JPH02131569A - Microchamber plate, cell detection method using the same, processing method and device, and cells - Google Patents

Abstract

(57) [Abstract] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a cell fusion device, and particularly to a microchamber plate for cell fusion and a particle discrimination method suitable for one-to-one fusion of different types of cells. and a particle processing device and a cell processing device.

[Conventional technology]

The conventional device supplies heterogeneous cells one pair at a time to compartments arranged in a lattice, as described in the literature (1986 Society for Precision Engineering Spring Conference Proceedings, p. 845-846), and then uses a suction nozzle to They were suctioned and fixed into a small opening in the compartment, and fusion was performed all at once under the same fusion conditions.

[Problem to be solved by the invention]

In the conventional technology described above, since a large number of cells are fused at once, it is difficult to reliably fuse all cells that have individual differences such as variations in size, cell membrane thickness, and activity, and it is difficult to reliably fuse all cells that have individual differences such as variations in size, cell membrane thickness, and activity. There was a problem in that it required a lot of effort to sort out the fused cells.

The purpose of the present invention is to independently grasp the state of cells with large individual differences, provide optimal fusion conditions, and reliably fuse a large amount of cells. The purpose of this method is to introduce genes, and also to make it possible to individually process the cells of f and R, such as understanding the state of the blood based on information such as its shape using blood.

[Means for solving the problem] In order to achieve the above purpose, an independent electrode is provided in each compartment, and a voltage etc. can be applied independently to each electrode and resistance etc. between the electrodes can be measured. A structured microchamber plate with multiple compartments to position and hold particles and cells was used. Furthermore, by measuring the behavior of particles and cells within the compartment as the potential between the electrodes that sandwich them, it has become possible to distinguish between particles and cells. We also obtained a device and a cell fusion device that enable individual processing and fusion by applying voltage between electrodes that sandwich particles or cells.

[Effect]

By applying voltage to a pair of electrodes provided in a compartment on a microchamber plate, a pair of cells existing between the electrodes can be brought into contact with each other, fused through the cell membranes in contact with each other, and furthermore, the electrodes can be The changes in the shape of the fused cells can be detected from the changes in the potential between the two, and the fusion pulse voltage can be strengthened or weakened or adjusted. Many compartments,
N from the compartments on the microchambers arranged on a grid.
By making +X an independent structure, it is possible to delicately control the fusion conditions that depend on the individual differences of cells present in each compartment, allowing for efficient fusion of a large number of cells. be able to. Furthermore, since the state of the cells existing in each compartment can be grasped independently, the desired cell arrangement can be determined without the need for labor-intensive visual inspection or other means.
It is possible to extract only the desired cells by knowing the location, and it is effective! It becomes possible to generate the image with high quality.

Furthermore, since the cells can be processed using laser beams or X-rays while understanding the behavior of the cells existing in the compartment, cell processing such as gene introduction can be performed easily and reliably. Furthermore, it is possible to determine whether or not there are air bubbles attached to the compartments of the microchamber that would pose an obstacle when positioning and holding cells in the liquid, making it possible to remove air bubbles in advance, allowing cells, etc. It is possible to provide a microchamber that can handle minute particles with high reliability.

〔Example〕

Hereinafter, one embodiment of the present invention will be described with reference to the first to 321st examples. Figure 1 shows the microchamber plate of the present invention.
FIG. 2 shows a cell fusion device using a microchamber plate, and FIG. 3 shows a wiring pattern 9'j of the electrodes of the microchamber plate. 1st, [Kozu ~ In Figure 3,
The numbers for common parts were the same.

FIG. 1 shows an enlarged cross-sectional view of a microchamber plate when handling +itl +J particles having a size of 20 to 100 Atm. The microchamber plate 901 has compartments 101-103, 201- formed in a lattice-like arrangement with a pitch of 770 μm by anisotropic etching on a 400 μm thick Si wafer.
It has 203. In order to electrically fuse a pair of cells, two different cells are suctioned and held in the compartments 101 to 103.

