Ice-Binding Protein Derived from Glaciozyma Can Improve the Viability of Cryopreserved Mammalian Cells

Abstract

Ice-binding proteins (IBPs) can inhibit ice recrystallization (IR), a major cause of cell death during cryopreservation. IBPs are hypothesized to improve cell viability after cryopreservation by alleviating the cryoinjury caused by IR. In our previous studies, we showed that supplementation of the freezing medium with the recombinant IBP of the Arctic yeast Glaciozyma sp. (designated as LeIBP) could reduce post-thaw hemolysis of human red blood cells and increase the survival of cryopreserved diatoms. Here, we showed that LeIBP could improve the viability of cryopreserved mammalian cells. Human cervical cancer cells (HeLa), mouse fibroblasts (NIH/3T3), human preosteoblasts (MC3T3-E1), Chinese hamster ovary cells (CHO-K1), and human keratinocytes (HaCaT) were evaluated. These mammalian cells were frozen in dimethyl sulfoxide (DMSO)/fetal bovine serum (FBS) solution with or without 0.1 mg/ml LeIBP at a cooling rate of -1°C/min in a -80°C freezer overnight. The minimum effective concentration (0.1 mg/ml) of LeIBP was determined, based on the viability of HeLa cells after treatment with LeIBP during cryopreservation and the IR inhibition assay results. The post-thaw viability of mammalian cells was examined. In all cases, cell viability was significantly enhanced by more than 10% by LeIBP supplementation in 5% DMSO/5% FBS: viability increased by 20% for HeLa cells, 28% for NIH/3T3 cells, 21% for MC3T3-E1, 10% for CHO-K1, and 20% for HaCaT. Furthermore, addition of LeIBP reduced the concentrations of toxic DMSO and FBS down to 5%. Therefore, we demonstrated that LeIBP can increase the viability of cryopreserved mammalian cells by inhibiting IR.

Marine Antifreeze Proteins: Structure, Function, and Application to Cryopreservation as a Potential Cryoprotectant

“…The TH activity of pLeIBP was about 0.42 °C, which was similar to that of LeIBP expressed in E. coli . The availability of large quantities of pLeIBP allowed us to use this protein in further application studies [65,66,67,143,144,145]. …”

“…However, cells are not always viable after thawing [145,159]. The freezing and thawing process during cryopreservation causes cryo-injury to cells (Figure 5).…”

“…This is because type III AFP is easy to produce recombinantly compared with other fish AFPs and because it has been studied longer than other marine-derived AFPs, such as LeIBP and FfIBP. The results listed in Table 1 also showed that hyperactive AFPs do not always ensure better cryopreservation efficiency [65,66,145]. For example, moderately active LeIBP protects mouse ovarian tissue more effectively than 10-fold hyperactive FfIBP [65].…”

“…The AFP concentration used in cryopreservations was also determined empirically (Table 1). The IRI endpoint, sometimes expressed as mg/mL, does not indicate the effective amount of AFP in cryopreservation, and the solubility of AFPs and the molar concentration in the freezing medium should also be considered [145]. Quite frequently, higher concentrations of AFPs lead to a decrease in the post-thaw survival of cryopreserved cells, which may be due to the formation of destructive needle-like ice at high AFP concentrations [65,66,68,69,92,162,163].…”

“…Some of the cell types tested for cryopreservation with the addition of AFPs include sperms [167,168,169,170,171,176,178,200], oocytes [66,70,83,177,181,183,185,190,198,199], human liver cells [173], RIN-5F insulin tumor cells [174], diatoms [143], red blood cells [18,144,197], muscle cells [162,179], gut cell [188], islet cells [193], E. coli [83], and human cell lines [145] including HeLa cells, NIH/3T3 cells, preosteoblasts (MC3T3-E1 cells), and human ketatinocytes (HaCaT cells). Thus, the addition of AFPs seems to mainly enhance the cryopreservation efficiency regardless of cell type and freezing method, with a handful of exceptions [68,89,90,162,164].…”