optimisation of exposure conditions for in vitro radiobiology experiments

7
SCIENTIFIC PAPER Optimisation of exposure conditions for in vitro radiobiology experiments Elizabeth Claridge Mackonis Natalka Suchowerska Pourandokht Naseri David R. McKenzie Received: 23 October 2011 / Accepted: 28 February 2012 / Published online: 28 March 2012 Ó Australasian College of Physical Scientists and Engineers in Medicine 2012 Abstract Despite the long history of using cell cultures in vitro for radiobiological studies, there is to date no study specifically addressing the dosimetric implications of flask selection and exposure environment in clonogenic assays. The consequent variability in dosimetry between labora- tories impedes the comparison of results. In this study we compare the dose to cells adherent to the base of three types of commonly used culture flasks or plates. The cells are exposed to a 6MV clinical photon beam using either an open or a half blocked field. The depth of medium in each flask is varied with the medium surrounding the flask either water or air. The results show that the dose to the cells is more affected by the scattering conditions surrounding the flasks than by the level of filling within the flask. It is recommended that water or a water equivalent phantom material is used to surround the flasks or plates to approximate full scatter conditions at the cell layer. How- ever for modulated fields, surrounding the 24 well plates with water-equivalent material is inadequate because of the large volume of air surrounding individual wells. Our results stress the importance of measuring the dose for new experimental configurations. Keywords Radiobiology Á Dosimetry Á In vitro Á Irradiation Á MV Introduction In vitro studies are extensively used in research. They are particularly valuable for hypothesis testing and evaluating the response of a chosen cell line to radiation and/or che- motherapy drugs. Consequently, an accurate knowledge of dose to the cells for in vitro studies is essential. Repro- ducibility of results across laboratories can only be accomplished if the dose to cells is accurately measured and reported. The use of different laboratory protocols for in vitro cell irradiations can have dosimetric consequences, making it invalid to compare results. For example, com- parisons between laboratories of the bystander effect have been difficult due to differences in experimental design [1]. Progress cannot be made until dosimetry has been elimi- nated as a source of variability. The increasing use of cell cultures to investigate the effect of spatially modulated radiation fields, requires clonogenic studies using adherent cell layers such that the position of the cells in the flask can be correlated to the dose received [2, 3]. However the requirements of good cell biology practice are sometimes incompatible with accurate and reproducible dosimetry. For example, good biology would require cells to remain at a constant tem- perature, which is often inconvenient when transporting cells for irradiation. Reproducible dosimetry requires the cells to be in conditions of electronic equilibrium. Sub- merging the flasks containing the cells in a water bath does not always achieve this, because air pockets are inherent in their design. Furthermore, there is a high risk of contami- nation if the flask cannot be made water tight. E. Claridge Mackonis (&) Á N. Suchowerska Sydney Cancer Centre, Royal Prince Alfred Hospital, Missenden Road, Camperdown, NSW 2050, Australia N. Suchowerska Á P. Naseri Á D. R. McKenzie School of Physics, University of Sydney, Sydney, NSW 2006, Australia 123 Australas Phys Eng Sci Med (2012) 35:151–157 DOI 10.1007/s13246-012-0132-6

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Page 1: Optimisation of exposure conditions for in vitro radiobiology experiments

SCIENTIFIC PAPER

Optimisation of exposure conditions for in vitroradiobiology experiments

Elizabeth Claridge Mackonis • Natalka Suchowerska •

Pourandokht Naseri • David R. McKenzie

Received: 23 October 2011 / Accepted: 28 February 2012 / Published online: 28 March 2012

� Australasian College of Physical Scientists and Engineers in Medicine 2012

Abstract Despite the long history of using cell cultures in

vitro for radiobiological studies, there is to date no study

specifically addressing the dosimetric implications of flask

selection and exposure environment in clonogenic assays.

The consequent variability in dosimetry between labora-

tories impedes the comparison of results. In this study we

compare the dose to cells adherent to the base of three

types of commonly used culture flasks or plates. The cells

are exposed to a 6MV clinical photon beam using either an

open or a half blocked field. The depth of medium in each

flask is varied with the medium surrounding the flask either

water or air. The results show that the dose to the cells is

more affected by the scattering conditions surrounding the

flasks than by the level of filling within the flask. It is

recommended that water or a water equivalent phantom

material is used to surround the flasks or plates to

approximate full scatter conditions at the cell layer. How-

ever for modulated fields, surrounding the 24 well plates

with water-equivalent material is inadequate because of the

large volume of air surrounding individual wells. Our

results stress the importance of measuring the dose for new

experimental configurations.

