CN113493774A - Culture medium for improving hybridoma cell fusion rate - Google Patents
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- 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
- C12N5/10—Cells modified by introduction of foreign genetic material
- C12N5/12—Fused cells, e.g. hybridomas
- C12N5/16—Animal cells
- C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
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- C12N2501/30—Hormones
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Abstract
The invention discloses a culture medium for improving the fusion rate of hybridoma cells, and relates to the technical field of hybridoma cell fusion. Thereby achieving the technical effects of promoting cell fusion, maintaining good growth state, obviously improving fusion efficiency compared with the common HAT (HT) culture medium, having better cell subcloning culture state, shortening culture time and simultaneously reducing the probability of cell pollution.
Description
Technical Field
The invention relates to the technical field of hybridoma cell fusion, in particular to a culture medium for improving the hybridoma cell fusion rate.
Background
In order to increase the fusion rate of hybridoma cell fusion and stabilize the growth of the fused cells, mouse peritoneal macrophages are usually added during fusion and subcloning, and are cultured together with hybridoma cells as feeder cells.
However, the applicant of the present invention finds that the prior art has at least the following technical problems:
this method is cumbersome, time consuming and increases the chance of cell contamination.
Disclosure of Invention
Technical problem to be solved
In order to overcome the defects of the prior art, the embodiment of the invention provides a culture medium for improving the fusion rate of hybridoma cells, and solves the technical problems that the existing procedures are complicated and time-consuming, and the probability of cell pollution is increased.
(II) technical scheme
The invention is realized by the following technical scheme: the invention provides a culture medium for improving the fusion rate of hybridoma cells, which is prepared by adding nutrient solution, oxaloacetic acid, human insulin and interleukin 6 with certain concentration into HAT and HT culture media.
Preferably, the nutrient solution is culture supernatant of SP2/0 cells, and when the cell density is 80% -90%, the culture supernatant is collected, namely the nutrient solution.
Preferably, the culture medium used by the SP2/0 cell is specifically formulated as follows: the 10% fetal bovine serum + 88% DMEM +1% glutamine +1% sodium pyruvate.
Preferably, the HAT culture medium is specifically formulated as follows: 15% fetal bovine serum +84% DMEM +1% glutamine + HAT.
Preferably, the HT medium is specifically formulated as follows: 15% fetal bovine serum +84% DMEM +1% glutamine + HT.
Preferably, 2% nutrient solution, 150 mg/L oxaloacetate, 2 mg/L human insulin and 50 ng/L interleukin 6 are added to the culture medium in volume percentage.
Preferably, the nutrient solution is stored at-20 ℃.
(III) advantageous effects
One or more technical solutions in the embodiments of the present application have at least one or more of the following technical effects:
according to the culture medium for improving the hybridoma cell fusion rate provided by the embodiment of the invention, the HAT (HT) culture medium is added with nutrient solution, oxaloacetic acid, human insulin and interleukin 6 at certain concentrations, so that cell fusion can be promoted and a good growth state can be maintained. The nutrient solution in the research is culture supernatant of SP2/0 cells, and the key technology is that a culture medium used for culturing SP2/0 cells is used for culturing SP2/0, when the cell density is 80-90%, the culture supernatant is collected to be the nutrient solution, and the nutrient solution is placed at-20 ℃ for long-term storage and is taken at any time, so that the nutrient solution is convenient and time-saving. Compared with the common HAT (HT) culture medium, the HAT (HT) culture medium added with nutrient solution, oxaloacetic acid, human insulin and interleukin 6 can obviously improve the fusion efficiency, has better cell subcloning culture state and shortens the culture time. In addition, in the conventional cell fusion process, abdominal macrophages of the mouse are taken as a trophoblast to provide nutrition for the fused hybridoma cells. However, in the process of taking the abdominal cavity macrophages, the mouse needs to be dissected under aseptic operation, a blank medium (DMEM) is injected into the abdominal cavity of the mouse by using an injector and then extracted, and the process has high requirements and has the risk of pollution. The culture medium provided by the embodiment of the invention does not need to add abdominal cavity macrophages, and a series of processes such as mouse dissection and the like are avoided, so that the probability of cell pollution is reduced, and the technical problems that the existing process is complicated and time-consuming, and the probability of cell pollution is increased are solved.
Drawings
FIG. 1 is a photograph taken 7 days after cell fusion in a control group according to an embodiment of the present invention;
FIG. 2 is a photograph taken 7 days after fusion of group 1 cells in the example of the present invention;
FIG. 3 is a photograph taken 7 days after fusion of group 2 cells in example 2 of the present invention;
FIG. 4 is a photograph taken 7 days after fusion of group 3 cells in the example of the present invention;
FIG. 5 is a photograph taken 7 days after the fusion of the group 4 cells in the example of the present invention.
