WO2016039397A1

WO2016039397A1 - Method and device for arranging cells using laser

Info

Publication number: WO2016039397A1
Application number: PCT/JP2015/075659
Authority: WO
Inventors: 吉川　研一; 浩章谷口; 太恵子太田; 橋本　周; 晋一朗米田; 葵 ▲吉▼田
Original assignee: 学校法人同志社
Priority date: 2014-09-10
Filing date: 2015-09-09
Publication date: 2016-03-17
Also published as: JPWO2016039397A1

Abstract

Provided are a method and a device for arranging cells using a laser, whereby cells can be arranged so that the cells are prevented from separating from each other. The method according to the present invention comprises: a step for capturing cells using a laser; a step for conveying the captured cells relative to other cells and contacting the cells in a polymer solution that contains a water-soluble polymer in an aqueous solvent; and maintaining the contact among the cells using a depletion effect of the polymer solution. The device according to the present invention comprises: a laser light source; a polymer solution that contains a water-soluble polymer in an aqueous solvent and can exert a depletion effect for maintaining contact among cells; a sample cell for housing the cells and a sample cell; a light condensing lens for condensing the laser in the sample cell; and a moving unit for relatively three-dimensionally moving the sample cell and the light condensing spot of the laser.

Description

Method and apparatus for arranging cells using laser

The present invention relates to a method and an apparatus for arranging cells using a laser.

The cell exhibits its function by taking various arrangements in the living body. Accordingly, it is strongly desired in research in the field of life science that the arrangement of cells in a living body can be reproduced in vitro.

In order to reproduce the arrangement of cells, a technique that can manipulate cells freely is necessary. One example of a technique for manipulating cells is laser trapping. Laser trapping is a technique for condensing a laser and capturing a minute object, which is a dielectric, at the condensing part of the laser, and is also called optical tweezers. According to laser trapping, cells can be captured in a non-contact manner and moved in three dimensions.

For example, Patent Literature 1-2 discloses that cells are manipulated by laser trapping to adhere cells together.

JP 2007-189998 A US Patent Publication No. 2010267105A1 JP 2006-296361 A Japanese Patent Laid-Open No. 03-297385

∙ If cells are simply brought into contact with each other by laser trapping, they will be separated immediately. As this factor, the electrostatic repulsion force by a cell membrane potential, the repulsion accompanying the fluctuation motion of a cell membrane, etc. are mentioned.

In the method disclosed in Patent Document 1, one cell is avidinized in advance and the other cell is biotinylated. Then, these cells are brought into contact with each other by laser trapping and adhered instantaneously and strongly by an avidin-biotin bond.
In the method disclosed in Patent Document 2, one cell is fixed on a chemically modified substrate in advance. The other cell is then transported to and brought into contact with one cell by laser trapping to form a cell pair on the substrate.

As described above, in the conventional method, in order to overcome the problem that the cells are immediately separated from each other, a process for treating the cells is necessary before the cells are brought into contact with each other by laser trapping.

An object of the present invention is to provide a method and an apparatus for arranging cells using a laser, which can obtain stable contact between cells without previously treating the cells.

The inventors focused on the depletion effect caused by the polymer solution. The inventors have found that stable contact between cells can be obtained by utilizing the depletion effect.

The present invention is a method of arranging cells using a laser, comprising:
Capturing cells with a laser;
In a polymer solution containing a water-soluble polymer in which the solvent is an aqueous solvent, transporting the captured cells relative to other cells and bringing them into contact;
And maintaining the contact of these cells by the depletion effect of the polymer solution.

Preferably, the cell contact by the depletion effect is maintained for 300 seconds or more.

Preferably, the method includes a step of placing the formed cell array in an environment in which the polymer solution does not exist.

Preferably, the method includes a step of conveying the formed cell array from the polymer solution to a liquid medium.

Preferably, the cells are arranged three-dimensionally.

Preferably, the concentration C of the water-soluble polymer in the polymer solution is 0.1C ^* ≦ C ≦ 10C ^* when the overlap concentration of the water-soluble polymer is C ^*. Preparing a molecular solution. Further preferably, the concentration C of the water-soluble polymer is 0.3C ^* ≦ C ≦ 3C ^* .

Furthermore, the present invention is an apparatus for arranging cells using a laser,
A laser light source for generating a laser;
A polymer solution that is an aqueous solvent, contains a water-soluble polymer, and can generate a depletion effect for maintaining contact of the cells;
A sample cell for containing the cell and the sample cell;
A condensing lens for condensing the laser from the laser light source in the sample cell;
And a moving unit for moving the sample cell and the condensing point of the laser relatively in three dimensions.

A plurality of the condensing lenses are provided,
The moving unit includes a condensing point moving mechanism for moving the condensing point of the laser in three dimensions with respect to the sample cell for each condensing lens.

Preferably, one of the horizontal directions of the sample cell is formed with a first inlet for introducing the polymer solution into the liquid storage space of the sample cell,
On the other side of the horizontal direction of the sample cell, a second inlet for introducing additional liquid that cannot cause a depletion effect into the liquid storage space is formed,
An exhaust port for discharging air in the liquid storage space is formed at the center of the sample cell.

