JP2013185903A - Method and apparatus for evaluating cell culture substrate, and method for manufacturing cell culture substrate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for evaluating a state of a functional layer consisting of poly-N-isopropylacrylamide as a temperature-responsive polymer, in a short time without any necessity of executing a cell culture.SOLUTION: The method for evaluating a cell culture substrate according to the present invention includes; a substrate; and a functional layer provided on the substrate and including organic compound whose cellular adhesiveness is changed by a predetermined stimulus. The method includes: a first measuring step of measuring a falling speed of droplet supplied to the surface of the functional layer of the inclined cell culture substrate; and a second measuring step of measuring a falling speed of droplet supplied to the surface of the functional layer of the inclined cell culture substrate under a condition where the cellular adhesiveness of the functional layer is changed from when measuring the first measuring step. The state of the functional layer is evaluated on the basis of the droplet falling speed obtained in the first measuring step and the droplet falling speed obtained by the second measuring step.

Description

  The present invention relates to a method and an apparatus for evaluating a cell culture substrate having a functional layer on the substrate, and a method for producing such a cell culture substrate.

  A technique called “cell sheet engineering”, in which cells are cultured in a sheet form and the cells are recovered in a sheet form simply by lowering the temperature without using an enzyme such as trypsin, has attracted attention in the field of regenerative medicine. The cell sheet obtained by this technology has already been confirmed to have a certain therapeutic effect in regenerative medicine such as the cornea and periodontal tissue, and clinical research and clinical trials are already underway in Europe. It is also possible to create a three-dimensional tissue model by laminating multiple types of cell sheets, and to create mature tissue with vascular tissue in vitro. Research and development and treatment based on this technology Is expected more and more in the future.

  Control of adhesion and desorption of the cultured cell sheet is performed, for example, via a functional layer such as poly-N-isopropylacrylamide (PIPAAm) fixed on a substrate. This polymer is a material whose hydration ability changes mainly in response to temperature. At a temperature lower than the critical dissolution temperature, the affinity for surrounding water is improved, and the polymer takes up water and swells, so that cells are swelled on the surface. It exhibits the property of making it difficult to adhere (cell non-adhesiveness), and the property that makes it easier for cells to adhere to the surface due to water detaching from the polymer at temperatures above the same temperature (cell adhesion) It is. It is known that cell deadhesion is affected by the immobilization density and chain length of the above PIPAAm. Therefore, in order to examine the quality of cell deadhesion, a method for evaluating the state of the functional layer Development is desired.

As a conventional method for evaluating a cell culture substrate provided with a functional layer on the substrate, (Patent Document 1) is known. In (Patent Document 1), cells are seeded on the surface of a cell culture substrate coated with a temperature-responsive polymer such as poly-N-isopropylacrylamide whose hydration power changes in a temperature range of 0 to 80 ° C. In 48 hours, temperature-responsive cell culture in which cells are detached and recovered from the substrate surface by temperature treatment without using protein hydrolase and quantified with a spectrometer using a reagent that reacts with living cells A method for evaluating a substrate is disclosed. The cultured cells are required to be attached on the surface of the base material in an individual state so as to be 100 to 10,000 cells / cm ² per surface area of the base material. That is, when cells are collected in a sheet state, the influence of the force of pulling the cells due to cell-cell adhesion cannot be ignored. Therefore, in (Cited document 1), by seeding on a substrate in individual states (sparse state) The result of eliminating the influence of cell-cell adhesion and reflecting only the interaction between the substrate and the cells is obtained.

  The evaluation method described in the above (Patent Document 1) has a problem in that it takes at least several hours to several tens of hours to perform the evaluation in order to perform cell culture.

JP 2009-82123 A

  Therefore, in view of the above-described conventional situation, the present invention eliminates the need for cell culture and can evaluate the state of the functional layer composed of PIPAAm and the like in a short time, and evaluation for implementing the method. An object is to provide an apparatus. Another object of the present invention is to provide a method for producing a cell culture substrate having a good functional layer state using the above evaluation method.

  The present inventors paid attention to the point that the repelling behavior of the liquid on the surface of the functional layer differs depending on the state of the functional layer containing an organic compound whose cell adhesion changes by a predetermined stimulus such as PIPAAm. And, under the conditions before and after the cell adhesiveness change, measure the speed of the droplet that falls on the surface of the functional layer, find that the state of the functional layer can be evaluated using those multiple velocity values as an index, The present invention has been completed. That is, the present invention includes the following inventions.

