In this context, we assessed the responses of cultured fibroblasts with respect to their differentiation into myofibroblasts using optimised cytokines (TGF-β1, IL-6 and IL-8) for scar formation in 2D (tissue culture plate, collagen type I coated plate) vs 3D collagen type I gel based constructs.
In triplicate, cells were plated in a 24-well collagen type I-coated plate.
One, named a type I TB′ plate in this study, is isostructural to the known θ phase with a large c value of about 0.64 nm, having a habit plane parallel to the matrix {1 0 0}α; the other, type II TB′, is characterized by a=0.41 and c=0.61 nm, having a habit plane inclined at about 20° with {1 0 0}α, while maintaining a coherent interface.
Type I assay plates are further supplemented with 40 ng/ml anhydrotetracycline to induce tetA expression without affecting growth rate.
However, no impairment of binding of podocytes to collagen type I coated plates was observed after incubation with monoclonal antibody IIH6.
Cells were harvested with trypsin/EDTA, plated at 5 × 10 cells per 24-well plastic plate or type I collagen-coated plate (for GLM-2 and GLM-4 cells) in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, and then treated with increasing doses of gefitinib (0.01, 0.1, 1 and 10 μ M) or trastuzumab (0, 1, 10, and 100 μg ml−1) on days 1 and 3.
Cells were harvested with trypsin/EDTA and were plated at 1 × 10 cells per 96-well plastic plate or type I collagen-coated plate (for GLM-2 and GLM-4 lines) in DMEM and treated with increasing doses of gefitinib (0, 0.1, 1 and 10 μ M), LY294002 (0, 1, 10 and 50 μ M) and U0126 (0, 1, 10 and 50 μ M) on day 1 in the presence of 10% FBS.
The digested cells were then plated onto type I collagen (BD Biosciences -coated plate and were cultured in DMEM containing 10% fetal bovine serum (FBS), streptomycin (100 µg/mL) and penicillin (100 U/mL).
Purified hepatic progenitors in a plating medium (DMEM/HAM F-12, 10% heat-inactivated FBS, 1 g/l human BSA fraction V, 1% L-glutamine) were plated onto a type I collagen-coated plate and incubated for 4 hours.
