Mesenchymal stem cell detachment with trace trypsin is superior to EDTA for in vitro chemotaxis and adhesion assays

Abstract

Trypsin is frequently used to dissociate mesenchymal stem cells (MSCs) for in vitro adhesion and chemotaxis assays. However, its potential impact on surface receptor degradation is poorly understood. The purpose of this study was to evaluate the effect of trypsin-EDTA exposure versus PBS-EDTA on MSC surface receptor integrity and function. Primary human MSCs were detached with PBS-EDTA alone, or Cell Dissociation Buffer followed by 30 s exposure to 0.05% w/v trypsin-EDTA (trace trypsin method, TT), or 0.25% w/v trypsin exposure for 2 or 5 min. Cells were characterized for surface integrity of β1 integrin (CD29) and PDGF Receptor (PDGF-R), and assessed in vitro for adhesion to atelocollagen-coated surfaces and migration to PDGF-BB. PBS-EDTA detachment fully preserved receptor integrity but routinely detached only half of the adherent cells and led to cell aggregates that failed to adhere evenly across the Transwell migration insert. Both CD29 and PDGF-R were significantly degraded by 0.25% trypsin detachment for 2 or 5 min compared to the TT method or PBS-EDTA (p < 0.05). Cells migrated optimally to PDGF-BB when detached with the TT method (3.1-fold vs α-MEM, p = 0.01). Cells attached optimally to atelocollagen when detached using the TT method or PBS-EDTA (6- to 10-fold vs 0.25% trypsin, p < 0.01). CDB followed by trace trypsin-EDTA exposure is recommended over PBS-EDTA to produce a single-cell MSC suspension that preserves receptor integrity and more reproducible receptor-mediated responses.

Keywords: Cell adhesion; Cell migration; Mesenchymal stem cells; Trypsin.