CN114891739A - Optimization method for separating and culturing pig bone marrow mesenchymal stem cells - Google Patents
Optimization method for separating and culturing pig bone marrow mesenchymal stem cells Download PDFInfo
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Abstract
The invention belongs to the technical field of cell culture, and discloses an optimization method for separating and culturing porcine mesenchymal stem cells, which comprises the steps of flushing the outside of a leg bone by PBS (phosphate buffer solution), and collecting bone marrow by using a whole bone marrow stem cell culture solution; transferring the washed whole bone marrow stem cell culture solution into a centrifuge tube for centrifugation; adding erythrocyte lysate, blowing, and standing; centrifuging the liquid after standing, and collecting a cell layer; the culture medium was added to the cell pellet, half of the medium was changed every three days, and the cells were passaged on day 9. The BMSCs are separated by adopting optimization operation, and the erythrocyte lysate is added into the marrow fluid, so that the erythrocytes accounting for a large proportion in the marrow fluid are crushed and lysed, and the cell purity is improved; the method of half-changing the liquid is adopted, and more cells are retained by means of a small amount of times. The optimized cell separation and culture method can improve the separation efficiency and purity of the BMSCs, avoid the influence on the activity of the BMSCs, and has simple and convenient operation and low cost.
Description
Technical Field
The invention belongs to the technical field of cell culture, and particularly relates to an optimization method for separating and culturing porcine bone marrow mesenchymal stem cells.
Background
At present, Mesenchymal Stem Cells (MSCs) can be isolated from various tissue sources, including bone marrow, fat, placenta, connective tissue, etc., wherein MSCs derived from bone marrow are called bone marrow-derived mesenchymal stem cells (BMSCs), have the characteristics of immunoregulatory function, capability of differentiating into various cells, high proliferation rate, and broad research and application prospects. The traditional whole bone marrow culture method utilizes that BMSCs have good adhesive capacity under the condition of low serum, and removes non-attached hybrid cells when replacing culture solution so as to realize the separation and purification of the BMSCs; percoll and Ficoll concentration gradient centrifugation are used for separating BMSCs according to different concentrations from other components; and according to cell surface markers, BMSCs are sorted by a flow cytometer, and high-purity BMSCs can be obtained by sorting by the flow cytometer, but the BMSCs have large influence on the vitality and differentiation capacity of cells, have large requirements on bone marrow and are harsh in experimental conditions. Because the content of BMSCs in bone marrow is low and the cell proportion is small, in order to ensure that the separated BMSCs have better activity and higher proliferation capacity, other cells are usually mixed in the BMSCs by adopting a traditional separation method, so that the separation purity of the BMSCs is not high, and the subsequent differentiation capacity of the BMSCs is also influenced.
Through the above analysis, the problems and defects of the prior art are as follows: in the traditional method for separating the bone marrow mesenchymal stem cells, other cells are usually mixed in the BMSCs, and the separation effect is not ideal.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides an optimization method for separating and culturing porcine bone marrow mesenchymal stem cells.
The invention is realized in this way, a pig bone marrow mesenchymal stem cell isolated culture optimization method, the pig bone marrow mesenchymal stem cell isolated culture optimization method includes:
adding a certain amount of erythrocyte lysate into the collected marrow fluid to promote erythrocyte lysis, adopting a semi-fluid-changing method, utilizing the characteristic that BMSCs have strong adhesiveness to a plastic culture dish in a low serum culture medium, gradually removing non-adherent hybrid cells in the fluid-changing process, and purifying the cells through passage.
Further, the optimization method for the separation culture of the pig bone marrow mesenchymal stem cells comprises the following steps:
step one, washing the outside of leg bones by PBS, and collecting bone marrow by using a whole bone marrow stem cell culture solution;
step two, transferring the washed whole bone marrow stem cell culture solution into a centrifuge tube for centrifugation;
adding erythrocyte lysate, blowing, and standing;
step four, centrifuging the liquid after standing in the step three, and collecting a cell layer;
step five, adding culture solution into the cell sediment, and replacing half of the culture medium every three days;
step six, cells were passaged on day 9.
Further, the collecting bone marrow in the first step comprises:
the thigh bone of the piglet is obtained, the outer part of the thigh bone is washed for multiple times by PBS containing 3 percent and 1 percent of streptomycin, the brittle bones at the two ends of the thigh bone of the piglet are cut off by an autoclaved scalpel in a sterile ultra-clean bench to expose red bone marrow, and the two ends of the bone marrow are punctured by a 20mL sterile injector head.
