JP2010051176A - Module, apparatus, and method for cell coupling - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a module, an apparatus, and a method for cell coupling which simply and accurately carry out cell coupling in a microchannel. <P>SOLUTION: The cell coupling module includes an ovum transport channel 12 for transporting an ovum 13 together with a transport medium, an ovum catching channel 16 that is connected to the ovum transport channel 12 and has a V-shape for mooring the ovum 13, a donor transport channel 14 that is connected to the ovum catching channel 16 and transport a donor cell 15 to be coupled with the ovum 13 moored to the ovum catching channel 16 together with the transport medium, and a carrying and transport channel 18 for transporting the ovum 13 and the donor cell 15 after coupling together with the transport medium. The ovum catching channel 16 has both the ends of the V-shape connected to the ovum transport channel 12 and the carrying and transport channel 18, the central bent part 16c of the V-shape has a width narrower than the widths at both ends of V-shape, and the donor transport channel 14 is connected to a part to which the ovum 13 of the ovum catching channel 16 is moored. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a cell coupling module, a cell coupling apparatus, and a cell coupling method, and more specifically, a cell capable of easily and reliably coupling cells together in producing a cloned embryo, cell fusion, and the like. The present invention relates to a coupling module, a cell coupling device, and a cell coupling method.

  In the field of biochemistry, a micro (chemical) chip has been developed in which a micro flow channel is formed on a glass substrate or a plastic substrate, and a chemical reaction occurs in the micro closed space. In particular, as an application of DNA PCR (Polymerase Chain Reaction), compared to the prior art, high-speed separation detection of several to tens of times is possible, and many products have been developed (for example, see Non-Patent Document 1). ). Currently, its application is centered on biochemical reactions, and technologies such as individual transfer of individual cells such as separation and processing of cells and individual processing have not been established.

  By the way, the establishment of clone technology is a technology that is expected to revolutionize the future medical and chemical industries. In the field of functional genomics, mice with specific genes (KO mice) have been created to investigate genetic effects and are used for various drug discovery and treatment studies. In the method for producing KO mice, chimera mice are mainly produced from ES cells, and KO mice are produced from their progeny. However, KO mice are produced directly from ES cells by a clone technique by a stable cloning technique. The method and the method of making a cloned mouse by KO of a gene in somatic cells become useful. In particular, in animal species and mouse strains in which ES cells have not been established, the creation of clones from somatic cells is very effective. On the other hand, the current nuclear transfer operation in cloning involves many manual operations during the transfer process (see, for example, Non-Patent Document 2), which is an obstacle to efficient research and reasonable evaluation. . For example, when comparing the effects of added chemicals on transplanted egg cells, the effects of the chemicals cannot be accurately evaluated if there is a large variation in ovum damage due to manual labor.

In this way, labor saving research and stabilization of experimental conditions are indispensable for the development of functional genomics technology, and in view of future industrialization, automation of work is important. Various studies have been made (for example, see Patent Document 1).
JP 2006-325429 A ANDREW J. DEMELLO, “Microfluidics: DNA amplification moves on”, Nature 422, 28-29 (06March 2003); doi: 10.1038 / 422028a. T. WAKAYAMA, ACF PERRY, M. ZUCCOTTI, KR JOHNSON, R. YANAGIMACHI, “Full-termdevelopment of mice from enucleated oocytes injected with cumulus cell nuclei”, Nature 394, 369-374 (23 July 1998); doi: 10.1038 / 28615.

  An object of the present invention is to provide a cell coupling module, a cell coupling device, and a cell coupling method capable of simply and accurately performing cell coupling in a microchannel.

  The cell coupling module of the present invention is a cell coupling module that couples a first cell and a second cell in a carrier medium in a microchannel, and the first cell together with the carrier medium A first transport channel for transporting the gas, a capture channel connected to the first transport channel and having a V-shape for mooring the first cells, and the capture channel connected to the capture channel A second transport channel for transporting the second cells for coupling to the first cells anchored in the flow channel together with a transport medium; and the first and second cells after coupling A third transport channel that transports the transport medium together with the transport medium, and the capture channel is connected to the first transport channel and the third transport channel at both ends of the V shape, and has a V shape. The center bend is narrower than the V-shaped ends. And Tsu, said second conveying passage, characterized in that the first cell of the trapping channel is connected to the part to be anchored.

  Since the cell can be restored to its original function without any obstacle even if it is deformed to some extent, the present invention uses this characteristic to make the first bent portion at the center of the V-shaped capture channel. By tethering the cells and transporting the second cells from the second transport channel, the second cells can be easily coupled to the first cells.

