JP6511340B2 - Evaluation method of functional layer - Google Patents

Description

  The present invention relates to a method of evaluating a functional layer for cell culture.

  Cells are cultured in sheets, and the technology called "cell sheet engineering" that recovers cells in sheets only by lowering the temperature without using enzymes such as trypsin is attracting attention in the field of regenerative medicine. . Cell sheets obtained by this technology have already been confirmed to have a certain curative effect in regenerative medicine, and clinical research and clinical trials have already been advanced. The control of adhesion and desorption of the cultured cell sheet is performed, for example, via a functional layer such as poly-N-isopropylacrylamide (PNIPAAm) immobilized on a substrate.

  Polymers such as PNIPAAm are materials whose hydration ability changes mainly in response to temperature, and at temperatures below the critical solution temperature, their affinity for surrounding water is improved, and the polymer takes in and swells liquids such as water. It has the property of making it difficult for cells to adhere to the surface (cell non-adhesion). On the other hand, at a temperature above the critical solution temperature, water is detached from the polymer, and the polymer shrinks to facilitate adhesion of cells to the surface (cell adhesion). Cell adhesion is known to be affected by the immobilization density and chain length of PNIPAAm.

  By controlling the hydrophobicity of the surface of the functional layer, control of cell adhesion, water repellency, stain resistance and the like is performed. In particular, in the control of cell adhesion, sufficient performance stability and reproducibility can not be obtained unless the amount of polymer and surface properties of the functional layer are strictly controlled. Therefore, in order to improve the performance stability and reproducibility of the functional layer, it is important to appropriately evaluate the surface properties and the like of the functional layer. As an example of the evaluation method of a functional layer, the inspection method of the cell culture base material provided with the functional layer containing PNIPAAm on a base material is disclosed (refer following patent document 1).

  The inspection method described in Patent Document 1 is characterized by including the following first inspection step and second inspection step. In the first test step, the falling velocity of the first droplet supplied to the surface of the functional layer of the inclined cell culture substrate is measured, and the function is performed based on the obtained falling velocity of the first droplet. Determine whether the condition of the sex layer is good or bad. In the second inspection step, the second droplet is supplied to the surface of the functional layer, and the contact area in which the second droplet and the functional layer come into contact is reduced in the process of evaporation of the supplied second droplet. The speed is measured, and the state of the functional layer is determined based on the obtained reduction speed.

JP, 2013-195399, A

  The method described in Patent Document 1 is useful in that it is not necessary to carry out cell culture, and the state of the functional layer can be inspected in a short time. However, the information obtained in one examination is limited to local information on the surface of the functional layer. Therefore, in order to evaluate the property of the whole surface, it is necessary to increase a test | inspection location, and the subject that evaluation requires time has a subject. Moreover, when a functional layer takes in and swells liquids, such as water, the subject that it is necessary to test after drying a functional layer occurs.

  This invention is made in view of the said subject, and provides the evaluation method of the functional layer which can evaluate the property of the whole surface of the functional layer which took in and swelled liquids, such as water, in a short time. The purpose is

  In order to achieve the above object, the method of evaluating a functional layer of the present invention is a method of evaluating a functional layer containing a functional polymer having cell adhesiveness, which comprises a swelling step of swelling the functional layer with a liquid A step of wetting the whole evaluation region of the surface of the functional layer after the swelling step with a liquid, and one end of the functional layer after the wetting step above the other end in the evaluation region Characterized in that it has a measuring step of measuring the liquidation time until the wet area wetted with the liquid decreases to a predetermined size, and an evaluation step of evaluating the surface property based on the liquidation time. I assume.

  According to the evaluation method of the functional layer of the present invention, it is possible to evaluate the property of the entire surface of the functional layer which has taken in and swelled a liquid such as water in a short time.

BRIEF DESCRIPTION OF THE DRAWINGS The typical perspective view which shows the functional layer which concerns on embodiment of this invention. It is a figure which shows the high-order structural change of the functional layer shown in FIG. 1, (a) is the aggregated state which shows hydrophobicity, (b) is a typical perspective view of the swollen state which shows hydrophilicity. BRIEF DESCRIPTION OF THE DRAWINGS The block diagram which shows schematic structure of the inspection apparatus of the functional layer which concerns on embodiment of this invention. FIG. 4 is a block diagram showing a modification of the inspection apparatus shown in FIG. 3A. The flowchart which shows each process of the evaluation method of the functional layer which concerns on embodiment of this invention. The flowchart which shows the evaluation procedure of a functional layer using the test | inspection apparatus shown to FIG. 3A. It is a figure which shows operation | movement of the test | inspection apparatus shown to FIG. 3A, (a) is a state which hold | maintained the dish horizontally, (b) is a schematic diagram which shows the state which inclined the dish to the setting angle. (A) is a sectional view and a plan view of a dish showing a state in which liquid is contained in a horizontal dish, and (b) a state in which the dish of (a) is inclined. (A) is a state in which the liquid is placed in a horizontal dish, (b) is a state in which the liquid volume in the dish is adjusted, (c) is a state immediately after tilting the dish in (a), (d) is Sectional drawing and a top view of a dish which show the state where predetermined time passed, respectively from the state of c). It is an image figure of the camera of the inspection apparatus shown to FIG. 3A, (a) is an image figure before image processing, (b) is an image process. (A) is a schematic cross-sectional view of a dish having a support layer between the bottom of the dish and the functional layer, (b) is selectively applied to the evaluation area of the surface of the functional layer shown in (a) Typical sectional drawing which shows the state which supplied the liquid. The typical perspective view which shows the modification of the evaluation method of a functional layer. Explanatory drawing explaining the measurement process in the modification shown in FIG. 11, and an evaluation process.

  Hereinafter, an embodiment of a method for evaluating a functional layer of the present invention will be described with reference to the drawings. In the following, first, the functional layer to be evaluated will be described, then, an embodiment of an inspection apparatus for inspecting the functional layer will be described, and finally, the functional layer of the present embodiment using the inspection apparatus The evaluation method of will be described.

