JP2512387B2 - Cell sorting method and cell collection method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for sorting cells and a method for collecting cells, and more particularly, to a method for sorting cells and a method for collecting cells utilizing a difference in the state of cell adhesion.

[0002]

2. Description of the Related Art Conventionally, cells are selected by contacting a cell with another cell or substance, observing a difference in adhesion between the cell and another cell or substance contacted with the cell, and selecting the cell. Sorting methods are known.

Heretofore, such cell sorting methods include a method using a swirling incubator and a plurality of micro-organisms.
Methods using pipettes have been proposed and discussed.

[0004] Among the above-mentioned cell sorting methods, a method using a swirling incubator means that a plurality of isolated cells are put in a container filled with a culture solution, placed in a swirling incubator, and subjected to the flow and vibration of the culture solution by swirling. The cells are brought into contact with each other, and after a certain period of time, the state of the cells placed in the container is observed. Based on the results of this observation, cells that are in an adhered state or cells that are not in an adhered state are selected.

[0005] The collection of the cells selected in this manner has been performed by suction using a micropipette or the like for collection.

In the method using a plurality of micro-pipettes, a first micro-pipette fixed to a mechanical first fine-movement device and a first micro-movement device to which the first micro-pipette is fixed And a second micro-pipette fixed to a second micro-motion device different from the first micro-motion device, wherein the first micro-motion device and the second micro-motion device use the first micro-pipet and the second micro-motion device. -The operation for cell sorting was performed while slightly moving the pipette.

That is, after selecting the first cell in the culture solution, the first micropipette is operated by the first micromotion device, and the first cell is suction-fixed to the tip of the first micropipette. . Next, when a second cell different from the first cell in the culture solution is selected, the second micropipette is operated by the second micromotion device, and the second micropipette is operated. The second cell is suction-fixed to the tip. Thereafter, further, the second micropipette is operated by the second micromotion device, and the second cells sucked and fixed to the tip of the second micropipette are transferred to the first micropipette.
The second cell is separated from the second micropipette while maintaining contact between the first cell and the second cell after a certain time, by contacting the first cell suction-fixed to the tip of the pipette. I do.

After the second cell is separated from the second micropipette, the first cell and the second cell adhere to each other depending on whether the second cell falls due to gravity. It was determined whether the cells were sorted or not.

The collection of the cells selected in this manner can be performed by operating the first micropipette with the first microtremor or by collecting the first cell from the first micropipette. After separation, the micro
The sample was collected by suction using a pipette or the like.

[0010]

However, in the above-described cell sorting method, there is a problem that individual selection of cells based on the state of adhesion may not be performed accurately. .

That is, in the above-described conventional cell sorting method using a swirling incubator, the cells are contacted with each other by chance due to the flow of a culture solution, vibration, and the like. For this reason, even if the density of the isolated cells in the culture solution, the speed and amplitude of the swirling of the swirling incubator, and the time of the swirling are adjusted, it is inevitable that the state of contact differs significantly between individual cells. Was.

As a result, whether the contact between the cells is one-to-one between the isolated cells, one-to-many between the isolated cells and a plurality of cell aggregates, or many-to-many between the plurality of cell aggregates Was difficult to identify.

However, in general, the state of adhesion between cells is
Because it depends on the number of cells in contact, if a cell cannot be contacted under certain conditions by accident, a particular isolated cell will identify other cells or substances with which it has contacted, It will not be possible to reveal the fact that it only shows adhesion to species of cells and substances. For this reason, in the method using a swirling incubator, it is actually extremely difficult to individually select cells using the state of adhesion as an index.

On the other hand, in the above-mentioned conventional cell sorting method using a plurality of micro pipettes, cells can be brought into contact with each other under specific conditions, not by accident. The point that the state can be examined is advantageous as compared with the method using a swirling incubator.

However, if cells are suction-fixed to the tip of the micropipette, an unnatural tension is applied to the cell membrane unless the suction pressure is delicately adjusted, causing damage or deformation to the suction-fixed cells, and It was to affect cell utilization.

