Dissociated and Explant Cultures of Myenteric Neurons of Pig Small Intestine

Abstract

The gastrointestinal tract of pig is a good and available (slaughterhouse material) model to study the enteric nervous system in relation to nutrition, inflammation, development and disease in general. In order to investigate the responses of the enteric nervous network to a variety of stimuli, e.g. growth factors, hormones, extracellular matrix components, but also noxious compounds in a controlled manner, an in vitro experimental set‐up is most appropriate. Methods to obtain in vitro cultures of enteric neurons of rodents, chicken and human are well described. This study attempted to use these methods on pig material. The muscle layer containing the myenteric plexus was dissected from pieces of fetal, neonatal and adult pig jejunum. They were rinsed in Hanks basal salt solution (BSS) in which antibiotics were added. Pieces of ±25 mm2 were transferred to BSS with 1 mg/ml collagenase and 1 mg/ml trypsin inhibitor and incubated for 60 min (5% CO2, 90% RH). Subsequently, they were vortexed for 20 s and ganglia were selected. The procedure was repeated ±4 times. Myenteric ganglia could then be plated or further dissociated in 1 mg/ml trypsin in BSS for 30 min in the incubator. Afterwards, the dissociated ganglia were centrifuged (5 min 1500 rpm) and the trypsin‐solution was replaced with Dulbecco's minimal essential medium (DMEM). The explants or dissociated myenteric ganglia were transferred on non‐coated or coated (poly‐L‐lysine, laminin, fibronectin, collagen or extracellular matrix gel) cover slips. After incubating for 45 min they were topped with 1 ml of DMEM with antibiotics and with or without fetal calf serum (5%). Medium was replaced twice a week. Using immunohistochemistry, both neurons (PGP9.5) and glial cells (S100) could be identified in both culture types. When cultivated under harsh conditions, the dissociated cultures gave rise to neurosphere‐like bodies, containing neurons and glial cells. Thus, the digestion and dissociation technique is applicable to pig material.