微流控法去除低温保护剂对卵母细胞发育的影响
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摘要
为了减小低温保护剂去除过程对卵母细胞造成的渗透损伤和毒性损伤,本文利用微流控芯片对猪二次减数分裂中期(MⅡ-stage)卵母细胞低温保护剂的去除方案进行了优化研究。首先分析了微流控去除方法中去除时间、去除液成分及浓度对卵母细胞存活率及体外发育情况的影响,然后将微流控去除方法与传统的一步法、两步法进行了比较。研究结果表明,微流控法中去除总时间为8 min时,卵母细胞存活率(95.99%±4.64%)及桑椹胚率(74.17%±1.18%)与新鲜细胞(98.53%±2.94%;78.22%±1.34%)相比,差异无统计学意义;1 mol/L蔗糖去除液最有利于卵母细胞低温保护剂去除后的存活及体外发育;微流控法去除低温保护剂后,卵母细胞的存活率、体外发育情况等,均好于传统去除方法。本文研究结果提示,以微流控法去除低温保护剂可减小对卵母细胞的损伤,从而可能进一步提高卵母细胞的低温保存效果。
In order to reduce osmotic damage and chemical toxicity of cryoprotectants(CPA) to oocytes during unloading process, the microfluidic chip was used to remove CPA from porcine MⅡ oocytes in this study. Firstly, the effects of unloading time, composition and concentration of diluting solutions of microfluidic method on survival rate and developmental capacity of oocytes were studied, then microfluidic method was compared with traditional one-step and two-step CPA unloading protocols. The results showed that when the total time is 8 minutes, the survival rate and morula rate of oocytes treated with microfluidic method could achieve 95.99% ± 4.64% and 74.17% ± 1.18%, respectively, which were not significantly different from fresh control group(98.53% ± 2.94%; 78.22% ± 1.34%). In addition, 1 mol/L sucrose diluting solutions were more beneficial than other solutions, and it was also showed that microfluidic method achieved better survival, cleavage rate of oocytes than traditional methods. Microfluidic CPA removal protocol can reduce the damage to oocytes during unloading process, and may further improve the cryopreservation effect of oocytes.
In order to reduce osmotic damage and chemical toxicity of cryoprotectants(CPA) to oocytes during unloading process, the microfluidic chip was used to remove CPA from porcine MⅡ oocytes in this study. Firstly, the effects of unloading time, composition and concentration of diluting solutions of microfluidic method on survival rate and developmental capacity of oocytes were studied, then microfluidic method was compared with traditional one-step and two-step CPA unloading protocols. The results showed that when the total time is 8 minutes, the survival rate and morula rate of oocytes treated with microfluidic method could achieve 95.99% ± 4.64% and 74.17% ± 1.18%, respectively, which were not significantly different from fresh control group(98.53% ± 2.94%; 78.22% ± 1.34%). In addition, 1 mol/L sucrose diluting solutions were more beneficial than other solutions, and it was also showed that microfluidic method achieved better survival, cleavage rate of oocytes than traditional methods. Microfluidic CPA removal protocol can reduce the damage to oocytes during unloading process, and may further improve the cryopreservation effect of oocytes.
引文
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2al-Hasani S,Kirsch J,Diedrich K,et al.Successful embryo transfer of cryopreserved and in-vitro fertilized rabbit oocytes.Hum Reprod,1989,4(1):77-79.
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4Maclellan L J,Carnevale E M,Coutinho da Silva M A,et al.Pregnancies from vitrified equine oocytes collected from superstimulated and non-stimulated mares.Theriogenology,2002,58(5):911-919.
5Chen H H,Shen Hong,Heimfeld S,et al.A microfluidic study of mouse dendritic cell membrane transport properties of water and cryoprotectants.Int J Heat Mass Transf,2008,51(23/24):5687-5694.
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10Fleming K K,Longmire E K,Hubel A.Numerical characterization of diffusion-based extraction in cell-laden flow through a microfluidic channel.J Biomech Eng,2007,129(5):703-711.
11Mata C,Longmire E,Mckenna D,et al.Cell motion and recovery in a two-stream microfluidic device.Microfluid Nanofluidics,2010,8(4):457-465.
12Mata C,Longmire E K,Mckenna D H,et al.Experimental study of diffusion-based extraction from a cell suspension.Microfluid Nanofluidics,2008,5(4):529-540.
13Hanna J,Hubel A,Lemke E.Diffusion-based extraction of DMSO from a cell suspension in a three stream,vertical microchannel Biotechnol Bioeng,2012,109(9):2316-2324.
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15Paynter S J,O'neil L,Fuller B J,et al.Membrane permeability of human oocytes in the presence of the cryoprotectant propane-1,2-diol.Fertil Steril,2001,75(3):532-538.
16Paynter S J,Borini A,Bianchi V,et al.Volume changes of mature human oocytes on exposure to cryoprotectant solutions used in slow cooling procedures.Hum Reprod,2005,20(5):1194-1199.
17周新丽,衣星越,周楠峰,等.用于低温保护剂混合的微流控芯片设计及优化.生物医学工程学杂志,2016,35(3):461-465.
18戴建军.冷冻保存对猪卵母细胞凋亡模式与基因表达的影响.南京:南京农业大学,2015.
19张德福,朱良成,刘东,等.猪卵母细胞冷冻保存研究.中国农业科学,2006,39(6):1233-1240.
20杨喜,吴静,刘丑生,等.细胞松弛素B、温度、离心处理对猪卵母细胞OPS玻璃化冷冻的影响.内蒙古大学学报:自然科学版,2013,(5):525-529.