Preferably, the concentration C of the aqueous polymer in the polymer solution is 0.1C ^* ≦ C ≦ 10C ^* when the overlapping concentration of the water-soluble polymer is C ^* . Further preferably, the concentration C of the water-soluble polymer is 0.3C ^* ≦ C ≦ 3C ^* .

By utilizing the depletion effect of the polymer solution, stable contact of the cells can be obtained without pretreatment of the cells. Arbitrary stable three-dimensional cell arrays can be obtained without fixing the cells to a scaffold such as a gel or a substrate. The arrangement of cells in the living body can be freely reproduced outside the living body.

It is a figure for demonstrating overlap density. It is a figure for demonstrating the stable cell contact by a depletion effect. It is a figure which shows schematic structure of the cell arrangement | sequence apparatus which concerns on one Embodiment of this invention. 4A is a perspective view showing an example of a sample cell in the apparatus of FIG. FIG. 4B is a cross-sectional view showing an example of use of the sample cell of FIG. 4A. It is a figure which shows the principal part of the cell arrangement | sequence apparatus which concerns on other embodiment. The experimental result of Experimental example 1 at the time of using an aqueous solvent is shown. 6A is an image showing a positional change of two NMuMGs with time, FIG. 6B is an image of NMuMG at a specific time, FIG. 6C is a schematic diagram corresponding to FIG. 6B, and FIG. FIG. The experimental result of Experimental example 1 at the time of using a PEG solution is shown. 7A is an image showing a change in position of two NMuMGs with time, FIG. 7B is an image of NMuMG at a specific time, FIG. 7C is a schematic diagram corresponding to FIG. 7B, and FIG. 7D describes an experimental operation. FIG. It is a graph which shows the relationship between the density | concentration of PEG of Experimental example 1, and the contact maintenance probability between cells. The experimental result of Experimental example 2 at the time of making a cell contact for several seconds is shown. 9A is an image showing a positional change of two NMuMGs with time, FIG. 9B is an image of NMuMG at a specific time, FIG. 9C is a schematic diagram corresponding to FIG. 9B, and FIG. 9D describes an experimental operation. FIG. The experimental result of Experimental example 2 at the time of making a cell contact for several minutes is shown. FIG. 10A is an image showing a change in position of two NMuMGs over time, FIG. 10B is an image of NMuMG at a specific time, FIG. 10C is a schematic diagram corresponding to FIG. 10B, and FIG. 10D describes an experimental operation. FIG. It is an image of the experimental result of Experimental example 3 which shows a mode that NMuMG is arranged three-dimensionally in a PEG solution. It is an image of the experimental result of Experimental example 4 which shows a mode that NMuMG is arranged three-dimensionally in a PEG solution. It is a schematic diagram corresponding to the image of FIG. 12B. It is an image of the experimental result of Experimental example 4 which shows a mode that NMuMG is arranged substantially linearly in a PEG solution. It is an image of the experimental result of Experimental Example 5 when erythrocytes are arranged in an aqueous solvent. It is an image of the experimental result of Experimental Example 5 when erythrocytes are arranged in a PEG solution. The image of the experimental result of Experimental example 6 is shown. FIG. 17A is an image showing the state of NMuMG in an aqueous solvent. FIG. 17B is an image showing the state of NMuMG in a dextran solution. The image of the experimental result of Experimental example 6 is shown. FIG. 18A is an image showing the appearance of red blood cells in an aqueous solvent. FIG. 18B is an image showing the state of red blood cells in a dextran solution. It is a figure for demonstrating the utilization example of this invention.

Hereinafter, an embodiment of a method and an apparatus for arranging cells using a laser according to the present invention will be described with reference to the drawings.

[Depletion effect by polymer solution]
In the present invention, the depletion effect of the polymer solution is used for cell arrangement.

“The depletion effect” is the following phenomenon. When two objects approach in the polymer solution, the polymer present between the two objects is eliminated. Then, a region where no polymer exists (that is, a region where the polymer is depleted) is formed between the two objects, while a region where the polymer exists is formed around the two objects. Thereby, the osmotic pressure works and the two objects are pushed from the surroundings to cause agglomeration. This phenomenon is called the depletion effect.

[Preparation of polymer solution]
As shown in FIG. 1A, when the polymer solution is a dilute solution having a low polymer concentration, the polymers do not contact each other and behave as one molecule. On the other hand, as shown in FIG. 1B, when the concentration of the polymer exceeds a certain standard, the polymers start to contact each other. The concentration at which the polymers start to come into contact with each other is referred to as the overlapping concentration C ^* . The overlap density C ^* is defined by the following formula 1.

Formula 1

In Formula 1, M is the molecular weight of the polymer, [rho is the specific gravity of the solvent, N _A is Avogadro constant, Rg is the radius of gyration of the polymer. The inertia radius Rg has a relationship represented by the following formula 2.

