微流控芯片用于卵母细胞冷冻保存的实验研究
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摘要
低温保存对卵母细胞造成渗透损伤、毒性损伤和冰晶损伤,使得细胞冻后质量难以提高.本文首次提出将微流控法添加-去除保护剂分别与三种冷冻载体(OPS、QC及Cryotop)搭配使用,对猪卵母细胞进行冷冻保存,并与传统冷冻法进行比较;然后,首次选用透明陶瓷和玻璃制作集成一体化芯片,对猪卵母细胞进行冷冻保存,以冷冻保存后的细胞存活率和发育率为判断依据,筛选出较好的方案;最后,对冻后卵母细胞的早期凋亡情况、胞内活性氧水平和线粒体膜电位水平进行分析.结果表明,微流控添加-去除保护剂组卵母细胞冻后存活率以及卵裂率都显著高于传统冷冻组,可以有效降低卵母细胞的早期凋亡率和胞内活性氧水平,减小线粒体损伤,提高细胞的冻后质量.透明陶瓷一体化芯片保存卵母细胞得到的存活率和卵裂率与传统OPS冷冻的保存结果无显著差异.微流控芯片技术为卵母细胞的低温保存提供新的思路,有较好的应用前景.
Cryopreservation will cause osmotic damage, toxic damage and ice crystal damage to oocytes, thus it is difficult to improve the quality of the frozen oocytes. In this paper, microfluidic and stepwise cryoprotectant(CPA)loading-unloading protocols were combined with three kinds of cryopreservation devices(OPS, QC and Cryotop)to cryopreserve the porcine oocytes for the first time. Moreover, PDMS-transparent ceramic and PDMS-glass integration microfluidic chips were employed for oocytes cryopreservation for the first time, the survival rate and development rate of cryopreserved oocytes were used to evaluate optimal cryopreservation protocol. Finally, early apoptosis rate, intracellular reactive oxygen species(ROS) and mitochondrial membrane potential of oocytes were analyzed. The results showed that the survival rate and cleavage rate of frozen oocytes by microfluidic loading-unloading of CPAs were significantly higher than traditional loading-unloading group. Microfluidic method could reduce the early apoptosis rate, intracellular ROS and mitochondrial damage, improve the quality of frozen oocytes. The survival rate and cleavage rate of oocytes cryopreserved by PDMS-transparent ceramic integrated chip were not significantly different from traditional OPS method. Microfluidic chip technology provides a new idea for oocytes cryopreservation and has good application prospects.
Cryopreservation will cause osmotic damage, toxic damage and ice crystal damage to oocytes, thus it is difficult to improve the quality of the frozen oocytes. In this paper, microfluidic and stepwise cryoprotectant(CPA)loading-unloading protocols were combined with three kinds of cryopreservation devices(OPS, QC and Cryotop)to cryopreserve the porcine oocytes for the first time. Moreover, PDMS-transparent ceramic and PDMS-glass integration microfluidic chips were employed for oocytes cryopreservation for the first time, the survival rate and development rate of cryopreserved oocytes were used to evaluate optimal cryopreservation protocol. Finally, early apoptosis rate, intracellular reactive oxygen species(ROS) and mitochondrial membrane potential of oocytes were analyzed. The results showed that the survival rate and cleavage rate of frozen oocytes by microfluidic loading-unloading of CPAs were significantly higher than traditional loading-unloading group. Microfluidic method could reduce the early apoptosis rate, intracellular ROS and mitochondrial damage, improve the quality of frozen oocytes. The survival rate and cleavage rate of oocytes cryopreserved by PDMS-transparent ceramic integrated chip were not significantly different from traditional OPS method. Microfluidic chip technology provides a new idea for oocytes cryopreservation and has good application prospects.