  Here, it is preferable that the capture channel has a tapered shape in which the width gradually decreases from the end connected to the V-shaped first transport channel toward the bent portion at the center of the V-shape. .

  In addition, when the diameter of the first cell is d, the width B of the V-shaped central portion of the capture channel is preferably in the range of 0.7d ≦ B <d, 0.8d More preferably, it is in the range of ≦ B ≦ 0.95d.

  Each of the first transport channel, the capture channel, the second transport channel, and the third transport channel may have a rectangular cross section. Thereby, a microchannel can be easily formed by lithography, for example. Further, when the cross section of the trapping channel is rectangular, the carrier medium can flow from the corner of the rectangle even when a substantially spherical cell is locked. Therefore, the trapping flow is completely captured by the first cell. It is possible to prevent the road from being blocked.

  The cell coupling module of the present invention is suitable when the first cell is an egg and the second cell is a donor cell.

  The cell coupling device of the present invention includes the above-described cell coupling module, a first liquid feeding pump that sends out the carrier medium for conveying the first cells to the first carrier channel, And a second liquid feed pump for feeding the transport medium for transporting the second cells to the second transport flow path.

  By adjusting the liquid feeding of the first liquid feeding pump, it is possible to control the movement and stop of the first cell. Further, by adjusting the liquid feeding of the second liquid feeding pump, it is possible to control the movement of the second cell, the coupling with the first cell, the stoppage, and the like.

  The cell coupling device according to the present invention includes an image sensor that captures an image in the capture channel, and the first liquid feed pump and the second liquid feed based on an image captured by the image sensor. It is possible to further comprise a controller for controlling the pump liquid supply.

  As described above, the configuration including the image sensor and the controller makes it possible to automate cell coupling.

  The cell coupling method of the present invention is a cell coupling method using the above-described cell coupling module, wherein the carrier medium is sent out to the first carrier channel and the first channel into the capture channel. Reducing the delivery of the carrier medium from the first conveying channel when the first cell reaches the bent portion at the center of the V shape of the capture channel and is anchored. Stop and send the carrier medium to the second carrier channel to couple the second cells to the first cells moored to the capture channel, the first carrier channel And / or sending the transport medium from the second transport flow path to transport the coupled first and second cells to a third transport flow path.

  In this way, the transfer of the transport medium in the first transport flow path is adjusted to move and moor the first cell, and the transport of the transport medium in the second transport flow path is adjusted to the second cell. Can be coupled to the anchored first cell, so that the cell can be easily coupled without providing a complicated mechanism.

  If the cell coupling module, the cell coupling device, and the cell coupling method of the present invention are used, the first cell can be adjusted by adjusting the delivery of the carrier medium in the first carrier channel without providing a complicated mechanism. Can be securely anchored to the central bent portion of the capture channel. Subsequently, the second cell can be reliably and easily coupled to the first cell by adjusting the delivery of the carrier medium in the second carrier channel. Thereby, cell coupling can be performed simply and reliably.

  Embodiments of the present invention will be described with reference to the drawings, but the present invention is not limited to the following embodiments. In the present embodiment, an operation when creating a cloned embryo from an egg will be described as an example. When producing a cloned embryo, after removing the zona pellucida of the ovum, the ovum is divided into two, and the donor cell is coupled to the one without the nucleus.

  FIG. 1 is a schematic configuration diagram of a cell coupling device according to an embodiment of the present invention. The cell coupling device of this embodiment includes a cell coupling module 10 in which a microchannel 11 is formed, an egg feeding pump 20 and a donor feeding pump 22 that deliver a carrier medium such as a culture medium and physiological saline. , A controller 24 for controlling the amount (speed) of feeding of the egg feeding pump 20 and the donor feeding pump 22, an image sensor 28 for taking an image of the microchannel 11, and monitoring of the image of the microchannel 11 And a monitor 26 for displaying and monitoring the operation status of the entire apparatus.

