# A High-Throughput In Vitro Radiobiology Platform for Megavoltage Photon Linear Accelerator Studies
## 4 Discussion  
Recent advances in the discovery and development of new anticancer drugs provide an opportunity to enhance the efficacy of radiotherapy. To quickly and efficiently screen a large number of potential radiation-drug combinations, there is a need for a high-throughput in vitro radiobiology platform [14]. In the past, a modulated Cobalt-60 beam was used to vary radiation dose across a 96-well tissue culture plate for radiobiology experiments with a physical lead wedge used to modulate the beam [15]. However, Cobalt-60 teletherapy machines have largely been superseded by more modern and sophisticated linear accelerators that have the capability of producing multi-energy photon and electron beams. Multileaf collimators with varying functional capabilities have replaced traditional lead wedges. Modern linear accelerators can modulate radiation beams, and modulated radiation beams have already been used for radiobiology experiments [16]. A multiwell tissue culture plate based irradiation technique using homogeneous radiation dose distribution was developed for in vitro experiments [17]. Although this helps to investigate a range of drug doses in a single multiwell tissue culture plate, this has not eliminated the need for different plates for different radiation doses. It is possible to conduct cell viability experiments in 96-well plates with smooth dose gradients of up to 10.6 Gy with spatially distributed radiation [18]. A step- and-shoot radiation delivery technique was also used to deliver different radiation doses to a 96-well plate using treatment table movements along with clinical radiotherapy equipment [19] and there is potential for further improvement and refinements of this application. Our high-throughput, in vitro radiobiology platform utilises widely available clinical radiotherapy facilities and IMRT, which has the ability to deliver different radiation doses to a single multiwell tissue culture plate. When cells are exposed to modulated radiation beams, due to the bystander effects of radiation, the proliferative capacity of cells is affected depending on experimental conditions [20; 21]. To address this issue, in addition to the dosimetric verification of accurate dose delivery, we validated our platform by demonstrating bioequivalence between radiation doses delivered with tissue culture plate IMRT and conventional, open-field, homogeneous irradiations. Another concern with using IMRT for radiobiological experiments is the potential risk of intrafraction DNA repair due to the prolonged radiotherapy fraction treatment delivery times in IMRT [21; 22; 23; 24], which potentially impacts the results of radiobiology experiments. This is an interesting area in radiobiology research that would need further exploration [25]. Our radiobiology platform has several advantages over previously described models. It allows testing of various radiation-drug dose combinations in a single multiwell tissue culture plate and the choice of three types of multiwell tissue culture plate allows our platform to be used for various in vitro radiobiology applications. As our platform uses widely available clinical radiotherapy facilities, it has the potential to enable more institutions to conduct radiobiology experiments. One limitation is that it is not possible to deliver the exact target radiation doses, although satisfactory mean target radiation doses were achieved. This is likely because of very steep radiation dose gradients given the dose range investigated. To address this limitation, one solution is to fit experimental results with linear regression modelling and derive results for target radiation doses. Another possible solution is to use the experimental result of each mean radiation dose as they were measured/calculated. Further technical advances in radiotherapy planning and equipment may help to overcome this limitation in the future. Air cavities can influence radiation interactions with matter and its dose deposition [26].