JP2003284544A - Apparatus for cell separation and classification and substrate for cell arrangement - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an apparatus for cell separation and classification, with which a great number of small-diameter cell-containing liquid drops are formed while controlling damage to cells and a substrate for cell adjustment. <P>SOLUTION: The apparatus for cell separation and classification is equipped with a cell arrangement part 1 with which a cell suspension containing a great number of cells is treated and cells are arranged at intervals and made to flow, a cell information detection part 3 with which the cells arranged in the cell arrangement part 1 are detected to detect information related to the cells and a cell classification 5 with which the cells are classified based on the information detected by the cell information detection part 3. The cell arrangement part 1 is equipped with a first route 11 for making the cell suspension 70 flow and a second route 12 which is connected to the first route 11 in the direction crossing the first route and makes a dividing liquid material 75 for dividing the flow of the cell suspension 70 of the first route 11. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a cell separation and sorting apparatus for separating and sorting cells contained in a cell suspension, and a cell alignment used for aligning cells in the cell separation and sorting apparatus. Substrate.

[0002]

2. Description of the Related Art Conventionally, there has been provided a cell separation / sorting device called a flow cytometer for separating and sorting cells. This cell separation and sorting device detects a cell alignment part that processes a cell suspension containing a large number of cells and arranges the cells to flow at an interval, and detects the cells aligned in the cell alignment part. A cell information detection unit that performs processing to detect information regarding cells and a cell selection unit that selects cells based on the information detected by the cell information detection unit are provided.

FIG. 6 schematically shows a cell alignment section 100 of a conventional cell separation and sorting apparatus. The cell alignment unit 100 is called a sheath flow type, and is provided coaxially with the first nozzle unit 101 for flowing the cell suspension 200 downward and the first nozzle unit 101 on the outer peripheral side of the first nozzle unit 101. And a second nozzle portion 102. The second nozzle portion 102 surrounds the cell suspension 200 from the outside with the sheath liquid 3
Flow 00 downwards. Thereby, the flow of the cell suspension 200 is narrowed down to align the cells. In this case, sheath fluid 3
When the pressure of 00 is increased, the channel diameter of the cell suspension 200 becomes smaller. Further, when the pressure of the sheath liquid 300 is lowered, the flow path diameter of the cell suspension 200 increases.

A cell alignment unit 100 adopting the above-mentioned sheath flow type is disclosed in FIG. 11 of Japanese Patent No. 2749906 (publication number: Japanese Patent Laid-Open No. 3-122548).

If the cells 400 of the cell suspension 200 are aligned by the cell aligning unit 100 as described above, the cell information detecting unit can favorably perform the detecting process on each cell 400. The advantage is that information about 400 can be better detected.

[0006]

By the way, according to the above-mentioned cell separation / sorting apparatus, the cell suspension 200 squeezed into a small diameter is used.
Ultrasonic vibration is applied to the flow of
It is supposed to form minute cell-containing droplets. However, since ultrasonic vibration is applied to the flow of the cell suspension 200 to form cell-containing droplets, the cells 400 contained in the cell-containing droplets tend to be damaged,
Not preferred.

The present invention has been made in view of the above circumstances, and a cell separation / selection device and a cell alignment substrate capable of generating a large number of cell-containing droplets having a small diameter while suppressing damage to cells. The challenge is to provide.

[0008]

In order to solve the above-mentioned problems, a cell separation and sorting apparatus according to the present invention treats a cell suspension containing a large number of cells to arrange the cells at intervals. The cell aligning part that is caused to flow, the cell information detecting part that performs detection processing on the cells aligned in the cell aligning part to detect information about the cells, and the cell information detecting part detects cells based on the information detected by the cell information detecting part. In the cell separation / sorting device including a cell sorting unit for sorting, the cell aligning unit communicates with a first passage through which a cell suspension containing cells flows and a first passage in a direction intersecting with the first passage. And a second passage through which the flow of the cell suspension is divided and a dividing liquid material for forming a large number of minute cell-containing droplets containing cells is caused to flow.

The liquid material for dividing flows into the second passage. In this state, the cell suspension containing a large number of cells flows through the first passage. When the cell suspension flowing in the first passage reaches the intersection of the first passage and the second passage, the cell suspension is continuously broken into fine microdroplets due to the dividing liquid material flowing in the second passage. To be done. As a result, a large number of minute cell-containing droplets are continuously generated. The large number of cell-containing droplets produced are
It flows downstream along the second passage.

The cell alignment substrate according to the present invention can be used in the above-described cell separation / sorting apparatus, and is a cell alignment substrate for aligning cells at intervals by flowing a cell suspension containing cells. Therefore, the cell alignment substrate communicates with the first passage through which the cell suspension containing cells flows and in a direction intersecting with the first passage, and also divides the cell suspension in the first passage to separate the cells. It is characterized by comprising a second passage through which the liquid material for separation that forms a large number of minute liquid droplets containing cells is contained.

The liquid material for dividing flows into the second passage. In this state, the cell suspension containing a large number of cells flows through the first passage. When the cell suspension flowing in the first passage reaches the intersection of the first passage and the second passage, the cell suspension is continuously broken into fine microdroplets due to the dividing liquid material flowing in the second passage. To be done. As a result, a large number of minute cell-containing droplets are continuously generated. The large number of cell-containing droplets produced are
It flows downstream along the second passage.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION A cell separation / sorting apparatus according to the present invention comprises a cell alignment section for treating a cell suspension containing a large number of cells and aligning and flowing the cells at intervals. A cell information detection unit that performs detection processing on the cells aligned by the alignment unit to detect information about the cells, and a cell selection unit that selects cells based on the information detected by the cell information detection unit . It is preferable that the number of cells contained in one cell-containing droplet is one. That is, one droplet / one cell is preferable. However, in some cases, a plurality of cells may be contained in one cell-containing droplet.

The cell alignment section according to the present invention communicates with a first passage through which a cell suspension containing cells flows, and in a direction intersecting with the first passage, and divides the cell suspension in the first passage. And a second passage through which a dividing liquid material for continuously forming a large number of minute cell-containing droplets flows. The second passage intersects the first passage. The term “cross” means that the first passage and the second passage communicate with each other. The crossing angle can be appropriately selected, but in general, it can be set to 90 degrees or in the vicinity of 90 degrees, but the crossing angle is not limited to this. The first passage and the second passage may intersect with each other in a T shape, in some cases, in a cross shape, or in a Y shape. The first passage and the second passage may have a groove shape.

