JP2006325429A - Automatic nuclear transplantation apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an automatic nuclear transplantation apparatus capable of uniformly, stably, and efficiently conducting nucleus transplantation, by automatizing all of nucleus transplantation operations conventionally conducted by hand in micro channels. <P>SOLUTION: This automatic nuclear transplantation apparatus includes modules for conducting processes of dividing, separating, and fusing an ovum, respectively, wherein the modules each have a transport system including the micro channel and are connected to each other, and therefore the ovum travels through the channels, so that the ovum to which a new nucleus is transplanted is automatically formed. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an apparatus for automating nuclear transplantation in fields requiring functionalization and evaluation of cells, such as medical fields such as regenerative medicine and pharmaceuticals, and agricultural, forestry and fisheries fields such as breed improvement. It is related with the apparatus which implement | achieves uniformization.

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.
Thus, labor-saving research and stabilization of experimental conditions through automation are indispensable for the development of functional genomics technology. Also, work automation is important in considering future industrialization.
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 an automatic nuclear transfer apparatus that performs nuclear transfer uniformly, stably, and efficiently by automating all nuclear transfer operations that have been performed manually in a microchannel. is there.

  The present inventors diligently studied the above problems, and found that the nuclear transfer operation can be automated by combining the microchannel systems of the modules that perform division, separation, and fusion. It came to be completed.

That is, the present invention
(1) It includes modules that perform the steps of cell division, separation, and fusion. Each module has a transport system that includes a microchannel, and is connected to the cell. A device that can create an egg transplanted with various nuclei,
(2) A module that divides a cell into two or more in the microchannel without destroying the cell membrane of the cell by locally narrowing the microchannel by an external driving device. , (1) automatic nuclear transfer device,
(3) It has a configuration of two channels in which the gradually narrowed channel and its outlet are connected to another wide channel, and the cells are pushed into the gradually narrowed channel to control the flow rate at the outlet. An automatic nuclear transfer device according to (1) or (2), comprising a module for dividing the cell into two or more without destroying the cell membrane of the cell,
(4) Any one of the items (1) to (3), characterized by having a module having a flow channel structure that discriminates the presence or absence of nuclei and separates and extracts only cells that do not contain nuclei in the flow channel. An automatic nuclear transfer device according to
(5) The automatic nucleus according to any one of (1) to (4), which has a module having a function of fixing or retaining cells in a flow path and exchanging surrounding liquid. Transplant device,
(6) It is characterized by having a control circuit for counting cells at the input end and / or the output end of each module when the flow paths are connected between the modules in order to transport and manage the cells between the modules in each step. The automatic nuclear transfer device according to any one of (1) to (5),
(7) Each module can be connected through a common bus for two types of control information signals such as the flow path through which the cells are transported and the in-module drive device, and the system can be easily changed by simply plugging in and replacing each module. The automatic nuclear transfer device according to any one of (1) to (6), which has a structure that can be produced;
(8) The automatic nuclear transfer apparatus according to any one of (1) to (7), wherein the cells are eggs.

(9) A method for producing a cell in which a new nucleus is transplanted, wherein the cell is automatically divided, separated and fused by moving the transport system of the microchannel. Method,
(10) The method includes a step of dividing the egg into two or more in the microchannel without destroying the cell membrane of the cell by locally narrowing the microchannel with an external driving device (9 ) Method of nuclear transfer according to paragraph
(11) By using a flow path that is gradually narrowed and two flow paths whose outlets are connected to another wide flow path, cells are pushed into the gradually narrowed flow path, and the flow rate at the outlet is controlled. A nuclear transfer method according to (9) or (10), comprising a step of dividing the cell into two or more without destroying the cell membrane of the cell;
(12) The nuclear transfer according to any one of (9) to (11), which includes a step of determining the presence or absence of nuclei and separating and extracting only cells that do not contain nuclei in the flow path. Method,
(13) The nuclear transfer method according to any one of claims 9 to 12, comprising a step of exchanging a surrounding liquid by fixing or retaining cells in the flow path.
(14) In order to manage the transport of cells between the processes, when connecting the transport system of the micro flow path between the processes, including detecting and counting the cells at the input end and / or the output end of each process (9) The nuclear transfer method according to any one of items (9) to (13), and (15) The cell according to any one of items (9) to (14), wherein the cell is an egg. The present invention provides a method for nuclear transfer.

