WO2018014692A1 - 清洗卵子或受精卵表面的方法 - Google Patents

清洗卵子或受精卵表面的方法 Download PDF

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WO2018014692A1
WO2018014692A1 PCT/CN2017/089204 CN2017089204W WO2018014692A1 WO 2018014692 A1 WO2018014692 A1 WO 2018014692A1 CN 2017089204 W CN2017089204 W CN 2017089204W WO 2018014692 A1 WO2018014692 A1 WO 2018014692A1
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egg
culture
culture solution
dish
fertilized
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French (fr)
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蔡立义
陆思嘉
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序康医疗科技（苏州）有限公司
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0608—Germ cells
    • C12N5/0609—Oocytes, oogonia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0604—Whole embryos; Culture medium therefor
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
    • Y02A40/81—Aquaculture, e.g. of fish

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  • the present invention is in the field of reproductive medicine, and in particular, the present invention relates to a method of washing the surface of an egg or fertilized egg.
  • IVF technology is a powerful technical tool against infertility. But for a variety of reasons, the success rate from embryo implantation to the birth of the fetus is not high (usually only about 40%). In addition to maternal health reasons, the quality of fertilized eggs is one of the important reasons for the failure of embryonic development.
  • PGS Preimplantation Genetic Screen
  • blastocyst a saclike structure composed of about 80–100 cells, called a blastocyst.
  • one to several trophoblast cells can be taken up by a capillary glass tube under a microscope for detection.
  • Studies have shown that a small amount of DNA is released into the culture medium (blastocyst culture medium) during early embryonic development in vitro (days 1-5). Although this mechanism is still unclear, the presence of embryo-derived DNA in blastocyst culture fluids provides a material basis for non-destructive testing of embryonic chromosome status.
  • the first generation of IVF used IVF for in vitro fertilization of eggs.
  • IVF in vitro fertilization of eggs.
  • only one sperm can be effectively fertilized, so that the egg becomes a fertilized egg, and other unnecessary ineffective sperm adhere to the surface of the fertilized egg. After they die, they are released into the in vitro culture solution. Therefore, the parent DNA will Interference is formed for subsequent NICS detection.
  • a large number of granulosa cells derived from the mother are inevitably attached to the surface of the egg cells extracted from the mother, and these granule cells are released into the culture solution, and therefore, the maternal DNA also interferes with the detection of the NICS.
  • the second-generation IVF used the intracytoplasmic sperm injection method (ICSI) to fertilize the eggs in vitro, the contamination caused by excess sperm was avoided. Moreover, before in vitro fertilization, the granulosa cells attached to the surface of the egg are routinely cleaned to partially expose the surface of the egg, providing a channel for the sperm injection needle, but the surface of the egg after conventional washing still adheres to more granule cells, for subsequent NICS detection forms interference.
  • ICSI intracytoplasmic sperm injection method
  • the present invention establishes a technical method of thoroughly removing granulosa cells and/or excess sperm from the surface of an egg (for ICSI) or fertilized egg (for IVF) to avoid interference with NICS detection.
  • the object of the present invention is to establish a technical method for completely removing the granulosa cells and/or excess sperm on the surface of the egg during the IVF (ICSF and ICSI) process, so that the subsequent embryo culture solution is suitable for NICS detection.
  • a method of washing an egg or a surface of a fertilized egg comprising the steps of:
  • the egg or fertilized egg is plucked with a rod-shaped tool having a third culture solution, wherein the third The culture medium contains protein; the granulosa cells and/or excess sperm are removed from the surface of the egg or fertilized egg, and are completely removed.
  • the rod-shaped tool with the third culture solution means that the rod-shaped tool has a third culture solution.
  • the rod-shaped tool is a stick or a stick.
  • the rod-shaped tool is made of any suitable material such as glass, plastic or the like.
  • the protein is a serum protein.
  • the untreated egg is an egg taken out of the mother for 2-3 hours.
  • the untreated fertilized egg is a fertilized egg that is fertilized in vitro for 4 hours or 16-20 hours.
  • the first culture solution mimics the composition of the human fallopian tube fluid and uses HEPES as a buffer component.
