Technique for cryopreservation of intestinal smooth muscle cells

Attempts were made to develop a simplified procedure for long-term cryopreservation of intestinal smooth muscle cells (ISMC). ISMC were collected from the ileum of Sprague-Dawley neonatal rats through cellular dissociation in trypsin. Cryopreservation method comprised of a rapid 1-step (protocol 1) and a slow 3-step (protocol 2) freezing of ISMC for 1week. Preparations were thawed and single ISMC were assessed via the comet assay and damaged DNA was quantified through comet tail moment. The control unfrozen ISMC exhibited DNA damage of 2.34+/-0.35 compared to ISMC cooled via protocol 2 (2.62+/-0.36) and protocol 1 (10.15+/-0.72). Thereafter, protocol 2 freezing method was adopted and ISMC were cryopreserved for 1-week, 1-month, and 4-months to analyse the temporal and long-term cryopreservation of ISMC. This revealed a DNA damage of 2.62+/-0.36 (1-week), 3.81+/-0.72 (1-month), and 5.1+/-0.9 (4-months). Gradual cooling is suitable for continuing storage of ISMC and although fluctuation in cryoinjury is observed with time this is considered to reflect cell-to-cell variability.