Characterization of Adipogenic Chemicals in Three Different Cell Culture Systems: Implications for Reproducibility Based on Cell Source and Handling

Abstract

The potential for chemical exposures to exacerbate the development and/or prevalence of metabolic disorders, such as obesity, is currently of great societal concern. Various in vitro assays are available to assess adipocyte differentiation, though little work has been done to standardize protocols and compare models effectively. This study compares several adipogenic cell culture systems under a variety of conditions to assess variability in responses. Two sources of 3T3-L1 preadipocytes as well as OP9 preadipocytes were assessed for cell proliferation and triglyceride accumulation following different induction periods and using various tissue culture plates. Both cell line and cell source had a significant impact on potencies and efficacies of adipogenic chemicals. Gene expression analyses suggested that differential expression of nuclear receptors involved in adipogenesis underlie the differences between OP9 and 3T3-L1 cells; however, there were also differences based on 3T3-L1 cell source. Induction period modulated potency and efficacy of response depending on cell line and test chemical, and large variations were observed in triglyceride accumulation and cell proliferation between brands of tissue culture plates. Our results suggest that the selection of a cell system and differentiation protocol significantly impacts the detection of adipogenic chemicals, and therefore, influences reproducibility of these studies.

Characterization of Adipogenic Activity of House Dust Extracts and Semi-Volatile Indoor Contaminants in 3T3-L1 Cells

“…3T3-L1 cells were obtained from Zenbio, Inc. at passage 8 (cat# SP-L1-F, lot# 3T3062104; Research Triangle Park, NC) and were maintained in Dulbecco’s Modified Eagle Medium – High Glucose (DMEM-HG; Gibco cat# 11995) supplemented with 10% bovine calf serum and 1% penicillin and streptomycin (Gibco cat# 15140) 36 . Cells were maintained in a sub-confluent state until differentiation, and each thaw was differentiated within 8 passages (p8–15), with no significant changes in control chemical response observed in that time.…”

“…Cells were induced to differentiate as described in detail previously 36 . Briefly, cells were seeded into 96-well tissue culture plates (Greiner cat # 655090) at approximately 30,000 cells per well in pre-adipocyte media.…”

“…Plates were assayed for triglyceride accumulation, DNA content, and cell viability (ATP production) ten days after induction of differentiation, as described previously 36 . Media was removed from wells of tissue culture plates and cells rinsed with phosphate-buffered saline before replacing with 200 μL of dye mixture (19 mL saline, 1 drop/mL NucBlue (Thermo# R37605), and 500 μL AdipoRed (Lonza# PT-7009)).…”

Unconventional oil and gas chemicals and wastewater-impacted water samples promote adipogenesis via PPARγ-dependent and independent mechanisms in 3T3-L1 cells

“…3T3–L1 cells were obtained from Zenbio, Inc. at passage 8 (cat# SPL1-F, lot# 3T3062104; Research Triangle Park, NC) and were maintained, differentiated, and maintained as described in detail previously (Kassotis et al, 2017a; Kassotis et al, 2017b). Cells were cultured in Dulbecco’s Modified Eagle Medium–High Glucose (DMEM-HG; Gibco cat# 11995) supplemented with 10% bovine calf serum and 1% penicillin and streptomycin, and were maintained in a sub-confluent state until differentiation; each thaw was differentiated within 5 passages (p9–13), with no apparent changes in control chemical response in this time.…”

“…Previous research by our laboratory and colleagues has demonstrated significant impacts on nuclear receptor activity from samples collected in surface and groundwater near UOG spill sites in Colorado, USA (CO) (Kassotis et al, 2014), downstream from an UOG wastewater injection disposal site in West Virginia, USA (WV) (Kassotis et al, 2016b), and downstream from an UOG wastewater spill in North Dakota, USA (ND) (Cozzarelli et al, 2017). Notably, antagonism of thyroid receptor beta (TRβ) and the androgen receptor (AR) are both pathways that can influence adipogenesis (Kassotis et al, 2017a; Kassotis et al, 2017b; Niemelä et al, 2008) suggesting a potential for metabolic disruption by UOG chemicals.…”