Goals of the paper• Drug-induced liver toxicity  acute liver failure and post-  market drug withdrawals   – Preclinical a...
Microscale Liver   Hepatocyte Cultures          Reusable stencil  ECM gets the liver cells to stick       Green = hepatocy...
Other Observations• 3T3-J2 fibroblasts were used as ECM because:   – Readily available, ease of propagation   – Lack of li...
Hepatocyte Morphology• Rapid loss of morphology in pure cultures• Micro-patterned co-culture retains morphology
Hepatocyte Viability• Albumin secretion  protein synthesis (stable in 3-6 days)• Urea synthesis  nitrogen metabolism (st...
DME Activities and Canalicular Transport• Phase I, II, III: CYPs, glucuronidation, sulfation, transporters• Tested over 3 ...
DME Expression Levels (91 genes)• Phase I: CYPs, FMOs, MAOs, aldehydre oxidase, epoxide  hydrolase• Phase II: UGTs, SULTs,...
Expression Levels• Nuclear receptors, transcription factors, transporters• DMEs• Compared fresh hepatocytes to micropatter...
Toxicity Screen• Relative toxicity (mitochondrial activity) corresponded to  relative hepatotoxicity• Troglitazone vs othe...
Time and Cocentration DependentToxicity of Troglitazone
CYP Induction: 3-4 d                       PXR                       CAR                 AhR
DDI Study and Species Differences
Conclusions• Method developed to test drug metabolism and toxicity   – Human hepatocytes in a micropatterned multiwell for...
Future Directions• Further scale it down to a 384-well format for high-throughput  screening• Get the cultures to survive ...
Microscale culture of human liver cells for drug development
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Microscale culture of human liver cells for drug development

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Microscale culture of human liver cells for drug development

  1. 1. Goals of the paper• Drug-induced liver toxicity  acute liver failure and post- market drug withdrawals – Preclinical animal models are inadequate – Current human liver models are limited • Liver slices, microsomes, cell lines, and hepatocytes• To develop a method for culturing human hepatocytes in a multi-well format• To test the viability and functionality of the hepatocytes – Measuring enzyme activity and expression levels
  2. 2. Microscale Liver Hepatocyte Cultures Reusable stencil ECM gets the liver cells to stick Green = hepatocytes Orange = fibroblasts 24 well plates 10,000 hepatocytes in 37 colonies888 repeating hepatic microstructures Stable for several weeks in culture
  3. 3. Other Observations• 3T3-J2 fibroblasts were used as ECM because: – Readily available, ease of propagation – Lack of liver specific gene expression – Induction of high levels of liver-specific functions – Human non-parenchymal cells did not stabilize to the same extent – Enhanced function and longevity• Micropatterned clusters outperformed random distribution
  4. 4. Hepatocyte Morphology• Rapid loss of morphology in pure cultures• Micro-patterned co-culture retains morphology
  5. 5. Hepatocyte Viability• Albumin secretion  protein synthesis (stable in 3-6 days)• Urea synthesis  nitrogen metabolism (stable immediately)• Rapid loss of both in the pure cultures
  6. 6. DME Activities and Canalicular Transport• Phase I, II, III: CYPs, glucuronidation, sulfation, transporters• Tested over 3 weeks
  7. 7. DME Expression Levels (91 genes)• Phase I: CYPs, FMOs, MAOs, aldehydre oxidase, epoxide hydrolase• Phase II: UGTs, SULTs, COMT, NATs, GSTs, other transferases• 0 and 8 weeks (pure heptocytes are inactive)
  8. 8. Expression Levels• Nuclear receptors, transcription factors, transporters• DMEs• Compared fresh hepatocytes to micropatterned co-culture
  9. 9. Toxicity Screen• Relative toxicity (mitochondrial activity) corresponded to relative hepatotoxicity• Troglitazone vs other glitazones
  10. 10. Time and Cocentration DependentToxicity of Troglitazone
  11. 11. CYP Induction: 3-4 d PXR CAR AhR
  12. 12. DDI Study and Species Differences
  13. 13. Conclusions• Method developed to test drug metabolism and toxicity – Human hepatocytes in a micropatterned multiwell format• Great stability – Maintains liver functions for several weeks – Better than current collagen/matrigel systems • Stability, hepatic function, robustness• Order of magnitude fewer hepatocytes required – Works for various fresh hepatocytes (age, gender, medical history) – Works with cryopreserved hepatocytes as well
  14. 14. Future Directions• Further scale it down to a 384-well format for high-throughput screening• Get the cultures to survive for several months to further improve ability to do long-term drug tests• Infect the cultures with infectious diseases to enable study of pathogen lifecycle and for drug discovery purposes• Hepregen (an MIT spinout): – has licensed the technology – established collaborative research agreements with big pharma (Merck, Novartis) for the development, validation, and evaluation – hopes to be operational around spring 2008

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