JP2007244378A - Method and apparatus for cell fixation and cell fixing substrate - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell fixing method and apparatus enabling the fixation of cells to a substrate without damaging the cell and provide a cell fixing substrate. <P>SOLUTION: The cell fixing method comprises the bonding a cell 4a to a substrate 2 by radiating light 10 containing a light component having a wavelength of 330-410 nm to the cell 4a contacting with the substrate 2. The invention further provides a substrate and apparatus for cell fixation, a method for examining the action of a drug to the cell using the method, etc., and a method for classifying multiple kinds of cells by the fixation level. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a cell immobilization substrate used for applications such as confirmation of the influence of a drug on cells such as animal cells, a cell immobilization method and apparatus applicable to the production thereof, and an inspection using the cell immobilization substrate The present invention relates to a method and a method for sorting cells.

In research on cells such as animal cells, cells are cultured under specific environmental conditions, and the influence of the environmental conditions on the cells is evaluated. For example, drug screening for confirming the influence of drugs on cells is a technology indispensable for the development of new drugs.
In this type of technology, a cell immobilization substrate in which cells are immobilized on a base material is used (see, for example, Patent Document 1).
Techniques for immobilizing cells on a substrate include a method for adhering cells to a substrate via an antibody that specifically binds to the target cell, and a method for immobilizing cells on a substrate via an organic compound film (For example, refer to Patent Document 2).
JP-A-2005-46121 JP-A-10-123031

However, in the prior art, in order to immobilize cells on a substrate, it is necessary to attach an antibody to the substrate in advance or to form an organic compound film, so there is an inconvenience that it takes time to immobilize cells.
In addition, when a cell immobilization substrate is used for drug screening or the like, it is required that the cell is in a physiologically normal state, and therefore it is necessary that the cell is not physiologically damaged during immobilization. It becomes.
In the technique of attaching cells using antibodies or organic compound membranes, it is difficult to accurately measure the effects of drugs on cells because these antibodies and organic compound membranes affect the physiological state of cells. was there.
In recent years, interest in tailor-made medicine taking into account individual differences in drug sensitivity has increased, and the use of cell-immobilized substrates has been studied. However, cost reduction is indispensable for popularizing tailor-made medicine in general. It is. For this reason, an efficient cell immobilization method is desired.
The present invention has been made in view of the above circumstances, and the objects thereof are as follows.
(1) Provided are a cell immobilization method and apparatus, a cell immobilization substrate, an inspection method, and a cell sorting method that can immobilize cells on a base material efficiently and without causing damage.
(2) Provided are a cell immobilization method and apparatus, a cell immobilization substrate, and an inspection method that enable accurate measurement in examining the action of a drug on cells.

The configuration of the present invention is as follows.
(1) A method of immobilizing cells on a substrate surface by adhesion, wherein the cells are adhered to the substrate by irradiating the cells with light in a state where the cells are in contact with the substrate surface And the light includes light having a wavelength of 330 to 410 nm.
(2) The cell immobilization method according to (1), wherein the irradiation energy of the light in the wavelength range is 1 to 100 J / cm ² .
(3) The cell immobilization method according to (1) or (2), wherein the light irradiation to the cells is performed in the presence of serum.
(4) The cell immobilization method according to any one of (1) to (3), wherein at least a surface of the base material is made of a non-photoresponsive material.
(5) The cell immobilization method according to (4), wherein at least the surface of the base material is made of polystyrene.
(6) A cell-immobilized substrate, wherein cells are immobilized on a substrate by the method according to any one of (1) to (5).
(7) An apparatus for immobilizing cells on the surface of a base material by adhesion, comprising irradiation means for irradiating light on an arbitrary region of the base material, the irradiation means comprising the cells on the surface of the base material By irradiating the cells with the light in contact with the cells, the cells can be adhered to the substrate, and the light includes light having a wavelength of 330 to 410 nm.
(8) The irradiating means includes a light source and a reflecting means, and the reflecting means reflects the light from the light source so as to irradiate an arbitrary region of the base material. (7) The cell immobilization device according to (7).
(9) A method for examining the action of a drug on the cell using the cell-immobilized substrate according to (6), wherein the drug is brought into contact with the cell and the action of the drug on the cell is detected. Inspection method characterized by that.
(10) A method of separating a part of cells from a plurality of types of cells, wherein the plurality of types of cells are guided to a substrate surface, and among these cells, the part of the cells are in contact with the substrate surface Then, the cells are selectively irradiated with light containing light having a wavelength of 330 to 410 nm to adhere the cells to the substrate, and then cells other than the cells are removed from the substrate surface. Cell sorting method.

In the cell immobilization method of the present invention, cells are adhered to the substrate surface by irradiating light containing light having a wavelength of 330 to 410 nm, so that the cells are sufficiently adhered and immobilized without damaging the cells. can do.
Therefore, it is possible to perform accurate measurement in examining the action of the drug on the cells.
In addition, since the cells can be adhered to the substrate without using inclusions such as antibodies, a substrate pretreatment step is not necessary. For this reason, a work process can be simplified and a cell can be fixed efficiently. Therefore, the manufacturing cost of the cell immobilization substrate can be reduced.

