Study on the growth promoting capacity of calf and fetal bovine serum for animal cells "in vitro" II: electrophoretic study and survey on the antiproteolytic activity of pools of calf and fetal bovine serum

Rizzo, Edda de; Vaz, Celidéia Aparecida Coppi; Mendes, Inácio França; Yano, Anatércia Ferreira Bonfim

doi:10.1590/S0036-46651984000200005

Abstracts

Calf serum and fetal bovine serum present great variability as to its growth promoting efficiency (GPE). As supplement of culture media to cultivate cells of animal origin they stimulate the "in vitro" multiplication and maintain cell viability. When fourteen lots of calf sera of variable GPE had the total protein contents as well as the percentages of serum fractions determined, no significant differences that could possibly explain the variability of the GPE were observed. Evaluation of the antiproteolytic activity of nineteen lots of calf serum and eighteen serum lots of younger calves showed that the former exhibited lower antiproteolytic titers (1:40 to 1:80) than the latter (1:80 to 1:160). Twelve lots of fetal bovine serum studied in parallel, showed the highest concentration of antiproteolytic factors, with titers equal to 1:320. Sera of bovine origin, but not fetal sera, are usually heat-inactivated, what was demonstrated to be responsible for the decrease of the antiproteolytic activity of 75% of the lots tested. This could explain the inability of certain heat-inactivated sera in promoting multiplication of some cells "in vitro", as verified with primary monkey kidney cells. The results obtained in this study indicated the convenience of submiting each lot of serum to be introduced in cell culture to previous determination of its characteristics, such as growth promoting efficiency, antiproteolytic activity and also toxicity, absence of extraneous agents, etc., in order to minimize the possibility of using serum lots of questionable quality, thus preventing not only the loss of cell lines, but also undesirable and sometimes expensive delays.

O soro de vitelas e o soro fetal bovino apresentam grande variabilidade no que se refere à sua capacidade promotora de crescimento (CPC) e são empregados no cultivo de células de origem animal, suplementando os meios de cultura, com a finalidade de estimular a multiplicação das células "in vitro", assim como manter a viabilidade das mesmas. Quando 14 lotes de soro de vitelas apresentando diferentes CPC tiveram seu teor total de proteínas, assim como as percentagens séricas presentes determinados, não foram observadas diferenças significativas que justificassem a variação daquela capacidade. Na avaliação da atividade antiproteolítica (AA) de 19 lotes de soros de vitelas e de 18 de soros de bezerros de tenra idade, foi constatado que os primeiros apresentavam títulos antiproteolíticos mais baixos (1:40 a 1:80) do que os registrados para os segundos (1:80 a 1:160). Doze lotes de soros fetais bovinos, avaliados em paralelo, exibiram concentração elevada de fatores antiproteolíticos, evidenciada por títulos de 1:320. A inativação a 56°C durante 30 min. a que são geralmente submetidos os soros de origem bovina (com exceção dos fetais) demonstrou ser responsável pela baixa da AA dos mesmos, o que talvez justifique a incapacidade que certos soros inativados têm, de promoverem a multiplicação de algumas células "in vitro", como é o caso das células primárias de rim de macaco. Os resultados obtidos salientam a conveniência de se determinar previamente, para cada lote de soro de origem bovina a ser introduzido em cultura celular, certas características, tais como a capacidade promotora de crescimento, a atividade antiproteolítica e também a ausência de agentes adventícios, toxicidade, etc, de modo a minimizar a possibilidade de, ao utilizar lotes de soro de qualidade questionável, incorrer em perda de linhagens celulares, assim como em indesejáveis atrasos.

