MEASURING APPARATUS, MEASURING APPARATUS OPERATION PROGRAM, AND MEASURING METHOD

Abstract

The measuring device comprises: a mounting section to which a flow path device including a first flow path, a second flow path having a communicating portion communicating with at least a portion of the first flow path, and a cell sheet in which a plurality of cultured cells are arranged in a bonded state, the cell sheet separating the first flow path and the second flow path at the communicating portion, is detachably mounted; and a detection section that optically distinguishes and detects the leakage status of each of the plurality of tracers that leak from the first flow path to the second flow path through defects that occur in the cell sheet when an evaluation liquid for evaluating the barrier properties of the cell sheet, the evaluation liquid containing a mixture of multiple types of tracers with different sizes, is sent to the first flow path.

Description

[Technical field]

[0001] The techniques disclosed herein relate to a measurement device, an operating program for the measurement device, and a measurement method. Regarding. [Background technology]

[0002] A flow channel device is known that includes a fine flow channel capable of transporting liquid and a cell sheet in which a plurality of cultured cells are arranged in a bonded state (see JP2018-522586A). Such a flow channel device is used, for example, to evaluate the barrier property of the cell sheet.

[0003] An evaluation liquid containing a tracer is used to evaluate the barrier property of a cell sheet. If a defect occurs in the cell sheet, the tracer contained in the evaluation liquid leaks through the defect. In this case, the barrier property of the cell sheet is evaluated as being decreased compared to a state without defects. In this way, the barrier property of the cell sheet is evaluated by detecting defects in the cell sheet.

[0004] As an example, the barrier properties of a cell sheet are evaluated to examine the efficacy or side effects of a drug. That is, the efficacy or side effects of a drug are examined by detecting defects on the cell sheet when the drug is brought into contact with the cell sheet. As another example, the barrier properties of a cell sheet are evaluated so that a manufacturer of a flow channel device can confirm that no defects have occurred in the cell sheet in the flow channel device during inspection before shipping the product. The barrier properties of a cell sheet are also evaluated during the development of the flow channel device 11 to inspect whether the cell sheet meets specifications, etc.

[0005] In JP2018-522586A, the method for evaluating the barrier properties of a cell sheet is as follows. First, the flow path device described in JP2018-522586A has two flow paths: a first flow path (perfusion channel) and a second flow path (gel channel) (paragraph 0155, Figures 1A and 1B). The first flow path and the second flow path are partially connected, and the connected portion is partitioned by a cell sheet.

[0006] In the method described in JP2018-522586A, an evaluation liquid for evaluating the barrier property of a cell sheet is injected into the first flow path of such a flow path device. A fluorescent probe is used as an example of a tracer in the evaluation liquid, and culture medium is used as a solvent for the fluorescent probe. If the cell sheet is free of defects and has perfect barrier properties, the fluorescent probe will not leak into the second flow path. On the other hand, if the cell sheet is defective, the fluorescent probe will leak into the second flow path. This leakage state is observed using a fluorescent microscope (paragraph 0155, Figures 1 to 3). JP2018-522586A also describes that multiple types of tracers (fluorescent probes) of different sizes may be used as the tracers (fluorescent probes) (paragraph 0155, Figure 16). Summary of the Invention [Problem to be solved by the invention]

[0007] There are various types of defects in cell sheets, such as defects caused by some cells, such as dead cells, dropping off from the cell sheet, defects caused by destruction of tight junctions between adjacent cells, and defects caused by partial detachment of the cell sheet. The size of these defects also varies. For example, defects caused by destruction of tight junctions are on the order of 1 nm (nanometer) to tens of nm, whereas defects caused by dropping off cells and partial detachment of the cell sheet are on the order of 1 μm (micrometer) to tens of μm.

[0008] When evaluating the barrier property of a cell sheet, there is a demand for not only simply detecting the presence or absence of defects, but also detecting more detailed defect states, such as the approximate size of defects occurring in the cell sheet and the approximate proportion of defects of different sizes present, because detecting such more detailed defect states allows for a more detailed evaluation of the barrier property of the cell sheet compared to simply detecting the presence or absence of defects.

[0009] As described above, JP2018-522586A describes that multiple types of tracers with different sizes may be used as tracers (fluorescent probes). However, JP2018-522586A does not clearly describe the purpose and method of using multiple types of tracers with different sizes.

[0010] It is conceivable that multiple types of defects of different sizes can be detected by using multiple types of tracers with different sizes as follows: First, an evaluation liquid containing a relatively large (large) size tracer is used to detect large size defects. The large size tracer penetrates only large size defects. Therefore, leakage of the large size tracer indicates the presence of a large size defect.