[Functional layer]
FIG. 1 is a schematic perspective view showing a functional layer 1 according to an embodiment of the present invention. The functional layer 1 of the present embodiment is, for example, a layer provided on a substrate 2 such as a resin film or a cell culture vessel, and made of a material containing a functional polymer having a function capable of culturing cells. is there. The film thickness t of the functional layer 1 may be, for example, 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.

  Examples of the material constituting the functional layer 1 include basic polymers such as polylysine, aminopropyltriethoxysilane, N- (2-aminoethyl)-as a material exhibiting adhesiveness to cells by electrostatic interaction, for example. Basic compounds such as 3-aminopropyltrimethoxysilane and condensates containing them can be mentioned. Moreover, as a material exhibiting adhesiveness with cells by biological properties, fibronectin, laminin, tenascin, vitronectin, RGD (arginine-glycine-aspartic acid) sequence-containing peptide, YIGSR (tyrosine-isoleucine-glycine-serine-arginine) Sequence-containing peptides, collagen, atelocollagen, gelatin and the like can be mentioned.

  Among them, in the case where a cell culture substrate is used to desorb the cells from the substrate after culturing the cells or to collect the cells in the form of a sheet, the function of changing cell adhesion by a predetermined stimulus is used. What has it is employable as a constituent material of functional layer 1. For example, as a constituent material of the functional layer 1, it is possible to include a stimulus-responsive polymer that changes from cell adhesion to non-cell adhesion by applying a stimulus. 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 suitable one for the cell sheet to be produced can be appropriately selected. . As a constituent material of the functional layer 1, in particular, a temperature responsive polymer is preferable because cell adhesion changes due to temperature change and stimulation is easily applied. In the present embodiment, the functional polymer contained in the functional layer 1 is a temperature responsive polymer.

  FIGS. 2 (a) and 2 (b) are schematic views showing a change in higher-order structure of the functional layer 1 shown in FIG. 2 (a) is a schematic perspective view of the state in which the temperature responsive polymer of the functional layer 1 is aggregated, and FIG. 2 (b) is a state in which the temperature responsive polymer of the functional layer 1 is swollen. It is a typical perspective view.

  As a temperature-responsive polymer contained in the functional layer 1, for example, a polymer that exhibits cell adhesion at a temperature at which cells C are cultured and exhibits cell non-adhesion at a temperature at which the prepared cell sheet is peeled off is used. Is preferred. Specifically, as a constituent material of the functional layer 1 of the present embodiment, it is preferable to use a temperature responsive polymer having the following properties. At a temperature above the critical dissolution temperature, as shown in FIG. 2 (a), the polymer shrinks by detaching water from the polymer, and the property of facilitating adhesion of the cell C to the surface of the functional layer 1 (cell adhesion Gender). At a temperature lower than the critical solution temperature, as shown in FIG. 2 (b), the affinity to the surrounding water is improved, and the polymer takes in a liquid such as water and swells to make it difficult to adhere the cell C to the surface. (Cell non-adherent) is shown. Such critical solution temperature is called lower critical solution temperature T. As a constituent material of the functional layer 1, it is preferable to use a temperature responsive polymer in which T is 0 ° C. to 80 ° C., more preferably 0 ° C. to 50 ° C. It is because the cell C can be cultured stably as T is 0 degreeC-80 degreeC.

  Suitable temperature responsive polymers include acrylic polymers or methacrylic polymers. More specifically, for example, poly-N-isopropylacrylamide (T = 32 ° C.), poly-N-n-propyl acrylamide (T = 21 ° C.), poly-N-n-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 Poly-N, N-diethylacrylamide (T = 32 ° C.) and the like can be mentioned.

  As monomers for forming these polymers, monomers that can be polymerized by irradiation 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 a species can be used. When one type of monomer is used alone, the polymer formed on the substrate 2 becomes a homopolymer, and when two or more monomers are used together, the polymer formed on the substrate 2 becomes a heteropolymer However, any form is applicable in the present invention.

  In addition, if it is necessary to adjust the lower limit critical solution temperature depending on the type of proliferating cells, or if it is necessary to adjust the balance of hydrophilicity and hydrophobicity of the functional layer 1, other monomers other than the above may be used. Further, copolymerization may be performed. Furthermore, the functional layer 1 may be configured using a graft or block copolymer of the above-described polymer and another polymer, or a mixture of the above-described polymer and another polymer. Moreover, it is also possible to crosslink in the range which the intrinsic property of a polymer is not impaired.

  The functional layer 1 containing a temperature responsive polymer or the like can be formed on the substrate 2 by appropriately employing a conventionally known method. For example, a coating composition comprising a monomer that forms the target stimulus-responsive polymer by polymerization and an organic solvent capable of dissolving the monomer is prepared, and this is coated on the surface of the substrate 2 according to a conventional coating method. Form a coating, and then polymerize the monomers in the coating by suitable means such as irradiation to form a polymer, and graft the surface between the substrate 2 and the polymer It can be formed by causing a reaction.

  The material of the base 2 supporting the functional layer 1 is not particularly limited. Specifically, for example, polyethylene terephthalate (PET), polystyrene (PS), polycarbonate (PC), TAC (triacetyl cellulose), polyimide (PI), nylon (Ny), low Resin materials such as high density polyethylene (LDPE), medium density polyethylene (MDPE), polyvinyl chloride, polyvinylidene chloride, polyphenylene sulfide, polyether sulfone, polyethylene naphthalate, polypropylene and acrylic resin, and inorganic materials such as glass and quartz The resin material is preferably used. The surface of the substrate 2 on which the functional layer 1 is provided can be a surface subjected to easy adhesion treatment. "Easy adhesion treatment" refers to, for example, treatment with an easy adhesive such as polyester, acrylic ester, polyurethane, polyethylene imine, silane coupling agent, perfluorooctane sulfonic acid (PFOS) and the like.

