WO2017188065A1 - Fluid flow control device and fluid flow control method - Google Patents

Abstract

The present invention provides a technique such that the flow of a fluid and a concentration gradient formed by the flow can be controlled in a desired direction. A chamber 11 is a closed space extending in a two-dimensional direction. The chamber 11 includes a specific region 111 in a part thereof. Both a first port and second port 121, 122 deliver the fluid into the chamber 11 in a direction crossing the extension direction of the chamber 11. Thus, a combined flow having a velocity component in at least one direction is generated at or near the specific region 111. Discharge ports 13 allow the fluid to be discharged from an edge part in the extension direction of the chamber 11 or the vicinity thereof.

Description

Fluid flow control device and fluid flow control method

The present invention relates to a technique for controlling the flow of fluid.

The development of so-called concentration gradient formation techniques with the development of microfluidic devices has advanced research on how cells detect concentration gradients. However, in the conventional microfluidic device, it is difficult to apply a drug stimulus and a concentration gradient in an arbitrary direction to an arbitrary part of a cell, and to smoothly switch the direction in time. The problem will be specifically described below.

1. As a method of administering drug stimulation to cells in a microchannel, a method using a T-shaped channel (see Patent Document 1 and Non-Patent Document 1 below), a stair-shaped mixer, or a Christmas tree-type branch flow There is a technique (see Patent Document 2 and Non-Patent Document 2 below) that forms a concentration gradient by distributing a laminar flow by a path. In these techniques, the direction of laminar flow, and hence the direction of the concentration gradient of the stimulant, is uniquely determined. The same problem applies to a system in which a stimulating substance administration port is provided on the ceiling of the chamber (see Patent Document 3 below).

2. Regarding the temporal switching of the gradient, in the three-branch type flow path, change the gradient direction by switching the stimulus inlet (see Non-Patent Document 3 below), and move the gradient position by changing the flow velocity. The technique to perform (refer the following nonpatent literature 4) is proposed. In addition, there is a technique (see Non-Patent Document 5 below) in which a Christmas tree-type branch channel is made into a two-layer type and the layer is switched between these layers. In these methods, the control of the gradient direction is limited to 180-degree reversal, and a transient disturbance of the stimulation concentration is unavoidably caused at the time of switching.

3. A method in which the stimulation angle is changed by forming a laminar flow by a plurality of inflow and outflow ports at the end of a radial flow channel formed by superimposing three linear flow channels (non-patent document 6 below) See also). However, this technique does not allow continuously variable adjustment of the stimulation angle due to the position of the inflow / outflow and its discreteness. The same problem also applies to a passive system that does not use laminar flow (a system in which stimulating substances are added from a plurality of exudation ports provided at the peripheral edge of a circular chamber, see Patent Document 4 below).

4. In a device of a system that is attached to a manipulator and is movable, the inflow and outflow ports are arranged in a dipole shape (see Non-Patent Document 7 below), and a laminar flow can be applied immediately above the cells. By arranging this device in an arbitrary direction, it is possible to change the angle at which the stimulus is applied, but the laminar flow direction is temporarily changed due to the instability of the velocity field caused by the arrangement and movement of the device. Will be disturbed.

In many cases, stimulation of physiologically active molecules such as hormones, growth factors, and attractants is important in terms of which side of the cell the stimulus is applied from. For example, in the case of nerves, the direction in which the axon extends depends on which side of the axon is attracted from which side. For cells that move like leukocytes, the direction of movement is determined by the difference between the front and rear ends of the cell and the angle at which the stimulus is applied. Movement toward or away from the stimulant concentration gradient is called chemotaxis movement. With the development and application of so-called gradient forming devices using microfluidic devices, understanding of how cell morphology and movement are controlled by concentration gradients of stimulating substances has advanced. However, on the other hand, how to change the direction of the cells is a problem that is almost untouched.

As described above, there is a demand for precisely observing how the direction of movement of a cell is changed by stimulation or whether the direction of extension of a cell is changed. Conventionally, assays for examining changes in the direction of cell motility against changes in the concentration gradient of chemotactic attractants have been performed by filling a micropipette with a stimulating substance and changing its position with a manipulator. (See Non-Patent Documents 8 and 9 below). In this method, the concentration gradient of the stimulating substance can be given in an arbitrary direction. However, in order to accurately switch the angle of the concentration gradient, an operator's skilled manual operation is required. In addition, in this method, since removal of the gradient is left to passive diffusion, quick switching is difficult in principle. In addition, since the flow of the solution due to the movement of the micropipette in the solution is inevitably generated, it is difficult to form a stable concentration gradient at a cell size (micrometer scale).

US Pat. No. 7,112,444 U.S. Pat. No. 7,314,070 U.S. Pat. No. 7,470,403 U.S. Pat. No. 8,216,526

Beebe, D. J., Mensing, G. A. & Walker, G. M. 2002 Physics and applications of microfluidics in biology. Annual review of biomedical engineering 4, 261-286. (Doi: 10.1146 / annure601.12 .125916) Jeon, N. L., Dertinger, S. K. W., Chiu, D. T., Choi, I. S., Stroock, A. D. & Whitesides, G. M. 2000 Generation of Solution and Surface Gradients Using Microfluidic Systems. Langmuir 16, 8311-8316. (Doi: 10.1021 / la000600b) Meier, B., Zielinski, A., Weber, C., Arcizet, D., Youssef, S., Franosch, T., Radler, J. O. & Heinrich, D. 2011 Chemotactic cell trapping in controlled alternating gradient Proc Natl Acad Sci USA 108, 11417-11422. (Doi: 10.1073 / pnas.1014853108) Nakajima, A., Ishihara, S., Imoto, D. & Sawai, S.wai2014 Rectified directional sensing in long-range cell migration. Nat Commun 5, 5367. (doi: 10.1038 / ncomms6367) Irimia, D., Liu, S.-Y., Tharp, W. G., Samadani, A., Toner, M. & Poznansky, M. C. 2006 Microfluidic system for measuring neutrophil migratory responses to fast gradesients . Lab Chip 6, 191-198. (Doi: 10.1039 / b511877h) Moorjani, S., Nielson, R., Chang, X. A. & Shear, J. B. 2010 Dynamic remodeling of subcellular chemical gradients using a multi-directional flow device. Lab Chip 10, 2139-2146. (Doi: 10.1039 / c004627b) Qasaimeh, M. A., Gervais, T. & Juncker, D. 2010 Microfluidic quadrupole and floating concentration gradient. Nat Commun 2, 464-464. (Doi: 10.1038 / ncomms1471) Swanson, J. A. & Taylor, D. L. 1982 Local and spatially coordinated movements in Dictyostelium discoideum amoebae during chemotaxis. Cell 28, 225-232. Andrew, N. & Insall, R. H. 2007 Chemotaxis in shallow gradients is mediated independently of PtdIns 3-kinase by biased choices between random protrusions. Nat Cell Biol 9, 193-1200. (Nc10.10)

Therefore, there is a need for a new method that can add a concentration gradient of a stimulating substance in an arbitrary direction and can switch the angle smoothly or discontinuously.

The present invention has been made based on the above situation. A main object of the present invention is to provide a technique capable of controlling a fluid flow and a concentration gradient formed thereby in a desired direction.

The means for solving the above-described problems can be described as the following items.

(Item 1)
An apparatus for controlling the flow of fluid,
It has a device body,
The device body includes a chamber, an inflow port, and an exhaust port,
The chamber is a closed space extended in a two-dimensional direction,
And the chamber includes a specific region in a part thereof,
The inflow port includes at least a first port and a second port;
The first port and the second port both supply fluid into the chamber in a direction that intersects the extension direction of the chamber, so that the first port and the second port are directed to at least one direction in or near the specific region. It is configured to form a composite flow with a velocity component,
The discharge port is configured to discharge the fluid from an end portion in the extension direction of the chamber or the vicinity thereof.

