[Isolation, culture and identification of sheep multinucleated chorionic trophoblast cells].

OBJECTIVE

To develop a set of methods for isolating and culturing sheep multinucleated chorionic trophoblast cells and identify them.

METHODS

Trophoblast cells were isolated and cultured by two-step digestion of trypsin and collagenase, and observed under an inverted phase-contrast microscope. They were further identified through conventional HE staining, immunohistochemical staining and transmission electron microscopy.

RESULTS

Under an inverted phase-contrast microscope, the obtained trophoblast cells exhibited epithelioid and sheet-like spreading growth with binuclear or multinuclear. The multinucleated trophoblast cytoplasm from the sheep placental cotyledons and the slides of the trophoblast cells were all stained brown and exhibited positive reactivity by immunohistochemical staining with cytokeratin antibodies. Also abundance of microvilli on surface of cells, together with intracytoplasmic vacuoles, microfilament and lipid droplets were observed under a transmission electron microscope.

CONCLUSION

Two-step digestion of trypsin and collagenase has been established for the isolation and cultivation of sheep multinucleated trophoblast cells, and using it, we obtained sheep multinucleated chorionic trophoblast cells with a high purity and biological activity.