Development of a flicking system for producing 3-dimensional cells in microbeads

Culturing cells on planar compliant substrates produces low yield of microtissues. A variety of microtechnologies was introduced and developed to encapsulate cells for producing microtissues. However, the size control of the microcapsules is challenging to the current technologies for cells encapsulation. In this research, we proposed a simple yet efficient technique to encapsulate cells leading to the growth of microtissues. The technique involved with the development of a flicking system which can be applied to encapsulate cells in calcium alginate microbeads at different flow rates and flicking speed. The microbeads size increased with the flow rate but decreased with the flicking speed. Flicking speed of 80 rpm and flow rate of 4 μl/min was chosen to use in the cells encapsulation. The cells encapsulated in the microbeads at cell density of 31.9 × 106 cells/ml (HaCaT) and 94.2 × 106 cells/ml (ORL-48) were cultured and observed for proliferation over a period of time. The microbeads had a smooth surface with spongy and porous surface texture in FE-SEM imaging. In nucleus (DAPI) staining, the encapsulated HaCaT and ORL-48 cells in microbeads grew from scatter individual cells to form cells aggregate and then microtissues. In live and dead cells stainings, majority (≈99 %) of the microtissues cultured in the calcium alginate microbeads were stained in green which indicated the cells were alive. Alginate lyase was used to dissolve the alginate shells in which, the intact microtissues was released. The microtissues obtained were characterised by a rough and uneven surface could be due to the extracellular matrix proteins adjoining the cells. The cells of microtissues were viable and able to proliferate and spread into 2D monolayer in replating experiment. The flicking system has produced microbeads with controllable size and allows the growth of microtissues. HaCaT and ORL-48 cells encapsulated in calcium alginate microbeads can integrate into microtissues after two weeks of culture