Micro-discharge treatment of cultured cells

I.E. Kieft1, J.L.V. Broers1,2, D.W. Slaaf1,2, F.C.S. Ramaekers2, E. Stoffels1

1Eindhoven University of TechnologyThe Netherlands2University Maastricht, The Netherlands

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

Plasma needle

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

Treatment of cells

Cells are just treated with plasmaWithin 20s cells detach from plastic petridish

Treatment of CHO K1 cells

15 min

4 hours 1 hour

Control

Cells can reattach within hours after treatment!

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

Causes of effects of treatmentUV light? Electric field?

Spectrum from He with N2.Other admixtures, more UV?

If only electric field present no effects, also known from literature

Polk (2000)

Cause of effectsHigh Temperature? Thermocouple: Temperature does not rise above 30 oC.

Skin treatment: No temperature rise

20

22

24

26

28

30

0 2 4 6 8 10

distance to needle (mm)

tem

pera

ture

(o C

) 0.15 W in He

Thermocouple:-no RF noise-also temperature risewith water

Cause of effectsRadicals?!Neutralization by antioxidants. Radicals attack DNA, proteins, lipids. Detection with fluorescent probe dissolved in liquid

picture made in situ

CM-H2DCFDA probe

probe reacts to: H2O2, HO., HOO., ONOO- and NO

Fluorescence measurementsRadicals of plasma treatment compared to NO released by NOR-1.

Radical flux is order of 1012 per second into liquid.Radical concentration in plasma ~1019 m-3.

0.0E+00

1.0E+04

2.0E+04

3.0E+04

4.0E+04

5.0E+04

6.0E+04

7.0E+04

8.0E+04

9.0E+04

0 5 10 15 20 25

NO (μM)

inte

nsity

(a.u

.)

0.0E+00

2.0E+14

4.0E+14

6.0E+14

8.0E+14

1.0E+15

1.2E+15

1.4E+15

1.6E+15

1.8E+15

0 1 2 3 4 5 6 7 8 9

time (min)ra

dica

ls (-

)

0.0E+00

1.0E+14

2.0E+14

3.0E+14

4.0E+14

5.0E+14

6.0E+14

7.0E+14

0 1 2 3 4 5 6

position (mm)

radi

cals

(-)

Cause of effects

0.0E+00

1.0E+14

2.0E+14

3.0E+14

4.0E+14

5.0E+14

6.0E+14

7.0E+14

0 1 2 3 4 5 6

distance (mm)

radi

cals

(-)

0.0E+00

5.0E+13

1.0E+14

1.5E+14

2.0E+14

2.5E+14

3.0E+14

3.5E+14

4.0E+14

4.5E+14

5.0E+14

0 0.2 0.4 0.6 0.8 1 1.2added oxygen (%)

radi

cals

(-)

Distance needle to liquid

Position needle to tubeOxygen admixture

position distance

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 % O2 factor 10 decrease

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 1012 per second

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