Le gras, c’est la vie - fat is life!

 Longer considered as an inert tissue for energy storage, adipose tissue (AT) appeared in recent decades to be one of the largest endocrine organs in the body. This complex tissue is mainly composed of adipocytes (storing triglycerides, important fuel source of our body) and the stromal vascular fraction (SVF) consisting of endothelial cells, pericytes, monocytes, macrophages, fibroblasts and adipose stem/progenitor cells (ASCs). The AT is a central metabolic organ in the regulation of systemic energy homeostasis by acting as a caloric reservoir. The white AT (WAT) stores extra energy as lipids obtained during food consumption and releases it as energy during food scarcity periods, whereas the brown AT (BAT) accumulates lipids for cold-induced adaptive thermogenesis. Besides these functions, AT has the ability to synthesize and release hormones, cytokines, and metabolites, termed as adipokines, that influence a variety of physiological and pathophysiological processes, as insulin sensitivity, inflammation, vascular functions and food intake. In response to changes in the nutritional status, WAT undergoes dynamic remodelling affecting adipose tissue-resident cells. In the context of obesity, metabolic disorder characterized by an excessive increase in white fat mass, WAT dynamic remodelling leads to a dysfunctional AT. Changes in the number (hyperplasia) and size (hypertrophy) of the adipocytes affect the microenvironment resulting in alterations in adipokine secretion, adipocyte death, local hypoxia, and fatty acid fluxes. In parallel, SVF cells in the WAT are also affected by these changes leading to exhaustion of ASC pool, uncontrolled inflammatory responses accompanied with onset of fibrosis and insulin resistance. As worldwide public health issue and consequence of our modern and western lifestyle, it is important to understand adipose tissue biology in order to find approach in the treatment of obesity.

In our lab, we are focusing our researches on ASCs. These stem cells bear a strong resemblance to bone marrow stem cells (BMSCs) as shown by their expression of common cell surface markers, their similar gene expression profiles, their self-renewal capacity and their similar differentiation potentials. These cells are of great interest for the field of regenerative medicine. Compared to BMSCs, ASCs can be obtained in large quantities at low risks. WAT is more abundant, easily accessible and provides higher yields of mesenchymal stem cells (MSCs) compared to BM. In this way, ASCs can appear as the preferred choice of adult stem cells for future clinical applications.

But how are ASCs obtained?

The initial methods to isolate cells from adipose tissue were pioneered by Rodbell, and Rodbell and Jones in the 1960s. They simply minced rat fat pads, washed extensively to remove contaminating hematopoietic cells, incubated the tissue fragments with collagenase, centrifuged the digest, obtaining finally different layers separating the floating population of mature adipocytes from the pelleted stromal vascular fraction (SVF). Nowadays, the method used in our lab follows the same principle. As shown in the figure, WAT obtained from human patient is under sterile conditions mechanically dissected to remove blood vessels and connective fibrous material and get smaller pieces of AT, then incubate with collagenase in order to digest the tissue and disaggregate cells. The dispersed obtained cells are then filtered to remove remaining connective tissue and centrifuged. From this centrifugation resulted the separation of SVF (pellet) from adipocytes (upper layer). At this step, adipocytes can be harvested and after several smooth washings used in cell culture. Concerning the SVF, the pellet is resuspended in erythrocyte lysis buffer in order to lyse red blood cells and then the SVF undergo several filtrations through cell strainer of defined size alternate with centrifugations. The resulting SVF is still composed of a heterogeneous cell population composed of ASCs, endothelial cells, immune cells and fibroblasts, but ASCs are known to adhere on plastic surfaces compared to the other cell types. Therefore, the cell suspension is seeded on plastic flask and after a few early passages of cells, the ASCs in culture survive and lead to homogeneity of the population.

by Camille BRUCKER