Design, Fabrication And Application Of 3D Multicomponent Organ-like Chip For Drug Screening

Cell-level test is one of the important parts in the current preclinical drug screening. Traditional cell culture platforms, such as 96-well plate, are crude and with it, it is not possible to establish a complex microenvironment in vivo. Therefore, the absorption, distribution, metabolism and excretion properties of the drug candidates are not involved in thepreclinical cell-level test. This makes the screening results unreliable and inaccurate. Microfluidic chip contains cell-, tissue- and organ-scale units which has the capacityof control and analysis fluid precisely, with the advantages of low reagent consumption, low cost and high throughput. Therefore, the microfluidic chip is considered as an idealplatform to rebuild and control the microenvironment. Most of the reported multi-organ chips still stay in a premature stage for concept proving with their design philosophy of integration of cells from different tissue in serial and/or multipled way. Transmembrane structure and sufficient drug test dates still lag behind.In this dissertation, a multilayer(3D), multi-cell(multicomponent) and multi-functional microfluidic organ-like chip for drug screening is presented. This device is composed of, from top to bottom, Caco-2 cells, HUVEC cells, primary hepatocytes, HUVEC cells, MCF-7 cells, combination of fat, lung and heart tissues, and dialysis membrane. In principle, the sequence of each unit follows the ADMEprocess of oral drug in vivo. Under a typical design, the oral drug is first absorbed by intestinal epithelium villi, then go through the capillary into the blood and feeds into liver, and then transported to the whole body by heart, and go through the capillary into each tissue, and finally excrete by kidney finally.In the first part of the thesis, design and fabrication of 3D multicomponent organ-like chip were presented. Then, functions of each biological unit in the chip were investigated. Absorption and barrier functions of Caco-2 and HUVEC cell layers were demonstrated by sodium fluorescence, propranolol and fluorescence-labeleddextranpermeation test. Metabolism ability of primary SD rat hepatocytes were tested by cyclophosphamide. LIVE/DEAD(?) Viability/Cytotoxicity Kit was used to analyze the vitality of fat, lung and heart tissues on chip.In the second part, a series ofclassicaldrugs were test on the organ-like chip, for their tissue distribution, drug-time curve, anti-tumor activity and hepatotoxicity properties. Epothilone B displayed a high anti-tumor activity on chip. Most of the results had the same behavior with those from the animal test. Therefore, this organ-like microfluidics was proved more suitable for cell-level drug screening than the traditional method. The test results can provide references for animal and clinical test, and the chip may increase drug screening efficiency.In the third part, for demonstrating the outstanding performances on chip, the anti-tumor effects of epothilone B was then researched. UTD1, a genetically engineered epothilone analog and a new microtubule inhibitor, lead to different cell destiny with different concentration. At a high concentration(800 nM), microtubule dynamics were totally destroyed and mitosis was completely blocked. P21 and Bax were not up regulated, and P53 was localized mainly in the cytoplasm. Caspase 7 was activated and starts downstream apoptotic signals. At the low concentration(50 nM), UTD1 disturbed mitosis, yet did not block it completely, thus it transformed MCF-7 and HT29 cells into euploid cells and activated the G0/G1 checkpoint and P53. Accumulation of protein is not necessary for the transcriptional activity of PS3, and a basal level of PS3 is sufficient for that. Subsequently P21, but not Bax is transcribed. These findings indicate P53 play a contrary role for different drug stress. P53 may be detrimental to the chemotherapy when the low concentration of tubulin-targeted drug is existed.This dissertation describes the design, fabrication and application research of 3D multicomponent organ-like chip for drug screening, and we found that the ADME test for a series of drugs in vitro with most of the results displayed same behaviors with the animal test. This proves the microfluidic technique has a huge potential for preclinical drug screening, and provides solid supports and may pave the way to the eventual establishment of "human-on-chip". Research of anti-tumor effects of UTD1 confirmed that varying disorder degrees lead to contrary P53 behavior and downstream signal response, contributing to further disclosure of the P53 involved signal pathway.