细胞生物学

Skin models play critical roles in understanding disease mechanisms and advancing therapeutic development. However, conventional systems based on 2D cell cultures, in vivo animal models, and ex vivo tissue explants are limited by insufficient physiological complexity, interspecies differences, and restricted accessibility, respectively. Advances in biofabrication technologies have enabled the engineering of 3D skin equivalents that better balance biological complexity and experimental scalability. Here, we present a biofabrication protocol inspired by the regenerative processes of wound healing to construct vascularized 3D organotypic skin models in a stepwise manner. The approach integrates bioprinting for precise spatial organization of cellular compartments with guided cell self-organization to achieve native-like tissue complexity and heterogeneity. Through a programmable culture strategy, tissue maturation proceeds sequentially through keratinocyte proliferation and collective migration, microchannel endothelialization, basal-to-suprabasal differentiation, and progressive extracellular matrix remodeling within a fibrin-based scaffold. The resulting tissue constructs comprise stratified epidermal layers positioned atop a vascularized, fibroblast-remodeled dermal matrix. Beyond reproducing key structural features of human skin, this protocol recapitulates cellular processes associated with tissue regeneration, providing a dynamic platform for investigating disease pathogenesis, progression, and therapeutic responses.

The airway epithelium interfaces with the external environment through its apical surface and with the extracellular matrix (ECM) through its basolateral surface. To model this organization in vitro, we developed a decellularized ECM-incorporated apical-out airway organoid (dECM-AoAO) platform in which human bronchial epithelial cells (HBECs) self-assemble around human lung-derived decellularized ECM microparticles (dECM-MPs). This configuration preserves apical-out polarity while enabling direct epithelial–ECM interactions. Here, we describe a protocol for the vacuum filtration and quantification of dECM-MPs, the generation of dECM-AoAOs, and ultimately, whole-mount immunofluorescence staining for organoid characterization.

Breast cancer is the most frequently diagnosed cancer in women, representing approximately 25% of all cancers in women worldwide. Both breast cancer research and histopathological diagnostics mainly show a two-dimensional planar view of the three-dimensional breast cancerous architecture. Recently, the application of optical tissue clearing, together with 3D microscopy, has been applied to visualize the complexity of whole tumor samples. Preliminary studies on whole-organ mouse mammary glands and tissues from human breast cancer patients subjected to optical tissue clearing and volumetric imaging have enabled the detection of previously unrecognized spatial cellular interactions and structural features within intact breast tissue. There is currently no standardized clearing workflow for breast and lymph node tissues. In this protocol, we optimized and validated the MASH (multiscale architectonic staining of human cortex) immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO)-like clearing and labeling pipeline for the investigation of formalin-fixed and paraffin-embedded (FFPE) breast tissue and lymph nodes obtained from breast cancer patients. This illustrates the application of the protocol in a new biological and clinical context, as human breast and lymph node tissues differ substantially from brain tissues in their composition, architecture, and optical properties. Whole FFPE tissue blocks are deparaffinized in liquid paraffin and xylene, bleached through methanol dehydration and a subsequent hydrogen peroxide incubation, and stained with a diverse set of small molecule dyes. As a next step, the tissues are delipidated and subjected to refractive index matching with ethyl cinnamate to reach optimal tissue transparency. Importantly, the applied dehydration and delipidation nicely preserve the morphology of the tissue, and the shrinkage is minimal. This allows reliable 3D imaging of large tissue samples within a timeframe of 10 days, providing clinicians and biomedical researchers with a more holistic view of the FFPE tissue sample and its spatial organization.

