3D-Zellkulturgerüste für die biomedizinische Grundlagenforschung

Transcription

1 3D-Zellkulturgerüste für die biomedizinische Grundlagenforschung Zoologisches Institut, Fakultät für Chemie- und Biowissenschaften, Zell- und Neurobiologie KIT University of the State of Baden-Württemberg and National Large-scale Research Center of the Helmholtz Association

2 Cell Differentiation: Tissue Engineering DeForest & Anseth (2012) Annu.Rev.Chem.Biomol.Eng. 3:

3 Cell Differentiation: 3D Culture Systems Baker & Chen (2012) J.Cell Sci 125: Greiner A.M., Richter B., Bastmeyer M. (2012) Micro-Engineered 3D Scaffolds for Cell Culture Studies. Macromolecular Biosciences 10: DOI: /mabi

4 Wish List Controllable 3D-Environment with: - defined geometry - adjustable mechanical properties - patterned matrix composition - multiple bio-functionalization - quantitative bio-functionalization - passivation (non-protein binding) - local application of soluble factors 4

5 Three-dimensionality Cell-cell adhesion / signaling Chemical matrix composition Growth factors Cell-matrix adhesion Physical matrix characteristics 5

6 Direct Laser Writing (DLW) 3D Martin Wegener Georg von Freymann Device: Photonic Professional (Nanoscribe GmbH) Details: 6

7 Direct Laser Writing (DLW) photoresist glass substrate 1 7

8 Direct Laser Writing (DLW) sample scanning 1 2 8

9 Direct Laser Writing (DLW) monomeric photoresist crosslinked polymer structure

10 Direct Laser Writing (DLW)

11 Direct Laser Writing (DLW)

12 Fabricated Structures 20 µm 12

13 3D Cellular Microenvironments: Different Cell Types Endothelium Connective Tissue: 13

14 3D Cellular Microenvironments: Cell Volume 3D MEF NRK LSM 2D AFM Fibroblast-like Epithelial-like 14 AM Greiner, F Klein, T Gudzenko, B Richter, T Striebel, BG Wundari, TJ Autenrieth, M Wegener, CM Franz and M Bastmeyer (2015) Cell type-specific adaptation of cellular and nuclear volume in micro-engineered 3D environments. Biomaterials 69:

15 3D Cellular Microenvironments: Analysis of Cell Migration Simplified well-defined quasi-3d cellular microenvironments with different mesh sizes Alexandra Greiner 15 AM Greiner, M Jäckel, AC Scheiwe, DR Stamow, TJ Autenrieth, J Lahann, CM Franz and M Bastmeyer Multifunctional polymer scaffolds with adjustable pore size and chemoattractant gradients for studying cell matrix invasion (2014) Biomaterials 35:

16 3D Cellular Microenvironments: Analysis of Cell Migration The nucleus is 5-10x stiffer than the surounding cytoskeleton The nucleus is the limiting factor for cell migration in tissues The nucleus is mechanically stabilized by a network of structural proteins (Lamins) that are connected to the cytoskleleton Enhanced cell migration because of softer nuclei is postulated in: embryogenesis metastasizing cancer cells Nuclear mechanics during cell migration P Friedl, K Wolf, and J Lammerding Current Opinion in Cell Biology 2011, 23:

17 AFM approach to measure the interphase nuclear stiffness in MEF WT and Lamin A (lmna) KO cells MEF lmna knockout (KO) cells have a softer interphase nucleus compared to MEF wildtype (WT) cells. 17

18 Invasion of WT and lmna KO fibroblast into 3D scaffolds on glass MEF lmna KO cells invade to a higher level into the 3D scaffold than MEF WT cells. Thus, cell invasion is dependent on the nuclear stiffness. 18

19 3D Cellular Microenvironments: Mouse Embryonic Stem Cells Impact of dimensionality and adhesion area on cell proliferation? Sarah Baur FN FN Mona Jaggy 19

20 Three-dimensionality Cell-cell adhesion / signaling Chemical matrix composition Growth factors Cell-matrix adhesion Physical matrix characteristics 20

21 Flexible 3D Cellular Microenvironments Primary Cardiomyocytes ORMOCER e: Anorganic-organic Hybridpolymer 21 F Klein, T Striebel, J Fischer, Z Jiang, CM Franz, G von Freymann, M Wegener and M Bastmeyer (2010). Elastic fully three-dimensional microstructure scaffolds for cell force measurements. Advanced Materials 22:

