CN111979188A - Placenta mesenchymal stem cell isolation culture amplification method - Google Patents
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Abstract
The invention discloses a separation, culture and amplification method of placenta mesenchymal stem cells, which comprises the steps of 1) treatment, 2) cleaning, 3) culture and 4) inoculation and passage.
Description
Technical Field
The invention relates to the technical field of stem cell isolated culture, in particular to a placenta mesenchymal stem cell isolated culture amplification method.
Background
Mesenchymal stem cells, MSCs) are important members of the stem cell family, originating from early-developing mesoderm and ectoderm, and were first found in bone marrow, and MSCs are a group of adult stem cells with high self-renewal capacity and multipotentiality. Umbilical cord, amnion, amniotic fluid, placenta and the like are used as wastes after fetal delivery, and compared with bone marrow mesenchymal stem cells, tissues derived from the placenta have wide sources, are not limited by theory, and have the advantage of easy amplification culture, and more attention is paid. With the continuous and deep research on mesenchymal stem cells such as umbilical cord, amnion, amniotic fluid and the like, the method has the advantages of immunoregulation, cell factor secretion, convenient material taking and the like, shows wider and wider application prospects in the fields of autoimmune diseases, regenerative medicine and the like, and becomes an ideal seed cell for cell therapy. At present, the adult stem cell which is the most clinically applied and researched after hematopoietic stem cells and is the most mature adult stem cell
The MSCs in the placenta tissue are mainly distributed in tissues derived from fetuses, such as placenta, chorion and the like, have strong plasticity and multidirectional differentiation potential, and can differentiate to three germ layers under different induction culture conditions. The placenta mesenchymal stem cells have the functions of hematopoietic support, low immunogenicity and immunosuppression, and can secrete various cytokines to repair damaged cells, so the placenta mesenchymal stem cells are ideal cells for stem cell treatment, tissue repair and regenerative medicine at present. The method has important significance for the exploration of the placenta mesenchymal stem cell separation culture method and the research on the cell biological characteristics thereof, and can lay a foundation for the future cell transplantation application. At present, two methods are generally adopted for separating the placenta mesenchymal stem cells, namely a direct adherence method of tissue blocks and enzyme digestion.
The economic cost of enzyme digestion separation is high, the digestion time is long, generally 4-6 hours are needed, the longest digestion time is 16 hours, the size of a tissue block can influence the digestion effect of cells, if the digestion is insufficient, a sufficient number of cells cannot be obtained, and the digestion time is too long, the damage of digestive enzymes to the cells is too large, and a large number of cells die. The tissue block culture method primary culture scheme is simple and easy to operate, and has obvious advantages in the aspects of shortening the operation time, reducing the cost, and being high in cell yield.
Disclosure of Invention
The invention provides a separation, culture and amplification method of placenta mesenchymal stem cells.
The scheme of the invention is as follows:
a separation culture and amplification method of placenta mesenchymal stem cells comprises the following steps:
1) cleaning up
After the fetus is delivered, obtaining placenta tissues under an aseptic condition, washing the surface of the placenta by using a cleaning solution, and cleaning and removing residual blood and other impurities;
2) treatment of
Gently peeling amnion from the washed placenta tissue, removing blood vessel tissue, repeatedly washing the placenta tissue in a buffer solution containing 100U/ml penicillin and 100ug/ml streptomycin to remove blood clots, and shearing the placenta tissue into pieces with the size of 1-3 cm by using an aseptic tissue shear3。
3) Culturing
Paving the cut placenta tissue blocks in a cell culture dish, and adding the placenta tissue blocks into a culture solution containing 10-20% fetal calf serum for culture after water is volatilized; the operation is characterized in that the tissue block is small, the tissue adherence is firm, the mesenchymal stem cells can climb out of the tissue block, and a large amount of primary cells can be obtained;
4) passage of inoculation
After the growth of the placenta primary separated mesenchymal stem cells reaches 80-90% and is fused, subculture can be carried out to obtain placenta mesenchymal stem cells, the cells are digested by 0.25% pancreatin/EDTA (ethylene diamine tetraacetic acid) according to 4000-8000 cells/cm2And (3) inoculating, wherein the placenta mesenchymal stem cells can reach 95% fusion 4-5 days after inoculation, and the placenta mesenchymal stem cells fused by more than 95% can be amplified to more than 20 generations. The digestion time is short, the damage to cells is small, the inoculation density can well control the growth cycle of the cells, and the placenta mesenchymal stem cells after passage according to the inoculation density can be amplified for more than 20 generations.
