JP2008271905A - Device for processing cell and method for the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To prevent the mixed presence of unnecessary cells in the introduction of genes at a high throughput rate. <P>SOLUTION: This device for processing the cells, having a confluence of a multiple number of fine flow passages for conveying each of cells or fine particles 4 and fusing each of the cells or particles 4 with each other, on the multiple number of fine flow passages is provided, by detecting the cells or fine particles 4 by bisecting sensors 6. The liquid-sending speed of the cells or particles 4 to the fine flow passages is controlled, based on whether the cells or fine particles 4 is detected. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a cell processing apparatus and a cell processing method for stimulating, modifying, or modifying cells, and more particularly to a cell processing apparatus and a cell processing method using a flow path.

  Conventionally, in order to process particles using a microchannel, a technique using a holding mechanism for holding particles has been considered (for example, see Patent Document 1). This retention mechanism can act to exceed the placement force exerted by the fluid flow. By carrying out like this, the particle | grains hold | maintained at the some holding | maintenance mechanism can be exposed to the chemical | medical solution of a flow path.

  In addition, a technique for detecting the concentration of a target object from the state of diffusion indicated in the profile obtained as a result of detecting the diffusion front surface has been considered (for example, see Patent Document 2).

Conventional gene transfer other than Patent Document 1 and Patent Document 2 includes DEAE dextran method, calcium phosphate method, lipofection method, electroporation method, microinjection method, and particle gun method.
JP-T-2005-521425 (1st page, 76th page, 77th page, FIG. 11C) Japanese translation of PCT publication No. 2003-500653 (first page, ninth page, FIG. 3A)

  Such a conventional technique uses and operates particles (including cells) as an analysis technology μTAS (micro total analysis system) that makes use of the characteristics of a microchannel such as originally detecting a substance or obtaining a concentration.

  For this reason, particle operations are often not well suited for operations for stimulating, modifying, or modifying cells.

  Originally, gene transfer (transfection) for modifying cells requires reliable and high-throughput gene transfer into individual cells. Therefore, in cell manipulation, it is necessary to introduce a gene into each cell without omission and to perform this manipulation with high throughput.

  In such a case, it is not always necessary to have a holding mechanism as described in Patent Document 1. Rather, it is necessary to perform cell manipulations at the throughput rate of the fluid flow without countering the placement force exerted by the fluid flow.

  Further, it is not necessary to detect a diffusion front having a cross section in the flow channel direction as described in Patent Document 2 to obtain a target concentration.

  In addition, the conventional gene transfer described above other than Patent Document 1 and Patent Document 2 does not perform a single operation for each cell. Therefore, after such an operation, there is a problem that cells into which genes are introduced and cells that are not so are mixed.

  Conventionally, in order to sort out such cells, it is essential to connect several drug resistance genes and fluorescent marker genes downstream of the target gene and to be able to select them later with drugs, fluorescent colonies, and flow cytometers. .

  The present invention has been made in view of the problems of the conventional techniques as described above, and a cell processing apparatus and a cell processing capable of preventing unnecessary mixing of cells at the time of gene introduction at a high throughput rate It aims to provide a method.

In order to achieve the above object, the present invention provides:
A cell processing apparatus for joining a plurality of particles on the plurality of flow paths by fusing together a plurality of flow paths for transporting particles containing cells,
Detecting means for detecting the particles on the flow path;
Control means for controlling the liquid feeding speed of the particles to the flow path based on whether or not the particles are detected by the detection means.

  Since the present invention is configured as described above, unnecessary cells can be prevented from being mixed at the time of gene introduction at a high throughput rate.

  Embodiments of the present invention will be described below with reference to the drawings.

  FIG. 1 is a diagram showing an embodiment of a flow path provided in the cell processing apparatus of the present invention.

  As shown in FIG. 1, this embodiment is a plurality of flow paths that transport different substances and merge (fuse) each other in a Y-shape or T-shape (for example, T-shaped path 3). Moreover, this junction is provided in multiple stages. And the sensor 1 which is a detection means which detects a substance is provided in the upstream, downstream, or both of a junction.

  At least one of these merging channels must be for carrying cells to be modified or modified. In addition, at least one of the other channels carries particles or cells (including viruses) containing nucleic acids such as DNA or RNA, peptides or proteins for modifying or modifying the cells. It must be.

