WO2020237739A1 - 一种高活力精子筛选微流控装置 - Google Patents

一种高活力精子筛选微流控装置 Download PDF

Info

Publication number
WO2020237739A1
WO2020237739A1 PCT/CN2019/091172 CN2019091172W WO2020237739A1 WO 2020237739 A1 WO2020237739 A1 WO 2020237739A1 CN 2019091172 W CN2019091172 W CN 2019091172W WO 2020237739 A1 WO2020237739 A1 WO 2020237739A1
Authority
WO
WIPO (PCT)
Prior art keywords
sperm
microfluidic device
nutrient solution
chamber
screening
Prior art date
Application number
PCT/CN2019/091172
Other languages
English (en)
French (fr)
Inventor
陆金春
Original Assignee
南京精竞生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 南京精竞生物科技有限公司 filed Critical 南京精竞生物科技有限公司
Publication of WO2020237739A1 publication Critical patent/WO2020237739A1/zh

Links

Images

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting

Definitions

  • the invention relates to the field of biotechnology, in particular to a microfluidic device for screening high vigor sperm.
  • the most common manifestations of abnormal spermatogenesis and maturation are oligospermia and asthenospermia, but these patients will always have some sperm with normal motility.
  • the techniques commonly used in laboratories to separate high-quality sperm are the upstream method and density gradient centrifugation. These operations It can cause damage to high-quality sperm, and the poor depth of the small holes on the microfluidic chip can achieve the purpose of natural screening of vigorous sperm; the high vigorous sperm screened out after centrifugation and concentration will still cause damage to the sperm, while the high vigorous sperm will pass When the separation layer reaches another small-volume chamber, it can play a role of natural concentration and avoid centrifugation. Therefore, the microfluidic device can screen and concentrate highly viable sperm.
  • the screened sperm can be directly subjected to in vitro fertilization (IVF) Or intraplasmic sperm injection (ICSI).
  • the present invention provides a high-viability sperm screening microfluidic device, which solves the problem that the existing sperm screening process cannot avoid the centrifugal operation after the sperm upstream, the density gradient centrifugation operation and some of the separation liquid The influence of ingredients The operation causes damage to sperm, and the problem of sperm activity cannot be guaranteed.
  • a high-motility sperm screening microfluidic device comprising a barrel, the inner cavity of the barrel is divided into two chambers inside and outside by a thin annular wall, and The inner chamber and the outer chamber are in a concentric structure.
  • the structure of the present invention is compact and reasonable. It uses the characteristics of sperm upstream and along the interface to avoid the potential adverse effects of various operations such as in vitro centrifugation and washing on the sperm, and can effectively concentrate Highly vigorous sperm.
  • This invention device can obtain the highly vigorous sperm required by assisted reproductive technology, which can greatly improve the success rate of in vitro fertilization.
  • This invention device is not only suitable for human reproduction such as the reproduction center of various medical institutions, but also suitable for animal breeding .
  • the inner chamber is filled with sperm nutrient solution
  • the outer chamber is provided with a semen sample
  • the semen sample just covers the upper surface of the inner chamber
  • the upstream sperm reaches the top of the outer chamber and swims along the interface to the inner chamber.
  • the chamber will eventually converge in the inner chamber.
  • the inner chamber has a radius of 2.5mm and a depth of 20mm
  • the outer chamber has a net annular radius of 2mm and a depth of 20mm
  • the thin annular wall between the inner and outer chambers has a thickness of 0.5mm and a height It is 20mm, the structure is simple and reasonable, and easy to realize.
  • the sperm nutrient solution is a combination of one or more of self-prepared sperm nutrient solution, commercial Earle's solution or BWW solution, and the semen samples are from male patients of infertile couples.
  • the sperm nutrient solution is composed of the following components during the self-preparation process: NaCl 0.721g, MgSO 4 ⁇ 7H 2 O 0.247g, fructose 1g, Na 2 HPO 4 3.581g, KH 2 PO 4 0.136g and Purified water 1000ml, sperm nutrient solution composition is reasonable, can effectively ensure the vitality of sperm, the effect is good.