  When the temperature-responsive polymer such as poly-N-isopropyl acrylamide (PNIPAAm) is contained in the functional layer 1, for example, when the total amount is too small, the functional layer 1 is subjected to the temperature of the culture condition of the lower critical solution temperature T or more. The cell adhesion is exhibited, but there is a risk that the temperature will be lower than T and the cell will not be detached. In addition, when the total amount of the thermoresponsive polymer contained in the functional layer 1 is too large, the functional layer 1 may become a defective product that does not exhibit cell adhesiveness at a temperature of culture conditions of T or more. Therefore, it is necessary to appropriately evaluate the properties of the functional layer 1 before cell culture, to identify unevenness in the surface of the functional layer 1 and differences in performance, and to know the performance of the functional layer 1.

  As a method of analyzing the functional layer 1, infrared spectroscopy (IR), ellipsometry, a method of measuring water contact angle or water contact angle, and measurement with an atomic force microscope in water (AFM in water), etc. Various methods are known. Although IR is excellent for measuring the total amount of the temperature responsive polymer contained in the functional layer 1 in a dry state, it is not suitable for measuring the properties of the functional layer 1 swollen with a liquid such as water, and is compared It can measure only the narrow area. Ellipsometry is excellent for measuring the properties of the functional layer 1 in a dry state, but is not suitable for measuring the properties of the functional layer 1 swollen with a liquid such as water, and can measure only a relatively narrow region. In the method of measuring the water contact angle or the underwater contact angle, although it is possible to measure the properties of the functional layer 1 in a state of being swollen with a liquid such as water, only relatively narrow regions can be measured. In addition, in this method, the measured value largely fluctuates due to the spread of water droplets on the surface of the functional layer 1, the roughness of the surface of the functional layer 1, fine deposits, and the like, and stable evaluation is difficult. Underwater AFM is excellent for measuring the properties of the functional layer 1 in a state of being swollen with a liquid such as water, but has problems in terms of simplicity of measurement, nondestructive evaluation and the size of the measurement area.

  As a result of investigation based on analysis of AFM in water, it is considered that the surface of the functional layer 1 is not a dense film, but aggregates of point-dispersed PNIPAAm etc. are gathered, depending on the density and size of the aggregates It can be thought that cell adhesion changes. In addition, aggregates such as PNIPAAm swell at a temperature lower than the lower limit critical solution temperature T and the property difference of aggregates tends to appear, so evaluation of the functional layer 1 in a state swollen with a liquid such as water is performed Is effective. The evaluation method of the functional layer 1 according to the embodiment of the present invention to be described later uses PNIPAAm by using an analysis method that directly reflects information of aggregates such as PNIPAAm on the functional layer 1 in a state of being swollen with water. The surface texture of the functional layer 1 can be evaluated more accurately than the conventional method of measuring only the total amount and thickness of the etc.

In addition, as a liquid for swelling the functional layer 1, not only water but an aqueous solution in which a salt such as NaCl or Na ₂ CO ₃ is dissolved in water can be used. If the functional layer 1 or the dish D is resistant, an organic solvent or an organic solvent mixed solution can be used.

[Inspection device of functional layer]
Next, an embodiment of an inspection device of the functional layer 1 used for the evaluation method of the functional layer 1 of the present invention will be described. FIG. 3A is a block diagram showing a schematic configuration of an example of an inspection apparatus 100 capable of performing the evaluation method of the functional layer 1 of the present embodiment. FIG. 3B is a block diagram showing a modification of the inspection apparatus 100 shown in FIG. 3A.

  The inspection apparatus 100 of the functional layer 1 of the present embodiment includes a rotary stage 101 that holds the dish D, and a control unit 102 that controls the rotary stage 101. In addition, the inspection apparatus 100 according to the present embodiment includes a camera 103 for imaging the functional layer 1 in the dish D, coaxial illumination 104 for illuminating an imaging area of the camera 103, and an image processing unit 105 for processing an image of the camera 103. And a display 106 for displaying the image processed by the image processing unit 105.

  The rotary stage 101 holds a base material such as the dish D provided with the functional layer 1, and can be inclined at a predetermined angle from a state where the dish D etc. is horizontally supported under the control of the control unit 102. Is configured as. The control unit 102 includes a switch 107 for starting or ending control of the rotation stage 101 and the image processing unit 105. For example, the control unit 102 first holds the rotary stage 101 holding the dish D etc. horizontally for a predetermined time, and then rotates it by a predetermined angle α to set the angle α to the predetermined time. It is possible to control to be maintained over and finally to rotate in the reverse direction by the same angle α to return to the horizontal state.

  The camera 103 continuously captures an image of the functional layer 1 on the bottom of the dish D under control of the image processing unit 105 and illumination by the coaxial illumination 104. The camera 103 and the coaxial illumination 104 may be disposed above the dish D and on the side of the opening of the dish D as shown in FIG. 3A, or when the dish D is transparent, as shown in FIG. 3B. It may be disposed on the bottom side of the dish D below D. By arranging the camera 103 and the coaxial illumination 104 on the bottom side of the dish D below the dish D, for example, when the dish D is inclined, a part of the functional layer 1 can be detected by the side wall of the dish D. It can prevent becoming a blind spot.

  The image processing unit 105 recognizes the position of the functional layer 1 from the image captured by the camera 103 under the control of the control unit 102, and sets at least a part of the surface of the functional layer 1 as a target for evaluating the surface property. It sets to evaluation area R1 (refer FIG.7 and FIG.8) which becomes. In the present embodiment, the lower part of the surface of the functional layer 1 is a non-evaluation area R2, and the upper part of the surface of the functional layer 1 excluding the non-evaluation area R2 is an evaluation area R1. Here, the evaluation area R1 is an area exposed on the liquid surface by putting a liquid W such as water in the dish D and tilting it at a predetermined angle α. The non-evaluation area R2 is an area which remains immersed in the liquid W even if the liquid W is put in the dish D and inclined at a predetermined angle α.

  In addition, when it is possible to expose the entire surface of the functional layer 1 on the liquid surface when the one end is disposed above the other end by inclining the functional layer 1 as described above The entire surface of the functional layer 1 can also be set as the evaluation region R1. When the rotary stage 101 is not used, one end of the functional layer 1 is disposed above the other end so that the surface is parallel to the vertical direction, and the entire surface is immersed in the liquid, and then the upper side The entire surface of the functional layer 1 can be set as the evaluation region R1 even when the entire surface is exposed to the liquid surface by pulling the surface.

