CN109880796B - Cell culture method for rapidly obtaining primary cells in vitro - Google Patents
Cell culture method for rapidly obtaining primary cells in vitro Download PDFInfo
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- CN109880796B CN109880796B CN201910212202.5A CN201910212202A CN109880796B CN 109880796 B CN109880796 B CN 109880796B CN 201910212202 A CN201910212202 A CN 201910212202A CN 109880796 B CN109880796 B CN 109880796B
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Abstract
The invention relates to the technical field of cell culture, and particularly provides a cell culture method for rapidly obtaining primary cells in vitro. The invention provides application of RM cells in cell culture, wherein the RM cells are used as feeder cells in the cell culture, so that the cost is low, the operation is simple, and the cost of the cell culture, particularly the primary cell culture, in basic research and clinical application is reduced to a great extent. RM cells are different from other feeder layer cells, are safe, have low potential risk and strong universality, and can be used for culturing various cells.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a cell culture method for rapidly obtaining primary cells in vitro.
Background
Cell culture refers to a method of simulating in vivo environment (sterile, proper temperature, pH value, certain nutritional conditions, etc.) in vitro to enable the cells to survive, grow, reproduce and maintain the main structure and function. The cell culture includes primary culture and subculture.
The primary culture is also called as primary culture, which refers to the primary culture of cells, tissues and organs directly taken out of the body, the primary culture has short in vitro time and similar genetic characters to the cells in the body, and is suitable for the research of cell morphology, function, differentiation and the like. More strictly, primary culture refers to culture before successful passage, when the cells retain the basic properties of the original cells, and if normal, still retain the diploid number. However, in practice, the cultured cells within the first to tenth generations are generally collectively referred to as primary cell culture.
The primary culture of cells is divided into feeder layer cell culture and pure cytokine culture. Feeder cells are monolayers of certain cells treated with mitotic blockers, and during the primary or primary culture phase, cells are generally dependent on feeder cells that secrete growth factors necessary for their survival and proliferation in vitro. The growth factors secreted by different types of feeder cells are slightly different, but all require that the feeder cells do not divide and proliferate during culture and remain metabolically active. Feeder cells are commonly used as growth promoters and differentiation inhibitors in cell culture. The culture mode of the feeder layer cells is relatively cheap and has stable effect, but the feeder layer cells in the culture process are generally tumor cells and have potential risks. Therefore, it is important to provide a novel feeder layer cell with strong versatility and a method for culturing the cell.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide an application of RM cells in cell culture, so as to alleviate the technical problems of poor universality, high cost, potential risk and low success rate of primary cell culture in the prior art.
The second purpose of the present invention is to provide a feeder layer cell to alleviate the technical problems of high cost and potential risks of feeder layer cells in the prior art.
The third objective of the present invention is to provide a method for preparing feeder layer cells, so as to alleviate the technical problems of high cost and complicated operation of the prior art.
The fourth purpose of the invention is to provide the application of the feeder layer cells in cell culture.
The fifth objective of the present invention is to provide a cell culture method to alleviate the problems of complex cell culture, high cost and low success rate in the prior art.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
use of RM cells in cell culture.
Further, the RM cells are feeder layer cells;
preferably, the cell culture is a cell primary culture;
preferably, the cell comprises a tumor cell or a normal cell;
preferably, the normal cells comprise normal epithelial cells;
preferably, the sources of RM cells include commercially purchased RM cells and legally obtained RM tissue specimens.
A feeder layer cell comprising RM cells for use as described above.
Further, the number of passages of the RM cells is 0-5.
According to the preparation method of the feeder layer cells, the RM cells subcultured for 0-5 times are treated by a mitotic blocker to obtain the feeder layer cells.
Further, the subculture medium is a DMEM medium containing 5-20% FBS;
preferably, the mitotic blocker is mitomycin;
preferably, the concentration of mitomycin is 1 to 20. mu.g/mL.
The feeder layer cell is applied to cell culture.