The cell suspension flowing through the first passage has a liquid material and a large number of cells contained in the liquid material. Examples of the liquid substance include cell buffer, physiological saline, isotonic solution of cells and the like, but those which are less likely to be clogged are preferable. In general, cells have hydrophilicity, and therefore, as the liquid material forming the cell suspension, those having hydrophilicity can be adopted. The cells contained in the cell suspension include cell constituents, cell-related substances, and organelles in addition to the cells themselves.
Blood cells (white blood cells, red blood cells, platelets, etc.), animal cells (cultured cells, isolated tissues, etc.), plant cells, microorganisms (bacteria, protozoa, fungi, etc.), marine organisms (plankton, etc.), sperm, yeast, mitochondria, nuclei , Proteins, nucleic acids such as DNA and RNA, antibodies and the like.

The average diameter of the cell-containing liquid droplets varies depending on the type of cells, but is 4 to 800 μm, especially 4
The thickness is, for example, 400 μm to 4 μm to 200 μm, but is not limited thereto.

The dividing liquid material flowing in the second passage is capable of dividing the cell suspension in the first passage to form a large number of minute cell-containing droplets containing one or a plurality of cells. I wish I had it. A liquid material having hydrophobicity can be adopted as the liquid material for disruption. Therefore, examples of the liquid material for fragmentation include oil and the like. Examples of the oil include sunflower oil, olive oil, cutting oil, linseed oil, silicone oil, mineral oil and the like. Since oil is rich in lubricity, it is possible to improve the passability of cell-containing droplets in the second passage.

According to the cell separation and sorting apparatus of the present invention, preferably, the first passage and the second passage can be formed in a common base. Examples of the material of the common base include resin such as acrylic resin, glass, and silicon. The common base can be substrate-shaped. Thus, the common base can be a common substrate. The passage width of the first passage and the passage width of the second passage must be large enough to allow the cell-containing droplets to pass therethrough, and although it varies depending on the type of cells, it is 1 to 800 μm, particularly 2 to 500 μm.
The thickness may be, for example, μm or 5 to 300 μm.

Of the second passages through which the liquid material for division flows, the passage width D2 near the portion intersecting the first passages is preferably larger than the passage width D1 of the first passages. The downstream side passage width D21 of the second passage is preferably a passage width in which adjacent cell-containing droplets flow one by one with an interval.

According to the cell separation / sorting apparatus of the present invention, preferably, when the cell-containing droplets flow at least on the downstream side of the second passage, between the cell-containing droplets adjacent to each other,
It is possible to adopt a form in which a liquid material for division (oil or the like) is present.

According to the cell separation / sorting apparatus of the present invention, preferably, the cell alignment unit (cell alignment substrate) has a cell information detection unit for one or a plurality of cells aligned by the cell alignment unit. It is possible to adopt a mode in which there is a detection position where the detection processing is performed in.

According to the cell separation / sorting apparatus of the present invention, it is possible to preferably adopt a mode in which a guide portion for guiding the cell-containing droplets flowing through the second passage to the detection position is provided. Examples of the guide portion include an inclined surface and a throttle portion that guide the cell-containing droplets flowing through the second passage to the detection position. If the guide portion is provided in this way, the cell-containing droplets that flow in alignment with the second passage are effectively guided to the detection position of the second passage. Therefore, since the position of the cell-containing droplet is defined in the passage width direction of the second passage, it is possible to improve the detection accuracy when the cell information detecting unit detects the information regarding the cells.

As the cell information detection unit according to the present invention,
Preferably, it is possible to adopt a mode in which electromagnetic waves are applied to the cell-containing droplets flowing through the detection position of the second passage to detect information about cells contained in the cell-containing droplets. Light can be used as the electromagnetic wave. As the light, a laser beam having excellent directivity is preferable. This is because the direction, wavelength, and intensity of light are highly constant. Examples of the laser beam include an argon laser, a helium neon laser, a helium neon laser, a helium cadmium laser, and a gallium aluminum laser. The information about cells may be any information that can be detected by applying electromagnetic waves. For example, when electromagnetic waves are applied and scattered light is received, information such as cell density and size can be obtained. When electromagnetic waves are applied as excitation light and the fluorescence state due to them is observed, information such as the expression status of cells can be obtained.

The cell information detecting section preferably applies an electromagnetic wave such as a laser beam to the cell-containing droplet flowing through the detection position of the second passage to detect fluorescence or the like from the cells contained in the cell-containing droplet. It is possible to adopt a mode in which information on cells is detected based on fluorescence or the like. In this case, a substance that emits fluorescence when excited can be preliminarily loaded on the cells.

According to the cell separation / sorting apparatus of the present invention, it is preferable that a gas is applied to the cell-containing droplets to perform a detaching operation to detach the cell-containing droplets from the cell outlet of the second passage by gas pressure. It is possible to adopt a form having a jet portion. In this case, gas can be applied to the cell-containing droplets in the second passage to effectively separate the cell-containing droplets from the cell outlet in the second passage by gas pressure. Since gas pressure is used,
The damage to the cells can be suppressed. The gas may be air, but may be an inert gas such as nitrogen gas or argon gas in some cases. The gas may be ejected from above toward the cell-containing droplets downward, but in some cases may be ejected from the side toward the cell-containing droplets.

According to the cell separation / sorting apparatus of the present invention, the cell-containing droplet is separated at least one of before and during the detaching operation for detaching the cell-containing droplet from the cell outlet of the second passage. It is possible to adopt a mode in which a liquid material is replenished in and a volume increasing portion for increasing the liquid portion of the cell-containing droplet is provided.