(1) A method for evaluating a cell culture substrate, comprising a substrate and a functional layer provided on the substrate and containing an organic compound whose cell adhesion is changed by a predetermined stimulus,
A first measurement step of measuring a falling speed of a droplet supplied to the surface of the functional layer of the inclined cell culture substrate;
The measurement of the first measurement step is to measure the falling speed of the droplet supplied to the surface of the functional layer of the inclined cell culture substrate under the condition that the cell adhesion of the functional layer changes. Two measurement steps;
Have
The cell culture substrate evaluation method for evaluating the state of the functional layer based on the droplet falling speed obtained in the first measuring step and the droplet falling speed obtained in the second measuring step .
(2) The method for evaluating a cell culture substrate according to the above (1), wherein the organic compound is a temperature-responsive polymer whose cell adhesiveness is changed by temperature change.
(3) An apparatus for carrying out the evaluation method according to (1) or (2) above,
A cell culture substrate is disposed, and a stage for tilting the cell culture substrate;
A droplet supply means for supplying droplets to the surface of the functional layer of the cell culture substrate disposed on the stage;
An imaging means for photographing the falling droplet;
A falling speed calculation unit that calculates the falling speed of the droplet based on the image taken by the imaging means;
A stimulus applying means for applying a predetermined stimulus to the cell culture substrate disposed on the stage;
An apparatus for evaluating a cell culture substrate.

(4) A step of providing a cell culture substrate by providing a functional layer containing an organic compound whose cell adhesion is changed by a predetermined stimulus on the substrate;
For the obtained cell culture substrate, a step of performing the evaluation method described in (1) above;
Have
The manufacturing method of the cell culture substratum which uses what the state of the functional layer was judged to be good as a result of evaluation.
(5) The organic compound has a lower critical solution temperature of 0 ° C. to 80 ° C., exhibits cell adhesion at a temperature equal to or higher than the lower critical solution temperature, and exhibits non-cell adhesion at a temperature lower than the lower critical solution temperature. A temperature responsive polymer,
A first measurement step in which the cell culture substrate is inclined at an angle of 40 ° at a temperature equal to or higher than the lower critical solution temperature, and the falling speed of the droplets supplied to the surface of the functional layer is measured;
A second measurement step in which the cell culture substrate is tilted at an angle of 60 ° at a temperature lower than the lower critical solution temperature, and the drop rate of the droplets supplied to the surface of the functional layer is measured;
Have
The drop rate of the droplet obtained in the first measurement step is 0.05 mm / msec to 0.10 mm / msec, and the drop rate of the droplet obtained in the second measurement step is 1.0 × 10 ³ mm. The method for producing a cell culture substrate according to the above (4), wherein the state of the functional layer is determined to be good when it is / msec to 1.0 × 10 ⁴ mm / msec.

  According to the present invention, it is not necessary to perform cell culture, and the temperature responsiveness of the cell culture substrate can be evaluated in a short time. In addition, it is possible to efficiently produce a cell culture substrate excellent in cell de-adhesiveness.

It is a figure for demonstrating the evaluation method of the cell culture substratum which concerns on this invention. It is a figure which shows one Embodiment of the evaluation apparatus of the cell culture substratum which concerns on this invention.

Hereinafter, the present invention will be described in detail with reference to the drawings. First, the structure of the cell culture substrate which is the evaluation target of the present invention will be described.
A cell culture substrate is configured by providing a functional layer containing an organic compound whose cell adhesion changes by a predetermined stimulus on a substrate (usually, one side of the substrate). In general, the substrate and the functional layer are in direct contact with each other, but an intermediate layer such as a primer layer for facilitating the fixation of the functional layer may be interposed as necessary.

  The cell culture substrate is used by being installed on the inner bottom surface of a cell culture container that contains cells and a medium. Therefore, the base material may also serve as the bottom plate of the cell culture container, or the laminate of the base material and the functional layer is later joined to the inner bottom surface of the container formed from the bottom plate and the side wall. May be. When it is in the form of a laminate to be subsequently joined to the container, the substrate is plate-like or film-like, and the side opposite to the side on which the functional layer is provided is joined to the bottom plate of the cell culture vessel A pressure-sensitive adhesive layer and a release sheet can be appropriately provided.

