CN1441652A - Microfluidic channel embryo and/or oocyte handling, analysis and biological evaluation - Google Patents

Microfluidic channel embryo and/or oocyte handling, analysis and biological evaluation Download PDF

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CN1441652A
CN1441652A CN 01812654 CN01812654A CN1441652A CN 1441652 A CN1441652 A CN 1441652A CN 01812654 CN01812654 CN 01812654 CN 01812654 A CN01812654 A CN 01812654A CN 1441652 A CN1441652 A CN 1441652A
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embryo
embryos
fluid
constriction
fluid passage
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戴维·J·毕比
伊恩·K·格拉斯哥
马修·B·惠勒
亨利·泽林格
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伊利诺伊大学受托管理委员会
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    • B01L2400/0415Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
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    • B01L2400/086Passive control of flow resistance using baffles or other fixed flow obstructions

Abstract

Microfluidic embryo scaled channels (14) for handling and positioning embryos provide the opportunity to evaluate and treat embryos in improved manners. Fluid flow is used to move and position embryos within microfluidic channels and channel geometrics may be used to place embryos at specific locations. Surface properties and compliance (deformation) properties of embryos are evaluated as a predictor of viability. The microfluidic channels provide the opportunity for fine controls of pressure to conduct various evalutions at forces slightly below which damage to embryos is known to occur. Measurement of the distance and/or which embryos roll in a same pressure gradient microfluidic channel provides information, with healthy embryos traveling slower or a shorter distance as they demonstrate more stiction to channel walls. Positioned at a constriction (14a, 14b, 24, 26), health embryos also appear to deform less than unhealthy embryos that are more readily pulled into a constriction. In addition, healthy embryos appear to resume their shape better. Fluid from microfluidic channels is easily collected downstream without altering the embryo environment, providing a better opportunity for chemical analysis of fluid chemical analysis than convention manual handling and sampling techniques. Zona pellucida removal of mammalian embryos is achieved as embryos are moved through flow to a precise location where lysing agent can be washed over the embryo to achieve zona removal. Cumulus removal is realized with a series of constrictions to cut cumulus followed by fluid flows to remove cut cumulus from the embryo.

Description

微流控通道胚胎和/或卵母细胞处理,分析和生物评价 Microfluidic channel embryos and / or oocytes processing, analysis and biological evaluation

技术领域 FIELD

本发明总的来说涉及胚胎的处理。 The present invention generally relates to the treatment of embryos. 本发明还涉及卵母细胞(未受精胚胎),和卵的处理。 The present invention further relates to the oocyte (unfertilised embryos), and treated eggs. 这里所用的胚胎包含卵母细胞,卵以及受精胚胎。 As used herein comprises oocytes embryos, egg fertilization and embryo. 本发明特别涉及胚胎的微流控处理,以用来培养、操作和分析。 The present invention particularly relates to microfluidic processing embryos, to be used to culture, manipulation and analysis.

背景技术 Background technique

辅助繁殖技术的工艺的重要性和使用频率不断增加,该技术中对来自哺乳动物生物源单独处理。 Assisted reproductive technology techniques importance and frequency of use is increasing, the art processing of a mammal from a biological source alone. 这些技术对通过独立的有性繁殖无法生孩子的人而言具有最直接的好处。 These technologies have the most direct benefits in terms of sexual reproduction by independent people can not have children. 农业也日益依赖这种辅助繁殖技术。 Agriculture is increasingly dependent on such aid reproductive technology. 胚胎操作用于家畜繁殖中,以便控制这样的事情,即使牛更快地遗传进化,并允许一个个体优异的母牛或公牛的遗传特征传给大量的后代,其后代数量超过通过独立有性繁殖所得的后代数量。 Embryo manipulation for livestock breeding in order to control such things, even faster bovine genetic evolution, and allow a superior individual cow or bull's genetic characteristics to pass a large number of offspring, which exceeds the number of progeny by sexual reproduction is independently the resulting number of offspring.

由于基因操作手术,无性繁殖系化和体外受精(IVF)技术的发展,家畜胚胎操作变得越来越常规化。 Due to genetic manipulation operation, development and clonal in vitro fertilization (IVF) technology, livestock embryo manipulation become more and more routine. 家畜胚胎操作的总目标是增加生产效率,特别是涉及繁殖、产奶量或特殊的奶成分的生产效率,降低脂肪含量,瘦肉组织生长并降低对特定病的易感性。 The overall objective is to increase embryo manipulation of livestock production efficiency, particularly to breeding, milk or milk components specific productivity, reduce fat, lean tissue growth and reduced susceptibility to a particular disease. 胚胎转移还用来引入或拯救有价值的种质(germplasm),繁殖稀少的饲养动物,例如濒于灭绝的外来物种。 Embryo transfer is also used to introduce or to save valuable germplasm (germplasm), rare breeding domestic animals, e.g. endangered exotic species.

由于成本和较低的成功率,给人以及家畜使用这些辅助繁殖技术带来明显的负担。 Because of lower costs and success rates, and giving livestock such auxiliary reproductive technologies bring significant burden. 对人的繁殖来说,这种成本和失败增加了感情和经济负担。 Human reproduction, this failure costs and increased emotional and economic burden. 另外,预防失败的安全措施常导致不希望的或很难处理的多胎,以及需要保养额外保存的胚胎,并且在以后的一些时间点作出的额外的困难决定。 In addition, security measures to prevent failure often leads to unwanted or difficult to deal with multiple births, and the need for additional maintenance to save embryos, and additional difficulties decision was made after some point in time. 家畜繁殖则主要关心费用问题。 Livestock breeding is mainly concerned with costs.

繁殖和测试技术以及其它胚胎处理技术的失败率主要归因于在执行这些技术时对胚胎而言效果显著的处理和操作。 Reproductive failure rates and testing techniques and other techniques embryos treated mainly due to handling operations and the implementation of these techniques in the embryo significant effect. 最近几年,动物繁殖技术得以提高,但在动物繁殖中采用的工艺并没有显著变化。 In recent years, animal breeding technology improved, but the process used in Animal Reproduction and did not change significantly. 精密镗孔的玻璃移液管仍是胚胎学家使用的其中一个基本的工具。 Precision bore glass pipette is still one of the basic tools used embryologist. 利用标准的皮氏培养皿,例如卵的体外成熟(IVM),体外受精和胚胎培养(EC)的程序中需要为每个程序若干次挑选和放置单独的卵和胚胎。 Using standard petri dishes, egg such as in vitro maturation (the IVM), in vitro fertilization and embryo culture (EC) program requires several separate pick and place the eggs and embryos for each program.

从一个皮氏培养皿至另一个皮氏培养皿的这种处理和移动明显存在潜在的损坏或污损。 From one petri dish to another Petri dish handling and moving this potential significantly damaged or defaced. 尽管如此,更重要的可能是在皮氏培养皿中稳定的胚胎无法模拟相应的自然界生物繁殖条件。 Nevertheless, it may be more important in a Petri dish stable embryo can not simulate the corresponding natural biological breeding conditions. 人们付出了一些努力,以便使皮氏培养皿中的胚胎借助皮氏培养皿的搅动而移动,但这是随意的方法。 People pay some effort, so that the embryos in a Petri dish with agitation to move the petri dish, but this method is arbitrary. 在此,由于手动皮氏培养皿进行传统的胚胎处理的方法需要相对大量的生物培养基,这也会因此产生费用问题。 Here, since the conventional manual Petri dishes for embryo method requires a relatively large amount of processing biological medium, which will therefore have cost implications. 牛胚胎用移液管和大的贵重的操纵器独立处理。 Bovine embryos precious transfusing liquid pipe manipulator and a large independent processing. 包括用于人胚胎培养的生长因子的大量生物培养基的使用使相应的体外程序花费更高。 Including a large amount of human embryo culture media of biological growth factor of the corresponding in vitro procedure more costly. 家畜生长刺激素例如每50微克花费超过200美元。 Livestock growth-stimulating hormone for example, every 50 micrograms spent more than $ 200.

当胚胎生长时,这种静态培养系统也不允许改变培养基内的环境。 When the growth of the embryo, this static culture system does not allow changes in the environmental medium. 具有流动的培养基的现有培养系统具有小至0.2至0.5毫升的培养室。 Existing culture medium having a flow system having a small 0.2 to 0.5 ml of the culture chamber. 然而,培养容积大于所需,且培养基补充太快。 However, the volume of the culture is greater than desired, and supplemented fast. 促进生长的内生生长因子被稀释和冲走。 Promote the growth of endogenous growth factors are diluted and washed away. 当使用贵重的生长因子例如IGF-II(每50微克200美元)时,所需要的大体积的培养基大大地增加了成本。 When using a valuable growth factors such as when a large volume of IGF-II (US $ 200 per 50 [mu] g), required to greatly increase the cost of the medium. 另外,现有的系统不能跟踪单独的胚胎。 Further, the conventional system can not follow a single embryo.

传统的处理技术在评价胚胎方面还能力有限。 Conventional processing techniques in the evaluation of the embryo further limited capacity. 能够对预植入胚胎包括胚胎、精子或卵子的单倍体核受精卵和卵母细胞进行评价将提供更佳的植入成功率。 Can be evaluated pre-implantation embryos fertilized oocytes and nuclear haploid cells include embryos, sperm or ovum implant will provide a better success rate. 当前,我们对大多数胚胎在其转移到容器中之前对其形态学进行评价。 Currently, the majority of our embryo before it is transferred to a container to evaluate its morphology. 形态学检查将挑选出具有严重缺陷的胚胎,但它不是生存能力的高度可靠的指示器。 Morphological examination would single out the embryo with severe defects, but it is not a highly reliable indicator of viability. 目前采用的是在一段时间内进行化学监测,但需要在一段时间内进行大量的测量。 It is currently used to monitor the chemical over time, but requires a large number of measurements over time. 结果是生存能力的更佳的预测器,大约80%数量级,但许多胚胎在监测程序中没有生存下来。 The result is a better predictor of viability, about 80% of the order, but many embryos do not survive in the monitoring program. 这样需要使用补充的胚胎。 This requires the use of supplementary embryos. 从而导致多胎和其它并发症,并伴有额外的劳动力成本。 Resulting in multiple births and other complications, and accompanied by additional labor costs.

传统的技术还提供去除透明带的苛刻的方法,这是制造嵌合体(chimeric)胚胎中关键的步骤。 The conventional technology also provides a method of removing harsh zona pellucida, which is manufactured chimera (chimeric) embryos critical step. 传统上,采用移液管口将一个胚胎从包含培养基的一个组织培养皿吸取至一个包含酸性培养基的培养皿中。 Traditionally, a pipette from a port to a tissue culture dish containing embryo medium to draw a petri dish containing the acidic medium. 胚胎留在培养基中一段时间(几十秒),然后用移液管口吸取至包含新鲜的培养基的培养皿中。 Embryos left in the medium for some time (tens of seconds), and then with a pipette suction opening to a fresh petri dish containing medium. 接下来,胚胎在移液管内进出冲刷几次,以便迅速冲散所有酸性培养基,并使之对细胞膜的损害最小。 Next, the embryo out of the pipette flushing several times, all the acidic medium in order to quickly break up and minimize the damage to the cell membrane. 移液管口的开口与胚胎大小几乎相同,因此,冲刷在胚胎上产生剪切应力。 Opening of the pipette opening and the embryo is almost the same size, therefore, shear stress on the flushing the embryo. 因此,移液管口操作的不精确将使胚胎受损的几率大大增加。 Thus, opening operation of the pipette inaccuracies will greatly increase the risk of fetal damage.

这样,需要一种改进的胚胎处理装置和方法,它以公知的胚胎处理技术解决上述问题。 Thus, a need for an improved apparatus and method for processing embryos, embryos that known processing techniques solve the above problems. 一种改进的胚胎处理装置和方法应提供一种改进的方法来模拟自然条件。 An improved apparatus and method for processing embryos should provide an improved method to simulate natural conditions. 还应提供一种构件模块,例如胚胎培育系统,胚胎分析系统,胚胎存储系统和类似的系统。 Member is also provided a module, such as embryonic cultivation system, the system analyzes the embryo, the embryo storage systems and similar systems. 较大和/或大功率和精确的仪器可基于该构件模块。 Larger and / or power and based on the precise instrument module member. 另外还需要改进对胚胎生存能力的评价。 Further evaluation of the need to improve embryo viability.

发明内容 SUMMARY

这些需要符合或超出现有的微流控胚胎处理装置和方法。 These need to meet or exceed conventional microfluidic apparatus and method for processing embryos. 本发明模拟胚胎的生物旋转。 Rotating biological simulation embryo present invention. 一个胚胎流动通道通过流体使嵌入其中的胚胎移动,并且其尺寸确定为与待处理的特定类型的一个或多个胚胎为相同数量级。 A flow passage through the fluid so that the embryo is embedded embryo movement therein, and is dimensioned to a particular type or more embryos to be treated the same order. 所确定的尺寸和流体连通产生了胚胎的模拟的生物旋转。 The determined size and in fluid communication with the rotating biological simulation produced embryos. 另外,在一个或多个胚胎周围流动的流体避免了其停滞,降低了一个或多个胚胎产生“褥疮”的可能性。 Further, in one or more embryos fluid flowing around it avoids stagnation, reduces the possibility of one or more embryos "bedsore" in.

