Alginate Hydrogel Microencapsulation Inhibits Devitrification and Enables Large‐Volume Low‐CPA Cell Vitrification

Abstract

Cryopreservation of stem cells is important to meet their ever-increasing demand by the burgeoning cell-based medicine. The conventional slow freezing for stem cell cryopreservation suffers from inevitable cell injury associated with ice formation and the vitrification (i.e., no visible ice formation) approach is emerging as a new strategy for cell cryopreservation. A major challenge to cell vitrification is intracellular ice formation (IIF, a lethal event to cells) induced by devitrification (i.e., formation of visible ice in previously vitrified solution) during warming the vitrified cells at cryogenic temperature back to super-zero temperatures. Consequently, high and toxic concentrations of penetrating cryoprotectants (i.e., high CPAs, up to ~8 M) and/or limited sample volumes (up to ~2.5 μl) have been used to minimize IIF during vitrification. We reveal that alginate hydrogel microencapsulation can effectively inhibit devitrification during warming. Our data show that if ice formation were minimized during cooling, IIF is negligible in alginate hydrogel-microencapsulated cells during the entire cooling and warming procedure of vitrification. This enables vitrification of pluripotent and multipotent stem cells with up to ~4 times lower concentration of penetrating CPAs (up to 2 M, low CPA) in up to ~100 times larger sample volume (up to ~250 μl, large volume).

Microfluidics for cryopreservation

“…For vitrification or low-CPA vitrification, cooling process is very simple compared to programmable slow freezing because the sample is directly immersed in liquid nitrogen for the fastest cooling available (Fahy, 1986b; Fahy et al, 1984; Huang et al, 2015; Wang et al, 2016) (Figure 1D (a)). Because no ice forms in either extra- or intracellular solution, no water or CPA transport across the cell membrane during freezing/thawing occurs (Fahy and Wowk, 2015) (Figure 1D (b).…”

“…Its extension form was further successfully used for the design of CPA equilibration for adherent endothelial cells (Davidson et al, 2015). Compared with programmed slow freezing, vitrification/low-CPA vitrification requires comparatively more CPA; therefore CPA addition and removal processes are more time consuming and costly (Choi et al, 2015a; Choi et al, 2015b; Huang et al, 2015; Wang et al, 2016). …”

“…Furthermore, preventing cytotoxicity from high CPA concentrations required to decrease critical cooling rates necessary for vitreous cryopreservation is of importance (Fahy and Wowk, 2015). Unprecedented successes in cell cooling and warming have been accomplished with emerging novel microfluidic platforms which have intrinsic advantages, especially for cell-specific manipulation and strong controllability for rapid thermal responses (Huang et al, 2015; Lai et al, 2015a; Pyne et al, 2014; Vom et al, 2013; Zhou et al, 2013; Zhou et al, 2015). …”

“…Cryopreservation of limited cells such as oocytes, sperm (azoospermia; severe oligozoospermia), and spermatogonic stem cells remains a challenge for traditional methods but cooling and warming on a chip may address novel needs. Also, microfluidics can be used for microscale encapsulation of cells, which allows cell vitrification with low CPA concentration (Huang et al, 2015; Zhang et al, 2013; Zhao, S.T. et al, 2014).…”

Applications and optimization of cryopreservation technologies to cellular therapeutics

“…Several studies have shown promising protective effects of biomaterials on cell cryopreservation [80]. In a more mechanistic study, alginate hydrogel was shown to inhibit devitrification during the warming process of vitrified stem cells [81]. Altogether, these beneficial properties make encapsulation an encouraging strategy for cell therapy and the cryopreservation process.…”