CN114015645A - Method for culturing granular cells - Google Patents
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
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- C12N2500/00—Specific components of cell culture medium
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
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Abstract
The invention discloses a method for culturing granular cells, which comprises the steps of cleaning and digesting granular cell tissues, then suspending the granular cell tissues by using an MEM culture system, filtering the granular cell tissues, then centrifugally separating filtrate, then suspending precipitates by using the MEM culture system, inoculating a culture flask for culturing, and finishing the culture of the granular cells; the MEM culture system comprises stem cell extracting solution, human serum albumin and MEM culture medium. In the prior art, a tissue digestion method is adopted to separate the granular cells growing by monoclonal adherence, so that the purified granular stem cells can be obtained, and can be successfully proliferated under the culture of a DMEM culture medium (containing 10% fetal calf serum, 100U/mL of penicillin and 100U/mL of streptomycin), but the activity is low.
Description
Technical Field
The invention belongs to the biotechnology, and particularly relates to a method for culturing granular cells.
Background
Human ovarian malignant teratoma is the most common ovarian malignant germ cell tumor, so that the patients have menstrual disorder, amenorrhea, infertility and premature ovarian failure, and the protection of the ovarian function of the patients is of concern (see: the problem related to the protection of the ovarian function after the ovarian cancer operation by chemotherapy). In the pharmaceutical experiment, toxicity to physiological functional cells needs to be analyzed while studying toxicity to malignant cells, and ovarian granulosa cells are often used for comparison as functional cells of the ovary. Therefore, granular cell culture is a basic means, and the prior art mostly adopts a DMEM medium containing 10% fetal calf serum to culture granular cells, but the cultured cells are weak in activity.
Disclosure of Invention
The invention discloses a method for culturing granular cells, which is simple in composition, and the obtained cells are strong in activity and passable after 7-day culture. The applicant discloses that the conventional culture medium modified by the existing stem cell exosome freeze-dried powder is used for culturing endometrial cells, a good technical effect is achieved, and the method needs to improve the culture of granular cells. Therefore, the invention provides a method for culturing granular cells, which can obtain good culture effect different from the culture of inner membrane cells.
The invention adopts the following technical scheme:
a method of culturing granulosa cells, comprising the steps of: cleaning and digesting granular cell tissues, then suspending the granular cell tissues by using an MEM culture system, filtering the granular cell tissues, then centrifugally separating filtrate, then suspending the precipitate by using the MEM culture system, and inoculating a culture flask for culture to finish the culture of granular cells; the MEM culture system comprises a stem cell extracting solution, human serum albumin and a MEM culture medium, and preferably, the stem cell extracting solution and the human serum albumin are added into the MEM culture medium to obtain the cell culture system.
The granular cell tissue is human granular cell tissue, and as common knowledge, the residual tissue of the ovum is collected and is the existing product. In the invention, the residual granular cell tissue of the collected ovum is centrifuged, the upper layer liquid is sucked out, the residual tissue is washed by using an MEM culture medium, then collagenase working solution is added for digestion until the tissue disappears, then an MEM culture system is added, the mixture is filtered, the filtrate is centrifuged and separated, the obtained precipitate is resuspended by using the MEM culture system, and then the suspended precipitate is inoculated into a culture flask for culture, thus completing the culture of granular cells. Preferably, after inoculation of the culture flask, a new MEM culture system is replaced every 3 days.
In the invention, the stem cell extracting solution is an existing product, see CN111759866A previously disclosed by the applicant of the invention, the extracting solution is prepared by adopting physiological saline for induction, the components are various and rich in various stem cell exocrine cytokines, extracellular matrix, exosome vesicles and the like, and the unexpected technical effect can be achieved by culturing the granular cells by using the composite components.
In the invention, in the MEM culture system, the volume percentage of human albumin is 1-5%, preferably 2.5-4.5%, more preferably 2-4%, and most preferably 3.5%; the volume fraction of the stem cell extracting solution is 0.5-2%, preferably 0.8-1.5%, and most preferably 1%; the balance being MEM medium.
