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

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 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.

Bioencapsulation Technologies in Tissue Engineering

“…To generate a liquid core, alginate hydrogel beads first must be coated with poly-L-lysine or chitosan before liquefying the center, which is a complex process (9). One-step fabrication of alginate coreshell microcapsules has been used to encapsulate embryonic stem cells with improved cell proliferation, aggregation and directed differentiation efficiency (7, 8). …”

“…Electrostatic spray and microfluidic channel/nozzle are the most common methods used for bioencapsulation, especially since they both show distinct potential for producing core-shell microcapsules (Fig. 3) (7, 8). The vibration nozzle technique is considered to be the most industrially up-scalable technique for microcapsule production, especially when the viscosity of the encapsulation solution is low (51).…”

“…This is sheared by the oil (continuous) flow. As a result of the immiscible nature of water and oil, droplets are formed (7, 47). Rapid exchange of the toxic oil phase in a microencapsulation chip is critical to maintaining a high cell survival rate (55).…”

“…In addition to using this technology for cardiac repair, biocapsules have been used for constructing beating cardiac tissue. Some of the most common bioencapsulation systems used in these cardiac applications are alginate-poly-L-lysine (62) and alginate core-shell microcapsules (7, 8). …”

“…( B ) A microfluidics device–based system: at the focusing junction, both the core (sodium carboxymethyl cellulose–containing cells) and shell (alginate) flows were sheared by the crossing oil flow (mineral oil containing calcium chloride) into droplets. Then, calcium cations diffused into the droplets and gelled alginate, thus forming the microcapsule shell (7)-Reproduced by permission of The Royal Society of Chemistry. H: height (or depth); W: width.…”