應用熱電致冷低溫顯微鏡探討九孔卵及豬卵母細胞之低溫凍結特性

Study on Characteristics of Intracellular Ice Formation Behavior for Haliotis Diversicolor Eggs and Porcine Oocytes by a Thermal Electric Cooled Cryomicroscope

楊江益(Chiang-Yi Yang)

指導教授 ： 林逹德

國立臺灣大學/生物資源暨農學院/生物產業機電工程學研究所/博士(2012年)

https://doi.org/10.6342/NTU.2012.01659

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原泡核期(germinal vesicle，GV)豬卵母細胞低溫凍結特性的研究及分析。熱電致冷低溫顯微鏡性能測試的結果為：在恒溫控制方面，溫度最低可達溫-55degreeC，最高溫可達70degreeC，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70degreeC/min；升溫速率最快為112degreeC/min。實驗所記錄到的溫度值與設定值之均方誤差均在0.4degreeC之內。九孔卵之細胞內凍結 (intracellular ice formation, IIF)實驗結果發現：在二甲基亞碸(dimethyl sulfoxide, DMSO)的濃度為2.0、2.5、3.0和4.0M等以及降溫速率為1.5、3、7和12degreeC/min等範圍內，九孔卵發生細胞內凍結的機率可以獲得有效的抑制。為降低溶液效應(solution toxicity effect, STE)的影響，選擇2.0M DMSO為懸浮溶液，並以1.5degreeC/min之降溫速率對九孔卵進行細胞膜滲透活性的觀察，其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原泡核期(germinal vesicle，GV)豬卵母細胞低溫凍結特性的研究及分析。熱電致冷低溫顯微鏡性能測試的結果為：在恒溫控制方面，溫度最低可達溫-55degreeC，最高溫可達70degreeC，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70degreeC/min；升溫速率最快為112degreeC/min。實驗所記錄到的溫度值與設定值之均方誤差均在0.4degreeC之內。九孔卵之細胞內凍結 (intracellular ice formation, IIF)實驗結果發現：在二甲基亞碸(dimethyl sulfoxide, DMSO)的濃度為2.0、2.5、3.0和4.0M等以及降溫速率為1.5、3、7和12degreeC/min等範圍內，九孔卵發生細胞內凍結的機率可以獲得有效的抑制。為降低溶液效應(solution toxicity effect, STE)的影響，選擇2.0M DMSO為懸浮溶液，並以1.5degreeC/min之降溫速率對九孔卵進行細胞膜滲透活性的觀察，其結果為累積細胞內凍結機率為15.8%，解凍後有48.8%的九孔卵可以恢復其細胞膜的滲透活性，當降溫速率加快，則恢復活性的比例減少。以相同條件進行冷凍保存卵解凍後的受精實驗，正規化存活率為23.7%(發育為浮游)。至於豬卵母細胞，本研究用24、12、6、3、及1.5degreeC/min的降溫速率，成熟培養液(NCSU-23)混合2.0M DMSO為懸浮溶液，從20degreeC等速率降溫至-50degreeC對GV期豬卵母細胞進行IIF實驗，發現各降溫速率對應的累積細胞內凍結機率分別為100%、100%，50.5%、54.3%及58.6%。GV期豬卵母細胞解凍後經44小時的體外成熟培養，以Hoechst 33258染色法進行檢驗，如果發育到第二次減數分裂(metaphase II)期則判定為存活。6、3、及1.5degreeC/min等降溫速率分別對應的正規化存活率為37.7+-4.6%, 45.0+-4.4%以及 45.4+-5.9%。實驗結果顯示傳統的低溫冷凍保存法可以成功地冷凍保存九孔卵及豬卵母細胞，本研究並提出應用電熱致冷低溫顯微鏡探討九孔卵及豬卵母細胞之低溫凍結特性以選取最佳冷凍條件的方法。

關鍵字

細胞內凍結 ； 九孔 ； 豬 ； 冷凍保存 ； TEC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 germinal vesicle (GV) stage porcine oocytes. The TEC cryomicroscope system can achieve accurate control of cooling rate up to 70degreeC/min and heating rate up to 112degreeC/min. The isothermal control can be achieved between 70degreeC and -55degreeC with root-mean-squared-error less than 0.4degreeC. According to the IIF experiments of small abalone eggs, IIF were well suppressed at the cooling rates of 1.5, 3, 7, 12degreeC/min in the presence of 2.0, 2.5, 3.0 and 4.0 M dimethyl sulfoxide (DMSO), respectively. Thus, 2.0 M DMSO was selected to be the suspension solution for considering the solution toxicity effect (STE)，and the eggs were cooled at 1.5 degreeC/min to perform osmotic observation. The results showed 48.8% of small abalone eggs was osmotically active after thawing and higher freezing rate reduced the percentage of osmotically active eggs. By separate experiment of fertility test with 1.5degreeC/min cooling rate in the presence of 2.0 M DMSO as cryopreservation protocol, a normalized hatching rate of 23.7% was obtained. As to IIF experiments of germinal vesicle (GV) stage porcine oocytes , five constant cooling rates of 24, 12, 6, 3 and 1.5degreeC/min were tested in experiments in freezing GV porcine oocytes from 20degreeC to -50degreeC in an NCSU-23 medium plus 2.0 M DMSO. The total cumulative probabilities of IIF temperature distribution were 100%, 100%, 50.0%, 54.3% and 58.6% at cooling rates of 24, 12, 6, 3 and 1.5degreeC/min, respectively. Further experiments were performed to examine the feasibility of using these protocols to cryopreserve GV porcine oocytes. After 44 h of in-vitro maturation in NCSU-23, the survival of thawed oocytes was checked. The survival was defined as porcine oocytes developed from the GV stage to the MII stage by using Hoechst 33258 staining. Normalized survival rates of 37.7+-4.6%, 45.0+-4.4% and 45.4+-5.9% were obtained for GV oocytes frozen at 1.5, 3 and 6degreeC/min, respectively. The experimental results indicate that slow freezing is a feasible approach for cryopreservation of small abalone eggs and GV porcine oocytes when cooling rate is properly selected. This study also demonstrated an efficient approach for investigating optimal cooling rates by assessing the IIF characteristics of small abalone eggs and GV porcine COCs by using a TEC cryomicroscope system.

並列關鍵字

IIF ； Abalone ； Porcine ； Cryopreservation ； TEC cryomicroscope

參考文獻

Chiang, Y. S. 2002. Development and application of a TEC-cryomicroscope. Thesis. National Taiwan University, Taiwan.

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