低温保护剂添加与去除过程对猪卵母细胞的渗透损伤
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摘要
在卵母细胞低温保存过程中,低温保护剂的添加与去除是必不可少的步骤,但此过程会对细胞造成致命的渗透损伤和毒性损伤。为了研究保护剂添加和去除联合过程对猪MII期细胞的损伤程度,本文设计并制作了一个适合卵母细胞低温保护剂连续添加与去除的微流体装置,分别采用微流控线性法和传统的分步法加载和去除低温保护剂,分析了两种方法对卵母细胞的体积变化以及存活率与发育潜能的影响。结果表明:微流控线性法加载和去除低温保护剂时,卵母细胞的体积变化明显小于分步法;线性法处理后细胞的存活率、卵裂率、囊胚率分别为95.3%、64.4%、19.4%,都显著高于传统的分步法(79.4%、43.6%、9.7%)(P<0.05)。因此,微流体装置用于卵母细胞低温保护剂添加与去除是有效的,能够显著减小细胞的渗透损伤,为卵母细胞低温保存技术提供了新思路。
Oocyte cryopreservation has become a practical tool in assisted reproductive technology and fertility preservation. Cryoprotective agents( CPAs) addition and removal are essential steps during oocytes cryopreservation,however,the processes of CPAs addition and removal may cause fatal osmotic and toxic injuries to oocytes. In order to study the injuries of porcine MII-stage oocytes during CPAs addition and removal combined processes and minimize osmotic and toxic injuries to oocytes,primarily,a microfluidic device for continuous loading and unloading of CPAs was designed and fabricated in this study. CPAs were loaded and unloaded with the microfluidic linear method and the conventional step-wise method,respectively. Then,the cell volume changes and the effects on the survival rate and developmental potential of oocytes were investigated. The results showed that the oocyte volume changes with the microfluidic device were obviously less than step-wise method. The survival,cleavage and blastocyst rate of oocytes were 95. 3%,64. 4%,and 19. 4%,respectively,which were significantly higher than the traditional step-wise method( 79. 4%,43. 6%,and 9. 7%)( P < 0. 05). In conclusion,microfluidic device can be used efficiently to load and unload of CPAs,and significantly reduce the osmotic shock to oocytes,which may open up a new path for oocyte cryopreservation.
Oocyte cryopreservation has become a practical tool in assisted reproductive technology and fertility preservation. Cryoprotective agents( CPAs) addition and removal are essential steps during oocytes cryopreservation,however,the processes of CPAs addition and removal may cause fatal osmotic and toxic injuries to oocytes. In order to study the injuries of porcine MII-stage oocytes during CPAs addition and removal combined processes and minimize osmotic and toxic injuries to oocytes,primarily,a microfluidic device for continuous loading and unloading of CPAs was designed and fabricated in this study. CPAs were loaded and unloaded with the microfluidic linear method and the conventional step-wise method,respectively. Then,the cell volume changes and the effects on the survival rate and developmental potential of oocytes were investigated. The results showed that the oocyte volume changes with the microfluidic device were obviously less than step-wise method. The survival,cleavage and blastocyst rate of oocytes were 95. 3%,64. 4%,and 19. 4%,respectively,which were significantly higher than the traditional step-wise method( 79. 4%,43. 6%,and 9. 7%)( P < 0. 05). In conclusion,microfluidic device can be used efficiently to load and unload of CPAs,and significantly reduce the osmotic shock to oocytes,which may open up a new path for oocyte cryopreservation.
引文
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[10]解政鼎,马学虎,艾丹亭,等.低温保护剂对神经干细胞球添加过程的模拟分析[J].化工学报,2013,64(11):3956-3967.(XIE Zhengding,MA Xuehu,AI Danting,et al.Analysis of cryoprotectant addition for neural stem cell sphere[J].Journal of Chemical Industry and Engineering,2013,64(11):3956-3967.)
[11]Mullen S E,Li M,Li Y,et al.Human oocyte vitrification:the permeability of metaphase II oocytes to water and ethylene glycol and the appliance toward vitrification[J].Fertility and Sterility,2008,89(6):1812-1825.
[12]Heo Y S,Lee H J,Hassell B A,et al.Controlled loading of cryoprotectants(CPAs)to oocyte with linear and com-plex CPA profiles on a microfluidic platform[J].Lab on a Chip,2011,11(20):3530-3537.
[13]Mata C,Longmire E K,Mckenna D H,et al.Experimental study of diffusion-based extraction from a cell suspension[J].Microfluidics and Nanofluidics,2008,5(4):529-540.
[14]Wang X,Al Naib A,Sun D W,et al.Membrane permeability characteristics of bovine oocytes and development of a step-wise cryoprotectant adding and diluting protocol[J].Cryobiology,2010,61(1):58-65.