本发明还允许生物培养基流体逐渐改变,与手工重复地将一个胚胎从在移液管或皮氏培养皿中的一种培养基转移到另一种培养基中相比较,这具有显著的优点。 The present invention also allows the biological fluid medium is gradually changed, and is repeatedly manually embryo from a pipette or one kind of medium petri dishes are transferred to another medium compared with significant advantages which . 培养基的逐渐改变避免了在局部环境种突然改变所带来的冲击。 Medium gradually changed to avoid the impact of sudden changes in the local environment caused by species. 本发明的微流控系统还允许一个胚胎与其它胚胎共同培养,一个或多个胚胎与胚胎上游的细胞共同培养,并保持对与对象胚胎分享共同的生物培养基的胚胎进行独立控制培养,从而确保试验胚胎具有与对象胚胎相同的环境条件。 The microfluidic system of the present invention also allows an embryo co-culture with embryonic other, one or more co-culture with embryonic upstream cell embryos, and maintain the biological medium share common object embryos cultured embryos be controlled independently, whereby ensure that the test subject has the embryo embryo same environmental conditions.

本发明的其它方面涉及微流控胚胎经处理以便进行操作、评价和定位胚胎的广义原理上的特殊使用。 Other aspects of the present invention relates to a microfluidic embryos treated for particular use in a broad sense operation principle, and positioning evaluation embryos. 一方面涉及使用一系列的逐渐收缩部来去除胚胎周围的材料。 In one aspect involves the use of a series of tapering part to the removal of material surrounding the embryo. 这一点从从卵母细胞去掉周围的堆积物(cumulus)得到证明。 This is evidenced from the surrounding remove deposits from the oocyte (cumulus). 最初的几个收缩部切除堆积物,然后在最终的小的收缩部该堆积物从卵母细胞上吸出,该收缩部的尺寸为可防止卵母细胞通过。 The first few constriction deposit removal, and then aspirated from the deposit oocytes little shrinkage in the final portion, the size of the constriction is prevented oocyte.

根据本发明的基本原理,胚胎评价也可实现。 According to the basic principles of the invention, evaluation of the embryo can be achieved. 在一个优选的评价中,对胚胎的表面性能和柔顺性(变形)进行评价。 In a preferred evaluation, the surface properties of the embryo and compliance (deformation) were evaluated. 微流控通道提供对压力进行微调控制的机会,以便在略低于公知的发生胚胎损害的力时进行不同的评价。 Microfluidic channels to provide the opportunity for fine control of pressure, for different evaluation when a force slightly lower than the known occurrence of damage to the embryo. 在一个相同压力梯度微流控通道中对胚胎滚动的距离和/或速度进行测量可提供的信息是,健康的胚胎表现为与通道壁具有更大的静摩擦,因而它们移动较慢或移动较短的距离。 Measurements may provide information embryonic rolling distance and / or speed of the same at a pressure gradient in the microfluidic channel, the performance of healthy embryos channel wall has a larger static friction, so that they move slower moving or shorter the distance. 当定位一在收缩部时,健康的胚胎液还表现为其变形小于不健康的胚胎,该不健康的胚胎更容易牵拉到收缩部内。 When positioned at a constriction, healthy embryos were also exhibit its deformation smaller than healthy embryo, the embryo unhealthy more easily pulled into the constriction. 另外,健康的胚胎表现为能够更好地恢复其形状。 In addition, the performance of healthy embryos are better able to recover its shape.

微流控通道中的流体在不改变胚胎环境的条件下很容易在下游收集,从而提供比传统的人工处理和取样技术更好的机会来进行流体化学分析。 Microfluidic channel downstream of the fluid is easily collected without changing the environmental conditions of the embryo, so as to provide better than traditional manual sampling techniques and processing opportunity to fluid chemical analysis. 另外,从微流控通道收集的全部流体已经过胚胎。 Further, all of the collected fluid from the microfluidic channel has been embryos. 这提供比围绕皮氏培养皿中的胚胎的流体停滞更好的评价信息。 This provides a ratio of the fluid surrounding the embryos in a Petri dish stagnation better evaluation information.

通过本发明,培养基可连续或周期性的经过胚胎,并在下游收集,这消除了在皮氏培养皿技术中所需要的额外处理。 By the present invention, the medium may be continuously or periodically through embryos, and collected downstream, which eliminates the additional processing techniques in a petri dish required. 本发明提供更一致的流体样品,因为流体可以相同的方式重复收集,反之,从皮氏培养皿中采集的样品可基于吸入培养基的移液管的放置而改变,即它与一个胚胎有多远或多近。 The present invention provides a more consistent fluid sample, as fluid may be collected in the same manner is repeated, on the contrary, from the collected samples in a Petri dish culture medium may be based on the placement of the suction pipette is changed, i.e. it is a multiple of embryo far or near.

使用透明的通道区域允许进行许多种类的光学分析。 A transparent region to allow passage of many types of optical analysis. 在透明区域处,可添加污点或颜料,以便目视检查透明区域。 In the area of ​​the transparent, stains or pigments may be added to visually inspect the transparent region. 该透明区域还提供使用图象分析装置的机会,因为微流控通道可构造成将一个胚胎精确地定位在一个位置,以便通过成像设备分析。 The transparent area also offers the opportunity to use the image analysis device, since the microfluidic channels can be configured to accurately position an embryo at a position so as analyzed by the image forming apparatus.

采用本发明的通道和收缩部来给胚胎精确地定位和/或流动操作还能够进一步提供一种去除哺乳动物胚胎的透明带的改进的方法。 Channel and the constriction of the present invention is employed to accurately position the embryo and / or flow operation can further provide an improved method of mammalian embryo zona pellucida removed. 胚胎通过流体移动到一个精确的位置,在此,细胞溶解剂可冲洗胚胎,以去掉透明带。 Embryo to move to an exact position by the fluid, in this case, cell lysis agent may be flushed embryos, to remove the zona pellucida.

附图说明 BRIEF DESCRIPTION

参考附图,并通过下面的详细描述,对本领域的普通技术人员来说,本发明的其它目的,特征和优点将变得更清楚,附图中:图1表示根据本发明构造的一种优选的微流控胚胎处理装置的横截面;图2(a)是表示用于胚胎定位的一种优选的窄的微流控通道收缩部的顶视图;图2(b)是表示用于胚胎定位的另一种优选的浅的微流控通道收缩部的横截面视图;图2(c)和2(d)是一种替代的优选的流体动力学收缩部的示意图;图2(e)是又一种替代的优选的流体动力学收缩部的示意图;图2(f)是一种替代的优选的机械收缩部几何外形; Referring to the drawings, and the following detailed description, one of ordinary skill in the art, other objects, features and advantages of the invention will become more apparent from the accompanying drawings in which: Figure 1 shows a preferred configuration of the invention according to cross-sectional embryos microfluidic processing apparatus; FIG. 2 (a) shows a top view of a narrow microfluidic channel constriction for positioning one kind of embryo preferred; FIG. 2 (b) is a diagram for locating the embryo another preferred cross-sectional view of a shallow microfluidic channel constriction; FIG. 2 (c) and 2 (d) is a schematic view of the preferred hydrodynamic an alternative constriction; FIG. 2 (e) is an alternative and preferred fluid dynamics schematic constriction; FIG. 2 (f) is an alternative preferred geometry portion of the mechanical contraction;

图2(g)表示例如用于带处理的图2(e)流体动力学收缩部中特定的流动模型。 FIG. 2 (g) represents, for example (e) constriction for hydrodynamic processing of FIG. 2 with a specific flow model.

图3(a)是根据本发明构造的一种优选的重力流驱动的微流控培养和测试装置的透视图;图3(b)是用于本发明的去除透明带过程的微流控通道的示意横截面;图4是根据本发明构造的胚胎分析装置的框图;图5(a)-5(c)表示根据本发明的优选的胚胎微流控通道嵌入和去除结构;图6(a)-6(b)表示根据本发明构造的优选的培养装置;图7(a)和7(b)示意性表示用于从卵母细胞国去除堆积物的狭窄的通道外形;图8(a)和8(b)表示用于去除堆积物的完整的原型装置;图8(c)至(f)表示在图8(a)和8(b)的原型试验中使用的步骤,以便从卵母细胞中去除堆积物;和图9(a)-9(d)表示用作胚胎生存能力的指示器的胚胎的变形评价。 FIG. 3 (a) is a perspective view of a preferred configuration of the present invention, gravity flow-driven culture and microfluidic testing device; FIG. 3 (b) is a microfluidic channel for the removal of the zona pellucida of the process of the present invention is a schematic cross-section; FIG. 4 is a block diagram showing a configuration of the embryo analysis apparatus according to the present invention; FIG. 5 (a) -5 (c) represents embryonic microfluidic channel and removal of the embedded structure of the preferred invention; Figures 6 (a ) -6 (b) shows a preferred configuration of the culture apparatus of the present invention; FIG. 7 (a) and 7 (b) schematically showing a narrow channel shape for removing deposits from the oocyte States; Figures 8 (a ) and 8 (b) represents a complete prototype device for removing deposits; steps used in FIG. 8 (a) and 8 (b) in FIG prototype test 8 (c) to (f) show in order from egg oocytes remove the deposit; and FIG. 9 (a) -9 (d) shows a modification of the evaluation of embryo viability of the embryo as the pointer.

具体实施方式 Detailed ways

本发明提供一种微流控胚胎处理装置,该装置减少了在胚胎的天然生物宿主外处理该胚胎时施加在其上的应力。 The present invention provides a microfluidic embryos processing apparatus which reduces the stress exerted on the processing embryo extraembryonic natural biological host. 该装置和方法在促进胚胎滑动和旋转的通道中,通过流体辅助移动,再现了模拟胚胎生物旋转。 The apparatus and method to promote embryo sliding and rotating channel, the fluid moves through the auxiliary reproducing a simulated biological embryonic rotation. 这里涉及的旋转可包含完全旋转或部分旋转。 This involves rotating may comprise rotating or partially complete rotation. 部分旋转也可称为摇动。 Rotating portion may also be referred to as a swing.

现在参考图1,图中所示是微流控胚胎处理装置10的横截面图,该装置包括一个胚胎传输网络12,该胚胎传输网络至少部分由基本上为胚胎级别的通道14形成。 Referring now to Figure 1, shown is a microfluidic processing embryos cross-sectional view of the apparatus 10, the apparatus comprising a transport network 12 embryo, the embryo transfer network is formed at least partially by a substantially level the embryo passage 14. 通道14内的胚胎16与通道14中的流体流一起移动,同时通道的紧密尺寸导致胚胎14以模拟的生物旋转运动移动。 14 embryos fluid passage 14 in the flow passage 16 and moved together, while the results in embryonic compact dimensions of the channel 14 to simulate the movement of a biological rotational movement. 十倍于胚胎大小的通道已用来形成滚动和滑动。 Ten times the size of the embryo channel is used to form rolling and sliding. 在生物宿主中,发育的胚胎在其初始发育阶段向着子宫移动,它们通过旋转和滑动附着在子宫上。 In a prokaryotic host, uterine move toward the developing embryo in its initial stages of development, they are attached to the uterus by rotation and sliding. 微流控通道14产生这种模拟运动。 Microfluidic channels 14 to produce an analog of such movement.

通道的尺寸对产生生物旋转很重要。 The dimensions of the channels is important to produce bio rotation. 高度是严格的尺度,并且业已发现约三倍于胚胎直径的高度可促使旋转。 Strict height dimension, and it has been found that about three times the height of the diameter of the embryo may cause rotation. 由于流体的流动也起作用,确定的该比率在某种程度上可改变,但已发现三比一的最大比率可产生旋转。 Since the flow of the fluid also acts to determine the ratio may vary to some extent, but it has been found that the maximum ratio of three to one rotation can be generated. 可以理解,通道宽度不那么重要。 It is understood that the channel width is not so important. 宽度可任意选择。 Width can be arbitrarily selected. 这样,如果胚胎保持在一个数量级时,那么宽度可小于胚胎直径的两倍。 Thus, if the embryo is maintained at an order of magnitude, the width may be less than two times the diameter of the embryo. 如果希望有多个胚胎,可使用更大宽度的通道。 If you want more embryos, using a larger channel width.

通道14的网络提供培养胚胎的手段,并且提供可将胚胎移动和放置在期望位置的手段。 Network channels 14 provides means for embryo culture, embryo and provides a means can be moved and placed in a desired position. 在发育的最初阶段,多数哺乳动物胚胎的大小在受精后头几天中通常保持恒定。 In the initial stages of development, the size of most mammalian embryos are usually kept constant at fertilization during the first few days. 这样,通道14的大小不会对其中培养的胚胎造成障碍。 Thus, the size of the channel 14 is not an obstacle to embryos cultured therein. 有利的是,胚胎16可保持移动和/或可具有围绕它的连续或脉冲式流体流,以避免对胚胎16的潜在的有害生物作用。 Advantageously, the holder 16 may move the embryo and / or continuous or pulsed fluid stream may have surrounding it, to prevent potentially harmful biological effects on the embryo 16.

包括通道的装置10的优选示例结构也在图1中显示。 Preferred exemplary structure includes a channel device 10 also shown in FIG. 1. 通过对合适材料,例如硅片18采用任何合适的显微机械加工技术可形成微流控通道14。 By suitable material, such as silicon wafer 18 by any suitable micromachining techniques 14 may be formed in the microfluidic channel. 所选材料必须能消毒并且不应对胚胎造成生物威胁。 The selected material must be disinfected and should not cause embryonic biological threats. 装置的通道14整个被覆层20密封。 14 the entire sealing layer 20 covering the passage means. 形成玻璃或其它透明材料的覆层有利于方便地视觉监控通道14中的胚胎。 Forming a glass or other transparent material coating layer facilitates the easy visual monitoring channel 14 embryos. 粘合剂22将覆层20粘到硅片18上。 The adhesive 22 adhered to the coating 20 on the wafer 18. 另外,覆层的材料可设计成能屏蔽掉对通道14内的胚胎有害的射线。 Further, the coating material can be designed to shield the passage 14 in the embryo harmful radiation.

