Stem cell-derived exosomes have gained increasing attention due to their therapeutic potential in various diseases. The successful acquisition of exosomes with the desired therapeutic efficacy requires both the appropriate cultivation of the source stem cells and the use of suitable isolation methods. In this context, serum-free culture approaches have emerged as a key strategy to enhance exosome yield. This chapter explains in detail how to properly grow stem cells, how to adapt them to serum-free culture conditions, and how to get exosomes from these cells.
T cell-mediated therapies are major breakthroughs in the treatment of melanoma. However, clinical hurdles in the application of T cell-mediated therapies in melanoma persist and limit their potential. As such, preclinical research efforts remain focused on their development and optimization. Preclinical research techniques include in vivo evaluation with xenograft mouse models, ex vivo patient tumor explant culture, and in vitro tumor spheroids and melanoma/T cell co-cultures. However, these models lack the appropriate tissue context of melanoma skin cancer. Here, we describe a technique for the incorporation of primary T cells and chimeric antigen receptor (CAR)-T cells with melanoma cells in human skin organoids derived from primary epidermal keratinocytes.
Mechanical properties play a critical role in regulating ovarian function, yet their spatial variation within intact tissue remains poorly understood due to the limitations of conventional mechanical testing techniques. Confocal Brillouin microscopy offers a non-contact and label-free approach for mapping mechanical properties of the intact ovarian tissue without perturbing tissue structure. In fresh ovarian tissue, Brillouin images reveal spatial heterogeneity in mechanical properties across follicles, surrounding stroma, and fluid-filled regions, suggesting underlying differences in cellular organization and extracellular matrix composition. These measurements capture both inter- and intra-follicular variations, providing insight into regional differences in mechanical properties within and between follicles. This chapter outlines the principles of Brillouin microscopy and its application to fresh ovarian tissue, highlighting its capability to resolve microscale mechanical variations in situ. This method provides a foundation for understanding the role of mechanical properties in ovarian function, with potential applications in disease models and reproductive health research.
This chapter presents a stepwise optimization of a Matrigel®-based in vitro angiogenesis assay using human umbilical vein endothelial cells (HUVECs) to achieve reproducible and quantifiable tube formation. Critical parameters-including culture surface, matrix composition and thickness, coating temperature and incubation time, growth factor-defined serum-free medium, and cell seeding density-were systematically evaluated. Optimal conditions were identified as 3 mg/mL Matrigel (100 μL/well), polymerized at 22 °C for 90 min, with HUVECs seeded at 20,000 cells/cm2 and cultured in a defined growth factor cocktail. Standardized imaging and ImageJ-based analysis enabled robust quantification of network features. This protocol improves consistency, stability, and comparability across angiogenesis studies.
Identifying therapies with potential clinical relevance is a critical task for intervening on pancreatic ductal adenocarcinoma (PDAC), the most common pancreatic malignancy, characterized by an extremely poor prognosis and expected to be among the most prevalent lethal tumors in the coming years. Unified therapeutic options are limited because PDAC tumors are complex at the genomic, epigenetic, and metabolic levels, often involving multiple and interacting pathways orchestrated by undruggable drivers. Inter-patient heterogeneity in drug response, accompanied by high recurrent rates of chemoresistance, makes PDAC an almost incurable tumor by pharmacological means. Resource-intensive approaches need to be implemented in order to identify effective therapies along the integration of PDAC tumor heterogeneity landscape. Large-scale technologies such as high-content screening (HCS) hold promise to uncover novel cures, especially when therapy response interrogation involves the incorporation of highly physiological-competent human PDAC models such as collections of patient-derived tumor organoids (PDOs). However, systematic and unbiassed drug combination-HCS applied directly on PDOs, intended to ensure the detection of effective pairs or multidrug combinations to preclinically inform on effective drug combinatorial regimens, is still missing. This chapter proposes a PDO-guided drug-synergy image-based HCS pipeline already implemented in a proof-of-concept study for PDAC therapy development, design and validation of drug-synergy screening, using collections of PDOs.
Repairing osteochondral defects remains a significant therapeutic challenge due to the complex hierarchical structure of the tissue, while existing treatment strategies are largely palliative rather than curative. For osteochondral tissue production, three-dimensional (3D) bioprinting has become a potent technique to produce biomimetic scaffolds with regulated architecture and cell distribution. This chapter provides a comprehensive and repeatable procedure for the production and biological assessment of 3D bioprinted gelatin methacryloyl (GelMA) scaffolds that incorporate mesenchymal stem cells (MSCs) obtained from bone marrow. The methodology outlines the techniques for photo-crosslinking, extrusion-based bioprinting, MSC expansion and encapsulation, and bioink preparation in order to guarantee cell viability and structural integrity. Comprehensive in vitro evaluation techniques are also described, such as differentiation evaluation related to osteogenic and chondrogenic commitment, cell viability, and proliferation analysis. This methodical approach offers researchers a useful foundation for creating MSC-laden GelMA constructs appropriate for use in osteochondral tissue engineering and additional translational studies.