  The channels other than the above are channels for simply adding factors related to cell growth, such as medium exchange, buffer exchange, and various growth factors, drugs for selecting drugs, signaling substances, and various labeled antibodies. May be included. Such a flow path may not have the sensor 1. The sensor 1 is not provided in the T-junction 3 shown in FIG.

  The length of the flow path may be long enough to satisfy the residence time required for cell division or cell modification. As the flow path having a long residence time, the meandering path 2 shown in FIG. 1 or a circulation flow path combined with the flow path switching valve can be used.

  Further, the position where the sensor 1 is provided is preferably in the vicinity of the three-way, and may be provided on either the upstream side or the downstream side of the junction point depending on the purpose. When the cell and the particle are brought into contact, or when the cell and the drug solution are brought into contact with each other, it is desirable to provide the upstream side. In addition, when the target substance is transferred into the nucleus by an external stimulus applying means for applying a stimulus from the outside after the cells and particles have fused, it is desirable to provide it on the downstream side.

  A cell handled in a microchannel such as the channel of this embodiment has a size of several microns to several hundred microns. Moreover, since it is a cell, in order to operate this, the inside of a flow path needs to be hydrophilic.

  On the other hand, the non-cellular particles handled in the microchannel have a size of several tens of nanometers to several microns.

  In order to make it easy to perform a single operation for these individual particles, it is desirable that the flow path width with respect to the particles be more than 1 time and less than 10 times the flow path. However, if the flow path width is extremely narrow, it becomes difficult to introduce a liquid containing particles into the flow path. Therefore, it is necessary to consider the liquid feeding pressure P with respect to the flow path diameter r. The condition at this time is expressed by the following equation.

P> 2T cos θ / r−ρgh (Formula 1)
Here, T is the surface tension, θ is the contact angle between the solution and the flow path, g is the acceleration of gravity, ρ is the density of the solution, and h is the height of the solution. If the flow path is installed horizontally (h = 0), it is not necessary to consider the second term of (Equation 1).

  In addition, electrophoresis or electroosmotic flow can be used as the liquid feeding means, but there are restrictions on the buffer composition, salt concentration, pH, etc., and liquid feeding with a pump is more desirable.

  In addition to the elastomer such as silicon substrate, glass, resin, PDMS, etc., the material of the channel can be a gel such as polyacrylamide gel that can pattern the channel with heat, ultraviolet rays, or the like.

  The form of the sensor 1 will be described. For the detection of particles, one that measures an electrical impedance change by a pair of electrodes or one that measures an optical light intensity change by a light receiving element can be used.

  From the viewpoint of detection sensitivity, it is desirable that the size of the sensor composed of the electrode or the light receiving element is equal to or smaller than the particle to be detected.

  In addition, a method of measuring a light intensity change of a light receiving element with a configuration of a general thermal lens measurement system or a confocal optical system using a laser beam with a pinhole is also good for small particle detection. In this case, it is necessary to provide a pinhole before the light receiving element.

  FIG. 2A is a diagram illustrating an example of a flow path using a two-divided sensor, and FIG. 2B is a diagram illustrating an output state of the two-divided sensor.

  In the Y-shaped channel shown in FIG. 2A, a two-divided sensor 6 is provided on the upstream side of the merging point in order to detect the position of the particle or cell with higher accuracy. In order to achieve contact between particles flowing from different flow paths, sensors must be provided in both flow paths.

  Two light intensity signals output from the two-divided sensor 6 are denoted by F and R, respectively. When F and R are led to the input (F) 9 of the differential amplifier circuit and the input (R) 10 of the differential amplifier circuit, respectively, an output signal 8 of the differential amplifier circuit is obtained. Thus, when the particle or cell 4 passes through the center of the two-divided sensor 6, a zero cross signal as shown by the broken line in FIG. 2B can be generated. For this reason, the position of the particle | grains or the cell 4 can be known correctly. This information is used for pump drive and flow rate control.

  Next, the timing control mode for mutual particle encounter will be described.

  When the particle or cell detection signal is received from the sensor 1, the liquid feeding to the flow path by the pump is stopped unless the particle or cell detection signal from the sensor 1 provided in the remaining one flow path is generated. After that, when a particle or cell detection signal is generated from the sensor 1 provided in the remaining one flow path, the position of the particle or cell in each flow path up to the merging point is calculated and flows through each flow path. Both pumps are driven so that the particles or cells meet each other.

  Next, the necessary properties of particles or cells flowing through the flow path will be described.