不象其它细胞倾向于浮在流体培养基中,相对大而重的胚胎沉在微流控通道14的底部。 Unlike other cells they tend to float in a fluid medium, large and relatively heavy embryos settle to the bottom 14 of the microfluidic channel. 所关心的典型的哺乳动物植入前胚胎是90至180μm直径的球体。 A typical mammalian preimplantation embryo of interest is a sphere of a diameter of 90 to 180μm. 每个胚胎中,细胞膜围绕每个细胞(卵裂球),透明带、糖蛋白膜或壳围绕整个细胞团块。 Each embryo cell membrane around each cell (blastomere), zona pellucida glycoprotein or shell around the entire cell clumps. 细胞在受精后头几天分裂几次,胚胎的体积保持恒定,在本发明的原理基础上构造的同一装置中,卵可受精和发育成胚泡。 In the first few days of fertilization cell division times, the volume of the embryo is kept constant, the same principles of the device on the basis of the present invention is constructed, and fertilized eggs can be developed into a blastocyst. 胚泡是胚胎植入子宫前的最后阶段。 Blastocyst is the last stage before the embryo into the uterus.

对制造这种装置和类似装置来说,同样重要的是其能够处理单个的胚胎或少量胚胎。 For manufacturing such devices and the like, it is also important that it can handle a small amount of a single embryo or embryos. 将胚胎定位到给定位置,移动到替代位置,并且围绕胚胎保持恒定或改变的生物状态都是根据本发明的基本原理提供的能力,并且本发明的受精、发育、试验结构和其它装置均依赖于部分或全部这些性能。 The embryo is positioned to a given position, moved to an alternate location, and ability to maintain a constant or altered biological states are provided in accordance with the basic principles of the invention around the embryo, and the fertilized, development, testing, and other structures of the present invention are dependent in some or all of these properties. 为了使胚胎能在发育期间连续移动,可产生长通道或形成一个圈。 In order to be able to move continuously in the embryo during development, it can produce a long form or a ring channel. 可供选择的是,在例如图6(a)和6(b)所示的发育站可发生胚胎的停置。 Alternatively, in the example of FIG. 6 (a) and 6 (b), the parking station embryo development may occur. 通过改变流体流,有限大小的隔室或通道也可用来使胚胎在其中前后翻转,如后面将进一步根据图6(a)和6(b)来对此进行讨论的那样。 By varying the fluid flow, the limited size of the compartments or channels can also be used in the embryo before and after the inversion which, as will be further discussed as this is performed in accordance with FIG. 6 (a) and 6 (b).

本发明利用收缩部提供独立胚胎的精确定位,其优选实施例如图2(a)和2(b)所示。 The present invention provides accurate positioning of the constriction using separate embryos, preferred embodiment example of FIG. 2 (a) and 2 (b) shown in FIG. 图2(a)是在微流控通道14中形成的狭窄的收缩部24的截面顶视图。 FIG 2 (a) is a sectional top view of a narrow constriction is formed in the microfluidic passageway 14 24. 许多原因导致需要例如在胚胎处理装置10中精确定位。 For example, many causes embryos require processing apparatus 10 is positioned precisely. 在装置中设置的分析仪器需要胚胎在电极、光电检测器、显微镜的焦点或其它类似传感器处精确定位。 Means provided in the analytical instrument is required embryos electrode, a photoelectric detector, the focus of the microscope or the like at a precise positioning sensors. 将胚胎传输到收缩部24允许这种所需的定位而不需借助反馈系统。 The embryos are transferred to the contraction portion 24 allowing such desired positioning without need to rely on a feedback system. 通过使生物流体培养基30反向流动,胚胎16即可简单地不受收缩部24约束。 By biological fluid medium reverse flow 30, the embryo 16 can not easily restrained shrinkage portion 24. 即使在保持在收缩部时,胚胎16也承受着围绕它的生物流体培养基的流动,因为流体30流经它并经过收缩部24。 Even when held in the constriction, the embryo 16 is also under biological fluid medium flowing around it, because the fluid 30 flowing through it and through the constriction 24. 这是有利的,因为滞流于流体中的胚胎具有更多的潜在可能产生“褥疮”,这是在胚胎处理技术中对低成功率的令人疑虑但还未证明的解释。 This is advantageous because stagnant fluid in the embryo has the potential to produce more "bed sores", which is in the embryonic process technology for the low success rate of cause concern but not yet proven explanation.

微流控通道14的侧壁部分28a,28b在期望的位置收缩,以防止胚胎16通过那里。 Sidewall portions 14 of the microfluidic channel 28a, 28b at a desired position contracts to prevent the embryo 16 therethrough. 收缩部24没有完全关闭微流控通道14,这样流体生物培养基30可经过定位在收缩部24处的胚胎16。 Contraction portion 24 is not completely closed microfluidic channel 14, so that the biological fluid through the medium 30 may be positioned in the embryo at the contraction portion 2416. 图2(b)显示出了替代的浅收缩部26的侧截面图,其中当胚胎16位于收缩部时,流体生物培养基30同样能经过。 FIG 2 (b) shows a side sectional view of an alternative shallow constriction 26, wherein when the embryo is located in the constriction 16, the same fluid 30 can pass biological media. 收缩部也可以为其它形状。 Contraction portion may have other shapes. 总的来说,能够防止胚胎通过,同时允许流体流经收缩部的任何,例如对称形状以及梳状都是可以接受的,以便在根据本发明的装置10中定位胚胎。 In general, the embryo can be prevented through, while allowing any such symmetric shape and the comb portion of the fluid flowing through the contraction is acceptable, so that the positioning device of the invention according to the embryo 10. 最好收缩部的尺寸设定成使胚胎定位并防止该胚胎通过,同时不必增加在装置10中使用的流体压力来移动胚胎。 Size is preferably set such that the constriction prevents the embryo and the embryo is positioned through, without having to increase the fluid pressure in the apparatus 10 used to move the embryo. 收缩部长度还应保持足够小,以避免流体控制出现问题,因为微流控通道的收缩部将具有比微流控通道14的无约束部分更高的单位长度流体阻力。 Minister of shrinkage should be kept small enough to avoid flow control problems, since the constriction of the microfluidic channel having a fluid resistance per unit length than the microfluidic channel unconstrained portion 14 higher.

在微流控通道14内的流体动力还可以用于定位胚胎。 Hydrodynamic within the microfluidic channel 14 may also be used to locate the embryo. 尽管图2(a)和2(b)所示的收缩部是物理收缩部,但在图2(c)和2(d)中,可实现有效的流体动态收缩,而不必采用物理障碍来阻止胚胎的通过。 Although FIG. 2 (a) and 2 (b), is a physical constriction constriction, but in FIG. 2 (c) and 2 (d) may be implemented effective fluid dynamic shrinkage without using a physical barrier to prevent embryo through. 在图2(c)和2(d)中,一个深度增加的微流控通道阱部分14a限定了一个位置,在该位置,胚胎16可利用流体动力学控制,即溢出和流过阱部分14a从而有意识地得到保持。 In FIG. 2 (c) and 2 (d), the increase of the depth of a microfluidic channel defining a well portion position 14a, in this position, the embryo 16 can be controlled using fluid dynamics, i.e., the overflow portion 14a, and flow through the well thus consciously maintained. 在图(c)中,高层流或非层流导致胚胎16留在阱部分内,因为在阱部分14a的前缘发生了分流。 In FIG. (C), the high-level non-laminar flow results in embryonic left in the well portion 16, since the split occurs at the leading edge of the trap portion 14a. 在(低)流速期间,流体流线符合包括阱部分14a的微流控通道14的轮廓,以便将胚胎冲出阱部分外。 During the (low) flow rate of the fluid flow lines follow the contour of the well comprises a microfluidic channel 14 of the portion 14a, so that the embryo out of the outer portion of the well. 对于特定的阱几何外形和尺寸,可分别计算保持胚胎和冲出胚胎的预定流速,或者可通过试验确定。 For a particular geometry and dimensions of the well can be separately calculated and maintained a predetermined flow rate out of the embryonic embryos, or can be experimentally determined. 图2(e)表示定位胚胎16的一个替代的策略。 FIG. 2 (e) locating the embryo showing an alternative strategy 16. 在图2(e)中,在微流控通道14的交叉处形成T形汇合处14b。 In FIG. 2 (e), a T-shaped junction 14b is formed at the intersection of the microfluidic channel 14. 在平衡的流体流条件下,如图2(e)所示,在胚胎位置没有流动,允许它保持就位。 Fluid flow in a balanced condition, as shown in FIG 2 (e), the no flow position of the embryo, allowing it to remain in place. 尽管没有图示,但具有锯齿形或其它小的物理形状可能增加图2(e)中胚胎16的稳定性。 Although not shown, having a sawtooth or other small shapes may increase the physical stability of FIG. 2 (e) of 16 embryos. 图2(f)中示出了另一种几何外形构造。 FIG 2 (f) shows another configuration geometry.

对于图2(a)-2(f)所示的几何外形和流动而言,有可能将胚胎定位在微流控通道的网络内的一个精确位置。 For the geometry of FIG. 2 and the flow shown in (a) -2 (f), it is possible to locate a precise position of the embryo within the network of microfluidic channels. 通过成像或其它装置以及对胚胎的处理来提供目测检查、胚胎去除、胚胎测试、分析胚胎的机会。 Provided by the imaging means and a processing or other visual inspection of the embryo, the embryo is removed, the test embryos, embryos opportunity analysis. 因此,采用本发明可实现对胚胎的许多机械操作,并且通过对胚胎的移动进行这种精确的定位有助于进行任何处理、分析或操作,同时本发明还能够操作和控制流体环境。 Accordingly, the present invention may be implemented using a number of mechanical operation of the embryo, and the embryo is performed by moving this precise positioning contribute any processing, analysis or operation, while the present invention is also capable of operating and control fluid environment.

利用收缩部和微流控通道流体来将胚胎定位在图2(a)-2(e)所示类似的装置中的特定的典型的处理技术是从胚胎中去除透明带。 Using constriction and fluid to the microfluidic channel is positioned in the embryos of FIG. 2 (a) -2 (e) shown in the specific exemplary processing apparatus is similar to the zona pellucida is removed from the embryo. 透明带是包围胚细胞的糖蛋白基质。 It is surrounded by the zona pellucida glycoprotein matrix blasts. 嵌合体,即具有两个DNA组的单体动物是通过去除透明带并将分离的胚细胞聚在一起来实现的。 Chimera, i.e., a monomer having groups of animals two DNA by removing the zona pellucida and the embryonic cells isolated poly together to achieve. 对于基因转录程序、试管受精(IVF)和细胞活体检查来说,变薄或去除带也很重要。 Procedures for gene transcription, in vitro fertilization (IVF) and cell biopsy, a thinning or removing tape are also important. 为去除透明带限定存放位置,例如如图2(f)所示。 To remove the zona pellucida defining a storage position, for example, in FIG. 2 (f) shown in FIG. 在图2(f)中收缩部顶面处为0.01数量级的低高宽比可能需要支撑,以避免塌陷。 In Figure 2 the top surface of the contraction portion (f) of the order of 0.01 for the low aspect ratio may need support to prevent collapse. 在一种原型装置中,这利用围绕收缩区的聚二甲基硅氧烷(PDMS)柱实现。 In a prototype apparatus, using a polydimethylsiloxane which surrounds the constriction (PDMS) column realized. 一个或多个胚胎靠在其上的收缩区可以成形为使胚胎聚在一起,在嵌合体成形时这是必要的。 Against which the one or more embryos in the constriction may be shaped such that the embryo together, when this chimera molding is necessary. V形放置表面以如下方式将胚胎聚在一起。 V-shaped surfaces disposed in a manner that the embryos together. 在如图2(f)所示的收缩部上游,微流控通道构造成使得酸性溶液的控制冲洗可导致该溶液流过停留的一个或多个胚胎。 In (f), upstream of the contraction portion in FIG. 2, the microfluidic channel configured such that the control of the acid solution may cause the solution was flushed through one or more embryos stay. 例如,酸性入口可t形汇合到主流内,以便导入图2(f)的停留区域。 For example, the acid may be t-shaped inlet merging into the mainstream in order to introduce the staging area in FIG. 2 (f) of. 在收缩部附近和上游,在短期内例如由细胞溶解栓提供进入主通道的酸性溶液,以实现去除透明带。 In the vicinity of the constriction and the upstream, in the short term, for example, dissolving the cells into the main channel stopper provided in an acidic solution, in order to effect removal of the zona pellucida. 对于微流控通道而言,在所需的时间段内可实现流动的精确控制。 For microfluidic channel, in the period of time required for precise control of the flow. 在原型装置中,与包括收缩部和“T”交叉通道在内的主微流控通道相连的注射器用来控制胚胎定位和使流体流过定位的胚胎。 In the prototype apparatus, the syringe connected to the main channel comprises a microfluidic constriction and "T" cross channel including embryos for controlling fluid flow through the positioning and the positioning of the embryo. 注射器提供对流体的精确控制,计算机控制的微型注射器进一步对流量控制和计时进行精确控制。 Injector provides precise control of the fluid, the micro computer controlled flow control injector further precise control and timing.