[0011] In addition, when there are no large-sized defects in the cell sheet, small-sized defects are detected using an evaluation liquid containing a small-sized tracer having a relatively small size. When there are no large-sized defects in the cell sheet, small-sized defects can be detected by gradually decreasing the size of the tracer.

[0012] In this way, by using multiple types of evaluation liquids each containing tracers of different sizes and switching them sequentially through the flow path device while detecting defects, it is possible to detect the size of defects that have occurred in the cell sheet.

[0013] However, this method of using a plurality of evaluation liquids containing tracers of different sizes has the problem that it requires replacement of the evaluation liquid, which is time-consuming and labor-intensive.

[0014] The technology disclosed herein aims to provide a measuring device, an operating program for the measuring device, and a measuring method to be used in evaluating the barrier properties of cell sheets that are capable of detecting multiple types of defects of different sizes that occur in cell sheets without requiring much time and effort compared to conventional techniques. [Means for solving the problem]

[0015] In order to achieve the above-mentioned object, the measuring device according to the disclosed technology includes a first flow path, a second flow path having a communicating portion communicating with at least a portion of the first flow path, and a cell sheet in which a plurality of cultured cells are arranged in a bound state, the cell sheet separating the first flow path and the second flow path at the communicating portion; an attachment portion to which a flow path device is detachably attached; and a detection portion that optically distinguishes and detects the leakage status of each of the plurality of tracers leaking from the first flow path to the second flow path through defects that occur in the cell sheet when an evaluation liquid for evaluating the barrier property of the cell sheet, the evaluation liquid being a mixture of multiple types of tracers with different sizes, is sent to the first flow path.

[0016] It is preferable that each of the multiple types of tracers is a fluorescent tracer that emits fluorescence in a different wavelength range, and the detection section detects the multiple types of fluorescence emitted by each of the multiple types of tracers.

[0017] It is preferable that the detection unit has an excitation light source that irradiates excitation light that excites each of the multiple types of tracers to produce fluorescence, an optical filter that separates the multiple types of fluorescence, and a light receiving unit that receives the fluorescence separated by the optical filter.

[0018] It is preferable that there is one excitation light source, and a light receiving unit is provided for each of the multiple types of fluorescence.

[0019] It is preferable that the excitation light source irradiates excitation light at a position downstream in the flow direction of the tracer leaking from the communicating portion in the flow path device attached to the mounting portion, where the excitation light is incident only on the second flow path out of the first flow path and the second flow path.

[0020] The leakage condition preferably includes a change in leakage amount over time.

[0021] It is preferable that the device further comprises a data processing section for performing data processing on the detection signal outputted by the detection section.

[0022] It is preferable that the data processing unit has a signal processing unit that performs at least one of a correction process for correcting the dark current of the detection unit, a removal process for removing overlapping portions of the wavelength ranges of multiple types of light from the detection signals corresponding to each type of light, and a conversion process for converting the detection signal output as a voltage signal into light intensity.

[0023] The data processing unit preferably has a concentration deriving unit that derives the respective concentrations of the plurality of types of tracers leaked from the first flow path to the second flow path based on the detection signal output by the detection unit.

[0024] The data processing unit preferably includes a defect state deriving unit that derives the state of a defect occurring in the cell sheet based on the respective concentrations of the multiple types of tracers.

[0025] It is preferable that the device further includes an output control unit that outputs the leakage state detected by the detection unit to a display in real time.

[0026] The cell sheet is cultured on a support, and the support is preferably a permeable membrane having pores with a diameter of 1 μm or more.

[0027] The particle size of each of the multiple types of tracers is preferably within the range of 1 nm to 100 μm.

[0028] The multiple types of tracers preferably include tracers with particle sizes on the order of 1 nm to several hundreds of nm, and tracers with particle sizes on the order of 1 μm to several tens of μm.

[0029] It is preferable that a supply mechanism is provided that supplies the evaluation liquid to the first flow path.

[0030] An operating program of a measuring device according to the disclosed technique is an operating program for evaluating the barrier property of a cell sheet using a flow path device including a first flow path, a second flow path having a communicating portion communicating with at least a portion of the first flow path, and a cell sheet on which a plurality of cultured cells are arranged in a bound state, the cell sheet separating the first flow path and the second flow path at the communicating portion, the operating program causing a computer to function as an acquisition unit that acquires a detection signal from a detection unit that optically distinguishes and detects the leakage status of each of a plurality of tracers leaking from the first flow path to the second flow path through defects occurring in the cell sheet when an evaluation liquid for evaluating the barrier property of the cell sheet, the evaluation liquid being a mixture of a plurality of types of tracers with different sizes, is sent to the first flow path, and a processing unit that executes an output control process to output the leakage status based on the acquired detection signal.

