CN114854690A - Additive for primary culture of cancer cells and culture medium and application thereof - Google Patents

Additive for primary culture of cancer cells and culture medium and application thereof Download PDF

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CN114854690A
CN114854690A CN202210418768.5A CN202210418768A CN114854690A CN 114854690 A CN114854690 A CN 114854690A CN 202210418768 A CN202210418768 A CN 202210418768A CN 114854690 A CN114854690 A CN 114854690A
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cancer cells
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武瑞琴
王正远
杨海
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Nanning Yunmifang Biomedical Technology Co ltd
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Abstract

The invention provides an additive for primary culture of cancer cells, a culture medium and application thereof, belonging to the technical field of medical treatment. The additive for primary culture of cancer cells is characterized by comprising at least three of cholera toxin, R-spondin, A-83-01 and Y-27632. The media used for primary culture of cancer cells included complete DMEM media and F12 nutrient mix; the F12 nutritional blend includes at least three of cholera toxin, R-spondin, A-83-01, and Y-27632. The invention develops a culture system independent of trophoblast cells by optimizing a screening culture method and a culture medium formula, has high success rate, greatly reduces the difficulty of large-scale preparation of the culture medium, and is easier to establish a stable quality control system, thereby realizing commercialization.

Description

Additive for primary culture of cancer cells and culture medium and application thereof
Technical Field
The invention relates to the technical field of detection kits, in particular to an additive for primary culture of cancer cells, a culture medium and application thereof.
Background
The primary tumor cells are just separated from the tissues, so that the biological characteristics of the primary tumor cells are not changed greatly, and the biological characteristics of the original tumor cells can be well maintained. Because of the genetic background difference, the pathogenesis and treatment sensitivity of different patients are individually different, the primary culture of autologous tumor cells is an important means for researching the tumor pathogenesis and the molecular biological characteristics of tumor cells of patients.
The artificial simulation of the in vitro survival environment of tumor cells is the basis of primary culture of the tumor cells, and the survival environment of the tumor cells needs to be mainly a culture medium except for environment factors such as sterility, temperature, pH value, humidity and the like, and provides nutrient substances required by growth of the cells. At present, the primary culture and the establishment of the cell line are most commonly carried out by a basic culture solution containing 10 percent of fetal calf serum. However, the success rate of the tumor cell culture technology for culturing primary tumor cells is very low. This is because the serum-containing medium composition is undefined; furthermore, it is possible to promote the growth of some cells (e.g. fibroblasts) and inhibit the growth of other cells (e.g. epidermal cells) by using serum which contains some substances toxic to the cells, and thus the growth of the cells is affected, or even the cells are killed. Mycoplasma and viruses are possibly brought in the serum drawing process, potential influence is generated on cells, and experiment failure or unreliability of experiment results can be caused. The serum also has the defects of large batch difference, unstable performance and the like. Serum-free culture is more beneficial to cell growth, batch-to-batch consistency is high, heterogeneity from serum can be eliminated, certainty is increased, and animal ethical disputes do not exist.
The development of some serum-free culture media for primary tumor cells in recent years improves the success rate of primary tumor cell culture. However, in the process of culturing primary tumor cells, the conventional serum-free culture medium still needs to introduce the irradiated trophoblast cells with different sources into a culture system, so that the establishment of the culture system is too complicated and inconvenient to operate, and simultaneously, the standard and large-scale production of the kit is challenged, the mass production is difficult, and simultaneously, the fibroblast which is a main mixed component in the primary tumor cells cannot be effectively inhibited and removed.
Disclosure of Invention
The invention aims to provide an additive for primary culture of cancer cells, a culture medium and application thereof, and develops a culture system independent of trophoblast cells and/or serum by optimizing and screening a culture method and a culture medium formula, wherein the culture system can realize one or more of the following purposes: the success rate is high; the difficulty in large-scale preparation of the culture medium is reduced; it is easy to establish a stable quality control system, and further commercialization is realized.
