1. Introduction

Over the last 20 years, the global incidence of malignant tumors has increased steadily^[1]. The current clinical experience has revealed significant differences in radiosensitivity among different types of tumors^{[2, 3]}, but also differences in radiosensitivity among different individuals with the same tumor type^[4-7]. This means that basic research on tumor radiotherapy is very important, as it provides an experimental basis and theoretical support for the personalized treatment of patients to increase the precision of specific radiotherapy treatments. For the radiosensitivity of tumor cells, the radiation source is used to irradiate cells, so that the cells receive a certain dose, so as to observe its radiosensitivity. Irradiation conditions are different for each research unit. In recent years, irradiation with a 6 MV X-ray linear accelerator has been conducted in most hospitals by using different irradiation methods. Yang et al.^[8] used 6 MV X-ray irradiation treatment on MDA-MB-231 cells during the logarithmic growth phase; the source-to-skin distance (SSD) was noted to be 100 cm, but the build-up and back-scattering materials were not stated.

Some studies have described the irradiation conditions and methods; however, the description was not sufficiently detailed. A study by Jin et al.^[9] described cells that were irradiated with 6 MV X-ray radiation with an SSD of 100 cm and an irradiation field of 25 × 25 cm². The build-up and back-scattering materials were not explained. In a study by Chen et al.^[10], a vertical beam irradiation method with 6 MV X-ray radiation, gantry angle of 180°, and 1.5 cm of build-up materials were adopted whereas Maria A. Zachari et al.^[11] irradiated cells with 6 MV X-ray radiation and a 10 × 10 cm² irradiation field. In summary, many studies on the radiosensitivity of various tumors have been reported; however, the specific irradiation conditions and methods differ between studies, which means that many irradiation experiments cannot be reproduced between different research centers. Meanwhile, it has become ever more important to establish a convenient and feasible cell irradiation method able to deliver an accurate dose.

The question of whether the cell receives the preset dose is of importance. Without other factors, and only considering the SCD (source-cell distance), build-up and back-scattering effect, cavity effect, and the convenience and feasibility of cell culture, three cell irradiation methods were designed for the most frequently used 96-well plates and 6-well plates by physical measurement. The human breast cancer cell line, MDA-MB-231, was used to verify the differences in the three cell irradiation methods on cell radiosensitivity by using cell proliferation and clonogenic assays. The aim of the present study was to establish and validate an accurate and convenient cell irradiation method, thereby providing an experimental basis and theoretical support for the study of radiosensitivity of tumor cells.

2. Materials and methods

2.2. Simulated "cell culture plate" system

The cell culture plate system was made up of ware cover, cavity, culture medium, and ware bottom. As the density of the solid water, which is often adopted as the clinical homogeneous phantom that is close to that of the cell culture plate, the system was built up and simulated by the solid water. The cavity was built of four pieces of polystyrene with a density merely equal to that of air was placed on the solid water. The well wall was ignored, as shown in Fig. 1. The specific parameters of the cell culture plate simulated by solid water are shown in Table 1.

Table 1Full-screen View

The specific parameters of cell culture plastic ware

	Ware height (cm)	Culture medium height (cm)	Cavity height (cm)
96-well plate	1.1	0.6	0.5
6-well plate	1.7	0.3	1.4

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Fig. 1Full-screen View

A cross section of the simulated "cell culture plate" system. A: Ware bottom; B: Culture medium; C: The phantom of building cavity; D: Cavity; E: Ware cover; F: The gap between the bed and the bottom of the cell culture plate. (A, B, and E are all built of solid water). In the real cell irradiation experiment, F is escapable and filled by using solid water. Thus, the gap is filled by using solid water in the simulation. C is built of polystyrene, in which the density is nearly equal to that of air, in order to build the cavity. G is used to put the plane-parallel ionization chamber.