  Here, non-cell particles containing DNA, RNA or other nucleic acids, peptides or proteins that contact the cell to be modified or modified must have the characteristic of being fused with the cell by endocytosis after contact. Don't be. As those having such characteristics, liposomes used in general lipofection methods in which nucleic acids such as DNA and RNA, peptides or proteins are encapsulated can be used. In the case of viruses, endocytosis easily occurs via a cell surface receptor. In addition, general nanoparticles of up to several hundred nanometers can cause endocytosis without having lipid membranes such as liposomes. Nanoparticles obtained by the conventional calcium phosphate method are considered to be similar to these nanoparticles. Furthermore, about several tens of nanoparticles or less, since the nuclear membrane pore size of the cell is about 25 nm, it is expected to easily reach the nucleus.

  Moreover, in the form of transfection, it is necessary to consider the case where the object can be achieved by introducing it into the cytoplasm, for example, RNAi, or the case where it must be transferred into the nucleus.

  When DNA must be transported into the nucleus, for example, as an external stimulus application means for transferring from the cytoplasm into the nucleus, after fusion of temperature, electrical pulse (electric stimulation), ultrasound, light, magnetism from the outside It is good to have means that can be added. For this reason, a means for detecting the fused particles may be provided. At this time, liposomes corresponding to such external stimuli, pH, light, temperature sensitive liposomes, liposomes encapsulating magnetic materials, magnetic nanoparticles, or virus particles can be used. In the case of magnetic particles, it is possible to take a technique of attracting with magnetic force, breaking through the nuclear film, and transferring the target substance into the nucleus. In addition, in the case of using ultrasonic waves, in addition to the purpose of introducing into the nucleus by instantaneous rupture of liposomes, a standing wave is created in the flow path by applying vibration, which corresponds to a node of the standing wave. There is a property that particles gather in the center of the channel. By utilizing this, when the particles are too small for the channel, the particles can be used only for the purpose of collecting the particles in the center of the channel.

  In the above, the form necessary for fusing one cell and a particle for modifying it has been described. However, by introducing a plurality of particles by combining a plurality of flow paths, more advanced transfection is performed. be able to.

  Conventional transfection, for example, is often transient, even though the gene has been introduced, and is extremely difficult to reach homologous recombination. The background of these problems is that the state of factors involved in overall signal transduction, such as the state of the cell cycle during gene transfer, importin, exportin, and the generation of nuclear translocation signals cannot be ignored. In the nucleus, factors related to the DNA methylation state, histone methylation, acetylation, and deacetylation cannot be ignored. On the other hand, many drugs that act on or relate to these factors have been reported in recent years, and it is possible to combine these drugs and perform a multi-step, high-level transfection form by using the present invention. Become.

  FIG. 3 is a diagram showing an embodiment in which a drug is supplied from another flow path to stimulate particles or cells.

As shown in FIG. 3, the present embodiment is a flow path that merges in a T shape. Particles or cells 4 are supplied from one channel, and a drug for stimulating the particles or cells 4 is supplied to one channel in a pulsed manner. These steps can be provided before or after transfection to allow more advanced transfection.
Example 1
(Preparation of flow path)
FIG. 4 is a diagram showing Example 1 in the flow path of the present invention.

In the first embodiment, as shown in FIG. 4, a quad photodiode sensor 11 using a single crystal Si substrate is used at the junction point of the Y-shaped flow path, and a second pin photodiode is provided downstream of the junction point. 11a is arranged. In this flow path, hydrogenated amorphous silicon and hydrogenated amorphous silicon carbide are deposited on a substrate to be an n layer by a plasma chemical vapor deposition (PCVD) method. At that time, the film thickness is set to 360 nm and 140 nm, respectively, and the Au electrode is patterned thereon. The film formation conditions are 1 × 10 ⁻⁷ Torr, RF power 20 mW / cm ² , 250 ° C., and 120 mTorr during deposition. SiO ₂ is laminated as a sealing film by a sputtering method, and a silicone mold release agent is applied by spin coating, a SU8 resist is applied, and exposed to form a flow path pattern. Next, after PDMS (polydimethylsiloxane) is poured into a mold and peeled, the SU8 structure inside is removed, and then the cured PDMS is covered again to form a flow path.
(Installation and operation)
FIG. 5 is a diagram showing an embodiment of the cell processing apparatus of the present invention.