As a preferred technical scheme, the buffer solution for washing in the step 2) is phosphate buffer solution or normal saline containing 100U/ml of penicillin and 100ug/ml of streptomycin.
As a preferable technical scheme, the culture solution of 10-20% fetal calf serum in the step 3) contains the alpha-MEM basic culture solution containing nucleoside and the fetal calf serum, which is more beneficial to the separation and proliferation of stem cells, and can better maintain the dryness and biological functions of the stem cells in the long-term amplification culture process.
As an optimal technical scheme, in the step 3), the culture solution of 10-20% fetal calf serum is replaced once every 3 days after cell culture in the cell culture dish, then the culture solution of 10-20% fetal calf serum is replaced every 2-3 days after wall adhesion, the placenta mesenchymal stem cells climb out of the tissue block after 4-5 days, and the placenta mesenchymal stem cells can reach 80-90% fusion after 7-10 days.
Preferably, the culture solution containing 10-20% fetal calf serum is added in the step 3) in a volume of 20-50 ml/dish.
As an improvement of the technical scheme, the method comprises the step of performing quality evaluation on the mesenchymal stem cells obtained in the step 4), wherein the quality evaluation comprises cell number, cell activity, cell surface markers, differentiation function, bacterial detection, mycoplasma detection and safety evaluation.
The production specification, the quality control system and the evaluation system of the placenta mesenchymal stem cells established by the method can quickly, simply and conveniently obtain the mesenchymal stem cells for clinical research and application.
Due to the adoption of the technical scheme, the placenta mesenchymal stem cell separation culture amplification method comprises the steps of 1) cleaning, obtaining placenta tissues under an aseptic condition, washing the surface of the placenta with a cleaning solution, and cleaning to remove residual blood and other impurities; 2) treating, gently peeling amnion, washing the peeled placenta in PBS containing double antibiotics (100U/ml penicillin, 100ug/ml streptomycin) to remove blood clot, and cutting into 1cm pieces with aseptic tissue scissors3Left and right size; 3) culturing, namely flatly paving the placenta tissue block in a cell culture dish, and adding the placenta tissue block into a culture solution containing 10-20% fetal calf serum for culturing after water is volatilized; 4) inoculating and subculturing, and performing primary separation culture of mesenchymal stem cells by using the placenta tissue massAfter 80-90% of fusion, the cells can be subcultured to obtain placenta mesenchymal stem cells, and the cells are digested by 0.25% pancreatin/EDTA (ethylene diamine tetraacetic acid) according to the ratio of 4000-8000 cells/cm2Inoculating, wherein the placenta mesenchymal stem cells can reach 95% fusion 4-5 days after inoculation, and the placenta mesenchymal stem cells fused by more than 95% can be amplified to more than 20 generations; the method for obtaining the placenta mesenchymal stem cells by the tissue mass separation method is simple, low in cost, good in cell activity and high in purity, the concentration of the finally obtained cells is high, the obtained mesenchymal stem cells are simple, the culture time is shortened, the cells can be seen to climb out of the tissue mass after being cultured for 4-5 days, the concentration of the finally obtained cells is high, the whole stem cell separation, preparation and culture process can quickly and efficiently obtain the high-purity mesenchymal stem cells from placenta tissues, the cell quality completely meets the national promulgated stem cell preparation quality control and preclinical research guide principle (trial), and the guarantee is provided for clinical application; the invention is characterized in that the placenta tissue is obtained, the amnion is cleaned and slightly stripped, the vascular tissue is removed, and the size of the tissue block is controlled to be 1cm3The cell culture dish is adopted for spreading, and the solution is replenished after the water is completely volatilized, so that the yield of the primary cells is obviously improved.
Drawings
FIG. 1 is the dissected placenta of example 3;
FIG. 2 placental tissue mass with tissue minced and spread
Fig. 3 is a map of placental mesenchymal stem cells of example 3 crawled out of a tissue mass;
fig. 4 is a flow assay of placental mesenchymal stem cells of example 3;
fig. 5 is a graph of identification of multipotentiality of placental mesenchymal stem cells according to example 4;
Detailed Description
In order to make up for the above disadvantages, the present invention provides a method for separating, culturing and amplifying placental mesenchymal stem cells to solve the above problems in the background art.
