Penn strep

Following informed consent and IRB approval at the authors′ institution, human AMSCs were isolated from lipo-aspirates obtained from a representative donor using previously described methods^{13 (link), 14 (link),49 (link)}. The donor-derived AMSCs selected in this study have previously been shown to express standard MSC markers and are capable of multi-lineage differentiation^{13 (link)–15 (link)}. In brief, donor lipo-tissue was aspirated and then digested using Type I collagenase (Worthington Biochemicals, Lakewood, NJ) for 1.5 hours at 37°C, centrifuged at 400g for 5 min, rinsed with phosphate buffered saline (Life Technologies, Grand Island, NY), and strained using 70μm cell strainers (BD Biosciences, San Jose, CA). Following this, the aspirate was treated with 154 mM NH₄Cl, 10 mM KHCO₃, and 0.1 mM EDTA for erythrocyte lysis. The remaining AMSCs were expanded in advanced minimum essential medium (Life Technologies, Grand Island, NY), supplemented with 5% (vol/vol) human platelet lysate (PLTMax; MillCreekLifeSciences, Rochester, MN), 2mM Glutamax (Life Technologies, Grand Island, NY), 2 U/mL heparin, and 1% Penn-Strep (100 U/mL penicillin, 100 μg/mL streptomycin; Cellgro, Corning, NY). All AMSCs used in experimentation were of passage 7.

Cerebral cortices from 1–3 day old New Zealand white rabbits were excised, meninges removed, and suspended in 5 mL of 0.5% trypsin for 15 min to digest the tissue. The trypsin was neutralized using DMEM medium (DMEM-4.5 g, glucose10% FBS and 1% Penn/Strep) (Cellgro, Manassas, VA USA). The tissue was minced using constant pipetting and the tissue suspension was filtered through a 40 μm nylon cell strainer (BD falcon, franklin lakes, NJ, USA) to remove tissue debris. The filtered cell suspension was centrifuged at 1000 rpm for 5 min at 4°C and the pellet was re-suspended in culture media and were seeded in T-175 flasks and left undisturbed for enhancing their attachment of the cells for a period of 7 days at 37°C and 5% CO₂ atmosphere. Later the cell debris and unattached cells in the culture were removed by gently changing the media every 2 days to grow and expand until 90% confluency for another week.

MC3T3-E1 Subclone 4 osteoblastic cell lines, acquired from ATCC (ATCC CRL-2594), were cultured in αMEM + Glutamax (GIBCO 32571–036) and supplemented with 10% fetal bovine serum (FBS, Sigma Aldrich) and 1% Penn/Strep (Corning). To stimulate procollagen synthesis and secretion, 100 μM ascorbic acid 2-phospate (Sigma Aldrich) was supplemented during transfection, 12–24 h before imaging.

Human EOC cell line A2780-CP20 (designated as CP20) was a kind gift from Dr. Anil Sood (MD Anderson Cancer Center, Houston, TX); OV90 was from American Type Culture Collection (Manassas, VA). EGFP (enhanced green fluorescent protein) stably expressing CP20 or OV90 cells were established by transfecting the cells with pcDNA3-EGFP vector (Watertown, MA) with FuGENE 6 Transfection Reagent (E2691, Promega, Madison, WI). All cell lines were cultured in RPMI 1640 (10-040-CV, Corning, NY) with 10% Fetal Bovine Serum (FBS, 16000-044, Life technologies, Carlsbad, CA) and 1% Penn-Strep (15140-122, Life technologies). Primary ovarian CAF TAF18 were isolated and identified in this laboratory [39 (link)], grown in DMEM:F12 (10-090, Corning, NY) with 15% FBS and 1% Penn-Strep, and used up to passage 7. HMEC, a kind gift from Dr. Xin Zhang (OUHSC Stephenson Cancer Center, Oklahoma City, OK), and HUVEC (Lonza, Walkersville, MD) were propagated in Endothelial Cell Growth Medium 2 (EGM, CC-3162) and used up to passage 7 [56 (link)]. When different types of cells were cultured together, the mixed cells were grown in a mixed medium prepared by combining equal volumes of the individual medium for each of the three cell types. All cells were maintained in a humidified 95% air and 5% CO2 atmosphere at 37 °C.

CiA:Oct4 MEF cell lines immortalized by infection of simian virus 40 large T antigen, were obtained and cultured as previously described (Hathaway et al., 2012 (link)). Briefly, cells were cultured at 37 °C 5% CO₂ conditions. Base media was either FluoroBrite DMEM Media (ThermoFisher, A1896701) for imaging, or DMEM (Corning, MT10013CV) for standard cell culture. Media was supplemented with 10% FBS (Gibco, Lot:1972526), 10 mM HEPES pH 7.5, 10 mM NEAA, 0.1% 1000 × 2-betamercaptoethanol (Gibco, 21,985,023), 1% 100× Penn-Strep (Corning, 30–002-CI). Additionally, l-Glutamine (Corning, 25005CI) at 4 mM was added to FluoroBrite media.