That is, if the suction pressure is applied too much to the cells, the cells will be destroyed, and it is necessary to perform the cell sorting operation again.

Incidentally, the adjustment of the suction pressure for sucking and fixing the cells to the tip of the micro pipette needs to be performed finely as appropriate in accordance with the capillary phenomenon which differs for each micro pipette. Have difficulty.

Even if the suction pressure of each micro pipette can be adjusted as described above, it is necessary to separate the second cells from the second micro pipette when examining the state of cell-cell adhesion. This separation operation also required fine adjustment of the suction pressure.

The adjustment of the suction pressure in this separation operation is as follows.
Adjustment of the suction pressure during suction fixation is more difficult, but insufficient adjustment of the suction pressure in this separation operation may cause unnecessary momentum or disturbance to the cells or the surrounding culture medium. However, even with cells showing an adhesive state, the adhesive state is released by such momentum or disturbance, and there is a fear that it is difficult to determine the formation of the adhesive.

As described above, the conventional cell sorting method as described above has an inherent problem that individual selection of cells based on the state of cell adhesion may not be performed accurately. Met.

The present invention has been made in view of such various problems of the prior art, and it is an object of the present invention to accurately select individual cells by using the state of cell adhesion as an index. It is an object of the present invention to provide a cell sorting method and a cell collection method which can be performed in the above.

[0022]

Means for Solving the Problems In order to achieve the above object, a cell sorting method according to the present invention comprises contacting any cell with another object and then contacting the cell with the object by the contact. Based on the state of adhesion, in the cell sorting method for sorting the cells, the cells are captured by irradiating the cells with light, and the light is moved relative to the object, thereby capturing the cells with the light. The cell is moved in the direction of the object, the cell is brought into contact with the object, the irradiation of the light is stopped after the contact between the cell and the object, the cell captured by the light Is released, and the cells are sorted based on the state of adhesion between the cells and the object due to the contact.

In the cell sorting method according to the present invention, if the cell and the object adhere to each other due to the contact between the cell and the object, the cell may be irradiated with light as necessary. By capturing the light and moving the light relative to the object, a force is applied to the cells captured by the light in a direction to separate the cells from the object, and the cells by the contact and the cells The cells were sorted based on the state of adhesion to the object.

On the other hand, the cell collection method according to the present invention comprises the steps of: after contacting an arbitrary cell with another object, sorting the cells based on the state of adhesion between the cell and the object due to the contact. Performing, in the cell collection method of collecting the selected cells at an arbitrary location, capturing and irradiating the cells with light, and moving the light relative to the object, thereby obtaining the light Move the cells captured by in the direction of the object, contact the cells and the object,
The irradiation of the light is stopped after the contact between the cell and the object, the capture by the light of the cell captured by the light is released, and the adhesion of the cell and the object by the contact is released. Based on, based on the sorting of the cells, further irradiating the selected cells with light and capturing the light, after moving the selected cells captured by the light to any location, stopping the irradiation of the light In addition, the capture of the selected cells captured by the light by the light is released, and the selected cells are collected at the arbitrary location.

Further, in the cell collection method according to the present invention, if the cells adhere to the object by contacting the cells with the object, the cells may be irradiated with light as necessary. By capturing and moving the light relative to the object, a force is applied in a direction to separate the cell captured by the light from the object, and the cell is contacted with the cell. Based on the state of adhesion to the object, sorting the cells, further irradiating the selected cells with light to capture, after moving the selected cells captured by the light to any location, The irradiation of the light was stopped, the capture of the selected cells captured by the light by the light was released, and the selected cells were collected at the arbitrary location.

[0026]

When an object is irradiated with condensed light, light pressure is generated in the object due to the reflection and refraction of light, and the object can be captured.

Therefore, when the cells are captured by irradiating the cells with light, the cells captured by the light can be relatively moved toward an arbitrary place. For this reason, it becomes possible to move a specific cell toward a target object and to contact the specific cell with the target object.

