CN112410285A - Culture method of human placenta mesenchymal stem cells - Google Patents
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Abstract
The invention discloses a method for culturing human placenta mesenchymal stem cells, which relates to the technical field of biological science, and has the technical scheme that: the method specifically comprises the following steps: s1, obtaining P4 generation human placenta mesenchymal stem cells, and carrying out subculture; s2, changing the cell culture solution every 2 days during subculture; and S3, carrying out cell passage and cryopreservation on the human placenta mesenchymal stem cells when the fusion degree of the human placenta mesenchymal stem cells reaches 80-90%, wherein the cells are counted during cryopreservation. The culture method of the human placenta mesenchymal stem cells can be used for culturing the human placenta mesenchymal stem cells, and is convenient for skillfully mastering the overall process, recovery, liquid change, passage and cryopreservation operation of cell culture; in addition, the method for culturing the human placental mesenchymal stem cells is optimized, the culture cost for culturing the human placental mesenchymal stem cells by adopting the method is low, and the culture operation process is simple and reliable.
Description
Technical Field
The invention relates to the technical field of bioscience, in particular to a method for culturing human placental mesenchymal stem cells.
Background
Mesenchymal Stem Cells (MSC), an adult stem cell with self-replicating ability and multipotentiality, has low immunogenicity and characteristics of homing to ischemic or damaged tissues, can home to a specific site after being infused into a host, and can directionally differentiate into cells of endodermal, mesodermal and ectodermal 3 germ layer-derived tissues under the influence of microenvironment, such as more than 10 mature cells of bone, cartilage, tendon, fat, liver, kidney, skin, muscle, nerve and even pancreas, thereby becoming ideal seed cells for organ repair in regenerative medicine.
The mesenchymal stem cells have wide clinical application prospect, and can be used for treating diseases such as nervous system diseases, liver and kidney injuries, autoimmune diseases, heart diseases, bone diseases, cartilage diseases, ischemic vascular diseases, diabetic complications, tumors and the like. They may also be used in tissue engineering and facial contouring. In addition, they can be co-transplanted with hematopoietic stem cells for the treatment of hematological disorders.
There are three main sources of mesenchymal stem cells: bone marrow, adipose tissue, and placental tissue delivered by the infant at birth, with the greatest amount of placenta remaining. After the baby is born, the mesenchymal stem cells remained in the placenta can be extracted and stored for a long time for the child for the need of the child every now and then, the valuable health resources are discarded because the stem cells are not understood. Placental Mesenchymal Stem Cells (MSCs) are a type of pluripotent stem cell that is derived from the mesoderm during embryonic development. In the normal tissue damage repair process of the body, MSC is an important cell bank involved in tissue regeneration. Under the action of special signals caused by tissue damage, the MSCs migrate to the damaged part, gather and proliferate locally, and differentiate along different paths according to different damage signals. MSCs are easy to separate and amplify, have strong in vitro multiplication capacity, and can still maintain the multidirectional differentiation capacity even if the MSCs are amplified by 1 hundred million times. Thus, MSC is a practical tissue repair seed cell.
In recent years, the use of placenta as a source of mesenchymal stem cell tissue has been increasingly studied. The placenta mesenchymal stem cells are used for treating graft-versus-host disease caused by hematopoietic stem cell transplantation at present and have proved to have a great clinical application prospect. The swedish scientist injects the placenta mesenchymal stem cells to dozens of patients receiving the allogeneic hematopoietic stem cell transplantation, and the safety of the placenta mesenchymal stem cell transplantation is proved; meanwhile, in treatment, the survival rate of the graft-versus-host disease patient can be obviously improved by injecting the placenta mesenchymal stem cells. Overall, the placental mesenchymal stem cells have advantages mainly in the following aspects: 1) the materials are easy to obtain, and the sources of the raw materials are sufficient, so that the regeneration of life resources is realized. 2) The strong differentiation capability can directionally induce and differentiate various stem cells such as mesenchymal stem cells, vascular stem cells, epithelial stem cells, neural stem cells, hepatic stem cells and the like. 3) Sufficient quantity, convenient use, strong multiplication capacity, and the number of cultured cells can reach 10 hundred million, and the cells can be used by multiple people for multiple times. 4) When the medicine is used in people, matching is not needed, immune rejection reaction can not be generated, and meanwhile, the closer the blood relationship is, the higher the bioavailability is, and the better the use effect is. 5) Has wide disease range, anti-aging, health recovery, cardiovascular and cerebrovascular system diseases, diabetes, liver and kidney injury, brain and spinal nerve injury, autoimmune diseases, graft-versus-host disease and other diseases.
