of ice recrystallization inhibitors (IRIs). These low molecular mass
(<300 AMUs) IRIs can be added prior to freezing of cells and
tissues to control ice growth and have been shown to increase postthaw viability and functional capacity [9–17]. This is an enabling
technology for many regenerative and cellular therapies, to treat
blood-related disorders, cancers, and various immune disorders
[18]. The IRI technology also has applications in the frozen food
industry where ice recrystallization in frozen foods (meats, ice
cream, doughs) compromises the structural integrity of cellular
membranes [19] decreasing the quality (taste, texture, etc.) and
decreasing nutrients after thawing. Proof of concept for the beneficial effect of inhibiting ice recrystallization in frozen food products
has been previously demonstrated using high molecular mass antifreeze proteins (AFPs) [20–22].
The ability of naturally occurring peptides to inhibit ice growth
was first reported over 50 years ago [23, 24] with the discovery of
antifreeze (glycol)proteins (AF(G)Ps) that were first isolated from
fish in the Antarctic ocean. It is because of these proteins that deepsea teleost fish are capable of surviving in sub-zero temperatures
[23]. Since that time, similar proteins have been reported in plants,
fungi, arthropods, yeast, and bacteria [25–31]. Applications of AF
(G)Ps are extensive due to their antifreeze properties that are
evident at low protein concentrations. However, isolation and purification of these proteins continues to be a challenge and limits their
commercial use. In an effort to address this issue, analogues of AF
(G)Ps have been synthesized with various modifications that maintain varying degrees of antifreeze activity. Reports of synthetic
antifreeze glycoproteins [13, 32–37], small molecule carbohydrates
[9–11, 38–43], polymers [44–49], and polyampholytes [50–52]
are all examples of such analogues.
Naturally occurring AF(G)Ps and many synthetic derivatives
typically exhibit two types of antifreeze activity. The first is thermal
hysteresis (TH) activity [53, 54], and the second is the ability to
inhibit ice recrystallization [34, 55]. Typically, the thermal hysteresis ability is not amenable to cryopreservation applications because
the binding of an AF(G)P to the surface of ice changes the habit of
the ice crystal resulting in extensive cellular damage at temperatures
below the freezing point [53, 56, 57].
In contrast, the ability to inhibit ice recrystallization is a very
desirable property for cryopreservation [58]. To determine the
extent of IRI activity a compound possesses, several assays have
been developed to assess changes in ice crystal size. Examples
include the capillary method [59], the gold nanoparticle assay
[60], high-throughput sapphire slide assay [61], splat cooling
assay (SCA) [62], and the sucrose sandwich assay (SSA)
[63]. These assays involve either observing the difference in morphology of ice crystals in the presence and absence of IRIs or
identifying concentrations at which IRI activity is optimal as well
272
Anna A. Ampaw et al.
(<300 AMUs) IRIs can be added prior to freezing of cells and
tissues to control ice growth and have been shown to increase postthaw viability and functional capacity [9–17]. This is an enabling
technology for many regenerative and cellular therapies, to treat
blood-related disorders, cancers, and various immune disorders
[18]. The IRI technology also has applications in the frozen food
industry where ice recrystallization in frozen foods (meats, ice
cream, doughs) compromises the structural integrity of cellular
membranes [19] decreasing the quality (taste, texture, etc.) and
decreasing nutrients after thawing. Proof of concept for the beneficial effect of inhibiting ice recrystallization in frozen food products
has been previously demonstrated using high molecular mass antifreeze proteins (AFPs) [20–22].
The ability of naturally occurring peptides to inhibit ice growth
was first reported over 50 years ago [23, 24] with the discovery of
antifreeze (glycol)proteins (AF(G)Ps) that were first isolated from
fish in the Antarctic ocean. It is because of these proteins that deepsea teleost fish are capable of surviving in sub-zero temperatures
[23]. Since that time, similar proteins have been reported in plants,
fungi, arthropods, yeast, and bacteria [25–31]. Applications of AF
(G)Ps are extensive due to their antifreeze properties that are
evident at low protein concentrations. However, isolation and purification of these proteins continues to be a challenge and limits their
commercial use. In an effort to address this issue, analogues of AF
(G)Ps have been synthesized with various modifications that maintain varying degrees of antifreeze activity. Reports of synthetic
antifreeze glycoproteins [13, 32–37], small molecule carbohydrates
[9–11, 38–43], polymers [44–49], and polyampholytes [50–52]
are all examples of such analogues.
Naturally occurring AF(G)Ps and many synthetic derivatives
typically exhibit two types of antifreeze activity. The first is thermal
hysteresis (TH) activity [53, 54], and the second is the ability to
inhibit ice recrystallization [34, 55]. Typically, the thermal hysteresis ability is not amenable to cryopreservation applications because
the binding of an AF(G)P to the surface of ice changes the habit of
the ice crystal resulting in extensive cellular damage at temperatures
below the freezing point [53, 56, 57].
In contrast, the ability to inhibit ice recrystallization is a very
desirable property for cryopreservation [58]. To determine the
extent of IRI activity a compound possesses, several assays have
been developed to assess changes in ice crystal size. Examples
include the capillary method [59], the gold nanoparticle assay
[60], high-throughput sapphire slide assay [61], splat cooling
assay (SCA) [62], and the sucrose sandwich assay (SSA)
[63]. These assays involve either observing the difference in morphology of ice crystals in the presence and absence of IRIs or
identifying concentrations at which IRI activity is optimal as well
272
Anna A. Ampaw et al.