In Vitro Assembly of 3D Bile Duct Networks within Decellularized Extracellular Matrix Hydrogels

Transcription

1 In Vitro Assembly of 3D Bile Duct Networks within Decellularized Extracellular Matrix Hydrogels Phillip Lewis, MS Department of Biomedical Engineering Northwestern University

2 In Vitro Assembly of 3D Bile Duct Networks within Decellularized Extracellular Matrix Hydrogels Liver and Biliary Tree Function and Tissue Engineering Decellularied Extracellular Matrix (decm) Hydrogels 3D Printing decm

3 In Vitro Assembly of 3D Bile Duct Networks within Decellularized Extracellular Matrix Hydrogels Liver has massive regenerative capacity, though it is not perfect >14,000 patients awaiting liver transplants in the US alone

4 Liver Anatomy & Physiology Liver function is grouped into 3 main categories: Metabolism Lipids (cholesterol), proteins, carbohydrates, drugs, etc. Synthesis Clotting factors, compliment factors, etc. Secretion Bile

5 Liver Anatomy & Physiology

6 Liver Tissue Engineering Strategies Takebe et al. Nature Uygun et al. Nature Medicine Sampaziotis et al. Nature Medicine

7 Intrahepatic Bile Duct Tissue Engineering Katsuda et al. Tissue Engineering Part A Roma et al. Clinical Science Mizuguchi et al. Molecular Pathology of Liver Diseases Dianat et al. Hepatology Auth et al. Hepatology Wade, Burdick. Materials Today. 2012

8 Intrahepatic Bile Duct Tissue Engineering Decellularized Extracellular Matrix (decm) Free from immunogenic cellular material Retains native organ structure Retains organ specific signaling components Can be processed into a gel Uygun et al. Nature Medicine ⁰C Mince Ly ophilize Pepsin 1M NaOH 0.1% SDS/dH 2 O Mill 0.01N HCl 10X PBS, dh 2 O 37 C

9 Intrahepatic Bile Duct Tissue Engineering x Z y y Z x

10 Intrahepatic Bile Duct Tissue Engineering Type 1 Collagen Matrigel Liver decm Day 7 Day μm

11 Intrahepatic Bile Duct Tissue Engineering

12 Intrahepatic Bile Duct Tissue Engineering Gel Structure Matrigel Duct Structure decm Credit: Jimmy Su Roma et al. Clinical Science Tanimizu et al. Molecular Biology of the Cell. 20

13 Intrahepatic Bile Duct Tissue Engineering HNF1β Intrahepatic cholangiocytes E-Cad Tight junctions DAPI - Nuclei SOX9 - Intrahepatic cholangiocytes E-Cad Tight junctions DAPI - Nuclei E-Cad Tight junctions Laminin Basement membrane protein DAPI - Nuclei Credit: Beatriz Sosa Pineda

14 Intrahepatic Bile Duct Tissue Engineering ZO1 Tight Junction Protein E-Cad Tight junctions DAPI - Nuclei CLDN7 Cholangiocyte tight junctions E-Cad Tight junctions CK19 Cytoskeletal protein Sox9 Intrahepatic Cholangiocytes DAPI - Nuclei Credit: Beatriz Sosa Pineda

15 Intrahepatic Bile Duct Tissue Engineering Bile Duct Function CLF Roma et al. Clinical Science Rifampicin CLF mcherry Cholangiocytes

16 Intrahepatic Bile Duct Tissue Engineering Duct Branch Structure Single nucleation point Tree Model Multicellular network assembly model

17 Analyzing Branch Structure Label different populations of the same cell line with 3 different fluorescent proteins mcherry egfp Azurite Observe duct structures over time 2D culture

18 Analyzing Branch Structure Label different populations of the same cell line with 3 different fluorescent proteins mcherry egfp Azurite Observe duct structures over time Single color controls 2 days in culture

19 Analyzing Branch Structure 2 Days

20 Analyzing Branch Structure 4 Days

21 Analyzing Branch Structure 6 Days

22 Analyzing Branch Structure 2 Days

23 Analyzing Branch Structure 4 Days

24 Analyzing Branch Structure 6 Days Red channel excluded

25 Analyzing Branch Structure 8 Days

26 Analyzing Branch Structure Branching model may be proximity dependent Cells beginning near one another will create multicolored ducts (assembly model) Cells in isolation will clonally expand (Tree model)

27 Intrahepatic Bile Duct Tissue Engineering How can we control duct formation? Other cell types will need to be incorporated Lee, Cho, et al. Biomacromolecules PCL (thermoplastic) frame for 3D structure

28 Controlling Duct Formation Advantages of PCL frame Can allow vascularization, nutrient diffusion upon implantation Disadvantages Mechanical property mismatch Slow degradation Large size scales Pati, Cho, et al. Nature Communications

29 Controlling Duct Formation 10% Gelatin 5% Gelatin 0.5% decm Other strategies to 3D printing decm hydrogels Blending with 3D printable gels (gelatin) Synthetically recreating ECM components Sacrificial support structures (fugitive) 5% Gelatin 0.5% decm 2.5% Gelatin 0.5% decm Increasing polymer weight fraction stifles duct formation Live/Dead Credit: Ming Yan Depth coded Live/Dead

30 Controlling Duct Formation Synthetic 3D printable ECM Synthetically recreating decm enhances translation potential IKVAV (Laminin mimetic) E2 (Filler) Peptide amphiphile gels cannot be 3D printed alone Tanimizu, et al. Molecular Biology of the Cell Laminin is essential to duct formation and cell polarization Shah, et al. PNAS

31 Controlling Duct Formation Synthetic 3D printable ECM Peptide amphiphiles can be combined with chemically modified gelatin 1:3 Gel:PA 1:1 Gel:PA 3:1 Gel:PA 1:0 Gel:PA Credit: Ming Yan

32 Controlling Duct Formation Synthetic 3D printable ECM As printed (2) Cros slinked (3) Credit: Ming Yan

33 Synthetic 3D-Printable ECM Day 4 Day 7 Day 14 CLF uptake Gelatin E2 IKVAV Credit: Ming Yan

34 Controlling Duct Formation How can we align duct growth? F 127 decm 4⁰C Liquid decm Gel Material extrusion can align ducts decmalone cannot be extruded as a gel Solution: sacrificial materials Pluronic F 127: reversible thermo responsive triblock copolymer Liquid at 4⁰C, gel at warmer temperatures 37⁰C 4⁰C Buffer wash

35 Controlling Duct Formation How can we align duct growth? decm 4⁰C Liquid F 127 decm Gel 37⁰C 4⁰C Buffer wash

36 Controlling Duct Formation How can we align duct growth? 2mm 1mm Width of strut influences duct alignment Hypothesized due to collagen fibril alignment

37 Conclusions and Future Directions Liver decm can lead to complex duct formation in vitro We can control duct formation using 3D printing Precise decm content needs to be determined Future strategies will incorporate multiple cell types Translatable cell sources such as hepatoblasts or stem cells

38 Acknowledgements Collaborating Labs Jason Wertheim, MD, PhD Richard Green, MD Gianfranco Alpini, PhD Beatriz Sosa Pineda, PhD Zheng Jenny Zhang, MD Ramille Shah Lab Members Prof. Ramille N. Shah Alexandra Rutz* Shannon Taylor Adam Jakus* Jimmy Su Danielle Duggins* Nick Geisendorfer Christina Robinson* *=Shah lab graduate Ming Yan Margaret Hammersly Max Greenberg Emma Gargus* Kelly Hyland* Chris Lee* Clayton Rische Lazura Krasteva