WO2024045482A1 - 一种早期胚胎的联合激活装置和方法 - Google Patents

一种早期胚胎的联合激活装置和方法 Download PDF

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Publication number
WO2024045482A1
WO2024045482A1 PCT/CN2023/073078 CN2023073078W WO2024045482A1 WO 2024045482 A1 WO2024045482 A1 WO 2024045482A1 CN 2023073078 W CN2023073078 W CN 2023073078W WO 2024045482 A1 WO2024045482 A1 WO 2024045482A1
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early
early embryos
liquid metal
capillary
activation
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French (fr)
Inventor
黄海波
梅世亮
苗胜
庞焱
姜德胜
陈立国
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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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/10—Petri dish
    • 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
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • 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
    • C12M25/10—Hollow fibers or tubes
    • 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
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
    • 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
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/08—Chemical, biochemical or biological means, e.g. plasma jet, co-culture

Definitions

  • the present application relates to the technical field of embryo engineering, and in particular to a joint activation device and method for early embryos.
  • early embryo activation is a common approach at various stages of the early embryo micromanipulation process.
  • cytoplasmic activation of the oocyte is accomplished by the entry of sperm and the induction of calcium oscillations.
  • PA parthenogenetic activation
  • ICSI intracytoplasmic sperm injection
  • somatic cell nuclear transfer artificial assisted activation of oocytes is essential.
  • artificial auxiliary activation methods are mainly divided into single activation and combined activation.
  • Commonly used single activations mainly include mechanical activation, electrical activation, and chemical activation (calcium ionophore A23187, ionomycin, ethanol, etc.); combined activation mainly involves simple physical activation or chemical activation first, and then protein synthesis inhibition.
  • the commonly used joint activation is to first electrically activate early embryos and then soak the early embryos in a solution containing chemicals to chemically activate early embryos.
  • the device is relatively crude and has low accuracy.
  • Electrical activation uses an activation chamber with two parallel plate electrodes to electrically activate a batch of early embryos. Due to the large distance between the macroscopic parallel plate electrodes, the electrical stimulation voltage applied to a single early embryo is also large and the distribution is not uniform. It is extremely easy to cause excessive electrical damage to the embryo, and it is impossible to achieve precise electrical stimulation of specific parts of the early embryo; it is impossible to use a set of activation devices to complete the combined activation process of electrical activation and chemical activation of the early embryo.
  • the purpose of this application is to provide a joint activation device and method for early embryos, which can be used to achieve joint stimulation including electrical stimulation and chemical stimulation of specific parts of early embryos, and to realize a set of activation devices to complete the stimulation of early embryos. joint activation, thereby increasing the activation rate of early embryos.
  • this application provides a joint activation device for early embryos, including:
  • Petri dishes used to store early-stage embryos
  • a cell suction mechanism is used to adjust the position and posture of the early embryo and fix the early embryo
  • a joint activation mechanism includes a capillary microneedle and a first needle holder, and the capillary microneedle is connected to the first needle holder; the capillary microneedle includes a liquid metal channel and a chemical substance channel; Liquid metal and wires are provided in the liquid metal channel.
  • the liquid metal is located at the tip of the capillary microneedle.
  • the wires are used to connect the excitation power supply.
  • the liquid metal and the wires constitute a liquid metal electrode for energizing the capillary microneedle.
  • the early embryo is electrically activated; the chemical substance channel is used to release chemical substances to chemically activate the early embryo.
  • the capillary microneedle includes a plurality of liquid metal channels and one chemical substance channel, and the plurality of liquid metal channels are arranged around one chemical substance channel.
  • the bottom of the culture dish is provided with an array of micropits, and the micropits are used to prevent movement of the early embryos.
  • the first needle holding instrument includes a first screw cap, a second screw cap, a first connection base, a third screw cap and a first fixed rod;
  • the first screw cap is threadedly connected to the second screw cap, the second screw cap and the third screw cap are both threadedly connected to the first connecting seat, and the first fixed rod is connected to the third screw cap.
  • the tail of the capillary microneedle penetrates the first screw cap and the second screw cap, the first connection seat is provided with a pneumatic drive inlet, and the chemical substance channel in the capillary microneedle is in contact with the pneumatic drive The entrance is connected.
  • the cell suction mechanism includes a cell suction needle and a second needle holder, the cell suction needle is connected to the second needle holder, and the second needle holder is provided with an air guide port, The air guide port is connected with the cell suction needle.
  • the diameter of the liquid metal channel is 7 ⁇ m.
  • the diameter of the chemical substance channel is 10 ⁇ m.
  • the inner diameter of the cell suction needle is 15 ⁇ m and the outer diameter is 100 ⁇ m.
  • the diameter of the micropits is 150 ⁇ m and the depth is 50 ⁇ m.
  • This application also provides a joint activation method for early embryos, which is applied to the joint activation device for early embryos, including:
  • control the joint activation mechanism After the cell suction mechanism fixes the early embryo, control the joint activation mechanism to continuously pass high-voltage direct current pulses to the target site of the early embryo to electrically activate the early embryo;
  • the joint activation mechanism is controlled to release chemical substances to chemically activate the early embryo.
  • This application provides a joint activation device for early embryos, including: a culture dish for storing early embryos; a cell suction mechanism for adjusting the position and posture of early embryos and fixing early embryos; a joint activation mechanism for joint activation
  • the mechanism includes a capillary microneedle and a first needle-holding device, and the capillary microneedle is connected to the first needle-holding device; the capillary microneedle includes a liquid metal channel and a chemical substance channel; liquid metal and wires are provided in the liquid metal channel, and the liquid metal is located in the capillary microneedle. The tip of the fine needle and the wire are used to connect the excitation power supply.