To achieve one or more of the above objects, the present invention provides the following technical solutions.
The present invention provides an additive for primary culture of cancer cells comprising at least three of cholera toxin, R-spondin (e.g., R-spondin 1, R-spondin 2, R-spondin 3, and R-spondin 4), A-83-01, and Y-27632; exemplary are cholera toxin, R-spondin, Y-27632, in a mass ratio of 1: (2-3): (2-5); cholera toxin, R-spondin and A-83-01, wherein the mass ratio is 1: (2-3): (1-4); cholera toxin, A-83-01 and Y-27632, wherein the mass ratio is 1: (1-4): (2-5); r-spondin, A-83-01 and Y-27632, wherein the mass ratio is 1: (2-4): (1-3).
Preferably cholera toxin, R-spondin, Y-27632, or cholera toxin, A-83-01 and Y-27632.
As a further improvement of the present invention, there are included cholera toxin, R-spondin, A-83-01, and Y-27632.
Preferably, the mass ratio of the cholera toxin to the R-spondin to the A-83-01 to the Y-27632 is 1: (2-3): (1-4): (2-5).
The present invention further provides a medium for primary culture of cancer cells comprising the above-described supplement for primary culture of cancer cells, preferably the medium comprises complete DMEM medium and F12 nutrient cocktail, more preferably the supplement is comprised in the F12 nutrient cocktail.
As a further improvement of the invention, the F12 nutrient mixture also comprises hydrocortisone, EGF, insulin, amphotericin B and gentamicin.
Preferably, the mass ratio of the hydrocortisone, the EGF, the insulin, the amphotericin B and the gentamicin is (2-4): (1-3): (2-5): (1-5): (3-5).
As a further improvement of the invention, the complete DMEM medium may or may not contain 5-10% serum.
As a further improvement of the invention, the volume ratio of the complete DMEM medium to the F12 nutrient mixture is (2-4): 1.
the present invention also provides a method for culturing cancer cells, which comprises the step of culturing cancer cells in the culture medium of the present invention.
In a further improvement of the present invention, the cancer cell is at least one selected from the group consisting of a uterine cancer cell, a cervical cancer cell, an ovarian cancer cell, a renal cancer cell, an intestinal cancer cell, a gastric cancer cell, a head and neck cancer cell, a lung cancer cell, a breast cancer cell, a bladder cancer cell, a skin cancer cell, a melanoma cell, a pancreatic cancer cell, and a bile duct cancer cell, and is preferably a uterine cancer cell or a cervical cancer cell.
The present invention also provides a method for screening a drug sensitive to a cancer patient, which comprises culturing cancer cells derived from the patient in the culture medium of the present invention, and detecting sensitivity of the cultured cancer cells to a candidate drug in vitro. If a cancer cell is found to be sensitive to a candidate drug (e.g., can kill or inhibit proliferation of a cancer cell) by in vitro testing, then the candidate drug is sensitive to the cancer patient; otherwise, it is not sensitive.
The invention further provides an application of the culture medium for primary culture of cancer cells in tumor drug sensitivity screening.