  The image processing unit 105 is an area where liquid is present on the surface of the functional layer 1 in the same area with respect to the size of the evaluation area R1, for example, based on the image of the camera 103 under control of the control unit 102. The ratio of the size of the wet area R11 (see FIG. 9) is calculated. In addition, the image processing unit 105 measures, for example, the time until the ratio of the size of the wet area R11 to the size of the evaluation area R1 decreases to a predetermined ratio, that is, the liquid pulling time under the control of the control unit 102. Do. In the inspection apparatus 100 of the present embodiment, for example, the time until the ratio of the size of the wet area R11 to the size of the evaluation area R1 becomes 40% is measured as the liquidation time.

  Furthermore, the image processing unit 105 evaluates the surface property of the functional layer 1 based on the liquid immersion time, for example, under the control of the control unit 102. More specifically, in the image processing unit 105 or the control unit 102, for example, the surface property of the functional layer 1 is good (OK) when the measured liquidation time is within a predetermined time range. When the liquid immersion time is outside the predetermined time range, the surface property of the functional layer 1 is judged and evaluated as being defective (NG). Further, the image processing unit 105 causes the display 106 to display, for example, the liquid dripping time, the determination result, the threshold of the size of the wet area R11, the liquid dripping time threshold (time threshold), and the like under the control of the control unit 102. . The inspection apparatus 100 according to the present embodiment displays, on the display 106, the number of pixels in which the size of the wet area R11 is 40% of the size of the evaluation area R1 as the area threshold.

[Method of evaluating functional layer]
Next, an embodiment of the evaluation method of the functional layer 1 of the present invention will be described. FIG. 4 is a flowchart showing steps S1 to S4 of the method for evaluating the functional layer 1 according to the embodiment of the present invention. FIG. 5 is a flowchart showing the procedures P1 to P9 of the method of evaluating the functional layer 1 using the inspection apparatus 100 shown in FIG. 3A. 6A and 6B are diagrams showing the operation of the inspection apparatus 100 shown in FIG. 3A. FIG. 6A shows a state where the dish D is held horizontally, and FIG. 6B shows the dish D inclined to the set angle α. It is a schematic diagram which shows the state. Hereinafter, procedures P1 to P9 for performing steps S1 to S4 of the evaluation method of the functional layer 1 of the present embodiment using the inspection apparatus 100 will be described.

  The evaluation method of the functional layer 1 of the present embodiment is an evaluation method of the functional layer 1 containing a functional polymer having cell adhesiveness, and as shown in FIG. 4, a swelling step S1 and a wetting step S2 , Measurement step S3 and evaluation step S4.

(Swelling process)
In the swelling step S1, as shown in FIG. 6A, for example, a dish D provided with a functional layer 1 containing a temperature responsive polymer at the bottom is prepared, and a liquid W such as water is put in the dish D The functional layer 1 is immersed in the liquid W, and the water temperature and the temperature of the functional layer 1 are brought to a temperature below the critical solution temperature T. Then, the dish D is set on the rotation stage 101 (procedure P1), the measurement start button of the switch 107 is pressed (procedure P2), and the inspection apparatus 100 is activated. The inspection apparatus 100 swells the functional layer 1 with a liquid W such as water by maintaining the rotary stage 101 holding the dish D horizontally for a predetermined time under the control of the control unit 102. Step S1 is performed.

(Evaluation area setting process)
In the evaluation method of the functional layer 1 of the present embodiment, an evaluation area setting step is performed after the swelling step S1 and before the wetting step S2. Note that the implementation of the evaluation area setting step is not essential, and can be omitted. In the evaluation area setting step, the inspection apparatus 100 controls the rotation stage 101 holding the dish D under the control of the control unit 102 from 90 ° or less shown in FIG. 6B from the horizontal state shown in FIG. For example, it is rotated by a preset predetermined angle α of about 30 °. Then, the angle α is maintained for a predetermined time of, for example, about 10 seconds.

  As a result, the functional layer 1 is inclined with respect to the horizontal plane, one end of the functional layer 1 is disposed above the other end, and the liquid W on the surface of the functional layer 1 moves downward. Then, the upper part of the surface of the functional layer 1 is exposed on the liquid surface, and the lower part of the surface of the functional layer 1 is immersed in the liquid W. In the evaluation area setting step, the inspection apparatus 100 captures the functional layer 1 by the camera 103, recognizes the position of the functional layer 1 by the image processing unit 105, and exposes the surface of the functional layer 1 exposed on the liquid surface. The evaluation area R1 (see FIGS. 7B and 8C) is set in the upper part, and the size of the evaluation area R1, for example, the number of pixels or the area is calculated.

(Wet process)
In the wetting step S2, the entire evaluation area R1 of the surface of the swollen functional layer 1 after the swelling step S1 is wetted with the liquid W. In the wetting step S2, the inspection apparatus 100 rotates the rotating stage 101 holding the dish D in the reverse direction under the control of the control unit 102, and from the inclined state shown in FIG. 6 (b) to FIG. 6 (a) Returning to the horizontal state shown, the functional layer 1 in the dish D is dipped again in the liquid. As necessary, the whole of the evaluation region R1 of the functional layer 1 is wetted with the liquid W by replenishing or removing the liquid W. In the case where the above-mentioned evaluation area setting step is omitted, the functional layer 1 is immersed in the liquid W in the above-mentioned swelling step S1, so the wetting step S2 ends simultaneously with the completion of the swelling step S1. The entire evaluation region R1 of the surface of the functional layer 1 is wetted with the liquid W. As necessary, after adjusting the liquid quantity so that only the area which becomes the lower side in the next measurement step S3 among the evaluation area R1 and the area other than the evaluation area R1 becomes wet with the liquid W, It moves to the next measurement step S3.