Study on the growth promoting capacity of calf and fetal bovine serum for animal cells "in vitro" II Electrophoretic study and survey on the antiproteolytic activity of pools of calf and fetal bovine serum

Estudo sobre a capacidade promotora de crescimento de soros de vitelas e de soros fetais bovinos para células de origem animal cultivadas "in vitro" II Estudo eletroforético e pesquisa da atividade antiproteolítica de soros de vitelas e de soros fetais bovinos

Edda de Rizzo^I; Celidéia Aparecida Coppi Vaz^II; Inácio França Mendes^I; Anatércia Ferreira Bonfim Yano^I

^ISeção de Cultura de Tecidos e Controle Instituto Butantan. P.O. Box 65, São Paulo, SP, Brazil

^IICentro de Pesquisas Imunoquímicas Instituto de Ciências Biomédicas da Universidade de São Paulo. P.O. Box 4365, São Paulo, SP, Brazil

SUMMARY

Calf serum and fetal bovine serum present great variability as to its growth promoting efficiency (GPE). As supplement of culture media to cultivate cells of animal origin they stimulate the "in vitro" multiplication and maintain cell viability. When fourteen lots of calf sera of variable GPE had the total protein contents as well as the percentages of serum fractions determined, no significant differences that could possibly explain the variability of the GPE were observed. Evaluation of the antiproteolytic activity of nineteen lots of calf serum and eighteen serum lots of younger calves showed that the former exhibited lower antiproteolytic titers (1:40 to 1:80) than the latter (1:80 to 1:160). Twelve lots of fetal bovine serum studied in parallel, showed the highest concentration of antiproteolytic factors, with titers equal to 1:320. Sera of bovine origin, but not fetal sera, are usually heat-inactivated, what was demonstrated to be responsible for the decrease of the antiproteolytic activity of 75% of the lots tested. This could explain the inability of certain heat-inactivated sera in promoting multiplication of some cells "in vitro", as verified with primary monkey kidney cells. The results obtained in this study indicated the convenience of submiting each lot of serum to be introduced in cell culture to previous determination of its characteristics, such as growth promoting efficiency, antiproteolytic activity and also toxicity, absence of extraneous agents, etc., in order to minimize the possibility of using serum lots of questionable quality, thus preventing not only the loss of cell lines, but also undesirable and sometimes expensive delays.

RESUMO

O soro de vitelas e o soro fetal bovino apresentam grande variabilidade no que se refere à sua capacidade promotora de crescimento (CPC) e são empregados no cultivo de células de origem animal, suplementando os meios de cultura, com a finalidade de estimular a multiplicação das células "in vitro", assim como manter a viabilidade das mesmas. Quando 14 lotes de soro de vitelas apresentando diferentes CPC tiveram seu teor total de proteínas, assim como as percentagens séricas presentes determinados, não foram observadas diferenças significativas que justificassem a variação daquela capacidade.

Na avaliação da atividade antiproteolítica (AA) de 19 lotes de soros de vitelas e de 18 de soros de bezerros de tenra idade, foi constatado que os primeiros apresentavam títulos antiproteolíticos mais baixos (1:40 a 1:80) do que os registrados para os segundos (1:80 a 1:160). Doze lotes de soros fetais bovinos, avaliados em paralelo, exibiram concentração elevada de fatores antiproteolíticos, evidenciada por títulos de 1:320. A inativação a 56°C durante 30 min. a que são geralmente submetidos os soros de origem bovina (com exceção dos fetais) demonstrou ser responsável pela baixa da AA dos mesmos, o que talvez justifique a incapacidade que certos soros inativados têm, de promoverem a multiplicação de algumas células "in vitro", como é o caso das células primárias de rim de macaco.

Os resultados obtidos salientam a conveniência de se determinar previamente, para cada lote de soro de origem bovina a ser introduzido em cultura celular, certas características, tais como a capacidade promotora de crescimento, a atividade antiproteolítica e também a ausência de agentes adventícios, toxicidade, etc, de modo a minimizar a possibilidade de, ao utilizar lotes de soro de qualidade questionável, incorrer em perda de linhagens celulares, assim como em indesejáveis atrasos.

Texto completo disponível apenas em PDF.

Full text available only in PDF format.

