Hepatic Microenvironment

hepatic

Cross Talk of HSC with Neighboring Cells
As HSCs exist in the liver sinusoid along with other functionally important cells, such as hepatocytes, Kupffer cells, endothelial cells and liver stem cells, it is necessary to study the effect of drug treatment on other neighboring cells. This is tested as described below.
Conditioned medium approach
Activated HSC cells are seeded in 24-well plate with 80% confluence and incubated for 12 hr in regular growth medium. Fresh medium is added to the dish and incubated for the next 12 hr. This is followed by collection of medium and serves as the conditioned medium (CM).

One set of HSCs is grown under the treatment of drug and the other with plain growth medium (control). Now, this CM is added to other cell types such as HUVEC (representing endothelial cells), hepatocytes, Kupffer cells, and liver stem cells to study the respective property and function of each cell type under the treatment of CM (Majumder et al 2007).
Trans-differentiation of primary HSC to activated HSC
Primary HSC transdifferentiate to myofibroblast-like cells when it is cultured on plastic (Geerts et al.,1989). To evaluate the efficacy of drug treatment in blocking the transdifferentiation of primary HSC, cells are plated on plastic and grown for 14 days. During growth, cells are treated with drug (standardization for individual drug) in each consecutive day. After completion of incubation for 14 days,cells are probed with alpha smooth muscle actin (a-SMA) antibody followed by probing with corresponding secondary antibody tagged with HRP(horseradish peroxidase) and developed with 3-30-diaminonenzidine (DAB)/H2O2 solution. Images are acquired with an inverted microscope. The number of a-SMA positive cells per field is counted (Majumder et al 2013).
Co-culture models
co cult
co cultra
This model mimics the situation of liver sinusoid where hepatic stellate cells and endothelial cell lineage exist very close to each other and can able to affect each other’s activity. We are using this model in order to find out the effect of hepatic stellate cells on endothelial cells.Figure 1 showing the parts of the co-culture chamber (membrane and internal parts). Figure 2 membrane is placed inside the chamber in order to grow cells upon the membrane. Figure 3 and 4 Membrane supporting part of the co-culture chamber is placed inside to hold the membrane in place. Figure 5 Upper part of the co-culture chamber is tightened in order to hold the membrane along with the membrane holder. Figure 6 the whole set up is placed in the 12-well tissue culture plate already plated with cells. In this set up, it is possible to grow two cell layers simultaneously as depicted in the upper right cartoon (Majumder et al 2007).
HSC perturbing liver endothelium
1) Endothelial ring formation assay:
endotheliyalEndothelial cells are seeded on collagen (collagen type I) coated 24-well plate with 60% cell density. After 7h of incubation, CM is added to the cells and then incubated for 12h. The number of formed rings is counted under bright field, phase contrast microscope. Only the complete ring structures created from 3 to 5 EC are counted as complete rings. (Majumder et al 2007). The source for the above figure is LRSU – AUKBC.
2) Wound healing assay:
wood healing assayWound is created by scratching the EC monolayer with a 1 mm wide sterile plastic scraper. Cells are washed with PBS, treated with CM and incubated for 8h. Bright-field images are taken with 4X magnifications under an inverted bright field microscope. The rate of wound healing is quantified from the images using Scion Image, Release Alpha 4.0 3.2 and Adobe Photoshop version 6.0. (Majumder et al 2013). The source for the above figure is LRSU – AUKBC.
3) Capillary tube formation of endothelial cells on matrigel: Slide chambers are coated with 100 µl Matrigel per chamber and incubated at 37°C for 30 min to promote gelling. Four hours after splitting, endothelial cells are treated with HSC derived CM and incubated further for 24h in matrigel coated wells and incubated at 370C/5% CO2 in humidified atmosphere. Each well is photographed at random with the use of a light microscope. The length of the tube is measured at 40x magnification with Scion image, and expressed as mm/mm2. All assays are done in triplicate in three independent experiments (Donovan et al 2001 and Majumder et al 2013).
4) Aortic ring assay: Slide chambers are coated with 100 µl Matrigel per chamber and incubated at 37°C for 30 min to promote gelling. Thoracic aortas are excised from 8- to 12-week-old Wistar rats (range 275–340 g), and the fibroadipose tissue is removed. The aortas are sectioned into 1-mm-long cross-sections, rinsed five times with EGM-2, placed on the matrigel-coated wells. MCDB-131 medium with 10% fetal bovine serum, supplemented with 10 ng/ml epidermal growth factor, 1 microgram/ml hydrocortisone along with HSC derived CM are added. After 5 days of incubation, vascular outgrowth is quantified by counting all sprouts from one ring. All assays are performed twice in triplicate (Burbridge and West, 2001 and Majumder et al 2013).
5) Ex vivo chick embryo area vasculosa assay:
ex vivo chickFourth day incubated eggs are broken and gently plated on a cellophane bed covered petri dishes under sterile conditions. Discs containing CM from activated HSC are placed on the egg yolks and incubated for another 12h.
Images are taken at 20X magnification using a CoolPix camera adapted to a stereo-microscope, after 0h, 6h and 12h of incubation. The quantification of angiogenesis is performed by using Angioquant software (Majumder et al 2013). The source for the above figure is LRSU-AUKBC.
6) Tracking immuno-modulation of activated HSC
Immuno-modulation

REFERENCES

1. Majumder S, Tamilarasan KP, Kolluru GK, Muley A, Nair CM, Omanakuttan A, Murty KV, Chatterjee S. Activated pericyte attenuates endothelial functions: nitric oxide-cGMP rescues activated pericyte-associated endothelial dysfunctions.Biochem Cell Biol. 2007 Dec;85(6):709-20.
2. 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.
3. Donovan, D.,Brown,N.J.,Bishop,E.T.,Lewis,C.E.,2001.Comparison of three in vitro human‘angiogenesis’assays with capillaries formed in vivo. Angiogenesis 4(2),113–121.
4. Burbridge, M.F.,West,D.C.,2001. Rat aortic ring: 3D model of angiogenesis in vitro. Methods Mol.Med.46,185–204.