Detailed Description
The embodiment of the invention provides a culture medium for improving the fusion rate of hybridoma cells, which is used for solving the technical problems of complicated and time-consuming processes and increased probability of cell pollution in the prior art.
The technical scheme provided by the invention has the following general idea: the culture medium is prepared by adding nutrient solution, oxaloacetic acid, human insulin and interleukin 6 with certain concentration into HAT and HT culture medium, thereby achieving the technical effects of promoting cell fusion and maintaining good growth state, obviously improving fusion efficiency, realizing better cell subcloning culture state, shortening culture time and simultaneously reducing the probability of cell pollution compared with common HAT (HT) culture medium.
HAT medium refers to H, A, T three components added to a basic medium, which is used for the cell fusion step, HT medium refers to H, T two components added to a basic medium, which is used for the subcloning step, and the experiments in the examples of the present application include the cell fusion test and the subcloning test.
The technical solutions of the present invention are described in detail below with reference to the drawings and specific embodiments, and it should be understood that the specific features in the embodiments and examples of the present invention are described in detail in the technical solutions of the present application, and are not limited to the technical solutions of the present application, and the technical features in the embodiments and examples of the present application may be combined with each other without conflict.
Example one
Preparation of culture Medium
The specific formulation of the culture medium used for SP2/0 cells was as follows: 10% fetal bovine serum + 88% DMEM +1% glutamine +1% sodium pyruvate.
The SP2/0 cell, namely the mouse myeloma cell, is a cell commonly used in the current cell fusion, and can be fused with a mouse B cell to form a hybridoma cell under a specific condition due to the unlimited proliferation capacity, and the hybridoma cell retains the amphiphilic characteristic, can secrete an antibody and can be proliferated indefinitely.
Preparing HAT culture medium: 15% fetal bovine serum +84% DMEM +1% glutamine + HAT or HT medium: 15% fetal bovine serum +84% DMEM +1% glutamine + HT. Adding the components with different concentrations in the table 1 into the culture medium respectively according to the volume percentage, wherein the concentration 1 represents that 10ml of nutrient solution is added into 1L of culture medium, and the concentration 2 represents that 20ml of nutrient solution is added; the unit of human insulin is mg/L, wherein 1 concentration represents that 1mg of human insulin is added into 1L of culture medium, and 2 concentration represents that 2mg of human insulin is added; interleukin 6 and oxaloacetate, and so on.
In order to screen the optimal experimental dosage of different components, the components of the culture medium are proportioned from low concentration to high concentration, and the total amount is divided into 4 gradients. See table 1 specifically:
table 1: concentration ratio of each component of culture medium
| Components | Concentration 1 | Concentration 2 | Concentration 3 | Concentration 4 |
| Nutrient solution (%) | 1 | 2 | 5 | 10 |
| Interleukin 6 (ng/L) | 20 | 50 | 100 | 150 |
| Human insulin (mg/L) | 1 | 2 | 5 | 10 |
| Oxaloacetic acid (mg/L) | 50 | 150 | 200 | 300 |
The components were added to the conventional medium at the concentrations shown in Table 1, as shown in Table 2:
table 2: grouping of culture media for experiments
| Group of | Control group | Group 1 | Group 2 | Group 3 | Group 4 |
| Composition (I) | Conventional culture medium: 15% FBS +84% DMEM +1% Glu + HAT (HT) | Conventional Medium + concentration 1 | Conventional Medium + concentration 2 | Conventional Medium + concentration 3 | Conventional Medium + concentration 4 |
Example two
Hybridoma cell fusion assay
5 blank mice were used for spleen and growth log phase SP2/0 cells, and PEG was used as fusion agent for cell fusion. The fused hybridoma cells were cultured in the above 5 media, each laid on 5 plates, at 37 deg.C in 5% CO2And (5) carrying out static culture under the condition. Cells were observed 5 days, 7 days, and 10 days after the fusion, and the number of cloning wells, the number of cloning clusters, and the state of the cells were used as reference indices to evaluate the quality of the medium. As shown in table 3:
TABLE 3 hybridoma cell fusion test results
In the experiment, the number of cloning wells, the number of cloning clusters and the state of cells are taken as evaluation parameters, and from the experimental result, the culture effects of the group 1 and the control group are basically consistent and inferior to those of other 3 groups; the other 3 groups were almost indistinguishable when evaluated from the standpoint of cell culture efficiency, but the group 2 was most preferable in view of production cost.
