Biology subjects

Ratti, F.

Publications and source records attributed to Ratti, F..

3 recordsLinked to original sources

bioRxiv2025

3D printing and bioprinting for miniaturized and scalable hanging-drop organoids culture

Three-dimensional (3D) cell culture systems rely on the manipulation of a biologically derived matrix, typically soluble Basement Membrane Extract (sBME), in which cells or cellular aggregates, such as organoids, are suspended. This matrix provides mechanobiological support, promoting cellular processes. However, the handling of sBME-based matrices containing cellular constructs poses significant challenges due to their rheological properties. We developed an integrated bioprinting system to surpass the conventional pipetting, seeding and culture in multiwell plates. The system combines a fluidic cartridge with innovative 3D-printed biocompatible culture tools designed to host and preserve high-throughput microcultures of Patient-Derived Organoids (PDOs) in sBME. The miniaturized hanging-drop configuration enables extended culture periods and high-throughput imaging screenings. This comprehensive approach overcomes common issues associated with sBME, including sedimentation of cellular aggregates, premature gelation, and structural collapse, which negatively impact culture quality and reproducibility throughout the entire 3D culture workflow, from seeding to culture maintenance, and post-culture analyses. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/678315v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@19f976eorg.highwire.dtl.DTLVardef@8eeefcorg.highwire.dtl.DTLVardef@1ebe6c7org.highwire.dtl.DTLVardef@7c4403_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- Miniaturized 3D hanging-drop matrix-embedded organoid culture in a 384-well plate - Custom cartridge enables homogeneous bioprinting of organoids in sBME-based matrix - 3D-printed tools support compact, scalable multiwell culture systems - System suited for miniaturized culture organoids for high-throughput drug screening - Scalable miniaturized culture system for extended periods of time

bioengineering↗

bioRxiv2025

Macroscopic Analyses of RNA-Seq Data to Reveal Chromatin Modifications in Aging and Disease

Regulation of gene expression is fundamental for proper cellular function, and is constrained by the local chromatin environment of each gene, which varies spatially along the chromosome and is shaped by epigenetic modifications. Epigenetic modifications induce changes in the local chromatin structure, which can influence gene expression, by affecting the accessibility of DNA to transcription factors. Such changes are particularly relevant in aging and genetic disorders like Hutchinson-Gilford Progeria Syndrome (HGPS) and Werner Syndrome (WRN), where altered chromatin structure contributes to disease pathology. In this study, we analyze RNA-seq data using macroscopic metrics designed to be explicitly sensitive to chromatin modifications. The first metric, intra-chromosomal gene correlation length, measures spatial correlations in gene expressions along the chromosome. The second metric employs an energy landscape model based on the Arrhenius equation to estimate the energetic barriers associated with chromatin state transitions. We apply these metrics to various aging-related datasets, demonstrating their sensitivity to changes in the chromatin structure and the interpretability of the resulting outputs. The intra-chromosomal gene correlation length is particularly effective in quantifying changes in RNA-seq profiles due to increased chromatin accessibility during aging (and conversely, reduced accessibility due to treatment). This metric not only accurately distinguishes cell states, but also provides insight into the direction of aging. For instance, our observations on the effects of anti-sense oligonucleotide (ASO) treatment align with the existing literature, demonstrating that ASO partially restores chromatin structure in diseased cells. They additionally quantify the more pronounced effects in HGPS compared to WRN. The barrier energy landscape further extends this capability by offering a framework for understanding the progressive degradation of the regulatory mechanisms. Together, these metrics provide robust screening tools that enhance our ability to exploit common measurements such as RNA-seq to derive new phenotypes such as chromatin dynamics on aging and disease, offering an alternative perspective that complements traditional analytical techniques and enriches our understanding of cellular states.

bioinformatics↗

bioRxiv2024

Development of a high-throughput 3D culture microfluidic platform for multi-parameter phenotypic and omics profiling of patient-derived organoids

Patient-derived organoids (PDOs) are poised to become central tools both in clinical practice, to preemptively identify patient optimal treatments, and in drug discovery, overcoming the limitations of cancer cell lines. However, the use of PDOs in these settings has been hampered by several bottlenecks, including sample requirements, assay time, and handling in the context of high-throughput assays. We developed a Microfluidic Platform for Organoids culture (MPO) that miniaturises and simplifies PDOs cultures in a 384-plate format. Both retrospective and prospective clinical studies demonstrate MPO predictive value and the straightforward implementation in the clinical setting. MPO allows subcellular phenotypic screenings, as imaging-based applications like Cell Painting, target engagement analyses, alongside the comprehensive definition of PDOs genomic, transcriptomic, proteomic, lipidomic, and metabolomic landscapes. Harnessing the pleiotropic capabilities of MPO, we uncovered the role of EZH2 inhibitors to prevent the long-term emergence of resistance to RAS inhibitors in metastatic colon cancer. In all, we demonstrate the potential of MPO to impact clinical practice, alongside exploration of the mechanisms underlying compound response and resistance.

cancer biology↗