JP6900217B2 - Method for preparing cultured cell specimens - Google Patents

Description

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

With the progress of cell culturing 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, studies, tests, measurements, etc. Has been done.

Japanese Unexamined Patent Publication No. 2003-093051

Depending on the type and content of tests and measurements performed using cultured cells, cells are cultured on the surface of a flat culture substrate according to a predetermined pattern (in other words, the shape is an arrangement / orientation mode). May be desired or needed.

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 changes, cell differentiation, etc., there is a problem that the shape of cells and tissues arranged in a specific space cannot be maintained, especially in long-term culture.

For example, in a biological test, as shown in FIG. 13, when an experiment is performed using a group of cells, the threshold value of environmental factors such as chemical substances is determined by the interaction between cells due to the conjugation between cells. There is a problem that it may be underestimated below the original threshold.

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

Therefore, the present invention provides a cultured cell that does not have a physical structure that interferes with various measurements and can produce a specimen in which mature cells and tissues arranged and oriented in a desired pattern are cultured. It is an object of the present invention to provide a method for producing a specimen. Specifically, for example, as shown in FIG. 14, by arranging and orienting cells in a predetermined pattern, interaction between cells is eliminated and the threshold value of environmental factors such as chemical substances is correctly set according to the original threshold value. It is an object of the present invention to provide a method for producing a cultured cell specimen that can be grasped.

In order to achieve such an object, in the method for producing a cultured cell specimen of the present invention, a region of a cell non-adhesive substrate is formed on the bottom surface of a culture vessel, and a template in which a cell culture spot is formed is a region of the cell non-adhesive substrate. A region of the cell adhesion substrate is formed on the upper surface of the region of the cell non-adhesion substrate corresponding to the bottom of each of the cell culture spots of the template, and then the medium and cells and tissues are placed in the culture vessel. At least one of the cells is charged and adhered to the area of the cell adhesion substrate in the cell culture spot for culturing, and after the cells and tissues are cultivated, the template is removed from the culture vessel to remove the cells. A population of cells or tissues cultured on the adhesion substrate is left, and a specimen is prepared in which the shape and arrangement of each population is maintained according to the sequence pattern of the cell culture spot of the template .

Therefore, according to this method for producing a cultured cell specimen, a physical structure (that is, a template) is utilized in the initial stage of culturing a living cell or tissue to invade a region of a cell non-adhesive substrate and an adherent molecule or the like. Long-term culture is performed in a state where the cells are contaminated, and even after the physical structure (template) is removed, due to the effect of the cell non-adhesive substrate, the population of cells / tissues that follow the physical structure (template) The shape is maintained and the arrangement and orientation of each group is maintained.

Further, 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 the magnetic force which is an external force, it is pulled up straight up and removed without tilting or swinging. In this case, even if the adhesion between the non-cell adhesive substrate and the template cannot be expected sufficiently, the magnetic template is attracted from below the culture vessel by using magnetic force to cause non-cell cells. The adhesive substrate and the mold can be well adhered and fixed to maintain the placement of the mold for a long period of time.

Further, 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 forming a region of a cell non-adhesive substrate, and the region of the cell adhesion substrate is formed by heating. It may be detached from the cell non-adhesive substrate. In this case, the region of the cell adhesion substrate (in other words, the range in which the cell adhesion substrate is formed) of the cell non-adhesion matrix is heated so that the region of each cell adhesion substrate is individually divided into the cell non-adhesion substrates. Peel off from.

In addition, the method for producing a cultured cell specimen of the present invention has the property of having cell or tissue adhesion as a substance forming a region of a cell adhesion substrate (in other words, a material that functions as a cell adhesion substrate). ) To non-adhesive properties of cells and tissues (in other words, materials that function as cell non-adhesive substrates), and cells and tissues from the area of cell adhesion substrates when heated or cooled. May be separated and collected. 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 region of the cell adhesion substrate (in other words, the range where the cell adhesion substrate is formed). Will be done.