Since the cell-containing droplets are minute, the effects of viscosity, friction, etc. are likely to be large. In this case, it is disadvantageous to separate the cell-containing droplet from the cell outlet of the second passage. In this respect, as described above, at least one of before the detachment operation and at the time of the detachment operation, if the liquid content of the cell-containing droplets is increased by replenishing the liquid content of the cell-containing droplets, the liquid content of the cell-containing droplets is increased. Since the volume is increased, the size of the cell-containing droplet is increased, which is advantageous for detaching the cell-containing droplet from the cell outlet of the second passage. Furthermore, since the amount of the liquid material of the cell-containing droplet is increased, the impact at the time of detachment is mitigated, which is advantageous in suppressing the damage of the cells of the cell-containing droplet.

[0027]

(First Embodiment) A first embodiment of the present invention will be described below with reference to FIGS. The cell separation / sorting apparatus according to the present embodiment is a cell suspension 70 containing a large number of cells.
The cell alignment substrate 1 that functions as a cell alignment portion that is processed by aligning cells to flow at intervals, a holding portion 10 that holds the cell alignment substrate 1, and the cell alignment substrate 1 A cell information detection unit 3 that performs a detection process on the one selected cell to detect information about the cell, and a cell selection unit 5 that selects cells based on the information detected by the cell information detection unit 3. I have it.

The cell alignment substrate 1 has a first passage 11 through which a cell suspension 70 containing cells flows, and the first passage 11 and a T passage.
It has the 2nd passage 12 which intersects in a letter shape and is open for free passage. The cell alignment substrate 1 is a common substrate having a first passage 11 and a second passage 12. The cell alignment substrate 1 is
It is formed of a resin such as an acrylic resin or a glass plate.

In the cell alignment substrate 1, the first passage 11
Is formed in a groove shape, extends linearly in the lateral direction, and has a first inlet port 13 on the upstream end side. First passage 11
Into the first inlet 13 of the
There is provided a first fluid source 21 that flows at. First fluid source 2
1 is formed by a micro pump.

In the cell alignment substrate 1, the second passage 12
Is formed in a groove shape and extends in a horizontal direction in a straight line, and connects the second inflow port 14 on the upstream end side, the cell outlet 15 on the downstream end side, and the second inflow port 14 and the cell outlet 15 And a communication passage 16. In the second inflow port 14 on the upstream side of the second passage 12, the dividing liquid material 75 is flowed at a constant flow rate V2.
A fluid source 22 is provided. The second fluid source 22 is formed by a micro pump.

According to this embodiment, the passage width D2 of the second passage 12 is set larger than the passage width D1 of the first passage 11 (D2> D1). The diameter of the cell outlet 15 is set larger than the diameter of the cell-containing droplet 77 immediately after being generated at the intersecting portion 12x. The reason is that the cell-containing droplet 77 can be easily dropped from the cell outlet 15. The passage width of the downstream communication passage 16d of the communication passage 16 of the second passage 12 is set to be the same as or slightly smaller than the diameter of the cell-containing droplet 77 immediately after generation. The liquid portion of the cell-containing droplet 77 flowing in the downstream communication passage 16d is slightly distorted while touching the inner wall surface of the downstream communication passage 16d. The downstream communication passage 16d has the same or substantially the same passage width over the entire length of the downstream communication passage 16d.

A detection position 17 is arranged between the second inlet 14 and the cell outlet 15 of the second passage 12. The detection position 17 is aligned in the aligned state on the cell alignment substrate 1
This is a position where the cell information detection unit 3 performs detection processing on the cells 78 of the individual cell-containing droplets 77.

According to this embodiment, the cell information detector 3 is provided near the detection position 17 of the second passage 12. The cell information detection unit 3 includes an optical fiber 30 whose tip 30a faces the detection position 17 of the cell alignment substrate 1, and an optical fiber 3.
A light projecting section 31 having a laser element projecting a laser beam for excitation (inspection light) as an electromagnetic wave to the other end 30c of 0.
And a light receiving unit 32 that receives the fluorescence emitted by the cells excited by the laser beam, and a detection unit 33 that detects information about the cell based on the light receiving signal from the light receiving unit 32. In this way, since the tip portion 30a of the optical fiber 30 is made to approach and face the cell-containing droplet 77 at the detection position 17 of the cell alignment substrate 1, the main body of the cell information detection unit 3 of the optical system is separated from the cell alignment substrate 1. Can be installed in different positions.

Further, in the present embodiment, as shown in FIGS. 1 and 2, the guide portion 1 for guiding the cell-containing droplet 77 flowing in the second passage 12 of the cell alignment substrate 1 in an aligned state to the detection position 17.
9 is provided. The guide portion 19 is the cell alignment substrate 1
Has a function of squeezing the dividing liquid material 75 (fluid) flowing through the second passage 12 of the second passage 1.
It is formed by a guide surface 19c (division liquid material squeezing means) which is inclined so that the passage width of the second passage 12 becomes narrower toward the downstream of 2. The guide surface 19c is the second passage 12
It is formed on a wall surface 12m on the side opposite to the first passage 11. This is because when the cell-containing droplet 77 flows, the swing displacement of the cell-containing droplet 77 in the passage width direction of the second passage 12 is suppressed as much as possible.

According to this embodiment, of the communication passages 16 of the second passage 12, the upstream communication passage 16 upstream of the guide portion 19 is provided.
The passage width D2 of u is set relatively large. However, of the communication passages 16 of the second passage 12, the passage width D21 of the upstream communication passage 16d on the downstream side of the guide portion 19 is equal to the passage width D2.
It is set smaller than 1 and contains one cell-containing droplet 77.
The width of the passage allows passage.

As described above, the cell sorting section 5 (see FIG. 1) is provided in the vicinity of the cell outlet 15 located on the downstream side of the detection position 17 in the second passage 12 of the cell alignment substrate 1. There is. As shown in FIG. 1, the cell sorting section 5
A collection plate 55 having a gas ejection part 50, a sorting plate 52 driven by a driving part 51 such as a motor in the X direction and the Y direction, and a large number of hole-shaped collection parts 54 for individually collecting cells (cell collection Means) and a drive unit 56 for moving the collecting plate 55 in the X direction and the Y direction, respectively. Since the drive unit 56 is controlled by the control system, any collecting unit 54 of the collecting plate 55 can be located below the cell outlet 15 of the second passage 12.