Exemplary or as a further development of the invention, the steps of the specific application may be as follows:
s1, separating tumor tissues:
s101, rapidly washing the excised tissue specimen with an ethanol solution, washing with a PBS (phosphate buffer solution) solution at the temperature of 2-5 ℃, and transferring to a sterile culture dish;
s102, removing residual adipose tissues;
s103, cutting a fresh specimen into small blocks with the diameter smaller than 10mm to obtain tissue fragments;
s2, primary cell culture:
s201, diluting a collagenase/hyaluronidase solution with a concentration of 10 times by using an F culture medium, and placing the solution in a tube;
s202, mixing the F culture medium/collagenase/hyaluronidase with dispase;
s203, transferring the tissue fragments obtained in the step S103 to a tube containing F culture medium/collagenase/hyaluronidase/dispase, and incubating for 1-3h on a swing platform at 35-40 ℃;
s204, after digestion, centrifuging the cells and removing supernatant;
s205, resuspending the cell pellet in the complete DMEM/F12 culture medium, filtering the cell suspension into a new centrifuge tube through a 100-mum cell filter, centrifuging the cells and discarding the supernatant;
s3, testing drug sensitivity:
s301, digesting the cells cultured in the step S205 into a single cell suspension, counting, diluting, and inoculating to a 96-well plate, wherein the total number of the cells in each well is 3000;
s302, carrying out adherent culture for 10-15h, adding a DMSO solution of the drugs to be detected into corresponding holes, wherein the highest administration concentration of each drug is 50Um, and diluting 5 concentrations 10 times below to obtain 6 administration concentrations;
s303, continuously culturing for 48-72 hours after administration, measuring the cell activity by a fluorescence method, and calculating the IC of each medicament 50 The value is obtained.
As a further improvement of the invention, the time for the ethanol solution to rapidly wash does not exceed 3 s; the concentration of the ethanol solution is 95-100%.
Cholera toxin is a protein consisting of two different subunits that are not covalently linked. Each toxin molecule has a subunit a and a plurality of subunits B.
R-spondin is a secreted protein known to be involved in the activation and regulation of Wnt signaling pathways, including R-spondin 1, R-spondin 2, R-spondin 3 and R-spondin 4. In the additive or the medium of the present invention, two or more of R-spondin may be used in combination. Of course, the R-spondin can be a variant of the R-spondin, e.g., an active fragment thereof, or a fusion protein of the R-spondin, provided that the activity of the R-spondin is possessed.
A-83-01 is a TGF-beta signaling inhibitor, capable of selectively inhibiting multiple kinases.
Y-27632 is a ROCK inhibitor and may be derived from Sigma without limitation.
The invention has the following beneficial effects: the invention develops a culture system independent of trophoblast cells by optimizing a screening culture method and a culture medium formula, has high success rate, greatly reduces the difficulty of large-scale preparation of the culture medium, and is easier to establish a stable quality control system, thereby realizing commercialization. Although the components of the additive of the present invention are known and may even be used for the culture of cells, such as stem cells, the inventors of the present invention have unexpectedly found that the use of the additive in cancer cells or tumor cells derived from a cancer or tumor patient can improve the success rate of the culture, which is of great benefit for the precise medical treatment or precise drug selection of cancer.
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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 only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a graph comparing the sensitivity of each tumor drug in example 8.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Comparative example
This example provides a medium for primary culture of cancer cells comprising 200mL of complete DMEM medium and 100mL of F12 nutrient mix; the F12 nutrient mix included 2mg hydrocortisone, 1mg EGF, 2mg insulin, 1mg amphotericin B, 3mg gentamicin, and 5mg of additives for primary culture of cancer cells.
Example 1
This example provides a medium for primary culture of cancer cells comprising 200mL of complete DMEM medium and 100mL of F12 nutrient mix; the F12 nutrient mixture included 2mg hydrocortisone, 1mg EGF, 2mg insulin, 1mg amphotericin B, 3mg gentamicin, and 5mg additives for primary culture of cancer cells;
the additive for primary culture of cancer cells comprises cholera toxin, R-spondin and Y-27632, and the mass ratio is 1: 2: 2.
example 2
This example provides a medium for primary culture of cancer cells comprising 400mL of complete DMEM medium and 100mL of F12 nutrient mix; the F12 nutrient mixture includes 4mg hydrocortisone, 3mg EGF, 5mg insulin, 5mg amphotericin B, 5mg gentamicin, and 5mg additives for primary culture of cancer cells;
the additive for primary culture of cancer cells comprises cholera toxin, R-spondin and Y-27632, and the mass ratio is 1: 3: 5.
