Chromosome Studies During Long-Term Cultivation of Epithelioid Cercopithecus and Cynomolgus Monkey Kidney Cell Lines 1

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1 Chromosome Studies During Long-Term Cultivation of Epithelioid Cercopithecus and Cynomolgus Monkey Kidney Cell Lines 1 JEAN FERGUSON and G. A. TOMKINS, Commonwealth Serum Laboratories, Parkville, Victoria, Australia SUMMARY-Cytogenetic studies were performed on epithelioid cells derived from Cercopithecus monkey kidneys and cynomolgus monkey embryo kidneys. The cells were grown in tissue culture for periods of up to 22 months during which, in some instances, more than 130 passages were made. Chromosome counts were performed at intervals, and increases in chromosome number were observed in all lines surviving for more than 50 to 60 passages. The diploid chromosome number was replaced with a range of chromosome numbers in the triploid region. Some changes were noted in chromosome morphology, but the chromosomes could still be clearly identified as belonging to the species of origin of the cell line. While these chromosomal changes were taking place, the cell cultures showed no apparent change in morphology or growth rate. There was no appreciable difference in the behaviqr of lines derived from kidneys of the two different monkey species. Epithelioid cell lines were of particular interest because of their widespread use for in vitro virus and biochemical studies and because of the implication of these cells as contaminants in the reported establishment of some cell lines.-j Nat Cancer Inst 33: , THE MAJORITY of cell cultures derived from human and monkey tissues and propagated in vitro for long periods fall into two classes: 1) cells with epithelioid morphology and heteroploid chromosome constitution that can be maintained in vitro indefinitely; 2) cells with fibroblast-like morphology and diploid chromosome constitution that diminish in growth rate after prolonged cultivation in oitro and usually fail to survive after approximately 50 passages. While the fibroblast lines maintain their single diploid chromosome number throughout their lifetime (1-3), the established epithelial lines exhibit a range of chromosome numbers around a mode approximately 50 percent higher than the diploid number of the tissue of origin of the cell (4-6). Earlier papers have described chromosomal shifts in such lines. These shifts have been accompanied by abrupt changes in morphology and cultural characteristics generally interpreted as a "transformation" of a few cells in the culture, which gives them a selective advantage resulting in their overgrowing the culture (7). More recently it has been suggest~d that at least some of these rapidly multiplying "trans- 1 Received January 17,