According to the method for producing a cultured cell specimen of the present invention, a physical structure (that is, a template) is utilized in the initial stage of culturing a living cell or tissue to invade a region of a cell non-adhesive substrate and an adherent molecule. It is possible to carry out long-term culture in a state where contamination such as is prevented, and even after the physical structure (template) is removed, due to the effect of the cell non-adhesive substrate, the cells / tissues that follow the physical structure (template) Since the shape of the population can be maintained and the arrangement / orientation of each population can be maintained, mature living cells and tissues in any shape and arrangement / orientation (in other words, a planar pattern) can be maintained. It becomes possible to prepare a specimen in which the cells are cultured.

In the method for producing a cultured cell specimen of the present invention, when a mold to which a magnetic material is added is used, the mold is removed by pulling it straight up without tilting or rocking. Therefore, it is possible to avoid the influence / damage on cells and tissues in the operation of removing the mold. In this case, also, by attracting the template from below the culture vessel using magnetic force, the cell non-adhesive substrate and the template can be well adhered and fixed, and the arrangement of the template can be maintained for a long period of time. , It becomes possible to more reliably prevent cell invasion into the region of the cell non-adhesive 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 region of the cell adhesion substrate among the cell non-adhesive substrates (in other words, in other words). By heating (the range where the cell adhesion matrix is formed), the region of each cell adhesion substrate can be individually detached from the cell non-adhesion substrate, so that a plurality of cells / tissues cultured in the same environment can be used. It becomes possible to easily separate and collect individual populations and analyze and evaluate each individual cell / tissue population.

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

It is a flowchart explaining an example of embodiment of the method of making the cultured cell specimen of this invention. It is a vertical cross-sectional view which shows typically the state in which the template is placed on the upper surface of the cell non-adhesive substrate in the method of preparing the cultured cell specimen of an embodiment. It is a top view which shows an example of the arrangement of a hole as a cell culture spot in a template. It is a conceptual diagram explaining the outline of the environmental factor (time-varying uniform magnetic field) exposure test by the specimen in which cells were cultured using the template shown in FIG. It is a vertical cross-sectional view which shows typically the state which the region of the cell adhesion substrate was formed in the hole of the template in the method of making the cultured cell specimen of an embodiment. It is a vertical cross-sectional view which shows typically the state in which the culture medium, the cell and the tissue were put in in the method of preparing the cultured cell specimen of an embodiment. It is a vertical cross-sectional view which shows typically the state at the time of culture in the method of making the cultured cell specimen of an embodiment. It is a vertical cross-sectional view which shows typically the state which the template was removed after culturing in the method of preparing the cultured cell specimen of an embodiment. It is a figure which shows the state of the boundary of the cell population immediately after removing the template in the specimen prepared in Example 1. FIG. (A) is a figure which shows the state of the specimen under the culture condition <1>. (B) is a figure which shows the state of the specimen under the culture condition <2>. It is a figure which shows the state of the boundary of the cell population on the 7th day after removing the template in the specimen of the culture condition <1> prepared in Example 1. FIG. It is a figure which shows the state of the boundary of the cell population on the 7th day after removing the template in the specimen of the culture condition <2> prepared in Example 1. FIG. It is a figure which shows the boundary of the cell population 46 days from the start of culture in the specimen prepared in Example 1. FIG. (A) is a figure which shows the state of the specimen under the culture condition <1>. (B) is a figure which shows the state of the specimen under the culture condition <2>. It is a conceptual diagram explaining a problem in a conventional biological test. It is a conceptual diagram explaining the concept of the method of solving the problem in the conventional biological test.

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 the method for producing a cultured cell specimen of the present invention.

In the method for producing a cultured cell specimen of the present embodiment, the region 2 of the cell non-adhesive substrate is formed in the culture vessel 1 (S1), and the template 3 in which the cell culture spot 3a is formed is the region of the cell non-adhesive substrate. Along with (S2) placed in 2, a region 4 of the cell adhesion substrate is formed in the cell culture spot 3a of the template 3 (S3), and the medium 5 and the cells 6 and the tissue 6 are placed in the culture vessel 1 on the region 4. At least one of the above is charged (S4), and the template 3 is removed (S7) after the cells 6 and the tissue 6 have been cultured (S6) (see FIG. 1).