In conclusion, the mesenchymal stem cells bring new hopes for future regenerative medicine, and the intensive research and clinical application of the mesenchymal stem cells will certainly benefit mankind in the near future. Therefore, the invention aims to design and provide a culture method of human placental mesenchymal stem cells.
Disclosure of Invention
The invention aims to provide a culture method of human placenta mesenchymal stem cells, which can be used for culturing the human placenta mesenchymal stem cells, observing the growth state of the human placenta mesenchymal stem cells in the culture process, and performing passage cryopreservation on the cultured cells, thereby facilitating the operation of skillfully mastering the whole process, recovery, solution exchange, passage and cryopreservation of cell culture; by the culture method, the operation of identifying whether the placenta mesenchymal stem cells have characteristics of the mesenchymal stem cells or not in the culture process is facilitated; in addition, the method for culturing the human placental mesenchymal stem cells is optimized, the culture cost for culturing the human placental mesenchymal stem cells by adopting the method is low, and the culture operation process is simple and reliable.
The technical purpose of the invention is realized by the following technical scheme: a method for culturing human placental mesenchymal stem cells specifically comprises the following steps:
s1, obtaining human placenta mesenchymal stem cells, extracting the mesenchymal stem cells from placenta tissues by adopting an enzyme direct digestion method, carrying out subculture to obtain P4 generation human placenta mesenchymal stem cells, and carrying out subculture on the obtained P4 generation human placenta mesenchymal stem cells by using a serum-free culture medium special for the stem cells;
s2, changing liquid for cells, observing the cells at regular time in the process of subculturing the P4 generation human placenta mesenchymal stem cells, and changing liquid for cells every 2 days;
s3, carrying out cell passage and cryopreservation, wherein when the fusion degree of the human placenta mesenchymal stem cells subjected to subculture reaches 80-90%, the human placenta mesenchymal stem cells are subjected to passage and cryopreservation; the specific method for freezing and storing the cells comprises the following steps:
1) irradiating the ultra-clean bench with ultraviolet light for 30min in advance, and placing serum-free culture medium, serum-containing culture medium and trypsin in a 37 deg.C constant temperature water bath for 15 min;
2) sucking out the culture medium in the culture flask, and cleaning the culture medium twice by adopting 2ml of PBS;
3) the PBS was aspirated and 1ml trypsin was added to the flask;
4) screwing the bottle cap of the culture bottle, shaking the culture bottle, beating the bottle body with palm, and observing the cell shape under an inverted microscope after trypsin reacts for 1-2 min;
5) adding 1ml of culture medium with serum into a culture bottle to terminate the reaction, sucking 15ml of cell suspension after the first blowing and beating mixing (about 10 times), transferring the cell suspension into a centrifuge tube, and centrifuging the cell suspension in the centrifuge tube by adopting a centrifuge;
6) discarding the supernatant in the centrifuge tube, adding 1ml of serum-free culture medium, performing blow-beating mixing (about 15 times), taking 900ul of cell suspension in a cryopreservation tube by using a pipette, adding 100ul of DMSO, performing blow-beating mixing again, sealing by using a sealing film, immediately placing in a programmed cooling box, keeping the temperature at-80 ℃, and after preserving in the programmed cooling box for one night, placing in a liquid nitrogen tank for preservation;
wherein the cells are counted while cryopreserving the human placental mesenchymal stem cells.
