CN113234663A - Method for culturing endometrial cells - Google Patents
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Abstract
The invention discloses a method for culturing endometrial cells, which comprises the steps of adding a stem cell exosome freeze-dried powder aqueous solution and human serum albumin into a DMEM/F12 culture medium to obtain a cell culture system; and (3) resuspending the endometrial cells obtained by separating endometrial tissues by using a DMEM/F12 culture medium, adding the endometrial cells into the cell culture system, then centrifugally separating, taking the precipitate, resuspending the precipitate by using the cell culture system, and inoculating a culture bottle for culturing to finish the culture of the endometrial cells. According to the method, the cell grows to the 7 th day, the cell density reaches 90%, the cell passage can be carried out, and the cell activity is good.
Description
Technical Field
The invention belongs to the biotechnology, and particularly relates to a method for culturing endometrial cells.
Background
The endometrium is composed of a single-layer columnar epithelium and a lamina propria, the causes of endometrial injury include the damage of the endometrium basal lamina and the myometrium caused by postpartum uterine curettage, spontaneous abortion, artificial abortion, endometrial ablation, pelvic tuberculosis, infection and the like, and researches suggest that when the endometrium basal lamina is damaged, the loss or the damage of endometrial cells can be caused. In the prior art, cells can be effectively separated from tissues, then cultured and then transfused back to the body, so that damage repair is realized. The process of cell culture is complex, and the prior art takes a basic culture medium as an essential component and adds proper additives to expect a good proliferation effect. In the prior art, cells are obtained from endometrial tissues, IL-8 and IFN-alpha 2b with different concentrations are added into a culture medium, and the proliferation condition of the cells is detected by an MTT method after glandular epithelial cells are cultured for 24 hours. Meanwhile, the intimal cells are cultured in a medium containing IL-8, and the influence of IL-8 on glandular epithelial cells and interstitial cells is observed by a transmission electron microscope after 24 hours. Cell proliferation was found to be significantly dose dependent on IL-8 levels and was most pronounced at IL-8 concentrations of 10 ng/mL. The observation of an electron microscope shows that under the action of 1ng/ml IL-8, the function of the intima cells is active, the secretion is vigorous, and the extracellular collagen production can be seen. IFN-alpha 2b has obvious inhibition effect on the proliferation of glandular epithelial cells, so the IFN-alpha 2b and IL-8 have influence on the endothelial cells. As is well known, the culture method has a definite relationship on the passability and the vitality of cells, the prior art generally adopts a basic culture medium, fetal calf serum, penicillin and streptomycin as a culture system, the cell density reaches 80 percent and can be passaged, but the problems of slow growth, poor passaging effect and the like still exist (Mol Hum Reprod, 2013, 19 (6): 361-.
Disclosure of Invention
The invention discloses a method for culturing endometrial cells, which is simple in composition, and the cells obtained by 7 days of culture are strong in activity and can be passed.
The invention adopts the following technical scheme:
a method of culturing endometrial cells comprising the steps of:
(1) adding the stem cell exosome freeze-dried powder aqueous solution and human serum albumin into a DMEM/F12 culture medium to obtain a cell culture system;
(2) and (3) resuspending the endometrial cells obtained by separating endometrial tissues by using a DMEM/F12 culture medium, adding the endometrial cells into the cell culture system, then centrifugally separating, taking the precipitate, resuspending the precipitate by using the cell culture system, and inoculating a culture bottle for culturing to finish the culture of the endometrial cells.
Endometrial tissue is routinely preserved in preservation solutions as a matter of common knowledge. The method comprises the steps of taking human endometrial tissue for centrifugal separation, sucking out upper-layer preservation solution, adding a DMEM/F12 culture medium and collagenase working solution, and digesting to obtain digestive juice; filtering the digestive juice with a filter screen, centrifuging the filtrate, and removing the supernatant to obtain the endometrial cells.
