JP5849792B2 - Method and apparatus for evaluating cell culture substrate, and method for producing cell culture substrate - Google Patents

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 (Patent 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 fact that the evaporation behavior of droplets supplied to the surface of the functional layer differs depending on the state of the functional layer for culturing cells, such as PIPAAm. Then, the rate of decrease in the contact area between the droplet and the functional layer during the process of evaporation of the droplet was measured, and it was found that the state of the functional layer could be evaluated using the velocity value as an index, thereby completing the present invention. That is, the present invention includes the following inventions.

(1) A method for evaluating a cell culture substrate having a substrate and a functional layer for culturing cells provided on the substrate,
Supplying droplets to the surface of the functional layer;
Measuring the rate of decrease of the contact area where the droplet and the functional layer contact in the process of evaporation of the supplied droplet;
Have
The method for evaluating a cell culture substrate, wherein the state of the functional layer is evaluated based on the measured decrease rate.
(2) The method further includes a step of measuring a change in the contact angle of the droplet in the process of evaporating the supplied droplet,
The change in the contact angle includes a section in which the contact angle decreases, a section in which the contact angle is maintained thereafter, and a section in which the contact angle decreases again thereafter. The method for evaluating a cell culture substrate according to the above (1), which is a rate of decrease in a section in which the contact angle is maintained.
(3) The method for evaluating a cell culture substrate according to the above (1) or (2), wherein the functional layer contains a temperature-responsive polymer whose cell adhesiveness changes due to temperature change.
(4) An apparatus for carrying out the evaluation method according to (1) above,
A stage on which the cell culture substrate is placed;
A droplet supply means for supplying droplets to the surface of the functional layer of the cell culture substrate disposed on the stage;
Evaporation induction means for evaporating the droplets;
Imaging means for photographing the evaporating droplets;
A reduction rate calculation unit that calculates a reduction rate of the droplet contact area based on an image captured by the imaging unit;
An apparatus for evaluating a cell culture substrate.
(5) An apparatus for carrying out the evaluation method according to (2) above,
A stage on which the cell culture substrate is placed;
A droplet supply means for supplying droplets to the surface of the functional layer of the cell culture substrate disposed on the stage;
Evaporation induction means for evaporating the droplets;
Imaging means for photographing the evaporating droplets;
A reduction rate calculation unit that calculates a reduction rate of the droplet contact area based on an image captured by the imaging unit;
A contact angle calculation unit that calculates a contact angle of a droplet based on an image photographed by an imaging unit;
An apparatus for evaluating a cell culture substrate.
(6) preparing a cell culture substrate having a substrate and a functional layer for culturing cells provided on the substrate;
For the prepared cell culture substrate, a step of performing the evaluation method according to any one of (1) to (3) 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.

  According to the present invention, it is not necessary to perform cell culture, and it is possible to evaluate the de-adhesiveness of cells on the cell culture substrate 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 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. It is a graph which shows the result of having measured the change of the contact angle and contact radius of a droplet in the process in which the droplet in Example 1 evaporates. It is a graph which shows the result of having measured the change of the contact angle and contact radius of a droplet in the process in which the droplet in Example 2 evaporates. It is a graph which shows the result of having measured the change of the contact angle and contact radius of a droplet in the process in which the droplet in Example 3 evaporates. It is a graph which shows the result of having measured the change of the contact angle and contact radius of a droplet in the process in which the droplet in Example 4 evaporates. It is the graph which compared the decreasing rate of the contact radius of the droplet measured in Examples 1-4. It is the graph which compared the contact angle difference of the droplet measured in Examples 1-4.

