A method for the study of steady-state kinetics in cell suspensions. evaluation of estrogen metabolism in HEC-1B cells

Abstract

A procedure has been developed for the superfusion of cells in suspension with pairs of metabolically related labeled compounds ([ 3H]-A and [ 14C]-B). Cells (approx. 6 × 10 6) are placed in a superfusion chamber limited by a cellulose ester membrane of 3μ pore size. The superfusion medium with the tracers is driven through capillary Teflon tubing into a chamber by a syringe pump (10 ml/h) and collected in test tubes immersed in an ice bath. The chamber is mounted on a Vortex mixer for agitation. The superfusion is conducted at 37°C inside a temperature controlled room. After a period of superfusion sufficiently long to produce an isotopic steady state (1–2 h). the concentrations of 3H- and 14C-labeled compounds A and B are measured in the superfusion medium, superfusate and superfused cells. Rates of entry, interconversion, exit and metabolism of A and B per cell are estimated from the data, using formulas derived on the basis of a suitable model. Application of the method to the study of the metabolism of estradiol and estrone in an endometrial adenocarcinoma eell line of human origin (HEC) demonstrated its feasibility. Constant flow rates were maintained without excessive pressure build-up. The cells appeared undamaged and vital at the end of the procedures. Superfusion of homogeneous cell suspensions offers the advantage of allowing uniform access of tracers, oxygen and nutrients to each cell, and provides an experimental design which closely approximates the model on which the calculations are based.