WO2021248637A1 - 面向突破发育阻滞的早期胚胎拟输卵管环境体外培养芯片 - Google Patents

面向突破发育阻滞的早期胚胎拟输卵管环境体外培养芯片 Download PDF

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WO2021248637A1
WO2021248637A1 PCT/CN2020/103146 CN2020103146W WO2021248637A1 WO 2021248637 A1 WO2021248637 A1 WO 2021248637A1 CN 2020103146 W CN2020103146 W CN 2020103146W WO 2021248637 A1 WO2021248637 A1 WO 2021248637A1
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culture
plate
cell culture
fallopian tube
hole
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English (en)
French (fr)
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黄海波
顾鸣伟
戴辞海
朱易辰
陈立国
刘吉柱
王阳俊
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Suzhou University
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Suzhou University
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    • 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
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
    • 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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/38—Caps; Covers; Plugs; Pouring means
    • 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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/48—Holding appliances; Racks; Supports

Definitions

  • the invention relates to the technical field of in vitro culture, in particular to an early embryonic oviduct environment in vitro culture chip for breaking through developmental block.
  • Oocytes contain important biological genetic information needed for the reproduction of life, and are the origin of life gestation.
  • the research on oocytes has always occupied a very important position in life science and medical research.
  • scientists have begun to study the in vitro culture of early embryos, and found that the in vitro culture of early embryos is very prone to developmental block and the survival rate has not been high.
  • the so-called developmental retardation means that the reconstructed egg cell embryo starts to divide after being activated and develops from the 4-cell stage to the 8-cell stage. This period happens to be the transitional period of maternal gene-embryonic gene.
  • the maternal mRNA in the embryonic cell is exhausted, and its own genome has not been activated yet.
  • the early embryo is extremely fragile, very sensitive to the external environment, and easily stops developing.
  • the inability of the existing in vitro embryo culture technology to provide a similar environment as that in the fallopian tube is the cause of a large number of developmental blockages in the process of in vitro embryo culture.
  • it is generally considered to be more effective to improve the composition of the culture medium, the co-cultivation of trophoblast somatic cells, dynamic culture and multi-ovum co-cultivation.
  • Studies on the co-culture of trophoblast somatic cells have shown that the co-culture of oviduct epithelial cells can reduce the endogenous ROS of mouse embryos during the maternal-embryonic gene transition period, promote the transition from maternal to zygotic, and overcome embryo development in vitro.
  • Dynamic culture can simulate the physical stimulation of the oviduct wall and cilia when the embryo is transported in the fallopian tube, such as using the shear force of the fluid to stimulate the embryo, which has a positive effect on the successful development of the embryo.
  • the survival rate of the embryo is And the training speed has been improved.
  • the autocrine or paracrine factors produced by the embryo will affect the development of the embryo and the rate of blastocyst formation.
  • the interaction between the fertilized egg and the fertilized egg is very important.
  • the fertilized egg co-cultured with multiple eggs has better performance than the fertilized egg cultured in isolation. Developmental potential.
  • the purpose of the present invention is to provide a chip for in vitro culture of early embryonic oviduct environment for breaking through developmental block.
  • An early embryonic fallopian tube environment in vitro culture chip for breaking through developmental block including a culture medium driving part and a culture chip body.
  • the culture driving part includes a hose track plate, a peristaltic pump, a hose and two hose interfaces ,
  • the peristaltic pump is installed on the hose track plate, the hose is partially arranged in the hose track plate, and two free ends of the hose are respectively connected with the two hose interfaces
  • the culture chip body includes a substrate, a positioning plate, a piping plate, a sealing plate, a cover plate, a fallopian tube epithelial cell culture rack, and a cumulus cell culturing rack.
  • the substrate, positioning plate, piping plate, and sealing plate are compact from bottom to top. Connected, the cover plate and the sealing plate can be opened and closed, and the fallopian tube epithelial cell culture rack and the cumulus cell culture rack are both installed on the piping plate.
  • the sealing plate is provided with a culture medium inlet hole, a culture medium outlet hole, and a sealing hole, and the two hose ports are respectively tightly fitted in the culture medium inlet hole and the culture medium outlet hole.
  • the cover plate and the sealing hole are hermetically matched.
  • the piping plate is provided with a culture medium inlet pool, a culture medium outlet pool, an inlet main channel, an outlet main channel, a culture tank, a fallopian tube epithelial cell culture rack placement groove, and a cumulus cell culture rack placement
  • the culture medium inlet pool and the culture medium outlet pool are respectively arranged corresponding to the culture medium inlet hole and the culture medium outlet hole, and the culture medium inlet pool and the culture medium outlet pool are respectively connected to the inlet main channel and the outlet
  • the main channel is connected, the inlet main channel and the outlet main channel are both connected to the culture tank through a shunt channel, the fallopian tube epithelial cell culture rack placement groove is arranged on the inlet main channel, and the cumulus cell
  • the culturing rack placement groove is arranged on the culturing tank.