Note that the compartments 201 to 203 are shown without cells. All compartments have suction holes 6 in compartment 103.
A suction hole having the same structure as 03 is formed. It has a structure in which cells can be positioned and held by suctioning from below the suction hole using a suction means (not shown).

These cells were placed in a container (not shown) (FIG. 2 906).
A predetermined number of cells are transferred to each compartment by a transport means (not shown) (902, 903 in FIG. 2) in an isotonic solution filled with a liquid. A set of electrodes 80 is provided in the compartment 103.
2,803 is formed, and is connected to a control system (FIG. 2, 905), not shown, via wiring 10.13. The other compartments 101, electrodes and wiring 11, 12 and 20-23 are connected to the control system.

FIG. 2 shows the microchamber plate 90 shown in FIG.
1 is an external view of a cell fusion device 904 using cell fusion device 1. 0.5 as an isotonic solution to maintain cell activity by equalizing osmotic pressure.
A mol of sorbitol solution is placed in a container 906. Assuming that the heterogeneous cells are A and B, means 902 for transporting the cell A
, has means 903 for transporting cells B. In order to transfer one cell A and one cell B each to the compartments of the microchamber plate 901, the transport means has a suction seat that sucks only one cell at a position corresponding to each compartment. There is. The transport means 902 and 903 can move in the x- and z-axis directions, respectively, and the pair of cells A and B are positioned and held by transferring the cells to the compartments of the microchamber plate. Wiring from electrodes provided independently in each compartment is connected to a control system 905.

The control j'J905 uses a power supply to electrically fuse the cells in the compartment of the microchamber plate, and grasps the state of the cells as a potential change between the electrodes sandwiching the cell, and controls the power supply according to the state of the cells. It is comprised of a processing circuit that performs processing such as adjusting the applied voltage and opening/closing, and a circuit that controls operations such as driving the transport means 902 and 903k to transfer cells to the microchamber plate.

In Figure 3, each compartment of the microchamber plate is provided independently? I! Poles 101-104, 201-20
4,301-304, 401-404 and wiring from these electrodes 10-14, 20-24.30-34.40
This is an example in which 44 is patterned into a planar shape.

Next, the process of electrical cell fusion will be described using the cells in the compartment 103 as an example in FIG. Transferred by conveyance means 1
fL, positioned cells 800 and 801 are suction holes 60
Due to the suction pressure from 3, they come into contact with each other directly above the suction hole. A pair of cells has a suction hole 603 extending longitudinally between the electrodes.
They are positioned so that they come in contact with each other to form a figure 8 shape, and their longitudinal direction coincides with the direction of the electric field. When a pulsed voltage is applied between the electrodes while maintaining contact, the pair of cells sandwiched between the electrodes begin to fuse from the part of the cell membrane that they contacted. As the fusion progresses, the cells that were in contact with each other,
It changes from a potbelly shape to an ellipsoid shape and then becomes a spherical body. The resistance, capacitance, and amount of charge between a pair of electrodes change as the shape of the cell changes.

Assuming that cells with radius rll r2 fuse and change to the size of radius r, the following equation holds true since the total volume of the cells is constant.

rE'+r2'=r, the maximum length of the cells in contact is 2.(rE+rz), and the maximum diameter of the cell after fusion is 2.r,=2.(r'+r2'). For example, when two cells with equal radius R are in contact, the longest distance is 4R. The longest distance shot after fusion is 2.52·R, which is 63% smaller than when the two m cells were in contact. Therefore, it can be detected as a potential change between the electrodes.