22 Flexible 3D Cellular Microenvironments Primary Cardiomyocytes in 3D-Ormocer -Structures 22

23 Flexible 3D Cellular Microenvironments Displacement: 0.81 ± 0.07 µm Primary cardiomyocytes in 3D-Ormocer -structures 23

24 Flexible 3D Cellular Microenvironments: Force Measurements Contraction force: nn Clemens Franz 24 F Klein, T Striebel, J Fischer, Z Jiang, CM Franz, G von Freymann, M Wegener and M Bastmeyer (2010). Elastic fully three-dimensional microstructure scaffolds for cell force measurements. Advanced Materials 22:

25 Three-dimensionality Cell-cell adhesion / signaling Chemical matrix composition Growth factors Cell-matrix adhesion Physical matrix characteristics 25

26 3D Cellular Microenvironments: Bio-functionalization in 3D, 1 st Strategy 1. Resist: PEG-derivate 2. Resist: Ormocere Polyethylene glycol diacrylate (PEG-DA) + 5% Pentaerythritol tetraacrylate (PETA) Anorganic-Organic-Hybridpolymer 26

27 3D Cellular Microenvironments: Bio-functionalization in 3D, 1 st Strategy Functionalization with Fibronectin 27 F Klein, B Richter, T Striebel, CM Franz, G von Freyman, M Wegener and M Bastmeyer (2011). Two-Component Polymer Scaffolds for Controlled Three-Dimensional Cell Culture. Advanced Materials 23:

28 3D Cellular Microenvironments: Bio-functionalization in 3D, 1 st Strategy 28

29 3D Cellular Microenvironments: Mechanical stimulation of fibroblasts Andrea Scheiwe 29 Scheiwe A., Frank S., Autenrieth T., Bastmeyer M. and Wegener M. (2015) Subcellular stretch-induced cytoskeletal response of single fibroblasts with 3D designer scaffolds. Biomaterials 44:

30 3D Cellular Microenvironments: Aerobics for fibroblasts 10 µm displacement 0.5 Hz frequency 15 min duration Chemical fixation Immunostaining 30

31 3D Cellular Microenvironments: Mechanical stimulation of fibroblasts Immunostaining for Actin, Paxillin, and phospho-focal Adhesion Kinase (pfak) 31

32 3D Cellular Microenvironments: Mechanical stimulation of fibroblasts Actin: n = 12 32

33 3D Cellular Microenvironments: Mechanical stimulation of fibroblasts n = 12 Actin Paxillin pfak 33

34 3D Cellular Microenvironments: Bio-functionalization in 3D, 2 nd Strategy Biocompatible Click Chemistry 34 Christopher Barner-Kowollik Guillaume Delaittre Thomas Pauloehrl Pauloehrl T., Delaittre G., Winkler V., Welle A., Bruns M., Börner H.G., Greiner A.M., Bastmeyer M. and Barner-Kowollik C. (2012) Adding spatial control to click chemistry: phototriggered Diels-Alder surface (bio)functionalization at ambient temperature. Angew Chem Int Ed 51: Beni Richter

35 3D Cellular Microenvironments: Bio-functionalization in 3D, 2 nd Strategy 1. DLW 2. Coating 3. UV+Click 4. Functionalize Si-photoenol R Phototriggered Diels-Alder cycloaddition 35 T Pauloehrl, G Delaittre, M Bastmeyer and C Barner-Kowollik (2012) Ambient Temperature Polymer Modification by In-Situ Phototriggered Deprotection and Thiol-Ene Chemistry. Polym. Chem. 3:

36 Bio-Molecular Tool Box 4. Functionalize 36

37 3D Cellular Microenvironments: Multiple Bio-functionalization 37 Richter B., Pauloehrl T., Kaschke J., Fichtner D., Fischer J., Greiner A.M., Wedlich D., Wegener M., Delaittre G., Barner- Kowollik C. and Bastmeyer M. (2013) Three-Dimensional Macroscaffolds Exhibiting Spatially Resolved Surface Chemistry. Advanced Materials 25:

38 Collaborators: Christopher Barner-Kowollik (KIT) Clemens Franz (KIT) Martin Wegener (KIT) Ulrich Schwarz (HD) Nina Ulrich (HD) Prof. Dr. Martin Bastmeyer, Zoologisches Institut, Zell- und Neurobiologie