  • the liquid metal and the wire constitute a liquid metal electrode for electrical activation of early embryos; the chemical substance channel is used to release chemicals for chemical activation of early embryos.
  • the capillary microneedles of this application can achieve precise joint activation of local parts of a single early embryo, such as the nuclear substance area, and reduce excessive electrical or chemical damage to other parts of the early embryo, thereby increasing the activation rate of early embryos; and a joint activation mechanism integrates It combines electrical activation and chemical activation to improve the convenience of combined activation of early embryos; the cell holding mechanism can quickly adjust the position of cells, shorten time, and reduce factors that lead to cell death or stagnant development due to adjustment of cell position.
  • the joint activation method of early embryos provided by the embodiments of the present application corresponds to the device and has the above effects.
  • Figure 1 is a structural diagram of a joint activation device for early embryos provided by an embodiment of the present application
  • Figure 2 is a structural diagram of a culture dish provided by an embodiment of the present application.
  • Figure 3 is a structural diagram of a joint activation mechanism provided by an embodiment of the present application.
  • Figure 4 is a tip structure diagram of a capillary microneedle provided by an embodiment of the present application.
  • Figure 5 is a schematic cross-sectional view of the tip of a capillary microneedle provided by an embodiment of the present application
  • Figure 6 is an internal structural diagram of a capillary microneedle provided by an embodiment of the present application.
  • Figure 7 is a process flow chart for preparing a capillary microneedle tip provided by an embodiment of the present application.
  • Figure 8 is a structural diagram of a cell suction mechanism provided by an embodiment of the present application.
  • Figure 9 is a flow chart of a joint activation method for early embryos provided by an embodiment of the present application.
  • the reference numbers are as follows: 1 is a culture dish, 2 is a joint activation mechanism, 3 is a cell suction mechanism, 4 is a cell, 101 is a micro pit, 201 is a capillary microneedle, 202 is the first screw cap, 203 is the second screw cap.
  • 204 is the first connecting seat
  • 205 is the third screw cap
  • 206 is the first fixed rod
  • 207 is the air pressure drive inlet
  • 208 is the wire outlet
  • 209 is the first annular washer
  • 210 is the second annular washer
  • 211 is Chemical pipeline
  • 2011 is the liquid metal channel
  • 2012 is the chemical substance channel
  • 2013 is the wire
  • 301 is the cell suction needle
  • 302 is the fourth screw cap
  • 303 is the fifth screw cap
  • 304 is the second connection seat
  • 305 is The sixth screw cap
  • 306 is the second fixed rod
  • 307 is the air guide port
  • 308 is the gas pipe
  • 309 is the third annular washer
  • 310 is the fourth annular washer
  • A is the tip of the capillary microneedle
  • B is the cell suction needle. The tip of the needle.
  • the principle of electrical activation is that under the action of a short high-voltage direct current pulse in early embryos, the stability of the phosphodiester bimolecular structure of the membrane changes, and many recoverable tiny holes are formed on the cell membrane, allowing extracellular Ca+ to enter the cell and causing The intracytoplasmic Ca+ concentration increases, and the increase in intracellular Ca2+ concentration causes the disappearance of CSF activity, cyclinB is rapidly degraded, MPF activity disappears, and cells are activated to complete the second meiosis and enter the next cell cycle.
  • Chemical activation mainly includes calcium ionophore A23187, ionomycin, ethanol activation, protein synthesis inhibitors and protein phosphorylation inhibitors.
  • CaA Calcium ionophore A23187 activates early embryos mainly through the release of intracytoplasmic Ca2+.
  • Ionomycin another calcium ionophore, also functions through the release of Ca2+ in early embryos. However, it does not directly promote the release of Ca2+ in early embryos, but first uses Ca2+ channels on the cell membrane to influx from the outside of the cell into the cell, and then activates Ca2+ in the endogenous endoplasmic reticulum to enter the cytoplasm of early embryos.
  • the activation of early embryos by ethanol mainly causes the formation of IP3 in the cell membrane. Through IP3, the receptor mediates the release of endogenous Ca2+, causing the early response of early embryo activation.
  • the protein cytokines maturation-promoting factor (MPF) and cell quiescence factor (CSF) are regulators of the cell cycle, and it is precisely because of their action that oocytes will be arrested in the MII phase. These factors are very sensitive to Ca2+. When the egg-activated cytoplasmic Ca2+ rises to a certain level, MPF and CSF will be inactivated or disappear.
  • the use of protein synthesis inhibitors to activate early embryos does not work because it can cause fluctuations in Ca2+ concentration, but because it can inhibit the synthesis of these protein-based cytokines, thereby causing early embryos to leave the MII stage and resume the second meiosis.
  • Protein phosphorylation inhibitors can prevent protein phosphorylation and thereby inhibit the activities of MPF and CSF. They also inhibit the discharge of the second polar body and ensure that the chromosomes of parthenogenetically activated oocytes are diploid.
  • the core of this application is to provide a device and method for joint activation of early embryos.
  • Figure 1 is a structural diagram of a joint activation device for early embryos provided by an embodiment of the present application. As shown in Figure 1, 1 is a culture dish, 2 is a joint activation mechanism, 3 is a cell suction mechanism, and 4 is a cell cells.
  • Petri dish 1 is used to store early embryos; cell suction mechanism 3 is used to adjust the position and posture of early embryos and fix early embryos; joint activation mechanism 2, joint activation mechanism 2 includes capillary microneedles 201 and a first needle holding device , the capillary microneedle 201 is connected to the first needle holder; the capillary microneedle 201 includes a liquid metal channel 2011 and a chemical substance channel 2012; the liquid metal channel 2011 is provided with liquid metal and a wire 2013, and the liquid metal is located at the tip of the capillary microneedle 201, The wire 2013 is used to connect the excitation power supply.
