Ice Recrystallization Inhibition Is Insufficient to Explain Cryopreservation Abilities of Antifreeze Proteins

Abstract

Antifreeze proteins (AFPs) and glycoproteins (AFGPs) are exemplary at modifying ice crystal growth and at inhibiting ice recrystallization (IRI) in frozen solutions. These properties make them highly attractive for cold storage and cryopreservation applications of biological tissue, food, and other water-based materials. The specific requirements for optimal cryostorage remain unknown, but high IRI activity has been proposed to be crucial. Here, we show that high IRI activity alone is insufficient to explain the beneficial effects of AF(G)Ps on human red blood cell (hRBC) survival. We show that AF(G)Ps with different IRI activities cause similar cell recoveries of hRBCs and that a modified AFGP variant with decreased IRI activity shows increased cell recovery. The AFGP variant was found to have enhanced interactions with a hRBC model membrane, indicating that the capability to stabilize cell membranes is another important factor for increasing the survival of cells after cryostorage. This information should be considered when designing novel synthetic cryoprotectants.

Figure 1

Cryopreservation of hRBC in the presence of AFGP_1–5. (a) Recovery of hRBC cryopreserved in PBS solutions with different concentrations of AFGP_1–5 thawed at 45 °C. (b) Recovery of hRBC cryopreserved in HES solutions (130 mg/mL) with different AFGP_1–5 concentrations thawed at 23 °C. Experiments were performed three times, and the error bars represent the standard deviation between the individual measurements.

Figure 2

(a) IRI inhibition activity of AFGP_1–5 and the different variants. The IRI activity of AFGP_1–5-ald, AFGP_1–5-car, and AFGP_1–5-ipp at 2 μg/mL is reduced by ∼13, 50, and 63%, respectively. Reprinted with permission from ref (37). Copyright 2021 ACS Publications. (b) Effects of AFGP_1–5 and different variants on the cell recovery of cryopreserved hRBCs in HES solution (130 mg/mL), thawed at 23 °C. Experiments were performed three times, and the error bars represent the standard deviation between the individual measurements.

Figure 3

Effect of different AF(G)Ps and variants on the cell recovery of cryopreserved hRBC in HES solution (130 mg/mL), thawed at 23 °C. Experiments were performed three times, and the error bars represent the standard deviation between the individual measurements.

Figure 4

Interactions of AFGP_1–5 with erythrocyte model membranes. (a) Difference in surface pressure Δπ versus time after the addition of AFGP_1–5-ipp and AFGP_1–5 into the PBS subphase of the DOPC/SM/DOPE monolayer. Δπ represents the change in the surface pressure after the addition of AFGP_1–5 (5 mg/mL) and of PBS buffer. (b) Schematic representation of AFGP_1–5-ipp and AFGP_1–5 proteins interacting with an RBC model membrane.