Further, the specific method of cell counting described in step S3 is:
a. irradiating the ultra-clean bench with ultraviolet light for 30min in advance, and placing serum-free culture medium, serum-containing culture medium and trypsin in a 37 deg.C constant temperature water bath for 15 min;
b. sucking out the culture medium in the culture flask, and cleaning the culture medium twice by adopting 2ml of PBS;
c. the PBS was aspirated and 1ml trypsin was added to the flask;
d. screwing the bottle cap of the culture bottle, shaking the culture bottle, beating the bottle body with palm, and observing the cell shape under an inverted microscope after trypsin reacts for 1-2 min;
e. adding 1ml of culture medium with serum into a culture bottle to terminate the reaction, sucking 15ml of cell suspension after blowing, beating and mixing (about 10 times), transferring the cell suspension into a centrifuge tube, and centrifuging the cell suspension in the centrifuge tube by adopting a centrifuge, wherein the centrifugation speed is 1000rpm, the temperature is 24 ℃, and the time is 8 min;
f. discarding the supernatant in the centrifuge tube, adding 1ml of culture medium, and mixing by blowing (about 15 times);
g. after the mixing, 10ul of cell suspension was taken by pipette into a blood cell counting plate and the blood cell counting plate was placed under an inverted microscope to count the cells, wherein the number of cells in 4 cells of 16 cells was counted and the average number was taken, and the cell density was the average number multiplied by 104。
Further, the rotation speed of the centrifugation in the step 5) in the step S3 is 1000rpm, the temperature is 24 ℃, and the time is 8 min.
Further, the specific method for cell counting described in step S3 further includes: after cell counting, the cell suspension was diluted with an appropriate amount of medium and re-inoculated in a 25T flask.
Further, after cell counting, the density of seeding was about 50%.
Further, the serum-containing culture medium described in step S3 is a DMEM medium containing serum.
In conclusion, the invention has the following beneficial effects: the culture method of the human placenta mesenchymal stem cells can be used for culturing the human placenta mesenchymal stem cells, observing the growth state of the human placenta mesenchymal stem cells in the culture process, and performing passage cryopreservation on the cultured cells, thereby facilitating the skillful mastering of the whole process, recovery, solution change, passage and cryopreservation operation of cell culture; by the culture method, the operation of identifying whether the placenta mesenchymal stem cells have characteristics of the mesenchymal stem cells or not in the culture process is facilitated; in addition, the method for culturing the human placental mesenchymal stem cells is optimized, the culture cost for culturing the human placental mesenchymal stem cells by adopting the method is low, and the culture operation process is simple and reliable.
Drawings
FIG. 1 is a flow chart in an embodiment of the invention;
fig. 2 is a bright field diagram of placental mesenchymal stem cells in an embodiment of the invention;
FIG. 3 is a diagram of immunofluorescence identification of placental mesenchymal stem cells in an embodiment of the present invention;
fig. 4 is a graph of growth of placental mesenchymal stem cells in an example of the invention.
Detailed Description
The invention is described in further detail below with reference to figures 1-4.
Example (b): a method for culturing human placental mesenchymal stem cells is disclosed, as shown in figure 1, and specifically comprises the following steps:
s1, obtaining human placenta mesenchymal stem cells, extracting the mesenchymal stem cells from placenta tissues by adopting an enzyme direct digestion method, carrying out subculture to obtain P4 generation human placenta mesenchymal stem cells, and carrying out subculture on the obtained P4 generation human placenta mesenchymal stem cells by using a serum-free culture medium special for the stem cells.
And S2, performing cell replacement, namely, regularly observing cells in the process of subculturing the P4 generation human placenta mesenchymal stem cells, and performing cell replacement every 2 days (since a serum-free culture medium cannot change color, the cell replacement is required every two days, the cells are required to be frequently observed, and the phenomenon that the cells start to die due to overlarge cell density is avoided).
And S3, carrying out cell passage and cryopreservation, and when the fusion degree of the human placenta mesenchymal stem cells subjected to subculture reaches 80-90%, carrying out passage and cryopreservation on the human placenta mesenchymal stem cells. The specific method for freezing and storing the cells comprises the following steps:
1) irradiating the ultra-clean bench with ultraviolet light for 30min in advance, and placing serum-free culture medium, serum-containing culture medium and trypsin in a 37 deg.C constant temperature water bath for 15 min.