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.
The cell culture substrate is configured by providing a functional layer for culturing cells 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 material constituting the functional layer is not particularly limited as long as it has a function of culturing cells. For example, basic materials such as polylysine, basic polymers such as polylysine, aminopropyltriethoxysilane, N- (2-aminoethyl) -3-aminopropyltrimethoxysilane, etc., as materials showing adhesion to cells by electrostatic interaction Examples thereof include compounds and condensates containing them. In addition, as materials exhibiting adhesiveness to cells due to biological characteristics, fibronectin, laminin, tenascin, vitronectin, RGD (arginine-glycine-aspartic acid) sequence-containing peptide, YIGSR (tyrosine-isoleucine-glycine-serine-arginine) Examples include sequence-containing peptides, collagen, atelocollagen, gelatin and the like.

  Among them, when a cell culture substrate is used to detach cells from the substrate after cell culture or to collect cells in a sheet form, the cell adhesion by a predetermined stimulus is used as a functional layer. Those having a function of changing can be adopted. For example, a stimulus-responsive polymer that changes from cell adhesiveness to non-cell adhesiveness upon application of a stimulus can be included. 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 a temperature-responsive polymer or the like can be formed on a substrate by appropriately adopting a conventionally known technique. 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 step of supplying a droplet to the surface of the functional layer, a step of measuring a decrease rate of a contact area where the droplet and the functional layer are in contact with each other in a process in which the supplied droplet evaporates, and It is characterized by having.

  Specifically, first, as shown in FIGS. 1 and 2, a droplet L is supplied to the surface of the functional layer 20 in the cell culture substrate 1 including the substrate 10 and the functional layer 20. Then, the droplet L is heated by an appropriate method such as indirectly heating the droplet L via the cell culture substrate 1, or directly heating the droplet L, or reducing the pressure of the atmosphere. Evaporation is performed, and a decrease in the contact area between the droplet L and the functional layer 20 in the evaporation process is observed by an imaging means such as a CCD camera. At this time, the inventors reflect the state of the functional layer 20 based on the value of the decrease rate because the decrease rate of the contact area between the droplet L and the functional layer 20 reflects the state of the functional layer 20. Has been found to be evaluable. As a value indicating the contact region between the droplet L and the functional layer 20, for example, an area value of a region occupied by the droplet L when the droplet L is observed from above can be employed. Alternatively, since the droplet L isotropically spreads in the horizontal direction on the functional layer 20 and can be regarded as a circular shape when viewed from above, the area value of the region such as the contact radius r corresponding to the area value of the contact region. It is good also as the decreasing speed of a contact area | region with the change speed of the value corresponding to. Note that water is usually used as the droplet L, but other liquids such as alcohol may be used in some cases. Specifically, ethanol, isopropyl alcohol, methanol or the like can be used. Further, the temperature when the droplet L is heated is appropriately set in consideration of the measurement efficiency. For example, when the droplet L is water, it is usually preferable to set the temperature to about 30 ° C to 60 ° C. More preferably, it is about 40 degreeC-50 degreeC.

  As shown in FIG. 1, for example, when the functional layer 20 contains a temperature-responsive polymer such as poly-N-isopropylacrylamide (PIPAAm) and its immobilization amount is small, the hydrophobicity of the surface of the functional layer 20 is reduced. In this case, since the influence of the interaction between the droplet L and the functional layer 20 is reduced, the droplet L maintains its shape (while maintaining a similar shape). As shown in (a) to (c) of 1, the contact radius r corresponding to the contact area decreases gently, that is, at a low speed. On the other hand, as shown in FIG. 2, for example, when the functional layer 20 contains a temperature-responsive polymer such as PIPAAm and the amount of immobilization thereof is large, the hydrophobicity of the surface of the functional layer 20 is relatively low. (Hydrophilicity increases). In this case, since the influence of the interaction between the droplet L and the functional layer 20 becomes large, the droplet L only decreases in the contact angle θ while maintaining the contact radius r in the initial stage of the evaporation process ( Process from (a) to (b) in FIG. Thereafter, when the evaporation of the droplet L further proceeds, the interaction force acting between the droplet L and the functional layer 20 cannot resist the surface tension of the droplet L, and the contact angle θ remains almost unchanged. The contact radius r corresponding to the contact area decreases rapidly, that is, at a high speed (the process from (b) to (c) in FIG. 2). The above description and FIGS. 1 and 2 exaggerate the tendency when the interaction between the droplet L and the functional layer 20 is weak and strong. The change in the contact angle θ proceeds in a complex manner.

