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.