As shown in FIG. 1, the gas ejection portion 50 faces the cell outlet 15 so as to eject air to the cell-containing droplet 77 which has flowed through the second passage 12 and reached the cell outlet 15. And an air supply source 5 for storing compressed air
0b and a valve 50c for opening and closing the ejection nozzle 50a.

The jet nozzle 50a is arranged above the cell outlet 15 of the second passage 12. When the valve 50c is opened, the ejection nozzle 50a ejects air from above toward the cell-containing droplet 77 that has reached the cell outlet 15. Therefore, the cell-containing droplet 77 that has reached the cell outlet 15 of the second passage 12 can be dropped and separated from the cell outlet 15 of the second passage 12 by air pressure.

In the cell alignment substrate 1, the second passage 12
An increasing passage 24 is connected to the cell outlet 15 of the cell. An increasing unit 25 capable of supplying a buffer solution is connected to the increasing passage 24. The volume increasing passage 24 increases the amount of the liquid portion of the cell-containing droplet 77 in the cell outlet 15 by sending the buffer solution functioning as a liquid toward the cell outlet 15 in the second passage 12, thereby increasing the cell-containing droplet. This is for increasing the size of 77.

The sorting plate 52 of the cell sorting section 5 is the second passage 1
It is provided below the second cell outlet 15. When the cells 78 contained in the cell-containing droplet 77 are target cells,
The sorting plate 52 collects the cell-containing droplets 77 containing the cells 78 in a predetermined collecting portion 54 of the collecting plate 55. The selection plate 52 is rotatably (movably) provided, and is rotationally driven by a drive unit 51 such as a drive motor. The selection plate 52 is held on the upper surface of the selection plate 52 and a through hole 57 penetrating in the thickness direction. And a waste liquid storage container 58. The through holes 57 and the waste liquid storage containers 58 are alternately arranged on the sorting plate 52.

In use, the liquid material for fragmentation 75 is fed from the second inlet 14 of the second passage 12 of the cell alignment substrate 1 to the cell outlet 15 by the second fluid source 22 at a constant flow rate V2.
To flow in the direction of arrow W2. In this state, the cell suspension 70 containing a large number of cells is directed from the first inlet 13 of the first passage 11 of the cell alignment substrate 1 toward the intersection 12x of the second passage 12 by the first fluid source 21. Constant flow velocity V1 in the first passage 11
To flow in the direction of arrow W1. As the dividing liquid material 75, oil such as sunflower oil having hydrophobicity can be used.

As described above, the cell suspension 70 reaches the intersection 12x between the first passage 11 and the second passage 12 while the liquid separating material 75 is flowing in the second passage 12. Therefore, the cell suspension 70 is subjected to a shearing force by the liquid material for disruption 75 and finely fractured. As a result, the cell suspension 7
From 0 to a large number of cell-containing droplets 77 are continuously generated.
The cell-containing droplet 77 is formed to have a size such that one cell is contained therein, and one cell-containing droplet 77 contains one cell or does not contain any cell. Or either.

The cell-containing droplets 77 flow in line with the disrupting liquid material 75 in a line toward the downstream side of the second passage 12, that is, toward the detection position 17 and the cell outlet 15 in the second passage 12. . At this time, the second cell alignment substrate 1
Since the guide portion 19 is provided in the passage 12, the cell-containing droplet 77 flowing in the second passage 12 is regulated in the passage width direction of the second passage 12, and the cell-containing droplet 77 is located downstream of the second passage 12. The communication passages 16d are arranged in a line. Therefore, the cell-containing droplets 77 are separated one by one, at regular intervals, or at substantially regular intervals, to the second passages 12.
Flows along the downstream communication passage 16d of the communication passage 16 of.

Therefore, the cells 78 contained in the cell-containing droplet 77 can reach the converging position of the cell information detecting section 3 favorably. Therefore, the laser beam as the detection light projected from the light projecting unit 31 of the cell information detecting unit 3 through the optical fiber 30 is focused on the cells 78 of the cell-containing droplet 77. As a result, the fluorescent substance previously carried on the cell 78 irradiated with the laser beam is excited. The excited fluorescence is received by the light receiving section 32 via the optical fiber 30. As a result, the cell information detection unit 3 detects whether the cell 78 of the cell-containing droplet 77 that has reached the detection position 17 is the target cell. When the detected cells 78 of the cell-containing droplet 77 are target cells, the control system outputs a target cell signal. When the detected cell 78 of the cell-containing droplet 77 is not the target cell, the control system outputs a non-target cell signal.

The cell-containing droplet 77 that has passed through the detection position 17 of the cell alignment substrate 1 flows along the downstream communication passage 16d of the second passage 12 toward the downstream thereof in the direction of the arrow W3,
The cell outlet 15 is reached. Cell exit 1 from detection position 17
The distance up to 5 is a fixed distance. In addition, the first fluid source 21
Since the flow velocity of the cell suspension 70 flowing from the second fluid source and the flow velocity of the liquid separating material 75 flowing from the second fluid source 22 are constant,
The flow velocity of the cell-containing droplet 77 flowing through the passage 12 may be considered to be constant. Therefore, the time required for the cell-containing droplet 77 determined to be the target cell to reach the cell outlet 15 is grasped by the control system.

Then, the cell-containing droplet 77 is transferred to the second passage 12
At the time of reaching the cell outlet 15 of
The control system causes the buffer solution of the increasing portion 25 to flow into the increasing passage 24. As a result, the cell-containing droplet 77 at the cell outlet 15 is replenished with the buffer solution (liquid substance), and the liquid portion of the cell-containing droplet 77 is increased. As a result, the size of the cell-containing droplet 77 increases at the cell outlet 15, and it becomes easy to avoid the influence of surface tension and the like, which is advantageous for separating the cell-containing droplet 77 from the cell outlet 15 of the second passage 12. Become.

After performing the volume increasing operation as described above, the control system applies air to the cell-containing droplet 77 at the cell outlet 15 of the second passage 12 from the nozzle 50a of the gas ejection portion 50, and the cell-containing liquid 77 is supplied with air pressure. A detaching operation is performed in which the droplet 77 is dropped from the cell outlet 15 to be detached. As a result, the cell-containing droplet 77 is effectively separated from the cell outlet 15.