  • two supports are provided on the sealing plate, the two supports are connected with the cover plate by a rotating shaft, and the cover plate is provided with a boss, and the boss is connected to the cover plate.
  • the sealing hole is sealed and matched.
  • a positioning array is provided on the positioning plate, the positioning array includes a plurality of positioning units, the plurality of positioning units are arranged in an array, and the plurality of positioning units extend into the culture chamber In the pool.
  • each of the positioning units includes a plurality of fan lobes, and the plurality of fan lobes form an open cylindrical structure.
  • the fallopian tube epithelial cell culture rack includes a hollow first cylinder, at least one first through hole group is arranged on the axial surface of the first cylinder, and the first through hole group includes A plurality of first through holes, the plurality of first through holes are arranged in a circular array, a plurality of second through hole groups are provided on the peripheral surface of the first cylinder, and the second through hole groups include a plurality of The second through holes are arranged in a circular array.
  • the cumulus cell culture rack includes a cumulus cell culture membrane scaffold and a cumulus cell culture membrane, and the cumulus cell culture membrane scaffold is connected to the surface of the cumulus cell culture membrane.
  • the cumulus cell culture membrane support includes a second cylinder and two support plates connected to the second cylinder, and the second cylinder closely fits with the culture tank. .
  • the cover plate, the sealing plate, the pipe plate and the positioning plate are respectively provided with a first threaded hole, a second threaded hole, a third threaded hole, and a fourth threaded hole.
  • the invention cleverly designs the in vitro culture chip, divides it into a culture medium driving part and a culture chip body, realizes a compact and reasonable layout of each structure, and integrates the three major factors of the construct cytotrophoblast 3D environment, dynamic culture, and multi-egg co-cultivation.
  • On the same chip there are fallopian tube epithelial cell culture racks and cumulus cell culture racks in the pipeline plate, which are used to construct the 3D environment of the somatic trophoblast.
  • the peristaltic pump is used to drive the flow of the medium to provide dynamic culture of oocytes. Environment, multiple oocytes are placed in a positioning array to achieve the effect of multi-egg co-cultivation.
  • Figure 1 is a schematic structural diagram of a preferred embodiment of the present invention.
  • FIG. 2 is a schematic diagram of the structure of the culture medium driving part of the preferred embodiment of the present invention.
  • FIG. 3 is a schematic diagram of the structure of the culture chip body of the preferred embodiment of the present invention.
  • FIG. 4 is a schematic diagram of the structure of the cover plate of the preferred embodiment of the present invention.
  • Figure 5 is a schematic diagram of the structure of the sealing plate of the preferred embodiment of the present invention.
  • Figure 6 is a schematic structural diagram of a piping plate according to a preferred embodiment of the present invention.
  • Fig. 7 is a schematic structural diagram of a positioning plate of a preferred embodiment of the present invention.
  • Fig. 8 is an enlarged schematic diagram of A in Fig. 7;
  • Figure 9 is a schematic structural diagram of a fallopian tube epithelial cell culture rack according to a preferred embodiment of the present invention.
  • Figure 10 is a schematic structural diagram of a cumulus cell culture rack according to a preferred embodiment of the present invention.
  • Figure 11 is a schematic structural diagram of a cumulus cell culture membrane according to a preferred embodiment of the present invention.
  • Figure 12 is a partial enlarged schematic diagram of the cumulus cell culture membrane of the preferred embodiment of the present invention.
  • an early embryonic oviduct-like environment in vitro culture chip for breaking through developmental block is used for in vitro culture of oocytes, including medium driving part 1 and culture chip body 2.
  • the culture medium driving part 1 includes a hose track plate 11, a peristaltic pump 12, a hose 13, and two hose ports 14.
  • the peristaltic pump 12 is installed on the hose track plate 11, and the hose 13 is partially arranged on the hose track plate 11. Wherein, the two free ends of the hose 13 are respectively connected to the two hose ports 14.
  • the culture chip body 2 includes a base plate 21, a positioning plate 22, a pipe plate 23, a sealing plate 24, a cover plate 25, and a fallopian tube epithelial cell culture rack 26 And the cumulus cell culture rack 27, the base plate 21, the positioning plate 22, the pipe plate 23, and the sealing plate 24 are tightly connected from bottom to top, the cover plate 25 and the sealing plate 24 can be opened and closed, the fallopian tube epithelial cell culture rack 26 and the egg
  • the mound cell culture racks 27 are all installed on the pipe plate 23.
  • the sealing plate 24 of the present invention is preferably provided with a culture medium inlet hole 241, a culture medium outlet hole 242, and a sealing hole 243.
  • the cover plate 25 and the sealing hole 243 are tightly matched with each other, and the two hose ports 14 are respectively closely matched.
  • the inner surface of the culture medium inlet hole 241 and the inner surface of the culture medium outlet hole 242 can be coated with impermeable paint.
  • the present invention preferably provides two supports 244 on the sealing plate 24.