FIG. 1 shows a cell array 1 which is an example of the cell immobilization substrate of the present invention.
In the cell array 1 shown here, four flow paths 3a to 3d are formed in the base material 2, and the first to fourth cells 4a to 4d are immobilized in the first to fourth flow paths 3a to 3d. Yes.
Although the constituent material of the base material 2 is not specifically limited, a synthetic resin, glass, a metal, a silicon, etc. can be mentioned.
Examples of suitable synthetic resins include polystyrene resins, silicone resins (eg, polydimethylsiloxane resins), acrylic resins (eg, polymethyl methacrylate resins), polyethylene resins, polypropylene resins, polycarbonate resins, and epoxy resins. Resins can be mentioned.
The base material 2 should just be what the surface consists of the said constituent material, and a surface layer part may consist of the said material, and the other part may be comprised with another material.
The base material 2 is preferably a material that can transmit irradiation light (described later).
In addition, although the material whose molecular structure etc. change with light is called photoresponsive material, the material (non-photoresponsive material) without photoresponsiveness can be used as a constituent material of the base material 2. Examples of the non-photoresponsive material include the aforementioned materials (synthetic resin, glass, metal, silicon, etc.).

The base material 2 can improve adhesiveness by surface treatment. As the surface treatment method, a treatment method capable of forming a polar functional group (eg, —OH, —NH ₂ , —COOH, etc.) on the surface of the substrate 2, for example, plasma treatment, ozone treatment, corona discharge treatment, flame treatment. Is preferred.
In particular, it is preferable to use TCPS (tissue culture polystyrene) which is polystyrene that has been subjected to plasma treatment or ozone treatment.
A coat layer made of a cell adhesion component may be provided on the surface of the substrate 2. As the cell adhesion component, for example, one or more of fibronectin, vitronectin and laminin can be used. By forming a coating layer made of fibronectin or the like, the adhesive strength of cells to the substrate 2 can be increased. The reason why cell coat strength is enhanced by the coat layer is that the higher-order structure of the membrane protein (eg, integrin) on the cell surface is changed by light and binds tightly to the ligand of the coat layer component (eg, fibronectin). Can be guessed.

The cross-sectional shape of the first to fourth flow paths 3a to 3d is not particularly limited, and can be, for example, a rectangle, a triangle, a trapezoid, a circle, a semicircle, an ellipse, or the like. In the illustrated example, the planar views of the flow paths 3a to 3d are linear and are formed substantially parallel to each other.
The flow paths 3a to 3d are preferably flow holes formed by, for example, disposing a cover material on a base on which grooves are formed. The flow paths 3a to 3d are preferably closed flow paths.

  As shown in FIG. 1, first to fourth cells 4 a to 4 d are fixed to the inner surface of the first channel 3 a side by side in the channel length direction. Similarly to the first channel 3a, the first to fourth cells 4a to 4d are also immobilized on the inner surfaces of the second to fourth channels 3b to 3d.

Next, a method for manufacturing the cell array 1 will be described.
FIG. 7 is a configuration diagram illustrating an example of an apparatus for irradiating the base material 2 with light.
This irradiation apparatus includes a holding base 21 (holding means) for holding the base material 2, an irradiation means 22 for irradiating light 10 on an arbitrary region of the base material 2, and an inverted microscope 23 (which can observe the base material 2). Observation means) and control means 24 such as a personal computer.
The irradiation unit 22 includes a light source (not shown) and a DMD 25 (Digital Micromirror Device) (reflection unit). DMD 25 is divided into a plurality of micromirrors. Each micromirror can be set at an angle independently by a signal from the control means 24, and can reflect the light 10 from the light source to irradiate the substrate 2 with a pattern 10 of light according to the signal. It has become. With this configuration, the DMD 25 can irradiate an arbitrary region of the substrate 2 with the light 10. For example, the light 10 can be irradiated only to a partial region of the surface of the substrate 2, or the light 10 can be irradiated to the entire region.
A general-purpose ultraviolet lamp or the like can be used as the light source.
The inverted microscope 23 can observe the cells on the substrate 2 with the observation light 26.

As shown in FIG. 2, the culture solution containing the first cells 4 a is introduced into the first to fourth flow paths 3 a to 3 d of the substrate 2.
As the culture solution, a cell culture medium that can improve the physiological state of the cells 4a to 4d can be used. As this culture medium, what added serum to the general purpose base culture medium can be illustrated. Examples of the base medium include D′ MEM, HamF12, HamF10, RPMI1640, and the like. These may be used alone or in combination of two or more thereof.
As the serum, one or more of FBS (Fetal Bovine Serum), FCS (Fetal Calf Serum), NCS (Newborn Calf serum), CS (Calf Serum), and HS (Horse Serum) can be used.
Moreover, a serum-free medium and a protein-free medium can also be used as the medium.
Examples of cells that can be used in the present invention include those derived from animals (for example, human cells), plants, and microorganisms.

