JP2008173018A - Method for culturing cell and substrate for cell culture - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for inoculating cells with small variation in the number of cells when the cells are inoculated in a plurality of culture vessels, or a means for uniformly inoculating cells in a culture vessel. <P>SOLUTION: Used is a substrate for cell culture having a cellular adhesiveness-variable surface in which cell-adhesive compartments are arranged in certain intervals, and the hydrophilicity and hydrophobicity of regions except the cell-adhesive compartments are varied by external stimulation to change the cellular adhesiveness. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a cell culture substrate for culturing cells. More specifically, without strict adjustment of the number of cells, there is little variation in the number of cells in a plurality of culture vessels, and a cell culture substrate that can be easily cultured at a uniform cell number and cell density, And a cell culture method using the same.

  Until now, studies have been widely conducted to identify the proliferation and differentiation state of cells and the substances produced by the cells by culturing the cells. In recent years, studies have been conducted in which patient-derived cells and pluripotent stem cells are cultured in vitro and the cells are actively used in medicine. In the use of cells in such research and medical fields, it is necessary to control proliferation and differentiation with high reproducibility in a plurality of experiments or a plurality of samples regardless of the skill of the person who operates the cells. However, cell kinetics is greatly influenced not only by the composition of the culture solution but also by the number and density of cells, so when seeding cells in a culture vessel, the number and density of cells according to the type of cells used Must be seeded under optimal conditions. For this purpose, it is necessary to strictly adjust the number of cells before seeding and to uniformly distribute cells on the culture substrate when seeding.

  However, in order to adjust the number of cells, an error occurs because a part of the cell suspension is counted and diluted. In addition, when seeding cells, the cell suspension is usually aspirated using a pipette and seeded in a culture vessel. Since many cells settle naturally in the suspension, many cells are When the seeding process is performed on a plurality of culture containers, the number of cells varies among the culture containers. Furthermore, when cells are cultured for adhesion, it is difficult to uniformly adhere the cells to the surface of the culture vessel, and the density of the cells increases particularly at the edge of the vessel and at the center due to shaking of the culture solution. Will be biased. If the cell density is uneven, cell differentiation will be promoted in the part where the cell density is high, which will cause bias in cell growth and differentiation in the culture vessel, and the reproducibility and reliability of the culture will be impaired. There is a problem that it ends up.

  As a method for uniformly seeding cells, a cell seeding method having a step of uniformly infiltrating cells into a culture substrate with respect to a porous cell culture substrate is disclosed (for example, see Patent Document 1). ). This is used for three-dimensional culture and is not a technique that can be used for normal two-dimensional culture. In addition, a technique for uniformly dispersing a cell suspension by giving a shape to an inner surface of a culture vessel is disclosed (for example, see Patent Document 2). According to this, the problem of many cells adhering to the inner surface of the culture vessel can be improved, but the bias to the center of the cell due to shaking of the culture solution or the process of seeding cells in multiple vessels There is no solution to the variation in.

  On the other hand, a culture vessel in which a plurality of cell adhesive regions are provided on a cell non-adhesive surface is disclosed (for example, Patent Document 3). Also, a method of immobilizing temperature-responsive polymers on the surface of a culture dish by an electron beam irradiation method (for example, Non-Patent Document 1), and cell non-adhesive properties around a cell adhesion region composed of temperature-responsive polymers A cell array substrate that is surrounded by the region is disclosed (for example, Patent Document 4). These are culture substrates used for culturing cells in a state where the cells are divided into independent compartments, and the cells cannot be cultured on the entire surface of the culture vessel which is the subject of the present invention.

By the way, as a technique for controlling cell adhesion and non-adhesion, a technique for controlling cell adhesion by temperature by using a culture substrate coated with a temperature-responsive polymer is disclosed (for example, non-patent). Literature 1, Patent Literature 4, Patent Literature 5). In addition, a technique for controlling cell adhesion / non-adhesion by light stimulation is disclosed (for example, Patent Document 6). These techniques are useful because they can change the adhesion of cells at any timing during cell culture.
JP 2005-34020 A JP-A-11-290062 JP 2000-41659 A JP 2003-33177 A JP 2003-310244 A JP 2005-210936 A Yamato M., et al., J. Biomedical Material Research, 55, 137 (2001)

  Therefore, an object of the present invention is to reduce the number of cells that are seeded for each culture container when seeding cells, and to easily realize uniform seeding of cells in each culture container. is there.

