Affordable Clinical Research Services for Global Liver Research Industry........Cell Based High Resolution Assays Hepatocytes and Liver Function Tests Stellate Cells Activation & Fibrogenesis Kupffer Cell Assay, Hepatic Sinusoidal Endothelial Assay

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1. Hepatic stellate cell viability
Trypan blue assay
Cell viability is measured following the trypan blue protocol. Activated HSCs are seeded in 12 well plates and incubated for 4 hr (standardization for individual drug) with drug. Next, the cells are treated with trypan blue (0.04 mg/ml) and incubated for another 15 min. Bright field images are taken using Olympus inverted microscope. The number of cells having a blue nucleus is counted and percentage of dead cells is being plotted in the form of a graph (Strober, 2001).
CMFDA based assay
Cell viability is measured using 5- chloromethylfluorescein diacetate probe (CMFDA). Here, activated HSCs are subjected to drug treatment for 4 hr (standardization for individual drug), followed by incubation with 5 μM CMFDA for 2 hr. Next, the cells are washed with PBS and images are taken using DP71 camera adapted to an Olympus IX71 microscope (Johnson et al 2013).
2. Measurement of Apoptosis
(i) PI Incorporation Assay
Activated HSC cells are treated with drug for 4 hr (standardization for individual drug), incubated with 1 μM of propidium iodide (PI) for 10 mins. To remove unbound PI, cells are washed with PBS gently and the fluorescent images are acquired with DP71 camera fitted with an Olympus IX71 microscope. The number of PI positive cells are determined for 5 different fields of 5 independent experiments and a graph is plotted. (Majumder et al 2011).
(ii) Annexin V-FITC Apoptosis Detection Kit
Activated HSCs are treated with drug for 4 hr (standardization for individual drug) and are being processed according to the protocol supplied by the manufacturer (Calbiochem, Germany). Fluorescence images of the cells are captured and the number of annexin V-FITC-positive cells are counted per field.

REFERENCES
1. Strober W. Trypan blue exclusion test of cell viability.Curr Protoc Immunol. 2001 May;Appendix 3:Appendix 3B.
2. Johnson S, Nguyen V, Coder D.Assessment of cell viability.Curr Protoc Cytom. 2013;Chapter 9:Unit9.2.
3. Majumder S, Siamwala JH, Srinivasan S, Sinha S, Sridhara SR, Soundararajan G, Seerapu HR, Chatterjee S. Simulated microgravity promoted differentiation of bipotential murine oval liver stem cells by modulating BMP4/Notch1 signaling. J Cell Biochem. 2011 Jul;112(7):1898-908.
Cell Proliferation – 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) based assay
Activated HSCs are grown at about 70% confluency in 24 well plate. Next cells are subjected to drug treatment for 8 hr (standardization for individual drug). After incubation MTT is added to medium to get a final concentration of 1mg/ml. After 2 hr incubation with MTT,cells are subjected to DMSO treatment. DMSO lyse the cells to release MTT based colour products from the cells. The optical density (O.D.) of the colour products is measured by using the ELISA microplate reader at 570nm. (Majumder et al 2007 and Vistica et al 1991).
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. Vistica DT, Skehan P, Scudiero D, Monks A, Pittman A, Boyd MR.Tetrazolium-based assays for cellular viability: a critical examination of selected parameters affecting formazan production.Cancer Res. 1991 May 15;51(10):2515-20.
Hepatic Stellate Cell Migration & Contraction
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.
boydensLower 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 assay

contraction assayContraction 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

Detailed methodology:

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).

Cell Permeability
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
cell permabilitywith 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

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. 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.
Collagen formation assay
Collagen forms the ground substance of connective tissue. It is composed of three amino- acids and stains strongly with acid red dyes due to the affinity of the cationic groups of the proteins for the anionic reactive groups of the acid dyes. Activated HSC produces a large amount of collagen, Source: LRSU-AUKBC which is one of the important markers for HSC activation.
Collagen formation assay is performed using the collagen specific dye, Sirius red, as previously described (Lee et al 1998 and Marotta et al 1985). A solution of 5 mg/ml concentration Sirius red is prepared by dissolving 0.1g of Sirius red in 100ml of saturated picric acid solution. Cells are treated with drug for 8h (standardization for individual drug) and then probed with acidic solution of Sirius red.
cpllagen After 1h of incubation, cells are washed with PBS and then observed under microscope followed by capturing bright field images (refer figure on the left side, source LRSU-AUKBC). Colorimetric measurement of collagen bound Sirius red is performed by lysing the cells in sodium hydroxide (NaOH) 0.1M for 30min at room temperature and by measuring the supernatant at 530 nm using ELISA microplate reader.
Western blot analysis to measure collagen type I: Cells are treated with drug followed by measurement of secreted collagen type 1 in media and within the cells. Total protein concentration is normalized using Biuret Assay. Proteins are detected by using 1:1000 dilution of collagen type 1 antibody and 1:1000 dilutions of goat anti-rabbit IgG conjugated to horseradish peroxidase. The blots are developed by using substrate TMB/H2O2. Loading control used is β-actin as house keeping gene. (Majumder et al 2014).

