Antifreeze proteins (AFPs) are biological antifreezes with unique properties, including thermal hysteresis (TH), ice recrystallization inhibition (IRI), and interaction with membranes and/or membrane proteins. These properties have been utilized in the preservation of biological samples at low temperatures. Here, we review the structure and function of marine-derived AFPs, including moderately active fish AFPs and hyperactive polar AFPs. We also survey previous and current reports of cryopreservation using AFPs. Cryopreserved biological samples are relatively diverse ranging from diatoms and reproductive cells to embryos and organs. Cryopreserved biological samples mainly originate from mammals. Most cryopreservation trials using marine-derived AFPs have demonstrated that addition of AFPs can improve post-thaw viability regardless of freezing method (slow-freezing or vitrification), storage temperature, and types of biological sample type.

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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.

A Gram-negative, non-motile, rod-shaped bacterial strain, designated MA1-6 T , was isolated from a sea squirt (Halocynthia roretzi) collected from the South Sea, Korea, and was characterized to determine its taxonomic position. Strain T grew optimally at pH 7.0-8.0, at 30 6C and in the presence of 2-3 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain MA1-6 T fell within the clade comprising Ruegeria species and exhibited 95.3-96.5 % similarity to the type strains of recognized Ruegeria species. Strain MA1-6 T contained Q-10 as the predominant ubiquinone and C 18 : 1 v7c as the major fatty acid, which is consistent with data for Ruegeria species. The major polar lipids detected in strain MA1-6 T andRuegeria atlantica KCTC 12424 T were phosphatidylcholine, phosphatidylglycerol, an unidentified aminolipid and an unidentified lipid. The DNA G+C content of strain MA1-6 T was 58.6 mol%.Differential phenotypic properties, together with phylogenetic distinctiveness, demonstrated that strain MA1-6 T can be distinguished from recognized Ruegeria species. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain MA1-6 T is considered to represent a novel species of the genus Ruegeria, for which the name Ruegeria halocynthiae sp. nov. is proposed; the type strain is MA1-6 T (5KCTC 23463 T 5CCUG 60744 T ).

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We used nine microsatellite DNA markers to estimate genetic variation among wild and cultured populations of the sea squirt Halocynthia roretzi. The loci were polymorphic, with 6-32 alleles, and allelic richness ranged from 6.0 to 26.1 in each population. The wild and the cultured populations had similar mean heterozygosities (H O and H E), allele numbers, and allelic richness. One cultured population with softness syndrome had a lower mean in the observed heterozygosity (H O = 0.57) and higher mean inbreeding coefficient (F IS = 0.261) than any other populations. This suggests that the loss of genetic variation in the diseased population might be due to increased inbreeding. A neighbor-joining tree and pairwise population estimates of F ST showed moderate genetic differentiation between the wild and the cultured populations. Additionally, the softness syndrome population was genetically divergent from wild populations, but it was genetically close to the cultured populations.