Alginate Microencapsulation Parameters Modulate Embryonic Stem Cell Aggregate Expansion and Phenotype

Pluripotent embryonic stem cells (ESCs) are of great interest in regenerative medicine due to their capacity for expansion and differentiation into all somatic cell types; however, most current processes are incapable of manufacturing sufficient quantities of homogeneous stem cell populations. Microencapsulation of ESCs within alginate capsules is a potential platform for scalable biomanufacturing, as the capsules shield cells from hydrodynamic forces found in suspension bioreactors while providing a controlled environment for differentiation. Despite the advantages of an encapsulation-based system, there has been a deficiency in examining the impact of encapsulation parameters on ESC behavior. Alginate is a common material used for encapsulation, but the composition of the polymer varies based on the source, which has led to variability in reported results. Therefore, the objective of this study was to systematically examine the impact of alginate composition, specifically the ratio of guluronic (G) and mannuronic (M) acid, on microencapsulated ESC expansion and phenotype.

Murine ESCs (D3 line) were formed into 500 cell aggregates using forced centrifugation prior to encapsulation in 1.5% alginate (10⁴ aggregates/mL) using a Nisco electrostatic bead generator. Two alginate compositions were examined: one with a higher content of G residues (>60%, termed High G) and one with a higher content of M residues (>50%, termed High M). Aggregates were cultured in serum-free N2B27 media for up to 14 days. Bead mechanical properties were determined using a microscale compression system, cell viability was determined using a LIVE/DEAD assay, cell number was assessed using a CyQuant assay, and gene expression was quantified via RT-PCR. Histological analysis was performed on cryosections for immunostaining. Conditioned media was collected from the cultures and analyzed using ELISAs for vascular endothelial growth factor (VEGF) and bone morphogenic protein-4 (BMP-4).

The elastic modulus of High G alginate beads was higher than High M alginate (8.2 kPa and 6.2 kPa, respectively; p = 0.011), indicating that the capsule types had divergent bulk mechanical properties. Aggregates in the High G alginate remained round whereas aggregates in the High M alginate exhibited an elongated shape. Though cell viability remained high over 14 days (>80%), decreased proliferation was observed in the encapsulated groups, chiefly in the stiffer High G alginate, compared to unencapsulated controls. Gene expression of the pluripotency markers Oct4 and Nanog decreased over time, although the encapsulated cells, particularly those in the stiffer High G alginate, exhibited a slower decay of Oct4 expression when compared with unencapsulated controls. The cells within the High M conditions exhibited increased gene and protein expression of the endoderm marker alpha-fetoprotein (AFP) and the cardiac markers myosin light chain (MLC-2v) and alpha-smooth muscle actin (α-SMA). Additionally, cells within High M alginate produced higher levels of VEGF and BMP-4 on a per cell basis than unencapsulated cells or cells within High G alginate.

Together, these findings suggest that alginate material properties can be used to control stem cell fate and aid in the development of robust and scalable stem cell bioprocessing strategies.