Formula 2

In Equation 2, n is the degree of polymerization of the polymer, b is the Kuhn length, υ is a scaling index, and 1/2 ≦ ν ≦ 3/5.

By using this overlap concentration C ^* as a guide, for example, a polymer solution capable of producing a depletion effect can be prepared by using the order of the overlap concentration C ^* as a guide. If the polymer concentration C is too low relative to the overlap concentration C ^* , the depletion effect does not occur. On the other hand, when the polymer concentration C becomes higher than the overlap concentration C ^* , the depletion effect occurs, but the viscosity of the polymer solution suddenly increases. As described below, the present invention is a cell with a laser for conveying a polymer solution, the viscosity of the solution when the concentration C of the polymer overlap concentration C ^* is too high than becomes extremely large, conveyance of the cell Can cause problems.

In the present invention, in view of depletion effects and cell transport, it is preferable to adjust the polymer solution so that the concentration C of the polymer is in the range of 0.1C ^{^*} ≦ C ≦ 10C ^*. A more preferable range of the concentration C of the polymer is 0.3C ^* ≦ C ≦ 3C ^* .

As described above, when the polymer concentration C exceeds the overlap concentration C ^* , the viscosity of the polymer solution suddenly increases. Therefore, if the polymer concentration C is gradually increased to find a place where the viscosity of the polymer solution starts to increase suddenly, the polymer concentration C can be obtained without calculating the overlap concentration C ^*. ^* It is possible to easily adjust the polymer solution which is about.

Since the present invention targets cells, the solvent of the polymer solution is an aqueous solvent. The aqueous solvent is water or a mixed solvent of water and a hydrophilic solvent. The hydrophilic solvent is, for example, a polyhydric alcohol such as ethylene glycol, dimethyl sulfoxide (DMSO), or the like, and it is preferable to allow the cells to remain alive in the polymer solution. The ratio of water in the aqueous solvent is, for example, 10% to 100% by weight percent, and is appropriately determined according to the type of the hydrophilic solvent.

The polymer used can produce a depletion effect as long as it is a water-soluble polymer. Examples of the water-soluble polymer include synthetic polymers such as polyethylene glycol and vinyl alcohol, natural polymers such as proteins (albumin, collagen, etc.), polysaccharides (dextran, starch, etc.).

Note that a substance necessary for culturing cells (for example, glucose or the like) may be added to the polymer solution. That is, a solution obtained by adding a water-soluble polymer to a liquid medium may be used as a polymer solution.

[Cell contact by depletion effect]
As shown in FIG. 2, when cells approach each other in the polymer solution adjusted as described above, aggregation of cells is caused by a depletion effect. Cells usually leave in a normal solution due to an electrostatic repulsion interaction caused by a cell membrane potential or a repulsion effect associated with a fluctuation motion of the cell membrane, but in this polymer solution, they continue to contact each other due to a depletion interaction. That is, due to the depletion effect of the polymer solution, the contact between cells is maintained ignoring the influence of the repulsive interaction between the cell membranes.

When the contact of cells is maintained by the depletion effect, the cells adhere to each other. As used herein, “adhesion” means that cells are in stable contact with each other so that they do not leave even when placed in an environment where a depletion effect cannot occur (eg, a normal liquid medium). (Hereinafter the same). It has been empirically known that the contact time until the cells reach an adhesive state is not sufficient in a few seconds, but a few minutes are necessary, and 300 seconds (5 minutes) or more is almost sufficient.

It is presumed that the cells are brought into an adhesion state in this way as follows.

From experimental observations in the following experimental examples, it is known that contact between cells due to the depletion effect is planar contact as shown in FIG. That is, the surface of the cell membrane of both cells is deformed flat and the cells are in contact with each other. The area of this plane contact is estimated to be several μm ² to several tens μm ² .

The above-mentioned repulsive interaction and depletion interaction act between cells in contact with each other, and these compete. As described above, unless the cell-to-cell distance is about the diameter of the polymer, a region in which the polymer is depleted does not occur between the cells, and thus no depletion interaction occurs between the cells. Therefore, the contact between cells due to the depletion effect is, on the microscopic scale, a gap of about several nanometers to several tens of nanometers, which is about the diameter of a polymer, on the order of an inertia radius Rg, between the cell membranes of both cells. It is thought that.

That is, it is considered that the cell contact due to the depletion effect occurs in a state where the flat surfaces of several μm ² to several tens of μm ² of the cell membrane face each other with a gap of several nm to several tens of nm. By providing such a gap, the fluidity of the cell membrane is ensured even in a contact state, that is, components such as proteins and lipids on the surface of the cell membrane are flowing. Due to this fluidity, the positive factors of one cell membrane (eg, a positively charged protein) and the other cell membrane are between the surfaces of a flat cell membrane within the few minutes that cell contact is maintained by the depletion effect. It is considered that the components are rearranged so that negative factors (for example, proteins having a negative charge) face each other. Such attractive interactions are thought to contribute not only to electrostatic interactions but also to various chemical interactions such as hydrophobic interactions and hydrogen bonds. Such rearrangement brings the cells into a more stable contact state, that is, a contact state.