The automatic nuclear transfer apparatus of the present invention can perform nuclear transfer stably and efficiently by automating all the steps of the nuclear transfer operation in the microchannel. Thereby, mass production of nuclear transplanted eggs is possible.
The automatic nuclear transfer device of the present invention uses not only chemical reactions in the current biochemical field (applications centered on mixing and reaction of liquids), but also a micro-channel system for individual transport of individual micro-organisms such as cells. By expanding the application to processing, it becomes possible to provide a large amount of automated processing to the field that handles cells, and the application field expands, and it is expected to have a great effect not only for bio research but also for industrialization Is done.

Hereinafter, preferred embodiments of the automatic nuclear transfer apparatus of the present invention will be described in detail with reference to the accompanying drawings. In the description of each drawing, the same reference numerals denote the same parts. In addition, although an egg (egg cell) is demonstrated below as an example of the cell to process, this invention is not limited to this, The automatic nuclear transfer apparatus of this invention is arbitrary animal cells, such as a somatic cell, and a plant cell. It can also be used for ES cells and the like.
FIG. 1 is a diagram showing a protocol for egg cell cloning. In FIG. 1, 1 is an egg, 2 is a nucleus, 3 is a zona pellucida, and 4 is a donor cell.
As can be seen from FIG. 1, the egg cell cloning is mainly performed by removing the zona pellucida 3, dividing the ovum 1 into two pieces, separating (sorting) the divided eggs with or without the nucleus 2, This includes an operation step of coupling and cell fusion with the donor cell 4. The automatic nuclear transfer apparatus of the present invention processes each process (work) conventionally performed manually by a module that performs each work exclusively.
With reference to FIG. 2, the automatic nuclear transfer apparatus of this invention is demonstrated.
FIG. 2 is a diagram showing an outline of the automatic nuclear transfer apparatus of the present invention. In FIG. 2, 5 is a supply port, 6 is a division processing module, 7 is a separation module, 8 is a fusion processing module, 9 is a take-out port, and 10 is a conveyance / electrical interface.
As is clear from FIG. 2, the automatic nuclear transfer apparatus of the present invention includes modules for performing the steps of egg division, separation, and fusion, and each module corresponding to each step of nuclear transfer (ie, supply port 5, The division processing module 6, the separation module 7, the fusion processing module 8, and the takeout port 9) are connected by a transport / electrical interface 10.
In the present invention, a module having a function of fixing or retaining (damming) cells in a flow path and exchanging liquid around the cells can be included. In the apparatus of the present invention, when attention is paid to two modules in series, it is useful when the atmosphere liquid is different between the previous module and the subsequent module in the work process. For example, the former module is an aqueous solution, and the latter module is a buffer.
Further, in the present invention, in order to carry and manage cells between modules in each process, a control circuit that detects and counts cells at the input end and / or output end of each module when the flow paths are connected between modules is provided. It is preferable.
The apparatus of the present invention may include all of the plurality of modules described above, or may include only a part thereof. Moreover, the above-mentioned plural / plural types of modules may be connected in any order according to the protocol, system change, and work purpose.
The transfer / electrical interface includes a transfer system of a micro flow path through which a cell such as an egg moves, and an information signal system for controlling an in-module drive device and the like.

The microchannel has a feature that it is easy to form a laminar flow. According to the feature, in the present invention, cells can be stably conveyed in an aligned state.
In the present invention, the microchannel may have any shape and diameter as long as the cells move by the liquid flow, but preferably has a diameter of 10 to 300 μm. Further, the flow rate in the microchannel may be arbitrary as long as the cells move by the liquid flow, but is preferably 10 to 500 μm / sec.
In addition, the liquid that allows cells to flow in the microchannel may be any liquid, but is preferably any aqueous solution or any buffer.
As a driving device for flowing a liquid containing cells into a micro channel, a syringe type pump such as a syringe, a tubing pump for flowing a liquid inside by crushing a tube from the outside, or the like can be used.
The transport system flow path between the modules can be connected by an arbitrary tube or the like, and the control information signal system can also be connected between the modules by an arbitrary electrical connector.
It is preferable that a transport / electrical interface including the transport system of the micro channel and the control information signal system can be connected between the modules through a common bus. By doing so, it is possible to easily change the type and order of work processes by easily plugging and replacing each module, so it is possible to easily cope with changes in protocols and systems, and other work in the bio field. It can be applied as an automation system.