At this time, the cell outlet 15 of the cell alignment substrate 1
When the cell 78 contained in the cell-containing droplet 77 separated from the target cell is the target cell, the control system controls the through hole 57 of the sorting plate 52 of the cell sorting section 5 to be located directly below the cell outlet 15. The sorting plate 52 is controlled. The control system also controls the desired collecting portion 54 of the collecting plate 55 so as to be located directly below the cell outlet 15. For this reason,
Cell-containing droplet 77 dropped from the cell outlet 15 and separated
Through the through hole 57 of the sorting plate 52 of the cell sorting unit 5,
It is received and collected by a desired collecting portion 54 of the collecting plate 55.

When the cells 78 of the cell-containing droplet 77 separated from the cell outlet 15 of the cell alignment substrate 1 are not the target cells, the waste liquid storage container 58 of the selection plate 52 is positioned directly below the cell outlet 15. The control system controls the selection plate 52 of the cell selection unit 5. Therefore, the cell outlet 15
The cell-containing droplet 77 that has fallen off from the separation plate 5 does not pass through the through hole 57 of the selection plate 52 of the cell selection unit 5,
It is received by the second waste liquid storage container 58.

As shown in FIG. 1, since the through holes 57 and the waste liquid storage containers 58 of the selection plate 52 are provided alternately, the selection plate 52 only needs to be rotated by a small angle. The moving distance can be reduced, and the selection plate 52
The responsiveness of can be improved.

By the way, since the cell-containing droplet 77 is minute, the influence of viscosity, friction, surface tension, etc. is likely to be large.
In this case, it is disadvantageous to separate the cell-containing droplet 77 from the cell outlet 15 of the second passage 12. In this respect, as described above, at least one of before and during the detaching operation, the cell-containing droplet 77 is replenished with a buffer solution which is a liquid substance, and the liquid portion of the cell-containing droplet 77 is increased. There is. Therefore, the size of the cell-containing droplet 77 is increased, which is advantageous for separating the cell-containing droplet 77 from the cell outlet of the second passage 12.

During the detachment operation, the cell-containing droplet 7
Even when 7 is dropped from the cell outlet 15 of the second passage 12, since the liquid portion of the cell-containing droplet 77 is increased, the impact can be mitigated, and the cell 78 of the cell-containing droplet 77 can be reduced. It is effective in suppressing the damage given to. Furthermore, since the amount of the liquid material of the cell-containing droplets 77 is increased, the shock absorbing property is improved, and therefore the collecting portion 54 of the collecting plate 55 is
When the cell-containing droplet 77 is received by the cell-containing droplet 7
This is advantageous in suppressing damage to the cells 78 contained in 7.

Further, according to this embodiment, the cell alignment substrate 1
Since the guide portion 19 is provided in the second passage 12 of
The cell-containing droplets 77 flowing through the second passage 12 of the cell alignment substrate 1 form a line and are effectively guided to the detection position 17 of the second passage 12. Therefore, the cells 78 of the cell-containing droplets 77 flowing through the second passage 12 can reach the light collection position from the cell information detection unit 3 favorably. Therefore, it is advantageous for the cell information detection unit 3 to detect the information regarding the cells 78 contained in the cell-containing droplet 77.

According to this embodiment, if the flow velocity of the cell suspension 70 flowing from the first fluid source 21 and the flow velocity of the liquid separating material 75 flowing from the second fluid source 22 are changed, they flow in line. The interval between the cell-containing droplets 77 can be adjusted.

According to this embodiment, the cells 78 are surrounded by the liquid material to form the cell-containing droplets 77, so that the downstream communication passage 16d on the downstream side of the second passage 12 has a narrow passage width. Even if there is, the advantage that the cells 78 are less likely to be clogged in the second passage 12 is obtained.

3A to 3C show the first passage 11
The form of an intersection portion 12x between the second passage 12 and the second passage 12 is shown. Figure 3
In the form shown in (A), the first passage 11 and the second passage 12 are orthogonal to each other in a T shape. In the form shown in FIG. 3 (B), the first passage 11 is inclined so as to be directed toward the downstream end of the second passage 12 (direction of arrow W2) toward the tip 11e. In the form shown in FIG. 3 (C), the first passage 11 is inclined so as to face the upstream direction (arrow WO direction) of the second passage 12 toward the tip 11e.

4A and 4B show the shape of the guide surface 19c of the guide portion 19 formed in the second passage 12. As shown in FIG. Figure 4
In the form shown in (A), the first passage 1 of the second passages 12
A guide surface 19c is formed on the wall surface 12n on the side where 1 is formed. In the form shown in FIG. 4 (B), the second passage 1
Of the two, the wall surface 12 on the side where the first passage 11 is not formed
A pair of guide surfaces 19c of the guide portion 19 are formed so as to face each other on both m and the wall surface 12n on the side where the first passage 11 is formed.

(Second Embodiment) A second embodiment of the present invention will be described below with reference to FIG. The cell separation / sorting apparatus according to the present embodiment has basically the same configuration as that of the first embodiment, and basically has the same operation effect.

The cell separating and sorting apparatus according to the present embodiment is for cell alignment as a cell aligning section which processes a cell suspension 70 containing cells to align and flow one cell at intervals. A substrate 1B, a holding unit 10 that holds the cell alignment substrate 1B, and a cell information detection unit 3B that performs detection processing on one cell aligned on the cell alignment substrate 1B to detect information about the cell. , And a cell selection unit 5B that selects cells based on the information detected by the cell information detection unit 3B.

The cell alignment substrate 1B has a first passage 11 through which a cell suspension 70 containing a large number of cells flows, and a second passage 12 communicating with the first passage 11 in a direction intersecting with the first passage 11.
Have and. The cell alignment substrate 1B is a common substrate having a first passage 11 and a second passage 12.

In the cell alignment substrate 1B, the first passage 1
1 is formed in the shape of a groove, is linearly extended in the lateral direction, and has a first inflow port 13 on the upstream end side. First passage 11
Into the first inlet 13 of the
There is provided a first fluid source 21 that flows at. Second passage 12
Is formed in a groove shape and extends in a horizontal direction in a straight line, and connects the second inflow port 14 on the upstream end side, the cell outlet 15 on the downstream end side, and the second inflow port 14 and the cell outlet 15 The communication passage 16 and the cell outlet 1 for cell recovery formed on the downstream side
5 and a cell outlet 18 for disposal formed on the downstream side. A second fluid source 22 is provided at the second inlet 14 of the second passage 12 to flow the liquid dividing material 75 at a constant flow velocity V2.

