應用熱電致冷低溫顯微鏡探討九孔卵與胚胎之細胞內凍結特性

Characterization of Intracellular Ice Formation Behavior of Small Abalone Eggs and Embryos by Using a TEC Cryomicroscope

楊江益(Chiang-Yi Yang) ； 林達德(Ta-Te Lin)

《台灣農學會報》 13卷6期 (2012/12) Pp. 544-563

https://doi.org/10.6730/JAAT.201212_13(6).0002

摘要

本研究之目的為研製溫度控制準確且易操作的熱電致冷低溫顯微鏡系統，並將其應用於九孔卵及胚胎低溫凍結特性的比較分析。熱電致冷低溫顯微鏡性能測試的結果為：在恒溫控制方面，溫度控制範圍為最低可達溫-55℃，最高溫可達70℃，恒溫溫度可在此範圍內任意設定。至於降溫速率方面最快為70℃/min；升溫速率最快為112℃/min。經實驗記錄到的溫度值與設定值之均方誤差在0.4℃之內。以熱電致冷低溫顯微鏡系統觀察九孔卵及胚胎在冷凍保存過程中的細胞內凍結現象（intracellular ice formation, IIF），實驗資料顯示在冷凍過程中，九孔卵及胚胎的細胞內凍結現象、發生凍結的時間、溫度及細胞內凍結累機機率等都可以被記錄及計算。在以2.0M二甲基亞為懸浮液，由20℃等速率降溫（不植冰）至-50℃的細胞內凍結觀察實驗中發現：九孔卵在降溫速率為24℃/min時，其細胞內凍結累積機率為100%，較低之降溫速率則開始有九孔卵未發生細胞內凍結；而九孔胚胎則為在降溫速率為6℃/min時，其細胞內凍結累積機率為100%，當降溫速率低於6℃/min時，則開始有胚胎未發生細胞內凍結。以九孔卵與胚胎之細胞內凍結觀察實驗結果，對九孔卵及胚胎進行冷凍保存的觀察及存活率分析，由20℃以1.5℃/min等速率自然降溫至-50℃後，維持在-50℃恒溫10 min，再以112℃/min回溫至28℃維持10min之程序，實驗結果顯示九孔卵的累積細胞內凍結機率為15.8%，解凍後的存活率為23.7%；九孔胚胎的累積細胞內凍結機率為40.5%，解凍後的存活率為26.8%，此結果顯示以此冷凍保存條件保存九孔卵或胚胎均為可行，且存活率在統計理論上並無顯著差異 (P ＞ 0.05)。此外，細胞內凍結累積機率分佈的統計分析結果，均符合韋伯分佈，韋伯分佈的統計資訊亦顯示九孔胚胎較卵容易發生細胞內凍結且變異性較大。

關鍵字

細胞內凍結 ； 九孔 ； 冷凍保存 ； 熱電致冷 ； 低溫顯微鏡

並列摘要

This study aimed to develop a cryomicroscope system based on thermoelectric cooling (TEC) and to use the TEC cryomicroscope system to investigate intracellular ice formation (IIF) behaviors of small abalone eggs and embryos. Compare with conventional cryomicroscope systems which use liquid nitrogen as the coolant, the TEC cryomicroscope system proposed here has the advantage of low cost and ease of operation. The system can achieve accurate control of the cooling rate up to 70℃/min and the heating rate up to 112℃/min. The isothermal control can be achieved between 70℃ and -55℃ with a root-mean-squared-error of less than 0.5℃. Experimental results showed that all the IIF temperatures of each individual oocyte or embryo could be recorded clearly and cumulative IIF probabilities could be calculated using the developed TEC cryomicroscope system. Experiments were performed to cool small abalone eggs and embryos from 20℃ to -50℃ at various cooling rates. The cumulative IIF probabilities of small abalone eggs were 100% when the cooling rates were higher than 24℃ /min. For small abalone embryos, cooling rates higher than 6℃/min resulted in 100% cumulative IIF probability. On the other hand, some eggs and embryos did not experience IIF with cooling rates lower than 24 ℃ /min and 6℃ /min for eggs and embryos, respectively. Further experiments were carried out to investigate the relationship between IIF behavior and survival after freezing at lower cooling rates. The results revealed that the total cumulative probability of IIF for eggs was 15.8% and the survival of eggs was 23.7% using a cooling rate of 1.5℃ /min. The total cumulative probability of IIF for embryos was 40.5% and the survival of embryos was 26.8%. There is no significant difference between the survival rates of eggs and embryos. Our results indicate that cooling small abalone eggs and embryos from 20℃ to -50℃ at 1.5℃/min in the presence of 2.0M DMSO is a feasible protocol for their cryopreservation. Furthermore, all the plots of cumulative probabilities of IIF temperature distribution showed a good agreement with Weibull distribution. Statistical information about Weibull parameters revealed that it was easier for embryos to form intracellular ice than it was for eggs during freezing when subject to the same cooling conditions.

並列關鍵字

intracellular ice formation ； small abalone ； cryopreservation ； thermal electric cooling ； cryomicroscopea