  • the two supports 244 are connected to the cover plate 25 by a rotating shaft 28, and the cover plate 25 is provided with The boss 251, as shown in FIG. 4, the boss 251 and the sealing hole 243 are hermetically fitted.
  • the sealing hole 243 is provided at the center of the sealing plate 24, the medium inlet hole 241 and the medium outlet hole 242 are respectively located on the left and right sides of the sealing hole 243, and the two supports 244 are both located on the rear side of the sealing hole 243.
  • the piping plate 23 of the present invention is preferably provided with a culture medium inlet tank 231, a culture medium outlet tank 232, an inlet main channel 233, an outlet main channel 234, a culture tank 235, and a fallopian tube epithelial cell culture rack placement groove 236.
  • the cumulus cell culture rack placement slot 237, the medium inlet pool 231 and the medium outlet pool 232 are respectively arranged corresponding to the medium inlet hole 241 and the medium outlet hole 242, the medium inlet pool 231 and the medium outlet pool 232 respectively It is connected to the inlet main channel 233 and the outlet main channel 234.
  • the inlet main channel 233 and the outlet main channel 234 are both connected to the culture tank 235 through the shunt channel 238.
  • the fallopian tube epithelial cell culture rack placement groove 237 is provided on the inlet main channel 234 ,
  • the cumulus cell culture rack placement slot 237 is set on the culture tank 235.
  • the culture tank 235 is located at the center of the duct plate 23.
  • the medium inlet pool 231 is coaxial with the medium inlet hole 241
  • the medium outlet pool 232 is coaxial with the medium outlet hole 242, which facilitates the flow of the medium.
  • the diameter of the culture tank 235 is 4 to 5 mm
  • the diameter of the medium inlet tank 231 is 2 to 3 mm
  • the diameter of the medium outlet tank 232 is 2 to 3 mm
  • the width of the main inlet channel 233 is 1.0 to 1.2 mm.
  • the length of the main inlet channel 233 is 9-10mm
  • the width of a single channel in the shunt channel 238 is 0.3-0.4mm
  • the length of the room is 15mm.
  • the fallopian tube epithelial cell culture rack 26 includes a hollow first cylinder 261. At least one first through hole group is provided on the axial surface of the first cylinder 261, and the first through hole group includes a plurality of first through holes.
  • each first through hole 262 extends through the two axial surfaces of the first cylinder 261 in the axial direction, a plurality of first through holes 262 are arranged in a circular array, and the peripheral surface of the first cylinder 261 is provided There are a plurality of second through hole groups, the plurality of second through hole groups are arranged at intervals along the axial direction, the second through hole group includes a plurality of second through holes 263, and the plurality of second through holes 263 are arranged in a circular array, The second through hole 263 communicates with the first through hole 262.
  • two first through hole groups are provided on the axial surface of the first cylinder 261, and the two first through hole groups extend in the radial direction.
  • the cumulus cell culture frame 27 includes a cumulus cell culture membrane support 271 and a cumulus cell culture membrane 272, and the cumulus cell culture membrane support 271 is connected to the surface of the cumulus cell culture membrane 272.
  • the cumulus cell culture membrane support 271 includes a second cylinder 2711, two support plates 2712 connected to the upper part of the second cylinder 2711, and a bottom part of the second cylinder 2711.
  • the two connected support blocks 2713 and the second cylinder 2711 are closely matched with the culture tank 235 to compress the volume of the culture tank 235, save the cost of the culture medium, and prevent the outflow of the culture medium.
  • the diameter of the cumulus cell culture membrane 272 is 3 to 4 mm and the thickness is 0.1 to 0.2 mm. It is further preferred that the cumulus cell culture membrane 272 includes a third cylinder 2721.
  • the side of the third cylinder 2721 is provided with two rows of channel assemblies, and each row of channel assemblies includes a plurality of parallel first channels 2722 and a plurality of parallel second channels. 2723, the first channel 2722 and the second channel 2723 are arranged vertically, and the plurality of first channels 2722 and the plurality of second channels 2723 are arranged in a vertical array.
  • the sizes of the first channel 2722 and the second channel 2723 are all 0.06 ⁇ 0.03mm, The distance between the two first passages 2722 in the same row and the distance between the two second passages 2723 are both 0.09 mm.
  • the upper and lower surfaces of the third cylinder 2721 have a plurality of square holes 2724 penetrating through them. The intersection of the passage 2723 communicates with the square hole 2724, and the size of the square hole 2724 is 0.15 ⁇ 0.15 mm.
  • the extended space of the sealed hole 243 can enclose the culture tank 235, the fallopian tube epithelial cell culture rack placing groove 236, and the cumulus cell culture rack placing groove 237.
  • the positioning plate 22 of the present invention is provided with a positioning array.
  • the positioning array includes a plurality of positioning units 221, which are arranged in an array, and the plurality of positioning units 221 extend into the culture tank. 235 within.
  • each positioning unit 221 includes a plurality of fan lobes 2211, and the plurality of fan lobes 2211 form an open cylindrical structure.
