A Novel Core-Shell Microcapsule for Encapsulation and 3D Culture of Embryonic Stem Cells

Abstract

In this study, we report the preparation of a novel microcapsule of ~ 100 μm with a liquid (as compared to solid-like alginate hydrogel) core and an alginate-chitosan-alginate (ACA) shell for encapsulation and culture of embryonic stem (ES) cells in the miniaturized 3D space of the liquid core. Murine R1 ES cells cultured in the microcapsules were found to survive (> 90%) well and proliferate to form either a single aggregate of pluripotent cells or embryoid body (EB) of more differentiated cells in each microcapsule within 7 days, dependent on the culture medium used. This novel microcapsule technology allows massive production of the cell aggregates or EBs of uniform size and controllable pluripotency, which is important for the practical application of stem cell based therapy. Moreover, the semipermeable ACA shell was found to significantly reduce immunoglobulin G (IgG) binding to the encapsulated cells by up to 8.2 times, compared to non-encapsulated cardiac fibroblasts, mesenchymal stem cells, and ES cells. This reduction should minimize inflammatory and immune responses induced damage to the cells implanted in vivo becasue IgG binding is an important first step of the undesired host responses. Therefore, the ACA microcapsule with selective shell permeability should be of importance to advance the emerging cell-based medicine.

Fig. 1

Typical phase images of microcapsules formed after coating (coating time: 3 min) plain alginate microbeads with chitosan either without mannitol washing before coating in 0.4 % chitosan solution (A) or with mannitol washing before coating in 0.1 % (B), 0.5 % (C), and 0.4 % (D) chitosan solution together with surface zeta potential (E) and ATR-FTIR spectra (F) showing successful preparation of the ACA (alginate-chitosan-alginate) microcapsules (liquid core): 1, sodium alginate; 2, chitosan; 3, plain alginate microbeads; 4, AC microcapsules; 5, ACA microcapsules; and scale bar (applicable to all micrographs), 100 μm

Fig. 2

Typical phase and merged fluorescence images of the cell-loaded ACA microcapsules (liquid core) showing live (green) and dead (red) ES cells at day 0 (A–B), after 7-day cutlture in complete ES cell medium (C–D), after 2-day culture in complete ES cell medium flowed by 5-day culture in regular culture medium (E–F) showing the formation of a single cell aggregate in each microcapsule, together with quantitative real time RT-PCR (qRT-PCR) data of four pluripotent genes (G) and four more differentiated genes (H) in the aggregated cells and control ES cells: Nestin gene for ectoderm; GSC (gooscoid) and T (brachyury) genes for mesoderm; AFP (α-fetoprotein) gene for endoderm; and scale bar (applicable to all micrographs), 100 μm

Fig. 3

Flow cytometry data (A) showing the percentage of IgG bound (positive) cells after incubating cardiac fibroblasts (differentiated cells), C3H10T1/2 mesenchymal stem cells, and R1 ES cells with FITC-IgG (160 kD) overnight and typical confocal fluorescence images of the middle plane through plain alginate microbeads (B) and ACA microcapsules (liquid core, C) after incubating with FITC-IgG (1 hr for B and 12 hr for C) together with that of the ACA microcapsules after incubating with FITC-dextran (4.4 kD) for 1 hr (D): The ACA shell is clearly visible as the dark ring in panel (D) for the ACA microcapsules with a liquid core, but not in (B) for the plain alginate microbeads. Scale bar (applicable to all micrographs): 100 μm

Scheme 1

A schematic illustration of the procedure for preparing ACA microcapsules with a liquid core loaded with cells by 1, electrospraying to produce cell loaded plain alginate microbeads (a solid microsphere of alginate hydrogel with no shell); 2, coating the microbeads with chitosan to form aliginate-chitosan (AC) microcapsules (with an AC shell); 3, coating the AC microcapsules further with alginate to form alginate-chitosan-alginate (ACA) microcapsules (with an ACA shell and solid-like core of alginate hydrogel loaded with cells); and 4, liquefying the alginate hydrogel core to give rise to the ACA microcapsules with a liquid core loaded with cells: The letters G and M represent the guluronate and mannuronate blocks in alginate, respectively.