2 620 FERGUSON AND TOMKINS formed" cultures may have been due to accidental contamination of cultures with other more rapidly growing cell lines (5, 8). In this paper we describe observations on the chromosome and cultural characters of epithelioid cell lines over a long period. Cell lines and sublines derived from the kidneys of two monkey species were examined for periods of up to 22 months in tissue culture, during which, in some instances, more than 130 passages were made. Chromosome counts were made at each passage after initiation of the cultures in the cynomolgus kidney cells and after the 41st passage in the Cercopithecus kidney cells. Cultures were observed for any change in growth rate or morphology of the cells. MATERIALS AND METHODS Cercopithecus monkey kidney cultures.-designated BS-C-1, these cultures were kindly supplied by Mrs. H. Hopps, Division of Biologics Standards, National Institutes of Health, Bethesda, Maryland (9). On arrival in this laboratory in December, 1961, the cells were in their 41st passage and appeared epithelioid. Cells were maintained in pyrex milk dilution (MD) bottles in medium 858 plus 20 percent fetal calf serum. Medium 858 was modified (70) by the addition of a solution designated DG at one tenth of the original formulas (71). The serum was heat-inactivated at 56 0 C for 30 minutes and then filtered through ultrafine sintered glass just before use. Medium was replaced 2 to 3 times per week and subcultures were made every 7 days. During subculture all reagents were preheated to 37 0 C. After removal of growth medium, the cell sheet was washed once with phosphatebuffered saline (72). One ml of 0.25 percent trypsin solution was added for 5 minutes and then discarded. Another 1 ml of trypsin solution was added for 20 minutes and cells were then loosened from the glass surface by being pipetted with 3 ml medium 858. The resulting cell suspension was divided between 2 MD bottles, each containing 8 ml medium 858 and 2.5 ml fetal calf serum. Cynomolgus monkey (Macaca irus) embryo kidney cultures.-designated ME(l) and ME(2), these cultures were isolated in this laboratory. Kidneys removed aseptically from 4- to 5-month embryos were dissected to yield cortex which, after being washed in phosphate-buffered saline, was minced finely with surgical scissors and then washed again. Tissue from one pair of kidneys was distributed between six 60 mm petri dishes. In each dish the tissue was gently agitated in 0.4 ml of warm trypsin (0.25%) and incubated at 37 0 C for 15 to 20 minutes. A volume of 2 ml of medium 858 was then added and the cell suspension transferred to an MD bottle containing 8 ml of medium 858 plus 2.5 ml of heat-inactivated, filtered fetal calf serum. At day 3 fresh growth medium was added. At day 8, epithelioid cells, covering more than half the cell surface, were removed with 1 ml trypsin and transferred to a fresh MD bottle containing medium 858 and 20 percent fetal calf serum. Growth medium was replaced 3 times weekly and cells were subcultured 1: 2 every 5 to 7 days. Chromosome investigations.-on cells grown on coverslips in 60 mm petri dishes as previously described (73), counts were made at a magnification of 1,000 with the use of a crosswire grid in the eyepiece. Mitotic figures showing no overlapping of the chromosomes were photographed and karyotypes made by chromosomes arranged in descending order of size and according to the position of the centromere. RESULTS Chromosomal Analysis and Cultural Characteristics of Cercopithecus Monkey Kidney Epithelioid Lines The original BS-C-1 culture was divided into 4 separate sublines designated #1, 2, 3, and 4. Number 1 has been cultured continuously since its arrival in this laboratory, while #2, 3, and 4 were initially stored at C (74) before being thawed and then kept in continuous cultivation. Chromosome counts were made regularly on each subline (table 1). When first examined at the 49th passage, #1 showed a majority of cells with chromosome numbers slightly below the diploid number of 60. When examined 5 passages later, there had been a shift away from the diploid number to numbers in the vicinity of 100 in the few cells counted. This chromosome range has been maintained throughout 70 subsequent passages. The second subline (#2) showed a gradual shift from cells with a diploid number of chromosomes to cells with a range of numbers around 110 (text- JOURNAL OF THE NATIONAL CANCER INSTITUTE

3 MONKEY KIDNEY CELL LINES: CHROMOSOMAL CHANGES 621 TABLE l.--changes in chromosome number during long-term cultivation of two epithelioid cell lines derived from monkey kidney cells Cell line BS-C-l ME (1) ME(2) Subline (No.) Diploid No. of tissue of origin fig. 1). Sublines #3 and 4 have shown only cells with a near-diploid chromosome number. Karyotypes were examined by arrangement of the chromosomes in descending order of size and according to the position of the centromere (fig. la). Karyotypes of cells with chromosome numbers in the region of 100 showed an increase in number in the medium and large chromosomes but not in the smallest (fig. lb). The four sublines of BS-C-l have been cultured over long periods. During this time, although #1 and 2 showed the chromosomal changes already described, there has been no change in the growth rate of the cells and no appreciable change in their morphology (figs. 3a and 3b). The third subline, #3, is now in the 60th passage since its isolation in tissue culture and shows no alteration of cultural characteristics. We have failed to maintain #4 in continuous culture. Although there was no apparent change in the environment, toward the 50th passage this subline had cells granular in appearance. The growth rate of the cultures decreased Chromosome examination Chromosome range predominantly until between 49th and 54th passage when chromosome numbers shifted to (see textfig. 1) Similar chromosome shift noted between 45th and 64th passage (see text-fig. 1) Chromosome range of seen between passages 35 and 56 j no chromosome shift noted as yet Chromosome range of seen between passages 22 and 51 Chromosome count of 42 until 20th passagej cells next examined between 28th and 35th passage when chromosome range was Two chromosome ranges, and 75-85, seen between 23d and 31st passage j 15-20% of cells showing the latter range Chromosome count of 42 until 20th passagej cells examined between 23d and 30th passage showed a range around a modal figure of 42 j later passages showed an increase in higher chromosome numbers, until at 64th passage there was a wide range of numbers between 30 and 90 (see text-fig. 2) Cultural characteristics Morphology of cells has remained unchanged throughout j passage of cells is continuing " " Gradual decrease in growth rate until cells failed to passage beyond 51 Gradual decrease in growth rate until cells failed to passage beyond 40 Gradual decrease in growth rate until cells failed to passage beyond 33 Morphology of cells has remained unchanged throughout j passage of cells is continuing and the cells failed to grow after the 51st transfer since their isolation in tissue culture. Chromosomal Analysis and Cultural Characteristics of Cynomolgus Monkey Kidney Epithelioid Lines The culture derived from the monkey embryo, ME(l), was divided into 2 sublines designated #1 and 2. Number 1 has been cultured continuously since its initiation in this laboratory, while #2 was stored after the 20th passage for a period at C before being thawed and kept in continuous culture. Chromosome counts were made regularly on these two sublines and on a line ME(2) derived from a second monkey embryo (see table 1). Both sublines of ME(l) revealed chromosome shifts to higher numbers. Number 1 showed all cells to have chromosome numbers in the 3n-4n region before the culture failed to grow after the 40th passage, while #2 showed a shift to higher chromosome numbers in 15 to 20 percent of the cells before failing to grow beyond the 33d passage. Chromo- VOL. 33, NO.4, OCTOBER 1964

