Mesenchymal stromal/stem cells (MSCs) are promising candidates for cell therapy and regenerative medicine. However, murine bone marrow-derived MSCs (mBMSCs) are much more difficult to isolate and purify because of their high heterogeneity and very limited numbers within bone morrow. To this end, this protocol was developed to establish an appropriate method for isolation and expansion of mBMSCs for preclinical research. Cells were dissociated and isolated mainly from compact bones via an enzyme digestion method to dissociate sufficient cell pools for further expansion ex vivo. Adherent cells collected were spindle-shaped with colony-forming units, and expressed a series of surface markers, including Oct4, CD29, and Sca-1 (stem cell antigen-1). Additionally, mBMSCs possessed potent capacity to differentiate into osteoblasts, adipocytes, and chondrocytes under appropriate culture conditions. Therefore, this study provides a standardized and reliable method for quick expansion of murine BMSCs. © 2026 Wiley Periodicals LLC. Basic Protocol: Enzymatic digestion method.
Biomaterial-based bioinks are increasingly utilized in bioprinting to engineer three-dimensional (3D) constructs with living cells for tissue engineering and disease modeling. Among various bioinks explored, alginate-based formulations stand out due to their good biocompatibility, mild gelation conditions, tunable mechanical properties, and ease of crosslinking via divalent cations such as calcium. Despite their widespread use, standardized protocols for preparing alginate-based bioinks and characterizing bioprinted constructs have not been well documented. Our laboratory has developed and validated reproducible methods for preparing a variety of alginate-based bioinks and printing cell-laden constructs tailored for diverse applications. In this article, we present detailed step-by-step protocols covering bioink preparation and rheological characterization, extrusion-based bioprinting of cell-laden constructs, post-printing culture and co-culture techniques, printability assessment, and live/dead and immunofluorescence assays. These protocols serve as a standardized framework for the fabrication and characterization of 3D bioprinted alginate-based cell-laden constructs, thereby facilitating translational research in tissue engineering, disease modeling, and preclinical therapeutic development. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 : Bioink preparation Basic Protocol 2 : Bioink characterization using rheology Basic Protocol 3 : Scaffold design and bioprinting Support Protocol : 3D-printing parameter determination Basic Protocol 4 : Printability and cell viability analyses, and immunofluorescence assay
Angiogenesis is crucial in tissue repair, wound healing, and embryo development. Maintaining a balance between pro-angiogenic and anti-angiogenic factors is crucial for tissue homeostasis, thereby preventing the development of pathological conditions. In addition, the tissue microenvironment constantly influences molecular signaling, potentially altering this delicate balance. The chorioallantoic membrane (CAM) assay has emerged as a dynamic, cost-effective, and ethically favorable alternative for studying angiogenesis. This work presents a detailed and accessible approach to the CAM assay, focusing on the evaluation of the angiogenic potential of applied stimuli (such as conditioned media, extracellular vesicles, or bioscaffolds) through various techniques, including hematoxylin and eosin, PicroSirius Red, Alcian Blue, and resorcin-fuchsin staining; immunofluorescence; immunohistochemistry; and scanning electron microscopy (SEM). Our protocol uses the in ovo CAM assay approach, utilizing a simple incubator setup that eliminates the need for expensive equipment. The primary objective is to provide a comprehensive methodology for researchers to efficiently analyze angiogenesis, offering insights into vessel morphology and protein expression. The experiment comprises three major steps: (1) egg opening, stimulation, and sample collection; (2) histological processing and paraffin blocking; and (3) microscopic analyses. The first and second procedures take approximately 15 days, after which each analysis can be conducted whenever the researcher judges appropriate. The expected results include several types of data concerning the proliferation rate, vessel presence and integrity, measurement of angiogenic-specific proteins, and investigation of structural proteins. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Angiogenesis quail and chicken chorioallantoic membrane assay analyses.
Measuring oxygen consumption allows for the role of mitochondrial function in biological phenomena and mitochondrial diseases to be determined. Although respirometry has become a common approach in disease research, current methods are limited by the necessity to process and measure tissue samples within 1 hr of acquisition. Detailed by Acin-Perez and colleagues, a new respirometry approach designed for previously frozen tissue samples eliminates these hurdles for mitochondrial study. This technique allows for the measurement of maximal respiratory capacity in samples frozen for long-term storage before testing. This protocol article describes the optimal tissue isolation methods and the combination of substrates to define electron transport chain function at high resolution in previously frozen tissue samples. © 2020 The Authors. Basic Protocol 1: Sample collection, storage, and homogenization for previously frozen tissue respirometry Basic Protocol 2: Running a Seahorse respirometry assay using previously frozen tissue samples Basic Protocol 3: Normalization to mitochondrial content for previously frozen tissue respirometry.
Incorporation of a stable-isotope metabolic tracer into cells or tissue can be followed at submicron resolution by multi-isotope imaging mass spectrometry (MIMS), a form of imaging secondary ion microscopy optimized for accurate isotope ratio measurement from microvolumes of sample (as small as ∼30 nm across). In a metabolic MIMS experiment, a cell or animal is metabolically labeled with a tracer containing a stable isotope. Relative accumulation of the heavy isotope in the fixed sample is then measured as an increase over its natural abundance by MIMS. MIMS has been used to measure protein turnover in single organelles, track cellular division in vivo, visualize sphingolipid rafts on the plasma membrane, and measure dopamine incorporation into dense-core vesicles, among other biological applications. In this article, we introduce metabolic analysis using NanoSIMS by focusing on two specific applications: quantifying protein turnover in single organelles of cultured cells and tracking cell replication in mouse tissues in vivo. These examples illustrate the versatility of metabolic analysis with MIMS. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Metabolic labeling for MIMS Basic Protocol 2: Embedding of samples for correlative transmission electron microscopy and MIMS with a genetically encoded reporter Alternate Protocol: Embedding of samples for correlative light microscopy and MIMS Support Protocol: Preparation of silicon wafers as sample supports for MIMS Basic Protocol 3: Analysis of MIMS data.
