In Vitro Cell. De'0. Biol. 28A:77-79, February 1992 © 1992 Tissue Culture Association 0883-8364/92 $01.50+0.00
Letter to the Editor ROUTINE ESTABLISHMENT OF PRIMARY ELASMOBRANCH CELL CULTURES
50 #g/ml was added to all media and the final osmolarity of the media measured 9 3 0 - 1 0 0 0 raPs. The flasks were placed at 26 ° C in either ambient air or 5% CO2. Half the conditioned medium was changed with fresh medium weekly. Of the eight tissue types tested for growth in vitro, heart atrium, kidney, testis, and brain cells could be routinely cultivated from all species examined. No marked differences in the extent and type of cell growth were noted between species. Growth was evident within one week following the plating of tissues into flasks, and extensive growth with mitotic activity was obtained after three weeks of cultivation (Fig. 1}. The morphology of kidney cells from the shark were seen as fibroblast-like (Fig. I a), epithelial-like (Fig. 1 b), and large epithelial-like (Fig. 1 c). The shark heart, kidney, and brain grew as epithelial-like cells (Fig. 1 d). Spleen, liver, and fin epidermis did not grow. Heart atrium and brain tissues could be grown by plating these tissues directly into medium following mincing. These two tissue types began to adhere to a flask surface within 10 minutes, and more than 90% of the tissue pieces adhered within 1 h of incubation. Kidney and testicular tissues required exposure to trypsin for successful cultivation, and only 50% of the tissue pieces remained adherent after 1 h of incubation. If the minced tissues were treated with trypsin for periods longer than 15 min they became gelatinous, the trypsin was difficult to remove, and the ceils subsequently died. Eagle's minimum essential medium, RPMI1640, and Medium 199 modified as described above, did not support the growth of primary cultures. The modified Opti-MEM I was superior to that of Ham's F-12 and Leibovitz's in both initiating and maintaining growth. Cultures initiated on Opti-MEM I and changed to Leibovitz's or Ham's F-12 media became granular and ceased growing (Table 1).
Dear Editor: Members of the class Elasmobranchiomorphi (Elasmobranchii/ Chondrichthyes of some authors), such as sharks, skates, and rays are interesting organisms for a number of reasons. Unlike most marine mammals and marine bony fishes, elasmobranchs have intracellular osmolarities nearly equal to that of seawater (11). This high osmolarity is maintained by concentrating urea to an average of 0.4 M. In order to counteract the destabilizing effects of the urea on proteins and other macromolecules, they utilize methylamine compounds and amino acids (10,17). Shark cartilage is a source of compounds that prevent the vascularization of animal and human tumors (8). The mechanism whereby sharks exhibit resistance to the development of malignant neoplasias (4) remains unknown, even though sharks possess oncogenes (12). It would be advantageous to employ cultivated elasmobrancb cells to address the areas of research described above on the cellular level and provide a means to isolate elasmobranch viruses. Garner (2) obtained limited growth in brain cells from a sharpnose shark (Rhizoprionodon porosus) after one month of culture. Our Laboratory (6) reported limited success in primary culture using a medium supplemented with urea and TMAO. Grogan and Lund (3) used urea and TMAO in the short term culture of elasmobranch immunocytes. In this paper, we describe a medium and procedures for the routine and rapid establishment of shark and sting ray primary cultures. Heart, kidney, brain, liver, testes, spleen, and fin epidermis tissues were aseptically resected from 20 nurse sharks (Ginglymostoma cirratum), 4 horn sharks (Heterodontus francisc 0, 1 silky shark (Carcharhinusfalciformis), and 8 yellow stingrays (Urolophus jamaicensis). The tissues were placed in phosphate buffered saline modified for use with shark cells (SPBS), which is Dulbecco's phosphate buffered saline to which 333 mM urea, 240 mM sodium chloride (NaCI) and 54 mM trimethylamine N-oxide (TMAO)/(Sigma Chemical Co., St. Louis, MO) are added. Samples from all tissues examined were minced and divided evenly. Half were placed directly onto the surface