Microfluidic Droplet-based Hydrogel Beads For 3D Tissue-like Structures Formation

With the development of mammal cell culture in vitro, numerous vital advances have been gotten in the understanding of life functions at celluar, tissue and organ levels. However, most of these results were obtained by 2D cell culture in which the cell environment is not consistent with the complex 3D environment in vivo of which the specificity is determined by the 3D cell arrangement. In addition, to some extent, the functions of organs are determined by the 3D cell arrangement. Thus, the development of 3D cell culture in vitro is vital in current cell biology researchs. Recently, with the ability of mimicing the structures and functions of tissues, the cell spheroids which are formed by the self-assembly of multiple cells, as one of the 3D cell culture methods, have been uesd in some reasarch areas such as drug screening and cancer therapy. Moreover, the non-spherical cell aggregates which are formed by the fusion of multiple cells have shown giant potentials in tissue engineening as the build blocks of the tissue construction in vitro. However, current methods of cell spheroids and cell aggregates formation have many drawbacks that restrict the development of their applications. In the present study, based on the microfluidic droplet, we designed and made two types of microfluidic chips that can generate different cell-laden hydrogel droplets Combined with the different gelation methods, the formation of cell spheroids and shape-controlled cell aggregates with uniform size in hydrogel microbeads were realized. Our results showed that the microfluidic droplet-based cell-laden hydrogel microbeads will provide a new method for 3D cell culture in vitro. The results obtained in the present work are as follows:1. In this study, a chip mould was successfully fabricated by adjusting the conditions of pre-bake, the exposure time and the developing time. On this basis, a microfluidic device with good mechanical properties and structural integrity, which was consisted of a cell scattering unit and a droplet formation unit, was fabricated through adjusting the ratio of between PDMS pre-polymer and curing agent, the curing temperature and time during the fabrication process. The results showed that the optimal conditions for the mould fabrication were 65 °C 2 min + 95 °C 4 min for the pre-bake, 75 s for the exposure time and 4 min for the developing time. The optimal conditions for the PDMS chip fabrication were 7:1 for ratio of between PDMS pre-polymer and curing agent, 85°C for the curing temperature and 40 min for the curing time. By using this microfluidic device, the formation of droplets with different size was realized. The ability of forming droplets with different concentration by on-chip changing the ratio between the aqueous flows was demonstrated by using the fluorescein, which establishes the foundation for the formation of cell spheroids in mixed hydrogel microbeads.2. By on-chip changing the flow rates of the two hydrogel solutions, the ratios between alginate and matrigel for mixed hydrogel microbeads formation were screened. The results showed the optimal ratio was 1:1. Using this condition, He La cell spheroids with uniform size were obtained by encapsulating and culturing He La cell in the corresponding mixed hydrogel microbeads. The observation of cytoskeleton by staining demonstrated the He La cells attached each other without empty gaps and formed a 3D cell-to-cell direct contact. The anticancer drug test showed that He La cell spheroids formed in the mixed hydrogel beads had more powerful resistance to drugs compared to conventional monolayer culture cells.3. The microfluidic droplet chip which had a cell scattering unit and a droplet formation unit was consisted of three layers: the fluidic layer, the PDMS film layer, and a glass slide. It was designed by using Auto CAD software and fabricated by soft lithography with PDMS. Combined with the external gelation method, the cell-laden Ca-alginate microbeads with uniform size were formed. By culturing the cells in microbeads, cell aggregates with high cell viability were obtained. By changing the concentration of alginate and Ca Cl2, human cervicalcarcinoma, human hepatocellular liver carcinoma and human umbilical vein endothelial cell aggregates with spherical, spindle- and branch-like shapes were successfully obtained in the corresponding Ca-alginate microbeads, which demonstrated the controllability and generalizability in this method.4. Cytoskeletal staining analysis and SEM observation showed that the cell aggregates were densely packed and interconnected. The retrival of the formed cell aggregates were realized by liquefying the Ca-alginate microbeads through chelation using sodium citrate. AO/PI staining and cell reculture process demonstrated that the cell aggregates maintained high viability and proliferation after being harvested from the Ca-alginate microbeads. The mechanism of the formation of the shape-controled cell aggregates was studied by observing the inner structure of Ca-alginate microbeads, which was accomplished by frist encapsulating Ca-alginate mirobeads formed in different concentration of alginate and Ca Cl2 into epoxy resin, and then cutting off them into slices and staining with methylene blue. The results showed the shape and size of inner structure was similar with those of formed cell aggregates, which indicated the mechanism of the formation of the shape-controled cell aggregates was the cell culture spatial constraints caused by the inner structure of the Ca-alginate microbeads.