CN113583962A - Induction culture medium and method for inducing and obtaining cancer cell persistent infection NDV - Google Patents
Induction culture medium and method for inducing and obtaining cancer cell persistent infection NDV Download PDFInfo
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Abstract
The invention relates to an induction culture medium and a method for inducing cancer cells to continuously infect NDV (Newcastle disease virus), wherein the induction culture medium comprises the following components: DMEM high-sugar medium, fetal calf serum, beta-mercaptoethanol, vitamin A, N2 cell culture additive and B27 cell culture additive. The invention can obviously improve the induction efficiency of cancer cells, the induction efficiency of the esophagus cancer cell EC9706 is improved by 99.9 times, and the induction efficiency of the stomach cancer cell BGC823 is improved by 260.75 times.
Description
Technical Field
The invention relates to an induction culture medium and a method for inducing cancer cells to continuously infect NDV, belonging to the technical field of biology.
Background
Oncolytic virus therapy is a method for treating cancer, and oncolytic virus can selectively kill tumor cells, has no obvious toxicity to normal tissues of human, and is theoretically a perfect material for treating cancer. However, oncolytic viruses are of many types, some of which have long been discovered, but viral oncolytic has not been the mainstream method for treating cancer so far, indicating that many problems still need to be solved. One very important reason is that the mechanisms by which cancer cells resist killing by oncolytic viruses are currently poorly understood.
Newcastle Disease Virus (NDV) is an oncolytic virus. The normal human body does not have anti-NDV antibody, so the infection is easy to succeed; NDV can selectively kill tumor cells, and has the advantages of light or unobvious damage to normal tissues of a human body and the like. However, when NDV is used for oncolytic therapy, the phenomenon that the oncolytic effect is weakened after multiple uses exists, and no research report reveals the mechanism of the phenomenon so far, so that NDV oncolytic therapy is not widely applied in clinic.
Cells sensitive to NDV may not have a mechanism for resisting virus and killing, while cancer cells insensitive to NDV have a mechanism for resisting virus and killing, but the differentiation degree is high, and it is difficult to find a proper reference cell to screen out which factors in the cells cause the resistance to virus and killing. Therefore, the lack of a suitable cell model has made it difficult to study cancer cells against the killing mechanism of oncolytic viruses.
There is a technology that can study which changes occur in a cell to affect the ability of the cell to obtain anti-viral killing by inducing the cell to produce anti-NDV virus. BHK-21 cells infected with NDV continuously are obtained by induction on BHK-21 cells at present, but other cells are not reported, and the most important reason is that the induction efficiency is extremely low, and in the case of BHK-21 cells which are easy to induce successfully, one million cells on average are only used for entering a continuous infection state after NDV infection. For common cancer cells, we try to find that the induction efficiency is lower after induction, and even the induction cannot be successfully induced. Based on such a problem, we tried to improve the induction efficiency, and finally formulated an induction medium which can improve the induction efficiency of cancer cells by about one hundred times and allow some cancer cells, which have not been reported, to obtain the persistent infection ability.
Disclosure of Invention
The invention provides an induction culture medium, which comprises the following specific scheme:
an induction culture medium, which consists of the following components: DMEM high-sugar medium, fetal calf serum, beta-mercaptoethanol, vitamin A, N2 cell culture additive and B27 cell culture additive.
Further, the volume ratio of the added fetal calf serum is set to be 2.5%, 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20% by changing the content of the fetal calf serum in the induction medium of the present invention. The highest induction efficiency was found when the fetal calf serum ratio was 12.5% and 15%. In view of cost, the volume concentration of fetal bovine serum in the induction medium is preferably 12.5%.
Furthermore, the induction culture medium added with beta-mercaptoethanol is compared with the induction culture medium without beta-mercaptoethanol, the influence on the induction efficiency of the cancer cells is detected, and the effect is found that the efficiency is not obviously improved with a control group after the beta-mercaptoethanol is added, but the stability of the cells successfully induced is increased in different batches of induction. Further, the amount of beta-mercaptoethanol added to the induction medium was determined to be 1mmol/L by a preferable experiment.
