JP2018161097A - Production method of culture cell test piece - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a test piece in which mature cells and tissues are cultured which are arranged and oriented in a desired pattern without having a physical structure which is detrimental to each kind of measurement.SOLUTION: A region 2 which has a cell non-adhesion matrix is formed in a culture vessel 1 (S1), a casting mold 3 in which a cell culture spot 3a is formed is installed in the region 2 having the cell non-adhesion matrix (S2) and inside of the cell culture spot 3a of the casting mold 3 has a region 4 having a cell adhesion matrix formed thereon (S3), then a medium 5 and at least one of a cell 6 and a tissue 6 are put into the culture vessel 1 (S4), and after performing culture of the cell 6 and the tissue 6 (S6), the casting mold 3 is detached (S7).SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method for producing a cultured cell specimen. More specifically, the present invention relates to a technique suitable for use in preparing a specimen in which biological cells and tissues are cultured in an arbitrary planar pattern on the surface of a flat culture substrate, for example.

  With the advancement of cell culture technology, for example, cells such as animal tissues and organs are cultured in vitro (for example, Patent Document 1), and the cultured cells are used for various tests, research, examinations, measurements, and the like. It has been.

JP 2003-093051 A

  Depending on the type and content of the test or measurement performed using the cultured cells, the cells are cultured on the surface of the flat culture substrate according to a predetermined pattern (in other words, the shape is an arrangement / orientation mode). Sometimes it is desirable or necessary.

  In the culture of living cells and tissues, it is necessary to control the living cells and tissues in a culture environment in the long-term culture (specifically, about several weeks to several months) required for functional differentiation and maturation. However, due to cell proliferation, morphological change, cell differentiation, and the like, there is a problem that the shape of cells and tissues arranged in a specific space cannot be maintained particularly in long-term culture.

  For example, in a bioassay, as shown in FIG. 13, when an experiment is performed using a group of cell populations, the threshold of environmental factors such as chemical substances is caused by the interaction between cells caused by cell junctions. There is a problem that it may be estimated lower than the original threshold.

  In addition, when various types of measurements are performed using cultured cells or tissues, if there is a physical structure on the culture substrate, it may interfere with measurement or become noise in measurement, resulting in measurement results. There is a problem that it will affect.

  Therefore, the present invention provides a cultured cell sample that can produce a specimen in which mature cells or tissues that have no physical structure that hinders various measurements and the like and are arranged and oriented in a desired pattern are cultured. It aims at providing the preparation method of a sample. Specifically, for example, as shown in FIG. 14, by arranging and orienting cells in a predetermined pattern, the interaction between cells is eliminated, and the threshold of environmental factors such as chemical substances is correctly set as the original threshold. It is an object of the present invention to provide a method for preparing a cultured cell specimen that can be grasped.

  In order to achieve such an object, the method for producing a cultured cell specimen of the present invention is such that a cell non-adherent substrate region is formed in a culture container, and a template in which a cell culture spot is formed is formed in the cell non-adherent substrate region. At the same time, a cell adhesion substrate region is formed in the cell culture spot of the template, and then at least one of the medium and the cells and tissues is put into the culture container to culture the cells and tissues. After that, the mold is removed.

  Therefore, according to this method for preparing cultured cell specimens, in the initial stage of culture of living cells and tissues, the physical structure (that is, the template) is used to enter the cell non-adherent substrate region, adhesion molecules, etc. After the long-term culture is carried out in a state that prevents contamination of the cells, and after the physical structure (template) is removed, the effect of the cell non-adhesive substrate causes the cell / tissue population that follows the physical structure (template) to The shape is maintained and the arrangement / orientation of each group is maintained.

  In addition, in the method for producing a cultured cell specimen of the present invention, a template to which a magnetic material is added may be used. In this case, since the mold is pulled up by using a magnetic force that is an external force, the mold is pulled straight up and removed without tilting or swinging. In this case, even when the adhesion between the cell non-adhesive substrate and the template cannot be expected sufficiently, the magnetic non-adherent template is attracted from the bottom of the culture vessel using magnetic force to The placement of the mold can be maintained over a long period of time by fixing the adhesive substrate and the mold in good contact.

  In addition, in the method for producing a cultured cell specimen of the present invention, a substance to which a temperature-responsive polymer is added is used as a substance for forming a cell non-adhesive substrate region, and the cell adhesion substrate region is changed by heating. You may make it peel from a cell non-adhesion substrate. In this case, the cell adhesion substrate region (in other words, the range where the cell adhesion substrate is formed) of the cell non-adhesion substrate is heated, so that each cell adhesion substrate region is individually divided into the cell non-adhesion substrate. Peel from.