Further, the capturing power of the object by the light can be easily and precisely controlled by adjusting the light output incident on the object, so that the necessary minimum capturing force that does not impair the cells to be captured can be easily obtained. In addition, by stopping the light irradiation, the cells can be released without giving any harmful vibration, so that unnecessary force or disturbance is not given to the cells or the surrounding culture solution.

[0029] Therefore, individual selection and collection of cells using the state of cell adhesion as an index can be performed accurately.

[0030]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a block diagram showing an embodiment of a cell sorting method and a cell collection method according to the present invention.

FIG. 1 shows a cell selection / collection device for carrying out the cell selection method and the cell collection method according to the present invention. The cell selection / collection device includes a laser unit 10, It comprises a microscope irradiation unit 20 and a sample container unit 30.

The laser section 10 includes a continuous wave YAG laser 11 for generating an infrared laser, a half-wave plate 12 for controlling the polarization direction of a laser beam emitted from the continuous wave YAG laser 11, and a half-wave plate. Wave plate 1
It comprises a polarizing beam splitter 13 for splitting the laser beam emitted from 2 into two beams, and a light source 14 for illumination.

In the laser section 10, a linearly polarized laser beam emitted from the continuous wave YAG laser 11 is used.
The polarization direction of the beam is arbitrarily controlled by a half-wave plate 12 and is incident on a polarization beam splitter 13. Further, the polarization direction is controlled by the polarization beam splitter 13, and the single laser beam is divided into two laser beams. One of the laser beams is superimposed on the light from the illumination light source 14 and is incident on the microscope irradiation unit 20, and the other laser beam is directly incident on the microscope irradiation unit 20.

The output ratio between the two laser beams emitted from the polarization beam splitter 13 is determined by the polarization direction of the polarization beam splitter 13.

The output of the laser beam emitted from the continuous wave YAG laser 11 can be appropriately controlled.

Next, the microscope irradiating unit 20 includes a first microscope 21 located above and a second microscope 22 located below.
In the first microscope section 21, a television camera 23 is provided.

The television camera 23 has a continuous oscillation YA
This is for confirming the optical path of the infrared laser generated by the G laser 11, and has sensitivity in the infrared region.

Then, as described above, one of the two laser beams emitted from the polarization beam splitter 13 is introduced into the optical path of the epi-fluorescence device of the first microscope 21 and the other laser beam. The laser beam is superimposed with light from the illumination light source 14 and guided to the optical path of the second microscope 22 for illumination.

However, the condenser lens 24 for illumination of the second microscope 22 is a high-precision objective lens because it is necessary to perform precise light collection.

Further, the sample container section 30 contains a sample container 3 containing a culture solution A for containing cells such as isolated cells.
1, a micropipette 32, and a collection container 33. The entire sample container section 30 is fixed to a fine movement stage 34 arranged on a base on which the first microscope 21 and the second microscope 22 are installed. Therefore, by moving the fine movement stage, the laser beam is put into the culture solution A filled in the sample container 31 at a position on the optical path of the laser beam emitted from the first microscope 21 and the second microscope 22. Cells can be selected and aligned.

The base end 3 of the micro pipette 32
2a is connected to a vacuum pump (not shown), and by operating this vacuum pump, cells contained in the culture solution A filled in the sample container 31 can be fixed by suction.

On the other hand, the collection container 33 is made of a material that transmits light, and a small amount of laser light is collected around the collection container 33.
Even if the beam is reflected or scattered, a laser
The light output by the beam is set so as not to be affected.

Next, referring to FIGS. 2A to 2D, a procedure for selecting and collecting cells using the above-described cell selection / collection apparatus will be described.

