Cell Culture
Figure 2
Live (green) and dead (red) stained 96-hour mono- and
co-culture A549 and fibroblast spheroids. Spheroids were exposed to high (34.5µM) and low (27.6µM) doses of
doxorubicin or vehicle control (no doxorubicin) for 48 hours in Corning 384-well spheroid microplates. Fibroblast (FB) monoculture displayed the most intense live staining upon low dose exposure to
doxorubicin, while A549 monoculture showed
increased cell death. The 9:1 ratio of FB to A549 cells also displayed a protective effect at the low-dose doxorubicin exposure. All cell types
displayed significant toxicity after high dose exposure. Images captured using an
EVOS fluorescent microscope at 10x objective. Scale bar = 400µm
microplate. Cell viability was assessed using CellTiter-Glo 3D cell viability assay and the lumi- nescent signal from samples were plotted as per- cent compared to vehicle control to generate dose response curves (Figure 3). Toxicity potency
(TC50) values were calculated and compared across the spheroid culture conditions (Table 1). Although the presence of FB and immune cells did not affect the potency of compounds carboplatin and cisplatin to A549 lung tumour cells, a right- shift in the potency of several other known chemotherapeutics was observed, demonstrating the protective effects of the presence of other cell types such as FB in a co-culture spheroid.
Co-culture with endothelial cells In this study, an epithelial tumour cell line was cultured in 96-well Corning spheroid microplates with and without the presence of GFP-expressing mouse endothelial C166 cells. Tumour spheroid viability was screened after treatment with vari- ous chemotherapeutics in both single tumour cell line culture and endothelial co-culture conditions. Dose-dependent responses of selected chemother- apeutics were compared, demonstrating the importance of including multiple cell types in 3D assays to more closely mimic the in vivo tumor microenvironment.
44
Spheroid formation HT-29 (ATCC® Cat. No. HTB-38) and C166-GFP (ATCC Cat. No. CRL-2583) were cultured in 2D format following ATCC’s protocols. Confluent cells were harvested and seeded into 96-well spheroid microplates (Corning Cat. No. 4520) at 2,000 total cells per well either in mono-culture or in co-culture with a seeding ratio of 1:1. Cells were seeded in 100µL per well of culture medium (IMDM [Corning Cat. No. 10-016-CM] supple- mented with 10% FBS [Corning Cat. No. 35-010- CV]). Cells were cultured in a humidified 37˚C,
5% CO2 incubator for 24 hours for the formation of a spheroid in each well. After 24 hours the spheroids were overlaid with 100µL per well of 1.76mg/mL Corning Matrigel matrix (Corning Cat. No. 354234) diluted in culture medium. The Corning spheroid microplates were centrifuged at 220 x g at 4˚C for two minutes and returned to a humidified 37˚C, 5% CO2 incubator for four days.
Cell viability assays
Five days after cell seeding, 50µL per well of com- pounds in culture medium containing 0.5% dimethyl sulfoxide (DMSO) (Corning Cat. No. 25- 950-CQC) were added. 50µL of culture medium containing 0.5% DMSO was used as vehicle con- trol. Spheroids were cultured for an additional 48
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