LMH suspension cell, serum-free domestication method and application

Abstract

The invention belongs to the technical field of biology, and discloses an LMH suspension cell, and the preservation unit of the LMH suspension cell is China General Microbiological Culture Collection Center. The preservation time is November 22, 2024; the preservation number is CGMCC NO: 46140; the classification name of the LMH suspension cells is a chicken liver cancer epithelial cell line, and the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The LMH suspension cell is successfully domesticated by an adherent cell in a serum-free culture medium, so that the exogenous pollution of the cell is successfully reduced or avoided. Meanwhile, the invention also provides a domestication method and application of the LMH suspension cell.

Description

Technical Field

[0001] The present application relates to the biological field, and specifically relates to a LMH suspension cell, a serum-free acclimation method and an application. Background Art

[0002] LMH adherent cells can be domesticated to obtain LMH suspension cells, which can be used for virus culture. For details, see the following literature:

[0003] Publication number CN118291370A, the subject is a patent application for a serum-free full suspension LMH cell and its domestication method and application;

[0004] Publication number CN113564127A, the subject is a patent application for a method for large-scale suspension culture of avian adenovirus.

[0005] The above techniques can be used to obtain LMH suspension cells suitable for virus propagation. However, the above schemes all adopt a low-serum acclimation method, more specifically, continuously reducing the serum concentration of the acclimation medium until it can survive in a serum-free medium.

[0006] The use of serum-containing culture medium for domestication poses potential safety risks and may cause exogenous contamination.

[0007] However, all existing LMH adherent cells need to adopt the above-mentioned low serum acclimation method without exception. Summary of the invention

[0008] The purpose of the present invention is to provide a strain of LMH suspension cells, which are successfully domesticated from adherent cells in a serum-free culture medium, and the exogenous contamination of the cells is successfully reduced or avoided.

[0009] At the same time, the invention also proposes a taming method and application of the LMH suspension cells.

[0010] To achieve the above-mentioned purpose, the present application discloses a strain of LMH suspension cells, the depository of which is the China General Microbiological Culture Collection Center; the deposit time is November 22, 2024; the deposit number is CGMCC NO:46140; the classification name of the LMH suspension cells is chicken hepatocarcinoma epithelial cell line, and the deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0011] A serum-free acclimation method for LMH suspension cells as described above comprises the following steps:

[0012] Step 1: Digest LMH adherent cells;

[0013] Step 2: Digested LMH adherent cells were treated with EXgrowthTM LMH serum-free medium was used for culture and subculture, and the seeding density of LMH adherent cells was not less than 1.5×10 6 cells / ml; the culture temperature was 36.9-37°C; after 3-5 generations of culture, LMH suspension cells were obtained after identification.

[0014] The LMH adherent cells used in the present invention are a strain of cells that can be successfully adhered to the wall without serum, obtained by the applicant through multiple screening and separation in the laboratory.

[0015] The LMH suspension cells of the present invention are acclimated without serum throughout the whole process, and will not introduce any exogenous contamination, thus having very high safety.

[0016] In the serum-free acclimation method for LMH adherent cells, in step 2, the time for each subculture is 72 to 96 hours; after each subculture, the LMH adherent cells are cultured in EXgrowth TM Dilute to a seeding density of no less than 1.5 × 10 6 cells / ml.

[0017] In the serum-free acclimation method for LMH adherent cells described above, during each passage, LMH adherent cells were grown in EXgrowth TM The dilution concentration in LMH serum-free medium is (1.5~2)×10 6 cells / ml.

[0018] In the above serum-free acclimation method for LMH adherent cells, in step 2, the qualified criteria are:

[0019] When LMH adherent cells are in EXgrowth TM The dilution concentration in LMH serum-free medium was 1.5 × 10 6 cells / ml, and cultured for 72 hours. TM The cell density of LMH adherent cells in LMH serum-free medium should not be less than 4×10 6 cells / ml, cultured for 96 hours, the resulting EXgrowth TM The cell density of LMH adherent cells in LMH serum-free medium should not be less than 6×10 6 cells / ml; and the cell viability exceeds 95%, it is considered to be qualified.

