OpenTox 2022 Virtual Conference

Spheroids production for toxicology evaluation of chemical substances Angie Castro*, Dayan Guerra, Andrés Pareja 

ABSTRACT:

Two-dimensional (2D) cell cultures, are used as an alternative for experimental animals in toxicity tests, but to approximate a biological system, 3D cultures have been developed, allowing scientists obtain biological relevance responses. This let to explore a variety of  strategies permitting to establish an optimal methodology in spheroids production, creating  different materials like hanging drop plate or low-attachment plate. Though these materials  make the spheroids production easier, their purchase is not as affordable as a traditional  microplate. Keratinocytes spheroids (HEK 001) were obtained using two different  techniques: hanging drop and low-attachment surface adding low-melting point agarose (1,5% w/v) in the well, then taken the plate for centrifugation, this helps spheroids  formation. The lactate dehydrogenase test was carried out using a commercial kit of LDH  (MAK006 – Lactate Dehydrogenase Activity Assay Kit), in monolayer and 3D cultures  (spheroids), this made possible a comparison between cell cultures. Spheroids were  successfully obtained, when using the low-attachment technique, taking into consideration HEK 001 low capacity for spheroids formation while using hanging drop technique, making  it viable to get spheroids in less time (3 – 4 days) unlike hanging drop (> 7 days). LDH test,  showed comparable results between monolayer and 3D culture, according to specific  characteristics of each technique. It is remarkable, the application of a low-cost technique  that permits spheroids obtention, helping to reach a deeper understanding in biological  behavior of 3D culture, that will improve toxicological tests.