Keywords Radiobiology � Dosimetry � In vitro �Irradiation � MV

Introduction

In vitro studies are extensively used in research. They are

particularly valuable for hypothesis testing and evaluating

the response of a chosen cell line to radiation and/or che-

motherapy drugs. Consequently, an accurate knowledge of

dose to the cells for in vitro studies is essential. Repro-

ducibility of results across laboratories can only be

accomplished if the dose to cells is accurately measured

and reported. The use of different laboratory protocols for

in vitro cell irradiations can have dosimetric consequences,

making it invalid to compare results. For example, com-

parisons between laboratories of the bystander effect have

been difficult due to differences in experimental design [1].

Progress cannot be made until dosimetry has been elimi-

nated as a source of variability.

The increasing use of cell cultures to investigate the

effect of spatially modulated radiation fields, requires

clonogenic studies using adherent cell layers such that the

position of the cells in the flask can be correlated to the

dose received [2, 3]. However the requirements of good

cell biology practice are sometimes incompatible with

accurate and reproducible dosimetry. For example, good

biology would require cells to remain at a constant tem-

perature, which is often inconvenient when transporting

cells for irradiation. Reproducible dosimetry requires the

cells to be in conditions of electronic equilibrium. Sub-

merging the flasks containing the cells in a water bath does

not always achieve this, because air pockets are inherent in

their design. Furthermore, there is a high risk of contami-

nation if the flask cannot be made water tight.

E. Claridge Mackonis (&) � N. Suchowerska

Sydney Cancer Centre, Royal Prince Alfred Hospital,

Missenden Road, Camperdown, NSW 2050, Australia

N. Suchowerska � P. Naseri � D. R. McKenzie

School of Physics, University of Sydney, Sydney,

NSW 2006, Australia

123

Australas Phys Eng Sci Med (2012) 35:151–157

DOI 10.1007/s13246-012-0132-6

Page 2: Optimisation of exposure conditions for in vitro radiobiology experiments

Most radiotherapy cancer treatments use megavoltage

photon beams, where the dose distribution depends

strongly on the composition and geometry of the materials

being irradiated. For radiation protection experiments, the

energy distribution which results from the experiment

geometry may also be important [4–6]. For in vitro studies,

there is a large choice of irradiation conditions (flasks, petri

dishes, multiwell plates, tubes) which result in different

radiation scattering conditions. Any air spaces above or

around the cells can compromise the conditions of elec-

tronic equilibrium, introducing uncertainty in the dose

delivered to the cells.

Currently there are no standard protocols for irradiating

cell cultures in vitro because of the variety of experimental

designs used. Some authors [2, 7–10] provide full details of

their experimental irradiation conditions, while others

simply state that ‘dosimetry’ had been performed, but

without detailed description [11]. Others calculate the dose

deviation from assumed full scatter conditions [12].Table 1

summarises the range of approaches to ‘dosimetry’ for in

vitro cell irradiations reported in the literature.

In this study, we report the measured dose in common

configurations for irradiating cell cultures exposed in

therapeutic mega-voltage photon beams. We measure the

dose to an adherent cell layer for a range of scatter con-

ditions. The effect of the following are addressed: the

medium surrounding the flask/plate (air, water, or water-

equivalent phantom), the culture conditions (for example,

the number and size of wells) and the level of filling of the

wells or the flask.

Method

The dose to the base of the flasks for the irradiation con-

ditions was measured using radiochromic film dosimetry

Table 1 Dosimetry for in vitro cell irradiations reported in the literature

Authors Flask/plate Irradiation setup Dosimetry

Seymour and Mothersill

[11]

T25 Co-60 beam at room temperature, no further

detail

None mentioned

Ling et al. [13] 34 mm cell

culture disks

Array of sources placed 12 mm above cell

layer

TLD and ionisation chamber measurements

Mu et al. [14] Lux 11 mm

tissue

culture disc

Custom phantom with growth medium and

controlled atmosphere, 35 cm from the

source

FeSO4- dosimetry

Suchoweska et al. [15] T75 Linac irradiation from beneath the flask in a

water bath. Full scatter.