This chapter provides a detailed, step-by-step protocol for the fabrication of architecturally defined brain organoid-like neural microtissues using a sequential digital light processing (DLP) bioprinting strategy. The method relies exclusively on DLP technology to fabricate complex heterogeneous structures through a layer-by-layer vat-switching technique. Central to this protocol is the formulation of a nanocomposite bioink, where reduced graphene oxide (rGO) nanoparticles and bacterial cellulose are mixed with gelatin methacryloyl (GelMA) matrix. This composition provides enhanced electrical conductivity, printability, and mechanical stability, promoting neural network maturation. We describe the complete workflow from induced pluripotent stem cell (iPSC) culture and neural induction, through bioink preparation and DLP printing parameter optimization, to long-term static culture and functional validation. This protocol addresses key limitations of conventional self-assembly methods, offering improved reproducibility, structural control, and physiological relevance for modeling neurodevelopment and disease.
Precise tracking of transplanted cells within mouse tissues is essential for evaluating engraftment, migration, and long-term persistence in transplantation and regenerative medicine research. Here, we present a practical and versatile cell-tracking workflow focusing on PKH26-based direct fluorescent labeling and GFP-mediated indirect genetic labeling. PKH26 provides rapid and robust membrane labeling that is useful for short-term tracing, while GFP enables stable and heritable fluorescence suitable for long-term monitoring of transplanted cells. This chapter provides optimized protocols, troubleshooting strategies, and critical technical considerations for implementing these complementary methods. Together, these approaches offer a flexible and reliable framework for evaluating the engraftment, survival, distribution, and behavior of transplanted cells in mouse models.
Induced pluripotent stem cells (iPSCs) have enabled significant advances in regenerative medicine; however, reprogramming methods using viral vectors pose risks to safety and genomic integrity in clinical applications. This protocol details a non-viral, integration-free approach for delivering plasmid DNA (pDNA) into somatic cells via solid lipid nanoparticles (SLNs). SLNs protect pDNA from enzymatic degradation and ensure high transfection efficiency in cells, thereby preventing permanent genetic alterations in the host genome.The protocol provides step-by-step guidance for the preparation of human fibroblasts, the sequential transcription cycles of SLN-pDNA encoding OSKM factors, and the induction and isolation of iPSC colonies. Furthermore, comprehensive characterization methods are described to confirm pluripotency, including monitoring GFP expression levels, quantitative real-time PCR, immunofluorescence staining, flow cytometry, alkaline phosphatase activity assays, and in vitro tri-lineage differentiation.iPSCs obtained while preserving genomic integrity can be safely used in advanced regenerative medicine applications, such as disease modeling, high-throughput drug screening, and personalized cell therapies. This approach enhances safety and efficacy in clinical settings and provides a valuable platform for the development of iPSC-based therapeutic and research applications.
The human hair follicle (HF) harbors distinct populations of epithelial stem and progenitor cells that are essential for hair homeostasis and epidermal wound healing. While the follicular niche, the anatomical equivalent of the murine bulge located at the insertion site of the arrector pili muscle, has historically been the primary target for the isolation of hair follicle stem cells (HFSCs), capturing the full epithelial stem and progenitor hierarchy within the outer root sheath (ORS) is critical for a comprehensive understanding of human hair follicle biology. As integrin α6 (CD49f) is a well-established surface marker associated with basal epithelial stem and progenitor states, and long-term proliferative potential of immature keratinocytes, we have exploited this marker to isolate an epithelial cell population corresponding to the keratinocyte basal layer of the ORS. This chapter describes a protocol for the enrichment of stem and progenitor cells from intact human hair follicles, combining manual microdissection of the entire hair follicle, enzymatic dissociation, and flow cytometry sorting based on CD49f expression.Functional validation using in vitro proliferation and clone-forming assays showed that the CD49fhigh fraction exhibits significantly greater proliferative and clonogenic capacity, compared to the ORS total and CD49flow populations. This method preserved cell viability and phenotypic integrity. Consequently, the isolated keratinocyte populations are suitable for a broad range of downstream uses and applications, including transcriptomic analyses such as single-cell RNA sequencing (scRNA-seq), functional assays, and regenerative studies. Altogether, this workflow provides a robust platform for future approaches dedicated to the treatment of alopecia, promotion of hair longevity, and for tissue engineering applications.
