3D Spheroid Models for Drug Testing and Histological Processing with Coconut Oil: An Experimental Study of In Vitro Analysis ^†

Introduction: Three-dimensional (3D) cell cultures are laboratory models composed of cells that grow in three dimensions and interact with each other and the extracellular matrix, which results in proliferation, differentiation, morphology, and functionality conditions similar to those in living tissues and makes these platforms adequate for drug testing. The spheroid, a 3D cell culture with a sphere shape, may be analyzed by optical microscopy through histological processing, but this process uses xylene, which is associated with organic damage. This study proposes an analysis of a method for human keratinocytes (HaCaT) spheroid production, cell viability after exposure to a new pharmacological product, and histological processing with coconut oil. Methodology: HaCaT cells were grown at 37 °C and 5% CO₂, transferred to a 96-well agarose-coated U-shaped-bottom microplate at agitation, and the spheroids were formed in seven days. Some spheroids were exposed to the new product at concentrations that varied from 1:25 to 1:3200, and their viability was observed through luminescence. The rest of them were fixed in 10% formalin, dehydrated in ethanol solutions, cleared in coconut oil, embedded in paraffin, sectioned, and stained in hematoxylin and eosin. Results: The spheroids demonstrated reproducibility and cell viability after the exposure to the new product. Histological analysis revealed regular cell clusters, compact formations, distinct borders, and morphology resembling that of skin epithelium. Conclusions: The spheroids were trustworthy models for preclinical testing. The new product was safe for HaCaT cells. The coconut oil was an adequate clearing agent.