(Measurement process)
In the measurement step S3, one end of the functional layer 1 after the wetting step S2 is disposed above the other end, and the liquid until the wet area R11 wet with the liquid W in the evaluation area R1 decreases to a predetermined size Measure the withdrawal time. More specifically, the inspection apparatus 100 controls the rotation stage 101 holding the dish D under control of the control unit 102 from 90 ° or less shown in FIG. 6B from the horizontal state shown in FIG. For example, it is rotated by a preset predetermined angle α of about 40 ° (procedure P3). Then, the angle α is maintained for a predetermined time of, for example, about 10 seconds (procedure P4). As a result, the functional layer 1 is inclined with respect to the horizontal plane, and one end of the functional layer 1 is disposed above the other end.

  In addition, the inspection apparatus 100 captures an image of the functional layer 1 with the camera 103, and starts measurement of the liquid draw time by the image processing unit 105 (procedure P5). The inspection apparatus 100 captures the wet area R11 (see FIGS. 7B and 8C) in the evaluation area R1 with the camera 103, and the image processing unit 105 measures the size of the wet area R11, for example, the number of pixels. Alternatively, the area is calculated (procedure P6), and it is determined whether or not the size of the wet area R11 has decreased to a predetermined threshold value, for example, 40% of the evaluation area R1 within a preset time (procedure P7) ).

  If it is determined that the size of the wet area R11 has decreased to the threshold value (arrow Y in FIG. 5) as a result of the determination in the procedure P7, the inspection apparatus 100 inclines the dish D to set one end of the functional layer 1 The image processing unit 105 or the control unit 102 measures the liquid dripping time until the size of the wet region R11 decreases to the threshold after being disposed above the other end (procedure P8). On the other hand, in a state where the dish D is inclined at the angle α, the dish D is automatically reduced if the size of the wet area R11 does not decrease to a predetermined threshold even after a preset time has elapsed (arrow N in FIG. 5). May be returned to the horizontal state and the process may return to step P5.

  Next, referring to FIGS. 7 and 8, functional layer 1 is provided on the entire bottom surface of dish D, and functional layer 1 is provided on a portion of the bottom surface of dish D. In the case, procedures P3 to P7 shown in FIG. 5 will be described in more detail.

  Fig.7 (a) is sectional drawing and the top view which show the state which put the liquid W in the horizontal dish D in which the functional layer 1 was provided in the whole bottom face. FIG.7 (b) is sectional drawing and the top view which show the state to which predetermined time passed, after making the dish D shown to Fig.7 (a) incline.

  In the dish D shown in FIG. 7, the functional layer 1 is provided on the entire bottom surface in the dish D. The evaluation region R1 of the functional layer 1 is set in the upper part of the surface of the functional layer 1 exposed on the liquid surface in the inclined state shown in FIG. Is set to the non-evaluation area R2. The surface of the side wall in the dish D is subjected to, for example, liquid repellency processing so as to exclude the liquid W from the periphery of the evaluation region R1 in the measurement step S3. Therefore, when the dish D is rotated to a predetermined angle in the above-described procedures P3 and P4 shown in FIG. 5 and one end of the functional layer 1 is disposed above the other end as shown in FIG. 7 (b) The side walls in the dish D exclude the liquid W from the periphery of the evaluation area R1 excluding the boundary with the non-evaluation area R2 immersed in the liquid W moved downward of the dish D.

  More specifically, the liquid W is prevented from adhering to the side wall of the dish D, and the liquid W present in the evaluation area R1 on the surface of the functional layer 1 is affected by the liquid W around the evaluation area R1. Can be minimized. Therefore, in the dish D shown in FIG. 7, the surface of the side wall in the dish D is treated to be liquid repellent, so that the liquid W is excluded from the periphery of the evaluation area R1 in the measurement step S3 and the liquid in the periphery of the evaluation area R1 The liquid immersion time can be measured without being affected by W.

  Fig.8 (a) is sectional drawing and the top view which show the state which put the liquid W in the horizontal dish D in which the functional layer 1 was provided in a part of bottom face. FIG. 8B is a cross-sectional view and a plan view showing a state after the liquid amount adjustment of the liquid W. FIG.8 (c) is sectional drawing and the top view which show the state immediately after making the dish D shown in FIG.8 (b) incline. Fig. 8 (d) is a cross-sectional view and a plan view showing the dish D after a predetermined time has elapsed from the state shown in Fig. 8 (c).

  In the dish D shown in FIG. 8, the functional layer 1 provided at the center of the bottom in the dish D is rectangular. More specifically, the functional layer 1 is formed in a square or rectangular shape in plan view at a position away from the surface of the side wall in the dish D. The evaluation region R1 of the functional layer 1 is set in the upper part of the surface of the functional layer 1 exposed on the liquid surface in a state where a predetermined time has elapsed after the inclination shown in FIG. The part to be immersed in the liquid W is set as the non-evaluation area R2.

  The bottom in the dish D around the functional layer 1 is subjected to liquid repellent treatment. Therefore, the liquid W is removed from the bottom of the dish D around the functional layer 1 by adjusting the amount of the liquid W, and the water repellent area of the bottom of the functional layer 1 and the dish D is used for the dish D. It is possible to wet the liquid W only in the lower area at the time of inclination. That is, in the dish D shown in FIG. 8, the bottom surface around the functional layer 1 in the dish D is subjected to liquid repellent treatment, so that the liquid W is excluded from the periphery of the evaluation area R1 in the measurement step S3 and the evaluation area The liquid immersion time can be measured without being affected by the liquid W around R1.

  Further, as shown in FIG. 3B, when the camera 103 and the coaxial illumination 104 are disposed on the bottom side of the dish D below the dish D, the functional layer 1 is formed by the side walls of the dish D when the dish D is inclined. Can be prevented from becoming a blind spot of the camera 103. Therefore, even when a plurality of dishes D are evaluated at one time, it is possible to measure the liquidation time without the visual field of the functional layer 1 being blocked by the side walls of the dishes D.

  Next, an example of the image processing performed in the measurement step S3 will be described with reference to FIGS. 8 (a) to 8 (d), 9 (a), and 9 (b). FIG. 9A is an image diagram showing an image of the camera 103 of the measurement apparatus of the present embodiment. FIG. 9B is an image diagram showing an image processed by the image processing unit 105 of the inspection apparatus 100 of the present embodiment.

