Design And Fabrication Of A Three Dimensional And Dynamic Cell Culture Micro-device

Cell culture in vitro is an important means to study the physiological characteristics of cells.The traditional cell culture can't reconstruct the real microenvironment of cells in vivo.Therefore,it has been an urgent problem to construct the three-dimensional and dynamic microenvironment for cells.Plant leaves have outstanding function for material's transportation.The characteristic that polygonal vein-mesh connects with multiple transport channels provides a way for the design of micro-device's micro-channel.Extracellular matrix(ECM)in vivo is a layered network structure which provides support,connectivity and protection for cells.In other words,ECM is an important attachment space for cell growth.It provides a reliable basis for cell growth on the three-dimensional scaffolds.This paper has designed and manufactured a micro-device which can realize the three-dimensional and dynamic cell culture.And the following studies are carried out:(1)Structure design of cell culture micro-devices.Based on characteristics of the transport channel "multi-in and multi-out" of the plant vein mesh structure,a cell culture micro-device was designed which composed of cell-feeding layer,intermediate connecting layer and three-dimensional culture layer.On the basis of layered structure characteristics of extracellular matrix,three-dimensional scaffolds were integrated in the 3D culture chamber.The three-dimensional culture layer is the main structure of micro-devices.It includes import and export of the culture solution,the micro-channels which achieve the "multi-in and multi-out" of the culture liquid,the polygon culture chamber and three-dimensional scaffolds which provide three-dimensional micro-environment in the culture chamber.The fluid simulation of cell culture micro-device was carried out.The structure owning excellent stability and uniformity was chosen as micro-device structures for cell culture.(2)Fabrication of cell culture micro-devices.The relationship between PCL three-dimensional scaffolds' diameter and working voltage,the movement speed of substrate,the diameter of printer head,the height of printer head,fluid supply speed,solution concentration and so on were researched.In order to provide reasonable parameters for the integration of 3D scaffolds in cell culture micro-device.The real height of three-dimensional scaffolds at different printing layers was measured.A three-layer PDMS micro-channel structure of cell culture micro-device was fabricated by soft etching technology molding technology.A three-dimensional PCL scaffolds was printed in the micro-device culture chamber by using electrohydrodynamic direct-writing method.The forming of 3D scaffolds on the micro-device's surface was observed and studied.The mask technology and biochip laser cutting technology were used to achieve the integration of three scaffolds in culture chamber.Plasma bonding technology was used to realize the bonding of three layers micro-device's structure.(3)Feasibility verification of micro-cell culture device.The inlet was set different feed speed.The liquid experiment in micro-device was carried out by fluorescence velocimetry.The results were compared with the simulation results to verify the stability and uniformity of the micro-device.At last,Hela cells were used as experimental subjects to conduct cell experiments in the cell culture micro-device and dish.The state of cells growth was observed.The results show that the cells cultured in micro-device have large space utilization and higher survival rate.Thus,the three-dimensional and dynamic cell culture micro-device is feasible.