  • a method for preparing a microfluidic device for screening high motility sperm specifically includes the following steps:
  • step S5. Perform demolding treatment on the male mold processed in step S4 to obtain a microfluidic device with channels.
  • the method for preparing a microfluidic device for screening high vigor sperm of the present invention has a reasonable method, convenient application, is suitable for large-scale industrial production, and has low production cost.
  • a working method of a microfluidic device for high motility sperm screening specifically includes the following steps:
  • T2 Use a sampler to take the sperm nutrient solution and fill it into the inner chamber, the total filling is 400 ⁇ l;
  • T5. Cover the surface of the device with a sealing film, that is, cover the openings of the 4 inner and outer chambers, and then place the device horizontally in a 37°C wet box;
  • This sperm nutrient solution containing highly viable sperm can be directly used for in vitro fertilization or to pick sperm from it for intracytoplasmic sperm injection.
  • the working method of the microfluidic device for screening high motility sperm of the present invention is reasonable and convenient in application. It can enrich sperm with normal morphology, low DNA fragmentation index (DFI) and good motility in vitro for IVF or ICSI.
  • DFI DNA fragmentation index
  • the invention provides a microfluidic device for screening high vigor sperm. Compared with the prior art, it has the following beneficial effects:
  • the high-viability sperm screening microfluidic device uses the characteristics of sperm upstream and along the interface to avoid the potential adverse effects of various operations such as in vitro centrifugation and washing in the existing assisted reproductive technology process on the sperm.
  • the setting is compact and reasonable, and can effectively concentrate highly vigorous sperm.
  • the microfluidic device for screening highly viable sperm can effectively enrich sperm with normal morphology, low DNA fragmentation index (DFI) and good viability for in vitro fertilization by using the difference in volume between the inner and outer chambers of the concentric structure.
  • IVF Intracytoplasmic Sperm Injection
  • ICSI Intracytoplasmic Sperm Injection
  • the high vigor sperm screening microfluidic device has a wide range of applications. It is not only suitable for human reproduction such as the reproduction center of various medical institutions, but also for animal breeding. This device can be used in both human reproduction and animal breeding.
  • Figure 1 is a top view of the microfluidic device of the present invention.
  • Figure 2 is a front view of the inside of the microfluidic device of the present invention.
  • a microfluidic device for screening high motility sperm which specifically includes the following embodiments:
  • a microfluidic device for screening high motility sperm comprising a cylinder body, the inner cavity of the cylinder body is divided into two chambers inside and outside by a thin annular wall, and the inner chamber and the outer chamber are in a concentric structure.
  • the chamber is filled with sperm nutrient solution
  • the outer chamber is provided with a semen sample, and the semen sample just covers the upper surface of the inner chamber, the radius of the inner chamber is 2.5mm, the depth is 20mm, and the net radius of the outer chamber is The thickness of the thin annular wall between the inner and outer chambers is 0.5mm and the height is 20mm.
  • the sperm nutrient solution is a self-prepared sperm nutrient solution, and the semen sample comes from a male patient of an infertile couple.
  • the solution is composed of the following components: NaCl 0.721g, MgSO 4 ⁇ 7H 2 O 0.247g, fructose 1g, Na 2 HPO 4 3.581g, KH 2 PO 4 0.136g and purified water 1000ml.
  • a preparation method of a microfluidic device for screening high motility sperm specifically includes the following steps:
  • step S5. Perform demolding treatment on the male mold processed in step S4 to obtain a microfluidic device with channels.
  • a working method of a microfluidic device for high motility sperm screening specifically includes the following steps:
  • T2 Use a sampler to take the sperm nutrient solution and fill it into the inner chamber, the total filling is 400 ⁇ l;
  • T3 Use a sampler to take the mixed semen sample and fill it into the outer chamber, the total filling is 1ml;