A separation culture and amplification method of placenta mesenchymal stem cells comprises the following steps:
1) cleaning up
After the fetus is delivered, obtaining placenta tissues under an aseptic condition, washing the surface of the placenta by using a cleaning solution, and cleaning and removing residual blood and other impurities;
2) treatment of
And (3) slightly stripping the amnion from the washed placenta tissue, and removing the blood vessel tissue. Repeatedly washing placenta in a buffer solution containing 100U/ml penicillin and 100ug/ml streptomycin to remove blood clots, and shearing placenta tissues into pieces with the size of 1-3 cm by using an aseptic tissue shear3。
3) Culturing
Spreading the minced placenta tissue blocks in a cell culture dish, and adding the placenta tissue blocks into a culture solution containing 10-20% fetal calf serum for culture after water is volatilized; the operation is characterized in that the tissue block is small, the tissue adherence is firm, the mesenchymal stem cells can climb out of the tissue block, and a large amount of primary cells can be obtained;
4) passage of inoculation
After the growth of the primary separated mesenchymal stem cells of the placenta tissue mass reaches 80-90% and is fused, subculture can be carried out to obtain the placenta mesenchymal stem cells, the cells are digested by 0.255% pancreatin/EDTA according to 4000-8000 cells/cm2And (3) inoculating, wherein the placenta mesenchymal stem cells can reach 95% fusion 4-5 days after inoculation, and the placenta mesenchymal stem cells fused by more than 95% can be amplified to more than 20 generations. The digestion time is short, the damage to cells is small, the inoculation density can well control the growth cycle of the cells, and the chorionic mesenchymal stem cells can be amplified for more than 20 generations according to the digestion of the cells after the inoculation density passage.
The buffer solution for washing in the step 2) is phosphate buffer solution or normal saline containing 100U/ml of double-antibody penicillin and 100ug/ml of streptomycin.
The culture solution of 10-20% fetal calf serum in the step 3) contains alpha-MEM basic culture solution containing nucleoside and fetal calf serum, which is more beneficial to separation and proliferation of stem cells, and can better maintain the dryness and biological functions of the stem cells in the long-term amplification culture process.
And 3) changing a culture solution of 10-20% fetal calf serum once every 3 days of cell culture in the cell culture dish in the step 3), then changing the culture solution of 10-20% fetal calf serum every 2-3 days of interval, allowing the chorion mesenchymal stem cells to climb out of the tissue block after adhering to the wall for 4-5 days, and allowing the placental mesenchymal stem cells to reach 80-90% fusion after 7-10 days.
And 3) adding 20-50 ml/dish of culture solution containing 10-20% fetal calf serum.
Further comprising the step of performing quality evaluation on the mesenchymal stem cells obtained in the step 4), wherein the quality evaluation comprises cell number, cell activity, cell surface markers, differentiation function, bacterial detection, mycoplasma detection and safety evaluation.
The production specification, the quality control system and the evaluation system of the placenta mesenchymal stem cells established by the method can quickly, simply and conveniently obtain the mesenchymal stem cells for clinical research and application.
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.
Example 1:
1) cleaning, namely obtaining placenta tissues under an aseptic condition after delivery of the fetus, washing the surface of the placenta by using a cleaning solution, and cleaning and removing residual blood and other impurities;
2) processing, gently peeling amnion from the washed placenta tissue, removing blood vessel tissue, repeatedly washing placenta in PBS containing 100U/ml penicillin and 100ug/ml streptomycin to remove blood clot, and shearing placenta tissue into 2cm pieces3。
3) Culturing, namely flatly paving the placenta tissue block in a cell culture dish, and adding the placenta tissue block into a culture solution containing 10-20% fetal calf serum for culturing after water is volatilized; the operation is characterized in that the tissue block is small, the tissue adherence is firm, the mesenchymal stem cells can climb out of the tissue block, and a large amount of primary cells can be obtained;
4) inoculating and subculturing, wherein after the growth of the primary separated mesenchymal stem cells of the placenta tissue mass reaches 80-90 percent of fusion, the cells can be subjected to subculture amplification to obtain the mesenchymal stem cells of the placenta, and 0.25 percent pancreatin/E is usedDigesting the cells with DTA at a concentration of 5000 cells/cm2And (3) inoculating, wherein the placenta mesenchymal stem cells can reach 95% fusion 4-5 days after inoculation, and the placenta mesenchymal stem cells fused by more than 95% can be amplified to more than 20 generations. The digestion time is short, the damage to cells is small, the inoculation density can well control the growth cycle of the cells, and the chorionic mesenchymal stem cells can be amplified for more than 20 generations according to the digestion of the cells after the inoculation density passage.
The culture solution of 10-20% fetal calf serum in the step 3) contains alpha-MEM basic culture solution containing nucleoside and fetal calf serum, which is more beneficial to separation and proliferation of stem cells, and can better maintain the dryness and biological functions of the stem cells in the long-term amplification culture process.