  Next, as shown in FIG. 2 and FIG. 3, the light 10 is irradiated to a part of the flow paths 3 a to 3 d using the irradiation device. In the example of illustration, the linear light 10 along a direction perpendicular | vertical with respect to the flow paths 3a-3d is irradiated. In the example of illustration, the light 10 is irradiated from the lower side of the base material 2, and permeate | transmits the bottom part of the flow paths 3a-3d from the lower surface (surface opposite to the surface with the cells 4a-4d) side, and reaches the cell 4a.

If the wavelength 10 of the light 10 irradiating the channels 3a to 3d is too short, the physiological state of the cells 4a to 4d is adversely affected. If the wavelength is too long, the cells 4a to 4d are not sufficiently adhered.
For this reason, the light 10 including light having a wavelength of 330 to 410 nm is used.
Light having a wavelength in the above range can sufficiently adhere the cells 4a to 4d to the base material 2 without damaging the cells 4a to 4d. Moreover, the extracellular matrix and membrane protein of the cells 4a to 4d are not adversely affected by light irradiation.
The light 10 may include light having a wavelength outside the range, but light having a wavelength less than the range (less than 330 nm) may adversely affect the physiological state of the cell. Is desirable to be low.

If the irradiation energy of the light 10 is too small, the adhesion of the cells 4a to 4d becomes insufficient, and if it is too large, the physiological state of the cells 4a to 4d is adversely affected. 100 J / cm ² (preferably 1 to 70 J / cm ² ) is suitable.
By making irradiation energy into the said range, the cells 4a-4d can fully be adhere | attached on the base material 2 without damaging the cells 4a-4d.
If the intensity of the light 10 is too small, the adhesion of the cells 4a to 4d becomes insufficient, and if it is too large, the physiological state of the cells 4a to 4d is adversely affected, so 0.01 to 1 W / cm ² is preferable.

In the flow channels 3a to 3d (hereinafter referred to as first irradiated portions 6a to 6d) irradiated with the light 10, the cells 4a to 4d in contact with the inner surfaces (bottom surfaces) of the flow channels 3a to 3d are flow channels. 3a-3d is strongly adhered and fixed to the inner surface.
The cells 4a to 4d strongly adhere to the flow paths 3a to 3d even when there is almost no temperature change of the substrate 2 due to the irradiation of the light 10.
As shown in FIG. 4, when the flow paths 3a to 3d are washed with the cleaning liquid, the unadhered first cells 4a are removed, and only the first cells 4a adhered to the first irradiated portions 6a to 6d are flow paths 3a to 3d. It remains in 3d. As the cleaning solution, for example, a phosphate buffer solution can be used.

Although the mechanism by which the cells 4a to 4d are adhered to the inner surfaces of the flow paths 3a to 3d by irradiation with the light 10 is unknown, the following estimation is possible.
The cells 4a to 4d contacting the flow paths 3a to 3d secrete extracellular matrix. The nature of the extracellular matrix changes as the molecular structure changes due to the irradiation of light 10, and the cells 4a to 4d are strongly adhered to the inner surfaces of the flow paths 3a to 3d.

Next, as shown in FIG. 5, the light 10 is irradiated to a position (second irradiation portions 7 a to 7 d) that is a part of the flow paths 3 a to 3 d and is different from the first irradiation portions 6 a to 6 d, and the second cells The culture solution containing 4b is passed through the flow paths 3a to 3d. In the illustrated example, the second irradiation portions 7a to 7d are upstream of the first irradiation portions 6a to 6d in the medicine flow direction (described later).
Thereby, the 2nd cell 4b is made to adhere and fix to the 2nd irradiation parts 7a-7d.
As shown in FIG. 6, only the second cells 4b adhered to the second irradiated portions 7a to 7d remain in the flow paths 3a to 3d by washing.

  Next, a position that is a part of the flow paths 3a to 3d and is different from the irradiated portions 6a to 6d and 7a to 7d (slightly upstream from the irradiated portions 7a to 7d in the illustrated example, hereinafter referred to as third irradiated portions 8a to 8d). 1) is irradiated with light 10, and a culture solution containing the third cells 4c is caused to flow through the flow paths 3a to 3d, and the third cells 4c are adhered and fixed to the third irradiated portions 8a to 8d.

Next, a position that is a part of the flow paths 3a to 3d and is different from the irradiated portions 6a to 6d, 7a to 7d, and 8a to 8d (in the illustrated example, slightly upstream from the irradiated portions 8a to 8d. Hereinafter, the fourth irradiated portion. 9a to 9d (refer to FIG. 1) is irradiated with light 10, and the culture solution containing the fourth cells 4d is caused to flow through the flow paths 3a to 3d, and the fourth cells 4d are adhered and fixed to the fourth irradiated portions 9a to 9d. Make it.
The first to fourth cells 4a to 4d may be different cells.
By the above operation, the cell array 1 in which the cells 4a to 4d are adhered to the four flow paths 3a to 3d is obtained (see FIG. 1).
Irradiation with the light 10 does not cause physiological damage to the cells 4a to 4d, and the cells 4a to 4d after irradiation are maintained in a normal state. The survival rate (viability) of the cells 4a to 4d after irradiation with the light 10 is, for example, 90% or more with respect to that before irradiation.

