Monoclonal cell of chicken liver cancer cell line and culture method thereof
By using a mixed culture medium of FBS and cell supernatant in chicken liver cancer cell culture and combining specific growth factors, the survival rate of monoclonal cells of chicken liver cancer cell lines was successfully improved, and the problem of inability to culture under traditional methods was solved, and the problem of failure to be cultivated under traditional methods was achieved. Efficient cell replication and knockout studies.
Patent Information
- Application Number
- CN202411440347.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-15
- Publication Date
- 2024-12-06
AI Technical Summary
The prior art cannot successfully culture monoclonal cells of chicken liver cancer cell lines, the survival rate of traditional methods is 0%, and there is a lack of effective methods for gene knockout studies.
A mixture of DMEM/F12 culture medium containing 18-22 wt% FBS and cell supernatant was used, and a single-cell culture medium was formed by adding growth factors such as epidermal growth factor, hepatocyte growth factor, transformed growth factor, etc., and passed through 37°C. , cultured under 5% CO2 conditions, improving the survival rate of cells.
The successful culture of monoclonal cells of chicken liver cancer cell line was achieved, with a survival rate of 70%, allowing chicken liver cancer cells to replicate efficiently and form cell lines suitable for gene knockout.
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Figure CN119082035A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the biological field, and in particular to a chicken liver cancer cell line monoclonal cell and a culture method thereof. Background Art
[0002] Chicken hepatoma cell line (LMH) is a liver cancer cell line that cannot replicate when the cell density drops to a certain ratio. LMH is often used as an in vitro infection vector for poultry viruses, providing a stable host infection model for scientific research on the pathogenicity of poultry viruses and disease prevention and control. Avian viruses that can infect LMH include Newcastle disease virus, avian influenza virus, avian leukosis virus, avian adenovirus, etc. With the in-depth study of viral pathogenicity, monoclonal cell lines that knock out or overexpress a certain host gene are needed.
[0003] In the prior art, the culture of monoclonal cell lines is mostly prepared by the following method: cells are continuously diluted in a culture medium until they are diluted into single cells, and then cultured in the culture medium to obtain a monoclonal cell line.
[0004] The above commonly used method encountered difficulties in the monoclonal culture of chicken liver cancer cell lines. It has been verified that the survival rate of monoclonal cells of chicken liver cancer cell lines cultured using traditional methods is 0.
[0005] In the existing literature and patent databases, there is no document disclosing a case of successfully culturing monoclonal cells of a chicken hepatoma cell line.
[0006] The problem that this program needs to solve: How to achieve monoclonal culture of chicken liver cancer cell lines. Summary of the invention
[0007] The purpose of the present invention is to provide a method for culturing monoclonal cells of a chicken liver cancer cell line. The method adopts the culture medium supernatant of the chicken liver cancer cell line and the culture medium added with growth factors, FBS and transferrin to mix, thereby achieving the successful cultivation of the monoclonal cells of the chicken liver cancer cell line, and the survival rate thereof reaches 70%.
[0008] At the same time, the present invention also provides chicken liver cancer cell line monoclonal cells obtained based on the method.
[0009] To achieve the above-mentioned purpose, the present application discloses a method for culturing monoclonal cells of a chicken liver cancer cell line, wherein a single chicken liver cancer cell is added to a single cell culture medium for culturing, wherein the single cell culture medium comprises a DMEM / F12 culture medium containing 18-22wt% FBS and a cell supernatant; the culture medium also contains transferrin with a final concentration of 8-12 μg / mL, a growth factor with a final concentration of 20-30 ng / mL, and a dual antibody;
[0010] The preparation method of the cell supernatant is as follows: chicken liver cancer cells are cultured in a DMEM / F12 culture medium containing 8-12 wt% FBS, and the supernatant is collected after a period of culture to obtain the cell supernatant.
