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Microscale Engineering of Tissue (Liver) Models for Compound Screening
Salman R. Khetani
Assistant Professor Department of Mechanical Engineering
School of Biomedical Engineering
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Fresh Hepatocytes
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Isolated Primary Hepatocytes Rapid Loss of Viability and Phenotypic Functions In Vitro
Albumin – marker of liver’s synthetic ability. Other functions decline as well (i.e. CYP450)
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The Liver Microenvironment Role of the Microenvironment in Modulating Hepatic Fates
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Khademhosseini A et al. PNAS 2006;103:2480-2487
Microscale Technologies For Tissue Engineering Semiconductor-Driven Tools for Biological Applications
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Micropatterned Co-Cultures (MPCCs) Role of Tissue Organization in Modulating Hepatic Functions
Khetani and Bhatia, Nature Biotechnology, 26(1), p120-126, 2007
HUMAN Hepatocytes – Stromal Support Cells
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MPCC Functionality and Longevity Mimicking Elements Of In Vivo Liver Physiology In Vitro
Other functions (i.e. CYP450 and conjugation enzymes, transporters) show similar kinetics/stability
Human and Monkey MPCCs – High levels of functions for ~1 month Rat MPCCs – High levels of functions for ~2 months
Urea Albumin
Khetani and Bhatia, Nature Biotechnology, 26(1), p120-126, 2007
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T-Cadherin Induces Hepatocyte Functions Cellular (CHO) and Purified (Recombinant) Protein Presentation
Khetani et al, FASEB J, 22(11), p3768-3775, 2008
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Up to 96-well standard tissue culture plates Compatible with fresh and cryopreserved
hepatocytes (human, rat, monkey) Amenable to automated fluid handlers Amenable to high content imaging
Miniaturized MPCCs HepatoPac™ by Hepregen Corporation (Medford, MA)
Prototype Development Funded by Deshpande Center at MIT
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Drug Metabolism in MPCCs Better Prediction of Drug Clearance & Metabolite Production
• Long-term continuous (up to 7 days) incubations allows for clearance of low turnover compounds and detection of secondary metabolites
• Tight well-to-well variability (CV < 15%) allows rank ordering of compounds based on intrinsic clearance
• With Pfizer (Groton, CT)
Wang W, Khetani S et al, Drug Metab Dispo, 38(10), p1900-1905, 2010
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• 96-well format MPCCs • Repeat dosing with drugs (45 drugs total) over 5 to 9 days • Cellular functions assessed:
• Albumin • Urea • ATP • Glutathione
• Hypotheses: • Repeat dosing improves sensitivity without loss of specificity • Functional assays are as sensitive as destructive toxicity endpoints for rapid (and non-destructive) hazard identification
• Human cultures are more sensitive than rat ones
Detection of Drug-Induced Liver Injury in MPCCs
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Detection of Drug-Induced Liver Injury in MPCCs
• Repeat dosing (2 vs. 4 doses) improves sensitivity without compromising specificity (~90%)
• Human cultures are more sensitive than rat ones (~70% vs. ~50%) for predicting human DILI hazards
• Albumin/urea are more sensitive than ATP and GSH for rapid hazard (and non-destructive) identification
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Day 10 Day 2 Day 10
Kupffer Macrophages in MPCCs
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Hepatitis C (HCV) A Global Epidemic
An estimated 170 million people worldwide are infected
Leading cause of liver transplants in U.S.
No vaccine available
Severe side effects of current therapies
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Replication of HCV in MPCCs for Several Weeks Primary human hepatocyte in MPCCs are susceptible to HCV
Ploss/Khetani et al, PNAS, 107(7), p3141-3145, 2010
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Type 2 Diabetes Mellitus (T2DM) A Global Epidemic
~285 million people affected and rising due to obesity. T2DM accounts for 90-95% of the cases
Difficulties maintaining glucose levels in the blood due to:
Reduced insulin output from pancreas AND
Insulin resistance in peripheral tissues
Complications include:
CVD, neuropathy, nephropathy, eye damage, foot damage, hearing problems etc.
