Quantitative analysis of rat adipose tissue cell recovery, and non-fat cell volume, in primary cell cultures

Background. White adipose tissue (WAT) is a complex, disperse, multifunctional organ which contains adipocytes, and a large proportion of fat, but also other cell types, active in defence, regeneration and signalling functions. Studies with adipocytes often require their isolation from WAT breaking up the matrix collagen fibres, but primary cultures of these cells could not be easily correlated to intact WAT, since often recovery and viability are unknown. Experimental design. Epididymal WAT of 4-6 young adult rats was used to isolate adipocytes with collagenase. Careful recording of lipid content of tissue, and all fraction volumes and weights, allowed us to trace the amount of initial WAT fat remaining in the cell preparation. Functionality was estimated by incubation with glucose and measurement of lactate production. Non-adipocyte cells were also recovered and their sizes (and those of adipocytes) were also measured. The presence of non-nucleated cells (erythrocytes) was also estimated. Results. Cell numbers and sizes were correlated from all fractions to intact WAT. Tracing the lipid content, the recovery of adipocytes in the final, metabolically active, preparation was in the range of 70-75%. Adipocytes were 7%, erythrocytes 68% and other stromal (nucleated cells) 24% of total WAT cells. However, their overall volumes were, 91%, 0.05%, and 0.2% of WAT. Non-fat volume of adipocytes was 2.5% of WAT. Conclusions. The methodology presented here allows for a direct quantitative reference to the original tissue of studies using isolated cells. We have found, also, that the "live cell mass" of adipose tissue is very small (about 25 µL/g for adipocytes and 2 µL/g stromal, plus about 1 µL/g blood). This fact, translates into an extremely high (with respect to the actual "live cytoplasm" size) metabolic activity, which make WAT an even more significant agent in the control of energy metabolism.