ACKNOWLEDGEMENTS

The Authors are indebt to Drs. Clélia Helena Martinez (Instituto Adolfo Lutz), and Walter Pinto Jr. (Unicamp) for kindly providing samples of calf and fetal bovine serum, respectively; to Mrs. Angela de Faria and Mr. Roberto Chaves for the typing 'work.

Recebido para publicação em 15/6/1983.

1. BALK, S. D. Calcium as a regulator of the proliferation of normal, but not transformed chicken fibroblasts in a plasma-containing medium. Proc. Nat. Acad. Sci. U.S.A. 68: 271-275, 1971.
2. BOTELHO, G. G. Proteína total e frações do soro sangüíneo de bovinos submetidos ao cloridrato de xilazina. Arq. Esc. Vet. U.F.M.G. 29: 43-48, 1977.
3. CHANG, R. S. Continuous subcultivation of epithelial-like cells from normal human tissues. Proc. Soc. Exp. Biol. Med. 87: 440-443, 1954.
4. CUNNINGHAM, D. D. & PARDEE, A. B. Transport changes rapidly initiated by serum addition to "contact inhibited" 3T3 cells. Proc. Nat. Acad. Sci. U.S.A. 64: 1049-1056, 1969.
5. DAVIES, P. F. & ROSS, R. Mediation of pinocytosis in cultured arterial smooth muscle and endothelial cells by platelet-derived growth factor. J. Cell Biol. 79: 663-671, 1978.
6. DE LUCA, C; HABEEB, A. F. S. A. & TRISTSCH, G. L. The stimulation of growth of mammalian cells "in vitro" by a peptide fraction from enzymatic digests of serum. Exp. Cell Res. 43: 98-106, 1966.
7. DULAK, N. C & TEMIN, H. M. A partially purified polypeptide fraction from rat liver cell conditioned medium with multiplication stimulating activity for embryo fibroblasts. J. Cell Physiol. 81: 153-160, 1973.
8. DULBECCO, R. Topoinhibition and serum requirement of transformed and untransformed cells. Nature 227: 802-806, 1970.
9. HADDEN, J. W.; HADDEN, E. M.; HADDOX, M. K. & GOLDBERG, N. D. Guanosine 3':5'-cyclic monophosphate: a possible intracelular mediator of mito-genic influences in lymphocytes. Proc. Nat. Acad. Sci. U.S.A. 69: 3024-3027, 1972.
10. HAUDENSCHILD, C. C; ZAHNISER, D.; FOLKMAN, J. & KLAGSBURN, M. Human vascular endothelial cells in culture: lack of response to serum growth factors. Exp. Cell Res. 98: 175-183, 1976.
11. HEALY, G. M. & PARKER, R. C. Cultivation of mammalian cells in defined media with protein and nonprotein supplements. J. Cell Biol. 30: 539-553, 1966.
12. HERSHKO, A.; MAMONT, P.; SHIELDS, R. & TOM-KINS, G. M. "Pleiotypic response". Nature New Bull. 232: 206-211, 1971.
13. HOFFMANN, R.; RISTOW, H. J.; VESER, J. & FRANK, W. Properties of two growth-stimulating proteins isolated from fetal calf serum. Exp. Cell Res. 80: 275-280, 1973.
14. HOLLEY, R. W.; BALDWIN, J. H. & KIERNAN, J. A. Control of growth of a tumor cell by linoleic acid. Proc. Nat. Acad. Sci. U.S.A. 71: 3976-3978, 1974.
15. HOLLEY, R. W. & KIERNAN, J. A. "Contact inhibition" of cell division in 3T3 cells. Proc. Nat. Acad. Sci. U.S.A. 60: 300-304, 1968.
16. HOLLEY, R. W. & KIERNAN, J. A. Growth control in cell cultures. CIBA Foundation Symposium (ed. J. Knight & G.E.W. Wolstenhome). London, Churchill, 1971, p. 3.
17. SEFTON, B. & RUBIN, H. Stimulation of glucose transport in cultures of density-inhibited chick embryo cells. Proc. Acad. Sci. U.S.A. 68: 3154-3157, 1971.
18. SEIFEST, W. E. & RUDLAND, P. S. Possible involvement of ciclyc GMP in growth control of cultured mouse cells. Nature 248: 138-140, 1974.
19. TEMIN, H. M.; PIERSON, R. W. & DULAK, N. C. Growth, nutrition and metabolism of cells in culture. In: G. H. Rothblat, V. J. Christofalo 1: 49-81. New York, Academic Press, 1972.
20. THORGEIRSSON, G. & ROBERTSON JR., A. L. Platelet factors and the human vascular wall. Part. 2. Such factors are not required for endothelial cell proliferation and migration. Atherosclerosis 31: 231-238, 1978.
21. TODARO, G. J.; LAZAR, G. K. & GREEN, H. The inhibition of cell division in a contact-inhibited mammalian cell line. J. Cell Comp. Physiol. 66: 325-333, 1965.
22. TUCHIYA, H. N.; MENDES, I. F.; MIYAKI, C. & RIZZO, E. de Study on the growth promotion capacity of calf serum for animal cells "in vitro". I-Test of 56 lots of calf serum against several cell lines and primary cell cultures. Rev. Inst. Med. trop. São Paulo 22: 281-285, 1980.
23. VILLELA, L. G.; SILVIA, E. & COSTA VAL, V. P. Effect of diet on the total protein content in female Gir blood serum. Arq. Esc. Vet. XXII: 113-117, 1970.
24 WALLIS, C; VER, B. & MELNICK, J. L. The role of serum and fetuin in the growth of monkey-cells in culture. Exp. Cell Res. 58: 271-282, 1969.

