Microtechnology Tools for Studying the Cellular Environment

Transcription

1 Microtechnology Tools for Studying the Cellular Environment Sangeeta N. Bhatia, MD, PhD Health Sciences and Technology/ Electrical Engineering & Computer Science Massachusetts Institute of Technology Brigham & Women s Hospital

2 Engineering Liver Tissue Cell Microenvironment -Source -Stable Phenotype Tissue Fabrication -Miniaturized -Macroscale with microscale features Cell-Based Therapies Structure/Function In Vitro Screening

3 Cues in the Microenvironment Cell-Cell Interactions Cell-Matrix Interactions Soluble Factors 3- D context Physical Forces

4 Effects of Co-Culture Albumin (μg/10 6 cells/hour) Freshly Isolated Hepatocytes Day Pure Hepatocytes 2 weeks later

5 Effects of Co-Culture Albumin (μg/10 6 cells/hour) In vivo levels Freshly Isolated Hepatocytes Day Co-Cultured Hepatocytes 2 weeks later

6 Photolithographic Micropatterning Bhatia+, J Biomed Mat Res, 1997; Bhatia+, J of Biom Science, 1998; Bhatia+, Biotech Prog, 1998; Bhatia +, FASEB J, 1999; Khetani+, Hepatology, 2004

7 Control of Cell-Cell Interactions Island size 36 μm 100 μm 490 μm Cocultures Hepatocytes

8 Micropatterned Co-Cultures Reveal Role of Tissue Organization Urea Synthesis [μg/day] Hepatocyte Island Diameter [μm] Micropatterned Hepatocytes Alone Micropatterned Co-Cultures * * * * * * Albumin Secretion [μg/day] heps HEPS-11 Micropatterned Hepatocytes Alone co-culture * * * * * * * CO-CULT11 Micropatterned Co-Cultures Molecular Mechanisms -Gap junctions -Fibroblast products Dynamics/Reciprocal Species-Specificity Miniaturize for ADME/T Bhatia+ FASEB J, 1999; Khetani+ Hepatology, 2004

9 Hui+, unpublished Reconfigurable Cell Culture 80 µm gap Contact 18 h contact Contact then gap Contact then removal Micromechanical Substrate

10 Effect of Cell Type on Co-Culture Albumin μg/day DAY 6 Hepatocyte/3T3-J2 Hepatocyte/NIH-3T3 Hepatocyte/NIH2-3T3 Hepatocyte/3T3-L1 Hepatocytes Only Urea μg/day ALBUMIN UREA 15 Khetani+ Hepatology, 2004

11 Miniaturized Liver in a Well: Stencil-Based Soft Lithographic Process Khetani+, submitted ~270 cells per island, 10K / well versus 150K / well in conventional systems

12 Micropatterned Co-Culture Secretion Phase I, II, III mrna profile Toxicity Drug-drug interaction Species-specific responses

13 Acute toxicity on Human Tissues

14 Cues in the Microenvironment Cell-Cell Interactions Cell-Matrix Interactions Soluble Factors 3- D context Physical Forces

15 Combinatorial Matrix Biology Flaim+ Nature Methods, 2005

16 Flaim+ Nature Methods, 2005 ECM Microarray Layout

17 Viable Living Cell Microarrays Flaim+ Nature Methods, 2005

18 Differentiation Modulated by Local ECM Flaim+ Nature Methods, 2005

19 ES Cell Microarrays Day 3, F-Actin, Day 1 Confocal 0.1% >> 17 % or 170X Flaim+ Nature Methods, 2005

20 Cues in the Microenvironment Cell-Cell Interactions Cell-Matrix Interactions Soluble Factors 3- D context Physical Forces

21 3-D Tissue Fabrication 3-D D Printing Kim/Griffith, Ann Surg 1998 Fused Deposition Molding Liver Microarchitecture Hutmacher, J Biomater Sci Polm Ed 2001

22 Using Light to Fabricate Tissues Photopolymerization PEGDA + cells + Stereolithography hν Photoinitiator + light Hydrogel + entrapped cells flow Add Layer flow 3 12 Hern/Hubbell JBMR 1998 Elisseef/Langer PNAS 1999

23 3-D Hepatic Tissue Scale-up Design Photograph Micrograph Liu+, unpublished

24 3-D Hepatic Tissue MTT Viability Static Unpatt Perfused Patt

25 DEP Cell Arraying top electrode Electric Field Magnitude +DEP DEP HIGH z x bottom electrode z x F DEP = K ƒ CM E 2 LOW y x 100 um y z x E-field at at bottom surface Albrecht+ (2004) Biophysical J

26 Immobilization in 3D by Photopolymerization 100 µm Albrecht+ (2005) Lab-On-A-Chip

27 Immobilization in 3D by Photopolymerization light 100 µm

28 Albrecht+ (2006) Nature Methods 3D Microenvironment

29 Effect of 3D cluster size on chondrocytes Albrecht+ (2006) Nature Methods

30 Varying Cell Type and Hydrogel Albrecht+ (2006) Nature Methods

31 Summary & Conclusions Microenvironment -StromalCells - Comb. of Matrix - Soluble Gradients -3D Enabling microtechnology tools Generalizable -organs - stem cells Cell-Based Therapies Structure/Function In Vitro Screening

32 Collaborators M. Mrksich, U of Chicago F. Gage, Salk Institute C. Chen, UPenn R. Sah, UCSD M. Sailor, UCSD P. Sharp, MIT E. Ruoslahti, Burnham Institute J. West, Rice U B. Ranscht, Burnham Institute Surface Logix, Inc. Members of LMRT Acknowledgements Post-doctoral Fellows: E. Hui, G. Underhill, D. Eddington, D. Albrecht, D. Min, S. Khetani, S. March Riera Graduate: C. Flaim, A. Derfus, G. von Maltzahn, T. Harris, A. Chen, S. Mittal Staff: K. Hudson, S. Kangiser, M. Akiyama, S. Katz Funding David and Lucile Packard Foundation, NIH NIDDK, NIH NCI NSF CAREER, NCI/NASA, Deshpande Center

33 Photopatterning Constructs 500µm focal plane 100µm Albrecht+ (2005) Lab-On-A-Chip

34 Co-Culture with T-cad CHO T-cadherin induces hepatocyte function sirna knockdown Culture on Acellular T-cad Khetani+, unpublished