The cell fusion module (fusion processing module) will be described.
As the cell fusion module, for example, the procedures and methods described in Nature. 1986 Mar 6-12; 320 (6057): 63-5. Nuclear transplantation in sheep embryos. Willadsen SM. Can be applied.
Specifically, a cell fusion module in which electrodes are installed with a minute flow channel interposed therebetween can be mentioned. It is preferable that the inlet and the outlet of the microchannel are respectively connected to other modules.
In the cell fusion module, an alternating electric field is applied between electrodes installed across a microchannel through which a cell containing no nucleus and a cell containing the nucleus flow. As a result, cells that do not contain nuclei and donor cells that contain nuclei that have flowed through the microchannel can be aligned in a direction parallel to the electric field. Thereafter, cell fusion can be performed by applying a DC pulse to the electrodes.
As a source of new nuclei at the time of nuclear transfer, any donor cell can be mentioned.
As another embodiment of the cell fusion module, a micromanipulator can be used, for example, Biol Reprod. 1987 Nov; 37 (4): 859-66. “Nuclear transplantation in the bovine embryo: assessment of donor nuclei and recipient. oocyte. ”Prather RS, Barnes FL, Sims MM, Robl JM, Eyestone WH, First NL.
Specifically, a metal needle serving as an electrode is placed at the tip of two opposed micromanipulators, and two cells (a cell that does not contain a nucleus and a donor cell that contains a nucleus) are located on the line between the two needles. Fusion can be performed by applying a direct-current pulse so that they are arranged side by side, that is, in a positional relationship of needle tip + cell not containing a nucleus + donor cell containing a nucleus + needle tip. At this time, the two cells are preferably in contact with each other, and the tip of the needle may not be in contact with the cell surface.

  The present invention is a device that automates the nuclear transfer operation by applying a micro operation technique and a transfer technique in a flow path, and enables the efficiency of cloning operations, high quality, mass production, and the like Applied to medical fields such as regenerative medicine and pharmaceuticals that require work related to analysis and functionalization of minute biomaterials, the field of agriculture, forestry and fisheries such as improvement and breeding of livestock, preservation of genetic resources such as rare varieties, and foods Is possible.

Hereinafter, preferred examples of each module used in the present invention will be described in detail based on examples, but the present invention is not limited to these examples.
Example 1
First, the first embodiment (corresponding to the item (2)) of the module that performs cell division will be described with reference to FIGS.
FIG. 3 is a cross-sectional view illustrating one embodiment of a split module. In FIG. 3, 15 is a cell (egg), 20 is a micro flow path, 31 is a flexible member, 31a is a wall surface deforming part, 32 is a member connected to a driving device, and L is a liquid flow. Fig.4 (a) is a cross-sectional view which shows embodiment of the division | segmentation module using a sharp blade. FIG.4 (b) is a longitudinal cross-sectional view which shows embodiment of the division | segmentation module using a sharp blade. In FIG. 4, 31b is a flexible member, and 40 is a sharp blade.
As apparent from FIG. 3, in this embodiment, the flow channel wall surface is deformed by the member 32 connected to the driving device such as a motor or a piezoelectric element with respect to the micro flow channel 20 formed by the flexible member 31. By forming the wall surface deformation portion 31a, the cells in the flow path can be crushed and divided without breaking the cell membrane of the egg. The compression may be performed from one side or from both sides.

Moreover, in this embodiment, as shown in FIGS. 4A and 4B, a method of cutting and dividing the cell wall by incorporating a sharp blade 40 into the wall surface is also possible. In this case, either one side or both sides may be excised.
Examples of the flexible member that forms the minute flow path include elastic members such as silicon rubber and PDMS (polydimethylsiloxane). Since these elastic members are excellent in adsorbability, it is preferable that the blade surface is adsorbed and the liquid in the flow channel does not flow out if the surface of the blade is sufficiently flat.

