PURPOSE: Despite the rapidly growing body of work on stem cell-based adipose tissue engineering, there remains much to be learned about the role of the scaffold and culture environments in directing the adipogenic differentiation of cells.  The purpose of this study was to examine how scaffold environments, with varying stiffness and multiple types of media conditions (traditional differentiation medium, adipocyte-conditioned medium, and transwell-based co-culture with mature adipocytes), impacted the adipogenic differentiation of human preadipocytes in vitro and in vivo.
METHODS: Aspirated human fat was washed, digested with Type I collagenase (1 mg/mL), centrifugated at 600 rpm for 10 min, and the stromal vascular fraction, which contained preadipocytes, was expanded in culture.  Preadipocytes at passage 4 and fresh adipocytes were used for various experiments.  Cells were photo-encapsulated in diacrylated polyethylene glycol (PEG-DA) or methacrylated hyaluronic acid (HA-MA) for three dimensional assays.  Media and cells were studied at time points from 4-16 days of post-differentiation.  By using biochemical assays for adipogenic markers (leptin, adiponectin, glycerol) and Oil Red O staining, differentiation was quantified.
Cell-scaffold constructs were then surgically placed in nude mice (n=20) for 4 weeks.  After harvesting the in vivo tissue samples, RT-PCR was performed to isolate DNA and standard agarose gel electrophoresis was performed on control and experimental samples.  Oil Red O staining was also performed on tissue samples.  
RESULTS: After 16 days of culture in a two dimensional environment, adipogenesis was greatest in the preadipocyte/adipocyte co-culture, while the application of adipocyte-conditioned medium was associated with the least adipogenic differentiation.  These media trends continued in three dimensional environments, where two scaffolds (3.4 and 8.0 kDa polyethylene glycol-based hydrogels) also emerged as most supportive of differentiation. Thus, by altering the media growth conditions and scaffold environment, the differentiation of preadipocytes may be manipulated. 
The results of the in vivo assays showed that after 4 weeks of implantation in the nude mouse, preadipocytes combined with 8 kDa PEG differentiated into adipocytes better than controls as evidenced by positive Oil Red O staining and the presence of adiponectin by PCR.
CONCLUSION: This controlled investigation of how combinations of scaffold and media variables affect the adipogenic differentiation of preadipocytes will help advance the field’s goal of engineering functional, viable adipose tissue.