Engineering a Better In Vitro Drug Metabolism Model

1. Engineering a Better In Vitro Drug Metabolism Model Olga Filippova, Munir Nahri, Akash Patel Group 1

2. Current In Vitro Tissue Models • Drug metabolism models • Animals, liver slices, isolated hepatocytes, enzymes • Predict in vivo drug metabolism by scaling in vitro data • Factors that effect in vitro model efficacy • Metabolism in other tissues • Equilibrium of drugs between blood and hepatocytes • Active transport through the sinusoidal membrane • Newest advancement – bioprinted microorgans INSERT DRUG METABOLISM PICTURE!!!

3. Maintaining Hepatocyte Function • Extracellular Matrix Interaction • Chemical composition • Dynamic composition alter cell activity • Mechanical regulation • Critical determination of cell cycle progression/differentiation • Cell-to-Cell Communication • Physical • Interactions between hepatocytes and non-parenchymal cells crucial to maintaining differentiated function • Chemical • Cytokines, Growth Factors, Paracrine and Autocrine

4. Objective and Specific Aims Objective: Create a better in vitro drug metabolism model Specific Aims 1. Improve hepatocyte survival • 3D, sinusoidal printed constructs • Better ECM composition through Matrigel • Co-culture with hepatocyte stellate cells 2. Improve drug metabolism specific function • Increase Cytochromes P450 (CYP) expression • Matrigel culture • Stellate cell communication

5. Experimental Design Cells • Sprague-Dawley rat harvested hepatocytes • Immortalized rat liver stellate cell line HSC-T6 Biomaterial • Alginate with hepatocytes and/or stellate cells • Matrigel with hepatocytes and/or stellate cells Construct Printing • Solid freeform fabrication of cell seeded biomaterial into 3D sinusoidal pattern INSERT SIDE VIEW OF PRINTING

6. Experimental Design Construct Characterization • Hepatocyte survival • Live/Dead cell assay for viability • Alamar blue assay for proliferation • Hepatocyte function • Northern blot for CYP mRNA expression • Nonpolar active drugs into polar inactivemetabolites Controls/Variables Alginate Alginate Hepatocytes Hepatocytes Stellate Cells Matrigel Matrigel Hepatocytes Hepatocytes Stellate Cells

7. Specific Aim 1: Improve Survival Rationale • Printed 3D sinusoidal construct better mimic in vivo hepatocyte microenvironment(Chang) • Matrigel composition (laminin, collagen IV) better recreates natural ECM(Kibbey) • Co-cultures with non-parenchymal cells show improved long term cell viability and activity of differentiated hepatocytes(Abu) • Stellate cells produce HGF, TGF-α, and EGF • Potent mitogens for hepatocytes

8. Specific Aim 1: Improve Survival Expected Results • Matrigel superior to alginate • Co-culture superior to hepatocytes only Pitfalls • Printing parameters negatively affect viability • Unique thermo-physical Matrigel properties Alternatives • Co-culture different cell types • Alter printing conditions

9. Specific Aim 2: Improve Function Rationale • Matrigel better mimics in vivo ECM microenvironment • Matrigel medium shown to reverse initial decrease in CYP expression after hepatocyte isolation(Gold) • Co-culture hepatocytes with stellate cells improve hepatocyte specific drug metabolism function • Better albumin and cytochrome CYP expressionZA, ZB • Prolonged CYP functionZB

10. Specific Aim 2: Improve Function Expected Results • CYP expression will increase in Matrigel culture • CYP expression will further increase in co-cultures Pitfalls • Increase in expression of only certain CYP • Initial drop in CYP expression still present Alternative solution • Use drug metabolism assay to asses CYP function • Use alternative assay to assess liver function

11. Journal Club Kocarek TA, Schuetz EG, and Guzelian PS. Expression of multiple forms of cytochrome P450 mRNAs in primary cultures of rat hepatocytes maintained on matrigel. Molecular Pharmacology 43, 1992, 328. Problem: Isolated hepatocyte lose CYP expression Method: Assess CYP expression of hepatocytes on different culture media Result: Spontaneous reexpression of CYP mRNA shown on Matrigel Conclusion: Matrigel better medium for drug metabolism model

12. Journal Club Experimental Setup • Rat hepatocytes plated on dishes coated with Matrigel or collagen I • 5 days • Northern blot mRNA analysis

13. Journal Club Results • Matrigel – mRNA spontaneously re-expressed after an initial decay • Drop - 1A2, 2E1, 3A1/2 • Rise – 2B1/2, 2C6, 2C7, 4A1 • Steady – 2D, 2A1/2

14. References • Iwatsubo T, Hirota N, Ooie T, Suzuki H, Shimada N, Chiba K, Ishizaki T, Green CE, Tyson CA, and Sugiyama Y. Prefiction of in vivo drug metabolism in the human liver from in vitro metabolism data. Pharmacol. Ther. 73, 1997, 147. • Venkatakrishnan K, von Moltke LL, and Greenblatt DJ. Human drug metabolism and the cytochromes P450: Application and relevance of in vitro models. J. Clin. Pharmacol. 41, 2001, 1149. • Zuber R, Anzenbacherova E, and Anzenbacher P. Cytochromes P450 and experimental models of drug metabolism. J. Cell. Mol. Med. 6, 2002, 189. • Iwahori T, Matsuura T, maehashi H, Sugo k, saito M, Hosokawa M, Chiba K, Masaki T, Aizaki H, Ohkawa K, and Suzuki T. CYP3A4 inducible model for in vitro analysis of human drug metabolism using a bioartificial liver. Hepatology 37, 2003, 665. • Kocarek TA, Schuetz EG, and Guzelian PS. Expression of multiple forms of cytochrome P450 mRNAs in primary cultures of rat hepatocytes maintained on matrigel. Molecular Pharmacology 43, 1992, 328. • ZA: Abu-Absi, SF, Hansen, LK, Hu, W. Three-dimensional co-culture of hepatocytes and stellate cells. Cytotechnology 45, 2004, 125. • ZB: Thomas, RJ, Bhandari, R, Barrett DA, Bennett, AJ, Fry, JR, Powe, D, Thomson, BJ, Shakesheff, KM. The effect of three-dimensional co-culture of hepatocytes and hepatic stellate cells on key heptocyte functions in vitro. • Chang R, Nam J, and Sun W. Direct cell writing of 3D microorgan for in vitro pharmacokinetic model. Tissue Engineering 14, 157, 2008. • Kibbey, M. C. 1994. “Maintenance of the EHS sarcoma and Matrigel preparation,” Journal of Tissue Culture Methods, Vol. 16, pp. 227-230.

15. Questions? Olga Filippova, Munir Nahri, Akash Patel Group 1