of 25 cm 2 cell culture flasks (Coming Works, Coming, NY) (14). The other half of each sample was treated for 15 min with 0.25% trypsin (DIFCO, Detroit, MI). The trypsin was removed by pipeting and the tissues were then placed into flasks (15). Culture medium was gently added after the tissues were allowed to adhere to the surface of the flask for 1 h (9). Eagle's minimum essential medium, Medium 199, RPMI164o, Leibovitz's L-15, Ham's F-12 and Opti-MEM I (Grand Island Biological Co., Grand Island, NY) were modified by the addition of 333 mM urea, 188 mM NaCI and 54 mM TMAO, with the pH adjusted to 7.1 Fetal bovine serum (FBS), shark serum, or skate serum, all heat inactivated at 56 ° C for 30 min, were added to a final concentration of 10% to the modified medium. Gentamicin at
TABLE 1 MEDIUM TYPE AND GROWTH OF SHARK CELLS~ Medium
Growth
Opti-MEM I Ham's F-12 Leibovitz's L-15 Eagle's MEM RMPI164o Medium 199
++++ ++ ++
Cardiac atrial tissue from nurse sharks was minced and divided equally into 25 cm2 flasks. After allowing the tissues to adhere for one hour, medium was gently added and the cultures placed at 26 ° C in an atmosphere of 5% CO2 in air. Media were modified by the addition of urea, NaC1, and trimethylamine N-oxide. Media was changed once a week. Growth represented by ( + + + + ) = confluent monolayer in 25 cm2 flasks within two months, to (-) = no growth after two months.
77
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HARTMANN ET AL.
Flc. 1. a-d. Nurse shark primary cultures: fragment (dark area) showing extensive cellular outgrowth after 21 days of culture in Opti-MEM 1 medium. ×420. Bar: 100 ttm. a. Fibroblast-like cells and mitotically active cells (arrow) from the kidney, b. Epithelial-like kidney cells, note the muhinucleate cells (arrow). c. Large epithelial-hke kidney cells, d. Mixture of fibroblastoid and epithehaloid cells from the brain. Note the macrophage-like cells (arrow).
Pooled serum from either sharks or rays was toxic to primary monolayer cultures and did not support the growth of initial explants. Fetal bovine serum, at a concentration of 10% in the medium, was essential for establishing cultures. Some lots of FBS were superior to others in their growth promoting effects. Some lots of urea appeared toxic to the cultures and thus cell culture grade urea, stored at - 2 0 ° C, was employed. Although entire monolayers of cells were grown as primary cultures in 25 em 2 flasks, the cultures could not be successfully passaged by using 0.25% trypsin, 0.04% ethylenediamine tetraacetate acid (EDTA), 0.25% trypsin and 0.04% EDTA, shaking to dislodge M phase cells, or mechanical scraping with a rubber-tipped pipette. The vast majority (approximately 70%) of cells died before attachment, and the viable cells that attached would not divide in the presence of fresh medium, conditioned medium, or fractions from conditioned medium harvested from rapidly growing primary cultures.
The requirement for TMAO in the culture and maintenance of shark cells is supported by previous research on elasmobranch cell culture (3,5,6). Elasmobranchs concentrate urea, TMAO, and NaCI to achieve an osmolality almost equal to that of sea water. Although urea is toxic to most vertebrates, Yancey and Somero (16) have shown that some shark enzymes require urea for optimal kinetic activity. It has subsequently been shown that TMAO is essential in counteracting the perturbing effects of urea on "non-urea" dependent enzymes in the elasmobranch system and on enzymes in the mammalian system (17). Shark brain was the only tissue capable of limited growth in media devoid of TMAO, but containing urea and NaCI. Garner (2) was able to obtain limited growth of shark brain cells after one month of culture in a medium devoid of TMAO. It is thus possible that shark brain cells can synthesize limited amounts of TMAO, or other methylamine compounds, or they may counteract the effects of urea by some novel means. It has been shown that liver and
79
ELASMOBRANCH PRIMARY CELL CULTURE kidney ceils of the Japanese eel can synthesize TMAO (1). The lack of TMAO in previous media used to cultivate elasmobranch cells (2,7,13) may account for the limited growth reported in those studies.