  The material of the substrate is not particularly limited. Specifically, polyethylene terephthalate (PET), polystyrene (PS), polycarbonate (PC), TAC (triacetyl cellulose), polyimide (PI), nylon (Ny), low density polyethylene (LDPE), medium density polyethylene (MDPE) ), Resin materials such as polyvinyl chloride, polyvinylidene chloride, polyphenylene sulfide, polyether sulfone, polyethylene naphthalate, polypropylene, acrylic resin, and inorganic materials such as glass and quartz, and resin materials are preferably used.

  The surface of the substrate on the side where the functional layer is provided can be a surface subjected to an easy adhesion treatment. “Easy adhesion treatment” refers to treatment with an easy adhesive such as polyester, acrylic ester, polyurethane, polyethyleneimine, silane coupling agent, perfluorooctane sulfonic acid (PFOS), and the like.

  The organic compound constituting the functional layer is not particularly limited as long as it has a function of changing cell adhesion by a predetermined stimulus. For example, a stimulus-responsive polymer that changes from cell adhesiveness to non-cell adhesiveness by applying a stimulus is preferably used. Examples of the stimulus responsive polymer include a temperature responsive polymer, a pH responsive polymer, an ion responsive polymer, a photoresponsive polymer, and the like, and a polymer suitable for the cell sheet to be produced can be appropriately selected. . In particular, a temperature-responsive polymer is preferable because cell adhesion changes due to temperature change and stimulation is easily applied.

  As the temperature-responsive polymer, for example, a polymer that exhibits cell adhesion at a temperature at which cells are cultured and exhibits cell non-adhesion at a temperature at which the produced cell sheet is peeled may be used. Specifically, at a temperature lower than the critical dissolution temperature, the affinity for surrounding water is improved, and the polymer takes in water and swells to show the property of making it difficult for cells to adhere to the surface (cell non-adhesiveness). It is preferable to use a material that exhibits a property (cell adhesion) that makes it easier for cells to adhere to the surface due to water detaching from the polymer at a temperature higher than the temperature. Such a critical solution temperature is called a lower critical solution temperature. A temperature-responsive polymer having a lower critical solution temperature T of 0 ° C. to 80 ° C., more preferably 0 ° C. to 50 ° C. may be used. This is because the cells can be stably cultured when T is 0 ° C to 80 ° C.

  Suitable temperature-responsive polymers include acrylic polymers or methacrylic polymers. More specifically, for example, poly-N-isopropylacrylamide (T = 32 ° C.), poly-Nn-propyl acrylamide (T = 21 ° C.), poly-Nn-propyl methacrylamide (T = 32 ° C.) Poly-N-ethoxyethyl acrylamide (T = about 35 ° C.), poly-N-tetrahydrofurfuryl acrylamide (T = about 28 ° C.), poly-N-tetrahydrofurfuryl methacrylamide (T = about 35 ° C.), and Examples include poly-N, N-diethylacrylamide (T = 32 ° C.).

  As a monomer for forming these polymers, a monomer that can be polymerized by irradiation with radiation can be used. Examples of the monomer include (meth) acrylamide compounds, N- (or N, N-di) alkyl-substituted (meth) acrylamide derivatives, (meth) acrylamide derivatives having a cyclic group, and vinyl ether derivatives. More than seeds can be used. When a single monomer is used alone, the polymer formed on the substrate is a homopolymer, and when multiple monomers are used together, the polymer formed on the substrate is a heteropolymer. Any form can be applied in the present invention.

  In addition, when it is necessary to adjust T depending on the type of proliferating cells, or when it is necessary to adjust the hydrophilic / hydrophobic balance of the functional layer, other monomers other than those described above may be further added and used in combination. Polymerization may be performed. Furthermore, you may comprise a functional layer using the graft | grafting or block copolymer of the said polymer and another polymer, or the mixture of the said polymer and another polymer. Moreover, it is also possible to crosslink within a range where the original properties of the polymer are not impaired.

  A functional layer containing an organic compound such as a temperature-responsive polymer can be formed on a substrate by appropriately employing a conventionally known method. For example, a coating composition containing a monomer that forms a target stimulus-responsive polymer by polymerization and an organic solvent capable of dissolving the monomer is prepared, and this is applied to the surface of a substrate according to a conventional coating method. A coating film is formed, and then the monomer in the coating film is polymerized by an appropriate means such as radiation irradiation to form a polymer, and a grafting reaction is performed between the surface of the substrate and the polymer. It can be formed by generating.