  Details of the cell coupling module 10 in this embodiment are shown in FIG. As shown in FIGS. 1 and 2, the cell coupling module 10 according to the present embodiment transports an ovum 13 (which means a nuclei having no nucleus among the bisected eggs, the same applies hereinafter) together with a transport medium. An egg transport channel 12 (first transport channel), an egg catching channel 16 having a V-shape following the egg transport channel 12, and a donor cell for transporting donor cells for coupling to the egg together with a transport medium It has a transport channel 14 (second transport channel) and an unloading transport channel 18 (third transport channel) for transporting the coupled eggs and donor cells together with the transport medium. In the present embodiment, the egg catching channel 16 is formed in a tapered shape whose width gradually decreases from the end connected to the V-shaped egg transport channel 12 toward the bent portion 16c at the center of the V-shape. In addition, it is formed in a tapered shape whose width gradually decreases from the end connected to the V-shaped carry-out conveyance channel 18 toward the bent portion 16c at the center of the V-shape. In the present embodiment, the micro flow path 11 includes an ovum transport flow path 12, an egg capture flow path 16, a donor transport flow path 14, and a carry-out transport flow path 18.

  The microchannel 11 in the present embodiment is created as follows. First, a coating film of a photo-curing agent (resist) for lithography is formed on the substrate, and then the portion of the shape of the microchannel 11 is irradiated with light and cured using a photomask. Furthermore, a convex pattern having the shape of the microchannel 11 is formed on the substrate by removing the unirradiated portion. Next, PDMS (polydimethylsiloxane) is applied on the substrate so as to cover this pattern, and after curing this, the cell coupling module 10 in which the concave microchannel 11 is formed is removed from the substrate. can get. The cross-sectional shape of the microchannel 11 created in this way is substantially rectangular, for example, 0.05 mm × 0.05 mm to 0.2 mm × 0.2 mm.

The shape of the microchannel 11 in this embodiment will be described with reference to FIG. Usually, the diameter (d) of the ovum 13 is usually about 80 to about 120 μm, but in this embodiment, an ovum with d = 100 μm was used. The width (W) of the ovum transfer channel 12 is not particularly limited as long as it is not large enough to prevent the movement of the ovum 13, but in this embodiment, W = 200 μm (= 2d). Further, the protruding length (A) of the egg capturing channel 16 from the side surface of the egg transport channel 12 is A = 100 μm (= d = 1/2 W). Further, one width (C ₁ or C ₂ ) of the V-shape was C ₁ = C ₂ = 200 μm (= W). Furthermore, the width (B) of the V-shaped central portion of the bent portion 16c of the egg catching channel 16 was set to B = 90 μm (0.9d). Further, the donor transport channel 14 is provided at a right angle to the convex side surface 16b of the egg catching channel 16, and is a line that is lowered from the concave top 16a of the egg catching channel 16 at a right angle to the side surface 16b. It is provided at a position where the distance D is minimized.

  In the present embodiment, as shown in FIG. 1, a carrier medium is sent from the egg feeding liquid pump 20 to the egg carrier flow path 12. The delivery amount of the carrier medium from the egg feeding pump 20 is 0.5 to 1 μl / min. In addition, a transport medium is sent from the donor liquid pump 22 to the donor transport flow path 14. The delivery amount of the carrier medium from the donor liquid pump 22 is 0.05 to 0.1 μl / min. The delivery amount of the carrier medium from the egg feeding pump 20 and the donor feeding pump 22 is controlled by the controller 24 based on the image of the egg catching channel 16 from the image sensor 28.

  A cell coupling method in the apparatus having the above configuration will be described. First, the carrier medium is sent out from the ovum feeding pump 20 and the donor feeding pump 22 to fill the entire microchannel 11 with the carrier medium. Next, the ovum 13 is introduced into the ovum transport channel 12 using a micropipette, a glass capillary tube, or the like. In addition, the egg 13 and the donor cell 15 which will be described later are previously treated with PHA (Phytohemagglutinin) in order to enhance adhesion.

  Next, the feeding of the carrier medium from the egg feeding pump 20 is started. When it is detected from the image from the image sensor 28 that the ovum 13 is locked to the central bent portion 16c of the ovum capturing flow path 16 as shown in FIG. Reduce or stop media delivery. Here, since the egg 13 has a restoring property that it returns to the original state even if it is deformed to some extent, even if it is deformed and held in the bent portion 16c as shown in FIG. Absent.

  Next, donor cells 15 are introduced into the donor transport channel 14 using a micropipette, a glass capillary tube, or the like. Subsequently, the delivery of the transport medium from the donor liquid pump 22 is started. When it is confirmed by the image from the image sensor 28 that the donor cell 15 has moved through the donor transport channel 14 and reached the ovum 13 and then has adhered to the ovum 13, the ovum solution is fed again. Delivery of the transport medium from the pump 20 is started. Thereby, the ovum 13 and the donor cell 15 adhered to each other are pushed out to the carry-out conveyance channel 18 while being deformed by the pressure of the conveyance medium. The extruded egg 13 and donor cell 15 are taken out from the carry-out transport channel 18 using a micropipette, a glass capillary tube, or the like. By the above, the coupling of the ovum 13 and the donor cell 15 using the cell coupling device of this embodiment is completed.