According to this embodiment, the passage width D2 of the second passage 12 near the intersection 12x is set larger than the passage width D1 of the first passage 11 (D2> D).
1). The passage width D21 of the downstream communication passage 16d of the communication passage 16 of the second passage 12 is set to be the same as or slightly smaller than the diameter of the cell-containing droplet 77 so that the cell-containing droplets 77 can flow one by one. There is. The diameter of the cell outlet 15 is the intersection 1
The diameter is set to be larger than the diameter of the cell-containing droplet 77 immediately after being generated by 2x. The reason is that the cell outlet 15
This is for facilitating the drop of the cell-containing droplet 77 from.

In other words, according to this embodiment, the second passage 1
Of the two communication passages 16, the passage width D2 of the upstream communication passage 16u on the upstream side of the guide portion 19 is set to be relatively large. However, in the communication passage 16 of the second passage 12, the passage width D21 of the upstream communication passage 16d on the downstream side of the guide portion 19 is set to be smaller than the passage width D2, and one cell-containing droplet 77 passes through. The width of the passage is defined as possible.

In the cell alignment substrate 1B, the second passage 1
2 is a detection position 1 between the second inlet 14 and the cell outlet 15.
7 is provided. The detection position 17 is a position where the cell information detection unit 3B performs detection processing on one cell aligned in the aligned state on the cell alignment substrate 1B.

Also in this embodiment, the cell information detecting section 3B is provided. The cell information detection unit 3B is a cell alignment substrate 1B.
31B facing the detection position 17 of the light source, a light receiving portion 32B for receiving the fluorescence emitted from the cells, and a light receiving portion 32B.
And a detection unit 33B that detects information related to cells based on the received light signal in. The light projecting unit 31B includes an LED element 35 that emits light and a lens 36 that condenses the light from the LED element 35. The light receiving section 32B is formed of a filter 37 that selects light of a specific wavelength and a light receiving element 38.

Further, in this embodiment, as in the first embodiment,
A guide portion 19 that guides the cell-containing droplet 77 flowing through the second passage 12 of the cell alignment substrate 1B to the detection position 17 is provided. The guide portion 19 is provided on the upstream side of the detection position 17 and has a function of squeezing the fluid flowing through the second passage 12 of the cell alignment substrate 1B toward the downstream side.
The guide surface 19c is formed so that the passage width of the second passage 12 becomes narrower toward the downstream side of the passage 12.

As shown in FIG. 5, the downstream side of the second passage 12 is branched into a Y shape, that is, the collection path 12s connected to the cell outlet 15 for cell collection and the cell outlet 18 for disposal.
It is branched to a waste liquid path 12t connected to (waste liquid discharge port).

Also in this embodiment, the cell sorting section 5B is provided. The cell sorting unit 5B includes sorting electrodes 60, 61, 62 (guide means for cell-containing droplets) formed on the bottom of the cell alignment substrate 1B, a gas ejection unit 50, and a large number of cells individually collected. It has a collection plate 55 (cell collection means) having a hole-shaped collection unit 54, and a drive unit 56 for moving the collection plate 55 in the X direction and the Y direction.

The sorting electrode 61 is provided on the back surface of the bottom of the collection path 12s connected to the cell outlet 15 for cell collection. The sorting electrode 62 is provided on the back surface of the bottom portion of the waste liquid path 12t connected to the cell outlet 18 for disposal. The sorting electrode 60 is provided on a branch portion between the recovery passage 12s and the waste liquid passage 12t, or on the back surface of the bottom portion upstream of the branch portion.

As in the first embodiment, the gas ejection portion 50 is
A jet nozzle 50a facing the cell outlet 15 so as to jet air to the cell-containing droplets 77 flowing through the second passage 12 and reaching the cell outlet 15, an air supply source 50b containing compressed air, and a jet nozzle 50a. Valve 50c for opening and closing
Have and.

The jet nozzle 50a is arranged above the cell outlet 15 of the second passage 12, and jets air from above onto the cell-containing droplet 77 that has reached the cell outlet 15. Therefore, the cell-containing droplet 77 that has reached the cell outlet 15 of the second passage 12 can be dropped and separated from the cell outlet 15 of the second passage 12 by air pressure.

In the cell alignment substrate 1B, the second passage 1
An increasing passage 24 communicates with the second cell outlet 15.
An increasing portion 25 for containing a buffer solution is connected to the increasing passage 24. The volume increasing passage 24 is for supplying a buffer solution functioning as a liquid material to the cell outlet 15 of the second passage 12 to increase the liquid portion of the cell-containing droplet 77 of the cell outlet 15. Sorting plate 52 of cell sorting unit 5
Are provided below the cell outlet 15 of the second passage 12.

In use, as in the first embodiment,
From the second inlet 14 of the second passage 12 of the cell alignment substrate 1B to the cell outlet 1 of the dividing liquid material 75 by the second fluid source 22.
Flow at a constant flow velocity V2 in the direction of arrow W2. In this state, the cell suspension 70 containing a large number of cells is supplied with the first fluid source 2
1, the cell alignment substrate 1B is caused to flow from the first inflow port 13 of the first passage 11 toward the intersection 12x with the second passage 12 in the first passage 11 at a constant flow velocity V1 in the arrow W1 direction.

As described above, the second of the cell alignment substrate 1B is used.
When the disrupting liquid material 75 flows in the passage 12, the cell suspension 70 containing cells flows in the intersection portion 12x between the first passage 11 and the second passage 12. Therefore, the cell suspension 70 is subjected to a shearing force by the liquid material for disruption 75 and finely fractured. As a result, a large number of cell-containing droplets 77 containing one cell 78 are continuously generated.