  • the liquid metal and the wire 2013 constitute liquid metal for electrical activation of early embryos; the chemical channel 2012 is used for releasing chemicals to chemically activate early embryos.
  • An embodiment of the present application provides a joint activation device for early embryos, including: a culture dish for storing early embryos; a cell suction mechanism for adjusting the position and posture of early embryos and fixing early embryos; a joint activation mechanism,
  • the joint activation mechanism includes a capillary microneedle and a first needle-holding device, and the capillary microneedle is connected to the first needle-holding device;
  • the capillary microneedle includes a liquid metal channel and a chemical substance channel; the liquid metal channel is provided with liquid metal and a wire, and the liquid metal Located at the tip of the capillary microneedle, the wire is used to connect the power supply.
  • the liquid metal and the wire constitute an electrode for electrical activation of early embryos; the chemical channel is used to release chemicals to chemically activate early embryos.
  • the capillary microneedles of this application can achieve precise joint activation of local parts of a single early embryo, such as the nuclear substance area, and reduce excessive electrical or chemical damage to other parts of the early embryo, thereby increasing the activation rate of early embryos; and a joint activation mechanism integrates It combines the functions of electrical activation and chemical activation to improve the convenience of combined activation of early embryos; the cell holding mechanism can quickly adjust the position of cells, shorten time, and reduce factors that lead to cell death or stagnant development due to adjustment of cell position. .
  • FIG. 2 is a structural diagram of a culture dish provided by the embodiment of the present application. As shown in Figure 2, the bottom of the culture dish 1 is provided with an array of micro pits 101. , the micropit diameter is 150 ⁇ m and the depth is 50 ⁇ m.
  • the culture dish 1 is equivalent to the operating table of the entire device.
  • the cells 4 are placed in the culture dish 1.
  • the micro-pits 101 at the bottom prevent the early embryos from flowing easily and are convenient for grabbing.
  • FIG 3 is a structural diagram of a joint activation mechanism provided by an embodiment of the present application.
  • the joint activation mechanism 2 includes a capillary microneedle 201 and a first needle holder.
  • the first needle holding instrument includes a first screw cap 202, a second screw cap 203, a first connection base 204, a third screw cap 205, a first fixed rod 206, Pneumatic drive inlet 207, wire outlet 208, first annular gasket 209, second annular gasket 210, chemical pipeline 211.
  • A is the needle tip part of the capillary microneedle 201 .
  • the first screw cap 202 is threadedly connected to the second screw cap 203, the second screw cap 203 and the third screw cap 205 are both threadedly connected to the first connecting seat 204, the first fixed rod 206 is threadedly connected to the third screw cap 205;
  • the tail of the microneedle 201 penetrates the first screw cap 202 and the second screw cap 203.
  • the first connection seat 204 is provided with a pneumatic drive inlet 207.
  • the chemical channel 2012 in the capillary microneedle 201 is connected to the pneumatic drive inlet 207 through a chemical pipeline 211. .
  • a through hole is provided on the side wall of the capillary microneedle 201 near the tail, and the corresponding second screw cap 203 is provided with a wire outlet 208 to facilitate the extraction of the wire 2013 from the liquid metal channel 2011.
  • the embodiment of the present application does not specifically limit the number of chemical substance channels 2012 and liquid metal channels 2011 in the capillary microneedle.
  • the capillary microneedle 201 may include multiple liquid metal channels 2011 and one chemical substance channel 2012.
  • the air pressure driven inlet 207 can be connected to a microinjection pump, which increases the gas pressure in the chemical substance channel 2012 to release the chemical in the chemical substance channel 2012.
  • Figure 4 is a tip structural diagram of a capillary microneedle provided by an embodiment of the present application.
  • Figure 5 is a schematic cross-sectional view of a tip of a capillary microneedle provided by an embodiment of the present application. Combining Figures 4 and 5, it includes 6 liquids Metal channel 2011 and a chemical substance channel 2012, six liquid metal channels 2011 are arranged around a chemical substance channel 2012; the diameter of the liquid metal channel 2011 is 7 ⁇ m, and the diameter of the chemical substance channel 2012 is 10 ⁇ m.
  • Figure 6 is an internal structural diagram of a capillary microneedle provided by an embodiment of the present application. As shown in Figure 6, a liquid metal channel 2011 is provided with liquid metal and a wire 2013.
  • the liquid metal is located at the tip of the capillary microneedle 201, and the wire 2013 Leading from the tip of the needle to the through hole near the tail of the needle, the wire 2013 is used to connect to the power supply.
  • the liquid metal and the wire 2013 form an electrode that can electrically activate the cells.
  • the first fixed rod 206 of the first needle holder can be connected to a mechanical arm (not shown in the figure), and the position adjustment of the joint activation mechanism is realized by controlling the mechanical arm.
  • the first annular gasket 209 and the second annular gasket 210 in the first needle holder are used to deform and fix the tail of the capillary microneedle 201 when it is squeezed by the screw cap.
  • the capillary microneedle 201 in the embodiment of the present application is detachably connected to the first needle holder, so that the capillary microneedle 201 can be easily replaced.
  • Figure 7 is a flow chart of a process for preparing the tip of a capillary microneedle provided by an embodiment of the present application.
  • the process for preparing the tip of a capillary microneedle in the joint activation mechanism will be introduced below with reference to Figure 7 process:
  • a capillary microneedle drawing instrument is used to heat and draw the glass tube to a set shape and size. Before stretching the glass tube, the glass tube is prefabricated. The shape of the glass tube is a chemical channel with a diameter of 1mm in the middle. There are six microtubes (liquid metal channels) with a diameter of 0.7 mm surrounding the chemical channel. The glass tube is heated and drawn by a needle puller into a 10 ⁇ m chemical channel and six 7 ⁇ m liquid metal channels.