(Item 2)
The control device according to item 1, wherein the first port and the second port are arranged at or near a position where a phase difference of about 120 degrees is centered on the specific region.

(Item 3)
The inflow port further includes a third port;
The third port is configured to supply a fluid into the chamber in a direction crossing the extension direction of the chamber,
The control device according to item 2, wherein the third port is arranged at or near the position where the phase difference of about 120 degrees with each of the first and second ports is centered on the specific region.

(Item 4)
The inflow port further comprises a controlled flow port;
The control device according to any one of items 1 to 3, wherein the controlled flow port is configured to supply a controlled flow to the specific region or the vicinity thereof.

(Item 5)
Furthermore, it has a control part,
The control unit is configured to control the flow rate or flow rate of the fluid flowing into the chamber from the inflow port,
Further, the control unit is configured to control the direction of the combined flow by controlling an inflow amount or a flow velocity of the fluid from the inflow port to the chamber. The control device described in 1.

(Item 6)
The control device according to item 5, wherein the control unit is configured to rotate the direction of the combined flow so that an angular velocity is constant.

(Item 7)
The control device according to item 5 or 6, wherein the control unit is configured to control an inflow amount or a flow rate of the fluid so that a concentration gradient profile in the combined flow is constant.

(Item 8)
In addition, it has a stimulus input unit,
As the fluid flowing into the chamber from the inflow port, a caged material is used,
Item 8. The control device according to any one of Items 1 to 7, wherein the stimulus input unit is configured to apply a stimulus for uncaging to the caged material that flows into the chamber and constitutes the combined flow. .

(Item 9)
The control device according to any one of items 1 to 8, wherein the specific region is a central portion of the chamber in the two-dimensional direction or a position in the vicinity thereof.

(Item 10)
The inflow port further includes a fourth port;
The chamber further includes a second specific region in a part thereof,
The second area is a position different from the specific area,
The fourth port is configured to supply a fluid into the chamber in a direction crossing the extension direction of the chamber, thereby having a velocity component in at least one direction at or near the second specific region. 10. The control device according to any one of items 1 to 9, wherein the control device is configured to form a flow.

(Item 11)
The control device according to any one of items 1 to 10, wherein the discharge port is configured to discharge the fluid at a substantially uniform speed at an end portion in the extension direction of the chamber.

(Item 12)
Furthermore, it has a fluid supply part and a fluid discharge part,
The fluid supply unit is configured to supply the fluid to the inflow port,
The control device according to any one of items 1 to 11, wherein the fluid discharge section is configured to collect the fluid from the discharge port.

(Item 13)
A method for controlling a flow of fluid using the control device according to any one of items 1 to 12,
By supplying a fluid from the first port and the second port into the chamber in a direction crossing the extension direction of the chamber, a velocity component in at least one direction is generated in or near the specific region. Forming a composite flow having,
Changing the direction or flow rate of the combined flow by changing the flow velocity or flow rate of the fluid in one or both of the first port and the second port.

According to the present invention, the flow of fluid and the concentration gradient formed thereby can be controlled in a desired direction.

