By Christine Gagliardi, Bruce A. Bunnell (auth.), Jeffrey M. Gimble, Bruce A. Bunnell (eds.)
During the prior decade, quite a lot of medical disciplines have followed using adipose-derived stem/stromal cells (ASCs) as an incredible device for learn and discovery. In Adipose-Derived Stem Cells: equipment and Protocols, specialists from the sector, together with individuals of the esteemed overseas Federation of Adipose Therapeutics and technology (IFATS), offer outlined and proven protocols which will additional codify the usage of those strong and obtainable cells. With chapters prepared round ways spanning the invention, pre-clinical, and medical approaches, a lot of the emphasis is put on human ASC, whereas extra strategies regarding small and massive animal species are integrated. As a quantity within the hugely winning tools in Molecular Biology™ sequence, the specific contributions comprise introductions to their respective subject matters, lists of the mandatory fabrics and reagents, step by step, quite simply reproducible laboratory protocols, and notes on troubleshooting and warding off identified pitfalls. accomplished and state-of-the-art, Adipose-Derived Stem Cells: equipment and Protocols serves as an important reference textual content for knowledgeable researchers in addition to new scholars at the route to extra exploring the remarkable capability of ASCs.
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Extra info for Adipose-Derived Stem Cells: Methods and Protocols
Metabolism 50, 407–13. Zuk, P. , De Ugarte, D. , Huang, J. , Alfonso, Z. , Fraser, J. , and Hedrick, M. H. (2002) Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 13, 4279–95. Zuk, P. , Futrell, J. , Katz, A. , Lorenz, H. , an\d Hedrick, M. H. (2001) Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 7, 211–28. , Lott, K. , Awad, H. , Hicok, K. , and Gimble, J. M. (2006) Clonal analysis of the differentiation potential of human adipose-derived adult stem cells.
Use a 100-mm filter (BD–Falcon) that is available for filtering small volumes. However, if it is desirable to collect the mature adipocytes at a later step, use a nylon filter (sterile) screen with a pore size of 250 mm. As has been reported (6), it is possible to separate small and large mature adipocytes for further analysis by using a 75-mm pore size filter at this step. Rather than aspirating the oil and yellow layer containing the adipocytes, carefully remove the oil and yellow layer and filter (BD-Falcon has filters of various pore sizes that work well for small volumes).
Transfer the medium containing the suspended cells from the flask to a sterile 50-ml conical tube. Centrifuge at 1,200 rpm (300 ´ g) for 5 min. Aspirate the supernatant and suspend the cells with a small volume of stromal medium (~2 ml). Proceed to cell counting by taking an aliquot of cells diluted in Trypan Blue (for a 1:4 dilution: add 25 ml of suspended cells to 75 ml of Trypan Blue). Count cells using the hemocytometer. After counting, you have several options: Cryopreservation Suspend the cell pellet in room-temperature freezing medium at a concentration of 2 × 106 cells/ml.