  • liquid metal from the tail of the capillary microneedle glass tube into six glass microtubes with a diameter of 7 ⁇ m.
  • a high-precision syringe is used to inject liquid metal from the tail of the capillary microneedle.
  • the liquid metal in this device is gallium-based liquid metal, but the liquid metal is not limited to gallium-based liquid metal.
  • the chemical substance channel needs to be blocked to prevent the liquid metal from flowing into the chemical substance channel.
  • the surface is quickly oxidized to form a thin solid oxide film of about 1 nm, which helps stabilize the shape of the liquid metal capillary microneedle electrode and prevents the liquid metal from flowing out from the tip of the capillary microneedle and affecting the Performance of capillary microneedle electrodes.
  • FIG 8 is a structural diagram of a cell suction mechanism provided by an embodiment of the present application.
  • the cell suction mechanism includes a cell suction needle 301 and a second needle holder.
  • the cell suction needle 301 and the second needle holder The second needle-holding device is connected with an air guide port 307, and the air guide port 307 is connected with the cell suction and holding needle 301.
  • the second needle holder includes a fourth screw cap 302, a fifth screw cap 303, a second connecting seat 304, a sixth screw cap 305, a second fixed rod 306, an air guide port 307, a gas pipe 308, a third annular gasket 309, Fourth annular washer 310.
  • B is the tip of the cell suction needle.
  • the embodiments of this application are effective for cell absorption.
  • the needle 301 is not specifically limited.
  • the tip of the cell suction needle 301 is made of a prefabricated glass hose with an inner diameter of 15 ⁇ m and an outer diameter of 100 ⁇ m.
  • the principle of negative pressure is used to suck the early embryos and keep the cells in a In a relatively stable state, the cell holding mechanism 3 can put down or hold the cells at any time. More importantly, when the cells are placed on the culture dish, the cell holding mechanism 3 can be moved to different positions around the cells, and then moved to different positions around the cells.
  • Cell suction can achieve the purpose of adjusting cell posture, which is very convenient and has high convenience and repeatability.
  • the second fixed rod 306 of the second needle holder can be connected to a mechanical arm (not shown in the figure), and the position adjustment of the cell suction mechanism 3 is realized by controlling the mechanical arm.
  • the third annular gasket 309 and the fourth annular gasket 310 in the second needle holder are used to deform and fix the tail of the cell suction needle 301 when it is squeezed by the screw cap.
  • the joint activation device for early embryos is described in detail.
