ESTABLISHMENT AND SUSPENSION ACCLIMATION OF CRFK ADHERENT CELL LINE, AND ITS APPLICATION

Abstract

Provided are establishment and suspension acclimation of a Crandell Reese Feline Kidney (CRFK) adherent cell line, and its application. The CRFK cell line, named CRFK-BLA, is deposited at the China General Microbiological Culture Collection Center (CGMCC), with the address being Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit number being CGMCC NO: 45703; the CRFK cell line was classified and named CRFK cells, and was deposited on Aug. 17, 2023. A serum-free complete suspension culture type CRFK cell line obtained by acclimation on the basis of CRFK-BLA is named CRFK-BLS, and is deposited at the China General Microbiological Culture Collection Center (CGMCC), with the address being Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit number being CGMCC NO: 45704; the CRFK cell line was classified and named CRFK suspension cells.

Claims

1. A Crandell Reese Feline Kidney (CRFK) cell line, wherein the CRFK cell line, named CRFK-BLA, is deposited at the China General Microbiological Culture Collection Center (CGMCC), with the address being Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC NO: 45703; and the CRFK cell line was classified and named CRFK cells, and was deposited on Aug. 17, 2023.

2. A serum-free complete suspension culture type CRFK cell line, wherein the serum-free complete suspension culture type CRFK cell line, named CRFK-BLS, is deposited at the China General Microbiological Culture Collection Center (CGMCC), with the address being Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC NO: 45704; and the CRFK cell line was classified and named CRFK suspension cells, and was deposited on Aug. 17, 2023.

3. (canceled)

4. (canceled)

5. A method for culturing virus vaccines using the CRFK cell line according to claim 1, the virus is selected from the group consisting of canine parvovirus, feline panleukopenia virus, feline calicivirus, feline infectious rhinotracheitis virus, and mink viral enteritis virus.

6. A method for culturing virus vaccines using the serum-free complete suspension culture type CRFK cell line according to claim 2, the virus is selected from the group consisting of canine parvovirus, feline panleukopenia virus, feline calicivirus, feline infectious rhinotracheitis virus, and mink viral enteritis virus.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1 shows the F10 generation (20) of feline kidney primary cells.

[0032] FIG. 2 shows a CRFK cell line at different generations (20), where A of FIG. 2 represents CRFK-F15 (15th generation); and B of FIG. 2 represents CRFK-F20 (20th generation).

[0033] FIG. 3 shows a virus culture sensitivity experiment (20) of a CRFK cell line, where A of FIG. 3 represents a CRFK-BLA control, B of FIG. 3 represents CRFK-BLA-FPV, C of FIG. 3 represents CRFK-BLA-FHV, D of FIG. 3 represents a CRFK control, E of FIG. 3 represents CRFK-FPV, and F of FIG. 3 represents CRFK-FHV.

[0034] FIG. 4 shows viral titers of CRFK and CRFK-BLA.

[0035] FIG. 5 shows the results of low-serum and suspension acclimation of a CRFK cell line (20), where A of FIG. 5 represents the adherent growth of CRFK-BLA in 2% FBS and DMEM, and B of FIG. 5 represents the suspension growth of CRFK-BLA in 0.5% FBS and complete suspension medium.

[0036] FIG. 6 shows the process of cell growth from monoclonal to monolayer, where A of FIG. 6 represents serum-free acclimation monoclonal CRFK-BLA, B of FIG. 6 represents single-cell proliferate into two cells through mitosis, C of FIG. 6 represents two cells proliferate into four cells through mitosis, D of FIG. 6 represents four cells divide into eight cells, E of FIG. 6 represents cells spread and proliferate on the matrix, and F of FIG. 6 represents single-cell form a continuous single-layer cell.

[0037] FIG. 7 shows the microscopic results (20) of a suspended CRFK-BLS cell line.

[0038] FIG. 8 shows the microscopic growth pattern of a suspended CRFK-BLS cell line.

[0039] FIG. 9 shows a virus sensitivity experiment of a CRFK-BLS cell line.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0040] The following are specific embodiments of the present disclosure to further illustrate the present disclosure, but should not be construed as limiting the scope of the present disclosure. Those skilled in the art should understand that the details and forms of the technical solutions of the present disclosure can be modified or replaced without departing from the spirit and scope of the present disclosure, but these modifications or replacements shall all fall within the protection scope of the present disclosure.

[0041] The experimental methods used in the following embodiments are conventional methods unless otherwise specified.