[0011] In the present invention, the role of the cell supernatant is to provide unknown nutrients to individual cells or signal transduction substances that stimulate cell proliferation; the role of the growth factor is to promote cell proliferation; when the culture medium supernatant and the culture medium added with growth factors, FBS, and transferrin are mixed, the successful culture of monoclonal cells of chicken liver cancer cell line is achieved, and the survival rate of the culture of monoclonal cells of chicken liver cancer cell line by traditional methods is increased from 0% to 70%.
[0012] In the above-mentioned chicken liver cancer cell line monoclonal cell culture method, the growth factor is one or more combinations of epidermal growth factor, hepatocyte growth factor, transforming growth factor, and fibroblast growth factor.
[0013] In the above-mentioned chicken liver cancer cell line monoclonal cell culture method, the volume ratio of the DMEM / F12 culture medium containing 18-22wt% FBS to the cell supernatant is 1:1.
[0014] In the above-mentioned chicken liver cancer cell line monoclonal cell culture method, the dual antibodies are penicillin and streptomycin.
[0015] In the above-mentioned chicken liver cancer cell line monoclonal cell culture method, a single chicken liver cancer cell is added to each 200 μL single cell culture medium for culture; the culture process is: culture in a cell culture incubator containing 5% CO2 at 37°C for a period of time.
[0016] In the above-mentioned chicken liver cancer cell line monoclonal cell culture method, the culture time is 12 to 16 days.
[0017] In the above-mentioned chicken liver cancer cell line monoclonal cell culture method, in the method for preparing the cell supernatant, the cells are cultured in a cell culture incubator containing 5% CO2 at 37°C, and the supernatant is collected after 48 hours of culture to obtain the cell supernatant.
[0018] In addition, the present invention also discloses a chicken liver cancer cell line monoclonal cell, which is prepared by any of the above methods.
[0019] The beneficial effects of this application are:
[0020] The present invention uses a culture medium supernatant of a chicken liver cancer cell line and a culture medium added with epidermal growth factor, FBS, and transferrin for mixing, thereby achieving successful culture of monoclonal cells of the chicken liver cancer cell line, with a survival rate of 70%, so that non-viable chicken liver cancer cells can be efficiently replicated to form a cell line that can be subsequently gene-knocked out. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 This is a microscope photograph of chicken liver cancer cell line monoclonal cell culture using the single cell culture medium of Example 1;
[0022] Figure 2 This is a microscope photo of monoclonal cell culture of chicken hepatoma cell line using traditional culture methods. DETAILED DESCRIPTION
[0023] The present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. In the description of the present invention, it should be noted that, if no specific conditions are specified in the embodiments, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.
[0024] Source of reagents:
[0025] LMH cell line: ATCC, catalog number: CRL-2117;
[0026] DMEM / F12 culture medium: Gibco, DMEM / F12 (1:1) culture medium catalog number: C11330500BT;
[0027] Fetal bovine serum: VivaCell BIOSCIENCES, Fetal Bovine Serum, catalog number: C04001-500;
[0028] Double antibody: Biosharp, penicillin-streptomycin solution (100×), catalog number: BL505A;
[0029] Transferrin: Sigma, catalog number: T8158-100MG;
[0030] Epidermal growth factor: Sigma, catalog number: E9644-5MG;
[0031] Hepatocyte growth factor: SinoBiological Company, catalog number: 10463-HNAS;
[0032] Transforming growth factor: SinoBiological Company, catalog number: 10804-H08H-B;
[0033] Fibroblast growth factor: SinoBiological Company, catalog number: GMP-10014-HNAE;
[0034] Trypsin: Gibco, 0.25% Trysin-EDTA (1×), catalog number: 25200-072.
[0035] Part I. Preparation of cell supernatant
[0036] The LMH cell line was plated at a concentration of 90% in a T75 culture flask, and the T75 culture flask had a DMEM / F12 medium; the DMEM / F12 medium contained 10 wt % fetal bovine serum, 100 U / mL penicillin and 0.1 mg / mL streptomycin;
[0037] The culture conditions were: culture in a cell culture incubator at 37°C with 5% CO2;
[0038] After 48 h of culture, the cell supernatant was collected into a sterile 50 ml centrifuge tube and stored at 4°C, or stored at -20°C for a long term.