  • the positioning array includes nine positioning units 221, and the nine positioning units 221 are arranged in a 3 ⁇ 3 array. It is further preferred that the positioning unit 221 in the middle of the nine positioning units 221 is located at the center of the positioning plate 22. It is further preferred that each positioning unit 221 includes eight fan lobes 2211. It is further preferred that the inner diameter of the positioning unit 221 is 200-240 ⁇ m, which matches the tip of a 0.5-10 ⁇ l pipette, the positioning unit 221 can accommodate 1-2 oocytes, and the outer diameter of the positioning unit 221 is 240-300 ⁇ m. The gap between the petals 2211 is 50-70 ⁇ m, and the height of each fan petal 2211 is 200-250 ⁇ m.
  • a first threaded hole 252, a second threaded hole 245, a third threaded hole 239, and a fourth threaded hole 222 are provided on the cover plate 25, the sealing plate 24, the piping plate 23, and the positioning plate 22, respectively.
  • the first threaded hole 252, the second threaded hole 245, the third threaded hole 239, and the fourth threaded hole 222 have the same size.
  • the second threaded hole 245, the third threaded hole 239, and the fourth threaded hole 222 are coaxial.
  • the first threaded hole 252, the second threaded hole 245, the third threaded hole 239, and the fourth threaded hole 222 are concentric, and bolts (not shown in the figure) can be used to connect the cover plate 25 and the sealing plate 24 tightly connected together.
  • the thickness of the substrate 21 is preferably 2 to 3 mm
  • the thickness of the positioning plate 22 is 2 to 3 mm
  • the thickness of the duct plate 23 is 5 to 6 mm
  • the thickness of the sealing plate 24 is 3 to 4 mm
  • the substrate 21 is made of glass material
  • the positioning plate 22 The pipe plate 23 and the sealing plate 24 are made of polydimethylsiloxane material.
  • the fallopian tube epithelial cell culture rack 26 is implanted with oviduct epithelial cells for culture, and the cumulus cell culture rack 27 is implanted with cumulus cells for culture.
  • a small amount of medium liquid is dropped into the positioning unit 221, and the oocytes are taken and put into In the positioning unit 221, the fallopian tube epithelial cell culture rack 26 is placed in the fallopian tube epithelial cell culture rack placing groove 236, the cumulus cell culture rack 27 is placed in the cumulus cell culture rack placing groove 237, the cover plate 25 is closed, and the cover plate is closed.