Next, an example of a method for examining the action of a drug on the cells 4a to 4d using the cell array 1 will be described.
As shown in FIG. 8, the 1st-4th chemical | medical agent containing liquid 11a-11d is flowed to the flow paths 3a-3d, respectively. The drug-containing liquids 11a to 11d are preferably liquids containing different drugs.
As a result, all of the first to fourth drug-containing liquids 11a to 11d come into contact with the first to fourth cells 4a to 4d, and therefore all combinations of the four types of drugs and the four types of cells. Sixteen assays can be performed simultaneously.

Next, as shown in FIG. 9, the reaction between the drug-containing liquids 11 a to 11 d and the cells 4 a to 4 d is detected using the detection means 12.
Although the detection method is not particularly limited, for example, there is a method in which drug-containing liquids 11a to 11d labeled with a fluorescent dye or a radioactive substance are used, and the amount of these taken into the cells 4a to 4d is detected by the intensity of fluorescence or the like. Can be adopted.
In addition, a method of introducing a GFP (Green Fluorescent Protein) gene into the cells 4a to 4d and detecting the amount of GFP produced based on the fluorescence intensity; using a label that emits fluorescence based on an enzyme activity such as esterase, Method of detecting cell viability by viability: A method of detecting physiological activity of a cell by staining a physiologically active substance produced by the cell with an antibody can also be employed.

In the cell immobilization method, the cells 4a to 4d are adhered to the inner surfaces of the flow paths 3a to 3d by irradiating light 10 including light having a wavelength of 330 to 410 nm, so that the cells 4a to 4d are not physiologically damaged. In addition, the substrate 2 can be sufficiently adhered and fixed.
Therefore, accurate measurement is possible when examining the action of the drug on the cells 4a to 4d.
Moreover, since the cells 4a to 4d can be adhered to the base material 2 without using an inclusion such as an antibody or an organic compound film, a pretreatment step of the base material 2 is not necessary. For this reason, a work process is simplified and the cells 4a-4 can be fixed efficiently. Therefore, the manufacturing cost of the cell array 1 can be reduced.

In addition, when inclusions such as antibodies are used, the inclusions may have some influence on the physiological state of the cells 4a to 4d. However, in the cell immobilization method, the cells 4a to 4d are free from inclusions. Since it adheres to the base material 2, the physiological state of the cells 4a to 4d is not adversely affected.
Therefore, accurate measurement is possible when examining the action of the drug on the cells 4a to 4d.
Furthermore, since the cells 4a to 4d are directly bonded to the base material 2, the number of work steps can be reduced, so that contamination hardly occurs.

In the inspection method using the cell array 1, by holding a plurality of types of cells 4a to 4d in a plurality of flow paths 3a to 3d, assays relating to all combinations of the cells 4a to 4d and the drug-containing liquids 11a to 11d are simultaneously performed. be able to. For this reason, many types of assay can be performed efficiently and the effect | action of many types of chemical | medical agent containing liquid 11a-11d can be investigated easily and at low cost.
Therefore, by making the cell array 1 using the user's cells 4a to 4d, it is possible to cope with the individual characteristics of the user. For example, in medical treatment, treatment according to individual patient characteristics (for example, drug sensitivity) is possible.

In the prior art, the operation of arranging the cells on the microarray chip is often performed by spotting in an open system, and it is difficult to prevent contamination. However, in the method using the cell array 1, a series of operations are performed. , All can be performed in the closed flow paths 3a to 3d.
Therefore, contamination can be prevented and an accurate assay can be performed in an aseptic environment.

In the above inspection method, different cells 4a to 4d are adhered to each of the flow paths 3a to 3d. However, in the present invention, in at least one of the plurality of flow paths, a plurality of cells arranged in the flow path is used. Two or more of them may be different from each other.
Further, in the above inspection method, different drug-containing liquids 11a to 11d are flowed through the flow paths 3a to 3d, respectively, but in the present invention, different drug-containing liquids may be flowed into at least two of the flow paths.

Next, an example of the cell sorting method of the present invention will be described.
As shown in FIG. 10, a substrate 32 such as a culture dish or a culture cuvette is prepared. As the constituent material of the base material 32, those listed as materials usable for the base material 2 can be used.
Cells 34 containing a plurality of different types of cells 34a to 34c are seeded and cultured on the surface of the substrate 32. The cells 34a to 34c grow on the surface of the substrate 32.
To this, for example, a polyclonal or monoclonal antibody labeled with a fluorescent dye or the like is added and allowed to bind to the cells 34a to 34c. Thereby, the position information of the cell to be sorted can be detected.

Next, the position information 35a to 35c of the cells 34a to 34c is taken into the control means 24 of the irradiation apparatus shown in FIG. 7, and the above-described light 10 is irradiated only to the target cells based on the position information 35a to 35c.
Since the cells irradiated with the light 10 are fixed to the base material 32, the cells 34a to 34c can be separately collected by recovering the cells not irradiated with the light 10 from the base material 32 by washing or the like. . For example, when recovering the cells 34a, only the cells 34a can be recovered by washing after irradiating only the other cells 34b and 34c with the light 10 to fix them to the substrate 32.

