In Vitro Cell. Dev. Biol.--Animal 36:617~19, November/December 2000 �9 2000 Society for In Vitro Biology 1071-2690/00 $10.00+0.00

Letter to the Editor

ESTABLISHMENT OF COLOSSOMA BRACHYPOMUM EMBRYO CELL LINE

Dear Editor: Numerous studies have been conducted to elucidate the impacts

of cold stress on warm-water fishes (Zou and Lou, 1998). The effects of low temperatures on the physiological functionality of the central nervous system (Miyata et al., 1995), cellular membrane (Vigh et al., 1993), isoenzymes (Baldwin et al., 1970), and metabolism (Chen et al., 1988) have been the focus of recent research. However, these studies were mostly carried out using tissues from various adult fish. While they provided important information on biochemical func- tions of individual organs of warm-water fish under low tempera- tures, the highly differentiated cell types in these tissues make it difficult to understand the cellular and molecular mechanisms of cold stress. A homogeneous cell type would be very useful for the study of cold stress at the cellular level.

Colossoma brachypomum is a typical warm-water fish that origi- nates in the tropical Amazon River, with a reputation of rich nutri- ents, low content of cholesterol, and rapid growth. Incapable of liv- ing a normal life under a temperature of 12 ~ C, this fish could not survive the winter temperatures in temperate regions, and thus farming of this species in this kind of region is very limited. In order to study the lethal mechanisms of low temperature in this fish, we have established a cell line, designated as WM-9001, from C. brachypomum blastulas, that was successfully passed and main- tained for 75 generations. We also determined the growth rates of the cell line at different temperatures and measured some aspects of biochemical and morphological changes in these cells upon cold stress. It is demonstrated that this stable cell line could serve as a model system for studying cold stress on warm-water fishes. Fur- thermore, the embryonic nature of this cell line makes it useful to screen for cold-tolerant mutants (Fryer and Lannan, 1994), and fur- ther use them as donors in nuclear transplantation or chimera pro- duction to generate cold-tolerant variants (Specksnijder et al., 1997; Hong et al., 1998).

Oosperms of C. brachypomum were kept in 28 ~ C water for 2.5 h to develop to the early blastula stage. The blastulas were then sterilized in 0.01% KMnO4 for 30 min, and in 70% ethyl alcohol for 10 s, then rinsed with Roswell Park Memorial Institute (RPMI) 1640 medium twice, and it was bathed in RPMI 1640 (GIBCO BRL, Grand Island, NY) medium containing 1000 U/ml penicillin and 1000 ~g/ml streptomycin. The egg membranes were carefully re- moved with sterilized forceps or by digestion with 0.25% trypsin (Sigma Chemical Co., St. Louis, MO). The blastulas were transferred to 25-ml culture flasks (about 20 blastulas per flask). About 8 h later, medium RPMI 1640, supplemented with 20% fetal bovine serum, without penicillin or streptomycin, was added to the flasks. The flasks were then incubated at 30 ~ C. Radiate growth from the embryonic tissues appeared after I d. In about 10 d the newly grown cells were covering the flasks. The cells were then digested with 0.25% trypsin in phosphate-buffered saline and then seeded. The

cell line was passed for a consecutive 25 generations in a 10-d interval. After 25 passages, cell growth was accelerated and passage intervals reduced to 5-7 d. Most of the cells grew to a fibroblast- like shape (Fig. la).

In order to obtain a chromosomal spread, WM-9001 cells were treated with 0.25 ~g/ml colcemid for 8 h. The metaphase cells were collected by stirring and stained with Giemsa. Of a total of 200 metaphase ceils counted, 71% had a chromosome number of 2n = 54 (Fig. lb). All the chromosomes showed median kinetochores. The mitotic index curve of the cell line at its 37th passages was deter- mined using the slide method at the culturing temperature of 30 ~ C. The mitotic index in metaphase was 40-60% and the cell dou- bling time was 19.71 h. The cell cycle analysis of WM-9001 was carried out with a flow cytometer, the Go + G, S and G2 + M phases were found to be 76.71, 12.97, and 10.92%, respectively.

