202 IN VITRO DIFFERENTIATION OF ADULT PORCINE ADIPOSE-DERIVED STEM CELLS AFTER LABELING WITH PKH26 AND FLOW CYTOMETRY

Abstract

The purpose of this study was to investigate the possible effects of the fluorescent dye PKH26 and flow cytometry on adult porcine adipose-derived stem cells (ADSCs) after exposing them to adipogenic and osteogenic differentiation conditions. Adipose tissue was isolated from swine (11 months of age) and digested with 0.075% collagenase at 37°C for 90 min. The digested adipose tissue was centrifuged at 200g for 10 min to obtain a cell pellet. The pellet was re-suspended with DMEM, and the ADSCs were plated onto 75 cm² flasks (5000-10 000 cells per cm²) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% gentamicin. Passage 3 cells were labeled with fluorescent dye (PKH26 red fluorescent cell linker kit; Sigma Chemical, St. Louis, MO, USA) and sorted by flow cytometry. After labeling and sorting, the sorted and unsorted (control group) cells were replated and exposed to adipogenic (1 µM dexamethasone, 0.5 mM isobutylmethylxantine, 10 µM insulin and 200°M indomethacin) and osteogenic (0.1 µM dexamethasone, 10 mM ±-glycerophosphate, and 50°M ascorbic acid) differentiation conditions when the cells were 90% confluent. Cells were evaluated based on morphology and specific staining properties. Adipogenic differentiation was confirmed by oil red O-positive staining of large lipid vacuoles, and osteogenic differentiation by Von Kossa staining 2 weeks after initiation of differentiation. The frequency of oil red O-positive colonies in both sorted and unsorted group was similar (15.0% vs. 13.2%, respectively). The number of osteogenic nodules, confirmed by the presence of calcium by Von Kossa staining, in the sorted and unsorted group was 17 and 184 per flask, respectively. In conclusion, this study demonstrates that adult porcine adipose-derived stem cells maintain their differentiation potential after labeling with fluorescent dye and sorting by flow cytometry. This should allow for more rapid evaluation of the differentiation potential of ADSCs in vitro.

This work was partially supported by the Council for Food and Agricultural Research (C-FAR) Sentinel Program, University of Illinois and CNPq, Brazil (M. Mello).