Adipocyte differentiation medium

Primary human pre-adipocytes (ATCC PCS-210-010) were differentiated into mature adipocytes in wells of 24-well-plates containing adipocyte differentiation medium (Cell Applications, Inc. San Diego, CA) in a 5% CO₂ atmosphere at 37° C. According to the product sheet from ATCC, these human pre-adipocytes are derived from de-differentiated mature adipocytes and provide an ideal culture model for the study of diabetes, obesity, metabolism, insulin sensitivity and adipose biology. These cells can be expanded in an undifferentiated state for future differentiation to mature adipocytes and show higher efficiency of adipogenesis compared to mesenchymal stem cells. In this study, the basic concentration of glucose in adipocyte differentiation medium from Cell Applications Inc. is 1.5mg/ml. The cells were then treated with increasing dose of glucose (2.0–3.5 mg/ml, Sigma-Aldrich, St. Louis, MO), or TNF-α (0–0.5 ng/ml, Sigma-Aldrich) for 24 hours. Total RNA was isolated from the cells using TRIzol (Life Technologies, Grand Island, NY) and RT was performed for further Quantitative Real-time-PCR analysis.

Primary normal human pre-adipocytes (ATCC PCS-210–010, American Type Culture Collection, Manassas, VA, USA) were differentiated into mature adipocytes in wells of 24-well-plates containing adipocyte differentiation medium (Cell Applications, Inc. San Diego, CA) in a 5% CO2 atmosphere at 37⁰ C^{29 (link)}. These cells can be expanded in an undifferentiated state for future differentiation to mature adipocytes and show higher efficiency of adipogenesis compared to mesenchymal stem cells. In this study, the cells were cultured in adipocyte differentiation medium with increasing doses of glucose (8.4mM to 19.3mM, Sigma-Aldrich, St. Louis, MO), or ADM (1×10^{− 10}M to 1×10^{− 8}M, Sigma-Aldrich) for 24 hours. Total RNA was isolated from the cells using TRIzol (Life Technologies, Grand Island, NY) and RT was performed for further Quantitative Real-time-PCR analysis.

Cryopreserved primary human liver cells were obtained from Massachusetts General Hospital (MGH, Boston, MA, USA, lot#HW54). Cells were plated after thawing on ECL Cell Attachment Matrix (Sigma) on 70% isopropanol-sterilized 15 mm glass coverslips (cs), at a density of approximately 250,000 cells/cs. Cells were cultured in vendor-recommended medium^{28 (link)} for 5–9 days before incorporation into systems. Medium was replaced fully every 48 h as previously described^{25 (link),28 (link)}.
Primary human cardiac preadipocytes (Cell Applications, Sigma) were plated and began differentiation into mature adipocytes approximately 2 weeks before incorporation into systems. Cells were plated onto 70% isopropanol-sterilized 15 mm glass coverslips at a density of 90,000 cells/cs in Preadipocyte Growth Medium (Cell Applications, Sigma). Preadipocytes were cultured in growth medium for 1–3 days until confluent, and medium was replaced with Adipocyte Differentiation Medium (Cell Applications, Sigma). Differentiation of cells to mature adipocytes was designated when cells exhibited extensive lipid droplet accumulation.
For experiments in monoculture, treatment was performed in-plate, and cells remained in plate for testing/readouts. Prior to functional or endpoint assays, two-organ systems were disassembled and the organ modules were transferred to well-plates.