JP2829005B2 - Micro-chamber plate, cell detection method, treatment method and apparatus using the same, and cell - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a cell fusion device, and more particularly to the use of a heterogeneous cell for one cell.
The present invention relates to a micro-chamber plate for cell fusion suitable for one-to-one fusion, a particle discriminating method, a particle processing device, and a cell processing device.

[Conventional technology]

The conventional device supplies a pair of heterogeneous cells to the grid-arranged compartments as described in the literature (Paper Collection, Spring Meeting of the Japan Society of Precision Engineering, Spring Meeting, 1987). And was fixed to the small opening of the compartment by suction, and fusion was performed at once under the same fusion conditions.

[Problems to be solved by the invention]

In the above prior art, since a large number of cells are fused at once, it is difficult to reliably fuse all cells having individual differences such as variations in size, thickness of cell membrane, and activity, and unfused cells. There was a problem that labor was required for selecting the fused cells. An object of the present invention is to independently grasp the state of a cell having a large individual difference, provide a fusion condition under optimal conditions, and reliably fuse a large number of cells. Another object of the present invention is to introduce a gene, and to enable individual processing of various cells, such as grasping the state of blood from information such as its shape using blood.

[Means for solving the problem]

In order to achieve the above purpose, independent electrodes are provided in each compartment, and voltage and the like are independently applied to each electrode, and particles and cells are positioned and held so that the resistance between the electrodes can be measured. A micro-chamber plate with a number of compartments was used. Also, by measuring the behavior of particles and cells in the compartment as the potential between electrodes sandwiching them, it was possible to determine the particles and cells. Also,
By applying a voltage between electrodes sandwiching particles and cells, a device and a cell fusion device capable of individually processing and fusion were obtained.

[Action] A pair of electrodes provided in the compartment on the micro-chamber plate is applied with a voltage to bring a pair of cells existing between the electrodes into contact with each other, or to fuse through a cell membrane in contact with each other, Can know the change in the shape of the fused cells from the change in the potential between the electrodes, and can increase or decrease the fusion pulse voltage. By making the electrodes from the compartments independent on a microchamber in which a large number of compartments are arranged on a lattice, the electrofusion conditions depending on the individual differences of cells in each compartment can be delicate. Since control becomes possible, a large number of cells can be efficiently fused. Furthermore, since the state of the cells present in each compartment can be grasped independently, it is possible to grasp and extract the location of the desired cells without the need for laborious visual observation or the like. Can be produced reliably and efficiently. Also, while grasping the behavior of cells existing in the compartment,
Since cells can be processed using laser beams or X-rays, cell processing such as gene transfer can be performed easily and reliably. Furthermore, since it is possible to determine the presence or absence of air bubbles in the liquid in the micro chamber for positioning the cells in the liquid, which may hinder the positioning and holding of the cells, it is possible to remove the air bubbles in advance, and to remove the fine particles such as the cells. It is possible to provide a micro-chamber that can handle highly reliable particles.

〔Example〕

An embodiment of the present invention will be described below with reference to FIGS. FIG. 1 is a micro-chamber plate of the present invention, FIG. 2 is a cell fusion device using the micro-chamber plate, and FIG. 3 is an example of a wiring pattern of electrodes of the micro-chamber plate. 1 to 3, the numbers of the common parts are the same.

FIG. 1 shows a bird's-eye view of an enlarged cross section of a microchamber plate in the case of handling cells having a size of 20 to 100 μm as particles. The micro-chamber plate 901 has compartments 101 to 103 formed in a 400 μm-thick Si wafer by anisotropic etching processing in a grid-like arrangement with a pitch of 770 μm.
201-203. In order to electrically fuse a pair of cells in the compartments 101 to 103, two heterogeneous cells are suctioned and held two by two. Note that the cells are not contained in the compartments 201 to 203. All compartments have suction holes 6 in compartment 103
A suction hole having the same structure as 03 is formed. The cells can be positioned and held by sucking from below the suction holes using suction means (not shown). These cells are placed in an isotonic solution filled in a container (not shown) (FIG. 2, 906) in a transport means (not shown) (FIG. 2).
902, 903) by a predetermined number of each compartment. A pair of electrodes 802 and 803 are formed in the compartment 103,
It is connected to a control system (not shown) (FIG. 2 905) via the wirings 10 and 13. From the other compartments 101, 102, 201 to 203, electrodes and wirings 11, 12, and 20 to 23 are connected to a control system so that a voltage can be applied and a potential can be measured independently of each other.

