Establishment of a long-term culture system for rat colon epithelial cells

Bartsch, Ingrid; Zschaler, Ingrid; Haseloff, Monika; Steinberg, Pablo

doi:10.1290/0404035

Cited by 4 publications

(10 citation statements)

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“…After connective tissue was carefully removed under microscope, the esophageal mucosa was cut into squares and the squares were incubated in a trypsin-EDTA solution (Mediatech, Manassas, VA) for 15 min at 37°C in a shaking water bath set to agitate at 100 rpm with the epithelium facing downward. The epithelial cells were separated from the tissue by scraping away the overlaying tissue with a small forceps, and the remaining cells were pipetted multiple times to dissociate large cell clumps (2). The tissue was transferred to a second petri dish containing a similar solution without trypsin and containing 5 g/ml soybean trypsin inhibitor.…”

Section: Methodsmentioning

confidence: 99%

HCl-activated neural and epithelial vanilloid receptors (TRPV1) in cat esophageal mucosa

Cheng¹,

Monte²,

Ma³

et al. 2009

American Journal of Physiology-Gastrointestinal and Liver Physi

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To test whether transient receptor potential channel vanilloid subfamily member-1 (TRPV1) mediates acid-induced inflammation in the esophagus, a tubular segment of esophageal mucosa was tied at both ends, forming a sac. The sac was filled with 0.01 N HCl (or Krebs buffer for control) and kept in oxygenated Krebs buffer at 37°C. The medium around the sac (supernatant) was collected after 3 h. Supernatant of the HCl-filled sac abolished contraction of esophageal circular muscle strips in response to electric field stimulation. Contraction was similarly abolished by supernatant of mucosal sac filled with the TRPV1 agonist capsaicin (10 Ϫ6 M). These effects were reversed by the selective TRPV1 antagonist 5Ј-iodoresiniferatoxin (IRTX) and by the plateletactivating factor (PAF) receptor antagonist CV9388. Substance P and CGRP levels in mucosa and in supernatant increased in response to HCl, and these increases were abolished by IRTX and by tetrodotoxin (TTX) but not affected by CV9388, indicating that substance P and CGRP are neurally released and PAF independent. In contrast, the increase in PAF was blocked by IRTX but not by TTX. Presence of TRPV1 receptor was confirmed by RT-PCR and by Western blot analysis in whole mucosa and in esophageal epithelial cells enzymatically isolated and sorted by flow cytometry or immunoprecipitated with cytokeratin antibodies. In epithelial cells PAF increased in response to HCl, and the increase was abolished by IRTX. We conclude that HCl-induced activation of TRPV1 receptors in esophageal mucosa causes release of substance P and CGRP from neurons and release of PAF from epithelial cells. smooth muscle; signal transduction; platelet-activating factor TRANSIENT RECEPTOR POTENTIAL channel vanilloid subfamily member-1 (TRPV1, also known as VR1) was originally described in primary sensory neurons as a receptor for capsaicin and related natural irritants (collectively referred to as vanilloids) (61). TRPV1 is a nonselective cation channel expressed by several cell types including sensory nerves. It is activated by heat, by the pungent ingredient of chili peppers, capsaicin, or by endogenous hydrogen ions released in tissues during inflammation (61) or present in gastric reflux. TRPV1 is now believed to function as a molecular integrator of noxious stimuli, including acids, heat, pollutants with negative electric charge, and endogenous proinflammatory substances (23). TRPV1 activation in primary afferent neurons evokes the sensation of burning pain and induces neurogenic inflammation by the release of substance P and calcitonin gene-related peptide (CGRP) (65, 66). Upregulated TRPV1 expression has been demonstrated in disease states such as inflammatory bowel disease (66) and irritable bowel syndrome (15) and, more recently, in esophageal submucosal neurons of esophagitis patients (5, 41).TRPV1-associated "neurogenic inflammation" has been examined in several systems, particularly in the airways, and to a lesser extent in the digestive tract. In the digestive tract most publications have ...

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Section: Methodsmentioning

confidence: 99%

HCl-activated neural and epithelial vanilloid receptors (TRPV1) in cat esophageal mucosa

Cheng¹,

Monte²,

Ma³

et al. 2009

American Journal of Physiology-Gastrointestinal and Liver Physi

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“…Several earlier studies demonstrated long-term propagation of primary epithelial cell cultures from rodent and human colon [1,3,4,6,7]. Some showed long-term replication potential and functional differentiation, suggesting that the cultures may have contained stem cell populations, although such characterization was not performed.…”

Section: Organ Culturementioning

confidence: 99%

“…However, replicating cells in the colon may undergo mutations after damage by toxic agents resulting in carcinogenesis. The high incidence of colon cancer has engendered considerable effort to develop in vitro models of colon epithelial cells [1][2][3][4][5][6][7] that have contributed to the current knowledge of mechanisms involved in colon cancer as well as normal colonic growth and differentiation. In contrast, there have been far fewer studies of small intestine.…”

Section: Introductionmentioning

confidence: 99%

Intestinal Epithelial Cells In Vitro

Chopra

Dombkowski

Stemmer

et al. 2010

Stem Cells and Development

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Recent advances in the biology of stem cells has resulted in signifi cant interest in the development of normal epithelial cell lines from the intestinal mucosa, both to exploit the therapeutic potential of stem cells in tissue regeneration and to develop treatment models of degenerative disorders of the digestive tract. However, the diffi culty of propagating cell lines of normal intestinal epithelium has impeded research into the molecular mechanisms underlying differentiation of stem/progenitor cells into the various intestinal lineages. Several short-term organ/organoid and epithelial cell culture models have been described. There is a dearth of long-term epithelial and/or stem cell cultures of intestine. With an expanding role of stem cells in the treatment of degenerative disorders, there is a critical need for additional efforts to develop in vitro models of stem/progenitor epithelial cells of intestine. The objective of this review is to recapitulate the current status of technologies and knowledge for in vitro propagation of intestinal epithelial cells, markers of the intestinal stem cells, and gene and protein expression profi les of the intestinal cellular differentiation.

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“…Though long-term cultures of primary colon epithelial cells (CEC) have been described, they remain a challenge (Chopra et al, 2010;Bartsch et al, 2004;Booth et al, 1995). Traditionally, the method referred to as organ culture was employed to study physiology and intestinal lineage outside of an organism.…”

Section: Colon Epithelial Cellsmentioning

confidence: 99%

“…Among a diversity of protocols of enzymatic isolation of epithelial cells, trypsin and collagen are predominantly used (Chopra et al, 2010). Apart from employing proteolysis, a variety of compositions of culture medium, application of growth factors and additional substances, like hydrocortisone or cholera toxin, culture conditioning with non-epithelial cells as well as the use of antimicrobial agents have been proposed in order to obtain several primary epithelial colon cell cultures of different species (Birkner et al, 2004;Deveney et al, 1996;Chopra & Yeh, 1981;Bartsch et al, 2004;Booth et al, 1995;Yeh & Chopra, 1980). Yet, since suggested concentrations of the respective substrates vary considerably between different authors, Chopra et al stated in their recent comprehensive review that the precise combination of each substrate and additive must be defined for each system (Chopra et al, 2010).…”

Section: Colon Epithelial Cellsmentioning

confidence: 99%