This embodiment is configured as shown in FIG. The flow path 24 is attached to the holder 7, and the flow path 24 and the syringes 12, 12 a and the petri dish 22 are connected by a tube 23. Here, the flow path 24 is the flow path described in FIG. The syringes 12 and 12 a are connected to the two flow paths 24. The four-divided sensor 11 leads the light intensity signals detected by the four divided sensors to the differential amplifier circuit in pairs as shown in FIG. 2 via the I / V conversion circuit 13, The A / D conversion circuit 14 performs A / D conversion. The second pin photodiode 11a provided on the downstream side of the junction point guides the detected light intensity signal to the A / D conversion circuit 14a via the I / V conversion circuit 13a, and the A / D conversion circuit 14a. Performs A / D conversion. The data A / D converted by the A / D conversion circuits 14 and 14a is used by the microcomputer 25 for ON / OFF determination of driving of the syringe pump 15 and an electric pulse application timing generation signal. Mouse myeloma cells previously diluted to a concentration of 0.5 × 10 ⁵ cells / ml with a medium and adjusted so that the concentration of polyethylene glycol is 20% are sucked into the syringe 12 and syringe pump 15 Attach to. Meanwhile, cells collected from mouse lymph nodes are adjusted to the same concentration and attached to the other syringe 12a. The syringe pump 15 is a control means that can be equipped with two syringes and can control driving independently of each other. Next, an electrode 16 obtained by performing gold vapor deposition on a previously flattened tungsten needle is observed with an actual microscope in an electrode insertion recess 21 provided on the side of the second pin photodiode 11a for detecting cells after fusion. Insert while. Next, the entire flow path 24 is illuminated by the halogen lamp 17 and the syringe pump 15 is driven. The first signal from the gas to the liquid enters the sensor, and when the particles are detected, the microcomputer 25 controls the flow rate so that the particles meet each other. When the fused particles are detected by the second pin photodiode 11a after joining, a pulse is applied to the electrode 16 to promote fusion in the nucleus.
(Example 2)
(Preparation of flow path)
FIG. 6 is a diagram showing Example 2 in the flow path of the present invention. FIG. 6A is a view of the present embodiment as viewed from above the flow path, and FIG. 6B is a view of the present embodiment as viewed from the flow path side.

In Example 2, as shown in FIG. 6, a glass substrate is used, and a pinhole 19 is disposed immediately below the flow path. The pinhole 19 has a size of about 400 nm by depositing aluminum on the back surface of the channel by sputtering. Similar to Example 1, a SU8 resist is applied and exposed to form a flow path pattern. Next, after PDMS is poured into a mold and peeled, the SU8 structure inside is removed, and then the cured PDMS is covered again to form a flow path. The flow path is filled with PC modifier (a product of AI Biochips Co., Ltd.) and the inside is hydrophilically treated. The processing method is according to Noh.
(Installation and operation)
The tube is connected in the same manner as in Example 1. As shown in FIG. 6 (b), red visible light with a collimator lens is irradiated to each pinhole 19 through the aperture 20 a of the semiconductor laser light source module 20 to the flow path. A part of the visible light emitted from the particle image plane passes through the pinhole 19 and is detected by the avalanche photodiode 18. The output of the avalanche photodiode 18 is A / D converted via an I / V conversion circuit, and the converted data is used for ON / OFF determination of syringe pump drive by a microcomputer. Mouse ES cells diluted and adjusted with a medium at a concentration of 0.5 × 10 ⁵ cells / ml in advance so as to be sufficiently discrete are sucked into a syringe and attached to a syringe pump. On the other hand, using Lipofectamine 2000 (trade name of Invitrogen Corporation), liposomes encapsulating plasmid DNA in which a GFP gene is linked downstream of the housekeeping gene are prepared. The production method follows the Noh book. After adjusting to the same concentration as before, liposomes are sucked into a syringe and attached to a syringe pump. Thereafter, the syringe pump is driven, and the flow rate is controlled so that cells and liposomes meet as in Example 1.

  As described above, in the present invention, since a single operation of each cell can be realized under a micro flow channel, the result is that a cell into which a gene is introduced and a cell that is not so are not mixed. Further, the throughput rate can be greatly improved as compared with the microinjection method.

  Therefore, the present invention solves the problem of throughput rate, which has been regarded as a conventional problem, and the problem that cells that have not undergone gene transfer coexist.