  • T5. Cover the surface of the device with a sealing film, that is, cover the openings of the 4 inner and outer chambers, and then place the device horizontally in a 37°C wet box;
  • This sperm nutrient solution containing highly viable sperm can be directly used for in vitro fertilization or to pick sperm from it for intracytoplasmic sperm injection.
  • a microfluidic device for screening high motility sperm comprising a cylinder body.
  • the inner cavity of the cylinder body is divided into two chambers inside and outside by a thin annular wall, and the inner chamber and the outer chamber are in a concentric structure.
  • the chamber is filled with sperm nutrient solution
  • the outer chamber is provided with a semen sample, and the semen sample just covers the upper surface of the inner chamber, the radius of the inner chamber is 2.5mm, the depth is 20mm, and the net radius of the outer chamber is The thickness of the thin annular wall between the inner and outer chambers is 0.5 mm and the height is 20 mm.
  • the sperm nutrient solution is commercial Earle's solution, and the semen samples are from male patients of infertile couples.
  • a preparation method of a microfluidic device for screening high motility sperm specifically includes the following steps:
  • step S5. Perform demolding treatment on the male mold processed in step S4 to obtain a microfluidic device with channels.
  • a working method of a microfluidic device for high motility sperm screening specifically includes the following steps:
  • T2 Use a sampler to take the sperm nutrient solution and fill it into the inner chamber, the total filling is 400 ⁇ l;
  • T5. Cover the surface of the device with a sealing film, that is, cover the openings of the 4 inner and outer chambers, and then place the device horizontally in a 37°C wet box;
  • This sperm nutrient solution containing highly viable sperm can be directly used for in vitro fertilization or to pick sperm from it for intracytoplasmic sperm injection.
  • a microfluidic device for screening high motility sperm comprising a cylinder body.
  • the inner cavity of the cylinder body is divided into two chambers inside and outside by a thin annular wall, and the inner chamber and the outer chamber are in a concentric structure.
  • the chamber is filled with sperm nutrient solution
  • the outer chamber is provided with a semen sample, and the semen sample just covers the upper surface of the inner chamber
  • the inner chamber has a radius of 2.5mm and a depth of 20mm
  • the outer chamber has a ring-shaped net radius of
  • the thickness of the thin annular wall between the inner and outer chambers is 0.5 mm and the height is 20 mm.
  • the sperm nutrient solution is BWW liquid
  • the semen sample comes from a male patient of an infertile couple.
  • a preparation method of a microfluidic device for screening high motility sperm specifically includes the following steps:
  • step S5. Perform demolding treatment on the male mold processed in step S4 to obtain a microfluidic device with channels.
  • a working method of a microfluidic device for high motility sperm screening specifically includes the following steps:
  • T2 Use a sampler to take the sperm nutrient solution and fill it into the inner chamber, the total filling is 400 ⁇ l;
  • T5. Cover the surface of the device with a sealing film, that is, cover the openings of the 4 inner and outer chambers, and then place the device horizontally in a 37°C wet box;
  • T6 Aspirate a semen sample from the exclusion chamber after 1 hour, and collect the sperm nutrient solution containing highly viable sperm in the inner chamber;
  • This sperm nutrient solution containing highly vigorous sperm can be directly used for in vitro fertilization or to pick sperm from it for intracytoplasmic sperm injection.
  • the shape and size of the inner and outer chambers can be improved without departing from the principle of the present invention, and the processing materials and methods can also be different. These improvements should be regarded as the protection scope of the present invention.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Sustainable Development (AREA)
  • Molecular Biology (AREA)
  • Cell Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Hematology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Dispersion Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Clinical Laboratory Science (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