First, the culture solution A filled in the sample container 31
A first microscope 21 and a second microscope 2 are generated by the continuous wave YAG laser 11 on any one sample cell B ₃ among the sample cells B _{1 to} B ₅ placed therein.
Irradiating a laser beam C emitted through 2;
Capturing the sample cell B _3. After that, the fine movement stage 34
To move the sample cell B ₃ captured by the laser beam C to the tip 32 b of the micropipette 32.
Move up to. Then, the vacuum pump is driven and a predetermined suction force is applied to the tip 32b of the micro pipette 32.
Then, the sample cell B ₃ captured by the laser beam C is fixed by suction (FIG. 2A).

Next, a sample cell B ₄ was used as a second sample cell.
Select, as above, in the sample cell B ₄ by irradiating a laser beam C emitted from the first microscope 21 and the second microscope 22, to capture the sample cell B _4. Thereafter, the fine movement stage 34 is moved to move the sample cell B ₄ captured by the laser beam C to the sample cell B ₃ suction-fixed to the tip 32 b of the micropipette 32, thereby moving the sample cell B _3. and contacting the sample cell B ₄ (Figure 2 (b)).

[0046] Then, laser while the beam remains C trapped, while the sample cell B ₃ for a predetermined time the sample cell B ₄
After contacting the stops the generation of the laser by the continuous wave YAG laser 11, releases the capture by laser beams C of the sample cell B _4. After releasing the capture of the sample cell B _{4 by} the laser beam C, the adhesion state between the sample cell B ₃ and the sample cell B ₄ is observed (FIG. 2).
(C)).

Based on the observation of the adhesion state between sample cell B ₃ and sample cell B ₄ , a sample in which adhesion was observed (or a sample in which adhesion was not observed) was determined as shown in FIGS. As described with respect to (b), after capturing by the laser beam C and moving to the collection container 33, the capture by the laser beam C is released and stored in the collection container 33 (FIG. 2 (d)). )).

When observing the adhesion state between the sample cell B ₃ and the sample cell B ₄ (FIG. 2C), when the adhesion between the sample cell B ₃ and the sample cell B ₄ is observed, Alternatively, the measurement of the adhesive force related to the adhesion may be performed as necessary. That is, the sample cell B ₄ is captured by the laser beam C generated by the continuous wave YAG laser 11,
Thereafter, the fine movement stage 34 is moved so that the sample cell B ₄
The manipulated to detach from the sample cell B _3. On this occasion,
By adjusting the output of a continuous wave YAG laser 11, examine the minimum optical power required to pull the sample cell B ₄ from a sample cell B _3, examine the adhesive strength of the sample cell (Figure 3).

As described above, based on a more detailed observation of the adhesion state between the sample cells B ₃ and B ₄ by examining the adhesive force of the sample cells, as described above with reference to FIG. After the desired sample cells are captured by the laser beam C and moved to the collection container 33, the capture by the laser beam C is released and accommodated in the collection container 33.

When the sample cells collected in the collection container 33 are used outside the sample container 31, the entire collection container 33 is taken out, or the inside of the collection container 33 is prepared using a separately prepared micropipette. The sample cells may be removed by suction.

That is, when the object is irradiated with the condensed light, light pressure is generated on the object due to the reflection and refraction of the light. Generally, this light pressure is extremely weak, but using a laser as a light source and condensing light near the diffraction limit not only provides sufficient power to manipulate cells in a culture solution, but also collects light. An object can be captured near the light spot. Further, by changing the position of the laser beam, the object can be manipulated three-dimensionally.

In particular, when an object is captured using two laser beams facing each other, such as the laser beam C emitted from the first microscope 21 and the second microscope 22 described above, the comparison can be made. Vertical movement, which is difficult to perform, can be relatively easily performed over a long distance of 1 mm or more.

Furthermore, the capturing power of the cells by the laser beam can be easily and precisely controlled by adjusting the light output incident on the cells, so that the minimum capturing power that does not impair the sample cells can be easily obtained. Can be obtained.

When examining the adhesion state, use a laser
By stopping the irradiation of the beam, the cells can be released without generating any harmful vibration that adversely affects the adhesion state of the cells.

