This paper reports the use of liquid marbles (LMs) as miniature bioreactors to produce three-dimensional (3D) spheroids including tumor-like spheriods from cancer cells and embryoid bodies (EBs) from stem cells. A liquid marble microbioreactor is prepared by placing a drop of cell suspension onto a polytetrafluoroethylene (PTFE) particle bed. Without the addition of growth factors, suspended EBs from liquid marbles exhibit spontaneous contraction. These results indicate that the liquid marble provides a suitable microenvironment to induce EB formation and spontaneous cardiac differentiation. The EBs were further plated onto gelatin-coated tissue culture dishes. Plated EBs express mature cardiomyocyte marker cardiac troponinT (cTnT), indicating that these EBs have differentiated into functional cardiomyocytes. The cardiomyocytes generated using this liquid marble approach could be useful for transplantation.
In this study, we developed a novel and rapid method to generate in vitro three-dimensional (3D) multicellular tumor spheroids using a surface acoustic wave (SAW) device. A SAW device with single-phase unidirectional transducer electrodes (SPUTD) on lithium niobate substrate was fabricated using standing UV photolithography and wet-etching techniques. To generate spheroids, the SAW device was loaded with medium containing human breast carcinoma (BT474) cells, an oscillating electrical signal at resonant frequency was supplied to the SPUDT to generate acoustic radiation in the medium. Spheroids with uniform size and shape can be obtained using this method in less than 1 minute, and the size of the spheroids can be controlled through adjusting the seeding density. The resulting spheroids were used for further cultivation and were monitored using an optical microscope in real time. The viability and actin organization of the spheroids were assessed using live/dead viability staining and actin cytoskeleton staining, respectively. Compared to spheroids generated using the liquid overlay method, the SAW generated spheroids exhibited higher circularity and higher viability. The F-actin filaments of spheroids appear to aggregate compared to that of untreated cells, indicating that mature spheroids can be obtained using this method. This spheroid generating method can be useful for a variety of biological studies and clinical applications.
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