Generation Of Molecular Gradients On Microfluidic Chip For Investigating The Guidance Of Hippocampal Neuron

Nervous system plays a key role in regulation of the normal activities of animals and can cause various diseases once the regulatory dysfunction. Neuron is the basic unit of structure and function in nervous system, and correctly orientated polarity of neuron and axonal growth are critical important for establishing neural pathways accurately. During the neuronal development, both the neuronal correct polarity and axonal growth along a specific way making connections with particular target cells are guided by the attractive and repulsive chemical gradients. In vitro, precise controlling the molecular pattern to investigate the regulation of guidance factors will help to elucidate the mechanisms of neuronal response to molecular gradient and understand the function and dysfunction of nervous system. Previous studies have found that there are various guidance factors expressed in vivo in different patterns, for example, forming different steepness of gradient in different brain area. During the neural development, quantitative investigation of the guidance of molecular gradients with varying steepness would contribute to precisely map the molecules expression in vivo. It is extremely sensitive for axons to response molecular gradients with different steepness, therefore, precisely measurement of axonal sensitivity in response to physiological gradients will contribute to accurately control axon growth trajectory and predict growth responses to varying environments. In addition, it is helpful to design effective regeneration treatment using growth factors.Microfluidic device with strong capacities of fluidic control and mass transfer is capable of flexible controlling microenvironment in vitro. Microfluidic chip with channels of micron-submicron containing nanolitre or microlitre liquid is close to the size of cells and subcellular structures, therefore, it is possible to reduce the substance transfer time and generate molecular gradients with high resolution. The design and fabrication of a gradient generator is simple, and multiple functional units can be integrated on a device to generate concentration gradients of different chemical substances. By now, although various of gradient generators have been used in the research of neuron guidance, establishment more delicate and intricate neuronal microenvironment based on microfluidic platform would facilitate the further investigation of growth factors expression and regulation of signaling pathways associated with neuronal development.Consideration of the present situations and challenges, therefore, the following researches combining the advantages of microfluidic chip used in gradients generation and cell analysis were carried out in this thesis:(1) We propose a laminar-based microfluidic device enabling simultaneous generation of multiple gradients with gradually changed slope on a single chip. This device, with two asymmetrically designed peripheral channels and opposite flow direction, could generate gradients with gradually changed slope in the center channel. Using this chip we quantitatively investigated the response of axon growth rate and growth direction to substrate-bound laminin gradients with different slopes.(2) We present a versatile and robust microfluidic device that can generate substrate-bound molecular gradients with evenly varying steepness on a single chip. Steepness with a high resolution that is less than 0.05%/mm can be generated and be highly and flexibly controlled by adjusting various parameters of the device. Using this device, we quantified the hippocampal axonal response to substrate-bound laminin and ephrin-A5 gradients with varying steepnesses.(3) We developed a multilayer microfluidic device to investigate quantitatively the response of axon growth rate to the gradient of soluble factor netrin-1. In this device, a porous membrane was used to compartment a gradient generation chip and an axon isolated culture chip to decrease the shear stress damage to neurons from flowed fluids.(4) We established a 3D microfluidic platform that integrated IKVAV molecule and diffused NGF gradient. IKVAV molecules were modified on the collagen material based on a photo-click reaction, followed by generation of a NGF gradient based on molecule diffusion. This proposed device is expected to investigate how IKVAV molecule to affect axon response to NGF gradients generated in a 3D microenvironment. In summary, we developed four novel molecular gradient generator for investigation of the hippocampal guidance.