Construction And Application Of 3D-Printed Microfluidic Chip Cell Analysis Platform

Cell analysis plays an important role in understanding life processes and biological mechanisms.As a cell analysis tool,microfluidic chips have the potential to revolutionize the way cell biology is studied because of their flexible structural design,low energy consumption,easy integration and automation.In this thesis,the flexible,direct and rapid prototyping 3D printing technology is used to produce microfluidic chips.The 3D printed microfluidic chips are combined with paper-based chips and PDMS membranes to build a series of small cell analysis platforms,which can be used for cell-based compound activity analysis and study the effect of oxygen/dissolved oxygen concentration gradient on cells and explore there mechanism.The research content of this thesis mainly includes the following parts:（1）Construction of hybridized cell analysis platform of 3D printed concentration gradient and paper-based microfluidic chipCompound activity tests based on cells are usually performed in porous cell culture plates.The configuration and distribution of different concentrations of compounds is complicated.Two-dimensional growth pattern of cells in cultured plates cannot simulate the three-dimensional growth in vivo.Moreover,the cultured plate is an open culture system,so the concentration of some volatile compounds changes with the extension of time,which brings uncertainty to the experiment.We built a cell analysis platform combining 3D printed and paper-based microfluidic chip.The 3D-printed chip consists of an upper and lower layer,the upper layer is for concentration gradient formation embedded with a"Christmas tree"network structure.The lower layer with culture chambers is for cell culture,and the paper-based chips were put into the culture chambers as the substrate for cell culture.The liquid from the upper layer could continuously add to paper-based chips.The design of the upper and lower layers makes concentration gradient formation layer and cell culture layer separated,which facilitates the planting of cells in the chip,and prevents cells from blocking the channels.Paper based chips were used as substrates for cell culture and analysis,which could provide three-dimensional growth and save the reagents used in cell staining analysis.When the injection flow rate of liquid was 2.5μL/min,the 3D printed microfluidic chip could form a good concentration gradient.The cell culture results showed that the cells cultured on the platform had good growth activity and morphology.（2）The effect of hydrogen sulfide on cancer cells and intracellular active molecules were studied on the hybridized platformHydrogen sulfide（H₂S）is an intercellular gas signaling molecule that plays an important role in many physiological and pathological processes.The effect of H₂S on cancer cell proliferation has been controversial.However,most of the experiments on H₂S were conducted in cultured plates.In the open culture system of cultured plates,H₂S is volatile compound,which brings instability and unreliability to the experiments.Therefore,more experimental evidences are needed to prove the effect of H₂S on cancer cells.The hybridized platform of 3D printing concentration gradient and paper-based microfluidic chip was used to study the effects of continuous low concentration H₂S on hepatocellular carcinoma cells SMMC-7721.Low concentration of H₂S continuously acting on cancer cells could inhibit cell proliferation by inducing cell apoptosis.H₂S could interact with intercellular NO to form biologically active intermediate polysulfides,leading to a series of physiological changes in cancer cells and eventually cell apoptosis.（3）Establishment and application of oxygen gradient platform for cell analysis with 3D printed microfluidic chipEnvironmental hypoxia caused by pollution is increasingly aggravated.In order to study the physiological response of cells in hypoxia environment,hypoxia perfusion chamber and hypoxia workstation are used to create hypoxia environment,however,these equipment are expensive and could not provide physiological hypoxic gradient.Air and nitrogen were injected into the 3D printed microfluidic chip to generate oxygen concentration gradient,and nitrocellulose membrane（NC membrane）paper-based chips were used as the substrate of cell culture in the 3D printed chip,so as to construct an oxygen gradient cell analysis platform.This platform could generate stable linear oxygen concentration gradient in a short time.Compared with the traditional PDMS oxygen concentration gradient cell culture chip which only relies on fluorescence imaging for cell analysis,the cells on our platform could be analyzed by more methods such as fluorescence imaging,flow cytometry,Western blot,q PCR and other methods.We used zebrafish embryonic cell line ZF4 as model to study the effects of hypoxia on cell cycle,intracellular signaling molecules and gene expression.The zebrafish cells were cultured on the oxygen gradient platform,the oxygen level in the cells showed a gradient change.Hypoxia caused the increase of reactive oxygen species in zebrafish cells and the accumulation of hypoxia inducible factor HIF-1αin the cells.HIF-1αfurther stimulated the transcription of intracellular vascular endothelial growth factor（VEGF）gene to generate more blood vessels for increasing oxygen supply.Meanwhile,hypoxia blocked the cell cycle process in G0/G1 phase,which stopped cell proliferation to maintain cell vitality under hypoxia condition.（4）Establishment of dissolved oxygen concentration gradient platform in 3D printed microfluidic chipWater hypoxia has become a serious environmental problem.In order to study the effects of hypoxia on aquatic animals,simple hypoxia platforms are urgently needed.We used 3D printing technology to make microfluidic chips in one step.Two-way liquid with different concentrations of dissolved oxygen passed through the network of"Christmas tree"structure to produce a gradient of dissolved oxygen.Transparent dimethylsiloxane（PDMS）membrane was used as the substrate for cell culture.The embryos and larvae of aquatic animals could be directly cultured in the growth chambers of the 3D printed chip.The dissolved oxygen concentration gradient generated in the 3D printed chip had a good linear correlation with theoretical dissolved oxygen concentration.The platform has good biocompatibility,and the zebrafish cells and embryos growed well on it for a long time.Using zebrafish whose embryos take a short time to develop and body is transparent as a model,we assessed the effect of different dissolved oxygen on its cells,embryos,and larvae.Hypoxia induces reactive oxygen species（ROS）production in zebrafish cells,embryos and larvae,ultimately leading to cell apoptosis and developmental impairment.Hypoxia also increased nitric oxide content in zebrafish cells,which might be a defensive strategy to overcome the adverse effect of hypoxia on fish cells.Hypoxia decreased the hatching rate of zebrafish embryos and inhibited their heart rate,but hypoxia had no significant effect on the heart rate of zebrafish larva.