Spheroid-based three-dimensional (3D) liver culture models, offering a desirable biomimetic microenvironment, are useful for recapitulating liver functions in vitro. However, a user-friendly, robust and specially optimized method has not been well developed for a convenient, highly efficient, and safe in situ perfusion culture of spheroid-based 3D liver models. Here, we have developed a biomimetic and reversibly assembled liver-on-a-chip (3D-LOC) platform and presented a proof of concept for long-term perfusion culture of 3D human HepG2/C3A spheroids for building a 3D liver spheroid model. On the basis of a fast and reversible seal of concave microwell-based PDMS–membrane–PDMS sandwich multilayer chips, it enables a high-throughput and parallel perfusion culture of 1080 cell spheroids in a high mass transfer and low fluid shear stress biomimetic microenvironment as well as allowing the convenient collection and analysis of the cell spheroids. In terms of reducing spheroid loss and maintaining cell morphology and viability in long-term perfusion culture, the cell spheroids in the 3D-LOC were more safe and efficient. Notably, the polarisation, liver-specific functions, and metabolic activity of the cell spheroids in 3D-LOC were also remarkably improved and exhibited better long-term maintenance over conventional perfusion methods. Additionally, a robust micromilling method that incorporates secondary PDMS coating techniques (SPCs) for fabricating V-shaped concave microwells was also developed. The V-shaped concave microwell arrays exhibited a higher distribution density and aperture ratio, making it easy to form large-scale and uniform-sized cell spheroids with minimum cell loss. In summary, the proposed 3D-LOC could provide a convenient and robust solution for the long-term safe perfusion culture of hepatic spheroids and be beneficial for a variety of potential applications including development of bio-artificial livers, disease modeling, and drug toxicity screening.

A microfluidic device for generating monodisperse superparamagnetic droplets and rapidly manipulating desired droplets into designated sub-microchannels by an external magnetic force is described. Superparamagnetic magnetite (Fe₃O₄) nanoparticles are synthesized by a chemical co-precipitation method. They are well dispersed in the water-phase to form a superparamagnetic fluid that is sheared into picoliter-volume monodisperse superparamagnetic droplets by the oil-phase in a T-junction PDMS microchannel. Superparamagnetic droplets always flow into sub-microchannel 1 due only to laminar flow without a magnetic field. But they are deflected from the direction of laminar flow by a perpendicular magnetic field. The results show that the deflection is proportional to the magnetic field gradient and magnetic nanoparticle concentration, and it is closely related to the magnet position. The observed experimental results make a good match with theoretical analysis. Single or bulk superparamagnetic droplets are successfully manipulated into the designated sub-microchannels 2 and 3 respectively, only by changing the positions of the magnet. Relatively high efficiency is obtained with more than 10 superparamagnetic droplets precisely manipulated per second. This simple and robust apparatus has wide applications in high throughput drug delivery/screening, immunoassay, cell research and synthesis of magnetic microparticles due to good biological compatibility and monodispersity of superparamagnetic droplets.