Research On Constructing A Dynamic Mechanical Microenvironment And Observing The Response Of Cells In Situ Based On Optical Means

Mechanical signal stimulation in biological microenvironment is one of the necessary conditions to maintain normal life activities.Changes in the intensity,frequency and mode of action of mechanical stimulation can trigger a series of physiological changes.However,most of the current studies on the regulation of cell morphological behavior by environmental signals mainly focus on chemical signals and the physical properties of the matrix environment(including hardness and microstructure),and there is insufficient research on the regulation mechanism of dynamic mechanical signals on cell behavior.The technical difficulties in this research are: 1.How to controllably introduce dynamic mechanical signals on a single cell;2.How to effectively evaluate the behavior of cells in response to dynamic mechanical signals.This project is based on optical methods combined with soft etching to prepare a microfluidic chip that can generate a reciprocating flow field,and build a three-dimensional culture environment(collagen three-dimensional structure)that simulates the tumor cell in vivo through microdroplet technology.The droplet is deformed by applying force to the droplet through the shear flow field and the graded micron confinement structure,which further induces the remodeling of the three-dimensional collagen structure in the droplet,thereby using the collagen network to mediate dynamic mechanical signal transmission to cells,to introduce dynamic mechanical signals with controllable frequency and intensity at the single-cell level.The specific research contents are as follows:(1)Based on the valve-controlled microfluidic technology,a chip which can produce reciprocating flow field is designed,and combined with the gradient micron confinement structure,the fluctuating shear force is introduced into the droplet.Then the dynamic process of droplet deformation under the influence of shear is simulated by COMSOL Multiphysics laminar phase field method.(2)The valve-controlled microfluidic chip with multi-layer structure is fabricated by optical means(including lithography,soft etching,etc.).The experimental conditions for generating micro-droplets were established based on the Y-shaped microchannel structure of the microfluidic chip.The feasibility of generating a reciprocating oscillating shear flow field with controllable amplitude and frequency in a chip using a microfluidic valve and a graded micron confinement structure was verified.(3)Based on the living cell optical imaging technology,the droplets and cell deformation in the shear flow field are tracked in real time,and the spatio-temporal correlation between dynamic mechanical signals and cell morphological behavior is established.In this thesis,a valve-controlled microfluidic chip which can produce repeated shear flow field is fabricated based on optical means,and a simulated three-dimensional collagen fiber matrix environment is constructed in the droplet to culture Hep G2 hepatoma cells.At the same time,the integrated microfluidic valve in the chip is used to produce a reciprocating oscillating shear flow field in gradient micron confinement structure to realize the repeated stretching of the droplet and its internal cells,in order to construct dynamic mechanical signal stimulation.It provides a new experimental platform for further research on the regulation mechanism of dynamic mechanical stimulation on cell morphological behavior,and has certain practical value in single cell scale detection of dynamic mechanical signal.