With reference to FIG. 5, a second embodiment (corresponding to the item (3)) of the module for dividing cells will be described.
FIG. 5 is a cross-sectional view showing a cell division module using a flow channel with a reduced diameter. In FIG. 5, L1 indicates a liquid flow and 50 indicates an outlet.
In the present embodiment, a micro-channel whose diameter is gradually narrower than the cell size as shown in FIG. 5 and a channel whose outlet 50 has a wide space are used, and the micro-stream gradually narrowing. By pushing the cells into the path, the cells can be divided without destroying the cell membrane. Since the force between the surfaces is dominant in the minute region, the larger the contact area between the cell and the channel wall, the more the cell can be retained. On the other hand, the cell region that has come out in a space wider than the outlet 50 is pulled by the fluid pressure of the liquid flow L1. That is, the cell can be pulled and divided by controlling the contact area between the liquid flow and the wall surface.
When the cell is an ovum, the diameter of the microchannel that gradually narrows from the input end side toward the outlet 50 is preferably 120 to 90 μm, which is about the diameter of the ovum. It is preferable that it is 50-30 micrometers which is the half or less of the diameter of an ovum.
In this embodiment, it is also possible to actively control the contact area between the cells and the wall surface with the above-described driving device or the like.

Next, a first embodiment (corresponding to the item (4)) of a module that performs cell separation and extraction will be described with reference to FIG.
FIG. 6A is a cross-sectional view showing a magnetic drive device having a sorting mechanism. FIG. 6B is a cross-sectional view taken along the line AA ′ of FIG. Reference numeral 61 denotes a magnetic body, 62 denotes an electromagnet, 63 denotes a loading port, 64 denotes a collection port, 65 denotes a discharge port, 66 denotes a power source, 67 denotes a mold, 68 denotes a passage, and 69 denotes an inner wall of the flow path. The input port 63 and the recovery port 64 are preferably connected to other modules, respectively.
A mechanism of a magnetic drive device that sorts only egg cells that do not contain nuclei in the flow path by driving a small magnetic body 61 in the flow path by an external magnetic field of the electromagnet 62 will be described. As shown in FIG. 6, a magnetic body 61 is installed in the magnetic drive device. The magnetic material is preferably a ferromagnetic material such as iron or nickel and its surface is treated so as not to rust in the solution. The shape of the magnetic material may be a cylinder or a quadrangular prism. An electromagnet 62 for generating a magnetic field is installed in the major axis direction of the magnetic wire as shown in FIG. The current supplied to these electromagnets 62 may be direct current or alternating current. For example, the magnetic drive device is set on the stage of an inverted optical microscope. Subsequent processing can be automated by electrifying the stage.
When sorting egg cells, it is necessary to introduce each egg cell into the sorting mechanism one by one, and any existing technique may be used.

FIG. 7 is a diagram showing an egg cell sorting process using the magnetic drive device. In the figure, F indicates the flow of cells. First, a solution in which an egg cell is dispersed is input from the input port 63. At this time, the magnetic body 61 is disposed at a position for closing the recovery port as shown in FIG. The solution flows through the passage 68 to both the recovery port 64 and the discharge port 65, and an object having a size larger than the gap between the magnetic body 61 and the micro flow path is the discharge port 65 on the side without the magnetic body. It flows to.
Here, when the egg cell 71 that does not include the target nucleus is selected from the egg cell flowing through the input port 63 based on a fluorescence reaction or the like, the egg cell 72 including the nucleus is discharged from the discharge port 65 as shown in FIG. To flow. When the egg cell 71 containing no nucleus is found, the magnetic material is driven to open the recovery port 64 as shown in FIG. 7C, and the egg cell 71 is flowed to the recovery port 64 and taken out. Immediately thereafter, the magnetic body is driven to the state shown in FIG.
In this embodiment, since actuator driving using a magnetic field is performed, (1) the influence on the cell is small, (2) energy is consumed only when the magnetic material is driven, and (3) a complicated control mechanism is provided. There is a merit that it is unnecessary. In general, the cells are not magnetized or weak, but the eggs are not moved by the magnetic field applied to drive the magnetic material.
The mold 67 is manufactured by microfabrication of the minute flow path of the magnetic drive device. For example, a magnetic drive device can be manufactured by molding with PDMS (polydimethylsiloxane). It can also be produced using glass.
FIG. 8 is an exploded view of the disposable magnetic drive microdevice.
As shown in FIG. 8, since the electromagnet 62 is attached to the outside of the microchip (including the disposable microfluidic chip 82, the disposable magnetic body 81, and the disposable cover glass 83), the magnetic drive device can be made disposable. Reference numeral 84 denotes a member for holding the electromagnet 62.