Further, by examining the minimum light output of the laser beam required to separate the sample cells showing the adhesion, the adhesive force of the sample cells can be examined. As a result, the adhesion state of the sample cells can be examined in more detail, so that the cell type can be specified more accurately.

Furthermore, the irradiation area of the condensed laser light can be limited to a very narrow area of about 1 μm in diameter, so that in the case of a normal biological sample, only one desired cell can be selectively used. Can be captured. In addition, it is also possible to capture a plurality of cell aggregates according to the combination of cells to be brought into contact with each other. And many-to-many of a plurality of cell aggregates can be arbitrarily set.

FIG. 4 shows the results of measuring the adhesive properties of hydra endodermal and ectodermal epithelial cells using the above-described cell selection / collection apparatus. As sample cells, isolated cells were used. In FIG. 4, "O" indicates a combination in which cell adhesion was observed, and "X" indicates a combination in which cell adhesion was not observed.

As can be understood from FIG. 4, it is apparent that selective adhesion occurs only between cells of the same kind. Therefore, based on FIG. 4, it is possible to determine the origin of any hydra epithelial cells, and select and collect them.

In the measurement of the adhesion characteristics of the endodermal and ectodermal epithelial cells of the above-mentioned hydra, the combination in which the adhesion was observed was again applied to the sample cells which had once adhered, as shown in FIG. An operation of irradiating a laser beam and separating the bonded portion was performed. As a result, the ectodermal hydra epithelial cells could easily detach the adherent part, whereas the endodermal hydra epithelial cells could detach the adherent part no matter how much the light output of the laser beam was increased. Did not.

That is, it can be seen that even in the sample cells in which the adhesion is observed, there is a clear difference in the adhesive strength depending on the cell type. Therefore, by examining the adhesive strength of the sample cells, the origin of any hydra epithelial cells can be determined more accurately, and the cells can be selected and collected.

In the above embodiment, a single continuous oscillation YAG laser was used as a laser source, and a single laser beam was divided to obtain two laser beams. However, the present invention is not limited to this. Alternatively, two laser sources may be provided so that a laser beam is obtained from each laser source.

When two laser sources are provided as described above, the control of the condensing state and the light output of the two laser beams can be performed more easily than the configuration shown in FIG. This is advantageous because it is possible to do so.

The laser beam has a continuous oscillation YA
Without limitation to an infrared laser beam by a G laser or the like, an appropriate one may be used depending on the cells to be captured.

Further, the collection container is not limited to a single container as shown in FIG. 1, but can be selectively collected by partitioning a part of the container with a light transmitting material.

[0065]

Since the present invention is configured as described above, it has the following effects.

Specific cells are captured by light irradiation,
The captured cells are brought into contact with the object, and the irradiation of light is stopped to release the cells without giving any harmful vibration. When the cells are opened, unnecessary force and disturbance are not applied to the cells or the surrounding culture solution.

Further, by examining the minimum light output of the laser beam required to separate the sample cells showing the adhesion, the adhesive force of the sample cells can be examined. As a result, the adhesion state of the sample cells can be examined in more detail.

For this reason, the state of cell adhesion is used as an index.
The individual sorting of cells can be performed accurately.

Furthermore, one-to-one between the individual cells from which the objects of individual selection are isolated, one-to-many between the isolated individual cells and a plurality of cell aggregates, or many-to-many among the plurality of cell aggregates. In addition, since it can be appropriately selected, cell sorting according to the adhesive property can be easily performed.

[Brief description of the drawings]

FIG. 1 is a configuration explanatory view of a cell sorting / collecting apparatus for carrying out a cell sorting method and a cell collecting method according to the present invention.

FIGS. 2 (a) to 2 (d) are explanatory diagrams showing operation procedures of a cell sorting method and a cell collection method according to the present invention.

FIG. 3 is an explanatory diagram showing an operation procedure for examining the adhesive force of a sample cell.