公开了一种高活力精子筛选微流控装置，包括筒体，所述筒体的内腔通过薄层环形壁分隔成内外两个腔室，且内腔室和外腔室为同心圆结构，内腔室置满精子营养液，外腔室置有精液样本，使精液样本刚好覆盖内腔室上表面。

Description

一种高活力精子筛选微流控装置 技术领域
本发明涉及生物技术领域，具体为一种高活力精子筛选微流控装置。
背景技术
世界范围内不孕不育的发病率约为15％-20％，而且有逐年递增趋势，据WHO预测，在不久的将来，生殖健康问题将是继心脑血管疾病和肿瘤后，威胁人类健康的第三大疾病，不孕不育发病率的增加，以及国家“二孩”政策的放开，大大推动了辅助生殖技术的发展，第一代试管婴儿技术——体外受精-胚胎移植技术(IVF-ET)自1978年开展以来，给许多不育不孕夫妇带来了福音；第二代试管婴儿技术——卵细胞质内单精子注射(ICSI)技术和第三代技术——植入前遗传学诊断(PGD)又进一步丰富和完善了辅助生殖技术，尽管如此，辅助生殖技术过程中许多操作均可能造成精子的损伤，最终导致胚胎不发育或流产，这也是导致试管婴儿技术成功率难以超过50％的重要原因之一，这些有损伤的操作包括：精子上游后的离心操作、密度梯度离心操作和分离液中某些成分的影响等，而要避免这些操作的影响，对高活力精子的筛选应尽可能避免离心操作而又起到浓缩作用。
精子发生与成熟异常最常见的表现为少精子症和弱精子症，但这些患者总会有部分活力正常的精子，实验室常用的分离优质精子的技术为上游法和密度梯度离心法，这些操作对优质精子会造成损伤，而通过微流控芯片上小孔深度差可达到自然筛选活力正常精子的目的；筛选出的高活力精子经过离心浓缩，仍会对精子造成损伤，而高活力精子越过分隔层到达另一小容积腔室时，可起自然浓缩的作用，避免离心操作，故通过此微流控装置可筛选高活力精子并进行浓缩，筛选出的精子可直接进行体外受精(IVF)或卵细胞质内单精子注射(ICSI)。
发明内容
(一)解决的技术问题
针对现有技术的不足，本发明提供了一种高活力精子筛选微流控装置，解决了现有的精子筛选过程中不能避免精子上游后的离心操作、密度梯度离心操作和分离液中某些成分的影响操作对精子造成损伤，无法保证精子活性的问题。
(二)技术方案
为实现以上目的，本发明通过以下技术方案予以实现：一种高活力精子筛选微流控装置，包括筒体，所述筒体的内腔通过薄层环形壁分隔成内外两个腔室，且内腔室和外腔室为同心 圆结构，本发明结构设置紧凑合理，利用精子上游和沿界面泳动特性，避免了体外离心、洗涤等各种操作对精子的潜在不利影响，并可有效浓缩高活力精子，利用此发明装置可获得辅助生殖技术所需的高活力精子，可以大大提高体外受精的成功率，此发明装置不仅适用于人类生殖如各医疗机构的生殖中心，而且适用于动物育种。
优选的，所述内腔室置满精子营养液，所述外腔室置有精液样本，且使精液样本刚好覆盖内腔室上表面，上游精子到达外腔室顶端后沿界面泳动至内腔室，将最终汇聚在内腔室。
优选的，所述内腔室半径为2.5mm，深度为20mm，所述外腔室环形净半径为2mm，深度为20mm，所述内外腔室之间的薄层环形壁厚度为0.5mm，高度为20mm，结构简单合理，易于实现。
优选的，所述精子营养液为自行配制精子营养液、商用Earle’s液或BWW液中的一种或多种的组合，且精液样本来自不育夫妇的男性患者。
优选的，所述精子营养液在自行配制过程中，由以下组分组成：NaCl 0.721g、MgSO 4·7H 2O 0.247g、果糖1g、Na 2HPO 4 3.581g、KH 2PO 4 0.136g和纯净水1000ml，精子营养液组分合理，可以有效保证精子的活力，效果好。
优选的，一种高活力精子筛选微流控装置的制备方法，具体包括以下步骤：
S1、按微流控装置设计图制备镍基、单晶硅或玻璃阳模；
S2、将聚甲基丙烯酸甲酯PMMA片材按阳模尺寸裁剪成小片，洗净、烘干；
S3、将步骤S2处理后的聚甲基丙烯酸甲酯PMMA片材装载至阳模上，压紧；
