LAUSR

Simple Summary Cancer spheroids are reproducible and relevant multicellular in vitro preclinical models. Thus, their use is required more and more for drug development processes in oncology in order to improve the prediction of anticancer drugs responses. Moreover, spheroid models allow for the reduction in animal experimentation, in accordance with the rule of Reduce, Refine, Replace (3Rs). In order to optimize and extend the use of these spheroid models, this works was focused on the development of an original methodology to keep these cancer spheroids in the long term. This innovative concept is based on a cold storage for up to 7 days of Triple-Negative Breast Cancer (TNBC) spheroids cultured in the synthetic serum-free OptiPASS® culture medium. Major spheroid characteristics could be preserved with this new conservation method, allowing their use in high throughput screening tests. Abstract Cancer spheroids are very effective preclinical models to improve anticancer drug screening. In order to optimize and extend the use of spheroid models, these works were focused on the development of a new storage concept to maintain these models in the longer term using the Triple-Negative Breast Cancer MDA-MB-231 spheroid models. The results highlight that the combination of a temperature of 4 °C and oxygen-free conditions allowed the spheroid characteristics of OptiPASS® serum-free culture medium to preserve the spheroid characteristics during 3-, 5- or 7-day-long storage. Indeed, after storage they were returned to normal culture conditions, with recovered spheroids presenting similar growth rates (recovery = 96.2%), viability (Live/Dead® profiles) and metabolic activities (recovery = 90.4%) compared to nonstored control spheroids. Likewise, both recovered spheroids (after storage) and nonstored controls presented the same response profiles as two conventional drugs, i.e., epirubicin and cisplatin, and two anti-PARP1 targeted drugs—i.e., olaparib and veliparib. This new original storage concept seems to induce a temporary stop in spheroid growth while maintaining their principal characteristics for further use. In this way, this innovative and simple storage concept may instigate future biological sample preservation strategies.