Further, the cell culture is a cell primary culture;
preferably, the cell comprises a tumor cell or a normal cell;
preferably, the normal cells comprise normal epithelial cells.
A cell culture method comprising co-culturing the feeder layer cells and target cells.
Further, the target cell includes a tumor cell or a normal cell;
preferably, the normal cells comprise normal epithelial cells;
preferably, the tumor cells comprise lung cancer cells, breast cancer cells, prostate cancer cells, kidney cancer cells or stomach cancer cells;
preferably, the cell culture is a cell primary culture.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides application of RM cells in cell culture, wherein the RM cells are used as feeder cells in the cell culture, so that the cost is low, the operation is simple, and the cost of the cell culture, particularly the cell primary cell culture, in basic research and clinical application is reduced to a great extent. RM cells are different from other feeder layer cells, are safe, have low potential risk and strong universality, and can be used for culturing various cells. The feeder layer cell culture method is simple and convenient, does not need to add other special factors, and has short time consumption and low cost. The cell culture method provided by the invention has high repeatability, high success rate and good safety, improves the cell culture efficiency, and is used for clinical tumor drug screening or tumor mechanism research and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a cell morphology map of RM cells in example 1;
FIG. 2 is a cell morphology map of RM cells in example 2;
FIG. 3 is a graph showing the results of treating RM cells with mitomycin at different concentrations in example 3, wherein mitomycin was used at 4. mu.g/mL, 8. mu.g/mL, 12. mu.g/mL, 16. mu.g/mL, 18. mu.g/mL and 20. mu.g/mL in the order from graph a to graph f;
FIG. 4 is a cultured cell morphology of feeder-free lung cancer cells in the experimental examples of the present invention;
FIG. 5 is a diagram showing the morphology of cells co-cultured with feeder cells in the test example of the present invention;
FIG. 6 is a cultured cell morphology of feeder-free breast cancer cells in an experimental example of the present invention;
FIG. 7 is a diagram showing the morphology of breast cancer cells co-cultured with feeder cells in the experimental examples of the present invention;
FIG. 8 is a diagram showing the morphology of cells co-cultured with prostate cancer cells and feeder cells in the experimental examples of the present invention;
FIG. 9 is a cell morphology of kidney cancer cells cocultured with feeder layer cells in the experimental example of the present invention;
FIG. 10 is a diagram showing the morphology of gastric cancer cells co-cultured with feeder cells in the experimental examples of the present invention;
FIG. 11 is a diagram showing the morphology of cells co-cultured with feeder layer cells in normal renal epithelial cells in an experimental example of the present invention;
FIG. 12 is a diagram showing the morphology of cells co-cultured with feeder layer cells in normal prostate epithelial cells in the experimental examples of the present invention;
FIG. 13 is a diagram showing the morphology of normal mammary epithelial cells cocultured with feeder cells in the experimental examples of the present invention;
FIG. 14 is a diagram showing the morphology of cells co-cultured with feeder layer cells in normal lung epithelial cells in the experimental examples of the present invention;
FIG. 15 is a diagram showing the morphology of p0 generation cells co-cultured with feeder layer cells in normal renal epithelial cells in an experimental example of the present invention;
FIG. 16 is a schematic diagram showing the co-culture of normal renal epithelial cells and feeder layer cells for p1 generation in the experimental examples of the present invention;
FIG. 17 is a cell morphology of p2 generation cocultured with feeder layer cells in kidney normal epithelial cells in the experimental example of the present invention.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.
In the present invention, all the embodiments and preferred methods mentioned herein can be combined with each other to form a new technical solution, if not specifically stated.
In the present invention, all the technical features mentioned herein and preferred features may be combined with each other to form a new technical solution, if not specifically stated.
In the present invention, the percentage (%) or parts means the weight percentage or parts by weight with respect to the composition, if not otherwise specified.
In the present invention, the components referred to or the preferred components thereof may be combined with each other to form a novel embodiment, if not specifically stated.