It is a block diagram for showing a schematic structure of a fluid flow control device in a first embodiment of the present invention. FIG. 2 is a schematic explanatory diagram of a device body used in the fluid flow control device of FIG. 1 in a plan view. FIG. 3 is a schematic cross-sectional view taken along line AA in FIG. 2. It is explanatory drawing for demonstrating the arrangement state of the inflow port and discharge port which are provided in the device main body of FIG. In this figure, the inflow port portion is shown on the upper side and the discharge port portion is shown on the lower side, and the positions of the first to third ports are indicated by broken lines in the lower chamber. It is described. It is explanatory drawing which displayed the explanatory drawing of FIG. 4 in three dimensions by the perspective. It is explanatory drawing for demonstrating the direction control of the stimulation flow using the apparatus of 1st Embodiment. FIG. 6A schematically shows the arrangement of channels in the chamber and the observation region (region surrounded by a rectangle). FIG. 6B shows the direction of the stimulation flow when liquid is fed from the channel 2. FIG. 6C shows a case where an equal amount is sent from channels 2 and 3. FIG. 6D shows a case where liquid is fed from the channel 3. It is explanatory drawing for showing the principle of the direction control of a stimulus flow. The direction of the stimulus flow from the channel 4 is controlled by controlling the liquid feeding from the i-th channel (here, channel 2) and the j-th channel (here, channel 3). The direction of the stimulation flow from channel 4 is determined by the superposition (v) of the flow (regulated flow) velocity (ie, v ₂ and v ₃ ) created by the liquid delivery from channel 2 and channel 3 at the position of channel 4. Determined. It is explanatory drawing for demonstrating the mode of the control flow in a chamber. 8A and 8B are photographs in which a buffer solution mixed with fluorescent beads having a diameter of 1 μm (Fluoresbrite® YG Carboxylate Microspheres 1.00 μm (15702), Polyscience) is fed, and the state of the regulated flow is visualized by the fluorescence of the beads. Show. This photograph was created by superimposing the maximum brightness values of 6 seconds continuous images taken with a confocal microscope. Small white circles in the picture indicate the positions of channels 1, 2, and 3. The channel 4 at the ceiling of the chamber center (white point) is closed in this example. FIG. 8A shows a case where the ratio p = 0.2 of the liquid feeding amounts from the channels 2 and 3 in the method of controlling the regulated liquid feeding amount. FIG. 8B shows the state of the regulated flow near the center of the chamber when the ratio p of the amount of liquid fed from the channels 2 and 3 is p = 0, 0.2, 0.5. 8C and 8D are graphs showing the direction of the regulated flow laminar flow and the evaluation of the flow velocity by the particle image velocimetry (PIV). The p dependence is shown by using measured values in the case of the controlled liquid supply amount constant control (white circle) and in the case of the controlled flow rate constant control (x). The average of the direction and flow rate of the regulated flow in the range of 70 μm × 70 μm at the center of the chamber was measured over 2 seconds at 30 millisecond intervals, and the time average ± standard deviation was calculated. FIG. 8C shows a theoretical curve (formula (1)) regarding the p dependence of the angle θ of the regulated flow, and FIG. 8D shows a theoretical curve (formula (2)) regarding the p dependence of the flow velocity. The average of the flow rates at p = 0 and 1 was taken as v _max of the theoretical curve (formula (2)). The broken line in FIG. 8D shows a case where the flow rate matches v _max regardless of the liquid feeding amount ratio p. A micro flow controller MFCS (Fluigent) was used for liquid feeding. The liquid supply amount Q _c per unit time when p = 0, 1 was 10 μL / min. It is a graph which shows the measured value of a stimulus flow. FIG. 9A shows a stimulation flow when the ratio p of liquid feed from channels 2 and 3 is p = 0, 0.3, 0.7, and 1 under controlled control of the feed amount (using a fluorescein solution for fluorescence measurement). ). FIG. 9B is a graph showing the direction dependency of the stimulation flow with respect to the liquid feeding amount ratio p. In the figure, circles indicate measured values, and the curves in the figure indicate theoretical curves (formula (1)) relating to the p dependence of θ. 9C and 9D are graphs showing concentration profiles formed in the direction perpendicular to the stimulation flow. FIG. 9C shows the case of the controlled liquid feed constant control, and FIG. 9D shows the case of the controlled flow rate constant control. In these figures, the horizontal axis indicates the position on the circumference having a radius of 400 μm from the center of the channel 4, and the vertical axis indicates the fluorescence value of fluorescein (stimulated flow). The graphs on the left side of these figures show the spatial profile of the concentration on the circumference when the liquid supply ratio p is changed from 0 to 1, and the graph on the right side shows the maximum concentration values. (That is, a graph in which concentration gradient curves are arranged at the same position for comparison of local maximum values). A micro flow control device (MFCS, Fluigent) was used for liquid feeding from channels 1, 2 and 3, and a syringe pump (Pump 11 Elite, Harvard Apparatus) was used for liquid feeding from channel 4. It is explanatory drawing which shows the example of a rotational wave stimulation. FIG. 10A is a confocal microscope image of a rotating wave when the amount of liquid fed in channels 1, 2, and 3 (CH1, 2, and 3) is changed with time. In this figure, the direction of the stimulus flow is rotated 360 degrees in 3 minutes. Here, a fluorescent substance is mixed in the stimulation solution, and the state of the stimulation flow is visualized by the fluorescence intensity. FIG. 10B is a graph showing a concentration gradient profile of the stimulation flow. This graph displays the fluorescence intensity (average fluorescence intensity in a width of 50 μm) on the circumference having a radius of 300 μm from the center of the liquid delivery port for stimulation. This graph also shows profiles at times 3:12, 3:42, 4:12, 4:42, 5:12, 5:42, and 6:12 (minutes: seconds), respectively. FIGS. 10C and 10D show the angle change state of the stimulus flow and its evaluation. These figures show the temporal change in the direction of stimulation flow (upper diagram) and the relationship between the angle and angular velocity of the stimulation flow (lower graph) on the circumference of a radius of 300 μm from the center of channel 4. . FIG. 10C shows a case where the liquid feeding ratio p is changed in proportion to time, and FIG. 10D shows a case where p is changed with time in accordance with the derived relational expression. The upper diagrams of FIGS. 10C and 10D show the fluorescence intensity in shades. FIG. 10E is a histogram for showing variations in angular velocity when the liquid feeding amount is changed in proportion to time and when time is changed according to the derived relational expression. It is a confocal microscope image of a stimulus flow, showing that the width of the stimulus flow is changed according to the amount of the stimulus solution delivered per unit time. The stimulation flow is visualized by the fluorescence of the fluorescent substance (fluorescein) mixed in the stimulation solution. Here, a bright part shows a fluorescent substance. Liquid was fed from channel 1 at 30 μL / min, and a regulated flow was formed from the upper right side of the visual field to the lower left side. The amount of the stimulation solution delivered from the channel 4 was 1.0 μL / min, 0.5 μL / min, 0.2 μL / min, and 0.1 μL / min from the left diagram. The scale bar indicates 500 μm. It is explanatory drawing for demonstrating formation of the stimulation flow by optical uncaging of a caged compound. 12A is a schematic diagram of stimulus flow formation by optical uncaging, FIG. 12B is a measurement example of a confocal microscope image, and FIG. 12C is a measurement example of a concentration gradient profile. A regulated flow of a caged compound of a fluorescent substance (CMNB caged fluorescein, F7103, Invitrogen) from the lower left side of the field of view to the upper right side was formed by the basic module, and the circular region (FIG. 12B) near the center of the field of view was irradiated with ultraviolet light. The fluorescence intensity of fluorescein released by uncaging was visualized. The concentration gradient of the liberated fluorescein was evaluated by the spatial distribution of the average fluorescence luminance in the region having a width of 10 μm on the circumference having a radius of 100 μm from the center of the ultraviolet light irradiation region (FIG. 12C). For light irradiation, a light stimulus forming apparatus (Mosaic, Photonic Instruments Inc.) was used. It is explanatory drawing for demonstrating the spatial profile of the stimulant substance density | concentration formed by the stimulus injection by optical uncaging, and its control. Here, a caged fluorescein solution was used as a stimulating solution, and fluorescence of fluorescein released by light irradiation was measured with a confocal microscope. FIG. 13A is a snapshot of the stimulation flow when the ratio of the amount of liquid fed from channels 2 and 3 is p = 0, 0.3, 0.7, 1 in the method of constant adjustment flow rate control. FIG. 13B is a graph showing the p dependence of the direction of the stimulation flow, in which the circles indicate actual measurement values, and the curve indicates the theoretical curve (formula (1)) regarding the p dependence of θ. FIG. 13C is a graph showing a spatial profile of the stimulating substance concentration formed in the direction perpendicular to the direction of the stimulating flow in the case of the controlled liquid supply constant control. The graph on the left side of the figure shows the spatial profile of the concentration on the circumference when the liquid supply ratio p is changed from 0 to 1, and the graph on the right side assumes the same position at the maximum concentration value. It is a graph of the case. FIG. 13D is a graph showing a spatial profile of stimulating substance concentration in the case of controlled flow rate constant control. Here, light irradiation was performed on a circular region having a radius of 34 μm located at the center of the chamber, and fluorescence of fluorescein was evaluated on a circumference having a radius of 100 μm from the center of the chamber. FIG. 13E is an explanatory diagram for explaining the dependency of the stimulus flow direction on the liquid feeding amount ratio p when the optical uncaging position is shifted from the chamber center. Here, the ratio p of the amount of liquid fed from channels 1 and 2 was controlled. The left diagram in FIG. 13E is an explanatory diagram for explaining the position where uncaging is performed, and the right diagram shows the relationship between the direction of the formed stimulation flow and the liquid delivery ratio p for each uncaging position. It is a graph shown in. It is a figure for demonstrating control of the stimulation flow by optical uncaging of a caged compound. FIG. 14A is an example (confocal microscope image) of controlling the width of the stimulus flow. The circles in these figures indicate the light irradiation areas, and the diameters thereof are 134 μm, 67 μm, 34 μm, and 17 μm. Using caged fluorescein as a simulated stimulant caged compound, a regulated flow from the upper right side of the visual field to the lower left side was formed in the basic module, and the circular region near the center of the visual field was irradiated with ultraviolet light. Here, the release of the fluorescent substance fluorescein due to uncaging is visualized as an increase in fluorescence brightness. It turns out that the width | variety of the stimulation flow formed changes with the magnitude | sizes of the area | region which irradiates light. FIG. 14B shows an example in which a relatively thin stimulation flow (half width of 83 μm) formed by optical uncaging is rotated. Here, the state of fluorescence brightness (confocal microscope image) of caged fluorescein uncaged by irradiation of ultraviolet light onto a circular region having a diameter of 67 μm is shown. The stimulus is visualized by measuring the fluorescence of the fluorescent substance released by uncaging. In addition, here, the stimulation flow is controlled to make one rotation in 3 minutes. The scale bar in the figure indicates 100 μm. It is a figure for demonstrating that a stimulus flow and its gradient stimulus can be shown to an observation field from a different direction by changing the optical uncaging position of a caged compound. FIG. 15A is a schematic diagram of stimulus flow control. FIG. 15B is a confocal microscope image of a stimulus flow (fluorescein released by uncaging) applied to the observation region from different directions. In this figure, a circle indicates an optical uncaging region. The position of the observation region in FIG. 15B is at the center of the triangle surrounded by the channels 1, 2, and 3 (square region in FIG. 15A). FIG. 15C is an enlarged view of the rectangular area in the image of FIG. 15B, and shows an enlarged image of the stimulus flow. The order of the image arrangement in FIG. 15C corresponds to FIG. 15B. FIG. 15D is a graph showing a gradient profile of the stimulation flow. FIG. 15D shows a luminance value profile (upper diagram in FIG. 15D) at a gradient in which the concentration increases toward the lower right (rectangular region in the upper diagram in FIG. 15C), and a gradient (FIG. 15C in which the concentration increases toward the upper right). It is a luminance value profile (lower figure of FIG. 15D) in the rectangular area in the lower figure. It is a schematic diagram of a multiplex channel system. FIG. 16A shows several arrangement examples of a stimulation liquid supply port (Stimulus inlet, S) and a buffer liquid supply port (Buffer inlet, B) in the multiplex flow channel system. The positional relationship of the liquid supply port (inflow port) in the first embodiment can be given as a module (1S / 3B, left figure in FIG. 16A), and a plurality of them can be arranged to give a stimulus to any position ( From left to right, 2S / 4B, 3S / 9B and 3S / 6B, 4S / 4B and 6S / 6B). FIG. 16B is a schematic diagram of a cell direction change experiment using a multiplex flow path system (3S / 6B). It is explanatory drawing which shows the example of mounting of the flow path of a duplex (2S / 4B type). FIG. 17A is an explanatory diagram showing a schematic flow path. FIG. 17B shows that the stimulation flow from channel 5 and channel 6 (fluorescein solution for fluorescence observation) is directed by feeding from channels 1, 2, and 3, and the sample (observation area, (Displayed by a square) shows the state of alternately loading (upper figure). The lower figure is a confocal microscope image, and here, the stimulus flow passing through the observation region (square in the upper figure) is switched every two minutes, and this is repeated in a cycle of four minutes. The scale bar in the figure indicates 200 μm. FIG. 17C shows an example in which the angle between alternating gradient stimuli can be selected from 0 degrees to about 180 degrees depending on the observation position in the sample chamber (square in the left figure) (in the right figure). Panels ROI1, ROI2, and ROI3 correspond to the rectangular observation areas 1, 2, and 3 in the left figure). The upper stage and the lower stage in the diagram on the right correspond to the stimulation flows from channels 5 and 6, respectively. The scale bar in the figure indicates 100 μm. It is a figure for demonstrating the migration of the cellular slime mold Dictyostelium discoideum cell with respect to the rotational wave stimulation of the chemotactic attractant cyclic AMP (cAMP). The figure on the left shows a snapshot of the stimulation flow of cAMP solution (containing fluorescein for fluorescence visualization). The figure on the right shows the migration trajectory of representative cells over 50 minutes (different concentrations indicate different cells). The broken line in the figure indicates the opening position of the ceiling that is the stimulation liquid feeding port. By rotating the stimulation flow, it can be seen that the gradient of cAMP moves and the cells move toward it. It is a figure for demonstrating the direction change movement of the neutrophil-like cell line HL60. The trajectory of HL60 cell migration towards the bacterial peptide fMLP is shown by the solid line in the lower figure. As shown in the upper figure, the stimulus alternates between a gradient in which the concentration of fMLP increases from the upper left of the visual field to the lower right and a gradient in which the concentration of fMLP increases from the lower left of the visual field to the upper right. .