[0020] In the serum-free acclimation method for LMH adherent cells, in step 1, the digestion method is: terminating the digestion after culturing the LMH adherent cell wall for 72 hours, and blowing and dispersing the cells to form single cells or 2-3 cell clusters.

[0021] Meanwhile, the present invention also discloses a LMH suspension cell, which is domesticated by the method as described above.

[0022] And, use of the LMH suspension cells as described above to culture viruses.

[0023] Finally, the present invention also discloses a method for culturing group I type 4 adenovirus, which comprises inoculating group I type 4 adenovirus with a culture medium containing the LMH suspension cells as described above.

[0024] The beneficial effects of this application are:

[0025] 1. The LMH adherent cells of the present invention are a specific strain of LMH adherent cells obtained through screening. The cells can be successfully domesticated in a serum-free culture medium, successfully reducing or avoiding exogenous contamination of the cells.

[0026] 2. The amount of adenovirus C4 antigen of group I type 4 cultured by domesticated LMH suspension cells of the present invention is 6 to 8 times that of adherent cells. The results of animal immune challenge test show that the protection rate of antigens harvested from suspension culture cells after 6 or 8 times dilution is higher than that of antigens prepared from the stock solution of adherent cell culture, which can greatly reduce the production cost when converted to large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a microscope photo of successfully domesticated LMH suspension cells;

[0028] Figure 2 This is a microscopic photograph of adenovirus type 4 (C4) group I cultured in LMH suspension cells;

[0029] Figure 3 Microscope photograph of group I adenovirus type 4 (C4) cultured on LMH adherent cells. DETAILED DESCRIPTION

[0030] 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.

[0031] Part I Source of Raw Materials

[0032] EXgrowth TM LMH serum-free medium (LMH SFM, catalog number: M0108), which does not contain any animal-derived ingredients, was developed by Shanghai Jiewei Pharmaceutical Technology Co., Ltd.

[0033] 199+10% FBS: 199 medium (Shanghai Jiewei Pharmaceutical Technology Co., Ltd., M0101) supplemented with 10% FBS (gibco or sigma);

[0034] DMEM / F12+10% FBS: DMEM / F12 medium (Shanghai Jiewei Pharmaceutical Technology Co., Ltd., M01013) supplemented with 10% FBS (gibco or sigma);

[0035] LMH adherent cells: a cell strain that can be successfully adhered to the wall without serum, obtained by our company through multiple screening and separation in the laboratory.

[0036] Part II Acclimation of LMH adherent cells

[0037] The LMH suspension cells are separated and preserved by our company. The preservation unit of the LMH suspension cells is the China General Microbiological Culture Collection Center; the preservation time is November 22, 2024; the preservation number is CGMCC NO:46140; the classification name of the LMH suspension cells is chicken hepatocarcinoma epithelial cell line, and the preservation address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0038] The domestication of LMH suspension cells of the present invention comprises the following steps:

[0039] Culture medium for adherent LMH cells: 199 + 10% FBS (gibco or sigma)

[0040] 2.1 Digestion

[0041] Preparation of LMH adherent cells: Ensure that LMH adherent cells are free of mycoplasma and exogenous contamination. Culture them in three T75 square flasks for 72 hours. Under the microscope, the cells are transparent, have clear outlines, and appear as a dense monolayer.

[0042] After digesting the LMH adherent cells in three T75 flasks, the digestion was terminated with DMEM / F12 + 10% FBS, and the cells were dispersed by blowing and dispersed, and they were in the state of single cells or 2-3 cell clusters. The number of LMH cells in one T75 flask was about 2×10 7 cells, that is, the number of cells in three T75 square bottles is about 6×10 7 cells, collect the digested cells into a 50 mL centrifuge tube to obtain 6 × 10 7 cells.

