(A) study on the establishment of continuous cell line (CMK) derived from Korean chipmunk kidney and its susceptibility to some viruses

의학과/박사[한글] A Study on the Establishment of Continuous Cell Line(CMK) derived from Korean Chipmunk Kidney and its Susceptibility to some Virusesunk Kidney and its Susceptibi Dong Hoon Hwang, M.D. Department of Medical Science, The Graduate School, Yonsei University (Directed by Drs. Joon Lew and Hyun Mo Kwak) Tissue culture techniques, originated by the pioneering works of Roux(1885) and Harrison(1970, have been one of the most fundamental methods in virological studies. Application of trypsin in dispersion of the cells from freshly ulbecco, 1952; Moscona, 1952; Younger, 1954 and in harvesting cells for subculture allowed to obtain uniform populations of sufficient density of cells and to achieve confluent cultures a few days after the initiation of tissue culture. It tissue culture experiences, the cells very commonly cease growing after a limited period of rapid growth during the subcultures of the primary ones. Therefore, it is generally accepted that cells should survive 12 to 20 generations before they can be considered a stain. Recently a many variety established cell lines have been developed(Cell culture Collection Committee, 1964). In spite of the rapid increase in the numbers of established cell lines dervied from various sources, the following observations strongly support the necessity of established new cell lines. i.e., it has been frequently shown that cell cultures derived from various sources differed significantly in their susceptibility to the same viruses. Vogt(1967) showed that Rous sarcoma virus, a model of defective virus(Hansfusa et al., 1963), could multiply and produce mature infective virus particles in susceptible cell culture, and accumulating evidences exist that various animal species and the cell lines derived from their tissues are contaminated with a variety of latent viruses(de Harven, 1964; Shipman, 1969; Smith et al., 1970; Lowy et al., 1971). Interestingly various tissues derived from human and primate, and domestic or laboratory animals have been exclusively used as the major sources of primary cultures and the subsequent development of cell lines. To the author's knowledge, neither continuous cell lines nor cloned cell lines have ever been established from wild, small mammals. In this study continuous cell line was established from the primary culture of chipmunk kidney cells through serial subculture over a period of one year. The cell line was tentatively named chipmunk kidney cells(CMK). The major characteristics of the continuous cell line were defined and the susceptibility to some visuses was tested as the preliminary screening for virus susceptibility of the cultures of chipmunk kidney cells. Materials and Methods A. Materials 1. Chipmunk kidneys: Korean chipmunks(Tamias sibiricus asiaticus, Gmelin) of both sex, age of less than one year, weighing 50 to 70gm of body weight, were used. Both kidneys were removed aseptically following sacrifice with ether anesthesia. 2. Growth medium for tissue culture: Growth medium consisted of NCTC 135(Difco, U.S.A.) and fetal bovine serum(Flow Labs, Inc., U.S.A.). The concentration of fetal bovine serum varied according to the level of subcultures; i.e., primary to 10th; 20%, 11th to 15th; 15% and 10% after the 16th subculture. Agar overlay medium for plaque assay of viruses consisted of 2X NCTC 135:5 parts, 2% Noble agar: 4 parts and fetal bovine serum: 1 part. 3. Miscellaneous solutions: a) Phosphate buffered saline(PBS, Dulbecco): pH 7.2, penicillin(100μ/ml) and streptomycin(100γ/ml) were added. b) Trypsin solution:0.25% trypsin(1;250, Difco, U.S.A.) in PBS was Seitz filtered and concentrations of antibiotics were increased i.e., penicillin 50μ/ml, and streptomycin 500γ/ml. c) Neutral red solution: 0.1% neutral red solution was made with PBS and autoclaved. d) Citric acid-crystal violet solution: 0.1% crystal violet solution was made with 0.1M citric acid solution and autoclaved. 4. Viruses: Four viruses are included for preliminary screening for viral susceptibility of the cultures of chipmunk kidney cells(Table 1). Viruses were stored at-50℃ before use and PBS was used as virus diluent. Table 1. Viruses ------------------------------------------------------------------------------- Virus Specification Remarks ------------------------------------------------------------------------------- Vaccinia virus vaccine lymph N.I.H., Korea. Japanese encephalitis virus, infected mouse brain Ditto. Nakayama Stain homogenate Influenza virus type A, infected allantoic fluid Dept. of Microbiology PR 8 strain Vesicular stomatitis virus tissue culture fluid Univ. of Calif., San Francisco, U.S.A ------------------------------------------------------------------------------- 5. Glasswares: For tissue culture purposes, Leighton tube(Pyrex) and prescription bottles(Neutraglass, Kimble Co, U.S.A) wre used. Haemosol(Meinecke & Co., Inc., U.S.A) was used for cleaning of glasswares, and all glasswares were sterilized by dry heat. 5. Chromosome studies: Chromosome spread was made oby the ethod of Merchant et al.