Parallel plate ionisation chamber

measurements

Sterzing et al. [9] Cryotubes Linac irradiation in a cylindrical water-

equivalent phantom

Pin-point ionisation chamber measurements

Bromley et al. [16] 6-well Perspex surrounding plate, linac irradiation

from beneath. Air in plate.

Farmer-type chamber and radiographic film

measurements (film above) for full scatter

conditions

Moiseenko et al. [8] 2 ml plastic

vials then

plated

Linac irradiation in an acrylic cylindrical

phantom

IC-10 ionisation chamber measurement

Claridge-Mackonis et al.[3]

T75 Flask in a water bath, linac irradiation from

beneath. Full scatter.

GafChromic film measurements

Bewes et al. [17] T75 Linac irradiation from side with cells in water

bath. Full scatter.

Thimble chamber measurements

Keall et al. [12] 4-well Linac irradiation from above. Water

equivalent material above and below flask

with air in flask

Ionisation chamber measurements (full scatter)

and calculations

Altman et al. [7] 6-well Customised phantom. Assessment of

irradiation setup before experiment

TLD and radiographic film measurements

(film below)

Gow et al. [18] T25 Co-60 and linac 20 MeV irradiation from

above with polystyrene buildup

None mentioned

Xing et al. [19] Not specified Cs-137 Not specified

Hehlgans et al. [20] Not specified 200 kV irradiation A duplex dosimeter measurement

Butterworth et al. [2] T75 and T25 Flask in water bath on water equivalent

phantom, linac irradiation from below

Radiochromic film measurements

and 2D array

152 Australas Phys Eng Sci Med (2012) 35:151–157

123

Page 3: Optimisation of exposure conditions for in vitro radiobiology experiments

(GAFCHROMICTM EBT and EBT2, International Spe-

cialty Products). To add further insight into the measure-

ments, Monte Carlo simulations of selected geometries

were carried out.

Irradiation and dosimetry

Three types of cell culture containers were considered: 24

well plates (Linbro Chemical Co, USA), 6 well plates and

T75 culture flasks (IwakiTM, Bibby-Sterlin Ltd., UK) were

used (Table 2). The T75 flasks and the multi-well plates

were constructed from polystyrene. The T75 flask had a

75 cm2 plating surface area, the 24 well plate had cylin-

drical wells 1.6 cm in diameter and the 6 well plate had

wells 3.5 cm in diameter. Each flask or plate was centred in

a 6MV photon beam produced by a Varian 21IXS linear

accelerator with two different beam arrangements: an open

field (30 9 30 cm) and a modulated field represented by a

half beam blocked to the central axis (30 9 15 cm) [16].

Radiochromic film was placed directly below the flask or

plates, at 100 cm from the source, to measure the dose

representative of that received by the cells. The film was

supported by 2 cm of Virtual WaterTM (Standard Imaging,

USA) and a 1 cm of polymethylmethylacrylate (PMMA)

on the treatment couch. For the 6 well plates, additional

disks of film were placed inside at the base of each well.

The linac gantry was positioned at 180�, exposing the

flasks from beneath. This arrangement placed the cell layer

at a depth of 3.2 cm in the irradiation phantom and at

100 cm from the radiation source.

Five different irradiation setups were used to investigate

the effect of the scattering material surrounding the flasks

or plates (Fig. 1). In the first set of irradiations, the film was

covered with Virtual WaterTM slabs (Fig. 1a) to achieve

full scatter conditions. In the second and third irradiations,

the flasks were placed in a PMMA box. This box was either

filled with water or air (Fig. 1b, c). As the 24-well and

6-well plates are not water tight, they could not be used for

irradiations where the bath was filled with water. The

fourth and fifth irradiations used Virtual WaterTM slabs to

cover or surround the flasks (Fig. 1d, e).

To identify the effect of the scattering material within

the flasks or wells, they were one quarter, one half, three

quarters or completely filled with water. This set-up reflects

the experimental practice of complete filling of the flasks

for example with PBS (phosphate buffer solution) or partial

filling of flasks for example with growth medium.

The dosimetric measurements were initially performed

with EBT GafChromic film. This type of film was replaced

by EBT2 GafChromic film when EBT was no longer

available. Calibration films were irradiated using full

scatter conditions for each set of measurements. After 24 h

the films were scanned on an EpsonTM 10000XL scanner

(Seiko Epson Corporation, Japan) with a consistent film

orientation at 150 dpi. Using Mephysto MC2 software

(PTW, Germany), the 16-bit grey-scale film images were

calibrated and dose profiles measured at 0.4 mm resolu-

tion. Profiles were measured along the centre of the rows of

wells for the 6- and 24-well plates and at the centre and

edges of the T75 flasks. A minimum of three replicate films

were used for each experimental condition.