From the above, it is presumed that the cells adhere when the contact of the cells is maintained for several minutes by the depletion effect.

In addition, when cells continue to contact each other for several hours or more, the cells themselves spontaneously form cell adhesion factors such as cadherin and integrin, so that more stable contact between cells can be obtained.

[Cell array device]
Next, an embodiment of an apparatus for arranging cells according to the present invention (hereinafter referred to as a cell arrangement apparatus) will be described. Referring to FIG. 3, the cell array device includes an optical system including at least a laser light source 1 for generating a laser and a condensing lens 2 (an objective lens in the present embodiment) for condensing the laser. Yes. In the optical system of the present embodiment, the beam expander 3, the galvano mirror 4A, the condensing

lenses

5A and 5B, the galvano mirror 4B, the condensing

lenses

5C and 5D, and the optical path from the laser light source 1 to the objective lens 2 The dichroic mirror 6A is arranged in this order.

Furthermore, the cell array device further includes a stage 7, a polymer solution 8, and a sample cell 9. The stage 7 is disposed above the condenser lens 2. In the polymer solution 8, the solvent is an aqueous solvent and contains a water-soluble polymer. In the polymer solution 8, the concentration C of the water-soluble polymer is adjusted using the overlapping concentration C ^* of the water-soluble polymer as a guide so that a depletion effect can be generated. A polymer solution 8 and a plurality of cells to be arranged are accommodated in the internal space of the sample cell 9. When the sample cell 9 is placed at a predetermined position on the stage 7, the sample cell 9 faces the emission side of the condenser lens 2. The stage 7 is made of a material that does not absorb the laser generated by the laser light source 1 or has an opening that allows the laser to pass therethrough. The sample cell 9 is also made of a material that does not absorb the laser at least at the portion where the laser is incident.

The laser from the laser light source 1 passes through the condenser lens 2 via the beam expander 3 and the like, is condensed by the condenser lens 2, and enters the sample cell 9 in a condensed state. With this configuration, the cells in the sample cell 9 can be captured in the vicinity of the laser condensing point (focal point).

The cell manipulation device includes a moving unit for moving the laser focusing point and the sample cell 9 relatively in three dimensions. The moving unit is used to manipulate cells in the polymer solution 8. The moving unit of the present embodiment moves the stage 7 supporting the sample cell 9 in the Z direction (laser optical axis direction) with respect to the laser condensing point, in the X direction perpendicular to the Z direction, and The stage moving mechanism 10 moves in the Y direction perpendicular to the Z direction and the X direction.

Although the moving unit is not shown, the moving point moving mechanism for moving the laser focusing point with respect to the sample cell 9 may be used. For example, the condensing point moving mechanism moves the condensing point in the Z direction with respect to the sample cell 9 by moving the condensing lens 2 in the Z direction, and the condensing point is moved by the rotation of the galvano mirrors 4A and 4B. It is the structure which moves to X direction and Y direction with respect to.

Further, the moving unit may be a combination of the stage moving mechanism 10 and the condensing point moving mechanism. As an example, the relative movement in the Z direction between the laser condensing point and the sample cell 9 is performed by a condensing point moving mechanism, and the relative movement in the X and Y directions between the laser condensing point and the sample cell 9 is moved on the stage. The structure performed by the mechanism 10 is mentioned.

The captured cells can be moved three-dimensionally in the polymer solution 8 by moving the focusing point and the sample cell 9 relatively by the moving unit while capturing the cells by the focused laser. it can. Thereby, the cells captured by the focused laser can be conveyed and brought into contact with other cells in the polymer solution 8 relatively.

The cell array device further includes an observation unit for observing cells in the sample cell 9. The observation unit includes a camera 11, an image processing device 12, a monitor 13, a recorder 14, a mercury lamp 15 (an example of an observation light source), an ND filter 16, an excitation filter 17,

dichroic mirrors

6A and 6B, a barrier filter 18, and the like. . Since the structure of this observation means is well known, its description is omitted.

The wavelength of the laser is not particularly limited, but it is preferable to select a wavelength that is less absorbed by the cells so as not to damage the cells. Moreover, it is preferable that the wavelength of the laser is selected such that the solvent (such as water) of the polymer solution is not heated. Therefore, generally, the range before and after including 1000 nm by infrared light, specifically, the range of 800 nm to 1200 nm is preferable, and the range of 900 nm to 1100 nm is more preferable. For example, the laser light source 1 is preferably an Nd: YAG laser and its fundamental wave (wavelength is 1064 nm) is preferably used. Note that when capturing a cell that absorbs less visible light, the cell can be easily captured without damaging the cell, even if a wavelength in the visible light region of 400 nm to 700 nm is selected.

The laser output is appropriately selected in consideration of the laser wavelength, the presence or absence of damage to the cells, the type and size of the cells, the type of solvent, the viscosity of the polymer solution 8, and the like. For example, when a laser wavelength in the range of 400 nm to 1500 nm is used, the laser output is preferably in the range of 1 mW to 1000 mW. For example, when using the fundamental wave of a Nd: YAG laser, the output is preferably in the range of 5 mW to 100 mW.