  • This application also provides corresponding embodiments of the joint activation method for early embryos.
  • Figure 9 is a flow chart of a joint activation method for early embryos provided by an embodiment of the present application. As shown in Figure 9, the joint activation method for early embryos includes:
  • Target sites in early embryos include nuclear material areas.
  • the specific process is as follows:
  • Early embryo fixation stage Use mouse or pig oocytes and sperm to fuse in vitro to form fertilized eggs. Place the early embryo on the culture dish, and then fix it with the cell holding mechanism 3 to prevent the early embryo from moving or rotating.
  • (2) Activation stage of early embryos Use joint activation mechanism 2 to activate early embryos.
  • a higher voltage DC pulse is continuously supplied, with a voltage of 3.2V and a pulse width of 50 to 100 ⁇ s.
  • the permeability of the cell membrane increases.
  • the chemical solution is released through the cell membrane and enters the early embryo, quickly activating a series of metabolic reactions in the early embryo.
  • the embodiments of the present application provide a joint activation method for early embryos, which electrically activates the cell membrane of early embryos, causing the stability of the phosphodiester bimolecular structure of the membrane to change, and forming many recoverable tiny holes on the cell membrane.
  • Calcium ionophores, ionomycin, etc. released by chemical activation enter cells through tiny holes and activate a series of metabolic reactions in early embryos, thereby greatly increasing the development and survival rate of embryos. This is of great significance to the in vitro development process of early embryos.

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Abstract

本申请提供一种早期胚胎的联合激活装置,包括:培养皿,用于存放早期胚胎;细胞吸持机构,用于调整早期胚胎的位姿,并固定早期胚胎;联合激活机构，该联合激活机构包括毛细微针和第一持针仪,毛细微针包括液体金属通道和化学物质通道;该液体金属通道内设有的液体金属和导线构成液态金属电极用于对早期胚胎进行电激活;该化学物质通道用于释放化学物质对早期胚胎进行化学激活。该联合激活机构集成了电激活和化学激活两种功能,提高对早期胚胎联合激活的便捷性,毛细微针可实现对单个早期胚胎局部部位如核物质区域的精准联合激活,减少对早期胚胎其他部位过度的电损伤或化学损伤,从而提升早期胚胎激活率。

Description

一种早期胚胎的联合激活装置和方法
本申请要求于2022年08月30日提交中国专利局、申请号为202211056138.4、发明名称为“一种早期胚胎的联合激活装置和方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本申请涉及胚胎工程技术领域，特别是涉及一种早期胚胎的联合激活装置和方法。
背景技术
在胚胎工程技术中，在早期胚胎显微操作流程的各个阶段进行早期胚胎激活是一种常用手段。在自然受精过程中，卵母细胞的细胞质激活是由精子的进入并诱导钙振荡来完成的。而在孤雌激活(PA)、胞浆内单精子注射(ICSI)、体细胞核移植中，卵母细胞的人工辅助激活是必不可少的。目前已知的人工辅助激活方式主要分为单一激活和联合激活。常用的单一激活主要有机械激活、电激活、化学激活(钙离子载体A23187、离子霉素(Ionomycin)及乙醇等)；联合激活主要是先单纯的物理激活或化学激活，之后再用蛋白质合成抑制剂、蛋白质磷酸化抑制剂或细胞松弛素B联合处理的一种激活方式。现在越来越多的研究证明联合激活比单一的电激活或化学激活效果好。
目前，常用的联合激活是先对早期胚胎进行电激活然后再将早期胚胎浸泡在包含化学物的溶液中以对早期胚胎进行化学激活，装置较为粗糙，精度很低。电激活是使用具有两块平行板电极的激活室来对一批早期胚胎进行电激活，由于宏观平行板电极的间距较大，针对单个早期胚胎的施加电刺激电压也较大并且分布不够均匀，极易对胚胎造成过度电损伤，并且无法完成对早期胚胎特定部位实施精准电刺激；无法用一套激活装置完成对早期胚胎的电激活和化学激活的联合激活流程。