FIG. 4 is a table showing an example of a selective adhesion state of isolated cells obtained by the cell sorting method according to the present invention.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 10 Laser part 11 Continuous oscillation YAG laser 12 Half wave plate 13 Polarized beam splitter 14 Light source for illumination 20 Microscope irradiation part 21 First microscope 22 Second microscope 23 Television camera 24 Condenser lens 30 Sample container part 31 Sample container 32 Micro-pipette 33 Collection container 34 Fine movement stage A Culture solution B ₁ sample cell B ₂ sample cell B ₃ sample cell B ₄ sample cell B ₅ sample cell C Laser beam

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical indication location G01N 1/04 G01N 1/04 H 1/28 15/14 C 15/14 G02B 21/32 G02B 21 / 32 G01N 1/28 J

Claims (6)

(57) [Claims] 1. A cell sorting method in which, after contacting an arbitrary cell with another object, the cell is sorted based on an adhesion state between the cell and the object due to the contact, By irradiating and capturing light, by moving the light relative to the object, the cells captured by the light are moved in the direction of the object, and the cells and the object are Contact, stop the irradiation of the light after the contact between the cells and the object, release the capture of the cells captured by the light by the light, the contact between the cells and the object by the contact A cell sorting method, wherein the cells are sorted based on an adhesion state. 2. A cell sorting method for sorting cells based on the state of adhesion between the cells and the object due to the contact after contacting an arbitrary cell with another object. By irradiating and capturing light, by moving the light relative to the object, the cells captured by the light are moved in the direction of the object, and the cells and the object are When the contact, the irradiation of the light is stopped after the contact between the cell and the object, when the capture of the cell captured by the light is released by the light, the cell and the object by the contact Selectively adhered to the cells by irradiating the cells with light, and moving the light relative to the objects, whereby the objects captured by the light Force in the direction away from Based on the state of adhesion between the cells and the object by said contact, cell sorting method and performing selection of said cell. 3. The cell according to claim 1, wherein the cell is an isolated cell or a plurality of cell aggregates, and the object is another isolated cell or another plurality of cell aggregates.
Item 4. The cell sorting method according to item 1. 4. After contacting an arbitrary cell with another object, the cells are sorted based on an adhesion state between the cell and the object due to the contact, and the selected cell is sorted into an arbitrary cell. In the cell collection method of collecting cells at a location, the cells are irradiated with light and captured, and the light is moved relative to the object, so that the cells captured by the light are captured by the object. Moving in the direction, contact the cell and the object, stop the irradiation of the light after the contact between the cell and the object, capture the cells captured by the light by the light. Release, based on the state of adhesion between the cell and the object by the contact, sorting the cells, further irradiating the selected cells with light and capturing, the selected cells captured by the light After moving to any place , Discontinue irradiation of the light,
A cell collection method, comprising: releasing the selected cells captured by the light from being captured by the light, and collecting the selected cells at the arbitrary location. 5. After contacting an arbitrary cell with another object, the cells are sorted based on the state of adhesion between the cell and the object due to the contact, and the selected cells are sorted into an arbitrary cell. In the cell collection method of collecting cells at a location, the cells are irradiated with light and captured, and the light is moved relative to the object, so that the cells captured by the light are captured by the object. Moving in the direction, contact the cell and the object, stop the irradiation of the light after the contact between the cell and the object, capture the cells captured by the light by the light. When released, if the cell and the object adhere to each other due to the contact, selectively irradiate and capture the cell with light, and move the light relative to the object. The cells captured by the light Applying force in a direction to separate from the object, based on the state of adhesion between the cell and the object by the contact, sorting the cells, further irradiating the selected cells with light, capturing, After moving the selected cells captured by the light to an arbitrary location, the irradiation of the light is stopped, the capture of the selected cells captured by the light by the light is released, and the selected cells are removed. A method for collecting cells, wherein the cells are collected at the arbitrary place. 6. The cell according to claim 4, wherein the cell is an isolated cell or a plurality of cell aggregates, and the object is another isolated cell or another plurality of cell aggregates.
Item 4. The cell collection method according to item 1.