口腔鳞状细胞癌-血管内皮细胞共培养微流控芯片的构建及验证OA

Objective To fabricate a microfluidic chip for the co-culture of oral squamous cell carcinoma cells and vascular endothelial cells,validate the interaction between the two cell types,and test the efficacy of combined chemotherapeutic and anti-angiogenic drugs.Methods A double-layer polydimethylsiloxane(PDMS)chip was fabricated,separated by a porous parylene membrane,with human umbilical vein endothelial cells(HUVECs)seeded in the upper chamber and human tongue squamous cell carcinoma cells(WSU-HN6)seeded in the lower chamber,respectively.Hydrodynamic analysis was performed to calculate the relative pressure,flow velocity,and shear stress within the chip.Monoculture(control)and co-culture conditions were separately established for upper-layer HUVECs and lower-layer HN6 cells:HUVECs were cultured alone or co-cultured with HN6 cells in the lower chip chamber,and HN6 cells were grouped in the same way in the upper chip chamber.For HUVECs,mRNA expression levels of vascular endothelial growth factor(VEGF),intercellular adhesion molecule 1(ICAM-1),and interleukin-8(IL-8)were detected by qRT-PCR,in the two groups,and vascular endothelial cadherin(VE-cadherin)protein expression was detected by immunofluorescence staining.Barrier function was assessed by transendothelial electrical resistance(TEER),while tube formation ability of HUVECs was evaluated using the tube formation assay.For HN6 cells,viability was determined by cell counting kit-8(CCK-8)assay.mRNA expression levels of VEGF,basic fibroblast growth factor(bFGF),neural cadherin(N-cadherin),epithelial cadherin(E-cadherin),and Vimentin were detected by qRT-PCR.Immunofluorescence staining was performed to detect the expression of epithelial-mesenchymal transition(EMT)-related markers.HN6 cell migration was evaluated by the scratch wound assay.HN6 cell apoptosis was detected by TUNEL staining.Results The medium flow rate at the chip inlet was set to 100 µm/s and was able to simulate in vivo physiological flow conditions.Hydrodynamic analysis confirmed that the flow conditions were consistent with physiological parameters.In co-culture group,HUVECs showed significantly elevated mRNA expression levels of VEGF and ICAM-1(P<0.01),compared to monoculture control group.Co-cultured HUVECs also significantly increased the number of tube junctions(P<0.05)and total tube length(P<0.01)in the tube formation assay,along with reduced trans-epithelial electrical resistance(TEER)as well as incomplete morphology of VE-cadherin.For HN6 cells,qRT-PCR results showed that,compared with monocultured HN6 cells,co-culture with HUVECs did not significantly affect cell viability but led to significant mRNA expression changes:VEGF and N-cadherin were significantly elevated(P<0.05),E-cadherin was reduced(P<0.05).The results of immunofluorescence staining confirmed corresponding protein-level changes during EMT-related markers,consistent with the qRT-PCR results:Vimentin(P<0.001),N-cadherin(P<0.01)protein expression levels significantly increased,and E-cadherin expression significantly decreased(P<0.05).Co-cultured HN6 cells also exhibited a significantly higher 24 h scratch wound healing rate than the monoculture group(P<0.01),which was inhibited by bevacizumab in a concentration-dependent manner.Bevacizumab showed no cytotoxicity on HN6 cells nor did it change their sensitivity to chemotherapeutic agents.Conclusion The double-layer microfluidic chip fabricated in this study can simulate the interaction between HN6 cells and HUVECs,thus enabling its use in drug screening.