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

Abstract

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 ºC) 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 µ g mL -1 ) and anti-mitotic cytosine arabinoside (6 µ M ) 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 α -actin, α -actinin and serotonin-5HT 3 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 5HT 3 receptors but not with antibodies to α -smooth muscle actin and α -actinin. Conversely, ISMC were stained with antibodies to α -smooth muscle actin and α -actinin but not with antibody to 5HT 3 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.

Establishment of Human Trophoblast Progenitor Cell Lines from the Chorion

“…Accordingly, we used trypsin to dissociate the colonies into single cell suspensions, which disrupted polarity and enabled differential adhesion, a method we and others have used to purify primary cells [59]. As shown in Figure 2A, the resulting cells grew as dispersed monolayers rather than as colonies.…”

Methods to Study the Myenteric Plexus of Rat Small Intestine

“…To culture enteric neurons, Lobo et al showed that it is possible to extract just the desired portion of the intestine, followed by 30 min of trypsinization and incubation of all the detached cells (Lobo et al 2007). Even though this procedure is relatively fast and easy, it results in a cell culture that depicts the composition of the entire intestine and not just enteric neurons.…”

NR4A1 modulates intestinal smooth muscle cell phenotype and dampens inflammation‐associated intestinal remodeling

“…The mixture was centrifuged at 1000 rpm for 10 min at 4°C, the pellet was resuspended in 10 ml of fresh media, and a differential adherence approach was used to selectively separate fibroblasts from SMCs. 33 Briefly, the cell mixture was placed in pre-warmed culture flasks for 20 min at 37°C, where fibroblasts adhered, and the suspension of non-adherent cells was placed in another pre-warmed culture flask whereby SMCs derived from the muscularis mucosae, circular layer, and longitudinal layer adhered. After 3 weeks in culture, SMCs were propagated in DMEM/ Nutrient Mixture F-12 Ham media containing l-glutamine, A/A, and SMC supplement pack (C-39363, PromoCell).…”