[Experimental study on fluorescent labeling and optimization method of purifying human stromal vascular fraction...

To find a kind of simple and effective method for purifying and labeling stromal vascular fraction cells (SVFs) so as to provide a theoretical basis for clinical application of SVFs.

The subcutaneous adipose tissue were harvested form volunteers. The adipose tissue was digested with 0.065%, 0.125%, and 0.185% type I collagenase, respectively. SVFs were harvested after digestion and counted. After trypan blue staining, the rate of viable cells was observed. SVFs was labeled by 1, l'-dioctadecyl-3, 3, 3', 3'-2-tetramethy-lindocyanine perchlorate (DiI). The fluorescent labeling and growth was observed under an inverted fluorescence microscope. MTT assay was used to detect cell proliferation.

The number of SVFs was (138.68 +/- 11.64) x 10(4), (183.80 +/- 10.16) x 10(4), and (293.07 +/- 8.31) x 10(4) in 0.065% group, 0.125% group, and 0.185% group, respectively, showing significant differences among 3 groups (P < 0.01). The rates of viable cells were 91% +/- 2%, 90% +/- 2%, and 81% +/- 2% in 0.065% group, 0.125% group, and 0.185% group, respectively, and it was significantly higher in 0.065% group and 0.125% group than in 0.185% group (P < 0.01), but no significant difference was found between 0.065% group and 0.125% group (P=0.881). Inverted fluorescence microscope showed that the cell membranes could be labeled by DiI with intact cell membrane, abundant cytoplasm, and good shape, but nucleus could not labeled. SVFs labeled by DiI could be cultured successfully and maintained a normal form. MTT assay showed that similar curves of the cell growth were observed before and after DiI labeled to SVFs.

The optimal collagenase concentration for purifying SVFs is 0.125%. DiI is a kind of ideal fluorescent dye for SVFs.