Figure 3: Re-plated MSC after 24 h of culture upon dissociation with a Trypsin and b enzyme-free dissociation buffer.

Mentions: As seen in Figure 2, the proportion of viable MSC that re-attached was significantly higher (p = 0.0004) upon dissociation with trypsin (82.1% ± 2.0%) compared to enzyme-free dissociation buffer (5.0% ± 0.2%). The same trend was observed after the dissociated MSC were subjected to freeze–thawing (68.4% ± 3.8% versus 2.8% ± 0.4%, respectively, p = 0.002). As seen in Figure 3, there was a higher proportion of attached cells 24 h after reseeding MSC dissociated by trypsin than with enzyme-free buffer. Virtually, all the non-attached cells were confirmed to be nonviable by manual trypan-blue staining (data not shown). Freeze–thawing significantly reduced the proportion of viable reattached MSC upon dissociation with trypsin (82.1% ± 2.0% versus 68.4% ± 3.8%, p = 0.01), but not with enzyme-free dissociation buffer (5.0 ± 0.6% versus 2.8% ± 2.1%, p = 0.17).

Heng BC, Cowan CM, Basu S - Biol Proced Online (2009)

Bottom Line: A significantly lower proportion of viable cells were obtained with enzyme-free dissociation buffers compared to trypsin.The proportion of viable cells that reattached was significantly lower for cells obtained by dissociation with enzyme-free dissociation buffer compared to trypsin.It was also demonstrated that exposure of trypsin-dissociated MSC to enzyme-free dissociation buffer for 1 h had no significant detrimental effect on cell viability.

Abstract: The dissociation of adherent mesenchymal stem cell (MSC) monolayers with trypsin and enzyme-free dissociation buffer was compared. A significantly lower proportion of viable cells were obtained with enzyme-free dissociation buffers compared to trypsin. Subsequently, the dissociated cells were re-seeded on new cell culture dishes and were subjected to the MTT assay 24 h later. The proportion of viable cells that reattached was significantly lower for cells obtained by dissociation with enzyme-free dissociation buffer compared to trypsin. Frozen-thawed MSC displayed a similar trend, yielding consistently higher cell viability and reattachment rates when dissociated with trypsin compared to enzyme-free dissociation buffer. It was also demonstrated that exposure of trypsin-dissociated MSC to enzyme-free dissociation buffer for 1 h had no significant detrimental effect on cell viability.