  In addition, the method for producing a cultured cell specimen of the present invention has the property of having cell or tissue adhesiveness by changing the temperature as a substance forming the cell adhesion substrate region (in other words, a material that functions as a cell adhesion substrate). ) To a property that does not have cell or tissue adhesiveness (in other words, a material that functions as a cell non-adhesive substrate), and is heated or cooled so that the cell or tissue is removed from the cell adhesion substrate region. May be collected separately. In this case, each cell group or each tissue group is individually separated from the cell adhesion substrate and recovered by heating or cooling the cell adhesion matrix region (in other words, the area where the cell adhesion matrix is formed). Is done.

  According to the method for producing a cultured cell specimen of the present invention, in the initial stage of culturing a living cell or tissue, a physical structure (that is, a template) is used to enter a cell non-adherent substrate region and an adhesion molecule. Long-term culture can be performed in a state in which contamination such as the above is prevented, and even after the physical structure (template) is removed, the effect of the cell non-adherent substrate allows the cells / tissues to follow the physical structure (template). Since the shape of the population can be maintained and the arrangement / orientation of each population can be maintained, living cells and tissues matured in any shape, arrangement / orientation (in other words, a planar pattern) It becomes possible to produce a specimen in which is cultured.

  In the method for preparing a cultured cell specimen of the present invention, when a template to which a magnetic material is added is used, the template is removed by pulling it straight up without tilting or swinging. Therefore, it is possible to avoid the influence and damage to the cells and tissues in the mold removal operation. In this case, since the template is attracted from below the culture vessel using magnetic force, the non-cell-adherent substrate and the template can be well adhered and fixed to maintain the template arrangement for a long time. In addition, it is possible to more reliably prevent cell intrusion into the region of the cell non-adherent substrate and contamination of adhesion molecules and the like.

  In the method for producing a cultured cell specimen of the present invention, when a cell non-adhesive substrate to which a temperature-responsive polymer is added is used, the cell adhesion substrate region of the cell non-adhesive substrate (in other words, By heating the cell adhesion substrate area), each cell adhesion substrate region can be detached from the cell non-adhesion substrate individually, so multiple cells / tissues cultured in the same condition environment It is possible to easily collect and collect individual populations and analyze and evaluate each individual cell / tissue population.

  In the method for preparing a cultured cell specimen of the present invention, when a cell adhesion substrate that changes from a cell adhesive property to a cell non-adhesive property due to a temperature change is used, the cell adhesion substrate region (in other words, cell Since each cell group and each tissue group can be separated from the cell adhesion substrate by heating or cooling the area where the adhesion matrix is formed), multiple cells / tissues cultured in the same condition environment It is possible to easily collect and collect individual populations and analyze and evaluate each individual cell / tissue population.

It is a flowchart explaining an example of embodiment of the preparation method of the cultured cell specimen of this invention. It is a longitudinal cross-sectional view which shows typically the state by which the casting_mold | template was installed in the upper surface of the cell non-adhesion substrate in the preparation methods of the cultured cell specimen of embodiment. It is a top view which shows an example of the arrangement | sequence of the hole as a cell culture spot in a casting_mold | template. It is a conceptual diagram explaining the outline of the environmental factor (uniform magnetic field which fluctuates with time) exposure test by the test body in which the template shown in FIG. 3 was used and the cells were cultured. It is a longitudinal cross-sectional view which shows typically the state by which the area | region of the cell adhesion substrate was formed in the hole of the casting_mold | template in the manufacturing method of the cultured cell specimen of embodiment. It is a longitudinal cross-sectional view which shows typically the state in which the culture medium and the cell and the structure | tissue in the manufacturing method of the cultured cell specimen of embodiment were thrown in. It is a longitudinal cross-sectional view which shows typically the state at the time of culture | cultivation in the preparation method of the cultured cell specimen of embodiment. It is a longitudinal cross-sectional view which shows typically the state from which the casting_mold | template was removed after culture | cultivation in the preparation method of the cultured cell specimen of embodiment. FIG. 3 is a diagram showing a boundary state of a cell population immediately after removing a template from a specimen produced in Example 1. (A) is a figure which shows the state of the specimen of culture condition <1>. (B) is a figure which shows the state of the test body of culture condition <2>. It is a figure which shows the state of the boundary of the cell population of the 7th day after removing the casting_mold | template in the test body of the culture conditions <1> produced in Example 1. FIG. It is a figure which shows the state of the boundary of the cell population of the 7th day after removing the casting_mold | template in the test body of culture conditions <2> produced in Example 1. FIG. FIG. 3 is a diagram showing the boundary of the cell population on the 46th day from the start of culture in the specimen prepared in Example 1. (A) is a figure which shows the state of the specimen of culture condition <1>. (B) is a figure which shows the state of the test body of culture condition <2>. It is a conceptual diagram explaining the problem in the conventional bioassay. It is a conceptual diagram explaining the view of the method of solving the problem in the conventional bioassay.