Main Biological Materials and Equipment Involved in Experiments

[0042] The virus species, including feline parvovirus HBX05 strain, feline herpesvirus BJS01 strain and feline calicivirus BJH13 strain, were isolated, identified and deposited by Taizhou Bioally Technology Co., Ltd.

[0043] Experimental animals: 10 days old female kittens without specific pathogens (negative for FPV, FHV, and FCV antigen tests).

[0044] Main reagents: viral genomic DNA/RNA extraction kit and RNA reverse transcription kit, purchased from Beijing TransGen Biotechnology Co., Ltd.; PCR and RT-PCR amplification kits, purchased from TaKaRa Bioengineering (Dalian) Co., Ltd.; thioglycolate fluid medium (TG), trypticase soy broth (TSB), mycoplasma liquid medium, and mycoplasma solid medium, purchased from Beijing Zhonghai Biotechnology Co., Ltd.; and high-glucose DMEM medium (ME105-001) and special-grade South American fetal bovine serum (S711-001S), purchased from Suzhou ShuangRu Biotechnology Co., Ltd.

[0045] A serum-free medium was prepared based on a DMEM medium by adding insulin, transferrin, cholesterol, sodium bicarbonate, HEPES, Pluronic F68 and other components (see the table below for specific components), and was filtered and sterilized through a 0.1 m microporous filter membrane.

TABLE-US-00001 Components Content (mg/L) L-asparagine monohydrate 750 L-isoleucine 400 L-phenylalanine 300 L-glutamic acid 300 L-threonine 350 L-cysteine hydrochloride monohydrate 350 L-lysine hydrochloride 500 L-proline 400 L-serine 700 L-arginine hydrochloride 350 L-histidine hydrochloride monohydrate 250 L-methionine 200 L-valine 400 L-aspartic acid 1500 L-leucine 700 L-tyrosine disodium salt hydrate 250 L-tryptophan 200 L-ascorbate 2-phosphate hemimagnesium salt hydrate 15 Choline chloride 80 Inositol 60 Para aminobenzoic acid 1.5 Vitamin B12 50 D-calcium pantothenate 6 Nicotinamide 80 Pyridoxine hydrochloride 32 Vitamin b1 hydrochloride 12 D-biotin 1.5 Folic acid 25 Riboflavin 1 Ethylenediaminetetraacetic acid tetrasodium salt 2.5 tetrahydrate Ferrous sulfate heptahydrate 2 2-hydroxypyridine nitrogen oxide 1.5 Anhydrous calcium chloride 10 Magnesium chloride hexahydrate 170.66107 Zinc sulfate heptahydrate 3 Reduced glutathione 0.6 Thioglycerol 6 Alpha-lipoic acid 1.3 Linoleic acid 0.04 D-glucose 6000 Hydroxyethyl piperazine ethylsulfonic acid (HEPES) 2000 Sodium bicarbonate 2300 Sodium pyruvate 140 Potassium chloride 400 Anhydrous disodium hydrogen phosphate 500 Sodium dihydrogen phosphate monohydrate 172.5 Phenol red 2

Embodiment 1: Establishment of CRFK-BLA Cell Line

[0046] Isolation and culture of feline kidney primary cells: the screened kittens without specific pathogens were painlessly euthanized and then soaked in a 75% alcohol solution; the abdominal cavity was cut open in a biosafety cabinet for taking out the kidneys, and the fascial layer of renal tissue was exfoliated; the obtained kidneys were cut into the size of rice grains with sterile scissors, and then rinsed for 3 times with sterile PBS buffer; 20 ml of trypsin with a concentration of 0.5 mg/ml was added, and the obtained mixture was placed in a constant temperature incubator at 37 C. for digestion for 1 h; the product was ground with a sterile grinder, and cell suspension was filtered with a 100-mesh screen; centrifugation was performed at 700 r/min for 5 min, and the product was washed for three times with a complete medium (DMEM containing 10% fetal bovine serum); and after being resuspended in 10 ml of the complete medium, the product was cultured at 37 C. in a constant temperature incubator containing 5% CO.sub.2, which was denoted as F1.