  • the first culture solution can be published in 1984 by Quinn et al. (Fertil Steril. 1984; 41: 202 or 1985; 44: 493.) preparation.
  • the culture solution uses HEPES as a buffer component to maintain pH stability for a long time in the air. No carbon dioxide gas environment is required for cultivation.
  • the culture can be purchased from various commercial companies (eg, SAGE media) or can be prepared by itself.
  • the first culture solution is an IM culture solution.
  • the first culture dish is a Falcon 353037 culture dish, a Corning 430166 culture dish or a Thermo Scientific 150288 culture dish.
  • the first culture dish is any commercially available standard cell culture surface treated cell culture dish. It can be sold by any other company, or it can be of any shape or size.
  • the hyaluronidase is commercially available from any cell culture reagent supplier.
  • the treatment time is 10 to 60 seconds; preferably, 20 to 40 seconds.
  • composition of the second culture solution is the same as that of the first culture solution, except that the second culture solution does not contain protein.
  • the second culture fluid is an M-IM culture fluid.
  • the second culture dish is a Falcon 351006 culture dish, a Corning 430589 Petri dish or a Thermo Scientific 150340 Petri dish.
  • the second culture dish is any commercially available surface treatment film that has not been surface treated. It can be sold by any other company, or it can be of any shape or size.
  • composition of the third culture solution is the same as that of the first culture solution, except that the third culture solution does not contain the buffer component HEPES.
  • the third culture solution can be published in 1984 by Quinn et al. (Fertil Steril. 1984; 41: 202 or 1985; 44: 493.) preparation.
  • the third culture solution differs from the first culture solution in that it has no buffer component (HEPES), so it must be used in a 5% carbon dioxide environment (in a carbon dioxide incubator).
  • HEPES buffer component
  • the culture can be purchased from various commercial companies (eg, SAGE media) or can be prepared by itself.
  • the third culture solution is a GM culture solution.
  • the granulosa cells and/or excess sperm are detached from the surface of the egg or fertilized egg and attached to the bottom surface of the culture dish.
  • Figure 1 shows an untreated egg that has just been removed from the mother, with a large number of granulosa cells attached to the surface.
  • Figure 2 shows the eggs treated by the step 3 washing, the surface granule cell adhesion is significantly reduced, but there are still granule cells attached.
  • Figure 3 shows the granule cells detached from the egg during the operation and attached to the bottom of the culture dish.
  • Figure 4 shows the eggs washed in steps 3 and 4 with the surface granule cells completely removed.
  • Figure 5 shows that the granulosa cells attached to the two egg zona pellucida were completely removed.
  • Figure 6 shows that there are still residual granule cells on the surface of the two eggs after treatment.
  • the proteins referred to herein are serum proteins.
  • the present invention provides a method of washing the surface of an egg or fertilized egg, the method comprising the steps of:
  • the hyaluronanase (available from any cell culture reagent supplier) is then added to treat the egg or fertilized egg for a period of time (e.g., 10-60 seconds; preferably, 20-40 seconds) to obtain the first Secondary treated egg or fertilized egg;
  • the egg or fertilized egg is then plucked with a rod-shaped tool with a third culture solution, wherein the third culture medium contains protein; the granulosa cells and/or excess sperm are removed from the surface of the egg or fertilized egg, thereby being completely removed clean.
  • the granulosa cells and/or excess sperm are detached from the surface of the egg or the fertilized egg and attached to the bottom surface of the culture dish.
  • the rod-shaped tool is a rod or a stick-like tool.
  • the rod-shaped tool can be made of any suitable material such as glass, plastic or the like.
  • the rod-shaped tool with the third culture solution means that the rod-shaped tool has a third culture solution.
  • the untreated egg may be an egg taken out from the mother for 2-3 hours.
  • the untreated fertilized egg may be a fertilized egg after 4 hours or 16-20 hours of in vitro fertilization.
  • the first culture solution simulates the components of the human fallopian tube fluid and uses HEPES as a buffer component.
  • the culture solution uses HEPES as a buffer component to maintain pH stability for a long time in the air. No carbon dioxide gas environment is required for cultivation.
