One-step microfluidic generation of pre-hatching embryo-like core–shell microcapsules for miniaturized 3D culture of pluripotent stem cells

TLDR

A novel core–shell microcapsule system is developed to mimic the miniaturized 3‑D architecture of pre‑hatching embryos with an aqueous liquid‑like core of embryonic cells and a hydrogel‑shell of zona pellucida. The system is fabricated using a non‑planar microfluidic flow‑focusing device that enables one‑step generation of alginate‑shell microcapsules with an aqueous core from two aqueous fluids. Mouse ES cells encapsulated in the liquid core survive >92 % and, after 7 days, ~20 cells form a single aggregate with higher pluripotency marker expression, and can be efficiently differentiated into beating cardiomyocytes, demonstrating the system’s advantage over conventional hanging‑drop methods.

Abstract

A novel core–shell microcapsule system is developed in this study to mimic the miniaturized 3D architecture of pre-hatching embryos with an aqueous liquid-like core of embryonic cells and a hydrogel-shell of zona pellucida. This is done by microfabricating a non-planar microfluidic flow-focusing device that enables one-step generation of microcapsules with an alginate hydrogel shell and an aqueous liquid core of cells from two aqueous fluids. Mouse embryonic stem (ES) cells encapsulated in the liquid core are found to survive well (>92%). Moreover, ~20 ES cells in the core can proliferate to form a single ES cell aggregate in each microcapsule within 7 days while at least a few hundred cells are usually needed by the commonly used hanging-drop method to form an embryoid body (EB) in each hanging drop. Quantitative RT-PCR analyses show significantly higher expression of pluripotency marker genes in the 3D aggregated ES cells compared to the cells under 2D culture. The aggregated ES cells can be efficiently differentiated into beating cardiomyocytes using a small molecule (cardiogenol C) without complex combination of multiple growth factors. Taken together, the novel 3D microfluidic and pre-hatching embryo-like microcapsule systems are of importance to facilitate in vitro culture of pluripotent stem cells for their ever-increasing use in modern cell-based medicine.