In Vivo Imaging Of Artificial Micro-tissue Based On Optical Clearing

Artificial micro-tissue is a widely used three-dimensional cell culture model,which is closer to the real physiological microenvironment of cells in the organism,and has become an important model for disease research and drug screening.The premise of artificial microtissue application is to accurately describe its model characteristics.However,the high scattering within tissues limits the penetration depth of light in the tissues,which greatly affects the imaging depth and imaging quality.Even artificial micro-tissue cannot obtain its three-dimensional structure information with high resolution.The development of tissue optical clearing technology has provided an important solution to the above problems.However,the current optical clearing methods used for artificial micro-tissue are mostly limited to fixed samples and are not suitable for imaging of artificial micro-tissue in vivo.This paper aims to develop a safe and efficient in vivo optical clearing method,which can realize the three-dimensional imaging of artificial micro-tissue in vivo.The main research contents are as follows:(1)The preliminary establishment of the optical clearing imaging method of artificial micro-tissue in vivo: Based on the basic principle of tissue optical clearing technology and the imaging requirements of living tissue,and considering the biocompatibility and physical and chemical properties of the reagents,such as transmission spectrum,osmotic pressure,refractive index,30% iodoxanol was selected as the optical clearing reagent for artificial micro-tissue in vivo.The results showed that the 30% iodoxanol had good compatibility with the commonly used fluorescence signals GFP and Hoechst 33342,and could effectively improve the imaging depth and imaging quality of artificial micro-tissue and enhance the visualization effect of the internal structure of the tissue.(2)Safety assessment of the optical clearing imaging method of artificial micro-tissue in vivo: The safety assessment of the optical clearing imaging method of artificial microtissue in vivo was carried out from the two-dimensional level and the three-dimensional level respectively,mainly including the effects on the morphology,activity and proliferation of cells and tissues.The results showed that 30% iodoxanol had a slight reversible effect on the morphology of 2D cells,but had no significant effect on the cell activity and proliferation.There was no significant effect on the morphology,activity and proliferation of threedimensional cell spheroids.It was proved that 30% iodoxanol had low cytotoxicity and did not significantly affect the physiological state,morphological characteristics and metabolic activities of the artificial micro-tissue.It had high safety for the artificial micro-tissue in vivo.(3)Observation of drug response in artificial micro-tissue in vivo based on optical clearing imaging: The effect of staurosporine was evaluated in terms of tumor micro-tissue morphology and cell activity.In vivo optical clearing imaging of artificial micro-tissue was applied to observe the response of tumor micro-tissue to staurosporine,combined with fluorescence microscopy imaging to quantitatively study the distribution of apoptotic cells in the tissue after drug treatment,and compared with the non-medicated treatment tumor micro-tissue.The results showed that staurosporine could effectively induce the apoptosis of tumor cells,and more information of apoptotic cells could be seen in the spheroids after optical clearing treatment,which made up for the lack of information caused by the opacity of the sample,and higher image quality could be obtained.In conclusion,the in vivo optical clearing imaging method used for artificial microtissue proposed in this paper can maintain the physiological state and cell activity of the tissue while being cleared,and improves the quality of in vivo imaging of artificial microtissue.It is expected to provide an important solution for the visualization of tissue structure and function in the fields of histopathological research,drug development and evaluation.