Research On The Construction Of Neuron Networks In Vitro Based On Microfluidic Technology

Building a neuron network in vitro,has the same type of cells and similar brain function,and analysis the function of neuron network under controlled conditions,is of great significance for the nervous system by learning,plasticity and information processing.In this paper,we focus on the research of neuron network in vitro construction,according to the research status of neuron network,a neuron network construction method based on microfluidic technology and microelectrode technology is proposed.On this basis,the structure and spontaneous are studied.Specific research content is divided into the following sections:1.The fabrication of micro devices based on neuron specific structure.The chip size is 10x10x5mm,and the structure comprises a liquid inlet,a liquid outlet and a cell culture chamber.The cell culture room is a 10x10 microarray network with a width of 100?m and a depth of 40?m.2.The optimal density of neuron network is applied to obtain the high quality of the neuron network.In the experiment,the chip 1x10~5cells/ml,5x104cells/ml and2x10~4cells/ml density were inoculated,and observe the growth of neuron network under the microscope.Finally complete the detection and dyeing experiments,the proliferation of neuron network.The results showed that the cell density of5x10~4cells/ml was the best,and the cell survival rate was over 99%.3.The neuron network was used to detect the morphological structure.In the experiment,the structure of the neuronal network was detected by using the tubulin antibody(Tubulin)and microtubule associated protein(MAP-2).The results showed that the neurons grew in the channel,the nucleus was large and round,the structure was clear,and the growth of the cells grew along the channel direction.Image J was used for quantitative neuron network.The average length of the neuron was measured to be 68.3?m,and the neuron structure was quantified by Sholl analysis.4.The fabrication of integrated micro devices based on 3D printing,micro fluidic technology and micro electrode technology,including micro electrode chip,PDMS.The MEA substrate is glass material,the size is 49x49x1mm,the number of microelectrodes is 60,and the structure is 8x8 arrays.5.Detection of spontaneous of neuron network by MEA system.The system hardware includes:Head stage,IFB,Temperature control system,PC and software.The spontaneous of neuron network has been tested,the waveform time history is less than 3ms,and amplitude is 50uv~2mv.There are three kinds of discharge patterns of neural networks,respectively,a single spike burst mode and burst mode,continuous release issued alternating single peak potential of continuous,experiment 24 channel detection modes for continuous mode potential outbreak.Neurons between the spike formation of conduction delay,and the pattern of electrodes can be accurately conduction,neuronal activity is not a single,clutter free sequence,but the result of coordination between neurons.