After applying a pulsed voltage for fusion, if the potential between the electrodes begins to change, then cell fusion has begun. As cell fusion progresses and forms a single sphere, the potential change between the electrodes becomes smaller. Therefore, immediately after applying a pulsed voltage for fusion to a pair of cells, the potential between the electrodes is measured,
By determining the rate of change, the starting point of fusion can be determined. Cells can be fused by continuing to apply a pulsed voltage at appropriate intervals until cell fusion begins, and when fusion has not yet started, by gradually increasing the applied pulsed voltage and changing the application conditions. After a potential change appears and the start of fusion is detected, application of the pulsed voltage is stopped in order to advance fusion. Further, in cases where the activity of the cell is weak, the cell may be damaged by the application of a pulsed voltage, but the mode of potential change at this time is a mode in which the potential changes rapidly and then becomes constant.

Therefore, it is possible to distinguish the moat from a moat whose potential changes gradually as the fusion progresses, and the point at which the cell is damaged can be ascertained.

If one cell in the compartment is found to be damaged, a high voltage is applied to destroy the remaining cells to prevent them from remaining unfused and multiplying during culture. Apply. The presence or absence of remaining cells and the state of destruction can also be detected from potential changes. By the method described below, it is possible to ensure that only desired fused cells remain. Note that the distance between the electrodes is 200 μm, and the distance between the electrodes is 200 μm. When fusing salad green cells using 5 mol of sorbitol solution, approximately 2 mol of sorbitol solution is used.
Cell fusion begins by applying a 5V, 150μs pulse voltage once or several times, and the fusion progresses from a figure-eight contact state, to a potbellied shape, to an ellipsoid shape, and then to a spherical shape in about 2 to 3 minutes. move on.

Since the wiring patterns from the electrodes in FIGS. 1 and 3 do not intersect with each other, patterning is simple, but there is a limit to the number of compartments that can be formed on the chamber plate. FIG. 4 shows an example in which an unlimited number of compartments can be formed on a chamber plate by making the wiring patterns intersect three-dimensionally. Wiring in the y direction 10.
A set of electrodes 11 to 67 are patterned corresponding to the intersections of wirings 1 to 7 in the X direction and 60. The connection method for applying a voltage to each electrode or measuring the potential between the electrodes is a known two-dimensional liquid crystal device or two-dimensional M
This is possible by applying a method of moving the OS image sensor or the like.

FIG. 5 is an enlarged cross-sectional view of a Si microchamber plate using the wiring method shown in FIG. 4.

Suction hole for suctioning and positioning cells (dimensions 10μm x 80μ
The cell holding groove plate 200 has a two-layered structure including a cell holding groove plate 200 having a cell retaining groove plate 200 and a side wall plate 201 forming a compartment corresponding to the position of the suction hole. In order to show the electrodes formed in the overlapping portion, the cell holding groove plate and the side wall plate are shown separated. Made of Au, thickness 300nm
Wirings 6, 7 and 10-30 correspond to the portions with the same numbers as the wiring pattern in FIG.

The intersecting portions of the intersecting wiring lines are electrically isolated by an insulating layer made of Sin2. Next, explanation will be given using the compartment 37.

An electrode 301 from the wiring 6 and an electrode 302 from the wiring 30 are formed in the longitudinal direction of the suction hole 300 of the compartment 37 .

In the lateral direction of the suction hole 300, protrusions 303 and 304 made of an insulator made of Sio2 are formed. Convex part 303 of a great manly body
, 304 are arranged across the suction hole, and the electrodes 301
It has a function of converging electric lines of force generated when a voltage is applied between and 302 at the center of the suction hole. Therefore, it is possible to efficiently apply voltage and all+ constant to a pair of cells that are positioned in contact with each other on the suction hole.