[0043] 2.2 F1 generation

[0044] Divide the above number into 6×10 7Cell suspension of about 10 cells was centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended with 15 mL of LMH SFM and pipetted evenly. The cells were counted and the cell suspension was transferred to a 125 mL or 250 mL shake flask. According to the counting results, 15 mL of serum-free medium can be added to ensure that the cell inoculation density is not less than 1.5 × 10 6 cells / ml(controlled at (1.5~2)×10 6 cells / ml to proceed to the next step);

[0045] The shaking flask was placed in a CO2 constant temperature shaker with a rotation speed of 125-130 rpm (shaking amplitude of 25 mm), a culture environment of 5 vol% CO2, and a culture temperature of 36.9-37°C, and was recorded as the F1 generation.

[0046] 2.3F2 generation

[0047] After the F1 generation LMH cells were cultured for 72 hours, the cells were counted and divided into two situations according to the cell density:

[0048] In one case, if the LMH cell density is higher than 2-3×10 6 cells / ml, dilute directly with LMH SFM without centrifugation, and keep the cell seeding density at about 1.5×10 6 cells / ml, and the shaking flask was placed in a 5 vol% CO2 shaking incubator and cultured continuously for 72-96 hr; this was recorded as the F2 generation.

[0049] In another case, if the cell density is less than 2×10 6 cells / ml, centrifuge and change medium, and continue culturing for 72-96 hours; this is recorded as the F2 generation.

[0050] 2.4 Multiple passages

[0051] Repeat the above 2.3 domestication steps for 3-5 generations, and basically achieve successful domestication (such as Figure 1 );

[0052] The standard for successful acclimation is: the LMH cell seeding density in the shake flask is 1.5×10 6 cells / ml, the cell density should not be less than 4×10 6 cells / ml; after 96 hours of culture, the cell density should not be less than 6×10 6 cells / ml; cell viability exceeds 95%, which means that LMH cells have been initially domesticated successfully; microscopic photos of successfully domesticated cells are shown in Figure 1 shown.

[0053] The process of cell acclimation generally requires 3-5 generations, and the single subculture cycle in the cell acclimation stage is 72-96 hours; the subculture ratio is 1:1.5-1:3, ensuring that the seeding density of the next generation of suspended LMH cells is maintained at about 1.5×10 6 cells / ml.

[0054] The successfully domesticated LMH cells were cryopreserved in time.

[0055] Domestication of other cells

[0056] There are also other LMH adherent cells in the laboratory of our company, which are kept in our own laboratory;

[0057] The above-mentioned acclimation method was used for acclimation. The acclimation results were as follows: 3 T75 adherent LMH cells were digested with trypsin, and then digested with DMEM / F12 + 10% FBS. The cells were dispersed by blowing, centrifuged at 1000 rpm for 5 min, and 6×10 7 cells, discard the supernatant, resuspend with 15mL LMH SFM and blow evenly, place the shaking bottle in a CO2 constant temperature shaker, rotate at 125-130rpm (shaker amplitude is 25mm), culture environment is 5vol% CO2, culture temperature is 36.9-37℃, and F1 generation acclimation is carried out. However, after 72 hours of culture, most of the cells are in agglomeration, the density of single living cells is low, and the viability is poor. After centrifugation and medium replacement, the number of dead cells increases, the number of living cells decreases, the cell agglomeration is serious, and the acclimation fails.

[0058] Part III. Propagation of adenovirus type 4 (C4)

[0059] When the LMH suspension cells successfully domesticated in the second part grow to a density of no less than 6×10 6 cells / ml, use fresh medium (EXgrowth TM LMH serum-free medium) was diluted to a 250 ml cell shake flask at a density of 2×10 6 cells / ml, and then inoculate group I type 4 adenovirus at a virus dose of 0.01 MOI, culture for 120 hours, harvest the virus liquid, and culture three batches continuously.

[0060] Three batches of adherent cells were cultured in parallel for comparative experiments.

[0061] The antigen titer was measured by agar amplification method, and the results of antigen determination for each batch are shown in Table 1. 50Only the number of live viruses can be detected, but the antigen titer cannot be accurately detected. The antigen titer can be detected more accurately by agar expansion method. Then, the harvested suspended virus liquid and the adherent cultured virus liquid were inactivated and prepared into vaccines. 10-day-old SPF chickens were immunized respectively, and 25 μl was injected subcutaneously into the neck of each chicken. After 21 days of immunization, the virus was challenged. All immunized chickens and control chickens were injected intramuscularly with 1 ml of group Ⅰ type 4 avian adenovirus liquid (containing 10 8.5 TCID 50 ). The challenge was observed for 5 days and the challenge protection rate was calculated. The test results are shown in Table 2.