This review presents a balanced synthesis of the efficacy-nanotoxicity window of nanomaterials-including carbon-based structures, metallic nanoparticles, and bioceramic composites-within the context of stem cell-assisted tissue engineering. It emphasizes that increases in conventional differentiation markers may not necessarily correspond to functional maturity or long-term safety. Therefore, a gradual shift from standard viability-based assays toward lineage-specific functional assessments is discussed. The nanotoxicological profile of these materials is examined across major tissue types, highlighting how similar physicochemical properties may yield beneficial or adverse outcomes depending on concentration, exposure time, and microenvironmental conditions. In bone tissue, the review considers the point at which enhanced mineralization may be accompanied by oxidative stress and mitochondrial strain. For cartilage, potential concerns related to ion and degradation product accumulation in avascular environments are addressed, including the possible induction of inflammatory signaling and hypertrophic markers such as collagen type X. In neural and cardiac applications, efforts to improve electrical conductivity are evaluated alongside potential electrophysiological alterations, including Ca2+ imbalance and rhythm disturbances at sub-cytotoxic levels. Vascular differentiation is discussed within the context of pro-angiogenic signaling and the risk of endothelial dysfunction. By comparing experimental systems ranging from 2D cultures to induced pluripotent stem cell (iPSC)-derived organoids, the review underscores the model-dependent and tissue-specific nature of nanotoxic responses. Overall, it outlines considerations for integrating physicochemical characterization with functional and metabolic endpoints to support more reliable evaluation of nanobiomaterials in regenerative medicine.
Fish epidermal keratocytes provide a powerful model system for investigating cell motility due to their rapid, persistent migration and stable morphology. While single-cell dynamics have been extensively characterized, recent studies have increasingly focused on collective migration. Because keratocytes cannot be maintained as continuous cell lines, experiments rely on primary cultures derived from fish scales. This chapter presents optimized protocols for culturing primary keratocytes, transiently introducing fluorescent probes using a custom electroporation system and performing high-resolution three-dimensional time-lapse imaging with confocal microscopy. These procedures enable visualization of dynamic molecular and cellular behaviors in both single-cell and collective migration contexts.
The skin, the largest organ of the human body, performs essential physiological functions including barrier protection, thermal regulation, and mechano-sensation. Despite its structural consistency, it exhibits marked regional heterogeneity, particularly under pathological conditions. Our recent investigations have revealed that in secondary lymphedema, the affected skin undergoes distinct pathomorphological changes, including hyperkeratosis, spongiosis, and altered keratinocyte polarity within the epidermis. These findings have prompted deeper studies into the molecular and epigenetic mechanisms contributing to the irreversible progression of this disease. This protocol describes a detailed, reproducible method for isolating and culturing primary keratinocytes from both full-thickness and split-thickness skin grafts obtained from lymphedematous regions. The workflow comprises tissue processing, enzymatic dissociation, selective enrichment of keratinocytes, and optimized culture conditions for downstream molecular analyses. Validation of isolated keratinocytes is performed using Western blotting, flow cytometry, and immunofluorescence histology. This approach preserves native phenotypic traits and epigenetic signatures in patient-derived keratinocytes, thereby enhancing the translational relevance of in vitro studies investigating the pathophysiology of secondary lymphedema.
Glioblastoma multiforme (GBM) is sustained by glioma stem cells (GSCs), a self-renewing tumor-initiating population that contributes to cellular heterogeneity, therapeutic resistance, and disease recurrence. Reproducing the complex tumor-microenvironment interaction of GBM in vitro remains a significant challenge. Stem cell-based three-dimensional (3D) organoid systems have emerged as physiologically relevant models that recapitulate tumor architecture, stemness, and dynamic cellular interactions compared with conventional two-dimensional cultures. In this chapter, readers are provided with standardized and reproducible methodological frameworks to generate, maintain, and validate GBM microenvironment-relevant 3D organoid models. By following the step-by-step protocols described, readers will be able to establish GBM organoids from single-cell suspension and microtumor fragments, while preserving tumor-initiating capacity and intratumoral heterogeneity. The chapter further enables readers to integrate neurospheres into cerebral organoid systems to model tumor-neural interactions within a tissue relevant context and to incorporate extracellular matrix (ECM)-informed strategies, including decellularized brain ECM (BdECM), to mimic brain-specific biochemical/mechanical cues. Readers will also be able to perform long-term (up to 60 days) cortical organoid culture with defined quality control (QC) and validation, including evaluation of structural organization, stemness markers expression, differentiation status, and reproducibility across batches. The chapter further provides practical problem-solving guidance to address common technical challenges during organoid establishment and maintenance. Collectively, these protocols offer a resilient, durable, and versatile toolkit to model GBM biology, interrogate tumor-microenvironment interactions, and evaluate therapeutic efficacy using clinically relevant 3D organoid systems.