因此，如何实现对早期胚胎的特定部位进行刺激，以及实现一套激活装置完成对早期胚胎的联合激活，提高早期胚胎激活率是本领域技术人员 亟需要解决的问题。
发明内容
本申请的目的是提供一种面向早期胚胎的联合激活装置和方法，用于实现对早期胚胎的特定部位进行包括电刺激和化学刺激在内的联合刺激，以及实现一套激活装置完成对早期胚胎的联合激活，从而提高早期胚胎激活率。
为解决上述技术问题，本申请提供一种早期胚胎的联合激活装置，包括：
培养皿，用于存放早期胚胎；
细胞吸持机构，用于调整所述早期胚胎的位姿，并固定所述早期胚胎；
联合激活机构，所述联合激活机构包括毛细微针和第一持针仪，所述毛细微针与所述第一持针仪连接；所述毛细微针包括液体金属通道和化学物质通道；所述液体金属通道内设有液体金属和导线，所述液体金属位于所述毛细微针尖端，所述导线用于连接激励电源，所述液体金属和所述导线构成液态金属电极用于对所述早期胚胎进行电激活；所述化学物质通道用于释放化学物质对所述早期胚胎进行化学激活。
优选地，所述毛细微针包括多个所述液体金属通道和一个所述化学物质通道，多个所述液体金属通道围绕一个所述化学物质通道设置。
优选地，所述培养皿的底部设有阵列的微坑，所述微坑用于防止所述早期胚胎的移动。
优选地，所述第一持针仪包括第一旋盖、第二旋盖、第一连接座、第三旋盖和第一固定杆；
所述第一旋盖与所述第二旋盖螺纹连接，所述第二旋盖、所述第三旋盖均与所述第一连接座螺纹连接，所述第一固定杆与所述第三旋盖螺纹连接；
所述毛细微针尾部贯穿所述第一旋盖和所述第二旋盖，所述第一连接座设有气压驱动入口，所述毛细微针中的所述化学物质通道与所述气压驱动入口连通。
优选地，所述细胞吸持机构包括细胞吸持针和第二持针仪，所述细胞吸持针与所述第二持针仪连接，所述第二持针仪上设有导气口，所述导气口与所述细胞吸持针连通。
优选地，所述液体金属通道的直径为7μm。
优选地，所述化学物质通道的直径为10μm。
优选地，所述细胞吸持针的内径为15μm，外径为100μm。
优选地，所述微坑直径为150μm，深度为50μm。
本申请还提供一种早期胚胎的联合激活方法，应用于所述的早期胚胎的联合激活装置，包括：
在细胞吸持机构固定早期胚胎之后，控制联合激活机构向所述早期胚胎的目标部位持续通入高压直流脉冲，以对所述早期胚胎进行电激活；
在所述早期胚胎经过电激活后，控制所述联合激活机构释放化学物质以对所述早期胚胎进行化学激活。
本申请所提供的一种早期胚胎的联合激活装置，包括：培养皿，用于存放早期胚胎；细胞吸持机构，用于调整早期胚胎的位姿，并固定早期胚胎；联合激活机构，联合激活机构包括毛细微针和第一持针仪，毛细微针与第一持针仪连接；毛细微针包括液体金属通道和化学物质通道；液体金属通道内设有液体金属和导线，液体金属位于毛细微针尖端，导线用于连接激励电源，液体金属和导线构成液体金属电极用于对早期胚胎进行电激活；化学物质通道用于释放化学物质对早期胚胎进行化学激活。本申请的毛细微针可实现对单个早期胚胎局部部位如核物质区域的精确联合激活，减少对早期胚胎其他部位过度的电损伤或化学损伤，从而提升早期胚胎激活率；并且一个联合激活机构集成了电激活和化学激活，提高对早期胚胎联合激活的便捷性；细胞吸持机构可快速调整细胞的位姿，缩减时间，减少因调整细胞位姿而导致细胞死亡或者停滞发育的因素。
本申请实施例所提供的一种早期胚胎的联合激活方法与装置对应，效果如上。
附图说明
为了更清楚地说明本申请实施例，下面将对实施例中所需要使用的附图做简单的介绍，显而易见地，下面描述中的附图仅仅是本申请的一些实施例，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为本申请实施例提供的一种早期胚胎的联合激活装置的结构图；
图2为本申请实施例提供的一种培养皿的结构图；
图3为本申请实施例提供的一种联合激活机构的结构图；
图4为本申请实施例提供的一种毛细微针的针尖结构图；
图5为本申请实施例提供的一种毛细微针的针尖横截面示意图；
图6为本申请实施例提供的一种毛细微针的内部结构图；
图7为本申请实施例提供的一种毛细微针针尖的工艺制备流程图；
图8为本申请实施例提供的一种细胞吸持机构的结构图；
图9为本申请实施例提供的一种早期胚胎的联合激活方法的流程图；
附图标记如下：1为培养皿、2为联合激活机构、3为细胞吸持机构、4为细胞、101为微坑、201为毛细微针、202为第一旋盖、203为第二旋盖、204为第一连接座、205为第三旋盖、206为第一固定杆、207为气压驱动入口、208为导线出口、209为第一环形垫圈、210为第二环形垫圈、211为化学物管道、2011为液体金属通道、2012为化学物质通道、2013为导线、301为细胞吸持针、302为第四旋盖、303为第五旋盖、304为第二连接座、305为第六旋盖、306为第二固定杆、307为导气口、308为气体管道、309为第三环形垫圈、310为第四环形垫圈、A为毛细微针的针尖、B为细胞吸持针的针尖。
具体实施方式
下面将结合本申请实施例中的附图，对本申请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申请一部分实施例，而不是全部实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下，所获得的所有其他实施例，都属于本申请保护范围。
目前，有关哺乳动物早期胚胎激活的研究已经做了很多，方法由原来的单一激活到现在的各种方法的联合使用，已取得了一定的进展。电激活是常用的激活方法，因该方法具有操作方便、激活率高和无化学毒副作用等特点而被广大研究者所青睐。电激活的原理是早期胚胎在短暂高压直流电脉冲的作用下，膜的磷酸二酯双分子结构的稳定性发生改变，细胞膜上形成很多可恢复的微小孔洞，使细胞外的Ca+进入细胞，并使胞质内Ca+浓度升高，细胞内Ca2+浓度的增加导致CSF活性消失，cyclinB被迅速降解，MPF活性消失，细胞被激活完成第二次减数分裂并进入下一细胞周期。化学激活主要包括钙离子载体A23187、离子霉素、乙醇激活、蛋白质合成抑制剂和蛋白质磷酸化抑制剂。钙离子载体A23187(CaA)主要通过胞质内Ca2+的释放来激活早期胚胎。离子霉素作为另一种钙离子载体，也是通过早期胚胎内Ca2+的释放而发挥功能的。但是它不是直接促进早期胚胎内Ca2+的释放，而是先动用细胞膜上Ca2+通道由细胞外向胞内涌入，然后又激活内源性内质网中的Ca2+进入早期胚胎胞质内。乙醇对早期胚胎的激活主要是引起细胞膜IP3的形成，通过IP3,受体介导内源性Ca2+释放，引起早期胚胎产生激活的早期反应。蛋白类细胞因子成熟促进因子(MPF)和细胞静止因子(CSF)是细胞周期的调节者，正是因为在二者的作用下卵母细胞才会停滞在MⅡ期。这些因子对Ca2+十分敏感，当卵激活胞质Ca2+升高到一定水平时，MPF和CSF便失活或消失。使用蛋白质合成抑制剂激活早期胚胎不是因为能引起Ca2+浓度波动而起作用，而是因为能抑制这些蛋白质类细胞因子的合成，从而使早期胚胎离开MⅡ期，恢复第二次减数分裂。蛋白质磷酸化抑制剂能阻止蛋白质的磷酸化进而抑制MPF和CSF的活性，同时也抑制了第二极体的排放，保证了孤雌激活卵母细胞的染色体为二倍体。
