Human IPSC Hydrogel Encapsulation for Efficient Production of Embryoid Bodies

Introduction:: Induced pluripotent stem cells (iPSCs) are autologous, can be attained with minimal invasion, and possess the ability to differentiate into nearly any cell type [1]. Consequently, iPSCs are a popular choice for regenerative cell-based therapies and tissue engineering applications. Prior to directing iPSCs toward a specific cell lineage, it is a common practice to form them into 3D spheroids known as embryoid bodies (EBs). EBs can be formed through various methods that rely on the forced aggregation of cells, including spontaneous self-aggregation in suspension culture and settling in low adhesion round-bottomed 96-well plates [2]. EBs can also be generated through the encapsulation of stem cells within hydrogels, such as alginate, which minimizes cell aggregation and promotes instead single cell proliferation into an EB [3]. Encapsulation often requires the use of a Rho-kinase inhibitor (ROCKi), Y-27632, to promote cell viability [4]. Encapsulated EBs can either be differentiated within the hydrogel or retrieved from the gel via decapsulation for additional processing [5]. One potential benefit of the hydrogel encapsulation method is that it may offer a means to substantially improve EB formation efficiency (i.e., the number of EBs per initial cell number) over conventional methods. This could enhance pilot scale cell manufacturing or high-throughput clinical or research operations. However, iPSC stemness can be impacted by the mechanical environment and cell responsiveness to the mechanical environment is known to be altered by ROCKi [6]. Here, we investigate the impact of alginate hydrogel encapsulation and ROCKi treatment on iPSC formation into EBs.


Materials and Methods:: Human iPSCs were grown to 80% confluency using E8 Flex stem cell culture medium. Two hours prior to lifting the cells, a media change was performed with the addition of 10 µM ROCKi. The cells were washed, then lifted using Accutase and incubated for 10 minutes. Dissociated iPSCs were then collected, centrifuged, and resuspended to achieve a suspension of 10⁶ cells/mL in media containing 10 µM ROCKi, and mixed with equal parts 1.2% (w/v) alginate. The solution was loaded into a syringe with a blunt 20G needle and added dropwise into a gelling bath containing 100 mM CaCl₂ for 5-10 minutes to form spherical alginate beads. Beads were then washed and transferred to culture media containing 10 µM ROCKi. The beads were cultured for one day before being split into groups that received either 0, 10, or 20 µM ROCKi over the span of 8 days (Fig. 1). Phase contrast images were collected on Days 2, 4, 6, and 8 for EB size analysis using FIJI. Decapsulated cells were enumerated using a Hoechst 33342 fluorometric assay. Significance (p < 0.05) was determined using a two-way ANOVA with post-hoc Tukey HSD tests.


Results, Conclusions, and Discussions:: Each alginate bead initially contained 1,680 ± 300 dispersed cells on the day of encapsulation (Day -1). By Day 8 of culture, significant differences in cell count were observed between the untreated and ROCKi treated groups: 24,300 ± 1,750 compared to 580,000 ± 30,000 (10 µM) cells per bead, respectively. EBs were observed in all groups by the end of Day 8. Within this same timeframe, the number and size of EBs per bead in ROCKi treated groups increased significantly (p < 0.05) from 0 to 235 ± 65, with each EB having an average diameter of 164 µm ± 8. The 10 and 20 µM ROCKi groups showed no significant difference in EB size or count. The 0 µM ROCKi group contained fewer EBs (37 ± 54 EBs per bead) with smaller diameters (115 µm ± 28.7) (Fig. 2). EB formation efficiency at Day 8 was approximately 3.0 %, 13.6%, and 14.3% for 0, 10, and 20 µM ROCKi, respectively. Thus, alginate encapsulation with ROCKi treatment seems to offer a method for increasing EB formation efficiency. For example, using the same initial number of cells, this method would produce 50 alginate beads and more than 10,000 EBs compared to the maximum of 96 produced in a 96 well plate (one EB per well) and would theoretically have a formation efficiency of 5.7%.


Generating EBs through encapsulation in alginate enabled the mass production of EBs but required the presence of ROCKi. While ROCKi promotes iPSC viability and proliferation, its effect on pluripotency markers is unclear, and other possible downstream effects have not yet been characterized. Consistent sizing of EBs is crucial when differentiating EBs toward specific cell lineages; thus, the use of encapsulation may later require precise sorting of decapsulated EBs to maximize the yield of usable EBs [7]. Regardless, increasing the efficiency of EB generation through encapsulation could lead to enhanced stem cell manufacturing practices for pilot scale research operations. Future work involves utilizing qPCR techniques to determine pluripotency and assess lineage bias towards any of the three germ layers as a function of method parameters.




Acknowledgements (Optional): :

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