A Novel Approach For Realizing Orthogonal Dual Gradients On Microfluidic Chip And Its Applications

It is well-known that the cellular microenvironment,including the extracellular matrix(ECM)as well as oxygen tension,profoundly affected cellular properties,such as division,metastasis,and differentiation.However,the synergistic effects of oxygen tension and extracellular matrix stiffness were rarely reported.In this study,we presented a robust microfluidic device capable of generating a stable and linear hydrogel stiffness gradient over a well-defined oxygen gradient,which could be numerically simulated and experimentally characterized.The developed device was constructed by two PDMS layers.The top layer contained a cell culture chamber for matrix stiffness generation,and NaOH and pyrogallol were used to scavenge oxygen and generate oxygen gradient in the bottom layer.A PDMS membrane was sandwiched between the two layers,which could exchange gas.In this study,human lung epithelial A549 cells were encapsulated in sodium alginate hydrogel with the stiffness gradient ranging from 58 kPa to 0.5 kPa.To quantitatively characterize the alginate concentration profiles,the fluorescent dye rhodamine B was introduced into the channel.It could be observed that the measured gradient was coordinated well with the numerical simulated result.By controlling various flow rates of chemical reagents with confined concentration,the lowest oxygen tension could reach to 1% and gradually increased to approximately 21%.According to the results,cellular viability showed no significant differences under different oxygen tensions,but exhibited a moderate decrease when the matrix stiffness reduced.Conversely,after the treatment of tirapazamine(TPZ),cells encapsulated in soft substrate showed greatly higher drug resistance than in rigid substrate,and an obvious increase of A549 cell apoptosis was observed due to the hypoxia-activated cytotoxicity of TPZ under low oxygen tensions.In conclusion,the developed microfluidic device provides an easy-to-operate platform which can generate orthogonal dual gradients of oxygen and matrix stiffness for 3D cell culture and cytotoxicity test.It exhibits good potential in the study of matrix stiffness-dependent cell responses and oxygen-sensitive drug cytotoxicity under 3D cell culture conditions.