  • the first culture solution can be prepared by reference to the literature published by Quinn et al., 1984 (Fertil Steril.) 1984; 41: 202 or 1985; 44: 493. It can also be purchased from various commercial companies (eg, SAGE media).
  • the first culture solution is an IM culture solution.
  • the first culture dish is any commercially available standard cell culture surface treated cell culture dish. It can be sold by any other company, or it can be of any shape or size.
  • the first culture dish is a Falcon 353037 culture dish, a Corning 430166 culture dish or a Thermo Scientific 150288 culture dish.
  • composition of the second culture solution is the same as that of the first culture solution, except that the second culture solution does not contain protein.
  • the second culture fluid is an M-IM culture fluid.
  • the second culture dish is any commercially available surface treatment film that has not been surface treated. It can be sold by any other company, or it can be of any shape or size.
  • the second culture dish is a Falcon 351006 culture dish, a Corning 430589 Petri dish or a Thermo Scientific 150340 Petri dish.
  • the composition of the third culture solution is the same as that of the first culture solution, except that the third culture solution does not contain buffer. Ingredients HEPES.
  • the third culture solution differs from the first culture solution in that it has no buffer component (HEPES), so it must be used in a 5% carbon dioxide environment (in a carbon dioxide incubator).
  • the third culture solution can be published in 1984 by Quinn et al. (Fertil Steril. 1984; 41: 202 or 1985; 44: 493.) It may be prepared or purchased from various commercial companies (eg, SAGE media).
  • the third culture solution is a GM culture solution.
  • ICSI's egg-clearing granulosa cell treatment is 2-3 hours after egg retrieval.
  • the untreated egg just taken out of the mother has a large number of granulosa cells attached to the surface, as shown in Figure 1.
  • the eggs with the remaining granulosa cells adhered to the surface were transferred to a Falcon 351006 culture dish containing serum-protein-free M-IM medium (ART-1023). At this time, the granule cells have a positive charge due to the negative neutralization of the protein in the culture solution. At the same time, the Falcon 351006 dish is made of untreated polystyrene and its surface is hydrophobic and negatively charged. Therefore, the granulosa cells attached to the surface of the egg adhere to the bottom surface of the culture dish (as shown in Figure 3).
  • a glass rod containing protein GM medium (ART-1526) (the protein-containing medium is used to neutralize the negative charge on the surface of the glass rod to prevent cells from sticking to the glass rod) and continuously move the egg.
  • the egg is rolled on the bottom surface of the dish. During the rolling process, the remaining granule cells on the surface of the egg adhere to the bottom surface of the culture dish and detach from the egg, and continue to push the egg to roll until the granule cells on the surface are completely removed.
  • Example 1 The method of Example 1 was employed except that the fertilized egg was used instead of the egg.
  • the treatment time for IVF to remove granulosa cells and excess sperm from the surface of the fertilized egg is after 4 hours (short-term fertilization) or 16-20 hours (conventional fertilization).
  • Example 1 The method of Example 1 was employed except that Corning's petri dish was used, and the culture solution was prepared according to the formulation in the literature (Fertil Steril. 1984; 41: 202, 1985; 44: 493.).
  • HTF culture medium containing human serum albumin and HEPES (according to the literature) and standard cells Culture the surface-treated culture dish (purchased from Corning; product number: 430166) instead of Falcon 353037 (polystyrene material, standard cell culture surface treatment) culture dish containing 1 ml of IM culture solution (ART-1520);
  • Example 1 The method of Example 1 was employed except that the M-IM culture medium containing no protein was replaced with the IM culture solution in step 4.4.
  • An advantage of the present invention is that it provides a means of thoroughly removing granulosa cells and/or excess sperm from the surface of the egg without causing any damage to the egg, fertilized egg or embryo, without touching the zona pellucida.
  • the main point is to remove granulosa cells and/or excess sperm from the surface of the egg (or fertilized egg) by using the cell surface, the composition of the culture solution (protein), and the charge relationship on the bottom surface of the cell culture dish.

