Rat hepatocyte is isolated through perfusion 2 step and incubated for 5 days.

Collagen is made at a ratio of rat tail collagen type 2: DDW: DMEM = 4:5:1 (collagen 1.2mg/mL), and is used at a pH of 7.2. Forty-five minutes before seeding, 100uL of collagen was placed on a 6well and seeded to 5 x 10^5 viable cells/mL.

Seeding medium : DMEM (5% FBS, 50 U/ml penicillin, 50 μg/ml streptomycin, 4 mg/L insulin, and 1 μM dexamethasone)

I replaced it with plating medium after 3 hours.

plating medium : DMEM (5% FBS, 4 mg/L insulin, 0.1 μM dexamethasone)

After 24 hours of seeding, sprinkle collagen and add culture medium after 45 minutes.

culture medium = DMEM + ITS™ (1% ITS™, 50 U/ml penicillin, 50 μg/ml streptomycin, and 0.1 μM dexamethasone

It is incubated for 5 days, and if morphology is confirmed with an optical microscope, bile duct formation is not complete.

What is the problem about my process?