Further, the collecting bone marrow in the first step further comprises:
the outer portion of the perforated pig leg bone was gently washed clean with 10mL of 1% streptomycin-containing PBS, and the perforated pig leg bone was placed in a new 10cm petri dish.
Further, the collecting bone marrow in the first step further comprises:
5mL of the whole bone marrow stem cell culture solution was extracted with a 1mL disposable sterile syringe, and the inside of the bone marrow was washed from one end of the leg bone to the other end, and the bone marrow was repeatedly washed 3 to 5 times.
Further, the centrifugation in the second step includes:
and transferring the washed whole bone marrow stem cell culture solution into a 15mL centrifuge tube, centrifuging for 5min at 1200r/min, and removing supernatant to obtain cell sediment.
Further, the erythrocyte lysate in the third step comprises:
and (4) adding the erythrocyte lysate with the volume 5 times that of the cell sediment in the step two, slightly blowing the cell sediment, and standing for 5 min.
Further, the centrifugation in step four comprises:
centrifuging the liquid after standing at the rotating speed of 1200r/min for 5min, and removing the supernatant to obtain cell sediment.
Further, in the fifth step, 10mL of stem cell culture solution is added into the cell sediment in the fourth step, the blown cell suspension is paved on a T75 cell culture bottle, and half liquid change is performed on the cells every 3 days.
Further, in step six, the passage is performed when the cell morphology of the BMSCs is significantly different from other types of cells at day 9, and the cell passage comprises:
sucking out all culture solution, adding preheated PBS, gently cleaning once, discarding the PBS, adding 2mL of pancreatin, and digesting for 1-3 min; placing the culture bottle under a microscope for observation, and adding 6-9 mL of culture medium containing serum or 1mL of serum to stop digestion when the cells become round and float; blowing and beating the cells by using a Pasteur pipette to enable the cells to fall off from the wall of the culture bottle, collecting liquid to a 15mL centrifuge tube, centrifuging for 5min at 1200r/min, and discarding supernatant; 10mL of stem cell culture medium was added to the cell pellet and the cell suspension was plated on a 10cm cell culture dish.
In combination with the technical solutions and the technical problems to be solved, please analyze the advantages and positive effects of the technical solutions to be protected in the present invention from the following aspects:
first, aiming at the technical problems existing in the prior art and the difficulty in solving the problems, the technical problems to be solved by the technical scheme of the present invention are closely combined with results, data and the like in the research and development process, and some creative technical effects are brought after the problems are solved. The specific description is as follows:
the mesenchymal stem cells with higher purity are separated from the bone marrow of the pig leg bone, and the better activity and the proliferation of the cells are ensured. Because mesenchymal stem cells in bone marrow occupy less, and meanwhile, the bone marrow contains a large amount of red blood cells and other types of mixed cells, BMSCs obtained by the traditional separation method have low purity, and the cells are generally sorted by a flow cytometer, which needs certain technical requirements and has high cost. Therefore, the BMSCs with higher purity can be separated simply and efficiently through the optimized experimental steps provided by the invention, and can be proliferated in large quantity, so that the subsequent other researches can be met; firstly, the step of cracking the red blood cells in the marrow fluid by adding the red blood cell lysate can quickly remove a large amount of red blood cells and improve the purity of the separated BMSCs; secondly, half liquid changing is carried out every three days after the cells are plated, instead of changing all the culture liquid once, so that BMSCs can be kept as much as possible while injecting the fresh culture liquid; thirdly, on the 9 th day after plating, the BMSCs are passaged after the cell morphology is displayed, and then the hybrid cells can be further removed, and the BMSCs can be purified.
The bone marrow fluid in the leg bone contains a large amount of red blood cells, which influences the separation of BMSCs, and a certain amount of red blood cell lysate is added into the collected bone marrow fluid to promote the red blood cell lysis, so that the separation efficiency of the BMSCs is improved; and then, by utilizing the characteristic that BMSCs have stronger adhesion to a plastic culture dish in a low-serum culture medium, non-adherent hybrid cells are gradually removed in the process of changing the liquid, so that the purpose of separating and purifying the BMSCs is achieved, and the BMSCs are simple to operate and are not easy to pollute. By adopting the semi-liquid changing method provided by the invention, more BMSCs can be reserved to a certain extent, so that the loss of a plurality of cells which are not adhered to the wall due to the fact that the culture solution is changed once is avoided.
