Inhibition of ice recrystallization and cryoprotective activity of wheat proteins in liver and pancreatic cells

Abstract

Efficient cryopreservation of cells at ultralow temperatures requires the use of substances that help maintain viability and metabolic functions post-thaw. We are developing new technology where plant proteins are used to substitute the commonly-used, but relatively toxic chemical dimethyl sulfoxide. Recombinant forms of four structurally diverse wheat proteins, TaIRI-2 (ice recrystallization inhibition), TaBAS1 (2-Cys peroxiredoxin), WCS120 (dehydrin), and TaENO (enolase) can efficiently cryopreserve hepatocytes and insulin-secreting INS832/13 cells. This study shows that TaIRI-2 and TaENO are internalized during the freeze-thaw process, while TaBAS1 and WCS120 remain at the extracellular level. Possible antifreeze activity of the four proteins was assessed. The "splat cooling" method for quantifying ice recrystallization inhibition activity (a property that characterizes antifreeze proteins) revealed that TaIRI-2 and TaENO are more potent than TaBAS1 and WCS120. Because of their ability to inhibit ice recrystallization, the wheat recombinant proteins TaIRI-2 and TaENO are promising candidates and could prove useful to improve cryopreservation protocols for hepatocytes and insulin-secreting cells, and possibly other cell types. TaENO does not have typical ice-binding domains, and the TargetFreeze tool did not predict an antifreeze capacity, suggesting the existence of nontypical antifreeze domains. The fact that TaBAS1 is an efficient cryoprotectant but does not show antifreeze activity indicates a different mechanism of action. The cryoprotective properties conferred by WCS120 depend on biochemical properties that remain to be determined. Overall, our results show that the proteins' efficiencies vary between cell types, and confirm that a combination of different protection mechanisms is needed to successfully cryopreserve mammalian cells.

Keywords: cryopreservation; ice recrystallization inhibition; mammalian cell; plant protein.

Figure 1

Internalization study of recombinant wheat proteins in hepatocytes. Confocal fluorescence microscopy images of hepatocytes after 30 min of incubation with Oregon green‐labeled recombinant wheat proteins TaIRI‐2, TaBAS1, WCS120, and TaENO (left panels). Lysosomes were stained with LysoTracker Red (middle panels). Merged images of labeled proteins and lysosomes are shown (right panels). Representative images (200×) are from at least three different preparations of proteins tested on five independent cell preparations.

Figure 2

Internalization study of recombinant wheat proteins in INS832/13 cells. Confocal microscopy images of INS832/13 cells after 30 min of incubation with Oregon green‐labeled recombinant proteins (left panels) and LysoTracker Red (middle panels). Merged images of labeled proteins and lysosomes are shown (right panels). Representative images (400×) are from at least three different preparations of proteins tested on five independent cell preparations.

Figure 3

Internalization study of recombinant wheat proteins in hepatocytes after thawing. Hepatocytes were incubated with LysoTracker Red and Oregon green‐labeled recombinant wheat proteins TaIRI‐2, TaBAS1, WCS120, and TaENO, and then were frozen. After thawing, lysosomes (middle panels) and fluorescent proteins (left panels) were detected by confocal microscopy. Merged images are shown (right panels). Representative images (200×) are from at least three different preparations of proteins tested on five independent cell preparations.

Figure 4

Internalization study of recombinant wheat proteins in INS832/13 cells after thawing. INS832/13 cells were incubated with LysoTracker Red and Oregon green‐labeled recombinant wheat proteins TaIRI‐2, TaBAS1, WCS120, and TaENO, and then were frozen. After thawing, lysosomes (middle panels) and fluorescent proteins (left panels) were detected by confocal fluorescence microscopy. Merged images are shown (right panels). Representative images (400×) are from at least three different preparations of proteins tested on five independent cell preparations.

Figure 5

Wheat proteins modify ice recrystallization. Light microscope images of ice crystals in the presence of different wheat proteins prepared in PBS following splat‐cooling assay. (A) PBS control solution, (B) WPE—3 mg/mL, (C) TaIRI‐2—3 mg/mL, (D) TaIRI‐2—20 mg/mL, (E) TaBAS1—3 mg/mL, (F) TaBAS1—10 mg/mL, (G) TaBAS1—127 mg/mL, (H) WCS120—3 mg/mL, (I) WCS120—18 mg/mL, (J) TaENO—3 mg/mL, (K) TaENO—100 mg/mL, (L) TaENO—303 mg/mL. Representative images are from three independent experiments.

Figure 6

IRI activity of wheat proteins. The y axis represents the percent mean grain size (% MGS) of ice crystals formed in the presence of proteins dissolved in PBS at the indicated concentrations, relative to the crystals formed in PBS alone. Data represent means ± SEM from three independent experiments. The % MGS for all protein concentrations is significantly different (P < 0.05) relative to PBS.

Figure 7

The wheat proteins do not possess thermal hysteresis capacity. Ice crystal morphologies of water in the presence of (A) WPE—2.5 mg/mL, (B) TaIRI‐2—3.3 mg/mL, (C) TaBAS1—10.0 mg/mL, (D) WCS120–10.0 mg/mL, or (E) TaENO—10.0 mg/mL.

Figure 8

Viability of rat hepatocytes following cryopreservation with different wheat proteins. Hepatocytes were cryopreserved for 7 days with different quantities of (A) wheat protein extract (WPE), or the recombinant proteins (B) TaIRI‐2, (C) TaBAS1, (D) WCS120, or (E) TaENO. Fresh cells served as controls and DMSO (15% + 50% fetal bovine serum (FBS)) as a reference cryoprotectant. Data for post‐thaw viability represent means ± SEM of at least three different preparations of proteins tested on three independent cell preparations. *(P < 0.05), **(P < 0.01), ***(P < 0.005): significant difference between cryopreserved cells, and fresh cell controls. ^▲(P < 0.05), ^▲▲(P < 0.01), ^▲▲▲(P < 0.005): significant difference between cells cryopreserved with protein versus DMSO.

Figure 9

Viability of INS832/13 cells after cryopreservation with wheat proteins. INS832/13 cells were cryopreserved for 7 days with different quantities of (A) wheat protein extract (WPE), (B) TaIRI‐2, (C) TaBAS1, (D) WCS120, or (E) TaENO. Fresh cells served as controls and DMSO (10% + 50% FBS) as a reference cryoprotectant. Data for post‐thaw viability represent means ± SEM of at least three different preparations of proteins tested on three independent cell preparations *(P < 0.05), **(P < 0.01), ***(P < 0.005): significant difference between cryopreserved cells and fresh cells. ^▲(P < 0.05), ^▲▲(P < 0.01), ^▲▲▲(P < 0.005): significant difference between cells cryopreserved with protein versus DMSO.