The cell array device may be configured as follows.
The sample cell 9 may have the following configuration. As shown in FIG. 4A, the sample cell 9 includes an upper plate 19 and a lower plate 20 that are spaced apart from each other in the vertical direction. Further, the sample cell 9 includes a plurality of spacers that are interposed between the upper plate 19 and the lower plate 20 and define the distance between the

plates

19 and 20. The spacer includes a first spacer 21 and a second spacer 22 that are spaced apart from each other in the first horizontal direction. Two second spacers 22 are provided and are spaced apart from each other in the second horizontal direction perpendicular to the first horizontal direction.

With the above arrangement, the space surrounded by the

plates

19 and 20 and the

spacers

21 and 22 becomes the liquid storage space 23 for storing the liquid. A first introduction port 24 for introducing the polymer solution 8 into the liquid storage space 23 is formed on one side in the second horizontal direction of the sample cell 9, and a depletion effect cannot be generated on the other side. A second introduction port 25 for introducing an additional liquid 27 (for example, a liquid medium) (see FIG. 4B) is formed. Then, the

second spacers

22 and 22 are arranged at a distance from each other, thereby exhausting the air in the liquid storage space 23 to one side in the first horizontal direction at the center of the sample cell 9. Is formed.

When the polymer solution 8 is introduced into the liquid storage space 23 from the first introduction port 24 and the additional liquid 27 is introduced into the liquid storage space 23 from the second introduction port 25, the liquid accommodation is performed as shown in FIG. 4B. The polymer solution 8 is accommodated in a region on one side of the space 23, and an additional liquid 27 is accommodated in the region on the other side. While the

liquids

8 and 27 are introduced, the air in the liquid storage space 23 is discharged from the exhaust port 26. Note that the difference in concentration of the water-soluble polymer between the polymer solution 8 and the additional liquid 27 is maintained for several hours unless stirring is performed.

In such a sample cell 9, if the additional liquid 27 is a liquid medium, the cells cultured in the liquid medium can be easily transported into the polymer solution 8 by the focused laser. Then, the cells adhered in the polymer solution 8 can be returned to the liquid medium again.

Further, the cell array device may be configured so that a plurality of cells can be independently operated by a plurality of focused lasers. For example, as shown in FIG. 5, the optical system includes a plurality of condensing lenses 2 (two in the embodiment) so as to face the sample cell 9. Further, the optical system is configured such that the laser passes through each condenser lens 2 and is condensed in the sample cell 9. This may be achieved such that a laser generated by one laser light source 1 is branched by an optical element, and each of the branched lasers passes through the condenser lens 2. Alternatively, this may be achieved by providing a plurality of laser light sources 1 for each condenser lens 2. In this case, the lasers of the laser light source 1 may have different wavelengths.

The moving unit includes the above-exemplified condensing point moving mechanism for moving the condensing point of the laser in three dimensions with respect to the sample cell 9 for each condensing lens 2. Thereby, the condensing points of a plurality of lasers can be independently moved in three dimensions with respect to the sample cell 9.

According to this configuration, since a plurality of cells can be operated independently at the same time, the cells can be arranged efficiently.

[Cell array]
Arrange cells in the following procedure.
A polymer solution 8 in which the concentration C of the water-soluble polymer is adjusted so that a depletion effect can be produced is prepared, and further, the above cell array device is prepared. Cells to be arranged and the polymer solution 8 are accommodated in the sample cell 9. Then, the sample cell 9 is placed on the stage 7.

Then, the laser irradiated from the laser light source 1 is condensed by the condenser lens 2 and is incident on the sample cell 9. The focusing unit of the laser and the sample cell 9 are relatively moved by the moving unit, and the cells are captured by the focused laser.

In a state in which the cells are captured, the focusing unit of the laser and the sample cell 9 are relatively moved by the moving unit, so that the cells are relatively conveyed and brought into contact with other cells in the polymer solution 8.

Then stop the laser irradiation. The contact of these cells is maintained by the depletion effect by the polymer solution 8 as described above. And if the contact is maintained for several minutes due to the depletion effect, these cells adhere to each other.

Further other cells are captured by the laser, conveyed relative to the cells already in contact, and brought into contact with the polymer solution 8. Stop laser irradiation. Thereafter, a cell sequence is formed by repeating the same steps. As described above, cell contact can be maintained by the depletion effect, and cells can be manipulated in three dimensions by laser trapping, so that an arbitrary three-dimensional cell array can be formed.

Then, after the cells reach an adhesive state, the cell arrangement may be moved from the polymer solution 8 and in an environment where the depletion effect cannot be generated, for example, in a normal liquid medium.

In arranging cells, a single cell may not be captured by a laser, but a plurality of cells may be captured if there is no problem in capturing power. That is, the cell group may be captured by a laser and conveyed to another cell or another cell group and brought into contact therewith. For example, when using the cell array device of FIG. 5, one cell is captured by one focused laser, the other cell is captured by the other focused laser, and at least one of the cells is captured. These cells may be brought into contact with each other by moving the focal point of the laser. The cells to be contacted may not be the same type, but may be different types.

