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.

Development of an intestinal cell culture model to obtain smooth muscle cells and myenteric neurones.

This paper reports on the development of an entirely new intestinal smooth muscle cell (ISMC) culture model using rat neonates for use in pharmacological research applications. Segments of the duodenum, jejunum and ileum were obtained from Sprague-Dawley rat neonates. The cell extraction technique consisted of ligating both ends of the intestine and incubating (37 degrees C) in 0.25% trypsin for periods of 30-90 min. Isolated cells were suspended in DMEM-HEPES, plated and allowed to proliferate for 7 days. Cell culture quality was assessed via a series of viability tests using the dye exclusion assay. In separate experiments, tissues were exposed to trypsin for varying durations and subsequently histological procedures were applied. Cell purification techniques included differential adhesion technique for minimizing fibroblasts. Selective treatments with neurotoxin scorpion venom (30 microg mL(-1)) and anti-mitotic cytosine arabinoside (6 microm) were also applied to purify respectively ISMC and myenteric neurones selectively. The different cell populations were identified in regard to morphology and growth characteristics via immunocytochemistry using antibodies to smooth muscle alpha-actin, alpha-actinin and serotonin-5HT3 receptors. Based on both viability and cell confluence experiments, results demonstrated that intestinal cells were best obtained from segments of the ileum dissociated in trypsin for 30 min. This provided the optimum parameters to yield highly viable cells and confluent cultures. The finding was further supported by histological studies demonstrating that an optimum incubation time of 30 min is required to isolate viable cells from the muscularis externae layer. When cell cultures were treated with cytosine arabinoside, the non-neuronal cells were abolished, resulting in the proliferation of cell bodies and extended neurites. Conversely, cultures treated with scorpion venom resulted in complete abolition of neurones and proliferation of increasing numbers of ISMC, which were spindle-shaped and uniform throughout the culture. When characterized by immunocytochemistry, neurones were stained with antibody to 5HT3 receptors but not with antibodies to alpha-smooth muscle actin and alpha-actinin. Conversely, ISMC were stained with antibodies to alpha-smooth muscle actin and alpha-actinin but not with antibody to 5HT3 receptors. The present study provides evidence that our method of dissociation and selectively purifying different cell populations will allow for pharmacological investigation of each cell type on different or defined mixtures of different cell types.