Glucose levels are maintained by the liver via storage (as polymer glycogen) and de novo production (gluconeogenesis).
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Gluconeogenesis (GNG) in MPCCs
Work of Mike Lehrer and Matt Davidson
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Glycogen Production in MPCCs
Work of Mike Lehrer and Matt Davidson
5mM Glucose, No Hormones
5mM Glucose + Insulin
5mM Glucose + Glucagon
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Patient-derived Induced Pluripotent Stem Cells
• iPSCs have been generated (~5 years ago) by expression of 4 transcription factors into somatic cells (i.e. skin)
• Human iPSCs can mimic human embryonic stem cells in all aspects of pluripotency and differentiation.
• Great potential for personalized medicine
• Functions in iPSC-derived human hepatocytes (iPSC-HHs) can be further improved (magnitude and longevity) using microscale engineering approaches.
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iPSCs-HHs in MPCCs: i-MPCCs
Work of Dustin Berger, Matt Davidson
Cells from Cellular Dynamics International
• i-MPH = micropatterned pure hepatocytes • i-MPCC = micropatterned co-cultures • CYP3A4-Glo from Promega: Contributions for CYP3A5 and CYP3A7? • Decline seen in sandwich cultures, which have ~5-10 fold lower activities
(on per cell basis) than MPCCs
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Toxicity Assessment in i-MPCCs
Work of Matt Davidson, Brent Ware
Cells from Cellular Dynamics International
• Diclofenac and amiodarone are hepatotoxins
• Aspirin and propranolol are relatively safe compounds
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Zonation in the Liver
• Zonation can lead to zone-specific drug toxicities in the liver
• Allows various hepatocytes in the liver to adapt to systemic changes in the body (i.e. fasting, feeding, disease)
• Very few model systems to study this in vitro.
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Modeling Oxygen In Vitro
Work of Dustin Berger
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Culture on Softer (More Tissue-Like) Substrates Chemo-mechanical Tuning of Polyelectrolyte Multilayers
Chen/Khetani et al, Biomaterials, 30, p1113-1120, 2009
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Engineering
• Microfabrication • Polymers • Computation/Modeling • High-throughput screening devices
• Multiplexed reporter systems
Overall Vision for Microfabricated Tissue Models Laboratory at CSU
Liver Disease
• Drugs • Industrial chemicals • Alcohol • Viruses • Nutrition
Cell Sources: Human, Animal, Stem Cells
Why? Prevention, Diagnosis and Treatment of Liver Disease
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Hepregen Research Team
Simon Aoyama Chitra Kanchagar Stacy Krzyzewski Amanda Moore Julianne Shi Jeannemarie Gaffney Okey Ukairo
Academic Collaborators
Charles Rice et al (Rockefeller) Sangeeta Bhatia et al (MIT) Mike Pagliassotti (CSU) Adam Chicco (CSU) Chuck Henry (Chemistry, CSU) Hugo Rosen (CU-Boulder) David Eddington (UIC) Neil Kaplowitz (USC)
Funding
Pfizer Corporate Partnership NIH Challenge Grant (PI: S.Khetani)
NSF SBIR Phase I and II (PI: S.Khetani) FDA SBIR Phase I and II (PI: S.Khetani)
CSU Mech. Eng Start-up funds
Acknowledgments
Industrial Collaborators
Scott Obach (Pfizer) Yvonne Will (Pfizer) Mike Aleo (Pfizer)
Microfabricted Tissue Models Lab (CSU)
Dustin Berger (PhD student) Brent Ware (PhD student) Christine Lin (Rotation PhD student) Matthew Davidson (ME student) Josh Pickrell (ME/SBME Undergraduate) Alison Bailey (CBE/SBME undergraduate)