Presentation Information
[2Ip07]Ethanol/Dry-Ice Rapid Freezing and Freeze-Drying that Suppresses Membrane Damage in Sea Urchin Gonads
*Munetsugu Ban1, Shuji Okura2, Mariko Ishikawa2, Naoji Kageyama2 (1. University of Yamanashi, 2. Pickles Corporation)
Keywords:
Sea urchin,Rapid freezing,Lyophilization
[Objective]Sea urchin gonads are highly susceptible to membrane damage during freezing, often resulting in leakage of internal contents and a substantial loss in quality. We examined whether rapid freezing using ethanol/dry ice for 2–3 seconds combined with freeze-drying could suppress membrane damage. The preservation of membrane structure was evaluated by histological and ultrastructural analyses.
[Methods]Gonads from the purple sea urchin Heliocidaris crassispina were rapidly frozen for 2–3 seconds using ethanol/dry ice. To remove residual ethanol, samples were placed on paper towels and stored at –30 °C for 3 days. Histological sections were prepared using standard paraffin embedding and HE staining. For transmission and scanning electron microscopy (TEM and SEM), samples were processed using conventional fixation, dehydration, and embedding methods, and observed using a TEM (JEM 2100F, JEOL) and an SEM (JSM-6510, JEOL), respectively. Freeze-drying was performed at –40 °C and 3.3 × 10⁻² mbar for 24 hours using a freeze-dryer (FreeZone 6 L, Labconco). The dried samples were stored under desiccation for one month, rehydrated with deionized water, and reanalyzed.
[Results]Histological analyses revealed that ice crystal formation was suppressed in the rapid-freezing group, and membrane damage in the gonads was reduced. Furthermore, rehydrated gonads after freeze-drying retained their membrane structures. Although TEM and SEM analyses showed minor structural disintegration in the gonadal membranes, the sequential treatment of ethanol/dry ice rapid freezing followed by freeze-drying appeared to be a promising method for suppressing membrane damage in sea urchin gonads.
[Methods]Gonads from the purple sea urchin Heliocidaris crassispina were rapidly frozen for 2–3 seconds using ethanol/dry ice. To remove residual ethanol, samples were placed on paper towels and stored at –30 °C for 3 days. Histological sections were prepared using standard paraffin embedding and HE staining. For transmission and scanning electron microscopy (TEM and SEM), samples were processed using conventional fixation, dehydration, and embedding methods, and observed using a TEM (JEM 2100F, JEOL) and an SEM (JSM-6510, JEOL), respectively. Freeze-drying was performed at –40 °C and 3.3 × 10⁻² mbar for 24 hours using a freeze-dryer (FreeZone 6 L, Labconco). The dried samples were stored under desiccation for one month, rehydrated with deionized water, and reanalyzed.
[Results]Histological analyses revealed that ice crystal formation was suppressed in the rapid-freezing group, and membrane damage in the gonads was reduced. Furthermore, rehydrated gonads after freeze-drying retained their membrane structures. Although TEM and SEM analyses showed minor structural disintegration in the gonadal membranes, the sequential treatment of ethanol/dry ice rapid freezing followed by freeze-drying appeared to be a promising method for suppressing membrane damage in sea urchin gonads.
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