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Abstract

一种清洗卵子或受精卵表面的方法，包括：步骤(1)将未经处理的卵子或未经处理的受精卵置于第一培养皿中，其中，所述第一培养皿经标准细胞培养表面处理且含有第一培养液，所述第一培养液含蛋白；然后加入透明质酸酶处理所述卵子或受精卵，从而得到经第一次处理的卵子或受精卵；(2)将经第一次处理的卵子或受精卵移入第二培养皿中，其中，所述第二培养皿未经表面处理且含有第二培养液，所述第二培养液不含蛋白；然后用带有第三培养液的棒状工具拨动所述卵子或受精卵，其中，所述第三培养液含蛋白；使颗粒细胞和/或多余精子脱离卵子或受精卵表面，从而被彻底清除干净。

Description

清洗卵子或受精卵表面的方法 技术领域
本发明属于生殖医学领域，具体地，本发明涉及一种清洗卵子或受精卵表面的方法。
背景技术
试管婴儿技术是一项对抗不孕不育的强大技术手段。但因各种原因，从胚胎植入子宫到胎儿出生的成功率不高(通常只有40％左右)。除母亲健康原因外，受精卵质量是导致胚胎发育失败的重要原因之一。
早期，在试管婴儿技术过程中，仅依靠显微镜下的形态观察，从多个胚胎中挑选2-3个相对“正常”者植入母亲子宫。而显微镜下的形态正常并无法反映染色体是否正常。错误地挑选形态正常而染色体异常的胚胎植入母亲子宫导致了很多试管婴儿受孕失败。
近年以来，人们建立了一些技术(统称为Preimplantation Genetic Screen，PGS)对体外培养胚胎的染色体状态进行检测，以筛选染色体正常的胚胎植入母亲子宫，从而提高受孕成功率。各种PGS方法检测所需的生物样本都是从体外培养胚胎中获得的一个至数个细胞，通过对这写细胞的检测可反映整个胚胎的染色体是否正常。胚胎活检有极体活检、卵裂球活检和囊胚滋养层细胞活检。PGS检测通常是活检卵裂球或囊胚滋养层细胞。但是，越来越多的研究表明卵裂期活检的安全性受到质疑，因此囊胚滋养层细胞活检逐渐成为PGS胚胎活检的主流。
当受精卵在体外培养液中生长发育5天时，胚胎是由约80–100个细胞组成的囊状结构，称为囊胚。此时，可在显微镜下，用毛细玻璃管吸取一个至数个滋养层细胞进行检测。已有研究表明，胚胎早期体外发育过程中(第1-5日)会将少量DNA释放入培养液(囊胚培养液)中。尽管对此机制尚不明了，但囊胚培养液中存在着胚胎来源的DNA提供了对胚胎染色体状态进行无损检测的物质基础。我们的前期发明《一种利用囊胚培养液检测胚胎染色体异常的方法》 (CN105368936A)正是建立了一套达成此目的技术方法(NICS)。但是，NICS方法容易受到一些常规试管婴儿体外胚胎培养过程中所忽略因素的干扰。
第一代试管婴儿(IVF)使用大量精子对卵子进行体外受精。然而对每个卵子而言，只有一个精子能够有效受精，使卵子成为受精卵，其它多余的无效精子会粘附在受精卵表面，它们死亡后释放入体外培养液中，因此，父源DNA会对后续的NICS检测形成干扰。另外，从母体提取的卵细胞表面不可避免地会附着大量母体来源的颗粒细胞，这些颗粒细胞向培养液中释放，因此，母源DNA也会对NICS检测形成干扰。
尽管二代试管婴儿采用了卵胞浆内单精子注射方法(ICSI)对卵子进行体外受精，避免了多余精子造成的污染问题。并且，体外受精前会对卵子表面附着的颗粒细胞做常规清洗以部分暴露卵子表面，为精子注射针提供通道，但常规清洗后的卵子表面仍会粘附有较多的颗粒细胞，对后续的NICS检测形成干扰。
因此，本发明建立了将卵子(针对ICSI)或受精卵(针对IVF)表面的颗粒细胞和/或多余精子彻底清除干净，以避免干扰NICS检测的技术方法。
发明内容
本发明的目的是建立在试管婴儿(IVF及ICSI)过程中彻底清除卵子表面的颗粒细胞和/或多余精子，使后续的胚胎培养液适用于NICS检测的技术方法。
