Dissociated and Explant Cultures of Myenteric Neurons of Pig Small Intestine
Identifieur interne : 001683 ( Istex/Corpus ); précédent : 001682; suivant : 001684Dissociated and Explant Cultures of Myenteric Neurons of Pig Small Intestine
Auteurs : C. Van Ginneken ; U. Rauch ; A. Weyns ; K. Sch FerSource :
- Anatomia, Histologia, Embryologia [ 0340-2096 ] ; 2005-12.
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.
Url:
DOI: 10.1111/j.1439-0264.2005.00669_121.x
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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.</div></front></TEI><istex><corpusName>wiley</corpusName><author><json:item><name>C. 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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. 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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. 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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 <i>in vitro</i> experimental set‐up is most appropriate. Methods to obtain <i>in vitro</i> 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 mm<sup>2</sup> were transferred to BSS with 1 mg/ml collagenase and 1 mg/ml trypsin inhibitor and incubated for 60 min (5% CO<sub>2</sub>, 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.</p></abstract></abstractGroup></contentMeta></header></component></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>Dissociated and Explant Cultures of Myenteric Neurons of Pig Small Intestine</title></titleInfo><titleInfo type="abbreviated" lang="en"><title>Abstracts</title></titleInfo><titleInfo type="alternative" contentType="CDATA" lang="en"><title>Dissociated and Explant Cultures of Myenteric Neurons of Pig Small Intestine</title></titleInfo><name type="personal"><namePart type="given">C.</namePart><namePart type="family">Van Ginneken</namePart><affiliation>Laboratory of Veterinary Anatomy & Embryology, Department of Veterinary Medicine, Faculty of Farmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, universiteitsplein 1, 2610 Antwerp, Belgium</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">U.</namePart><namePart type="family">Rauch</namePart><affiliation>Lehrgebiet Biotechnologie, Fachbereich Informatik und Mikrosystemtechnik, Fachhochschule Kaiserslautern, Standort Zweibrücken, Amerikastrasse 1, D‐66482 Zweibrücken, Germany</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">A.</namePart><namePart type="family">Weyns</namePart><affiliation>Laboratory of Veterinary Anatomy & Embryology, Department of Veterinary Medicine, Faculty of Farmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, universiteitsplein 1, 2610 Antwerp, Belgium</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">K. ‐H.</namePart><namePart type="family">Schäfer</namePart><affiliation>Lehrgebiet Biotechnologie, Fachbereich Informatik und Mikrosystemtechnik, Fachhochschule Kaiserslautern, Standort Zweibrücken, Amerikastrasse 1, D‐66482 Zweibrücken, Germany</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="abstract" displayLabel="abstract"></genre><originInfo><publisher>Blackwell Verlag GmbH</publisher><place><placeTerm type="text">Berlin, Germany</placeTerm></place><dateIssued encoding="w3cdtf">2005-12</dateIssued><copyrightDate encoding="w3cdtf">2005</copyrightDate></originInfo><language><languageTerm type="code" authority="rfc3066">en</languageTerm><languageTerm type="code" authority="iso639-2b">eng</languageTerm></language><physicalDescription><internetMediaType>text/html</internetMediaType></physicalDescription><abstract lang="en">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.</abstract><relatedItem type="host"><titleInfo><title>Anatomia, Histologia, Embryologia</title></titleInfo><genre type="journal">journal</genre><identifier type="ISSN">0340-2096</identifier><identifier type="eISSN">1439-0264</identifier><identifier type="DOI">10.1111/(ISSN)1439-0264</identifier><identifier type="PublisherID">AHE</identifier><part><date>2005</date><detail type="volume"><caption>vol.</caption><number>34</number></detail><detail type="supplement"><caption>Suppl. no.</caption><number>s1</number></detail><extent unit="pages"><start>53</start><end>53</end></extent></part></relatedItem><identifier type="istex">2B9D4FD4D2BAFF25F4CB6EF44A0D5FD37D487C9E</identifier><identifier type="DOI">10.1111/j.1439-0264.2005.00669_121.x</identifier><identifier type="ArticleID">AHE669_121_121</identifier><recordInfo><recordContentSource>WILEY</recordContentSource><recordOrigin>Blackwell Verlag GmbH</recordOrigin></recordInfo></mods></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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