FIG. 2 shows the micro-chamber plate 90 shown in FIG.
1 is an external view of a cell fusion device 904 using 1. A 0.5 mol sorbitol solution is placed in the container 906 as an isotonic solution in order to maintain the activity of the cells by adjusting the osmotic pressure. Assuming that the heterologous cells are A and B, it has means 902 for transporting cell A and means 903 for transporting cell B. The transfer means has a structure having a suction seat for sucking only one cell at a position corresponding to each cell in order to transfer each cell A and B to the cell of the microchamber plate 901 one by one. I have. The transport means 902 and 903 can move in the x and z axis directions, respectively.
By transferring the cells to the compartment of the micro-chamber plate, the pair of cells A and B is positioned and held. Wiring from electrodes provided independently in each compartment is connected to a control system 905. The control system 905 is a power supply for electrically fusing cells in the compartment of the microchamber plate,
A processing circuit that grasps the state of the cell as a potential change between the electrodes sandwiching the cell, adjusts the applied voltage according to the state of the cell, performs processing such as opening and closing, and drives the transport means 902, 903 to drive the micro chamber plate. And a circuit for controlling the operation of transferring the cells to the other.

FIG. 3 shows electrodes 101 to 104, 201 to 204, 301 to 304, 401 provided independently in the compartments of the microchamber plate.
~ 404, and wiring from these electrodes 10 ~ 14,20 ~ 24,30 ~ 3
This is an example in which 4,40 to 44 are patterned into a planar shape.

Next, the process of electrical cell fusion will be described with reference to FIG. 1 using cells in the compartment 103 as an example. The cells 800 and 801 that have been transferred and positioned by the transporting means come into contact with each other immediately above the suction holes by the suction pressure from the suction holes 603. The pair of cells are in contact with each other by suction holes 603 formed in the longitudinal direction between the electrodes to form an eight shape, and are positioned so that the longitudinal direction coincides with the electric field direction. When a pulsed voltage is applied between the electrodes while maintaining the contact, a pair of cells sandwiched between the electrodes starts to fuse from the contacted cell membrane portion. The cells that have been in contact with the progress of the fusion become spheres from daruma to ellipsoid. The resistance, the capacitance, and the amount of charge between a pair of electrodes change as the shape of the cell changes.

Assuming that cells of radii r ₁ and r ₂ fuse and change to the size of radius r ₃ , since the total volume of cells is constant,
The following equation holds.

The maximum length of _{^{r 1 3 + r 2 3 =}} r 3 3 contacted cell with _{2 · (r 1 + r 2} ), the maximum diameter of the cells after fusion _{2 · r 3 = 2 · (} r 1 3 + r 2 3) ^1/3 . For example, the longest distance when two cells of equal radius R are in contact is 4R. The radius of the sphere after fusion is (2) ^1/3 · R = 1.26 · R, and the longest distance is 2.52 · R
And 63% smaller than when two cells were in contact. Therefore, it can be detected as a potential change between the electrodes.

If a potential change between the electrodes starts after a pulsed voltage is applied for fusion, cell fusion has started at that time. When cell fusion progresses into one sphere, the potential change between the electrodes becomes smaller. Therefore, immediately after the pulse voltage for fusion is applied to a pair of cells, the potential between the electrodes is measured and the rate of change is obtained, whereby the start point of fusion can be grasped. Until the cell fusion starts, the pulsed voltage is continuously applied at an appropriate cycle, and when the fusion does not start, the applied pulsed voltage is gradually increased and the application condition is changed, whereby the cells can be fused. After a potential change appears and the start of fusion is detected, the application of the pulsed voltage is stopped to advance fusion. When the activity of the cells is weak, the cells may be damaged by application of a pulsed voltage. At this time, the mode of the potential change shows a mode that becomes constant after a steep change. Therefore, it is possible to distinguish from a mode of a gradual potential change that appears with the progress of fusion, and it is possible to know the time of cell breakage.