在本发明的第一方面中，提供了一种清洗卵子或受精卵表面的方法，其特征在于，包括步骤：
(1)将未经处理的卵子或未经处理的受精卵置于第一培养皿中，其中，所述第一培养皿经标准细胞培养表面处理且含有第一培养液，所述第一培养液含蛋白；
然后加入透明质酸酶处理所述卵子或受精卵，从而得到经第一次处理的卵子或受精卵；
(2)将经第一次处理的卵子或受精卵移入第二培养皿中，其中，所述第二培养皿未经表面处理且含有第二培养液，所述第二培养液不含蛋白；
然后用带有第三培养液的棒状工具拨动所述卵子或受精卵，其中，所述第三 培养液含蛋白；使颗粒细胞和/或多余精子脱离卵子或受精卵表面，从而被彻底清除干净。
在另一优选例中，所述带有第三培养液的棒状工具是指棒状工具上蘸有第三培养液。
在另一优选例中，所述棒状工具为棒或棍。
在另一优选例中，所述棒状工具为玻璃的、塑料的等任何适合的材料制成的。
在另一优选例中，所述蛋白为血清蛋白。
在另一优选例中，所述未经处理的卵子是从母体取出2-3小时后的卵子。
在另一优选例中，所述未经处理的受精卵是在体外受精4小时或16-20小时后的受精卵。
在另一优选例中，所述第一培养液模拟人输卵管液的成分，且用HEPES做缓冲成份。
在另一优选例中，所述第一培养液可参照Quinn等人于1984年公开发表(《生育与不孕》(Fertil Steril.)1984；41:202或1985；44:493.)的文献制备。
该培养液用HEPES做缓冲成份，在空气中可较长时间地保持pH值的稳定性。培养时无需二氧化碳气体环境。
该培养液可从各商业公司(如，SAGE media)购买，也可以自行配制。
在另一优选例中，所述第一培养液为IM培养液。
在另一优选例中，所述第一培养皿为Falcon 353037培养皿、康宁公司的430166培养皿或塞默飞公司的150288培养皿。
在另一优选例中，所述第一培养皿为任意的市售的经标准细胞培养表面处理的细胞培养皿。可以是任何其它公司出售的，也可以是任何形状或尺寸的。
在另一优选例中，所述透明质酸酶可从任何细胞培养试剂供应商购得。
在另一优选例中，步骤(1)中，所述处理的时间为10-60秒；较佳地，为20-40秒。
在另一优选例中，所述第二培养液的成分与第一培养液相同，区别仅在于第二培养液不含有蛋白。
在另一优选例中，所述第二培养液为M-IM培养液。
在另一优选例中，所述第二培养皿为Falcon 351006培养皿、康宁公司的430589培养皿或塞默飞公司的150340培养皿。
在另一优选例中，所述第二培养皿为任意的市售的未经表面处理的细胞表面膜。可以是任何其它公司出售的，也可以是任何形状或尺寸的。
在另一优选例中，所述第三培养液的成分与第一培养液相同，区别仅在于第三培养液不含缓冲成分HEPES。
在另一优选例中，所述第三培养液可参照Quinn等人于1984年公开发表(《生育与不孕》(Fertil Steril.)1984；41:202或1985；44:493.)的文献制备。
所述第三培养液与第一培养液的区别在于其中没有缓冲成份(HEPES)，所以必须在5％二氧化碳环境(二氧化碳培养箱中)下使用。
该培养液可从各商业公司(如，SAGE media)购买，也可以自行配制。
在另一优选例中，所述第三培养液为GM培养液。
在另一优选例中，所述颗粒细胞和/或多余精子从卵子或受精卵表面脱离后附于培养皿底部表面。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了刚从母体中取出的未经处理的卵子，表面附着有大量颗粒细胞。
图2显示了经步骤3清洗处理过的卵子，其表面颗粒细胞附着明显减少，但仍有颗粒细胞附着。
图3显示了操作过程中颗粒细胞脱离卵子，附着于培养皿底。
图4显示了经步骤3和步骤4清洗处理过的卵子，其表面颗粒细胞被彻底清除。