Hereinafter, a fluid flow control device according to a first embodiment of the present invention (hereinafter sometimes simply referred to as “control device” or “device”) will be described with reference to the accompanying drawings.

(Configuration of the first embodiment)
The control apparatus according to the present embodiment includes a device body 10. Further, this apparatus additionally includes a fluid supply unit (sometimes referred to as “liquid feeding unit”) 20, a fluid discharge unit (sometimes referred to as “drainage unit”) 30, and a control unit 40. (See FIG. 1).

(Device body)
The device body 10 includes a chamber 11, an inflow port 12, and an exhaust port 13 (see FIGS. 2 and 3). The device body 10 further includes an inflow channel 14 and an exhaust channel 15 (see FIG. 4).

(Chamber)
The chamber 11 is a closed space extended in a two-dimensional direction (see FIG. 3). More specifically, the chamber 11 is a flat and substantially cylindrical space formed inside the device body 10.

The chamber 11 includes a specific region 111 in a part thereof (see FIG. 2). In this embodiment, the specific area is an area to be observed. Further, in this embodiment, a substantially central portion of the chamber 11 is the specific region 111. The specific area 111 does not mean a partitioned area, but means that a part of the space in the chamber 11 is grasped as such, and an appropriate position can be recognized as the specific area 111 according to the purpose.

(Inflow port)
Inflow port 12 of this embodiment is provided with the 1st port 121, the 2nd port 122, and the 3rd port 123 (refer to Drawing 2 and Drawing 3).

Each of the first to third ports 121 to 123 supplies fluid into the chamber 11 in a direction (downward in FIG. 3) that intersects the extending direction of the chamber 11 (eg, the left-right direction in FIG. 3). Thus, a composition flow having a velocity component in at least one direction is formed in the specific region 111 or in the vicinity thereof. The method for forming the composite flow will be described later. Here, in the present specification, the fluid is not limited to a liquid, but may be a fluid having fluidity that can be an object of the present invention, for example, a fluid material such as a sol or gel. However, in the following description, liquid is basically assumed as the fluid.

The first port 121 and the second port 122 are arranged at or near the position where the specific region 111 is the center and the phase difference is about 120 degrees. Further, the third port 123 of the present embodiment is arranged at a position where the phase difference is about 120 degrees with respect to the first and second ports 121 and 122 or in the vicinity thereof with the specific region 111 as the center. That is, the first port 121 to the third port 123 are arranged at equal intervals on one virtual circumference.

The inflow port 12 of this embodiment further includes a controlled flow port 120. The controlled flow port 120 is configured to supply the controlled flow to the specific region 111 or the vicinity thereof (see FIGS. 2 and 3). More specifically, the controlled flow port 120 of the present embodiment can supply the controlled flow into the chamber 11 downward in FIG. 3, that is, in a direction parallel to the first to third ports 121 to 123. It is like that. Here, in this embodiment, a controlled flow that is a flow of a so-called stimulating substance is used as the controlled flow. The stimulating substance is a chemotactic attractant for the purpose of examining the chemotaxis of cells, for example.

(Discharge port)
The discharge port 13 is configured to discharge the fluid from an end portion in the extending direction of the chamber 11 or the vicinity thereof. Further, the discharge port 13 is configured to discharge the fluid at a substantially uniform speed at the end in the extending direction of the chamber 11.

More specifically, the discharge port 13 of the present embodiment is discretely formed on the side surface (left and right surfaces in FIG. 3) of the chamber 11 over substantially the entire circumference. Therefore, there are actually a plurality of discharge ports 13, but in this specification, the same reference numerals 13 are assigned to the respective discharge ports. The intervals between the discharge ports 13 are substantially uniform. Further, it is preferable that the distance between the specific region 111 and the discharge port 13 is as long as possible.

(Inflow channel)
The inflow channel 14 (see FIGS. 4 and 5) is a path for individually supplying the fluid supplied from the fluid supply unit 20 to the ports 120 to 123 constituting the inflow port 12. In this specification, the same reference numeral 14 is assigned to each inflow channel. Each inflow channel 14 is formed by a groove formed in the device body 10 in this embodiment. However, the specific configuration of the inflow channel 14 is not limited to this, and for example, a pipe capable of conveying a microfluid may be used.

(Discharge channel)
The discharge channel 15 (see FIG. 4) is a path for collecting the fluid supplied into the chamber 11 from the discharge port 13 and sending it to the fluid discharge unit 30. In this embodiment, the discharge channel 15 is formed by a groove formed in the device body 10, similarly to the inflow channel 14. However, as the discharge channel 15, for example, a pipe capable of feeding a microfluid may be used. In this embodiment, the shape of the discharge channel 15 is set so that the path lengths from the respective discharge ports 13 to the fluid discharge portions 30 are substantially equal (that is, the suction pressures at the respective discharge ports 13 are substantially equal). It is set.

(Fluid supply part)
The fluid supply unit 20 is configured to supply fluid to the inflow port 12.