It is a figure which shows one form of the flow path provided in the cell processing apparatus of this invention. (A) is a figure which shows an example of the flow path using a 2 split sensor, (b) is a figure which shows the mode of the output of a 2 split sensor. It is a figure which shows one form which supplies a chemical | medical agent from another flow path in order to stimulate a particle | grain or a cell. It is a figure which shows Example 1 in the flow path of this invention. It is a figure which shows one Embodiment of the cell processing apparatus of this invention. It is a figure which shows Example 2 in the flow path of this invention.

Explanation of symbols

1 sensor 2 meandering path 3 T-shaped path 4 particle or cell 5 drug 6 split sensor 7 holder 8 output signal of differential amplifier circuit 9 input of differential amplifier circuit (F)
10 Input of differential amplifier circuit (R)
11 Quadrant sensor 11a Second pin photodiode 12, 12a Syringe 13, 13a I / V conversion circuit 14, 14a A / D conversion circuit 15 Syringe pump 16 Electrode 17 Halogen lamp 18 Avalanche photodiode 19 Pinhole 20 Semiconductor laser Light source module 21 Electrode insertion recess 22 Petri dish 23 Tube 24 Flow path 25 Microcomputer

Claims (18)

A cell processing apparatus for combining a plurality of flow paths for transporting particles containing cells together and fusing the particles transported on the plurality of flow paths to each other,
Detecting means for detecting the particles on the flow path;
A cell processing apparatus comprising: control means for controlling a liquid feeding speed of the particles to the flow path based on whether the detection means detects the particles. The cell processing apparatus according to claim 1,
When the particle is detected by the detection unit on one of the plurality of channels, the control unit moves to the channel until the particle is detected by the detection unit on the other channel. The cell processing apparatus characterized by stopping liquid feeding. The cell processing apparatus according to claim 1 or 2,
The cell processing apparatus, wherein the detection means detects the particles on the flow path by detecting a change in light intensity or an electrical impedance change in the flow path. The cell processing apparatus according to claim 1 or 2,
The cell processing apparatus, wherein the detection means is a light receiving element. The cell processing apparatus according to claim 4, wherein
The cell processing apparatus, wherein the light receiving element is one of a sensor, a two-divided sensor, and a four-divided sensor. The cell processing apparatus according to claim 1 or 2,
The cell processing apparatus, wherein the detection means is a pinhole. The cell processing apparatus according to any one of claims 1 to 6,
A cell processing apparatus comprising an external stimulus applying means for applying a stimulus to the particles on a flow path where the flow paths merge. The cell processing apparatus according to claim 7,
The cell processing apparatus characterized in that the external stimulus applying means applies a stimulus of light, temperature, ultrasonic wave, magnetism, or electrical stimulus from the outside of the flow path where the flow paths merge. . The cell processing apparatus according to claim 7 or 8,
The cell processing apparatus, wherein the external stimulus applying means applies the stimulus based on whether or not particles are detected by the detecting means. The cell processing apparatus according to any one of claims 1 to 9,
A cell processing apparatus, wherein a drug is supplied in pulses to the particles. The cell processing apparatus according to any one of claims 1 to 10,
The cell processing apparatus, wherein the particles are liposomes containing DNA or RNA nucleic acids, peptides or proteins. A cell processing method in a cell processing apparatus for combining a plurality of flow paths for transporting particles containing cells into one flow path and fusing the particles transported on the plurality of flow paths to each other. And
Processing to detect the particles on the flow path;
A cell processing method comprising: controlling a liquid feeding speed of the particles to the flow channel based on whether or not the particles are detected. The cell processing method according to claim 12,
When the particles are detected on one of the plurality of channels, the liquid supply to the channel is stopped until the particles are detected on another channel. A characteristic cell processing method. The cell processing method according to claim 12 or 13,
A cell processing method comprising a process of detecting a change in light intensity or an electrical impedance change on the flow path. The cell processing method according to any one of claims 12 to 14,
A cell processing method comprising a step of applying a stimulus to the particles on a flow path where the flow paths merge. The cell processing method according to claim 15,
A cell processing method comprising: applying light, temperature, ultrasonic waves, magnetism, or electrical stimulation from the outside of the flow path where the flow paths merge. The cell processing method according to claim 15 or 16,
A cell processing method comprising a process of applying the stimulus based on whether particles are detected on the flow path. The cell processing method according to any one of claims 12 to 17,
A cell processing method comprising a step of supplying a drug in pulses to the particles.

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