[0039] Part II Preparation of Single Cell Culture Medium
[0040] Example 1
[0041] The single cell culture medium consisted of DMEM / F12 medium containing 20 wt% FBS and the cell supernatant in the first part at a volume ratio of 1:1;
[0042] The single cell culture medium contained transferrin at a final concentration of 10 μg / mL, hepatocyte growth factor at a final concentration of 25 ng / mL, and double antibodies at a final concentration of 100 U / mL penicillin and 0.1 mg / mL streptomycin.
[0043] Example 2
[0044] The method is substantially the same as Example 1, except that the concentration of transferrin is 8 μg / mL, the concentration of hepatocyte growth factor is 30 ng / mL, and the DMEM / F12 medium contains 18 wt % FBS.
[0045] Example 3
[0046] The method is substantially the same as Example 1, except that the concentration of transferrin is 12 μg / mL, the concentration of hepatocyte growth factor is 20 ng / mL, and the DMEM / F12 medium contains 22 wt% FBS.
[0047] Example 4
[0048] It is generally the same as Example 1, except that the growth factor is epidermal growth factor.
[0049] Example 5
[0050] The method is substantially the same as Example 1, except that the growth factor is fibroblast growth factor.
[0051] Example 6
[0052] The method is substantially the same as Example 1, except that the growth factor is composed of hepatocyte growth factor and epidermal growth factor in a weight ratio of 1:1.
[0053] Example 7
[0054] The method is substantially the same as Example 1, except that the growth factor is composed of hepatocyte growth factor and fibroblast growth factor in a weight ratio of 1:1.
[0055] Example 8
[0056] The method is substantially the same as Example 1, except that the growth factor is composed of epidermal growth factor and fibroblast growth factor in a weight ratio of 1:1.
[0057] Part III Monoclonal Cell Culture of Chicken Hepatoma Cell Line
[0058] The specific method for culturing monoclonal cells of chicken hepatoma cell line is as follows:
[0059] Step 1: LMH cells to be passaged (2*10 6 500 μL of trypsin was used to cover the cells and then the trypsin was discarded. After adding 2 mL of single cell culture medium to neutralize the excess trypsin, the cells were filtered into single cells using a 300-mesh filter according to the requirements of the flow cytometer (Beckman) to remove cell clumps;
[0060] Step 2: Add 200 μL of prepared single-cell culture medium to each 96-well plate and sort using a flow cytometer. Each well of the 96-well plate is punched with 1 cell by flow cytometry, and a total of 2 96-well plates are sorted;
[0061] Step 3: Place the 96-well plate in a cell culture incubator at 37°C with 5% CO2;
[0062] Step 4: Observe the cell morphology in each well for about 14 days, observe the cell morphology, etc., and record the cell survival rate.
[0063] The single cell culture medium refers to the above-mentioned examples and comparative examples.
[0064] The culture results of the single cell culture medium of Example 1 can be seen Figure 1 .
[0065] The traditional cultivation methods are:
[0066] Step 1: LMH cells to be passaged (2*10 6 ) Cover the cells with 500 μL of trypsin and discard the trypsin, add 2 mL of culture medium to neutralize the excess trypsin, and filter the cells into single cells using a 300-mesh filter according to the requirements of a flow cytometer (Beckman), and filter out cell clumps;
[0067] Step 2: Add 200 μL of prepared culture medium to each 96-well plate and sort using a flow cytometer. Each well of the 96-well plate is punched with 1 cell by flow cytometry, and a total of 2 96-well plates are sorted;
[0068] Step 3: Place the 96-well plate in a cell culture incubator at 37°C with 5% CO2;
[0069] Step 4: Observe the cell morphology in each well for about 14 days, observe the cell morphology, etc., and record the cell survival rate.
[0070] The culture medium in step 1 and step 2 is DMED / F12 medium to which 10% FBS, 100 U / mL penicillin and 0.1 mg / mL streptomycin are added.
[0071] The results of the traditional culture method can be seen Figure 2 .
[0072] pass Figure 2 It can be seen that traditional methods cannot achieve the survival and replication of LMH cells.