[0031] The measurement method according to the disclosed technology uses a flow path device including a first flow path, a second flow path having a communicating portion communicating with at least a portion of the first flow path, and a cell sheet in which a plurality of cultured cells are arranged in a bound state, the cell sheet separating the first flow path from the second flow path at the communicating portion, and includes a liquid delivery step of delivering an evaluation liquid for evaluating the barrier property of the cell sheet, the evaluation liquid being a mixture of multiple types of tracers of different sizes, to the first flow path, and a detection step of optically distinguishing and detecting the leakage status of each of the multiple tracers leaking from the first flow path to the second flow path through defects occurring in the cell sheet. Effect of the Invention

[0032] According to the technology disclosed herein, it is possible to provide a measuring device, an operating program for the measuring device, and a measuring method used for evaluating the barrier properties of cell sheets that are capable of detecting multiple types of defects of different sizes that occur in cell sheets without requiring much time and effort compared to conventional methods. [Brief description of the drawings]

[0033] [Figure 1] FIG. 1 is an overall configuration diagram of an apparatus for evaluating the barrier property of a cell sheet. [Diagram 2] FIG. 2 is an exploded perspective view of the flow path device. [Diagram 3] FIG. 2 is a cross-sectional view of a flow path device. [Figure 4] FIG. 2 is a longitudinal sectional view of a flow path device. [Diagram 5] FIG. 4 is a plan view showing an overlap of a first flow path and a second flow path. [Figure 6] FIG. [Figure 7] FIG. 2 is an explanatory diagram of the diameter of a hole in a permeable membrane and the particle size of a tracer. [Figure 8] FIG. 1 is an explanatory diagram of a cell sheet formed on a permeable membrane. [Figure 9] FIG. 1 is an explanatory diagram of defects occurring in a cell sheet. [Figure 10] 10A is an explanatory diagram showing a state in which the evaluation liquid is sent to the first flow path, and FIG 10B is an explanatory diagram showing the flow of the evaluation liquid when there is no defect in the cell sheet, and FIG 10B is an explanatory diagram showing the flow of the evaluation liquid when there is a defect in the cell sheet. [Figure 11] FIG. [Figure 12] 12A and 12B are explanatory diagrams showing the configuration of optical components of a detection unit, with Fig. 12A being a plan view and Fig. 12B being a side view. [Figure 13] Fig. 13 is an explanatory diagram showing filter characteristics, Fig. 13A shows the characteristics of the first filter, Fig. 13B shows the characteristics of the second filter, and Fig. 13C shows the characteristics of the third filter. [Figure 14]14A shows the wavelength of the fluorescence emitted by each tracer, and FIG 14B shows the wavelength of the fluorescence emitted by the second tracer, and FIG 14C shows the wavelength of the fluorescence emitted by the third tracer. [Figure 15] FIG. 2 is an explanatory diagram showing the relationship between the wavelength of fluorescence emitted by each tracer and the characteristics of each filter. [Figure 16] 5 is an explanatory diagram of detection signals output from each light receiving unit. FIG. [Figure 17] FIG. 2 shows a schematic configuration diagram of a computer functioning as a control device. [Figure 18] FIG. 2 is an explanatory diagram of functional blocks of a control device. [Figure 19] FIG. 2 is an explanatory diagram of signal processing. [Figure 20] FIG. 11 is an explanatory diagram of a concentration derivation process. [Figure 21] FIG. 13 is an explanatory diagram showing the correspondence between the size of defects that each tracer can penetrate. [Figure 22] 1 is a graph showing the relationship between the total area of ​​defects on a cell sheet and the index (concentration) of the leakage amount of each tracer. [Diagram 23] FIG. 13 is an explanatory diagram of an outline of a defect state derivation process. [Figure 24] FIG. 4 is a diagram illustrating details of a defect state derivation process. [Diagram 25] FIG. 13 is an explanatory diagram of a measurement result display screen. [Figure 26] 13 is a flowchart showing a procedure of a measurement process. [Figure 27] 10 is a flowchart showing the procedure of a measurement process in a comparative example. [Figure 28] FIG. 1 is an explanatory diagram showing an example of forming cell sheets on both sides of a permeable membrane. [Figure 29] FIG. 29 is an explanatory diagram showing a state in which a defect has occurred in the cell sheet in the example of FIG. 28. [Diagram 30] 1A and 1B are explanatory diagrams showing an example of a detection section having a rotating filter unit. [Diagram 31] 31 is an explanatory diagram showing the characteristics of each filter of the rotary filter unit of FIG. 30. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] As shown as an example in FIG. 1, the measuring device 10 is used to evaluate the barrier properties of a cell sheet 20 (see also FIGS. 8 and 9) formed in a flow path device 11. The cell sheet 20 is formed by seeding and culturing in the flow path device 11. The flow path device 11 on which the cell sheet 20 is formed is set in the measuring device 10 in a freely attachable and detachable manner. Then, in order to evaluate the barrier properties of the cell sheet 20 in the set flow path device 11, the measuring device 10 measures data that serves as an index for the evaluation. Examples of data that serves as an index for the evaluation of the barrier properties include the presence or absence of defects occurring in the cell sheet 20, as well as the state of the defects, including the proportion of defects of different sizes.