21田见晖,刘国世,曾申明,等.电脉冲激活对体外成熟猪卵母细胞卵裂率和囊胚率的影响.农业生物技术学报,2004,12(4):401-407.
22赵金凤.猪卵母细胞孤雌激活方法的研究.哈尔滨:东北农业大学,2010.
23郝子悦.猪卵母细胞体外成熟及孤雌激活的研究.扬州:扬州大学,2008.
24罗婷,蒙丽娜,刘晓华,等.哺乳动物附植前胚胎细胞凋亡的研究进展.中国畜牧兽医,2012,39(11):125-128.
25Martino A,Songsasen N,Leibo S P.Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling.Biol Reprod,1996,54(5):1059-1069.
26Crowe J H,Carpenter J F,Crowe L M.The role of vitrification in anhydrobiosis.Annu Rev Physiol,1998,60(1):73-103.
27李群,袁勤生.海藻糖的性质及应用.中国生化药物杂志,1995,(5):231-233.
2al-Hasani S,Kirsch J,Diedrich K,et al.Successful embryo transfer of cryopreserved and in-vitro fertilized rabbit oocytes.Hum Reprod,1989,4(1):77-79.
3Fuku E,Kojimat T,Shioya Y,et al.In vitro fertilization and development of frozen-thawed bovine oocytes.Cryobiology,1992,29(4):485-492.
4Maclellan L J,Carnevale E M,Coutinho da Silva M A,et al.Pregnancies from vitrified equine oocytes collected from superstimulated and non-stimulated mares.Theriogenology,2002,58(5):911-919.
5Chen H H,Shen Hong,Heimfeld S,et al.A microfluidic study of mouse dendritic cell membrane transport properties of water and cryoprotectants.Int J Heat Mass Transf,2008,51(23/24):5687-5694.
6Kobel S,Valero A,Latt J,et al.Optimization of microfluidic single cell trapping for long-term on-chip culture.Lab Chip,2010,10(7):857-863.
7Lecault V,White A K,Singhal A,et al.Microfluidic single cell analysis:from promise to practice.Curr Opin Chem Biol,2012,16 (3/4):381-390.
8Song Y S,Moon S,Hulli L,et al.Microfluidics for cryopreservation.Lab Chip,2009,9(13):1874-1881.
9Hubel A.Advancing the preservation of cellular therapy products.Transfusion,2011,51(Suppl 4):82S-86S.
10Fleming K K,Longmire E K,Hubel A.Numerical characterization of diffusion-based extraction in cell-laden flow through a microfluidic channel.J Biomech Eng,2007,129(5):703-711.
11Mata C,Longmire E,Mckenna D,et al.Cell motion and recovery in a two-stream microfluidic device.Microfluid Nanofluidics,2010,8(4):457-465.
12Mata C,Longmire E K,Mckenna D H,et al.Experimental study of diffusion-based extraction from a cell suspension.Microfluid Nanofluidics,2008,5(4):529-540.
13Hanna J,Hubel A,Lemke E.Diffusion-based extraction of DMSO from a cell suspension in a three stream,vertical microchannel Biotechnol Bioeng,2012,109(9):2316-2324.
14Heo Y S,Lee H J,Hassell B A,et al.Controlled loading of cryoprotectants(CPAs)to oocyte with linear and complex CPA profiles on a microfluidic platform.Lab Chip,2011,11(20):3530-3537.
15Paynter S J,O'neil L,Fuller B J,et al.Membrane permeability of human oocytes in the presence of the cryoprotectant propane-1,2-diol.Fertil Steril,2001,75(3):532-538.
16Paynter S J,Borini A,Bianchi V,et al.Volume changes of mature human oocytes on exposure to cryoprotectant solutions used in slow cooling procedures.Hum Reprod,2005,20(5):1194-1199.
17周新丽,衣星越,周楠峰,等.用于低温保护剂混合的微流控芯片设计及优化.生物医学工程学杂志,2016,35(3):461-465.
18戴建军.冷冻保存对猪卵母细胞凋亡模式与基因表达的影响.南京:南京农业大学,2015.
19张德福,朱良成,刘东,等.猪卵母细胞冷冻保存研究.中国农业科学,2006,39(6):1233-1240.
20杨喜,吴静,刘丑生,等.细胞松弛素B、温度、离心处理对猪卵母细胞OPS玻璃化冷冻的影响.内蒙古大学学报:自然科学版,2013,(5):525-529.
21田见晖,刘国世,曾申明,等.电脉冲激活对体外成熟猪卵母细胞卵裂率和囊胚率的影响.农业生物技术学报,2004,12(4):401-407.
22赵金凤.猪卵母细胞孤雌激活方法的研究.哈尔滨:东北农业大学,2010.
23郝子悦.猪卵母细胞体外成熟及孤雌激活的研究.扬州:扬州大学,2008.
24罗婷,蒙丽娜,刘晓华,等.哺乳动物附植前胚胎细胞凋亡的研究进展.中国畜牧兽医,2012,39(11):125-128.
25Martino A,Songsasen N,Leibo S P.Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling.Biol Reprod,1996,54(5):1059-1069.
26Crowe J H,Carpenter J F,Crowe L M.The role of vitrification in anhydrobiosis.Annu Rev Physiol,1998,60(1):73-103.
27李群,袁勤生.海藻糖的性质及应用.中国生化药物杂志,1995,(5):231-233.