By Christine Gagliardi, Bruce A. Bunnell (auth.), Jeffrey M. Gimble, Bruce A. Bunnell (eds.)
During the previous decade, a variety of medical disciplines have followed using adipose-derived stem/stromal cells (ASCs) as a tremendous software for examine and discovery. In Adipose-Derived Stem Cells: tools and Protocols, specialists from the sector, together with participants of the esteemed foreign Federation of Adipose Therapeutics and technological know-how (IFATS), offer outlined and validated protocols with a view to additional codify the usage of those robust and available cells. With chapters geared up round techniques spanning the invention, pre-clinical, and scientific approaches, a lot of the emphasis is put on human ASC, whereas extra ideas concerning small and massive animal species are incorporated. As a quantity within the hugely winning tools in Molecular Biology™ sequence, the precise contributions comprise introductions to their respective themes, lists of the required fabrics and reagents, step by step, conveniently reproducible laboratory protocols, and notes on troubleshooting and averting recognized pitfalls. finished and state of the art, Adipose-Derived Stem Cells: tools and Protocols serves as a necessary reference textual content for skilled researchers in addition to new scholars at the route to additional exploring the great capability of ASCs.
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Additional resources for Adipose-Derived Stem Cells: Methods and Protocols
May promote premature adipogenesis. Signs of deterioration, such as granularity around the nucleus, cytoplasmic vacuolations, and/or detachment of the cells from the plastic surface, may indicate inadequate or toxic medium, microbial contamination, or senescence of the primary cells. It is important to not overexpose the cells to the trypsin/ EDTA solution. This could decrease the cell viability. If the adipocytes are detaching, do not dry the well when changing the medium since adipocytes tend to float when new medium is added.
24 Yu et al. Resuspend the LAF cell pellet in erythrocyte lysis buffer. Let stand at room temperature for 5 min. Filter the LAF cell suspension through a 100-mm filter. Load cells onto a Ficoll density gradient. Centrifuge for 20 min at 800 × g at room temperature. Aspirate cells from gradient interface. Wash with PBS prewarmed to 37°C. Filter LAF cell suspension through 100-mm filter. Count nucleated cell number using Trypan Blue staining and a hemocytometer. Suspend LAF cells in Stromal medium.
Under standard conditions, the cells develop fibroblast-like morphology with increasing passages. The greatest number of ASCs is obtained from cultures plated at low densities. Low plating density and the use of DMEM-Ham’s F12 can facilitate ASC differentiation. The media composition also influences gene expression, and use of antioxidants and low calcium concentrations can increase the growth rate and life span of ASCs. Increasing knowledge on the molecular mechanisms regulating ASC proliferation and differentiation will continue to contribute to improved isolation and culture procedures.