If one cell is found to be damaged in the compartment, a high voltage is applied to destroy the remaining cells in order to prevent the remaining cells from remaining unfused and growing during culture. . The presence or absence and destruction of the remaining cells can also be detected from the potential change. By the above method, it is possible to reliably leave only the desired fused cells. The distance between the electrodes is 20
When the cells of salad vegetables are fused with 0 μm and 0.5 mol of sorbitol solution, cell fusion starts only by applying a pulse voltage of about 25 V, 150 μs about once or several times, and a figure-shaped contact is made. From the state, the fusion progresses into a sphere in about 2 to 3 minutes after passing through a ball and an ellipsoid.

Since the wiring patterns from the electrodes in FIGS. 1 and 3 do not cross each other, the patterning is simple, but the number of compartments that can be formed on the champer plate is limited.
FIG. 4 shows an embodiment in which wiring patterns can be formed on a chamber plate without limitation on the number of compartments by making the wiring patterns three-dimensionally cross each other. Wiring in y direction 10,6
A set of electrodes 11 to 67 is patterned corresponding to the intersections of the 0 and the wirings 1 to 7 in the x direction. A connection method for applying a voltage to each electrode or measuring a potential between the electrodes can be applied to a known method for driving a two-dimensional liquid crystal device, a two-dimensional MOS image sensor, or the like.

FIG. 5 is a bird's-eye view of an enlarged cross section of a microchamber plate made of Si using the wiring method shown in FIG.
Suction hole for locating cells by suction (dimensions: 10 μm × 80 μm)
And a side wall plate 201 forming a compartment corresponding to the position of the suction hole. In order to show the electrodes formed on the overlapping portion, the state where the cell holding groove plate and the side wall plate are separated is shown. Au 300nm thick wiring 6, 7 and
Reference numerals 10 to 30 correspond to portions having the same numbers as the wiring patterns in FIG. Intersecting portions of the intersecting wires are electrically insulated by an insulating layer made of SiO ₂ . Next, description will be made using the compartment 37. The electrode 3 from the wiring 6 extends in the longitudinal direction of the suction hole 300 in the compartment 37.
An electrode 302 is formed using 01 and a part of the wiring 30 as a direct electrode. In the lateral direction of the suction hole 300, protrusions 303 and 304 of an insulator made of SiO ₂ are formed. Insulating protrusion 303,3
Reference numeral 04 is disposed with the suction hole interposed therebetween, and has a function of converging lines of electric force generated when a voltage is applied between the electrodes 301 and 302 at the center of the suction hole. Therefore, efficient voltage application and measurement can be performed on a pair of cells positioned in contact with the suction hole.

Next, an embodiment in which the state or behavior of a cell existing between a pair of electrodes is electrically detected will be described with reference to FIGS. FIG. 6 is an enlarged view of an electrode portion according to the embodiment of the present invention.
2 shows a state in which heterologous cells 603 and 604 are in contact with each other and positioned. These electrodes and cells are isotonic
Immersed in 605. Literature (Cell Engineering Vol.3, No.6, 1988, p.4
0.5 mol as shown in 97-505 p.505, p.502)
The specific resistance of the isotonic solution of sorbitol is about 1 kΩ · mm, and the specific resistance of the cell membrane is about 100 MΩ · mm. Usually, the thickness of the cell membrane is about 10 nm, so that the resistance in the thickness direction is about 1 kΩ. In this embodiment, the distance between the electrodes 601 and 602 is 200 μm, and the resistance is about 0.2 kΩ. Therefore, the width of the opposing electrode is made smaller than the diameter of the cell or the insulator (303, 304 in FIG. 5).
) To provide a structure in which the electric field concentrates on the cells, it is possible to measure the resistance existing in the shape of the cells existing between the electrodes. Power supply 60 between electrodes 601 and 602
From step 6, a constant voltage is applied in a pulse form, and the resistance existing in the thickness of the cell membrane is obtained from the detection processing circuit 607 and output to a control system (corresponding to FIG. 905) not shown. The reason why the low voltage is applied in a pulsed manner is to prevent electrolysis of the isotonic liquid. 7 and 8 show an example of a temporal change in the resistance value existing in the shape of the cell obtained after the measurement process. FIG. 7 shows the resistance characteristics of the fusion process. FIG. 8 shows the process of cell destruction. In both figures, the horizontal axis represents time t, and the vertical axis represents resistance value r.