  Hereinafter, the configuration of the present invention will be described in detail based on an example of an embodiment shown in the drawings.

  1 to 8 show an example of an embodiment of a method for producing a cultured cell specimen of the present invention.

  In the method for producing a cultured cell specimen of this embodiment, a cell non-adherent substrate region 2 is formed in the culture vessel 1 (S1), and the template 3 on which the cell culture spot 3a is formed is a cell non-adherent substrate region. 2 (S2) and a cell adhesion substrate region 4 is formed in the cell culture spot 3a of the template 3 (S3). Then, the culture medium 1 and the cells 6 and 6 are transferred to the culture vessel 1. Is inserted (S4), and after the cells 6 and tissue 6 are cultured (S6), the template 3 is removed (S7) (see FIG. 1).

  The size of the culture vessel 1 (also referred to as a petri dish) takes into account the size of the mold 3 used in combination with the culture vessel 1 and the amount of cells / tissues 6 that are put into the culture vessel 1 and cultured. In addition, the volume of the medium 5 that enables long-term culture (in other words, the amount of the medium 5 that does not require replacement of the medium 5 during the long-term culture) can be secured at least. The Note that the depth of the culture vessel 1 is set to be equal to or larger than the dimension in which the template 3 is immersed in the culture medium 5 in the cell / tissue culture state in which the template 3 is installed in the culture vessel 1 and the culture medium 5 is introduced. .

  In carrying out the method for producing a cultured cell specimen, first, a region 2 of a cell non-adherent substrate is formed in the culture vessel 1 (S1; FIG. 2).

  The cell non-adherent substrate region 2 is a region formed by a substrate that does not have the adhesiveness of living cells or tissues, and is therefore a region of the substrate in which the proliferation and differentiation of the living cells are not induced.

  The substance used as the cell non-adhesive substrate is not limited to a specific substance, and a substance that suppresses the adhesion of cells and tissues or a substance that has been subjected to a predetermined treatment is appropriately selected. Specific examples of the cell non-adhesive substrate include, for example, agarose gel, bovine serum albumin (BSA), 2-methacryloyloxyethyl phosphorylcholine polymer, polyethylene glycol, organic fluorine compounds, and these as ends. A silane coupling agent or the like can be used.

  Specifically, the region 2 of the cell non-adhesive substrate is, for example, when an agarose gel is used to form the region 2, for example, after dropping an agarose solution prepared to about 1 to 2% into the culture vessel 1 For example, it is formed by air drying for about 1 hour. The type of agarose is not limited to a specific type, but L (low melting point type) and S (standard type) are preferable.

  For example, when bovine serum albumin (BSA) is used to form the region 2, the region 2 of the cell non-adhesive substrate is prepared, for example, in a BSA solution of about 0.1 to 1%. Is added to the culture vessel 1 and the culture vessel 1 is sealed with a paraffin film and then incubated at room temperature for 12 hours or more.

  The region 2 of the cell non-adhesive substrate is formed as a flat film (in other words, a layer) at least in a portion (in other words, a range) where the mold 3 is placed, and is flat over the entire bottom surface of the culture vessel 1. It is preferably formed as a film (layer).

  A glass plate (not shown) separate from the culture vessel 1 is placed on the bottom of the culture vessel 1, and a cell non-adhesive substrate region 2 is formed as a film or layer on the upper surface of the glass plate. You may be made to do.

  Next, the mold 3 is placed on the surface of the region 2 of the cell non-adhesive substrate (S2; FIG. 2).

  The template 3 is a member for forming (in other words, specifying or inducing) the adhesion pattern of the cells / tissues 6 on the substrate, and the region of the cell non-adhesive substrate formed at the bottom of the culture vessel 1 2 is configured such that a hole 3a (ie, a through hole) in which the lower surface and the upper surface are in contact with the upper surface of the plate 2 is formed in a plate-shaped member as a cell culture spot.

  The material of the mold 3 is not limited to a specific type, and an appropriate material is appropriately selected in consideration of, for example, that it cannot be altered by the culture medium 5 put into the culture vessel 1. The Specific examples of the material of the mold 3 include, for example, hydrogels such as silicon resin, polydimethylsiloxane (also referred to as “PDMS”), acrylic resin, glass, parylene, and agarose. Can be used.