[0035] As an example, the measuring device 10 is used by a manufacturer of the flow channel device 11 to evaluate the barrier properties of the cell sheet 20 formed in the flow channel device 11 when the flow channel device 11 is developed.

[0036] The measurement apparatus 10 includes a detection apparatus 12 and a control apparatus 13. The detection apparatus 12 includes a mounting portion 16 and a detection portion 17. The mounting portion 16 has the flow path device 11 detachably mounted thereon.

[0037] As described later in detail, when an evaluation liquid 21 for evaluating the barrier property of the cell sheet 20 is delivered to the flow path device 11, the detection unit 17 optically detects the leakage state of the fluorescent tracer TR (see Figs. 8 and 9) leaking through defects occurring in the cell sheet 20. The evaluation liquid 21 is a fluorescent tracer TR contained in a solvent such as a buffer. A tracer is a substance used to track the flow of a fluid such as a liquid, or a specific substance. The fluorescent tracer TR is a tracer that emits fluorescence, and specifically, a fluorescent substance itself or a substance labeled with a fluorescent substance is used as the fluorescent tracer TR. The detection unit 17 detects the leakage state by detecting the fluorescence FL emitted by the leaked fluorescent tracer TR.

[0038] The control device 13 controls the operation of the detection unit 17. Furthermore, the control device 13 acquires the detection signal output by the detection unit 17 and performs data processing on the acquired detection signal. As a result, various measurement results based on the detection signal are output. The control device 13 is composed of, for example, a computer.

[0039] The measurement device 10 further includes a supply mechanism 22 for supplying a liquid such as the evaluation liquid 21 to the flow path device 11 mounted on the mounting portion 16. The supply mechanism 22 includes a supply tube 22A, a discharge tube 22B, and a pump 22C. The supply tube 22A and the discharge tube 22B are attached to the flow path device 11 mounted on the mounting portion 16.

[0040] The flow path device 11 has two flow paths: a first flow path 25 and a second flow path 26. Two supply tubes 22A and two discharge tubes 22B are provided, and are attached to the first flow path 25 and the second flow path 26, respectively.

[0041] A pump 22C is connected to the supply tube 22A attached to the first flow path 25. The pump 22C is, for example, a syringe pump. The pump 22C supplies the evaluation liquid 21 to the first flow path 25 through the supply tube 22A. As described below, the first flow path 25 and the second flow path 26 are partially connected to each other, and the cell sheet 20 separates the first flow path 25 and the second flow path 26 at this connected portion.

[0042] When no defect occurs in the cell sheet 20, the communicating portion between the first flow path 25 and the second flow path 26 is sealed by the cell sheet 20. Therefore, when no defect occurs in the cell sheet 20, when the evaluation liquid 21 is sent to the first flow path 25, the fluorescent tracer TR contained in the evaluation liquid 21 does not leak into the second flow path 26. The evaluation liquid 21 supplied to the first flow path 25 passes through the first flow path 25 and is then discharged from the discharge tube 22B.

[0043] In contrast, if a defect occurs in the cell sheet 20, the fluorescent tracer TR in the evaluation liquid 21 supplied to the first flow path 25 leaks through the defect into the second flow path 26. If a defect occurs in the cell sheet 20, a leakage liquid 21A, which is a part of the evaluation liquid 21, flows into the second flow path 26. The leakage liquid 21A contains the fluorescent tracer TR. After passing through the second flow path 26, the leakage liquid 21A is discharged from the discharge tube 22B.

[0044] The supply tube 22A attached to the second flow path 26 is used to supply a buffer 30 (see FIG. 3, etc.) to the second flow path 26. For example, TBS (Tris Buffered Saline) and HBSS (Hanks' Balanced Salt Solution) are used as the buffer 30. The buffer 30 is the same as the buffer used as the solvent for the evaluation liquid 21 described above.

[0045] As shown in Fig. 2, the flow path device 11 includes a first flow path member 31, a second flow path member 32, a holding plate 33, a reinforcing plate 34, and a permeable membrane 36. The first flow path member 31 has a first flow path 25 formed on its lower surface, which is the lower side in Fig. 2. The second flow path member 32 has a second flow path 26 formed on its upper surface, which is the upper side in Fig. 2. The first flow path member 31 and the second flow path member 32 are assembled such that the first flow path 25 and the second flow path 26 are disposed opposite each other.

[0046] The first flow path member 31 is made of a transparent resin material having elasticity, such as PDMS (polydimethylsiloxane), for example, and the second flow path member 32 is made of a transparent resin material having rigidity, such as COP (cycloolefin polymer), for example.