Specifically, as the fluid supply unit 20, various devices that can be used for supplying a microfluid, such as a syringe pump or a liquid-feeding pressure control device, can be used. The fluid supply unit 20 is connected to an end of the inflow channel 14 (end opposite to the inflow port 12), and can supply fluid to the inflow channel 14. The fluid supply unit 20 of the present embodiment can set the flow rate or flow rate of the fluid to each inflow channel 14 for each port in accordance with control from the control unit 40.

(Fluid discharge part)
The fluid discharge unit 30 is configured to collect the fluid supplied into the chamber 11 from the discharge port 13 via the discharge channel 15. Specifically, various pumps for drainage can be used as the fluid discharge unit 30. Here, the fluid discharge unit 30 may collect the fluid isotropically from the chamber 11 so that the fluid already supplied to the chamber 11 does not disturb the flow of the fluid supplied to the chamber 11 thereafter. preferable. The fluid discharge unit 30 is not limited to a unit that actively discharges like a pump, but is a unit that collects a discharged fluid passively (that is, without performing active suction). Also good.

(Control part)
The control unit 40 is configured to control the flow rate or flow velocity of the fluid flowing into the chamber 11 from the inflow port 12 for each port. More specifically, the control unit of the present embodiment is configured to control the direction of the combined flow in the chamber by controlling the inflow amount or flow velocity of the fluid from the inflow port 12 to the chamber 11. . The operation in the control unit 40 will be described later.

(Operation of this system)
Hereinafter, the operation principle and control method of this embodiment will be described.

First, as an example, an observation target having adhesiveness such as a cell is adhered and fixed to a specific region (rectangular region in FIG. 6A) 111 near the center of the chamber 11. The fixing method of the observation target is not particularly limited, and may not be fixed as long as observation is possible. In the following description, the specific area may be referred to as an “observation area”.

Next, the control unit 40 inputs a controlled flow (hereinafter sometimes referred to as “stimulation” or “stimulated flow”) from the ceiling at the center of the chamber 11 (that is, the controlled flow port 120) by the fluid supply unit 20. To do.

At the same time or before and after this, the control unit 40 sends the liquid feeding amount from the first to third ports 121 to 123 surrounding the controlled flow port 120 to the liquid feeding amount from the fluid supply unit 20 to each inflow channel 14. Adjust by adjusting. This controls the direction and width of the laminar flow formed in the chamber, that is, the spatial direction profile of the stimulus flow and concentration. The method for this can be expressed as follows.
A method in which the beam-like stimulation flow and the concentration gradient formed thereby are directed in any direction in two dimensions by controlling the flow rate of the liquid feeding from each port.
A method of dynamically changing the amount of liquid delivered and changing the direction of the stimulation flow over time.
In this case, a method for adjusting the amount of liquid delivered to keep the rate of change in the stimulus flow direction constant.

(Basic principle)
In the following description, of the four inflow ports (hereinafter sometimes referred to as “liquid feeding ports”) 12 provided on the ceiling of the device body 10, the first to third ports 121 to 123 (hereinafter simply referred to as “channel 1,” 2 and 3 ”) constitutes the buffer feeding port, and the controlled flow port 120 (hereinafter sometimes simply referred to as“ channel 4 ”) constitutes the stimulation solution feeding port. And

Switch the liquid supply from channels 1, 2, and 3 while keeping the liquid supply volume from channel 4 constant. The liquid feeding from the channels 1, 2, and 3 forms a flow field toward a discharge port (hereinafter also referred to as “waste liquid port”) 13 (see FIG. 4) provided at the peripheral edge of the chamber 11.

Here, in particular, attention is paid to the vicinity of the center of the chamber 11 (the specific region 111 in FIG. 6A) where the cells to be observed are placed, and the flow direction in the vicinity of the channel 4 provided there is considered. When liquid is sent from only one of channels 1, 2, and 3, the direction of the stimulation flow is only by the flow (regulated flow) from the liquid feeding channel for adjusting laminar flow (channel 2 in the example of FIG. 6B). Determined. Next, when liquid is fed from both channels 2 and 3, the angle between the straight line that passes through channels 2 and 4 and the straight line that passes through channels 3 and 4 (120 degrees) depends on the ratio of the feed volume from both channels. Can be directed in any direction (FIG. 6C). In this case, the combined flow can be directed in an arbitrary direction depending on the ratio of the liquid feeding amounts of the channels 2 and 3. When liquid is fed only from channel 3, the flow is directed in a direction rotated 120 degrees from the direction formed from channel 2 alone (FIG. 3D). Such an operation is performed not only on the combination of the channels (2, 3) but also on the channels (3, 1) and (1, 2), so that the stimulation flow from the channel 4 is 120 degrees × 3 = It is expected to be directed in all directions of 360 degrees. In this specification, the combined flow includes a case where the flow is formed by liquid feeding from only one channel.

(Method of determining the direction and magnitude of the velocity of the regulated flow and stimulus flow)
The flow (regulated flow) from the i-th and j-th ports (in this example, any two of the first to third ports) for laminar flow adjustment is controlled flow port 120 (channel 4 in FIG. 7). It is assumed that a controlled flow having a velocity vector v is realized by combining in the vicinity of (Fig. 7). Let the flow velocity contribution from each channel at the position of channel 4 be represented by vectors v _i and v _j , keeping their sum constant (v _max ) while changing their absolute values from 0 to v _max. I will do it. Now the relative ratio of the jth flow velocity is p, i.e.

で与えられる。ここで、tan^-1は[0,180]度、流速比pは[0, 1]を定義域とする。φ＝１２０度（基本モジュール）の場合、調節流を目的の方向θ（図７）に向けるには、

となる。

Given in. Here, tan ^-1 is defined as [0,180] degrees, and the flow rate ratio p is defined as [0, 1]. In the case of φ = 120 degrees (basic module), to direct the regulated flow in the desired direction θ (FIG. 7),

It becomes.

It is expected that the direction of the stimulation flow from the channel 4 can be determined by forming the regulation flow in this way. As an actual operation, control the relative ratio of the pumped volume (the pumped volume Q _j from the j-th channel) / (sum of the pumped volumes Q _i + Q _j from the i-th and j-th channels). We will adjust the flow rate ratio _{(p = Q j / (Q} i + Q j)) by. However, here, Q _i and Q _j are the amounts of liquid fed per unit time from the i-th and j-th channels. There are mainly the following two methods for changing the flow rate ratio, that is, the liquid feeding ratio p.

1) Control p while keeping the sum of the amount of liquid delivered from the i-th and j-th channels, that is, the adjusted amount of liquid Q _sum (= Q ₂ + Q ₃ ) at a constant value Q _c ( Constant control ", _Qsum = _Qc ). In this case, the flow rate of the regulated flow at the center of the chamber is expressed by equation (2).

2) Control the adjusted liquid supply amount Q _sum together with the liquid supply amount ratio p so as to keep the flow rate of the adjusted flow at the center of the chamber 11 constant (“control of constant adjustment flow rate”). In order to realize this control, in order to cancel out the dependence on the liquid supply ratio p in the equation (2), the adjusted liquid supply Q _sum corresponds to p,

を保つように制御する。

Control to keep.

In any of the control methods, Q _sum = Q _c when the liquid feed ratio p is p = 0 and p = 1. The above discussion holds true for any combination of channel (2, 3) and channel (3, 1) and channel (1, 2).