[0047] After being cultured for 24 h, the primary cells were washed for 3 times with preheated PBS and then transferred into a new complete medium for culturing; after a monolayer was fully grown, the cells were rinsed with 0.125% trypsin to flush out cells in poor conditions; after culture medium was discarded, 0.125% trypsin was added again for digestion; and after being observed under a microscope to disperse into single cells, the cells were added into a complete medium immediately and passaged at a ratio of 1:3. Culturing was continued in this way until F10. After 10 consecutive generations of isolation and culture, the feline kidney primary cells were in good conditions. The feline kidney primary cells from the generation F10 that had grown well to a monolayer were observed under a microscope. The cells are rhombus-shaped and oblong-shaped, and have clear outlines (see FIG. 1).

[0048] The monolayer feline kidney primary cells in good conditions were taken and rinsed with 0.125% trypsin; after culture medium was discarded, 0.125% trypsin was added again for digestion; after being observed under a microscope to disperse into single cells, the cells were added into a complete medium immediately and passaged at a ratio of 1:3; and the product was cultured at 37 C. in a cell incubator containing 5% CO.sub.2. Passage was carried out continuously to F45 according to the above method, and the cell morphology and status were observed. The cell morphology of each passage was normal, and was rhombus-shaped and oblong-shaped, with clear outline (see FIG. 2); and the cell growth rate was stable. It shows that the feline kidney primary cells may be used as a continuous cell line after being subjected to continuous screening and culture. They are named CRFK-BLA and belong to a spontaneous permanent cell line.

[0049] The monolayer feline kidney primary cells in good conditions were taken and rinsed with 0.125% trypsin; after culture medium was discarded, 0.125% trypsin was added again for digestion; after being observed under a microscope to disperse into single cells, the cells were added into a complete medium immediately to prepare cell suspension for counting; centrifugation was performed at 800 r/min for 8-10 min, the supernatant was discarded, cryoprotectant (DMEM:fetal bovine serum:DMSO=6:3:1) was added, the obtained mixture was gently blown and evenly mixed, and the cell concentration was adjusted to 2.010.sup.6/ml; and the product was transferred into cryopreservation tubes, with 1.0 ml for each tube, and then labeled and stored in liquid nitrogen for later use.

CRFK-BLA Cell Line Purity Test

[0050] Cell resuscitation: CRFK-BLA cells frozen in liquid nitrogen were taken out, then placed in a water bath at 37 C. to thaw rapidly, and centrifuged at 800 r/min for 8-10 min. The obtained cells were added into a complete serum medium, gently blown to resuspend, and then transferred into a T25 cell flask; and the product was cultured at 37 C. in a cell incubator containing 5% CO.sub.2.

[0051] Sterility test: CRFK-BLA cells were taken and tested according to Appendix 3306, Volume III, Chinese Veterinary Pharmacopoeia, 2020 edition.

[0052] Mycoplasma test: CRFK-BLA cells were taken and tested according to Appendix 3308, Volume III, Chinese Veterinary Pharmacopoeia, 2020 edition.

[0053] Exogenous virus test: CRFK-BLA cells were taken and tested according to Appendix 3305, Volume III, Chinese Veterinary Pharmacopoeia, 2020 edition.

[0054] After resuscitation, the CRFK-BLA cell bank cells F10, F20 and F30 were tested for purity. All passages of cells passed the test and were free of bacteria, mycoplasma and exogenous virus contamination.

[0055] After the cells F20 were isolated, purified and amplified, they were named CRFK-BLA and deposited at the China General Microbiological Culture Collection Center (CGMCC), with the address being Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC NO: 45703; and the cells were classified and named CRFK cells, and were deposited on Aug. 17, 2023.

Embodiment 2: Virus Sensitivity Test for CRFK-BLA Cell Line

[0056] Feline parvovirus (HBX05 strain), feline herpesvirus (BJS01 strain) and feline calicivirus (BJH13 strain) were respectively inoculated into CRFK cells and deposited strain CRFK-BLA cells in T25 cell bottles according to a volume ratio; ({circle around (1)} FPV: simultaneous inoculation, inoculated into 10 ml of CRFK cell suspension containing 2% bovine serum at a ratio of 5%; {circle around (2)} FHV: inoculated into CRFK cells that have grown into a monolayer at a ratio of 0.1%; {circle around (3)} FCV: inoculated into CRFK cells that have grown into a monolayer at a ratio of 0.1%); at the same time, normal cells were used as a control; and the cells were placed at 37 C. in an incubator containing 5% CO.sub.2 for culture and observation. Whether the CRFK-BLA cell line was sensitive to all virus strains was observed; and when the cytopathic lesions reached 80%, the virus liquid was collected and enabled to be subjected to freeze thawing for three times, and then the virus TCID.sub.50 was determined. The results showed that obvious cytopathic lesions were seen after CRFK-BLA cells were exposed to the virus (see FIG. 3). After simultaneous inoculation of the FPV HBX05 strain, the typical lesions, such as swelling, rounding and drawing of cells, could be observed under a microscope. After inoculation of the FHVBJS01 strain, the cells showed round shrinkage and shedding lesions under a microscope. After inoculation of the FCV BJH13 strain, the typical lesions, such as cell agglomeration, shedding and grape clustering, could be observed under a microscope. The virus titers of the viruses cultured with the CRFK-BLA were higher than those of the viruses cultured with the CRFK cells (FIG. 4).