(1960b) and stained with 2% orcein solution(Matheson Coleman & Bell, U.S.A.). 6. frozen storage and recultivation experiment: Trypsin-dispersed cells were suspended in the storage medium(growth medium containing 10% glycerin) and 1.5 to 2ml of cell suspension in storage medium was put into lyophilization ampoule, and the ampoules were sealed and kept frozen at -15℃ and -50℃. During frozen storage, reculture experiement was performed at 1 week intervals. 7. Mycoplasma detection experiment: Diphasic medium and solid medium of Deeb and Kenny(1967) were used for isolation of Mycoplasmas from the trypsin-dispersed cell suspension. 8. Preparation of electronmicrograph: Cells grown in small plastic Petri dish(in CO^^2 incubator) were directly fixed with 3% glutaraldehyde and 1% osmium tetroxide and embedded with Epon 812. Thin sections were made with MT-2 ultramicrotome and stained with uranyl acetate and lead citrate. Electronmicrograph was take with Hitachi electronmicroscope(HU-11, E-Ⅰ model). 9. Viral susceptibility tests: Cytopathogenic effect(CPE) was tested with monolayer cultures grown in Leighton tubes, and plaque formation was carried out with monolayer cultures grown in prescription bottles. Virus adsorption was allowed for 1 hr. at 37℃ and plaques were examined by neutral red staining after 5 to 7 days of incubation at 37℃. Conclusion 1. A continuous cell line has been established from Korean chipmunk(Tamias sibiricus asiaticus, Gmelin) kidney cells by subculture technique in tissue culture. 2. The cells maintained the morphology of epithelial cells during subcultures from the primary to the 25th passages. No apparent changes have resulted in the growth pattern of established continuous cell line from those of the primary cultures. 3. Chromosome study with the cells of primary culture confirmed other's reports of the chromosome number of Korean chipmunk(2n=38). During subculture occurrence of aneuploidy became apparent. 4. Electronmicrographs of the continuous cell line revealed neither virus particles nor virus-line particles in the cytoplasm and nucleus of cells. Growth experiments also failed to detect the presence of Mycoplasma in the cells. 5. Vaccinia virus and Japanese encephalitis virus infection of cells of the 2nd and the 20th subculture produced both characteristic cytopathogenic effects and plaques respectively. But influenza virus type A, PR 8 stain and vesicular stomatitis virus failed to produce either cytopathogenic effects of plaques in this system. [영문] Tissue culture techniques, originated by the pioneering works of Roux(1885) and Harrison(1970, have been one of the most fundamental methods in virological studies. Application of trypsin in dispersion of the cells from freshly ulbecco, 1952; Moscona, 1952; Younger, 1954 and in harvesting cells for subculture allowed to obtain uniform populations of sufficient density of cells and to achieve confluent cultures a few days after the initiation of tissue culture. It tissue culture experiences, the cells very commonly cease growing after a limited period of rapid growth during the subcultures of the primary ones. Therefore, it is generally accepted that cells should survive 12 to 20 generations before they can be considered a stain. Recently a many variety established cell lines have been developed(Cell culture Collection Committee, 1964). In spite of the rapid increase in the numbers of established cell lines dervied from various sources, the following observations strongly support the necessity of established new cell lines. i.e., it has been frequently shown that cell cultures derived from various sources differed significantly in their susceptibility to the same viruses. Vogt(1967) showed that Rous sarcoma virus, a model of defective virus(Hansfusa et al., 1963), could multiply and produce mature infective virus particles in susceptible cell culture, and accumulating evidences exist that various animal species and the cell lines derived from their tissues are contaminated with a variety of latent viruses(de Harven, 1964; Shipman, 1969; Smith et al., 1970; Lowy et al., 1971). Interestingly various tissues derived from human and primate, and domestic or laboratory animals have been exclusively used as the major sources of primary cultures and the subsequent development of cell lines. To the author's knowledge, neither continuous cell lines nor cloned cell lines have ever been established from wild, small mammals. In this study continuous cell line was established from the primary culture of chipmunk kidney cells through serial subculture over a period of one year. The cell line was tentatively named chipmunk kidney cells(CMK). The major characteristics of the continuous cell line were defined and the susceptibility to some visuses was tested as the preliminary screening for virus susceptibility of the cultures of chipmunk kidney cells. Materials and Methods A. Materials 1. Chipmunk kidneys: Korean chipmunks(Tamias sibiricus asiaticus, Gmelin) of both sex, age of less than one year, weighing 50 to 70gm of body weight, were used. Both kidneys were removed aseptically following sacrifice with ether anesthesia. 