Furthermore, the addition of vitamin A is beneficial to the improvement of induction, and the addition amount of the vitamin A in the induction medium is determined to be 1 mu mol/L through a preferable experiment.
Furthermore, the N2 cell culture additive and the B27 cell culture additive can promote the improvement of the induction efficiency, and the addition amount of the N2 cell culture additive and the B27 cell culture additive in the induction medium is determined to be 1% through a preferable experiment.
The culture method of the induction culture medium comprises the following steps:
71 taking 800ml of DMEM high-sugar culture medium, adding 125ml of fetal calf serum, about 7 mu L of beta-mercaptoethanol, 2.865mg of vitamin A, 10ml of N2 cell culture additive and 10ml of B27 cell culture additive, and then fixing the volume to 1L to obtain a mixed solution;
and (5) sterile filtering the 72 mixed solution to obtain an induction culture medium, sealing, storing at 4 ℃, and effectively preserving within 30 days.
A method for inducing and obtaining cancer cells to be continuously infected with NDV is to use the induction culture medium provided by the invention and comprises the following steps:
after digestion of 51 cells, cells were resuspended with the induction medium and plated onto cell culture plates;
52 after the cells adhere to the wall, replacing a serum-free DMEM high-sugar culture medium, infecting NDV, and continuing replacing the induction culture medium for induction culture after 30 min;
and (3) after 3-5 days, replacing the induction culture medium once, and then replacing the induction culture medium according to the cell amount if the PH changes, and expanding and culturing the cells to obtain the cancer cells continuously infected.
Preferably, NDV in step 52 is La Sota strain.
The invention can obviously improve the induction efficiency of the cancer cells, and can induce various cancer cells to ensure that the various cancer cells obtain the continuous infection capability.
Detailed Description
Example 1:
induction of esophageal cancer cells EC9706 persistent infection with NDV:
preparing an induction culture medium, taking 800ml of a DMEM high-glucose culture medium, adding 125ml of fetal bovine serum, about 7 mu L of beta-mercaptoethanol, A2.865mg of vitamin, 10ml of N2 cell culture additive and 10ml of B27 cell culture additive, and then fixing the volume to 1L to obtain a mixed solution; the mixed solution is aseptically filtered to obtain an induction culture medium, and the induction culture medium is sealed and then stored at 4 ℃ and is effective within 30 days.
EC9706 cells were digested by conventional procedures and transferred to 6 48-well culture plates, 2 ten thousand cells per well, 3 of which were cultured with conventional medium (90% DMEM high sugar medium, 10% fetal bovine serum) and 3 of which were prepared with induction medium, each well finally containing 1ml of induction medium; after the cells adhere to the wall the next day, the cells are replaced by serum-free DMEM high-sugar medium, infected with NDV (La Sota strain, 10MOI), and are continuously replaced by conventional culture solution or induction culture medium for induction culture after 30 min. After about 3-5 days, after most of the cells die, the induction culture medium can be replaced once, the induction culture medium can be not replaced before the solution color changes (about to 7 days), after the solution color changes, the PH changes, a part of cells which are visible under a microscope are resistant to virus killing and can proliferate again, and then the induction culture medium is replaced periodically according to the cell amount, the cells are expanded and cultured, and the cancer cells which are continuously infected are obtained. Compared with the conventional culture solution group and the induction culture medium group, the induction effect is different. At about 15 days after induction, the number of clones was counted after clone formation, and the results showed that 3, 1 and 6 clones were produced from each plate in the conventional culture medium group and 359, 163 and 577 clones were produced in the induction medium group, respectively, suggesting that the induction medium was able to induce the production of persistently infected cells with high efficiency, which was 99.9 times higher than that in the conventional culture medium of the control group.
