Preparation of cultured smooth muscle cells from human myometrium for X-ray microanalysis

Methodological aspects of the use of X-ray microanalysis in physiological and pharmacological experiments on cultured myometrial cells were investigated. Cultured human myometrial cells were grown from biopsies after detaching the fibroblasts. Of the cultured cells, 95-98% showed desmin-like immunoreactivity. Transmission electron microscopy showed that subcultured cells were different from myometrial cells in situ. The effects of washing the cells to remove external salt-rich medium were investigated. All solutions removed the external medium, resulting in lower concentrations of Na and Cl. In the cells washed with 0.3 M mannitol, most of the elemental concentrations were significantly lower than in their unwashed counterparts and those washed in the other solutions. In cells washed in either 0.15 M ammonium acetate or distilled water, no significant differences in P and K compared with their unwashed counterparts were found. There were also no significant differences between cells washed in ammonium acetate and in distilled water. In subsequent experiments ammonium acetate was used. Incubation of cells in standard Ringer's solution resulted in an increase in Na and Cl, and a decrease in K, concomitantly with an increase in Ca. Although Ringer's solution per se can elicit changes in diffusible elements in the cells, physiological and pharmacological effects of oxytocin could still be detected in Ringer's solution. However, effects of oxytocin were different when the experiment was done in culture medium, instead of in Ringer's solution.