2. Growth medium for tissue culture: Growth medium consisted of NCTC 135(Difco, U.S.A.) and fetal bovine serum(Flow Labs, Inc., U.S.A.). The concentration of fetal bovine serum varied according to the level of subcultures; i.e., primary to 10th; 20%, 11th to 15th; 15% and 10% after the 16th subculture. Agar overlay medium for plaque assay of viruses consisted of 2X NCTC 135:5 parts, 2% Noble agar: 4 parts and fetal bovine serum: 1 part. 3. Miscellaneous solutions: a) Phosphate buffered saline(PBS, Dulbecco): pH 7.2, penicillin(100μ/ml) and streptomycin(100γ/ml) were added. b) Trypsin solution:0.25% trypsin(1;250, Difco, U.S.A.) in PBS was Seitz filtered and concentrations of antibiotics were increased i.e., penicillin 50μ/ml, and streptomycin 500γ/ml. c) Neutral red solution: 0.1% neutral red solution was made with PBS and autoclaved. d) Citric acid-crystal violet solution: 0.1% crystal violet solution was made with 0.1M citric acid solution and autoclaved. 4. Viruses: Four viruses are included for preliminary screening for viral susceptibility of the cultures of chipmunk kidney cells(Table 1). Viruses were stored at-50℃ before use and PBS was used as virus diluent. Table 1. Viruses ------------------------------------------------------------------------------- Virus Specification Remarks ------------------------------------------------------------------------------- Vaccinia virus vaccine lymph N.I.H., Korea. Japanese encephalitis virus, infected mouse brain Ditto. Nakayama Stain homogenate Influenza virus type A, infected allantoic fluid Dept. of Microbiology PR 8 strain Vesicular stomatitis virus tissue culture fluid Univ. of Calif., San Francisco, U.S.A ------------------------------------------------------------------------------- 5. Glasswares: For tissue culture purposes, Leighton tube(Pyrex) and prescription bottles(Neutraglass, Kimble Co, U.S.A) wre used. Haemosol(Meinecke & Co., Inc., U.S.A) was used for cleaning of glasswares, and all glasswares were sterilized by dry heat. 5. Chromosome studies: Chromosome spread was made oby the ethod of Merchant et al.(1960b) and stained with 2% orcein solution(Matheson Coleman & Bell, U.S.A.). 6. frozen storage and recultivation experiment: Trypsin-dispersed cells were suspended in the storage medium(growth medium containing 10% glycerin) and 1.5 to 2ml of cell suspension in storage medium was put into lyophilization ampoule, and the ampoules were sealed and kept frozen at -15℃ and -50℃. During frozen storage, reculture experiement was performed at 1 week intervals. 7. Mycoplasma detection experiment: Diphasic medium and solid medium of Deeb and Kenny(1967) were used for isolation of Mycoplasmas from the trypsin-dispersed cell suspension. 8. Preparation of electronmicrograph: Cells grown in small plastic Petri dish(in CO^^2 incubator) were directly fixed with 3% glutaraldehyde and 1% osmium tetroxide and embedded with Epon 812. Thin sections were made with MT-2 ultramicrotome and stained with uranyl acetate and lead citrate. Electronmicrograph was take with Hitachi electronmicroscope(HU-11, E-Ⅰ model). 9. Viral susceptibility tests: Cytopathogenic effect(CPE) was tested with monolayer cultures grown in Leighton tubes, and plaque formation was carried out with monolayer cultures grown in prescription bottles. Virus adsorption was allowed for 1 hr. at 37℃ and plaques were examined by neutral red staining after 5 to 7 days of incubation at 37℃. Conclusion 1. A continuous cell line has been established from Korean chipmunk(Tamias sibiricus asiaticus, Gmelin) kidney cells by subculture technique in tissue culture. 2. The cells maintained the morphology of epithelial cells during subcultures from the primary to the 25th passages. No apparent changes have resulted in the growth pattern of established continuous cell line from those of the primary cultures. 3. Chromosome study with the cells of primary culture confirmed other's reports of the chromosome number of Korean chipmunk(2n=38). During subculture occurrence of aneuploidy became apparent. 4. Electronmicrographs of the continuous cell line revealed neither virus particles nor virus-line particles in the cytoplasm and nucleus of cells. Growth experiments also failed to detect the presence of Mycoplasma in the cells. 5. Vaccinia virus and Japanese encephalitis virus infection of cells of the 2nd and the 20th subculture produced both characteristic cytopathogenic effects and plaques respectively. But influenza virus type A, PR 8 stain and vesicular stomatitis virus failed to produce either cytopathogenic effects of plaques in this system.restrictio