REFERENCES 1. Daikoku, T.; Murata, M.; Sakaguchi, M. Effects of intraperitoneally injected and dietary trimethylamine on the biosynthesis of trimethylamine oxide in relation to seawater adaptation of the eel, Anguilla japonica and the guppy, Poecilia reticulata. Comp. Biochem. Physiol. 89A:261-264; 1988. 2. Garner, W. D. A. Elasmobranch tissue culture: in vitro growth of brain explants from a shark (Rhizoprionodon) and dogfish (Squalus). Tissue and Cell 20:759-761; 1988. 3. Grogan, E. D;; Lund, R. A culture system for the maintenance and proliferation of shark and sting ray immunoctyes. J. Fish Biol. 36:633-642; 1990. 4. Harshberger, J. C. Activities report of registry of tumors in lower animals, 1965-1973. Washington, DC: Smithsonian Institution; 1974. 5. Hartmann, J. X.; Bissoon, L. M. Cultivation of kidney and heart cells from nurse and horn sharks. In Vitro 23(3):56A; 1987. (Abstract) 6. Hartmann, J. X.; Poyer, J. C. Growth parameters and requirements of embryonic cells from sharks. In Vitro 25(3):20A; 1989. (Abstract) 7. Jones, R. T.; Hudson, E. A.; Sato, T. Explant culture of shark tissues. In Vitro 19:258; 1983. (Abstract) 8. Lee, A.; Langer, R. Shark cartilage contains inhibitors of tumor angiogenesis. Science 221:1185-1187; 1983. 9. Noga, E. J. Primary tissue culture from organ fragments: a simplified method. Experimentia 35:181-182; 1979. 10. Perlman, D. F.; Goldstein, L. Nitrogen metabolism. In: Shuttleworth, T. J., ed. Physiology of elasmobranch fishes. New York: SpringerVerlag; 1988:253-273.
11. Shuttleworth, T. J. Salt and water balance--extrarenal mechanisms. In: Shuttleworth, T. J., ed. Physiology of elasmobranch fishes. New York: Springer-Verlag; 1988:171-194. 12. Sorg, M. J. An analysis of DNA sequences homologous to retroviral oncogenes in the shark (subclass, elasmobranchii). Melbourne: Florida International Univ. 1988: Thesis. 13. Wolf, K.; Quimby, M. C. Fish cell and tissue culture. In: Hoar, W. S.; Randall, D. J., eds. Fish physiology, Vol. 3. New York: Academic Press; 1969:253-305. 14. Wolf, K.; Quimby, M. C. Primary monolayer culture of fish cells initiated from minced tissues: procedure 41125. In: Evans, V. J.; Perry, V. P.; Vincent, M., eds. TCA Manual 2. Maryland: Tissue Culture Association; 1976a:445-448. 15. Wolf, K.; Quimby, M. C. Primary monolayer culture of fish cells initiated from trypsinized tissues: procedure 41541. In: Evans, V. J.; Perry, V. P.; Vincent, M., eds. TCA Manual 2. Maryland: Tissue Culture Association; 1976b:453-456. 16. Yancey, P. H.; Somero, G. N. Urea-requiring lactate dehydrogenases of marine elasmobranch fishes. J. Comp. Physiol. 125:135-141; 1978. 17. Yancey, P. H.; Somero, G. N. Counteraction of urea destabilization of protein structure by methylamine osmoregulatory compounds of elasmobranch fishes. Biochem. J. 183:317-323; 1979. James X. Hartmann Lionel M. Bissoon
James C. Poyer
Department of Biological Sciences Florida Atlantic University Boca Raton, Florida 33431 (Received 21 October 1991)
Letter to the Editor ROUTINE ESTABLISHMENT OF PRIMARY ELASMOBRANCH CELL CULTURES
50 #g/ml was added to all media and the final osmolarity of the media measured 9 3 0 - 1 0 0 0 raPs. The flasks were placed at 26 ° C in either ambient air or 5% CO2. Half the conditioned medium was changed with fresh medium weekly. Of the eight tissue types tested for growth in vitro, heart atrium, kidney, testis, and brain cells could be routinely cultivated from all species examined. No marked differences in the extent and type of cell growth were noted between species. Growth was evident within one week following the plating of tissues into flasks, and extensive growth with mitotic activity was obtained after three weeks of cultivation (Fig. 1}. The morphology of kidney cells