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.
Highlights
Bone marrow-derived mesenchymal stem cells (MSC) have demonstrated tremendous potential in the emerging field of regenerative medicine [1,2,3,4]
Confluent monolayers of MSC cultured within 12-well cell culture dishes (≈1.0–1.5×105 cells per well, surface area≈ 4.8 cm2) were dissociated with either 0.05% (w/v) Trypsin–EDTA (0.53 mM EDTA×4 Na, Cat no. 25300-054; Gibco BRL, Gaithersburg, MD, USA) or enzyme-free phosphate-buffered saline (PBS)-based cell dissociation buffer (Cat no. 13151014; Gibco BRL, Gaithersburg, MD, USA)
After freeze–thawing, there was no significant reduction in the viability of MSC dissociated either with trypsin (93.2%±3.2% versus 90.8%±2.8%, p90.05) or enzyme-free dissociation buffer (68.7%±5.0% versus 68.7%±7.1%, p90.05)
Summary
Bone marrow-derived mesenchymal stem cells (MSC) have demonstrated tremendous potential in the emerging field of regenerative medicine [1,2,3,4]. It is imperative to minimize animal and human-derived products within MSC culture [7, 8]. 162 Heng, Cowan, and Basu the digestive enzyme trypsin, which is routinely used to dissociate adherent MSC monolayers into single-cell suspensions during serial passages. It must be noted that enzymatic cell dissociation inevitably results in some degradation of surface proteins and glycoproteins. Enzyme-free cell dissociation is instead sometimes preferred to preserve the structural integrity of membrane surface proteins for ligand binding flow cytometry and immunohistochemistry [11, 12]
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