| The liver is the largest internal organ in the body and performs multiple functions including metabolic,synthetic,immunologic,and detoxification processes.Liver tissue engineering has evolved rapidly in the last three decades,which is driven by two unmet demands.The first demand derived from clinical liver transplantation,which is the only effective treatment for patients with severe liver dysfunction or terminal liver failure.However,the well-recognized therapeutic option is limited by liver donor shortage,high medical cost,and immunological rejection.This situation necessitates the creation of large-scale liver tissue constructs for the replacement and/or transplantation treatment of patients suffering liver failure.The second demand rises from pharmaceutical industries which rely on small-scale liver models to proceed in vitro drug development and screening.With the attempt to satisfy the aforementioned two needs,I developed two microfluidics-based methods to in vitro engineer the liver,i.e.,a microfluidics-based biomimetic method to establish a liver lobule-like liver chip for the analysis of drug toxicity and drug-drug interactions and a pneumatic-aided micromolding strategy to fabricate liver lobule-like microtissue as a building block for next-step bottom-up liver tissue engineering.All the results indicated that these two methods could facilitate the development of multiscale liver tissue engineering,as well as liver physiology and/or pathophysiology studies.The followings are the obtained results of the present thesis:1.In the first part of this work,I used AutoCAD software to design a multilayer microfluidic chip with a liver lobule-mimicking pattern inspired from the natural microstructure of the liver.Utilizing the photolithographic processes,the two molds,i.e.,a mold for the fluidic layer and a mold for the control layer,were respectively obtained.Adopting the multilayer soft lithography method reported by Quake group,I successfully fabricated a biomimetic microfluidic chip.The chip consisted of four layer,i.e.,the fluidic layer,the control layer,a thin adhesive/support layer and a glass slide(from top to bottom).In the fluidic layer,there was an embedded microchamber with multiple arrays of pillars.While in the control layer,there is an integrated pneumatic-aided microvalve system(PμS),which could facilitate the controllable inoculation and localization of hepatocytes and endothelial cells.It is worth mentioning that the binding process of the fluidic and control layers needs 12 h bake in an 80 °C oven.This is because that the contact area of the two layer was limited due to the arrangement of the pillar arrays in the fluidic layer.The actuation of the pneumatic-aided microvalve system was tested with various air pressure being applied.The result showed that 18 psi is enough to successfully act.2.Following a programmed process,hepatocytes(HepG2)and endothelial cells(Human Aortic Endothelial Cells,HAECs)were respectively infused into the fluidic microchamber and formed a liver lobule-like pattern with the help of the pneumatic-aided microvalve system(PμS).As observed under a confocal microscope,the 3D liver chip demonstrated a liver lobule-like morphology and composed of a radially patterned hepatic cord network and an intrinsic hepatic sinusoid-like network.The hepatic enzyme assay showed that the 3D biomimetic liver chip maintained high basal CYP-1A1/2 and UGT activities,responded dynamically to enzyme induction/inhibition,and preserved great hepatic capacity of drug metabolism.3.Using the established 3D biomimetic liver chip,the potential adverse reactions of drug induced liver injury were successfully analyzed via drug-drug interactions of clinical pharmaceuticals.The obtained results showed that pre-dosed pharmaceuticals(i.e.,omeprazole(OME),ciprofloxacin(CPFX),rifampicin(RIF)and probenecid(PBD))which disturbed CYP-1A1/2 and/or UGT activities would alter the toxic effect of a subsequently administrated drug(i.e.,acetaminophen(APAP)).All these results together validated the usefulness of the fabricated 3D biomimetic liver lobule-like liver chip for the in vitro toxicological studies.4.In the second part of this work,I proposed a novel and versatile approach,referred as to pneumatic-aided micro-molding(PAM),to flexibly fabricate microscale hydrogels(microgels)with precise manipulation of multiple types of cells and microstructure of hydrogels.This idea was achieved by using multiple types of strategically-designed pneumatic microvalves.By application of the PAM approach,various cell types(i.e.,HepG2 cells,NIH 3T3 fibroblasts and A549 cells)were encapsulated in different kinds of hydrogels(i.e.,collagen,gelatin and gelatin)that had well-defined geometries(i.e.,triangle,square and circle).In addition,cell-laden microgels with single or multiple micro-channels were fabricated,of which the shape,number and organization could be finely tuned by redesigning the configurations of the applied microvalves.Furthermore,multi-compartmental or multi-componential microgels with multiple cell types and composite hydrogel structures were constructed using a two-step process of PAM strategy.5.Finally,I applied the PAM strategy to fabricate a 3D liver lobule-like cell-laden microtissue.As well,the obtained images showed that the liver lobule-like microtissue was made up of a radial orchestration of hepatic cord-like and sinusoid-like structures.The resultant microtissue resembled the architecuaral complexity of the natural liver lobule.In addition,the liver lobule-like microtissue was leveraged for the assessment of acetaminophen-induced liver toxicity and hepatoprotective effect of glutathione.Taken together,the pneumatic-aided micro-molding(PAM)strategy may be a compelling and valuable tool in bottom-up tissue engineering. |
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