Cell Culture
of assessing compound action, spheroids can be readily analysed by imaging using light, fluores- cence and confocal microscopy – a major advan- tage over other, more complex, 3D cell culture models, including organ-on-a-chip approaches. As interest grows in these areas, technologies are now being developed to enable mass production of uni- formly-sized 3D spheroids, making them highly amenable for in vitro high throughput and toxicity screening applications.
Despite the advantages of spheroid culture, nonetheless, limitations of current technologies to enable simple and reliable assays have impeded the rapid adoption of 3D models for high-throughput screening (HTS). Indeed, conventional methods for 3D cell culture are time-consuming, display increased variability and lack the throughput required for automated HTS. Below, we describe applications of high-through- put screening using 3D cell culture to assess cell viability. In addition, we highlight the importance of including multiple cell types in 3D assays to more accurately assess potency of chemotherapeu- tic drug candidates.
Co-culture with cells of the microenvironment
Understanding the complex interactions between cancer cells and other cell types in the tumour microenvironment, such as fibroblasts, endothelial cells and immune cells, is critical to predict thera- peutic efficacy. This is important as many factors, including the secretion of cytokines and extracellu- lar matrices, may impact drug resistance and sensi- tisation1-3. 3D cultures develop hypoxic cores and demonstrate gradients of various soluble factors and a diffusion profile for drugs similar to tumours.
As shown in the following studies, the presence of primary cell types, as well as endothelial cells together with tumour cells, in a spheroid can affect the potency of chemotherapeutic compounds. A549 cells, a human lung carcinoma cell line, were cultured in Corning spheroid microplates with and without co-culture conditions, including primary human lung fibroblasts (FB) and peripher- al blood mononuclear cells (PBMC). Tumour spheroid viability was screened after treatment with various chemotherapeutics in both single cul- ture and co-culture conditions. Dose-dependent responses of selected chemotherapeutics were com- pared, demonstrating the impact that including multiple cell types in 3D assays can have on exper- imental results.
Mono- and co-culture spheroid formation A549 cells (ATCC® Cat. No. CCL-185) and nor- mal human lung fibroblasts (FB; Lonza Cat. No. CC-2512) were subcultured following vendors’ protocols. A549 cells were cultured in F-12K (Corning Cat. No. 10-025-CV) supplemented with 10% Fetal Bovine Serum (FBS) (Corning Cat. No. 35-010-CV) and FB were cultured in supplemented Fibroblast Basal Medium FGM-2 (FB medium) (Lonza Cat. No. CC-3132). Confluent cells were harvested and seeded into 384-well spheroid microplates (Corning Cat. No. 3830), adding 20µL of FB media with 2,000 cells per well. For chemotherapeutic dose response assays, A549 cells and FB were seeded at 2,000 cells per well in monoculture and in co-culture conditions with a 9:1 ratio of FB to A549 cells. Spheroid microplates were covered with breathable membrane sealing tape (Corning Cat. No. 3345) and pulse cen- trifuged at 130 x g prior to culture in a humidified
Table 1: Toxicity potency (TC50) values of chemotherapeutic compounds applied to mono-, co- and tri-culture spheroids
42 Drug Discovery World Winter 2017/18
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