In FIG. 7, the resistance value r ₁ (approximately 0.2 kΩ) of the isotonic solution alone is at time t ₁ when no cell is present between the electrodes, and one cell at time t ₂ when only one cell is positioned. The resistance value r ₂ (about ₂ kΩ) depending on the thickness of the cell, and the resistance value r ₃ depending on the film thickness of two cells during the time t ₃ during which only two cells are positioned.
(About 4 kΩ), at the time t ₄ during which cell fusion has begun and cell fusion is proceeding, the membranes at the sites in contact with each other become thinner, and as the fusion progresses, the resistance value decreases to r ₄ , As a result, hybrid cells are formed. The time t ₅ shows the resistance value r ₄ when dependent as hybrid cells.

FIG. 8 is an example of the characteristics indicating the number of cells and the presence or absence of breakage. Until time t ₁ ~t ₃ is the same as Figure 7. Change in the resistance value as in the change between the one of the two was present cells to failure times t ₃ and t ₄ becomes steeper. Therefore, it is possible to cell fusion is changed gradual resistance (7 corresponds to FIG t ₄₎ as compared to identify and process in progress. time
resistance r ₆ of t ₆ (approximately equal to r ₂₎ is a pair of cells 1
This is the resistance value of the cell when only one cell is damaged and one cell remains. Further, when the remaining one cell is damaged, the time t ₇
A resistance value r ₇ (approximately equal to r ₁ ) as shown in FIG. It is clear that the resistance value of each substance depends on the shape of the electrode, the concentration of the isotonic solution, the concentration of the electrolyte, the type of the cell, etc., but once the conditions are determined, there is no large variation. Further, it is easy to consider electrical characteristics such as capacitance and dielectric constant as an application of the detection processing circuit.

Although the above-described embodiment deals with cells of 20 to 100 μm, it can be easily applied to cells and particles other than this size by appropriately designing the compartments and suction holes of the micro-chamber plate. Clearly what you can do. Processing of the suction holes on the order of μm can be realized by applying a patterning technique or an etching technique used in a semiconductor process. Further, as the material and the like of the micro-chamber plate, glass, resin, ceramics, and the like other than Si can be used according to the processing size and accuracy.

Further, in addition to the cell fusion device shown in the present invention, a predetermined number of cells may be put into a compartment to perform a chemical treatment while grasping the state thereof, or may be used as a cell treatment device for performing a culture treatment and the like. is there.

In addition, a microchamber plate in which cells are positioned is placed in a liquid in which a predetermined gene is suspended, and a laser beam or X-ray squeezed finely is applied to a cell wall to open micropores, and the gene is inserted into cells. It can be used as an introduction device.

Further, if blood is used as particles, the number of blood cells positioned in the compartment can be counted, and the number of blood cells per unit flow rate can be measured to determine the blood cell concentration. Also, by applying a voltage to the blood cells positioned in the compartment,
It can be used as a blood cell processing apparatus that can determine the deformability of blood cells from the degree of shape change due to the voltage and can be used for diagnosis.