Next, a second embodiment of the module for performing separation and extraction of cells will be described with reference to FIG.
A mechanism of a magnetic drive device that sorts only egg cells that do not contain nuclei in the flow path by driving a small magnetic body in the flow path by an external magnetic field will be described. FIG. 9 shows an outline of a magnetic drive device having a sorting mechanism. The width of the microchannel driven by the magnetic body may be large enough to allow egg cells to pass in addition to the width of the magnetic body. A magnetic body 61 is installed in the magnetic drive device as shown in FIG. Ferromagnetic materials such as iron and nickel are suitable for the magnetic material, and the surface is preferably treated so as not to rust in the solution. The shape of the magnetic material may be a cylinder or a quadrangular prism. An electromagnet 62 for generating a magnetic field is installed in the minor axis direction of the magnetic wire as shown in FIG. The current supplied to these electromagnets 62 may be direct current or alternating current. For example, the magnetic drive device is set on the stage of an inverted optical microscope. Subsequent processing can be automated by electrifying the stage.

When sorting egg cells, it is necessary to introduce each egg cell into the sorting mechanism one by one, and any existing technique may be used. FIG. 10 shows an egg cell sorting process. In the figure, F indicates the flow of cells. First, a solution in which an egg cell is dispersed is input from the input port 63. At this time, the magnetic body is disposed at a position where the recovery port 64 is closed as shown in FIG. The solution flows through the passage 68 to both the recovery port 64 and the discharge port 65, but an object having a size larger than the gap between the magnetic body 61 and the micro flow path is directed to the port on the side where the magnetic body 61 is not present. And flow. The egg cells 71 that do not contain the target nucleus are selected from the egg cells flowing through the input port 63 based on a fluorescent reaction or the like. The egg cell 72 including the nucleus flows to the discharge port 65 as shown in FIG. When the egg cell 71 containing no nucleus is found, the magnetic body 61 is driven to open the recovery port 64 and the egg cell is flowed to the recovery port 64 and taken out as shown in FIG. Immediately thereafter, the magnetic body is driven to the state shown in FIG. In this example, the actuator is driven using a magnetic field, so that (1) the influence on the cell is small, (2) energy is consumed only when the magnetic material is driven, and (3) no complicated control mechanism is required. There are merits. In general, cells are not magnetized or are weak, but the eggs are not moved by the magnetic field applied to drive the magnetic material.
Further, as compared with the first embodiment of the cell separation / extraction module, since the driving distance required for switching the port is small, the power required for driving the magnetic material is small.
For the micro flow path, the mold 67 is manufactured by micro processing. For example, a magnetic drive device can be manufactured by molding with PDMS (polydimethylsiloxane). It can also be produced using glass.
Further, since the electromagnet is attached to the outside of the microchip, the magnetic drive device can be made disposable as in the first embodiment of the module for separating and extracting cells as described above.

Next, a first embodiment (corresponding to the item (5)) of a module for exchanging liquid around a cell will be described with reference to FIG.
A cell mechanism for fixing or retaining a cell in a magnetic drive device by an external magnetic field in a flow path and exchanging surrounding liquid is shown. FIG. 11 shows an outline of a magnetic drive device having a filtering mechanism. In the figure, reference numeral 130 denotes a reagent introduction port for introducing a liquid to be exchanged. A magnetic body 61 is installed in the magnetic drive device as shown in FIG. The selection of the magnetic body 61, the installation position of the electromagnet 62, and the selection of the power source 66 are the same as in the first embodiment of the cell separation and extraction module. For example, the magnetic drive device is set on the stage of an inverted optical microscope. Subsequent processing can be automated by electrifying the stage.
FIG. 12 shows the egg cell filtering process. In the figure, L represents the flow of the solution, and S represents the flow of the liquid to be exchanged. First, the egg cell selected by the sorting mechanism is input from the input port 63. At this time, the magnetic body 61 is disposed at a position where the recovery port 64 is opened as shown in FIG. When the egg cell flows, the magnetic body 61 is driven to block the flow path and block the egg cell as shown in FIG. At this time, the egg cell is blocked by the magnetic body 61, but the solution flows through the gap between the magnetic body 61 and the micro flow path. After damaging the cells, as shown in FIG. 12C, a solution to be exchanged is flowed from the reagent charging port 130, and the liquid around the egg cell 15 is exchanged. When recovering the processed egg cell, the magnetic body 61 is driven to open the flow path, and the egg cell 15 is flowed to the recovery port 64 and taken out. In this example, since the actuator is driven using a magnetic field, as in the first embodiment of the cell separation and extraction module, (1) the influence on the cell is small, and (2) the energy is supplied only when the magnetic material is driven. There is an advantage that (3) a complicated control mechanism is unnecessary. In general, the cells are not magnetized or weak, but the eggs are not moved by the magnetic field applied to drive the magnetic material.
For the micro flow path, the mold 67 is manufactured by micro processing. For example, a magnetic drive device can be manufactured by molding with PDMS (polydimethylsiloxane). It can also be produced using glass. Further, since the electromagnet 62 is attached to the outside of the microchip, the magnetic drive device can be made disposable as in the first embodiment of the cell separation and extraction module.

