Light-induced Cell Alignment And Harvest For Anisotropic Cell Sheet Technology

The development of tissue engineering technology provides a potential solution for the donor shortage in organ transplantation.Cell sheet technology is an important part of "bottom-up" tissue engineering strategy,and has been applied in cell nondestructive harvest,thin-layer tissue replacement and cell-dense three-dimensional?3D?tissue construction.However,the physiological functions of most natural tissues are closely related to the hierarchical structure of cells and extracellular matrix?ECM?,such as muscle,bone and ligament,blood vessel and nervous system.To build functionalized bionic tissue,well-organized orientation of cells and ECM is indispensable and an effective cell sheet transfer system is necessary.The light-induced cell sheet technology could harvest cells noninvasively based on the photoresponsive characteristics of TiO₂.In this article,light-induced cell alignment,light-induced anisotropic cell sheet harvest and photo-crosslinkable hydrogel have been integrated into the technology,and related material and biological mechanisms have been explored.First,photofunctionalization was demonstrated on TiO₂ nanodots film?TNF?,where proper UV pretreatment could improve the adhesion of proteins and cells.Micropatterned photofunctionalizaiton was obtained via photomask,and cells would display directional extension and arrangement on these micropatterned surfaces.Surface hydroxyl group features controlled by TiO₂ photofunctionalization initiated a hierarchical signal processing thus guided the selective protein adsorption as well as the subsequent cell behaviors.The direction remained during cell proliferation and anisotropic cell sheet?ACS?could be harvested via another illumination process.In order to further understand and optimize this technology,we introduced the"Fast Fourier transform" image processing method to obtain the quantitative data of the cell arrangement.The mathematical relationship between cell sizes and widths of micropattern determined the subsequent cell arrangement,and different types of anisotropic cell sheets had been obtained by this theory.By inhibiting cytoskeleton related proteins and pathways,we thought the myosin II/ROCK pathway mediated cell filopodia retraction was an important mechanism of cell sensing and cell alignment onmicropatterned surfaces.Further UV treatment allowed ACS detachment from TNF surface while simultaneously solidified the GelMA.The detached ACS carried by GelMA could be transferred easily.Moreover,two ACS were successfully stacked into a 3D bilayer construct with controllable orientation of individual layer and maintained cell alignment for more than 7 days.Interestingly,the anisotropic HFF-1 cell sheets could further induce HUVECs to form anisotropic capillary-like networks via upregulating VEGFA and ANGPT1 and producing anisotropic ECM.This developed integrated-functional ACS technology therefore provides a novel route to produce complex tissue constructs with well-defined orientations and may have a profound impact on regenerative medicine.