| OBJECTIVE:T-cell-based therapies are a promising way to treat a variety of diseases,especially cancer.The key to T cell therapy lies in the activation and rapid expansion of functional T cells in vitro.However,T cell expansion in vitro is usually carried out in specialized manufacturing centers,which is a lengthy and costly process.Therefore,how to efficiently and rapidly propagate autologous T cells isolated from patients in vitro has become one of the most critical factors affecting T cell therapy.Based on this,a flow-focused microfluidic chip was designed in this experiment.Jurkat cells were used as model cells to generate cell-loading calcium alginate microgel,and discussed the effect of the three-dimensional culture model based on microgel on the expansion of Jurkat cells in vitro.METHODS:Auto CAD software was used to design the microchannel structure of the droplet microfluidic chip;PDMS droplet microfluidic chips were prepared by soft etching;Real-time monitoring by fluorescence microscope was used to explore the water phase and oil phase that could stably generate droplets,and to determine the relationship between droplet diameter and two-phase flow velocity;The relationship between initial cell concentration and cell encapsulation rate was determined by counting the number of encapsulated cells in droplet;Different concentrations of crosslinkers were set to determine their effects on the formation of microgel;The proliferation of Jurkat cells in the microgel was detected by CCK-8 cell proliferation kit;The survival rate of Jurkat cells in microgel was determined by Calcein AM/PI staining kit;The cytokine secretion of Jurkat cells in the microgel was detected by enzyme-linked immunosorbent assay.RESULTS:The flow-focusing PDMS droplet microfluidic chip designed in this experiment can stably generate droplet with controllable size;Within a certain range,when the flow velocity of water phase remains unchanged,the droplet diameter decreases gradually with the increase of oil phase flow velocity,When the flow velocity of oil phase remains unchanged,the droplet diameter increases with the increase of the flow velocity of water phase;The larger the initial cell concentration was,the more cells were encapsulated in the droplet,and the cell encapsulation rate in the droplet was consistent with Poisson distribution;When the concentration of crosslinker was 5%,the droplet could be completely gelation,and the gelation time was about 2.5 min;Compared with macrogel and ordinary culture plate,the number of cells in microgel was more on the 7th day of culture,and the growth rate was relatively higher,and the cell survival rate was more than 75%;Jurkat cells were able to secrete more cytokines in three-dimensional culture within microgel,and the secretion of IFN-γ and TNF-α were 14.3% and 4.3% higher than those in culture plates on the 7th day.CONCLUSIONS:Calcium alginate microgel based on drop-microfluidic chips can be used as a three-dimensional culture model for T cells,providing a controllable and non-interfered microenvironment for T cells,thus promoting the proliferation of T cells and cytokine secretion in vitro. |
