By Christine Gagliardi, Bruce A. Bunnell (auth.), Jeffrey M. Gimble, Bruce A. Bunnell (eds.)
During the prior decade, a variety of medical disciplines have followed using adipose-derived stem/stromal cells (ASCs) as an enormous software for study and discovery. In Adipose-Derived Stem Cells: tools and Protocols, specialists from the sphere, together with individuals of the esteemed overseas Federation of Adipose Therapeutics and technology (IFATS), supply outlined and confirmed protocols on the way to additional codify the usage of those strong and obtainable cells. With chapters prepared round methods spanning the invention, pre-clinical, and medical methods, a lot of the emphasis is put on human ASC, whereas extra recommendations related to small and massive animal species are integrated. As a quantity within the hugely winning equipment in Molecular Biology™ sequence, the special contributions contain introductions to their respective subject matters, lists of the mandatory fabrics and reagents, step by step, with no trouble reproducible laboratory protocols, and notes on troubleshooting and heading off recognized pitfalls. complete and state-of-the-art, Adipose-Derived Stem Cells: equipment and Protocols serves as a necessary reference textual content for skilled researchers in addition to new scholars at the route to extra exploring the wonderful power of ASCs.
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Additional info for Adipose-Derived Stem Cells: Methods and Protocols
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.
3. 1. Obtaining the Adipose Tissue All procedures are performed in a biosafety hood. Ensure the muASC isolation procedure is initiated within 20 min of tissue extraction, especially if a large number of animals are being completed in 1 day. Warm buffers (at least one to two of 500 ml KRB, PBS, or PBS with 1% BSA) along with the Collagenase solution and Stromal media in the 37°C water bath (see Note 1). Place bench protector down in hood. Sacrifice animal using anesthesia or CO2 asphyxiation as approved by the AVMA Guidelines on Euthanasia.
Centrifuge a 20-mL aliquot in a microcentrifuge tube at 1,200 rpm (300 × g) for 5 min. Remove the supernatant and resuspend the pellet in 20 mL of red cell lysis buffer. Incubate for 5 min at room temperature. Add 20 mL of Trypan Blue and count the cells with a hemocytometer. Plate the cells at the appropriate density in complete stromal media and incubate at 37°C and 5% CO2. Change the media after 24 h to remove nonadherant cells. Media should be changed about every 3 days. 3. Aspirate the media from the flasks and rinse the cells with PBS.
Adipose-Derived Stem Cells: Methods and Protocols by Christine Gagliardi, Bruce A. Bunnell (auth.), Jeffrey M. Gimble, Bruce A. Bunnell (eds.)