A Novel Method of Human Adipose-derived Stem Cell Isolation With Resultant Increased Cell Yield.

Background: Adipose tissue represents an abundant and easily accessible source of multipotent cells, which may serve as excellent building blocks for tissue engineering. We have developed a novel protocol for isolating adipose-derived stromal cells (ASCs) from human lipoaspirate. The aim of this study was to compare our new method for ASC isolation to a previously published standard protocol. Methods: Human ASC isolation was performed using two methods to compare cell yield, cell viability, cell proliferation and regenerative potential. In brief, the new (NM) and conventional methods (CM) of ASC isolation differ in two key areas: the collagenase digestion buffer constituents and the use of an orbital shaker. The osteogenic and adipogenic potential of ASCs isolated using both protocols were assessed in vitro and gene expression analysis was performed. To assess the ability of isolated ASCs to generate bone in vivo, we created critical sized calvarial defects in mice, which were treated with ASCs loaded onto HA-PLGA scaffolds. In addition, to test the ability of isolated ASCs to enhance adipogenesis, ASCs were added to lipoaspirate and placed beneath the scalp of immunocompromised mice. Fat graft volume retention was subsequently assessed by serial computed tomographic volumetric scanning. Results: The NM results in a 10-fold increased yield of ASCs compared to CM. Resulting ASCs harvested using the NM demonstrate significantly increased cell viability and proliferation in vitro (*p

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