S4、利用压片机在加热、加压下将PMMA封合12-15分钟，通水冷却至室温；
S5、将步骤S4处理后的阳模进行脱模处理，即可得到带有通道的微流控装置。
本发明所述的一种高活力精子筛选微流控装置的制备方法，方法合理，应用便捷，适合大规模工业化生产，生产成本低。
优选的，一种高活力精子筛选微流控装置的工作方法，具体包括以下步骤：
T1、用去离子水冲洗内、外腔室后，于37℃烘箱中烘干；
T2、用加样器取精子营养液灌装至内腔室，灌装总量为400μl；
T3、用加样器取混匀的精液样本灌装至外腔室，灌装总量为1ml；
T4、同法取精子营养液和另外三个混匀的精液样本，分别灌装至其他三个内外腔室；
T5、用密封膜封盖装置表面，即封盖4个内外腔室的开口，然后将装置水平置于37℃湿盒中；
T6、1小时后吸除外腔室中精液样本，并收集内腔室中含有高活力精子的精子营养液；
T7、此含高活力精子的精子营养液可直接用于体外受精或从中挑取精子进行卵细胞质内单精子注射。
本发明所述的一种高活力精子筛选微流控装置的工作方法，方法合理，应用方便，可以在体外富集形态正常、DNA碎片化指数(DFI)低、活力好的精子用于IVF或ICSI。
(三)有益效果
本发明提供了一种高活力精子筛选微流控装置。与现有技术相比具备以下有益效果：
(1)、该高活力精子筛选微流控装置，通过利用精子上游和沿界面泳动特性，避免了现有辅助生殖技术过程中体外离心、洗涤等各种操作对精子的潜在不利影响，结构设置紧凑合理，并可有效浓缩高活力精子。
(2)、该高活力精子筛选微流控装置，通过利用同心圆结构内外腔室体积的差异，可有效富集形态正常、DNA碎片化指数(DFI)低、活力好的精子用于体外受精(IVF)或卵细胞质内单精子注射(ICSI)，可获得辅助生殖技术所需的高活力精子，以大大提高体外受精的成功率。
(3)、该高活力精子筛选微流控装置，适用范围广，不仅适用于人类生殖如各医疗机构的生殖中心，而且适用于动物育种，人类生殖和动物育种中均可使用此装置。
附图说明
图1为本发明微流控装置的俯视图；
图2为本发明微流控装置内部的正视图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
请参阅图1-2，本发明实施例提供三种技术方案：一种高活力精子筛选微流控装置，具体包括以下实施例：
实施例1
一种高活力精子筛选微流控装置，包括筒体，所述筒体的内腔通过薄层环形壁分隔成内外两个腔室，且内腔室和外腔室为同心圆结构，内腔室置满精子营养液，所述外腔室置有精液样本，且使精液样本刚好覆盖内腔室上表面，内腔室半径为2.5mm，深度为20mm，所述外腔室环形净半径为2mm，深度为20mm，所述内外腔室之间的薄层环形壁厚度为0.5mm，高度为20mm，精子营养液为自行配制精子营养液，且精液样本来自不育夫妇的男性患者，精子营养液在自行配制过程中，由以下组分组成：NaCl 0.721g、MgSO 4·7H 2O 0.247g、果糖1g、Na 2HPO 4 3.581g、KH 2PO 4 0.136g和纯净水1000ml。
一种高活力精子筛选微流控装置的制备方法，具体包括以下步骤：
S1、按微流控装置设计图制备镍基、单晶硅或玻璃阳模；
S2、将聚甲基丙烯酸甲酯PMMA片材按阳模尺寸裁剪成小片，洗净、烘干；
S3、将步骤S2处理后的聚甲基丙烯酸甲酯PMMA片材装载至阳模上，压紧；
S4、利用压片机在加热、加压下将PMMA封合12-15分钟，通水冷却至室温；
S5、将步骤S4处理后的阳模进行脱模处理，即可得到带有通道的微流控装置。
一种高活力精子筛选微流控装置的工作方法，具体包括以下步骤：
T1、用去离子水冲洗内、外腔室后，于37℃烘箱中烘干；
T2、用加样器取精子营养液灌装至内腔室，灌装总量为400μl；
T3、用加样器取混匀的精液样本灌装至外腔室，灌装总量为1ml；
T4、同法取精子营养液和另外三个混匀的精液样本，分别灌装至其他三个内外腔室；
