Automated Patch Clamping Systems Design Using Novel Materials Sandra - PowerPoint PPT Presentation

Introduction Background Design Work Conclusions Forschungszentrum Karlsruhe in der Helmholtz-Gemeinschaft Automated Patch Clamping Systems Design Using Novel Materials Sandra Wilson [a,b] , Alexander Welle [c] , Eric Gottwald [c] , Areles Molleman [d] ,, Wilhelm Pfleging [e] , Paul Kirby [b] , Jeremy Ramsden [b] [a] Institute for Microsystems Technology, Forschungszentrum Karlsruhe, DE [b] School of Applied Sciences, Cranfield University, UK [c] Institute for Biological Interfaces, Forschungszentrum Karlsruhe, DE [d] School of Life Sciences, University of Hertfordshire, UK [e] Institute for Materials Research 1, Forschungszentrum Karlsruhe, DE 04.10.2007 4M Annual Conference: Borovets, Bulgaria 1

Introduction Background Design Work Conclusions Forschungszentrum Karlsruhe in der Helmholtz-Gemeinschaft Project Context • Can 4M technologies & MST be used to produce a high throughput and automated cell measurement technique which presently is rather cumbersome? • Cells are surrounded by membranes, patch patch clamping is a method clamping where a patch of the membrane is sucked into a pipette so current measurements can be made. 04.10.2007 4M Annual Conference: Borovets, Bulgaria 2

Introduction Background Design Work Conclusions Forschungszentrum Karlsruhe in der Helmholtz-Gemeinschaft Outline • Introduction and Background – Importance of ion transport measurements – Transport across cells – H igh T hroughput S ystems ( HTS) • Design Considerations • New Process – Materials selection – Laser holes – Surface modification • Conclusions / Further Work 04.10.2007 4M Annual Conference: Borovets, Bulgaria 3

Introduction Background Design Work Conclusions Forschungszentrum Karlsruhe in der Helmholtz-Gemeinschaft Importance of Ion Channel Measurements • Understanding how and what effects drug transport into a cell, in research for identifying precursor factors leading to disease • Patch clamping for measuring electrical activity of cells as ions cross between inside and outside of the cell through their membranes. • Applications: – drug screening – proteomics and understanding cell function – ion channel function in hereditary diseases (epilepsy, Alzheimer's) Ca ++ in heart disease • Na + in pain management • K + in cell activation • 04.10.2007 4M Annual Conference: Borovets, Bulgaria 4

Introduction Background Design Work Conclusions Forschungszentrum Karlsruhe in der Helmholtz-Gemeinschaft Cell Membrane phospholipid bilayer distribution of main ions found in mammal cells extracellular medium Ion Intracellular Extracellular Range (mM) Range (mM) ( o ) ( i ) hydrophilic Na + 5-20 130-160 K + 130-160 4-8 hydrophobic Ca 2+ 0.5-1 1.2-4 Mg 2+ 10-20 1-5 Cl - 1-60 100-140 - HCO 3 1-3 20-30 [ ] RT ion ln = E o [ ] Equilibrium State (resting potential): intracellular medium zF ion i How does a cell manage to control the flow? 04.10.2007 4M Annual Conference: Borovets, Bulgaria 5

Introduction Background Design Work Conclusions Forschungszentrum Karlsruhe in der Helmholtz-Gemeinschaft Ion Channels • ‘Controlled Leak’- Ion Channels /Transporters K+ channel Na+/K+ pump sodium- glucose transporter extracellular membrane intracellular – Ion channels – Transporters: • pumps • exchangers 04.10.2007 4M Annual Conference: Borovets, Bulgaria 6

Introduction Background Design Work Conclusions Forschungszentrum Karlsruhe in der Helmholtz-Gemeinschaft Traditional Patch Clamping Ag/AgCl measurement electrode ‘KCl‘ filled pipette 2-3M 1G � seal Pipette opening diameter: 1-4µm 04.10.2007 4M Annual Conference: Borovets, Bulgaria 7