According to the invention, the stem cell exosome freeze-dried powder is added into ultrapure water to obtain a stem cell exosome freeze-dried powder aqueous solution, the stem cell exosome freeze-dried powder is an existing product and is obtained by drying a stem cell extracting solution, the extracting solution is prepared by adopting physiological saline for induction, the components are various and rich in various stem cell exosome cytokines, extracellular matrixes, exosome vesicles and the like, and the unexpected technical effect can be achieved by culturing the endometrial cells with the composite components.
In the invention, the volume of the human serum albumin is 3-6% of that of DMEM/F12 culture medium, preferably 4-5%, and most preferably 5%; the dosage ratio of the stem cell exosome freeze-dried powder to the DMEM/F12 culture medium is 1-10 mg: 100mL, preferably 3-7 mg: 100mL, and most preferably 5 mg: 100 mL; the dosage ratio of the stem cell exosome freeze-dried powder to water is 1-10 mg: 5mL, preferably 4-6 mg: 5mL, and most preferably 5 mg: 5 mL.
In the invention, the centrifugal separation parameter is 1000-2000 r/m for 3-8 min, preferably 1500 r/m for 5 min. When the culture bottle is inoculated, the inoculation density is 103/cm2～105/cm2Preferably 104/cm2。
In the invention, the culture bottles are T150 cell culture bottles, 20-30 ml of the cell culture system is added into each culture bottle, and preferably, 25ml of the cell culture system is added into each culture bottle.
In the present invention, the culture environment is 37 ℃ and 5% CO2The incubator of (1); the cell culture system was replaced every 3 days.
As a preferred technical scheme, the method takes human endometrial tissue for centrifugal separation, sucks out upper-layer preservation solution, adds DMEM/F12 culture medium and collagenase working solution, and digests to obtain digestive juice; filtering the digestive juice by using a filter screen, centrifuging the filtrate, removing the supernatant, re-suspending the precipitate by using a DMEM/F12 culture medium to obtain a re-suspension, adding the re-suspension into the cell culture system according to the volume ratio of 1: 8-12, then performing centrifugal separation, re-suspending the precipitate by using the cell culture system, and inoculating a culture bottle for culture to finish the culture of the endometrial cells.
The invention firstly dilutes and dissolves the previously developed stem cell extract freeze-dried powder with pure water as an additive composition, forms a culture system together with a basic culture medium and albumin, does not need to add other reagents or cytokines and the like, limits the proportion of each component, is used for culturing the endometrial cells, can grow by adhering to the wall in the cell growth process, can reach 80-90% of growth density after being cultured for 7 days, meets the passage requirement, and has good proliferation activity of the endometrial cells measured by a CCK-8 method. According to the kit, a DMEM/F12 culture medium, human serum albumin, stem cell exosome freeze-dried powder and ultrapure water form a culture system, endometrial cells are cultured by using the culture system for the first time, the fact that the endometrial cells can be subcultured after 7 days of culture is unexpectedly found, and cell viability detection results show that the cultured cells are good in viability, so that the kit disclosed by the invention has practical application value.
Drawings
FIG. 1 is a diagram of cells obtained after 7 days of culture in example one;
FIG. 2 is a cell map of comparative example I after 7 days of culture;
FIG. 3 is a cell map after 7 days of culture of comparative example II;
FIG. 4 is a diagram of cells after 7 days of six cultures in example;
FIG. 5 is a diagram of cells obtained after 7 days of culture in the seventh example;
FIG. 6 is a diagram of cells obtained after 7 days of the tenth culture in example.
