Synthemax 2 sc substrate

Cell migration analysis was performed on GSCs plated as an adherent monolayer in the presence of 1 μg/mL Synthemax II-SC substrate (Corning). GSCs were pretreated with SM for 6 h prior to performing scratches. Cells migrating from the leading edge were photographed using phase-contrast microscopy. Distance was measured in a 10 × field using ImageJ (National Institutes of Health).

After 10 days of blasticidin selection, polyclonal cell populations were collected with accutase and 1,000 cells seeded into 6-cm dishes precoated with Synthemax II-SC Substrate (Corning, 3535) at a concentration of 5 μg/cm² in mTeSR-E8 medium supplemented with 10× CloneR (Stem Cells, 05888). The medium was changed every day with blasticidin-supplemented mTeSR+Plus medium (10 μg/m). After 10 days, visible colonies were manually transferred into 12-well plates precoated with Vitronectin XF. Surviving monoclonal cell lines were expanded and used in later analyses.

H9 (WA-09) human embryonic stem cells were obtained from WiCell (Madison, WI) and, after initial feeder-based expansion on a layer of mitotically inactive human fibroblasts (D551), routinely cultured under feeder-free conditions on Synthemax II-SC substrate (Corning)-coated cell culture plates (as described previously [46 (link)]).

Adult human retinal pigment epithelial (ahRPE) cells were obtained from The Eye Bank for Sight Restoration (New York, NY, USA). Primary RPE cells were isolated from one donor with written informed consent for research purpose signed by the next of kin. A cryovial of 5.0 x 10⁵ cells was shipped to the Veneto Eye Bank Foundation in dry ice. After arrival, the cryovial was thawed in a 37°C water bath for 3 minutes. Thawed cell suspension was centrifuged at 259 x g for 5 minutes. The cell pellet was re-suspended in base medium for ahRPE cells containing DMEM/F12 and αMEM solution with 2% FBS plus additional supplements [18 (link)]. For initial cell thawing and plating, base medium was supplemented with FBS to achieve a final concentration of 10% (v/v) FBS. The cells where then plated in Synthemax^™ II-SC Substrate (Corning)-coated 24-well culture plate (Corning) at a density of ~ 1.0 x 10⁵/1.9 cm² and maintained in a humidified incubator kept at 37°C in 5% CO₂. The media was changed every 4^th day. Cells were cultured for a minimum of 4 weeks. For gene expression analysis, ahRPE cells were plated either on dDM unfolded over PET TC inserts with 1.0 μm pore size, 12-well format (6.0 x 10⁵ cells/dDM) or Synthemax^™ II-coated Sarstedt transwell inserts 1.0 μm pore size, 24-well format (2.0 x 10⁴ cells/insert).

The human embryonal carcinoma NTERA-2 (NT2) cell line was obtained from ATCC. NT2, 293T, and HeLa cells were cultured in DMEM high glucose with GlutaMAX (Invitrogen) supplemented with 10% fetal bovine serum (FBS; HyClone, Piscataway, NJ). Large scale culture of NT2 cells were described (Fong et al., 2011 (link)). Mouse ES cell line D3 was purchased from ATCC (Manassas, VA) and adapted to feeder-free condition as described (Fong et al., 2011 (link)). Differentiation of D3 cells was induced by maintaining cells in LIF-free ES cell medium containing 2–5 mM all-trans retinoic acid (Sigma Aldrich) for up to 7 days. Human ES cell line H9 (WiCell, Madison, WI) was maintained in feeder-independent conditions, using Synthemax SC-II Substrate (Corning) and grown in TeSR-E8 (Stemcell Technologies, Canada). Media was changed daily and cell cultures were passaged using Dispase (Stemcell Technologies), according to the manufacturer's protocol.