In the invention, the centrifugal separation parameter is 1500-2500 rpm/separation center for 1-10 minutes, preferably 2000 rpm/separation center for 5 minutes. When the culture bottle is inoculated, the inoculation density is 105Bottle-108Bottle, preferably 106Bottle, bottle is the existing product, such as corning company T75 cell culture bottle.
In the present invention, the culture environment is 37 ℃ and 5% CO2The incubator of (1); the culture bottles are T75 cell culture bottles, 10-30 ml of the MEM culture system is added into each culture bottle, and preferably 20ml of the MEM culture system is added into each culture bottle.
The granulosa cells play an important role in oocyte growth, follicular development and maturation as important functional cells in follicles, and the prior art shows that the addition of the granulosa cells in immature oocyte culture can restore the meiotic process or promote the expression of EGF-like proteins and the maturation of oocytes. Therefore, obtaining a large amount of high-quality ovarian granulosa cells in the research is the first condition for basic experimental research. The prior art has many reports about the culture of human ovarian granulosa cells, but the obtained cells are not satisfactory in terms of quantity and activity; in addition, in the prior art, a DMEM/F12 culture medium, human serum albumin, stem cell exosome freeze-dried powder and ultrapure water form a culture system for culturing endometrial cells, and the system is directly used for culturing ovarian granulosa cells, so that the culture effect is found to be poor. The invention creatively adopts the stem cell extract solution to replace the freeze-dried powder, greatly simplifies the preparation process, avoids the performance influence caused by freeze-drying-redissolution, and discloses a culture system with remarkable effect on ovarian granulosa cell culture through the change of the addition amount.
Detailed Description
All reagents of the invention are existing products, and specific tissue, cell processing method and testing method are conventional technologies.
In the present invention, the cell count test is a conventional method, and the number and the viability of the cells are detected by using a Countstar cell counter and trypan blue staining. Cell count and cell viability assays are common indicators of cell biological properties. When the cells are digested and passaged, the cells are attached to the wall after being suspended for a short time, then enter a latent period, then enter a logarithmic growth phase of cell mass division and proliferation, and stop proliferating after reaching a saturation density, and enter a plateau phase.
On the 7 th day of the culture, cell counting and cell viability detection by CCK8 (Shanghai Jingan' Biotech Co., Ltd.) were carried out, and the cells cultured in each culture system up to the 7 th day were cultured, and the cells were digested at 1X 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.
Referring to CN111759866A, primary human umbilical cord mesenchymal stem cells were cultured in cell number 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 micron, wherein the filtrate is a stem cell extract solution and is used for culturing the granular cells.
Freeze-drying the above stem cell extract solution by vacuum freeze dryer to obtain stem cell exosome freeze-dried powder, wherein the specific freeze-drying method is CN 113234663A; and taking 1mL of stem cell exosome freeze-dried powder corresponding to the stem cell extracting solution for the third comparative example.
The granular tissues come from hospitals, are washed by 0.9 percent of normal saline and are stored in conventional preservation solution, and the culture of the granular cells is not influenced by different human body sources; the granulosa cells are used as important functional cells in follicles and play an important role in the growth, follicular development and maturation of oocytes, and the prior art shows that the addition of the granulosa cells in the culture of immature oocytes can restore or promote the expression of EGF-like proteins and the maturation of oocytes through the meiosis process; therefore, obtaining a large amount of high-quality ovarian granulosa cells in the research is the first condition for basic experimental research. The invention obtains granular cells from the existing granular tissues through enzymolysis and digestion, and then the granular cells with good proliferation activity are obtained through the culture system formed by mixing MEM culture medium (Gibco), human serum albumin (Jetbelin) and stem cell extraction stock solution.
