Peg dithiol

3D hydrogels were prepared with a 20 kDa 4-arm PEG-MALeimide (PEG-MAL, Jenkem Technology, Plano, TX) at 10 wt % solution with 2 mM of cell adhesion peptide RGD (see the Supporting Information) and cross-linked at a 1:1 ratio with 1 kDa linear PEG-dithiol (Sigma-Aldrich) in sterile 2 mM triethanolamine (pH 7.4). Briefly, the MCTS pellet obtained from each method was resuspended in the PEG-RGD solution, and casting of hydrogels was done by mixing PEG-RGD-MCTS with the cross-linker at a ratio of 10:1. One microliter of the cross-linking solution was placed on the bottom of the plate, and then 9 µL of PEG-RGD-MCTS solution was added with vigorous mixing. Volumes of the hydrogels were limited to 10 µL to avoid oxygen and nutrient diffusion limitations. Gelation proceeded for 5 min^{41 (link)} at 37 °C to ensure complete polymerization before the addition of culture medium. MCTS from the three MCTS formation methods were transferred to 3D PEG-MAL hydrogels, by using similar seeding densities. PolyNIPAAM MCTS were encapsulated at a ratio of one 150 µL polyNIPAAM gel to nine 10 µL PEG-MAL hydrogels, microwell MCTS at a ratio of 4 cm², 1 mL to nine 10 µL PEG-MAL hydrogels, and suspension MCTS at a ratio of 9 cm², 3 mL to nine 10 µL PEG-MAL hydrogels. MCTS created via either polyNIPAAM, microwells or suspension were transferred with cut pipet tips to minimize shear stress.

Hyaluronic acid (60,000 Da, Genzyme Corporation, Cambridge, MA). Adipic dihydrazide, NHS-acrylate, acryloyl chloride, triethylamine, N-boc-ethylenediamine, 4-(4,6-dimethoxy[1 (link),3 (link),5 (link)]triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), vanillin, potassium carbonate, ethyl-4-bromobutyrate, dimethylformamide (DMF), nitric acid, ethanol, sulfuric acid, trifluoroacetic acid, methanol, and acetic acid were purchased from Fisher Scientific and used without purification. Picoline borane complex and PEG dithiol (MW 1000 Da) were purchased from Sigma Aldrich and used without purification. Ac-FKGGGERC-NH₂ (K peptide), Ac-GCRDGPQGIWGQDRCG-NH₂, NQEQVSPLRGDSPGNH2 (Q-RGD peptide), and FITC-labeled NQEQVSPLRGDSPG-NH2 were purchased from Genscript (Piscataway, NJ) and used without purification. 4-arm PEG-vinylsulfone (PEG-VS, MW 40kDa) and 4-arm PEG-maleimide (PEG-MAL, MW 20kDa) were purchased from JenKem Inc. and used without further purification.