ComparisonofEnzymaticandNonEnzymaticMeans3

MTT Assay on Reattached Cells

As seen in Fig.  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 Fig.  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).

Fig. 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.

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

Effects of Prolonged Exposure to Enzyme-free Dissociation Buffer

As seen in Fig.  4 , there was no significant change in cell viability after exposure of trypsin dissociated MSC to enzyme-free dissociation buffer for 1 h, (95.8%?±?1.8% versus 91.8%?±?3.9%, p?>?0.05)

Fig. 4 Proportion of viable MSC (in free suspension) after dissociation with trypsin and incubation for 1 h in enzyme-free dissociation buffer.

Discussion

To avoid the proteolytic effect of trypsin, commercially available enzyme-free cell dissociation buffers are sometimes utilized to preserve the structural integrity of membrane surface proteins for ligand binding flow cytometry and immunohistochemistry (11, 12). Nevertheless, enzyme-free dissociation buffers are not commonly used for the routine serial passage of various primary cell cultures and highly adherent cell lines; despite their potential to be “more gentle” on cells and avoid the potentially damaging proteolytic effects of trypsin on cell surface proteins. To our knowledge, there has not yet been any study to date that systematically investigates and compares the dissociation of confluent MSC monolayers with trypsin and enzyme-free dissociation buffer.

The results of our study showed that the immediate cell viability (trypan blue) was significantly higher upon dissociation with trypsin, compared to enzyme-free dissociation buffer. This was not because exposure of MSC to the enzyme-free dissociation buffer itself had any detrimental effect on cell viability, as demonstrated by the control experiment (Fig.  4 ). Neither is it because of the sudden removal of magnesium and calcium ions through EDTA chelation; because the MSC monolayers were washed with calcium and magnesium free PBS prior to dissociation, and the trypsin solution itself contains EDTA (0.53 mM EDTA?×?4 Na) in Ca⁺²- and Mg⁺²-free Hanks balanced salt solution. The lower cell viability with enzyme-free dissociation buffer compared to trypsin is thus manifested immediately upon dissociation and is caused neither by deprivation of free calcium ions, nor by prolonged exposure to the enzyme-free buffer per se. Future work is needed to uncover the underlying mechanisms behind these observations. In any case, it can be inferred that dissociation with the enzyme-buffer is more detrimental to MSC viability compared to trypsin. TrypLE? Express was not examined in this study because of the lack of information on the proprietary protease used in it. It is claimed by the manufacturer to strongly resemble trypsin and was therefore expected to produce results comparable to that obtained by Trypsin.

Additionally, the MTT assay showed a stark decrease in the proportion of viable reattached MSC upon dissociation with the enzyme-free buffer compared to trypsin (Figs.  2 and 3 ). Since natural tissue remodeling in vivo requires the breakdown of extracellular matrix by proteolytic enzymes (14–16), so as to enable cellular detachment, migration, and reattachment, it is plausible that the proteolysis of extracellular matrix and adhesion proteins may serve as a natural impetus or signal for cell reattachment to a new substratum. This is probably recapitulated in vitro through the proteolytic action of trypsin. With enzyme-free dissociation buffer, extracellular matrix and adhesion proteins synthesized by the cultured MSC are not digested away. Hence, there is a lack of natural impetus to reattach to a new substratum upon reseeding on new culture dishes. This could possibly explain the stark decrease in the proportion of viable reattached MSC upon dissociation with the enzyme-free buffer compared to trypsin (Figs.  2 and 3 ).

The observation that freeze–thawing did not lead to an immediate significant decrease in cell viability (Fig.  1 ), but resulted in a significant drop in the proportion of viable reattached MSC (Fig.  2 ) is not surprising, considering the fact that post-cryopreservation apoptosis in MSC is well-documented (17). Apoptosis takes a prolonged duration to manifest and would not be evident immediately after freeze–thawing.

In conclusion, our results show that the dissociation of MSC by enzyme-free buffer, as opposed to trypsin, causes a significant decrease in cell viability and reattachment of the dissociated MSC and, hence, is clearly unsuited for routine serial passage and propagation of MSC as used. Additionally, the data presented in this study would also have implications for the re-plating of dissociated MSC after cell-sorting, since enzyme-free cell dissociation is commonly used in fluorescence activated cell sorting (FACS) and magnetic affinity cell sorting (MACS ). Further studies are recommended to optimize the use of such buffers to dissociate MSCs for use in routine culture or for cell sorting purposes.

Acknowledgment  The work was supported entirely by grants from Abbott Vascular.