And 3) replacing the culture solution of 10% fetal calf serum once every 3 days after cell culture in the cell culture dish in the step 3), replacing the culture solution of 10% fetal calf serum every 2-3 days, allowing the placenta mesenchymal stem cells to climb out of the tissue block after adhering to the wall for 4-5 days, and allowing the placenta mesenchymal stem cells to reach 80-90% fusion after 7-10 days.
In the step 3), 20ml of culture solution containing 10% fetal calf serum is added in each dish.
Further comprising the step of performing quality evaluation on the mesenchymal stem cells obtained in the step 4), wherein the quality evaluation comprises cell number, cell activity, cell surface markers, differentiation function, bacterial detection, mycoplasma detection and safety evaluation.
Example 2:
a separation culture and amplification method of placenta mesenchymal stem cells comprises the following steps:
1) cleaning, namely obtaining placenta tissues under an aseptic condition after delivery of the fetus, washing the surface of the placenta by using a cleaning solution, and cleaning and removing residual blood and other impurities;
2) treating, gently peeling amnion, removing blood vessel tissue, repeatedly washing placenta in PBS containing 100U/ml penicillin and 100ug/ml streptomycin to remove blood clot, and shearing chorion tissue into pieces of 3cm in size with aseptic tissue scissors2。
3) Culturing, namely flatly paving the placenta tissue block in a cell culture dish, volatilizing water, and adding the volatilized placenta tissue block into a culture solution containing 10-20% fetal calf serum for culturing; the operation is characterized in that the tissue block is small, the tissue adherence is firm, the mesenchymal stem cells can climb out of the tissue block, and a large amount of primary cells can be obtained;
4) inoculating and subculturing, wherein after the growth of the primary separated mesenchymal stem cells of the placenta tissue mass reaches 80-90% and is fused, passage amplification culture can be carried out to obtain the mesenchymal stem cells of the placenta, the cells are digested by 0.25% pancreatin/EDTA according to the ratio of 3000-5000 cells/cm2And (3) inoculating, wherein the placenta mesenchymal stem cells can reach 95% fusion 4-5 days after inoculation, and the placenta mesenchymal stem cells fused by more than 95% can be amplified to more than 20 generations. The digestion time is short, the damage to cells is small, the inoculation density can well control the growth cycle of the cells, and the placenta mesenchymal stem cells after passage according to the inoculation density can be amplified for more than 20 generations.
The cleaning solution in the step 1) contains phosphate buffer solution of penicillin and streptomycin.
The culture solution of 10-20% fetal calf serum in the step 3) contains alpha-MEM basic culture solution containing nucleoside and fetal calf serum, which is more beneficial to separation and proliferation of stem cells, and can better maintain the dryness and biological functions of the stem cells in the long-term amplification culture process.
And 3) replacing the culture solution of 10% fetal calf serum once every 3 days after cell culture in the cell culture dish in the step 3), replacing the culture solution of 10% fetal calf serum every 2-3 days, allowing the placenta mesenchymal stem cells to climb out of the tissue block after adhering to the wall for 4-5 days, and allowing the placenta mesenchymal stem cells to reach 80-90% fusion after 7-10 days.
In the step 3), a culture solution containing 10% fetal calf serum is added in a volume of 50 ml/dish.
Further comprising the step of performing quality evaluation on the mesenchymal stem cells obtained in the step 4), wherein the quality evaluation comprises cell number, cell activity, cell surface markers, differentiation function, bacterial detection, mycoplasma detection and safety evaluation.
Example 3:
after screening infectious diseases and the like, signing an informed consent and a donation protocol, aseptically collecting placenta tissues after delivery, flushing the surface with Phosphate Buffer Solution (PBS) containing penicillin and streptomycin under an aseptic condition, and cleaning to remove blood and other impurities remained in blood vessels.
Cutting cross incision with umbilical cord root as cross center, slightly peeling amnion, removing blood vessel tissue, placing placenta (shown in figure 1) in PBS containing double antibody (100U/ml penicillin, 100ug/ml streptomycin), repeatedly washing to remove blood clot, cutting placenta tissue into pieces of 1cm in size by aseptic tissue scissors3Size tissue mass (see fig. 2).
The tissue is spread in a cell culture dish and after the water is evaporated, complete culture solution containing 10-20% fetal calf serum is added.
Changing liquid every 2-3 days after cell culture, wherein the cells can be seen to climb out of the tissue block after adherence for 4-5 days, and the placenta mesenchymal stem cells can be fused by 80-90% after 7-10 days;
digesting the cells with 0.25% trypsin/EDTA at 3000-2Inoculation, and the cell can reach 95% fusion passage 4-5 days after inoculation.
