Isolation and Culturing of Primary Murine Chondroprogenitor Cells: A Mammalian Model of Chondrogenesis.

Embryonic limb bud-derived micromass cultures are valuable tools for investigating cartilage development, tissue engineering, and therapeutic strategies for cartilage-related disorders. This collection of fine-tuned protocols used in our laboratories outlines step-by-step procedures for the isolation, expansion, and differentiation of primary mouse limb bud cells into chondrogenic micromass cultures. Key aspects covered in these protocols include synchronized fertilization of mice (Basic Protocol 1), tissue dissection, cell isolation, micromass formation, and culture optimization parameters, such as cell density and medium composition (Basic Protocol 2). We describe techniques for characterizing the chondrogenic differentiation process by histological analysis (Basic Protocol 3). The protocols also address common challenges encountered during the process and provide troubleshooting strategies. This fine-tuned comprehensive protocol serves as a valuable resource for scientists working in the fields of developmental biology, cartilage tissue engineering, and regenerative medicine, offering an updated methodology for the study of efficient chondrogenic differentiation and cartilage tissue regeneration. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Synchronized fertilization of mice Basic Protocol 2: Micromass culture of murine embryonic limb bud-derived cells Basic Protocol 3: Qualitative assessment of cartilage matrix production using Alcian blue staining.

Isolation and Micromass Culturing of Primary Chicken Chondroprogenitor Cells for Cartilage Regeneration.

Much of the skeletal system develops by endochondral ossification, a process that takes place in early fetal life. This makes the early stages of chondrogenesis, i.e., when chondroprogenitor mesenchymal cells differentiate to chondroblasts, challenging to study in vivo. In vitro methods for the study of chondrogenic differentiation have been available for some time. There is currently high interest in developing fine-tuned methodology that would allow chondrogenic cells to rebuild articular cartilage and restore joint functionality. The micromass culture system that relies on embryonic limb bud-derived chondroprogenitor cells is a popular method for the study of the signaling pathways that control the formation and maturation of cartilage. In this protocol, we describe a technique fine-tuned in our laboratory for culturing limb bud-derived mesenchymal cells from early-stage chick embryos in high density (Basic Protocol 1). We also provide a fine-tuned method for high-efficiency transient transfection of cells before plating using electroporation (Basic Protocol 2). In addition, protocols for histochemical detection of cartilage extracellular matrix using dimethyl methylene blue, Alcian blue, and safranin O are also provided (Basic Protocol 3 and Alternate Protocols 1 and 2, respectively). Finally, a step-by-step guide on a cell viability/proliferation assay using MTT reagent is also described (Basic Protocol 4). © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Micromass culture of chick embryonic limb bud-derived cells Basic Protocol 2: Transfection of cells with siRNA constructs using electroporation prior to micromass culturing Basic Protocol 3: Qualitative and quantitative assessment of cartilage matrix production using dimethyl methylene blue staining and image analysis Alternate Protocol 1: Qualitative assessment of cartilage matrix production using Alcian blue staining Alternate Protocol 2: Qualitative assessment of cartilage matrix production using safranin O staining Basic Protocol 4: Measurement of mitochondrial activity with the MTT assay.