10 g of human granular tissue remaining after collecting ova was centrifuged at 1500rpm for 5 minutes, the upper follicular fluid was aspirated, 12mL of MEM medium (Gibco) was added, 3mL of 0.3% collagenase working solution (Sigma) was added, and digestion was carried out at 37 ℃ for 45 minutes to obtain a digestion solution which was used in the following examples and comparative examples and was repeatedly prepared.
Example a method of culturing granulosa cells, comprising the steps of:
1mL of the stem cell extract solution was added to 95.5mL of MEM medium, and 3.5mL of human serum albumin was added to obtain a cell culture system.
Adding 15mL cell digestive juice into 10mL cell culture system, mixing, filtering with 100uM cell filter screen, centrifuging at 2000 rpm for 5 min, discarding supernatant, resuspending obtained cell precipitate with 15mL cell culture system, and mixing according to 10mL6Cell/flask density inoculum flasks were added to T75 cell culture flasks as day 0 of cell growth at 37 deg.C with 5% CO2The cell culture system (15 ml) was renewed every 3 days, and the number of cells reached 7.2X 10 by the time of day 76Taking a picture of the cells, and observing that the growth state of the cells is good; after 7 days of culture, the cell activity was measured by the CCK8 method, and the absorbance at 12 hours, 24 hours, 36 hours, and 48 hours was 1.253、1.862、2.264、2.291。
Comparative example 1
Adding 3.5mL of human albumin into 95.5mL of MEM (minimum medium volume) medium, and adding Vascular Endothelial Growth Factor (VEGF) and Hepatocyte Growth Factor (HGF) to obtain a control culture system, wherein the Vascular Endothelial Growth Factor (VEGF) and the Hepatocyte Growth Factor (HGF) are both 10 ng/mL.
Referring to the method of example one, the control culture system of comparative example one was substituted for the cell culture system of example one, and the number of cells reached 4.2X 10 by day 7 after the cells were grown without change6When the cells were photographed, the growth morphology of the cells was poor, and the absorbance at 12 hours, 24 hours, 36 hours, and 48 hours after 7 days of culture was 0.645, 1.103, 1.345, and 1.581, respectively.
Comparative example No. two
Human serum albumin (3.5 mL) was added to 95.5mL of MEM medium to obtain a blank culture system.
Referring to the method of example one, the blank culture system of comparative example two was substituted for the cell culture system of example one, and the number of cells reached 3.1X 10 by day 7 of cell growth with the rest unchanged6When the cells were photographed, the cells had poor growth morphology, and the absorbance at 12 hours, 24 hours, 36 hours, and 48 hours after 7 days of culture was 0.489, 0.761, 0.894, and 1.124, respectively.
Human serum albumin (4.5 mL) was added to 96.5mL of MEM to obtain a blank culture system, and the number of cells reached 3.8X 10 in accordance with the method of example one6And (4) respectively.
Comparative example No. three
And mixing the stem cell exosome freeze-dried powder with 1mL of ultrapure water, adding the mixture into 95.5mL of MEM culture medium, and adding 3.5mL of human serum albumin to obtain a contrast cell culture system.
Referring to the method of example one, the comparative culture system of comparative example three was substituted for the cell culture system of example one, and the number of cells reached 6.2X 10 by day 7 after the cells were grown without change6Absorbance of the culture solution at 12 hours, 24 hours, 36 hours and 48 hours after 7 days of cultureRespectively 0.899, 1.308, 1.789, 1.998.
Existing methods
Referring to the method of example one, the cell culture system of example one was replaced with the existing culture system (DMEM medium containing 10% fetal calf serum, penicillin 100U/mL, streptomycin 100U/mL), and the remaining cells were unchanged until the number of cells reached 6.5X 10 by day 76After 7 days of culture, the absorbance at 12 hours, 24 hours, 36 hours and 48 hours was 0.881, 1.257, 1.972 and 2.001, respectively.
Example two
0.5mL of the stem cell extract solution was added to 95mL of MEM medium, and 4.5mL of human serum albumin was added to obtain a cell culture system.