本申请的核心是提供一种早期胚胎的联合激活装置和方法。
为了使本技术领域的人员更好地理解本申请方案，下面结合附图(图1-图8)和具体实施方式对本申请作进一步的详细说明。
图1为本申请实施例提供的一种早期胚胎的联合激活装置的结构图，如图1所示，1为培养皿、2为联合激活机构、3为细胞吸持机构、4为细 胞。培养皿1，用于存放早期胚胎；细胞吸持机构3，用于调整早期胚胎的位姿，并固定早期胚胎；联合激活机构2，联合激活机构2包括毛细微针201和第一持针仪，毛细微针201与第一持针仪连接；毛细微针201包括液体金属通道2011和化学物质通道2012；液体金属通道2011内设有液体金属和导线2013，液体金属位于毛细微针201尖端，导线2013用于连接激励电源，液体金属和导线2013构成液态金属用于对早期胚胎进行电激活；化学物质通道2012用于释放化学物质对早期胚胎进行化学激活。
本申请实施例所提供的一种早期胚胎的联合激活装置，包括：培养皿，用于存放早期胚胎；细胞吸持机构，用于调整早期胚胎的位姿，并固定早期胚胎；联合激活机构，联合激活机构包括毛细微针和第一持针仪，毛细微针与第一持针仪连接；毛细微针包括液体金属通道和化学物质通道；液体金属通道内设有液体金属和导线，液体金属位于毛细微针尖端，导线用于连接电源，液体金属和导线构成电极用于对早期胚胎进行电激活；化学物质通道用于释放化学物质对早期胚胎进行化学激活。本申请的毛细微针可实现对单个早期胚胎局部部位如核物质区域的精确联合激活，减少对早期胚胎其他部位过度的电损伤或化学损伤，从而提升早期胚胎激活率；并且一个联合激活机构集成了电激活和化学激活两种功能，提高对早期胚胎联合激活的便捷性；细胞吸持机构可快速调整细胞的位姿，缩减时间，减少因调整细胞位姿而导致细胞死亡或者停滞发育的因素。
本申请实施例对培养皿1形状和规格不作具体限定，图2为本申请实施例提供的一种培养皿的结构图，如图2所述，培养皿1的底部设有阵列的微坑101，微坑直径为150μm，深度为50μm。培养皿1相当于整个装置的操作台，细胞4放在培养皿1里，底部的微坑101使早期胚胎不能轻易的流动，方便抓取。
图3为本申请实施例提供的一种联合激活机构的结构图，如图3所示，联合激活机构2包括毛细微针201和第一持针仪。第一持针仪包括第一旋盖202、第二旋盖203、第一连接座204、第三旋盖205、第一固定杆206、 气压驱动入口207、导线出口208、第一环形垫圈209、第二环形垫圈210、化学物管道211。A为毛细微针201的针尖部分。第一旋盖202与第二旋盖203螺纹连接，第二旋盖203、第三旋盖205均与第一连接座204螺纹连接，第一固定杆206与第三旋盖205螺纹连接；毛细微针201尾部贯穿第一旋盖202和第二旋盖203，第一连接座204设有气压驱动入口207，毛细微针201中的化学物质通道2012通过化学物管道211与气压驱动入口207连接。当然，在毛细微针201的侧壁靠近尾部设有通孔，对应的第二旋盖203设有导线出口208，方便液体金属通道2011中的导线2013引出。本申请实施例对毛细微针中的化学物质通道2012和液体金属通道2011的个数不作具体限定，毛细微针201可以包括多个液体金属通道2011和一个化学物质通道2012。气压驱动入口207可以与显微注射泵连接，显微注射泵增加化学物质通道2012的气体压强，以使化学物质通道2012中的化学物释放出来。图4为本申请实施例提供的一种毛细微针的针尖结构图，图5为本申请实施例提供的一种毛细微针的针尖横截面示意图，结合图4和图5，包括6个液体金属通道2011和一个化学物质通道2012，6个液体金属通道2011围绕一个化学物质通道2012设置；液体金属通道2011的直径为7μm，化学物质通道2012的直径为10μm。图6为本申请实施例提供的一种毛细微针的内部结构图，如图6所示，液体金属通道2011内设有液体金属和导线2013，液体金属位于毛细微针201的针尖，导线2013从针尖至靠近针尾的通孔引出，导线2013用于连接电源，液体金属与导线2013构成电极可对细胞进行电激活。为了便于联合激活机构2的位置调整，第一持针仪的第一固定杆206可以与机械臂(图中未示出)连接，通过控制机械臂实现联合激活机构的位置调整。第一持针仪中的第一环形垫圈209和第二环形垫圈210用于在受到旋盖的挤压时发生形变固定毛细微针201的尾部。本申请实施例中的毛细微针201与第一持针仪可拆卸连接，可以方便更换毛细微针201。
为了更加清楚地了解上述联合激活机构的结构，图7为本申请实施例提供的一种毛细微针针尖的工艺制备流程图，下面结合图7介绍联合激活机构中毛细微针的针尖的工艺制备流程：
(1)拉制玻璃管形成毛细微针。作为优选方案，用毛细微针拉针仪加热并拉制玻璃管至设定形状和尺寸，对玻璃管拉伸之前，玻璃管为预制的，玻璃管形状为中间有直径1mm的化学物质通道，围绕化学物质通道周围有六个直径为0.7mm的微管(液态金属通道)。玻璃管被拉针仪加热并拉制成一个10μm的化学物质通道和六个7μm的液态金属通道。
(2)从毛细微针玻璃管尾部向六根直径为7μm的玻璃微管注入液态金属。作为优选方案，用高精度注射器从毛细微针尾部注入液态金属，本装置中液态金属为镓基液态金属，但液态金属不限于镓基液态金属。在注入液态金属时需要将化学物质通道堵塞，避免液态金属流入化学物质通道。
(3)将液态金属甩向毛细微针尖端。作为优选方案，利用离心机高转速产生的巨大离心力将液态金属甩向毛细微针尖端，直至在显微镜下观察液态金属填满针尖同时没有气泡，在离心机工作前，设置离心机的转速为6500r/min，持续时间1-2分钟，也可以根据毛细微针的直径进行转速和时间的调整。
(4)修剪毛细微针电极尖端。作为优选方案，利用锻针仪修剪毛细微针尖端，使得修剪后的尖端直径满足要求，经过锻针仪截断及打磨，最后尖端形成10μm的化学物质通道和7μm的液态金属通道。在液态金属氧化凝固前，毛细微针玻璃管尾部引出六根导线，从毛细微针玻璃管尾部引出导线以用于连接电源，形成液态金属毛细微针电极。液态金属暴露于空气中表面很快被氧化形成一层薄薄的约1nm的固态氧化膜，这有助于稳定液态金属毛细微针电极的形状，避免液态金属从毛细微针尖端处流出，影响毛细微针电极的性能。