[0047] The first flow path member 31 is formed with an injection path 37 and a discharge path 38 connected to the first flow path 25. The first flow path member 31 is also formed with an injection path 37 and a discharge path 38 connected to the second flow path 26. That is, two injection paths 37 and two discharge paths 38 are provided corresponding to each of the first flow path 25 and the second flow path 26. The injection paths 37 and the discharge paths 38 are through paths that penetrate the first flow path member 31 in the up-down direction, i.e., in the thickness direction.

[0048] The holding plate 33 is disposed on the upper surface of the first flow path member 31. An inlet 33A and an outlet 33B are formed in the holding plate 33 at positions corresponding to the injection path 37 and the discharge path 38, respectively, of the first flow path member 31. A supply tube 22A and a discharge tube 22B are connected to the inlet 33A and the discharge port 33B, respectively.

[0049] The reinforcing plate 34 reinforces the rigidity of the flow path device 11. The reinforcing plate 34 is disposed between the first flow path member 31 and the second flow path member 32. In the reinforcing plate 34, a slit 34A is formed in a position corresponding to the first flow path 25 and the second flow path 26, the slit 34A being elongated along the direction in which the evaluation liquid 21 flows in the first flow path 25. The slit 34A is disposed in a communication portion between the first flow path 25 and the second flow path 26. In addition, an opening 34B is formed in the reinforcing plate 34. The opening 34B is disposed in a position corresponding to the connection portion 26B (see FIG. 5) formed at both ends of the second flow path 26 of the second flow path member 32. In addition, the opening 34B is connected to an injection path 37 and a discharge path 38 formed for the second flow path 26 in the first flow path member 31.

[0050] The permeable membrane 36 is disposed between the first flow path member 31 and the reinforcing plate 34, and is disposed at a position corresponding to the slit 34A. The permeable membrane 36 functions as a support for the cell sheet 20, and the cell sheet 20 is seeded and cultured on the permeable membrane 36. In addition, the permeable membrane 36 allows the fluorescent tracer TR contained in the evaluation liquid 21 to permeate (see FIGS. 8 and 9, etc.).

[0051] The holding plate 33, the first flow path member 31, the reinforcing plate 34, and the second flow path member 32 each have a screw hole 42 for inserting a bolt 41 therethrough. The holding plate 33, the first flow path member 31, the reinforcing plate 34, and the second flow path member 32 are stacked in this order to assemble the flow path device 11. Then, in the assembled state, the flow path device 11 is fastened with the bolt 41 and the nut 43.

[0052] 3 to 5, in an assembled state of the flow channel device 11, the straight line portions 25A and 26A (see FIG. 5) of the first flow channel 25 and the second flow channel 26 face each other. FIG. 3 is a transverse cross-sectional view of the flow channel device 11, FIG. 4 is a longitudinal cross-sectional view of the flow channel device 11, and FIG. 5 is a plan view showing the overlap of the first flow channel 25 and the second flow channel 26.

[0053] As shown in Fig. 4, the width W1 of the straight portion 25A of the first flow path 25 is, for example, about 200 µm, and the width W2 of the straight portion 26A of the second flow path 26 is, for example, about 400 µm. As shown in Fig. 5, the straight portion 25A and the straight portion 26A partially overlap. A slit 34A is disposed at this position, and a portion corresponding to the slit 34A becomes a communicating portion between the first flow path 25 and the second flow path 26. In addition, since the first flow path 25 and the second flow path 26 have a flow path with a width on the order of microns, the flow path device 11 is also called a micro flow path device or a microfluidic device.

[0054] The cell sheet 20 is formed on the surface of the permeable membrane 36 on the first flow path 25 side. The cell sheet 20 is formed at the communicating portion between the straight portion 25A and the straight portion 26A, and separates the first flow path 25 and the second flow path 26 at the straight portion 25A and the straight portion 26A.

[0055] In Figure 3, as indicated by the dashed double-dotted arrow, if a defect occurs in the cell sheet 20, a portion of the evaluation liquid 21 in the first flow path 25 leaks through the permeable membrane 36 and the slit 34A into the second flow path 26 as leakage liquid 21A containing the fluorescent tracer TR (Figures 8 and 9, etc.).

[0056] 5, at the upstream end and downstream end of straight portion 25A of first flow path 25, connection portions 25B are formed which are connected to injection path 37 and discharge path 38. Also, at the upstream end and downstream end of straight portion 26A of second flow path 26, connection portions 26B are formed which are connected to injection path 37 and discharge path 38.