[15]Wang L,Liu J,Zhou G B,et al.Quantitative investigations on the effects of exposure durations to the combined cryoprotective agents on mouse oocyte vitrification procedures[J].Biology of Reproduction,2011,85(5):884-894.
[16]Herrid M,Billah M,Malo C,et al.Optimization of a vitrification protocol for hatched blastocysts from the dromedary camel(Camelus dromedarius)[J].Theriogenology,2016,85(4):585-590.
[17]Song Y S,Moon S,Hulli L,et al.Microfluidics for cryopreservation[J].Lab on a Chip,2009,9(13):1874-1881.
[18]Guan N,Blomsma S A,van Midwoud P M,et al.Effects of cryoprotectant addition and washout methods on the viability of precision-cut liver slices[J].Cryobiology,2012,65(3):179-187.
[2]Chen C.Pregnancy after human oocyte cryopreservation[J].Lancet(London,England),1986,327(8486):884-886.
[3]Karlsson J M,Szurek E A,Higgins A Z,et al.Optimization of cryoprotectant loading into murine and human oo-cytes[J].Cryobiology,2014,68(1):18-28.
[4]李维杰,周新丽,吕福扣,等.纳米低温保护剂提高卵母细胞玻璃化保存效果的机理初探[J].制冷学报,2014,35(1):114-118.(LI Weijie,ZHOU Xinli,LYU Fukou,et al.Preliminary study on the mechanism of promoting the survival rate of vitrified oocytes with nano-cryoprotectants[J].Journal of Refrigeration,2014,35(1):114-118.)
[5]Kuwayama M.Highly efficient vitrification for cryopreservation of human oocytes and embryos:The Cryotop method[J].Theriogenology,2007,67(1):73-80.
[6]Chian R C,Wang Y,Li Y R.Oocyte vitrification:advances,progress and future goals[J].Journal of Assisted Reproduction and Genetics,2014,31(4):411-420.
[7]李维杰,王利红,刘宝林,等.新型卵母细胞载体降温速率的实验研究[J].制冷学报,2016,37(1):114-118.(LI Weijie,WANG Lihong,LIU Baolin,et al.Experimental study on the cooling rate of a new type of oocyte carriers[J].Journal of Refrigeration.2014,37(1):114-118.)
[8]王欣,华泽钊,刘宝林,等.Me2SO浓度及预处理条件对组织工程化真皮活性的影响[J].上海理工大学学报,2007,29(4):311-314.(WANG Xin,HUA Zezhao,LIU Baolin,et al.Effects of Me2SO and loading conditions on the cell viability of tissue-engineered dermal replacement[J].Journal of University of Shanghai for Science and Technology,2007,29(4):311-314.)
[9]Clark N A,Swain J E.Oocyte cryopreservation:searching for novel improvement strategies[J].Journal of Assisted Reproduction and Genetics,2013,30(7):865-875.
[10]解政鼎,马学虎,艾丹亭,等.低温保护剂对神经干细胞球添加过程的模拟分析[J].化工学报,2013,64(11):3956-3967.(XIE Zhengding,MA Xuehu,AI Danting,et al.Analysis of cryoprotectant addition for neural stem cell sphere[J].Journal of Chemical Industry and Engineering,2013,64(11):3956-3967.)
[11]Mullen S E,Li M,Li Y,et al.Human oocyte vitrification:the permeability of metaphase II oocytes to water and ethylene glycol and the appliance toward vitrification[J].Fertility and Sterility,2008,89(6):1812-1825.
[12]Heo Y S,Lee H J,Hassell B A,et al.Controlled loading of cryoprotectants(CPAs)to oocyte with linear and com-plex CPA profiles on a microfluidic platform[J].Lab on a Chip,2011,11(20):3530-3537.
[13]Mata C,Longmire E K,Mckenna D H,et al.Experimental study of diffusion-based extraction from a cell suspension[J].Microfluidics and Nanofluidics,2008,5(4):529-540.
[14]Wang X,Al Naib A,Sun D W,et al.Membrane permeability characteristics of bovine oocytes and development of a step-wise cryoprotectant adding and diluting protocol[J].Cryobiology,2010,61(1):58-65.
[15]Wang L,Liu J,Zhou G B,et al.Quantitative investigations on the effects of exposure durations to the combined cryoprotective agents on mouse oocyte vitrification procedures[J].Biology of Reproduction,2011,85(5):884-894.
[16]Herrid M,Billah M,Malo C,et al.Optimization of a vitrification protocol for hatched blastocysts from the dromedary camel(Camelus dromedarius)[J].Theriogenology,2016,85(4):585-590.
[17]Song Y S,Moon S,Hulli L,et al.Microfluidics for cryopreservation[J].Lab on a Chip,2009,9(13):1874-1881.
[18]Guan N,Blomsma S A,van Midwoud P M,et al.Effects of cryoprotectant addition and washout methods on the viability of precision-cut liver slices[J].Cryobiology,2012,65(3):179-187.