  The above object can be achieved by the following.

  (1) (i) a step of seeding cells on a substrate having a plurality of regions that are cell-adhesive and a region that is non-cell-adhesive; (ii) the cell is non-adhesive by applying an external stimulus to the substrate A method for culturing cells, comprising the step of imparting cell adhesion to a region, and (iii) culturing the seeded cells in the region imparted with cell adhesion.

  (2) The cell culture method according to (1), comprising a step of shaking the culture substrate following the step (i).

  (3) The cell culture method according to (1) or (2), which comprises a step of removing unadhered cells from the substrate following the step (i).

  (4) Following the step (i), including a step of collecting the culture solution, removing the cells in the collected culture solution, and returning the cells to the cell culture substrate again (1) to ( The cell culture method according to any one of 3).

  (5) A cell culture substrate comprising a cell adhesive region composed of a plurality of compartments arranged at regular intervals, and a cell adhesive variable region whose cell adhesiveness can be changed by external stimulation.

  (6) The cell culture substrate according to (5), wherein the cell adhesion variable region is coated with a stimulus-responsive polymer.

  (7) The cell culture substrate according to (5) or (6), wherein a section of the cell adhesive region is coated with at least one cell adhesive substance.

(8) The cell culture substrate according to any one of (5) to (7), wherein an area of one section of the cell adhesive region is 1 μm ² or more and 10,000 μm ² or less.

(9) The cell culture substrate according to any one of (5) to (8), wherein the cell adhesive region sections are arranged in an amount of 1 to 10 ⁵ per 1 cm ^{2 of the} substrate.

  (10) A cell culture method using the cell culture substrate according to any one of (5) to (9), the step of keeping the cell adhesion variable surface non-adherent, and cells on the substrate A cell comprising seeding and initial adhesion of cells to the cell adhesion region, removing the cells not initially adhered, and changing the cell adhesion variable surface to cell adhesion. Culture method.

  (11) The cell culture method according to (10), wherein cells equal to or greater than the number of the cell adhesive compartments are seeded.

  (12) The method for culturing cells according to (10) or (11), comprising a step of shaking the culture substrate after seeding the cells.

  (13) The culture solution is collected after a certain time after seeding the cells, the cells in the collected culture solution are removed, and then returned to the cell culture substrate again. (10) to (12) The cell culture method according to any one of the above.

  As a result of intensive studies, the present inventors have changed the hydrophilicity / hydrophobicity of a cell adhesive region composed of a plurality of compartments arranged at regular intervals and a region excluding the cell adhesive compartment by external stimulation, and cell adhesion. This has been solved by using a cell culture substrate having a cell adhesion variable surface whose properties change. When cell culture is performed using the cell culture substrate of the present application, a cell suspension that retains the number of cells necessary for the desired seeding density is maintained by keeping the cell adhesion variable surface non-adherent during cell seeding. After seeding and completing initial adhesion of cells to the cell adhesion area, cells that have not adhered are removed, the cell adhesion variable surface is changed to cell adhesion, and culturing is continued, so that the cells are cultured on the culture substrate. It is possible to grow or migrate by infiltrating the entire surface of the cell, and it is not necessary to strictly adjust the cell suspension, and culture with uniform initial conditions is possible.

  The present invention is described in detail below.

  In the cell culture method of the present invention, (i) a step of seeding cells on a substrate having a plurality of cell-adhesive regions and a non-cell-adhesive region, (ii) applying an external stimulus to the substrate, A step of imparting cell adhesiveness to the non-cell-adhesive region, and (iii) a step of culturing the seeded cells in the region imparted with cell adhesiveness. By seeding the cells in the step (i), the cells adhere to the cell adhesive region on the substrate. By setting the cell adhesion region at a desired position, the cells can be adhered to the desired position on the substrate. By imparting cell adhesion to the non-adhesive region in the step (ii), cells that could not adhere in the step (i) can be adhered, and the cell adhesive region on the substrate as shown in (iii) And it becomes possible to culture | cultivate a cell to a non-adhesive area | region. Through the above steps, cell culture at a desired position is possible. Furthermore, the number of cells can be adjusted.