  The film thickness of the functional layer is, for example, preferably in the range of 0.5 nm to 300 nm, and particularly preferably in the range of 1 nm to 100 nm, but is not limited thereto.

  Next, the cell culture substrate evaluation method and evaluation apparatus of the present invention will be described. The evaluation method of the present invention includes a first measurement step for measuring a falling speed of a droplet supplied to the surface of a functional layer of a tilted cell culture substrate, and a functional layer at the time of measurement in the first measurement step. And a second measurement step of measuring the falling speed of the droplets supplied to the surface of the functional layer of the inclined cell culture substrate under the condition that the cell adhesiveness changes.

  Here, “when measuring in the first measurement step is measured under a condition in which the cell adhesion of the functional layer changes” specifically depends on the type of organic compound constituting the functional layer, The first and second measurements are performed under two conditions including critical conditions (temperature, pH, light irradiation conditions, etc.) in which the state of the functional layer changes from cell adhesiveness to cell nonadhesiveness. For example, when the functional layer is composed of a temperature-responsive polymer having a lower critical solution temperature, the first measurement step is performed at a temperature equal to or higher than the lower critical solution temperature at which the functional layer exhibits cell adhesion. The second measurement step is performed at a temperature lower than the lower critical solution temperature showing cell non-adhesiveness. In addition, regarding the measurement under two conditions, it is arbitrary which one is used as the first or second measurement step.

  As shown in FIG. 1, the cell culture substrate 1 including the substrate 10 and the functional layer 20 is tilted at an angle α, and droplets L are supplied to the surface of the tilted functional layer 20. Since the droplet L falls on the functional layer 20, the fall speed at that time is measured using an imaging means such as a camera. Note that water is usually used as the droplet L, but other liquids such as alcohol may be used in some cases. The functional layer 20 is obtained in the first and second measuring steps because the properties such as hydrophilicity and hydrophobicity of the surface change with the boundary of the critical condition, and the velocity of the falling droplet L also changes accordingly. From the two falling speeds, it can be confirmed that the functional layer is functioning properly, and the quality of the cell de-adhesion can be evaluated. For example, as the functional layer becomes hydrophilic and the detachability of the cells increases, the falling speed generally decreases. Furthermore, the falling speed depends on the amount of the functional layer immobilized. That is, for example, in the case of a temperature-responsive polymer, the falling speed decreases as the amount of immobilization on the base material increases, so that the amount of immobilization can be determined from the value of the falling speed. At that time, since the measurement is performed under the two conditions of the first and second measurement steps, the reliability and accuracy of the estimated immobilization amount are increased.

  In FIG. 1, the amount of the droplet L is not particularly limited as long as an appropriate falling speed value can be obtained. For example, it can be set to about 1 μL to 1 mL, and is preferably set to the same amount in the first and second measurement steps. In addition, the angle α at which the cell culture substrate 1 is tilted is appropriately adjusted so that the measured falling speed has an appropriate value. Specifically, it can be set to 1 ° to 90 °. The angle α is preferably set to the same value in the first and second measurement steps, but the fall speeds of the first and second measurement steps are greatly different and the correlation between the angle α and the fall speed is clear. The angle α in one process may be smaller (larger) than the other so that the falling speed can be measured with high accuracy in both the first and second measurement processes.

By using the above evaluation method in the cell culture substrate production process, it is possible to efficiently produce a cell culture substrate in which the state of the functional layer (immobilization amount, cell de-adhesiveness) is good. . As an example, the functional layer has a lower critical solution temperature of 0 ° C. to 80 ° C., exhibits cell adhesion at a temperature equal to or higher than the lower critical solution temperature, and cell non-adhesive at a temperature lower than the lower critical solution temperature. The cell culture substrate containing a temperature-responsive polymer having the following first and second tilting of the cell culture substrate at a predetermined angle under a temperature condition not lower than the lower critical dissolution temperature and measuring the drop falling speed: 2 The measurement process is carried out, and the drop rate of the droplet obtained in the first measurement process is different from the drop rate of the droplet obtained in the second measurement process. It is good to confirm that the behavior of hydrophilicity and hydrophobicity is reflected. More specifically, for example, the drop rate of the droplet obtained in the first measurement step in which the cell culture substrate is inclined at an angle of 40 ° is 0.05 mm / msec to 0.10 mm / msec, and When the drop rate of the droplet obtained in the second measurement step in which the cell culture substrate is inclined at an angle of 60 ° is 1.0 × 10 ³ mm / msec to 1.0 × 10 ⁴ mm / msec It can be determined that the state of the functional layer is good. Such a determination operation can be performed in a very short time because the conventional cell culture is not performed. Furthermore, in manufacturing a plurality of cell culture substrates, cell culture is performed using several cell culture substrates in advance, and the cell culture results and the falling speed obtained by the first measurement step and the second measurement step are determined. The suitable range (or threshold value) of each falling rate is obtained by examining the correlation with the value, and the falling rate values of other cell culture substrates produced thereafter are included in the above preferred range (or threshold value). Whether or not the product is good or bad may be determined based on whether or not the product is good. Even in this case, since cell culture only needs to be performed at the beginning, the efficiency of mass production of the cell culture substrate can be greatly improved as a whole.