  Further, when the change in the contact angle θ of the droplet L during the process of evaporating the droplet L is measured, the interval in which the contact angle decreases in the initial stage of the evaporation process and the substantially constant contact angle thereafter are maintained. In many cases, the evaporation proceeds through a section where the contact angle decreases and thereafter a section where the contact angle decreases again. The reason for evaporation through a plurality of sections exhibiting different behaviors is not clear, but the force due to the interaction between the droplet L and the functional layer 20, the surface tension of the droplet L, gravity, etc. are comprehensive. This is considered to be the result of acting on In this case, it is preferable to employ the contact region decrease rate in a section in which a substantially constant contact angle is maintained as the value of the contact region decrease rate based on the evaluation of the state of the functional layer 20. Here, “substantially constant” refers to a state in which the absolute value of the change rate of the contact angle in the preceding and following sections is relatively small and can be regarded as constant. In this section, there is little influence of the volume reduction of the droplet L due to evaporation, and there is a correlation between the interaction between the droplet L and the functional layer 20 (that is, the state of the functional layer to be evaluated) and the reduction rate of the contact area. This is because it is considered the best. Of course, when the above three sections are not clearly observed, or when a simpler evaluation is performed, the reduction rate calculated by another method such as the average reduction rate of the contact area over the entire evaporation process is used. You may evaluate it.

  The amount of the droplet L is not particularly limited as long as the evaporation process of the droplet L can be appropriately observed. For example, it can be about 1 μL to 1 mL. When evaluating a plurality of cell culture substrates, it is preferable that the supplied droplets L have the same amount between the cell culture substrates.

  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. . Specifically, for example, cell culture is performed in advance using several cell culture substrates, and a correlation between the cell culture result and the reduction rate of the contact area is examined to determine a suitable range (or threshold value) of the reduction rate. The quality of the product can be determined based on whether or not the value of the decrease rate of other cell culture substrates to be mass-produced thereafter is included in the above preferable range (or threshold). In this case, in the mass production process of the cell culture substrate, since conventional cell culture is not performed for the evaluation, the evaluation can be performed in an extremely short time, and the mass production process of the cell culture substrate is greatly efficient. Can be achieved. As an example, when the substrate is made of polystyrene, the functional layer contains poly-N-isopropylacrylamide (PIPAAm), which is a temperature-responsive polymer, and 1 μL of water is supplied to the surface of the functional layer and evaporated at 40 ° C. If the decrease rate of the contact radius r in the section maintaining a substantially constant contact angle is within the range of 0.015 μm / msec to 0.020 μm / msec, from the viewpoint of the immobilized amount of PIPAAm and the cell debonding property It can be determined that the state of the functional layer is good.

  FIG. 3 shows an embodiment of an apparatus for carrying out the above-described evaluation method. This evaluation apparatus includes a stage 11 on which the cell culture substrate 1 is disposed, and a droplet such as a syringe for supplying the droplet L to the surface of the functional layer of the cell culture substrate 1 disposed on the stage 11. The supply means 12, the evaporation inducing means 13 for evaporating the droplets L, the imaging means 14 for photographing the evaporating droplets L, and the contact area reduction rate based on the images photographed by these imaging means. And a reduction rate calculation unit 15a for calculation. In the case of measuring the change in the contact angle of the droplet L during the evaporation process, the contact angle calculation unit 15b that calculates the contact angle of the droplet L based on the photographed image can be further provided. Examples of the evaporation induction means 13 include a heater for indirectly or directly heating the droplet L, a vacuum pump for reducing the atmospheric pressure, and the like.

