Preparation And Application Of Transparent Paper For 3D Cell Culture

Three dimensional(3D)cell culture has been developed rapidly,because it can accurately simulate the microenvironment in vivo,and is of great significance for the study of cell life activities.Cellulose paper as a kind of three dimensional cell culture platform was been reported.Cellulose paper has the characteristics of abundant sources,low cost,good biocompatibility,and high porosity.In addition,it is able to provide enough space for cell growth,and can be implemented through the stack and break up the paper simple transformation of 2D and 3D environment.Therefore,paper-based cell culture platform has been widely used in drug screening,building disease models and repairing tissue defects.However,due to the light scattering caused by the anisotropic arrangement of the fibers in the paper,cells cannot be directly observed clearly through the paper,resulting in low resolution of the cell images and limited the application of paper in cell culture.Additionally,most paper is not suitable for cell culture given that cellulose does not possess cell adhesion moieties specific to cells.Long-term exposure of cellulosic paper to medium can also break the bonds between fibers and lead to cell loss.Therefore,this study aims to prepare a cellulose paper for cell culture with good cell adhesion,high mechanical strength and high underwater transparency.And the 3D paper-based cell culture platform was developed to analyze the growth and migration of cells from the spatial scale.This study can broaden the application of paper-based materials in biomedicine.The main research contents are as follows:(1)Comparison of the preparation process and properties of different transparent paper.TEMPO oxidized transparent paper,regenerated cellulose paper and nanocellulose transparent paper were prepared by the method of TEMPO oxidation and dissolution regeneration using cotton fiber and microcrystalline nanocellulose(MCC)as raw materials.And the pore structure,transmittance and mechanical property of several kinds of paper were analyzed and compared.The results showed that the transparent paper prepared by the method of TEMPO oxidation had a porous structure(with an aperture of 10?30?m).However,the long fibers could be clearly observed under an optical microscope,and light transmittance of the paper was poor,which obstructed the cell observation.And it was easy to absorb water and swell,leading to the destruction of the paper structure.The regenerated cellulose paper possesses larger aperture,and the underwater light transmittance was 80%.However,fibers can still be observed under the optical microscope.The paper prepared with MCC cannot observe the fiber structure under the optical microscope,and it has excellent underwater transparency which transmittance>90%.The paper can keep the original shape and structure without significant changes after being soaked in water for a long time.Therefore,MCC is an appropriate raw material for the preparation of transparent paper and the dissolution regeneration method can maintain the structural of the material in water for a long time.(2)Study of preparation and property of transparent paper for cell culture.Porous cellulose paper was prepared by dissolving regenerated microcrystalline nanocellulose and adding porogens,and its pore structure,light transmittance,mechanical strength and biocompatibility were tested.In this study,paper with porosity of 77%,79%and 90%and pore sizes of 23 ?m,36 ?m and 46 ?m were prepared.It was found that the porous cellulose paper appeared white in air,and its transmissibility could reach above 95%under water.The mechanical strength of untreated porous cellulose paper were poor which the tensile strength was less than 1 MPa and the structure was destroyed after stirring in water 2 days.The tensile strength increased by 3 times after using PVA treated,and the paper structure did not change significantly after stirring in water 7 days.The CCK-8 and cell staining results showed that cells retain more than 95%cell activity after culturing in the environment containing transparent paper 1/3/5/7 days,indicating that the porous cellulose paper has good biocompatibility.(3)Constructing and feasibility study of 3D paper-based cell culture platform.In order to enhance the cell adhesion,the porous cellulose paper was treated with gelatin.The results showed that the cell density was significantly higher than that of the paper without treatment and the cell adhesion was improved.Cells were incubated on 1/2/3/4/5 layer paper-based cell culture platform respectively,and observed directly under the optical microscope and the fluorescence microscope after staining.The results showed that cells could also be observed directly and clearly by optical microscope in the single layer of paper prepared in this study compared with the traditional filter.At the same time,the resolution of fluorescence microscope images of cells on the transparent paper was significantly higher than that on filter paper.And the 3D environment was constructed by stacking multiple layers of paper.The cells of the single layers of paper were observed by optical microscope and fluorescence microscope,and analyzing the growth of cells on a spatial scale.It was found that cells were able to grow and migrate along the thickness of the paper,and the amount of migration was related to the initial cell inoculation density and the thickness of the paper-based platform.