Rat tail collagen type 1

Full-thickness 3D tissue-engineered oral mucosa models were manufactured by air/liquid interface culture of TR146 keratinocytes seeded onto fibroblast-populated collagen gels. A solution of 10 × DMEM, 8.5% (v/v) FBS, 2 mM L-glutamine, reconstitution buffer (22 mg mL⁻¹ sodium bicarbonate and 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), and 5 mg mL-^{1 (link)}
rat tail type I collagen (R & D system, UK) was prepared and neutralized by 1-M sodium hydroxide to pH=7.4 in an ice-cold environment by keeping everything on ice. Normal oral fibroblasts were added to the solution at a concentration of 500,000 cells/model, and 1 mL of the resultant cell-containing collagen mixture was transferred to cell culture transwell inserts (0.4 µm pore size, Millipore), incubated at 37°C for 2 hours until solidified, and then completely submerged in complete DMEM for 3 days. Subsequently, 1×10⁶ keratinocytes were seeded onto each model and kept in submerged culture for three days, after which the oral mucosal models were raised to air/liquid interface and cultured for a further 7 days.

To produce the engineered connective tissue layer, a solution of 10× concentrated DMEM, supplemented with FBS 8.5% (v/v); L-glutamine 2 mM; and reconstitution buffer, consisting of sodium bicarbonate (22 mg/mL) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (20 mM), was mixed with 5 mg/mL rat-tail type I collagen (R & D system, United Kingdom) and neutralized by 1 M sodium hydroxide to pH 7.4, while keeping everything on ice. Then 1 mL of the mixed cell suspension was added to the neutralized collagen solution at concentrations of 2 × 10⁵ fibroblasts and 1 × 10⁵ THP-1 monocytes per model (to simulate inflammatory status in the connective tissue layer), and 1 mL of the cell-populated collagen solution was transferred into cell culture inserts (0.4 µm pore size, Millipore) and incubated at 37 °C for 2 h until gel formation. The substrates were then submerged fully in complete DMEM for 3 days. Finally, 1 × 10⁶ keratinocytes were added onto each model and maintained in a submerged culture for 3 days in Green’s medium, and then were slightly raised to the air–liquid interface and further cultured for 3 days to induce some epithelial differentiation before insertion of the implants.

A solution of 5 mg/mL rat-tail type I collagen (R & D system, Minneapolis, MN, USA), concentrated DMEM (×10), 8.5% FCS (v/v), 2 mM L-glutamine 2, and reconstitution buffer including 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and 22 mg/mL sodium bicarbonate was prepared and neutralized by a 1 M sodium hydroxide solution to pH = 7.4 at a cold temperature by keeping the solution on ice. Collagen hydrogels were mixed with fibroblasts (5 × 10⁵/mL) and THP-1 monocytes (1 × 10⁵ /mL), and 1 mL of the cell-containing collagen mixture was transferred to Millipore tissue culture inserts with a 0.4 µm pore size, incubated at 37 °C for 2 h for solidification, and then submerged in complete DMEM for 3 days.
A total of 1 × 10⁶ keratinocytes were seeded onto the surface of each model and further cultured in a submerged condition for 3 days. The oral mucosal models were then raised to an air–liquid interface and further cultured for 7 days.