4 622 FERGUSON AND TOMKINS Diploid Bs~-H.l ; 10~ o III -' -' ~ :s BS-C ci z Passage 49..,----~--~~--_.~.a~.~.~~~~-~+ =7. SOl &; I' 200 approlt '.. L.Pass. S' I r LL -l11=2 : l~ [. m ~.L_ n ~.~ Passage 82 Passage 126.., 1'- Possage.5 ~ ~~-- Passage 51 ~g.l... "'"... ~ M so NO. OF CHROMOSOMES some counts on ME(2) cells revealed a gradual shift of numbers to a wide range between 30 and 90 (text-fig. 2). Since cynomolgus monkey chromosomes, with no acrocentric structures, are difficult to arrange into clearly defined groups, karyotypes were made by the chromosomes arranged in descending order of size and according to position of centromere (fig. 2a). Karyotypes of cells with chromosome numbers in the region of 70 showed each pair of chromosomes in the diploid set to be represented by 3 or even 4 chromosomes (fig. 2b). Cultivation for an indefinite period was attempted with ME(1) and ME(2) cells. While the two sublines of ME(l) failed to survive beyond the 40th and 33d passage, respectively, the ME(2) cell cultures are currently in their 64th passage. ME(1) cells showed a slowing down of growth rate and an increase in granularity before failing to Passage 56 I approx. TEXT-FlGURE l.--chromosome counts during serial passage of BS-C-l cells. passage. Their morphology was epithelioid throughout. ME(2) cells have shown no apparent change in growth rate or in their epithelioid morphology (figs. 4a and 4b). DISCUSSION In recent years techniques have become available for the study of the chromosome number and karyotype of cells grown in tissue culture, so that it is now possible to examine with more detail the history of the emergence of heteroploid cell lines. The earliest reports on the establishment of heteroploid cell lines noted the sudden appearance in cultures of morphologically distinct epithelioid cells with the property of a much increased growth rate. These cells were derived from human tissues of normal and carcinomatous origin; HeLa cells were derived in 1953 (75) from a human cervical JOURNAL OF THE NATIONAL CANCER INSTITUTE

5 MONKEY KIDNEY CELL LINES: CHROMOSOMAL CHANGES 623 M~ DiPlloid 10 Pa III ~..J ~ ~ ci z I SO l)o 2lJO Poasage 21 I n~,. I I..wa :I.~,. 30 ~ " SO I I Paaage21 a a, Paaage 35 a am, a n D 8m. NO. 80 OF CHROMOSOMES carcinoma, while in 1954 Chang (16) established lines from tissues, such as those of normal human conjunctiva and appendix. There followed many reports of continuous cell lines being established, and it was suggested that these cell lines arose from a few cells which "transformed" to meet the conditions of the environment and rapidly overgrew the original cell type (7). Attempts to discover whether a particular aspect of the cultural environment contributed to this "transformation" have not been successful and the emergence of these cell lines still has to be considered a chance occurrence. When accurate methods for the study of chromosomes in tissue culture became available in 1956 (17, 18), it was possible to examine in detail the chromosome complement of the heteroploid cell lines already established. It was found that most lines showed a range of chromosome numbers with a mode in the vicinity of the triploid figure. For a particular cell line this range could vary somewhat according to cultural conditions. Saksela a 90 Passage 58 Opprllll. 1 TEXT-FIGURE 2.-Chromosome counts during serial passage of ME(2) cells. demonstrated that the chromosome range of HeLa cells could vary when cultured in different samples of human sera or when cultured in heat-inactivated sera (19). It was also demonstrated that cloning of a cell with a chromosome number away from the mode resulted in a population of cells with again a range of numbers, but this time around a new mode (20). The chromosome numbers in these heteroploid epithelioid cultures are in marked contrast to those found more recently when fibroblast cells, derived from human and monkey tissues, were cultivated for long periods in vitro. Under the particular conditions of culture, fibroblast-like cells could be subcultured for approximately 50 passages without change in karyotype. In this study we used these same culture methods to determine whether ~pithelioid cells could also be cultivated for long periods without change in chromosome number and growth characteristics. In view of reported variation of chromosome VOL. 33, NO.4, OCTOBER 1964