  In the present invention, various labeling methods can be employed in addition to a method using a fluorescently labeled antibody. For example, a method of introducing a gene of a luminescent enzyme such as luciferase into a cell can be mentioned. Further, in the separation of cells having different forms, only the target cells can be irradiated with light under a microscope, and the cells can be separated and collected without labeling.

According to the present invention, a desired cell can be selected from cells cultured on a substrate, immobilized, and patterned. That is, unlike conventional patterning using a culture substrate on which a cell adhesion substance is previously supported according to a pattern, cells to be immobilized can be selected after culturing.
Since the cells remain normal after light irradiation, the above method is used to purify cells with certain properties, such as cells with high ability to produce physiologically active substances and cells that have been stably introduced after gene transfer. However, it can be applied to operations such as subsequent culture and growth.

Next, application examples of the cell sorting method of the present invention will be described. In the following description, the same reference numerals are given to the above-described configurations, and the description thereof is omitted.
When cells are purely cultured, it is necessary to prevent contamination with germs, but when germs are contaminated, they can be removed as follows.
As shown in FIG. 11, the specific cells 44 are cultured on the surface of the base material 32. When other cells 45a and 45b are mixed in the base material 32, the positional information 44a of the specific cell 44 obtained by using the fluorescent labeling method or the like is taken into the control means 24 of the irradiation apparatus shown in FIG. Based on the information 44a, only the specific cells 44 are irradiated with the light 10 and immobilized on the base material 32, and then the mixed cells 45a and 45b can be detached from the base material 32 and removed by washing or the like.
In addition, when the specific cell 44 and the mixed cells 45a and 45b can be visually identified, the irradiation pattern of the light 10 can be determined by observation with a microscope without labeling.

  The adhesion of cells by light may become weaker over time. For example, a cell adhered to a substrate by light irradiation may be able to be peeled again by being left for a predetermined time after the light irradiation is completed. For this reason, the cell once adhere | attached on the base material for the purpose of various bacteria removal etc. can be separately collect | recovered by the said classification method.

FIG. 12 is a process diagram showing a method for patterning a cell growth region.
The first cells 46 are grown on the entire surface of the base material 32, the first region 48 is irradiated with the light 10 based on the pattern 47, and the first cells 46 located in the first region 48 are fixed to the base material 32, The first cells 46 in the other region (second region 49) are removed by washing or the like. In the illustrated example, the first region 48 is formed in a plurality of strips substantially parallel to each other.
By growing the second cells 50 in the second region 49 from which the first cells 46 have been removed, the first region 48 where the first cells 46 are present and the second region 49 where the second cells 50 are present are: They are alternately arranged in a predetermined direction (left and right direction in the figure).
Such patterning of the proliferation region of cells is effective when analyzing information transmission between cells or producing a physiologically active substance produced in the presence of multiple types of cells.

FIG. 13 shows an example of an apparatus that can be used for sorting cells.
The cell sorting apparatus shown here includes a cell culture means having a base material to which cells can adhere, a means for supplying a medium to the cell culture means, a means for irradiating the base material with light, and a cell position on the base material. It has a means for detecting, a means for separating cells detached by light irradiation, and a washing liquid supply means is added if necessary.

Hereinafter, the configuration of the cell sorting apparatus will be described in detail.
This cell sorting apparatus includes a cell culture cuvette 51 (cell culture means) having a substrate 52 to which cells can adhere, a medium reservoir 53 (medium supply means) for supplying a medium to the cuvette 51, and a substrate 52 for the cuvette 51. The projector 54 for irradiating the light 10 described above (irradiation means), a color CCD camera 55 (cell position detection means) for detecting the position of the cell and sending the position information signal to the control means 57, and a plurality of collection containers 56a are switched. A separation / recovery means 56 provided as possible, and a control means 57 for controlling the operation of each of these means are provided.
This apparatus may be provided with a cleaning liquid supply means (not shown) for supplying a cleaning liquid to the cell culture cuvette 51 as necessary.

The cell culture cuvette 51 is a container having a bottom made of a base material 52. The cell culture cuvette 51 can irradiate the base material 52 with light 10 from the outside and observe cells on the surface of the base material 52.
The cell culture cuvette 51 includes a main body 51a having a base 52, an introduction path 51b for introducing a medium into the main body 51a, and a lead-out path 51c for deriving the medium. Opening / closing means 58 such as a valve is provided in the paths 51b and 51c. As a constituent material of the base material 52, those listed as materials usable for the base material 2 can be used.
The projector 54 is an irradiating unit that irradiates light of a pattern corresponding to a signal from the control unit 57, and can irradiate an arbitrary region on the surface of the substrate 52.
The projector 54 includes a light source (not shown) and an optical conversion unit (not shown) that converts an irradiation area of light from the light source into the pattern. A DMD, a transmissive liquid crystal panel, or the like can be used as the optical conversion unit.

It is desirable that the control means 57 controls the taking-in of the cell position information, the setting of the light irradiation from the projector 54 based on the information, and the supply and stop of the culture medium or washing solution to the cell culture cuvette 51.
The color CCD camera 55 preferably has a resolution, optical magnification, and sensitivity sufficient to identify individual cells or cell groups. In addition, when a plurality of antibody-supported fluorescent dyes are used to identify cell types, it is desirable that colors can be identified.
In the above description, the process of seeding and culturing cells in a cell culture cuvette has been described. However, the present invention can also be applied to a process in which cells are simply introduced into a cuvette and separated and collected. This process is effective for separating and collecting each cell type in, for example, a tissue containing a plurality of types of cells.

