ComparisonofEnzymaticandNonEnzymaticMeans2

MTT Assay on Reattached Cells

MSC were seeded in 12-well cell culture dishes with 5.0?×?10⁴ cells per well (≈4.8 cm²). After 5 to 6 days of culture, confluent MSC monolayers were attained (1.17?×?10⁵ cells per well), and these were then dissociated with either trypsin or enzyme-free dissociation buffer. Some wells containing intact confluent MSC monolayers were retained as the control and were not dissociated with either trypsin or enzyme-free cell dissociation buffer. The dissociated cell suspension from each well were collected and placed within microcentrifuge tubes and subjected to centrifugation at 500×g for 5 min. The supernatant was then discarded, and the cell pellet was reconstituted in 1.0 ml of cell culture media (MSCGM? bullet kit, Cat no. PT-3001; Lonza, Walkersville, MD, USA) prior to being re-seeded onto fresh 12-well cell culture dishes. After 24 h of culture, the unattached cells were washed off with PBS, and the reattached cells were then subjected to the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (13). Briefly, this involved placing 1.0 ml of 1 mg/ml MTT constituted in culture media within each well, following by incubation for 3 h at 37°C in the dark. After incubation, the MTT solution was removed, and the stained cells were washed two times in PBS followed by air-drying. The MTT-formazan products were extracted in the dark at room temperature with 0.25 ml of DMSO in each well. One hundred microliters aliquots of the supernatant in each well were then transferred into a 96-well flat-bottomed cell culture plates, and the absorbance was measured spectrophotometrically at 570 nm using a SpectraMax M5 modular microplate reader (Molecular Devices Corporation, Sunnyvale, CA, USA). From the absorbance values, the percentage of reattached viable cells (after dissociation with trypsin and cell-free dissociation buffer) can then be computed by dividing the MTT absorbance values obtained after dissociation with the absorbance reading for the nondissociated control (after correction for 100 μl DMSO blanks).

Effects of Cryopreservation on Cell Viability and Reattachment

Trypan blue exclusion and MTT assays were repeated on frozen–thawed MSC that had been dissociated either with trypsin or enzyme-free dissociation buffer. The cryopreservation solution was composed of DMEM supplemented with 10% (v/v) fetal calf serum (FCS) and 10% (v/v) DMSO. The dissociated MSC were suspended in 1 ml of cryopreservation solution within cryovials and were subjected to slow cooling within a ?80°C refrigerator, through the use of isopropanol freezing containers (Nalgene, Rochester, NY, USA). After 2 h, the frozen cell-suspension within cryovials were immersed and stored in the vapor phase of liquid nitrogen for 1 h, prior to quick thawing within a water bath at 37°C.

Effects of Prolonged Exposure to Enzyme-free Dissociation Buffer

MSC dissociated with trypsin were exposed to enzyme-free dissociation buffer at 37°C (2.0?×?10⁵ cells per ml) for 1 h within 15 ml polypropylene tubes (Becton-Dickinson) that do not allow cell attachment. Viability of MSC in free suspension, before and after exposure to the enzyme-free dissociation buffer was assessed by the trypan blue exclusion assay, utilizing an automated cell counter.

Statistical Analysis of Data

There were three replicates for each experimental group, and the results from each data set were expressed as mean?±?standard derivations. Differences between data sets were assessed by the paired Student’s t test, with a value of p?<?0.05 being considered significantly different.

Results

Cell Viability Assessment

The proportion of viable MSC was significantly higher (p? =?0.002) upon dissociation with trypsin (93.2%?±?3.2%) compared to enzyme-free dissociation buffer (68.7%?±?5.0%), as seen in Fig.  1 . The same trend was observed after the dissociated MSC were subjected to freeze–thawing (90.8%?±?2.8% versus 68.7%?±?7.1%, respectively, p? =?0.007). Immediately 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%, p?>?0.05) or enzyme-free dissociation buffer (68.7%?±?5.0% versus 68.7%?±?7.1%, p?>?0.05).

Fig. 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.