The pig bone marrow mesenchymal stem cell separation culture optimization method provided by the invention also has the following advantages:
the PBS containing the streptomycin is used for washing the outside for multiple times, so that the sterility is ensured, and the influence of external pollution on the subsequent cell culture is eliminated. Washing bone marrow with a whole bone marrow stem cell culture solution to ensure that BMSCs to be separated are in a culture solution suitable for growth at the beginning;
obtaining bright red cell sediment (containing a large amount of red blood cells) after first centrifugation, and removing the supernatant to remove impurities in the supernatant;
thirdly, red blood cell lysate is added to lyse red blood cells in the cell sediment of the previous step, the purity of BMSCs separation is improved, white cell sediment is obtained after centrifugation, and the third step can be repeated if red cells exist;
retaining more BMSCs by adopting a semi-liquid changing mode, wherein the cell morphology of the BMSCs is gradually shown in the process and is obviously different from other types of cells;
fifthly, after the cell morphology of the BMSCs is displayed, the cells are further purified by means of passage, and the BMSCs are proliferated.
Secondly, considering the technical scheme as a whole or from the perspective of products, the technical effect and advantages of the technical scheme to be protected by the invention are specifically described as follows:
the BMSCs are separated by adopting optimization operation, and the erythrocyte lysate is added into the marrow fluid, so that the erythrocytes accounting for a large proportion in the marrow fluid are crushed and lysed, and the purity of the cells can be improved; on the other hand, a semi-liquid changing method is adopted, and more cells are reserved by means of a small amount of times; the optimized method can improve the separation efficiency and purity of the BMSCs, avoid the influence on the activity of the BMSCs, and has simple and convenient operation and low cost.
Third, as an inventive supplementary proof of the claims of the present invention, there are also presented several important aspects:
(1) the technical scheme of the invention fills the technical blank in the industry at home and abroad:
there are many methods for separating BMSCs, but few methods are used in combination. The method for separating the BMSCs of the pigs comprises the steps of firstly adding an erythrocyte lysate to remove erythrocytes in bone marrow fluid, then further removing mixed cells by the adherence characteristic of the BMSCs, retaining the BMSCs as much as possible by using a half-liquid-changing method, and finally purifying the cells by passage and enabling the BMSCs to proliferate. The traditional single method is adopted, the separation effect of the BMSCs is not ideal, the method provided by the invention integrates multiple methods, optimizes detailed operation, and can more simply and conveniently separate BMSCs with higher purity and better activity from bone marrow.
(2) The technical scheme of the invention overcomes the technical prejudice whether:
in the traditional method, BMSCs are separated only by using density gradient centrifugation, so that the impurity cells including red blood cells cannot be well removed, and it is thought that the activity of other cells may be influenced while red blood cells are lysed by a red blood cell lysate. The optimized operation of the invention increases the use of erythrocyte lysate, improves the separation purity of BMSCs, and simultaneously, the separated BMSCs have better activity, can proliferate in large quantity and can also carry out subsequent adipogenic induced differentiation experiments.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a flow chart of a method for optimizing the isolation and culture of mesenchymal stem cells of pig bone marrow provided by an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides an optimization method for the isolation and culture of the porcine mesenchymal stem cells, and the invention is described in detail with reference to the attached drawings.
First, an embodiment is explained. This section is an explanatory embodiment expanding on the claims so as to fully understand how the present invention is embodied by those skilled in the art.
As shown in fig. 1, the optimization method for isolation and culture of the pig bone marrow mesenchymal stem cells provided by the embodiment of the present invention includes the following steps:
s101, washing the outer part of a leg bone by PBS, and collecting bone marrow by using a whole bone marrow stem cell culture solution;
s102, transferring the washed whole bone marrow stem cell culture solution into a centrifuge tube for centrifugation;
s103, adding erythrocyte lysate, blowing, and standing;
s104, centrifuging the liquid after standing in the S103, and collecting a cell layer;
s105, adding a culture solution into the cell sediment, and replacing half of the culture medium every three days;
s106, cells were passaged on day 9.
