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Volume: Barrett's Esophagus
Chapter: Pathophysiology

What information can cell or organ culture bring on the mechanism of the proliferation response of Barrett's esophagus to acid?

G. Triadafilopoulos, R.C. Fitzgerald (Palo Alto)

The introduction and utilization of organ and cell culture techniques have dramatically enhanced translational research in Barrett's esophagus and have led to a better understanding of the mechanism of proliferation in this metaplastic epithelium [1]. Both techniques rely on harvesting of biopsy explants or brush cytology samples during endoscopy performed for Barrett's esophagus surveillance. In addition, human adenocarcinoma (TE7 and OE-33) and squamous carcinoma (OE-21) cell lines have been used with similar pattern of results.


Organ culture of Barrett's esophageal biopsies

The ex vivo organ culture model described is used due to the unavailability of a Barrett's esophagus cell line [2]. Endoscopic mucosal samples from normal esophagus (squamous controls), Barrett's esophagus and duodenum (columnar epithelial controls) are obtained from individuals undergoing endoscopic surveillance. The mucosal samples are immediately divided into two parts. One part is formalin-fixed for histopathological assessment; the other part is maintained in supplemented tissue culture medium for subsequent experimental use.

All specimens are independently analyzed in order to categorize normal esophageal mucosa, esophageal inflammation, metaplasia, degrees of dysplasia and esophageal adenocarcinoma. In order to confirm the presence of specialized intestinal metaplasia (IM), alcian blue staining is performed on all Barrett's esophagus samples. Morphological assessment by H&E stain is performed to ensure histologic integrity of organ culture tissues for each time point up to 24 hours.

Organ culture experiments are conducted for 24 hours with collection time points of 1 hour, 6 hours, 18 hours, and 24 hours. In order to assess the effect of luminal factors on the mucosal explants, tissues are exposed to acid (pH 3.5), bile acids (1mM) either continuously or as a 1-hour pulse, followed by incubation in pH and osmolality-controlled media. Depending on the experiment, tissues are exposed to acid alone (pH 3.5), bile acids alone (pH 7.4), acid + bile acids in combination (pH 3.5), or control media (pH 7.4).

In order to assess cell proliferation, 1 µCi/ml 3H-thymidine is added to the culture medium followed by incubation for up to 24 hours. Since 3H-thymidine incorporation is a measure of total DNA synthesis and does not discriminate between cellular compartments, proliferating nuclear cell antigen (PCNA) immunohistochemistry is also separately performed in order to examine the specific population of Barrett's esophagus the manifests proliferation changes by PCNA staining.

Protein extraction and immunoblot analysis may also be performed in order to assess both PCNA (a marker of proliferation) and Villin expression as a marker of intestinal cell differentiation in Barrett's esophagus epithelia treated with bile acids ± acid in a continuous (24 hours) or 1-hour pulse fashion.

Cell culture of Barrett's esophageal cells

After being withdrawn from the endoscope, the cytology brush is brushed on to chamber slides. The cells are then left for several minutes to adhere to the slide and 0.5 ml of primary culture medium is added to the chamber. The medium contains MCDB 153 modified by the addition of 10% fetal calf serum, 0.4 µg/ml hydrocortisone, 20 ng/ml epidermal growth factor, 10 mol/L cholera toxin, 150 µg/ml bovine pituitary extract, 100 U/ml penicillin and 100 µg/ml streptomycin. The cells are incubated at 37° C with 5% CO2 and used for intracellular pH studies within 24 hour of collection [3].

Cell culture of Barrett's adenocarcinoma cell lines

TE7 and OE-33 cells are derived from patients with Barrett's associated adenocarcinoma and have been used as in vitro models for Barrett's esophagus. Another cell line, OE-21 may be sub-cloned from an esophageal squamous cell carcinoma and used as control. The OE-21 cell line has a multi-layered, stratified, non-keratinizing phenotype and OE-33 has a single layer, mucin-secreting, low-moderate goblet cell density phenotype. All cells are seeded at 4 x 104/cm2 and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 1 mM glutamine and grown as monolayers in 10 cm tissue culture dishes. For intracellular pH (pHi) experiments, cells are sub-cultured onto 4-well Lab-Tek coverglass chamber slides [4].