去除透明带的一种替代的机械方法涉及对透明带的机械破坏。 A method of removing the zona pellucida of alternative mechanical methods involve mechanical disruption of the zona pellucida. 这是例如使带通过本发明的微流控通道来实现的,该微流控通道包括一种机械结构,以便在透明带上刻划或切割。 This belt is for example achieved by microfluidic channels of the present invention, the microfluidic channels comprises a mechanical structure, so as to bring the transparent score or cut. 本发明的精确的流量控制还有可能形成一种层流方法,以便对带“刻划”。 Precise flow control is also possible according to the present invention forms a laminar flow method, in order to strip "scoring." 在图2(e)的“T”形汇合处,两种不同溶液的两个分离流束在“T”形处汇合流入一个如图2(g)所示的单一通道,这里的虚线指流束分离。 Flows into a 2 (g) as shown in FIG single channel in FIG. 2 (e) of the "T" shaped confluence of the two streams separate two different solutions meet at "T" shaped, the broken line here refers stream beam splitter. 这些流束可以在单一通道内因层流而保持分离。 These streams may be kept separate in a single internal layer flow passage. 一个流束(左边)包含例如使透明带稀薄的酸。 A stream (left) comprising for example a transparent thin strip acid. 驱动压力控制在两种溶液之间的交界面的横向位置,这样,胚胎16的透明带由酸“刻划”。 Lateral position of the driving pressure is controlled at the interface between the two solutions, so that the zona pellucida of the embryo 16 "scored" by the acid.

包括如图2(a)所示的收缩部,用于定位胚胎的培养和试验装置31在图3(a)中示出。 As shown in FIG comprising a constriction (a) 2, and for positioning the embryo culture test apparatus 31 (a) is shown in FIG. 装置31具有在微流控通道14的网络32中的流体流,该流体的流动基于在若干流体容器34内的流体30的液面,由重力驱动。 Means in the fluid stream 31 having a network 32 of the microfluidic channel 14, the flow of the fluid based on the fluid level in the fluid reservoir 34 of a plurality 30, driven by the force of gravity. 用于驱动流体30的任何适当的装置均被认为与本发明的基本原理相符合,例如泵送,但图3(a)中所示的重力法对于简单和效率而言最优。 Any suitable means for driving the fluid 30 that are aligned with the underlying principles of the invention, e.g. pumping, but as shown in FIG gravimetric 3 (a) is optimal for simplicity and efficiency. 流动的方向由容器34中流体的液面简单控制。 The direction of flow of fluid from the container 34 in a simple control level. 这样,例如保持在收缩部24以便通过适当的仪器培养或检查的胚胎16通过首先调节液面得到定位,从而使其从入口36移动到收缩部24,并且当流体流反向经过收缩部24时使胚胎释放。 Thus, for example, held in the contraction portion 24 for contraction portion 24 by a suitable culturing or inspection instrument 16 embryos obtained by first adjusting the positioning level, so that it moves from the inlet 36 to the contracted portion 24, and the fluid flows back up through the the embryo is released. 通过流体流使胚胎移动到出口38来实现胚胎16的去除。 Fluid flow through the outlet 38 is moved to the embryo to effect removal of 16 embryos.

在移动经过网络32的微流控通道14期间,如上所述,一个或多个胚胎滚动和滑动,以模拟胚胎朝向哺乳动物宿主的体内子宫的自然移动。 During movement through the microfluidic channel 14 of network 32, as described above, one or more embryos rolling and sliding, to simulate the natural movement of the embryo toward the body of the uterus of a mammalian host. 这种期望的移动方式可借助于适当的表面活性剂例如BSA(牛的血清白蛋白)。 This desired movement by means of a suitable manner, for example, surfactants (bovine serum albumin) BSA. 该表面活性剂有助于促进胚胎滚动时的某种程度上的滑动。 The surfactant to some extent help promote embryo rolling slides.

图3(a)还示出了本发明提供有平行附加微流控处理和培养装置31a而具有的附加优点。 FIG. 3 (a) also shows an additional advantage of the present invention there is provided an additional parallel processing microfluidic devices 31a and has a culture. 附加装置31a具有与装置31相似的结构,但可具有更少甚至单个微流控通道。 Attachment means 31 and 31a has a similar structure, but may have fewer or even a single microfluidic channel. 理想化地讲,它们的结构是相同的。 Ideally speaking, their structure is the same. 装置31a的重要特征是它与主装置的入口36和出口38共享共同的流体源。 Important features of the apparatus 31a, it is an inlet 36 and an outlet of the master device 38 share a common fluid source. 装置31a中处理的胚胎与主装置31中的胚胎生物隔离,但它们通过共享相同的流体源、压力和/或相同的生物培养基条件而具有相同的生物学条件。 Embryos Embryo isolation and biological host device 31 in the processing apparatus 31a, they share the same fluid source through a pressure and / or the same biological media conditions and with the same biological conditions. 在示例的使用中,附加装置31a因此可能形成重要的控制培养,其中通过试验胚胎的发育或发育缺乏可确认主装置31中建立的条件是合适或是不合适。 In the example used, the additional device 31a thus formed may be important control culture wherein development of embryos experimentally confirmed that lack or developmental conditions established in the main apparatus 31 is suitable or unsuitable.

图4是胚胎分析装置的方框图。 FIG 4 is a block diagram embryo analysis device. 在图4的装置中,微流控通道14的网络32将胚胎移到一个或多个分析站40a,40b或40c。 In the apparatus of FIG. 4, the microfluidic network controller 32 of the passage 14 to move one or more embryos analysis stations 40a, 40b or 40c. 胚胎经过如上所述那样的收缩部定位于给定分析站。 After the embryo as described above constrictions positioned in a given analysis station. 分析站可包括能获得关于胚胎的信息的任何仪器,同时通过形成收缩部使胚胎定位于对于分析站中特定仪器来说合适的检测点。 Analysis station may comprise any apparatus capable of obtaining information on the embryo, the embryo while positioned in a suitable detection point for the analysis station for a specific instrument by forming a constriction. 胚胎经过一个或多个出口38a,38b移出装置,这样可替代地将胚胎引导至采取停靠区形式的培养站、微流控通道14的附加长度段或为了使胚胎在培养期间连续移动而形成的环状微流控通道中。 Embryo through one or more outlets 38a, 38b out of the apparatus, so that the embryos may alternatively be guided to take the form of a docking station area of ​​culture, an additional microfluidic channel lengths 14 or for embryo culture during the continuous movement of the formed annular microfluidic channel.

用于本发明装置中的入口和出口可包括用于胚胎嵌入或移去的任何传统方式或结构。 Inlet and outlet means for use in the present invention may comprise any conventional manner or embryo structures embedded or removable. 但是,嵌入和移去的附加优选结构如图5(a)-5(c)中所示。 However, embedding the additional and preferred removable structure shown in FIG. 5 (a) -5 (c) of. 在图5(a)中,使用与微流控通道14流体连通的阱42。 In FIG. 5 (a), the well fluid 14 using the microfluidic channel 42 in communication. 阱42中的流体最好还包括重力供送,它有助于驱动通道14中的微流控流体。 Preferably, the fluid in the well 42 further comprises a gravity feed, it helps to drive channel microfluidic fluid 14. 胚胎16置于阱42内,并且与生物培养基一起移动到通道14中,或者在未建立流体条件下简单地独立沉在通道14中。 16 embryos placed in the well 42, and moves with the biological medium into the passage 14, the fluid or in an independent condition is not simply sink in the channel 14. 可采用第二类似的阱42,使用移液管44或相似装置移去胚胎。 The second well 42 can be similar, using a pipette 44 or similar means for removing the embryo. 移液管也可用来嵌入胚胎。 The pipette may also be used to embed embryos. 在图5(b)中,在通道14末端处的悬挂滴46用来嵌入和移去胚胎。 In FIG. 5 (b), the suspension dropwise at the end of the channel 4614 for embedding and removing the embryo. 通过表面张力保持悬挂滴46。 Remains suspended by the surface tension drops 46. 在胚胎嵌入之后,在该点可添加流体,或通过装置中的流体流动吸入胚胎。 After the embryo is embedded, fluid can be added at this point, fluid flow through the embryonic or inhalation device. 可供选择的是,装置可倾斜,以促使胚胎离开悬挂滴46。 Alternatively, the means may be inclined to cause the droplets 46 suspended away from the embryo. 在图5(c)中,与通道14直接连通的漏斗形孔48用来嵌入和移去胚胎。 In FIG. 5 (c), the funnel-shaped aperture 14 in direct communication with the channel 48 to insert and remove the embryo. 漏斗形帮助移液管44或相似装置定位。 Help pipette funnel or similar device 44 is positioned. 小直径孔48处的表面张力将防止流体漏出,但通道14中的压力必须不超过征服表面张力并导致流体漏出这一点。 The surface tension at the small-diameter hole 48 will prevent fluid from leaking, but the pressure in the channel 14 must not exceed the surface tension of the conquer and this causes the fluid to leak out. 嵌入的胚胎将沉入通道14中,同时当胚胎接近孔时将流体从孔中抽出即可移去胚胎。 Embryos will sink embedded in the channel 14, while when the embryo near the fluid withdrawn from the bore hole to remove the embryo. 当然,图5所示的技术均可相互结合或与传统技术相结合,以便将胚胎嵌入给定处理装置和从其中移去。 Of course, the technique shown in FIG. 5 may be bonded to each other or in combination with the conventional art, in order to embed the embryos from a given process and apparatus wherein removal. 另外,可用移动盖或翼片作为抗污染和/或蒸发的保护装置覆盖阱42或孔48。 Further, movement of the cover or flap can be used as a protective means contamination and / or evaporation of the cover 42 or the well bore 48.

现在参考图6(a)和6(b),图中示出了根据本发明的胚胎培养装置50。 Referring now to FIG. 6 (a) and 6 (b), there is shown a culture apparatus 50 according to the present invention embryos. 培养装置50中的流体培养基流在培养基入口52和培养基出口54之间的任一方向流动。 Fluid culture medium in the device 50 in a medium inlet 52 and flow in either direction between the medium flow outlet 54. 装置包括若干陷阱或隔室56。 Means includes a plurality of compartments or trap 56. 如图6(b)最佳所见,陷阱56包括被浅区58分隔的深区。 FIG 6 (b) best seen, the trap 56 includes the deep region 58 separated by shallow zone. 入口52和出口54之间的流体流过浅区,并且经过深区,以便在深区隔室56中前后移动胚胎。 Between the inlet 52 and outlet 54 fluid flows through the shallow region and the deep region through, so as to move back and forth in the 56 embryos in the deep chamber segment. 胚胎经过通道孔60嵌入和移去,其中通道孔60可通过图5(a)至5(c)中的任何优选方法形成。 Embryonic fitted through the passage hole 60 and removed, wherein the passage holes 60 may be any preferred method (c) forming (a) through 5 in FIG. 5. 在装置50中,本领域的普通技术人员会这样想到,胚胎可在隔室56中前后移动,以便模拟生物学旋转,可与培养基中的其它胚胎享有相同的培养基条件,并且可容易地移去和嵌入。 In the apparatus 50, those of ordinary skill in the art would expect such, the embryo can move back and forth in the compartment 56, so as to simulate biological rotation, with the other embryo culture medium have the same conditions, and can be readily removal and embedding. 虽然图6(a)和6(b)示出的是顶部装载实施例,以便将胚胎放置在间隔中,但装置也可在底部装载的情况下工作,基本上与图6(a)和6(b)中所示相反。 Although FIG. 6 (a) and 6 (b) shows a top loading embodiment, so that the embryo is placed in the gap, but the device can also operate in the case of loading at the bottom, substantially the FIG 6 (a) and 6 Instead shown in (b). 在这种底部装载布置中,胚胎仍保持在深部分但不经过浅部分。 In such an arrangement the bottom loading, the embryo remains in a deep portion and not through the shallow portion. 替代实施例可包括代替浅收缩部的缺口,在此胚胎不能经过缺口,但流体流可在其间产生,并且缺口的深度可与胚胎保持隔室的深度相同。 Alternate embodiments may include a shallow notch in place of constriction, the embryo can not pass through this gap, the fluid flow may be generated therebetween, and the depth of the notch depth may remain the same compartment and embryos.

如图3(a)所示的样机装置已生产出并经过了试验。 As shown in FIG 3 (a) prototype device shown have been produced and subjected to test. 出于完整性的原因,这里描述了典型的样机。 The sake of completeness, here describes a typical prototype. 本领域的技术人员可以理解,通过任何其它传统的微制造技术,可完成制造样机的方式。 Those skilled in the art will be appreciated by any other conventional microfabrication techniques, the prototype can be manufactured complete. 本领域的技术人员还估计,生产装置的制造可显著不同,并且样机装置的具体数字尺寸和条件不对本发明的保护范围起限制作用。 Those skilled in the art also estimated manufacturing apparatus may be significantly different, and in particular the size and condition of the digital prototype device does not protect limiting the scope of the present invention.

在典型的样机通道中,1Pa/mm的压力梯度导致培养基以约10- 10m3/s(100nl/s)数量级的流量流动,平均速度是1至2mm/s。 In a typical prototype passage, the pressure 1Pa / mm the gradient medium to result in approximately 10- 10m3 / s (100nl / s) flow rate of the order of magnitude of the flow, the average speed is 1 to 2mm / s. 在这些流动条件下;胚胎沿着通道底部滚动;流体将在通道的相同区域中以1/3至1/2范围的速度运动。 Under these flow conditions; Embryo roll along the bottom of the channel; the fluid at a rate of 1/3 to 1/2 of the range of motion in the region of the same channel. 通过向连接到通道末端的阱处施加压力,胚胎可输送到(和保留在)包括培养隔室和恢复阱的特定位置。 By applying pressure to the well connected to the end of the channel, it may be delivered to the embryo (and retained) and a recovery compartment comprises culturing a particular location of the well. 胚胎填充在通道的重要位置,因此大大改变培养基的流动。 Embryonic filling an important position in the passage, thus greatly change the flow of the medium. 培养基经过通道的流动为层流。 After the culture medium flow channel is a laminar flow.

