24- 27 For pluripotent stem cells, however, encapsulating them in a miniaturized 3D aqueous liquid-like space might be desired for maintaining their stemness.Īlginate has been one of the most widely used natural biomaterials for cell encapsulation because of its excellent biocompatibility and mild condition of gelling through ionic crosslinking that is not harmful to living cells. In recent years, both macro and microencapsulation of cells in solid-like hydrogels of various biomaterials for 3D culture have been studied intensively. 15- 23 Although useful for research purpose, these culture methods suffer difficulty to scale up, cell damage due to shear stress, and/or limited control of aggregate size and shape, which hinders the clinical application of pluripotent stem cells. 13- 14 Hanging drop, low-attachment plates, and micro-patterned wells have been commonly used to provide a bulk and open 3D liquid environment for culturing pluripotent stem cells. The former is non-physiological, which has been shown to result in altered gene and protein expression in many non-pluripotent or non-stem cells compared to 3D culture. As a result, pluripotent stem cells have been commonly cultured either on 2D substrates overlapped with or in 3D suspension of bulk liquid culture medium.

S1), 8- 12 an aqueous liquid microenvironment allowing ES cells to form aggregates without much resistance might be desired for maintaining their pluripotency (or stemness). 5- 7Īs ES cells naturally form aggregates in their native home of a pre-hatching stage embryo consisting of a miniaturized (~70-150 μm dependent on species) aqueous 3D core of embryonic cells and a hydrogel shell known as the zona pellucida ( Fig. The pluripotency of ES cells, however, is regulated by their niche or culturing microenvironment that is still elusive today. 1- 4 This is because pluripotent stem cells are capable of self-renewal to maintain pluripotency that allows them being coaxed (or directed) to differentiate into any desired type of somatic cells under an appropriate cue. Pluripotent stem cells such as embryonic stem (ES) cells have been intensively explored in recent years as a promising cell source for tissue regeneration, which is particularly valuable for treating diseases due to the loss of static or permanent cells (e.g., cardiomyocytes) that do not replicate much at all in response to injury. Considering its wide availability, easiness to set up and operate, reusability, and high production rate, the novel coaxial electrospray technology together with the microcapsule system is of importance for mass production of ES cells with high pluripotency to facilitate translation of the emerging pluripotent stem cell-based regenerative medicine into the clinic. The higher pluripotency is further suggested by their significantly higher capability of differentiation into beating cardiomyocytes and higher expression of cardiomyocyte specific gene markers on average after directed differentiation under the same conditions. Quantitative analyses of gene and protein expression indicate that ES cells cultured in the miniaturized 3D liquid core of the core-shell microcapsules have significantly higher pluripotency on average than the cells cultured on 2D substrate or in the conventional 3D alginate hydrogel microbeads without a core-shell architecture.

Approximately 50 murine embryonic stem (ES) cells encapsulated in the core with high viability (92.3 ± 2.9%) can proliferate to form a single ES cell aggregate of 128.9 ± 17.4 μm in each microcapsule within 7 days.

A novel coaxial electrospray technology is developed to generate microcapsules with a hydrogel shell of alginate and an aqueous liquid core of living cells using two aqueous fluids in one step.

Core shell needle