The bone marrow collection in step S101 provided in the embodiment of the present invention includes: obtaining the thigh bone of a piglet, flushing the outside of the thigh bone by PBS (phosphate buffer solution) containing streptomycin, cutting the brittle bones at the two ends of the thigh bone of the piglet by a scalpel subjected to autoclaving in an aseptic ultra-clean bench to expose red bone marrow, and puncturing the two ends of the bone marrow by a 20mL aseptic injector head; washing the outer part of the perforated pig leg bone with 10mL of PBS containing 1% streptomycin, gently washing the outer part of the perforated pig leg bone, and placing the perforated pig leg bone in a new 10cm culture dish; 5mL of the whole bone marrow stem cell culture solution was extracted with a 1mL disposable sterile syringe, and the inside of the bone marrow was washed from one end of the leg bone to the other end, and the bone marrow was repeatedly washed 3 to 5 times.
The centrifugation in step S102 provided in the embodiment of the present invention includes: and transferring the washed whole bone marrow stem cell culture solution into a 15mL centrifuge tube, centrifuging for 5min at 1200r/min, and removing supernatant to obtain cell sediment.
The erythrocyte lysate added in step S103 provided by the embodiment of the present invention includes: adding erythrocyte lysate with 5 times volume of cell precipitate, gently blowing, and standing for 5 min.
The centrifugation in step S104 provided in the embodiment of the present invention includes: centrifuging the liquid after standing at the rotating speed of 1200r/min for 5min, and removing the supernatant to obtain cell sediment.
In step S105 provided in the present invention, 10mL of stem cell culture solution is added to the cell pellet, the cell suspension is spread in a T75 cell culture flask, and half-replacement of the cell is performed every 3 days.
And II, application embodiment. In order to prove the creativity and the technical value of the technical scheme of the invention, the part is the application example of the technical scheme of the claims on specific products or related technologies.
BMSCs of a one-month-old female Bama pig are separated by using the technical scheme of the invention, when the separated cells are just plated (marked as day 0), the cells are in a single-cell suspension state under an optical microscope, half liquid change is carried out every three days, a plurality of floating cells exist in a culture solution, the cells are mostly circular, fibrous cells appear when the cells are cultured to day 7, the confluence degree of the fibrous cells reaches about 80% when the cells are cultured at day 9, and subculture is carried out at the moment. After passage, the round cells are obviously reduced, and the fibrous cells are increased along with the prolonging of the culture time, mostly distributed in an arrangement shape, and sometimes in a vortex shape.
Isolated BMSCs from bama pigs were analyzed for surface antigens using flow cytometry: upon incubation of antibody CD44, a total of 81.7% of the cells in BMSCs were detected to express CD 44; upon incubation of antibody CD90, 88.3% of the total cells in BMSCs were detected to express CD 90; upon incubation of antibody CD105, a total of 89.3% of the cells in BMSCs were detected to express CD 105. In conclusion, the purity of the BMSCs of the Bama pigs separated and purified by the method is about 86.4 percent.
After BMSCs are full of the cells, adipogenic induction culture is carried out, lipid droplets can be observed in the cells on the 1 st to 2 nd days of induction, and the lipid droplets are continuously enlarged and become round along with the increase of the induction time. The cell lipid drops after induced differentiation can be observed to be obvious through oil red O staining, and the expression level of the adipogenic marker genes of the cells after induced differentiation is detected through real-time fluorescent quantitative PCR (qPCR), so that the expression level of the genes is found to be remarkably higher than that of the cells without induced differentiation. Therefore, the BMSCs cells of the Bama pigs separated and purified by the method have better activity and can be used for a adipogenesis induction experiment.
The technical scheme of the invention can simply, conveniently and efficiently separate the pig BMSCs, has low cost, and the separated cells have good activity and can be used for other subsequent experiments.
And thirdly, evidence of relevant effects of the embodiment. The embodiment of the invention achieves some positive effects in the process of research and development or use, and has great advantages compared with the prior art, and the following contents are described by combining data, diagrams and the like in the test process.
1. Principle of experiment
Marrow fluid in leg bones contains a large amount of red blood cells, which influences the separation of BMSCs, and a certain amount of red blood cell lysate is added into the collected marrow fluid to promote the lysis of the red blood cells, thereby improving the separation efficiency of the BMSCs. And then, by utilizing the characteristic that BMSCs have stronger adhesion to a plastic culture dish in a low-serum culture medium, non-adherent hybrid cells are gradually removed in the process of changing the liquid, so that the purpose of separating and purifying the BMSCs is achieved, and the BMSCs are simple to operate and are not easy to pollute. By adopting a half-liquid-changing method, more BMSCs can be reserved to a certain extent, so that the loss of a plurality of cells which are not adhered to the wall due to the replacement of the culture solution only once is avoided.