样机微流控通道的网络已在如图3(a)所示的装置中制出:在3英寸<100>的硅片中蚀刻沟槽,然后粘合玻璃覆层以形成通道。 Prototype microfluidic channel network has been formed out of (a) shown in FIG. 3 means: a 3 inch & lt; 100 & gt; etching a trench in a silicon wafer, a glass cladding layer and an adhesive to form the channel. 通过在弹性体中采用微模制技术已制造出包括微流控通道的装置。 By using micro-molding techniques have been manufactured in the elastomer means comprises a microfluidic channel. 其它塑料和技术也可能合适,包括例如热塑材料的注模。 Plastics and other techniques may be suitable, including, for example, injection molded thermoplastic material. 典型的通道网络包括几个分支微流控通道,该通道在靠近装置的中心处交叉。 A typical network comprising several branches channel microfluidic channel, which intersect near the center of the device. 长度范围从1.5至2.5cm的分支在顶部为160至200μm深,250至350μm宽。 A length ranging from 1.5 to 2.5cm at the top of the deep branch 160 to 200μm, 250 to 350μm wide. 生产样机装置的第一步包括使用传统的照相平版印刷技术在氮化硅(SiN)覆层上做图案。 The first step comprises the production of prototype devices using conventional photolithographic techniques to make a pattern on the silicon nitride (SiN) coating. 用氢氧化钾(KOH)溶液各向异性蚀刻微流控通道。 With potassium hydroxide (KOH) anisotropic etch solution microfluidic channel. 或以传统方式采用碳化物补强钻头,或采用超声波在玻璃覆层中钻孔。 Or in a conventional manner using a drill carbide reinforcement, or ultrasonic drilling a hole in the glass coating. 使用UV可固化环氧树脂(NOA 61,Norland Products,Inc.New Brunswick,NJ)将玻璃覆层粘到晶片上,或在450℃环境中使用500V将Pyrex(派莱克斯耐热玻璃)7740覆层阳极化粘合到晶片上。 A UV curable epoxy resin (NOA 61, Norland Products, Inc.New Brunswick, NJ) glass coating adhered to the wafer, or 500V to a Pyrex (heat resistant Pyrex glass) coated at 7740 deg.] C environment 450 anodized layer is adhered onto the wafer. 在阳极化粘合前,利用缓冲氧化物浸蚀剂(BOE)可去掉氮化物涂层。 Before bonding anodization, using a buffered oxide etchant (BOE) may be eliminated nitride coatings. 在通道网络的每个分支的端部,或者利用环氧树脂(快粘5分钟环氧树脂,或5小时固化环氧树脂胶;均由GC Electronics,Rockford,IL制造),或者利用硅有机粘合剂(由纽约Waterford的通用电气公司制造的RTV 108和RTV 118,或DowCornign Corp.,Midland,MI生产的Sylgard牌184),将玻璃阱粘合到玻璃覆层上。 End of each branch of the channel network, or an epoxy resin (epoxy tack five minutes, five hours, or cured epoxy; GC Electronics, Rockford, IL manufactured by), or silicon organic binder agent (RTV 108 and RTV, manufactured by general Electric company of Waterford, New York 118, or DowCornign Corp., Midland, MI production Sylgard card 184), the well adhered to the glass cladding glass.

在样机装置中,象图2(a)(“窄”)和2(b)(“浅”)的那些收缩部已制出并已经过试验。 In the prototype apparatus, as in FIG. 2 (a) ( "narrow") and 2 (b) those of the constriction ( "shallow") has been tested and has produced out. 利用单一掩膜和蚀刻操作可构造如图2(a)所示的“窄”收缩部的通道。 Using a single masking and etching operations may be configured as shown in FIG 2 (a) channel "narrow" contracted portion shown in FIG. 如图2(b)所示,具有“浅”收缩部的通道需要两个掩膜和两个蚀刻操作。 As shown in FIG 2 (b), it includes a "shallow" contraction passage portion requires two masks and two etching operations.

对样机装置中除5分钟环氧树脂外的所有元件材料作试验,以便测量胚胎的生物相容性。 All elements of the prototype device material than 5 minutes for testing an epoxy resin, in order to measure the biocompatibility of the embryo. 在使用本发明时,本领域的技术人员可以理解,可从备选材料中选择那些用于样机装置的材料,但生物相容性必须总是建立在已有的数据和/或测试上。 When using the present invention, those skilled in the art will be appreciated, for those materials may be selected from the candidate prototype device material, a biocompatible but must always be based on existing data and / or testing. 虽然许多材料已知与某些细胞相容或有毒,但在调查微制造技术中所用的材料与胚胎的相容性方面几乎没有做什么工作。 Although many materials are known to be compatible or toxic to certain cells, but in terms of compatibility survey microfabrication techniques and materials used in the embryo almost did not do any work. 所选材料也可根据哺乳动物的类型而改变,其中被处理的特定胚胎来自该哺乳动物。 The selected material may also vary depending on the type of mammal, the particular embryo from which the mammal to be treated.

在样机试验中,双细胞老鼠的胚胎(B6SJL/F2)随机分配,并且在基质上培养,在培养基M16(Sigma,St.Louis,MO)中具有牛血清白蛋白(BSA;4mg/ml;Sigma),覆盖有矿物油(Sigma)。 In prototype testing, a double mouse ES cells (B6SJL / F2) were randomly assigned, and cultured on a substrate, with bovine serum albumin (BSA in the culture medium M16 (Sigma, St.Louis, MO); 4mg / ml; Sigma), covered with mineral oil (Sigma). 所有胚胎均在37℃下5%CO2空气环境中培养96小时。 All embryos were cultured at 37 ℃ 5% CO2 air atmosphere for 96 hours. 每24小时测试胚胎的发育率。 Test embryo development rate per 24 hours. 针对每种材料将胚胎中达到胚泡阶段的百分比与对比组的百分比相比。 The percentages are compared for each material of the comparative group embryo reaches the blastocyst stage. 达到胚泡阶段的老鼠胚胎在胚胎转移前的最后可能阶段可能没有发育的障碍。 Obstacles to reach the blastocyst stage mouse embryos at the last possible stage before embryo transfer may not be development. 不能保证没有副作用,除非胚胎也转移给受者老鼠,并且一直监控到后代出生,出于实用和经济原因在胚泡阶段共同总结试验。 We can not guarantee that no side effects, unless you also transfer the embryo into the recipient mice, and has been monitoring the offspring born, for practical and economic reasons common tests are summarized in the blastocyst stage. 多数被测材料证明与老鼠胚胎相容,包括硅片,SiN涂层,NOA 61和RTV 118。 Most of the material being tested to prove compatible with mouse embryos, including silicon, SiN coatings, NOA 61, and RTV 118. 一些材料,例如5分钟环氧树脂尚未被测试,因为它仅用于理论装置中,以证明本发明的机械和流体原理,不可能用于产品装置中。 Some materials, for example epoxy Not tested 5 minutes, since it is only a theory apparatus, and to show the fluid mechanical principles of the invention, the product can not be used in the apparatus.

我们进行试验,以测试样机装置的几个方面。 We conducted experiments to test several aspects of the prototype device. 不同试验需要带不同通道结构的装置。 Different test device with a different channel structure needs. 在所有试验中,卤素灯泡经光纤照亮通道,这可以在立体显微镜下观察到。 In all tests, a halogen bulb illuminated via fiber channel, which can be observed under a stereoscopic microscope. 量筒和秒表用来确定流率。 Graduated cylinder and a stopwatch is used to determine the flow rate. 因为流体不可压缩,通道任何部分中的平均流体速度正是流率除以横截面面积。 Since the incompressible fluid, the average fluid velocity is the flow rate of the portion of any channel divided by the cross-sectional area.

给定流率的胚胎运动速度的测量发生在简单的直线通道中,该通道29mm长,162μm深和160-380(底-顶)μm宽。 A given flow rate measuring embryo motion speed occur in a simple linear path, 29mm length of the channel, 162μm deep, and 160-380 (bottom - top) [mu] m wide. 压力梯度变化,并且在每个设定下测量胚胎速度。 Pressure gradient, and the embryo was measured at each speed setting. 通道填满磷酸盐缓冲剂盐(PBS),有和没有BSA。 Fill channel phosphate buffer salt (PBS), with and without BSA. 培养基的长颈瓶连接通道。 Flask medium connecting channel. 通过调整长颈瓶的高度,使用微米头转化阶段,压力差最终调节到0.05Pa内。 By adjusting the height of the flask, using the first conversion stage m, the pressure difference finally adjusted to 0.05Pa. 通过在试验之间接管使压头至零,从而使长颈瓶相互连接。 By taking test between the ram to zero, so that the flask is connected to each other. 微制样机装置在过氧化氢/氢氧化铵/去离子水溶液中清洁,并且在进行试验前新移液管尖粘有环氧树脂。 System prototype micro means of hydrogen peroxide / ammonium hydroxide / deionized water cleaning, prior to performing the test and a new pipette tip with an epoxy adhesive. 在使用BSA之前进行所有使用PBS而无BSA的试验。 All tests performed without BSA in PBS BSA prior to use. 一旦通道填满培养基,老鼠的胚胎就放置在通道入口处的入口阱中。 Once the media filled channel, mouse embryos placed on the inlet of the inlet passage in the well.

我们进行试验,以观察通道尺寸和形状对胚胎传输的影响。 We tested to observe the effect of the size and shape of the channel transfer embryo. 对这些试验来说,一个装置构造有一个长的连续通道,该通道具有11个部分,每个部分都具有如下四个深度之一:140,164,194和210μm。 For these tests, a device configured with a long continuous channel, the channel having a portion 11, each portion having a depth of one of the following four: 140,164,194 and 210μm. 在每种深度下,通道具有2或3个不同宽度。 In each depth, channel 2 or 3 having different widths. 在晶片的表面处测得的宽度其范围从275至480μm。 The width of the surface of the wafer measured range from 275 to 480μm. 在最窄部分,胚胎被几何收缩,以便V形-槽上运动,而在其它区域中则沿着具有平坦-底部的通道运动。 In the narrowest portion, is geometrically embryos contracted, so that the V-shaped - groove movement, while in the other areas along with flat - bottom channel motion. 观察运动速度和旋转特性,并且比较不同部分。 Observation movement speed and the rotational characteristic and comparing different parts.

在收缩部处对胚胎的观察发生在几个装置中,该收缩部为窄和浅型收缩部。 Portion of the shrinkage occurs in the observation of several embryos apparatus, the constriction is narrow and shallow type constriction. 胚胎实际直接引导到特定收缩部处。 Actual embryo guided directly to a specific portion of the contraction. 通过添加或减少流体改变每个阱中培养基的高度,调整通道网络的每个分支中的压力梯度。 By adding or reducing the pressure gradient change the height of each branch fluid medium in each well, adjust the channel in the network. 压力头调整1至8mm(10至80Pa)。 Adjusting a pressure head to 8mm (10 to 80Pa).

正当胚胎放置在培养基中沉在容器底部时,置于微流控通道中的胚胎沉积在底部。 Just place the embryo in a medium sink at the bottom of the container, placed in the microfluidic channel embryos deposited on the bottom. 在所有试验中,当培养基流动时,胚胎转动并且在流动方向上沿着通道底部滑动。 In all tests, when the medium flows, and slides along the bottom of the embryo is rotated in the direction of flow channel. 通常它们还保持与通道的其中一个侧壁接触。 They also typically held in contact with one of the sidewalls of the channel. 在培养基(磷酸盐缓冲剂盐)中无任何表面活性剂的最初试验中,胚胎出现转动而不会沿着通道底部滑动。 In (phosphate salt buffer) without any surfactant initial test medium, embryo occurs without slipping along the bottom of the channel is rotated. 在培养基容纳有BSA(4mg/ml)的后来的试验中,胚胎滑动或转动,同时沿着底部滑动。 Later tests containing BSA (4mg / ml) in the culture medium, embryo slide or rotate while sliding along the bottom.

试验揭示,在通道中的胚胎的运动速率依赖于培养基速度。 Tests revealed that, in the channel depends on the rate of movement of the embryo culture medium speed. 在围绕它们的流体的速度低于50μm/s时,有时它们粘在通道底部。 When the fluid surrounding them slower than 50μm / s, they may stick to the bottom of the channel. 对PBS和PBS/BSA这两种培养基来说,0.16Pa/mm的压力梯度驱动流体以大约380μm/s的平均速度经过通道。 Against PBS and PBS / BSA these two media, the pressure 0.16Pa / mm gradient driven fluid at an average speed of about 380μm / s through the channel. 胚胎以187-250μm/s(380μm/s的49至66%)转动。 Embryonic rotated at 187-250μm / s (380μm / 49 to 66% s in). 当培养基更快流动时,胚胎更快转动,并在转动时滑动。 When the medium flows faster, faster rotation of the embryo, and slides while rotating. 已观察到此时胚胎运动的实际速度和从一个胚胎到下一个胚胎的粘性变化的趋势与在相同通道中同一时间、在几乎相同的路线中运动的胚胎会快25%。 At this time, the trend has been observed that the actual speed of movement of the embryo and embryo from one to the next change in viscosity of an embryo in the same channel at the same time, the movement at nearly the same route embryo 25% faster. 在所观察到的范围中,150至1000μm/s的速度与压力梯度呈线性关系。 In the observed range, velocity and pressure of 150 to 1000μm / s linear gradient.

试验结果表明,通道尺寸和形状的作用与由原因推出结果的预测匹配。 The results showed that the effect of the size and shape of the channel introduced by reason predicting the outcome of matches. 对给定的流率来说,平均流体速度和胚胎速度在有较小横截面的通道中较大。 For a given flow rate, the average fluid velocity and speed of embryo passage larger in cross-section smaller. 相反,对给定的压力梯度来说,平均流体速度和胚胎速度在具有较大横截面面积的通道中较大。 Conversely, for a given pressure gradient, the average fluid velocity and speed of embryo having a larger cross-sectional area of ​​the larger passage. 在这两种情况下,胚胎在V形-槽上比在具有平坦-底部的通道上运动得更慢。 In both cases, the V-shaped embryos - groove having a flat ratio of - moving more slowly on the bottom of the channel. 胚胎在V形-槽中比在平坦-底部通道上还更可能楔入并且粘附。 V-shaped embryos - are also more likely wedged and adhered on the bottom of the channel - the ratio of the flat groove.

