Adipose tissue explant culture using PDMS flow chambers: an alternative to static explant culture.

Abstract

As obesity rates continue to rise, it is important that we can effectively study adipose tissue to understand its physiological contribution in individuals with obesity. Unfortunately, due to the fragility and buoyancy of adipose tissue, culture remains challenging. Ex vivo culture of tissue explants is possible, however after 48 hours explants often display declining viability, increased inflammation, and de-differentiation. Other common approaches include differentiation of preadipocytes and adipocyte isolation by enzymatic dissociation, however these methods are time-consuming and fail to recapitulate the structure and cellular network within adipose tissue. Given these shortcomings, we developed a novel explant culture method using polydimethylsiloxane (PDMS) flow chambers attached to a micro peristaltic pump. This approach reduces air interface while enabling media perfusion, time-resolved measurements of secreted factors, and easy incorporation of treatments. Using our chambers, we assessed viability with resazurin and lactate dehydrogenase (LDH) assays, physiology by measuring glycerol release, architecture by confocal imaging, and retention of adipose gene expression by qPCR. Explants remained viable for over 72 hours. Resazurin reduction was at 84 ± 9% of baseline, and LDH release remained low. Isoproterenol treatment resulted in 2.7 ± 0.5-fold increased glycerol release, while insulin returned release to baseline. Confocal imaging showed preserved architecture, while qPCR of human tissue with insulin and dexamethasone supplementation showed maintained expression of PPARG and FABP4 over 72 hours. Overall, our results suggest PDMS flow chambers are a suitable method for adipose explant culture that requires minimal processing, making this system a viable option for translational research.