Abstract
The successful implementation of human embryonic stem cells (hESCs)-based technologies requires the production of relevant numbers of well-characterized cells and their efficient long-term storage. In this study, cells were microencapsulated in alginate to develop an integrated bioprocess for expansion and cryopreservation of pluripotent hESCs. Different three-dimensional (3D) culture strategies were evaluated and compared, specifically, microencapsulation of hESCs as: i) single cells, ii) aggregates and iii) immobilized on microcarriers. In order to establish a scalable bioprocess, hESC-microcapsules were cultured in stirred tank bioreactors.The combination of microencapsulation and microcarrier technology resulted in a highly efficient protocol for the production and storage of pluripotent hESCs. This strategy ensured high expansion ratios (an approximately twenty-fold increase in cell concentration) and high cell recovery yields (>70%) after cryopreservation. When compared with non-encapsulated cells, cell survival post-thawing demonstrated a three-fold improvement without compromising hESC characteristics.Microencapsulation also improved the culture of hESC aggregates by protecting cells from hydrodynamic shear stress, controlling aggregate size and maintaining cell pluripotency for two weeks.This work establishes that microencapsulation technology may prove a powerful tool for integrating the expansion and cryopreservation of pluripotent hESCs. The 3D culture strategy developed herein represents a significant breakthrough towards the implementation of hESCs in clinical and industrial applications.
Highlights
Human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells constitute an exciting emerging field
The percentage of populated microcapsules was very low (,10%, data not shown). These results indicate that the microencapsulation of single cells is not a suitable strategy for expanding hESCs
Efficient culture strategies are urgently needed to accelerate the transition of hESCs to clinical and industrial applications
Summary
Human pluripotent stem cells, including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) constitute an exciting emerging field. The inherent capacity of these cells to grow indefinitely (self-renewal) and to differentiate into any mature cell of the human body (pluripotency) makes them extremely attractive for regenerative medicine, tissue engineering, drug discovery and toxicology [1]. The establishment of effective and robust protocols for large-scale expansion, storage and distribution of hESCs is imperative for the development of high quality therapeutic products and/or functional screening tools. The inadequacy of conventional 2D culture systems in mimicking the in vivo microenvironments of stem cell niches has proven a constant shortcoming in both basic biology and tissue engineering studies [3]. The inherent variability, lack of environmental control and low production yields associated with these culturing approaches are the main drawbacks hampering the development of efficient, scalable and cost-effective stem cell expansion processes (reviewed in [4]). The low cell recovery yields and the high rates of uncontrolled differentiation obtained after cryopreservation [5] limit the use of the 2-D systems in clinical and/or industrial applications
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