The cell culture system of example one was replaced with the method of example one, and the number of cells reached 6.5X 10 by day 7 after the cells were grown without change6When the cells reached the standard of cell passage and cultured for 7 days, the absorbance at 12 hours and 48 hours was 1.061 and 2.116, respectively.
EXAMPLE III
2mL of the stem cell extract solution was added to 97mL of MEM medium, and 1mL of human serum albumin was added to obtain a cell culture system.
Referring to the method of example one, the cell culture system of example one was replaced, and the number of cells reached 6.1X 10 by day 7 when the cells were grown6After 7 days of culture, the absorbance at 12 hours and 48 hours was 0.878 and 2.008, respectively.
Example four
1mL of the stem cell extract solution was added to 96.5mL of MEM medium, and 2.5mL of human serum albumin was added to obtain a cell culture system.
The cell culture system of example one was replaced with the method of example one, and the number of cells reached 6.8X 10 by day 7 after the cells were grown without change6After 7 days of culture, the absorbance at 12 hours and 48 hours was 1.199 and 2.201, respectively.
EXAMPLE five
With reference to the method of example one, centrifuge at 2000 rpm for 5 minutesCentrifuging at 2500 rpm for 3 min, maintaining the rest, and allowing the cells to grow to 7 days until the cell number reaches 7.0 × 106After 7 days of culture, the absorbance at 12 hours and 48 hours was 1.195 and 2.279, respectively.
The compositions of the above examples and comparative culture systems and the culture results were as follows:
in the table, the raw materials are by volume.
In the prior art, a tissue digestion method is adopted to separate granular cells growing by monoclonal adherence, so that purified granular stem cells can be obtained and can be successfully proliferated under the culture of a DMEM culture medium (containing 10% fetal calf serum, 100U/mL of penicillin and 100U/mL of streptomycin); but the cell viability was slightly poor. The culture method disclosed by the invention has the advantages that the cells grow to the 7 th day, the cell number is high, the cell passage can be carried out, the proliferation activity of the granular cells is measured by using the conventional CCK-8 method, the growth curves are S-like, and the cell activity is high.
Claims (10)
1. A method of culturing granulosa cells, comprising the steps of: cleaning and digesting granular cell tissues, then suspending the granular cell tissues by using an MEM culture system, filtering the granular cell tissues, then centrifugally separating filtrate, then suspending the precipitate by using the MEM culture system, and inoculating a culture flask for culture to finish the culture of granular cells; the MEM culture system comprises stem cell extracting solution, human serum albumin and MEM culture medium.
2. The method for culturing granulosa cells according to claim 1, wherein the stem cell extract solution is induced from mesenchymal stem cells using physiological saline.
3. The method for culturing granular cells according to claim 2, wherein the MEM culture system is replaced with a new one every 3 days after the culture flask is inoculated.
4. The method for culturing granular cells according to claim 1, wherein the MEM cell culture system is obtained by adding a stem cell extract solution and human serum albumin to the MEM medium.
5. The method for culturing granulosa cells according to claim 4, wherein the percentage by volume of human serum albumin in the MEM culture system is 1-5%; the volume fraction of the stem cell extracting solution is 0.5-2%; the balance being MEM medium.
6. The method for culturing granulosa cells according to claim 5, wherein the percentage by volume of human serum albumin in the MEM culture system is 2-4%; the volume fraction of the stem cell extracting solution is 0.8-1.5%; the balance being MEM medium.
7. The method for culturing granulocytes of claim 1, wherein the centrifugation is performed at 1500-2500 rpm for 1-10 minutes.
8. The method for culturing granular cells according to claim 1, wherein the seeding density is 10 when the culture flask is seeded5Bottle-108A bottle.
9. The application of the stem cell extracting solution in the culture of granular cells or the preparation of a granular cell culture system.
10. Granulosa cells cultured by the method for culturing granulosa cells according to claim 1.
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