  A magnetic material may be added to the material forming the mold 3. In this case, in the process of S7, which will be described later, the mold 3 is removed using a magnetic force that is an external force, and the mold 3 is pulled straight up and removed without tilting or swinging. Therefore, it is possible to avoid the influence and damage to the cells / tissues 6 in the operation of removing the mold 3. In this case, even if the adhesion between the cell non-adhesive substrate and the template 3 cannot be sufficiently expected, the magnetic template 3 is attracted from the lower side of the culture vessel 1 using magnetic force. This enhances the adsorption force of the template 3 to the cell non-adherent substrate so that the cell non-adherent substrate and the template 3 can be well adhered and fixed to maintain the arrangement of the template 3 over a long period of time. It becomes possible to more reliably prevent cell intrusion into the region 2 of the adhesion substrate and contamination of adhesion molecules and the like.

  The magnetic material added to the material forming the template 3 is not limited to a specific type. For example, it is appropriate to consider the influence on the proliferation / differentiation of cells / tissues 6. Are appropriately selected. As the magnetic material to be added, for example, various magnetic particles can be selected.

  Although the template 3 is not limited to a specific size or shape, considering that a generally round cell culture container in which the template 3 is to be placed is widespread, for example, the diameter is 5 It is thought that a disk-shaped thing of about 10 cm 2 is easily adapted.

  The hole 3a (through hole) formed in the template 3 as a cell culture spot is not limited to a specific size or shape, and for example, cultured cells required for each specimen depending on the contents of various measurements and the like. -It is formed in a size or shape that matches the desired shape as a group of tissues.

  Specifically, the hole 3a as the cell culture spot is, for example, a void / hole penetrating through with a minimum dimension (inner difference) of about 0.01 to 2 mm and a maximum dimension (inner difference) of about 2 to 3 mm. It may be formed as a gap. Note that only one hole 3a may be formed as a cell culture spot in one template 3, or a plurality of holes 3a may be formed.

  The arrangement and orientation of the holes 3a (through holes) formed in the template 3 as a cell culture spot are not limited to a specific pattern, and may be required for each specimen depending on the contents of various measurements, for example. To a desired arrangement / orientation of the cultured cell or tissue population to be set.

  Specifically, the arrangement and orientation of the holes 3a (through holes) as the cell culture spots are specifically, for example, specimens used for an application test of a time-varying uniform magnetic field as an example of an environmental factor exposure test. It is conceivable that concentric circles are set as shown in FIG.

  By installing and culturing the template 3 shown in FIG. 3, a specimen is prepared in which a plurality of concentric groups of cells / tissues 6 are arranged and oriented according to the pattern of the holes 3a as the cell culture spots of the template 3. It can be considered that the test is performed by applying a time-varying uniform magnetic field to the specimen as shown in FIG.

As shown in FIG. 4, in the method in which a magnetic field that varies uniformly in the vertical direction from the upper surface of the culture vessel 1 (specimen) is incident, the current density induced in the culture medium 5 follows the following mathematical formula 1, so that the culture is performed. A concentric induced current intensity distribution is formed that is proportional to the radial distance from the center of the container 1 (ie, r = 0). In FIG. 3, a broken line (reference numeral 9) represents an induced current intensity distribution, and the induced current intensity becomes stronger toward the outside.
(Formula 1) J = σπfBr
Here, J: induced current density [A / m ² ], σ: medium conductivity [S / m], π: circumference, f: frequency [Hz], B: magnetic flux density [T], r: radius [M] is represented respectively.

  A time-varying uniform magnetic field as shown in FIG. 4 is applied to a specimen in which a group of a plurality of concentric cells / tissues 6 are arranged and oriented by cell culture using the template 3 shown in FIG. Application reduces the variation in the induced current intensity applied to the group of cells / tissues 6 (the smaller the size of the hole 3a, the smaller the variation in the induced current intensity) and the cellular response to different induced electric field strengths. Evaluation can be performed simultaneously, and an analysis of a relationship between a difference in stimulation direction and intensity and a difference in influence on cells can be efficiently and easily performed. In addition, evaluation with a single type of cell and a plurality of types depending on whether a single type of cell or multiple types of cells are seeded in each of the holes 3a as cell culture spots formed in the template 3 You will be able to both perform assessments on your network of cells.

  It is preferable that the lower surface of the mold 3 is adsorbed to the upper surface of the region 2 of the cell non-adhesive substrate. Specifically, for example, when the template 3 is formed of polydimethylsiloxane (PDMS), when the template 3 is brought into contact with the region 2 of the cell non-adhesive substrate, a molecule that acts on PDMS and the cell non-adhesive substrate. PDMS is adsorbed and fixed by the interatomic force.

  Note that a film may be formed on the surface of the mold 3 with a substance that does not have cell or tissue adhesiveness.

  A plurality of molds 3 may be installed in one culture vessel 1.