  In the measurement step S3, as shown in FIG. 8A, for example, the amount of liquid W in the dish D is reduced from the state in which the whole of the functional layer 1 is immersed in the liquid W with the dish D in a horizontal state. I will let you. As a result, as shown in FIG. 8B, the liquid W is removed from the periphery of the functional layer 1 by the liquid repellency of the bottom surface in the dish D. Although the amount and arrangement of the liquid W are not particularly limited, for example, a pipette or the like is used, and among the liquid repellent areas in the bottom surface of the functional layer 1 and the dish D, only the area which becomes lower when the dish D is inclined. It is preferable to adjust so that the liquid W is retained on the surface and becomes wet with the liquid W.

  Next, it is in the state of being inclined to the predetermined angle α shown in FIG. 8C by the procedure P3 shown in FIG. 5, and finally the state in which only the non-measurement region R2 shown in FIG. become. The inclination angle α of the functional layer 1 at this time is not particularly limited as long as the liquid W in the dish D does not overflow to the outside, but it is an angle at which the property of the functional layer 1 can be determined most accurately. It is preferable to set. The inclination angle α of the functional layer 1 can be set to, for example, 5 ° or more and 50 ° or less, preferably about 30 °, with respect to the horizontal surface.

  As shown in FIG. 8 (b), before tilting the dish D, the liquid W on the bottom of the surrounding dish D except for the lower end of the liquid-repellent processed functional layer 1 has already been removed, and the functional layer 1 is The whole of the evaluation area R1 on the surface is covered with the liquid W and is in a wet state, that is, a wet area R11. Thereafter, as shown in FIG. 8C, the dish D is inclined to cause the action of gravity in a state in which water is excluded from the surroundings except the lower end of the evaluation region R1 by the liquid repellency of the bottom surface of the dish D. Thus, the liquid W in the evaluation region R1 of the functional layer 1 moves downward, and the state of the liquid dripping is photographed by the camera 103.

  As described above, in the present embodiment, the liquid W is disposed in the water-repellent area of the bottom surface in the dish D before the dish D is inclined, in the area under the functional layer 1 when the dish D is inclined. ing. Thereby, when the dish D is inclined, the liquid W is prevented from staying on the functional layer 1 at the boundary between the functional layer 1 and the water repellent region on the bottom surface of the dish D. The liquid W can be smoothly drained.

  At this time, as shown in FIG. 9A, the wet region R11 in the wet state in which the liquid W is present in the evaluation region R1 of the surface of the functional layer 1 and the liquid W is pulled to the surface of the functional layer 1 The image is taken by the camera 103 with the exposed area R12 exposed. In addition, there may be a case where the cloudy area R13 can be confirmed between the wet area R11 and the liquid area R12. It can be estimated that the cloudy area R13 is an area where the liquid W on the surface of the functional layer 1 is drawn but the functional layer 1 sufficiently holds the liquid W. Since the cloudy area R13 is reduced along with the reduction of the wet area R11, a correlation with the wet area R11 is recognized, but the behavior of the cloudy area R13 does not necessarily coincide with the behavior of the wet area R11, and the functionality Affected by the surface texture of layer 1 and other parameters. Therefore, in the measurement step S3, the functional layer 1 may be evaluated using the cloudy area R13 as in the wet area R11.

  In the measurement step S3 of the present embodiment, the image processing unit 105 performs, for example, image processing such as binarization processing as shown in FIG. 9B in the image of the camera 103 shown in FIG. 9A. Then, the cloudy area R13 is excluded to make the outer edge of the wet area R11 clear. Then, for example, the image processing unit 105 and the control unit 102 measure a liquid dripping time until the image processing wet region R11 decreases to a predetermined size with respect to the evaluation region R1 of the functional layer 1. The size of the wet area R11, which is a threshold for measuring the liquid transfer time, is not particularly limited, but can be set in the range of 0% to 80% of the area of the evaluation area R1 or the number of pixels, for example. 30%, 40%, or 50% of the area or the number of pixels, etc. can be set to a size required to accurately determine the properties of the functional layer 1.

(Evaluation process)
In the evaluation step S4, the property of the surface of the evaluation region R1 of the functional layer 1 is evaluated based on the liquidation time measured in the measurement step S3. Specifically, the inspection apparatus 100 compares the liquidation time measured in the measurement step S3 by the control unit 102 and the image processing unit 105 with the preset lower limit time and upper limit time. As a result of comparison, if the liquidation time is equal to or more than the lower limit time and equal to or less than the upper limit time, the image processing unit 105 determines and evaluates that the surface property of the evaluation region R1 of the functional layer 1 is good (OK). If the pulling time is shorter than the lower limit time or longer than the upper limit time, it is evaluated and determined as failure (NG). The image processing unit 105 and the control unit 102 display the determination result and the like on the display 106 (procedure P8). By the above, the evaluation method of functional layer 1 of this embodiment is completed.

  As described above, in the evaluation method of the functional layer 1 of the present embodiment, the information obtained by one evaluation is not limited to the local information of the surface of the functional layer 1, and the information of the functional layer 1 is not It is possible to obtain information of a wide range of evaluation area R1 on or near the entire surface. Therefore, the property of the surface of the functional layer 1 can be obtained by a single evaluation, and the evaluation time can be shortened as compared with the prior art. In addition, even when the functional layer 1 is swollen by taking in the liquid W such as water, the evaluation can be performed without drying the functional layer 1. Therefore, according to the evaluation method of the functional layer 1 of the present embodiment, it is possible to evaluate the property of the surface of the functional layer 1 which has taken in and swelled the liquid W such as water in a short time.

  That is, in the evaluation method of the functional layer 1 of the present embodiment, the property of the functional layer 1 swollen with the liquid W such as water can be favorably measured, and the simplicity of the measurement is excellent. The entire surface of or near the wide surface can be evaluated at one time. Furthermore, the evaluation method of the functional layer 1 of the present embodiment enables evaluation of properties including not only the amount of PNIPAAm but also the crosslink density by evaluation of the surface property, and excellent in measurement reproducibility, nondestructive inspection and There is an advantage that nondestructive evaluation is possible.

