The Use of Silk as a Scaffold for Mature, Sustainable Unilocular Adipose 3D Tissue Engineered Systems

Abstract

There is a critical need for monitoring physiologically relevant, sustainable, human adipose tissues in vitro to gain new insights into metabolic diseases. To support long term culture, a 3D silk scaffold assisted culture system was developed that maintained mature unilocular adipocytes ex vivo in co-culture with pre-adipocytes, endothelial cells, and smooth muscle cells obtained from small volumes of liquefied adipose samples. Without the silk scaffold, adipose tissue explants could not be sustained in long term culture (3 months) due to their fragility. Adjustments to media components were used to tune lipid metabolism and proliferation, in addition to responsiveness to an inflammatory stimulus. Interestingly, patient specific responses to TNFα stimulation were observed, providing a proof of concept translational technique for patient specific disease modeling in the future. In summary, our novel 3D scaffold assisted approach is required for establishing physiologically relevant, sustainable, human adipose tissue systems from small volumes of lipoaspirate, making this methodology of great value to studies of metabolism, adipokine-driven diseases, and other diseases where the roles of adipocytes are only now becoming uncovered.

Variability in responses observed in human white adipose tissue models

“…It has been used to image the large unilocular lipid droplet of mature adipocytes (Le et al, 2007), which is the hallmark of their in vivo phenotype. In prior work (Abbott, Wang, et al, 2016), we found that our adipose tissue system maintained unilocular mature adipocytes with destructive imaging techniques. To validate that this phenotype could be detected with non-destructive methods, we used CARS imaging in the current study demonstrating that there was not a significant effect of time in culture on the unilocular lipid areas of adipocytes ( Figure 1b).…”

“…Although there is a consistent ratio between the number of adipose tissue stromal cells and adipocytes (unrelated to BMI), during adipose expansion, cell size precedes the increase in fat cell number (van Harmelen et al, 2003); therefore, normalizing by DNA content in the current study may have added an additional confounding factor. Furthermore, different populations of cells may proliferate more than others (i.e., fibroblasts over adipocytes), although our prior work (Abbott, Wang, et al, 2016) demonstrates that these cell types are present throughout culture. These factors likely contributed to the large variation in lipolytic responses and glucose uptake observed in the current study.…”

“…2.3 | Seeding the scaffolds with adipose tissue Scaffolds were seeded as described previously (Abbott, Wang, et al, 2016). Subcutaneous adipose tissue from abdominoplasty surgeries was processed on the same day of surgery with institutional review board approval (Protocol #0906007).…”

“…However, much of the research on inhibition of insulin signalling with TNFα has been performed in animal models (Hotamisligil, Shargill, & Spiegelman, 1993;Uysal, Wiesbrock, Marino, & Hotamisligil, 1997), or with differentiated murine cells (Hotamisligil, Murray, Choy, & Spiegelman, 1994;Peraldi, Hotamisligil, Buurman, White, & Spiegelman, 1996). Our model contains human cells in co-culture with stromal vascular cells (endothelial cells, stem cells, and pericytes; Abbott, Wang, et al, 2016) that could account for the observed differences. In addition, human plasma TNFα levels and insulin resistance are not always correlated.…”

“…The goal of the current study is to establish whether our previously characterized seeding method for maintaining a human adipose tissue depot ex vivo (Abbott, Wang, et al, ) could be used to recapitulate human responses to stimuli known to alter adipocyte metabolism in vitro. Due to their large and fragile nature, ex vivo culture of mature adipocytes has been limited and most human in vitro culture systems differentiate human adipose derived stem cells (Cheung et al, ; Jaikumar et al ., ; Luo & Choong, ; Wittmann et al ., ).…”