Human villous placenta in monolayer culture : vital mitochondrial studies
A protocol adapted for the propagation of monolayer cultures from human villous placenta is described. Keratin immunofluorescent localization was utilized to characterize trophoblast cells in sub-cultivated cultures derived from first trimester placentae. The appearance of autofluorescent particles in long-term first trimester placental cultures is reported. The monolayer culture system was employed to study living mitochondria. Using the vital fluorescent dye rhodamine 123, fluorescence microscopy of living first trimester human placental cells and choriocarcinoma cells was carried out. The length and intracellular distribution of mitochondria are described for normal placental cells, somatic hybrids of normal cells, and choriocarcinoma cells. In normal first trimester placental cells, quantitative relationships between the projected cell surface areas and numbers/lengths of their mitochondria are described by regression equations. From the equations, it is deduced that the total (aggregate) length of mitochondria per cell is directly proportional to the 0.86 +/- 0.07 power of the cell's projected surface area. The number of mitochondria per cell is also a direct function of the cellular surface area, with an exponent of 0.82 +/- 0.07; whilst the total length of mitochondria per unit projected cell surface area is an inverse function (exponent = -0.14 + 0.07) of the cell surface area. The reaction of living mitochondria in cultured first trimester placental cells to a number of chemical agents including colchicine was investigated. In the presence of colchicine, it appears that the distribution of mitochondria becomes more restricted to the perinuclear cytoplasm and that the mean length of the organelles is reduced. However, the total length of all mitochondria in colchicine-treated cells was not significantly different from that in similar cells which were not exposed to the drug. Experiments investigating the recovery of cells from colchicine-mediated changes in their mitochondrial orientation and length is presented.