Biology subjects

Lopez-Meza, J. E.

Publications and source records attributed to Lopez-Meza, J. E..

3 recordsLinked to original sources

bioRxiv2026

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

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

cell biology↗

bioRxiv2026

A universal resazurin-based viability assay for prokaryotic and eukaryotic cells in 2D and 3D cultures

In vitro cytotoxicity assessments frequently rely on staining-based methods that indirectly estimate viable cell numbers indirectly. A major limitation of many such techniques is their endpoint nature, requiring cell lysis or irreversible processing that precludes longitudinal monitoring of cellular responses following treatment. An ideal assay for evaluating cell viability and proliferation should be simple, rapid, cost-effective, reproducible, and highly sensitive, while also enabling accurate quantification with minimal interference from test compounds. The resazurin reduction assay satisfies these criteria, offering a sensitive and economical alternative to conventional tetrazolium-based methods. Although both assay types depend on the metabolic reduction of a dye by viable cells, they differ mechanistically. Tetrazolium salts (e.g., MTT) are reduced by cellular dehydrogenases to insoluble formazan crystals that require solubilization before to detection. In contrast, resazurin--a cell-permeable, non-fluorescent blue dye--is reduced to resorufin, a highly fluorescent compound detectable without additional processing steps. This property renders the resazurin assay broadly applicable to viability testing in eukaryotic cells cultured in both 2D and 3D formats, as well as in bacterial systems. Here, we present a streamlined, universal protocol for implementing the resazurin reduction assay across diverse experimental models, emphasizing its practicality, reproducibility, and adaptability for real-time viability monitoring. Key featuresO_LIReal-time, non-destructive monitoring: Enables longitudinal studies by allowing repeated measurements of the same samples over hours without toxicity or disruption. C_LIO_LIStreamlined workflow: A simple "add-incubate-read" protocol eliminates the need for cell lysis, washing, or extraction, saving time and reducing variability. C_LIO_LIBroad sample compatibility: Versatile and reliable for use with 2D monolayers, 3D spheroids, organoids, and bacterial cultures. C_LIO_LIHigh sensitivity: Fluorescent detection of resorufin provides exceptional sensitivity, enabling accurate quantification of even small viable cell populations. C_LIO_LILow background and minimal interference: A clean fluorescent readout reduces the risk of signal artifacts, offering a more reliable alternative to traditional colorimetric assays. C_LIO_LICost-effective and accessible: Utilizes standard laboratory plate readers and commercially available reagents, making it an economical choice for any lab. C_LIO_LIScalable for high-throughput screening: Easily adaptable to various plate formats, supporting both small-scale experiments and large-scale automated screening applications. C_LI Graphical overview O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/718248v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@82bcecorg.highwire.dtl.DTLVardef@14164aforg.highwire.dtl.DTLVardef@395118org.highwire.dtl.DTLVardef@fb1349_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

A simple and cost-effective method for generating spheroids from triple-negative breast cancer cell line (MDA-MB-231)

Breast cancer (BC) is the most frequently diagnosed malignancy in women and a leading cause of cancer-related mortality worldwide. Molecular classification based on estrogen receptor (ER), progesterone receptor (PR), HER2, and Ki67 expression guides prognosis and therapy, with triple-negative breast cancer (TNBC)--lacking ER, PR, and HER2 representing 15-20% of cases. TNBCs aggressive behavior, early recurrence, and limited treatment options underscore the need for improved models to develop targeted therapies. While monolayer (2D) cultures have advanced cancer research, they poorly replicate the three-dimensional (3D) tumor microenvironment (TME), leading to translational gaps. 3D spheroids address these limitations by recapitulating cell-cell/matrix interactions, metabolic gradients, and hypoxic cores, offering a physiologically relevant platform for studying metastasis, drug resistance, and therapeutic screening. Here, we present a simple, cost-effective method for generating spheroids. This protocol is applicable across different cell types, bridging the gap between traditional 2D cultures and in vivo studies. 3D cell culture opens the door to personalized medicine and drug discovery. Graphical overview O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/694917v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1b11259org.highwire.dtl.DTLVardef@2bc537org.highwire.dtl.DTLVardef@1a0c4f2org.highwire.dtl.DTLVardef@1e213ae_HPS_FORMAT_FIGEXP M_FIG C_FIG Key featuresO_LIEmploys a cost-effective, lab-made agarose coating to create ultra-low attachment surfaces in standard 96-well plates. C_LIO_LISpecifically optimized for generating consistent spheroids from the aggressive MDA-MB-231 triple-negative breast cancer cell line. C_LIO_LIGenerates measurable spheroids within 96 hours using only basic cell culture equipment and an orbital shaker. C_LIO_LIProvides a clear workflow from spheroid formation to quantitative size analysis using freely available (ImageJ) and common (GraphPad Prism) software. C_LI

cell biology↗