Abstract

Introduction: Myocardial infarction (MI) results from the death of cardiomyocytes (CM) following obstruction of blood flow and diminished oxygen supply to the tissue (hypoxia). Human adipose tissue-derived stem cells (hADSCs) used in pre-clinical models can replace damaged CM, however, this has not been replicated in human clinical trials due to early loss of hADSCs. We hypothesize that coupling of hADSCs to dying CMs may account for part of this loss. Furthermore, cell culturing is essential aspect of any in-vitro experiment. Through multiple trials we sought to maximize the efficiency of our culturing procedures in order to best facilitate the work in our lab.

Methods: Four aliquots of hADSCs were cultured and the effects of various reagents and culturing practices were investigated. To examine cell coupling, hADSCs will be cultured for different lengths of time with fluorescent dyes that are either permeable or impermeable to the cell membrane. We will assess the time course of coupling between hADSCs under both normoxic and hypoxic conditions by using fluorescent-activated cell sorting (FACS).

Results: Our previous studies demonstrate that adult mesenchymal stem cells possess membrane proteins (connexins) that contribute to cell-cell coupling. The proposed studies will address the functional significance of connexins related to hADSC coupling. The results of our hADSC culturing trials found we can optimize our cultures to facilitate these experiments through several procedural and reagent modifications. Key modifications to culturing protocols include, 1. decreased time spent in culture, 2. utilization of pure, established cell lots, 3. using smaller flasks.