Growths of the WM-9001 cells at different temperatures were measured. Muhiple flasks of WM-9001 cells with the same initial density of 2 • 10S/ml were incubated at 30 ~ C for 2 h for the adhesion to take place. The flasks were then divided into eight groups and incubated at 5, 10, 15, 20, 25, 30, 35, and 40 ~ C, respectively. The number of cells in each group was counted after 5 d of incubation. The proliferation rates at different temperatures are shown in Fig. 2. Below 10 ~ C, the cells were damaged, shed gradually, and died out finally. At 15 ~ C, cells did not divide and few of them shed from the wall. At 20 ~ C, growth was slow, whereas at 20-35 ~ C growth became rapid. At 40 ~ C, ceils stopped prolif- eration. We obtained the cell growth curves at 20, 27, 30, and 37 ~ C, respectively, with the initial cell density of 6 X 104/ml (Fig. 3), indicating that the growth of the WM-9001 cell was rapid at 27-30 ~ C, especially at 37 ~ C.

Comparisons of the temperature-dependent growth patterns of the WM-9001 cell line and that of the ZC-7901 cell line, established from the lip cell of grass carp, Ctenopharyngodon ideUus, a eury- thermal fish species (Zhang and Yang, 1981), showed that the two have different survival and optimal growth temperatures. As shown above, the WM-9001 cells grew optimally at 30-37 ~ C, and stopped growing until the temperatures reached 40 ~ C. On the other hand, the ZC7901 cells grew optimally at 27-31 ~ C, stopped dividing at 35 ~ C, and died out above 37 ~ C. The ceils of WM-9001 did not survive for 5 d at temperatures below 10 ~ C, whereas the ZC7901 cells survived several mo at 4 -10 ~ C. Interestingly, there is a co- incidence between the temperature-dependent growth characteris- tics of the WM-9001 cell and the survival rate of adult C. brachy- pomum at different temperatures. The C. brachypomum grew at 21-32 ~ C, with optimal growth temperatures ranging from 28-31 ~ C (Helfand et al., 1982; Chen et al., 1988). Most of them live abnormally and lose body balance when water temperature is below 12 ~ C. Mortality rises rapidly at 10 ~ C. The parallelism between the temperature-dependent growth of the WM-9001 cell line and the

6 1 7

618 MING AND SUNHONG

FIG. 1. (a) The embryonic cell (39 passages) of C. brachypomum. (b) The chromosome of the embryonic cell. Bar, a, 75 Ixm; b, 10 tzm.

I ' ~ 14

I2

If)

8

4

2

0

5 I 0 15 21) 25 :18 35 40

Temperature (~

FIG. 2. The WM-9001 growth (40 passages) at different temperatures.

35

3O

~25

~ 2 0

2 1 0

~5 r t i i k i ~ i

1 2 3 4 5 6 7 I0 13 16 Time (d )

FIG. 3. Growth curve of the WM-9001 cell.

survival pattern of the adult fish might indicate the two share similar biochemical mechanisms upon cold stress, and the WM-9001 cells could be used as a model to study the molecular and cellular mech- anisms underlying cold stress on this fish.

The WM-9001 cells were subjected to the cold-stress tempera- ture of 8 ~ C. In 3 d, the cells demonstrated a clear contour with a large number of stored particles accumulated in the cytoplasm. In 6 d, the cells shed in large quantity with a trypan blue staining refusal rate of 78.5% and failed to recover when incubated at 30 ~ C. The fornlazan optical density value (3-[4,5-dimethyhhiazole-2- yl]-2,5-biphenyltetrazolium bromide activity) reduced to 64.6% in 3 d and to 50% in 10 d. The bioluminescence of the cells doubled in 3 d, and the enzyme superoxide dismutase activity was elevated. When the ZC7901 cells were subjected to the same temperature (8 ~ C), on the other hand, the above biochemical and morphological changes were not found. The biochemical and morphological chang- es might result from changed activities of some metabolic enzymes or cold-stress proteins rather than from the direct damages of the cell membranes (Helfand et al., 1982). In addition, the biolumi- nescence method is a convenient and nondestructive way of deter- mining the presence of cold stress in cells.

