In Vitro Cell. Dev. Biol. 31:338-339. May 1995 © 1995 Society for In Vitro Biology 1071-2690/95 $05.00 + 0.00

Letter to the Editor CULTURE OF HUMAN ESOPHAGEAL ENDOSCOPIC BIOPSIES

Dear Editor: Many groups have attempted to establish a system to determine

human tumor response to cytotoxic therapy in vitro (1). This group has established an optimized primary culture system (2) which has successfully been applied to the culture of surgical specimens of squamous cell carcinomas and adenocarcinomas of the esophagus (3). However, these surgical specimens are taken at esophagectomy when a patient may have already received chemoradiotherapy. As with Trifillis et al. (4) our goal was to culture human epithelium cells from small amounts of tissue available from, in our case, endoscopic biopsy material. We report here the successful culture of epithelial cells from pretreatment endoscopic biopsies taken from patients with suspected esophageal carcinoma.

Fresh endoscopic biopsies were obtained from 10 patients with histologically confirmed esophageal carcinoma. Three patients had squamous cell carcinomas and seven had adenocarcinomas.

The biopsies were placed in supplemented growth medium, RPMI 1640 (GIBCO, Grand Island, NY) and brought immediately to the laboratory for culture. Growth medium (500 ml) RPMI 1640 was supplemented with 60 ml fetal bovine serum (GIBCO). 40 ml horse serum {GIBCO), 5 ml penicillin-streptomycin solution (5000 IUhnl,

5000 p,g/ml GIBCO), 0.5 ml hydrocortisone (2.5 mg/ml, Glaxo, Uxbridge, England), 0.5 ml insulin (100 1U/ml, Novo, Newcastle Upon Tyne, United Kindom), 5 ml Fungisone (amphotekicin B 250 UG/ml, GIBCO). In the laboratory- the biopsies were cut into explants of approximately 1 m m 2, placed in a solution of trypsin/collagenase (Sigma, St. Louis, MO, type IV) 1 ml/mg and allowed to digest at 37 ° C for 10 rain. The digested explants were then plated singly in Nun- clon 25-cm 2 flasks with 2 ml of the supplemented growth medium, and placed in a Forma Scientific incubator at 37 ° C and 5% CO2.

The explants became attached to the surface of the culture flasks after approximately 24 h, and after 2 to 3 days in culture epithelium- like cells could be seen migrating from the explant. A total of 14 days in culture was allowed, after which time the cultures were re- moved from the incubator, fixed in 10% buffered formalin, and im- munocytochemical examination carried out.

Nine of ten biopsies grew successfully in vitro. The epithelial na- ture of the cells constituting the outgrowth was confirmed using a universal cytokeratin antibody (Amersham, Bucks., UK) (Fig. 1). The population of proliferating ceils was determined using an antibody (Ki67, Dako, Glostrup Denmark), to nuclear membrane antigen (Fig. 2). Labeling by all antibodies was developed using peroxidase-con- jugated anti-mouse IgG (Vectastain, ABC kit) and diaminobenzal-

FiG. I. Cellular outgrowth from an esophageal endoscopic biopsy, cul- tured for 14 days at 37 ° C, stained for expression of cytokeratin..Note the dark reaction product in the cytoplasm of the cellular outgrowth.

FIG. 2. Dark staining (Ki67 positive) focus on a cellular outgrowth from an esophageal endoscopic biopsy.

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CULTURE OF HUMAN ESOPHAGEAL ENDOSCOPIC BIOPSIES 339

FIG. 3. Characterization of malignant nature of cellular outgrowth from esophageal endoscopic biopsy. Note the variation in the cell size and shape, and lack of orientation of cellular growth.

dehyde (DAB). The malignant nature of the cultured cells from the biopsies can be characterized by the variation in the cell size and shape and lack of orientation of the cells (Fig. 3).

To conclude, cultures of human tumor esophageal epithelium can be established from endoscopic biopsies. The ability to culture these

pretreatment endoscopic biopsies enhances the clinical relevance of the system as an individual predictor of patient response to cytotoxic

therapy.

REFERENCES

1. Hoffman, V.; Berens, M. E.; Martz, G., editor. Predictive drug testing on human tumor cells. New York: Springer-Verlag; 1984.

2. Mothersill, C.; Sheridan, M.; Harney, J., et al. Development of an opti- mized in vitro system for measurement of human tumor response to cytotoxie agents. In: Spier, Griffith, Butterworth: Animal cell culture technology', eds. Oxford, Britain; 1993.

3. Mothersill, C.; Seymour, C. B.; O'Brien, A., et al. Proliferation of normal and malignant human epithelial cells post irradiation. Aeta Oncoh 30:851458; 1991.

4. Trifillis, A. L.: Jacobs, S.; Cui, X., et al. Culture and characterization of normal epithelium from cystoscopic biopsies of human bladder. In Vitro Cell. Dev. Biol. 29A:908-911; 1993.

Mary- Sheridan Iona M. Reid

Carmel Mothersill Thomas P. J. Henness)

Department of Physics Room 114

Dublin Institute of Technology Kevin St. Dublin 8

Ireland: and Department of Surgery

St. James's Hospital Dublin 8

Ireland (1. M. R., T. P. J. H.)

Received 28 November 1994)