Predehydration and Ice Seeding in the Presence of Trehalose Enable Cell Cryopreservation

Abstract

Conventional approaches for cell cryopreservation require the use of toxic membrane-penetrating cryoprotective agents (pCPA), which limits the clinical application of cryopreserved cells. Here, we show intentionally induced ice formation at a high subzero temperature (> −10 °C) during cryopreservation, which is often referred to as ice seeding, could result in significant cell injury in the absence of any pCPA. This issue can be mitigated by predehydrating cells using extracellular trehalose to their minimal volume with minimized osmotically active water before ice seeding. We further observe that ice seeding can minimize the interfacial free energy that drives the devastating ice recrystallization-induced cell injury during warming cryopreserved samples. Indeed, by combining predehydration using extracellular trehalose with ice seeding at high subzero temperatures, high cell viability or recovery is achieved for fibroblasts, adult stem cells, and red blood cells after cryopreservation without using any pCPA. The pCPA-free technology developed in this study may greatly facilitate the long-term storage and ready availability of living cells, tissues, and organs that are of high demand by modern cell-based medicine.

Advances in present‐day frozen dough technology and its improver and novel biotech ingredients development trends—A review

“…In a recent study by Huang et al (2017), combined treatment of predehydration using extracellular trehalose and ice seeding at high subzero temperature resulted in high cell viability of fibroblasts, adult stem cells, and red blood cells. Ice seeding minimized free energy that drives ice recrystallization-induced cell injury during thawing of cryopreserved cells (Huang et al, 2017).…”

“…In a recent study by Huang et al (2017), combined treatment of predehydration using extracellular trehalose and ice seeding at high subzero temperature resulted in high cell viability of fibroblasts, adult stem cells, and red blood cells. Ice seeding minimized free energy that drives ice recrystallization-induced cell injury during thawing of cryopreserved cells (Huang et al, 2017). In yeast (S. cerevisiae), ice-seeding temperatures enhance growth and survival in the log phase of growth during freezing process; in prolonged storage, this was more important than the presence of trehalose (Nakamura et al, 2009).…”

Developmental competence of in vitro matured ovine oocytes vitrified in solutions with different concentrations of trehalose

“…Aqueous solution of trehalose exhibits the highest glass transition temperature (Tg) among other sugars (Chen, Fowler, & Toner, 2000). Trehalose is also believed to decrease potential intracellular ice formation by increasing the viscosity of cytoplasm (Jain & Roy, 2009), through enhancing oocyte's dehydration by osmotic pressure (Huang et al, 2017;Wright et al, 2004). Trehalose has been previously used for cryopreservation of oocyte and embryo in different domestic species, such as porcine (Somfai et al, 2015), goat (Begin, Bhatia, Baldassarre, Dinnyes, & Keefer, 2003), buffalo (Abd-Allah, 2009) and bovine (Park & Yu, 2008).…”

The Use of Chitooligosaccharides in Cryopreservation: Discussion of Concept and First Answers from DSC Thermal Analysis

“…However, the beneficial use of saccharides to substitute penetrating CPA compounds has been proven in recent years. Indeed, saccharides have been used for cell cryopreservation in slow-freezing [37][38][39][40][41][42][43][44][45][46][47][48][49][50][51] or vitrification procedures [16-18, 39, 52-55]. A nonexhaustive overview from the literature of the use of the saccharides is displayed in Table 1.…”

“…For the cryopreservation of cat embryos, Gómez et al [67] were able to obtain interesting survival rates (>80%) by combining propylene glycol (1.4 M), sucrose (0.125 M), and dextran 10% (w/w). Slow-freezing procedures for some weak cellular systems have been proposed, without recourse to CPA penetrants [43]. This strategy is based on the induction, prior to ice formation, of cellular dehydration using osmotically active extracellular compounds (e.g., trehalose).…”