图5显示了两个卵子透明带上附着的颗粒细胞被彻底清除。
图6显示了处理后的两个卵子表面仍有颗粒细胞残留。
具体实施方式
本发明人经过广泛而深入的研究，意外发现了一种彻底清洗卵子或受精卵表面的方法。在此基础上，发明人完成了本发明。
本文中所提及的蛋白为血清蛋白。
清洗卵子或受精卵表面的方法
本发明提供了一种清洗卵子或受精卵表面的方法，所述方法包括步骤：
(1)将未经处理的卵子或未经处理的受精卵置于第一培养皿中，其中，所述第一培养皿经标准细胞培养表面处理且含有第一培养液，所述第一培养液含蛋白；
然后加入透明质酸酶(可从任何细胞培养试剂供应商购得)处理所述卵子或受精卵一段时间(如10-60秒；较佳地，为20-40秒)，从而得到经第一次处理的卵子或受精卵；
(2)将经第一次处理的卵子或受精卵移入第二培养皿中，其中，所述第二培养皿未经表面处理且含有第二培养液，所述第二培养液不含蛋白；
然后用带有第三培养液的棒状工具拨动所述卵子或受精卵，其中，所述第三培养液含蛋白；使颗粒细胞和/或多余精子脱离卵子或受精卵表面，从而被彻底清除干净。
其中，所述颗粒细胞和/或多余精子从卵子或受精卵表面脱离后附于培养皿底部表面。
所述棒状工具为棒或棍类似工具。所述棒状工具可以为玻璃的、塑料的等任何适合的材料制成的。所述带有第三培养液的棒状工具是指棒状工具上蘸有第三培养液。
未经处理的卵子或未经处理的受精卵
所述未经处理的卵子可以是从母体取出2-3小时后的卵子。
所述未经处理的受精卵可以是在体外受精4小时或16-20小时后的受精卵。
第一培养液
所述第一培养液模拟人输卵管液的成分，且用HEPES做缓冲成份。该培养液用HEPES做缓冲成份，在空气中可较长时间地保持pH值的稳定性。培养时无需二氧化碳气体环境。
所述第一培养液可参照Quinn等人于1984年公开发表(《生育与不孕》(Fertil Steril.)1984；41:202或1985；44:493.)的文献制备。也可从各商业公司(如，SAGE media)购买。
在另一优选例中，所述第一培养液为IM培养液。
第一培养皿
所述第一培养皿为任意的市售的经标准细胞培养表面处理的细胞培养皿。可以是任何其它公司出售的，也可以是任何形状或尺寸的。
在另一优选例中，所述第一培养皿为Falcon 353037培养皿、康宁公司的430166培养皿或塞默飞公司的150288培养皿。
第二培养液
所述第二培养液的成分与第一培养液相同，区别仅在于第二培养液不含有蛋白。
在另一优选例中，所述第二培养液为M-IM培养液。
第二培养皿
所述第二培养皿为任意的市售的未经表面处理的细胞表面膜。可以是任何其它公司出售的，也可以是任何形状或尺寸的。
在另一优选例中，所述第二培养皿为Falcon 351006培养皿、康宁公司的430589培养皿或塞默飞公司的150340培养皿。
第三培养液
所述第三培养液的成分与第一培养液相同，区别仅在于第三培养液不含缓冲 成分HEPES。所述第三培养液与第一培养液的区别在于其中没有缓冲成份(HEPES)，所以必须在5％二氧化碳环境(二氧化碳培养箱中)下使用。
在另一优选例中，所述第三培养液可参照Quinn等人于1984年公开发表(《生育与不孕》(Fertil Steril.)1984；41:202或1985；44:493.)的文献制备或可从各商业公司(如，SAGE media)购买。
在另一优选例中，所述第三培养液为GM培养液。
本发明技术方案的有益效果是：
1.彻底清除卵子(或受精卵)表面的颗粒细胞，避免了试管婴儿(IVF及ICSI)过程中NICS检测可能遭受的颗粒细胞和/或多余精子污染。
2.避免了胚胎透明带剥除的操作，简化了技术操作步骤，避免了透明带剥除操作可能带来的胚胎损伤风险。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例1.
