Three-Dimensional Cell Scaffolds Fabricated By Femtosecond Laser And Its Applications In Neural Network Construction

Three-dimensional(3D)cell culture is close to the native cell growth environment,which can better exert the functions of cells themselves and the connection between cells.Therefore,it is of great significance to develop 3D cell scaffolds suitable for 3D cell culture.In recent years,various 3D cell scaffolds have emerged successively,and have been widely used in the fields of biochip integration,vascular tissue generation,and 3D neural network construction.Neuron is a kind of cell with special axon and dendrite structure,that can be guided to form complex neural networks through custommade 3D scaffolds,which have aroused strong interest by researchers.However,due to the limited flexibility and precision of fabrication techniques,the currently reported 3D neural cell scaffolds are difficult to guide single cells,which greatly restricts the development of in vitro modeling of biological neural networks.Accordingly,this paper proposes to fabricate 3D neural cell scaffolds using femtosecond laser two-photon polymerization technology.This technology can break through the optical diffraction limit and achieve sub-micron processing resolution,so it is suitable for the preparation of high-precision 3D cell scaffolds with controllable morphology.Based on femtosecond laser processing technology,this paper prepared two kinds of 3D neural cell scaffolds,namely micropillar array and microtube array.The construction of 3D neural circuits and accelerated growth of neural cells are realized by these two scaffolds respectively.The main contents of this paper are as follows:(1)The directional induction and differentiation of neural stem cells into neurons and the extraction and culture of primary fetal rat hippocampal neurons are realized.For neurons induced to differentiate from neural stem cells,the effects of serum concentration and protein type on the ratio of neuronal differentiation are studied.For differentiated and extracted neurons,the cell state,cell activity,cell function and cell morphology are characterized by live-cell imaging,immunofluorescence imaging,calcium fluorescence imaging,and scanning electron microscopy imaging.(2)3D micropillar scaffolds are prepared,and the guiding effects of their spacing and height on the growth morphology and orientation of neurons are studied.Regardless of linear or circular arrangement,the scaffold can realize the directional growth of neurons to form 3D neural circuits.Under the guidance of the circular scaffolds,the proportion of neurons forming autologous neurons can reach more than 50%.By calcium imaging technology,the transmission of synaptic signals in the formed 3D neural circuits is analyzed,confirming the successful formation of functional 3D neural networks.(3)3D porous hollow microtubes are prepared,and the effects of their inner diameter,wall thickness and length on the accelerated directional growth of neuronal neurites are studied.The microtubes simulate the axonal myelin sheath,which can accelerate the growth velocity of neurites by up to 10 times.In addition,in response to the problem of directional connection of neural networks,this paper further prepared a magnetic 3D porous hollow microtube,which achieved selective directional connection of neural clusters with the aid of a magnetic field.Meanwhile,immunofluorescence technology and calcium imaging technology confirmed the successful connection of the two neural clusters,that is,a functional neural network is formed.In this paper,based on femtosecond laser processing technology,microarrays are proposed to promote the acceleration of neural growth and neural circuit formation in vitro,which provides a new method and idea for 3D biological neural network modeling,and further expands the application of femtosecond laser technology.