EXAMPLE III
Subclone culture
In order to verify whether the culture medium supports the good growth state of the subcloned cells, the test is specially added in the process of preparing the interleukin 6 monoclonal antibody. 5 media were added dropwise to 5 96-well plates, one medium and one plate, 100 uL/well, one day before the assay. Taking positive cells after the first subclone screening, wherein the state of the positive cells is required to be good and the cell density reaches 80-90%, and performing monoclonal sorting by adopting a limiting dilution method. The cells were counted and then diluted to theoretically 1 cell per 100uL of the dilution, 100uL per well was added dropwise to a 96-well plate prepared in advance at 37 ℃ with 5% CO2And (5) carrying out static culture under the condition. Observation of cell status, single clone formation of a ViewThe time taken by the field density and the positive rate are used as reference to evaluate the quality of the culture medium. After the second subcloning was completed, the experimental procedure was repeated once more and the results were recorded. As shown in table 4:
TABLE 4 subclone culture test results
The subcloning experiment was repeated twice, and the cell state, the time taken for a single clone to form a visual field, and the positive rate were used as evaluation parameters, and the results showed that: each group had no difference in cell status and positive rate, but from the aspect of cell growth rate, 4 groups added with nutrients were faster than the control group, but group 1 was only faster by about 1 day, and the effect was not obvious. However, group 2, group 3 and group 4 can save the time cost of 2-3 days; in view of production cost, group 2 is the most preferable.
One or more technical solutions in the embodiments of the present application have at least one or more of the following technical effects:
according to the culture medium for improving the hybridoma cell fusion rate provided by the embodiment of the invention, the HAT (HT) culture medium is added with nutrient solution, oxaloacetic acid, human insulin and interleukin 6 at certain concentrations, so that cell fusion can be promoted and a good growth state can be maintained. The nutrient solution in the research is culture supernatant of SP2/0 cells, and the key technology is that a culture medium used for culturing SP2/0 cells is used for culturing SP2/0, when the cell density is 80-90%, the culture supernatant is collected to be the nutrient solution, and the nutrient solution is placed at-20 ℃ for long-term storage and is taken at any time, so that the nutrient solution is convenient and time-saving. Compared with the common HAT (HT) culture medium, the HAT (HT) culture medium added with nutrient solution, oxaloacetic acid, human insulin and interleukin 6 can obviously improve the fusion efficiency, has better cell subcloning culture state and shortens the culture time. In addition, in the conventional cell fusion process, abdominal macrophages of the mouse are taken as a trophoblast to provide nutrition for the fused hybridoma cells. However, in the process of taking the abdominal cavity macrophages, the mouse needs to be dissected under aseptic operation, a blank medium (DMEM) is injected into the abdominal cavity of the mouse by using an injector and then extracted, and the process has high requirements and has the risk of pollution. The culture medium provided by the embodiment of the invention does not need to add abdominal cavity macrophages, and a series of processes such as mouse dissection and the like are avoided, so that the probability of cell pollution is reduced, and the technical problems that the existing process is complicated and time-consuming, and the probability of cell pollution is increased are solved.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (7)
1. A culture medium for improving the fusion rate of hybridoma cells, which is characterized in that: the culture medium is HAT and HT culture medium added with nutrient solution, oxaloacetic acid, human insulin and interleukin 6 in certain concentration.
2. The culture medium for increasing the fusion rate of hybridoma cells according to claim 1, wherein the culture medium comprises: the nutrient solution is culture supernatant of SP2/0 cells, and when the cell density is 80-90%, the culture supernatant is collected, namely the nutrient solution.
3. The culture medium for increasing the fusion rate of hybridoma cells according to claim 2, wherein: the specific formula of the culture medium used by the SP2/0 cell is as follows: the 10% fetal bovine serum + 88% DMEM +1% glutamine +1% sodium pyruvate.
4. The culture medium for increasing the fusion rate of hybridoma cells according to claim 3, wherein the culture medium comprises: the HAT culture medium comprises the following specific formula: 15% fetal bovine serum +84% DMEM +1% glutamine + HAT.
5. The culture medium for increasing the fusion rate of hybridoma cells according to claim 3, wherein the culture medium comprises: the HT medium comprises the following specific formula: 15% fetal bovine serum +84% DMEM +1% glutamine + HT.
6. The culture medium for increasing the fusion rate of hybridoma cells according to claim 2, wherein: 2% nutrient solution, 150 mg/L oxaloacetate, 2 mg/L human insulin and 50 ng/L interleukin 6 are added to the culture medium according to volume percentage.
7. The culture medium for increasing the fusion rate of hybridoma cells according to claim 2, wherein: and storing the nutrient solution at-20 ℃.
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