Hereinafter, an example of the operation of the cell sorting apparatus will be described.
By opening / closing the opening / closing means 58, the medium is supplied from the medium reservoir 53 to the cell culture cuvette 51, and the cells are seeded and cultured.
At this time, target cells may be fluorescently labeled in advance or may be labeled with a fluorescent antibody after culturing. The position of the cell on the substrate 52 in the cell culture cuvette 51 is detected by the color CCD camera 55, and the position information signal is input to the control means 57.
Thereby, the target cell to be sorted is identified by the fluorescent label, and the light 10 is irradiated to cells other than the target cell by the projector 54. The setting of the irradiation position may be automatic control by the control means 57 or may be performed manually while observing cells.
The cells irradiated with the light 10 adhere to the base material 52, but the cells not irradiated with the light 10 can be detached from the base material 52. Therefore, the cells are selectively supplied to the culture cuvette 51 by supplying a culture medium or a washing solution. Then, it is removed from the base material 52 and collected in the collection container 56 a of the separate collection means 56.
The sorting and collecting means 56 is provided so that a plurality of collecting containers 56a can be switched. Therefore, it is easy to collect the sorted cells in different collecting containers 56a.

(Example 1)
A plate-like base material (96 holes (well)) made of polystyrene (TCPS) whose surface was plasma-treated was prepared, and an average of 100 animal cells were seeded in each well and cultured for 23 hours. Then, using the irradiation apparatus shown in FIG. 7, light was irradiated from the bottom surface side of the well under several wavelength conditions and energy conditions. As a cell, CHO-K1 was used.
The surface was washed with a phosphate buffer containing 1 mM EDTA, and the amount of the remaining cells was visually confirmed to determine the quality of cell adhesion induced by light irradiation. Tables 1 and 2 indicate the degree of cell adhesion, the residual rate after performing a certain washing operation necessary and sufficient for removing unirradiated cells (◎), 50% or more and less than 80% (○ ), 20% or more and less than 50% (Δ), or less than 20% (×).
Cell proliferation can be confirmed by freezing 3 days after light irradiation, adding CyQUAUT that emits fluorescence with an intensity proportional to the number of cells, measuring the fluorescence intensity with a plate reader, and comparing it with a non-irradiated sample. Judged. In Tables 1 and 2, the degree of proliferation is shown as any of 90% or more (◯), 50% or more and less than 90% (Δ), and less than 50% (×) with respect to the non-irradiated reference conditions.

As shown in Table 1, in Test Example 1 using light having a wavelength of 313 nm, a result indicating that most cells were killed was obtained.
In Test Example 2 using light with a wavelength of 334 nm, cell adhesion to the substrate surface could be observed. Although no cell death was observed, some effects on proliferation were observed.
In Test Example 3 using light with a wavelength of 365 nm, the cell adhesiveness increased without affecting the proliferation.
From the results of Test Examples 1 to 3, it can be considered that when the wavelength is shorter than that of Test Example 2, the damage of the cells is increased by the light irradiation with the intensity capable of enhancing the cell adhesiveness.
Among Test Examples 4 and 5 using light having a wavelength of 405 nm, in Test Example 4 in which the irradiation energy was 70 J / cm ² , it was considered that the cell damage was small because no effect was observed on cell proliferation. The cell adhesion was slightly low. In Test Example 5 in which the irradiation energy was larger than that in Test Example 4, the cell adhesiveness was increased, but a slight effect on the proliferation was observed.
From this fact, it can be considered that when the wavelength is much greater than those in Test Examples 4 and 5, sufficient cell adhesiveness is difficult to obtain within a range of strength that does not significantly affect cell proliferation.
In Test Example 6 using light having a wavelength of 436 nm, cell adhesion was insufficient.
From the above, it can be seen that irradiation with light having a wavelength of 330 to 410 nm is suitable for obtaining sufficient cell adhesion without adversely affecting cell growth.

As shown in Table 2, in Test Example 7 in which the irradiation energy was 0.6 J / cm ² , cell adhesion was insufficient, and in Test Example 12 in which the irradiation energy was 120 J / cm ² , the proliferation was inferior. became.

The effect of light irradiation energy on cell proliferation was examined as follows.
CHO-K1 cells were cultured on the substrate surface, irradiated with light having a predetermined pattern, and then washed with a phosphate buffer containing 1 mM EDTA. The wavelength of light was 365 nm.
FIG. 14 is a graph showing the change over time in the number of cells after light irradiation. The vertical axis represents the number of cells, and the horizontal axis represents the elapsed time after light irradiation. Irradiation energy of the light was 30 J / cm ² and 120 J / cm ^2. For comparison, the test results when no light irradiation is performed are also shown (0 J / cm ² ).
When the irradiation energy was 30 J / cm ² , the same growth as when no light irradiation was performed was obtained, but when the irradiation energy was 120 J / cm ² , the growth was inferior.