  The cell culture substrate of the present invention has a cell adhesion region comprising a plurality of compartments arranged at predetermined intervals on a base material, and a property other than each cell adhesion region is made hydrophilic / hydrophobic by external stimulation. And at least a region having a cell adhesion variable surface capable of switching between cell adhesion and non-adhesion. By arranging the cell adhesion regions at regular intervals, cells can be initially adhered on the substrate at regular intervals. As a result, when cells infiltrate and proliferate on the entire surface of the culture substrate, it is advantageous because they can be uniformly propagated.

  The substrate for cell culture according to the present invention is not particularly limited, but those having good cell adhesion, those capable of introducing a functional group for promoting cell adhesion, and good adhesion substances The thing which can be carry | supported can be mentioned. In addition, any material and shape can be used as long as they can stably fix and carry a polymer that forms a surface that changes hydrophilicity / hydrophobicity by an external stimulus. Specifically, a glass plate, a silicon substrate, a metal and ceramic substrate, polystyrene, polyethylene terephthalate, polymethyl methacrylate, and a plastic plate, a plastic sheet, and a polymer film made of a polymer compound capable of providing other forms. , Etc. can be suitably used. In addition, in order to improve the adhesion of the cell adhesion region, or to immobilize the adhesive substance and the substance that changes hydrophilicity / hydrophobicity by external stimulation, a part or the whole of the substrate is treated with a chemical substance or irradiated with radiation. You may perform the process to irradiate. The base material may be a so-called petri dish with a wall-like structure provided on the outer periphery, or a sheet. In the case of a petri dish, the culture solution can be directly put. On the other hand, if it is in the form of a sheet, the sheet can be placed in a culture vessel and cultured if necessary.

  The substrate can be subjected to various treatments as necessary. Specifically, it is possible to perform chemical physical treatment such as cleaning to remove undesired substances, irradiation with radiation such as ultraviolet rays, and corona discharge. Moreover, you may apply | coat a polymer material, a silane coupling agent, etc. to a part or whole surface of a base material as needed.

  Any cell adhesive region can be used as long as cells adhere well and can be cultured. For example, a substrate exhibiting cell adhesiveness may be selected in advance, and a cell adhesive variable surface may be formed so that the substrate surface is exposed at regular intervals, or a functional group having good cell adhesiveness may be formed in the cell adhesive region. It may be introduced on the substrate surface. In order to adhere cells, at least one kind of cell adhesive substance may be fixed to the surface of the cell adhesion region. The cell adhesion substance is not particularly limited, but polylysine, collagen, vitronectin, fibrin, fibronectin, laminin, other cell adhesion proteins, polypeptides and the like are preferably used. For immobilization of the cell adhesive substance to the substrate, biological affinity may be used, either through a covalent bond or through electrostatic attraction. When immobilized via a covalent bond, the adhesive substance can be immobilized with a strong force, and since the binding force is not easily affected by cells or culture medium, it can be stably immobilized on the substrate. .

  The cell adhesion variable region surface can be prepared by chemically binding the end of a highly branched chain having a functional group whose hydrophilicity / hydrophobicity is changed by an external stimulus, directly or via a trunk polymer. The polymer used in the present invention may be a homopolymer or a copolymer. Here, the external stimulus can include heat or light.

