Pre adipocyte growth medium

Chemicals and reagents were purchased from the following sources: polydimethylsiloxane (PDMS) -Sylgard® 184 from Dow Corning (MI, USA); polyethylene terephthalate (PET) membrane from Corning Transwell (USA); PBS from Biowest (USA); human pre-adipocytes (HPAd) cat # 802s-05a, pre-adipocyte growth medium, adipocyte differentiation medium, adipocyte maintenance medium, and adipocyte starvation medium from Cell Applications (USA); iMDM medium from Thermo Fisher Scientific (USA); Lipid A (LPA) from E.coli serotype R515 (TLR grade) from Alexis Biochemials (SA, USA); TNF-α, IL-6 and IL-8 ELISA kits, CD11b-FITC (clone ICRF44), CD45-PE (clone: 2D1), CD3-PerCP-Cy5.5 (cline: HIT3A), CD19-PE-Cy7 (clone: SJ25C1), CD1c-APC-Cy7 (clone: L161), and CD14-PacBlue (clone: HCD14) from Biolegend (USA); CD56-APC (clone: TULY56) and calceine-AMfrom ThermoFisher Scientific (USA) ;2-NBDG glucose uptake cell-based assay kit from Cayman Chemical (USA); Hoechst 33342 from Molecular Probe (OR, USA); Bradford reagent and 0.4% trypan blue, metformin hydrochloride and docosapentaenoic acid (DPA) from Sigma Aldrich (USA); Other chemicals were of analytical grades from Sigma Aldrich (USA).

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.