  Next, a cell adhesion substrate region 4 is formed in the hole 3a as a cell culture spot of the template 3 (S3; FIG. 5).

  The cell adhesion substrate region 4 is a substrate region having adhesion of living cells and tissues, and is therefore a substrate region in which proliferation and differentiation of the living cells are induced.

  The substance used as the cell adhesion substrate is not limited to a specific substance. For example, the cell or tissue adhesion is induced after considering the type and type of the cell / tissue 6 to be cultured. A substance or a substance obtained by subjecting a predetermined substance to a predetermined treatment is appropriately selected. Specifically, for example, laminin, collagen, fibronectin, polyethyleneimine (PEI), polylysine, Puramatrix, antibody, etc. can be used as the cell adhesion substrate.

  Specifically, for example, when polyethyleneimine (PEI) is used for forming the region 4, the region 4 of the cell adhesion substrate is refrigerated by dropping a PEI solution prepared to about 0.1%, for example. For example, it is formed by rinsing with sterilized water after being stored overnight (for example, about 4 ° C.).

  For example, when poly-L-lysine (PLL) is used to form the region 4, the PLL solution prepared to about 0.1% is used as the cell adhesion substrate region 4, for example. It is formed by dripping and storing overnight in a refrigerator (for example, about 4 ° C.) and then washing with sterile water (for example, about once).

  The cell adhesion substrate region 4 is formed at the bottom of each hole 3a formed as a cell culture spot in the template 3 in a state where the template 3 is placed (in other words, adsorbed) on the upper surface of the cell non-adhesion substrate region 2. A flat film (in other words, a layer) is formed on the upper surface of the region 2 of the corresponding cell non-adhesive substrate.

  The cell adhesion substrate region 4 may be formed so as to be laminated on the upper surface of the cell non-adhesion substrate region 2 at a portion corresponding to the bottom of the hole 3a. It may be formed by modifying at least the upper surface side portion (in other words, at least the surface layer portion) of the region 2 or replacing it with at least the upper surface side portion of the region 2 of the cell non-adhesive substrate.

  Here, the cell adhesion substrate region 4 formed in the hole 3a as the cell culture spot is seeded with the same type of cell / tissue 6 after forming the cell adhesion substrate region 4 of a different type for each hole 3a. Then, by exposing to environmental factors after culturing, environmental factors under the same conditions can be applied to cells / tissues 6 having different adhesion environments. This makes it possible to observe the difference in response depending on the adhesion mode (for example, covalent bond, antigen-antibody reaction, other binding reaction, etc.) and adhesion strength between the cell / tissue 6 and the cell adhesion substrate. In addition, the efficiency of the evaluation experiment can be improved and the error between samples can be reduced.

  Next, the culture medium 5 and the cells / tissues 6 are put into the culture vessel 1 (S4; FIG. 6).

  The culture medium 5 (also referred to as a liquid culture medium or a culture solution) is not limited to a specific type. For example, the culture medium 5 has the type / property and fluidity of the cell / tissue 6 to be cultured and has a hole 3a in the mold 3. In consideration of the possibility of entering the vehicle, an appropriate one is appropriately selected.

  In consideration of the culture period required for functionally maturation of the cells / tissue 6, the medium 5 is introduced at least in an amount sufficient to enable the culture over the culture period. In addition, it adjusts so that the casting_mold | template 3 installed in the bottom part of the culture container 1 may be immersed in the culture medium 5. FIG.

  Here, environmental factors and chemicals may be added to the culture medium 5. This makes it possible to evaluate the response of different cell / tissue 6 populations at the same time under the same culture conditions.

  The cells / tissues 6 are put in a single separated state or in an aggregated state using, for example, a pipette 7.

  The cells / tissues 6 are not limited to specific types, and those designated and specified for each specimen are appropriately selected according to the contents of various measurements and the like.

  For the cell / tissue 6, for example, a desired amount (number) to be bonded as an initial state to each of the holes 3 a as cell culture spots formed in the mold 3 is considered according to the contents of various measurements and the like. Thus, for example, an amount sufficient to allow the desired amount of adhesion is introduced.

  In addition, regarding the introduction of the medium 5 and the cells / tissues 6, the cells / tissues 6 may be introduced into the culture medium 5 after the culture medium 5 is introduced into the culture vessel 1, or the cell culture spots. Alternatively, the culture medium 5 may be introduced into the culture vessel 1 after the cells / tissues 6 are (individually) introduced into the holes 3a.

  The cells / tissue 6 put into the culture vessel 1 adheres to the cell adhesion substrate region 4 in the hole 3a formed as a cell culture spot in the template 3.

  Subsequently, the medium 5 is replaced (S5).