Mosaic animals are highly valuable for investigating complex biological processes and cell lineages in vivo. Traditional mosaic techniques in Drosophila, such as the FRT/Flp system, rely on exogenous site-specific recombination sequences, preventing their application to unmodified mutant chromosomes or wild-derived strains. Mosaic analysis by gRNA-induced crossing-over (MAGIC) overcomes this limitation by utilizing the CRISPR/Cas9 system to generate targeted double-strand breaks (DSBs) that induce somatic homologous recombination in precursor cells. Here, we describe a comprehensive protocol for applying MAGIC with a newly developed, genome-wide MAGIC kit. This protocol utilizes optimized gRNA-markers with the Qtg2.1 scaffold for high-efficiency clone induction, alongside improved fluorescent labeling strategies for both positive MAGIC (pMAGIC) and negative MAGIC (nMAGIC). The procedure details the genetic crossing schemes, temporal induction of clones, and tissue processing for diverse Drosophila cell types. This method enables convenient mosaic analysis across all chromosomes and allows for the study of pericentromeric genes, deficiency chromosomes, and species-specific alleles in interspecific hybrids.

Calcium signaling is a universal, versatile process in which ionized or free calcium (Ca²⁺) acts as a second messenger to regulate various cellular activities, including hormone secretion, contraction, proliferation, gene expression, and apoptosis. Changes in the cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_cyt) in hepatocytes play a central role in mediating the actions of insulin, glucagon, catecholamines, and other hormones on carbohydrate, lipid, and protein metabolism in the liver. Ratiometric chemical Ca²⁺ indicators are fluorescent dyes that change their emission or excitation spectrum upon binding to calcium, allowing for precise, quantitative measurements of changes in the intracellular Ca²⁺ concentration. They enable calibration by calculating the ratio of two fluorescence intensities, correcting for artifacts such as uneven dye loading, photobleaching, and cell volume variations. Fura-2 acetoxymethyl ester (AM) (hereinafter referred to as Fura-2), a ratiometric and sensitive indicator dye, is a popular fluorescent Ca²⁺ reporter for measuring intracellular calcium. Here, we describe a comprehensive and detailed protocol for Ca²⁺ imaging of the H4IIE cell line and primary rodent hepatocytes in vitro via the chemical reporter Fura-2, which can also be employed on a wide variety of cell types. Unlike previously published protocols, this protocol addresses the challenge of facilitating the attachment of liver cell lines and primary hepatocytes to glass coverslips for imaging using an inverted fluorescence microscope. Our protocol describes two different loading/labeling strategies for Fura-2 dye: one is cost-effective but requires skillful pipettor handling, and the second one is easy but expensive as it needs a large volume of Krebs-Ringer HEPES (KRH)-Fura-2 solution. If the coverslips are handled properly, the cost-effective coverslip-only loading approach produces similar quality results as the large volume method. Finally, we describe a simple and user-friendly procedure to analyze Ca²⁺ signals over time using Microsoft Excel’s functional equations.

Acid ceramidase (aCDase) is a lysosomal amidase that catalyzes the hydrolysis of sphingolipids (SphL), including ceramides and glucosylceramides. Altered expressions of aCDase are associated with several pathological conditions, such as cancer, inflammation, pain, and pulmonary disorders. aCDase activity is reduced in Farber disease, spinal muscular atrophy with progressive myoclonic epilepsy, diabetes, and cardiovascular disease. Recent reports suggest that aCDase inhibition may be an emerging strategy for treating several SphL-related neurodegenerative conditions, such as Krabbe, Gaucher, and Parkinson’s disease, due to its role in the accumulation of glycosphingolipids. Therefore, the development of a tissue-based aCDase activity assay has potential applications in clinical diagnostics and drug discovery, enabling the evaluation of the onset and progression of disease from biological samples of patients, drug-target engagement analysis, and identification of biomarkers. Here, we report a detailed protocol for detecting aCDase activity in tissue lysates, using Rbm14-12 as a specific fluorogenic substrate for aCDase. Assay protocol optimization, including a procedure for the preparation and storage of tissue lysates and the identification of optimal protein tissue lysate amounts and substrate concentrations based on kinetic enzymatic parameter analyses, is described.