REFERENCES

1. Marotta M, Martino G – Sensitive spectrophotometric method for the quantitative estimation of collagen. Anal Biochem.;150:86-90, 1985.
2. Lee, D.A., Assoku, E.,Doyle,V.,1998. A specific quantitative assay for collagen synthesis by cells seeded in collagen-based biomaterials using sirius red F3B precipitation. J.Mater.Sci.Mater.Med.9(1),47–51.
3. Majumder S, Gajalakshmi P, Chatterjee S. “Use of stem cells to block the activation of hepatic stellate cells in diseased liver” Edited by Somasundaram I in a book entitled “Stem cell therapy for organ failure” – Springer Verlag  Publications.2014 : 221-232.
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.
Hepatic Stellate Cell Live Imaging
We also have state of the art facility to extend liver drug discovery research to the next levels of activities.  State of the art live imaging platform coupled with on stage CO2 incubator and hypoxia chamber is available to track live cells.
1. Confocal Microscopy: High end optical images with a complete analysis of activated HSC cells under the treatment of liver drugs is performed using immunostaining technique.

2. Flow cytometry and cell sorting platform: The effect of the drugs on cellular targets is carried out using flow cytometry and FACS.

REFERENCES

1. Yin C, Evason KJ, Asahina K, Stainier DY.Hepatic stellate cells in liver development, regeneration, and cancer. J Clin Invest. 2013 May;123(5):1902-10.
2. Mederacke I, Hsu CC, Troeger JS, Huebener P, Mu X, Dapito DH, Pradere JP, Schwabe RF.Fate tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis independent of its aetiology. Nat Commun. 2013;4:2823.
Hepatic Stellate Cell Genomics
1. Sanger sequencing: DNA sequencing in activated HSCs under the treatment of drug is performed using Sangers dideoxy method.
2. Next Generation Sequencing: High throughput sequencing using an illumina miseq machine is carried out with complete analysis and interpretation in hepatic stellate cells under the treatment of drug.
3. Analysis of big data: Big data analytics is the steps of analysing large data sets containing a variety of data types to unravel hidden patterns and unknown correlations. The findings and its interpretations may lead to new horizons of drug discovery; developing a a more effective drug for liver diseses.

REFERENCES

1. Okuyama H, Shimahara Y, Kawada N.The hepatic stellate cell in the post-genomic era.Histol Histopathol. 2002 Apr;17(2):487-95.
2. El Taghdouini A, Sørensen AL, Reiner AH, Coll M, Verhulst S, Mannaerts I, Øie CI, Smedsrød B, Najimi M, Sokal E, Luttun A, Sancho-Bru P, Collas P, van Grunsven LA.Genome-wide analysis of DNA methylation and gene expression patterns in purified, uncultured human liver cells and activated hepatic stellate cells.Oncotarget. 2015 Sep 29;6(29):26729-45.
Hepatic Stellate Cell Secretome & Proteomics
Secretome : The secretome is the bulk of secreted organic molecules and inorganic components by biological cells, tissues, organs, and organisms. However, the term is usually referred as the secreted proteins of a proteome.
The concept of secretome is very critical to Hepatic Stellate Cell Biology as the HSC define sthe liver patho-physiology and pathologies by cross talking to other partners in the HSC microenvironment.
Proteomics: The gene expression profile is validated by using Mass Spectrometry. Identification of proteins in HSCs under the treatment of drugs is performed using ICP-MS.

REFERENCES

1. Kristensen DB, Kawada N, Imamura K, Miyamoto Y, Tateno C, Seki S, Kuroki T, Yoshizato K.Proteome analysis of rat hepatic stellate cells.Hepatology. 2000 Aug;32(2):268-77.
2. Zhang H, Wu P, Chen F, Hao Y, Lao Y, Ren L, Sun L, Sun W, Wei H, Chan DW, Jiang Y, He F.SILAC-based quantitative proteomic analysis of secretome between activated and reverted hepatic stellate cells.Proteomics. 2014 Sep;14(17-18):1977-86.
3. Ji J, Yu F, Ji Q, Li Z, Wang K, Zhang J, Lu J, Chen L, E Q, Zeng Y, Ji Y.Comparative proteomic analysis of rat hepatic stellate cell activation: a comprehensive view and suppressed immune response.Hepatology. 2012 Jul;56(1):332-49.
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