Next, an example in which the state and behavior of cells existing between a pair of electrodes are electrically detected will be described using FIGS. 6 to 8. 6th
The figure is an enlarged view of the f1 pole of the embodiment of the present invention, and shows an electrode 601.
, 602, different types of cells 603 and 604 are shown in contact with each other and positioned. These electrodes and cells are isotonic′/F! Immerse yourself in i605. Literature (Cell Engineering Vol. 3, No. 6, 1988, p. 497-
505 p. 500, p. 502) As shown in h, 0. The specific resistance of an isotonic solution of 5 mol of sorbitol is about 1 kΩ・mm, and the specific resistance of the cell membrane is about 100,
&Ω・mm. Normally, the thickness of a cell membrane is about 10 nm, so the resistance in the thickness direction is about 1 kΩ. The distance between the electrodes 601 and 602 of the present invention is 200 μm, and the resistance thereof is about 0.2 kΩ. Therefore, either make the width of the opposing electrodes smaller than the diameter of the cell, or
304) to create a structure in which the world concentrates on cells, it is possible to measure the resistance that exists in the shape of the cells that exist between the electrodes. electrodes 601 and 60
A constant voltage is applied in pulse form from a power supply 606 between 2 and the detection processing circuit 607 calculates the resistance existing in the thickness of the cell membrane and outputs it to a control system (not shown) (corresponding to 905 in FIG. 2). . Note that this is to prevent electrolysis of the isotonic solution by applying a low voltage in a pulsed manner.

FIGS. 7 and 8 show examples of temporal changes in resistance values present in the shape of cells obtained after the measurement process. Figure 7 shows the resistance characteristics of the fusion process. Moreover, FIG. 8 shows the cell destruction process. In both figures, the horizontal axis represents time, and the vertical axis represents resistance value r.

In the 7th M, cells? 1! Time t that does not exist between poles
, is the resistance value of only the isotonic solution r, (approximately 0.2 kΩ), and the time when only one cell is positioned, and 2 is the resistance value rz (approximately 2 kΩ) that depends on the membrane thickness of one cell. , 2 cells
At time t4 when only two cells are positioned, the resistance value r3 (approximately 4 kΩ) depends on the membrane thickness of two cells, and at time t4 when cell fusion has started and cell fusion is progressing, they are in contact with each other. As the membrane at the site becomes thinner and fusion progresses, the resistance value decreases to r4, and as a result, a hybrid cell is formed.

At time t, the resistance value r when existing as a hybrid cell
4 is shown.

FIG. 8 is an example of characteristics showing the number of cells and the presence or absence of damage. The period from time t1 to time t is the same as in FIG. It takes time for one of the two existing cells to break, and the change in resistance value becomes steep, as in the case with Toshi 4. Therefore, it is possible to distinguish and process the gradual change in resistance value during cell fusion (corresponding to Fig. 7). The resistance value r, (approximately equal to r2) at time t6 is the resistance value of the cell when only one of the pair of cells is damaged and only one remains. Furthermore, when the remaining one cell is also damaged, the resistance value rt (approximately r1
). It is clear that the resistance value of each substance depends on the shape of the electrode, the concentration of isotonic solution, the concentration of -1 solute, the type of cells, etc., but once the conditions are determined, large variations can be eliminated. do not have. Furthermore, it is easy to consider electrical characteristics such as capacitance and dielectric constant as applications of the detection processing circuit.

The examples described above deal with cells of 20 to 100 μm, but cells and particles with sizes other than this can easily be handled by appropriately designing the compartments and suction holes of the microchamber plate. It is clear that it can be applied to micro-chamber plate materials etc. The processing of suction holes on the μm order can be realized by applying patterning and etching technologies used in semiconductor processes. Depending on the accuracy, it is also possible to use glass, resin, ceramics, etc. other than Si.

Furthermore, in addition to the cell fusion device shown in the present invention, it can also be used as a cell processing device, such as placing a predetermined number of cells in a compartment and performing chemical treatment or culturing while monitoring their condition. be.

In addition, a microchamber plate with cells positioned in it is placed in a solution with a predetermined gene suspended in it, and the cell wall is irradiated with a narrowly focused laser beam or It can also be used as an introduction device.