  The stimuli-responsive polymer as used in the present invention is hydrophilic at the lower critical solution temperature or lower, becomes non-cell-adhesive, becomes hydrophobic at the lower critical solution temperature or higher, and becomes cell-adhesive. Examples thereof include a temperature responsive polymer, a photoresponsive polymer, and the like. Examples of temperature-responsive polymers include (meth) acrylamide compounds such as acrylamide and methacrylamide, N-ethylacrylamide (lower critical dissolution temperature of homopolymer 72 ° C.), Nn-propyl acrylamide (21 ° C.). N-propyl methacrylamide (27 ° C), N-isopropylacrylamide (32 ° C), N-isopropylmethacrylamide (43 ° C), N-cyclopropylacrylamide (45 ° C), N-cyclopropyl Methacrylamide (at 60 ° C), N-ethoxyethylacrylamide (at about 35 ° C), N-ethoxyethylmethacrylamide (at about 45 ° C), N-tetrahydrofurfurylacrylamide (at about 28 ° C), N-tetrahydrofur N-alkyl substitution such as furyl methacrylamide (about 35 ° C.) N) such as acrylamide derivatives, N, N-dimethyl (meth) acrylamide, N, N-ethylmethylacrylamide (lower critical dissolution temperature of homopolymer 56 ° C.), N, N-diethylacrylamide (32 ° C.) N-dialkyl-substituted (meth) acrylamide derivatives, 1- (1-oxo-2-propenyl) -pyrrolidine (56 ° C.), 1- (1-oxo-2-propenyl) -piperidine (about 6 ° C.), 4- (1-oxo-2-propenyl) -morpholine, 1- (1-oxo-2-methyl-2-propenyl) -pyrrolidine, 1- (1-oxo-2-methyl-2-propenyl) -piperidine, (Meth) acrylamide derivatives having a cyclic group such as 4- (1-oxo-2-methyl-2-propenyl) -morpholine, methyl vinyl ether (lower critical solution temperature of homopolymer) 35 ° C) or the like, or when it is necessary to adjust the critical lysis temperature depending on the cell type, when it is necessary to enhance the interaction between the coating substance and the support, For the purpose of adjusting the hydrophobic balance, copolymerization with monomers other than those described above, graft polymerization or copolymerization of polymers, or a mixture of polymers or copolymers may be used. Moreover, it is also possible to crosslink within a range that does not impair the original properties of the polymer. The photoresponsive polymer includes a polymer that undergoes a photoisomerization reaction, such as an absorbent polymer having an azobenzene group, and a copolymer of a vinyl derivative of triphenylmethane leuco hydroxide and an acrylamide monomer. A temperature-responsive polymer having a functional group capable of photoion dissociation as in the above, or a temperature-responsive polymer in which hydrophobic interaction is photochanged, such as N-isopropylacrylamide gel containing spirobenzopyran, can be used.

Is not particularly limited on the size of the cell adhesive region, in 1μm ² ~10000μm ^2, more preferably of 4μm ² ~900μm ² size, preferably the size of the cells can be adhered only one each compartment . The shape of the cell adhesion region is not particularly limited, but a circular shape is preferable. In addition, the density of the cell adhesive region can be appropriately selected according to the cell to be used and the purpose of cell culture, but the cell adhesive region has 1 to 10 ⁶ compartments per 1 cm ^{2 of the} substrate, more preferably 10 to 10 ⁵ are preferred.

  FIG. 1 is an example schematically showing a cell culture substrate of the present invention. A cell adhesive region 2 is formed on the surface of the substrate 1, and a cell adhesive variable surface 3 is formed so as to surround the cell adhesive region 2.

  As the cells that can be used in the present invention, any cells can be used according to the purpose as long as they are cultured in an adherent state. For example, nerve cells, hepatocytes, fibroblasts, myoblasts, smooth muscle cells, cardiomyocytes, skeletal muscle cells, stem cells, mesodermal stem cells, embryonic stem cells, glial cells, embryonic stem cells, hematopoietic stem cells, mast cells , Whole cells such as adipocytes, neural stem cells, etc. (for example, primary cultured cells isolated from animals such as humans, mice, rats, etc., and their transformed cells or non-transformed cells); cell masses are appropriately selected Can be used.

