AM was donated with written informed consent by mothers seronegative for the human immunodeficiency and hepatitis B and C viruses at the time of cesarian section. The AM was stored in 15% dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO) and phosphate-buffered saline (PBS) at −30°C until use. Denuded AM was prepared as described previously.
15 Membranes were rinsed in PBS, spread onto the upper chambers of a six-well insert (Transwell; Corning, Corning, NY), and air dried at room temperature. Fibrin-coated inserts were prepared as described previously.
13 Briefly, cell culture inserts (Corning) were coated with 250 μL fibrin (3 mg/mL; Bolheal; Astellas, Tokyo, Japan) and stored at 4°C. After the fibrin had polymerized 2 hours later, the inserts were kept in storage until use.
The procedures for harvesting and culturing the oral mucosal tissues were as described by Satake et al.
16 All tissue donors were informed of the purpose of the study and received advice on oral hygiene and treatment for tooth decay before the oral mucosal biopsy. After sterilizing the oral cavity, the inferior buccal mucosa was excised with an 8-mm-diameter biopsy punch (KAI Industries Co., Ltd., Gifu, Japan) with the donor under local anesthesia. Specimens were removed from the submucosal connective tissue by dissection with scissors. The mucosal epithelium was cut into small pieces and then washed several times in sterile Ca
2+ and Mg
2+-free PBS to remove blood and adipose tissue. The specimens were then submerged in Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 mixture at a ratio of 1:1 (vol/vol) (Invitrogen Corp., Carlsbad, CA) with 5 g/mL gentamicin (Invitrogen), 0.25 g/mL amphotericin B (Sigma), 100 μg/mL streptomycin (Wako Pure Chemical, Osaka, Japan), and 100 units/mL penicillin (Wako). The basal cells of the oral mucosal epithelial cells were harvested after enzymatic treatment with 1.2 IU Dispase II (Roche Diagnostics, Indianapolis, IN) at 37°C for 1 hour and 0.05% trypsin-0.53 mM EDTA solution (Invitrogen-Gibco) at room temperature for 10 minutes. The cell suspension was washed in DMEM/F12 medium (1:1 mixture) with 10% fetal bovine serum (FBS), to remove debris and small pieces of residual material. A single-cell suspension of basal cells from the oral mucosal epithelium was resuspended in conditioned medium for oral mucosal epithelium (DMEM/F12 supplemented with 10 ng/mL epidermal growth factor [EGF], Invitrogen-Gibco; 10 μg/mL insulin, Wako; 0.5 μg/mL hydrocortisone, Sigma; 1 μM isoproterenol, Sigma; 2 nM triiodothyronine, Sigma; 100 μg/mL streptomycin, Wako; 100 units/mL penicillin, Wako; and 4% autologous serum), followed by seeding (1.0–2.0 × 10
5 cells/well) onto human denuded AM- or fibrin-coated culture plate inserts in a six-well plate (Transwell; Corning) containing mitomycin-C (MMC; Sigma) treated with 3T3 fibroblasts (2.5 × 10
5 cells/well). The culture was submerged in this medium until confluence, after which the medium was changed (DMEM/F12 supplemented with 10 ng/mL EGF, 10 μg/mL insulin, 0.5 μg/mL hydrocortisone, 100 μg/mL streptomycin, 100 units/mL penicillin, and 4% autologous serum). The culture was exposed to air by lowering the level of the medium at the end of the culture period. Aprotinin (300 KIU/mL; Wako) was added to the medium to prevent fibrin digestion during culture. A histopathologic examination revealed that the cultivated oral mucosal epithelial sheets consisted of five to six cell layers. Aprotinin was discontinued in conjunction with cell growth (
Fig. 1).