example 3
This example provides a medium for primary culture of cancer cells comprising 300mL of complete DMEM medium and 100mL of F12 nutrient mix; the F12 nutrient mixture includes 3mg hydrocortisone, 2mg EGF, 3.5mg insulin, 3.5mg amphotericin B, 4mg gentamicin, and 5mg additives for primary culture of cancer cells;
the additive for primary culture of cancer cells comprises cholera toxin, R-spondin and Y-27632, and the mass ratio is 1: 2.5: 3.5.
example 4
Compared with the example 3, the additive for primary culture of the cancer cells comprises cholera toxin, R-spondin and A-83-01, wherein the mass ratio of the cholera toxin to the R-spondin to the A-83-01 is 1: 2.5: 3, other conditions are not changed.
Example 5
Compared with the example 3, the additive for primary culture of the cancer cells comprises cholera toxin, A-83-01 and Y-27632, wherein the mass ratio is 1: 3: 3.5, other conditions are not changed.
Example 6
Compared with the example 3, the additive for the primary culture of the cancer cells comprises cholera toxin, R-spondin, A-83-01 and Y-27632, wherein the mass ratio of the cholera toxin to the R-spondin to the A-83-01 to the Y-27632 is 1: 2.5: 3: 3.5, and other conditions are not changed.
Example 7
The complete DMEM medium additionally contained 10% serum compared to example 6, and the other conditions were not changed.
Example 8
A method for using a culture medium for primary culture of cancer cells in tumor drug sensitivity screening comprises the following specific steps:
s1, separating tumor tissues:
s101, rapidly washing the uterine cancer cell tissue excised specimen with 97% ethanol solution for not more than 3s, washing with 4 ℃ PBS solution, and transferring to a sterile culture dish;
s102, removing residual adipose tissues;
s103, cutting a fresh specimen into small blocks with the diameter smaller than 10mm to obtain tissue fragments;
s2, primary cell culture:
s201, diluting a 10-fold concentration collagenase/hyaluronidase solution by using an F culture medium, and placing the solution in a tube;
s202, mixing the F culture medium/collagenase/hyaluronidase with dispase;
s203, transferring the tissue fragments obtained in the step S103 to a tube containing F culture medium/collagenase/hyaluronidase/dispase, and incubating for 3 hours on a swing platform at 37 ℃;
s204, after digestion, centrifuging the cells at 4 ℃ for 5min at 100G, and removing supernatant;
s205, resuspending the cell pellet in the culture medium for primary culture of cancer cells prepared in example 3, filtering the cell suspension into a new centrifuge tube through a 100-mum cell filter, centrifuging the cells at 4 ℃ for 5min at 100G, and discarding the supernatant;
s3, testing drug sensitivity:
s301, digesting the cells cultured in the step S205 into a single cell suspension, counting, diluting, and inoculating to a 96-well plate, wherein the total number of cells in each well is 3000;
s302, after 12-hour adherent culture, adding a DMSO solution of drugs to be tested (including carboplatin, cisplatin, paclitaxel, fluorouracil, gemcitabine, cyclophosphamide, adriamycin, docetaxel, pemetrexed, vinorelbine and irinotecan) into corresponding holes, wherein the highest administration concentration of each drug is 50Um, and diluting 5 concentrations 10 times below to obtain 6 administration concentrations;
s303, continuously culturing for 64 hours after administration, measuring the cell activity by a fluorescence method, and calculating the IC of each medicament 50 The value is obtained.
The results are shown in FIG. 1. As can be seen from fig. 1, doxorubicin and irinotecan are relatively sensitive.
Test example 1: primary culture period and cell number statistics of cervical cancer primary cells
Cervical cancer primary cells were obtained from the sample cervical cancer tissue sample according to the method before step S2 of example 8. For the cervical cancer primary cells obtained, the culture media in examples 1 to 7 and the comparative example medium were used, and cultured at a viable cell density of 1X 10 4 Per cm 2 The cells were inoculated and cultured, digested and counted after the cells were expanded to 85%, and the number of days of culture until digestion was recorded as one culture cycle. The cells obtained by amplification were subjected to different generations of amplification, and after digestion of each generation, the corresponding culture cycles were counted and recorded, the results are shown in table 1.
