Développement de microenvironnements 3D mimétiques de la crypte intestinale par Bioimpression

Transcription

1 Développement de microenvironnements 3D mimétiques de la crypte intestinale par Bioimpression J. Creff, A. Besson, R. Courson, S. Souleille, E. Trévisiol, J. Foncy, L.Malaquin Equipe ELiA - LAAS CNRS Equipe Cycle Cellulaire et Cancer - CRCT Matching Day «Techno & Cancer» - 14 septembre 2017 LAAS, Toulouse LAAS-CNRS / Laboratoire d analyse et d architecture des systèmes du CNRS Laboratoire conventionné avec l Université Fédérale de Toulouse Midi-Pyrénées

2 Microenvironment models for cell culture What is needed : 3D topography Porosity Stiffness Cell heterogeneity Environment control Vascular network

3 Microenvironment models for cell culture Pati, F.; Gantelius, J.; Svahn, H. A. 3D Bioprinting of Tissue/Organ Models. Angew. Chemie Int. Ed. 2016, 55, Drawbacks of recent tissue/organ models Multistep fabrication methods Limited cell viability and functionality Missing complex and hierarchical tissue structure Missing 3D structure in the case of biochips Large variation in results obtained from animal studies Incapability of animal models to reproduce features of human tissues and organs Tightening controls on use of animals for scientific experimentation D. Ingber, Lab On chip 2012

4 Bioprinting Murphy, S. V., & Atala, A. (2014). 3D bioprinting of tissues and organs. Nature biotechnology Wang, J., et al. (2014). Phage Nanofibers Induce Vascularized Osteogenesis in 3D Printed Bone Scaffolds. Advanced Materials. d e Kolesky, D. B. et al. (2014). 3D Bioprinting of Vascularized, Heterogeneous Cell Laden Tissue Constructs. Advanced Materials, 26(19), Mannoor, M. S. et al. (2013). 3D printed bionic ears. Nano letters, 13(6),

5 What a 3D printer can do Macro Micro Nano Cell and Tissue Engineering MEMs Microfluidics

6 Architectures for Cell Colonization Multi photons Lithography 3D printing of artificial scaffolds Accardo, A.et al. Multiphoton Direct Laser Writing and 3D Imaging of Polymeric Freestanding Architectures for Cell Colonization. Small 2017, (IP- Dip resist Nanoscribe) Seeding of N2A (Neuroblastoma Cells) A. Accardo Collaboration with Inserm TONIC U1214 Two scaf

7 3D Printing projects at LAAS MultiFAB Platform LAMP Light Assisted Microfluidic Printing High resolution 3D printing DILASE 3D Patnership with Kloé (Montpellier) Patent filed (LAAS CNRS TTT) Technical specifications : Technical specifications : - Targeted Resolution X,Y : 5um Targeted Resolution Z : 5-100um Laser Wavelength : 405 nm (50 mw) Samples size (10 x 10 x 5 cm - X,Y,Z) - Multimaterial (Microfluidic injection) Matrix or Cell printing Targeted Resolution X,Y : 20um Targeted Resolution Z : 5-500um Laser Wavelength : 405 nm (50 mw) Samples size (10 x 10 x 5 cm - X,Y,Z)

8 Tumor progression: influence of microenvironment In Vitro models for intestinal epithelium : CRCT ( A.Besson) Institut Curie (J.L. Viovy) / IRSD (A. Ferrand, N. Vergnolle) Van der Flier L.G. and Clevers H., Annue. Rev. Physiol, 2009 Tian H. et al, Nature, 2011 Van Landeghem L. et al, Am. J Physiol. Gastrointest. Liver Physiol, 2012 Clevers H., Cell, 2013 Barker N., Nature, 2014 p57 as an inhibitor of CDK (cyclin dependant kinase) Stem cell fate and proliferation

9 In Vitro models for intestinal epithelium 3D printing DS3000 (PU/PA Biocompatible photoresist)

10 In Vitro models for intestinal epithelium 3D printing DS3000 (PU/PA Biocompatible photoresist) PEG-DA (700) +Irgacure Acrylic acid

11 In Vitro models for intestinal epithelium 3D printing Improve adhesion : hydrogel mix PEG-DA acrylic acid + collagen 1 coating Ø With acrylic acid: cellular layer uniform and confluent Ø Without acrylic acid: weak adhesion, round cells and no confluence Ø Best adhesion: 40% PEG % acrylic acid mix

12 Selecting the right material to mimic ECM - PEGDA Hydrogels Β-catenin Phalloidin Hoescht/β-cat Hoescht/β-cat/ Phalloidin 40%PEG-DA % acrylic acid + Laminin 50µg/mL 40%PEG-DA % acrylic acid + fibronectin 250 µg/ml 40%PEG-DA % acrylic acid 40%PEG-DA700 + fibronectine 250µg/mL 40%PEG-DA700 Hoechst 33342

13 In Vitro models for intestinal epithelium 3D printing Improve adhesion : hydrogel mix PEG-DA acrylic acid + collagen I coating Two-photon confocal imaging A) B) Light sheet fluorescence microscopy (S. Allart, CPTP - Inserm) (LSFM) (J. Rouquette ITAV) 500µm

14 In Vitro models for intestinal epithelium Light sheet fluorescence microscopy Vili 500µm 200µm Crypt Structures (40 % PEGDA 700, 30% acrylic acid, 30% water/medium, 0,01% Irgacure et 1% fluorescent nanoparticles (300nm ).

15 Towards intestine epithelium Caco 2 cell culture ( J+4 ) Two-photon confocal imaging

16 Perspectives Intestinal model : Mimicking cell heterogenity (J. Creff, A. Besson CRCT, A. Ferrand, D. Hamel IRSD) Red particules contamination à Multimaterial printing à Cell viability à Cell lines / Human samples à Integration in microfluidic devices Hauteur des structures 100 µm In vitro models of bon marrow microenvironments. (F. Deschaseaux, L. Casteilla StromaLab, W. han, J. Camonis, Inst. Curie)

17 Acknowledgements LAAS CNRS E. Trévisiol, C. Vieu, J. Foncy, E. Dague (EliA Group) R. Courson (TEAM) S. Souleille, C. Blatché, X. Dollat, J. roux (I2C) CRCT A. Besson, J. creff IRSD U1220 A. Ferrand, N. Vergnolle Equipex LEAF A.M. Gué Cancéropôle GSO Oncodevice Project MultiFAB Project (Région Occitanie, Feder) HoliFAB Project (H2020) Stromalab L. Casteilla, F. Deschaseaux CPTP Platform (S. Allart) ITAV Platform (J. Rouquette)