*3.1. 2D Models*

White adipocytes have large unilocular lipid droplets in their cytoplasm. This causes the mature adipocytes to float in media when cultured using 2-dimensional (2D) approaches. Unable to meet their nutritional requirements, the floating adipocytes will lyse in a matter of days [69]. Techniques have been developed to combat this unique problem, including a method termed "ceiling culture" where flasks are filled with media and adipocytes attach to the ceiling of the cell culture flask [69–71]. However, cells cultured in this way do not function the same as in vivo adipocytes and lose their lipid stores [72]. As an alternative to culturing mature adipocytes ex vivo, ASCs are di fferentiated on 2D substrates to an immature multilocular phenotype (full di fferentiation cannot be achieved in these systems as lipid laden cells will detach from the surface).

Two-dimensional culture systems have been used to model cellular changes during obesity and adipose tissue fibrosis. Many systems use macrophages to mimic the inflammatory e ffects associated with obesity that trigger adipose tissue fibrosis [53,73–77]. One model system used media that was conditioned by macrophages from an obese human patient with or without supplementation of lipopolysaccharide (LPS), to study the secretory e ffects on ASCs from a healthy patient. The results indicated that ASCs have a lower capacity to di fferentiate in the presence of macrophage byproducts. Additionally, in the presence of LPS the ASCs became proinflammatory and secreted higher concentrations of inflammatory factors [77]. Following a similar experimental setup, another study found that ASCs exposed to factors secreted by M1 macrophages increased ECM remodeling. The ASCs had a proinflammatory phenotype, increased proliferation and migration, but a

decreased ability to differentiate [53]. Together, these results indicate that the presence of secretory factors from M1 polarized macrophages primes ASCs to develop a proinflammatory phenotype that is pro-fibrotic, rather than pro-adipogenic.

Another study corroborated these results with a co-culture model. Proinflammatory macrophages (CD14+) and human ASCs (hASCs) were investigated to determine how stem cell differentiation was affected by their interaction. Consistently, there was a significant decrease in differentiation after 14 days in the presence of proinflammatory macrophages, with a decrease in adiponectin, CEBPβ, and GLUT4 and an increase in IL6 gene expression [74]. These results have an increased physiological relevance compared to similar studies [5,75,78] because both cell types were sourced from the same patient. It was proposed that the cytokine levels would be similar to native human adipose tissue because the same cell source was used. This suggests that 2D models could be used to study other fibrotic mechanisms related to ASC commitment and proinflammatory factors.