In the present invention, unless otherwise stated, the numerical range "a-b" represents a shorthand representation of any combination of real numbers between a and b, where a and b are both real numbers. For example, a numerical range of "6 to 22" means that all real numbers between "6 to 22" have been listed herein, and "6 to 22" is simply a shorthand representation of the combination of these values.
The "ranges" disclosed herein may have one or more lower limits and one or more upper limits, respectively, in the form of lower limits and upper limits.
In the present invention, unless otherwise specified, the individual reactions or operation steps may or may not be performed in sequence. Preferably, the reaction processes herein are carried out sequentially.
Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.
The present invention provides the use of RM cells in cell culture. RM cells (human embryonic villi cells) refer to RM tissue specimens obtained legally, and cultured cells are called RM cells, which become feeder RM cells after being treated with mitotic blockers. The RM tissue sample can be, but is not limited to, the RM tissue sample remaining from the hospital chromosome test. It should be noted that the RM cells referred to in the present invention are not derived from embryos having a potential to develop into humans, and may be RM cells purchased from commercial sources.
RM cells are feeder layer cells in cell culture. RM cells are used as feeder cells in cell culture, so that the cost is low, the operation is simple, and the cost of cell culture, particularly cell primary culture, in basic research and clinical application is reduced to a great extent. RM cells are different from other feeder layer cells, are safe, have low potential risk and strong universality, and can be used for culturing various cells.
In some embodiments, the cell culture is a cell primary culture.
In some embodiments, the cell comprises a tumor cell or a normal cell, wherein the normal cell comprises a normal epithelial cell. Both tumor cells and normal cells can realize in-vitro culture and amplification by using RM cells, so that the cell culture efficiency is improved, and the universality is high.
A feeder layer cell comprising the RM cell described above.
In some embodiments, the number of passages of the RM cells is 0-5. Since the RM cells within 6 generations can maintain good growth activity per se, the number of passages of the RM cells is not preferably more than 5.
A method for preparing feeder layer cells comprises treating RM cells subcultured for 0-5 times with mitotic blocker to obtain feeder layer cells. The feeder layer cell culture method is simple and convenient, does not need to add other special factors, and has short time consumption and low cost.
In some embodiments, the subculture medium is DMEM medium containing 5-20% FBS.
In some preferred embodiments, the RM cells are derived from legally obtained RM tissue specimens, digested with trypsin, resuspended in 10% FBS-containing DMEM media, inoculated into a T25 flask, placed at 37 deg.C and 5% CO2Cultured in an incubator. For P0 generation cells, the growth rate was slower, and every 2 days, whether the cells were observed under an inverted microscopeWhen the cells climb out, the liquid is changed to change the floating dead cells and the tissue residues which are not attached to the wall. Because the cells in the P0 generation grow slowly, the cells can be passaged when the cells grow to about 60 percent, and the cells after the P0 generation are subjected to expansion passage when the cells grow to about 90 percent according to the normal cell passage requirement.
In some embodiments, the mitotic blocker is mitomycin.
In some embodiments, the concentration of mitomycin is from 1 to 20. mu.g/mL.
The invention provides application of the feeder layer cells in cell culture.
In some embodiments, the cell culture is a primary cell culture, the cells comprising tumor cells or normal cells, wherein the normal cells comprise normal epithelial cells.
A cell culture method comprising co-culturing the feeder layer cells and target cells. The cell culture method provided by the invention has high repeatability, high success rate and good safety, improves the cell culture efficiency, and is used for clinical tumor drug screening or tumor mechanism research and the like.
In some embodiments, the target cell comprises a tumor cell or a normal cell, wherein the normal cell comprises a normal epithelial cell. The cell culture method is suitable for various cells, has strong universality, and can stably and massively amplify tumor cells such as but not limited to lung cancer cells, breast cancer cells, prostate cancer cells, kidney cancer cells or stomach cancer cells and the like, and normal epithelial cells such as but not limited to lung epithelial cells, breast epithelial cells, prostate epithelial cells, kidney epithelial cells or stomach epithelial cells and the like.
