culture medium, and the cell density in the freezing medium is 8 multiplied by 107each/mL, respectively filling the cryopreservation liquid into 2mL of cryopreservation tubes, wherein each tube is 1mL, performing cryopreservation in liquid nitrogen to construct an adipose mesenchymal stem cell bank, and marking the cryopreservation date and a cell information file so as to facilitate retrieval and query;
the composition of the purification medium is as follows: basal medium DMEM/F12, each ingredient added to DMEM/F12 medium at final concentration: 25 mug/mL of glutathione, 11.5ng/mL of D-limonene, 7.2ng/mL of ranolazine, 8.9ng/mL of jack bean protein A, 5 mug/mL of vitamin C, 4.5ng/mL of sodium selenite and 10 mug/mL of L-arginine.
Comparative example 1
Comparative example 1 provides a method for constructing an adipose-derived mesenchymal stem cell bank, which is different from example 1 in that ranolazine in a purification medium is omitted, and the rest is the same as example 1.
Comparative example 2
Comparative example 2 provides a method for constructing an adipose-derived mesenchymal stem cell bank, which is different from example 1 in that D-limonene in a purification medium is omitted, and the rest is the same as example 1.
Comparative example 3
Comparative example 3 provides a method for constructing an adipose-derived mesenchymal stem cell bank, which is different from example 1 in that the purification of canavalin a in a culture medium is omitted, and the rest is the same as example 1.
Comparative example 4
Comparative example 4 provides a method for constructing an adipose-derived mesenchymal stem cell bank, which is different from example 1 in that ranolazine, D-limonene, and canavalin a in a purification medium are omitted, and the rest is the same as example 1.
Test examples
Cell viability assay: the viability of the cells was statistically counted by trypan blue staining method using P3-substituted adipose-derived mesenchymal stem cells before cryopreservation, and the results are shown in table 1.
TABLE 1
Group of
Survival rate
Example 1
99.28%
Comparative example 1
97.46%
Comparative example 2
84.97%
Comparative example 3
86.72%
Comparative example 4
79.83%
As can be seen from Table 1, the survival rate of the cells in example 1 is higher than that of comparative examples 1 to 4, and the cell activity of the cell bank is further satisfied, and the cell activity is reduced to different degrees by adjusting the composition of the purified culture medium in comparative examples 1 to 4.
And (3) detecting the purity of the cells: the results of the flow cytometry of the collected P3 generation are shown in Table 2.
TABLE 2
Positive rate of detection index
CD105
CD90
CD73
CD59
CD44
CD13
CD34
CD14
Example 1
99.89%
99.94%
99.82%
99.95%
99.76%
99.87%
0.32%
0.18%
Comparative example 1
98.05%
97.69%
98.21%
97.96%
97.64%
98.09%
2.04%
2.11%
Comparative example 2
99.85%
99.92%
99.87%
99.91%
99.79%
99.83%
0.33%
0.17%
Comparative example 3
99.80%
99.91%
99.93%
99.88%
99.81%
99.72%
0.35%
0.21%
Comparative example 4
97.86%
97.73%
98.19%
97.92%
97.71%
98.11%
2.05%
2.09%
As can be seen from table 2: the cell purity in the example 1, the comparative example 2 and the comparative example 3 is higher, the positive antigen expression rate on the cell surface is higher than 99 percent, and the negative antigen expression rate is lower than 1 percent. The purity of the cells in comparative example 1 and comparative example 4 is somewhat reduced compared to example 1 because ranolazine is omitted in comparative example 1 and comparative example 4, and the addition of the component can improve the purity of the adipose mesenchymal stem cells.
And (3) detecting the cell proliferation capacity: taking the P3 generation adipose-derived mesenchymal stem cells harvested in example 1 and comparative examples 1 to 4, decomposing, adding a purified culture medium, blowing into a cell suspension, and adjusting the density of the cell suspension to 1 × 105each/mL, inoculated in 12-well plates, incubated at 37 ℃ with 5% CO2The culture was continued for 7 days in the incubator of (1), and 3 wells were stained with trypan blue and the cells were counted, and the results of the average measurement are shown in Table 3.
TABLE 3
Group of
Number of cells (. times.10)5One)
Example 1
26.24
Comparative example 1
22.51
Comparative example 2
17.48
Comparative example 3
19.65
Comparative example 4
15.49
As can be seen from table 4, the number of harvested cells was the largest in example 1 after the same culture process, indicating that the proliferation activity of the adipose tissue-derived mesenchymal stem cells was the best in example 1.
Detecting the osteogenesis capacity of the adipose-derived mesenchymal stem cells: the P3 generation cells harvested in example 1, comparative examples 1 to 4 were seeded in osteogenic induction medium (DMEM/F12 medium + 10% FBS + dexamethasone 80nM + insulin 2nM + glycerol phosphate 10 mM) in 12-well plates at a cell density of 1X 105one/mL, 5% CO at 37 ℃2The culture box is used for induction culture, the total amount of liquid is changed every 3 days in the culture process, the culture medium is removed after the culture is carried out for 14 days, after the cleaning of PBS, 4% paraformaldehyde is used for fixing at 4 ℃ overnight, the paraformaldehyde is removed, the PBS is used for cleaning for 2 times, 0.5% alizarin red is used for dyeing for 10min, the dyeing solution is removed, the washing is carried out for 3 times, the dyeing condition is observed by a microscope, the percentage of calcified area is counted, and the result is shown in Table 4.
TABLE 4
Group of
Area of calcification
Example 1
42.17%
Comparative example 1
38.41%
Comparative example 2
30.77%
Comparative example 3
32.59%
Comparative example 4
28.84%
As can be seen from table 4, the adipose-derived mesenchymal stem cells of example 1 had better osteogenic ability, and the cells of comparative examples 1 to 4 had a different degree of reduction in osteogenic ability than those of example 1.
In conclusion, the adipose-derived mesenchymal stem cells in the cell bank have the advantages of good activity, high purity, strong proliferation capacity and differentiation potential, and can fully meet various scientific researches and clinical applications of the adipose-derived mesenchymal stem cells.
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.