At a desired time point, culture media were removed, and gel discs containing 3D encapsulated cells were washed with HBSS twice. Gel digestion and cell lysis were done simultaneously using 50 mM EDTA in ribonuclease (RNase)–free water for less than 1 min. The samples were then directly added to TRIzol (Thermo Fisher Scientific). Chloroform (200 μl) was added per 1 ml of TRIzol to perform phase separation, followed by centrifugation for 15 min at 12,500 rpm, 4°C. The top layer was collected to a new tube, and RNA was precipitated with 250 μl of isopropanol and 250 μl of solution of 0.8 M sodium citrate and 1.2 M sodium chloride for at least 15 min at 4°C. Samples were then centrifuged at 12,500 rpm for 15 min at 4°C. The supernatant was removed, and the precipitated RNA was washed with 75% EtOH, followed by centrifugation for 5 min at 7500 rpm, 4°C. EtOH was then removed, and the purified RNA was resuspended in 15 μl of RNase-free water. RNA concentration was quantified by the NanoDrop spectrophotometer. Complementary DNA (cDNA) was reverse transcribed by SuperScript-III reverse transcriptase (Thermo Fisher Scientific). Quantitative polymerase chain reaction (qPCR) was performed in the ViiA7 qPCR system with Power SYBR Green master mix (Applied Biosystems). For each qPCR experiment, samples were analyzed in triplicates with 50 ng of cDNA per well. Relative gene expression was computed by the delta-delta Ct method by comparing Ct values to reference gene (GAPDH). See table S1 for the list of primers for qPCR.

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