6 624 FERGUSON AND TOMKINS ranges within one cell line, we compared several sublines of two types of cell. For these studies we used four sublines of a diploid epithelioid cell line isolated by Hopps et at. from Cercopithecus monkey kidney (diploid No. = 60) and compared it with three sublines isolated in our own laboratory from cynomolgus monkey kidney (diploid No. = 42) (21). From our experiments it can be seen that when an epithelioid line was maintained beyond 50 to 60 passages a chromosomal shift occurred. The phenomena have been observed in the two species with a markedly different diploid number. A gradual shift occurred to a range of chromosome numbers in the triploid region. There was no indication of the diploid cells being overgrown by a particular cell with a chromosome number in the triploid range and an altered morphology. Rather, there appeared to be a gradual change of chromosome numbers with no apparent alteration of morphology, growth rate, or cultural requirements of the cells. Karyotypes of cells with higher than the diploid number of chromosomes were examined to see if any particular chromosomes were involved in the duplication process. The three sublines of cells derived from cynomolgus monkey kidneys showed an increase in number of most of the chromosomes of the set, while with the BS-C-l cells the increase in chromosome number appeared to be confined to the medium and large chromosomes. While chromosomal analyses revealed marked changes taking place within the nucleus, no change in the cultural appearance or behavior of the cell was observed. The rapid growth rate found in the long-established heteroploid lines, such as in the HeLa cell, was not observed in our cultures. Recently Hopps et at. have shown (9) that in long-term culture the chromosomes of BS-C-1 cells changed from the diploid to near-tetraploid region without any appreciable change in the sensitivity of the cells to simian virus 40. In all our studies, lines emerged with chromosome morphology resembling that of the species of origin. There was, therefore, no reason to believe that the increased chromosome number was due to contamination with other cell lines. Such possible contaminations were reported by Clausen and Syverton (5) and Brand and Syverton (8) who characterized cell lines according to chromosome morphology and species-specific hemagglutination reactions. Using these tests they were able to show, for example, that heteroploid cell lines, supposedly derived from rabbit tissue, resembled human cells but not rabbit cells. It has been suggested that trypsin plays a part in altering the behavior of cells in tissue culture. Ludovici et at. (22) reported a more rapid transformation of cells in fibroblast-like cultures when a trypsin-antibiotic mixture was used for removal of cells from the glass during serial passage. Barski and Cassingena (23) found a difference in tumorproducing ability in normal mouse lung cells that had been subcultured over long periods with and without the aid of trypsin. A cell line transmitted by trypsin produced sarcomas in isologous mice, while another cell line, produced from the same explant but transmitted only by mechanical dispersion, showed no ability to produce sarcomas. REFERENCES (1) PuCK, T. T., CmClURA, S. J., and ROBINSON, A.: Genetics of somatic mammalian cells. III. Long term cultivation of euploid cells from human and animal cells. J Exp Med 108: , (2) HAYFLICK, L., and MOORHEAD, P. S.: The serial cultivation of human diploid cell strains. Exp Cell Res 25: , (3) FERGUSON, J., and WANSBROUGH, A.: Isolation and long-term culture of diploid mammalian cell lines. Cancer Res 22: , (4) Hsu, T. C., and MOORHEAD, P. S.: Mammalian chromosomes in vitro. VII. Heteroploidy in human cell strains. J Nat Cancer Inst 18: , (5) CLAUSEN, J. J., and SYVERTON, J. T.: Comparative chromosomal study of 31 cultured mammalian cell lines. J Nat Cancer Inst 28: , (6) NAKANISHI, Y. H., FERNANDES, M. V., MIZUTANI, M., and POMERAT, C. M.: On the chromosome numbers of human amnion cells in primary and strain cultures. Texas Rep BioI Med 17: , (7) WESTWOOD, J. C. N., and TrrMus, D. H. J.: Transformation in tissue culture cell lines: the possible genetic mechanism. Brit J Exp Path 38: , (8) BRAND, K. G., and SYVERTON, J. T.: Results of speciesspecific hemagglutination tests on "transformed," non transformed, and primary cell cultures. J Nat Cancer Inst 28: , (9) Hopps, H. E., BERHEIN, B. C., NISALAK, A.,TJIo, J. H., and SMADEL, J. E.: Biologic characteristics of a continuous kidney cell line derived from the African Green Monkey. J Immun 91: , (10) WHITE, J.: Personal communication. JOURNAL OF THE NATIONAL CANCER INSTITUTE

