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Nuclear Instruments and Methods in Physics Research A282 (1989) 22-27 North-Holland, Amsterdam

THE PREPARATION OF BIOLOGICAL TARGETS FOR HEAVY-ION EXPERIMENTS UP TO 20 MeV/ u W. KRAFT-WEYRATHER, G. KRAFT, S. RITTER, M. SCHOLZ and J.A. STANTON Gesellschaft für Schwerionenforschung, Planckstr. 1, 6100 Darmstadt, FRG

The radiobiological effects of heavy charged particles are of fundamental interest for the understanding of radioprotection problems in manned space flights and for the increasing use of heavy ions in radiotherapy . Therefore, an increasing number of radiobiological experiments are performed at heavy-ion accelerators . In these experiments biological targets such as, for instance, cultures of living mammalian cells have to be irradiated under sterile conditions and atmospheric pressure . The experimental solution of these and other biology-specific problems is discussed and a general outline of the experiments is given.

1. Introduction Biophysical investigations using particle accelerators have been of growing interest in the last years. Besides needs for basic research on the interaction of swift heavy particles with living material, the results of these experiments are needed for various applications in radiotherapy and space research [1] . Radiobiological experiments at heavy-ion accelerators pose stringent conditions on the preparation of biological targets. A biological target is always made of living material such as whole cells or cellular components. The major problem, however, is not producing the living cells for a target (these can be grown in a cell culture laboratory), but the technical problems in constructing a holder and creating an environment that makes it possible to expose living material to the beam without any additional impact on cell viability. Biological targets used at GSI range from different types of cells, for instance mammalian cells, yeast cells and bacteria spores to intra- and extracellular DNA, and finally to multicellular targets like eggs and seeds. This paper is focussed on the preparation of mammalian cells and DNA in solution as targets for heavyion irradiation, both of which represent extreme examples of biological targets . 2. Physical requirements for heavy-ion irradiation Low-energy accelerators, like the GSI UNILAC, having maximum energies of only 20 MeV/u for all ions impose several physical restrictions which lead to drastic limitations in target size. The first problem is the limited particle range : ions with energies up to 20 MeV/u have a range which is smaller than 0.5 mm in water or plastic material. This 0168-9002/89/$03 .50 © Elsevier Science Publishers B.V. (North-Holland Physics Publishing Division)

makes it impossible to expose samples in tissue culture flasks having a wall thickness of 1 mm or more. Therefore cells have to be exposed inside open Petri dishes. However, it is not sufficient that the particle beam just reaches the biological material. The individual ions should also exhibit well defined parameters of energy loss and ion energy: Radiobiological effect curves are measured as a function of the dose, i.e. the energy deposited in the target . The energy deposition of particle radiation is given by the energy loss, dE/dx. In radiobiology, the energy which is absorbed by the target is called the linear energy transfer (LET) and equals the energy loss of all emitted electrons contributing to energy deposition. Then dE/dx = LET~, and the dose can be described as the energy deposition of one particle as given by the LET multiplied by the density of particles per square unit . As 1 Gray (Gy) = 6.25 x 1015 eV/g, the dose can be

calculated as

dose(Gy) =1 .6 x 10-9 x LET(keV/pm) x particle fluence(part/en? ) . In the energy range considered in this paper, the contribution of the nuclear stopping to the biologically relevant effects is negligible . Recent measurements of the cross section for various types of biological damage like inactivation, chromosome aberration, and even DNA breaks [2] have illustrated that for heavy ions having LET values greater than 100 keV/Wm the energy deposition alone is not a good parameter to describe the experimental findings (fig. 1). Cross sections depend not only on the LET but more importantly on the particle velocity. For instance, for uranium ions between 4 and 15 MeV/u the LET is

W. Kraft-Weyrather et al. / Preparation of biological targets

N

E

23

100

C O u Gl H N 10 H O LJ

. . .I 100

I

.I

I

1000

I

LET IkeVINm]

.

. . . .I 10000

.

. . . . . .I

Fig. 1 . The inactivation cross section for V 79 Chinese hamster cells is given as a function of linear energy transfer (LET) . For LET _< 100 keV/psm all data points show one common curve independent of atomic number. For LET ~- 200 keV/Wm the a-LET curve separates from this common curve in order of increasing atomic number [2]. nearly constant but the cross sections for all biological effects increase by more than a factor of 3 . This is due to the increased diameter of the particle track which depends on the velocity and therefore on the energy of the primary particle. For experiments it is therefore necessary that the variation of both, energy loss and ion energy, is small over the traversal of the biological target . Together these conditions lead to a limitation of the target thickness to 50 Wm or less for heavy ions at UNILAC energies. For comparison, the thickness of a cell monolayer is in the order of 10 Wm. 3 . Biological conditions Taking into account that biological targets are always living targets with different intrinsic properties like different growth kinetics, sensitivity, etc ., several special problems arise . 3.1 . Sterility The main danger to biological targets is from crosscontaminations with other microcultures such as the infection of mammalian cells by bacteria, viruses, or fungi. To illustrate : the doubling time for V 79 Chinese Hamster cells is approximately 10-12 h whereas the doubling time for bacteria is 20-30 min . It is evident that a mammalian culture will be overgrown by an infection of bacteria within 2 days. Therefore all handling has to be performed in sterile flow hoods with sterilized materials . Antibiotics as well as fungicids have to be used too . This also represents a major problem for

