Optimization of agarose–gelatin double embedding to minimize sectioning artifacts in multicellular spheroids

Processing methods that do not induce shape distortion are essential for the analysis of tissue morphology. Although the use of agarose–gelatin double embedding to reduce shape distortion has been suggested, the effect of matrix concentration has not been addressed. Therefore, combinations of agarose (1–3%) and gelatin (1–10%) were evaluated using multicellular spheroids without extracellular matrix as a model of mechanical fragility. In this study, a blend of 2% agarose and 5% gelatin effectively prevented distortion. This protocol can enhance the morphological analysis of delicate tissues. Multicellular spheroids with poor mechanical strength can be damaged during histological processing. Although placing tissues in support materials is a known countermeasure, optimal conditions have not been defined. Changes in agarose and gelatin concentration conditions affected tissue distortion in this study. The optimized hydrogel blend preserves tissue architecture, thereby improving the quality of microscopic analysis.