from the shark were seen as fibroblast-like (Fig. I a), epithelial-like (Fig. 1 b), and large epithelial-like (Fig. 1 c). The shark heart, kidney, and brain grew as epithelial-like cells (Fig. 1 d). Spleen, liver, and fin epidermis did not grow. Heart atrium and brain tissues could be grown by plating these tissues directly into medium following mincing. These two tissue types began to adhere to a flask surface within 10 minutes, and more than 90% of the tissue pieces adhered within 1 h of incubation. Kidney and testicular tissues required exposure to trypsin for successful cultivation, and only 50% of the tissue pieces remained adherent after 1 h of incubation. If the minced tissues were treated with trypsin for periods longer than 15 min they became gelatinous, the trypsin was difficult to remove, and the ceils subsequently died. Eagle's minimum essential medium, RPMI1640, and Medium 199 modified as described above, did not support the growth of primary cultures. The modified Opti-MEM I was superior to that of Ham's F-12 and Leibovitz's in both initiating and maintaining growth. Cultures initiated on Opti-MEM I and changed to Leibovitz's or Ham's F-12 media became granular and ceased growing (Table 1).
Dear Editor: Members of the class Elasmobranchiomorphi (Elasmobranchii/ Chondrichthyes of some authors), such as sharks, skates, and rays are interesting organisms for a number of reasons. Unlike most marine mammals and marine bony fishes, elasmobranchs have intracellular osmolarities nearly equal to that of seawater (11). This high osmolarity is maintained by concentrating urea to an average of 0.4 M. In order to counteract the destabilizing effects of the urea on proteins and other macromolecules, they utilize methylamine compounds and amino acids (10,17). Shark cartilage is a source of compounds that prevent the vascularization of animal and human tumors (8). The mechanism whereby sharks exhibit resistance to the development of malignant neoplasias (4) remains unknown, even though sharks possess oncogenes (12). It would be advantageous to employ cultivated elasmobrancb cells to address the areas of research described above on the cellular level and provide a means to isolate elasmobranch viruses. Garner (2) obtained limited growth in brain cells from a sharpnose shark (Rhizoprionodon porosus) after one month of culture. Our Laboratory (6) reported limited success in primary culture using a medium supplemented with urea and TMAO. Grogan and Lund (3) used urea and TMAO in the short term culture of elasmobranch immunocytes. In this paper, we describe a medium and procedures for the routine and rapid establishment of shark and sting ray primary cultures. Heart, kidney, brain, liver, testes, spleen, and fin epidermis tissues were aseptically resected from 20 nurse sharks (Ginglymostoma cirratum), 4 horn sharks (Heterodontus francisc 0, 1 silky shark (Carcharhinusfalciformis), and 8 yellow stingrays (Urolophus jamaicensis). The tissues were placed in phosphate buffered saline modified for use with shark cells (SPBS), which is Dulbecco's phosphate buffered saline to which 333 mM urea, 240 mM sodium chloride (NaCI) and 54 mM trimethylamine N-oxide (TMAO)/(Sigma Chemical Co., St. Louis, MO) are added. Samples from all tissues examined were minced and divided evenly. Half were placed directly onto the surface of 25 cm 2 cell culture flasks (Coming Works, Coming, NY) (14). The other half of each sample was treated for 15 min with 0.25% trypsin (DIFCO, Detroit, MI). The trypsin was removed by pipeting and the tissues were then placed into flasks (15). Culture medium was gently added after the tissues were allowed to adhere to the surface of the flask for 1 h (9). Eagle's minimum essential medium, Medium 199, RPMI164o, Leibovitz's L-15, Ham's F-12 and Opti-MEM I (Grand Island Biological Co., Grand Island, NY) were modified by the addition of 333 mM urea, 188 mM NaCI and 54 mM TMAO, with the pH adjusted to 7.1 Fetal bovine serum (FBS), shark serum, or skate serum, all heat inactivated at 56 ° C for 30 min, were added to a final concentration of 10% to the modified medium. Gentamicin at