Detailed Description
Referring to CN111759866A, primary human umbilical cord mesenchymal stem cells were cultured at a cell density of 106cells/T150 flask, inoculated in serum-free MSC medium (ScienCell Corp.), then at 37 ℃ with 5% CO2Performing primary culture in the incubator, and changing the culture solution every other day; when the cell growth density is close to 90%, removing the culture medium in the culture bottle by using a disposable 10mL pipette, adding 20mL of physiological saline into each bottle for rinsing, and repeatedly rinsing for 3 times; adding 25mL of physiological saline (0.9%), adding no other substances, inducing culture, and culturing at 37 deg.C with 5% CO2The incubator is used for induction culture; the induction culture time is 72 hours; then centrifuging at 1500rpm for 10 minutes, and collecting supernatant; filtering the supernatant with a filter membrane of 0.1 micrometer to obtain dry cell extractive solution, and lyophilizing with vacuum lyophilizer to obtain dry cell exosome lyophilized powder. The freeze-drying process of the vacuum freeze-drying machine is as follows: subpackaging the dry cell extracting solution into penicillin bottles with the concentration of 5 ml/bottle, and pre-freezing overnight at-80 ℃; precooling for 3 hours by a freeze dryer, cooling to-55 ℃, and putting a sample; reducing the air pressure to 800Pa, maintaining the temperature at-55 ℃ for 2 hours; reducing the air pressure to 120Pa, raising the temperature to-40 ℃, and reducing the pressure and raising the temperature for 2 hours; the air pressure is maintained at 120Pa, the temperature is maintained at minus 40 ℃, and the time is 15 hours; reducing the air pressure to 100Pa, raising the temperature to-35 ℃, and reducing the pressure and raising the temperature for 2 hours; the air pressure is maintained at 100Pa, the temperature is maintained at-35 ℃, and the time is 5 hours; the air pressure is maintained at 100Pa, the temperature is increased to-25 ℃, and the time of the temperature increase process is 1 hour; the air pressure is maintained at 100Pa, the temperature is maintained at-25 ℃, and the time is 2 hours; the air pressure is maintained at 100Pa, the temperature is increased to 20 ℃, and the time of the temperature increase process is 1 hour; the air pressure is maintained at 100Pa, the temperature is maintained at 20 ℃, and the time is 2 hours; maintaining the air pressure at 100Pa, and raising the temperature to 30 ℃; reducing the air pressure to 10Pa, maintaining the temperature at 30 ℃, and maintaining the pressure and the temperature for 2 hours; and (5) recovering normal temperature and air pressure, and finishing freeze-drying to obtain the stem cell exosome freeze-dried powder.
The endometrial tissue comes from hospitals, is washed by 0.9% physiological saline and is stored in conventional preservation solution, and the culture of endometrial cells is not influenced by different human sources; the endometrial cells are used for autologous cell transplantation and improving the function of the autologous endometrium. The invention obtains endometrial cells from endometrial tissue by enzymolysis and digestion, and then the endometrial cells are cultured by a culture system consisting of DMEM/F12 culture medium (Gibco), human serum albumin (Jatebain), stem cell exosome freeze-dried powder and ultrapure water, so that the endometrial cells with good proliferation activity are obtained.
Taking 5 g of human endometrial tissue, centrifuging for 5 minutes at 1500rpm, sucking out upper layer preservation solution, adding 10mL of DMEM/F12 culture medium, adding 10mL of 0.5% collagenase working solution (Sigma), digesting for 60 minutes at 37 ℃ to obtain digestive juice, filtering the digestive juice by a 100-micron filter screen, centrifuging the filtrate for 5 minutes at 1500rpm, discarding supernatant, and resuspending cells to 5mL by using DMEM/F12 culture medium to obtain cell suspension, which is used for the following examples and comparative examples, and can not be repeatedly prepared.
Example a method of culturing endometrial cells, comprising the steps of:
adding 5mg of dry cell exosome freeze-dried powder into 5mL of ultrapure water for dissolving, then adding into 100mL of DMEM/F12 culture medium, and adding 5mL of human serum albumin (the volume of the human serum albumin is 5% of that of the DMEM/F12 culture medium) to obtain a cell culture system.