In some embodiments, the cell culture is a cell primary culture.
The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
EXAMPLE 1 culture of RM cells
Sources of materials in this exampleComprises the following steps: legal RM tissue specimens that can be used as feeder layers were collected. Soaking in PBS containing 1% double antibody (streptomycin/penicillin) for 2min, discarding PBS, cutting into small tissue blocks with small surgical scissors, transferring into 15mL centrifuge tube, adding appropriate amount of trypsin, digesting at 37 deg.C for 5-10 min until tissue blocks are obviously reduced and pancreatin suspension becomes turbid, and adding serum-containing culture medium to stop digestion. Centrifuging at 400g for 5min to obtain cell precipitate, resuspending in DMEM medium containing 10% FBS, inoculating into T25 culture flask, standing at 37 deg.C and 5% CO2Cultured in an incubator. The morphology of the cells is shown in FIG. 1.
EXAMPLE 2 passage expansion of RM cells
And for the P0 generation cells, the growth speed is slow, whether the cells climb out or not is observed under an inverted microscope every 2 days, and when the cells climb out, liquid changing treatment is carried out to change floating dead cells and non-adherent tissue residues. Since the cells of the P0 generation grow slowly, the cells can be passaged when the cells grow to about 60%, the cells are digested with 0.125% of trypsin, and passaged at a ratio of 1:3 to expand the cells. Cells after P0 passage are expanded and passed at a ratio of 1:3 by 0.125% of digested cells when the cells grow to about 90% according to the normal cell passage requirement. The cell morphology of RM cells is shown in figure 2.
EXAMPLE 3 preparation of feeder layer RM cells
RM cells used for preparing feeder layer cells need to maintain good growth activity within 6 generations, so that when the RM cells are expanded to P5, the RM cells are not expanded again. When the cells of the P5 generation grow to 90%, the culture medium is discarded, the cells are moistened by PBS and treated for 2h at 37 ℃ by DMEM culture solution containing mitomycin with the final concentration of 4 ug/mL, the culture solution is sucked by a pipette, PBS is added for moistening and washing for 2 times, 0.125% of trypsin is used for digesting the cells, 400g is used for centrifuging for 5min, precooled frozen stock solution of 10% DMSO and 90% FBS is used for re-suspending the cell sediment, the cell sediment is placed in a freezing storage box and stored in a refrigerator at minus 80 ℃ for one month for standby, and the cell sediment needs to be transferred to a liquid nitrogen tank for long-term storage.
The results of treating feeder RM cells with mitomycin at different concentrations are shown in FIG. 3, where a-f are the results of treatments with mitomycin at concentrations of 4. mu.g/mL, 8. mu.g/mL, 12. mu.g/mL, 16. mu.g/mL, 18. mu.g/mL, and 20. mu.g/mL, respectively.
EXAMPLE 4 Collection and processing of target specimens
Collecting fresh tumor tissues and paracarcinoma tissues of cancer patients in Shenzhen people hospital, signing an informed consent with all samples, adding a tissue preservation solution, and taking back on ice for later use. The tissue was placed in a 6 cm petri dish, pre-cooled PBS containing diabase (streptomycin/penicillin) was added, the tissue was repeatedly washed 3-5 times up and down, cut into 1 mm × 1 mm × 1 mm tissue pieces with surgical scissors, transferred to a 15mL centrifuge tube containing 4mL of mixed digestive enzymes, and transferred to a 37 ℃ incubator for digestion for 0.5 h-3 h, and the degree of digestion of the tissue was observed every half an hour. When the tissue mass is obviously reduced and the digestive enzyme solution is obviously turbid, 400g and 5min are observed, cell precipitates are collected by centrifugation at 4 ℃, 10 mL of DMEM medium is used for resuspension, 70-um cell sieves are used for filtration, filtrate is collected into a new centrifuge tube, and 400g and 5min are used for collecting cells by centrifugation at 4 ℃.
