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

PeerJ

article

Floriana Rotondo¹  María del Mar Romero¹  Ana Cecilia Ho-Palma¹  Xavier Remesar¹  José Antonio Fernández-López¹  Marià Alemany¹ 

[1] Department of Biochemistry and Molecular Biomedicine, Faculty of Biology, University of Barcelona;Institute of Biomedicine, University of Barcelona;CIBER OBN

关键词: Adipocyte;    Adipose tissue;    Stromal cells;    Cell culture;    Cell volume;    Cell viability;    Primary cell cultures;   

DOI  :  10.7717/peerj.2725

学科分类：社会科学、人文和艺术（综合）

来源: Inra

【 摘 要 】

BackgroundWhite adipose tissue (WAT) is a complex, diffuse, multifunctional organ which contains adipocytes, and a large proportion of fat, but also other cell types, active in defense, regeneration and signalling functions. Studies with adipocytes often require their isolation from WAT by breaking up the matrix of collagen fibres; however, it is unclear to what extent adipocyte number in primary cultures correlates with their number in intact WAT, since recovery and viability are often unknown.Experimental DesignEpididymal WAT of four 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 glucose uptake and lactate, glycerol and NEFA excretion rates up to 48 h. Non-adipocyte cells were also recovered and their sizes (and those of adipocytes) were measured. The presence of non-nucleated cells (erythrocytes) was also estimated.ResultsCell 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%. Cells showed even higher metabolic activity in the second than in the first day of incubation. Adipocytes were 7%, erythrocytes 66% and other stromal (nucleated cells) 27% of total WAT cells. However, their overall volumes were 90%, 0.05%, and 0.2% of WAT. Non-fat volume of adipocytes was 1.3% of WAT.ConclusionsThe methodology presented here allows for a direct quantitative reference to the original tissue of studies using isolated cells. We have also found that the “live cell mass” of adipose tissue is very small: about 13 µL/g for adipocytes and 2 µL/g stromal, plus about 1 µL/g blood (the rats were killed by exsanguination). These data translate (with respect to the actual “live cytoplasm” size) into an extremely high metabolic activity, which make WAT an even more significant agent in the control of energy metabolism.

【 授权许可】

CC BY