A 2-tailed Student’s t test was performed to test the

significance of the differences in measured dose. An

analysis of the errors in the measured doses was performed

following the method set out in the IAEA Technical Report

Series No. 398 [21]. Results were considered to be

Table 2 Shows the percentage difference in dose to the cells relative to the dose for full scatter conditions, for the irradiation setups of Fig. 1

Flasks/plate surrounded

by water (%)

Flasks/plate

in air (%)

Flasks/plate surrounded

by Virtual Water (%)

Flasks/plate covered

by Virtual Water (%)

T75

-2 -7 -3 -4

6 wells

N/A -9 ?1 -2

24 wells

N/A -13 -1 -3

All flasks in this table were filled with water. The combined uncertainty of each measurement is 2.4 %

Australas Phys Eng Sci Med (2012) 35:151–157 153

123

Page 4: Optimisation of exposure conditions for in vitro radiobiology experiments

significant if the t-test showed a difference (P \ 0.05) and

the dose difference was greater than the combined uncer-

tainty (Table 3).

Monte Carlo simulation

The NRC version of Electron–Gamma-Shower Monte

Carlo method (EGSnrc) was used to calculate the dose to

the cell layer for a range of irradiation setups. The spec-

trum for a 6MV photon beam produced by a Varian 21EX

linear accelerator was kindly supplied by M. Williams,

Illawarra Cancer Care Centre. Electron and photon trans-

port parameters were selected to include pair production,

the photoelectric effect, Rayleigh scattering, Compton

scattering and Bremsstrahlung. The scattering properties of

each component material were provided by the EGSnrc

software. The scattering properties of Solid Water 457 [22]

were used for the Virtual Water in the experimental setup.

The global cutoff energies for electrons and photons were

0.521 and 0.001 MeV respectively. The incident field was

perpendicular to the phantom surface, as shown in Fig. 1e.

The phantom consisted of a 3 cm thick slab of Solid Water

upon which the simulated square culture flask of dimen-

sions 2.5 9 2.5 9 2.3 cm3 was positioned. A flask wall

thickness and a cell layer thickness of 1 mm each were

used. Simulated flask contents were: filled with water, half

filled with water (10 mm depth) or quarter filled with water

(5 mm depth). When partial filling of the flask was used,

the remaining contents of the flask were assumed to be air.

The number of histories per data point was varied from

2 9 108 to 5 9 109 to achieve an acceptable level of

uncertainty. Quoted uncertainties are standard deviations for

each data point, except where the mean of the entire data set

has been calculated. In such cases the uncertainties are the

standard deviations of the data points from the mean.

Results

The dose profiles measured using film from below the cell

layer for the 24 well plate irradiated with an open field

(Fig. 2) show the insignificant effect of different depths of

medium inside the wells (the mean dose difference is less

Surrounding material either water or air

Water

Cells

Air

Solid

h0.1cm

3cm

2.3cm

2.5cm

20cm

17cm

Cell layer

6 MV

(b)(a)

(e)(d)

(c)

Flask Virtual Water

PMMA Water Couch‘tennis racquet’

(f)

Fig. 1 The five different irradiation designs used in this study with a

T75 flask and the geometry used in the Monte Carlo simulation. For

the experimental setups, the scattering materials are supported by the

carbon fibre tennis racquet couch top and a PMMA sheet and the

flasks are irradiated from below on central axis. The arrows show the

position of the film under the flask. a full scatter conditions in Virtual

Water, used as a reference, b the flask placed in a water bath, c the

flask surrounded by air, d the flask ‘‘covered’’ with Virtual Water

slabs, e the flask surrounded by Virtual Water slabs f the configuration

for the Monte Carlo simulation

Table 3 The error analysis showing an example of the uncertainty analysis performed

Component Type Std. Unc. (%) Comment

Noise in film readout A 1.7 From noise observed in uniform region of the film

Stability of scanner B 0.8 Based on ±2 % with triangular distribution

Height of flask/plate and film at linac B 0.1 Based on 1 mm error with rectangular distribution

Positioning of film on scanner B 0.4 Based on maximum 1 cm difference resulting in maximum

1 % readout difference (triangular distribution)

