1. Micro-discharge treatment of cultured cells I.E. Kieft 1, J.L.V. Broers 1,2, D.W. Slaaf 1,2, F.C.S. Ramaekers 2, E. Stoffels 1 1 Eindhoven University of Technology The Netherlands 2 University Maastricht, The Netherlands

2. Plasma set-up -13.56 MHz -~300 V p-p -~50 mW dissipation in plasma -carrier gas: helium -flow 1 to 2 liter/min -length needle ~10 cm

3. Plasma needle Plasma needle is a hand-operated tool. Plasma is in order of mm.

4. Treatment of cells Cells are just treated with plasma Within 20s cells detach from plastic petridish

5. Treatment of CHO K1 cells 15 min 4 hours 1 hour Control Cells can reattach within hours after treatment!

6. Effects on cells Cells detach from surface and from each other. Cells remain alive after treatment Small percentage of cells undergoes apoptosis Effects tested on CHO K1, 3T3 and NSCLC MR65. The effects are similar. Bacteria are more sensitive to plasma treatment than cells

7. Causes of effects of treatment UV light? Electric field? Spectrum from He with N 2. Other admixtures, more UV? If only electric field present no effects, also known from literature Polk (2000)

8. Cause of effects High Temperature? Thermocouple: Temperature does not rise above 30 o C. Skin treatment: No temperature rise Thermocouple: -no RF noise -also temperature rise with water

9. Cause of effects Radicals?! Neutralization by antioxidants. Radicals attack DNA, proteins, lipids. Detection with fluorescent probe dissolved in liquid picture made in situ CM-H 2 DCFDA probe probe reacts to: H 2 O 2, HO., HOO., ONOO - and NO

10. Fluorescence measurements Radicals of plasma treatment compared to NO released by NOR-1. Radical flux is order of 10 12 per second into liquid. Radical concentration in plasma ~10 19 m -3.

11. Cause of effects Distance needle to liquid Position needle to tube Oxygen admixture positiondistance

12. Cause of effects Conlusions from radical tests: -Distance of tip needle-surface liquid > 2mm  radical concentration less than half -Sticking out needle from perspex tube has little effect -Adding more oxygen  less radicals 1 % O 2  factor 10 decrease

13. Conclusions Plasma needle is a new and promising technique Treatment can be very localized and cells remain alive after treatment Effects on cells are likely to be caused by radicals Radicals diffusing into liquid can be easily detected with fluorescent probe Radical flux is on the order of 10 12 per second

14. Future of plasma treatment Plasma treatment can be used for sterilization of wounds acceleration of wound healing localized removal of cells (e.g. skin cancer) caries treatment in dentistry