Abstrakt
Introduction: Atherosclerosis is one of the leading causes of death worldwide. Obesity-induced changes in adipose tissue are a source of systemic subclinical inflammation, one of the main processes participating in atherogenesis. Data obtained on animal models are of limited use due to significant interspecies differences in lipid metabolism. The newest biotechnology, 3D organoid cultures based on human sample processing, could offer a new study model for adipose tissue macrophages and adipose tissue.
Objective: Establishment of self-organized 3D spheroids derived from adipose stromal vascular fraction.
Methods: Visceral adipose tissue was harvested during a kidney removal surgery (from the area outside of Gerota's fascia) intraoperatively during a living kidney donor nephrectomy. Samples were cleaned from all visible blood vessels, fibrous tissue, and residual blood, digested in collagenase solution, and stromal vascular fraction was isolated. Digested samples were immediately cooled, filtered, centrifuged, and seeded to ultra-low attachment plates. The Dulbecco's Modified Eagle Medium (DMEM) was supplemented with fetal calf serum, penicillin, streptomycin, amphotericin, and dipeptide L-alanyl-L-glutamine and was regularly changed during 15 days of lasting differentiation. A hormone cocktail (insulin, dexamethasone, rosiglitazone, 3-isobutyl-1-methylxanthine/IBMX) was used as a medium supplement for inducing adipogenic differentiation. The spheroids were analyzed by histologic, immunohistochemistry, and flow cytometry.
Results: Our analyses detected viable organized spheroids, and not only the spectrum of cells, including macrophages (CD68), stem cells (CD34), epithelial were demonstrated, but also a newly made extracellular matrix (collagen, carbohydrate molecules) confirming the metabolic activity of the spheroids.
Conclusions: Our methodological approach succeeded in forming organoids containing all main cell types in the stromal vascular fraction of adipose tissue. This investigation is a promising step in establishing a suitable in vitro model for studies of human adipose tissue changes concerning cardiovascular disease pathogenesis and drug treatment.