Identification and Characterization of Monolayer Cultures of Sheep Trophoblast Cells Maintained in Bicameral Culture Chambers

Abstract

Enriched epithelial cell and fibroblast fractions were isolated from ovine placentomes by isopycnic centrifugation of collagenase/DNAse-dispersed cells through a density gradient of 45% Percoll. The epithelial cells formed confluent monolayers when plated onto filters impregnated with a 50-microns layer of Matrigel in medium containing 10% fetal bovine serum. These cells were maintained in dual environment culture chambers in serum-free medium for at least 12 days. The epithelium had a polarized appearance similar to that found in vivo only when cells were plated at high density (10(7)/cells/cm2). The epithelial monolayer consisted predominantly of a single population of uninucleate cells with intracellular features similar to those previously described for ovine trophoblast both in vivo and in vitro. These cells stained positively with an antiserum to alpha-keratin, a marker specific to epithelial cells, and no staining was observed with antisera raised against binucleate cells or leucocyte-common antigen. Binucleate cells were detected by microscopy and immunostaining in the pellet of cells obtained from the Percoll gradient but were rarely seen in the epithelium. The epithelial monolayer excluded 3H-inulin, added to the basal chamber, from the apical chamber, thus demonstrating the formation of a permeability barrier similar to that found in vivo. The maintenance of a monolayer of pure ovine trophoblast cells in vitro, which retain the characteristics of the epithelium in vivo, will enable the study of many cellular functions of the trophoblast.

Matrix-Metalloproteinases-2 and -9 Production in Bovine Endometrial Cell Culture.

“…It was noted that MMP-2 production was similar both in non-collagen coated and collagen coated dishes. It is known that cell polarity affects protein secretion,but it was reported that cells could be correctly polarized after high-density plating [22,23]. It is possible that MMP-2 is produced by endometrial epithelial cells [17], although some previous reports indicated that endometrial epithelial cells do not produce MMP-2 [10,24].…”

Expressão das conexinas 32 e 43 em células trofoblásticas da placenta bovina em cultura celular

“…Dupla coloração para a conexina 32 (FITC) e núcleo (iodeto de propídeo). (barra = 30 µm) A proposta de realizar cultivo de células trofoblástica apóia-se na afirmação de Ralph et al (1989), na qual a manutenção de uma camada de células trofoblástica em cultura retém as mesmas características do epitélio in vivo possibilitando o estudo de muitas funções celulares do trofoblasto. A vantagem do cultivo de células in vitro é estar livre das variações sistêmicas do organismo, bem como de interferências sobre a homeostase, ou das condições de estresse ao qual está exposto um organismo vivo em qualquer modelo ou situação experimental.…”