2) The medium in the flask was aspirated and washed twice with 2ml of PBS (phosphate buffered saline).
3) The PBS was aspirated and 1ml trypsin was added to the flask.
4) Screwing the bottle cap of the culture bottle, shaking the culture bottle, beating the bottle body with palm, and observing the morphology of the cells under an inverted microscope after the trypsin reacts for 1-2 min (the digestion time of the trypsin is about 2min, and the digestion time can be mastered by observing the cell morphology change under the microscope).
5) Adding 1ml of culture medium with serum into a culture bottle to terminate the reaction (or terminating the reaction by using trypsin inhibitor), performing first blowing and beating mixing, then sucking 15ml of cell suspension and transferring the cell suspension into a centrifuge tube, and performing centrifugal operation on the cell suspension in the centrifuge tube by using a centrifugal machine, wherein the blowing and beating mixing times are about 10 times.
6) Discarding the supernatant in the centrifuge tube, adding 1ml of serum-free culture medium, blowing and mixing again, wherein the number of times of blowing and mixing is about 15, then taking 900ul of cell suspension in a cryopreservation tube by adopting a pipette gun, adding 100ul of DMSO (dimethyl sulfoxide/cryopreservation protective agent), blowing and mixing again, sealing by using a sealing film, immediately placing in a programmed cooling box, keeping at-80 ℃, and after preserving in the programmed cooling box for one night, placing in a liquid nitrogen tank for preservation.
Wherein the cells are counted while cryopreserving the human placental mesenchymal stem cells.
The specific method of cell counting in step S3 is:
a. irradiating the ultra-clean bench with ultraviolet light for 30min in advance, and placing serum-free culture medium, serum-containing culture medium and trypsin in a 37 deg.C constant temperature water bath for 15 min.
b. The medium in the flask was aspirated and washed twice with 2ml PBS.
c. The PBS was aspirated and 1ml trypsin was added to the flask.
d. And screwing the bottle cap of the culture bottle, shaking the culture bottle, beating the bottle body with palm, and observing the cell shape under an inverted microscope after trypsin reacts for 1-2 min.
e. Adding 1ml of culture medium with serum into a culture bottle to terminate the reaction, sucking 15ml of cell suspension after blowing and beating mixing (the blowing and beating mixing times are about 10 times), transferring the cell suspension into a centrifuge tube, and centrifuging the cell suspension in the centrifuge tube by adopting a centrifuge, wherein the centrifugation speed is 1000rpm, the temperature is 24 ℃, and the time is 8 min.
f. The supernatant in the centrifuge tube was discarded, 1ml of the medium was added, and the mixture was subjected to pipetting (the number of pipetting was about 15 times).
g. After the mixing, 10ul of cell suspension was taken by pipette into a blood cell counting plate and the blood cell counting plate was placed under an inverted microscope to count the cells, wherein the number of cells in 4 cells of 16 cells was counted and the average number was taken, and the cell density was the average number multiplied by 104。
The rotation speed of the centrifugation in step 5) in step S3 was 1000rpm, the temperature was 24 ℃, and the time was 8 min.
The specific method of cell counting in step S3 further includes: after cell counting, the cell suspension was diluted with an appropriate amount of medium and re-inoculated in a 25T flask.
After cell counting, the density of the inoculated cells is about 50%, and the cells are prevented from being too low to be favorable for cell growth. As shown in fig. 2, it reflects the growth of human placental mesenchymal stem cells seeded in 25T culture flasks. The result is: adherent cells can be seen 24 hours after the human placenta mesenchymal stem cells are inoculated, the shape is a fibroblast-like shape, and the cells grow in a vortex shape. The cells grow fast but are affected by the seeding density. The density started to increase rapidly at 50%, day 2.
The serum-containing medium in step S3 was DMEM medium (a medium containing various amino acids and glucose) containing serum.