2. Experimental procedure (see Table 1)
TABLE 1 Experimental procedure
3. The concrete steps of the experiment
(1) The femoral bone of the piglet is obtained, PBS containing 3 percent and 1 percent of streptomycin is used for washing the outer part of the femoral bone for multiple times, the brittle bones at the two ends of the femoral bone are cut off to expose red bone marrow by an autoclaved scalpel in a sterile ultra-clean bench, the two ends of the bone marrow are punctured by a 20mL sterile injector head, then 10mL PBS containing 1 percent of streptomycin is used for washing the outer part of the punctured femoral bone to be cleaned lightly, and the punctured femoral bone is placed in a new 10cm culture dish.
(3) 5mL of the whole bone marrow stem cell culture solution was extracted with a 1mL disposable sterile syringe, and the inside of the bone marrow was washed from one end of the leg bone to the other end, and the bone marrow was repeatedly washed 3 to 5 times.
(4) And transferring the washed whole bone marrow stem cell culture solution into a 15mL centrifuge tube, centrifuging at 1200r/min for 5min, and removing the supernatant.
(5) Adding erythrocyte lysate with 5 times volume of precipitate, slightly blowing and beating the cell precipitate, and standing for 5 min.
(6) The liquid after standing is centrifuged for 5min at the rotating speed of 1200r/min, and the supernatant is discarded.
(7) 10mL of stem cell culture medium was added to the cell pellet, and the blown cell suspension was plated in a T75 cell culture flask.
(8) Half-changes were made every 3 d.
(9) Cell morphology of day 9 BMSCs was clearly distinct from other cell types, passaged: sucking out all culture solution, adding preheated PBS for gentle washing once, discarding the PBS, adding 2mL of pancreatin for digestion for 1-3 min, placing the culture bottle under a microscope for observation, adding 6-9 mL of serum-containing culture medium (or 1mL of serum) when the cells become round and float to stop digestion, blowing and beating the cells from the wall of the culture bottle by using a Pasteur pipette, collecting the liquid to a 15mL centrifuge tube, centrifuging at 1200r/min for 5min, discarding the supernatant, adding 10mL of stem cell culture solution into cell sediment, and paving the cell suspension in a 10cm cell culture dish.
4. Experimental equipment and reagent
(1) The main experimental equipment: scalpel, sterile syringe, 10cm culture dish, beaker, T75 cell culture bottle, normal temperature centrifuge, 15mL centrifuge tube, CO 2 An incubator;
(2) the main experimental reagents are as follows: penicillin streptomycin, PBS, whole bone marrow stem cell culture solution and erythrocyte lysate.
The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. An optimization method for separating and culturing porcine mesenchymal stem cells is characterized by comprising the following steps:
adding a certain amount of erythrocyte lysate into the collected marrow fluid to promote erythrocyte lysis, adopting a semi-fluid-changing method, utilizing the characteristic that BMSCs have strong adhesiveness to a plastic culture dish in a low serum culture medium, gradually removing non-adherent hybrid cells in the fluid-changing process, and purifying the cells through passage.
2. The method for optimizing isolated culture of porcine mesenchymal stem cells according to claim 1, wherein the method for optimizing isolated culture of porcine mesenchymal stem cells comprises the following steps:
step one, washing the outside of leg bones by PBS, and collecting bone marrow by using a whole bone marrow stem cell culture solution;
step two, transferring the washed whole bone marrow stem cell culture solution into a centrifuge tube for centrifugation;
adding erythrocyte lysate, blowing, and standing;
step four, centrifuging the liquid after standing in the step three, and collecting a cell layer;
step five, adding culture solution into the cell sediment, and replacing half of the culture medium every three days;
step six, cells were passaged on day 9.
3. The method for optimizing the isolated culture of the porcine bone marrow mesenchymal stem cells according to claim 2, wherein the collecting bone marrow in the first step comprises:
the thigh bone of the piglet is obtained, PBS containing 3% and 1% streptomycin is used for washing the outer part of the thigh bone for multiple times, the brittle bones at the two ends of the pig thigh bone are cut off to expose red bone marrow in a sterile super clean bench by using an autoclaved scalpel, and the two ends of the bone marrow are punctured by using a 20mL sterile injector head.