7 MONKEY KIDNEY CELL LINES: CHROMOSOMAL CHANGES 625 (71) HEALY, G. M., FISHER, D. C., and PARKER, R. C.: Nutrition of animal cells in tissue culture. X. Synthetic medium No Proc Soc Exp BioI Med 89: 71-77, (12) DULBECCO, R., and VOGT, M.: Plaque formation and isolation of pure lines with poliomyelitis viruses. J Exp Med 99: , (73) FERGUSON, J.: Chromosome studies of human cells in tissue culture. Med J Aust 49 (1): 40-43, (74) ---: Long term storage of tissue culture cells. AustJ Exp BioI Med Sci 38: ,1960. (75) SCHERER, W. F., SYVERTON, J. T., and GEY, G. 0.: Studies on the propagation in vitro of poliomyelitis viruses. IV. Viral multiplication in a stable strain of human malignant epithelial cells (strain HeLa) derived from an epidermoid carcinoma of the cervix. J Exp Med 97: , (76) CHANG, R. S.: Continuous subcultivatioj' of epitheliallike cells from normal human tissues. Proc Soc Exp BioI Med 87: , (77) TJIO, J. H., and LEVAN, A.: The chromosome number of man. Hereditas (Lund) 42: 1-6, (78) FORD, C. E., and HAMERTON, J. L.: The chromosomes of man. Nature (London) 178: , (79) SAKSELA, E.: The effect of different human sera on the karyologic picture of HeLa cells. A contribution to the knowledge of environmental influences on cell populations. Acta Path Microbiol Scand Suppl 153: 1-73, (20) CHU, E. H. Y., and GILES, N. M.: Comparative chromosomal studies on mammalian cells in culture. I. The HeLa strain and its mutant clonal derivatives. J Nat Cancer Inst 20: , (27) CHU, E. H. Y., and BENDER, M. A.: Chromosome cytology and evolution in primates. Science 133: , (22) LUDOVICI, P. P., ASHFORD, C., and MILLER, N. F.: Studies on chemically induced and "spontaneous" alterations of morphology and growth potential in human cell culture. Cancer Res 22: , (23) BARSKI, G., and CASSINGENA, R.: Malignant transformation in vitro of cells from C57BL mouse normal pulmonary tissue. J Nat Cancer Inst 30: , VOL. 33, NO.4, OCTOBER 1964

8 JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 33 PLATE 116 FIGURE 1a.-Chromosomes of diploid BS-C-l cells arranged in groups according to size and to position of centromere. FIGURE 1 b.-chromosomes of heteroploid BS-C-l cells arranged in groups according to size and to position of centromere. FERGUSON AND TOMKINS

9 PLATE 117 JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 33 FIGURE 2a.-Chromosomes of diploid ME(2) cells arranged in groups according to size and position of centromere. FIGURE 2b.-Chromosomes of heteroploid ME(Z) cells arranged in groups according to size and position of centromere. 628 FERGUSON AND TOMKINS

10 JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 33 PLATE 118 FIGURE 3a.-Appearance of BS-C-l cells at 46th passage. FIGURE 3b.-Appearance of BS-C-l cells at 136th passage. FERGUSON AND TOMKINS 629

11 PLATE 119 JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 33 FIGURE 4a.-Appearance of ME(2) cells at 22d passage. FIGURE 4b.-Appearance of ME(2) cells at 58th passage. 630 FERGUSON AND TOMKINS