Biology subjects

Cervantes-Rivera, R.

Publications and source records attributed to Cervantes-Rivera, R..

5 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↗

bioRxiv2020

The Vibrio cholerae cytotoxin MakA induces noncanonical autophagy resulting in the spatial inhibition of canonical autophagy

Autophagy plays an essential role in the defence against many microbial pathogens as a regulator of both innate and adaptive immunity. Among some pathogens, sophisticated mechanisms have evolved that promote their ability to evade or subvert host autophagy. Here, we describe a novel mechanism of autophagy subversion mediated by the recently discovered Vibrio cholerae cytotoxin, MakA. pH-dependent endocytosis of MakA by host cells resulted in the formation of a cholesterol-rich endolysosomal membrane aggregate in the perinuclear region. Aggregate formation induced the noncanonical autophagy pathway driving unconventional LC3 lipidation on endolysosomal membranes. Subsequent sequestration of the ATG12-ATG5-ATG16L1 E3-like enzyme complex required for LC3 lipidation at the membranous aggregate resulted in an inhibition of both canonical autophagy and autophagy-related processes including the unconventional secretion of IL-1β. These findings identify a novel mechanism of host autophagy subversion and immune modulation employed by V. cholerae during bacterial infection.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗

bioRxiv2019

Complete genome sequence and annotation of the laboratory reference strain Shigella flexneri serovar 5a M90T and genome-wide transcription start site determination

BackgroundShigella is a Gram-negative facultative intracellular bacterium that causes bacillary dysentery in humans. Shigella invades cells of the colonic mucosa owing to its virulence plasmid-encoded Type 3 Secretion System (T3SS), and multiplies in the target cell cytosol. Although the laboratory reference strain S. flexneri serotype 5a M90T has been extensively used to understand the molecular mechanisms of pathogenesis, its complete genome sequence is not available, thereby greatly limiting studies employing high-throughput sequencing and systems biology approaches.\n\nResultsWe have sequenced, assembled, annotated and manually curated the full genome of S. flexneri 5a M90T. This yielded two complete circular contigs, the chromosome and the virulence plasmid (pWR100). To obtain the genome sequence, we have employed long-read PacBio DNA sequencing followed by polishing with Illumina RNA-seq data. This provides a new pipeline to prepare gapless, highly accurate genome sequences. Furthermore, we have performed genome-wide analysis of transcriptional start sites and determined the length of 5 untranslated regions (5-UTRs) at typical culture conditions for the inoculum of in vitro infection experiments. We identified 6,723 primary TSS (pTSS) and 7,328 secondary TSS (sTSS). The S. flexneri 5a M90T annotated genome sequence and the transcriptional start sites are integrated into RegulonDB (http://regulondb.ccg.unam.mx) and RSAT (http://embnet.ccg.unam.mx/rsat/) to use its analysis tools in S. flexneri 5a M90T genome.\n\nConclusionsWe provide the first complete genome for S. flexneri serotype 5a, specifically the laboratory reference strain M90T. Our work opens the possibility of employing S. flexneri M90T in high-quality systems biology studies such as transcriptomic and differential expression analyses or in genome evolution studies. Moreover, the catalogue of TSS that we report here can be used in molecular pathogenesis studies as a resource to know which genes are transcribed before infection of host cells. The genome sequence, together with the analysis of transcriptional start sites, is also a valuable tool for precise genetic manipulation of S. flexneri 5a M90T. The hybrid pipeline that we report here combining genome sequencing with long-reads technology and polishing with RNAseq data defines a powerful strategy for genome assembly, polishing and annotation in any type of organism.

microbiology↗