Peg mal

To form the hydrogel network, 2, 4, or 8-arm PEG-VS, PEG-Ac and PEG-Mal (JenKem Technology, Beijing, China) macromer powder was dissolved in sterile Dulbecco’s Phosphate Buffered Saline(DPBS) (pH 7.4, Gibco, USA) solution and 0.4 mg/100μL of photo-initiator Irgacure 2959 (Ciba, Basel, Switzerland, MW=224.3) to create a solid concentration of 5% w/v. Furthermore, a co-monomer, N-vinyl-2-pyrrolidone (NVP) (Sigma-Aldrich, USA), was added in some compositions at a final concentration of 0.1%, as NVP has been shown to enhance the gelation mechanism without impacting the cell compatibility [35 (link)]. To form the gels, the precursor solution (PEG, Irgacure 2959 and NVP (in some compositions)) was exposed to UV light at a constant intensity (1090 μW/cm² at a distance of 4 cm) for a designated duration.

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.