[0062] Table 1 Results of agar expansion in each batch of C4 suspension culture and adherent culture

[0063] Group Batch 1 Batch 2 Batch 3 Suspension culture 48 48 64 Adherent culture 8 6 8

[0064] Table 2C4 suspension culture and adherent culture challenge results

[0065] Group Number of attacks Number of cases Number of protections Protection rate / % Suspension culture-stock solution 10 0 10 100 Suspension culture - 6-fold dilution 10 0 10 100 Suspension culture - diluted 8 times 10 1 9 90 Adherent culture-stock solution 10 2 8 80 Blank control 10 10 0 -

[0066] Microscope photos of C4 suspension culture and adherent culture show Figure 2 and Figure 3 ;

[0067] Figure 2 This is a microscopic photograph of adenovirus type 4 (C4) group I cultured in LMH suspension cells;

[0068] Figure 3 This is a microscopic photograph of adenovirus type 4 (C4) of group I cultured on LMH adherent cells;

[0069] The above results show that the amount of C4 antigen in LMH suspension cell culture is 6 to 8 times that of adherent cells. The results of animal immune challenge test show that the protection rate of antigens harvested from suspension culture cells after 6 or 8 times dilution is higher than that of antigens prepared from the stock solution of adherent cell culture, which can greatly reduce production costs when converted to large-scale production.

Claims

1. A LMH suspension cell strain, characterized in that: The depository of the LMH suspension cells is the China General Microbiological Culture Collection Center; the deposit time is November 22, 2024; the deposit number is CGMCC NO:46140; the classification name of the LMH suspension cells is chicken hepatocarcinoma epithelial cell line, and the deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. A serum-free acclimation method for LMH suspension cells as claimed in claim 1, characterized in that: The steps include: Step 1: Digest LMH adherent cells; Step 2: Digested LMH adherent cells were treated with EXgrowth TM LMH serum-free medium was used for culture and subculture, and the seeding density of LMH adherent cells was not less than 1.5×10 6 cells / ml; the culture temperature was 36.9-37°C; after 3-5 generations of culture, LMH suspension cells were obtained after identification.

3. The serum-free acclimation method for LMH adherent cells according to claim 2, characterized in that: In step 2, each passage lasts for 72 to 96 hours; after each passage, the LMH adherent cells are grown in EXgrowth TM Dilute to a seeding density of no less than 1.5 × 10 6 cells / ml.

4. The serum-free acclimation method for LMH adherent cells according to claim 2, characterized in that: During each passage, LMH adherent cells were grown in EXgrowth TM The dilution concentration in LMH serum-free medium is (1.5~2)×10 6 cells / ml.

5. The serum-free acclimation method for LMH adherent cells according to claim 2, characterized in that: In step 2, the criteria for qualification are: When LMH adherent cells are in EXgrowth TM The dilution concentration in LMH serum-free medium was 1.5 × 10 6 cells / ml, and cultured for 72 hours. TM The cell density of LMH adherent cells in LMH serum-free medium should not be less than 4×10 6 cells / ml, cultured for 96 hours, the resulting EXgrowth TM The cell density of LMH adherent cells in LMH serum-free medium should not be less than 6×10 6 cells / ml; and the cell viability exceeds 95%, it is considered to be qualified.

6. The serum-free acclimation method for LMH adherent cells according to claim 2, characterized in that: In the step 1, the digestion method is: after culturing the LMH adherent cell wall for 72 hours, the digestion is terminated, and the cells are dispersed by blowing to form a single cell or a cluster of 2 to 3 cells.

7. A LMH suspension cell, characterized in that The method is domesticated according to claim 2 or 3.

8. Use of the LMH suspension cells as claimed in claim 7 to culture viruses.

9. A method for culturing adenovirus type 4 of group I, characterized in that: The culture medium containing the LMH suspension cells as claimed in claim 7 is used to inoculate the group I type 4 adenovirus.

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