Publication Dates

Publication in this collection
12 Apr 2013
Date of issue
Apr 1984

History

Received
15 June 1983

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

[1] 1. BALK, S. D. Calcium as a regulator of the proliferation of normal, but not transformed chicken fibroblasts in a plasma-containing medium. Proc. Nat. Acad. Sci. U.S.A. 68: 271-275, 1971.

[2] 2. BOTELHO, G. G. Proteína total e frações do soro sangüíneo de bovinos submetidos ao cloridrato de xilazina. Arq. Esc. Vet. U.F.M.G. 29: 43-48, 1977.

[3] 3. CHANG, R. S. Continuous subcultivation of epithelial-like cells from normal human tissues. Proc. Soc. Exp. Biol. Med. 87: 440-443, 1954.

[4] 4. CUNNINGHAM, D. D. & PARDEE, A. B. Transport changes rapidly initiated by serum addition to "contact inhibited" 3T3 cells. Proc. Nat. Acad. Sci. U.S.A. 64: 1049-1056, 1969.

[5] 5. DAVIES, P. F. & ROSS, R. Mediation of pinocytosis in cultured arterial smooth muscle and endothelial cells by platelet-derived growth factor. J. Cell Biol. 79: 663-671, 1978.

[6] 6. DE LUCA, C; HABEEB, A. F. S. A. & TRISTSCH, G. L. The stimulation of growth of mammalian cells "in vitro" by a peptide fraction from enzymatic digests of serum. Exp. Cell Res. 43: 98-106, 1966.

[7] 7. DULAK, N. C & TEMIN, H. M. A partially purified polypeptide fraction from rat liver cell conditioned medium with multiplication stimulating activity for embryo fibroblasts. J. Cell Physiol. 81: 153-160, 1973.

[8] 8. DULBECCO, R. Topoinhibition and serum requirement of transformed and untransformed cells. Nature 227: 802-806, 1970.

[9] 9. HADDEN, J. W.; HADDEN, E. M.; HADDOX, M. K. & GOLDBERG, N. D. Guanosine 3':5'-cyclic monophosphate: a possible intracelular mediator of mito-genic influences in lymphocytes. Proc. Nat. Acad. Sci. U.S.A. 69: 3024-3027, 1972.