The specific method for cell identification of the cultured cells is as follows:
1. the cultured cells were resuspended with trypsin and then seeded into 96-well plates at a density of 4000 cells per well.
2. After culturing the cells for 48h, the medium was aspirated.
3. After washing twice with PBS, cells were fixed with 4% paraformaldehyde for 20 min.
4. Washing with PBS for 5min 3 times.
5. 100ul of 5% sheep serum and 0.3% Triton-X100 blocking solution was added to each well, and the 96-well plate was incubated in a wet box at 37 ℃ for 30 min.
6. Adding a primary antibody. The antibodies were: CD44, CD45, CD 90. The antibodies were diluted in 2% sheep serum at a dilution factor of 1: 100.
The antibody dark bottom fraction was added to a 96-well plate at 30ul per well. Incubate at 4 ℃ for 18h in a wet box. The grouping is as follows in table 1.
TABLE 1
| Grouping | Antibodies | Hole(s) |
| Negative control | No, 2% sheep serum | 1 |
| CD45+CD90 | 3 | |
| CD44 | 3 |
7. PBS wash was performed 3 times for 5min each.
8. And (4) hatching a secondary antibody. Antibodies were G & R488, G & M594, respectively. Both antibodies were diluted together in 2% sheep serum at a 1:200 dilution. Add 30ul per well to a 96 well plate. Incubate for 1h at 37 ℃ in a wet box.
9. PBS wash was performed 3 times for 5min each.
10. The PBS was blotted dry. Add 10ul of DAPI staining solution per well.
11. The immunofluorescence identification result of the placenta mesenchymal stem cells is shown in figure 3 after the photograph under a fluorescence microscope.
As shown in fig. 3, mesenchymal stem cells have no specific surface antigen, express specific surface antigens of stromal cells and stromal cells such as CD29, CD44, CD71, CD90, etc., and do not express antigens such as CD45, CD34, etc. CD90, CD44 and CD45 were selected for experiments, and from the experimental results in FIG. 3, it can be seen that the cultured placental stem cells are CD90+ CD44+ CD 45-cells, which meet the characteristics of mesenchymal stem cells.
As shown in fig. 4, growth curves of the placental mesenchymal stem cells P4, P5, and P6 generations were examined by seeding 2000 cells per well of a 96-well plate. As can be seen from FIG. 4, the cells of the P4 and P5 generations rapidly grew in the first 3 days, the growth rate was the fastest from day 3 to day 4, the cells grew to about 80% after day 4, the growth rate became slow, and the plateau was reached on day 5. The growth rate of the P6 generation cells was slightly slower, and the plateau phase was reached on day 6.
The culture method of the human placenta mesenchymal stem cells can culture the human placenta mesenchymal stem cells, observe the growth state of the human placenta mesenchymal stem cells in the culture process, and perform passage cryopreservation on the cultured cells, thereby facilitating the skillful mastering of the whole process, recovery, liquid change, passage and cryopreservation operation of cell culture. By the culture method, the operation of identifying whether the placenta mesenchymal stem cells have characteristics of the mesenchymal stem cells or not in the culture process is facilitated. In addition, the method for culturing the human placental mesenchymal stem cells is optimized, the culture cost for culturing the human placental mesenchymal stem cells by adopting the method is low, and the culture operation process is simple and reliable.
The present embodiment is only for explaining the present invention, and it is not limited to the present invention, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present invention.