The liquid feeding control described above can be performed relatively easily by using a commercially available syringe pump with a programmable liquid feeding speed or a pressure control type micro flow control device as the fluid supply unit 20. An example of an actual flow (synthetic flow) formed in the chamber 11 is shown in FIG. In this example, a buffer containing fluorescent beads is fed from the channels 2 and 3 of the device body 10 (basic module) using a pressure control type micro flow control device as the fluid supply unit 20, and the chamber 11 (sample The state of the regulated flow in the chamber) was visualized. Here, the liquid supply port (controlled flow port 120) of channel 4 is closed. When the relative ratio p ((amount of fluid delivered from channel 3 Q ₃ ) / (sum of fluids delivered from channel 2 and channel 3 Q ₂ + Q ₃ )) is p = 0, the above discussion As expected in FIG. 8, the regulated flow (that is, the combined flow in the specific region 111) at the position of the channel 4 (the center of the chamber 11 and indicated by a white dot in FIG. 8) is directed rightward in FIG. When p = 0.2 or 0.5, the direction changes to the upper right. Using the particle image velocimetry (PIV), the direction and flow velocity of the regulated flow were evaluated from the time change of the position of the fluorescent beads (Fluoresbrite (R) YG Carboxylate Microspheres 1.00μm (15702), Polyscience). (FIGS. 8C and D). According to the method of constant adjustment liquid supply amount control, when the liquid supply amount ratio p is changed from 0 to 1, the direction of the adjustment flow changes 120 degrees (FIG. 8C). The p dependence of the regulated flow direction θ is in good agreement with the measured value and the theoretical value (formula (1)) (FIG. 8C). Further, the flow velocity shows non-monotonic dependence on p, and takes a minimum value at p = 0.5 (FIG. 8D). A good agreement with the theoretical formula (formula (2)) is also observed in the p dependence of the flow velocity (FIG. 8D). Even in the method of controlling the regulated flow rate constant, the p dependence of the regulated flow direction is in good agreement with the theoretical formula (Equation (1)) as before (FIG. 8C). Further, as expected, the flow velocity at that time is kept almost constant regardless of the value of p (FIG. 8D). In the case of controlled liquid feed constant control, the average flow rate when p is changed from 0 to 1 is 240 μm / sec, and the average variation (standard deviation / average) is 25%. In the controlled flow rate constant control, the average value of the flow rate is 330 μm / sec, and the average variation is 2%. As the fluid supply unit 20 in the above-described example, a micro flow control device MFCS manufactured by Fluide was used. In this example, the liquid supply amount Q _c per unit time when p = 0, 1 was 10 μL / min.

Next, FIG. 9 shows a state in which the stimulating substance solution is fed from the channel 4 while feeding the buffer from the channels 2 and 3 of the basic module to control the direction of the regulation flow. As expected from the basic behavior confirmed in Fig. 8, when the relative ratio of the pumping volume is p = 0, the stimulation flow is directed to the right, p = 0.3 is the upper right, and p is 0.7. When p = 1, the direction is changed to the upper left direction (FIG. 9A). When the liquid feeding ratio p is changed from 0 to 1, the direction of the stimulation flow changes by 120 degrees (FIG. 9B). The measured p dependence of the direction θ of the stimulation flow is in good agreement with the theoretical formula (formula (1)) as in the basic operation in FIG. 8 (FIG. 9B). In the case of the controlled liquid supply constant control, the mountain-shaped concentration gradient profile formed by the stimulus flow and the diffusion of the stimulus substance therefrom greatly changes depending on p (FIG. 9C). When p is close to 0 or 1, the maximum value is low, and when p is 0.5 or close to it, the maximum value is high (FIG. 9C). On the other hand, in the case of the controlled flow rate constant control, the spatial profile of the stimulating substance concentration hardly depends on the angle (FIG. 9D). In the case of constant controlled liquid supply control, the maximum value (fluorescence luminance value) of the concentration profile when p is changed from 0 to 1 is 0.49 on average, and the average variation (standard deviation / average value) is 14 The average concentration gradient in the range of 20% to 80% of the peak fluorescence luminance was 0.021 μm ⁻¹ , and the variation in slope (standard deviation / average value) was 18%. In contrast, in the case of the controlled flow rate constant control, the average maximum concentration was 0.40, the variation was 9%, the average gradient slope was 0.0018 μm ⁻¹ , and the gradient variation was 11%. In this way, in the case of the controlled flow rate constant control, it is possible to suppress the variation in the spatial profile of the concentration gradient in both the concentration peak value and the concentration gradient. In the above example, a micro flow control device (MFCS, Fluigent) is used for liquid feeding from channels 1, 2 and 3, and a syringe pump (Pump 11 Elite, Harvard Apparatus) is used for liquid feeding from channel 4. (The same applies hereinafter). That is, the control unit 40 of the present embodiment can control the inflow amount or flow rate of the fluid so that the concentration gradient profile in the combined flow is constant.

(Method of changing the direction of stimulation flow at a constant speed)
By continuously changing the combination of the liquid feeding speeds from the channels 1, 2, and 3 with time, the direction of the stimulation flow is rotated, and traveling wave type stimulation can be realized. Further, at this time, it is shown that the traveling speed of the traveling wave can be kept constant with respect to the normal direction of the gradient by dynamically controlling the liquid feeding amount based on the following consideration.

Based on the discussion on the basic principle described above, the direction of fluid flow in the vicinity of channel 4 with respect to the minute change of p is

In accordance with the above-mentioned argument, the channel is maintained while maintaining the angular velocity by temporally changing the liquid feeding ratio p in the order of the combination of channel (1, 2), channel (2, 3), channel (3, 1). FIG. 10 shows that the direction of the stimulus flow from 4 can be rotated once. By controlling the feeding of the channels 1, 2, and 3, first, the stimulus directed to the lower right direction was rotated once counterclockwise (FIG. 10A). The concentration distribution of the physiologically active substance created by the stimulation flow and its gradient has a mountain shape when viewed in the vertical direction of the flow. Corresponding to the rotation of the direction of the stimulation flow, the direction of the mountain-shaped concentration gradient changes (FIG. 10B). FIG. 10C shows a comparison of changes in the direction of the stimulation flow when the liquid feeding ratio p is changed to be proportional to time and when it is changed according to the derived relational expression (formula (3)). D). When the liquid feeding ratio p is changed linearly with time, the direction of the stimulation flow can be rotated, but the rotational speed changes depending on the direction and is not constant (FIG. 10C). On the other hand, when the liquid supply amount is changed according to the derived relational expression, a stimulation flow rotating at a constant angular velocity is realized as expected from the theoretical expression (FIG. 10D). Comparing the variations in angular velocities in the respective methods (FIG. 10E), when p is changed in proportion to time, two peaks having peaks at places higher and lower than the expected average speed, respectively. It has a distribution and the angular velocity varies from 2.02 ± 0.64 rad / min (average ± standard deviation) to about 30%. On the other hand, when time is changed according to the derived relationship, the distribution has a peak near the expected angular velocity, and the variation is 2.05 を ± 0.17 rad / min, and the variation is suppressed to 10% or less. As described above, according to the control unit of the present embodiment, the control unit 40 can rotate the direction of the combined flow so that the angular velocity is constant.

(Control method when the magnitude of stimulus flow is different)
The width of the stimulation flow can be adjusted by changing the amount of liquid fed per unit time from the liquid feed port of channel 4 (FIG. 11). A beam-like stimulation flow is formed by constricting the fluid by the adjustment flow from the upper right side to the lower left side of the field of view. A wide stimulation flow can be formed when the amount of stimulation flow is increased, and a wide stimulation flow is formed when the amount of delivery is reduced. The direction of the stimulation flow can be controlled by the above-described method even for the stimulation flows having different widths.

(Second Embodiment: When using stimulus injection by optical uncaging)
Next, a control device according to a second embodiment of the present invention will be described with further reference to FIG. In the description of the second embodiment, elements that are basically the same as those of the apparatus of the first embodiment described above are denoted by the same reference numerals, thereby avoiding redundant description.