Embodiment 3: Serum-Free Acclimation of CRFK-BLA Cell Line

[0057] The serum content in a medium was reduced by the gradient of a stepwise adaptation method.

[0058] Preparation of CRFK cells: inoculating the resuscitated CRFK cells (a deposited strain of CRFK-BLA) into a DMEM medium containing 8% fetal bovine serum, and DMEM was used as a base medium, with 8% fetal bovine serum being added for incubation (pH value: 6.8-7.2); and the product was incubated at 37 C. in a cell incubator containing 5% CO.sub.2. After the cells adhered to the wall and the monolayer confluent degree in a culture flask reached 90% or more, culture medium was discarded, and the product was digested and dispersed with 0.125% EDTA-trypsin, and then was subcultured for 5 generations according to the conventional method.

[0059] Gradient descent serum culture of CRFK cells: the obtained adherent culture type CRFK cells were cultured in each of DMEM mediums containing 6%, 4%, and 2% fetal bovine serum in sequence for 6 generations, subcultured at a ratio of 1:3, and then incubated at 37 C. in a cell incubator containing 5% CO.sub.2.

[0060] Low-serum adherent culture of CRFK cells in complete suspension medium: adherent culture was carried out on the cells obtained in the last generation in the previous step by using a complete suspension medium (as described in the above formula) containing 2% fetal bovine serum; after the cells adhered to the wall and the monolayer confluent degree in a culture flask reached 90% or more, culture medium was discarded; the product was digested and dispersed with 0.125% EDTA-trypsin, subcultured in a ratio of 1:2, and incubated at 37 C. in a cell incubator containing 5% CO.sub.2; and continuous passage culture was carried out for 5 generations to obtain a low-serum adherent culture type CRFK cell line adapted to the complete suspension medium.

[0061] Low-serum suspension culture of CRFK cells in complete suspension medium: the cell density obtained in the last generation in the previous step was adjusted to 110.sup.6 cells/mL by using a complete suspension medium containing 2% fetal bovine serum, and the cells were incubated at 37 C. and 120 r/min in a shaker containing 5% CO.sub.2; when the cell density reached 410.sup.6 cells/mL, a complete suspension medium containing 1% fetal bovine serum was used to culture the cells in flasks in a ratio of 1:4; when the cell density reached 410.sup.6 cells/mL, a complete suspension medium containing 0.5% fetal bovine serum was used to culture the cells in flasks in a ratio of 1:3; after five consecutive passages, acclimation was performed to obtain a low-serum complete suspension culture type CRFK cell line; and the obtained cell line was incubated at 37 C. and 120 r/min in a shaker containing 5% CO.sub.2.

[0062] After continuous gradient serum reduction, the self-isolated CRFK-BLA cell line was able to normally grow adhering to the wall and passage in the DMEM medium containing 2% fetal bovine serum (see A of FIG. 5). The cell morphology of each passage was normal, and was rhombus-shaped and oblong-shaped, with clear outlines. After being suspended and acclimated in the low-serum complete suspension medium, the CRFK-BLA cell line could grow normally in the complete suspension medium containing 0.5% fetal bovine serum (see B of FIG. 5). Under a microscope, it appeared single and transparent, with clear cell outline and high transparency. It shows that the low-serum complete suspension culture type CRFK-BLA cell line can be obtained through acclimation.

Embodiment 4: Acclimation Method for CRFK-BLA Single-Cell Suspension

[0063] Single-cell screening using a 96-well plate: the low-serum complete suspension culture type CRFK-BLA cell line was centrifuged, and then added into a serum-free complete suspension medium (as described in the above formula); the suspended cells were blown and counted, and the cells were diluted with the serum-free complete suspension medium so as to prepare 10 cells/mL cell suspension; and the cell suspension was added into a 96-well cell culture plate at 100 l/well, and then incubated at 37 C. in a cell incubator containing 5% CO.sub.2.