  • the boss 251 on the 25 is in a sealed fit with the sealing hole 243 on the sealing plate 24, and the liquid is prevented by screwing the bolts into the first threaded hole 252, the second threaded hole 245, the third threaded hole 239, and the fourth threaded hole 222.
  • another A hose interface 14 is fixed in the culture medium inlet hole 241, and the peristaltic pump 12 is turned on to connect it to the mains power, so that the culture medium flows in the in vitro culture chip and has a dynamic culture effect on the oocytes.

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Abstract

一种面向突破发育阻滞的早期胚胎拟输卵管环境体外培养芯片，包括培养基驱动部分和培养芯片本体，培养基驱动部分包括软管轨道板、蠕动泵、软管和两个软管接口，蠕动泵安装在软管轨道板上，软管部分设置于软管轨道板中，软管的两自由端分别与两个软管接口相连接，培养芯片本体包括基板、定位板、管道板、密封板、盖板、输卵管上皮细胞培养架和卵丘细胞培养架，基板、定位板、管道板、密封板从下往上依次紧密连接，盖板与密封板可开合连接，输卵管上皮细胞培养架和卵丘细胞培养架均安装在管道板上。

Description

面向突破发育阻滞的早期胚胎拟输卵管环境体外培养芯片 技术领域
本发明涉及体外培养技术领域，尤其涉及一种面向突破发育阻滞的早期胚胎拟输卵管环境体外培养芯片。
背景技术
卵母细胞包含生命体繁殖时所需的重要生物遗传信息，是生命孕育的起源，对卵母细胞的研究在生命科学与医学研究中一直占据着非常重要的位置。随着科学技术的发展，科学家们开始了对早期胚胎进行体外培养的研究，发现早期胚胎的体外培养极易遇到发育阻滞而导致存活率一直不高。所谓发育阻滞是指卵细胞重构胚从被激活后开始分裂，在4细胞期发育到8细胞期间。此时期正好是母体基因-胚胎基因过渡期，胚胎细胞中母源mRNA消耗殆尽，其自身基因组尚未被激活。在此阶段，早期胚胎极为脆弱，对外界环境非常敏感，极易停止发育。
现有的体外胚胎培养技术无法提供如同输卵管内的相近环境是导致体外胚胎培养过程中出现大量发育阻滞现象的原因。为了帮助卵母细胞在体外胚胎培养过程中通过发育阻滞，改进培养液成分、滋养层体细胞共同培养、动态培养和多卵协同培养被普遍认为是比较有效的。在滋养层体细胞共培养方面的研究表明，输卵管上皮细胞共培养能够降低母体-胚胎基因过渡期小鼠胚胎的内源性ROS，促进母型向合子型过渡，能够克服胚胎体外发育阻滞。动态培养能够模拟胚胎在输卵管中运输时，受到输卵管壁与纤毛的物理刺激，比如利用流体的剪切力刺激胚胎，对胚胎的成功发育起到积极的作用，与静态培养比较，胚胎的存活率和培养速度均有所提高。胚胎自身产生的自分泌或旁分泌因子会影响胚胎发育及其囊胚形成率，受精卵与受精卵之间的相互 交流非常重要，多卵共培养的受精卵比孤立培养的受精卵具有更好的发育潜能。
动态培养体系已经进行了非常多的研究并取得了具有相对静态培养体系明显优势性的实验结果。然而在实际应用中，静态培养体系依然占据了统治性的地位，操作的复杂性、装置的落后、使用不便阻碍了动态培养体系相关技术的应用推广。然而，随着卵细胞操作技术的发展趋势，静态培养体系的成功率已经难以满足需求。
因此，针对上述技术问题，有必要提供一种面向突破发育阻滞的早期胚胎拟输卵管环境体外培养芯片。
发明内容
针对现有技术不足，本发明的目的在于提供一种面向突破发育阻滞的早期胚胎拟输卵管环境体外培养芯片。
为了实现上述目的，本发明一实施例提供的技术方案如下：
一种面向突破发育阻滞的早期胚胎拟输卵管环境体外培养芯片，包括培养基驱动部分和培养芯片本体，所述培养基驱动部分包括软管轨道板、蠕动泵、软管和两个软管接口，所述蠕动泵安装在所述软管轨道板上，所述软管部分设置于所述软管轨道板中，所述软管的两自由端分别与所述两个软管接口相连接，所述培养芯片本体包括基板、定位板、管道板、密封板、盖板、输卵管上皮细胞培养架和卵丘细胞培养架，所述基板、定位板、管道板、密封板从下往上依次紧密连接，所述盖板与所述密封板可开合连接，所述输卵管上皮细胞培养架和卵丘细胞培养架均安装在所述管道板上。
作为本发明的进一步改进，所述密封板上设置有培养基入口孔、培养基出口孔、密封孔，两个所述软管接口分别紧密配合于所述培养基入口孔、培养基出口孔中，所述盖板与所述密封孔密封配合。