Film sheet differences B 1.4 Based on maximum ±7 cGy difference between calibration films,

a triangular distribution and 2 Gy dose

Linac dose stability B 0.4 Based on a maximum difference of 1 % and a triangular

distribution

Combined uncertainty 2.4

154 Australas Phys Eng Sci Med (2012) 35:151–157

123

Page 5: Optimisation of exposure conditions for in vitro radiobiology experiments

than the combined uncertainty of 2.4 %). The dose profile for

other flask or plate designs also did not vary significantly

with medium depth. Note that the dose profile for the 24 well

plate does not appear to have features associated with the

individual wells. The dose to the cells relative to full scatter

conditions, for open field irradiation are summarised in

Table 2.

Figure 3 summarises the measured dose for the 3 flask and

plate designs with different levels of filling. No significant

difference was observed (greater than ± 2.4 %) between

any of the measurements for a given irradiation setup.

For the 6-well plate, the dose in the medium at the bottom

of the wells was compared to the dose beneath the wells for a

half-blocked beam irradiation (Fig. 4a). The dose profiles

measured inside the wells were consistent in shape with the

dose below the wells. In both the open and shielded regions

of the field, the dose distribution changes significantly

(P \ 0.01 and difference[2.4 %) when the air surrounding

the flasks or plates is replaced by water. This is consistent

with Fig. 3 and is seen for all levels of filling.

Figure 4 shows the dose distributions for the 6-well plate

in air compared to full scatter conditions. The dose distri-

butions for the 6-well plate surrounded or covered with

Virtual Water are shown in Fig. 5a and compared to the full

scatter results obtained in Virtual Water in the absence of a

flask or plate. No significant difference between full scatter

conditions and the conditions created using Virtual Water

around the plate were found (differences \2.4 %). This

result was also observed for the T75 flask. However, in the

blocked region of the field (Fig. 5b), the dose profiles below

the 24-well plates are higher than for the full scatter condi-

tions (P \ 0.01). There was a measured dose difference from

full scatter conditions of at least 10 cGy averaged from 20 to

70 mm from the edge of the blocked region.

Monte Carlo simulation results

The results of the Monte Carlo simulations are shown in

Table 4. The calculated dose below the cell layer, at the

position of the film, is compared to the experimental results

measured with film placed below the wells. The calcula-

tions show that the difference in dose due to flask filling is

very small (\1 %) and agrees with the experimental results

which show no significant difference. However the dose is

significantly decreased, by approximately 3 %, when air

replaced water as the surrounding medium. The calculated

reduction in dose is less than is observed experimentally.

The results of Table 4 confirm that the use of film below

the well to measure the dose to the cell layer results in a

negligible error (\1 %).

Fig. 2 The measured dose profiles below the 24-well plate as a

function of distance from the beam centre. The plate is surrounded by

air. The level of well filling has a negligible effect on the dose

deposited. The thick horizontal lines demonstrate the positions of the

wells. Fully filled corresponds to a depth of 1 cm of medium

Fig. 3 The percentage deviation in dose from full scatter conditions

for all the flask and plate types. No significant differences were

observed within measurement uncertainty for the same irradiation

setup as the level of medium was changed. However the measured

dose was strongly dependent on the choice of surrounding scattering

material (air or water). The 1 cm depth is approximately equivalent to

a full well for the 24-well or 6-well plates

Fig. 4 Measured dose profiles across the wells of the 6-well plate for

a half beam blocked field from film placed either below or at the

bottom, inside the wells. The profiles are measured with the flask

surrounded by air. The inset shows the central region in more detail

Australas Phys Eng Sci Med (2012) 35:151–157 155

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Page 6: Optimisation of exposure conditions for in vitro radiobiology experiments

Discussion

Our results show that the single most important factor that

determines the dose received by a layer of cells is the

scattering material surrounding the culture flask or plate.

This result applies for both an open field and for the open

portion of a half-beam blocked field. Depending on the

flask or plate design, experimental measurements show that

the dose to the cell layer is reduced by between 7 and

13 % ± 2.4 % when the surrounding medium is changed

from water to air. Based on the cell survival curves for

primary fibroblast (AGO-1522) and human prostate cancer

(DU-145) cells [2], this could result in a survival fraction

error of approximately 7 and 10 % respectively. Monte

Carlo simulation confirms that when the flask is surrounded

by air, a dose deficit occurs both in the film layer and in the

adherent cell layer. The reduction in dose is caused by the

removal of back scattered radiation. The results also show

that blocks of water-equivalent phantom material can

provide equivalent scattering conditions to liquid water

despite unavoidable gaps around the perimeter of the flask

or plate. These results agree with the calculations of Keall

et al. [12] reporting a perturbation in dose of \3 % when

flasks are covered with water-equivalent material leaving

air inside and to the sides of the culture flasks.