  FIG. 2 shows an embodiment of an apparatus for performing the above-described evaluation method. In this evaluation apparatus, a cell culture substrate 1 is disposed, a stage 11 for tilting the cell culture substrate 1, and a droplet on the surface of the functional layer of the cell culture substrate 1 disposed on the stage 11. The droplet supply means 12 such as a syringe for supplying L, the imaging means 13a and 13b for photographing the falling droplet L, and the falling speed of the droplet L are calculated based on the images photographed by these imaging means. And a falling speed calculation unit 14.

The cell culture substrate 1 on the stage is illuminated with light from the light source 15 that has passed through the diffusion plate 16. The imaging means 13a, 13b only needs to be able to catch the falling droplets. Specifically, a high-speed camera equipped with a CCD or the like is preferably used. As shown in FIG. 2, by using a plurality of imaging means 13a and 13b and photographing from above and the side of the stage, for example, the falling speed can be measured with higher accuracy, but only one of the imaging means is available. It doesn't matter. The stage 11 is provided with a driving device for tilting the cell culture substrate at a set angle, and a predetermined stimulus such as a heater, a cooling device, or a light source for changing the cell adhesion of the functional layer. Stimulus imparting means 17 for imparting is connected to the stage 11 or provided separately. The droplet supply means 12 is set so that a set amount of droplets L can be dropped. The falling speed calculation unit has a function of analyzing the image acquired by the imaging unit and calculating the falling speed of the droplet. When calculating the speed, for example, focusing on the forward end L ₁ or the rear end L ₂ of the droplet L in FIG. 1, it can be determined from the displacement amount of the end portion.

  Next, the present invention will be described in more detail based on examples and comparative examples, but the present invention is not limited thereto.

-Manufacture of cell culture substrate Isopropyl alcohol containing 2% poly-N-isopropylacrylamide (Aldrich) and containing N-isopropylacrylamide at a final monomer concentration of 30%, 35% and 40% by weight, respectively. Three types of solutions were prepared by dissolving in (IPA). Subsequently, a polystyrene film sheet (PS) is used as a base material, the above three kinds of N-isopropylacrylamide solutions are coated on the PS, electron beam irradiation is performed using an electron beam irradiation apparatus, and a polycrystal is applied to the PS surface. A functional layer made of -N-isopropylacrylamide (PIPAAm) was formed by graft polymerization. The PS to which poly-N-isopropylacrylamide was bonded was washed with ion exchange water at 5 ° C. and then dried to obtain three types of cell culture substrates. Samples 1 to 3 are cell culture substrates corresponding to monomer concentrations of 30%, 35% and 40% by weight, respectively. As a comparative example, the same measurement as in samples 1 to 3 was performed on a sheet made of only PS.

First measurement step Samples 1 to 3 are placed in order on the stage of a contact angle measurement device DM-501 (trade name, manufactured by Kyowa Interface Science Co., Ltd.), and the heater temperature is set to the lower critical solution temperature of poly-N-isopropylacrylamide ( T = 32 ° C.) or higher, and the tilt angle of the stage was set to 40 °. Then, 1 microliter of pure water was dripped at the surface of the samples 1-3 with the micro syringe. After dropping, the image was taken from the side of the falling droplet with a CCD camera at a measurement interval of 100 msec, and the falling speed of the droplet (referred to as the first falling speed) was calculated from the displacement of the front end of the droplet.