2.3 Irradiation methods

The 6 MV X-ray was used, the depth of the maximum dose is 1.5 cm below the water and D_20/10=0.585. The thickness of the irradiated material required is 1.5 cm for the 6 MV X-ray beam. The simulated "cell culture plate" system was irradiated at five different preset doses: 2, 4, 6, 8, and 10 Gy, by the three methods. Each measurement was repeated five times.

i. Method G0B0F40 The 6 cm solid water (including the thickness of ware bottom and the gap between the bed and the bottom of the cell culture plate) was placed on the below the plane-parallel ionization chamber. This solid water is used as a back-scattering material. There were no additional build-up materials. As shown in Fig. 2 (a).

Fig. 2Full-screen View

The three irradiation methods for the simulated "cell culture plate" system. (a) G0B0F40 (A certain thickness of solid water (H) is placed on the below simulated "cell culture plate" system, to construct a 6 cm thickness of the back-scattering materials). (b) G0B1.5F40 (A certain thickness of solid water (I) is placed on the above simulated "cell culture plate" system, to construct a 1.5 cm thickness of the back-scattering materials. A certain thickness of solid water (H) is placed on the below simulated "cell culture plate" system, to construct a 6 cm thickness of back-scattering materials). (c) G180B1.5F40 (A certain thickness of solid water (I) is placed on the below simulated "cell culture plate" system, to construct a 1.5 cm thickness of the back-scattering materials). Note: H stands for the additional back-scattering materials; I stands for the additional adding build-up materials; G stands for gantry degree; B stands for the thickness of build-up materials; and F stands for field size.

ii. Method G0B1.5F40 The 6 cm solid water (including the thickness of ware bottom and the gap between the bed and the bottom of the cell culture plate) was placed below the plane-parallel ionization chamber. This solid water is used as a back-scattering material. Solid water of 1.5 cm thickness was then placed over the plane-parallel ionization chamber (including the thickness of ware cover and the height of culture medium). A certain thickness of solid water was placed on the above simulated "cell culture plate" system, in order to construct a 1.5 cm thickness of build-up materials, as shown in Fig. 2 (b).

iii. Method G180B1.5F40 Solid water of 1.5 cm thickness (including the thickness of ware bottom and the gap between the bed and the bottom of the cell culture plate) was placed below the plane-parallel ionization chamber. A certain thickness of solid water was placed on the below the simulated "cell culture plate" system, in order to construct a 1.5 cm thickness of the build-up materials, as shown in Fig. 2(c).

3. Results

3.2 Comparison of cell verification test

3.2.1 Comparison of proliferation of cells

MDA-MB-231 cells were irradiated with 4 Gy and the cell growth curves were measured by CCK-8 assay after irradiation using Method G0B0F40, G0B1.5F40, and G180B1.5F40, as shown in Fig. 3(a). There was no significant difference in the survival rate of MDA-MB-231 cells at 24 h after attachment between the three methods. The cells entered the logarithmic growth phase at 48 h after attachment. The difference appeared at 72 h after attachment and it became larger as time progressed. The OD value with Method G0B0F40 was highest, followed by Method G180B1.5F40, and the lowest for Method G0B1.5F40, which indicated that the actual irradiated dose of cells was lower than that with the other two methods. Method G0B1.5F40 had the lowest OD value and the lowest cell survival rate; the actual irradiated dose of cells in this method was higher than that of Method G0B0F40 and Method G180B1.5F40. It was also confirmed that the actual irradiated dose of cells for Method G0B1.5F40 was the highest overall, followed by Method G180B1.5F40 and then Method G0B0F40, in which the actual irradiated dose of cells was the lowest.

Fig. 3Full-screen View

(a). The cell growth curves for the irradiated MDA-MB-231 cells with Method G0B0F40, Method G0B1.5F40, and Method G180B1.5F40 (n = 6 per group, values represent the mean ± SD); (b). The comparison of the surviving fraction of the MDA-MB-231 cells after the three methods. All data are presented as the mean ± SD from four replicate wells.