In the shielded portion of the half blocked field, when air

surrounded the flask a dramatic reduction in dose was

observed compared to full scatter conditions (67 and 69 %

for the shielded wells in Fig. 4). For the T75 and 6-well

flasks, blocks of water-equivalent phantom material can

provide equivalent scattering conditions to liquid water in

the shielded portion of the field. However this does not

apply for the 24-well plates where the dose in the shielded

half of the field was still significantly lower due to the

larger proportion of air surrounding the wells gaps leading

to decrease in scatter. For experiments investigating cells

survival in the shielded parts of a modulated field, these

findings emphasise the importance of dosimetric validation

of experimental designs including the flask with the exact

configuration of the surrounding scattering medium.

There is a small discrepancy between the doses pre-

dicted by the Monte Carlo simulation below the flask

compared to the measured values. The lower dose observed

experimentally may be explained by the differences in

geometry between simulation and experiment. In the

idealised geometry of the simulation, only a single well or

Fig. 5 a The dose profiles below the wells of the 6-well plate when it

is surrounded by Virtual Water or covered by Virtual Water. b The

dose profiles below the wells of the 24-well plate, when it is

surrounded by Virtual Water or covered by Virtual Water. In each

plot, the thick horizontal lines demonstrate the positions of the wells.

The dose profile for full scatter conditions is shown for comparison

Table 4 The dose to film placed below a flask and to cell layers adherent to the bottom of a flask calculated using Monte Carlo simulation for the

geometry shown in Fig. 1e

Surrounding material Flask filling Monte Carlo

film layer (%)

Experimental film

layer (%)

Monte Carlo cell

layer (%)

Water Full 0.0 ± 0.1 -2 ± 2 0.5 ± 0.1

Water 1.0 cm -0.7 ± 0.1 2 ± 2 0.2 ± 0.1

Water 0.5 cm -0.4 ± 0.1 2 ± 2 -0.2 ± 0.1

Air Full -3.4 ± 0.1 -7 ± 2 -3.5 ± 0.1

Air 1.0 cm -3.2 ± 0.1 -6 ± 2 -3.2 ± 0.1

Air 0.5 cm -3.3 ± 0.1 -6 ± 2 -3.2 ± 0.1

The Monte Carlo results are compared with the experimental measurements for a T75 flask. The results are shown as a percentage deviation from

full scatter conditions

156 Australas Phys Eng Sci Med (2012) 35:151–157

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Page 7: Optimisation of exposure conditions for in vitro radiobiology experiments

flask is modelled with no neighbouring wells. Therefore,

the lack of lateral scatter to the film due to the air gaps

between the wells was not modelled.

For the conditions used in this study, the filling of flasks or

plates plays an insignificant role in determining the dose to

the cell layer. No significant difference was found for dif-

ferent filling of any of the flasks or plates in either the open or

half-beam blocked fields for any configuration of surround-

ing material. Monte Carlo simulation confirms that when the

flask is surrounded by water, only a very small change (a

maximum of 0.7 %) in dose occurs in both the film layer and

the adherent cell layer when the flask filling level is changed.

Taken together, these results allow the experimenter to

choose the level of flask filling to suit experimental needs.

Conclusion

We present the results of dosimetric measurements and

Monte Carlo simulations for typical experimental designs

used in in vitro exposures of cells. The results show that the

material surrounding a flask or plate is an important factor

determining the dose to the cells, while the level of filling

of the flask is relatively unimportant.

We show that Virtual Water can be used instead of

liquid water as a surrounding material, without affecting

the dose to the cell layer, except in shielded regions of

modulated fields. As an example we have shown that even

by surrounding the 24-well plate with water-equivalent

material it is not possible to assume that dose profiles for

modulated fields will replicate those for full scatter con-

ditions. It is therefore essential to perform dosimetric

measurements as a precursor to radiobiological experi-

ments. These measurements should be performed at the

location of the cells, particularly where air gaps are

inherent in the plate design.

Acknowledgments The authors acknowledge funding from the

NSW Cancer Council in support of this research.

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