TABLE 1 MEDIUM TYPE AND GROWTH OF SHARK CELLS~ Medium
Growth
Opti-MEM I Ham's F-12 Leibovitz's L-15 Eagle's MEM RMPI164o Medium 199
++++ ++ ++
Cardiac atrial tissue from nurse sharks was minced and divided equally into 25 cm2 flasks. After allowing the tissues to adhere for one hour, medium was gently added and the cultures placed at 26 ° C in an atmosphere of 5% CO2 in air. Media were modified by the addition of urea, NaC1, and trimethylamine N-oxide. Media was changed once a week. Growth represented by ( + + + + ) = confluent monolayer in 25 cm2 flasks within two months, to (-) = no growth after two months.
77
78
HARTMANN ET AL.
Flc. 1. a-d. Nurse shark primary cultures: fragment (dark area) showing extensive cellular outgrowth after 21 days of culture in Opti-MEM 1 medium. ×420. Bar: 100 ttm. a. Fibroblast-like cells and mitotically active cells (arrow) from the kidney, b. Epithelial-like kidney cells, note the muhinucleate cells (arrow). c. Large epithelial-hke kidney cells, d. Mixture of fibroblastoid and epithehaloid cells from the brain. Note the macrophage-like cells (arrow).
Pooled serum from either sharks or rays was toxic to primary monolayer cultures and did not support the growth of initial explants. Fetal bovine serum, at a concentration of 10% in the medium, was essential for establishing cultures. Some lots of FBS were superior to others in their growth promoting effects. Some lots of urea appeared toxic to the cultures and thus cell culture grade urea, stored at - 2 0 ° C, was employed. Although entire monolayers of cells were grown as primary cultures in 25 em 2 flasks, the cultures could not be successfully passaged by using 0.25% trypsin, 0.04% ethylenediamine tetraacetate acid (EDTA), 0.25% trypsin and 0.04% EDTA, shaking to dislodge M phase cells, or mechanical scraping with a rubber-tipped pipette. The vast majority (approximately 70%) of cells died before attachment, and the viable cells that attached would not divide in the presence of fresh medium, conditioned medium, or fractions from conditioned medium harvested from rapidly growing primary cultures.
The requirement for TMAO in the culture and maintenance of shark cells is supported by previous research on elasmobranch cell culture (3,5,6). Elasmobranchs concentrate urea, TMAO, and NaCI to achieve an osmolality almost equal to that of sea water. Although urea is toxic to most vertebrates, Yancey and Somero (16) have shown that some shark enzymes require urea for optimal kinetic activity. It has subsequently been shown that TMAO is essential in counteracting the perturbing effects of urea on "non-urea" dependent enzymes in the elasmobranch system and on enzymes in the mammalian system (17). Shark brain was the only tissue capable of limited growth in media devoid of TMAO, but containing urea and NaCI. Garner (2) was able to obtain limited growth of shark brain cells after one month of culture in a medium devoid of TMAO. It is thus possible that shark brain cells can synthesize limited amounts of TMAO, or other methylamine compounds, or they may counteract the effects of urea by some novel means. It has been shown that liver and
79
ELASMOBRANCH PRIMARY CELL CULTURE kidney ceils of the Japanese eel can synthesize TMAO (1). The lack of TMAO in previous media used to cultivate elasmobranch cells (2,7,13) may account for the limited growth reported in those studies.
