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Diffusion Kurtosis Image in assessment of 3D Cell Culture1Master's Program of Biomedical Informatics and Biomedical Engineering, Feng Chia University, Taichung, Taiwan, 2Department of Automatic Control Engineering, Feng Chia University, Taichung, Taiwan, 3Center for Molecular Medicine, China Medical University Hospital, Taichung, Taiwan, 4Department of Radiology, China Medical University Hospital, Taichung, Taiwan
Synopsis
A non-Gaussian kurtosis model has been shown to take into account tissue heterogeneity and two relative imaging biomarkers namely, the kurtosis coefficient and the corrected diffusion coefficient can be quantified. In this study, 3D cell culture with hydrogels ECM was used to investigate whether DKI may provide information on these microenvironmental parameters and the microenvironment-associated metastatic propensity of tumors. Our results demonstrated DKI-MRI may provide the potential biomarkers on the change of microenvironment in the application of 3D cell culture experiment.
Introduction
Diffusion-weighted imaging (DWI) methods is sensitivity to microscopic motion, which is due to Brownian motion of water molecules, base on Einstein's original concept that the diffusion water molecules follow a Gaussian distribution1. DWI can provide information on the microstructure and composition of tissues. However, it is a poor model to describe the diffusion in biological tissue due to the limitation of diffusion space, especially in high b values. The diffusion of water molecules confined within the intra-axonal spaces is expected to be restricted. Hence, the diffusion probability distribution of water molecules in the barriers within the tissue, and therefore a diffusion distribution with kurtosis K>0 is present. A non-Gaussian kurtosis model has been shown to take into account tissue heterogeneity and two parameters namely, the kurtosis coefficient (K) and the corrected diffusion coefficient DK can be quantified. This approach is technically demanding, time-consuming and requires the acquisition of high and very high b-values2. Therefore, the diffusion kurtosis image (DKI) will be applied to malignant tumors which usually have a higher cellularity and generally present with restricted water diffusion, the ADC values is lower and Dk is higher when compared to benign lesions3. DWI has been widely applied to the field of tumor diagnosis in clinic4-7. Recently, 3D cell culture with synthetic extracellular matrix (ECM) has been more widely used instead of the 2D culture platform which been demonstrated that cells behave more natively when cultured in three-dimensional environments8.In this study, 3D cell culture with hydrogels ECM was used to investigate whether DKI may provide information on these microenvironmental parameters and the microenvironment-associated metastatic propensity of tumors.Materials and Methods:
MR scan: All images were performed by a 3 Tesla MR scanner (GE Signa HDx, GE) using an 8 channels head array coil. DWI images were obtained with diffusion gradients (b factors : 0, 50, 250, 500, 750, 1000, 1250, 1500, 1750, 2000, 2250 sec/mm2) applied in each of three orthogonal directions. EP-DWI acquisitions (TR/TE =4100/101.3) were performed. We used the pancreas cancer cell line PANC1 in our experiments, which cultured in DMEM/F12 (10% fetal bovine serum, 1x P/S, 15mM HEPES Buffer, 2.5mML-Glutomine, 1.2g/L Sodium Bicarbonate). Cells and culture medium were mixed with 0.3 % agar concentration. Three different cell concentrations with three repetition (6.4x105 , 3.2x106 and 6.4x106 cells/ml) were filled into 9 wells (Figure 1). To avoid air bubbles influence during MRI scan, we used 2.4% agar to fill all space in plate. Data analysis: All MR data were digitally transferred from the MR unit console to a personal computer and processed with software developed in house by using Matlab. The parameters maps were generated by using a pixel-by-pixel computation according to the following monoexponential equation based on MRI diffusion principles : Sb/So = exp(−b(ADC)) and the DKI data is modeled according to the following equation based on the DKI theory: S/So= exp(−bD + b²D²K/6). Here ADC is the apparent diffusion coefficient by monoexponential equation, D is the diffusion coefficient by DKI model, and Dk is diffusion kurtosis. Mean ADCs, D, and Dk of all pixels within ROI were calculated for comparison among different cell density.Result
A 3D cell culture in 9-well dish was shown on Figure 1. Figure 2 illustrated the DWI of 3D cell culture and the selected ROI on one well. The microscopic photos in the wells were showed on Figure 3. ADC, D and Dk of different cell density measured mean and SD were illustrated on Figure 4. The ADC measured by monoexponential equation were 2.520 ± 0.021, 2.385 ± 0.009, and 2.140 ± 0.079 (10-3 mm2/s), for 6.4x105 , 3.2x106 and 6.4x106 cells/ml, respectively. The D measured by DKI equation were 2.658 ± 0.011, 2.549 ± 0.007, 2.319 ± 0.070 (10-3 mm2/s), for 6.4x105, 3.2x106 and 6.4x106 cells/ml, respectively. The Dk measured by DKI equation were 0.292 ± 0.028, 0.302 ± 0.026, 0.333 ± 0.023, for 6.4x105, 3.2x106 and 6.4x106 cells/ml, respectively.Discussion
In this study, 3D cell cultures with different cell density were prepared for evaluate the capability of DKI in the application of 3D cell culture experiment. Due to the thick layer, it is difficult to monitor the cell growth layer by layer using microscopy in the 3D cell culture experiment. DKI may support a monitor tool for the cell growth of 3D cell culture. Our results showed that ADC and D were a negative correlation with cell concentration and the kurtosis (Dk) was increased with high cell density. The reason might be the water diffusion is reduced because of smaller extracellular space.Conclusion
DKI may provide the potential biomarkers on the change of microenvironment in the application of 3D cell culture experiment.Acknowledgements
The study was supported partly from the Ministry of Science and Technology, R. O. C. under the Grant No. MOST 105-2221-E-035 -049 -MY2.References
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