The culture solution of 20% fetal calf serum contains alpha-MEM basic culture solution containing nucleoside and fetal calf serum.
The placenta simple mesenchymal stem cells obtained in the example 3 are subjected to mesenchymal stem cell dryness maintenance, and the surface antigens of the stem cells cultured by the three culture solutions are analyzed in a surface marker flow mode, so that the obtained MSCs have good homogeneity. All express CD73, CD105, CD 90; does not express CD45, CD34, CD11b, CD19and HLA-DR. The result is that the placenta mesenchymal stem cells P10 flow-batch detection shows that the expression of CD73, CD90 and CD105 is positive, and the expression of Negative-CD 45, CD34, CD11b, CD19and HLA-DR is Negative, and the figure is shown in figure 4.
Example 4:
performing multi-directional differentiation potential identification on the placental mesenchymal stem cells of example 3, wherein fig. 5A is an osteogenic differentiation control, and fig. 5B is alizarin red staining after osteogenic differentiation; FIG. 5C is a adipogenic differentiation control, and FIG. 5D is oil red "O" staining after adipogenic differentiation; fig. 5E is a chondrogenic differentiation control and fig. 5F is an allothrin blue staining after chondrogenic differentiation.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (7)
1. A placenta mesenchymal stem cell separation culture amplification method is characterized by comprising the following steps:
1) treatment of
After delivery of the fetus, flushing the placenta tissue with normal saline, peeling the amnion, and removing the vascular tissue as much as possible;
2) cleaning of
Repeatedly washing placenta with removed blood vessel tissue in buffer solution containing 100U/ml penicillin and 100ug/ml streptomycin for 3 times;
3) culturing
Shearing the cleaned placenta tissue by using sterile tissue, and shearing into pieces with the size of 1-3 cm3Spreading the cut tissue blocks in a cell culture dish, standing for 30-60 minutes, and adding the tissue blocks into a culture solution containing 10-20% fetal calf serum for culture after water is volatilized;
4) passage of inoculation
After the growth of the placenta tissue primary separation culture mesenchymal stem cells reaches 80-90% and is fused, subculture can be carried out to obtain placenta mesenchymal stem cells, the cells are digested by 0.25% pancreatin/EDTA (ethylene diamine tetraacetic acid) according to 4000-8000 cells/cm2And (3) inoculation, wherein after 4-5 days of inoculation, 95% of the mesenchymal stem cells can be fused, and more than 95% of the mesenchymal stem cells can be expanded to more than 20 generations.
2. The isolated culture and expansion method of placental mesenchymal stem cells according to claim 1, wherein the culture and expansion method comprises: the buffer solution for washing in the step 2) is phosphate buffer solution or normal saline containing 100U/ml of double-antibody penicillin and 100ug/ml of streptomycin.
3. The isolated culture and expansion method of placental mesenchymal stem cells according to claim 1, wherein the culture and expansion method comprises: the culture solution of 10-20% fetal calf serum in the step 3) contains alpha-MEM basic culture solution containing nucleoside and fetal calf serum.
4. The isolated culture and expansion method of placental mesenchymal stem cells according to claim 1, wherein the culture and expansion method comprises: and 3) replacing the culture solution of 10-20% fetal calf serum once after 3 days of cell culture in the cell culture dish in the step 3), replacing the culture solution of 10-20% fetal calf serum every 2-3 days later, allowing the placenta mesenchymal stem cells to climb out of the tissue block after 4-5 days of wall attachment, and allowing the placenta mesenchymal stem cells to reach 80-90% fusion after 7-10 days.
5. The isolated culture and expansion method of placental mesenchymal stem cells according to claim 1, wherein the culture and expansion method comprises: and 3) adding 20-50 ml/dish of culture solution containing 10-20% fetal calf serum.
6. The isolated culture and expansion method of placental mesenchymal stem cells according to claim 1, wherein the culture and expansion method comprises: further comprising the step of performing quality evaluation on the mesenchymal stem cells obtained in the step 4), wherein the quality evaluation comprises cell number, cell activity, cell surface markers, differentiation function, bacterial detection, mycoplasma detection and safety evaluation.
7. The isolated culture expansion method of placental mesenchymal stem cells according to any one of claims 1 to 6, wherein the culture expansion method comprises: the production specification, the quality control system and the evaluation system of the placenta mesenchymal stem cells established by the method can quickly, simply and conveniently obtain the mesenchymal stem cells for clinical research and application.
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