Cardiogenesis of Embryonic Stem Cells with Liquid Marble Micro‐Bioreactor

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Abstract
A liquid marble micro‐bioreactor is prepared by placing a drop of murine embryonic stem cell (ESC) (Oct4B2‐ESC) suspension onto a polytetrafluoroethylene (PTFE) particle bed. The Oct4B2‐ESC aggregates to form embryoid bodies (EBs) with relatively uniform size and shape in a liquid marble within 3 d. For the first time, the feasibility of differentiating ESC into cardiac lineages within liquid marbles is being investigated. Without the addition of growth factors, suspended EBs from liquid marbles express various precardiac mesoderm markers including Flk‐1, Gata4, and Nkx2.5. Some of the suspended EBs exhibit spontaneous contraction. These results indicate that the liquid marble provides a suitable microenvironment to induce EB formation and spontaneous cardiac mesoderm differentiation. Some of the EBs are subsequently plated onto gelatin‐coated tissue culture dishes. Plated EBs express mature cardiac markers atrial myosin light chain 2a (MLC2a) and ventricular myosin light chain (MLC2v), and the cardiac structural marker α‐actinin. More than 60% of the plated EBs exhibit spontaneous contraction and express mature cardiomyocyte marker cardiac troponin T (cTnT), indicating that these EBs have differentiated into functional cardiomyocytes. Together, these results demonstrate that the liquid‐marble technique is an easily employed, cost effective, and efficient approach to generate EBs and facilitating their cardiogenesis.

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