The size of the culture vessel 1 (also called a petri dish) takes into consideration the size of the template 3 used in combination with the culture vessel 1 and the amount of cells / tissues 6 put into the culture vessel 1 and cultured. After that, it is set so that at least a volume capable of accommodating an amount of medium 5 that enables long-term culture (in other words, an amount of medium 5 that does not require replacement of medium 5 during long-term culture) can be secured. To. 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 medium 5 in the cell / tissue culture state in which the template 3 is placed in the culture vessel 1 and the medium 5 is charged. ..

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

The region 2 of the cell non-adhesive substrate is a region formed by a substrate having no adhesiveness of living cells or tissues, and therefore is a region of a substrate on which proliferation or differentiation of living cells is 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 or tissues or a substance that has been subjected to a predetermined treatment is appropriately selected. Specific examples of the cell non-adhesive substrate include agarose gel, bovine serum albumin (BSA), 2-methacryloyloxyethyl phosphorylcholine polymer, polyethylene glycol, organic fluorine compound, and these terminals. A silane coupling agent or the like can be used.

Specifically, for the region 2 of the cell non-adhesive substrate, for example, when an agarose gel is used for the formation of 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.

Region 2 of the cell non-adhesive substrate, or specifically, for example, when bovine serum albumin (BSA) is used for the formation of the region 2, a BSA solution prepared to be about 0.1 to 1%, for example. Is dropped onto 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.

Region 2 of the cell non-adhesive substrate is formed as a flat film (in other words, a layer) at least in the portion (in other words, the range) where the template 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 container 1 is placed on the bottom of the culture container 1, and a region 2 of the cell non-adhesive substrate is formed on the upper surface of the glass plate as, for example, a film or a layer. It may be done.

Next, the template 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, identifying or inducing) an adhesion pattern of cells / tissues 6 on the substrate, and is a region of the cell non-adhesive substrate formed at the bottom of the culture vessel 1. Holes 3a (that is, through holes) in which the lower surface and the upper surface in contact with the upper surface of No. 2 are open are formed as cell culture spots on a plate-shaped member.

The material of the mold 3 is not limited to a specific type, and an appropriate material is appropriately selected, for example, considering that it cannot be altered depending on the medium 5 charged into the culture vessel 1. To. Specific examples of the material of the mold 3 include silicon resin, polydimethylsiloxane (also referred to as “PDMS”), acrylic resin, glass, parylene, and hydrogels such as 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 described later, the mold 3 is removed by using the magnetic force which 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 / damage on the cells / tissues 6 in the removal operation of the mold 3. In this case, even if the adhesion between the cell non-adhesive substrate and the template 3 cannot be expected sufficiently, the magnetic template 3 is attracted from below the culture vessel 1 by using magnetic force. As a result, the adsorption force of the template 3 to the cell non-adhesive substrate can be enhanced to allow the cell non-adhesive substrate and the template 3 to be in close contact with each other and fixed, so that the arrangement of the template 3 can be maintained for a long period of time. It becomes possible to more reliably prevent cell invasion of the adhesive substrate into region 2 and contamination of adhesive molecules and the like.

The magnetic material added to the material forming the template 3 is not limited to a specific type, and is appropriate after considering, for example, the influence on the growth and differentiation of cells / tissues 6. Is appropriately selected. As the material having magnetism to be added, for example, various magnetic particles can be selected.

Although the mold 3 is not limited to a specific size or shape, for example, considering that a circular one is widely used as a cell culture container in which the mold 3 is placed, for example, the diameter is 5 It is considered that a disk-shaped one of about 10 cm is easy to fit.

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 is, for example, a cultured cell required for each specimen according to the contents of various measurements and the like. -It is formed in a size and shape that matches the desired shape as a group of tissues.