In addition, for a micro-chamber plate in which particles are positioned and handled in a liquid, it is not preferable for air bubbles to adhere to the compartments or the suction holes in operating the particles. It is possible to detect the presence or absence of air bubbles in the compartment by removing the air bubbles in the compartment by measuring the potential between the electrodes in the absence of air bubbles in each compartment in advance, and to consider data processing. Therefore, a highly reliable microchamber can be provided.

〔The invention's effect〕

According to the present invention, since the states of cells and particles can be grasped individually and independently, conventionally, cell fusion has been difficult to automate due to the large individual difference of cells and having to rely on manual labor. Can be automatically performed in large quantities, which can contribute to the great development of cell engineering. In addition, since the arrangement of the compartments on the microchamber plate is known, it is easy to address and control the particles and cells in the compartment, and the behavior of the particles and cells after performing various treatments can be improved. By individually loading the data into a computer, it can contribute to cell and genetic engineering follow-up studies that have been considered impossible.

[Brief description of the drawings]

1 is a bird's-eye view of an enlarged cross section of the first embodiment of the present invention, FIG. 2 is an external view of the device of the first embodiment, FIG. 3 is a wiring pattern diagram of the first embodiment, FIG. Is a wiring pattern diagram of the second embodiment, FIG. 5 is a bird's-eye view of an enlarged cross section of the second embodiment,
FIG. 6 is an enlarged view of the electrode part of the embodiment of the present invention, FIG. 7 is a view showing a resistance characteristic in a fusion process, and FIG. 8 is a view showing a damage process of a cell. Description of reference numerals 10 to 21: wiring, 101 to 203: compartment, 603: suction hole, 8
00,801… Cell, 802,803… Electrode, 901… Micro chamber plate

──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventor Hiroyuki Obita 1-280 Higashi Koigakubo, Kokubunji-shi, Tokyo Inside the Central Research Laboratory, Hitachi, Ltd. (56) References JP-A-63-269977 (JP, A) (58) ) Surveyed field (Int.Cl. ⁶ , DB name) C12M 1/00

Claims (9)

(57) [Claims] 1. A substrate, a plurality of independent compartments formed on the surface of the substrate, holes penetrating from the bottom of the compartment to the bottom of the substrate, provided in the compartments and independently controllable for each compartment. A microchamber plate for cell treatment, comprising a pair of electrodes. 2. The micro-chamber plate according to claim 1, wherein said pair of wirings have a matrix configuration. 3. A method for positioning predetermined cells in each of a plurality of compartments of a micro-chamber plate in a liquid, by using a pair of electrodes provided in the compartments and electrical information obtained by a voltage applied thereto. A method for detecting cells, comprising detecting the presence and state of cells in the compartment for each compartment. 4. A method of positioning a predetermined cell in each of a plurality of compartments of a micro-chamber plate in a liquid, by using a pair of electrodes provided in the compartment and electrical information obtained by a voltage applied thereto. Detecting the presence or absence and state of cells in the compartment for each compartment, processing the cells present in the compartment, and treating the cells with electrical information obtained by a voltage applied to the pair of electrodes. Detecting the state of each cell in each compartment where cells are present. 5. A means for positioning a predetermined cell in each of a plurality of compartments of a microchamber plate in a liquid;
Means for detecting the presence or absence and state of cells in the compartment for each compartment by electrical information obtained by a pair of electrodes provided in the compartment and voltage applied thereto, A cell processing apparatus comprising: a processing unit; and a unit configured to detect a state of a cell after the processing in each compartment where the cell is present, based on electrical information obtained by a voltage applied to the pair of electrodes. 6. The cell processing apparatus according to claim 5, wherein the cells in the compartment are a combination of two different cells, and the cell treatment is a fusion treatment of two different cells or a cell destruction treatment. 7. The cell processing apparatus according to claim 5, wherein the processing of the cells is introduction of a gene that floats in a liquid. 8. The cell processing apparatus according to claim 6, further comprising means for irradiating a laser beam for making micropores in the outer skin of the cell for introducing the gene into the cell. 9. The cell processing apparatus according to claim 6, further comprising X-ray irradiating means for making micropores in the outer skin of the cell for introducing the gene into the cell.