Tumor models that closely imitate in vivo conditions are becoming increasingly popular in drug discovery and development for the screening of potential anti-cancer drugs. Multicellular tumor spheroids (MCTSes) effectively mimic the physiological conditions of solid tumors, making them excellent in vitro models for lead optimization and target validation. Out of the various techniques available for MCTS culture, the liquid-overlay method on agarose is one of the most inexpensive methods for MCTS generation. However, the reliable transfer of MCTS cultures using liquid-overlay for high-throughput screening may be compromised by a number of limitations, including the coating of microtiter plates (MPs) with agarose and the irreproducibility of uniform MCTS formation across wells. MPs are significantly prone to edge effects that result from excessive evaporation of medium from the exterior of the plate, preventing the use of the entire plate for drug tests. This manuscript provides detailed technical improvements to the liquid-overlay technique to increase the scalability and reproducibility of uniform MCTS formation. Additionally, details on a simple, semi-automatic, and universally applicable software tool for the evaluation of MCTS features after drug treatment is presented.

• MeSH
• buněčné kultury metody   MeSH
• buněčné sféroidy MeSH
• HCT116 buňky MeSH
• lidé MeSH
• nádorové buňky kultivované MeSH
• reprodukovatelnost výsledků MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• audiovizuální média MeSH
• časopisecké články MeSH

Spheroid cultures of cancer cells reproduce the spatial dimension-induced in vivo tumor traits more effectively than the conventional two-dimensional cell cultures. With growing interest in spheroids for high-throughput screening (HTS) assays, there is an increasing demand for cost-effective miniaturization of reproducible spheroids in microtiter plates (MPs). However, well-to-well variability in spheroid size, shape, and growth is a frequently encountered problem with almost every culture method that has prevented the transfer of spheroids to the HTS platform. This variability partly arises due to increased susceptibility of MPs to edge effects and evaporation-induced changes in the growth of spheroids. In this study, we examined the effect of evaporation on the reproducibility of spheroids of tumor and nontumor cell lines in 384-well plates, and show that culture conditions that prevent evaporation-induced medium loss result in the formation of uniform spheroids across the plate. Additionally, we also present a few technical improvements to increase the scalability of the liquid-overlay spheroid culturing technique in MPs, together with a simple software routine for the quantification of spheroid size. We believe that these cost-effective improvements will aid in further improvement of spheroid cultures for HTS drug discovery.

• Klíčová slova
• edge effect, evaporation, liquid-overlay culture, reproducibility, spheroids,
• MeSH
• antitumorózní látky izolace a purifikace   terapeutické užití   MeSH
• buněčné kultury metody   MeSH
• buněčné sféroidy účinky léků   MeSH
• léky antitumorózní - screeningové testy metody   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• objevování léků metody   MeSH
• rychlé screeningové testy metody   MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antitumorózní látky MeSH