From FIG. 14 and Table 2, by setting the irradiation energy of light to 1 to 100 J / cm ² (preferably 1 to 70 J / cm ² ), cell adhesion could be enhanced without affecting the proliferation. I understand that.

(Example 2)
A cell array 61 was prepared as follows (see FIGS. 15 and 16).
A base material 62 made of polystyrene (TCPS) having a plasma-treated surface and coated with a silicone resin that inhibits cell adhesion was prepared except for five circular regions 66 having a diameter of 200 μm. The channel 63 was formed in a rectangular cross section having a width of 600 μm and a depth of 200 μm.
A culture solution containing MDCK cells stained with CMTPX emitting red fluorescence was introduced into the channel 63 and cultured for 5 hours 30 minutes.
Next, by using the irradiation apparatus shown in FIG. 7 to irradiate a part of the flow path 63 with light, MDCK which is the first cell in two places (first irradiation part 64) out of the five circular areas 66. Cells 65 were adhered and fixed, and other non-immobilized cells were removed by washing.
FIG. 15 is a fluorescence micrograph of the flow path 63 after the first cells (MDCK cells 65) are fixed to the first irradiated portion 64.
After culturing for 2 hours, a culture solution containing CHO cells 68 stained with CMFDA emitting green fluorescence was introduced into the channel 63 and cultured for 5 hours 30 minutes.
Then, by using the irradiation device shown in FIG. 7 again, a part of the flow path is irradiated with light so that the remaining three circular regions 66 (second irradiation portions 67) have CHO cells 68 as second cells. The cells were adhered and fixed, and the other cells were removed by washing.
FIG. 16 is a fluorescence micrograph of the channel 63 after the second cells (CHO cells 68) are immobilized on the second irradiated portion 67. FIG. It can be seen that the MDCK cell 65 and the CHO cell 68 are immobilized at different positions in the same flow path 63.
The wavelength of light used for immobilizing MDCK cells 65 and CHO cells 68 was 365 nm, the intensity was 0.026 W / cm ² , and the irradiation time was 150 seconds (irradiation energy 3.9 J / cm ² ).

Example 3
A substrate 72 made of polystyrene (TCPS) having a plasma-treated surface was prepared, and MDCK cells 73 were uniformly seeded on the surface and cultured for 4 hours. Using the irradiation apparatus shown in FIG. 7, the base material was irradiated with light on the region 74 and the rectangular region 75 forming the characters “AIST”. The wavelength of light was 365 nm, the intensity was 0.08 W / cm ² , and the irradiation time was 10 minutes (irradiation energy 48 J / cm ² ).
FIG. 17 is a photograph of the surface of the substrate 72 after washing with a phosphate buffer containing 1 mM EDTA. It can be seen that the cells 73 were immobilized only in the regions 74 and 75 that were irradiated with light.

Example 4
CHO-K1 cells were uniformly seeded on a polystyrene substrate coated with fibronectin (No. 354457, manufactured by BD Biosciences), and cultured for 24 hours. The irradiation apparatus shown in FIG. 7 was used to irradiate light having a predetermined pattern. The wavelength of light was 365 nm, and the irradiation energy was 18 J / cm ² .
After acting on a phosphate buffer containing 1 mM EDTA for 10 minutes, the substrate surface was washed according to Example 3 using this phosphate buffer.
FIG. 18 is a photograph of the substrate surface after cleaning. It can be seen that the cells were immobilized in the region irradiated with light, and the cells were almost completely removed in the non-irradiated region, resulting in a pattern with very high contrast.

(Example 5)
In accordance with Example 4, a linear pattern composed of CHO-K1 cells was formed on the substrate surface. A photograph of the substrate surface is shown in FIG. FIG. 19 (b) shows a photograph of the surface of the base material after the cells were cultured for 24 hours.
From this figure, it can be seen that the cells that had undergone light irradiation maintained sufficient viability thereafter and proliferated beyond the irradiation region.

(Example 6)
According to Example 4, CHO-K1 cells were cultured on the substrate surface, irradiated with light having a predetermined pattern, and immediately after that, using a phosphate buffer solution (PBS) containing 1 mM EDTA, Was washed (PBS flow rate during washing: 2 m / s) to form a predetermined pattern (reference numeral 81 in FIG. 20).
The cells having the pattern were subsequently cultured for 8 hours, and then the substrate surface was washed under the above washing conditions (PBS flow rate during washing: 2 m / s). As a result, most cells were removed from the substrate surface. The code | symbol 82 of FIG. 20 shows the base-material surface after washing | cleaning.
This result shows that the adhesive strength of the cells by light irradiation weakened with time.
This shows that cell adhesion and detachment can be easily manipulated.

(Example 7)
A linear pattern was formed in the same manner as in Example 5 except that HeLa cells were used. A photograph of the substrate surface is shown in FIG. The wavelength of light was 365 nm, and the irradiation energy was 3.5 J / cm ² .

(Example 8)
A linear pattern was formed in the same manner as in Example 5 except that HepG2 cells were used. A photograph of the substrate surface is shown in FIG. The wavelength of light was 365 nm, and the irradiation energy was 3.0 J / cm ² .