图8为本申请实施例提供的一种细胞吸持机构的结构图，如8所示，细胞吸持机构包括细胞吸持针301和第二持针仪，细胞吸持针301与第二持针仪连接，第二持针仪上设有导气口307，导气口307与细胞吸持针301连通。第二持针仪包括第四旋盖302、第五旋盖303、第二连接座304、第六旋盖305、第二固定杆306、导气口307、气体管道308、第三环形垫圈309、第四环形垫圈310。B为细胞吸持针的针尖。本申请实施例对细胞吸 持针301的不作具体限定，细胞吸持针301的针尖使用预制的玻璃软管制造，内径为15μm，外径为100μm，在工作时利用负压的原理将早期胚胎吸住，使细胞处于一个相对稳定状态，细胞吸持机构3可随时放下或者吸持细胞，更重要的一点是，在细胞被放置在培养皿上时，将细胞吸持机构3移动到细胞周围不同的位置，然后再将细胞吸持，实现调整细胞位姿的目的，非常便利的，具有很高的便利性和重复性。为了便于细胞吸持机构3的位置调整，第二持针仪的第二固定杆306可以机械臂(图中未示出)连接，通过控制机械臂实现细胞吸持机构3的位置调整。第二持针仪中的第三环形垫圈309和第四环形垫圈310用于在受到旋盖的挤压时发生形变固定细胞吸持针301的尾部。
在上述实施例中，对于早期胚胎的联合激活装置进行了详细描述，本申请还提供早期胚胎的联合激活方法对应的实施例。
图9为本申请实施例提供的一种早期胚胎的联合激活方法的流程图，如图9所示，早期胚胎的联合激活方法包括：
S10：在细胞吸持机构固定早期胚胎之后，控制联合激活机构向早期胚胎的目标部位持续通入高压直流脉冲，以对早期胚胎进行电激活。
S11：在早期胚胎经过电激活后，控制联合激活机构释放化学物质以对早期胚胎进行化学激活。
上述方法可以是通过与细胞吸持机构和联合激活机构相连接的控制器进行控制。早期胚胎的目标部位包括核物质区等。具体的过程如下：
(1)早期胚胎固定阶段：利用小鼠或者猪的卵母细胞与精子进行体外融合，形成受精卵。将早期胚胎放置在培养皿上，再用细胞吸持机构3将其固定，防止早期胚胎发生移动或者旋转。
(2)早期胚胎的激活阶段：使用联合激活机构2对早期胚胎进行激活，电激活过程中，持续通入较高压直流脉冲，电压大小为3.2V，脉宽为50～100μs，当早期胚胎受到电激活后，细胞膜的通透性增加，此时释放化学物质溶液通过细胞膜进入早期胚胎，快速激活早期胚胎内的一系列代谢反应。
(3)早期胚胎调整位姿阶段：当使用联合激活机构2激活早期胚胎的一侧细胞膜后，应该改变细胞的姿态，此时应使用细胞吸持机构3(负压的原理)调整早期胚胎的姿态，在早期胚胎被放置在培养皿上时，将细胞吸持机构3移动到早期胚胎周围不同的位置，然后再将早期胚胎吸持，实现调整胚胎位姿的目的，调整完后再重复胚胎激活阶段的操作。
本申请实施例所提供的一种早期胚胎的联合激活方法，电激活早期胚胎的细胞膜，导致膜的磷酸二酯双分子结构的稳定性发生改变，细胞膜上形成很多可恢复的微小孔洞，此时化学激活释放的钙离子载体，离子霉素等通过微小孔洞进入细胞，激活了早期胚胎中一系列的代谢反应，达到胚胎的发育成活率大大增加的目的，对于早期胚胎体外发育过程具有重大意义。
以上对本申请所提供的一种早期胚胎的联合激活装置和方法进行了详细介绍。说明书中各个实施例采用递进的方式描述，每个实施例重点说明的都是与其他实施例的不同之处，各个实施例之间相同相似部分互相参见即可。对于实施例公开的装置而言，由于其与实施例公开的方法相对应，所以描述的比较简单，相关之处参见方法部分说明即可。应当指出，对于本技术领域的普通技术人员来说，在不脱离本申请原理的前提下，还可以对本申请进行若干改进和修饰，这些改进和修饰也落入本申请权利要求的保护范围内。
还需要说明的是，在本说明书中，诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。在没有更多限制的情况下，由语句“包括一个……”限定的要素，并不排除在包括所述要素的过程、方法、物品或者设备中还存在另外的相同要素。

Claims (10)

  1. 一种早期胚胎的联合激活装置，其特征在于，包括：
    培养皿，用于存放早期胚胎；
    细胞吸持机构，用于调整所述早期胚胎的位姿，并固定所述早期胚胎；
    联合激活机构，所述联合激活机构包括毛细微针和第一持针仪，所述毛细微针与所述第一持针仪连接；所述毛细微针包括液体金属通道和化学物质通道；所述液体金属通道内设有液体金属和导线，所述液体金属位于所述毛细微针尖端，所述导线用于连接激励电源，所述液体金属和所述导线构成液态金属电极用于对所述早期胚胎进行电激活；所述化学物质通道用于释放化学物质对所述早期胚胎进行化学激活。
  2. 根据权利要求1所述的早期胚胎的联合激活装置，其特征在于，所述毛细微针包括多个所述液体金属通道和一个所述化学物质通道，多个所述液体金属通道围绕一个所述化学物质通道设置。
  3. 根据权利要求1所述的早期胚胎的联合激活装置，其特征在于，所述培养皿的底部设有阵列的微坑，所述微坑用于防止所述早期胚胎的移动。
  4. 根据权利要求1所述的早期胚胎的联合激活装置，其特征在于，所述第一持针仪包括第一旋盖、第二旋盖、第一连接座、第三旋盖和第一固定杆；
    所述第一旋盖与所述第二旋盖螺纹连接，所述第二旋盖、所述第三旋盖均与所述第一连接座螺纹连接，所述第一固定杆与所述第三旋盖螺纹连接；
    所述毛细微针尾部贯穿所述第一旋盖和所述第二旋盖，所述第一连接座设有气压驱动入口，所述毛细微针中的所述化学物质通道与所述气压驱动入口连通。
  5. 根据权利要求1所述的早期胚胎的联合激活装置，其特征在于，所述细胞吸持机构包括细胞吸持针和第二持针仪，所述细胞吸持针与所述第二持针仪连接，所述第二持针仪上设有导气口，所述导气口与所述细胞吸持针连通。
  6. 根据权利要求1所述的早期胚胎的联合激活装置，其特征在于，所 述液体金属通道的直径为7μm。
  7. 根据权利要求1所述的早期胚胎的联合激活装置，其特征在于，所述化学物质通道的直径为10μm。
  8. 根据权利要求5所述的早期胚胎的联合激活装置，其特征在于，所述细胞吸持针的内径为15μm，外径为100μm。
  9. 根据权利要求3所述的早期胚胎的联合激活装置，其特征在于，所述微坑直径为150μm，深度为50μm。
  10. 一种早期胚胎的联合激活方法，其特征在于，应用于权利要求1-9任一项所述的早期胚胎的联合激活装置，包括：
    在细胞吸持机构固定早期胚胎之后，控制联合激活机构向所述早期胚胎的目标部位持续通入高压直流脉冲，以对所述早期胚胎进行电激活；
    在所述早期胚胎经过电激活后，控制所述联合激活机构释放化学物质以对所述早期胚胎进行化学激活。
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