Trypsinized, activated HSC cells are used for migration assay using Boyden’s chamber, which is a two-chamber transwell system. A collagen coated 8-µm pore size polycarbonate membranes separate the upper and lower chambers. HSC cells are loaded in the upper well with and without the drugs.
Lower well is filled with DMEM or with drugs. The chambers are then incubated at 37°C/5% CO2 for 2h. After the incubation, endothelial cells on the polycarbonate membrane are fixed and stained with propidium iodide, a fluorescent nuclear probe. Activated HSC migration activity is then quantified as the number of migrated cells to the lower surface of the membrane. Boyden’s chamber can be used for studying chemokinesis and chemotaxis properties of the cells (Majumder et al 2007).
Contraction of activated HSC cells is performed as described (Rockey et al 1993). Contraction of the HSC monolayer is induced by adding 20% FBS to the cells followed by tracking the monolayer for 5 mins (Refer figure on the left side).
Source: LRSU – AUKBC
Individual wells of a 24-well culture plate is pre-incubated with PBS containing 1% Bovine Serum Albumin (BSA,500 ml/well) for 1h at 370C and then washed twice with PBS and allowed to air dry.Collagen gels are prepared by mixing 60% type I rat tail collagen, 10% 10X DMEM, 10% 0.2M HEPES(4-(2-hydroxyethyl)- 1- piperazineethanesulfonicacid), and 20% DMEM at 40C to make a final concentration of collagen of 2.4mg/ml.The solution is added to the culture wells and incubated for 1h at 370C to gelatinize. Activated hepatic stellate cells are layered on top of the collagen lattice at a concentration of 2.5X105 cells/ml and serum starved for 24h. Cells are then treated with drug for 4 h (standardization for individual drug), followed by induction of the contraction of the monolayer by addition of 20% FBS,while control sets are performed without FBS. Cells are incubated for 15min,and then images are taken using a Nikon CoolPix camera adapted to a stereomicroscope. Images are analysed using ImageJ software to measure the area covered by the HSC coated collagen lattice at 0 and 15 min upon FBS stimulation. The index of relaxation is calculated by considering the starting collagen lattice area as 100% and recalculating the area of the lattice at 15 min upon FBS stimulation (Majumder et al 2013).
Endothelial cells are seeded onto the collagen- coated polycarbonate membrane in the permeability chamber and incubated for 24 h at 37° C/5% CO2. The permeability chambers
with endothelial cells are incubated in HSC-CM along with and without drug under study for 8 h. After the incubation, trypan blue (0.004%) is added in the upper half of the permeability chamber and incubated for 1 h at 37°C and 5% CO2. The solution from the lower chamber is collected to measure the optical density at 580 nm using a Varian Cary 4000 UV-Vis spectrophotometer (Majumder et al 2007).
Source: LRSU – AUKBC
2. Rockey DC, Housset CN, and Friedman SL. Activation-dependent contractility of rat hepatic lipocytes in culture and in vivo. J Clin Invest 1993 92: 1795–1804.
3. Majumder S., Piguet, A.C., Dufour, J.F., Chatterjee, S. Study of the cellular mechanism of Sunitinib mediated inactivation of activated hepatic stellate cells and its implications in angiogenesis. Eur J Pharmacol. 2013 5;705(1-3):86-95.