A surface‐tethered spheroid model for functional evaluation of 3T3‐L1 adipocytes

Abstract

In order to effectively treat obesity, it must be better understood at the cellular level with respect to metabolic state and environmental stress. However, current two-dimensional (2D) in vitro cell culture methods do not represent the in vivo adipose tissue appropriately due to the absence of complex architecture and cellular signaling. Conversely, 3D in vitro cultures have been reported to have optimal results mimicking the adipose tissue in vivo. The main aim of this study was to examine the efficacy of a novel conjugate of a genetically engineered polymer, elastin-like polypeptide (ELP) and a synthetic polymer, polyethyleneimine (PEI), toward creating a 3D preadipocyte culture system. We then used this 3D culture model to study the preadipocyte differentiation and adipocyte maintenance processes when subjected to various dosages of nutritionally relevant free fatty acids with respect to total DNA and protein content, cell viability, and intracellular triglyceride accumulation. Our results showed that 3T3-L1 preadipocytes cultured on the ELP-PEI surface formed 3D spheroids within 72 h, whereas the cells cultured on unmodified tissue culture polystyrene surfaces remained in monolayer configuration. Significant statistical differences were discovered between the 3D spheroid and 2D monolayer culture with respect to the DNA and protein content, fatty acid consumption, and triglyceride accumulation, indicating differences in cellular response. Results indicated that the 3D culture may be a more sensitive modeling technique for in vitro adipocyte culture and provides a platform for future evaluation of 3D in vitro adipocyte function.

Adipogenic differentiation of human adipose-derived stem cells grown as spheroids

“…Attributing 0% conversion to the neat ELP fluorescence and 100% conversion to the neat PEI fluorescence, we calculated the conversion for the ELP-PEI conjugation reaction to be 15 mol%, with the remaining 85 mol% material being the unreacted ELP. Based on our previous research which showed a 5 mol% ELP-PEI solution induced uniform spheroid formation [20,21], we further diluted the reaction product solution with more ELP to obtain a 5 mol% ELP-PEI solution with the remaining 95% material being neat ELP. As shown in Figure 1b, the turbidity of the ELP solution increased due to coacervation as the solution was heated and exhibited an inverse phase transition temperature (T t ) at 37.6 ± 0.9 °C (Fig.…”

“…For this research, we used discarded adipose tissue from a patient undergoing elective abdominoplasty or liposuction to study the impact of 3-D spheroid culturing technique on primary stromal vascular preadipocyte (hASC) differentiation and overall functionality. hASCs were cultured using similar methods pioneered with H35 rat hepatoma and 3T3-L1 preadipocyte model cells [20,21], though using maintenance media optimized for hASCs [26,32]. The experimental timeline is shown in Figure 2.…”

“…Conjugate of ELP with backbone structure Valine-Proline-Glycine-Valine-Glycine (molecular weight = 17,000 Da) and PEI (molecular weight = 800 Da, Sigma-Aldrich, St. Louis, MO) was prepared using carbodiimide chemistry as described elsewhere [20,21]. The phase transition temperatures of ELP and ELP-PEI solutions (5 mg/mL) were determined on 3 replicates (n = 3) using a Cary 100 spectrophotometer equipped with a temperature controller by measuring the absorbance at 350 nm as the solutions were heated from 20 to 80 °C at the rate of 1 °C/min.…”

“…Media were sonicated at 40°C for 45 minutes, cooled to 4 °C, and supplemented with 0.2 U/mL insulin, 100 units/mL penicillin, and 100 μg/mL streptomycin. Fatty acid loading efficacy was verified by gas chromatography analysis [20]. hASCs were exposed to control or fatty-acid containing maturation medium for up to 10 days after differentiation.…”

Opportunities and challenges in three-dimensional brown adipogenesis of stem cells

“…Compared to 2D culture, 3D culture for adipogenesis in general could: 1) deposit an extensive ECM network (Grayson et al, 2004), 2) recapitulate adipose stem cell microenvironments (Yang et al, 2010), 3) enhance adipogenic differentiation, exhibiting more mature adipogenesis of stem cells (Gerlach et al, 2012), efficient lipid accumulation and in vivo-like organogenesis (Daquinag et al, 2013), expression of adipocyte-specific markers (Hong et al, 2005; Neubauer et al, 2005; Stacey et al, 2009) as well as VEGF (Girandon et al, 2011), and increase secretion of adipokines such as leptin (Kang et al, 2005), 4) be more sensitive to insulin-stimulated glucose uptake and drug treatment (Brännmark et al, 2014; Turner et al, 2014), 5) allow long-term survival (Neuss et al, 2008a), and 6) be feasible to manipulate cellular microenvironments for adipogenesis (Daya et al, 2007). …”