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

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

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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

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Forschungszentrum Karlsruhe

in der Helmholtz-Gemeinschaft

04.10.2007 4M Annual Conference: Borovets, Bulgaria 1

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

Introduction Background Design Work Conclusions

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SLIDE 2

Forschungszentrum Karlsruhe

in der Helmholtz-Gemeinschaft

04.10.2007 4M Annual Conference: Borovets, Bulgaria 2

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 clamping is a method where a patch of the membrane is sucked into a pipette so current measurements can be made.

Introduction Background Design Work Conclusions

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Forschungszentrum Karlsruhe

in der Helmholtz-Gemeinschaft

04.10.2007 4M Annual Conference: Borovets, Bulgaria 3

  • Introduction and Background

– Importance of ion transport measurements – Transport across cells – High Throughput Systems (HTS)

  • Design Considerations
  • New Process

– Materials selection – Laser holes – Surface modification

  • Conclusions / Further Work

Outline

Introduction Background Design Work Conclusions

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Forschungszentrum Karlsruhe

in der Helmholtz-Gemeinschaft

04.10.2007 4M Annual Conference: Borovets, Bulgaria 4

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

Background Introduction Design Work Conclusions

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Forschungszentrum Karlsruhe

in der Helmholtz-Gemeinschaft

04.10.2007 4M Annual Conference: Borovets, Bulgaria 5

Cell Membrane

phospholipid bilayer

hydrophilic hydrophobic extracellular medium

distribution of main ions found in mammal cells How does a cell manage to control the flow?

20-30 1-3 HCO3

  • 100-140

1-60 Cl- 1-5 10-20 Mg2+ 1.2-4 0.5-1 Ca2+ 4-8 130-160 K+ 130-160 5-20 Na+ Extracellular Range (mM) (i) Intracellular Range (mM) (o) Ion

Equilibrium State (resting potential):

[ ] [ ]

i

  • ion

ion zF RT E ln =

intracellular medium Background Introduction Design Work Conclusions

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Forschungszentrum Karlsruhe

in der Helmholtz-Gemeinschaft

04.10.2007 4M Annual Conference: Borovets, Bulgaria 6

Ion Channels

  • ‘Controlled Leak’- Ion Channels /Transporters

K+ channel Na+/K+ pump sodium- glucose transporter

extracellular membrane intracellular – Ion channels – Transporters:

  • pumps
  • exchangers

Background Introduction Design Work Conclusions

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Forschungszentrum Karlsruhe

in der Helmholtz-Gemeinschaft

04.10.2007 4M Annual Conference: Borovets, Bulgaria 7

Traditional Patch Clamping

1G seal ‘KCl‘ filled pipette 2-3M Ag/AgCl measurement electrode Pipette opening diameter: 1-4µm Background Introduction Design Work Conclusions

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SLIDE 8

Forschungszentrum Karlsruhe

in der Helmholtz-Gemeinschaft

04.10.2007 4M Annual Conference: Borovets, Bulgaria 8

HTS Systems

Introduction Background

  • Info. not available

CHO, Jurkat HEK CHO,Jurkat HEK 293, CHO, Jurkat Neuro 2A Cell types 48 48 1-16 1-20

  • No. cells/run

cell suspension vacuum suction cell suspension vacuum pressure cell suspension vacuum suction cell suspension vacuum suction Cell Placement

Si/ SiOx

quartz glass borosilicate glass quartz glass Materials Hole Array SophionBioscience, ThinXXS US/DE Flyion Tuebingen, DE Nanion, Munich, DE Cytocentrics Rostock, DE Design

Design Work Conclusions

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Forschungszentrum Karlsruhe

in der Helmholtz-Gemeinschaft

04.10.2007 4M Annual Conference: Borovets, Bulgaria 9

Design Considerations

Materials

  • biocompatible, clean, good surface finish (Ra < 0.5 µm)
  • dielectric to form ‘Gigaseal‘
  • capable of small hole (1-5µm) forming
  • preferably optically translucent for combination with other

measurement techniques e.g. Ca fluoroscopy Cell Placement

  • method of moving or placing a cell over a hole
  • cell held in place during measurement
  • consider cell perfusion (nutrition)

Layout

  • multiple sites for parallel rapid measurement
  • allow the measurement of single cells/networks
  • measuring and counter electrode placement

Introduction Background Design Work Conclusions

Polymers Surface Modification Planar array

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SLIDE 10

Forschungszentrum Karlsruhe

in der Helmholtz-Gemeinschaft

04.10.2007 4M Annual Conference: Borovets, Bulgaria 10

Design

Introduction Background 2µm hole

electrolyte (KCl, 2M) polymer foil (5- 50µm) modified surface (diameter 25µm) 2-3µm hole cell, cultured

  • n polymer

modified surface (hydrophobic)

hole fabrication – laser drilling top surface modification for controlled cell adhesion bottom surface selective hydrophobic Work Conclusions Design

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SLIDE 11

Forschungszentrum Karlsruhe

in der Helmholtz-Gemeinschaft

04.10.2007 4M Annual Conference: Borovets, Bulgaria 11

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

Introduction Background Design Conclusions Work * Exitech PS2000 and an ATL 193nm laser

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SLIDE 12

Forschungszentrum Karlsruhe

in der Helmholtz-Gemeinschaft

04.10.2007 4M Annual Conference: Borovets, Bulgaria 12

Surface Modification/ Cell Array

Introduction Background Conclusions Design Work

Laser beam λ < 193 nm

O2 O2 O2 C=O C=O O−CH3 O2 O2 O2 O2 CH CH . . .

Polystyrene foil

formation of radicals, cracking of C-C bonds, separation of side chains/main chains, formation of new bonds (π), reaction with ambient gas or liquid, formation or coupling of new chemical groups

Laser fluence 5mJ/cm² optimized pulse number 400 pulses

Laser-assisted modification of polystyrene surfaces for cell culture applications, Wilhelm Pfleging, Michael Bruns, Alexander Welle, Sandra Wilson Journal of Applied Surface Science, Vol. 253, Issue 23, 30 Sept. 2007; p. 9177-9184

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Forschungszentrum Karlsruhe

in der Helmholtz-Gemeinschaft

04.10.2007 4M Annual Conference: Borovets, Bulgaria 13

Complete System

Cell perfusion & reference electrode Electrolyte delivery Measurement electrodes (exit rear) Introduction Background Design Work Conclusions Bioreactor: www.fzk.de/ibg

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SLIDE 14

Forschungszentrum Karlsruhe

in der Helmholtz-Gemeinschaft

04.10.2007 4M Annual Conference: Borovets, Bulgaria 14

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)

Introduction Background Design Work Conclusions

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Forschungszentrum Karlsruhe

in der Helmholtz-Gemeinschaft

04.10.2007 4M Annual Conference: Borovets, Bulgaria 15

Questions?

Introduction Background Design Work Conclusions