Furthermore, if blood is used as the particles, by counting the number of blood cells positioned in the compartment, the number of blood cells per unit flow rate can be measured, and the blood cell concentration can be determined.

It is also possible to use the device as a blood cell processing device that can apply a voltage to blood cells positioned in a compartment, determine the deformability of the blood cells from the degree of shape deformation caused by the voltage, and use it for diagnosis.

Note that for a microchamber plate that positions and handles particles in a liquid, it is undesirable for air bubbles to adhere to compartments or suction holes in terms of particle manipulation. By measuring the potential between the electrodes in each compartment in advance, it is possible to detect the presence or absence of air bubbles, remove air bubbles, and take other considerations in data processing, making it possible to create extremely reliable microchambers. can be provided.

〔Effect of the invention〕

According to the present invention, the states of cells and particles can be grasped individually and independently, so cell fusion, which was conventionally difficult to automate due to the large individual differences between cells, had no choice but to rely on human hands. Since it has become possible to create a system that can automatically perform large quantities of cell engineering, it can contribute to the major development of cell engineering. In addition, since the arrangement of the compartments on the microchamber plate is known, it is easy to manage the addresses of particles and cells in the compartments, and to check the behavior of particles and cells after various treatments. By individually importing and processing them into a computer, it is possible to contribute to follow-up research on cells and genetic engineering, which was previously considered impossible.

[Brief explanation of the drawing]

FIG. 1 is an enlarged cross-sectional view of the first embodiment of the present invention, FIG. 2 is an external view of the device of the first embodiment, and FIG. 3 is a wiring pattern diagram of the first embodiment. 4 is a wiring pattern diagram of the second embodiment, and FIG. 5 is an enlarged cross-sectional bird's-eye diagram of the second embodiment.
FIG. 6 is an enlarged view of the electrode portion of the embodiment of the present invention, FIG. 7 is a diagram showing the resistance characteristics in the fusion process, and FIG. 8 is a diagram showing the cell breakage process. Explanation of symbols 10-21... Wiring, 101-203... Compartment, 603
・Suction hole, 800, 801...Cell, 802, 8
03...Electrode, 901...Microchamber plate 〈/Kushoku 2wa 2ρ/', Ichitake Taji 2, 3 Ichimarusha/Rada〆θ. ．． {-,%ll Gogo, 5th agony jlJ, MeDgukanneitoconvexp 4th torture/ρ 2ρ . ? Ku4ρ Taρ z0 2nd by Otsu 1 Otsu I Otsu

Claims (1)

[Claims] 1. A microchamber plate characterized by having a plurality of compartments each having a pair of electrodes therein, and means for independently applying a voltage between the pair of electrodes for each compartment. . 2. The microchamber plate according to claim 1, further comprising means for detecting electrical resistance, capacitance, or accumulation between the pair of electrodes. 3. Particles characterized by specifying the presence or absence or shape of particles in the compartment from the electrical resistance, capacitance, or charge between the pair of electrodes detected using the microchamber plate according to claim 2. Discrimination method. 4. A particle processing device comprising the microchamber plate according to claim 2, further comprising means for positioning the particles between the pair of electrodes. 5. The particle processing device according to claim 4, wherein the particles are cells. 6. A cell fusion device comprising the microchamber plate according to claim 5, further comprising cell fusion means positioned between the pair of electrodes. 7. A cell characterized by being fused and cultured using the cell fusion device according to claim 6. 8. A device for introducing genes into cells, characterized in that the microchamber plate according to claim 2 is further provided with means for making micropores in the cells existing in the compartment. 9. The device for gene introduction into cells according to claim 8,
An apparatus for introducing genes into cells, characterized in that a laser beam is used as a means for opening the micropores. 10. The gene introduction device into cells according to claim 8, wherein X-rays are used as means for opening the micropores. 11. A blood cell processing apparatus according to claim 5, wherein the cells are blood cells, and a means for determining the activity and deformability of the blood cells is added.