Optimisation of exposure conditions for in vitro radiobiology experiments

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Abstract. Despite the long history of using cell cultures in vitro for radiobiological studies, there is to date no study specifically addressing the dosimetric ...
Australas Phys Eng Sci Med (2012) 35:151–157 DOI 10.1007/s13246-012-0132-6

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 laboratories 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. However 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.

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

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 chemotherapy drugs. Consequently, an accurate knowledge of dose to the cells for in vitro studies is essential. Reproducibility 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, comparisons between laboratories of the bystander effect have been difficult due to differences in experimental design [1]. Progress cannot be made until dosimetry has been eliminated 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 temperature, which is often inconvenient when transporting cells for irradiation. Reproducible dosimetry requires the cells to be in conditions of electronic equilibrium. Submerging 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 contamination if the flask cannot be made water tight.

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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 electronic 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 conditions. The effect of the following are addressed: the medium surrounding the flask/plate (air, water, or waterequivalent 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 conditions 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 waterequivalent 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] Butterworth et al. [2]

Not specified T75 and T25

200 kV irradiation Flask in water bath on water equivalent phantom, linac irradiation from below

A duplex dosimeter measurement Radiochromic film measurements and 2D array

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(GAFCHROMICTM EBT and EBT2, International Specialty Products). To add further insight into the measurements, Monte Carlo simulations of selected geometries were carried out.

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 resolution. 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

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 cylindrical 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

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 (%)

-2

-7

-3

-4

N/A

-9

?1

-2

N/A

-13

-1

-3

T75

6 wells

24 wells

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

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significant if the t-test showed a difference (P \ 0.05) and the dose difference was greater than the combined uncertainty (Table 3).

(a)

(b)

(c)

(d) Flask

(e)

Virtual Water

PMMA

Water

Couch ‘tennis racquet ’

20cm

17cm

Surrounding material either water or air Solid Water Cells Air

2.5cm Cell layer 0.1cm

2.3cm h

3cm

(f) 6 MV

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

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 spectrum 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 transport 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 dimensions 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

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

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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

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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 distributions 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 conditions (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

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

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 calculations 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. 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

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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

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 predicted 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

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

Water

Full

Monte Carlo film layer (%) 0.0 ± 0.1

Experimental film layer (%) -2 ± 2

Monte Carlo cell layer (%) 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 Air

1.0 cm 0.5 cm

-3.2 ± 0.1 -3.3 ± 0.1

-6 ± 2 -6 ± 2

-3.2 ± 0.1 -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

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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 different filling of any of the flasks or plates in either the open or half-beam blocked fields for any configuration of surrounding 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 conditions. It is therefore essential to perform dosimetric measurements as a precursor to radiobiological experiments. 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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