4. The method for optimizing isolated culture of porcine bone marrow mesenchymal stem cells according to claim 2, wherein the collecting bone marrow in the first step further comprises:
the outer portion of the perforated pig leg bone was gently washed clean with 10mL of 1% streptomycin-containing PBS, and the perforated pig leg bone was placed in a new 10cm petri dish.
5. The method for optimizing isolated culture of porcine bone marrow mesenchymal stem cells according to claim 2, wherein the collecting bone marrow in the first step further comprises:
5mL of the whole bone marrow stem cell culture solution was extracted with a 1mL disposable sterile syringe, and the inside of the bone marrow was washed from one end of the leg bone to the other end, and the bone marrow was repeatedly washed 3 to 5 times.
6. The method for optimizing isolation and culture of porcine bone marrow mesenchymal stem cells according to claim 2, wherein the centrifugation in the second step comprises:
and transferring the washed whole bone marrow stem cell culture solution into a 15mL centrifuge tube, centrifuging for 5min at 1200r/min, and removing supernatant to obtain cell sediment.
7. The method for optimizing the isolated culture of the porcine bone marrow mesenchymal stem cells according to claim 2, wherein the erythrocyte lysate in the third step comprises:
and (4) adding the erythrocyte lysate with the volume 5 times that of the cell sediment in the step two, slightly blowing the cell sediment, and standing for 5 min.
8. The method for optimizing the isolated culture of porcine bone marrow mesenchymal stem cells according to claim 2, wherein the centrifugation in the fourth step comprises:
centrifuging the liquid after standing at the rotating speed of 1200r/min for 5min, and removing the supernatant to obtain cell sediment.
9. The method for optimizing isolation and culture of the porcine bone marrow mesenchymal stem cells according to claim 2, wherein in the fifth step, 10mL of the stem cell culture solution is added into the cell sediment in the fourth step, the blown cell suspension is spread in a T75 cell culture flask, and half liquid change is performed on the cells every 3 days.
10. The method for optimizing isolation and culture of porcine bone marrow mesenchymal stem cells according to claim 2, wherein in the sixth step, the cell morphology of the BMSCs at day 9 is significantly different from that of other types of cells by carrying out the passage, and the passage comprises:
sucking out all culture solution, adding preheated PBS, gently cleaning once, discarding the PBS, adding 2mL of pancreatin, and digesting for 1-3 min; placing the culture bottle under a microscope for observation, and adding 6-9 mL of culture medium containing serum or 1mL of serum to stop digestion when the cells become round and float; blowing and beating the cells by using a Pasteur pipette to enable the cells to fall off from the wall of the culture bottle, collecting liquid to a 15mL centrifuge tube, centrifuging for 5min at 1200r/min, and discarding supernatant; 10mL of stem cell culture medium was added to the cell pellet and the cell suspension was plated on a 10cm cell culture dish.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102807966A (en) * | 2012-08-16 | 2012-12-05 | 博雅干细胞科技有限公司 | Method for freezing and thawing placental whole cells and separating and expanding stem cells |
| CN109456944A (en) * | 2018-11-02 | 2019-03-12 | 南京大学 | The mesenchymal stem cell and its preparation method and application of dual-gene modification |
| CN112458047A (en) * | 2020-11-30 | 2021-03-09 | 博雅干细胞科技有限公司 | Method for separating placenta mesenchymal stem cells and serum-free culture medium used by same |
| CN114939128A (en) * | 2022-06-02 | 2022-08-26 | 尧舜泽生物医药(南京)有限公司 | Application of bone marrow mesenchymal stem cell exosome in repairing articular cartilage defect |
-
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102807966A (en) * | 2012-08-16 | 2012-12-05 | 博雅干细胞科技有限公司 | Method for freezing and thawing placental whole cells and separating and expanding stem cells |
| CN109456944A (en) * | 2018-11-02 | 2019-03-12 | 南京大学 | The mesenchymal stem cell and its preparation method and application of dual-gene modification |
| CN112458047A (en) * | 2020-11-30 | 2021-03-09 | 博雅干细胞科技有限公司 | Method for separating placenta mesenchymal stem cells and serum-free culture medium used by same |
| CN114939128A (en) * | 2022-06-02 | 2022-08-26 | 尧舜泽生物医药(南京)有限公司 | Application of bone marrow mesenchymal stem cell exosome in repairing articular cartilage defect |
Non-Patent Citations (1)
| Title |
|---|
| 蔡金宏;林春博;杨渊;: "间充质干细胞分离方法的研究与进展" * |
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