  In order to form a cell-adhesive section and a cell-adhesive variable region on the cell culture substrate of the present invention, there is no particular limitation as long as these regions can be formed. A material that covers these regions may be applied and physically adsorbed, or may be fixed using electron beam irradiation, ultraviolet irradiation, plasma treatment, corona treatment, or the like. In that case, patterning can be performed by immobilizing a target substance only at a specific portion by using a mask. Alternatively, after the entire surface is coated with a material that covers the cell adhesion variable region, only a part is scraped by laser ablation, so that the scraped portion can be made a cell adhesion region. Furthermore, when the support and the coating material have an appropriate reactive functional group, an appropriate reactive functional group is patterned on the substrate in advance, and there are generally used organic reactions such as radical reaction, anion reaction, and cation reaction. The target material can be fixed using

  In order to form cell-adhesive compartments or cell-adhesive variable regions, a coating solution is prepared by dissolving or dispersing a material covering the surface in an appropriate solvent. A method of forming a film by coating on a sheet is mentioned. Examples of the coating method include a roll coater method, a blade coater method, an air knife coater method, a gate roll coater method, a bar coater method, a size press method, and a shim sizer method. Further examples include a spray coating method, a gravure coating method, a curtain coater method, a screen printing method, a flexographic printing method, and an offset printing method.

  In addition, the above-described materials can be arranged using a droplet discharge unit. The droplet discharge means is capable of discharging a droplet having a volume of 100 nl or less, more preferably a droplet of 1 nl or less, and includes a micropipette, a micro dispenser, and a discharge device using an ink jet method. It is done. In view of the fact that the discharge device can be manufactured at low cost and fine droplets can be discharged, a discharge device using an inkjet method can be preferably used. Furthermore, among the ink jet methods, the thermal ink jet method and the piezo ink jet method can be preferably used, and the ejection device based on the thermal ink jet method can easily perform fine processing of the ejection port and can arrange the bioactive substance at a high density. . Further, since the ejection device based on the piezo ink jet method generates ejection energy by displacement of the piezoelectric element, it is possible to stably eject the bioactive substance without applying thermal stress to the bioactive substance.

  The produced cell culture substrate can be stored in a container for a long time. If the container is sealed together with a desiccant or an oxygen scavenger, it can be stored more preferably and stably. It can also be simply laminated with a film or the like.

  Next, a method for culturing cells will be described. By using the cell culture substrate of the present invention, uniform cell seeding can be performed easily. The cell culture substrate may be sterilized by irradiating the cell culture substrate with radiation or ultraviolet rays or washing with an alcohol solution, if necessary, before culturing the cells. Thereby, it is possible to prevent the culture from being adversely affected by unwanted microorganisms.

  As the cell suspension used when seeding cells, a suspension obtained by adding a desired serum component to a culture solution suitable for the cells to be seeded is used. Since the initial cell density is determined by the number of cell adhesion regions present on the cell culture substrate, a cell culture substrate having an adhesion region density according to the purpose is selected. As the number of cells in the cell suspension, a cell suspension containing a number of cells equal to or greater than the total number of cell adhesion region sections on the cell culture substrate to be used is used. At this time, the number of cells in the cell suspension preferably includes twice as many cells as the total number of cell adhesion regions, more preferably four times or more. It is not necessary to adjust the number of cells.

  Prior to seeding the cells, the cell adhesive variable surface 4 around the cell adhesive surface 2 is kept hydrophilic and non-cell adhesive (FIG. 2A). Next, the cell suspension is seeded on a cell culture substrate, and the culture of the cells 5 is started (FIG. 2B). Even after sowing, the cell adhesion variable surface is kept hydrophilic and kept in a non-adhered state and allowed to stand for an appropriate time. As a result, cells adhere only to the cell adhesion region. At this time, it is preferable to periodically shake the cell culture substrate. Thereby, a larger number of cell adhesion regions and cells can be brought into contact with each other, and targeted seeding of uniform cells can be promoted. Since the time required for initial adhesion varies depending on the cells used, it can be adjusted according to the cells. After the initial adhesion of the cells to the cell adhesion region is completed, the culture solution on the cell culture substrate is collected, and the non-adhered cells are removed (FIG. 2C). Preferably, after recovering the culture solution, the cell culture substrate is washed once or several times with a phosphate buffer or a cell culture solution. Non-adherent cells can be completely removed, improving the reliability of uniform seeding. Next, a culture solution containing no cells is added. As this culture solution, a solution obtained by removing cells from the culture solution used when collecting non-adherent cells can be used. Thereby, the culture solution can be effectively used. Next, the cell-adhesive variable surface is changed to hydrophobic by external stimulation, and the cell-adhesive property is changed to a state where adhesion is possible (FIG. 2D). By further culturing the cells for the necessary time in this state, the cells infiltrate the cell adhesion variable surface, and culture with a uniform density is performed (FIG. 2E).