Next, a second embodiment (corresponding to the item (5)) of the module for exchanging liquid around the cell will be described with reference to FIG.
A cell mechanism for fixing or retaining a cell in a magnetic drive device by an external magnetic field in a flow path and exchanging surrounding liquid is shown. FIG. 13 shows an outline of a magnetic drive device having a sorting mechanism. In addition to the width of the magnetic body 61, the width of the micro flow path driven by the magnetic body 61 may be large enough to allow egg cells to pass through. The selection of the magnetic body 61, the installation position of the electromagnet 62, and the selection of the power source 66 are the same as in the second embodiment of the cell separation and extraction module. For example, the magnetic drive device is set on the stage of an inverted optical microscope. Subsequent processing can be automated by electrifying the stage.
FIG. 14 shows the egg cell filtering process. First, the egg cell 15 selected by the sorting mechanism is input from the input port 63. At this time, the magnetic body 61 is disposed at a position where the recovery port 64 is opened as shown in FIG. When the egg cell 15 flows, the magnetic body 61 is driven to block the flow path and block the egg cell as shown in FIG. At this time, the egg cell 15 is blocked by the magnetic material, but the solution flows through the gap between the magnetic material 61 and the minute flow path. After damming the particles, as shown in FIG. 14C, a solution to be exchanged is flowed from the reagent introduction port 130, and the liquid around the egg cell is exchanged. When recovering the processed egg cell, the magnetic material is driven to open the flow path, and the egg cell 15 is flowed to the recovery port 64 and taken out. In this example, since the actuator is driven using a magnetic field, as in the first embodiment of the cell separation and extraction module, (1) the influence on the cell is small, and (2) the energy is only applied when the magnetic material is driven. (3) No complicated control mechanism is required. In general, there is no magnetism in the egg cell, so the magnetic field applied to drive the magnetic material does not move or affect the egg cell. Further, as compared with the first embodiment of the cell separation / extraction module, since the driving distance required for opening and closing the filter can be reduced, the power required for driving the magnetic material can be reduced.
For the micro flow path, the mold 67 is manufactured by micro processing. For example, a magnetic drive device can be manufactured by molding with PDMS (polydimethylsiloxane). It can also be produced using glass. Further, since the electromagnet is attached to the outside of the microchip, the magnetic drive device can be made disposable as in the first embodiment of the cell separation and extraction module.

Next, with reference to FIG. 15, one embodiment (corresponding to the item (6)) for detecting and counting the input / output of cells in the module when cells are transferred between the modules will be described.
FIG. 15 is a cross-sectional view showing a module input / output management circuit. In FIG. 15, 151 is an optical waveguide, 152 is a light emitting circuit, 153 is a light receiving circuit, 154 is a counter circuit, and R is light irradiation.
Each module is connected by a flow path such as a tube, through which a biological tissue such as a cell 15 is conveyed. In order to carry and manage these cells, it is preferable that the input / output terminals of each module have a circuit that counts the cells and checks the number and properties of the introduced cells. As shown in FIG. 15, an optical waveguide 151 is arranged on the side surface of the microchannel 20, the light from the light emitting circuit 152 such as a photodiode is checked by an arbitrary light receiving circuit 153, and the passed cells are counted by the counter circuit 154. . Further, if light from the light emitting circuit 152 is used as excitation light and fluorescence is received by the light receiving circuit 153, it is possible to count cells stained with fluorescence by an arbitrary method. It is also possible to selectively check and manage only.