Adding 1mL cell suspension into 10mL cell culture system, mixing, centrifuging at 1500rpm for 5 min, discarding supernatant, and resuspending the obtained cell precipitate to 1mL with cell culture system according to 10%4/cm2The density of (2) into T150 cell culture flasks, 25ml of the cell culture system was added to each flask as the 0 th day of cell growth at 37 ℃ with 5% CO2The cell culture system is updated every 3 days, when the cell grows to 7 days, the cell density reaches 90 percent, cell passage can be carried out, cell photographing is carried out, and the cell growth state is observed, which is shown in figure 1; further, after 7 days of the culture, the absorbance at 12 hours, 24 hours, 36 hours, and 48 hours was 1.863, 2.162, 2.240, and 2.280, respectively.
Comparative example 1
Adding human serum albumin into 100mL of DMEM/F12 culture medium, and adding Vascular Endothelial Growth Factor (VEGF) and Hepatocyte Growth Factor (HGF) to obtain a control culture system, wherein the volume of the human serum albumin is 5% of that of the DMEM/F12 culture medium, and the volume of the Vascular Endothelial Growth Factor (VEGF) and the Hepatocyte Growth Factor (HGF) are both 20 ng/mL.
Adding 10mL of control culture system into 1mL of cell suspension, mixing, centrifuging at 1500rpm for 5 min, discarding supernatant, and resuspending the obtained cell precipitate to 1mL with cell culture system according to 10%4/cm2The density of (2) was inoculated into T150 cell culture flasks, and 25ml of a control culture system was added to each flask as the 0 th day of cell growth at 37 ℃ with 5% CO2The control culture system is updated every 3 days, when the cell growth reaches 7 days, the cell density reaches 65 percent and does not reach the cell passage standard, the cell is photographed, the cell growth form is not good, see figure 2, in addition, after 7 days of culture, the absorbance in 12 hours and 48 hours is respectively 0.866 and 1.056.
Comparative example No. two
Human serum albumin was added to 100mL of DMEM/F12 medium to obtain a blank culture system, wherein the volume of human serum albumin was 5% of the volume of DMEM/F12 medium.
Adding 1mL cell suspension into 10mL blank culture system, mixing, centrifuging at 1500rpm for 5 min, discarding supernatant, resuspending the obtained cell precipitate to 1mL with blank culture system, and performing 10mL4/cm2The density of (2) was inoculated into T150 cell culture flasks, and 25ml of a blank culture system was added to each flask as the 0 th day of cell growth at 37 ℃ with 5% CO2The blank culture system is updated every 3 days, when the cell grows to 7 days, the cell density reaches 40%, the cell passage cannot be carried out, the cell is photographed, the cell growth state is observed, see figure 3, in addition, after 7 days of culture, the absorbance in 12 hours and 48 hours is 0.325 and 0.421 respectively.
The cell density was measured by a conventional method using a microscope. Cell count and cell viability assays are common indicators of cell biological properties. When the cells are digested and passaged, after the cells are suspended for a short time, the cells are attached to the wall generally for 1 to 2 hours, then enter a latent period and then enter a logarithmic growth phase of cell mass division and proliferation to reach the goal ofAfter saturation, the cells stop proliferating and enter a plateau phase. In the above experiment, the cell density of the example cell grown to day 7 reached 90%, and cell passage was allowed; comparative example one cells grown to day 7, cell density reached 65%, cell passage was not possible; the cells of comparative example two were grown to day 7, and the cell density was only 40%, and cell passaging was not possible. On day 7 of culture, cells were photographed under a microscope model olympus CKX53, digital camera Mshot, 40-fold magnification. CCK8 (Shanghai Jingan's Biotech Co., Ltd.) was used to examine cell viability, cells cultured in each culture system up to day 7 were cultured, and cells were digested at 1 × 103And inoculating a 96-well plate for each cell/well, using a corresponding culture medium, and adding a CCK8 method for detecting the absorbance at 12h, 24h, 36h and 48h respectively, wherein the absorbance is in direct proportion to the cell activity.