  In the cell coupling device of the present embodiment, the coupling between the ovum 13 and the donor cell 15 is performed only by controlling the delivery amount of the carrier medium by the ovum feeding pump 20 and the donor feeding pump 22. It is possible to perform the coupling operation easily and reliably without using a simple mechanism. Moreover, since it is not necessary to use a liquid feeding pump 20 for eggs and a liquid feeding pump 22 for donors that have particularly high liquid feeding accuracy, it is possible to construct a coupling device at low cost.

  In the embodiment described above, the case where the first cell is a divided egg and the second cell is a donor cell has been described, but the present invention is not limited to this, and two This can be applied to the operation of coupling cells. In that case, it is necessary to change the widths of the first transport channel, the second transport channel, the capture channel, and the third transport channel according to the sizes of the first cell and the second cell. Needless to say.

  In the present embodiment, the case where the cross-sectional shape of the microchannel 11 is rectangular has been described. However, the present invention is not limited to this, and the cross-section may be, for example, a circle other than a rectangle.

  The cell coupling module, cell coupling device and cell coupling method of the present invention are useful for cell fusion, clonal livestock production, and the like, and thus can be used in the bio industry, livestock industry and the like.

It is a schematic block diagram of the cell coupling apparatus which concerns on one Embodiment of this invention. It is a top view of the cell coupling module in FIG. It is explanatory drawing which shows the cell coupling method in the cell coupling module of FIG.

Explanation of symbols

10 Cell Coupling Module 11 Micro Channel 12 Egg Transfer Channel (First Transfer Channel)
13 Ovum (first cell)
14 Donor transport channel (second transport channel)
15 Donor cells (second cells)
16 Oocyte capture channel (capture channel)
16c Bending part 16b Side face 16a Top part 18 Unloading conveyance flow path (3rd conveyance flow path)
20 Oocyte Delivery Pump 22 Donor Delivery Pump 24 Controller 28 Image Sensor 26 Monitor

Claims (8)

A cell coupling module for coupling a first cell and a second cell in a carrier medium in a microchannel,
A first transport channel for transporting the first cells together with the transport medium;
A capture channel connected to the first transport channel and having a V-shape for anchoring the first cells;
A second transport flow path for transporting the second cells together with a transport medium for coupling to the first cells tethered to the capture flow path connected to the capture flow path;
A third transport flow path for transporting the first and second cells after coupling together with a transport medium;
The capture channel has V-shaped ends connected to the first transfer channel and the third transfer channel, respectively, and the central bent portion of the V-shape is narrower than the V-shaped ends. The cell coupling module is characterized in that the second transport channel is connected to a portion of the capture channel where the first cells are anchored.   2. The cell according to claim 1, wherein the capture channel has a tapered shape with a width that gradually decreases from an end connected to the V-shaped first transport channel toward a bent portion at the center of the V-shape. Coupling module.   The cell cup according to claim 1 or 2, wherein a width B of a V-shaped central portion of the capture channel is in a range of 0.7d≤B <d, where d is the diameter of the first cell. Ring module.   The cell coupling according to any one of claims 1 to 3, wherein each of the first transport channel, the capture channel, the second transport channel, and the third transport channel has a rectangular cross section. module.     The cell coupling module according to any one of claims 1 to 4, wherein the first cell is an egg and the second cell is a donor cell. The cell coupling module according to any one of claims 1 to 5,
A first liquid feed pump for delivering the carrier medium for conveying the first cells to the first carrier channel;
A cell coupling device, comprising: a second liquid feeding pump that delivers the transport medium for transporting the second cells to the second transport channel. An image sensor that captures an image in the capture channel;
The cell according to claim 6, further comprising: a controller that controls liquid feeding of the first liquid feeding pump and the second liquid feeding pump based on an image captured by the image sensor. Coupling device. A cell coupling method using the cell coupling module according to any one of claims 1 to 5,
Transporting the first medium into the capture channel by delivering the transport medium to the first transport channel;
Reducing or stopping the delivery of the transport medium from the first transport channel when the first cell reaches the bent portion at the center of the V shape of the capture channel and is anchored;
Sending the carrier medium to the second carrier channel to couple the second cells to the first cells anchored in the capture channel;
Feeding the transport medium from the first transport channel and / or the second transport channel and transporting the first and second cells after coupling to the third transport channel. A cell coupling method characterized.

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