细胞生物学

Human induced pluripotent stem cell (hiPSC)-derived liver organoids have emerged as valuable models for studying human liver development. However, existing organoid systems often lack developmentally matched cell populations, particularly fetal hepatic stellate cells (HSCs), limiting their ability to recapitulate key developmental processes. Current approaches for generating HSCs rely on primary cells, immortalized cell lines, or hiPSC differentiation methods that frequently produce activated HSC-like cells and often require cell sorting. Here, we describe an efficient protocol for generating expandable fetal-like HSCs from hiPSCs through a stepwise differentiation strategy that mimics embryonic HSC development. The resulting cells can be robustly expanded while maintaining characteristic molecular and functional features of fetal HSCs. This protocol provides a reproducible and scalable source of fetal-like HSCs without cell sorting and supports the generation of multicellular liver organoids containing developmentally relevant stromal components. Beyond the validation of the protocol in studies of liver maturation and vascularization, it can be applied to investigations of HSC biology and congenital liver diseases.

Platelets originate from megakaryocytes, whose generation involves a series of biological processes including directed differentiation, proliferation, polyploidization, and maturation of hematopoietic stem cells. Abnormalities in megakaryocyte development and maturation can lead to quantitative and functional defects in platelets, thereby contributing to hemostatic or thrombotic disorders as well as the development of malignancies. Investigating megakaryocyte development and maturation and platelet production can provide important theoretical foundations for the diagnosis and treatment of thrombocytopenia, thrombotic diseases, and myeloproliferative neoplasms. Currently, there are three main clinical sources of hematopoietic stem cells (HSCs): bone marrow (BM), peripheral blood (PBSC), and umbilical cord blood (UCB). Among these, umbilical cord blood (UCB)-derived HSCs, due to their higher differentiation efficiency and stronger proliferative capacity, are the preferred starting cell source for studying megakaryocyte (MK) development and maturation and the mechanisms of platelet production. This article describes a detailed protocol covering all necessary steps for isolating CD34⁺ hematopoietic stem cells from umbilical cord blood, followed by in vitro induction culture with stem cell factor (SCF) and thrombopoietin (TPO) to generate mature megakaryocytes that highly express early megakaryocyte markers (CD41a, CD61) and late maturation markers (CD42a, CD42b). This protocol provides an effective tool for studying megakaryocyte development and platelet production and holds potential value for application in research on megakaryocyte-related diseases.

Amyotrophic lateral sclerosis (ALS) is characterized by early and spatially restricted pathology in motor axons, including distal degeneration and accumulation of aggregation-prone proteins such as TDP-43. However, a major limitation in the field has been the lack of approaches that enable robust, quantitative, and compartment-specific analysis of these early axonal events, particularly in human-relevant systems. Here, we describe an integrated experimental and analytical framework that enables quantitative dissection of axonal degeneration and protein aggregation, specifically within distal motor axons. By combining compartmentalized human co-cultures with a dedicated image analysis strategy, this approach enables selective and quantitative analysis of pathological processes specifically within axons, independent of surrounding tissues such as muscle and other cellular compartments. This framework captures both structural degeneration and protein aggregation dynamics at subcellular resolution, enabling spatially resolved quantitative analysis of disease-relevant changes along axons. Importantly, the analytical framework is not limited to TDP-43 but is broadly applicable to diverse aggregation-prone proteins, thereby providing a generalizable platform to study axonal pathology across neurodegenerative diseases. Together, this work provides a scalable approach for investigating axonal pathology as an early and measurable feature of neurodegeneration, with potential applications in mechanistic studies and therapeutic targeting in ALS and related disorders.

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.

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.

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.

Isolation of adult mouse ventricular myocytes is essential for studying cardiac physiology and cellular function. Traditional methods commonly rely on Langendorff perfusion systems, which provide continuous retrograde coronary perfusion but require specialized equipment and can be complex to operate. Here, we describe a simplified Langendorff-based protocol that uses a syringe pump–driven system to achieve constant-flow retrograde aortic perfusion during enzymatic digestion. The setup incorporates an inline heater for precise temperature control and uses widely available laboratory components, enabling consistent delivery of digestion enzymes. This approach maintains stable perfusion despite changes in coronary resistance and reduces variability associated with conventional gravity-driven systems. The protocol yields high-quality adult ventricular myocytes suitable for downstream functional analyses, including electrophysiology, contractility, and calcium imaging. Compared with traditional systems, this method is more accessible, reduces technical complexity, and improves reproducibility, facilitating adoption in laboratories without dedicated isolated-heart perfusion infrastructure.

Satellite cells are adult skeletal muscle stem cells that play essential roles in muscle regeneration. Understanding their behavior is critical for elucidating the mechanisms of muscle repair and advancing muscle regenerative therapies. This requires efficient methods for genetic manipulation in these cells. Retroviral-mediated gene delivery is commonly used for stable transgene expression in immortalized cell lines. However, existing approaches are not optimized for primary satellite cells, often resulting in variable efficiency and inconsistent outcomes. Here, we describe an optimized protocol for satellite cell isolation and culture, as well as retroviral production and infection of primary satellite cells that achieves high transduction efficiency. The satellite cell isolation procedure enriches for myofiber fragments prior to satellite cell release, thereby reducing contamination by non-myogenic cells and improving cell purity. Another key feature of this protocol is the concentration of retroviral particles and their resuspension in satellite cell growth medium prior to infection, which minimizes satellite cell exposure to packaging cell-conditioned medium. Compared to standard approaches, this protocol improves both infection efficiency and reproducibility. It is readily adaptable to a wide range of downstream applications, including microscopies, biochemical assays, and molecular biology analyses.

Polyploid hepatocytes are one of the unique features of the liver. Some polyploid hepatocytes have chromosomes in a single nucleus (e.g., 1x4n, 1x8n), while others separate their chromosomes into two nuclei (e.g., 2x2n, 2x4n). In ploidy research, hepatocytes are typically sorted according to their cellular ploidy, revealing their contribution to tumorigenesis and cellular senescence. However, the conventional sorting method fails to distinguish 1x4n from 2x2n, or 1x8n from 2x4n cells, leaving it unclear whether hepatocytes with the same cellular ploidy but different nuclear configurations are identical or phenotypically different. Here, we describe a detailed protocol for fractionating mononucleated and binucleated hepatocytes. First, we present the method for isolating primary mouse hepatocytes and staining them with the DNA dye Hoechst 33342. Flow cytometry is then used to detect fluorescence differences between mononucleated and binucleated hepatocytes. This protocol enables the discrimination of hepatocyte subpopulations with identical cellular ploidy, providing a useful tool to investigate the functional heterogeneity of polyploid hepatocytes.

In vitro cytotoxicity assessments frequently rely on staining-based methods that indirectly estimate viable cell numbers. 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 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 resazurin-based reduction assay across diverse experimental models, emphasizing its practicality, reproducibility, and adaptability for real-time viability monitoring.