Abstract
It is sometimes desirable to preserve mammalian cells by hypothermia rather than freezing during short term transplantation. Here we found an ability of hypothermic (+4°C) preservation of fish antifreeze protein (AFP) against rat insulinoma cells denoted as RIN-5F. The preservation ability was compared between type I–III AFPs and antifreeze glycoprotein (AFGP), which could be recently mass-prepared by a developed technique utilizing the muscle homogenates, but not the blood serum, of cold-adapted fishes. For AFGP, whose molecular weight is distributed in the range from 2.6 to 34 kDa, only the proteins less than 10 kDa were examined. The viability rate was evaluated by counting of the preserved RIN-5F cells unstained with trypan blue. Significantly, either AFPI or AFPIII dissolved into Euro-Collins (EC) solution at a concentration of 10 mg/ml could preserve approximately 60% of the cells for 5 days at +4°C. The 5-day preserved RIN-5F cells retained the ability to secrete insulin. Only 2% of the cells were, however, preserved for 5 days without AFP. Confocal photomicroscopy experiments further showed the significant binding ability of AFP to the cell surface. These results suggest that fish AFP enables 5-day quality storage of the insulinoma cells collected from a donor without freezing.
Highlights
Cells obtained by either cultivation or extraction from human or animal tissues are used in the fields of regenerative medicine and livestock farming [1]
We evaluated a ratio of the secreted amount of insulin after the 120 hrs of preservation versus the amount measured before the preservation, which was examined as another survival rate
Purity of the AFPI –III and antifreeze glycoprotein (AFGP) samples provided from Nichirei Foods Inc. was confirmed by SDS-PAGE (Figure 2)
Summary
Cells obtained by either cultivation or extraction from human or animal tissues are used in the fields of regenerative medicine and livestock farming [1]. For short-term transplantations occurring within 1–5 days, it is desirable to preserve the cells without freezing. We generally handled such cells as assemblies of several to millions, and the percentage of viable cells in the total number (i.e., survival rate) is generally improved by soaking them in a preservation solution comprising inorganic salts, glycerol, sugars, etc., such as lactated Ringer [2], Euro-Collins (EC) [3], and the University of Wisconsin (UW) solutions [4]. The above solutions were initially developed to preserve specific organs, but are used for virtually every type of cell and organ. We examined whether the performance of a cellpreservation solution is improved by addition of fish antifreeze protein (AFP), for which a general cell-membrane protection ability has been recognized in the last two decades [7]
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