作为本发明的进一步改进，所述管道板上设置有培养基入口池、培养基出口池、入口主通道、出口主通道、培养仓池、输卵管上皮细胞培养架放置槽和卵丘细胞培养架放置槽，所述培养基入口池、培养基出口池分别与所述培养基入口孔、培养基出口孔相对应设置，所述培养基入口池、培养基出口池分别与所述入口主通道、出口主通道相连通，所述入口主通道、出口主通道均通过分流通道与所述培养仓池相连通，所述输卵管上皮细胞培养架放置槽设置于所述入口主通道上，所述卵丘细胞培养架放置槽设置于所述培养仓池上。
作为本发明的进一步改进，所述密封板上设置有两个支座，所述两个支座与所述盖板通过转轴相连接，所述盖板上设置有凸台，所述凸台与所述密封孔密封配合。
作为本发明的进一步改进，所述定位板上设置有定位阵列，所述定位阵列包括多个定位单元，所述多个定位单元呈阵列排布，所述多个定位单元伸入所述培养仓池内。
作为本发明的进一步改进，每个所述定位单元包括多个扇瓣，所述多个扇瓣形成开放的圆柱状结构。
作为本发明的进一步改进，所述输卵管上皮细胞培养架包括空心的第一圆柱体，所述第一圆柱体的轴面上设置有至少一个第一通孔组，所述第一通孔组包括多个第一通孔，所述多个第一通孔呈圆形阵列排布，所述第一圆柱体的周面上设置有多个第二通孔组，所述第二通孔组包括多个第二通孔，所述多个第二通孔呈圆形阵列排布。
作为本发明的进一步改进，所述卵丘细胞培养架包括卵丘细胞培养膜支架和卵丘细胞培养膜，所述卵丘细胞培养膜支架与所述卵丘细胞培养膜表面相连接。
作为本发明的进一步改进，所述卵丘细胞培养膜支架包括第二圆柱体、 与所述第二圆柱体相连接的两个支板，所述第二圆柱体与所述培养仓池紧密配合。
作为本发明的进一步改进，所述盖板、密封板、管道板、定位板上分别设置有第一螺纹孔、第二螺纹孔、第三螺纹孔、第四螺纹孔。
本发明的有益效果是：
本发明对体外培养芯片进行巧妙地设计，分成培养基驱动部分和培养芯片本体，实现各结构紧凑合理地布局，将构建体细胞滋养层3D环境、动态培养、多卵协同培养三大因素集成在同一芯片上，在管道板中设有输卵管上皮细胞培养架和卵丘细胞培养架，用于构建体细胞滋养层3D环境，使用蠕动泵驱动培养基流动，用于对卵母细胞提供动态培养的环境，将多个卵母细胞放在定位阵列中，达到多卵协同培养的效果，不但可以更加逼近卵母细胞发育的真实自然环境，同时还可以研究各因素之间的内在联系，最终达到更好的培养效果，突破早期胚胎发育阻滞，具有外形美观、功能齐全、效率高、提升体外培养成功率、便携等诸多优点。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明中记载的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明的优选实施例的结构示意图；
图2为本发明的优选实施例的培养基驱动部分的结构示意图；
图3为本发明的优选实施例的培养芯片本体的结构示意图；
图4为本发明的优选实施例的盖板的结构示意图；
图5为本发明的优选实施例的密封板的结构示意图；
图6为本发明的优选实施例的管道板的结构示意图；
图7为本发明的优选实施例的定位板的结构示意图；
图8为图7中A的放大示意图；
图9为本发明的优选实施例的输卵管上皮细胞培养架的结构示意图；
图10为本发明的优选实施例的卵丘细胞培养架的结构示意图；
图11为本发明的优选实施例的卵丘细胞培养膜的结构示意图；
图12为本发明的优选实施例的卵丘细胞培养膜的局部放大示意图；
图中：1、培养基驱动部分，11、软管轨道板，12、蠕动泵，13、软管，14、软管接口，2、培养芯片本体，21、基板，22、定位板，221、定位单元，222、第四螺纹孔，2211、扇瓣，23、管道板，231、培养基入口池，232、培养基出口池，233、入口主通道，234、出口主通道，235、培养仓池，236、输卵管上皮细胞培养架放置槽，237、卵丘细胞培养架放置槽，238、分流通道，239、第三螺纹孔，24、密封板，241、培养基入口孔，242、培养基出口孔，243、密封孔，244、支座，245、第二螺纹孔，25、盖板，251、凸台，252、第一螺纹孔，26、输卵管上皮细胞培养架，261、第一圆柱体，262、第一通孔，263、第二通孔，27、卵丘细胞培养架，271、卵丘细胞培养膜支架，272、卵丘细胞培养膜，2711、第二圆柱体，2712、支板，2713、支块，2721、第三圆柱体，2722、第一通道，2723、第二通道，2724、正方形孔，28、转轴。
具体实施方式
为了使本技术领域的人员更好地理解本发明中的技术方案，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，都应当属于本发明保护的范围。
如图1、图2、图3所示，一种面向突破发育阻滞的早期胚胎拟输卵管环境体外培养芯片，用于卵母细胞的体外培养，包括培养基驱动部分1和培养芯片本体2，培养基驱动部分1包括软管轨道板11、蠕动泵12、软管13和两个软管接口14，蠕动泵12安装在软管轨道板11上，软管13部分设置于软管轨道板11中，软管13的两自由端分别与两个软管接口14相连接，培养芯片本体2包括基板21、定位板22、管道板23、密封板24、盖板25、输卵管上皮细胞培养架26和卵丘细胞培养架27，基板21、定位板22、管道板23、密封板24从下往上依次紧密连接，盖板25与密封板24可开合连接，输卵管上皮细胞培养架26和卵丘细胞培养架27均安装在管道板23上。