Specifically, the hole 3a as a cell culture spot has a penetrating void / hole having a minimum dimension (inner transfer) of about 0.01 to 2 mm and a maximum dimension (inner transfer) of about 2 to 3 mm. It is possible that it is formed as a gap. In addition, 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 mode of arrangement / orientation of the holes 3a (through holes) formed in the template 3 as the cell culture spot is not limited to a specific pattern, and is required for each specimen according to the contents of various measurements, for example. The mode is set according to the desired arrangement and orientation of the cultured cell or tissue population.

The mode of arrangement / orientation of the holes 3a (through holes) as cell culture spots is specifically a specimen used for a time-varying uniform magnetic field application test as an example of an environmental factor exposure test. It is conceivable that the cells are set concentrically as shown in FIG.

By installing and culturing the template 3 shown in FIG. 3, a specimen in which a plurality of concentric cell / tissue 6 populations are arranged and oriented according to the pattern of holes 3a as cell culture spots of the template 3 is prepared. As shown in FIG. 4, it is conceivable to apply a time-varying uniform magnetic field to the specimen to perform the test.

As shown in FIG. 4, in the method of injecting a magnetic field that fluctuates in a uniform time in the vertical direction from the upper surface of the culture vessel 1 (specimen), the current density induced in the medium 5 follows the following formula 1, so that the culture is performed. A concentric induced current intensity distribution proportional to the radial distance is formed from the central portion (that is, r = 0) of the container 1. In FIG. 3, the broken line (reference numeral 9) represents the 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: pi, f: frequency [Hz], B: magnetic flux density [T], r: radius. Represents [m] respectively.

A time-varying uniform magnetic field as shown in FIG. 4 to a specimen in which a plurality of concentric cell / tissue 6 populations are arranged and oriented by cell culture using the template 3 shown in FIG. The application reduces the variation in the induced current intensity applied to the population of cells / tissues 6 (note that the smaller the size of the hole 3a, the smaller the variation in the induced current intensity) and the cell response to different induced magnetic field strengths. It can be evaluated at the same time, and the analysis of the relationship between the difference in stimulation direction and intensity and the difference in the effect on cells can be performed efficiently, easily and appropriately. In addition, evaluation with a single type of cell is performed and a plurality of types are evaluated depending on whether a single type of cell is seeded or a plurality of types of cells are seeded in each of the holes 3a as a cell culture spot formed in the template 3. You will be able to do both evaluations on the network of cells.

It is preferable that the lower surface of the mold 3 is adsorbed on 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, the molecule that acts on the PDMS and the cell non-adhesive substrate. PDMS is adsorbed and fixed by the intermolecular force.

A film may be formed on the surface of the mold 3 by a substance having no adhesiveness between cells and tissues.

Further, a plurality of molds 3 may be installed in one culture container 1.

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

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

The substance used as the cell adhesion substrate is not limited to a specific substance, and the cell or tissue adhesion is induced after considering, for example, 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. As the cell adhesion substrate, specifically, for example, laminin, collagen, fibronectin, polyethyleneimine (PEI), polylysine, Puramatrix, antibody and the like can be used.

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, for example, about 0.1%. It is formed by storing it overnight at (for example, about 4 ° C.) and then washing it with sterile water (it is preferable to wash it four times or more because it is highly toxic).

Region 4 of the cell adhesion substrate, or specifically, for example, when poly-L-lysine (PLL) is used for the formation of the region 4, a PLL solution prepared to be about 0.1%, for example, is used. It is formed by dropping and storing in a refrigerator (for example, about 4 ° C.) overnight and then washing with sterile water (for example, about once).

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

The region 4 of the cell adhesion substrate may be formed so as to be laminated on the upper surface of the region 2 of the cell non-adhesive substrate at the portion corresponding to the bottom of the hole 3a, or the region 4 of the cell non-adhesive substrate may be formed. 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 cell non-adhesive substrate region 2.

Here, regarding the region 4 of the cell adhesion substrate formed in the hole 3a as the cell culture spot, the same type of cell / tissue 6 is seeded after forming the region 4 of the cell adhesion substrate of a different type for each hole 3a. By exposing to environmental factors after culturing, it is possible to apply environmental factors under the same conditions 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 the difference in 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 the samples can be reduced.