[0064] Single-cell screening using a 24-well plate: the cells were cultured for 48-72 h, several culture wells with only 1 cell/well were selected from the 96-well cell culture plate, and the single cell lines that grow well were selected; and the selected cell lines were transferred to a 24-well plate, and then incubated at 37 C. in a cell incubator containing 5% CO.sub.2.

[0065] Single-cell screening using a 6-well plate: the cells were culturing for 48-72 h, and the single cell lines that grow well were selected from the 24-well cell culture plate; and the selected cell lines were transferred to a 6-well plate, and then incubated at 37 C. in a cell incubator containing 5% CO.sub.2.

[0066] Single-cell adherent amplification culture: the cells were cultured for 48-72 h, and the single cell lines that grow well were selected from the 6-well cell culture plate; and the selected cell lines were transferred to a 25 cm.sup.2 culture flask, and then incubated at 37 C. in a cell incubator containing 5% CO.sub.2.

[0067] Single-cell suspension culture: when the cells grew to 90%, the cells were digested according to the above method, and then transferred to a shake flask to continue culturing. The cell density was adjusted to 510.sup.5 cells/mL, the cells were then incubated at 37 C. and 120 r/min in a shaker containing 5% CO.sub.2, and acclimation was performed to obtain a serum-free complete suspension culture type CRFK cell line (see FIG. 6). This cell line could grow normally in the serum-free medium, with unchanged cell morphology and clear outline, taking on the shapes of rhombus and oblong. After being transferred to a shake flask for culturing, the CRFK cell line was subcultured at the density of 510.sup.5 cells/mL, and was subjected to continuous passage culture in a serum-free medium for 5 generations. The CRFK cell line achieved normal suspension growth in the complete suspension medium (see FIG. 7). Under a microscope, it appeared single and transparent, with clear cell outline and high transparency, and was named CRFK-BLS.

[0068] After the serum-free complete suspension culture type CRFK cells obtained by acclimation were isolated, purified and amplified, they were named CRFK-BLS and deposited at the China General Microbiological Culture Collection Center (CGMCC), with the address being Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC NO: 45704; and the cells were classified and named CRFK suspension cells, and were deposited on Aug. 17, 2023.

[0069] The established CRFK-BLS cell line was passaged and subcultured at the density of 110.sup.6 cells/mL, and was stained with trypan blue every 24 h; the numbers of dead cells and living cells were counted with a hemocytometer, and the cell viability was calculated and displayed (see FIG. 8); and after 96 h of suspension culture, the cells reached a maximum density of 4.510.sup.6 cells/mL, with a viability of 900.5% and a doubling time of 25.370.35 h. It shows that the growth stability of this monoclonal cell line (CRFK-BLS) is better.

[0070] Virus sensitivity test: the established CRFK-BLS cell line was passaged and subcultured at the density of 110.sup.6 cells/mL. When the cell density reached 2.010.sup.6 cells/mL, feline parvovirus (HBX05 strain), feline herpesvirus (BJS01 strain) and feline calicivirus (BJH13 strain) were inoculated into CRFK-BLS suspension culture shake flasks according to certain volume ratios {circle around (1)} FPV: inoculated at a ratio of 5%; {circle around (12)} FHV: inoculated at a ratio of 0.1%; {circle around (3)} FCV: inoculated at a ratio of 0.1%); at the same time, normal cells were used as a control; and the cells were cultured at 37 C. and 120 r/min in a shaker containing 5% CO.sub.2. Whether the CRFK-R cell line was sensitive to all virus strains was observed. Virus liquid was collected and enabled to be subjected to freeze thawing for 3 times, and then the virus TCID.sub.50 (see FIG. 9) was determined. It is confirmed that this monoclonal cell line (CRFK-BLS) is more conducive to FPV and FHV culture than traditional adherent CRFK cells.

[0071] The foregoing description of the embodiments is provided to facilitate the understanding and use of the present disclosure by those of ordinary skill in the art. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and that the general principles described herein can be applied to other embodiments without the need for creative labor. Therefore, the present disclosure is not limited to the above-described embodiments. According to the principle of the present disclosure, improvements and modifications made by those skilled in the art without departing from the scope of the present disclosure shall fall within the protection scope of the present disclosure.