[10] 10. HAUDENSCHILD, C. C; ZAHNISER, D.; FOLKMAN, J. & KLAGSBURN, M. Human vascular endothelial cells in culture: lack of response to serum growth factors. Exp. Cell Res. 98: 175-183, 1976.

[11] 11. HEALY, G. M. & PARKER, R. C. Cultivation of mammalian cells in defined media with protein and nonprotein supplements. J. Cell Biol. 30: 539-553, 1966.

[12] 12. HERSHKO, A.; MAMONT, P.; SHIELDS, R. & TOM-KINS, G. M. "Pleiotypic response". Nature New Bull. 232: 206-211, 1971.

[13] 13. HOFFMANN, R.; RISTOW, H. J.; VESER, J. & FRANK, W. Properties of two growth-stimulating proteins isolated from fetal calf serum. Exp. Cell Res. 80: 275-280, 1973.

[14] 14. HOLLEY, R. W.; BALDWIN, J. H. & KIERNAN, J. A. Control of growth of a tumor cell by linoleic acid. Proc. Nat. Acad. Sci. U.S.A. 71: 3976-3978, 1974.

[15] 15. HOLLEY, R. W. & KIERNAN, J. A. "Contact inhibition" of cell division in 3T3 cells. Proc. Nat. Acad. Sci. U.S.A. 60: 300-304, 1968.

[16] 16. HOLLEY, R. W. & KIERNAN, J. A. Growth control in cell cultures. CIBA Foundation Symposium (ed. J. Knight & G.E.W. Wolstenhome). London, Churchill, 1971, p. 3.

[17] 17. SEFTON, B. & RUBIN, H. Stimulation of glucose transport in cultures of density-inhibited chick embryo cells. Proc. Acad. Sci. U.S.A. 68: 3154-3157, 1971.

[18] 18. SEIFEST, W. E. & RUDLAND, P. S. Possible involvement of ciclyc GMP in growth control of cultured mouse cells. Nature 248: 138-140, 1974.

[19] 19. TEMIN, H. M.; PIERSON, R. W. & DULAK, N. C. Growth, nutrition and metabolism of cells in culture. In: G. H. Rothblat, V. J. Christofalo 1: 49-81. New York, Academic Press, 1972.

[20] 20. THORGEIRSSON, G. & ROBERTSON JR., A. L. Platelet factors and the human vascular wall. Part. 2. Such factors are not required for endothelial cell proliferation and migration. Atherosclerosis 31: 231-238, 1978.

[21] 21. TODARO, G. J.; LAZAR, G. K. & GREEN, H. The inhibition of cell division in a contact-inhibited mammalian cell line. J. Cell Comp. Physiol. 66: 325-333, 1965.

[22] 22. TUCHIYA, H. N.; MENDES, I. F.; MIYAKI, C. & RIZZO, E. de Study on the growth promotion capacity of calf serum for animal cells "in vitro". I-Test of 56 lots of calf serum against several cell lines and primary cell cultures. Rev. Inst. Med. trop. São Paulo 22: 281-285, 1980.

[23] 23. VILLELA, L. G.; SILVIA, E. & COSTA VAL, V. P. Effect of diet on the total protein content in female Gir blood serum. Arq. Esc. Vet. XXII: 113-117, 1970.

[24] 24 WALLIS, C; VER, B. & MELNICK, J. L. The role of serum and fetuin in the growth of monkey-cells in culture. Exp. Cell Res. 58: 271-282, 1969.

Brasil

Brasil

Study on the growth promoting capacity of calf and fetal bovine serum for animal cells "in vitro" II: electrophoretic study and survey on the antiproteolytic activity of pools of calf and fetal bovine serum

Estudo sobre a capacidade promotora de crescimento de soros de vitelas e de soros fetais bovinos para células de origem animal cultivadas "in vitro" II: estudo eletroforético e pesquisa da atividade antiproteolítica de soros de vitelas e de soros fetais bovinos

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