引文
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[3]Shi W Q,Zhu S E,Zhang D,et al.Improved development by Taxol pretreatment after vitrification of in vitro matured porcine oocytes.Reproduction,2006,131(4):795-804
[4]Rojas C,Palomo M J,Albarrac觔N J L,et al.Vitrification of immature and in vitro matured pig oocytes:study of distribution of chromosomes,microtubules,and actin microfilaments.Cryobiology,2004,49(3):211-220
[5]Fujihira T,Kishida R,Fukui Y.Developmental capacity of vitrified immature porcine oocytes following ICSI:effects of cytochalasin B and cryoprotectants.Cryobiology,2004,49(3):286-290
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[7]Galeati G,Spinaci M,Vallorani C,et al.Pig oocyte vitrification by cryotop method:effects on viability,spindle and chromosome configuration and in vitro fertilization.Animal Reproduction Science,2011,127(1-2):43-49
[8]Kuwayama M,Vajta G,Kato O,et al.Highly efficient vitrification method for cryopreservation of human oocytes.Reproductive Biomedicine Online,2005,11(3):300-308
[9]Kuwayama M.Highly efficient vitrification for cryopreservation of human oocytes and embryos:the Cryotop method.Theriogenology,2007,67(1):73-80
[10]Song Y S,Moon S J,Hulli L,et al.Microfluidics for cryopreservation.Lab on A Chip,2009,9(13):1874-1881
[11]杨云,周新丽,戴建军,等.微流控线性加载低温保护剂减少猪MⅡ期卵母细胞的渗透损伤.生物化学与生物物理进展,2016,43(6):616-623Yang Y,Zhou X L,Dai J J,et al.Progress in Biochemistry and Biophysics,2016,43(6):616-623
[12]衣星越.微流控装置用于卵母细胞低温保护剂及冷冻保存的实验研究[D].上海:上海理工大学.2016Yi X Y.Shanghai:University of Shanghai for Science and Technology.2016
[13]Kondo E,Wada K,Hosokawa K,et al.Cryopreservation of adhered mammalian cells on a microfluidic device:Toward ready-to-use cell-based experimental platforms.Biotechnology&Bioengineering,2016,113(1):237-240
[14]Li S,Liu W,Lin L.On-chip cryopreservation of living cells.Journal of the Association for Laboratory Automation,2010,15(2):99-106
[15]Zou Y,Yin T,Chen S,et al.On-chip cryopreservation:a novel method for ultra-rapid cryoprotectant-free cryopreservation of small amounts of human spermatozoa.Plos One,2013,8(4):e61593
[16]Vajta G,Holm P,Kuwayama M,et al.Open pulled straw(OPS)vitrification:a new way to reduce cryoinjuries of bovine ova and embryos.Molecular Reproduction&Development,1998,51(1):53-58
[17]Guérin P,El M S,Ménézo Y.Oxidative stress and protection against reactive oxygen species in the pre-implantation embryo and its surroundings.Human Reproduction Update,2001,7(2):175-189
[18]李忌,郑荣梁.活性氧诱导细胞凋亡.中国细胞生物学学报,1997,1(3):116-119Li J,Zheng R L.Chinese Journal of Cell Biology,1997,1(3):116-119
[19]Tatone C,Di E G,Vento M,et al.Cryopreservation and oxidative stress in reproductive cells.Gynecological Endocrinology,2010,26(8):563-567
[20]Zhao X M,Du W H,Wang D,et al.Effect of cyclosporine pretreatment on mitochondrial function in vitrified bovine mature oocytes.Fertility&Sterility,2011,95(8):2786-2788
[21]Luz H K M,Wanderley L S,Faustino L R,et al.Role of antioxidants agents in germ cells and embryos cryopreservation.Acta Scientiae Veterinari,2011,39(2):23-26
[22]Ou X H,Li S,Wang Z B,et al.Maternal insulin resistance causes oxidative stress and mitochondrial dysfunction in mouse oocytes.Human Reproduction,2012,27(7):2130-2145
[23]林翠英,王世鄂.线粒体与卵母细胞和早期胚胎发育.解剖学研究,2008,30(5):385-388Lin C Y,Wang S E.Anatomy Research,2008,30(5):385-388
[24]Lei T,Guo N,Tan M H,et al.Effect of mouse oocyte vitrification on mitochondrial membrane potential and distribution.Journal of Huazhong University of Science and Technology(Medical Sciences),2014,34(1):99-102
[2]Moussa M,Shu J,Zhang X,et al.Cryopreservation of mammalian oocytes and embryos:current problems and future perspectives.Science China Life Sciences,2014,57(9):903-914