T5、用密封膜封盖装置表面，即封盖4个内外腔室的开口，然后将装置水平置于37℃湿盒中；
T6、1小时后吸除外腔室中精液样本，并收集内腔室中含有高活力精子的精子营养液；
T7、此含高活力精子的精子营养液可直接用于体外受精或从中挑取精子进行卵细胞质内单精子注射。
实施例2
一种高活力精子筛选微流控装置，包括筒体，所述筒体的内腔通过薄层环形壁分隔成内外两个腔室，且内腔室和外腔室为同心圆结构，内腔室置满精子营养液，所述外腔室置有精液样本，且使精液样本刚好覆盖内腔室上表面，内腔室半径为2.5mm，深度为20mm，所述 外腔室环形净半径为2mm，深度为20mm，所述内外腔室之间的薄层环形壁厚度为0.5mm，高度为20mm，精子营养液为商用Earle’s液，且精液样本来自不育夫妇的男性患者。
一种高活力精子筛选微流控装置的制备方法，具体包括以下步骤：
S1、按微流控装置设计图制备镍基、单晶硅或玻璃阳模；
S2、将聚甲基丙烯酸甲酯PMMA片材按阳模尺寸裁剪成小片，洗净、烘干；
S3、将步骤S2处理后的聚甲基丙烯酸甲酯PMMA片材装载至阳模上，压紧；
S4、利用压片机在加热、加压下将PMMA封合12-15分钟，通水冷却至室温；
S5、将步骤S4处理后的阳模进行脱模处理，即可得到带有通道的微流控装置。
一种高活力精子筛选微流控装置的工作方法，具体包括以下步骤：
T1、用去离子水冲洗内、外腔室后，于37℃烘箱中烘干；
T2、用加样器取精子营养液灌装至内腔室，灌装总量为400μl；
T3、用加样器取混匀的精液样本灌装至外腔室，灌装总量为1ml；
T4、同法取精子营养液和另外三个混匀的精液样本，分别灌装至其他三个内外腔室；
T5、用密封膜封盖装置表面，即封盖4个内外腔室的开口，然后将装置水平置于37℃湿盒中；
T6、1小时后吸除外腔室中精液样本，并收集内腔室中含有高活力精子的精子营养液；
T7、此含高活力精子的精子营养液可直接用于体外受精或从中挑取精子进行卵细胞质内单精子注射。
实施例3
一种高活力精子筛选微流控装置，包括筒体，所述筒体的内腔通过薄层环形壁分隔成内外两个腔室，且内腔室和外腔室为同心圆结构，内腔室置满精子营养液，所述外腔室置有精液样本，且使精液样本刚好覆盖内腔室上表面，内腔室半径为2.5mm，深度为20mm，所述外腔室环形净半径为2mm，深度为20mm，所述内外腔室之间的薄层环形壁厚度为0.5mm，高度为20mm，精子营养液为BWW液，且精液样本来自不育夫妇的男性患者。
一种高活力精子筛选微流控装置的制备方法，具体包括以下步骤：
S1、按微流控装置设计图制备镍基、单晶硅或玻璃阳模；
S2、将聚甲基丙烯酸甲酯PMMA片材按阳模尺寸裁剪成小片，洗净、烘干；
S3、将步骤S2处理后的聚甲基丙烯酸甲酯PMMA片材装载至阳模上，压紧；
S4、利用压片机在加热、加压下将PMMA封合12-15分钟，通水冷却至室温；
S5、将步骤S4处理后的阳模进行脱模处理，即可得到带有通道的微流控装置。
一种高活力精子筛选微流控装置的工作方法，具体包括以下步骤：
T1、用去离子水冲洗内、外腔室后，于37℃烘箱中烘干；
T2、用加样器取精子营养液灌装至内腔室，灌装总量为400μl；
T3、用加样器取混匀的精液样本灌装至外腔室，灌装总量为1ml；
T4、同法取精子营养液和另外三个混匀的精液样本，分别灌装至其他三个内外腔室；
T5、用密封膜封盖装置表面，即封盖4个内外腔室的开口，然后将装置水平置于37℃湿盒中；
T6、1小时后吸除外腔室中精液样本，并收集内腔室中含有高活力精子的精子营养液；
T7、此含高活力精子的精子营养液可直接用于体外受精或从中挑取精子进行卵细胞质内单精子注射。
本发明在不脱离本发明原理的前提下，内外腔室的形状和尺寸均可进行改进，加工材料和方式亦可不同，这些改进均应视为本发明的保护范围。
需要说明的是，在本文中，诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。