Example two
Dissolving 1mg of dry cell exosome freeze-dried powder in 5mL of ultrapure water, adding the dissolved dry cell exosome freeze-dried powder into 100mL of DMEM/F12 culture medium, and adding 5mL of human serum albumin (the volume of the human serum albumin is 5% of that of the DMEM/F12 culture medium) to obtain a cell culture system. Adding 1mL cell suspension into 10mL cell culture system, mixing, centrifuging at 1500rpm for 5 min, discarding supernatant, and resuspending the obtained cell precipitate to 1mL with cell culture system according to 10%4/cm2The density of (2) into T150 cell culture flasks, 25ml of the cell culture system was added to each flask as the 0 th day of cell growth at 37 ℃ with 5% CO2The cell culture system is updated every 3 days, when the cell growth reaches the 7 th day, the cell density reaches 75%, the cell is photographed, the cell growth state is observed, and in addition, the absorbance in 12 hours after the cell culture is carried out for 7 days is 1.062.
EXAMPLE III
Dissolving 10mg of dry cell exosome freeze-dried powder in 5mL of ultrapure water, adding the dissolved dry cell exosome freeze-dried powder into 100mL of DMEM/F12 culture medium, and adding 5mL of human serum albumin (the volume of the human serum albumin is 5% of that of the DMEM/F12 culture medium) to obtain a cell culture system. Taking 1mL of cell suspension, adding 10mL of cell culture system, mixing, and rotating at 1500rpmCentrifuging for 5 min, discarding the supernatant, and resuspending the obtained cell pellet to 1ml with cell culture system according to 104/cm2The density of (2) into T150 cell culture flasks, 25ml of the cell culture system was added to each flask as the 0 th day of cell growth at 37 ℃ with 5% CO2The cell culture system was renewed every 3 days, when the cell density reached 80% by the time of the growth of the cells to day 7, the cells were photographed and the growth state of the cells was observed, and further, after the culture for 7 days, the absorbance at 12 hours was 1.657.
Example four
Dissolving 3mg of dry cell exosome freeze-dried powder in 5mL of ultrapure water, adding the dissolved dry cell exosome freeze-dried powder into 100mL of DMEM/F12 culture medium, and adding 5mL of human serum albumin (the volume of the human serum albumin is 5% of that of the DMEM/F12 culture medium) to obtain a cell culture system. Adding 1mL cell suspension into 10mL cell culture system, mixing, centrifuging at 1500rpm for 5 min, discarding supernatant, and resuspending the obtained cell precipitate to 1mL with cell culture system according to 10%4/cm2The density of (2) into T150 cell culture flasks, 25ml of the cell culture system was added to each flask as the 0 th day of cell growth at 37 ℃ with 5% CO2The cell culture system is renewed every 3 days, when the cells grow to 7 days, the cells can be passaged, the cells are photographed, and the growth state of the cells is observed, and in addition, after the cells are cultured for 7 days, the absorbance is 1.589 after 12 hours.
EXAMPLE five
Adding 7mg of dry cell exosome freeze-dried powder into 5mL of ultrapure water for dissolving, then adding into 100mL of DMEM/F12 culture medium, and adding 5mL of human serum albumin (the volume of the human serum albumin is 5% of that of the DMEM/F12 culture medium) to obtain a cell culture system. Adding 1mL cell suspension into 10mL cell culture system, mixing, centrifuging at 1500rpm for 5 min, discarding supernatant, and resuspending the obtained cell precipitate to 1mL with cell culture system according to 10%4/cm2The density of (2) into T150 cell culture flasks, 25ml of the cell culture system was added to each flask as the 0 th day of cell growth at 37 ℃ with 5% CO2The culture is carried out in the incubator (2),the cell culture system was renewed every 3 days, and when the cells grew to day 7, passaging was allowed, photographing was performed on the cells, and the state of cell growth was observed, and further, after 7 days of culture, the absorbance was 1.722 at 12 hours.
