MONOLAYER CELL-CULTURE OF FRESHLY ISOLATED ADIPOCYTES USING EXTRACELLULAR BASEMENT-MEMBRANE COMPONENTS

Cell biological techniques requiring cells attached to surfaces, such as monolayer cell culture, microspectrofluorometry, and confocal micro scopy, have not been readily available for use on adipocytes because t hey float and tend to lyse when attached to charged non-biological sur faces. A new method for attaching freshly isolated rodent adipocytes t o thermanox plastic surfaces using Matrigel (a defined mixture of extr acellular matrix components that resembles the basal lamina surroundin g adipocytes in vivo) is described. The method takes advantage of an u nusual physical characteristic of Matrigel, i.e., that it is a liquid at cold temperatures and a hydrated gel at higher temperatures. To att ach the isolated cells, chilled thermanox plastic coverslips were coat ed with a thin uniform layer of ice-cold Matrigel and inverted into wa rm floating adipocytes. Adipocytes floated up against the liquid Matri gel and became immediately attached when the Matrigel changed to a gel in response to the warmth of the cells and media. Cell volume measure ments of the attached versus freshly isolated cells indicate no signif icant difference in the centroid cell volume of the attached cells. Th is indicates that the method does not select for small or large cells. Adipocytes maintained for 6 days in culture did not display any chang e in their size or differentiated microscopic appearance. The relative concentrations of major proteins in silver-stained SDS-PAGE gels and several differentiation state-dependent proteins, including ATP-citrat e lyase, carbonic anhydrase III (CA III), adipocyte lipid binding prot ein (ALBP), and pyruvate carboxylase, were examined. No significant ch ange was observed in the relative concentrations of these proteins whe n the Matrigel-cultured adipocytes were compared to freshly isolated c ells. In addition, Matrigel-cultured adipocytes retained their ability to respond to insulin, as shown by the positive effects of insulin on ATP-citrate lyase concentrations and the negative effects of insulin on ALBP and CA III concentrations. The results of these experiments in dicate that adipocytes on Matrigel may maintain many of their differen tiated characteristics. Attachment of adipocytes to Matrigel-coated co verslips also allowed spectrofluorometric measurements to be made usin g methods normally used for attached cells. Intracellular pH was measu red in a spectrofluorometer equipped with a perfusion chamber using th e fluorescent pH probe, BCECF. BCECF-loaded adipocytes displayed brief alkalinization in response to NH4Cl exposure and rebound acidificatio n upon its withdrawal, indicating that the Matrigel does not interfere with dye loading or intracellular pH regulation. This method should b e useful for any number of long-term studies on isolated adipocytes or cell biological techniques that require a surface attached cell.