the UNILAC experiments where different groups are performing experiments with all of these organisms at the same time. Therefore, cross-infections are very likely, so careful handling of samples is required. 3 .2. Tissue-compatible materials There are very few materials which are suitable for mammalian cell cultures : special plastics like Teflon, special glasses, gold, and some kinds of stainless steel, whereas most of the other metals, especially the heavier ones, are of extreme cell toxicity. All parts of the irradiation facility that come into contact with cells or growth media have to be tested for cell toxicity before use. 3.3. Humidity and atmospheric conditions To prevent cells from drying and subsequently dying, a certain humidity, mainly a thin film of special liquids (growth media, buffers) is necessary. To maintain this film during the exposure time, the surrounding air has to be saturated with water vapour. Irradiation of living material is only possible at atmospheric pressure. Low pressure and especially high vacuum would lead to exploding cells . In addition, for many experiments a regulated temperature and controlled atmosphere (air, NZ , O Z ) are necessary. 4 . Irradiation facility The experimental setup [3] used at GSI for biophysical experiments consists of two parts : a vacuum chamI . PROBLEMATICS IN HEAVY-ION EXPERIMENTS

W. Kraft-Weyrather et al. / Preparation of biological targets

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Elm

Ti window target target wobbler

-electro magnet

wobbler motor

JIME

in

beam

Zn S-smm carbon foil cassette

Faraday cup

target Fig. 2. Schematic view of the exposure setup BIBA. The beam pipe contains the beam control system, consisting of a ZnS screen as a visual control, a Faraday cup, and a secondary electron monitor (carbon foils) . The biological samples are stored in a cassette and lifted in front of the exit window for exposure [3].

Fig. 3. Electron microscope image of a monclayer of V 79 Chinese Hamster cells grown on a Petri dish. Cells in interphase are stretched out, but before division the cells round up (upper left).

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W. Kraft-Weyrather et al / Preparation of biological targets

ber housing the beam monitor system, which is connected to the vacuum system of the accelerator, and the irradiation chamber, which has to be maintained at atmospheric pressure (fig. 2) . Inside the monitor system, the intensity of the beam and its homogeneity can be monitored . A ZnS screen observed by means of a TV system is used to give an optical control of the homogeneity of the beam before the experiment . During the exposure of biological samples, the emission of secondary electrons of a carbon foil is used to measure the beam intensity. This transmission detector is calibrated by exposing suitable track detectors like glass, mica, or CR39 instead of the biological sample and counting the individual tracks. These detectors are used throughout the biophysical experiments and also represent permanent records of the beam properties after irradiation . At high particle energies (E >_ 10 MeV/Wm), when the energy loss of the primary beam is less critical, an ionisation chamber mounted at the atmospheric side of the exit window is also used for beam monitoring. The exit window for the beam from vacuum to atmospheric pressure consists of a 5 Rm Ti foil of 50 mm in diameter, supported by a grid of stainless steel. Cells are grown as monolayers on 35 mm diameter Petri dishes made of plastic with a specially conditioned surface, allowing the attachment of the cells (fig . 3). The Petri dishes are pressed into gold covered rings of magnetic stainless steel. Up to 19 of these samples can be vertically inserted in a teflon magazine filled with

Mylar foil DNA solution gold covered backing

filling holes

Fig. 4. Schematic cross section through the DNA samples. A thin film of DNA solution is injected in the space between a Mylar foil and the gold-plated backing. growth medium . For irradiation the samples are lifted by an electromagnet to their position in front of the Ti window. They are still covered by a thin growth medium film, which is sufficient to maintain the required humidity during irradiation time (up to 2 min) . In order to wash out the shadows of the supporting grid and to guarantee a homogeneous irradiation over the whole target area, the samples are moved by a wobbler system in a kind of Lissajous figure . This system makes it possible to irradiate many samples in a relatively short time. (Typically, irradiation of one magazine filled with mammalian cell samples is 20 min.) In addition, the possibility of storing the samples in the cassette with growth medium for several hours prevents the cells from drying in the case of unexpected events like machine breakdowns.