Claims (7)

  1. 一种高活力精子筛选微流控装置，包括筒体，其特征在于：所述筒体的内腔通过薄层环形壁分隔成内外两个腔室，且内腔室和外腔室为同心圆结构。
  2. 根据权利要求1所述的一种高活力精子筛选微流控装置，其特征在于：所述内腔室置满精子营养液，所述外腔室置有精液样本，且使精液样本刚好覆盖内腔室上表面。
  3. 根据权利要求1所述的一种高活力精子筛选微流控装置，其特征在于：所述内腔室半径为2.5mm，深度为20mm，所述外腔室环形净半径为2mm，深度为20mm，所述内外腔室之间的薄层环形壁厚度为0.5mm，高度为20mm。
  4. 根据权利要求2所述的一种高活力精子筛选微流控装置，其特征在于：所述精子营养液为自行配制精子营养液、商用Earle’s液或BWW液中的一种或多种的组合，且精液样本来自不育夫妇的男性患者。
  5. 根据权利要求4所述的一种高活力精子筛选微流控装置，其特征在于：所述精子营养液在自行配制过程中，由以下组分组成：NaCl 0.721g、MgSO 4·7H 2O 0.247g、果糖1g、Na 2HPO 43.581g、KH 2PO 4 0.136g和纯净水1000ml。
  6. 根据权利要求1-5任一项所述的一种高活力精子筛选微流控装置，其特征在于：其制备方法具体包括以下步骤：
    S1、按微流控装置设计图制备镍基、单晶硅或玻璃阳模；
    S2、将聚甲基丙烯酸甲酯PMMA片材按阳模尺寸裁剪成小片，洗净、烘干；
    S3、将步骤S2处理后的聚甲基丙烯酸甲酯PMMA片材装载至阳模上，压紧；
    S4、利用压片机在加热、加压下将PMMA封合12-15分钟，通水冷却至室温；
    S5、将步骤S4处理后的阳模进行脱模处理，即可得到带有通道的微流控装置。
  7. 根据权利要求1-5任一项所述的一种高活力精子筛选微流控装置，其特征在于：其工作方法具体包括以下步骤：
    T1、用去离子水冲洗内、外腔室后，于37℃烘箱中烘干；
    T2、用加样器取精子营养液灌装至内腔室，灌装总量为400μl；
    T3、用加样器取混匀的精液样本灌装至外腔室，灌装总量为1ml；
    T4、同法取精子营养液和另外三个混匀的精液样本，分别灌装至其他三个内外腔室；
    T5、用密封膜封盖装置表面，即封盖4个内外腔室的开口，然后将装置水平置于37℃湿盒中；
    T6、1小时后吸除外腔室中精液样本，并收集内腔室中含有高活力精子的精子营养液；
    T7、此含高活力精子的精子营养液可直接用于体外受精或从中挑取精子进行卵细胞质内单精子注射。
PCT/CN2019/091172 2019-05-30 2019-06-13 一种高活力精子筛选微流控装置 WO2020237739A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910465724.6A CN110172390A (zh) 2019-05-30 2019-05-30 一种高活力精子筛选微流控装置
CN201910465724.6 2019-05-30

Publications (1)

Publication Number Publication Date
WO2020237739A1 true WO2020237739A1 (zh) 2020-12-03

Family

ID=67696882

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/091172 WO2020237739A1 (zh) 2019-05-30 2019-06-13 一种高活力精子筛选微流控装置

Country Status (2)

Country Link
CN (1) CN110172390A (zh)
WO (1) WO2020237739A1 (zh)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010056755A2 (en) * 2008-11-11 2010-05-20 Craig H Randall Microfluidic embryo and gamete culture systems
CN201678676U (zh) * 2010-04-30 2010-12-22 山东省计划生育科学技术研究所 内套管式分离筛选精子管
CN202131314U (zh) * 2011-06-28 2012-02-01 深圳市博锐德生物科技有限公司 一种用于体外受精挑选精子的培养皿
WO2015077333A1 (en) * 2013-11-20 2015-05-28 Brigham And Women's Hospital , Inc. System and method for sperm sorting
CN105154320A (zh) * 2015-10-12 2015-12-16 深圳市博锐德生物科技有限公司 透明质酸及其盐或衍生物包被的单精子选择装置及应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4129722B2 (ja) * 2002-04-05 2008-08-06 ニプロ株式会社 精子選択採取用シャーレ
KR101855191B1 (ko) * 2016-09-01 2018-05-04 을지대학교 산학협력단 난자 및 초기 배아 배양용기
CN107475078A (zh) * 2017-10-12 2017-12-15 南京精竞生物科技有限公司 一种基于微流控技术的精子筛选与体外受精仿生装置