In the first embodiment, the controlled flow port for sending the controlled flow to the chamber 11 was provided. On the other hand, in 2nd Embodiment, the controlled flow port is abbreviate | omitted and the stimulus input part 50 is provided in the device main body 10 instead. In FIG. 12, the chamber 11 is not shown, and a virtual circle connecting the channels 1 to 3 is shown for convenience of explanation.

A caged material is used as the fluid flowing into the chamber 11 from the inflow port 12 in the second embodiment.

The stimulus input unit 50 is configured to apply a stimulus for uncaging to the caged material that flows into the chamber 11 and constitutes the combined flow. Specifically, the stimulus input unit 50 is a mirror or an optical fiber (not shown) for applying a light stimulus to the caged substance. A more specific operation of the second embodiment will be further described below.

(Stimulation method and stimulus flow formation and direction control)
The formation and direction control of the stimulus flow described in the first embodiment is a stimulus flow formed by activating (uncaging) a caged compound in which a physiologically active substance is bound to a photodegradable protective group by light irradiation. It can also be applied to. In this case, while the channel 4 of the basic module is closed, a solution in which a caged compound of a stimulating substance is mixed is introduced from the channels 1, 2, and 3, and a regulated flow (synthetic flow) according to the method described in the first embodiment. Is controlled (see FIG. 12A). The setting of the light irradiation region for uncaging can be performed by a method using a pinhole or a commercially available nanomirror system that can change the light irradiation region. By irradiating light in the center of the sample chamber and uncaging the caged compound, it is possible to form a beam-like stimulation flow and its concentration gradient from the irradiation position toward the downstream side of the regulated flow (see FIGS. 12B and 12C).

An example in which the direction of the stimulation flow is controlled according to the second embodiment is shown in FIG. In the center of the chamber (corresponding to the specific region 111) (channel 4 is closed), optical uncaging was performed, and the basic operation shown in the description of the first embodiment was performed. When the relative ratio of the liquid feeding amount is p = 0, the stimulus flow turns to the right, p = 0.3 turns to the upper right, p = 0.7 goes up, and p = 1 changes the direction to the upper left (FIG. 13A). When the ratio p of the liquid delivery amount is changed from 0 to 1, the direction of the stimulation flow changes by 120 degrees, and the measured p dependency of the direction θ of the stimulation flow is in good agreement with the theoretical formula (Equation (1)). It was seen (FIG. 13B). The spatial profile of the stimulating substance concentration in the case of the controlled liquid supply constant control shows a small maximum value at p = 0, 1, and a large maximum value as p approaches 0.5 (FIG. 13C). In the case of constant control flow rate control, the spatial profile of the stimulant concentration is kept more constant (FIG. 13D). In the case of constant control flow rate control, when the p is varied from 0 to 1, the peak fluorescence intensity value of the concentration profile is 2.94 on average and the average variation (standard deviation / average value) is 39% The average slope gradient in the range of 20% to 80% of the peak fluorescence brightness was 0.045 μm ⁻¹ , and the slope variation (standard deviation / average value) was 41%. On the other hand, in the case of the controlled flow rate constant control, the average value of peak fluorescence brightness was 1.76, the variation was 5%, the average gradient slope was 0.027 μm ⁻¹ , and the gradient variation was 6%. As described above, in the case of the controlled flow rate constant control, it is possible to greatly suppress the variation of the concentration spatial profile from the viewpoint of both the concentration peak value and the concentration gradient. In addition, even when the position of optical uncaging is shifted from the center, the direction of the stimulus flow can be controlled without greatly deviating from the theoretical value at least within the range of 300 μm from the center (FIG. 13E, from the chamber center). Distance of 100 μm, 140, 200, 300).

In the stimulus injection method by uncaging, the width of the stimulus flow is determined by the size of the light irradiation region (FIG. 14A). In this method, a stimulation flow having an arbitrary width in the range of 1 μm to 1 μm can be formed in a single device. Further, in the second embodiment, the direction of the stimulation flow can be controlled over time as in the first embodiment (FIG. 14B). In this way, even when stimulating using uncaging, a rotational wave of the stimulating flow can be formed based on the above-described method for controlling the direction of the regulated flow.

(Presentation of stimulus flow from any direction and its gradient by optical uncaging at multiple locations)
Since the stimulus flow formed in the basic module is always directed outward from the center of the chamber, the direction of the stimulus flow is uniquely determined at each location in the chamber. On the other hand, by performing stimulus injection by optical uncaging from a plurality of positions (regions) away from the center of the chamber, it is possible to input stimulus flows from different directions to the place of interest (that is, the specific region 111). Is possible. FIG. 15A shows the operation procedure when the optical uncaging is switched at two locations in the basic module and the direction of the stimulus flow formed from each location is controlled. As an execution example, a case where a stimulation flow from the lower left side of the chamber to the upper right side and a stimulation flow from the upper left side of the chamber 11 to the lower right side are input to the center of the chamber is shown (FIGS. 15B to 15D). Since two or more light irradiation positions can be provided, a stimulus flow and a gradient from an arbitrary direction can be presented at the position of interest.

Other configurations and advantages of the control device of the second embodiment are basically the same as those of the first embodiment described above, and thus further description is omitted.

(Third embodiment: Multiplex channel system)
Next, a control device according to a third embodiment of the present invention will be described. In the description of the third embodiment, elements that are basically the same as those of the apparatus of the first embodiment described above are denoted by the same reference numerals, thereby avoiding redundant description.

In the above-described second embodiment, the method of presenting the stimulus flow and the gradient from an arbitrary direction at the position of interest using optical uncaging has been described. The applicable range of this method is limited to the case where a caged compound of a physiologically active substance can be used and the optical uncaging position can be set in the microscope observation region. As an alternative method when these requirements are not satisfied or when light irradiation is not preferable, a multiplex system in which a plurality of basic modules are arranged can be considered (FIG. 16A). As described above, the basic module (the first embodiment) is composed of one stimulus feeding port (Stimulus inlet, S) and three buffer feeding ports for control arranged at the vertices of a triangle surrounding the stimulus feeding port (Stimulustiminlet, S). (Buffer inlet, B) (FIG. 16A left, 1S / 3B). A diamond-shaped duplex channel system (second from the left in FIG. 16A, 2S / 4B) in which two triangles are arranged in the chamber 11 and three in this chamber 11 (Fig. 16A). 16 is the third from the left, 3S / 9B and 3S6B), and the configuration of a channel system (FIG. 16 right, 4S / 4B and 6S6B) having more modules can be considered. In these multiplex flow path systems, the above-described stimulation flow direction control is performed in each module, and by combining a plurality of these, the stimulation flow and its gradient can be presented from different directions to the position of interest in the chamber 11. (FIG. 16B).

As an example of a multiplex channel system, a duplex (2S / 4B type) channel combining two basic modules is shown (FIG. 17). In this duplex channel, the control liquid supply port (second port 122 and third port 123 in this example) of the basic module is formed in units of two modules (first to third ports and controlled flow port). The liquid supply ports are arranged in a diamond shape (FIG. 17A). By using this flow path and controlling the direction of the aforementioned stimulus flow, gradient stimuli that alternated from different directions were formed (FIG. 17B). Furthermore, as shown in FIG. 17C, by appropriately selecting the three observation positions 1 to 3 (corresponding to the first to third specific regions 111 to 113) in the sample chamber, the directions in which the two gradient stimuli appear can be changed. A stimulation system having an arbitrary angle from 0 degrees to 180 degrees can be realized. ROI 1 to 3 in FIG. 17C show the concentration gradients (that is, gradient stimulation) observed in the first to third specific regions 111 to 113. In addition, although the position of the 2nd specific area | region 112 in FIG. 17C is set to the substantially same position as the 1st specific area | region 111 in FIG. 17B, this is an example to the last. The position of each specific region can be appropriately set according to various factors such as each port, chamber shape, observation purpose, and the like.