The embryonic nature of the WM-9001 cells is apparent. First, we found that the WM-9001 cells had 83.3% of average survival rate in the secondary culture, while the survival rates of other cell lines driven from somatic tissues, such as snout, caudal fin, and anal fin of the same fish were merely 1.2-6.7% (data not shown). Second, the cell lines derived from snout, caudal fin, and anal fin were 40, 45, and 47 passages, respectively. In addition, compared to the somatic cell lines, the WM-9001 cells demonstrated faster growth rates, less serum consumption, and better growth at 37 ~ C. The WM-9001 cells grew well and successfully passed in the min- imal essential medium while the somatic cells failed. All these in- dicated that the WM-9001 cell line has embryonic characteristics. The developmental potential of this cell line is currently under in- vestigation.

A C K N O W L E D G M E N T S

We thank Dr. Ye Wei (Panyu Tilapia Fishery in Guangzhou), Mr. Fu Yun, Dr. He Jiangguo (Parasitology teaching research group of Zhongshan Uni- versity), Ms. Weng Shaopin, and Ms. Ye Qiaozhen for their support. This research was supported by the Scientific Foundation of Zhejiang province (394339).

REFERENCES

Baldwin, J.; Hochacha, 13. W. Functional significance of isoenzymes in thermal acelimatization--acetylcholinesterase form trout brain. Biochem. J. 116:883487; 1970.

Chen, J. D.; Yew, E H.; Li, G. C. Thermal adaptation and heat shock response of tilapia ovary cells. J. Cell Physiol. 134(2):189-199; 1988.

Fryer, J. L.; Lannan, C. N. Three decades offish cell culture: a current listing of cell lines derived from fishes. J. Tissue Cult. Methods 16(2):87- 94: 1994.

Helfand, S. L.; Werkeister, J.; Roder, J. C. Chemiluminescence response of human natural killer cells. I. The relationship between target cell building, chemiluminescence and cytolysis. J. Exp. Ned. 156(2):492- 505; 1982.

Hong, Y.; Winkler, C.; Sehartl, M. Production of medaka fish chimeras from a stable embryonic stem cell line. Proc. Natl. Aead. Sei. USA 95(7):3679-3684; 1998.

Miyata, S.; Ishiyama, M.; Shido, O., et ah Central mechanism of neural ac- tivation which cold acclimation of rats using Fos immunohistoehem- istry. Neurosei. Res. 22,209-22,218; 1995.

ESTABLISHMENT OF COLOSSOMA EMBRYO CELL LINE 619

Speksnijder, J. E.; Hage, W. J.; Lanser, P. H., et al. In vivo assay for the developmental competence of embryo-derived zebra fish cell lines. Mol. Mar. Biol. Biotechnol. 6(1):21-32; 1997.

Vigh, L.; Los, D. A.; Horvath, I.; et al. The primary signal in the biological perception of temperature: Pd-catalyzed hydrogenation of membrane liquids stimulated the expression of the des A gene in Synechocystis PCC6803. Proc. Natl. Acad. Sci. USA 90:9090-9094; 1993.

Zhang Nian-ci; Yang Guang-zhi. The establishment of strain ZC-7901 and substrain ZC-7901S1 of Grass carp (Ctenopharyngodon idenllus) lip cell. Acta Biol. Exp. Sin. 14(1):101-106. 1981

Zou Shuming; Lou Yundong. The mechanisms of cold acclimatization and cold-toterance breeding in fish, J. Shanghai Fish. Univ. 7(8):231-237; 1998.

Zhang Ming ~ Chen Sunhong

College of Life Sciences

Zhej iang University

Wensan Road, Hangzhou, 310012

People's Republ ic of China

(Received 7 February 2000)

1Towhom con'espondence should be addressed.