Comparison of enzymatic and non-enzymatic means of dissociating adherent monolayers of mesenchymal stem cells

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.

Figure 1

Proportion of viable MSC (as determined by trypan blue exclusion assay) upon dissociation with trypsin and enzyme-free dissociation buffer, before and after freeze–thawing in 10% (v/v) DMSO.

Figure 2

Proportion of viable reattached MSC upon dissociation with trypsin and enzyme-free dissociation buffer, with and without freeze–thawing in 10% (v/v) DMSO. This was assessed by MTT assay, 24 h after re-plating the dissociated cells.

Figure 3

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

Figure 4

Proportion of viable MSC (in free suspension) after dissociation with trypsin and incubation for 1 h in enzyme-free dissociation buffer.