一、试剂和耗材
1.试剂
1.1含80U/ml的透明质酸酶的HEPES-HTF培养液(Hyaluronidase 80U/ml in HEPES-HTF)(规格：SAGE，产品号：ART4007-A)
1.2M-IM培养液:Quinn's Advantage含HEPES培养液(Quinn's Advantage Medium with HEPES)(规格：SAGE，产品号：ART-1023)
1.3IM培养液:Quinn's Advantage加蛋白受精培养液(Quinn's AdvantageTM Protein Plus Fertilization(TIF)Medium)(规格：SAGE，产品号： ART-1520)
1.4GM培养液:Quinn's Advantage加蛋白卵裂培养液(Quinn's Advantage Protein Plus Cleavage Medium)(规格：SAGE，产品号：ART-1526)
2.耗材
2.1Falcon 351006培养皿
2.2Falcon 363037培养皿
2.3巴斯德吸管
3.操作时机
ICSI的卵子清除颗粒细胞处理是在取卵后2－3小时。
刚从母体中取出的未经处理的卵子，表面附着有大量颗粒细胞，如图1所示。
4.操作程序：
4.1在卵子清洗前，准备好剥卵用吸管(100-120μm)，移卵用吸管(180-200μm)及一根末端烧圆的小玻棒。
4.2
在1个Falcon 353037(聚苯乙烯材质，标准细胞培养表面处理)培养皿中加入1ml IM培养液(ART-1520)；
1个Falcon 351006(聚苯乙烯材质，无处理)培养皿中加入2ml不含蛋白的M-IM培养液(ART-1023)。
4.3把从母体中取出的未经处理的卵子先放入含有IM培养液(ART-1520)的Falcon 353037培养皿中，再加入1ml 80U/ml的透明质酸酶的HEPES-HTF培养液，反复吹吸，持续30秒。在这一步骤中，因培养液中含有带负电的蛋白质，中和了颗粒细胞表面的正电荷。同时，聚苯乙烯材质的培养皿经细胞培养表面处理后，其表面成亲水的电荷中性状态。因此，卵子与其表面附着的颗粒细胞会悬浮在液体培养液中。透明质酸酶的消化作用弱化了颗粒细胞与卵子之间的粘附作用。在吹打的冲洗作用下，部分颗粒细胞即可脱离与卵 子的附着。
经上述步骤3清洗处理过的卵子，其表面颗粒细胞附着明显减少，但仍有颗粒细胞附着。如图2所示。
4.4将表面粘附有剩余颗粒细胞的卵子移入含有不含血清蛋白的M-IM培养液(ART-1023)的Falcon 351006培养皿中。此时，因培养液中不存在蛋白质的负电中和作用，颗粒细胞带有正电荷。同时，Falcon 351006培养皿为未经处理聚苯乙烯材质，其表面为疏水的负电状态。因此，卵子与其表面附着的颗粒细胞就会粘附于培养皿底部表面(如图3所示)。此时，用蘸过含蛋白GM培养液(ART-1526)的玻璃小棒(含蛋白培养液的作用是中和玻璃棒表面的负电荷，避免细胞黏附于玻璃棒上)不断拨动卵子，使卵子在培养皿底部表面滚动。在滚动过程中，卵子表面的剩余颗粒细胞就会粘附于培养皿底部表面与卵子脱离，持续推动卵子滚动，直至其表面的颗粒细胞被彻底清除干净。
经上述步骤3和4清洗处理过的卵子，其表面颗粒细胞被彻底清除，如图4所示。
4.5去除完颗粒细胞后，用吸管(先吸一点含蛋白的培养液)把卵子转移到GM培养液中，放入37℃，5％CO2，5％O2培养箱内培养，随后即可进入常规的单精子注射(ICSI)、胚胎体外培养(IVF及ICSI)和NICS检测流程。
实施例2
采用实施例1的方法，不同之处在于用受精卵代替卵子。而IVF清除受精卵表面颗粒细胞和多余精子的处理时间是在加精4小时(短时受精)或16－20小时(常规受精)之后。