EXAMPLE six
Adding 5mg of dry cell exosome freeze-dried powder into 5mL of ultrapure water for dissolving, then adding into 100mL of DMEM/F12 culture medium, and adding 5mL of human serum albumin (the volume of the human serum albumin is 5% of that of the DMEM/F12 culture medium) to obtain a cell culture system.
Adding 1mL cell suspension into 10mL cell culture system, mixing, centrifuging at 1600 rpm for 4 min, discarding supernatant, and resuspending the obtained cell precipitate to 1mL with cell culture system according to 10%4/cm2The density of (2) into T150 cell culture flasks, 25ml of the cell culture system was added to each flask as the 0 th day of cell growth at 37 ℃ with 5% CO2The cell culture system is updated every 3 days, when the cells grow to 7 days, the cells are photographed, the growth state of the cells is observed, and the cells can be passaged according to the figure 4.
EXAMPLE seven
Adding 5mg of dry cell exosome freeze-dried powder into 5mL of ultrapure water for dissolving, then adding into 100mL of DMEM/F12 culture medium, and adding 5mL of human serum albumin (the volume of the human serum albumin is 5% of that of the DMEM/F12 culture medium) to obtain a cell culture system.
Adding 1mL cell suspension into 10mL cell culture system, mixing, centrifuging at 1300 rpm/min for 8 min, discarding supernatant, and resuspending the obtained cell precipitate to 1mL with cell culture system according to 10%4/cm2The density of (2) into T150 cell culture flasks, 25ml of the cell culture system was added to each flask as the 0 th day of cell growth at 37 ℃ with 5% CO2The cell culture system is updated every 3 days, when the cells grow to 7 days, the cells are photographed, the growth state of the cells is observed, and the cells can be passaged according to the figure 5.
Example eight
Dissolving 5mg of dry cell exosome freeze-dried powder in 10mL of ultrapure water, adding the dissolved dry cell exosome freeze-dried powder into 100mL of DMEM/F12 culture medium, and adding 5mL of human serum albumin (the volume of the human serum albumin is 5% of that of the DMEM/F12 culture medium) to obtain a cell culture system.
Adding 1mL cell suspension into 10mL cell culture system, mixing, centrifuging at 1500rpm for 5 min, discarding supernatant, and resuspending the obtained cell precipitate to 1mL with cell culture system according to 10%4/cm2The density of (2) into T150 cell culture flasks, 25ml of the cell culture system was added to each flask as the 0 th day of cell growth at 37 ℃ with 5% CO2The cell culture system is updated every 3 days, cell passage can be carried out when the cells grow to 7 days, cell photographing is carried out, and the growth state of the cells is observed.
Example nine
Dissolving 5mg of dry cell exosome freeze-dried powder in 3mL of ultrapure water, adding the dissolved dry cell exosome freeze-dried powder into 100mL of DMEM/F12 culture medium, and adding 5mL of human serum albumin (the volume of the human serum albumin is 5% of that of the DMEM/F12 culture medium) to obtain a cell culture system.
Adding 1mL cell suspension into 10mL cell culture system, mixing, centrifuging at 1500rpm for 5 min, discarding supernatant, and resuspending the obtained cell precipitate to 1mL with cell culture system according to 10%4/cm2The density of (2) into T150 cell culture flasks, 25ml of the cell culture system was added to each flask as the 0 th day of cell growth at 37 ℃ with 5% CO2The cell culture system is updated every 3 days, cell passage can be carried out when the cells grow to 7 days, cell photographing is carried out, and the growth state of the cells is observed.
Example ten
Adding 5mg of dry cell exosome freeze-dried powder into 5mL of ultrapure water for dissolving, then adding into 100mL of DMEM/F12 culture medium, and adding 5mL of human serum albumin (the volume of the human serum albumin is 5% of that of the DMEM/F12 culture medium) to obtain a cell culture system.