  After the cells / tissues 6 are introduced, the medium 5 is exchanged after the time when the cells / tissues 6 adhere to the cell adhesion substrate region 4 in the hole 3a as a cell culture spot.

  The medium 5 is replaced when, for example, an appropriate time has elapsed after the cells / tissues 6 have been introduced in consideration of the type of cells / tissues 6 to be cultured.

  In addition, before the culture medium 5 is introduced into the culture container 1, the culture medium 5 is introduced into the culture container 1 after the cells / tissues 6 are directly introduced and seeded into the holes 3a as cell culture spots. In some cases, the medium 5 may not be replaced.

  Then, the cells / tissues 6 are cultured over a predetermined culture period (S6; FIG. 7).

  The culture period is appropriately set to an appropriate length in consideration of, for example, the conditions required for each specimen depending on the types of cells / tissues 6 to be cultured and the contents of various measurements.

  Here, one feature of the present invention is that it can be cultured for a long period of time so that the cells / tissues 6 can be functionally matured.

  Specifically, the culture period of the cell / tissue 6 may be set in a range of, for example, about 3 weeks to 3 months. In the present invention, culturing over a period of about 3 weeks or more is referred to as “long-term culturing”.

  During the culture period, cells / tissues 6 are prevented from entering the upper surface of the non-adherent substrate region 2 around the template 3 and the upper surface of the non-adherent substrate region 2 to which the lower surface of the template 3 is adhered. Or cell adhesion molecules via the medium 5 are removed, so that the cells / tissues 6 do not adhere.

  On the other hand, cells / tissues 6 are adhered to the region 4 of the cell adhesion substrate in the hole 3a formed as a cell culture spot on the template 3 to proliferate / differentiate.

  Here, since the upper surface of the hole 3a formed as a cell culture spot in the template 3 is opened and open to the medium 5, the cells adhered to the region 4 of the cell adhesion substrate in the hole 3a The tissue 6 is continuously and sufficiently supplied with nutrients from the medium 5.

  After a predetermined culture period has elapsed, the mold 3 is removed (S7; FIG. 8).

  By removing the template 3 (in other words, peeling off), a specimen in which a group of cells / tissues 6 patterned in a desired manner is formed on the substrate is obtained.

  According to the above-described process, the cell / tissue 6 adheres to a portion where the cell non-adhesive substrate in the culture vessel 1 is exposed or a portion where the upper surface of the cell non-adhesive substrate is in contact with the lower surface of the mold 3. Without attaching cells / tissues 6 only to the cell adhesion substrate region 4 in the hole 3a of the template 3, each of the groups of cells / tissues 6 has no bond at the adjacent portion, and each The cell / tissue 6 population is functionally sufficiently mature as a result of long-term culture.

  Even after the template 3 is removed, the shape and arrangement of each functionally mature group of cells / tissues 6 is maintained by the action of the non-cell-adherent substrate.

  Then, environmental factors that are exposed to cells / tissues 6 (specifically, specimens to which the cultured cells / tissues 6 are attached) by performing various measurements with the mold 3 removed. Thus, accurate measurement or the like can be performed in a state in which the spatial distribution of the environmental factor is not hindered, that is, the desired spatial distribution with respect to the environmental factor.

  When various experiments and measurements are performed using the cultured cells / tissue 6, the entire culture container 1 containing the cells / tissue 6 may be used as a specimen, Further, the cell non-adhesive substrate region 2 to which the cells / tissues 6 are adhered may be peeled off from the bottom of the culture vessel 1 and used as a specimen, or placed on the bottom of the culture vessel 1. When the cell non-adherent substrate region 2 is formed on the upper surface of the glass plate, the glass plate to which the cell non-adherent substrate region 2 is attached may be used as a specimen. .

  Here, a temperature-responsive polymer is added to a substance used for forming the region 2 of the cell non-adherent substrate, and the molecular structure is changed by heating (specifically, for example, the cell non-adherent substrate is melted). The cell adhesion substrate region 4 may be detached from the cell non-adhesion substrate. In this case, each of the cell adhesion substrate regions 4 (in other words, the range in which the cell adhesion substrate is formed) is locally heated, so that each cell adhesion substrate region 4 individually becomes a cell non-adhesion substrate. It is possible to separate and collect a plurality of cell / tissue 6 groups cultured in an environment under the same conditions individually, and analyze and evaluate each cell / tissue 6 group. It becomes possible.

  The temperature-responsive polymer added to the substance used for forming the region 2 of the cell non-adherent substrate is not limited to a specific type. For example, the substance used for forming the region 2 of the cell non-adherent substrate In consideration of the influence on the properties of the cells and the influence on the proliferation / differentiation of the cells / tissues 6, an appropriate one is appropriately selected. Specifically, for example, poly (N-isopropylacrylamide) can be selected as the temperature-responsive polymer to be added.