3.2.1 Comparison of the clonogenic capacities of cells

As can be seen from Fig. 3(b), obvious differences in the surviving fraction were found for cells after the three different irradiation methods. For methods G0B0F40, G180B1.5F40, and G0B1.5F40, SF(%) was 66.31±7.10, 31.52±5.27, and 25.12±5.21, respectively. In addition, the surviving fraction of the cells for Method G0B1.5F40 was significantly lower than that of Method G0B0F40 and slightly lower than that of Method G180B1.5F40. This showed that the actual irradiated dose of cells was highest with Method G0B1.5F40, and closest to the preset dose. When using this method, the radiosensitivity applied to the cell was accurate.

4. Discussion

It is not clear if the actual irradiated dose for the cells reaches the preset dose, as the results from different irradiation methods are contradictory. Although many research articles have reported data on cellular radiosensitivity, the published descriptions of the irradiation methods tend to be imprecise^{[12, 13]}. In this study, the results showed that no matter what type of cell culture plate was used, the best irradiation method, in which the irradiated dose was closest to the preset dose, occurred after our Method G0B1.5F40 above. For method G0B1.5F40, there was a 1.5 cm thickness of build-up materials (including the thickness of the ware cover and the height of culture medium) on the upside of the cell, the irradiated dose of cells is at the point of maximum dose, and the 6 cm thickness of the back-scattering materials (including the thickness of ware bottom and the gap between the bed and the bottom of the cell culture plate) at the back of the cell increases back-scattering. In this case, a millimeter of difference in the height of the culture medium has little impact on the experiment. For Method G0B0F40, although the 6 cm thickness of solid water (including the thickness of the ware bottom and the gap between the bed and the bottom of the cell culture plate) can increase back-scattering, there is insufficient build-up of materials (for 6 MV, the thickness of the build-up materials is 1.5 cm) on the upside. In this case, the actual irradiated dose of cells falls within the range of the high energy X-ray dose build-up region, and the actual irradiated dose is low. The millimeter of difference in the height of the culture medium in the actual experiment leads to a larger difference in the actual irradiated dose of cells. In Method G180B1.5F40, although there is a 1.5 cm thickness of build-up materials (including the thickness of ware bottom and the gap between the bed and the bottom of the cell culture plate), there was no material in the emission direction of the X-ray to increasing back-scattering, which results in a lower actual irradiated dose of cells than the preset dose. This occurs because the actual cell irradiation is not feasible. Therefore, it is important to add a 1.5 cm thickness of build-up materials (including the thickness of ware cover and the height of the culture medium) into the area above the cells after irradiation of the cells with 6 MV X-ray. In addition, a certain thickness of back-scattering material should be added into the area under the tissue culture plastic ware. In a study by Zheng et al. ^[14], who used a 6 MV X-ray linear accelerator, it was suggested that at least 5 cm thickness of solid water should be added as the back-scattering material. Therefore, in this paper, a 6 cm thickness of solid water was used as the back-scattering material. The thickness of back-scattering material influences the cell dose, but this is not discussed in this paper.

The dose differences of the 96-well plates were generally less than that of the 6-well plate for methods G0B0F40 and G180B1.5F40. For Method G0B0F40, the thickness of the build-up materials was 0.6 cm in the 96-well plate and 0.3 cm in the 6-well plate. Thus, the measured dose was closer to the preset dose: the dose difference was smaller for the 96-well plate, but not for the 6-well plate. For Method G180B1.5F40, the thickness of back-scattering material was 0.6 cm in the 96-well plate and 0.3 cm in the 6-well plate. In a 5 cm thickness of back-scattering material range^[13], the thickness of the back-scattering material is thicker, there are more back-scattering photons, and the back-scattering effect of the dose was larger. Thus, the measured dose was closer to the preset dose, the dose difference was smaller for the 96-well plate, but not for the 6-well plate. This also demonstrated that it was important to add the appropriate thickness of the build-up and back-scattering materials for cell irradiation according to the energy of the linear accelerator.

