Microencapsulation Technology: A Powerful Tool for Integrating Expansion and Cryopreservation of Human Embryonic Stem Cells

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.

A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation

“…Several hydrogel materials-such as alginate (24,27), agarose (28), and hyaluronic acid (29)-have been previously investigated for hPSC expansion or differentiation. However, to date thermoreversible materials have not been studied in hPSC culture, despite the fact that they offer a number of promising features for GMP-compatible, large-scale culture.…”

“…An attractive approach for scaling up production is to move cell culture from 2D to 3D (9,17), and accordingly several 3D suspension systems have been probed for hPSCs production: cell aggregates (18)(19)(20)(21), cells on microcarriers (22,23), and cells in alginate microencapsulates (24) (SI Appendix, Table S1). Although these approaches have some attractive aspects, they also highlight significant challenges for 3D hPSC culture (9) (SI Appendix, Table S1) including the following: (i) the use of components from human or animal tissue (e.g., Matrigel, serum, and/ or albumin), which limit reproducibility and/or scalability, pose risks for pathogen and immunogen transfer (18)(19)(20)(21)(22)(23)(24), and are thus problematic for good manufacturing practice (GMP) cell production (25); (ii) substantial cell agglomeration that can in some cases lead to differentiation and/or death (22,23); (iii) shear forces in agitated cultures that can compromise cell viability (18)(19)(20)(21)(22)(23); (iv) limited cell expansion rates and cell yields per volume (18)(19)(20)(21)(22)(23)(24); and (v) unclear potential for long-term serial expansion. As an example, in a recent culture of hPSCs within alginate hydrogel microspheres in mouse embryonic fibroblast conditioned medium, 5% of the encapsulated single hPSCs remained viable after 7 d, and an ∼10-to 20-fold expansion to a peak density of 3 × 10 6 cells per mL occurred after 20 d (24).…”

Robust Expansion of Human Pluripotent Stem Cells: Integration of Bioprocess Design With Transcriptomic and Metabolomic Characterization

“…CRL-2429, ATCC collection,), inactivated with mitomycin C (Sigma-Aldrich, Steinheim, Germany, https://www.sigmaaldrich.com), in knockout Dulbecco's modified Eagle's medium (DMEM) culture medium (knockout DMEM supplemented with 20% [vol/vol] knockout serum replacement, 1% [vol/vol] minimum essential medium nonessential amino acids, 0.1 mM 2-mercaptoethanol, 2 mM GlutaMAX, 1% [vol/vol] penicillin/streptomycin, 0.5% [vol/vol] gentamycin; all from Life Technologies, Paisley, UK, https://www.lifetechnologies.com) and 10 ng/ml basic fibroblast growth factor (Peprotech, Neuilly-Sur-Seine, France, https://www.peprotech.com), as reported previously [19]. hESC-C propagation was performed as described by Serra et al [20]. Extracellular components were also evaluated for the expansion of hESC-C: Matrigel Technologies) and MEF-CM [7], both supplemented with 10 mM Rock inhibitor (Calbiochem; EMD Millipore, Billerica, MA, http://www.emdmillipore.…”

Applications and optimization of cryopreservation technologies to cellular therapeutics

“…Similar to the production process, recovery could be achieved by transferring these cell constructs into a bioreactor which provides a dynamic environment aiming to mimic the perfusion condition which cells are subjected to in vivo, with more effective mass transfer properties [75]. A number of different types of bioreactors exist (fluidised bed, rotary cell culture system) that can optimally support cell recovery and metabolism whilst minimising cell damage [76][77][78][79]. Such systems can be readily scaled-up for fast recovery of large volumes of cryopreserved cell therapies.…”