Viability of human chorionic villi in organ culture

Abstract

The method and technique of organ culture of human chorionic villi was elaborated in our laboratory. In this report, placental specimens obtained at 15 to 18 weeks of gestation were studied in organ culture for 7 days in terms of the maintenance of morphological integrity and the preservation of functions. The morphological aspect of the viability of the various villous elements with special emphasis on the trophoblast cells was described histologically and ultrastructurally. The functional aspect of the viability was discussed by analysis of the suppressive effect of the cultivated villi on plasminogen activator (PA) secretion by OK-432 elicited mouse peritoneal macrophages, by analyses of the activity of delta 5-3 beta-hydroxysteroid dehydrogenase coupled with delta 5-delta 4-isomerase (HSD) and of the radioactivity of [125I]-Iododeoxyuridine ( [125I]-IUdR) retained in the DNA of the trophoblast cells. Specimens of normal placenta were obtained at the time of induced abortion. The gestational ages were 15, 16 and 18 weeks. Specimens for organ culture were prepared under sterile conditions within one hour after expulsion of the placenta. Through gross dissection, the villi were isolated and minced into fragments of approximately 1 to 2 mm3. Incubation was carried out at 37 degrees C in a conventional static chamber with a gas mixture of 95% air, 5% CO2. The culture dishes and culture media were renewed every day. Data are the mean values of the duplicate incubations. In the first series of organ culture, placental fragments were removed at the end of each day of incubation, washed thoroughly and transferred to the new dishes with a culture medium freed from fetal bovine serum, where further incubation was performed for 24 hours. After this, placental fragments were fixed in 4% neutral buffered formalin, processed through paraffin embedding, serially sectioned at 5 micron and stained by periodic-acid Schiff (PAS) procedure. Culture medium obtained in this series at each end of incubation was put into a "macrophage plate" with the addition of plasminogen in the concentration of 2 IU/m1, in which OK-432 elicited mouse peritoneal macrophages with the capacity to secrete PA were cultured. Reaction was terminated at 24 hours unless otherwise indicated. Ten microliters of the medium in the "macrophage plate" was applied to the fibrin plate, and reaction was performed for 18 hours. PA activity was expressed by plasmin activity converted from plasminogen measured by the single radial immuno-diffusion method.(ABSTRACT TRUNCATED AT 400 WORDS)