  In other words, the evaluation method of the functional layer 1 according to the present embodiment uses the liquid holding power of the entire surface of the functional layer 1 completely swollen with the liquid W such as water, for example, the water holding power as an index. It is possible to obtain information derived from the nature of the hydrophilic substance that the functional layer 1 has on the surface. This makes it possible to obtain information including local unevenness and overall nonuniformity of the surface of the functional layer 1 at one time.

  When an aqueous solution or the like in which a salt is dissolved in water is used as the liquid W for swelling the functional layer 1, the functional layer 1 becomes difficult to swell when the concentration of the salt of the liquid W becomes high. Therefore, the swelling characteristics of the functional layer 1 with respect to the concentration of the salt can be obtained by changing the concentration of the salt in the liquid W and measuring the liquidation time of the functional layer 1. Moreover, the liquid of a predetermined salt concentration is measured by measuring the liquidation time of each functional layer 1 with respect to the liquid W which the salt concentration prepared to the predetermined value using the several functional layer 1 from which specification differs. The swelling characteristics of each functional layer 1 with respect to W can be obtained.

  Moreover, in the evaluation method of the functional layer 1 of this embodiment, the evaluation area setting step is performed after the swelling step S1 and before the wetting step S2, and the position of the functional layer 1 is recognized for each dish D. An evaluation area R1 is set in the upper part of the surface of the functional layer 1 exposed on the liquid surface. As a result, it is possible to accurately measure the liquidation time regardless of the positional deviation of the functional layer 1 for each dish D or the difference in the amount of water in the dish D.

  Further, in the measurement step S3, not only the wet area R11 shown in FIG. 9A but also the size of the cloudy area R13, ie, the area, the number of pixels, and the density are measured, and a judgment criterion of good or bad is provided. Accuracy can be improved. Clouding region R13 is considered to be related to the liquid holding power of functional layer 1. Therefore, for example, when the time until the cloudy area R13 decreases to a predetermined size is longer than a predetermined time, it is determined that the amount of the temperature responsive polymer such as PNIPAAm is excessive, thereby determining the defective product. The sensitivity can be improved.

  As mentioned above, although the embodiment of the present invention has been described in detail using the drawings, the specific configuration is not limited to this embodiment, and there are design changes and the like within the scope of the present invention. Also, they are included in the present invention.

  For example, in the above-described embodiment, in the measurement step S3, in order to exclude the liquid W from the periphery of the evaluation region R1 of the functional layer 1, an example in which the liquid repelling treatment is performed on the surface or the bottom of the side wall in the dish D is described did. However, if the liquid W can be removed from the periphery of the evaluation area R1 of the surface of the functional layer 1 by another method, it is not necessary to perform the liquid repellent treatment on the dish D.

  FIG. 10A is a schematic cross-sectional view of a dish D having a support layer 3 between the bottom surface of the dish D and the functional layer 1. As a material of the support layer 3, for example, a material similar to dimethylpolysiloxane (PDMS) or dish D can be used. Thus, by providing the support layer 3 between the dish D and the functional layer 1, the functional layer 1 is separated from the bottom surface of the dish D and the edge of the functional layer 1 floats, that is, the free end. Can be Thereby, in the measurement step S3 described above, the liquid W is removed from the periphery of the evaluation area R1 on the surface of the functional layer 1 without subjecting the bottom surface of the dish D to liquid repellency, and the liquid dripping time is measured. Can.

  In the example shown in FIG. 10A, by making the area of the support layer 3 smaller than the area of the functional layer 1, the gap G is formed between the peripheral portion of the functional layer 1 and the bottom of the dish D. It forms and it is in the state of the free end which floated the edge of the functional layer 1. Thereby, in the measurement step S3 described above, the liquid W can be more effectively removed from the periphery of the evaluation area R1 on the surface of the functional layer 1. In addition, if the thickness of the functional layer 1 is sufficiently thick, the edge of the functional layer 1 is floated even without providing the gap G between the peripheral portion of the functional layer 1 and the bottom surface of the dish D In the state, the liquid W can be effectively removed from the periphery of the evaluation area R1 on the surface of the functional layer 1.

  FIG. 10B is a schematic cross-sectional view showing a state in which the liquid W is selectively supplied to the evaluation region R1 of the surface of the functional layer 1 shown in FIG. 10A. In the wetting step S2 of the embodiment described above, a part of the surface of the functional layer 1 is used as the evaluation area R1, but in the wetting step S2 of this modification, the entire surface of the functional layer 1 is used as the evaluation area R1. The liquid W may be selectively supplied to the evaluation area R1 and the non-evaluation area R2 using a liquid supply nozzle or the like. This is a functional layer having no support layer 3 between it and the bottom surface of the dish D as in the rectangular functional layer 1 shown in FIG. The same applies to case 1. Thus, by selectively supplying the liquid W to the evaluation region R1 of the surface of the functional layer 1 in the wetting step S2, the liquid dripping time is stably measured without being affected by the shape or property of the outer peripheral portion. can do. Moreover, when the dish D is inclined, it is possible to prevent the lower end portion of the functional layer 1 from being immersed in the liquid W, and it is possible to set substantially the entire surface of the functional layer 1 as the evaluation region R1. .

  Alternatively, the liquid W may be supplied to a plurality of locations on the surface of the functional layer 1. By measuring the liquid immersion time at a plurality of points on the surface of the functional layer 1, it becomes possible to evaluate the unevenness, variation, etc. of the partial water holding power of the surface of the functional layer 1 in the dish D. .

  In addition, for example, the process of producing the functional layer 1 including a temperature responsive polymer such as PNIPAAm includes a washing process of performing high-pressure water washing of the functional layer 1. Therefore, the swelling step S1 of swelling the functional layer 1 with the liquid W, that is, water is performed in the washing step of the functional layer 1, and subsequently, water is selectively supplied to the evaluation region R1 of the surface of the functional layer 1 By performing the wetting step S2 of wetting the whole of the evaluation region R1 with water, it is possible to efficiently incorporate the evaluation method of the functional layer 1 of the present embodiment into the manufacturing process of the functional layer 1.