Tumor mechanical microenvironment, particularly extracellular matrix stiffness, plays a critical role in regulating cancer cell behavior, including proliferation, quiescence, and drug resistance. Conventional 2D culture or stiff 3D scaffolds fail to recapitulate the physiological soft (normal) or pathologically stiff (tumoral) mechanical niches. Here, we present a detailed protocol for establishing a tunable 3D tumor spheroid culture system using sodium alginate–based hydrogels crosslinked with calcium ions at different concentrations to achieve soft or stiff conditions that mimic normal colon and colorectal cancer tissues, respectively. We describe the step-by-step procedures for fabricating stiffness-tunable hydrogels, culturing colorectal cancer spheroids, releasing spheroids for downstream analysis, and performing immunohistochemical staining on intact spheroids. This protocol enables the reproducible investigation of mechanosensitive pathways and drug resistance mechanisms in a physiologically relevant 3D context.

The conventional Ploton silver method employs a high-concentration 50% (w/v; 2.943 mol/L) silver nitrate solution for histological staining and characterization of the osteocyte lacuno-canalicular system (LCS). However, it is limited by prolonged staining times (55 min) and by risks of LCS ultrastructural damage and/or incomplete impregnation. To address these limitations, we developed the Wu–Wang silver nitrate staining method, which uses a 1 mol/L silver nitrate solution under elevated temperature (50–70 °C) to achieve rapid, effective, and high-contrast visualization of the osteocyte LCS within 10 min. We further demonstrate that this novel method enables robust LCS visualization across multiple vertebrate species. Compared with the Ploton method, the Wu–Wang method substantially reduces staining time and overcomes staining limitations inherent to prolonged exposure to concentrated silver nitrate solutions. This rapid and efficient staining method supports more accurate quantitative analysis of LCS morphology and facilitates systematic investigation of osteocyte and LCS morphogenesis, as well as the pathological mechanisms underlying bone and joint disease.

Progressive neurodegeneration linked to the accumulation of misfolded proteins is a hallmark of several neurodegenerative disorders, including Parkinson’s disease, Huntington’s disease, and Alzheimer’s disease. Dysfunction in the protein homeostasis machinery correlates with pathology. The chaperone protein DNAJB6 is expressed in neurons and oligodendrocytes and has been shown to play a key role in preventing amyloid aggregation by binding to amyloidogenic proteins and facilitating their refolding or degradation, in cooperation with other chaperones. Here, we describe a simple and feasible assay that enables high-throughput screening for DNAJB6 activity in a plate reader format. We use genetically engineered HEK293 cells that stably express DNAJB6 fused to either CFP or YFP. These cells can be plated into multi-well plates, and the fluorescence resonance energy transfer (FRET) signal can be measured for analysis of DNAJB6 dimerization, which is linked to DNAJB6 activity. The protocol can be used for drug screening and to identify compounds that increase DNAJB6 dimerization, and can serve as a starting point for finding new medicines that act through modulating DNAJB6 activity.

Ribonucleoprotein (RNP) condensates are membraneless organelles that exist alongside many RNA-driven processes, such as transcription and splicing. Despite their ubiquity, the biological necessity of forming a condensed phase remains unclear, particularly because the same RNP components exist both within these organelles and in the surrounding dilute phase. Most current methods for studying biochemical interaction dynamics within condensates rely on in vitro reconstitution of minimal factors or low-throughput single-molecule studies. However, RNP condensates are complex organelles containing tens to hundreds of proteins and hundreds to thousands of different RNAs. Here, we describe a scalable, high-throughput fluorescence microscopy–based approach to analyze protein–protein interaction networks, allowing for the rigorous assessment of dynamic, process-critical interactions within RNP condensates from live cells. This method takes advantage of fluorescence lifetime imaging (FLIM) and phasor plot analysis to automate segmentation of condensate-localized fluorescence signals. Using suitable FLIM–Förster resonant energy transfer (FLIM-FRET) fluorescent pairs fused to proteins of interest, protein–protein interactions can be actively monitored throughout various conditions via changes in fluorescence lifetime. Results from this assay yield valuable insight into the organization and assembly of essential factors for different condensate-associated processes to infer the functional consequences of RNP granule partitioning. Although this protocol is tailored for studying protein interactions within condensates, the design and execution framework can be adapted to investigate protein–protein interactions across a wide variety of compartments within different biological systems.