PriCells: Barrett's esophageal epithelial and fibroblast primary cultures              

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% CO^₂ in air at 37℃.
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.