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

The medium 5 (also referred to as a liquid medium or a culture medium) is not limited to a specific type, and has, for example, the type, properties, and fluidity of the cells / tissues 6 to be cultured, and has the hole 3a of the template 3. Appropriate ones are appropriately selected after considering that they can enter the culture medium.

The medium 5 is charged with at least an amount sufficient to enable culturing over the culturing period, taking into consideration the culturing period required for functional maturation of the cells / tissues 6. The mold 3 placed at the bottom of the culture vessel 1 is adjusted so as to be immersed in the medium 5.

Here, an environmental factor or a drug may be added to the medium 5. This makes it possible to evaluate the response of different cell / tissue 6 populations at once under the same culture conditions.

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

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

For the cells / tissues 6, for example, a desired amount (number) of the cells / tissues 6 to be adhered to each of the holes 3a as cell culture spots formed in the template 3 as an initial state is taken into consideration according to the contents of various measurements and the like. Then, for example, an amount sufficient to enable the desired amount of adhesion is added.

Regarding the charging of the medium 5 and the cells / tissues 6, the cells / tissues 6 may be charged into the medium 5 after the medium 5 is charged into the culture vessel 1, or the cell culture spot. The medium 5 may be charged into the culture vessel 1 after the cells / tissues 6 are (individually) charged into the hole 3a.

The cell / tissue 6 put into the culture vessel 1 adheres to the region 4 of the cell adhesion substrate 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 charged, the medium 5 is exchanged waiting for the time when these cells / tissues 6 adhere to the region 4 of the cell adhesion substrate in the hole 3a as the cell culture spot.

The medium 5 is exchanged, for example, after considering the type of cells / tissues 6 to be cultured, when an appropriate time elapses after the cells / tissues 6 are added.

Before the medium 5 was charged into the culture vessel 1, the cells / tissues 6 were directly charged / seeded into the hole 3a as the cell culture spot, and then the medium 5 was charged into the culture vessel 1. 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 culturing period is appropriately set to an appropriate length, for example, considering the conditions required for each specimen according to the type of cells / tissues 6 to be cultivated and the contents of various measurements.

Here, one of the features of the present invention is that it is possible to culture the cells / tissues 6 for a long period of time to the extent that they can be functionally matured.

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

During the culture period, the invasion of cells / tissues 6 is prevented from the upper surface of the cell non-adhesive substrate region 2 around the template 3 and the upper surface of the cell non-adhesive substrate region 2 to which the lower surface of the template 3 is adhered. The cells / tissues 6 do not adhere to each other because the cell adhesion molecules are removed or the cell adhesion molecules are removed via the medium 5.

On the other hand, cells / tissues 6 adhere to the region 4 of the cell adhesion substrate in the hole 3a formed as a cell culture spot in the template 3, and proliferation / differentiation is performed.

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

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

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

According to the above step, the cells / tissues 6 adhere to the portion of the culture vessel 1 where the cell non-adhesive substrate is exposed or the portion where the upper surface of the cell non-adherent substrate and the lower surface of the mold 3 are in contact with each other. Since the cell / tissue 6 adheres only to the region 4 of the cell adhesion substrate in the hole 3a of the template 3, each of the cell / tissue 6 populations independently has no binding at the adjacent portion, and each of them The cell / tissue 6 population is functionally sufficiently mature as a result of long-term culture.

Further, even after the template 3 is removed, the shape and arrangement of each of the functionally mature cell / tissue 6 populations are maintained by the action of the cell non-adhesive substrate.

Then, by performing various measurements with the template 3 removed, environmental factors such as exposure to cells / tissues 6 (specifically, specimens to which cultured cells / tissues 6 are attached) are exposed. It is possible to perform accurate measurement and the like without disturbing the spatial distribution of the above, that is, with the desired spatial distribution intended for environmental factors.