[3]Shi W Q,Zhu S E,Zhang D,et al.Improved development by Taxol pretreatment after vitrification of in vitro matured porcine oocytes.Reproduction,2006,131(4):795-804
[4]Rojas C,Palomo M J,Albarrac觔N J L,et al.Vitrification of immature and in vitro matured pig oocytes:study of distribution of chromosomes,microtubules,and actin microfilaments.Cryobiology,2004,49(3):211-220
[5]Fujihira T,Kishida R,Fukui Y.Developmental capacity of vitrified immature porcine oocytes following ICSI:effects of cytochalasin B and cryoprotectants.Cryobiology,2004,49(3):286-290
[6]Ogawa B,Ueno S,Nakayama N,et al.Developmental ability of porcine in vitro matured oocytes at the meiosisⅡstage after vitrification.J Reprod Dev,2010,56(3):356-361
[7]Galeati G,Spinaci M,Vallorani C,et al.Pig oocyte vitrification by cryotop method:effects on viability,spindle and chromosome configuration and in vitro fertilization.Animal Reproduction Science,2011,127(1-2):43-49
[8]Kuwayama M,Vajta G,Kato O,et al.Highly efficient vitrification method for cryopreservation of human oocytes.Reproductive Biomedicine Online,2005,11(3):300-308
[9]Kuwayama M.Highly efficient vitrification for cryopreservation of human oocytes and embryos:the Cryotop method.Theriogenology,2007,67(1):73-80
[10]Song Y S,Moon S J,Hulli L,et al.Microfluidics for cryopreservation.Lab on A Chip,2009,9(13):1874-1881
[11]杨云,周新丽,戴建军,等.微流控线性加载低温保护剂减少猪MⅡ期卵母细胞的渗透损伤.生物化学与生物物理进展,2016,43(6):616-623Yang Y,Zhou X L,Dai J J,et al.Progress in Biochemistry and Biophysics,2016,43(6):616-623
[12]衣星越.微流控装置用于卵母细胞低温保护剂及冷冻保存的实验研究[D].上海:上海理工大学.2016Yi X Y.Shanghai:University of Shanghai for Science and Technology.2016
[13]Kondo E,Wada K,Hosokawa K,et al.Cryopreservation of adhered mammalian cells on a microfluidic device:Toward ready-to-use cell-based experimental platforms.Biotechnology&Bioengineering,2016,113(1):237-240
[14]Li S,Liu W,Lin L.On-chip cryopreservation of living cells.Journal of the Association for Laboratory Automation,2010,15(2):99-106
[15]Zou Y,Yin T,Chen S,et al.On-chip cryopreservation:a novel method for ultra-rapid cryoprotectant-free cryopreservation of small amounts of human spermatozoa.Plos One,2013,8(4):e61593
[16]Vajta G,Holm P,Kuwayama M,et al.Open pulled straw(OPS)vitrification:a new way to reduce cryoinjuries of bovine ova and embryos.Molecular Reproduction&Development,1998,51(1):53-58
[17]Guérin P,El M S,Ménézo Y.Oxidative stress and protection against reactive oxygen species in the pre-implantation embryo and its surroundings.Human Reproduction Update,2001,7(2):175-189
[18]李忌,郑荣梁.活性氧诱导细胞凋亡.中国细胞生物学学报,1997,1(3):116-119Li J,Zheng R L.Chinese Journal of Cell Biology,1997,1(3):116-119
[19]Tatone C,Di E G,Vento M,et al.Cryopreservation and oxidative stress in reproductive cells.Gynecological Endocrinology,2010,26(8):563-567
[20]Zhao X M,Du W H,Wang D,et al.Effect of cyclosporine pretreatment on mitochondrial function in vitrified bovine mature oocytes.Fertility&Sterility,2011,95(8):2786-2788
[21]Luz H K M,Wanderley L S,Faustino L R,et al.Role of antioxidants agents in germ cells and embryos cryopreservation.Acta Scientiae Veterinari,2011,39(2):23-26
[22]Ou X H,Li S,Wang Z B,et al.Maternal insulin resistance causes oxidative stress and mitochondrial dysfunction in mouse oocytes.Human Reproduction,2012,27(7):2130-2145
[23]林翠英,王世鄂.线粒体与卵母细胞和早期胚胎发育.解剖学研究,2008,30(5):385-388Lin C Y,Wang S E.Anatomy Research,2008,30(5):385-388
[24]Lei T,Guo N,Tan M H,et al.Effect of mouse oocyte vitrification on mitochondrial membrane potential and distribution.Journal of Huazhong University of Science and Technology(Medical Sciences),2014,34(1):99-102