电渗流动下的流体还导致胚胎在通道中转动。 Fluid under electroosmotic flow also results in rotation of the embryo passage. 由于存在压力驱动的滴流流动,胚胎沿着通道底部以近似10μm/s转动。 Due to the trickle flow pressure driven embryos rotated approximately 10μm / s along the bottom of the channel. 接通电压导致老鼠胚胎沿着通道底部以快于20μm/s,至少30μm/s的速度向着有负极的阱转动。 Turn-on voltage results in mouse embryos along the bottom of the channel faster than 20μm / s, at least 30μm / s speed trap is rotated toward the negative electrode. 使电压极性反向,胚胎以近似10μm/s的速度在相反方向上转动。 The voltage polarity is reversed, the speed of embryos at approximately 10μm / s is rotated in the opposite direction. 不使用例如BSA的表面活性剂,所以只有少量或根本无滑动。 Without the use of BSA such as surfactants, so that only a small amount or no sliding. 如果用来移动胚胎,必须在仔细控制的条件下施以电辅助,以避免对培养基进行不希望的加热。 If used to move the embryo must be subjected to electric auxiliary under carefully controlled conditions, to avoid undesirable heating of culture medium.

采用Fluent/UNS 4.2(Fluent Inc.,Lebanon,新罕布什尔州)建立计算流体动态模型,并且采用Quickfield(Tera Analysis Inc.,Tarzana,加拿大)对具有恒定横截面面积的样机微流控通道进行2-维有限元分析,验证所观察的流率和流动模型。 Using Fluent / UNS 4.2 (Fluent Inc., Lebanon, NH) to establish a fluid dynamic model calculation, and the use of a 2-dimensional Quickfield (Tera Analysis Inc., Tarzana, Canada) prototype microfluidic channel has a constant cross-sectional area is finite element analysis, to verify the flow rate of the flow model and observed. 胚胎模型建立成为一个刚性球体。 Embryo model to become a rigid sphere. 前面已说过在典型的条件下胚胎没有出现变形。 No embryos previously mentioned deformation occurs under typical conditions. 为了分析层流,将通道的1或2mm部分啮合(mesh)成10000至30000个四角形元件。 In order to analyze the laminar flow, the passage of the engagement portion 1 or 2mm (mesh) into 10000-30000 quadrilateral elements. 一旦验证,采用计算机模型来确定流动速度变化图,设计具有较低压降的收缩部,以观察收缩部处滞留的胚胎上的力,并且分析相似通道中的电驱动流动。 Upon verification, the use of a computer model to determine the flow velocity profile, designed to have a low pressure drop constriction, to observe the shrinkage force of the retention portion of the embryo, and a similar analysis is electrically driven flow channel. 但是,将胚胎与通道阱的粘接和胚胎的变形结合分析将明显变得更复杂,并且我们并不期望那样做。 However, deformation of the adhesive and the channel well and embryos embryos binding analysis becomes significantly more complex, and we do not expect.

如上所述,在胚胎速度的直通道试验中,在0.16Pa/mm的压力梯度下,培养基具有380μm/s的平均速度。 As described above, the straight path velocity tests in embryos, under a pressure gradient 0.16Pa / mm, the medium having an average velocity 380μm / s of. 有限元分析确定在这些条件下中心线速度是815μm/s。 Finite element analysis determined under these conditions is the centerline velocity 815μm / s. 当在通道中运动时,胚胎正切于底部和一个壁。 When the motion in the channel, tangential to the embryo and a bottom wall. 考虑100μm直径的圆正切通道的底和一个壁。 A bottom wall and a diameter of a circle tangent to consider 100μm channel. 流体经过该圆运动的平均速度在胚胎不存在时是480μm/s。 The average velocity of the fluid through a circular movement in the absence of the embryo is 480μm / s. 但是,胚胎仅以187-250μm/s转动,在PBS和PBS/BSA培养基中都会快39-52%。 However, only embryos 187-250μm / s rotation, are 39-52% faster in PBS and PBS / BSA medium. 速度变化图促进胚胎向前和沿着壁滚动,这证实了目测观察。 Velocity profile fetal mouse to scroll forward and along the wall, which confirmed visually observed. 总而言之,胚胎转动速度为流体在相同区域的横截面中流动的速度的1/3至1/2。 In summary, embryos rotational speed of the fluid flowing in the same cross-sectional area of ​​1/3 to 1/2 of the speed.

收缩部大大增加了通道中的流体阻力。 Greatly increased constriction resistance of the fluid passage. 标准分析公式可帮助估计阻力,但收缩部的截面形状随位置变化。 Standard analytical formulas can help assess the resistance, but the cross-sectional shape with the change in position of the contraction portion. 在设计有掩膜并且蚀刻晶片之前分析收缩部的三维模型。 In the design and analysis of the three-dimensional model of the mask portion of the contraction before the etching of the wafer. 从有限元分析获得的信息导出最佳尺寸的收缩部。 Finite element analysis information obtained from the derived optimum size constrictions. 采用可根据装置的几何形状确定各自的大小的浅收缩部折衷了对小的流体阻力和坚固的结构的需求。 It may be determined using a shallow constriction respective sizes compromise the need for small fluid resistance and rugged construction of the device depending on the geometry. 典型的收缩部具有20μm的最小深度。 Typical constriction has a minimum depth of 20μm.

对在陷阱处的胚胎布局的研究表明,约10-8至10-7N数量级的横向力迫使胚胎向侧面移动,并且在入口斜面处离开侧面而进入浅收缩部。 Study of embryos at the layout of the trap indicates that the order of from about 10-8 to 10-7N lateral forces the embryo moves to the side, at the inlet side and exits into the shallow bevel constrictions.

试验揭示了微流控传输的几个有趣的特性,例如胚胎之间的速度变化并且趋于沿着通道底部转动、经常正切侧壁等。 Test revealed several interesting features microfluidic transmission such as speed variation between the embryo and tends to rotate along the bottom of the channel, often sidewall of the tangent. 但是,试验确实揭示了几个其他重要的论点。 However, the test does reveal several other important points. 电渗流动对帮助胚胎移动是有用的,并且可帮助形成用于胚胎处理和受精的栓子。 Electroosmotic flow is useful for helping embryo movement, and helps to form emboli and fertilized embryos treated. 流体流动的电控制必须仔细管理,因为高压可以几种途径伤害胚胎。 Electrical control fluid flow must be managed carefully, because high pressure can damage the embryo in several ways. 即使通道部分中胚胎离开了电场,施加的能量也会加热培养基(焦耳加热)使其超过生理温度,并且电解产物改变了PH值。 Even embryo passage section left field, also the application of energy heating medium (Joule heating) it is more than physiological temperature, and the PH value of the electrolysis product is changed. 注意胚胎需要约0.029渗摩(Osmol),即相对高的传导性。 Note that it takes about 0.029 osmoles embryos (Osmol), i.e., a relatively high conductivity. 而且,EOF在有表面活性剂的通道中降解,但胚胎在有例如BSA表面活性剂的培养基中更好。 Further, the EOF in the passage of a surfactant degradation, but for example, in the embryo culture of surfactants BSA better.

通过如图3(a)所示的重力供送装置或通过泵产生流体压力的装置提供电辅助微流控自由传输具有重要的优点。 Apparatus by gravity as shown in (a) in FIG. 3 or feed means by fluid pressure generated electric auxiliary pump providing a microfluidic free transmission has important advantages. 培养基可容易地随时间改变,以便适合发育中的胚胎的改变需求。 The medium can be easily changed over time to suit the changing demands of the developing embryo. 逐渐改变培养基的成分避免了经常伴随从具有不同培养基的一个皮氏培养皿到第二个皮氏培养皿传输而突然地改变环境而产生施加到胚胎上的应力。 Gradually changing the components of the medium are often accompanied by avoiding sudden environmental change from a Petri dish with a different medium to a second petri dish on the transmission and stress applied to the embryos. 本发明的胚胎的微流控处理比用移液管传输物理更严格,并且明显比包括某些刺穿外膜的传统实际操作的许多技术损伤少。 Embryos microfluidic present invention are more stringent than the physical transmission process using a pipette, and significantly less damage than many techniques including conventional practice certain of piercing the outer membrane.

可以预料,在如图3(a)所示的处理装置中对流体流动进行控制,进而控制胚胎的定位将通过大型自动装置中的程序控制仪器来处理。 It is anticipated that, in FIG. 3 (a) processing apparatus shown in controlling fluid flow, and thus control the positioning of the embryo will be handled by large-scale automatic process control instrument means. 在本发明的胚胎处理装置内的传感条件基础上可采用结合报警和警告的装置。 Sensing conditions in the embryonic processing apparatus of the present invention may be employed in combination apparatus on the basis of alarms and warnings. 在相似方式中,本发明的处理装置采用传统监控胚胎的仪器。 In a similar manner, the processing apparatus according to the present invention using conventional equipment to monitor the embryo. 本领域普通技术人员将基本上认识到,微流控胚胎处理装置因此形成一基本构件模块,许多有用的装置都基于此,并且这些装置将结合到本发明的实质中。 One of ordinary skill in the art will recognize that substantially the microfluidic processing apparatus embryos thus substantially forming a module member, many useful devices are based on this, and these devices will be incorporated into the substance of the present invention.

一些特定装置已表明了胚胎和卵母细胞的操作、试验和处理。 Have shown that a certain device operations, testing and handling of the embryos and oocytes. 微流控通道的特定几何形状示意描述在图7(a)和7(b)中,并说明能从卵母细胞上去除堆积物。 The particular geometry of the microfluidic channel is schematically depicted in FIG. 7 (a) and 7 (b), and instructions from oocytes to remove the deposit. 所述几何形状是微流控通道的一系列逐渐收缩部分,它们位于弯曲部分中。 The geometry of the tapering portion is a series of microfluidic channels that are located in the curved portion. 虽然图7(a)和7(b)具有弯曲的收缩部,但收缩部不是必须弯曲的。 Although FIG. 7 (a) and 7 (b) having the curved shrinking portion, but not necessarily, the curved portion of the contraction. 收缩部的内表面最好具有齿状或锯齿状的突起,以便在其经过时帮助切割堆积物。 The inner surface of the contraction portion preferably has a toothed or serrated projections to help cut through when it was deposited. 系列中的最后部分具有隔开一定距离的突起,以便避免损坏卵母细胞,前面部分具有间隔逐渐变小的突起,以便在流体压力下迫使卵母细胞经过收缩部时切割周围堆积物部分。 The last part of the series having spaced apart projections, in order to avoid damage to the oocytes, the front portion having a tapered projections spaced so as to force the oocytes under fluid pressure passes through the constriction deposit around the cutting portion. 在图7(a)中,多个收缩部(弯曲部分)的每一个都分别具有等距的突起,同时先前部分的下游部分采用更小间隔。 In FIG. 7 (a), each of the plurality contracted portions (curved portions) are respectively equidistant projection, while the downstream portion of the previous section using smaller intervals. 也可以构造成单一一部分,其中该部分中的突起逐渐靠近在一起,以便达到相同的目的,即在多个位置处更深地切割堆积物而不损坏胚胎。 It may be configured as a single part, wherein the protrusion portion is gradually close together, in order to achieve the same purpose, i.e. deeper cut without damaging the embryo was deposited at a plurality of positions. 图7(a)和7(b)所示的几何形状用于从牛的卵母细胞中移去堆积物。 FIG. 7 (a) and 7 (b) is shown the geometry for removing oocytes from bovine deposits. 在样机装置中,微流控通道的直部分是500微米宽。 In the prototype apparatus, the straight portion of the microfluidic channel is 500 microns wide. 所述装置使用了五个收缩部。 The device uses five constriction. 第一并且是最大的部分包括一些突起,这些突起的间隔从300微米逐渐减少到第五收缩部的50微米宽。 The first and largest part comprises a number of projections, these projections spaced 300 microns from 50 microns in width gradually reduced to a fifth of the contraction portion. 前四个收缩部在堆积物卵母细胞复合体经过时沿着流体流施力切割(已观察到“mohawk(溜冰之一种花式)”切割)堆积物。 Four front cutting portion along the fluid flow contraction force at the time the deposit after oocyte complexes (It has been observed "Mohawk (one kind of fancy skating)" cleavage) deposit. 在第五收缩部处一半堆积物被吸走。 It is sucked in the fifth contraction portion halfway deposits. 流体反向流动几次,以便重新定位卵母细胞,直到另一半堆积物经过最后的收缩部被抽掉。 Reverse flow of fluid several times to reposition the oocyte until the other half of the deposit after the final portion is deprived of contraction. 这样,适当尺寸的收缩部和几何形状可用来定位和限制胚胎和周围的结构。 Thus, an appropriate size and geometry of the constriction can be located and embryos and surrounding structures limit.

