23.2.7 Surface-Initiated Activator Generated by Electron Transfer Atom Transfer Radical Polymerization (SI-AGET ATRP) of OEGMEMA The macromonomer OEGMEMA (8 g, 16.7 mmol), water (7.5 mL), methanol (7.5 mL), copper(II) bromide (0.08 mmol, 17.9 mg) and 202-Bipyridine (0.24 mmol, 37.5 mg) were added to a Schlenk flask, sealed, and stirred for 30 min. Then, the flask was degassed with three freeze-vacuum-thaw cycles, and backfilled with nitrogen. Next, the mixture was transferred by syringe to a reaction flask containing micropatterned glass slides under vacuum. To start the reaction, L-ascorbic acid (0.8 mmol, 140.9 mg) in deionized water was injected into the flask. The reaction continued for 16 h to generate micropatterned thick PEGMEMA brushes. Polymerization was stopped by adding air, and the slides were rinsed with ethanol, water, and ethanol sequentially before drying under nitrogen. Prior to cell seeding, the slides were washed with ethanol for 2 h. 23.2.8 Adsorption of Adhesion Ligands Once the glass substrates were fabricated, they were coated with DMEM (Life Technologies) containing 83μg/mL Matrigel (WiCell) overnight at 37 C. Since PEGMEMA is bioinert, the Matrigel could only adhere to the gold-coated feature regions on the glass slides. For PDMS substrates, a PDMS mold with the intended features was coated overnight at 37 C with DMEM containing 83μg/mL Matrigel. Afterwards, the Matrigel-coated PDMS mold was dried with N2 and placed on top of a sacrificial polyvinyl alcohol (PVA) film [11]. After 1 h, the mold was removed and the PVA film was placed on top of the PDMS substrates in such a way that the Matrigel was sandwiched between the PVA film and the PDMS substrate. The PVA film plus PDMS substrate was incubated at 37 C for 20 min and then washed with PBS to dissolve the PVA film but still leave the Matrigel lanes on the PDMS substrates. The remaining areas between Matrigel lanes on the PDMS substrates were backfilled with Pluronic F127 (Sigma-Aldrich) for an additional hour to prevent cell outgrowth from the lanes. Both the glass slides and PDMS substrates were rinsed with PBS prior to cell seeding. 23.2.9 HESC-CM Seeding and Culture After zeocin purification, the pure population of hESC-CMs was washed with PBS and dissociated from their original culture dish via 5 min exposure to 0.25 % Trypsin (Life Technologies). Trypsin was used to ensure break up of the significant amount of cell–cell binding and extracellular matrix deposits that formed in these cultures during the differentiation process. These cells, referred to as pre-seed, were then seeded into new Matrigel-coated wells using 20 % serumcontaining media. After 10 days the pre-seed hESC-CMs were again exposed to 0.25 % Trypsin for 5 min and then seeded onto the micropatterned substrates using 20 % serum-containing media. Prior experimentation had resulted in better isolation of single cells using the pre-seed method, as opposed to seeding the hESC-CMs directly from the differentiation culture plates which resulted in more CM aggregates than single cells. The hESC-CMs were seeded at a density of 30,000 cells/cm2 which was enough cells to cover the substrate while avoiding excess “piling up” of seeded cells in multilayers. The following day, the media was replaced with B27-supplemented RPMI media containing 8 μg/mL Matrigel and changed every 3 days thereafter. 23.2.10 Immunofluorescence After 5 days of culture the samples were stained and imaged. The cells were washed once with PBS and then exposed to 4 % paraformaldehyde (PFA) (Electron Microscopy Sciences) for 15-min at room temperature. The cells were washed again with PBS and then treated with 0.1 % Triton (Sigma) for 6 min at room temperature to permeabilize the cell membrane. Again, the cells were washed with PBS and treated for 30 min with a blocking solution consisting of PBS, 2 % FBS, 0.1 % Triton, 11.2 mg/mL glycine, and 50 mg/mL BSA. Afterwards, phalloidin conjugated to tetramethylrhodamine B isothiocyanate (TRITC) (Sigma) was applied at a 50 mg/mL concentration to label actin filaments and DAPI was applied at a 1:1000 dilution to label nuclei. Cells were washed with PBS and transferred to coverslips, where they were mounted using ProLong 164 B.N. Napiwocki et al.
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