Spheroid Culture System Confers Differentiated Transcriptome Profile and Functional Advantage to 3T3-L1 Adipocytes

Abstract

This study highlights functional differences between 2-D monolayer and 3-D spheroid 3T3-L1 adipocyte culture models and explores the underlying genomic mechanisms responsible for the different phenotypes present. The spheroids showed higher triglyceride accumulation than the monolayer culture and further increase with larger spheroid size. Whole transcriptome analysis indicated significant differential expression of genes related to adipogenesis, including adipocytokine signaling, fatty acid metabolism, and PPAR-γ signaling. Spheroids also showed downregulation of matrix metalloproteinases (MMPs), integrin, actin-cytoskeleton associated genes, and Rho/GTPase3 expression relative to 2-D monolayer, indicating suppression of the Rho-ROCK pathway and thereby promoting adipogenic differentiation. When exposed to linoleic acid (500 μM) and TNF-α (125 ng/mL) to promote chronic adiposity, linoleic acid treatment resulted in increased intracellular triglycerides and subsequent TNF-α treatment resulted in significantly altered adipocytokine signaling, fatty acid metabolism, and PPAR signaling, in addition to upregulation of multiple MMPs in spheroids vs. monolayer. Overall, 3-D spheroids showed enhanced adipogenic phenotype as indicated by triglyceride synthesis and transcriptome changes while retaining sensitivity to a pro-inflammatory stimulus. The 3-D spheroid culture thus may provide a simple, convenient, and sensitive in vitro model to study adipocyte response to metabolic stresses relevant to clinical pathologies.

Comparison of the formation, adipogenic maturation, and retention of human adipose‐derived stem cell spheroids in scaffold‐free culture techniques

“…In our previous work, which focused more on the biological aspects of hASC culture (Turner, Harris, et al, 2014;Turner et al, 2015;Turner et al, 2017;Turner et al, 2018), we have established the relevancy of spheroid size and cellular adiposity by using Oil-Red-O staining and RT-PCR to further confirm the adipogenic differentiation of hASCs.…”

“…ELP‐PEI block copolymers are a class of multifunctional polymers that have been shown to exhibit the inverse phase transition behavior of ELP as well as the mild polyelectrolyte nature of the PEI. Previous work with ELP‐PEI has utilized a low molecular weight block of PEI (<1,200 g/mol) which, used in low concentrations in surface coatings, have been shown to elicit cellular aggregations into three‐dimensional structures (Clark & Janorkar, 2018; Gurumurthy et al, 2017; Penfornis et al, 2017; Turner, Harris, et al, 2014; Turner et al, 2015; Turner et al, 2017; Turner et al, 2018; Turner, Weeks, et al, 2014; Weeks et al, 2017). The ELP‐PEI800 copolymer coating has been used in the past to prepare 3D spheroid cultures of several different cell types, including rat hepatocytes, mouse 3T3‐L1 preadipocytes, H35 rat hepatoma, human osteosarcoma, and hASCs (Clark & Janorkar, 2018; Gurumurthy et al, 2017; Penfornis et al, 2017; Turner, Harris, et al, 2014; Turner et al, 2015; Turner et al, 2017; Turner et al, 2018; Turner, Weeks, et al, 2014; Weeks et al, 2017).…”

“…Previous work with ELP‐PEI has utilized a low molecular weight block of PEI (<1,200 g/mol) which, used in low concentrations in surface coatings, have been shown to elicit cellular aggregations into three‐dimensional structures (Clark & Janorkar, 2018; Gurumurthy et al, 2017; Penfornis et al, 2017; Turner, Harris, et al, 2014; Turner et al, 2015; Turner et al, 2017; Turner et al, 2018; Turner, Weeks, et al, 2014; Weeks et al, 2017). The ELP‐PEI800 copolymer coating has been used in the past to prepare 3D spheroid cultures of several different cell types, including rat hepatocytes, mouse 3T3‐L1 preadipocytes, H35 rat hepatoma, human osteosarcoma, and hASCs (Clark & Janorkar, 2018; Gurumurthy et al, 2017; Penfornis et al, 2017; Turner, Harris, et al, 2014; Turner et al, 2015; Turner et al, 2017; Turner et al, 2018; Turner, Weeks, et al, 2014; Weeks et al, 2017). Specifically, for the mouse 3T3‐L1 preadipocyte and hASC cultures, 3D spheroids formed atop the ELP‐PEI800 coating have been shown to be a better in vitro model compared to the 2D monolayer in terms of accumulation of triglycerides and sensitivity of cellular responses to nutrients and cytokines (Turner, Harris, et al, 2014; Turner et al, 2015; Turner et al, 2017; Turner et al, 2018).…”

“…ELP (Valine‐Proline‐Glycine‐Valine‐Glycine) 40 with molecular weight of 17,000 g/mol was produced using genetically modified E. coli and purified using inverse phase transition temperature cycling (Gurumurthy et al, 2017; Turner, Harris, et al, 2014; Turner et al, 2015; Turner et al, 2017; Turner et al, 2018). ELP‐PEI conjugates were synthesized by carbodiimide activation of the c‐terminus carboxyl group on the ELP for 15 minutes and dropwise addition of the activated ELP (MES buffer, pH 6.2, 30 mg ELP) to a PEI solution (MES buffer, pH 6.8, 10:1 PEI to ELP molar ratio) to react at 4°C for a minimum of 8 hr.…”

“…Such fatty spheroids become more buoyant over time, lift off of the culture surface and are removed during the next media change. To minimize the loss of such physiologically relevant fatty spheroids and to provide a proven, long‐term culture method for mature adipocytes in vitro, we investigated the use of elastin‐like polypeptide‐polyelectrolyte coated surfaces (Gurumurthy et al, 2017; Turner, Garrett, Didion, & Janorkar, 2018; Turner, Gurumurthy, Bailey, Elks, & Janorkar, 2017; Turner, Harris, Purser, Baker, & Janorkar, 2014; Turner, Tang, Weiss, & Janorkar, 2015). Elastin‐like polypeptides (ELPs) conjugated to polyethyleneimine (PEI) have been used in coatings as a 3D culture method for many different cell types (Clark & Janorkar, 2018; Gurumurthy et al, 2017; Penfornis et al, 2017; Turner, Weeks, McMurphy, & Janorkar, 2014; Weeks et al, 2017).…”