  By the above method, uniform cell seeding is possible even in a plurality of cell culture containers without strictly adjusting the number of cells.

  The present invention will be described in more detail with reference to the following examples. These examples are specific examples shown for a deeper understanding of the present invention, and the present invention is not limited to these specific examples. It is not limited to examples. Unless otherwise indicated, “%” is based on mass.

<Production of cell culture substrate>
Cell culture with a cell adhesion area density and a cell adhesion area surface shown in Table 1 using a polystyrene substrate as a base material and poly-N-isopropylacrylamide as a material for coating the cell adhesion variable surface. A substrate was prepared. First, an isopropyl alcohol solution of 30% N-isopropylacrylamide monomer was applied on a polystyrene substrate at a rate of 0.01 ml / cm ² . Next, the substrate was irradiated with an electron beam having an intensity of 0.25 MGy, the support surface was coated with poly-N-isopropylacrylamide, and the surface was washed with purified water. Next, using ArF excimer laser (pulse laser with a wavelength of 193 nm), a part of poly-N-isopropylacrylamide was cut into a circular shape with a diameter of 10 μm to expose the polystyrene layer, thereby forming a cell adhesion region. . Further, in Examples 2 to 6, a drawing solution prepared by dissolving the cell adhesion substance solution shown in Table 1 in an aqueous solution containing 5% isopropanol at a concentration of 0.5% was prepared so that the drawing solution can be used. Using a Canon inkjet printer PIXUS950i remodeled in the above, the cell adhesion substance solution was adsorbed on the exposed polystyrene layer.

<Cell culture>
In advance, the cell culture substrate prepared above was allowed to stand in a 27 ° C. environment to keep the cell adhesiveness variable surface non-adhesive. Next, the cells shown in Table 2 are seeded so that the seeded cell density per substrate area is obtained, and the cells are allowed to stand at 5% CO ₂ atmosphere / 27 ° C. for 4 hours to adhere the cells only to the cell adhesion region. I let you. At this time, the substrate was lightly shaken every other hour. Next, the culture solution was gently collected and rinsed once with PBS. After the cells that had not adhered to the collected culture solution were removed through a 2 μm filter, they were returned to the culture substrate. Subsequently, the culture substrate was left in a 37 ° C. environment in a 5% CO ₂ atmosphere to change the cell adhesion variable surface to cell adhesion, and further cultured for 48 hours. As a comparative example, cells were seeded under the conditions shown in Table 2 on a polystyrene dish having the same area as the cell culture substrate used in the examples, that is, a substrate not including the cell adhesion variable region, and 5% CO _2. Culturing was performed for 48 hours at 37 ° C. under atmosphere. For Example 6 and Comparative Example 6, 48 hours after seeding, the culture medium was replaced with a differentiation medium (Minimum Essential Medium (MEM) medium containing 5% horse serum (HS)), and the culture was further continued for 48 hours. In order to confirm reproducibility, 5 samples were cultured for each condition. (Abbreviations in the table are as follows: FCS: fetal bovine serum, DMEM: Dulbeco's Modified Eagle's minimum essential medium, RMPI: Rosewell Park Memorial Institute)

<Evaluation>
The cells after 5 hours from the start of the culture were visually inspected for the whole surface of the substrate using a phase-contrast inverted microscope. In Examples 1 to 6, the cells were uniformly dispersed on the entire surface of the culture substrate and were arranged at a density almost the same as the density of the adhesion region, but all the five samples under the same conditions had almost the same result. On the other hand, in Comparative Examples 1-6, the density of cells on the substrate surface was uneven in any dish, and in particular, there were many cells in the center of the dish and many cells in the peripheral part. In addition, the deviation in cell density was not constant among the five samples under the same conditions, and the deviation and density differed depending on the dish.

In addition, when the number of cells per unit area (9 mm ² ) was measured in any 5 regions of the culture substrate and the ratio of the number of cells in each region to the average number of cells in 5 regions was determined, Was in the range of 0.8 to 1.2, but in the comparative example, the value was less than 0.8 or greater than 1.2 in at least one region, and the variation was large.

