Figure 1. Schematic describing the overall workflow of the study. Tumor cells are expanded on culture plastic and passage with Trypsin (1). The cells are then suspended in hydrogel solution. 10mL droplets are pipetted into a well plate and crosslinked with UV light for 2 seconds (2). After 24 hours, drug treatment is started (3). Constructs are then analyzed for viability and Cx43 protein expression (4).
Previous work from our lab and others has shown that cancer cells grown in 3D hydrogels retain more properties of in vivo tumor cells (Devarasetty et al., 2020; Forsythe et al., 2019; Mazzocchi et al., 2018; Votanopoulos, Forsythe, et al., 2019; Votanopoulos, Mazzocchi, et al., 2019). For that reason, we have chosen to test the effect of the combinatorial treatments on cells grown in 3D hydrogel constructs instead of on traditional flat 2-dimension cell culture plastic. GBM tumor cells are encapsulated within the hydrogel solution, which is comprised of thiolated-HA, thiolated-gelatin, and polyethylene glycol diacrylate (PEGDA) in a 2:2:1 ratio. Six organoids were generated for each treatment group, including four organoids used for ATP assay and two for LIVE/DEAD imaging. Drug treatment proceeds for 7 days at which point, cell viability is assessed, or the samples are fixed for immunostaining. The hydrogel is also optically transparent which allows for analysis via confocal microscopy. See Figure 1 for a detailed outline of the workflow used in the studies presented here.