Axioloids are three-dimensional (3D) structures derived from pluripotent stem cells (PSCs) that model key aspects of human somitogenesis and early axis development in vitro. These mesoderm-based aggregates recapitulate essential morphogenetic features of the segmentation process, including axial elongation, sequential formation of epithelial somites with proper rostrocaudal patterning, and oscillatory activity of the segmentation clock. They further reproduce the spatiotemporal organization characteristic of early axial development, such as opposing FGF/WNT and retinoic acid signaling gradients and anteroposterior HOX gene expression patterns similar to those observed in vivo. Together, these attributes highlight axioloids as a robust and versatile platform for studying human axial development and congenital disorders of the spine and axial skeleton. In this chapter, we provide a concise, step-by-step protocol for generating axioloids, along with key developmental readouts and troubleshooting strategies to address common problems and issues encountered. This protocol provides a reproducible framework for producing and characterizing axioloids as an in vitro model of human axial development and disease.
Advanced in vitro developmental models, including embryo-like structures, organoids, and organ-on-chip platforms, have emerged as powerful systems for studying human development under controlled conditions. These models are enabled by advances in stem cell biology and bioengineering, allowing multicellular self-organization that recapitulates selected aspects of embryonic patterning and tissue formation. Key engineering parameters-such as stem cell source, signaling control, geometric confinement, matrix composition, and microfluidic regulation-define the boundary conditions that guide developmental outcomes. These systems are best understood as engineered abstractions rather than complete replicas of human development, reflecting trade-offs between reproducibility, complexity, and biological fidelity. Despite challenges related to variability, standardization, and ethical considerations, these platforms provide valuable tools for mechanistic studies, disease modeling, and drug discovery and support the transition toward human-relevant, animal-free research approaches.
Skin epidermal permeability is a critical barrier function, and its disruption is implicated in various dermatological conditions and in secondary lymphedema, leading to increased fluid loss and dermal complications. Many techniques exist to assess this permeability. However, these methods frequently require specialized instruments or lack the spatial resolution needed to identify specific cellular layers involved in barrier dysfunction. Here, we describe a step-by-step protocol for an immunofluorescence-based dye penetration assay that provides high spatial resolution for visualizing the permeation of fluorescent probes through specific epidermal strata in a mouse lymphedema model.
Many noncoding RNAs, especially long noncoding RNAs (lncRNAs), can bind to DNA sequences in specific regulatory regions in genomes, thereby regulating gene expression by recruiting epigenomic modification enzymes to their DNA-binding sites (DBSs). Experimental identification of DBSs genome-wide for the abundant lncRNAs in genomes of humans, mice, and other model animals is infeasible, making accurate and fast computational prediction of lncRNA:DNA-binding indispensable. Multiple methods and programs have been developed. This chapter describes LongTarget and its variant, Fasim-LongTarget, including how to use them step by step to predict DNA-binding domains (DBDs) and DBSs in multiple situations. Both the online and offline versions are described. In addition, we describe how to use the LongMan database, which stores orthologous sequences of human and mouse lncRNAs in other mammals, to explore species-specific lncRNAs and lncRNA:DNA binding. We also demonstrate how to evaluate predicted DBDs and DBSs using publicly available data in the UCSC Genome Browser.
Noncoding RNAs (ncRNAs) are involved in a variety of processes in the cell nucleus, including transcriptional control, shaping 3D genome, and assembly/maintenance of functional nuclear compartments. However, the specific functions of most of the currently identified ncRNAs remain unclear. To gain further insight into the role of ncRNA in the eukaryotic genome functioning, it is important to determine genome-wide association patterns of various ncRNAs with chromatin. To address this question, a panel of RNA-DNA proximity ligation-based approaches have been developed, which have allowed deciphering RNA-chromatin interactions at the genome-wide level. An important drawback of all these techniques, however, is that they do not reveal the proteins involved in RNA-DNA interactions. In this chapter, we describe RedChIP, a method combining RNA-DNA ligation with chromatin immunoprecipitation to identify RNA-chromatin interactions mediated by a particular protein. We present a detailed protocol for RedChIP, focusing on the technical nuances and subtleties of the experimental procedure, and discuss algorithms for processing and analyzing the sequencing data. RedChIP can be used to identify RNAs associated with genomic regions occupied by any protein of interest, enabling disclosure of ncRNAs involved in recruiting various protein complexes to chromatin.
Predicting a joint secondary structure formed by two RNAs plays a key role in understanding mechanisms and functions of complex RNA-RNA interactions. Here we describe a practical procedure of a fast and accurate computational method for predicting joint structures based on integer programming.