Example 2
An experiment of sorting fine particles according to the first embodiment of the cell separation and extraction module was performed. An iron wire having a diameter of 200 μm and a length of 6 mm was used as a magnetic material, and polystyrene particles having a diameter of 50 μm were used as an object for sorting. The driving distance of the iron wire required for port switching was 3 mm. Observation was performed using an inverted optical microscope at a magnification of 4 times.
A result is demonstrated according to FIG. As shown in FIGS. 7A and 7B, the recovery port was blocked by the iron wire, so the particles flowed to the discharge port. In the case of the target particles, the target particles could be selected by driving the iron wire, opening the recovery port and closing the discharge port. The driving speed of the iron wire is 90 mm / sec when 100 V is applied (power consumption: about 4.3 W). In this experimental system, the port can be switched 30 times per second. It was possible to sort egg cells at a higher speed.

Example 3
An experiment of sorting fine particles according to the second embodiment of the cell separation and extraction module was performed. An iron wire having a diameter of 200 μm and a length of 10 mm was used as a magnetic material, and polystyrene particles having a diameter of 50 μm were used as an object for sorting. The driving distance of the iron wire necessary for port switching was set to 0.1 mm. It observed using the same microscope as Example 2 by 4 time magnification.
A result is demonstrated according to FIG. As shown in FIGS. 10 (a) and 10 (b), the recovery port was blocked by the iron wire, so the particles flowed to the discharge port. In the case of the target particles, the target particles can be selected as shown in FIG. 10C by driving the iron wire, opening the recovery port and closing the discharge port. In this experimental system, when 35V is applied to the electromagnet (energy required for one drive: about 19 mJ), it is possible to switch the port 30 times per second. It was possible to sort egg cells.

Example 4
An experiment of exchanging liquid around microparticles according to the first embodiment of the pericellular liquid exchange module was performed. An iron wire having a diameter of 200 μm and a length of 6 mm was used as a magnetic material, and polystyrene particles having a diameter of 50 μm were used as an object for sorting. The driving distance of the iron wire required for opening and closing the filter was 3 mm. It observed using the microscope similar to Example 2 by 10 time magnification.
A result is demonstrated according to FIG. While the liquid flowed through the gap, as shown in FIG. 12C, it succeeded in damaging the polystyrene particles as egg cells flowing by the iron wire in the microchannel. The driving speed of the iron wire was 90 mm / second when 100 V was applied (power consumption: about 4.3 W), and the filter could be opened and closed 30 times per second in this experimental system.

Example 5
Experimental results of the liquid exchange around the microparticles according to the second embodiment of the pericellular liquid exchange module were performed. An iron wire having a diameter of 200 μm and a length of 10 mm was used as a magnetic material, and polystyrene particles having a diameter of 50 μm were used as objects for sorting. The driving distance of the iron wire necessary for opening and closing the filter was 0.1 mm. It observed using the same microscope as Example 2 by 4 time magnification.
A result is demonstrated according to FIG. As shown in FIG. 14 (b), the flow path was closed with an iron wire and the particles were flowed. As a result, the liquid flowed through the gap, while the liquid flowed with the iron wire as shown in FIG. 14 (c). The egg cells were successfully dammed in the microchannel. In this experimental system, it was possible to withstand a flow rate of about 7800 μm / sec when 5 V was applied to the electromagnet (power consumption: about 12 mW), and thus high-speed reagent replacement with low power consumption was possible.