如图5所示，本发明优选密封板24上设置有培养基入口孔241、培养基出口孔242、密封孔243，盖板25与密封孔243密封配合，两个软管接口14分别紧密配合于培养基入口孔241、培养基出口孔242中。为防止培养基流出，可在两个软管接口14外表面、培养基入口孔241内表面、培养基出口孔242内表面涂防渗透涂料。
为了便于盖板25与密封板24的开合连接，本发明优选密封板24上设置有两个支座244，两个支座244与盖板25通过转轴28相连接，盖板25上设置有凸台251，如图4所示，凸台251与密封孔243密封配合。
进一步优选密封孔243设于密封板24的中心位置，培养基入口孔241、培养基出口孔242分别位于密封孔243的左右两侧，两个支座244均位于密封孔243的后侧。
如图6所示，本发明优选管道板23上设置有培养基入口池231、培养基出口池232、入口主通道233、出口主通道234、培养仓池235、输卵管上皮细胞培养架放置槽236和卵丘细胞培养架放置槽237，培养基入口池231、培养基出口池232分别与培养基入口孔241、培养基出口孔242相对应设置，培 养基入口池231、培养基出口池232分别与入口主通道233、出口主通道234相连通，入口主通道233、出口主通道234均通过分流通道238与培养仓池235相连通，输卵管上皮细胞培养架放置槽237设置于入口主通道234上，卵丘细胞培养架放置槽237设置于培养仓池235上。
进一步优选培养仓池235位于管道板23的中心位置。
进一步优选培养基入口池231与培养基入口孔241同轴心，培养基出口池232与培养基出口孔242同轴心，便于培养基的流动。
本发明优选培养仓池235的直径为4～5mm，培养基入口池231的直径为2～3mm，培养基出口池232的直径为2～3mm，入口主通道233的宽度为1.0～1.2mm，入口主通道233的长度为9～10mm，分流通道238中单个通道宽度为0.3～0.4mm，入口主通道233与其中一个分流通道238连接处、出口主通道234与另一个分流通道238连接处之间的长度为15mm。
如图9所示，输卵管上皮细胞培养架26包括空心的第一圆柱体261，第一圆柱体261的轴面上设置有至少一个第一通孔组，第一通孔组包括多个第一通孔262，每个第一通孔262沿轴向延伸贯穿第一圆柱体261的两轴面，多个第一通孔262呈圆形阵列排布，第一圆柱体261的周面上设置有多个第二通孔组，多个第二通孔组沿轴向间隔设置，第二通孔组包括多个第二通孔263，多个第二通孔263呈圆形阵列排布，第二通孔263与第一通孔262相连通。本实施例中，第一圆柱体261的轴面上设置有两个第一通孔组，两个第一通孔组沿径向延伸。
如图10所示，卵丘细胞培养架27包括卵丘细胞培养膜支架271和卵丘细胞培养膜272，卵丘细胞培养膜支架271与卵丘细胞培养膜272表面相连接。
如图11、图12所示，进一步优选卵丘细胞培养膜支架271包括第二圆柱体2711、与第二圆柱体2711的上部相连接的两个支板2712以及与第二圆柱体2711的底部相连接的两个支块2713，第二圆柱体2711与培养仓池235紧 密配合，压缩培养仓池235的体积，节省培养基成本，同时防止培养基的流出。
进一步优选卵丘细胞培养膜272的直径为3～4mm、厚度为0.1～0.2mm。进一步优选卵丘细胞培养膜272包括第三圆柱体2721，第三圆柱体2721的侧面设置有两排通道组件，每排通道组件包括平行的多个第一通道2722和平行的多个第二通道2723，第一通道2722与第二通道2723垂直设置，多个第一通道2722与多个第二通道2723呈垂直阵列分布，第一通道2722、第二通道2723的大小都为0.06×0.03mm，同一排的两个第一通道2722之间间距、两个第二通道2723之间间距均为0.09mm，第三圆柱体2721的上下表面贯穿有多个正方形孔2724，第一通道2722与第二通道2723交汇处与正方形孔2724相连通，正方形孔2724的大小为0.15×0.15mm。
进一步优选密封孔243的延伸空间能够包络住培养仓池235、输卵管上皮细胞培养架放置槽236和卵丘细胞培养架放置槽237。
如图7、图8所示，本发明优选定位板22上设置有定位阵列，定位阵列包括多个定位单元221，多个定位单元221呈阵列排布，多个定位单元221伸入培养仓池235内。
进一步优选每个定位单元221包括多个扇瓣2211，多个扇瓣2211形成开放的圆柱状结构。
进一步优选定位阵列包括九个定位单元221，九个定位单元221呈3×3阵列排布。进一步优选九个定位单元221中处于中间的那个定位单元221位于定位板22的中心位置。进一步优选每个定位单元221包括八个扇瓣2211。进一步优选定位单元221的内径为200～240μm，与0.5-10μl移液枪枪头匹配，定位单元221内可容纳1-2个卵母细胞，定位单元221外径为240～300μm，相邻扇瓣2211之间间隙为50～70μm，每个扇瓣2211高度为200～250μm。
本发明优选盖板25、密封板24、管道板23、定位板22上分别设置有第 一螺纹孔252、第二螺纹孔245、第三螺纹孔239、第四螺纹孔222。第一螺纹孔252、第二螺纹孔245、第三螺纹孔239、第四螺纹孔222大小一样，第二螺纹孔245、第三螺纹孔239、第四螺纹孔222同轴心，当盖板25处于合上状态时，第一螺纹孔252、第二螺纹孔245、第三螺纹孔239、第四螺纹孔222同轴心，可用螺栓(图中未示出)将盖板25与密封板24紧密连接在一起。