移去堆积物的完整样机装置在图8(a)和(b)中描述,而图8(c)-(f)说明了采用图8(a)和8(b)所示样机从卵母细胞去掉堆积物过程中使用的步骤。 Means for removing a deposit complete prototype of (a) and (b) described in FIG. 8, and FIG. 8 (c) - (f) illustrate using Figure 8 (a) and 8 (b) from the prototype shown oocytes step cell deposit is removed during use. 聚丙烯阱粘在装载端口,以提供图8(a)中所示的入口处的较大的流体储存池。 Well polypropylene glued load port, to provide a larger fluid inlet of the storage tank shown in (a) in FIG. 8. 丙烯酸注射器连接组件退出端口,这样标准的注射器或其他配件可连接到装置。 Acrylic syringe assembly connected to exit ports, such a standard syringe or other accessories may be connected to the device. 注射器允许手工压力控制,或注射器泵可用作精确的流体控制器,普列克丝玻璃和PDMS原型允许用于胚胎贯穿通道网络的定位,并且在试验期间使卵母细胞在期望位置处停靠。 Allow manual pressure control syringe, or a syringe pump fluid controller can be used as accurate, glass and PDMS Puliekesi prototype allow for positioning through-passage embryonic network, and the oocyte during the test stops at a desired position. 另外,样机装置提供完整的视觉通路(对胚胎分析很重要),能够快速形成模型,并容易与未来的分析传感器构成一体。 Further, means for providing a complete prototype of the visual pathways (analysis is very important for the embryo), the model can be formed quickly and easily with integral sensor future analyzes. 通过使用漏斗形入口阱(图8(b)中的流入)简化了在装置中装载卵母细胞。 By using a funnel-shaped inlet well (inflow (b) in FIG. 8) simplifies the loading apparatus oocytes. 漏形模制成在通道的入口处该漏斗半的尖部与通的头部相连。 Shaped molded at the inlet of the drain passage of the tip of the funnel through the half head is connected. 这种宽漏斗结构允许卵母细胞复合体容易嵌入。 This arrangement allows a wide funnel-oocyte complexes easily fitted. 卵母细胞典型地沉入漏斗底部。 Oocytes are typically settle in the bottom hopper. 倾斜阱引导复合体进入在漏斗底部的通道入口。 Inclined guide composite trap inlet passage into the bottom of the hopper. 该装载方法简化了处理过程,因为它不需要精确的横向定位。 Approach simplifies the loading process, because it does not require precise lateral positioning. 为了将堆积物细胞处理成允许完全去除堆积物的结构,合成物经过两个收缩区(图8(c))。 In order to deposit the cells treated structure to allow complete removal of deposits, the composition after two constriction (FIG. 8 (c)). 这些变窄的区域迫使堆积物在卵母细胞的前后形成两个主块,如图8(d)中所示。 These narrowed regions are formed to force the deposit before and after the two main blocks oocyte, as shown in FIG. 8 (d). 两个收缩的堆积物制约区(图8(a))分别是200和150μm宽。 Two deposits shrinkage restriction region (FIG. 8 (a)) are respectively 200 and 150μm wide. 堆积物受到损坏,并且在限制区中集拢,然后流到移去端口(见图8(d)-8(f))),该端口包括在原型装置的微流控通道中的弯曲处彼此九十度放置的两个微小通道。 Deposits damaged, and rounded up in a restricted area and then to the port is removed (see FIG. 8 (d) -8 (f))), at the port comprises a curved microfluidic channel prototype of each other ninety placed two tiny channels. 端口允许堆积物进入(图8(e)和8(f)),但端口对于卵母细胞来说太小。 Port allows deposits to enter (FIG. 8 (e) and 8 (f)), but the port is too small oocyte. 使用流体流动控制,首先通过一个端口,然后通过另一个端口,从卵母细胞上吸走堆积物。 Using a fluid flow control, through a first port, and then through another port, from oocytes aspirated deposits.

本发明的流控通道也用来通过分析胚胎的机械特性实现胚胎健康评价的新颖方法。 Flow control channels of the invention are also used to implement the novel method healthy embryos evaluated by analyzing the mechanical properties of the embryo. 特定的机械特性证明,区分健康胚胎包括病人胚胎的表面特性和变形特性。 Demonstrate specific mechanical characteristics, surface properties to distinguish between healthy embryos and embryos deformation characteristics include patient. 胚胎在变形到没有永久损坏的一点后趋于恢复其形状,这被认为是健康的指示器,因为胚胎主动地保持其形状。 Embryo at one point that there is no permanent damage after deformation tends to recover its shape, which is considered an indicator of health because the embryo actively maintain its shape. 胚胎通过它们的薄膜有选择地运输离子,并且透明带的蛋白质恒定地重定位它们自己,以抵抗外力。 Embryos by their membrane selectively transport ions, and zona pellucida proteins constant reposition themselves, to resist external force. 离子和其他基质/代谢物运进和运出胚胎细胞是胚胎健康的功能。 Ions and other substrates / metabolites into and out of a healthy embryo is an embryonic cell function. 不健康的胚胎运输离子的能力降低,并且在变形到避免永久损坏的点后恢复其形状的相应能力降低。 Ability to transport ions unhealthy embryos decreases, to avoid the deformation and permanent damage to their respective ability to restore the shape point is lowered. 压力的控制和能够在收缩部处定位保证了胚胎能够在控制避免健康胚胎永久变形的情况下变形。 Pressure control and can be positioned at the contraction portion of the embryos to ensure that the case can be deformed in the control healthy embryos to avoid permanent deformation. 已实施的变形试验保持压力梯度低于0.1Pa/m(1mm的水/cm),并且流速等于或低于每秒几毫米。 Holding the test has been modified embodiment of the pressure gradient is less than 0.1Pa / m (1mm water / cm), and the flow rate is equal to or less than a few millimeters per second.

图9(a)-9(d)示出了用作胚胎生存能力的指示器的胚胎变形评价。 FIG. 9 (a) -9 (d) shows a modification of the evaluation of embryo viability of the embryo as the pointer. 收缩部的尺寸确定为胚胎经过时由于流体压力而变形。 Dimensional shrinkage portion when the fluid pressure is determined after embryo deformed. 健康的胚胎能在经过收缩部后较好地恢复图9(d)所示的形状。 Healthy embryos can better shape recovery (d), FIG. 9 after the constriction. 替代地,胚胎可在收缩部变形,该收缩部的尺寸防止其通过。 Alternatively, the embryos can be deformed in the shrinking, the size of the portion is prevented by the contraction. 在收缩部变形一段时间后,流体反向流动,反向和停止流动,或停止流动,使胚胎能恢复其形状。 After a period of contraction deformed, reverse flow of fluid, and the reverse flow is stopped, or the flow is stopped, the embryo can recover its shape.

通过对微流控通道提供的压力进行精确控制还能够估计胚胎与通道阱的静摩擦,因为健康的胚胎更有粘性。 Accurately controlled by the pressure of the microfluidic channel is also available to estimate the static friction of the embryos and the channel well, since more viscous healthy embryos. 这样,当与不健康的胚胎比较时,健康的胚胎可定量测量到,它更缓慢地下降到通道。 Thus, when compared to healthy embryo, the embryo may be healthy quantitatively measured, it drops to channel more slowly. 可供选择的是,可类似地比较运动的距离,在相同的流动和压力条件下健康胚胎运动更短的距离。 Alternatively, it is, can similarly compare the movement distance, the movement is shorter healthy embryos under the same flow and pressure conditions distance.

通过应用本发明的微流控通道的装置能更好地进行胚胎的流体分析。 Fluid could be better embryo by means of microfluidic channels of the present invention is applied to analysis. 从本发明的微流控通道培养装置的下游通道收集流体。 From a culture device downstream passage microfluidic channels of the present invention collect fluid. 根据按照本发明形的装置,收集到的流体已流经胚胎,因为本发明的装置避免了出现停滞的条件,并且微流控通道的尺寸使得通道中的流体经过胚胎附近。 The apparatus according to the present invention shaped, having passed through the fluid collected embryos, since the device according to the present invention avoids stagnation conditions, and the size of the microfluidic channel such that the control fluid passage through the vicinity of the embryo. 当下游流体被收集时,可能会期望添加相同量的上游流体,以便保持压力和微流控装置中的流动,由此可收集流体样本。 When the downstream fluid is collected, it may be desirable to add the same amount of the fluid upstream, in order to maintain the pressure and flow in the microfluidic device, whereby a fluid sample can be collected. 可编程的注射器泵可用来去掉流体样本,并且输入额外的上游流体。 Programmable syringe pump may be used to remove a fluid sample, and enter additional upstream fluid. 可供选择的是,可收集胚胎的所有下游流体,然后例如采用化学或光学分析来分析该流体。 Alternatively, all embryos were collected downstream of the fluid, for example, chemical or optical analysis is then employed to analyze the fluid.

本发明的系统可用于完整的卵母细胞成熟、受精和胚胎发育而无需严格的操作技术。 The system of the present invention may be used intact oocyte maturation, fertilization and embryo development without rigorous technique. 由于能够控制流体和定位胚胎,因而提供了卵母细胞成熟,与精液接触,然后模拟生物学发育的机会,所有这些都在本发明的单一装置中完成。 Since positioning control fluid and the embryo, thus providing oocyte maturation in contact with the semen, and the opportunity to simulate biological development, all of which are completed in a single apparatus of the present invention. 成熟(IVM),受精(IVF)和发育(EC)需要不同的介质,精子获能成熟和冲洗过程。 Maturation (IVM), fertilization (IVF) and developmental (EC) require a different medium, sperm capacitation process of maturation and rinse. 使用微流控通道的本发明的系统允许这些条件的快速改变和精确控制,允许在几小时或几天的时间中改变流体培养基的成分,以便模拟分泌物和成长因子在女性生殖道中的累积和胚胎移动到生殖道的不同部分。 Use of the microfluidic channel system of the present invention allows these conditions change rapidly and precisely controlled, allowing to change the composition of the fluid medium at the time of hours or days in order to simulate the growth factor secretion and accumulation in the female reproductive tract embryos and moved to a different part of the genital tract. 可以控制培养基,以便使其缓慢地流经每个胚胎(或卵母细胞)以提供新鲜供送的营养剂。 The medium may be controlled so that it slowly through each embryos (or oocytes) to provide a supply of fresh nutrients. 例如每小时一次的周期性流动与连续流动相比,将能够有限度地建立有益的自动分泌和副分泌因子,同时不会使无用的产物积累。 Periodically, for example hourly flow compared to continuous flow, will be able to automatically establish a useful and paracrine factors secreted limited degree, and will not cause unwanted product accumulation. 所有现有的IMF,IVF和EC技术可在本发明的简单装置中运用,这样不必在顺序进行的IMF,IVF和EC过程或各个过程的任何一个期间处理或转移给定的卵母细胞。 All existing IMF, IVF and EC techniques may be used in a simple apparatus of the present invention, the process or transferred to a given oocyte during any IMF, IVF, and EC is not necessary in this process sequence or the respective processes.

本发明的系统可减少多精入卵率,使用较少的精虫数量,并接合游动技术以选择最具活力的精子。 The system of the present invention may reduce the rate of polyspermy, using less number of sperm and sperm joined swimming techniques to select the most dynamic. 在典型的人类IVF过程中,在0.5至1.0ml的培养基滴液中,卵被50,000至100,000个精子包围。 In a typical human IVF process, in a medium of 0.5 to 1.0ml of the dropping, the egg is surrounded by 50,000 to 100,000 spermatozoa. 该数量可减少,因为微流控通道提供使精子和卵母细胞结合到一起的流动控制。 This number can be reduced since the microfluidic channels to provide sperm and oocytes coupled to the flow control together.

在家畜的IVF中,多精入卵率较高,例如猪约为40-70%。 In IVF of livestock, egg polyspermy higher rates, e.g., about 40-70% pig. 根据本发明,微流控通道导致精子非常靠近卵母细胞经过,例如约30μm。 According to the present invention, the microfluidic channel leads to very close to the sperm after oocyte, for example, about 30μm. 另外,可通过控制培养基流动率来控制精子靠近卵母细胞的时间间隔。 Further, the sperm may be controlled close to the time interval oocyte culture medium by controlling the flow rate. 替代地,根据本发明定位的停靠卵母细胞可保持非常靠近比较小的精子流体包的嵌入点,在减少多精入卵的机会的同时提供了靠近卵母细胞的精子的高浓度。 Alternatively, according to the present invention positioned docked oocytes can be kept very close to the relatively small fluid bag sperm embedded points, there is provided a high concentration of spermatozoa near the oocyte in reducing the chance of polyspermy simultaneously. 分离的栓子或弹丸可传递一至几个精子,以建立对输卵管中的状态的状况模拟,在那里任一时刻都有少量精子。 Isolated emboli or bolus may be transferred to a number of sperm to establish simulate the fallopian tube in a state, where any one time has a small amount of sperm. 该能力以及用于定位胚胎的具有T-交叉点或其他物理或有效的收缩通道允许将一至几个精子传递至卵细胞附近,以减少多精入卵。 This capability for positioning and having a T- intersection embryos or other physical or effective shrinkage channel to allow a transfer of several sperm to egg near to reduce polyspermy. 另外,胚胎数量级的通道的几何形状能够增加卵细胞和精子之间的接触。 Further, the geometry of the channels of the order of embryos increased contact between the egg and the sperm. 精子和侧壁的“弹起”增加了精子和卵细胞之间的有效接触。 Sperm and sidewall "bounce" increased effective contact between sperm and egg cells.

低温贮藏是另一项技术,该技术有利于根据本发明对胚胎的定位和精确控制围绕胚胎的流体环境。 Cryopreservation is another technique that is conducive to a fluid environment surrounding the embryo and precise control according to the positioning of the embryo of the present invention. 可以向在本发明的装置中定位或移动的胚胎传递低温,之后可反向应用低温贮藏工序。 May be transferred to the location or movement of the cryogenic device of the present invention, in the embryo, may be applied in reverse after cryopreservation step. 然后,如上所述,流体的改变可用来促成成熟,受精和发育。 Then, as described above, it can be used to bring about change in the fluid maturation, fertilization and development.