TABLE 1
Figure BDA0003605983940000101
As is clear from the above table, the culture medium prepared in example 7 of the present invention had a short culture period and the average number of amplified products was 88.5 ten thousand.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (10)

1. An additive for primary culture of cancer cells comprising at least three of cholera toxin, R-spondin, A-83-01, and Y-27632; preferably cholera toxin, R-spondin, Y-27632.
2. The additive for primary culture of cancer cells according to claim 1, comprising cholera toxin, R-spondin, a-83-01, and Y-27632.
3. A medium for primary culture of cancer cells, comprising the supplement for primary culture of cancer cells of any one of claims 1-2, preferably the medium comprises complete DMEM medium and F12 nutrient mixture, more preferably the supplement is comprised in the F12 nutrient mixture.
4. The culture medium for cancer cell primary culture according to claim 3, wherein the F12 nutrient mixture further comprises hydrocortisone, EGF, insulin, amphotericin B, gentamicin.
5. The medium for primary culture of cancer cells according to claim 4, characterized in that said complete DMEM medium with or without 5-10% serum.
6. The medium for primary culture of cancer cells according to claim 3, wherein the volume ratio of the complete DMEM medium and the F12 nutrient mixture is (2-4): 1.
7. the medium for primary culture of cancer cells according to any of claims 3-6, wherein the medium does not contain fibroblasts.
8. Use of a culture medium according to any one of claims 3 to 7 for the primary culture of cancer cells in a tumor drug susceptibility screen.
9. The use according to claim 8, characterized in that the specific method is as follows:
s1, separating tumor tissues:
s101, rapidly washing the excised tissue specimen with an ethanol solution, washing with a PBS (phosphate buffer solution) solution at the temperature of 2-5 ℃, and transferring to a sterile culture dish;
s102, removing residual adipose tissues;
s103, cutting a fresh specimen into small blocks with the diameter smaller than 10mm to obtain tissue fragments;
s2, primary cell culture:
s201, diluting a 10-fold concentration collagenase/hyaluronidase solution by using an F culture medium, and placing the solution in a tube;
s202, mixing the F culture medium/collagenase/hyaluronidase with dispase;
s203, transferring the tissue fragments obtained in the step S103 to a tube containing F culture medium/collagenase/hyaluronidase/dispase, and incubating for 1-3h on a swing platform at 35-40 ℃;
s204, after digestion, centrifuging the cells and removing supernatant;
s205, resuspending the cell pellet in complete DMEM/F12 medium according to any one of claims 3-7, filtering the cell suspension through a 100 μm cell filter into a new centrifuge tube, centrifuging the cells and discarding the supernatant;
s3, testing drug sensitivity:
s301, digesting the cells cultured in the step S205 into a single cell suspension, counting, diluting, and inoculating to a 96-well plate, wherein the total number of the cells in each well is 3000;
s302, carrying out adherent culture for 10-15h, adding a DMSO solution of the drugs to be detected into corresponding holes, wherein the highest administration concentration of each drug is 50Um, and diluting 5 concentrations 10 times below to obtain 6 administration concentrations;
s303, continuously culturing for 48-72 hours after administration, measuring the cell activity by a fluorescence method, and calculating the IC of each medicament 50 The value of the one or more of the one,
optionally, the ethanol solution is rapidly rinsed for no more than 3 s; the concentration of the ethanol solution is 95-100%.
10. Use of the additive of any one of claims 1-2 in the preparation of a culture medium for primary culture of cancer cells.
CN202210418768.5A 2022-04-20 2022-04-20 Additive for primary culture of cancer cells and culture medium and application thereof Pending CN114854690A (en)

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