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bioRxiv · 10.64898/2026.08.05.743094

A robust approach for preserving and sectioning fragile 3D spheroids for high-quality histological analysis

Cervantes-Rivera, R.·Figueroa Ortiz, S. J.·Romero Rosas, A. Z.·Sanchez Orozco, A.·Herrera-Vargas, M. A.·Melendez-Herrera, E.·Lopez-Rodriguez, M.·Ochoa-Zarzosa, A.·Lopez-Meza, J. E.
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Abstract

Three-dimensional (3D) spheroid models have become essential in cancer biology, drug screening, and tissue engineering. However, their small size, fragile structure, and tendency to disintegrate during routine histoprocessing present persistent technical challenges. Conventional paraffin embedding often results in tissue fragmentation, loss of spatial orientation, and poor section quality, whereas cryosectioning often compromises cellular morphology. Here, we present a robust, cost-effective protocol for preserving and sectioning fragile 3D spheroids, resulting in high-quality histological sections with intact architecture and excellent cellular detail. The method involves optimized handling and embedding procedures that stabilize spheroids during standard formalin fixation, paraffin infiltration, and microtomy, eliminating mechanical distortion and preserving spherical integrity for consistent sectioning. We demonstrate successful application across different cell line spheroids, with subsequent compatibility with hematoxylin and eosin (H&E) staining protocols. Compared to conventional methods, our approach significantly reduces sample loss, improves inter-section reproducibility, and preserves fine structural features such as necrotic cores, proliferative zones, and extracellular matrix components. This protocol provides a reliable, accessible solution for routine histological analysis of fragile 3D spheroids, facilitating more accurate morphological and molecular assessment in translational research settings. Key featuresO_LIMaintains spheroid integrity: Prevents mechanical distortion, fragmentation, and loss of spatial orientation during processing. C_LIO_LISignificantly reduces sample loss: Decreases failure rate compared to traditional methods, conserving valuable samples. C_LIO_LIBroad spheroid compatibility: Works effectively with primary tumor-derived, stem cell-derived, and co-culture spheroid models. C_LIO_LIEnables high-quality sectioning and staining: Delivers consistent, reproducible sections that are fully compatible with H&E, IHC, and IF. C_LI Graphical overview O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/743094v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1049254org.highwire.dtl.DTLVardef@122e817org.highwire.dtl.DTLVardef@b37b1corg.highwire.dtl.DTLVardef@15775e2_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Cervantes-Rivera, R., Figueroa Ortiz, S. J., Romero Rosas, A. Z., Sanchez Orozco, A., Herrera-Vargas, M. A., Melendez-Herrera, E., Lopez-Rodriguez, M., Ochoa-Zarzosa, A., Lopez-Meza, J. E.. 2026-08-11. A robust approach for preserving and sectioning fragile 3D spheroids for high-quality histological analysis. https://doi.org/10.64898/2026.08.05.743094

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