Example 9
Except for using MDCK cells, a pattern was formed in which the portion from which the cells were removed formed the letter “S” in the same manner as in Example 5. A photograph of the substrate surface is shown in FIG. The wavelength of light was 365 nm, and the irradiation energy was 24 J / cm ² .

  From the results of Examples 7 to 9, it can be seen that the present invention was applicable to a plurality of types of cells.

(Example 10)
As shown below, a pattern was formed using two types of cells.
In accordance with Example 4, a honeycomb-like pattern was formed on the culture substrate using CHO-K1 cells. A photograph of the substrate surface is shown in FIG.
Next, in the same manner, HeLa cells were adhered in the form of dots at the center of the hexagons forming the pattern. A photograph of the substrate surface is shown in FIG.
Thus, the cell of the predetermined pattern was able to be added to the base material with which the cell was already adhere | attached by light irradiation.

It is a block diagram which shows an example of the cell fixed substrate of this invention. It is process drawing which shows the manufacturing method of the cell fixed substrate shown in FIG. It is explanatory drawing explaining that a cell adhere | attaches on a base material in manufacture of the cell fixed substrate shown in FIG. FIG. 3 is a process diagram following FIG. 2. It is process drawing following a previous figure. It is process drawing following a previous figure. It is an example of the cell fixing device which can be used for the cell fixing method of this invention. It is explanatory drawing which shows an example of the method of using the cell fixed substrate shown in FIG. It is explanatory drawing which shows the method of detecting reaction of a cell and a chemical | medical agent using the cell fixed board | substrate shown in FIG. It is explanatory drawing which shows an example of the cell classification method of this invention. It is explanatory drawing which shows the other example of the cell classification method of this invention. It is explanatory drawing which shows the other example of the cell classification method of this invention. It is a block diagram which shows an example of the apparatus which can be used for the cell classification method of this invention. In an Example, it is a graph which shows the test result regarding the influence which the irradiation energy of light has on cell growth property. In an Example, it is a photograph of the flow path which fixed the cell in the predetermined position by light irradiation. In an Example, it is the photograph of the flow path which fixed another kind of cell in the predetermined position by the same process following the previous figure. In an Example, it is a photograph of the base-material surface which fixed the cell by irradiation of light. In an Example, it is a photograph of the base-material surface which fixed the cell by irradiation of light. In an Example, it is a photograph of the base-material surface which fixed the cell by irradiation of light. In an Example, it is a photograph of the base-material surface which fixed the cell by irradiation of light. In an Example, it is a photograph of the base-material surface which fixed the cell by irradiation of light. In an Example, it is a photograph of the base-material surface which fixed the cell by irradiation of light. In an Example, it is a photograph of the base-material surface which fixed the cell by irradiation of light. In an Example, it is a photograph of the base-material surface which fixed the cell by irradiation of light. In an Example, it is a photograph of the base-material surface which fixed the cell by irradiation of light.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Cell array (cell fixed board | substrate) 2, 32, 52 ... Base material, 3a-3d ... 1st-4th flow path, 4a-4d, 34, 34a-34c, 44, 46.50 ... Cell, 6a to 6d, 7a to 7d, 8a to 8d, 9a to 9d ... irradiated portion, 11a to 11d ... first to fourth drug-containing liquids, 12 ... detecting means, 22 ... irradiating means, 25 ... DMD (reflecting means).

Claims (10)

A method of immobilizing cells on a substrate surface by adhesion,
The cells are adhered to the substrate by irradiating the cells with light while the cells are in contact with the substrate surface,
The cell immobilization method, wherein the light includes light having a wavelength of 330 to 410 nm. Cell immobilization method according to claim 1, the irradiation energy of the light in the wavelength range characterized in that it is a 1~100J / cm ^2.   The cell immobilization method according to claim 1 or 2, wherein the cells are irradiated with light in the presence of serum.   The cell immobilization method according to any one of claims 1 to 3, wherein at least a surface of the base material is made of a non-photoresponsive material.   The cell immobilization method according to claim 4, wherein at least a surface of the base material is made of polystyrene.   A cell-immobilized substrate, wherein cells are immobilized on a substrate by the method according to any one of claims 1 to 5. An apparatus for fixing cells to a substrate surface by adhesion,
Irradiating means for irradiating light to an arbitrary region of the base material, and the irradiating means irradiates the cell with the light while the cell is in contact with the surface of the base material. A cell immobilization device characterized in that it can be adhered to a material, and the light includes light having a wavelength of 330 to 410 nm.   The irradiating means includes a light source and a reflecting means, and the reflecting means is configured to irradiate an arbitrary region of the base material by reflecting the light from the light source. The cell immobilization apparatus according to claim 7.   A method for examining the action of a drug on the cell using the cell-immobilized substrate according to claim 6, wherein the drug is brought into contact with the cell and the action of the drug on the cell is detected. Inspection method. A method of separating a part of cells from a plurality of types of cells,
The plurality of types of cells are guided to the surface of the substrate, and with some of the cells in contact with the surface of the substrate, the cells are selectively irradiated with light including light having a wavelength of 330 to 410 nm. The cell is adhered to the substrate, and then cells other than the cell are removed from the substrate surface.

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