The Formation And Evaluation Of The Three-dimensional Cell Spheroids Based On Microfluidic Technology

It is important to construct a solid tumor model in vitro for further study of cancer pathological mechanism and development of anticancer drugs.In recent years,the rapid development of microfluidic technology provides a new platform for cell biology research,such as cell capture,localization culture,sorting,stimulation and analysis,due to its advantages of small size,low sample consumption,high throughput,integration and fast transfer speed of mass and heat.In this paper,based on microfluidic technology,we constructed multicellular spheroids by the surface anti-adsorption modification method.The optimal conditions for spheroids were explored,and the factors in the growth and proliferation of the cell aggregations were studied,and the availability of spheroids as a drug screening model was evaluated.Firstly,we fabricated a double-layer PDMS chip which contains fluid channels and cell culture well array by micro-fabrication technology.There were mainly the snake-shaped multi-channel shunt structures in the upper layer of the chip used for fluid flow.It can generate liquid phases with a certain concentration gradient and guide them into the cell culture area.The bottom layer of the chip was the cell culture area,and 12microwells structures formed the culture array for the construction and culture of cell spheroids.Human lung cancer cells(A549)were used to investigate the factors in the spheroids by changing the modification conditions on the chip surface,and determine the optimal modification conditions for enhancing the anti-cell adsorption on the chip surface.Under the most suitable modification conditions,due to the dual action of microwells limitation and surface anti-adsorption,a three-dimensional spheroid with a diameter of about 130?m can be formed.And the spheroid structure was stable with a good activity.It can be used for drug screening.In order to meet the requirements of the reliable results and large sample of the drug testing capacity,we designed microfluidic chips that can generate high-throughput and homogeneous cell spheroids.The chip had double layers.The upper layer was the main channel of the fluid,and two groups of baffles were set in the channel to reduce the fluid flow rate and uniform fluid flow.The bottom layer was the cell culture array with 80 microwells.The optimal modification conditions were used to find a stable and uniform way to construct high-throughput cell spheroids.In the process of cell aggregation and proliferation,the effect of the initial cell concentration and culture time on cell aggregation and growth status were investigated by measuring the sizes of the spheroids.At the same time,the uniformity of the cell spheroids model was evaluated by its size.The result showed that the initial concentration of cells was 1.0×10~6 cells/mL,while the cells grew and proliferated well,and the equivalent diameter of spheroids was about 150?m.And the size of the cell aggregation showed a trend of decreasing first and then increasing with culture time.Under the optimized conditions,the spheroids in the chip showed good uniformity,and the size error between cell spheroids was less than 15?m.Finally,under the action of different concentrations of the anticancer drugs,the degree of damage to cell spheroids was evaluated.The results showed that the survival rate of the spheroids was about 81%without drug action,while the survival rate of the spheroids was only 21%after high concentration of drugs treatment.It is preliminarily verified that this chip can generate high-throughput and homogeneous spheroids,which can be used as a solid tumor model for drug screening.