Example 2:
induction of gastric cancer cell line BGC823 persistent infection NDV:
digesting BGC823 cells by conventional operation, transferring the cells to 6 culture plates with 48 wells, 2 ten thousand cells per well, wherein 3 plates were cultured with conventional medium (90% DMEM high sugar medium, 10% fetal bovine serum), and the other 3 plates were cultured with the prepared induction medium, each well finally containing 1ml of induction medium; after the cells adhere to the wall the next day, the cells are replaced by serum-free DMEM high-sugar medium, infected with NDV (La Sota strain, 10MOI), and are continuously replaced by conventional culture solution or induction culture medium for induction culture after 30 min. After about 3-5 days, after most of the cells die, the induction culture medium can be replaced once, the induction culture medium can be not replaced before the solution color changes (about to 7 days), after the solution color changes, a part of the cells which are seen under a microscope are resistant to virus killing and proliferate again, and then the induction culture medium is replaced periodically according to the cell amount, the cells are expanded and cultured, and the cancer cells which are continuously infected are obtained. Compared with the conventional culture solution group and the induction culture medium group, the induction effect is different. At about 15 days after induction, the number of clones was counted after clone formation, and the results showed that 0, 3 and 1 clone was produced per plate in the conventional culture medium group and 324, 461 and 258 clones were produced in the induction medium group, respectively, suggesting that the induction medium was able to induce the production of persistently infected cells with high efficiency, which was 260.75 times higher than that in the conventional culture medium of the control group.
Claims (9)
1. An induction culture medium, which is characterized by comprising the following components: DMEM high-sugar medium, fetal calf serum, beta-mercaptoethanol, vitamin A, N2 cell culture additive and B27 cell culture additive.
2. The induction medium of claim 1, wherein: the volume ratio concentration of the fetal bovine serum in the induction culture medium is 12.5%.
3. The induction medium of claim 1, wherein: the concentration of beta-mercaptoethanol in the induction culture medium is 1 mmol/L.
4. The induction medium of claim 1, wherein: the concentration of vitamin A in the induction medium was 1. mu. mol/L.
5. The induction medium of claim 1, wherein: the volume ratio concentration of the N2 cell culture additive in the induction medium is 1%.
6. The induction medium of claim 1, wherein: the volume ratio concentration of the B27 cell culture additive in the induction medium is 1%.
7. The induction medium according to claim 1, wherein the culture method of the induction medium comprises the following steps:
71 taking 800ml of DMEM high-sugar culture medium, adding 125ml of fetal calf serum, about 7 mu L of beta-mercaptoethanol, 2.865mg of vitamin A, 10ml of N2 cell culture additive and 10ml of B27 cell culture additive, and then fixing the volume to 1L to obtain a mixed solution;
and (72) sterile filtering the mixed solution to obtain an induction culture medium.
8. A method for inducing persistent NDV infection of cancer cells, comprising the steps of using the induction medium of any one of claims 1-6, and:
after digestion of 51 cells, cells were resuspended with the induction medium and plated onto cell culture plates;
52 after the cells adhere to the wall, replacing a serum-free DMEM high-sugar culture medium, infecting NDV, and continuing replacing the induction culture medium for induction culture after 30 min;
and (3) after 3-5 days, replacing the induction culture medium once, and then replacing the induction culture medium according to the cell quantity if the PH changes, so as to expand and culture the cells, thereby obtaining the cancer cells with continuous infection.
9. The method of claim 8 for inducing persistent infection of cancer cells with NDV, wherein: in step 52, NDV is a La Sota strain.
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| AU2015352351A1 (en) * | 2014-11-27 | 2017-06-15 | Koninklijke Nederlandse Akademie Van Wetenschappen | Culture medium |
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| CN111876377A (en) * | 2020-07-07 | 2020-11-03 | 湖南师范大学 | Stem cell cardiomyocyte induced differentiation method based on autophagy and application thereof |
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| AU2015352351A1 (en) * | 2014-11-27 | 2017-06-15 | Koninklijke Nederlandse Akademie Van Wetenschappen | Culture medium |
| CN105087476A (en) * | 2015-08-31 | 2015-11-25 | 中国医学科学院北京协和医院 | Induced differentiation method of 3T3-L1 preadipocytes line |
| CN108531453A (en) * | 2017-03-01 | 2018-09-14 | 中国科学院动物研究所 | A method of converting non-neuronal cell to neuronal cell |
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