[0057] As shown in FIG. 6, the permeable membrane 36 is a porous membrane in which a large number of holes 36A are formed. The permeable membrane 36 is made of a hydrophobic polymer that is soluble in a hydrophobic organic solvent, for example. Examples of the hydrophobic polymer include polymers such as polystyrene, polyacrylate, polymethacrylate, polyacrylamide, polycarbonate, and polypropylene. These polymers may be used alone or in combination of two or more. In addition, the material of the permeable membrane 36 is not limited to a hydrophobic polymer, and various materials can be selected from the viewpoint of cell adhesiveness, etc.

[0058] As shown in FIG. 7, the diameter D of the holes 36A of the permeable membrane 36 is a size that allows the fluorescent tracer TR contained in the evaluation liquid 21 to pass through. In this example, the diameter D is 1 μm or more. The permeable membrane 36 in this example is a honeycomb film in which a plurality of holes 36A are arranged in a honeycomb pattern. Of course, a film in which a plurality of holes 36A are arranged in a square pattern may also be used. The diameter D of the holes 36A is, for example, about 5 μm to about 20 μm, and is about 20 μm in this example. The aperture ratio is, for example, about 20% to about 50%, and is about 50% in this example. The aperture ratio may be 50% or more.

[0059] The evaluation liquid 21 contains multiple types of fluorescent tracers TR that differ in size, specifically, particle size. In this example, three types of fluorescent tracers TR1, TR2, and TR3 that differ in size are contained. In this specification, when it is necessary to distinguish between the three types of fluorescent tracers TR1 to TR3 that differ in size, a symbol with a number added to the symbol TR, such as TR1 to TR3, is used, but when it is not necessary to distinguish between the three types of fluorescent tracers TR that differ in size, the number is omitted and the symbol TR is simply used.

[0060] Each of the fluorescent tracers TR1 to TR3 emits fluorescence when irradiated with excitation light. The wavelength range of the fluorescence differs for each of the three fluorescent tracers TR1 to TR3, which have different sizes, as described below.

[0061] The size (particle size) of each of the three types of fluorescent tracers TR1 to TR3 is, for example, in the range of 1 nm to 100 μm. In this example, since the diameter D of the hole 36A is 20 μm, the fluorescent tracer TR used is also less than 20 μm.

[0062] Specifically, the fluorescent tracer TR1 has a size of several nm to several tens of nm, and is the smallest of the three types. The fluorescent tracer TR3 has a size of about 6 μm to 8 μm, and is the largest of the three types. The fluorescent tracer TR2 has a size of about 200 nm to 300 nm, which is an intermediate size between the fluorescent tracer TR1 and the fluorescent tracer TR2. Thus, in this example, the fluorescent tracers TR1 to TR3 of different sizes include the fluorescent tracer TR1 having a size (particle size) on the order of 1 nm to several tens of nm, the fluorescent tracer TR2 having a size (particle size) on the order of several hundreds of nm, and the fluorescent tracer TR3 having a size (particle size) on the order of 1 μm to 10 μm.

[0063] As the fluorescent tracer TR, for example, fluorescent molecules with a size (particle size) of 1 nm, dextran with various sizes (particle size) from several nm to 100 nm, and fluorescent beads with various sizes (particle size) from 10 nm to 100 μm can be used. As the dextran, for example, fluorescein, alexa, and tetramethylrhodamine can be used. In addition, red blood cells labeled with a fluorescent dye can be used as the fluorescent tracer TR. The size (particle size) of the red blood cells is about 6 μm to about 8 μm. The combination of sizes of the fluorescent tracer TR contained in the evaluation liquid 21 is appropriately selected from these fluorescent tracers.

[0064] 8, the cell sheet 20 is formed by arranging a plurality of cells 20A in a bound state on a permeable membrane 36. The cell sheet 20 is formed, for example, as follows. First, during manufacture of the flow path device 11, a culture medium and the cells 20A are supplied to the permeable membrane 36 through the first flow path 25. As a result, the cells are cultured on the permeable membrane 36, and a plurality of cells 20A are arranged on the permeable membrane 36 in a bound state. The cultured cell sheet 20 is adsorbed to the permeable membrane 36.

[0065] The first flow channel 25 of the flow channel device 11 is simulated as, for example, a blood vessel. In this case, for example, endothelial cells that constitute the inner wall of a blood vessel are formed as the cell sheet 20. In the endothelial cells, tight junctions 20B are formed between the cells 20A. Tight junctions are proteins that are expressed when the cells 20A adhere to each other. In a cell sheet having tight junctions like the cell sheet 20, the cells 20A are formed without any gaps between them. The cell sheet 20 having such tight junctions 20B has high barrier properties when no defects are generated.