As the cells are protected from contamination, the culture medium cannot be filled generally; a certain gap should be left between the upper surface of the culture medium and the culture plastic ware cover, and it's inescapable in the process of cell culture. This gap will cause the cavity effect of X-ray irradiation, which has been reported in several studies ^{[15, 16]}. A study by Xiao et al.^[17] has suggested that the cavity effect was lower when the air cavity was located in the deeper region within the tissues than when it is located in the shallower region within tissues. For the method G0B1.5F40 and G180B1.5F40, there is a 1.5 cm thickness of build-up materials, so the cavity effect was lower than that of G0B0F40. A study by Wang et al.^[18] suggested that when the area of the radiation field was greater than that of sagittal interface of the nasopharynx cavity, the nasopharyngeal cavity has very little influence on the dose distribution of the coaxial penetrating field, and it can therefore be neglected. In this study, a 40 × 40 cm² radiation field was adopted for the three methods^[19] and the area of the radiation field was greater than that of cavity, so the cavity effect can be ignored and the actual measured dose would be closer to the preset dose; that is, the cavity effect can be ignored for method G0B1.5F40. Thus, Method G0B1.5F40 represents the optimal method for cell irradiation.

In summary, when the cell will be irradiated, we must pay attention to the influence of the build-up effect and cavity effect in the future.

According to the experience of proliferation and the colony formation assays for MDA-MB-231 cells from literature reports^[20-22], the cell proliferation assay and clonogenic assay of MDA-MB-231 cells was performed to verify the difference of the three methods in this paper. For the cell proliferation assay, we found no significant difference in the survival rate of cells at 24 h after cell attachment for the three irradiation methods. This mainly resulted from the fact that cell apoptosis was not clear at this point and the obvious effect of radiotherapy on cell viability was small. After 48 h, the radiotherapy started to exert an effect on the cells. The dose was higher and the cell survival rate was lower at 72 h after cell attachment. The cell survival rate was highest in Method G0B0F40, because the actual irradiated dose of cells was lowest, followed by Method G180B1.5F40, and then Method G0B1.5F40. Compared with the other two methods, the actual measured dose in Method G0B1.5F40 was closest to the preset dose and the dose was highest, which resulted in the lowest cell survival rate. The cell growth curves demonstrated that there were significant differences for the three cell irradiation methods after 72 h. For the cell clonogenic assay, we can see that SF was highest for Method G0B0F40, second highest for Method G180B1.5F40, and lowest for Method G0B1.5F40 after 9 days. This is the dose of Method G0B1.5F40 that was higher than that of other methods, its dose was closest to the preset dose. This confirmed that method G0B1.5F40 was the optimal method for cell irradiation. From the above two experiments, which showed no significant dose difference in the cell experiments after 48 h for the three methods, but the dose difference was obvious in cell experiments (e.g., the clonogenic assay) after 48 h for the three methods. Each cell experiment lasts for a different time; for example, the cell clonogenic assay usually take longer than 9 days. Thus, method G0B1.5F40 should be chosen at the time of cell experiment. Therefore, an accurate cell irradiation method must be established before conducting a cell radiosensitivity test. An irradiation dose that is too high or too low will affect the experimental results, which will be detrimental to the comparison of the relevant experimental results of authors from different research laboratories.

In this study, three different irradiation methods were used to measure the physical doses of the cell for the simulated "cell culture plate" system. It was demonstrated through the growth curve and colony forming efficiency of MDA-MB-231 cells that Method G0B1.5F40 was the best cell irradiation method. In the cell culture process, the SCD, build-up and back-scattering effect, cavity effect, and the feasibility and convenience of the operation should be fully considered with the linear accelerator. The most accurate and effective cell irradiation method should always be used, which will reduce the dose differences and instability factors and provide a greater accuracy and comparability for laboratories conducting research into cellular radiosensitivity.