本发明优选基板21厚度为2～3mm，定位板22厚度为2～3mm，管道板23厚度为5～6mm，密封板24厚度为3～4mm，基板21采用玻璃材料制成，定位板22、管道板23、密封板24采用聚二甲基硅氧烷材料制成。
本发明工作原理如下：
将输卵管上皮细胞培养架26上植入输卵管上皮细胞进行培养，将卵丘细胞培养架27上植入卵丘细胞进行培养，在定位单元221中滴入少量培养基液体，取卵母细胞放入定位单元221中，将输卵管上皮细胞培养架26放置于输卵管上皮细胞培养架放置槽236，将卵丘细胞培养架27放置于卵丘细胞培养架放置槽237，盖板25合上，使盖板25上的凸台251与密封板24上的密封孔243密封配合，通过将螺栓旋入第一螺纹孔252、第二螺纹孔245、第三螺纹孔239、第四螺纹孔222防止培养基液体流出，将一个软管接口14固定在培养基出口孔242中，从培养基入口孔241注满培养基液体，待培养芯片本体2和软管13中都已经注满培养基液体时，将另一个软管接口14固定在培养基入口孔241中，打开蠕动泵12开关，将其接入市电，从而使得培养基在体外培养芯片中流动，对卵母细胞产生动态培养的作用。
对于本领域技术人员而言，显然本发明不限于上述示范性实施例的细节，而且在不背离本发明的精神或基本特征的情况下，能够以其他的具体形式实现本发明。因此，无论从哪一点来看，均应将实施例看作是示范性的，而且是非限制性的，本发明的范围由所附权利要求而不是上述说明限定，因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。 不应将权利要求中的任何附图标记视为限制所涉及的权利要求。
此外，应当理解，虽然本说明书按照实施方式加以描述，但并非每个实施方式仅包含一个独立的技术方案，说明书的这种叙述方式仅仅是为清楚起见，本领域技术人员应当将说明书作为一个整体，各实施例中的技术方案也可以经适当组合，形成本领域技术人员可以理解的其他实施方式。

Claims (10)

  1. 一种面向突破发育阻滞的早期胚胎拟输卵管环境体外培养芯片，其特征在于，包括培养基驱动部分和培养芯片本体，所述培养基驱动部分包括软管轨道板、蠕动泵、软管和两个软管接口，所述蠕动泵安装在所述软管轨道板上，所述软管部分设置于所述软管轨道板中，所述软管的两自由端分别与所述两个软管接口相连接，所述培养芯片本体包括基板、定位板、管道板、密封板、盖板、输卵管上皮细胞培养架和卵丘细胞培养架，所述基板、定位板、管道板、密封板从下往上依次紧密连接，所述盖板与所述密封板可开合连接，所述输卵管上皮细胞培养架和卵丘细胞培养架均安装在所述管道板上。
  2. 根据权利要求1所述的面向突破发育阻滞的早期胚胎拟输卵管环境体外培养芯片，其特征在于，所述密封板上设置有培养基入口孔、培养基出口孔、密封孔，两个所述软管接口分别紧密配合于所述培养基入口孔、培养基出口孔中，所述盖板与所述密封孔密封配合。
  3. 根据权利要求1所述的面向突破发育阻滞的早期胚胎拟输卵管环境体外培养芯片，其特征在于，所述管道板上设置有培养基入口池、培养基出口池、入口主通道、出口主通道、培养仓池、输卵管上皮细胞培养架放置槽和卵丘细胞培养架放置槽，所述培养基入口池、培养基出口池分别与所述培养基入口孔、培养基出口孔相对应设置，所述培养基入口池、培养基出口池分别与所述入口主通道、出口主通道相连通，所述入口主通道、出口主通道均通过分流通道与所述培养仓池相连通，所述输卵管上皮细胞培养架放置槽设置于所述入口主通道上，所述卵丘细胞培养架放置槽设置于所述培养仓池上。
  4. 根据权利要求2所述的面向突破发育阻滞的早期胚胎拟输卵管环境体外培养芯片，其特征在于，所述密封板上设置有两个支座，所述两个支座与所述盖板通过转轴相连接，所述盖板上设置有凸台，所述凸台与所述密封孔密封配合。
  5. 根据权利要求1所述的面向突破发育阻滞的早期胚胎拟输卵管环境体 外培养芯片，其特征在于，所述定位板上设置有定位阵列，所述定位阵列包括多个定位单元，所述多个定位单元呈阵列排布，所述多个定位单元伸入所述培养仓池内。
  6. 根据权利要求5所述的面向突破发育阻滞的早期胚胎拟输卵管环境体外培养芯片，其特征在于，每个所述定位单元包括多个扇瓣，所述多个扇瓣形成开放的圆柱状结构。
  7. 根据权利要求1所述的面向突破发育阻滞的早期胚胎拟输卵管环境体外培养芯片，其特征在于，所述输卵管上皮细胞培养架包括空心的第一圆柱体，所述第一圆柱体的轴面上设置有至少一个第一通孔组，所述第一通孔组包括多个第一通孔，所述多个第一通孔呈圆形阵列排布，所述第一圆柱体的周面上设置有多个第二通孔组，所述第二通孔组包括多个第二通孔，所述多个第二通孔呈圆形阵列排布。
  8. 根据权利要求1所述的面向突破发育阻滞的早期胚胎拟输卵管环境体外培养芯片，其特征在于，所述卵丘细胞培养架包括卵丘细胞培养膜支架和卵丘细胞培养膜，所述卵丘细胞培养膜支架与所述卵丘细胞培养膜表面相连接。
  9. 根据权利要求8所述的面向突破发育阻滞的早期胚胎拟输卵管环境体外培养芯片，其特征在于，所述卵丘细胞培养膜支架包括第二圆柱体、与所述第二圆柱体相连接的两个支板，所述第二圆柱体与所述培养仓池紧密配合。
  10. 根据权利要求2所述的面向突破发育阻滞的早期胚胎拟输卵管环境体外培养芯片，其特征在于，所述盖板、密封板、管道板、定位板上分别设置有第一螺纹孔、第二螺纹孔、第三螺纹孔、第四螺纹孔。
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