In vitro culture and maintenance of trophoblastic vesicles: Observations on the development of contractile structures with concurrent vessel development

Abstract

The use of trophoblastic vesicles as a model for the study of maternal recognition of pregnancy, embryo development and cell to cell interaction has increased in recent years. In this report, we describe the long-term culture of trophoblastic vesicles derived from enzymatically dispersed porcine blastocysts. Day 12 to 14 porcine embryos were enzymatically dispersed to form single cell suspensions, then allowed to plate in 25 cm 3 tissue-culture flasks. Vesicles formed 3 to 14 d following plating and ranged in size from 0.1 to 4.0mm in diameter. Vesicles larger than 1.5 mm at formation were able to float free in the medium for up to 65 days, at which time vesicles were histologically sectioned. Contractile elements formed in association with dense cell areas, and were found with tubular vascular vessels. Movement of cells that resembled eosinophilic cells was observed in the vessels and pulsed in synchrony with contractions of the primitive heart. Histological examination gave evidence of primitive blood cells and cardiac tissue, and no evidence of neural tissue was found, indicating the probability that the contractions were induced by permeable membrane depolarization. It is hypothesized from the observations reported in this paper that the cell differentiation required to form the contractile region and the vessel are under the control of the dense cell area. This indicates that trophoblastic vesicles fromed by the enzymatic dispersal of porcine blastocysts is a possible model for the study of induced cell differentiation and formation of the cardiac organ system in vitro.