Reply: Mechanical Micronization of Lipoaspirates
First, Bertheuil et al. pointed out that we did not determine whether the adipose-derived stem/stromal cells obtained from the squeezed fat or emulsified fat meet classic criteria of adipose-derived stem/stromal cells, such as colony-forming ability, multilineage differentiation capacity, and the presence of mesenchymal markers (CD105, CD73, and CD90).2 Although we did not show the detailed data of the function of cultured cells because of the limitation of figure numbers and text volume, we confirmed the function of adipose-derived stem/stromal cells as we did in many previous publications. In general, cultured adipose-derived stem/stromal cells did not show large differences in function among a variety of methods for harvest, isolation, and storage. According to our examination, CD90 is generally positive in any type of mesenchymal lineage cell, and CD73 and CD105 are also known to be relatively positive in cultured adipose-derived stem/stromal cells as we have shown previously.2,3 Thus, we do not think that these three surface antigens are definitive markers of adipose-derived stem/stromal cells.
Regarding the second comment that stromal vascular fraction after emulsification of the fat (fluid fraction) contained functional adipose-derived stem/stromal cells that fulfilled all the above criteria,4 it is true that the fluid fraction contained isolated adipose-derived stem/stromal cells; unfortunately, the quantity of isolated adipose-derived stem/stromal cells has been very small in all types of mechanical isolation so far. Actually, it was first published by us in 2006 that even the fluid portion of liposuction aspirates contain stromal vascular fraction, including adipose-derived stem/stromal cells.3 Based on our study for emulsification and squeeze, it should be emphasized that these mechanical methods are not as efficient for stromal vascular fraction isolation as enzymatic digestion at all. We could not perform flow cytometric analysis of isolated cells in the fluid portion without cell culture expansion in this study, because the cell number was too small. Instead, some fluid obtained from mechanical processing has numerous microtissue fractions containing adipose-derived stem/stromal cells (but not isolated adipose-derived stem/stromal cells). Thus, it is not appropriate to refer to them as “stromal vascular fraction,” as we proved that filtrated fluid of emulsified fat was filled with extracellular matrix fragments by scanning electron microscopy.1 According to the article by Bertheuil et al.,4 the liquid portion of emulsified fat provided only one-seventh to one-twelfth viable nucleated cells from 1 g of lipoaspirate compared with enzymatically digested stromal vascular fraction. After cell culture, adipose-derived stem/stromal cells can grow also from microtissue fragments that are seeded as well.
In conclusion, fluid products obtained from mechanical processing of lipoaspirates may be used therapeutically, but we need to understand what and how many cells or tissue fragments are really contained in the final product before use, and the clinical value of fluid products remains unknown. Depending on mechanical processing methods, most of the adipose-derived stem/stromal cells remain in the macrotissues, which can be easily removed from the fluid products by filtration. It will be of great interest to see further optimization of mechanical processing protocols in the future. We need to investigate biological components of each product and carefully evaluate their therapeutic effects in animals and human.