Antifreeze Protein Prolongs the Life-Time of Insulinoma Cells during Hypothermic Preservation

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.

Effects of Three Different Types of Antifreeze Proteins on Mouse Ovarian Tissue Cryopreservation and Transplantation

“…It is believed that many freeze-tolerant organisms inhabiting cold environments have developed AFPs to ensure their survival [ 30 ]. Because AFPs are effective at inhibiting IR, they are beneficial for the cryopreservation of cells and tissues [ 20 , 31 – 33 ]. The current study compared the cryoprotective efficacy of three types of recombinant AFPs in the vitrification of mouse OT.…”

“…Although these IBPs have different TH activities, and they all inhibit IR, which cannot be quantitated. A variety of studies have reported diverse effects by AFPs on the hypothermal preservation of an insulinoma cell line [ 20 ], Escherichia coli [ 21 ], red blood cells [ 11 ], bovine sperm [ 22 ], and mouse oocytes [ 23 ]. Through these previous studies, we could assume that AFPs also have preventive effects of cryodamage even in the cryopreservation of OT.…”

Ice Crystal Growth in Sucrose Solutions Containing Kappa‐ and Iota‐Carrageenans

“…AFP I is a low-molecular-weight protein (< 30 kDa) obtained from the barfin plaice (bp), also called the native bpAFP [19] with a molecular weight of 3.5 kDa [20]. AFGP was provided by Nichirei Co. Ltd. as a purified sample of AFGP from Eleginus gracilis [21] with an assumed molecular weight of 15 kDa [20]. The TH activity of AFP I was stated in 0.1 M ammonium bicarbonate (pH 7.9) at a concentration of 10 mg mL -1 .…”

Microfluidic Cold-Finger Device for the Investigation of Ice-Binding Proteins

“…The ability of IBPs to prevent freezing and freeze injury in nature has current and potential uses. These include applications in the food industry (15); cryopreserva-tion and chill-storage of cells (16)(17)(18), tissues, and organs for transplantation (19); cryosurgery; preventing frost damage to plants (20); and anti-icing (21).…”