ComparisonofEnzymaticandNonEnzymaticMeans1

Comparison of Enzymatic and Non-Enzymatic Means of Dissociating Adherent Monolayers of Mesenchymal Stem Cells

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.

Key Words: Dissociation - Enzyme - Mesenchymal - Stem cells - Trypsin

Introduction

Bone marrow-derived mesenchymal stem cells (MSC) have demonstrated tremendous potential in the emerging field of regenerative medicine (1–4). Nevertheless, a major challenge faced in the clinical application of MSC is the need for adequate cell numbers for achieving optimal efficacy in transplantation therapy. Hence, MSC need extensive ex vivo proliferation within prolonged durations of in vitro culture (5, 6). To avoid pathogenic transmission, it is imperative to minimize animal and human-derived products within MSC culture (7, 8). One key animal-derived product is the digestive enzyme trypsin, which is routinely used to dissociate adherent MSC monolayers into single-cell suspensions during serial passages. Alternative enzyme-free methods for dissociating adherent cell monolayers have been developed, such as the various commercially available enzyme-free dissociation buffers (9, 10) that work on the principle of chelating free calcium and magnesium ions in solution; as well as recombinant trypsin-like proteolytic enzymes produced from bacterial fermentation (i.e., TrypLE? Express commercially available from Gibco, Gaithersburg, MD, USA). However, it must be noted that enzymatic cell dissociation inevitably results in some degradation of surface proteins and glycoproteins. Hence, 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).

This study compares the dissociation of adherent MSC monolayers with either enzyme-free dissociation buffer or trypsin, the most commonly used enzyme for the dissociation of in vitro cultured cells. The viability of the newly dissociated cells was assessed by a simple trypan blue exclusion assay through the use of an automated cell counter. Subsequently, the dissociated MSC was re-plated on new cell culture dishes and subjected to the MTT assay 24 h later, to assess the re-attachment of viable cells. Additionally, similar assays were conducted on frozen–thawed MSC that were dissociated either with trypsin or enzyme-free dissociation buffer.

Materials and Methods

Cell Viability Assessment

Bone marrow-derived human MSC (Cat no: PT-2501, batch no: 6F4382, cryopreserved at the second passage) were purchased from Lonza (Walkersville, MD, USA). Cryopreserved MSC were thawed and cultured up to five passages upon purchase from Lonza (Walkersville, MD, USA), prior to being utilized for this study. 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 then 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. 13151-014; Gibco BRL, Gaithersburg, MD, USA). According to the manufacturer’s specification, the enzyme-free cell dissociation buffer is a membrane-filtered, isotonic, and enzyme-free aqueous formulation of salts, chelating agents, and cell-conditioning agents constituted in Ca²⁺- and Mg²⁺-free PBS. Prior to cell dissociation, both trypsin and the enzyme-free cell dissociation buffer were pre-warmed to 37°C within a water bath. The confluent monolayers of MSC cultured within 12-well cell culture dishes were then washed two times with Ca²⁺-free PBS, prior to the addition of 1 ml trypsin solution or enzyme-free cell-dissociation buffer within each well. These were then placed within the cell culture incubator and subjected to gentle pipetting every 2–3 min. On average, dissociation of confluent MSC monolayers with trypsin takes approximately 5–6 min with gentle pipetting; while with enzyme-free cell dissociation buffer, the corresponding duration is approximately 15 to 16 min. All pipeting was carried out with an automated pipet pump (VWR, Brisbane, CA, USA), and care was taken to ensure that the pipeting force was similar for both experimental groups, by utilizing the same settings and similar volume pipettes. The dissociated cell suspension from each well of the 12-well dish [(1.17?±?0.06)?×?10⁵ cells in 1 ml] were then placed in 1.5 ml microcentrifuge tubes and subjected to centrifugation at 500×g for 5 min. The supernatant was discarded, and the cell pellet was reconstituted in 0.5 ml PBS (with Ca²⁺), placed within accessory sample vials (Cat no. 383721, Beckman-Coulter, Fullerton, CA, USA) and analyzed for cell viability with the trypan blue exclusion assay, by utilizing an automated cell counter (Vi-Cell? XR analyzer, Cat no. 383556; Beckman Coulter, Fullerton, CA, USA) and Vi-CELL? XR Quad Pak Reagent Kit (Cat no. 383198; Beckman-Coulter, Fullerton, CA, USA). The automated cell counter mixes the cell suspension with an equal volume of 0.4% (w/v) trypan blue solution (500 μl), and automatically accounts for the fold dilution with trypan blue solution. For each experimental group, there were three replicate readings.