  The cell culture substrate 1 on the stage 11 is illuminated with light from the light source 16 that has passed through the diffusion plate 17. The imaging means 14 only needs to be able to catch the evaporated droplets, and specifically, a high-speed camera equipped with a CCD or the like is preferably used. As shown in FIG. 3, the droplet L can be photographed from the side of the stage 11 and the decreasing speed of the contact radius r of the droplet L can be measured. In addition, an imaging means is installed above or below the stage 11. Then, the droplet L may be observed in a plane, and the area of the contact region of the droplet L may be measured. When the image pickup means 14 is on the side of the stage 11, the reduction speed of the contact area and the contact angle of the droplet L can be measured simultaneously with one image pickup means, and the configuration of the apparatus becomes simple. The droplet supply means 12 is set so that a set amount of droplets L can be dropped. Furthermore, the decrease rate calculation unit 15a and the contact angle calculation unit 15b have a function of analyzing an image acquired by the imaging unit and calculating a decrease rate and a contact angle of the contact area.

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

Example 1
-Production of cell culture substrate 2% poly-N-isopropylacrylamide (Aldrich), N-isopropylacrylamide was dissolved in isopropyl alcohol (IPA) to a final monomer concentration of 30% by weight, and the solution was prepared. Prepared. Subsequently, a circular dish made of polystyrene for cell culture (product name: TCPS, manufactured by Becton Dickinson) was used as a substrate, and the N-isopropylacrylamide solution was coated on the inner bottom surface of the circular dish, and an electron beam irradiation apparatus The functional layer made of poly-N-isopropylacrylamide (PIPAAm) was formed on the inner bottom surface of the circular dish by graft polymerization. Thereafter, the circular dish was washed with ion exchange water at 5 ° C. and dried to obtain a cell culture substrate.

・ Evaluation of cell culture substrate The produced cell culture substrate was placed on the stage of a contact angle measuring device DM-501 (trade name, manufactured by Kyowa Interface Science Co., Ltd.), and the heater temperature was the lower limit of poly-N-isopropylacrylamide. The temperature was set to 40 ° C., which is higher than the critical dissolution temperature (T = 32 ° C.). Then, 1 microliter of pure water was dripped at the surface of the functional layer with the micro syringe. After dropping, the droplets were photographed from the side with a CCD camera at a measurement interval of 5000 msec, and the contact radius and contact angle of the droplets were calculated by software FAMAS (trade name, manufactured by Kyowa Interface Science Co., Ltd.). The measurement results are shown in FIG.

(Example 2)
The contact radius and contact angle of the droplets were calculated in the same manner as in Example 1 except that the functional layer was formed using a solution in which the final monomer concentration was 35% by weight. The measurement results are shown in FIG.

(Example 3)
The contact radius and contact angle of the droplets were calculated in the same manner as in Example 1 except that the functional layer was formed using a solution in which the final monomer concentration was 40% by weight. The measurement results are shown in FIG.

Example 4
As a cell culture substrate, a polystyrene circular dish (trade name: TCPS, manufactured by Becton Dickinson) for cell culture was used. In the same manner as in Example 1, the contact radius and contact angle of the droplet were calculated. The measurement results are shown in FIG.

  As shown in FIGS. 4 to 7, in all of Examples 1 to 4, the contact radius is maintained at a substantially constant value in the section (section 1) in which the contact angle decreases after the measurement is started, and the substantially constant contact angle is maintained. The contact radius decreased in the section to be maintained (section 2), and the contact radius further decreased in the section in which the contact angle decreased again (section 3). The decrease rate of the contact radius is calculated from the change of the contact radius in the section 2, and the result is shown in FIG. Further, a difference Δθ between the contact angle at the measurement start time (static contact angle) and the substantially constant contact angle (average value) in the section 2 is calculated, and the result is shown in FIG.