  As an example of a heating method for separating the cell adhesion substrate region 4 from the cell non-adhesion substrate region 2 formed by using a substance to which a temperature-responsive polymer is added, laser irradiation is merely an example. Can be mentioned.

  In addition, since the temperature changes as a substance forming the region 4 of the cell adhesion substrate, it has no cell or tissue adhesion due to the property of having cell or tissue adhesion (in other words, a material that functions as a cell adhesion substrate). A substance that transforms into a property (in other words, a material that functions as a cell non-adherent substrate) is used, and the cells / tissues 6 are separated and recovered from the region 4 of the cell adhesive substrate by heating or cooling. May be. In this case, each cell / tissue 6 is individually removed from the cell adhesion substrate by locally heating or cooling each of the cell adhesion substrate regions 4 (in other words, the range where the cell adhesion substrate is formed). It becomes possible to separate and collect a group of a plurality of cells / tissues 6 cultured in an environment of the same condition individually and collect and analyze / evaluate each group of cells / tissues 6 become.

  The substance used to form the cell adhesion substrate region 4 that changes from cell adhesion to cell non-adhesion by temperature change is not limited to a specific type. For example, proliferation / differentiation of cells / tissues 6 An appropriate one is appropriately selected in consideration of the influence on the above. Specifically, for example, poly (N-isopropylacrylamide) can be selected as a substance whose cell adhesiveness changes due to temperature change.

  As a method of giving a temperature change in order to separate the cell / tissue 6 from the region 4 of the cell adhesion substrate formed by using a substance whose cell adhesiveness changes due to a temperature change, as an example, in the case of heating, Laser irradiation is used, and when cooling, a Peltier element is used.

  According to the method for producing a cultured cell specimen configured as described above, the physical structure (ie, template 3) is utilized to the region 2 of the cell non-adherent substrate in the initial stage of culture of living cells and tissues. After the long-term culture is performed in a state in which cell invasion and contamination of adhesion molecules and the like are prevented, and the physical structure (template 3) is removed, the physical structure (template) is removed due to the effect of the cell non-adhesive substrate. The shape of the group of cells / tissues 6 according to 3) is maintained, and the arrangement / orientation of each group is maintained. Therefore, it is possible to produce a specimen in which living cells and tissues matured in an arbitrary shape, arrangement and orientation (in other words, a planar pattern) are cultured.

  According to the method for producing a cultured cell specimen configured as described above, a group of cells / tissues 6 is also adapted to correspond to the exposure intensity distribution of various environmental factors such as chemical substances, radiation, and electromagnetic fields. Can be arranged and oriented, so that dose response and time response can be evaluated simultaneously with a plurality of samples in correspondence with each exposure intensity.

  According to the method for producing a cultured cell specimen configured as described above, the cells / tissues 6 are contained in the culture vessel 1 having a capacity capable of accommodating the medium 5 (liquid medium, culture solution) corresponding to long-term culture. By culturing the medium, it is not necessary to perform a medium exchange operation. This makes it possible to perform long-term culture without affecting or damaging the cells / tissues 6 due to the medium exchange. According to the knowledge of the inventors, by storing the amount of the medium 5 corresponding to the long-term culture in the culture vessel 1, the growth / reproduction of the cells / tissue 6 caused by the waste discharged from the cells / tissue 6 is achieved. The waste products are dispersed in the medium 5 to such an extent that the inhibition of differentiation is sufficiently reduced. Also from this point, it is not necessary to replace the medium 5.

  Although the above-described embodiment is an example of a preferred embodiment for carrying out the present invention, the embodiment of the present invention is not limited to the above-described embodiment, and the present invention is not limited to the scope of the present invention. The invention can be variously modified.

  An embodiment in which the operational effects of the method for producing a cultured cell specimen of the present invention are verified will be described with reference to FIGS.

  As an example of the present invention, a template is placed on the upper surface of a cell non-adhesive substrate region formed at the bottom of a culture vessel, and a cell adhesion substrate region is formed in a hole as a cell culture spot of the template. After injecting the culture medium and cells into the culture container and culturing, the template was removed to prepare a specimen (sample).

The conditions of this example were as follows.
Cell non-adherent substrate: Agarose gel Cell adhesive substrate: Polyethyleneimine (PEI)
Mold material: Polydimethylsiloxane (PDMS)
Medium: Neurobasal medium (+ 2% B-27 supplement, 1% Glutamax supplement, 1% Pen-Strep) (cultured in 20 ml, half medium exchange once every 2 to 3 days)
Cells: Neurons collected from rat cerebral cortex Culture conditions <1>: Cultured for 7 days with template attached,
Thereafter, the template is removed and cultured for 39 days. Cultivation condition <2>: The template is attached and cultured for 21 days.
Thereafter, the mold is removed and cultured for 25 days.