Example 5 Co-culture of feeder layer RM cells and Primary cells
Taking out the prepared feeder layer RM cells from a liquid nitrogen tank or a refrigerator at minus 80 ℃, quickly placing the cells into a water bath kettle at 37 ℃ for quick shaking until the frozen solution is completely melted, transferring the cell suspension into a 15mL centrifuge tube containing 4mL of DMEM medium, centrifugally collecting cell precipitates at 400g for 5min, re-suspending the cell precipitates with 10% of FBS-containing DMEM medium, and carrying out 2 x 10 resuspension4/cm2The cell suspension was seeded into 24-well plates. The feeder layer RM cells can grow adherently after 3-5 h, and the feeder layer RM cells which grow adherently can be placed in an incubator for later use. The tissue cells treated in example 4 were resuspended in F medium and seeded into adherent-growing feeder RM cells at 37 ℃ in 5% CO2The growth state of the cells was observed every day and recorded by photographing under an inverted microscope.
The F culture medium comprises 73.7 mL of complete culture medium DMEM, 25 mL of F12 nutrient mix, 0.1 mL of hydrocortisone/EGF mix (25 ug/mL of hydrocortisone final concentration, EGFP 0.125ug/mL), 0.1 mL of insulin, 1 mL of penicillin/streptomycin/amphotericin B mixed solution and 0.1 mL of factor Y per 100 mL. Wherein the complete medium 100 mL DMEM includes 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin/amphotericin B mixture.
Test examples
The results of the co-culture of lung cancer cells with feeder cells and the control group with feeder cells free but otherwise identical conditions are shown in FIG. 4 (without feeder cells) and FIG. 5 (with feeder cells), respectively.
The results of the co-culture of breast cancer cells with feeder cells and the control group without feeder cells are shown in FIG. 6 (without feeder cells) and FIG. 7 (with feeder cells), respectively.
The results of co-culturing prostate cancer cells, kidney cancer cells, and stomach cancer cells with feeder layer cells of the present invention are shown in FIGS. 8 to 10.
The results of co-culturing kidney normal epithelial cells, prostate normal epithelial cells, breast normal epithelial cells, and lung normal epithelial cells with feeder layer cells of the present invention are shown in FIGS. 11 to 14, respectively.
The invention also subcultures the kidney normal epithelial cells and feeder layer cells, and the shapes of the kidney normal epithelial cells of p0-p2 generation are shown in figures 15-17.
The experimental results show that the feeder layer RM cells have good effect when applied to cell culture, particularly cell primary culture, have strong universality as feeder layer cells, simple operation, low cost, good repeatability, high success rate and guaranteed safety, and are suitable for large-scale use.
While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
Claims (3)
- Use of RM cells, which are human embryonic villi cells, which are tumor cells or normal cells, in cell culture;the normal cell is a kidney normal epithelial cell, a prostate normal epithelial cell, a breast normal epithelial cell or a lung normal epithelial cell;the tumor cell is lung cancer cell, breast cancer cell, prostate cancer cell, renal cancer cell or gastric cancer cell;the RM cells serve as feeder layer cells in cell culture;the cell culture is cell primary culture.
- 2. The use according to claim 1, wherein said source of RM cells comprises commercially available RM cells or legally obtained RM tissue specimens.
- 3. A cell culture method comprising co-culturing feeder cells with target cells;treating RM cells subjected to subculture for 0-5 times by using mitomycin of 1-20 mu g/mL to obtain feeder layer cells; the RM cell is a human embryonic villus cell;the subculture medium is a DMEM medium containing 5-20% FBS;the target cell is a tumor cell or a normal cell;the normal cell is a kidney normal epithelial cell, a prostate normal epithelial cell, a breast normal epithelial cell or a lung normal epithelial cell;the tumor cell is lung cancer cell, breast cancer cell, prostate cancer cell, renal cancer cell or gastric cancer cell;the cell culture is cell primary culture.
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