FIG. 1 is a diagram showing a clone processing protocol. FIG. 2 is a diagram showing an outline of the modularized automatic nuclear transfer apparatus of the present invention. FIG. 3 is a cross-sectional view illustrating one embodiment of a split module. FIG. 4 is a cross-sectional view showing an embodiment of a split module using a sharp blade. FIG. 5 is a cross-sectional view showing a cell division module using a flow channel with a reduced diameter. FIG. 6A is a plan sectional view of an egg cell sorting mechanism (electromagnets are arranged on both sides of the magnetic body in the long axis direction). FIG. 6B is a cross-sectional view taken along the line A-A ′ of the egg cell sorting mechanism (electromagnets arranged on both sides in the long axis direction of the magnetic material). FIG. 7 is a plan sectional view of an egg cell sorting process (electromagnets are arranged on both sides of the magnetic body in the long axis direction). FIG. 8 is an exploded view of the disposable magnetic drive microdevice. FIG. 9A is a plan view of an egg cell sorting mechanism (electromagnets are arranged on both sides in the short axis direction of the magnetic body). FIG. 9B is a cross-sectional view of the egg cell sorting mechanism (electromagnets are arranged on both sides of the magnetic body in the short axis direction). FIG. 10 is a plan view of an egg cell sorting process (electromagnets are arranged on both sides of the magnetic body in the short axis direction). FIG. 11A is a plan view of an egg cell filtering mechanism (electromagnets are arranged on both sides of the magnetic body in the long axis direction). FIG. 11B is a cross-sectional view of the egg cell filtering mechanism (electromagnets are arranged on both sides of the magnetic body in the long axis direction). FIG. 12 shows an egg cell filtering process (electromagnets are arranged on both sides of the magnetic body in the long axis direction). FIG. 13A is a plan view of an egg cell filtering mechanism (electromagnets are arranged on both sides of the magnetic body in the short axis direction). FIG. 13B is a cross-sectional view of the egg cell filtering mechanism (electromagnets are arranged on both sides of the magnetic body in the short axis direction). FIG. 14 is a plan view of an egg cell filtering process (electromagnets are arranged on both sides in the short axis direction of the magnetic material). FIG. 15 is a cross-sectional view showing a module input / output management circuit.

Explanation of symbols

1 Ovum 2 Nucleus 3 Zona pellucida 4 Donor cell 5 Supply port 6 Split processing module 7 Separation module 8 Fusion processing module 9 Removal port 10 Transport / electrical interface

Claims (15)

  It includes modules that perform cell division, separation, and fusion steps. Each module has a transport system that includes a microchannel, and is connected to move cells through the channel so that new nuclei are automatically created. A device that can produce transplanted cells.   2. A module that divides a cell into two or more in the micro channel without destroying a cell membrane of the cell by locally narrowing the micro channel with an external driving device. The automatic nuclear transfer device described.   The cell has a configuration in which the channel is gradually narrowed and two channels in which the outlet is connected to another wide channel, the cells are pushed into the gradually narrowed channel, and the flow rate at the outlet is controlled to control the cell 3. An automatic nuclear transfer apparatus according to claim 1, further comprising a module that divides the cell into two or more without destroying the cell membrane.   The automatic nuclear transfer according to any one of claims 1 to 3, further comprising a module having a flow channel structure that determines the presence or absence of a nucleus and separates and extracts only cells that do not contain a nucleus in the flow channel. apparatus.   The automatic nuclear transfer device according to any one of claims 1 to 4, further comprising a module having a function of fixing or retaining cells in a flow path and exchanging surrounding liquid.   2. A control circuit that counts cells at an input end and / or an output end of each module when the flow paths are connected between the modules in order to transport and manage the cells between the modules in each step. The automatic nuclear transfer apparatus of any one of -5.   2. Each module has a structure in which a flow path for transporting cells and a control information signal system can be connected through a common bus, and each module can be easily inserted and removed, and can be easily changed. The automatic nuclear transfer apparatus of any one of -6.   The automatic nuclear transfer device according to any one of claims 1 to 7, wherein the cell is an egg.   A method for producing a cell in which a new nucleus is transplanted, wherein the cell is automatically divided, separated and fused by moving the cell through a transport system of a microchannel.   10. The method of dividing a cell into two or more in the micro channel without destroying the cell membrane of the cell by locally narrowing the micro channel with an external driving device. Nuclear transfer method.   The cell membrane of the cell is formed by pushing the cells into the gradually narrowed channel and controlling the flow rate at the outlet by using the gradually narrowed channel and two channels whose outlets are connected to another wide channel. The nuclear transfer method according to claim 9 or 10, comprising a step of dividing the cell into two or more without destroying the cell.   The method for nuclear transfer according to any one of claims 9 to 11, further comprising the step of determining the presence or absence of nuclei and separating and extracting only cells that do not contain nuclei in the flow path.   The nuclear transfer method according to any one of claims 9 to 12, comprising a step of exchanging a surrounding liquid by fixing or retaining the cells in the flow path.   In order to manage the transport of cells between each process, it includes detecting and counting cells at the input end and / or the output end of each process when connecting the transport system of the micro flow path between each process. The nuclear transfer method according to any one of claims 9 to 13. The nuclear transfer method according to any one of claims 9 to 14, wherein the cell is an egg.