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. 1987;23(11):750–754. doi: 10.1007/BF02623675

Monolayer culture of cells originating from apreimplantation bovine embryo

D A Stringfellow 1, B W Gray 1, L H Lauerman 1, M S Thomson 1, P J Rhodes 1, R C Bird 1
PMCID: PMC7088839  PMID: 3680102

Summary

The objective of this study was to establish a method by which trophectodermal cells originating from individual preimplantation bovine embryos could be perpetuated in monolayer culture. A single, Day-11 bovine embryo collected nonsurgically from a mixed-breed beef cow was cultured in Ham's F10 medium supplemented with fetal bovine serum, sodium pyruvate, insulin and epidermal growth factor. After 13 d in culture the embryo had adhered to the surface of the plastic culture vessel and a monolayer covering 0.3 cm2 had developed in the manner of a tissue explant. The monolayer was successfully dispersed using trypsin-EDTA and the cells were passaged Expansion to a 25-cm2 flask was achieved by the 4th passage. By passaging cultures at a dilution ratio of 1∶2, cells were maintained for 38 passages before growth slowed. Transfers beyond the 44th passage were unsuccessful. The cell line, designated BE-13, was successfully frozen and thawed at the 9th, 12th, 15th, and 20th passages. The cell line contains both mono- and binucleate cells with a prominent rough endoplasmic reticulum characteristic of ruminant trophoblast cells. Susceptibility to eight bovine viruses was demonstrated. Such cell lines may provide inexpensive systems for the study of trophoblast metabolism and for investigation of the role of the trophoblast in the pathogenesis of selected bovine abortifacient diseases. Because of their range of viral susceptibility, these cells might also be useful for diagnostic purposes.

Key words: bovine trophoblast, preimplantation embryo, monolayer culture

Footnotes

Published as publication no. 1891 College of Veterinary Medicine, Auburn University, Alabama 36849. This work was funded in part by an Auburn University Faculty Research Grant-in-aid. Preliminary results of the study were presented in abstract form at the 1987 Annual Conference of the International Embryo Transfer Society.

References

  • 1.Anderson T. D., Cheville N. F., Meador V. P. Pathogenesis of placentitis in the goat inoculated withBrucella abortus. II Ultrastructural studies. Vet. Pathol. 1986;23:227–239. doi: 10.1177/030098588602300302. [DOI] [PubMed] [Google Scholar]
  • 2.Ansell J. D., Snow M. H. L. The development of trophoblastin vitro from blastocysts containing varing amounts of inner cell mass. J. Embryol. Exp. Morphol. 1975;33:177–185. [PubMed] [Google Scholar]
  • 3.Bartol F. F., Roberts R. M., Bazer F. W., et al. Characterization of proteins produced in vitro by preattachment bovine conceptuses. Biol. Reprod. 1985;32:681–693. doi: 10.1095/biolreprod32.3.681. [DOI] [PubMed] [Google Scholar]
  • 4.Cole R. J., Paul J. Properties of cultured preimplantation mouse and rabbit embryos, and cell strains derived from them. In: Wolstenholm G. E. W., O'Conner M., editors. Preimplantation stages of pregnancy. Boston: Little, Brown and Company; 1965. pp. 82–112. [Google Scholar]
  • 5.Gardner R. L. An investigation of inner cell mass and trophoblast tissues following their isolation from mouse blastocysts. J. Embryol. Exp. Morphol. 1972;28:279–312. [PubMed] [Google Scholar]
  • 6.Godkin J. D., Bazer F. W., Lewis G. S., et al. Synthesis and release of polypeptides by pig conceptuses during the period of blastocysts elongation and attachment. Biol. Reprod. 1982;27:977–987. doi: 10.1095/biolreprod27.4.977. [DOI] [PubMed] [Google Scholar]
  • 7.Godkin J. D., Bazer F. W., Roberts R. M. Protein production by cultures established from day 14–16 sheep and pig conceptuses. J. Reprod. Fertil. 1985;72:377–382. doi: 10.1530/jrf.0.0740377. [DOI] [PubMed] [Google Scholar]
  • 8.Masters R. A., Roberts R. M., Lewis G. S., et al. High molecular weight glycoproteins released by expanding preattachment sheep, pig, and cow blastocysts in culture. J. Reprod. Fertil. 1983;66:571–583. doi: 10.1530/jrf.0.0660571. [DOI] [PubMed] [Google Scholar]
  • 9.Rajamahendran R., Canseco R. S., Guazdauskas F. C., et al. Observations on in vitro development of bovine morula in Ham's F10 and Dulbecco's phosphate buffered saline supplemented with normal steer serum. Theriogenology. 1985;24:369–374. doi: 10.1016/0093-691X(85)90229-8. [DOI] [PubMed] [Google Scholar]
  • 10.Shaffer S. J., Wright R. W., Jr. Attachment and trophoblastic outgrowth of swine blastocysts in vitro. J. Anim. Sci. 1978;46:1712–1717. doi: 10.2527/jas1978.4661712x. [DOI] [PubMed] [Google Scholar]
  • 11.Shemesh M., Milaguir F., Ayalon N., et al. Steroidogenesis and prostaglandin synthesis by cultured bovine blastocysts. J. Reprod. Fertil. 1979;56:181–185. doi: 10.1530/jrf.0.0560181. [DOI] [PubMed] [Google Scholar]
  • 12.Siekevitz P., Palade G. E. A cytochemical study on the pancreas of the guinea pig. IV Chemical and metabolic investigations of the ribonucleoprotein particles. J. Biophys. Biochem. Cytol. 1959;5:1–10. doi: 10.1083/jcb.5.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Steven D. H. Interspecies differences in the structure and function of trophoblast. In: Loke Y. W., editor. Biology of trophoblast. New York: Elsevier; 1983. pp. 120–128. [Google Scholar]
  • 14.Wright R. W., Andersen G. B., Cupps P. T., et al. Successful culturein vitro of bovine embryos to the blastocyst stage. Biol. Reprod. 1976;14:157–162. doi: 10.1095/biolreprod14.2.157. [DOI] [PubMed] [Google Scholar]

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