As described above, according to the present invention, any stable three-dimensional cell array can be formed by the laser trapping and depletion effects. The arrangement of cells in a living body can be reproduced outside the living body. As a result, changes in cell dynamics in vivo can be observed in vitro, and gene expression, protein dynamics, and the like at that time can be analyzed. In addition, since cells can be arranged without using a scaffold such as a gel or a substrate, the possibility of foreign matters entering the cell arrangement is extremely low.

The preferred embodiments according to the present invention have been described above, but the present invention is not limited to the above-described examples.

An experimental example is shown below. In the following experiments, a cell array device having the same configuration as that shown in FIG. 3 was used. As a laser light source, an Nd: YAG laser (wavelength: 1064 nm) was used. D-MEM (manufactured by Wako Co., Ltd.) was used as the liquid medium in the following several experiments. In the drawings relating to the following experimental examples, the x mark represents the focal point of the laser.

[Experimental Example 1]
A PEG solution containing polyethylene glycol (hereinafter referred to as PEG) and a liquid medium was prepared. For PEG, the average molecular weight is M = 50,000, the average degree of polymerization is n≈1,000, and the Kuhn length is b≈0.76 nm. Assuming that the scaling index is ν = 1/2, from equation 2, the radius of inertia is Rg≈10 nm. Assuming that the specific gravity of the liquid medium is ρ≈1 g / ml, from Equation 1, the overlapping concentration is C ^* ≈20 mg / ml to 30 mg / ml. The PEG solution was adjusted so that the concentration of PEG was C = 40 mg / ml. In addition, a liquid medium (aqueous solvent) not containing PEG was prepared. Mouse mammary epithelial cells (NMuMG) were used as the cells. The output power of the laser is 40 mW to 80 mW.

As shown in FIG. 6 and FIG. 7, in each of the aqueous solvent and the PEG solution, one cell was captured by a laser, and the stage was moved in one direction to be brought into contact with the other cell. After contacting the cells for several seconds, the stage was moved in the other direction at 10 μm / sec.

In the aqueous solvent, as shown in FIG. 6, when the stage was moved after contacting the cells, the cell pairs were separated from each other. Although not shown, it was also confirmed that with an aqueous solvent, when the irradiation of the laser to the cells was stopped after the cells contacted, the cell pairs separated from each other with time.
On the other hand, in the PEG solution, as shown in FIG. 7, even when the stage was moved after cell contact, the cell pair was not separated from each other, and the cell contact was maintained.

That is, in the aqueous solvent, the cells were separated because the depletion effect did not occur, whereas in the PEG solution, the contact between the cells was maintained because the depletion effect occurred.

Moreover, the same thing as the above was performed several times for each of the PEG solution concentrations. FIG. 8 shows the probability that contact between cells was maintained at that time. As shown in FIG. 8, it was confirmed that when the PEG concentration was increased, the stability of cell contact due to the depletion effect was increased. When the concentration of PEG exceeded 40 mg / ml, cell manipulation became difficult with the set laser output due to the high viscosity of the PEG solution.

[Experiment 2]
Using the sample cell of FIG. 4, a liquid medium (aqueous solvent) was accommodated in one area of the sample cell, and a PEG solution composed of the liquid medium and PEG was accommodated in the other area. The outputs of PEG, cells, and laser are the same as in Experimental Example 1. The concentration of PEG is C = 40 mg / ml.

As shown in FIGS. 9 and 10, cells were captured in a PEG solution with a laser, the stage was moved, and the cells were brought into contact with the other cells to form a cell pair. After contact for a certain time, the stage was moved at 10 μm / sec, and the cell pair was transported toward the aqueous solvent.

FIG. 9 shows the state in which the cells were brought into contact with the PEG solution for about 2 seconds and then transported to the aqueous solvent. FIG. 10 shows that the cells were brought into contact with each other in the PEG solution for about 290 seconds after stopping the laser irradiation. FIG. 2 shows a state where the laser is irradiated again and conveyed to an aqueous solvent.
As shown in FIG. 9, in the case of transport after contact for about 2 seconds, the cell pairs are separated from each other during transport from the PEG solution to the aqueous solvent, and only one of the cells captured by the laser is transported to the aqueous solvent. It was.
On the other hand, as shown in FIG. 10, in the case of transport after contact for about 290 seconds, the cell pair was transported into the aqueous solvent without leaving each other. It was also confirmed that the cell pairs were not separated from each other even after the laser irradiation was stopped in the aqueous solvent. That is, it was confirmed that the cell pair was in an adhesive state.

From the above, it was found that cells can be adhered to each other during the contact by maintaining the contact of the cells by the depletion effect. It was also found that contact for several seconds was not sufficient for cell adhesion, and contact for several minutes was sufficient.