Introduction Background Design Work Conclusions Forschungszentrum Karlsruhe in der Helmholtz-Gemeinschaft HTS Systems Design Cytocentrics Nanion, Munich, Flyion SophionBioscience, DE ThinXXS US/DE Rostock, DE Tuebingen, DE Hole Array Materials quartz glass borosilicate glass quartz glass Si/ SiOx Cell cell suspension cell suspension cell suspension cell suspension Placement vacuum suction vacuum suction vacuum vacuum suction pressure No. cells/run 1-20 1-16 48 48 Cell types CHO, Jurkat CHO,Jurkat CHO, Jurkat Info. not available Neuro 2A HEK HEK 293, 04.10.2007 4M Annual Conference: Borovets, Bulgaria 8

Introduction Background Design Work Conclusions Forschungszentrum Karlsruhe in der Helmholtz-Gemeinschaft Design Considerations Materials • biocompatible, clean, good surface finish (Ra < 0.5 µm) • multiple sites for parallel rapid measurement • method of moving or placing a cell over a hole Polymers • allow the measurement of single cells/networks • cell held in place during measurement • dielectric to form ‘Gigaseal‘ • consider cell perfusion (nutrition) • measuring and counter electrode placement • capable of small hole (1-5µm) forming Surface Modification • preferably optically translucent for combination with other Planar array measurement techniques e.g. Ca fluoroscopy Layout Cell Placement 04.10.2007 4M Annual Conference: Borovets, Bulgaria 9

Introduction Background Design Work Conclusions Forschungszentrum Karlsruhe in der Helmholtz-Gemeinschaft Design cell, cultured modified on polymer surface (diameter 25µm) polymer foil (5- 2µm 50µm) hole 2-3µm hole modified electrolyte surface (KCl, 2M) (hydrophobic) � hole fabrication – laser drilling � top surface modification for controlled cell adhesion � bottom surface selective hydrophobic 04.10.2007 4M Annual Conference: Borovets, Bulgaria 10

Introduction Background Design Work Conclusions Forschungszentrum Karlsruhe in der Helmholtz-Gemeinschaft Hole Production • 193nm (ArF) laser* to produce 2µm holes with modified surfaces for cell growth into the hole. Pulse length 20ns , 0.3 • J/cm², 100 pulses • 50µm PS foil * Exitech PS2000 and an ATL 193nm laser 04.10.2007 4M Annual Conference: Borovets, Bulgaria 11

Introduction Background Design Work Conclusions Forschungszentrum Karlsruhe in der Helmholtz-Gemeinschaft Surface Modification/ Cell Array Laser beam λ < 193 nm O 2 O 2 O 2 O 2 O 2 O 2 O 2 O − CH 3 C=O CH . . . CH C=O Polystyrene foil Laser fluence 5mJ/cm² optimized pulse number 400 pulses formation of radicals, cracking of C-C bonds, separation of side chains/main chains, formation of Laser-assisted modification of polystyrene surfaces for cell culture applications, Wilhelm Pfleging, Michael Bruns, Alexander Welle, Sandra Wilson new bonds ( π ), reaction with ambient gas or liquid, formation or coupling of new chemical groups Journal of Applied Surface Science, Vol. 253, Issue 23, 30 Sept. 2007; p. 9177-9184 04.10.2007 4M Annual Conference: Borovets, Bulgaria 12

Introduction Background Design Work Conclusions Forschungszentrum Karlsruhe in der Helmholtz-Gemeinschaft Complete System Cell perfusion & reference electrode Measurement electrodes (exit rear) Electrolyte delivery Bioreactor: www.fzk.de/ibg 04.10.2007 4M Annual Conference: Borovets, Bulgaria 13

Introduction Background Design Work Conclusions Forschungszentrum Karlsruhe in der Helmholtz-Gemeinschaft Conclusions/ Further Work • Concept for polymer patch clamping array has been designed, developed and is principally viable • Successfully modified the surface of polystrene for culturing of networks of PC-12 cells (array of 100 cells) • Optimized parameters required for drilling 2 – 5µm holes in PS • Successful culturing of cells to seal over hole area Further Work • ‘seal’ resistance?? • Bio-reactor modification for separate microfluidic channels under the cells • Planar electrode array to be developed • Interfacing to patch clamping software ( commercially available) 04.10.2007 4M Annual Conference: Borovets, Bulgaria 14

Introduction Background Design Work Conclusions Forschungszentrum Karlsruhe in der Helmholtz-Gemeinschaft Questions? 04.10.2007 4M Annual Conference: Borovets, Bulgaria 15