  FIG. 11 is a schematic perspective view showing a modified example of the evaluation method of the functional layer 1 described above. FIG. 12 is an explanatory view for explaining the measurement step S3 and the evaluation step S4 in the modification shown in FIG. The swelling step S1 shown in FIG. 4 may be performed by immersing the functional layer 1 formed on the substrate P in the liquid W, as shown in FIG. In this case, the substrate P is supported perpendicularly to the horizontal plane, and the substrate P is immersed in the liquid W in a state where the angle of the functional layer 1 to the horizontal plane is 90 °, and the functional layer 1 is treated with the liquid W Swell. Further, since the functional layer 1 is immersed in the liquid W, the wetting step S2 of wetting the whole of the evaluation region R1 of the surface of the functional layer 1 after the swelling step S1 with the liquid W is simultaneous with the end of the swelling step S1. finish. In the present modification, substantially the entire surface of the functional layer 1 can be used as the evaluation region R1.

  In the measurement step S3, the substrate P is pulled out of the liquid, and the functional layer 1 in a state in which the entire evaluation region R1 is wetted by the liquid W is pulled up onto the liquid surface. Thus, the wet region R11 of the evaluation region R1 on the surface of the functional layer 1 has a rectangular shape as shown in FIG. 12B from the state where it spreads over the entire evaluation region R1 shown in FIG. It is gradually reduced while being kept, and as shown in FIG. 12C, the length L along the horizontal direction approaches 0 and finally disappears. In this modification, in the measurement step S3, it is possible to measure the time until the length L of the wet region R11 along the horizontal direction decreases to a predetermined length as the liquidation time. In addition, in the measurement step S3, the time until the length L of the wet region R11 along the horizontal direction becomes zero may be measured as the liquidation time. This makes it possible to more easily measure the liquidation time as compared to the case where the area or the number of pixels of the wet region R11 is measured.

[Example]
Poly-N-isopropylacrylamide (PNIPAAm) is dissolved in isopropyl alcohol to a final concentration of 2% by weight and polyethylene glycol diacrylate (PEGDA) to a final concentration of 0.12 or 0.15% by weight to prepare a coating solution. did. The above coating solution was coated on a polystyrene film substrate with a No. 2 miyabar. After the coating liquid was dried, electron beam irradiation was performed to fix PNIPAAm on the surface of the substrate. The electron beam was irradiated at 110 kV · 120 kGy or 110 kV · 150 kGy to prepare samples 1 to 3 of a cell culture film having a functional layer containing PNIPAAm on a substrate. Thereafter, the prepared sample was washed and allowed to dry naturally.

  The sample after washing was cut out in a rectangular shape of 3.5 cm × 4.5 cm, and the sample was fixed to a metal plate with the edge of the functional layer floating through the support layer made of dimethylpolysiloxane (PDMS) . Then, as shown in FIG. 11, the sample is immersed in the water together with the metal plate for 30 minutes, and then the metal plate together with the sample is pulled up, and the functionality as shown in FIGS. 12 (a) to 12 (c). The liquid dripping time until the width of the wetting area which keeps drawing rectangular shape on the surface of the layer becomes 0 was measured with a stopwatch.

The washed sample was cut into a 38 mm 、 circle, and a 38 mm ハ ウ ジ ン グ housing was joined with an adhesive to prepare a cell culture dish (dish). Bovine vascular endothelial cells (obtained from JCRB cell bank) were prepared and seeded at 5.0 × 10 ⁴ cells / cm ² in a dish. At this time, the medium used was DMEM (manufactured by Sigma) containing 10% FBS. The culture is carried out in a CO ₂ incubator at 37 ° C., 5% CO ₂ conditions, and the incubator is taken out from the incubator on the 6th day of inoculation and placed in a room temperature environment to observe the state of cells and measure the time required for cell sheet recovery did. Table 1 shows the results.

  As shown in Table 1, the functional layer of sample 1 has a liquiding time of 2.51 seconds, and it takes 38 minutes to leave the incubator and peel off the cell sheet, and it takes some time to peel off the cells. It cost me a lot. The functional layer of sample 2 had a solutioning time of 3.78 seconds, and the time required for peeling of the cell sheet was 7 minutes, indicating that it was sufficiently practical. The functional layer of sample 3 had a liquid immersion time of 5.78 seconds, and the cells did not adhere sufficiently to the functional layer and did not become confluent. Therefore, for example, the quality of the functional layer can be evaluated by setting the lower limit of the liquid transfer time to 3.00 seconds and the upper limit of the liquid transfer time to 4.00 seconds or less.

1 Functional Layer L Length of Wetted Region along Horizontal Direction R1 Evaluation Region R11 Wetted Region S1 Swelling Step S2 Wetting Step S3 Measuring Step S4 Evaluation Step W Liquid

Claims (7)

A method of evaluating a functional layer comprising a functional polymer having cell adhesion, comprising:
A swelling step of swelling the functional layer with a liquid;
Wetting the whole evaluation area of the surface of the functional layer after the swelling step with a liquid;
Measuring step of arranging one end of the functional layer after the wetting step above the other end, and measuring the liquidation time until the wet area wetted with the liquid in the evaluation area is reduced to a predetermined size When,
An evaluation step of evaluating the surface property based on the liquid transfer time;
The evaluation method of the functional layer characterized by having. In the wetting step, immersing the evaluation area in a liquid;
The method for evaluating a functional layer according to claim 1, wherein in the measurement step, the liquid is removed from the periphery of the evaluation area, and the liquid draw time is measured.   The method for evaluating a functional layer according to claim 1, wherein in the wetting step, a liquid is selectively supplied to the evaluation area.   The said evaluation area | region is a rectangle, The evaluation method of the functional layer of Claim 2 or Claim 3 characterized by the above-mentioned.   5. The functional layer according to claim 4, wherein the time taken for the length of the wet area along the horizontal direction to decrease to a predetermined length is measured as the liquidation time in the measurement step. Evaluation method.   The method for evaluating a functional layer according to claim 5, wherein, in the measurement step, a time until the length of the wet area becomes 0 is measured as the liquidation time.   The said functional polymer is a temperature-responsive polymer, The evaluation method of the functional layer as described in any one of the Claims 1-6 characterized by the above-mentioned.

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