“Experimental Example 3”
As shown in FIG. 11, five cells were arranged three-dimensionally in a PEG solution.
The same PEG solution as in Experimental Example 2 was used. The concentration of PEG is 40 mg / ml. Regarding the relative movement of the cells in Experimental Example 3, the relative movement of the captured cells in the XY plane is performed by the stage moving mechanism, and the relative movement of the captured cells in the Z direction is performed by the movement of the condenser lens. It was. The cell and laser outputs are the same as in Experimental Examples 1 and 2.

First, four cells were arranged in a PEG solution in a rectangular shape on the XY plane by conveying the cells with a laser (see images af in FIG. 11, schematic diagrams a ′ to f ′). Next, another cell was captured by a laser, moved in the X direction, the Y direction, and the Z direction so as to be positioned directly above the center of the square cell array (images g and h in FIG. 11, g). (See schematic diagram of 'h'). In the images of g and h, the array of cells that are blurred in black is below the cells being captured.

Next, the captured cells were moved in the Z direction and brought into contact with the array of square cells (see the image of i in FIG. 11 and the schematic diagram of i ′). In the image i, white and blurred cells are captured and are located above the cell array. The three-dimensional cell arrangement in the PEG solution was maintained over time.

[Experimental Example 4]
As shown in FIGS. 12 and 13, six cells were sterically arranged in a PEG solution.
A liquid medium and a polymer solution containing PEG were used. PEG has an average molecular weight of M = 7,500, an average degree of polymerization of n≈174, and a Kuhn length of b≈0.76 nm. Assuming that the scaling index is ν = 1/2, from Equation 2, the radius of inertia is Rg≈4.1 nm. Assuming that the density of the liquid medium is ρ≈1 g / ml, from Equation 1, the overlap concentration is C ^* ≈40 mg / ml to 50 mg / ml. The PEG solution was adjusted so that the PEG concentration was C = 75 mg / ml. The cell is NMuMG. The output power of the laser is 0.2W.

By conveying the cells with a laser, five cells were arranged in an annular shape on the XY plane as shown in FIG. 12A. This arrangement is shown in FIG. 12A. Next, as shown in FIGS. 12B and 13, another cell was captured by a laser and moved in the X, Y, and Z directions, and brought into contact with the annular array from the Z direction. The three-dimensional cell arrangement in the PEG solution was maintained over time.

From the above, it was found that the contact of cells can be maintained by utilizing the depletion effect of the polymer solution, whereby cells can be selectively and three-dimensionally arranged in the polymer solution.

Note that, as shown in FIG. 14, two cells that are in contact with each other due to the depletion effect were captured by the laser, and were transported to and brought into contact with the other two cells that were in contact with each other due to the depletion effect. Further, it was confirmed that the substantially linear cell arrangement was maintained even after the laser irradiation to the cells was stopped.

[Experimental Example 5]
As shown in FIG. 15, cells were arrayed using equine erythrocytes instead of NMuMG.

A PEG solution consisting of PEG and water was prepared. PEG is the same as in Experimental Example 4. Since the specific gravity of water is ρ = 1 g / ml, the overlap concentration is C ^* ≈40 mg / ml to 50 mg / ml. The PEG solution was adjusted so that the concentration of PEG was C = 200 mg / ml. Moreover, NaCl aqueous solution (aqueous solvent) which does not contain PEG was prepared. The concentration of NaCl is 0.9 mg / ml. The laser output is 0.2W.

As shown in the images at t = 0 s and t = 13 s in FIG. 15, the cells were repeatedly transported by a laser in an aqueous solvent to arrange four cells. Then, laser irradiation was stopped and the state of the arranged cells was observed. As is apparent from the images at t = 13 s, t = 26 s, and t = 39 s, it was confirmed that the cell arrangement in the aqueous solvent collapsed over time.
As shown in FIG. 16, the cells were repeatedly transported by laser in the PEG solution, and the cells could be arranged in an annular shape. In addition, it was confirmed that an annular array was maintained over time.

[Experimental Example 6]
As the water-soluble polymer, dextran, which is a natural polymer, was used instead of PEG, which is a synthetic polymer. The laser output was 80 mW. NMuMG was used for the cells.

A dextran solution containing a liquid medium and dextran was prepared. Dextran has an average molecular weight of M = 200,000. The concentration of dextran is 50 mg / ml. Moreover, the liquid culture medium (aqueous solvent) which does not contain dextran was prepared.

As shown in FIG. 17A, the cells were captured by a laser in an aqueous solvent (image at t = 0), contacted with other cells, and then the laser irradiation to the cells was stopped (image at t = 5), and the time of the cells The progress was observed. As shown in the image at t = 180, it was confirmed that the contact area of the cells became smaller with time, that is, the cells gradually started to separate.

As shown in FIG. 17B, cells were captured by a laser in a dextran solution (image at t = 0), contacted with other cells, and then the laser irradiation to the cells was stopped (image at t = 5), and the time of the cells The progress was observed. As shown in the image at t = 180, the cells were in contact with each other over time.