以上已展示和描述本发明的各种实施例,但应理解,其他的修改、替代和替换显然是本领域的普通技术之一。 Above have been shown and described various embodiments of the present invention, it is to be understood that other modifications, substitutions and alternatives are apparent to one of ordinary skill in the art. 这些修改、替代和替换可以在不背离由权利要求限定的本发明的实质和范围的情况下可做出。 Such modifications, substitutions and alterations can be made without departing from the spirit and scope of the invention as defined by the appended claims.

本发明的各种特征在权利要求中提出。 Various features of the present invention are set forth in the appended claims.

Claims (15)

1.一种评价胚胎的方法,该方法的步骤包括:将胚胎放在一个大约胚胎级别的流体通道中;在流体通道中形成流体流;和当胚胎处于该流体通道内时,评价胚胎的特性。 1. A method for the evaluation of the embryo, the method comprising the step of: the embryos in a level of about embryonic fluid passage; a fluid flow in the fluid passage; and when the embryo is within the fluid passage, the characteristics evaluation of embryos .
2.根据权利要求1所述的方法,其特征在于所述形成流体流的步骤使胚胎在流体通道内移动,所述评价胚胎特性的步骤包括测量胚胎在流体通道内移动的速度。 2. The method according to claim 1, characterized in that the rate of formation of the embryo within the fluid passageway moving step moves the embryo fluid flow within the fluid passageway, the evaluating step comprises measuring embryo properties.
3.根据权利要求1所述的方法,其特征在于所述形成流体流的步骤使胚胎在流体通道内移动,所述评价胚胎特性的步骤包括测量胚胎在流体通道内移动的距离。 3. The method according to claim 1, wherein said step of forming the embryo moving fluid stream within the fluid passageway, the evaluating step comprises measuring characteristic embryo embryo moves from within the fluid passage.
4.根据权利要求1所述的方法,其特征在于所述评价步骤包括在流体通道内的胚胎下游的流体通道中获得流体样品,以及对流体样品进行化学分析。 4. The method according to claim 1, wherein the evaluating step includes a fluid passage within the fluid passageway downstream of the embryos obtained in a fluid sample, and chemical analysis of the fluid sample.
5.根据权利要求1所述的方法,其特征在于所述流体通道包括收缩部,该收缩部的尺寸为可使胚胎在经过时变形,所述评价步骤评价在胚胎经过收缩部后恢复其形状的趋势。 5. The method according to claim 1, wherein said fluid passage comprises a constriction, the size of the constriction can deform when passing through the embryo, the embryo in the evaluation step evaluation recover its shape after the constriction the trend of.
6.根据权利要求1所述的方法,其特征在于所述流体通道包括收缩部,该收缩部的尺寸可防止胚胎通过,所述形成流体流的步骤形成流体流,以便使胚胎移动到收缩部,然后通过流体压力略微变形一段较短的时间,所述评价步骤评价在胚胎变形一段较短的时间后恢复其形状的趋势。 6. The method according to claim 1, wherein said fluid passage comprises a constriction, the size of the embryo can be prevented by the constriction, the fluid flow step of forming a fluid stream to move to shrink the embryo portion then slightly distorted short period of time by the fluid pressure, the evaluation step evaluate the tendency to restore its shape after deformation embryonic a short time.
7.一种处理胚胎的方法,其步骤包括:将胚胎放在一个大约胚胎级别的流体通道中;在流体通道中形成流体流;和当胚胎定位在该流体通道内时,处理胚胎以改变胚胎的特性。 A method of treating embryos, comprising the steps of: the embryos in a level of about embryonic fluid passage; a fluid flow in the fluid passage; and when the embryo is positioned in the fluid passage, the process to change the embryos Embryo It features.
8.根据权利要求7所述的方法,其特征在于所述流体通道包括一系列收缩部,该收缩部包括彼此的间距逐渐减小的若干突起,所述处理步骤包括在流体通道中操纵流体流,以便使胚胎经过一个或多个收缩部,并去除堆积物。 8. The method according to claim 7, wherein the fluid channel comprises a series of contraction portion, the contraction portion includes a plurality of spacing projections is gradually reduced, said process comprising the step of actuating a fluid flow in the fluid passage , so that the embryo through one or more constrictions, and remove the deposit.
9.根据权利要求8所述的方法,其特征在于一系列收缩部中的最后一个收缩部的尺寸能够阻碍胚胎通过,所述处理步骤包括操纵流体通道中的流体流,以便使胚胎经过一系列收缩部中其它一些收缩部,然后当胚胎定位在一系列收缩部中的最后一个收缩部时,操纵流体流,以便吸掉堆积物。 9. The method according to claim 8, characterized in that the size of the last portion of the contraction portion in a series of contractions can be hindered by the embryo, the process comprising the step of actuating a fluid flow in the fluid passage, so that the embryos through a series of other constriction constriction, and then when the embryo is positioned in a series of constrictions in the last constriction actuating fluid stream to siphoning off deposits.
10.根据权利要求7所述的方法,其特征在于流体通道包括其尺寸为可防止胚胎通过的收缩部,所述处理步骤包括改变流体通道中的流体,以便使酸性溶液流过定位于收缩部的胚胎,以去掉透明带。 10. The method according to claim 7, characterized in that the fluid passage comprises a constriction prevents a size of the embryo by a process comprising the step of changing the fluid passage, so that the acid solution flows through the constriction is positioned embryos, to remove the zona pellucida.
11.根据权利要求10所述的方法,其特征在于通过改变流体通道中的流体,以便使酸性溶液流过位于收缩部的两个胚胎,以去掉透明带,然后改变流体通道中的流体,以便在两个胚胎上促进嵌合体培养,从而,所述处理步骤用来形成嵌合体。 11. The method according to claim 10, characterized in that by changing the fluid passage, so that the acidic solution flow through the two constrictions located embryos, to remove the zona pellucida, and then change the fluid passage, so that promoting chimeras embryos cultured in two, whereby said processing step used to form chimeras.
12.一种微流控胚胎处理装置,它包括:一种胚胎输送网络(32,50),该胚胎输送网络具有生物培养基,以便使嵌入其中的胚胎移动,所述输送网络包括大约胚胎级别的胚胎流体通道(14)和用于将流体输入该输送网络内的开口(36,52)。 A microfluidic embryos processing apparatus comprising: one kind of embryonic transport networks (32, 50), which has biological media delivery network embryo, the embryo in order to move embedded therein, the transport network comprising a level about embryos embryonic fluid passage (14) and a fluid inlet opening (36, 52) within the transport network.
13.根据权利要求12所述的装置,其特征在于还包括在两个流体通道的交叉处形成的输送线路的t形汇合处(14b),和用于在一个单独的流体通道位置将流体输入输送网络中的第二开口。 13. The apparatus according to claim 12, wherein the junction further comprises a t-shaped transmission line formed at the intersection of two fluid passages (14b), for fluid input and a separate fluid channel position a second opening in the transport network.
14.根据权利要求1所述的装置,其特征在于还包括在所述流体通道内形成的阱(14a),它用来在所述流体通道内在预定的流速期间,保持胚胎,并允许在所述流体通道内,在小于所述预定流速的期间使胚胎排出。 14. The apparatus according to claim 1, characterized by further comprising a well (14a) formed within said fluid passage, which is used in the internal passage of the fluid during a predetermined flow rate, to maintain the embryo, and allowing the within said fluid passage, during less than the predetermined discharge flow rate of the embryo.
15.一种微流控胚胎受精装置,它包括:一种胚胎输送网络(32,50),该胚胎输送网络具有生物培养基,以便使嵌入其中的胚胎移动,所述输送网络包括大约胚胎级别的胚胎流体通道(14),以及用于将流体输入该输送网络内的开口(36,52);和在两个流体通道的交叉处形成的输送网络中的T形汇合处(14b),以及用于在一个单独的流体通道位置将精子流体输入输送网络中的第二开口。 15. A microfluidic device fertilized embryo, comprising: one kind of embryonic transport networks (32, 50), which has biological media delivery network embryo, the embryo in order to move embedded therein, the transport network comprising a level about embryos embryonic fluid passage (14), and a fluid inlet opening (36, 52) within the transport network; and a T-shaped junction in the transport network is formed at the intersection of two fluid passages (14b), and for a single fluid channel fluid into a second position opening the sperm transport network.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101835886A (en) * 2007-08-24 2010-09-15 智能生物系统有限公司 Mesoscale bioreactor platform for perfusion
WO2011160430A1 (en) * 2010-06-25 2011-12-29 Capitalbio Corporation Integrated microfluidic device for single oocyte trapping
CN103232967A (en) * 2009-09-30 2013-08-07 韦尔豪泽Nr公司 Method of singulating embryos
CN103517676A (en) * 2010-09-27 2014-01-15 奥克索金股份有限公司 Apparatus, method, and system for automated imaging and evaluation of embryos, oocytes, and stem cells
CN104254596A (en) * 2012-04-16 2014-12-31 康奈尔大学 Automated intracytoplasmic sperm injection assisted fertilization system

Families Citing this family (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2003224817B2 (en) 2002-04-01 2008-11-06 Fluidigm Corporation Microfluidic particle-analysis systems
WO2004101743A2 (en) * 2003-05-06 2004-11-25 Bellbrook Labs, Llc Three dimensional cell cultures in a microscale fluid handling system
EP1641411A1 (en) * 2003-06-06 2006-04-05 The Regents Of The University Of Michigan Integrated microfluidic sperm isolation and insemination device
GB0321158D0 (en) * 2003-09-10 2003-10-08 Central Research Lab Ltd Apparatus and method for handling cells,embryos or oocytes
EP1609850A1 (en) 2004-06-24 2005-12-28 Biovir v/Jacob Mollenbach Culture dish for culturing biological cells
JP2006055072A (en) * 2004-08-20 2006-03-02 Fujitsu Ltd Apparatus and method for conveying control
WO2006101051A1 (en) * 2005-03-18 2006-09-28 Effector Cell Institute, Inc. Cell observation aiding instrument and method of cell observation therewith
GB0508983D0 (en) 2005-05-03 2005-06-08 Oxford Gene Tech Ip Ltd Cell analyser
US20070090166A1 (en) * 2005-10-18 2007-04-26 Shuichi Takayama Microfluidic cell culture device
US20070084706A1 (en) * 2005-10-18 2007-04-19 Shuichi Takayama Microfluidic cell culture device and method for using same
TW200920841A (en) * 2007-09-25 2009-05-16 Cytyc Corp Microfluidic apparatus for manipulating imaging and analyzing cells of a cytological specimen
KR100891487B1 (en) * 2007-11-01 2009-04-01 한국과학기술원 Enucleation system of oocyte or ovum which based on microfluid mechanics
WO2009079474A1 (en) * 2007-12-14 2009-06-25 The University Of North Carolina At Chapel Hill Methods, systems, and computer readable media for facilitating automation of blastocyst microinjection
CN102112593A (en) * 2008-08-01 2011-06-29 智能生物系统公司 A chamber of a bioreactor platform
GB2464300A (en) * 2008-10-10 2010-04-14 Univ Dublin City Microfluidic multiplexed cellular and molecular analysis device and method
GB0821636D0 (en) * 2008-11-26 2008-12-31 Ucl Business Plc Device
US20120196358A1 (en) * 2009-06-01 2012-08-02 Fred Burbank Device for removing cumulus from oocytes
EP2316565A1 (en) * 2009-10-26 2011-05-04 Fei Company A micro-reactor for observing particles in a fluid
AU2015203738B2 (en) * 2013-03-14 2017-10-05 Inguran, Llc Apparatus and methods for high throughput sperm sorting
US9757726B2 (en) 2013-03-14 2017-09-12 Inguran, Llc System for high throughput sperm sorting
US10371622B2 (en) 2013-03-14 2019-08-06 Inguran, Llc Device for high throughput sperm sorting
AU2013202635B2 (en) * 2013-03-14 2015-10-29 Inguran, Llc Apparatus and methods for high throughput sperm sorting
KR101959208B1 (en) * 2017-05-24 2019-03-19 한국기계연구원 Droplet based biochip, a method for producing a tumor, and a method for deciding a metastasis tumor
WO2019195620A1 (en) * 2018-04-04 2019-10-10 The General Hospital Corporation Microfluidic systems and methods to denude mammalian oocytes

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3207424B2 (en) * 1992-05-01 2001-09-10 トラスティーズ・オブ・ザ・ユニバーシティ・オブ・ペンシルベニア Handling of fluids in micromachined analyzer
WO1999061888A2 (en) * 1998-05-22 1999-12-02 California Institute Of Technology Microfabricated cell sorter
US6193647B1 (en) * 1999-04-08 2001-02-27 The Board Of Trustees Of The University Of Illinois Microfluidic embryo and/or oocyte handling device and method

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101835886A (en) * 2007-08-24 2010-09-15 智能生物系统有限公司 Mesoscale bioreactor platform for perfusion
CN103232967A (en) * 2009-09-30 2013-08-07 韦尔豪泽Nr公司 Method of singulating embryos
WO2011160430A1 (en) * 2010-06-25 2011-12-29 Capitalbio Corporation Integrated microfluidic device for single oocyte trapping
US9445840B2 (en) 2010-06-25 2016-09-20 Tsinghua University Integrated microfluidic device for single oocyte trapping
CN103517676A (en) * 2010-09-27 2014-01-15 奥克索金股份有限公司 Apparatus, method, and system for automated imaging and evaluation of embryos, oocytes, and stem cells
CN104254596A (en) * 2012-04-16 2014-12-31 康奈尔大学 Automated intracytoplasmic sperm injection assisted fertilization system
CN104254596B (en) * 2012-04-16 2016-09-07 康奈尔大学 Automation intracytoplasmic sperm injection accessory pollination system

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IL152650D0 (en) 2003-06-24
WO2001088087A2 (en) 2001-11-22
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