  In this example, in order to verify the maintenance performance of the desired cell shape specified by the shape of the mold hole in plan view and the shape of the cell adhesion substrate region in the hole, the sample prepared under the culture condition <1> and the culture For each of the samples prepared under the condition <2>, the state in which nerve fibers extending from the nerve cells in the cell adhesion substrate region enter the cell non-adhesion substrate region was observed.

  As a result of the observation, FIG. 9 shows the state immediately after removing the template (that is, the seventh day from the start of the culture for the sample of the culture condition <1> and the 21st day from the start of the culture for the sample of the culture condition <2>). 7 days after removal of the template (that is, the 14th day from the start of the culture for the sample of the culture condition <1>, and the 28th day from the start of the culture for the sample of the culture condition <2>) The results shown in FIG. 10 (culture condition <1>) and FIG. 11 (culture condition <2>) are obtained for the state of (eye), and further, the results shown in FIG. It was.

  From the results shown in FIG. 9, immediately after the removal of the template, the boundary between the cell adhesion substrate region and the cell non-adhesion substrate region is clear regardless of the culture condition <1> or the culture condition <2>. It was confirmed that no cell adhesion or invasion into the area was observed.

  From the results shown in FIG. 10, under the culture condition <1>, it can be seen that nerve fibers have invaded from the cell adhesion substrate region to the cell non-adhesion substrate region 7 days after removing the template. It was confirmed.

  On the other hand, from the results shown in FIG. 11, under the culture condition <2>, the boundary between the cell adhesion substrate region and the cell non-adhesion substrate region is clear even after 7 days from the removal of the template. It was confirmed that no invasion of cells (nerve fibers) into the area of the substrate was observed.

  From the results shown in FIG. 12, it can be seen that, after 46 days from the start of the culture, the neurite invades into the region of the cell non-adherent substrate continuously and further advances under the culture condition <1> ( (A)) On the other hand, in the culture condition <2>, the boundary between the cell adhesion substrate region and the cell non-adhesion substrate region is clear, and cells (nerve fibers) enter the cell non-adhesion substrate region. It was confirmed that the cell group maintained the same shape as the hole shape of the template.

  From the results shown in FIG. 9 to FIG. 12, the nerve cells are invaded into the region of the cell non-adherent substrate by physically blocking the extension of the nerve fibers at the initial stage of the culture in which the nerve cells form a network structure with a template. It was confirmed that the growth of active nerve fibers could be suppressed and the formation and maturation of the neural network inside the template could be promoted. As a result, for the sample (culture condition <2>) that has been cultured for a long time (21 days) in the template, the nerve fibers entered the non-adherent substrate region even in the long-term culture (25 days) after removing the template. It was thought that this was able to be suppressed.

  From the above results, according to the present invention, in the initial stage of culturing of living cells, long-term culturing is performed in a state where a template is used to prevent entry of cells into the non-adherent substrate region and contamination of adhesion molecules and the like. After that, it was confirmed that a population of living cells matured in an arbitrary shape and maintained in a clear shape boundary could be produced by the effect of the cell non-adhesive substrate although the template was removed. It was.

DESCRIPTION OF SYMBOLS 1 Culture container 2 Cell non-adhesion substrate area 3 Template 3a Cell culture spot, hole 4 Cell adhesion substrate area 5 Medium 6 Cell, Tissue 7 Pipette

Claims (4)

  A cell non-adhesive substrate region is formed in the culture container, and a template in which a cell culture spot is formed is placed in the cell non-adhesive substrate region, and the cell adhesive substrate region in the cell culture spot of the template A culture medium, and at least one of a culture medium and cells and tissues is put into the culture container, and the template is removed after the cells and tissues are cultured. A method for producing a cell specimen.   The method for producing a cultured cell specimen according to claim 1, wherein a template to which a magnetic material is added is used as the template.   A substance to which a temperature-responsive polymer is added is used as a substance for forming the cell non-adherent substrate region, and the cell adhesive substrate region is separated from the cell non-adherent substrate by heating. The method for producing a cultured cell specimen according to claim 1.   A substance that changes from a property having adhesiveness to the cells or the tissue to a property having no adhesiveness to the cells or the tissue by changing the temperature as a substance that forms the region of the cell adhesion substrate is used, The method for producing a cultured cell specimen according to claim 1, wherein the cells and the tissue are separated and recovered from the region of the cell adhesion substrate by heating or cooling.