[0073] Part IV: Results Statistics and Analysis
[0074] The single cell culture medium of the second part is applied to the single cell culture process of the third part, and the survival rate is calculated;
[0075] The statistical results refer to Table 1;
[0076] Table 1 Monoclonal cell survival rate
[0077] Survival rate Example 1 70％ Example 2 67％ Example 3 65％ Example 4 35％ Example 5 15％ Example 6 60％ Example 7 54％ Example 8 25％ Traditional methods 0％
[0078] Result analysis:
[0079] 1. It can be seen from Examples 1 to 5 that the use of a culture medium composed of DMEM / F12 culture medium and cell supernatant is the basis for the successful culture of single cells; the use of hepatocyte growth factor is a relatively important means. Compared with epidermal growth factor and fibroblast growth factor, the survival rate of hepatocyte growth factor is increased by 30% to 50%;
[0080] 2. The importance of hepatocyte growth factor is further demonstrated through Examples 6 to 8. Compared with the single cell culture medium without hepatocyte growth factor, the single cell culture medium containing hepatocyte growth factor can basically reach or approach the single cell culture medium using hepatocyte growth factor.
[0081] 3. As an important breakthrough of the present invention, the present invention overcomes the difficulty that the traditional method cannot successfully culture monoclonal cells of chicken liver cancer cell line.
[0082] In summary, the unique innovation of this case lies in: using a mixture of basal culture medium with added growth factors and cell supernatant as a single cell culture medium, providing nutrients to single cells or signal transduction substances that stimulate cell proliferation through cell supernatant, promoting cell proliferation through growth factors, so that chicken liver cancer cells that cannot survive can replicate efficiently and form a cell line that can be subsequently knocked out. At the same time, through the screening of growth factors, a hepatocyte growth factor that is particularly suitable for chicken liver cancer cells was obtained, which plays a pivotal role in improving the survival rate of single cells.
Claims
1. A method for culturing monoclonal cells of chicken hepatoma cell lines, characterized in that: A single chicken liver cancer cell is added to a single cell culture medium for culture, wherein the single cell culture medium comprises a DMEM / F12 culture medium containing 18-22wt% FBS and a cell supernatant; the single cell culture medium also contains transferrin with a final concentration of 8-12 μg / mL, a growth factor with a final concentration of 20-30 ng / mL, and a dual antibody; The preparation method of the cell supernatant is as follows: chicken liver cancer cells are cultured in a DMEM / F12 culture medium containing 8-12 wt% FBS, and the supernatant is collected after a period of culture to obtain the cell supernatant.
2. The method for culturing monoclonal cells of chicken hepatoma cell line according to claim 1, characterized in that: The growth factor is one or more combinations of epidermal growth factor, hepatocyte growth factor, transforming growth factor, and fibroblast growth factor.
3. The method for culturing monoclonal cells of chicken hepatoma cell lines according to claim 1, characterized in that: The volume ratio of the DMEM / F12 culture medium containing 18-22wt% FBS to the cell supernatant is 1:
1.
4. The method for culturing monoclonal cells of chicken hepatoma cell line according to claim 3, characterized in that: The dual resistance is penicillin and streptomycin.
5. The method for culturing monoclonal cells of chicken hepatoma cell line according to claim 1, characterized in that: A single chicken liver cancer cell was added to each 200 μL single cell culture medium for culturing. The culturing process was as follows: culturing in a cell culture incubator containing 5% CO 2 at 37° C. for a period of time.
6. The method for culturing monoclonal cells of chicken hepatoma cell line according to claim 5, characterized in that: The culture time is 12 to 16 days.
7. The method for culturing monoclonal cells of chicken hepatoma cell lines according to claim 1, characterized in that: In the preparation method of the cell supernatant, the cells are cultured in a cell culture incubator containing 5% CO2 at 37°C, and the supernatant is collected after 48 hours of culture to obtain the cell supernatant.
8. A chicken liver cancer cell line monoclonal cell, characterized in that: The method is prepared by any one of claims 1 to 7.