HEAVY ION BEAM TEFLON MAGAZIN

24 h

WITH STOCK-CULTURE

PETRI DISH

PROBES

1:10 1 :1001 :1000 T RYPSI NIZED COUNTED

6~Ë

DILUTED PLATED FOR

I

COLONY FORMING TEST INCUBATED

7d, 37° C

STAINING, COUNTING OF THE ( > 50

COLONIES CELLS)

Fig. 5. Experimental scheme for inactivation measurements [3]. Cells are grown over longer periods in stock culture. 24 h before irradiation they are plated in Petri dishes. For the exposure to the particle beam, the samples are stored in a magazine and lifted for a controlled time into the beam . After exposure, the cells are removed by trypsination and processed in different ways. For the colony test, cells are diluted according to their expected survival and incubated again for 7 d. Each cell with a integer genome is able to grow to a visible colony. I. PROBLEMATICS IN HEAVY-ION EXPERIMENTS

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W. Kraft-Weyrather et al. / Preparation of biological targets

Fig. 6. Ring chromosome and normal chromosomes. The ring chromosome is the result of an exchange figure measured 24 h after the exposure of V 79 Chinese Hamster cells to 74 MeV/u At ions.

For irradiation of cells in suspension or of DNA solutions, it is necessary to prepare a thin layer of liquid containing the cells or the DNA . For this purpose a chamber was constructed with a depth of 50 gm (fig. 4) . The chamber is covered by a 12 Wm thick Mylar foil . Biological suspensions are injected into the chamber through a thin hole from the back of the target, using a syringe. Each sample contains 20 pl solution, which can be collected after exposure by injecting another nonmixing liquid like glycerin or by mechanical pressure at the foil.

ferent endpoints being studied : for inactivation experiments cells are removed by trypsin solution, counted, diluted, and plated for colony forming. After 1 week each surviving cell will form a colony of more than 50 cells that can be stained and counted . (For details see ref. [6] .) For other endpoints like measurements of chromosome aberrations (fig. 6), cell cycle changes, mutation, etc., cells are reincubated after irradiation for several hours, days, or weeks before the results can be measured . The result of such a measurement can be drawn as a Fuence-effect curve, i .e . a survival curve (fig. 7) or as an induction curve of aberrations . From these

5. Experimental scheme A typical experiment is shown schematically in fig. 5 : Cells which are grown in a stock culture are seeded into 35 mm diameter Petri dishes . For optimal homogeneity during irradiation cells are centerplated over an area of 20 mm in diameter [4] . To allow cell attachment they are incubated for 4-36 h, depending on the special conditions of the experiment . For experiments with synchronized cells or with cells infected with viruses, the required time between plating and irradiation is fixed to within one hour or less . For instance green monkey cells which have been infected with SV40 virus have to be exposed (36 t 1) after infections [5] . After irradiation cells are treated according to the dif-

2

4

6

B

10

PARTICLE FLUENCE

l

12x106 [aii 2

Fig. 7. Fluence effect curve for the inactivation of V 79 Chinese Hamster cells exposed to different ions .

W. Kraft- Weyrather et al. / Preparation of biological targets

curves cross sections can be calculated as shown in fig . 1.

6. Summary The exposure of biological materials imposes very different conditions on target preparations than the fabrication of targets for atomic and nuclear physics experiments . Targets of living cells have to be handled under sterile conditions, they have to be exposed at atmospheric pressure under air or other controlled gas mixtures at a defined temperature. The exposure times have to be short and for the transfer between cell-culture laboratory and irradiation facility the same strict criteria have to be applied. These conditions are difficult to meet, but the experience at GSI shows that fast exposure of biological material is possible when the necessary facilities are installed .

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Thiel . They also want to thank the operating group of the UNILAC, Darmstadt .

References [1] Extended Abstracts of the 3rd Workshop of Heavy Charged Particles in Biology and Medicine, GSI Darmstadt, GSIReport-87-11 (1987) . [2] G. Kraft, Nucl . Sci. Appl . 3 (1987) 1 . [3] G. Kraft, H.W . Daues, B. Fischer, U . Kopf, H .P . Liebold, D . Quis, H . Stelzer, J . Kiefer, F . Schöpfer, E. Schneider, K. Weber, H . Wulf and H . Dertinger, Nucl . Instr. and Meth. 168 (1980) 175 . [4] H . Wulf, W. Kraft-Weyrather, H .G . Miltenburger, E.A. Blakely, C.A . Tobias and G. Kraft, Radiat. Res. 104 (1985) 122. [5] R. Roots, G. Kraft and E. Gosschalk, Int . J. Radiat . Oncol . Biol. Phys. 1 1 (1985) 259. [6] R.I . Freshney, Culture of animal cells (Alan R. Liss, New York, 1983).

Acknowledgements The authors acknowledge the excellent technical assistance provided by G. Lenz, H. Penninger and S .

I . PROBLEMATICS IN HEAVY-ION EXPERIMENTS