REFERENCES 1. Daikoku, T.; Murata, M.; Sakaguchi, M. Effects of intraperitoneally injected and dietary trimethylamine on the biosynthesis of trimethylamine oxide in relation to seawater adaptation of the eel, Anguilla japonica and the guppy, Poecilia reticulata. Comp. Biochem. Physiol. 89A:261-264; 1988. 2. Garner, W. D. A. Elasmobranch tissue culture: in vitro growth of brain explants from a shark (Rhizoprionodon) and dogfish (Squalus). Tissue and Cell 20:759-761; 1988. 3. Grogan, E. D;; Lund, R. A culture system for the maintenance and proliferation of shark and sting ray immunoctyes. J. Fish Biol. 36:633-642; 1990. 4. Harshberger, J. C. Activities report of registry of tumors in lower animals, 1965-1973. Washington, DC: Smithsonian Institution; 1974. 5. Hartmann, J. X.; Bissoon, L. M. Cultivation of kidney and heart cells from nurse and horn sharks. In Vitro 23(3):56A; 1987. (Abstract) 6. Hartmann, J. X.; Poyer, J. C. Growth parameters and requirements of embryonic cells from sharks. In Vitro 25(3):20A; 1989. (Abstract) 7. Jones, R. T.; Hudson, E. A.; Sato, T. Explant culture of shark tissues. In Vitro 19:258; 1983. (Abstract) 8. Lee, A.; Langer, R. Shark cartilage contains inhibitors of tumor angiogenesis. Science 221:1185-1187; 1983. 9. Noga, E. J. Primary tissue culture from organ fragments: a simplified method. Experimentia 35:181-182; 1979. 10. Perlman, D. F.; Goldstein, L. Nitrogen metabolism. In: Shuttleworth, T. J., ed. Physiology of elasmobranch fishes. New York: SpringerVerlag; 1988:253-273.
11. Shuttleworth, T. J. Salt and water balance--extrarenal mechanisms. In: Shuttleworth, T. J., ed. Physiology of elasmobranch fishes. New York: Springer-Verlag; 1988:171-194. 12. Sorg, M. J. An analysis of DNA sequences homologous to retroviral oncogenes in the shark (subclass, elasmobranchii). Melbourne: Florida International Univ. 1988: Thesis. 13. Wolf, K.; Quimby, M. C. Fish cell and tissue culture. In: Hoar, W. S.; Randall, D. J., eds. Fish physiology, Vol. 3. New York: Academic Press; 1969:253-305. 14. Wolf, K.; Quimby, M. C. Primary monolayer culture of fish cells initiated from minced tissues: procedure 41125. In: Evans, V. J.; Perry, V. P.; Vincent, M., eds. TCA Manual 2. Maryland: Tissue Culture Association; 1976a:445-448. 15. Wolf, K.; Quimby, M. C. Primary monolayer culture of fish cells initiated from trypsinized tissues: procedure 41541. In: Evans, V. J.; Perry, V. P.; Vincent, M., eds. TCA Manual 2. Maryland: Tissue Culture Association; 1976b:453-456. 16. Yancey, P. H.; Somero, G. N. Urea-requiring lactate dehydrogenases of marine elasmobranch fishes. J. Comp. Physiol. 125:135-141; 1978. 17. Yancey, P. H.; Somero, G. N. Counteraction of urea destabilization of protein structure by methylamine osmoregulatory compounds of elasmobranch fishes. Biochem. J. 183:317-323; 1979. James X. Hartmann Lionel M. Bissoon
James C. Poyer
Department of Biological Sciences Florida Atlantic University Boca Raton, Florida 33431 (Received 21 October 1991)