[0066] 9, there are various types of defects DF in the cell sheet 20, such as defect DF1 caused by destruction of tight junctions 20B between adjacent cells 20A, defect DF2 caused by some cells 20A, such as dead cells, dropping off from the cell sheet 20, and defect DF3 caused by partial detachment of the cell sheet 20. The defect DF3 caused by partial detachment of the cell sheet 20 is caused, for example, by a portion of the cell 20A that is incompletely adsorbed to the permeable membrane 36 coming into contact with the liquid flowing through the first flow path 25.

[0067] When the cell sheet 20 is provided in a fine first flow channel 25, as in the flow channel device 11, the fluid resistance of the liquid flowing in the first flow channel 25 is large. Therefore, when the liquid comes into contact with the cell sheet 20, the cell sheet 20 is likely to peel off.

[0068] These defects DF1 to DF23 vary in size. For example, the size of defect DF1 caused by the destruction of tight junctions 20B is on the order of several nm to several tens of nm, whereas the size of defect DF2 caused by the detachment of cells 20A is on the order of several hundreds of nm, and defect DF3 caused by partial detachment of cell sheet 20 is on the order of 1 μm to 100 μm. Of defects DF1 to DF3, defect DF1 is the smallest in size (small size). Defect DF3 is the largest in size (large size). Defect DF2 is of medium size. Here, as with the fluorescent tracer TR, when defects DF1 to DF3 are collectively referred to without distinguishing between each of the defects DF1 to DF3, they are simply referred to as defect DF without adding a number.

[0069] The small-sized defect DF1 is of a size that allows the small-sized fluorescent tracer TR1 to pass therethrough, the medium-sized defect DF2 is of a size that allows the medium-sized fluorescent tracer TR2 to pass therethrough, and the large-sized defect DF3 is of a size that allows the large-sized fluorescent tracer TR3 to pass therethrough.

[0070] As shown in Fig. 10, when evaluation liquid 21 containing three different sizes of fluorescent tracers TR1 to TR3 is flowed through first flow path 25, if no defects DF are formed in cell sheet 20 as shown in Fig. 10A, leakage of leakage liquid 21A containing fluorescent tracers TR1 to TR3 does not occur into second flow path 26. In contrast, as shown in Fig. 10B, if a defect DF is formed in cell sheet 20, leakage liquid 21A containing at least the small size of fluorescent tracer TR1 among the three sizes of fluorescent tracers TR1 to TR3 leaks out depending on the size of the defect DF.

[0071] Reference numeral 46 denotes a detection position where the detection unit 17 detects the leakage state of the leaking liquid 21A containing the fluorescent tracer TR. The detection position 46 is downstream in the flow direction of the fluorescent tracer TR leaking from a position corresponding to the slit 34A, which is a communicating portion, and is set at a position where the excitation light is incident only on the second flow path 26 out of the first flow path 25 and the second flow path 26.

[0072] As described later, the detection unit 17 has an excitation light source 56 (see FIG. 11) that irradiates excitation light. That is, the excitation light source 56 irradiates excitation light at a position that is downstream in the flow direction of the evaluation liquid 21 leaking from the slit 34A corresponding to the communicating portion and the defect DF of the cell sheet 20 in the flow path device 11 mounted on the mounting unit 16, and is incident only on the second flow path 26 out of the first flow path 25 and the second flow path 26.

[0073] 11, the detection unit 17 includes a first detector 51, a second detector 52, a third detector 53, a first lens barrel 54, a second lens barrel 55, an excitation light source 56, a shutter unit 57, and an objective lens 58. The excitation light source 56 is a laser light source such as a semiconductor laser, and emits, as excitation light, excitation light LB (see FIG. 12) having a central wavelength of, for example, 476 nm.

[0074] The shutter section 57 is disposed in front of the irradiation direction of the excitation light source 56. The shutter section 57 moves the shutter 57A, which blocks the excitation light, between an open position and a closed position. As an example, the shutter 57A is an electrically operated shutter. When the shutter 57A is in the open position, the excitation light LB is incident on the first barrel section 54. On the other hand, when the shutter 57A is in the closed position, the excitation light LB is blocked by the shutter 57A, and the incidence of the excitation light LB is prohibited.

[0075] Fig. 12 mainly shows the configuration of optical components inside the first barrel 54. Fig. 12A is a plan view of the inside of the first barrel 54. Fig. 12B is a side view of the inside of the first barrel 54. Optical components such as dichroic mirrors DM0 to DM3 and a condenser lens 59 are provided inside the first barrel 54. The dichroic mirrors DM0 to DM3 are optical filters that reflect or transmit light in a specific wavelength range.

[0076] In Fig. 13, as shown in Fig. 13A, the dichroic mirror DM1 has a transmission characteristic of reflecting light with a wavelength of less than 505 nm and transmitting light with a wavelength of 505 nm or more. As shown in Fig. 13B, the dichroic mirror DM2 has a transmission characteristic of reflecting light with a wavelength of less than 567 nm and transmitting light with a wavelength of 567 nm or more. As shown in Fig. 13C, the dichroic mirror DM3 has a transmission characteristic of reflecting light with a wavelength of less than 650 nm and transmitting light with a wavelength of 650 nm or more.