Second measurement step Samples 1 to 3 are placed in order on the stage of a contact angle measuring device DM-501 (trade name, manufactured by Kyowa Interface Science Co., Ltd.), and the heater temperature is set to the lower critical solution temperature of poly-N-isopropylacrylamide ( T = 32 ° C.) and the tilt angle of the stage were set to 60 °. Then, 1 microliter of pure water was dripped at the surface of the samples 1-3 with the micro syringe. After dropping, the image was taken from the side of the falling droplet with a CCD camera at a measurement interval of 100 msec, and the falling speed of the droplet (referred to as the second falling speed) was calculated from the displacement of the front end of the droplet.
The measurement results are shown in Table 1.

  As shown in Table 1, since the first falling speed and the second falling speed are different in all samples 1 to 3, it was confirmed that a functional layer was formed on the surface of the cell culture substrate. On the other hand, in the case of PS alone, the first falling speed and the second falling speed did not change because there was no functional layer. In addition, since the value of the second falling speed was smaller than the first falling speed, it became clear that the functional layer became hydrophilic and exhibited cell non-adhesiveness at a temperature lower than the lower critical solution temperature. Furthermore, the values of the first and second falling speeds reflect the monomer concentration of the functional layer (PIPAAm immobilization amount), and it was found that the higher the monomer concentration, the lower the falling speed. Sample 1 and sample 2 showed no difference in the second falling speed, but a difference was seen in the first falling speed, so it was confirmed that both were distinguishable.

  In the present invention, since the falling speed is measured under two temperature conditions, the amount of immobilized PIPAAm can be determined with higher resolution.

DESCRIPTION OF SYMBOLS 1 Cell culture base material 10 Base material 11 Stage 12 Droplet supply means 13a, 13b Imaging means 14 Falling speed calculation part 15 Light source 16 Diffusion plate 17 Stimulation means 20 Functional layer L Droplet L ₁ Front end L ₂ Rear end α angle

Claims (5)

A cell culture substrate evaluation method comprising: a substrate; and a functional layer that is provided on the substrate and includes an organic compound that changes cell adhesion by a predetermined stimulus,
A first measurement step of measuring a falling speed of a droplet supplied to the surface of the functional layer of the inclined cell culture substrate;
The measurement of the first measurement step is to measure the falling speed of the droplet supplied to the surface of the functional layer of the inclined cell culture substrate under the condition that the cell adhesion of the functional layer changes. Two measurement steps;
Have
The cell culture substrate evaluation method for evaluating the state of the functional layer based on the droplet falling speed obtained in the first measuring step and the droplet falling speed obtained in the second measuring step .   The method for evaluating a cell culture substrate according to claim 1, wherein the organic compound is a temperature-responsive polymer whose cell adhesiveness is changed by temperature change. An apparatus for carrying out the evaluation method according to claim 1 or 2,
A cell culture substrate is disposed, and a stage for tilting the cell culture substrate;
A droplet supply means for supplying droplets to the surface of the functional layer of the cell culture substrate disposed on the stage;
An imaging means for photographing the falling droplet;
A falling speed calculation unit that calculates the falling speed of the droplet based on the image taken by the imaging means;
A stimulus applying means for applying a predetermined stimulus to the cell culture substrate disposed on the stage;
An apparatus for evaluating a cell culture substrate. A step of providing a cell culture substrate by providing a functional layer containing an organic compound whose cell adhesion changes by a predetermined stimulus on the substrate;
A step of performing the evaluation method according to claim 1 for the obtained cell culture substrate;
Have
The manufacturing method of the cell culture substratum which uses what the state of the functional layer was judged to be good as a result of evaluation. A temperature response in which an organic compound has a lower critical lysis temperature of 0 ° C. to 80 ° C., exhibits cell adhesion at a temperature equal to or higher than the lower critical lysis temperature, and exhibits cell non-adhesion at a temperature lower than the lower critical lysis temperature. A functional polymer,
A first measurement step in which the cell culture substrate is inclined at an angle of 40 ° at a temperature equal to or higher than the lower critical solution temperature, and the falling speed of the droplets supplied to the surface of the functional layer is measured;
A second measurement step in which the cell culture substrate is tilted at an angle of 60 ° at a temperature lower than the lower critical solution temperature, and the drop rate of the droplets supplied to the surface of the functional layer is measured;
Have
The drop rate of the droplet obtained in the first measurement step is 0.05 mm / msec to 0.10 mm / msec, and the drop rate of the droplet obtained in the second measurement step is 1.0 × 10 ³ mm. The method for producing a cell culture substrate according to claim 4, wherein the state of the functional layer is determined to be good when the ratio is / msec to 1.0 x 10 ⁴ mm / msec.

Images