Microscale Engineering of Tissue (Liver) Models for Compound Screening

Salman R. Khetani

Assistant Professor Department of Mechanical Engineering

School of Biomedical Engineering

Fresh Hepatocytes

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

1 2 3 4 5 6 7 9 10 12 14

Day

Albu

min

(µg/

106 ce

lls/h

our)

2 weeks later

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)

The Liver Microenvironment Role of the Microenvironment in Modulating Hepatic Fates

Khademhosseini A et al. PNAS 2006;103:2480-2487

Microscale Technologies For Tissue Engineering Semiconductor-Driven Tools for Biological Applications

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

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

T-Cadherin Induces Hepatocyte Functions Cellular (CHO) and Purified (Recombinant) Protein Presentation

Khetani et al, FASEB J, 22(11), p3768-3775, 2008

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

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

• 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

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

Day 10 Day 2 Day 10

Kupffer Macrophages in MPCCs

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

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

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).

Gluconeogenesis (GNG) in MPCCs

Work of Mike Lehrer and Matt Davidson

Glycogen Production in MPCCs

Work of Mike Lehrer and Matt Davidson

5mM Glucose, No Hormones

5mM Glucose + Insulin

5mM Glucose + Glucagon

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.

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

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

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.

Modeling Oxygen In Vitro

Work of Dustin Berger

Culture on Softer (More Tissue-Like) Substrates Chemo-mechanical Tuning of Polyelectrolyte Multilayers

Chen/Khetani et al, Biomaterials, 30, p1113-1120, 2009

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

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)