Information on the mechanism of proliferation in Barrett's esophagus

Ion exchangers in Barrett's esophagus and adenocarcinoma

Ion exchangers (Na+-independent Cl-/HCO3 exchanger and sodium-hydrogen exchanger) for acid loading are located in the baso-lateral surface of the cell. It is well established that the sodium-hydrogen exchanger (NHE) controls intracellular pH (pHi) and cell volume in many cell systems. NHE is an acid extruder in response to pulses of extra-cellular acid exposure. At the end of an acid reflux event, enhanced NHE activity leads to an intracellular alkaline overshoot effect that accelerates cells into S phase of cell cycle. Using the ex vivo organ culture model described above, we have shown that enhanced NHE activity is necessary for the hyper-proliferative effect of acid pulses on Barrett’s esophagus [5] (Figure 1).

Figure 1. A proposed model for the role of NHE in the hyperproliferative response of BE to an acid pulse (From Ref. 5). An acid pulse results in increased NHE activity. After acid clearance, a process aided by esophageal motility and secretions, neutralization of esophageal pH occurs. This leads to transient cytoplasmic alkalinization until the activity of NHE is reset. This cytoplasmic alkalinization results in a transition from G to S phase of the cell cycle and hence triggers a proliferative response.

Acid loading in Barrett's esophagus

In short-term cell incubation systems (see above), acid loading is greater in Barrett's than squamous esophageal cells (?pHi -0.22 ± 0.08 versus -0.13 ± 0.01) and maximal in Barrett's adenocarcinoma cells. Barrett's cells utilize Na+-independent Cl-/HCO3 exchange and sodium-hydrogen exchange for acid loading; in contrast squamous esophageal cells acidify by Na+-independent Cl-/HCO3 exchange. Using these cell line systems, repeated acid exposure does not attenuate acid-loading in Barrett's adenocarcinoma cells (TE7) in culture and the cells recover fully their pHi, in contrast to squamous cells (OE-21). Overall, the mechanisms for pHi control in Barrett's esophagus resemble the known mechanisms for pHi control of gastric epithelial cells [6].

The role of protein kinase C activation

Upregulation of NHE activity in response to acute pH changes in Barrett's esophagus is thought to result from alterations in NHE phosphorylation that are mediated, at least in part, by protein kinase C (PKC). In organ culture experiments using the PKC inhibitor bisindolylmaleimide (BIM) the acid-induced cell proliferation was inhibited while the use of the PKC activator phorbol 12-myristate 13 acetate (PMA) did not increase proliferation further, suggesting that cell proliferation was maximally stimulated under these conditions. Therefore, PKC may be a mechanism for the acid-pulse activation of NHE in Barrett's esophagus [5].


Cell and organ culture techniques have allowed experiments that elucidate the mechanism(s) of proliferation in Barrett's esophagus. In such experiments, acid pulses activate NHE in Barrett's esophagus through a PKC-dependent mechanism. The enhanced proliferative response and better physiological pHi control of Barrett's cells may confer a survival advantage compared to squamous esophageal cells.


1. Fitzgerald RC, Triadafilopoulos G. Recent developments in the molecular characterization of Barrett's esophagus. Dig Dis 1998;16:63-80.

2. Fitzgerald RC, Omary MB, Triadafilopoulos G. Dynamic effects of acid on Barrett's esophagus: an ex vivo proliferation and differentiation model. J Clin Invest 1996;98:2120-2128.

3. Fitzgerald RC, Omary MB, Triadafilopoulos G. Acid modulation of HT29 cell growth and differentiation. An in vitro model of Barrett's esophagus. J Cell Sci 1997;110:663-671.

4. Fitzgerald RC, Farthing MJ, Triadafilopoulos G. Novel adaptation of brush cytology technique for short-term primary culture of squamous and Barrett's esophageal cells. Gastrointest Endosc 2001;54:186-189.

5. Fitzgerald RC, Omary MB, Triadafilopoulos G. Altered sodium-hydrogen exchange activity is a mechanism for acidinduced hyperproliferation in Barrett's esophagus. Am J Physiol 1998;275:G47-55.

6. Fitzgerald RC, Farthing MJ, Triadafilopoulos G. Characterization of intracellular pH control in Barrett's esophagus: evidence for an adaptive cellular response to gastroesophageal reflux. Gastroenterology 2000;118:A223.

Publication date: August 2003 OESO©2015