The generated cell-containing droplets 77 flow toward the downstream of the second passage 12 of the cell alignment substrate 1B together with the dividing liquid material 75. At this time, since the guide portion 19 is provided in the second passage 12 of the cell alignment substrate 1B, the cell-containing droplet 77 flowing in the second passage 12 is restricted in the passage width direction of the second passage 12, The cell-containing droplets 77 flow in a line at equal intervals along the downstream communication passage 16d of the communication passage 16 of the second passage 12 in a line. Therefore, the cells contained in the cell-containing droplet 77 flowing through the second passage 12 are
The detection position 17 of the cell information detection unit 3B can be reached. That is, the cells contained in the cell-containing droplet 77 are
The light collection position of the cell information detection unit 3B can be satisfactorily reached.

Therefore, the light projecting section 31 of the cell information detecting section 3B
The detection light emitted from B emits the cells 7 of the cell-containing droplet 77.
Focus on 8. Therefore, the fluorescent substance previously carried by the cell 78 is excited. The excited fluorescence is received by the light receiving unit 32B through the filter 37. As a result, the cell information detection unit 3 determines whether the cell 78 of the cell-containing droplet 77 that has reached the detection position 17 is the target cell. When the detected cells 78 of the cell-containing droplet 77 are target cells, the control system outputs a target cell signal.
When the detected cell 78 of the cell-containing droplet 77 is not the target cell, the control system outputs a non-target cell signal.

According to this embodiment, the sorting electrode 60 provided upstream of the branch portion between the waste liquid path 12t and the recovery path 12s charges the cell-containing droplet 77.

If the cells 78 of the cell-containing droplet 77 flowing through the second passage 12 are the target cells, the cell information detecting section 3
When detected by B, a voltage is applied to the selection electrode 61 by the control system. As a result, the cell-containing droplet 77
Is guided toward the collection path 12s by the electrostatic attraction of the sorting electrode 61, that is, in the direction of arrow W5 to the cell outlet 15 for cell collection. Further, the increasing portion 25 controlled by the control system causes the buffer solution contained therein to increase in the passage 24.
Shed on. Thereby, the cell outlet 15 of the cell alignment substrate 1B
A buffer solution (liquid substance) is replenished to the cell-containing droplet 77 in (1) to increase the liquid portion of the cell-containing droplet 77. As a result, the size of the cell-containing droplet 77 at the cell outlet 15 for cell recovery is increased, which is advantageous for separating the cell-containing droplet 77 from the cell outlet 15 of the second passage 12 of the cell alignment substrate 1B. . Therefore, the selection electrodes 60 and 61 described above can function as a guide unit that guides the cell-containing droplets containing the target cells 78 to the collection path 12s for collecting cells.

After performing the volume increasing operation as described above, the control system supplies air to the cell-containing droplet 77 at the cell outlet 15 of the second passage 12 of the cell alignment substrate 1B from the nozzle 50a of the gas ejection unit 50. A cell-containing droplet 77 is applied by air pressure.
A detachment operation is performed in which the cells are dropped from the cell outlet 15 to be detached.

According to this embodiment, when the cells 78 contained in the cell-containing droplet 77 are the target cells, the predetermined collecting portion 54 of the collecting plate 55 is located right below the cell outlet 15. For this reason, the cell-containing droplets 77 that have fallen out of the cell outlet 15 and are separated are received by the desired collecting portion 54 of the collecting plate 55 and collected.

As a result of the detection by the cell information detecting section 3 described above, when the cell 78 of the cell-containing droplet 77 is detected not to be the target cell, a voltage is applied to another sorting electrode 62 by the control system. , The cell-containing droplet 77
Is electrostatically attracted toward the waste liquid path 12t by the sorting electrode 62, that is, in the direction of the arrow W6, and reaches the cell outlet 18 (waste liquid discharge port) not through the cell outlet 15 but through the waste liquid path 12t. It falls from (waste liquid discharge port) and is received by a waste liquid storage container (not shown).

Therefore, when the cells 78 of the cell-containing droplet 77 are not the target cells, the above-mentioned selection electrodes 60 and 62 can function as a guiding means for guiding the cell-containing droplet 77 to the disposal route.

The cell-containing droplet 77 containing cells which are not the target cells may be naturally dropped from the cell outlet 18 for disposal, or air may be applied from a nozzle of another gas ejecting portion and air pressure may be applied to the cell outlet. A detaching operation may be performed in which the 18 cell-containing droplets 77 are dropped and detached.

(Others) According to the above-described first and second embodiments, the first fluid source 21 and the second fluid source 22 are micro pumps, but the present invention is not limited to this, and a fluid tank may be used. Further, a micro cylinder device that pushes out with a piston may be used. The dividing liquid material 75 is not limited to oil, but a fluorine-based inert liquid (registered trademark: Fluorinert)
Can also be used. According to the first embodiment, the cell information detection unit 3 has the tip end at the detection position 17 of the cell alignment substrate 1.
An optical fiber 30 facing the optical fiber 30 and a light projecting section 3 having a laser element projecting a laser beam on the other end of the optical fiber 30.
1, the light receiving unit 32 that receives fluorescence, and the detection unit 33 that detects information about cells based on the light reception signal of the light receiving unit 32, but the present invention is not limited to this.

According to the above-described first and second embodiments, the cell-containing droplets 77 containing one cell 78 are aligned with the dividing liquid material 75 in a line at equal intervals. Although it flows toward the downstream of the second passage 12 of the substrate 1B for use, the cell-containing droplet 77 may be formed into a plurality of cells 7 depending on the case.
A form containing 8 may be used.

According to the above embodiment, the downstream communication passage 16
The passage width of d is set to be the same as or slightly smaller than the diameter of the cell-containing droplet 77 immediately after generation, but the present invention is not limited to this, and the passage width of the downstream communication passage 16d is set to the cell-containing droplet 7 immediately after generation.
It may be slightly larger than the diameter of 7.

According to the above-described first and second embodiments, the gas ejection unit 50 includes the ejection nozzle 50a for ejecting air to the cell-containing droplet 77, the air supply source 50b for containing compressed air, and A valve 50 for opening and closing the ejection nozzle 50a
However, the present invention is not limited to this, and a micro pump that ejects a gas such as air to the cell-containing droplet 77 may be used.