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010056755A2 (en) * 2008-11-11 2010-05-20 Craig H Randall Microfluidic embryo and gamete culture systems
CN201678676U (zh) * 2010-04-30 2010-12-22 山东省计划生育科学技术研究所 内套管式分离筛选精子管
CN202131314U (zh) * 2011-06-28 2012-02-01 深圳市博锐德生物科技有限公司 一种用于体外受精挑选精子的培养皿
WO2015077333A1 (en) * 2013-11-20 2015-05-28 Brigham And Women's Hospital , Inc. System and method for sperm sorting
CN105154320A (zh) * 2015-10-12 2015-12-16 深圳市博锐德生物科技有限公司 透明质酸及其盐或衍生物包被的单精子选择装置及应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LI, JINGCHUN ET AL.: "Study on a New Sperm Separator by Sperm Flowing Upstream in Animals", CHINESE JOURNAL OF ANIMAL SCIENCE, vol. 53, no. 12, 31 December 2017 (2017-12-31), DOI: 20191206183943X *

Also Published As

Publication number Publication date
CN110172390A (zh) 2019-08-27

Similar Documents

Publication Publication Date Title
Bhartiya et al. Novel insights into adult and cancer stem cell biology
CN107475078A (zh) 一种基于微流控技术的精子筛选与体外受精仿生装置
Rappaport et al. Establishment of cleavage furrows by the mitotic spindle
CN103469309B (zh) 一种组织匀浆法分离活细胞构建细胞库的方法
CN104762257B (zh) 一种从脐带制备间充质干细胞的方法
CN106038597B (zh) 间充质干细胞在制备治疗急性肺损伤制剂中的应用
Strug et al. Making more womb: clinical perspectives supporting the development and utilization of mesenchymal stem cell therapy for endometrial regeneration and infertility
CN102719396A (zh) 人体脂肪干细胞快速提取方法及其专用人脂肪组织消化剂
CN106265740A (zh) 脐带间充质干细胞联合黄芪多糖在制备治疗高血糖和糖尿病肾病药物中的应用
CN109337863A (zh) 一种预测化合物对脂肪性肝病毒性的体外模型构建方法
WO2020237739A1 (zh) 一种高活力精子筛选微流控装置
CN103416393A (zh) 一种植物线虫临时玻片的制作方法
CN104990780A (zh) 一种制作细胞块的专用离心管和方法
Xu et al. Merits and challenges of iPSC-derived organoids for clinical applications
Nakahama et al. Generation of cardiomyocytes from pluripotent stem cells
CN1876161A (zh) 治疗乳腺增生的乳癖康药物制剂及制法和质量控制方法
CN112326964A (zh) 筛选靶细胞的方法、试剂盒及其用途
CN112326952A (zh) 筛选细胞的方法、试剂盒及其用途
CN204988821U (zh) 一种制作细胞块的专用离心管
CN107858330B (zh) 一种小鼠骨髓造血干/祖细胞体外衰老细胞模型的建立方法
CN108715829A (zh) 一种提高人类体外受精胚胎发育率的方法
Lowell et al. Regeneration of complete hydra from isolated epidermal explants
CN204342767U (zh) 体外受精培养筛
CN110592005A (zh) 间充质干细胞的分离方法
Kitala et al. Isolation, culturing and preparation for transplantation of amniotic mesenchymal stem cells Repetitive and reproducible laboratory, technical protocol.

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19930585

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19930585

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 23/06/2022)

122 Ep: pct application non-entry in european phase

Ref document number: 19930585

Country of ref document: EP

Kind code of ref document: A1