  From the results of FIGS. 8 and 9, the cell culture substrates of Examples 1 to 4 showed significant differences in both the contact radius reduction rate and the contact angle difference. In particular, the cell culture substrates of Examples 1 to 3 that differ only in the amount of PIPAAm immobilized could be clearly distinguished. Since the cell culture substrate of Example 3 has a larger amount of immobilized PIPAAm than Examples 1 and 2 and the interaction between the droplet L and the functional layer is strong, the contact angle θ is higher in Example 3. The contact radius is maintained until it becomes smaller, and it is considered that the difference Δθ between the static contact angle at the start of measurement and the substantially constant contact angle in the section 2 is increased.

Next, the cell culture substrate prepared in Examples 1 to 4 was filled with DMEM medium containing 10% FBS, and 3TS Swiss Albino cells were seeded under conditions of 6.25 × 10 ³ cells / cm ² . After culturing at 37 ° C. for 18 hours, the cells were transferred to a low-temperature incubator at 20 ° C. After incubation for 30 minutes, the state of detached and floating cells was observed. As a result, the cell culture substrate of Example 3 had good cell detachability (removability), whereas the cell culture substrate of Examples 1 and 2 did not exfoliate part of the cells. Was not good. In the cell culture substrate of Example 4, no cells were detached. From these results, it was found that a cell culture substrate with good cell de-adhesiveness can be efficiently produced by using the decrease rate and contact angle difference of the droplet contact area in the process of droplet evaporation as an index. It was.

DESCRIPTION OF SYMBOLS 1 Cell culture base material 10 Base material 11 Stage 12 Droplet supply means 13 Evaporation induction means 14 Imaging means 15a Decrease speed calculation part 15b Contact angle calculation part 16 Light source 17 Diffusion plate 20 Functional layer L Droplet r Contact radius θ Contact angle

Claims (6)

A cell culture substrate evaluation method comprising a substrate and a functional layer for culturing cells provided on the substrate,
Supplying droplets to the surface of the functional layer;
Measuring the rate of decrease of the contact area where the droplet and the functional layer contact in the process of evaporation of the supplied droplet;
Have
The method for evaluating a cell culture substrate, wherein the state of the functional layer is evaluated based on the measured decrease rate. Measuring the change in the contact angle of the droplet in the process of evaporating the supplied droplet;
The change in the contact angle includes a section in which the contact angle decreases, a section in which the contact angle is maintained thereafter, and a section in which the contact angle decreases again thereafter. The method for evaluating a cell culture substrate according to claim 1, wherein the rate is a decrease rate in a section where the contact angle is maintained.   The method for evaluating a cell culture substrate according to claim 1 or 2, wherein the functional layer contains a temperature-responsive polymer whose cell adhesiveness is changed by temperature change. An apparatus for carrying out the evaluation method according to claim 1,
A stage on which the cell culture substrate is placed;
A droplet supply means for supplying droplets to the surface of the functional layer of the cell culture substrate disposed on the stage;
Evaporation induction means for evaporating the droplets;
Imaging means for photographing the evaporating droplets;
A reduction rate calculation unit that calculates a reduction rate of the contact area based on an image captured by the imaging unit;
An apparatus for evaluating a cell culture substrate. An apparatus for carrying out the evaluation method according to claim 2,
A stage on which the cell culture substrate is placed;
A droplet supply means for supplying droplets to the surface of the functional layer of the cell culture substrate disposed on the stage;
Evaporation induction means for evaporating the droplets;
Imaging means for photographing the evaporating droplets;
A reduction rate calculation unit that calculates a reduction rate of the contact area based on an image captured by the imaging unit;
A contact angle calculation unit that calculates a contact angle of a droplet based on an image photographed by an imaging unit;
An apparatus for evaluating a cell culture substrate. Preparing a cell culture substrate having a substrate and a functional layer for culturing cells provided on the substrate;
For the prepared cell culture substrate, a step of performing the evaluation method according to any one of claims 1 to 3,
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.

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