According to the second embodiment, the sorting electrode 61 is provided in the collection passage 12s connected to the cell outlet 15 for cell collection, and the sorting electrode 62 is connected to the waste liquid passage 12t connected to the cell outlet 18 for disposal. Is provided in the electrode 6 for sorting
0 is provided at the branch portion of the recovery passage 12s and the waste liquid passage 12t or at the upstream of the branch portion, but it is not limited to this. In short, the sorting electrode collects the cell-containing droplet 77. It may be provided at a position where it can be guided to the route 12s or the waste liquid route 12t. The cell alignment substrate 1 may be attachable / detachable or fixed to the cell separation / selection device.

In addition, the present invention is not limited to the above-described embodiments, but can be implemented with various modifications without departing from the scope of the invention.

The following technical idea can be understood from the above description.

(Additional Item 1) A cell aligning section which treats a cell suspension containing a large number of cells and causes the cells to align and flow at intervals, and the cells aligned in the cell aligning section. The cell separation / selection device comprising a cell information detection unit that performs detection processing to detect information about cells and a cell selection unit that selects cells based on the information detected by the cell information detection unit. The alignment unit has a detection position at which the cell information detection unit performs a detection process on the cells of the cell-containing droplets aligned by the cell alignment unit,
The cell separation / sorting device, wherein a guide part for guiding the cell-containing droplets flowing through the second passage to the detection position is provided in the cell alignment part. In this case, since the cell-containing droplet flowing through the second passage is guided to the detection position by the guide portion, the detection processing of the cell of the cell-containing droplet can be performed well. The guide portion can be formed by a throttle portion that narrows the passage width of the second passage.

(Additional Item 2) A cell aligning section which treats a cell suspension containing a large number of cells to flow the cells in an aligned manner at intervals, and to the cells aligned in the cell aligning section. A cell separation / selection device comprising a cell information detection unit that performs detection processing to detect information about cells, and a cell selection unit that selects cells based on the information detected by the cell information detection unit. A cell separation / sorting device comprising: a gas jetting unit for applying a gas to a droplet and performing a detaching operation for detaching the cell-containing droplet from the cell outlet of the second passage by gas pressure. In this case, since the cell-containing droplet is separated from the cell outlet of the second passage by gas pressure such as air pressure, it is possible to suppress damage to the cells at the time of separation.

(Additional Item 3) A cell aligning portion which is prepared by treating a cell suspension containing a large number of cells so that the cells are aligned at an interval and flowing, and the cells aligned by the cell aligning portion. A cell separation / selection device comprising a cell information detection unit that performs detection processing to detect information about cells, and a cell selection unit that selects cells based on the information detected by the cell information detection unit. At least one of before and during the detaching operation for detaching the droplet from the cell outlet of the second passage, the liquid content is replenished to the cell-containing droplet to increase the liquid portion of the cell-containing droplet. A cell separation and sorting apparatus, which is provided with an increasing unit. In this case, since the size of the cell-containing droplet can be increased, the cell-containing droplet can be favorably separated from the cell outlet.

[0094]

As described above, according to the present invention, it is possible to provide a cell separation / selection device and a cell alignment substrate that can generate cell-containing droplets while suppressing damage to cells.

[Brief description of drawings]

FIG. 1 is a perspective view schematically showing a cell separation / selection device.

FIG. 2 is a configuration diagram schematically showing a cell separation / selection device.

FIG. 3 is a plan view showing a form of an intersection of a first passage and a second passage.

FIG. 4 is a plan view showing a form of a guide portion in a second passage of the cell alignment substrate.

FIG. 5 is a configuration diagram schematically showing a cell separation / selection device.

FIG. 6 is a configuration diagram schematically showing a cell alignment unit according to a conventional technique.

[Explanation of symbols]

In the figure, 1 is a cell alignment substrate (cell alignment part), and 11 is a first
Passage, 12 is second passage, 12x is intersection, 17 is measurement position, 19 is guide, 19c is guide surface, 24 is increase passage, 25 is increase passage, 25 is increase passage, 3 is cell information detector, 70 is cell suspension Liquid, 75 is a liquid material for disruption, 77 is a cell-containing droplet, 78
Is a cell, 5 is a cell sorting unit, 50 is a gas ejection unit, 52 is a sorting plate, 55 is a collecting plate, 57 is a through hole, 58 is a waste liquid storage container, and 60 to 62 are sorting electrodes.

Claims (6)

[Claims] 1. A cell aligning unit that treats a cell suspension containing a large number of cells and causes the cells to align and flow at intervals, and a sensing process for the cells aligned in the cell aligning unit. In a cell separation and sorting device comprising: a cell information detecting unit that detects information about cells by performing a cell sorting unit that sorts cells based on the information detected by the cell information detecting unit; A first passage through which a cell suspension containing cells flows, and a flow path of the cell suspension in the first passage which is connected to the first passage in a direction intersecting with the first passage and which divides the flow of the cell suspension, A cell separation / sorting device, comprising: a second passage through which a dividing liquid material that forms a large number of cell-containing droplets flows. 2. The cell separation / sorting device according to claim 1, wherein the cell alignment section includes a common base section having the first passage and the second passage. 3. The cell alignment unit according to claim 1 or 2, wherein the cell alignment unit has a detection position at which the cell information detection unit performs a detection process on the cells of the cell-containing droplets aligned by the cell alignment unit. A cell separation / sorting device having a guide part for guiding the cell-containing droplets flowing through the second passage to the detection position. 4. The detachment operation according to claim 1, wherein gas is applied to the cell-containing droplets and the cell-containing droplets are detached from the cell outlet of the second passage by gas pressure. A cell separation / sorting device, characterized in that a gas jetting portion is provided. 5. The method according to any one of claims 1 to 4, wherein at least one of before the detaching operation for detaching the cell-containing droplet from the cell outlet of the second passage and at the time of the detaching operation, A cell separation / sorting device, comprising: an increasing portion for replenishing a liquid substance in the cell-containing droplets to increase a liquid portion of the cell-containing droplets. 6. A cell alignment substrate for aligning cells at intervals by flowing a cell suspension containing cells, wherein the cell alignment substrate comprises a cell suspension containing cells. A liquid material for disruption, which communicates with the passage in a direction intersecting with the first passage and divides the cell suspension in the first passage to form a large number of small cell-containing droplets containing cells. And a second passage through which the cells flow.