Adding 1mL cell suspension into 10mL cell culture system, mixing, centrifuging at 3000 rpm for 5 min, discarding supernatant, and collecting cell precipitateThe cell culture system was resuspended to 1ml, according to 104/cm2The density of (2) into T150 cell culture flasks, 25ml of the cell culture system was added to each flask as the 0 th day of cell growth at 37 ℃ with 5% CO2The cell culture system is renewed every 3 days, when the cells grow to 7 days, the cells are photographed, and the growth state of the cells is observed, see fig. 6.
In the prior art, a tissue digestion method is adopted to separate the endometrial cells growing by monoclonal adherence, so that the purified endometrial stem cells can be obtained, and the endometrial stem cells can be successfully differentiated into osteoblasts and smooth muscle cells under the induction of an osteogenesis induction culture medium and a smooth muscle induction culture medium. The proliferation activity of the endometrial cells is measured by a CCK-8 method, a growth curve graph is drawn, growth curves are S-like, the growth speed is gentle in the first 2 days, the cell proliferation speed is accelerated in 4-7 days, the growth curves are in an exponential growth phase, the cell proliferation speed is gentle again after 9-12 days, the cell number is in a plateau phase, and the cell number is relatively stable. According to the culture method disclosed by the invention, the cells grow to the 7 th day, the cell density reaches 90%, the cell passage can be carried out, and the cell activity is high.
Claims (10)
1. A method of culturing endometrial cells, comprising the steps of:
(1) adding the stem cell exosome freeze-dried powder aqueous solution and human serum albumin into a DMEM/F12 culture medium to obtain a cell culture system;
(2) and (3) resuspending the endometrial cells obtained by separating endometrial tissues by using a DMEM/F12 culture medium, adding the endometrial cells into the cell culture system, then centrifugally separating, taking the precipitate, resuspending the precipitate by using the cell culture system, and inoculating a culture bottle for culturing to finish the culture of the endometrial cells.
2. The method for culturing endometrial cells according to claim 1, wherein the endometrial cells are obtained by separating endometrial tissue from human endometrial tissue by centrifugation, sucking out the upper layer of preservation solution, adding a DMEM/F12 culture medium and collagenase working solution, and digesting to obtain a digestive juice; filtering the digestive juice with a filter screen, centrifuging the filtrate, and removing the supernatant to obtain the endometrial cells.
3. The method for culturing endometrial cells according to claim 1, wherein the lyophilized powder of stem cell exosomes is added to ultrapure water to obtain an aqueous solution of lyophilized powder of stem cell exosomes.
4. The method for culturing endometrial cells according to claim 1, wherein the volume of human serum albumin is 3-6% of the volume of DMEM/F12; the dosage ratio of the stem cell exosome freeze-dried powder to the DMEM/F12 culture medium is 1-10 mg: 100 mL.
5. The method for culturing endometrial cells according to claim 4, wherein the volume of human serum albumin is 4-5% of the volume of DMEM/F12; the dosage ratio of the stem cell exosome freeze-dried powder to the DMEM/F12 culture medium is 3-7 mg: 100 mL.
6. The method for culturing endometrial cells according to claim 1, wherein the centrifugation is performed at 1000 to 2000 rpm for 3 to 8 minutes.
7. The method for culturing endometrial cells according to claim 1, wherein the density of the inoculated culture flask is 103/cm2～105/cm2。
8. The method for culturing endometrial cells according to claim 1, wherein the culture flask is a T150 cell culture flask, and 20-30 ml of said cell culture system is added to each culture flask.
9. The method for culturing endometrial cells according to claim 1, wherein the culturing is performed in an environment of 5% CO at 37 ℃2The incubator of (1); the cell culture system was replaced every 3 days.
10. Endometrial cells cultured by the method for culturing endometrial cells according to claim 1.
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