Claims (6)
1. A method for culturing human placental mesenchymal stem cells is characterized by comprising the following steps: the method specifically comprises the following steps:
s1, obtaining human placenta mesenchymal stem cells, extracting the mesenchymal stem cells from placenta tissues by adopting an enzyme direct digestion method, carrying out subculture to obtain P4 generation human placenta mesenchymal stem cells, and carrying out subculture on the obtained P4 generation human placenta mesenchymal stem cells by using a serum-free culture medium special for the stem cells;
s2, changing liquid for cells, observing the cells at regular time in the process of subculturing the P4 generation human placenta mesenchymal stem cells, and changing liquid for cells every 2 days;
s3, carrying out cell passage and cryopreservation, wherein when the fusion degree of the human placenta mesenchymal stem cells subjected to subculture reaches 80-90%, the human placenta mesenchymal stem cells are subjected to passage and cryopreservation; the specific method for freezing and storing the cells comprises the following steps:
1) irradiating the ultra-clean bench with ultraviolet light for 30min in advance, and placing serum-free culture medium, serum-containing culture medium and trypsin in a 37 deg.C constant temperature water bath for 15 min;
2) sucking out the culture medium in the culture flask, and cleaning the culture medium twice by adopting 2ml of PBS;
3) the PBS was aspirated and 1ml trypsin was added to the flask;
4) screwing the bottle cap of the culture bottle, shaking the culture bottle, beating the bottle body with palm, and observing the cell shape under an inverted microscope after trypsin reacts for 1-2 min;
5) adding 1ml of culture medium with serum into a culture bottle to terminate the reaction, sucking 15ml of cell suspension after the first blowing and beating mixing (about 10 times), transferring the cell suspension into a centrifuge tube, and centrifuging the cell suspension in the centrifuge tube by adopting a centrifuge;
6) discarding the supernatant in the centrifuge tube, adding 1ml of serum-free culture medium, performing blow-beating mixing (about 15 times), taking 900ul of cell suspension in a cryopreservation tube by using a pipette, adding 100ul of DMSO, performing blow-beating mixing again, sealing by using a sealing film, immediately placing in a programmed cooling box, keeping the temperature at-80 ℃, and after the cells are stored in the programmed cooling box for one night, placing in a liquid nitrogen tank for preservation;
wherein the cells are counted while cryopreserving the human placental mesenchymal stem cells.
2. The method of claim 1, wherein the human placental mesenchymal stem cells are cultured by: the specific method of cell counting described in step S3 is:
a. irradiating the ultra-clean bench with ultraviolet light for 30min in advance, and placing serum-free culture medium, serum-containing culture medium and trypsin in a 37 deg.C constant temperature water bath for 15 min;
b. sucking out the culture medium in the culture flask, and cleaning the culture medium twice by adopting 2ml of PBS;
c. the PBS was aspirated and 1ml trypsin was added to the flask;
d. screwing the bottle cap of the culture bottle, shaking the culture bottle, beating the bottle body with palm, and observing the cell shape under an inverted microscope after trypsin reacts for 1-2 min;
e. adding 1ml of culture medium with serum into a culture bottle to terminate the reaction, sucking 15ml of cell suspension after blowing, beating and mixing (about 10 times), transferring the cell suspension into a centrifuge tube, and centrifuging the cell suspension in the centrifuge tube by adopting a centrifuge, wherein the centrifugation speed is 1000rpm, the temperature is 24 ℃, and the time is 8 min;
f. discarding the supernatant in the centrifuge tube, adding 1ml of culture medium, and mixing by blowing (about 15 times);
g. after the mixing, 10ul of cell suspension was taken by pipette into a blood cell counting plate and the blood cell counting plate was placed under an inverted microscope to count the cells, wherein the number of cells in 4 cells of 16 cells was counted and the average number was taken, and the cell density was the average number multiplied by 104。
3. The method of claim 1, wherein the human placental mesenchymal stem cells are cultured by: the rotation speed of the centrifugation in the step 5) in the step S3 is 1000rpm, the temperature is 24 ℃, and the time is 8 min.
4. The method of claim 2, wherein the human placental mesenchymal stem cells are cultured by: the specific method for cell counting described in step S3 further includes: after cell counting, the cell suspension was diluted with an appropriate amount of medium and re-inoculated in a 25T flask.
5. The method of claim 4, wherein the human placental mesenchymal stem cells are cultured by: the density of the inoculation was 50%.
6. The method of claim 1, wherein the human placental mesenchymal stem cells are cultured by: the serum-containing culture medium described in step S3 is a DMEM medium containing serum.
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| CN105950550A (en) * | 2016-07-21 | 2016-09-21 | 四川新生命干细胞科技股份有限公司 | Mesenchymal stem cell serum-free medium and cell isolation and cultivation methods |
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