Furthermore, the same thing was performed using erythrocytes instead of NMuMG. A dextran solution containing NaCl, dextran and water was prepared. Dextran has an average molecular weight of M = 200,000. The concentration of dextran is C = 50 mg / ml. And the NaCl aqueous solution (aqueous solvent) which does not contain dextran was prepared. In both solutions, the concentration of NaCl is 0.9 mg / ml.

As shown in FIG. 18A, when the laser irradiation was stopped in the aqueous solvent, the cells separated from each other with time. On the other hand, as shown in FIG. 18B, the cells were kept in contact with each other even after the laser irradiation was stopped in the dextran solution.

Thus, it was found that cell contact can be maintained by the depletion effect even when a natural polymer such as dextran is used in addition to a synthetic polymer such as PEG.

From each of the above experimental examples, it was found that by utilizing the depletion effect, the contact of cells can be maintained ignoring the influence of the repulsive interaction between cells, and the cells can be adhered during the contact. . It was also found that by using the depletion effect and laser trapping, an arbitrary three-dimensional cell array can be formed without using a scaffold.

[Production of brown adipose tissue]
The present invention may be applicable to the production of cellular tissue.
As an example, the production of a three-dimensional brown adipose tissue having a vascular system using a bio-3D printer (for example, manufactured by Cyfuse) and the present invention can be mentioned.
By producing a three-dimensional structure of cells with a bio 3D printer and culturing the cells, it is possible to produce a cell tissue composed of blood vessels and brown adipocytes arranged around them as shown in FIG. 19A. It has been. If this can be realized, according to the present invention, as shown in FIG. 19A, a three-dimensional structure is produced in which cells are transported to the cell tissue by a laser focused in the polymer solution 8 and branched from the blood vessels of the cell tissue. However, it is considered that by culturing this, a capillary vessel branched from the blood vessel can be produced. Thereby, it is considered that a three-dimensional brown adipose tissue having a vascular system as shown in FIG. 19B could be produced.

Brown adipocytes are being shown to have a stronger effect of reducing the increased glucose in the blood than insulin. Therefore, if the production of a three-dimensional brown adipose tissue having a vascular system can be realized by using the present invention, it will lead to the development of a novel treatment method for type 2 diabetes.

[Induction of differentiation of iPS cells and ES cells]
In cell differentiation from iPS cells and ES cells, the arrangement of cells is important. For example, by using the present invention, it is possible to conduct research to clarify how the efficiency of differentiation into specific cells changes when iPS cells or ES cells are arbitrarily arranged and differentiation is induced.

DESCRIPTION OF SYMBOLS 1 Laser light source 2 Condensing lens 7 Stage 8 Polymer solution 9 Sample cell 10 Stage moving mechanism

Claims

A method of arranging cells using a laser,
Capturing cells with a laser;
In a polymer solution containing a water-soluble polymer in which the solvent is an aqueous solvent, transporting the captured cells relative to other cells and bringing them into contact;
Maintaining the contact of these cells by the depletion effect of the polymer solution.
2. The method according to claim 1, wherein the contact of the cells by the depletion effect is maintained for 300 seconds or more.
3. The method according to claim 1, further comprising a step of placing the formed cell array in an environment in which the polymer solution does not exist.
The method according to claim 1, further comprising a step of conveying the formed cell array from the polymer solution to a liquid medium.
The method according to any one of claims 1 to 4, wherein the cells are arranged three-dimensionally.
The polymer solution is adjusted so that the concentration C of the water-soluble polymer in the polymer solution is 0.1C * ≦ C ≦ 10C * when the overlapping concentration of the water-soluble polymer is C *. The method according to claim 1, comprising a step of adjusting.
The method according to claim 6, wherein the concentration C of the water-soluble polymer is 0.3C * ≦ C ≦ 3C * .
A device for arranging cells,
A laser light source for generating a laser;
A polymer solution that is an aqueous solvent, contains a water-soluble polymer, and can generate a depletion effect to maintain contact of the cells;
A sample cell for containing the cell and the sample cell;
A condensing lens for condensing the laser from the laser light source in the sample cell;
An apparatus comprising: a moving unit for moving the sample cell and the condensing point of the laser relatively in three dimensions.
A plurality of the condensing lenses are provided,
The apparatus according to claim 8, wherein the moving unit includes a condensing point moving mechanism for moving the condensing point of a laser in three dimensions with respect to the sample cell for each of the condensing lenses.
A first inlet for introducing the polymer solution into the liquid storage space of the sample cell is formed in one of the horizontal directions of the sample cell,
On the other side of the horizontal direction of the sample cell, a second inlet for introducing additional liquid that cannot cause a depletion effect into the liquid storage space is formed,
The apparatus according to claim 8 or 9, wherein an exhaust port for discharging air in the liquid storage space is formed at a central portion of the sample cell.
The concentration C of the aqueous polymer in the polymer solution is 0.1C * ≦ C ≦ 10C *, where C * is an overlapping concentration of the water-soluble polymer. Item 11. The apparatus according to any one of Items 10.
12. The apparatus according to claim 11, wherein the concentration C of the water-soluble polymer is 0.3C * ≦ C ≦ 3C * .