结果发现，IVF受精卵表面的颗粒细胞和多余精子可以被彻底清除。
实施例3
采用实施例1的方法，不同之处在于使用康宁公司的培养皿，并根据文献(Fertil Steril.1984；41:202,1985；44:493.)中的配方自制培养液。
用含人血清白蛋白及HEPES的HTF培养液(根据文献自配)和经标准细胞 培养表面处理的培养皿(购自：康宁；产品号：430166)代替含1ml IM培养液(ART-1520)的Falcon 353037(聚苯乙烯材质，标准细胞培养表面处理)培养皿；
用不含蛋白，但含有HEPES的HTF培养液(根据文献自配)和未经表面处理的培养皿(购自：康宁；产品号：430589)代替含2ml不含蛋白的M-IM培养液(ART-1023)的Falcon 351006(聚苯乙烯材质，无处理)培养皿。
结果表明：两个卵子透明带上附着的颗粒细胞被彻底清除，如图5所示。
对比例1
采用实施例1的方法，不同之处在于在步骤4.4中用IM培养液代替了不含蛋白的M-IM培养液。
结果导致颗粒细胞与培养皿底粘附不牢，无法从卵子表面脱离。处理后的两个卵子表面仍有颗粒细胞残留，如图6所示。
本发明的优点是在对卵子、受精卵或胚胎不产生任何损伤，不触动透明带的情况下提供了一种彻底清除卵子表面颗粒细胞和/或多余精子的方法。其要点在于利用细胞表面、培养液成份(蛋白质)和细胞培养皿底部表面的电荷关系将颗粒细胞和/或多余精子从卵子(或受精卵)表面粘除。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种清洗卵子或受精卵表面的方法，其特征在于，包括步骤：
    (1)将未经处理的卵子或未经处理的受精卵置于第一培养皿中，其中，所述第一培养皿经标准细胞培养表面处理且含有第一培养液，所述第一培养液含蛋白；
    然后加入透明质酸酶处理所述卵子或受精卵，从而得到经第一次处理的卵子或受精卵；
    (2)将经第一次处理的卵子或受精卵移入第二培养皿中，其中，所述第二培养皿未经表面处理且含有第二培养液，所述第二培养液不含蛋白；
    然后用带有第三培养液的棒状工具拨动所述卵子或受精卵，其中，所述第三培养液含蛋白；使颗粒细胞和/或多余精子脱离卵子或受精卵表面，从而被彻底清除干净。
  2. 如权利要求1所述的方法，其特征在于，所述未经处理的卵子是从母体取出2-3小时后的卵子。
  3. 如权利要求1所述的方法，其特征在于，所述未经处理的受精卵是在体外受精4小时或16-20小时后的受精卵。
  4. 如权利要求1所述的方法，其特征在于，所述第一培养液模拟人输卵管液的成分，且用HEPES做缓冲成份。
  5. 如权利要求1所述的方法，其特征在于，所述第一培养皿为Falcon 353037培养皿、康宁公司的430166培养皿或塞默飞公司的150288培养皿。
  6. 如权利要求1所述的方法，其特征在于，步骤(1)中，所述处理的时间为10-60秒。
  7. 如权利要求1所述的方法，其特征在于，所述第二培养液的成分与第一培养液相同，区别仅在于第二培养液不含有蛋白。
  8. 如权利要求1所述的方法，其特征在于，所述第二培养皿为Falcon 351006培养皿、康宁公司的430589培养皿或塞默飞公司的150340培养皿。
  9. 如权利要求1所述的方法，其特征在于，所述第三培养液的成分与第一培养液相同，区别仅在于第三培养液不含缓冲成分HEPES。
  10. 如权利要求1所述的方法，其特征在于，所述颗粒细胞和/或多余精子从卵 子或受精卵表面脱离后附于培养皿底部表面。
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