In the example of FIG. 17B, channels 1, 2, and 4 correspond to the first to third ports 121 to 123, and channel 5 corresponds to the controlled port 120. Similarly, channels 3, 2, and 4 correspond to the first to third ports 121 to 123, and channel 6 corresponds to the controlled port 120. Here, the channel 3 can also be recognized as the fourth port 124.

The configuration and advantages other than those described above in the third embodiment are basically the same as those in the first embodiment, and thus detailed descriptions thereof will be omitted.

(advantage)
In the configuration (basic module) of the first embodiment, from the buffer liquid supply port (first to third ports 123) arranged so as to surround the stimulation liquid supply port (controlled flow port 120) in the center of the chamber 11. By controlling the liquid feeding amount, the direction of the laminar flow (synthetic flow) from the center of the chamber 11 toward the peripheral portion can be directed in an arbitrary direction within a range of 360 degrees. Furthermore, the direction of the stimulus flow from the center of the circular chamber 11 toward the peripheral portion can be changed while keeping the angular velocity constant by controlling the amount of liquid fed from each port in an angle-dependent manner. Changing the direction of the stimulation flow means that the direction of the concentration gradient of the stimulating substance formed by the stimulation flow is changed. The concentration distribution of the physiologically active substance created by the stimulation flow and its gradient has a mountain shape when viewed in the vertical direction of the flow (FIGS. 9C and D). For this reason, by repeatedly rotating the stimulation flow 360 degrees, it is possible to periodically give a single traveling wave stimulus to the cells. FIG. 18 shows an example in which a rotational wave stimulus that makes one turn in 6 minutes is given to a cellular slime mold that is a typical example of a moving cell. Cellular slime molds are moving in the direction opposite to the traveling direction of the rotation wave. In slime mold cells, it is considered that the attracting substances that are formed in a self-organizing manner in the population gather together to form multi-cells. By artificially generating a field that mimics it, it is possible to analyze in detail the perception of the attractive field of cells and the chemotaxis mechanism. In general, it can be said that the place where the attracting substance or physiologically active substance in the tissue is created is dynamic both temporally and spatially. Such a dynamic and complex field can be mathematically represented as a superposition of waves with specific wavelengths and time periods. A rotating wave having a specific wavelength and period that can be generated by the devices of the above-described embodiments can be regarded as the simplest case of such a field. By analyzing the chemotaxis and response characteristics of cells to rotational wave stimulation of various wavelengths and cycles, the time space of concentration gradients for the chemotaxis of cellular slime molds, neutrophils, and even cancer cells Evaluate responsiveness to changes and analyze how cells determine the timing and direction of movement. These analyzes are expected to provide useful information for understanding the mechanisms of cell information processing and chemotaxis in the field of physiologically active substances in tissues.

As in the third embodiment, by combining two or more basic modules, a plurality of laminar flows in different directions can be alternately introduced. This makes it possible to analyze how a cell changes its extension direction and movement direction when a gradient stimulus is applied from a different direction from the beginning. As an application example of such a multiplex flow path system, a cell turning experiment is considered in which stimulation input positions are prepared at a plurality of positions surrounding the cell, and gradient stimulation in various directions as viewed from the cell is given ( FIG. 16B). FIG. 19 shows an experimental example in which two gradient stimuli directed in different directions were alternately applied to cells using a duplex channel. The neutrophil-like cells (HL60) of the immune system move in a zigzag direction while moving in the direction of climbing the concentration gradient formed by the stimulus flow and following the change in the direction of the gradient. The state of going is captured. Such cell reorientation is thought to be important for immune cells to move to the damaged site during inflammatory reaction, and cancer cells for understanding invasive metastasis. Provides a useful tool. By combining with live cell imaging under a microscope, the apparatus of each embodiment can be used for intracellular molecular dynamics analysis and mutant analysis.

Note that the content of the present invention is not limited to the above embodiment. In the present invention, various modifications can be made to the specific configuration within the scope of the claims.

DESCRIPTION OF SYMBOLS 10 Device main body 11 Chamber 111 Specific area | region 12 Inflow port 120 Controlled flow port 121 1st port 122 2nd port 123 3rd port 124 4th port 13 Exhaust port 14 Inflow channel 15 Exhaust channel 111 Specific area | region 20 Fluid supply part 30 Fluid Discharge unit 40 Control unit 50 Stimulus input unit

Claims (13)

An apparatus for controlling the flow of fluid,
It has a device body,
The device body includes a chamber, an inflow port, and an exhaust port,
The chamber is a closed space extended in a two-dimensional direction,
And the chamber includes a specific region in a part thereof,
The inflow port includes at least a first port and a second port;
The first port and the second port both supply fluid into the chamber in a direction that intersects the extension direction of the chamber, so that the first port and the second port are directed to at least one direction in or near the specific region. It is configured to form a composite flow with a velocity component,
The discharge port is configured to discharge the fluid from an end portion in the extension direction of the chamber or the vicinity thereof. The control device according to claim 1, wherein the first port and the second port are arranged at or near a position where a phase difference of about 120 degrees is centered on the specific region. The inflow port further includes a third port;
The third port is configured to supply a fluid into the chamber in a direction crossing the extension direction of the chamber,
The control device according to claim 2, wherein the third port is arranged at or near a position where a phase difference of about 120 degrees with each of the first and second ports is centered on the specific region. The inflow port further comprises a controlled flow port;
The control device according to any one of claims 1 to 3, wherein the controlled flow port is configured to supply a controlled flow to the specific region or the vicinity thereof. Furthermore, it has a control part,
The control unit is configured to control the flow rate or flow rate of the fluid flowing into the chamber from the inflow port,
The control unit is configured to control a direction of the combined flow by controlling an inflow amount or a flow velocity of the fluid from the inflow port to the chamber. The control device according to item. The control device according to claim 5, wherein the control unit is configured to rotate the direction of the combined flow so that an angular velocity is constant. The control device according to claim 5, wherein the control unit is configured to control an inflow amount or a flow rate of the fluid so that a concentration gradient profile in the combined flow is constant. In addition, it has a stimulus input unit,
As the fluid flowing into the chamber from the inflow port, a caged material is used,
The control according to any one of claims 1 to 7, wherein the stimulus input unit is configured to apply a stimulus for uncaging to the caged material that flows into the chamber and constitutes the combined flow. apparatus. The control device according to any one of claims 1 to 8, wherein the specific region is a central portion of the chamber in the two-dimensional direction or a position in the vicinity thereof. The inflow port further includes a fourth port;
The chamber further includes a second specific region in a part thereof,
The second specific area is a position different from the specific area,
The fourth port is configured to supply a fluid into the chamber in a direction crossing the extension direction of the chamber, thereby having a velocity component in at least one direction at or near the second specific region. The control device according to any one of claims 1 to 9, wherein the control device is configured to form a flow. The control device according to any one of claims 1 to 10, wherein the discharge port is configured to discharge the fluid at a substantially uniform speed at an end portion in the extension direction of the chamber. Furthermore, it has a fluid supply part and a fluid discharge part,
The fluid supply unit is configured to supply the fluid to the inflow port,
The control device according to any one of claims 1 to 11, wherein the fluid discharge section is configured to collect the fluid from the discharge port. A method for controlling a flow of fluid using the control device according to any one of claims 1 to 12, comprising:
By supplying a fluid from the first port and the second port into the chamber in a direction crossing the extension direction of the chamber, a velocity component in at least one direction is generated in or near the specific region. Forming a composite flow having,
Changing the direction or flow rate of the combined flow by changing the flow velocity or flow rate of the fluid in one or both of the first port and the second port.

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