  Next, after the work shown in <Cell culture>, the cells were treated with 10% formalin for 15 minutes and methanol for 15 minutes and fixed, and then the cells were multiplied by 1000 times with the fluorescent dye TOTO-3 (Molecular Probes). Dilute and react for 30 minutes to fluorescently stain the DNA. Next, an infrared imaging system Odyssey (manufactured by Aloka) was used to detect a wavelength of 700 nm, and a fluorescence image of the entire culture substrate was captured. In Example 6 and Comparative Example 6, cells were reacted with a mouse anti-myosin heavy chain antibody for 1 hour as a primary antibody, washed 3 times with PBS, and then anti-mouse IgG IRDye 800 (manufactured by Rockland Immunochemicls) as a secondary antibody. For 1 hour. Odyssey was used to detect a wavelength of 800 nm to evaluate the expression of myosin heavy chain, which is an indicator of muscle differentiation. Using the captured image, the substrate was divided into 25 sections, and the amount of DNA in each section and the expression level of myosin heavy chain in Example 6 and Comparative Example 6 were evaluated. As a result, in Examples 1 to 6, the cell density was almost constant regardless of the compartment, but in Comparative Examples 1 to 6, the cell density varied greatly depending on the compartment. Moreover, when the expression level of the myosin heavy chain per unit area was compared between Example 6 and Comparative Example 6, the variation in Example 6 was less than that in Comparative Example 6.

It is a figure for demonstrating an example of the substrate for cell cultures of this invention. It is a figure for demonstrating an example of the cell culture method using the cell culture substrate of this invention.

Explanation of symbols

1 Substrate 2 Cell adhesion region 3 Cell adhesion variable surface 4 Cellular variable surface (non-adhesion state)
5 Cell 6 Cellular variable surface (adhesion state)

Claims (13)

(I) seeding cells on a substrate having a plurality of regions that are cell-adhesive and a region that is non-cell-adhesive;
(Ii) applying an external stimulus to the substrate and imparting cell adhesion to the non-cell-adhesive region; and (iii) culturing the seeded cells in the region imparted with the cell adhesion;
A cell culture method comprising the steps of:   The cell culture method according to claim 1, further comprising a step of shaking the culture substrate following the step (i).   The cell culture method according to claim 1 or 2, further comprising a step of removing unadhered cells from the substrate following the step (i).   4. The method according to claim 1, further comprising a step of recovering the culture solution after the step (i), removing the cells in the recovered culture solution, and returning the cells to the cell culture substrate again. A cell culture method according to claim 1. A cell culture substrate comprising a cell adhesion region comprising a plurality of sections arranged at regular intervals and a cell adhesion variable region whose cell adhesion can be changed by external stimulation. 6. The cell culture substrate according to claim 5, wherein the cell adhesion variable region is coated with a stimulus-responsive polymer. The cell culture substrate according to claim 5 or 6, wherein one section of the cell adhesive region is coated with at least one cell adhesive substance. The cell culture substrate according to any one of claims 5 to 7, wherein an area of one section of the cell adhesive region is 1 µm ² or more and 10,000 µm ² or less. The cell culture substrate according to any one of claims 5 to 8, wherein the cell adhesive region sections are arranged in a range of 1 to 10 ⁵ per 1 cm ^{2 of the} substrate. A cell culturing method using the cell culture substrate according to any one of claims 5 to 9, wherein the cell adhesion variable surface is kept non-cell-adhesive, and cells are seeded on the substrate to adhere to the cell. A cell culture method comprising the steps of initial adhesion of cells to a sex region, a step of removing cells not having the initial adhesion, and a step of changing a cell adhesion variable surface to cell adhesion. The cell culture method according to claim 10, wherein cells equal to or greater than the number of the cell adhesive compartments are seeded. The cell culture method according to claim 10 or 11, comprising a step of shaking the culture substrate after seeding the cells. The culture solution is collected after a predetermined time after seeding the cells, the cells in the collected culture solution are removed, and then returned to the cell culture substrate again. Cell culture method.

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