1. Biopsy specimens for tissue culture were immediately placed on ice in primary cell culture system.
2. Within 4 hours from the time of the biopsy, the specimens were minced into approximately 2-mm3 fragments.
3. Primary cell cultures were initiated by maintaining the cells in primary epithelial cell system
primary epithelial medium
10% fetal bovine serum (FBS)
0.4 μg/mL hydrocortisone
20 ng/mL epidermal growth factor
10−10 mol/L cholera toxin
140 μg/mL bovine pituitary extract
20 μg/mL adenine, 100 U/mL penicillin
100 μg/mL streptomycin
0.25 μg/mL amphotericin B
4 mmol/L glutamine
5 μg/mL insulin
5 μg/mL transferring
5 ng/mL selenium
4. Cells were maintained in a humidified atmosphere containing 5% CO2 in air at 37 °C.
5. Esophageal
fibroblast cultures from the biopsy specimens were isolated by allowing
the fibroblasts to overgrow the epithelial cell culture for 4–6 weeks.
6. After
differential trypsinization with primary , the fibroblast cultures were
maintained in primary fibroblast cell culture system.
7. Epithelial
cells and fibroblasts were detached with trypsin, and aliquots of the
cell suspensions (30 000 cells/mL) were placed in each well of four-well
chamber slides for 72 hours.
8. Because only a limited number of
epithelial cells could be grown as a primary culture from a single
patient, experiments used cells pooled from several patients.
9. Cells were pooled from consecutive patients without selection or segregation.
10. For any given experiment, pooled cultured cells were obtained from two or three patients.
11. Cell cultures derived from both dysplastic and nondysplastic tissues were combined.
12. Cultures
of Barrett's esophageal epithelial cells were characterized by direct
light microscopy after cytochemical staining with hematoxylin and eosin,
periodic acid-Schiff, and Alcian blue (pH 2.5) and immunostaining with a
panel of monoclonal anti-keratin antibodies.
13. In addition, Barrett's esophageal epithelial cells were also immunostained with Mab-Das-1 (a Barrett's specific antibody).
14. Fibroblasts were immunostained with a mouse monoclonal antibody to vimentin and smooth muscle α-actin.
Reference
Buttar NS, Wang KK, Anderson MA, Dierkhising RA,
Pacifico RJ, Krishnadath KK, et al. The effect of selective
cyclooxygenase-2 inhibition in Barrett's esophagus epithelium: an in
vitro study. J Natl Cancer Inst. 2002; 94: 422–429.
Palanca-Wessels
MC, Barrett MT, Galipeau PC, Rohrer KL, Reid BJ, Rabinovitch PS. Genetic
analysis of long-term Barrett's esophagus epithelial cultures
exhibiting cytogenetic and ploidy abnormalities. Gastroenterology. 1998;
114: 295–304.
Garewal HS, Leibovitz A, Sampliner RE, Ramsey L,
Hendrix MJ, Sloan D. Tissue culture of epithelial cells from esophageal
specialized columnar epithelium (Barrett's esophagus). Dig Dis Sci.
1992; 37: 532–536.
Hybbinette S, Bostrom M, Lindberg K. Enzymatic
dissociation of keratinocytes from human skin biopsies for in vitro cell
propagation. Exp Dermatol. 1999; 8: 30–38.