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

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

The temperature-responsive polymer added to the substance used for the formation of region 2 of the cell non-adhesive substrate is not limited to a specific type, for example, the substance used for the formation of region 2 of the cell non-adhesive substrate. An appropriate one is appropriately selected after considering the influence on the properties of the cell / tissue 6 and the influence on the proliferation / differentiation of the cell / tissue 6. Specifically, for example, poly (N-isopropylacrylamide) can be selected as the temperature-responsive polymer to be added.

As a heating method for peeling 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, it does not have cell or tissue adhesion due to its property of having cell or tissue adhesion (in other words, a material that functions as a cell adhesion substrate) due to changes in temperature as a substance forming region 4 of the cell adhesion substrate. A substance that transforms into properties (in other words, a material that functions as a cell non-adhesive substrate) is used so that cells / tissues 6 can be separated and recovered from region 4 of the cell adhesion substrate by heating or cooling. You may. In this case, each cell / tissue 6 is individually removed from the cell adhesion substrate by locally heating or cooling each region 4 of the cell adhesion substrate (in other words, the range in which the cell adhesion substrate is formed). It has become possible to separate and easily separate and collect a plurality of cell / tissue 6 populations cultured in the same environment, and analyze / evaluate each individual cell / tissue 6 population. become.

The substance used for forming the region 4 of the cell adhesion substrate, which changes from cell adhesion to cell non-adhesion due to a temperature change, is not limited to a specific type, and is, for example, proliferation / differentiation of cells / tissues 6. Appropriate ones are appropriately selected after considering the influence on the cells. Specifically, for example, poly (N-isopropylacrylamide) can be selected as the substance whose cell adhesion changes with temperature change.

As a method of giving a temperature change in order to separate cells / tissues 6 from the region 4 of the cell adhesion substrate formed by using a substance whose cell adhesion changes with a temperature change, only one example is the case of heating. Is laser irradiation, and when cooling, a Perche element is used.

According to the method for producing a cultured cell specimen constructed as described above, the physical structure (that is, template 3) is utilized in the initial stage of culturing living cells and tissues to the region 2 of the cell non-adhesive substrate. Long-term culture was performed in a state where cell invasion and contamination of adherent molecules were prevented, and even after the physical structure (template 3) was removed, the physical structure (template) was affected by the effect of the cell non-adhesive substrate. The shape of the cell / tissue 6 population according to 3) is maintained, and the arrangement / orientation of each population is maintained. Therefore, it is possible to prepare a specimen in which mature living cells and tissues are cultured in an arbitrary shape, arrangement, and orientation (in other words, a planar pattern).

According to the method for producing a cultured cell specimen constructed as described above, and a population of cells / tissues 6 corresponding to the exposure intensity distribution of various environmental factors such as chemical substances, radiation, and electromagnetic fields. Can be arranged and oriented, so that the 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 a culture vessel 1 having a capacity capable of accommodating a medium 5 (liquid medium, culture medium) corresponding to long-term culture. By culturing the medium, the operation of medium exchange is not required, which enables long-term culture without affecting or damaging the cells / tissues 6 associated with the medium exchange. According to the findings of the inventors, by storing the medium 5 in an amount corresponding to long-term culture in the culture vessel 1, the proliferation of cells / tissues 6 caused by the waste products discharged from the cells / tissues 6 The waste products are dispersed in the medium 5 to the extent that the inhibition of differentiation is sufficiently reduced, and from this point as well, replacement of the medium 5 becomes unnecessary.

Although the above-described embodiment is an example of a suitable mode for carrying out the present invention, the embodiment of the present invention is not limited to the above-mentioned one, and the present invention is not limited to the above-mentioned embodiment and does not deviate from the gist of the present invention. The invention can be modified in various ways.

Examples of verifying the action and effect of the method for producing a cultured cell specimen of the present invention will be described with reference to FIGS. 9 to 12.

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

The conditions of this example were as follows.
Non-cell adhesion substrate: Agarose gel Cell adhesion substrate: Polyethylenimine (PEI)
Mold material: Polydimethylsiloxane (PDMS)
Medium: Neurobasal medium (+ 2% B-27 supplement, 1% Glutamax supplement, 1% Pen-Strep) (cultured in 20 ml, half volume exchanged once every 2 to 3 days)
Cells: Nerve cells collected from rat cerebral cortex Culturing conditions <1>: Incubate for 7 days with a template attached.
After that, culture for 39 days with the mold removed. Culture condition <2>: Incubate for 21 days with the mold attached.
After that, culture for 25 days with the mold removed.

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

As a result of observation, FIG. 9 shows the state immediately after the template was removed (that is, 7 days from the start of culture for the sample under culture condition <1>, and 21 days from the start of culture for the sample under culture condition <2>). 7 days after removing the template (that is, 14 days from the start of culture for the sample under culture condition <1>, 28 days from the start of culture for the sample under culture condition <2>. The results shown in FIGS. 10 (culture conditions <1>) and 11 (culture conditions <2>) were obtained for the state of (eyes), and the results shown in FIG. 12 were obtained for the state 46 days after the start of culture. It was.

From the results shown in FIG. 9, immediately after the mold was removed, the boundary between the cell adhesion substrate region and the cell non-adhesion substrate region was clear under both the culture conditions <1> and the culture conditions <2>, and the cell non-adhesion substrate 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 the nerve fibers invaded from the cell adhesion substrate region to the cell non-adhesion substrate region 7 days after the template was removed. It was confirmed that.

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 was clear even 7 days after the template was removed, and the cells were non-adherent. It was confirmed that the invasion of cells (nerve fibers) into the area of the substrate was not observed.

From the results shown in FIG. 12, it can be seen that 46 days after the start of culture, under the culture condition <1>, the invasion of the nerve protrusions into the region of the cell non-adherent substrate continues and further progresses ( 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) invade the cell non-adhesion substrate region. It was confirmed that the cell group retained the same shape as the hole shape of the template.

From the results shown in FIGS. 9 to 12, the elongation of nerve fibers in the early stage of culture in which nerve cells form a network structure is physically blocked by a template to invade the region of the cell non-adhesive substrate. It was confirmed that it can suppress the elongation of active nerve fibers and promote the formation and maturation of the neural network inside the template. As a result, for the sample (culture condition <2>) that was subjected to long-term culture (21 days) in the template, nerve fibers invaded the region of the cell non-adhesive substrate even in the long-term culture (25 days) after removal of the template. It was considered that this could be suppressed.

From the above results, according to the present invention, in the initial stage of culturing living cells, long-term culturing is performed in a state where a template is used to prevent cell invasion into the region of non-adhesive matrix of cells and contamination of adherent molecules and the like. After that, it was confirmed that although the template was removed, the effect of the cell non-adhesive substrate made it possible to generate a population of living cells that matured in any shape and that the boundary of the shape was maintained with a clear boundary. It was.

1 Culture vessel 2 Cell non-adhesion substrate area 3 Template 3a Cell culture spot, hole 4 Cell adhesion substrate area 5 Medium 6 cells, tissue 7 Pipet

Claims (4)

A region of cell non-adhesive substrate is formed on the bottom surface of the culture vessel,
The template on which the cell culture spot is formed is placed in the region of the cell non-adhesive substrate, and the cell adhesion substrate is placed on the upper surface of the region of the cell non-adhesive substrate corresponding to the bottom of each of the template in the cell culture spot. Region is formed,
Then, at least one of the medium and cells and tissues is put into the culture vessel and adhered to the region of the cell adhesion substrate in the cell culture spot for culturing.
After the cells and tissues have been cultured, the template is removed from the culture vessel to leave a population of the cells or tissues cultured on the cell adhesion substrate, and the cell culture of the template remains. A method for producing a cultured cell specimen, which comprises producing a specimen in which the shape and arrangement of each group are maintained according to a spot arrangement pattern. 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 forming the region of the cell non-adhesive substrate, and the region of the cell adhesion substrate is separated from the cell non-adhesive substrate by heating. The method for producing a cultured cell specimen according to claim 1. As a substance forming the region of the cell adhesion substrate, a substance that changes from the property of having the adhesiveness of the cell or the tissue to the property of not having the adhesiveness of the cell or the tissue by changing the temperature 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.

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