[0077] Although not shown in FIG. 13, the dichroic mirror DM0 has a transmission characteristic of reflecting light with a wavelength less than 900 nm and transmitting light with a wavelength of 900 nm or more (see FIG. 15). The dichroic mirror DM0 does not require wavelength selection for the fluorescence FL1 to FL3 and LB, so a specularly reflective mirror may be used. However, if a reflective mirror is used, infrared rays in the wavelength range of 900 nm or more will also be reflected. If infrared rays enter the first detector 51 to the third detector 53, this is not preferable as it will cause a rise in temperature. For this reason, the dichroic mirror DM0 is used instead of a reflective mirror.

[0078] 12A, the excitation light LB that passes through shutter 57A and enters first barrel 54 has a central wavelength of 476 nm and is therefore reflected by dichroic mirror DM1. Dichroic mirror DM1 reflects the excitation light LB toward dichroic mirror DM0. Dichroic mirror DM0 reflects the incident excitation light LB toward objective lens 58 disposed at the downstream end of second barrel 55.

[0079] The objective lens 58 causes the excitation light LB to be incident on the detection position 46 (see FIG. 10) of the flow path device 11 mounted on the mounting portion 16. The objective lens 58 focuses the excitation light LB so that the spot diameter of the excitation light LB fits within the width W2 (400 nm in this example) of the second flow path 26.

[0080] When there is leakage of the fluorescent tracers TR1 to TR3 into the second flow path 26, the fluorescent tracers TR1 to TR3 emit fluorescence FL1 to FL3 in the respective wavelength ranges by irradiating the detection position 46 with excitation light LB. In this example, there is one excitation light source 56, and the fluorescent tracers TR1 to TR3 emit fluorescence FL1 to FL3 in different wavelength ranges, respectively, in response to one type of excitation light LB.

[0081] FIG. 14 shows an example of the wavelength range of the fluorescence FL1 to FL3 of each of the fluorescent tracers TR1 to TR3. As shown in FIG. 14A, the fluorescence FL1 emitted by the small-sized fluorescent tracer TR1 has a wavelength range of about 500 nm to about 700 nm. As shown in FIG. 14B, the fluorescence FL2 emitted by the medium-sized fluorescent tracer TR2 has a wavelength range of about 570 nm to about 640 nm. As shown in FIG. 14C, the fluorescence FL3 emitted by the large-sized fluorescent tracer TR3 has a wavelength range of about 660 nm to about 850 nm.

[0082] 15, the dichroic mirror DM1 transmits all of these three types of fluorescence FL1 to FL3. The dichroic mirror DM2 transmits only the fluorescence FL2 and FL3 out of the three types of fluorescence FL1 to FL3. The dichroic mirror DM3 transmits only the fluorescence FL3.

[0083] 12, the fluorescence FL1 to FL3 emitted from the fluorescent tracers TR1 to TR3 is incident on the dichroic mirror DM0. In addition to the fluorescence FL1 to FL3, the excitation light LB reflected by the flow path device 11 and re-entering the objective lens 58 is also re-entered on the dichroic mirror DM0.

[0084] The fluorescence FL1 to FL3 and the excitation light LB are reflected by the dichroic mirror DM0 and enter the dichroic mirror DM1. The dichroic mirror DM1 reflects the excitation light LB and transmits the fluorescence FL1 to FL3. In this example, the dichroic mirror DM1 is configured with two mirrors, and the fluorescence FL1 to FL3 that transmits through the first dichroic mirror DM1 also transmits through the second dichroic mirror DM1. The reason that the dichroic mirror DM1 is configured with two mirrors is that a laser beam with high optical intensity is used as the excitation light LB, and this laser beam must be reliably cut off.

[0085] A dichroic mirror DM2 is disposed after the dichroic mirror DM1. The dichroic mirror DM2 transmits the fluorescence FL2 and FL3, while reflecting the fluorescence FL1 toward the first detector 51. The reflected fluorescence FL1 is collected by a collecting lens 59. The collected light beam is incident on the light receiving section 51A in the first detector 51. In this way, the fluorescence FL1 is separated from the fluorescence FL2 and FL3 by the dichroic mirror DM2.

[0086] The fluorescence FL2 and FL3 that pass through the dichroic mirror DM2 are incident on the dichroic mirror DM3. The dichroic mirror DM3 transmits the fluorescence FL3 while reflecting the fluorescence FL2 towards the second detector 52. The reflected fluorescence FL2 is collected by the collecting lens 59. The collected light beam is incident on the light receiving section 52A in the second detector 52. In this way, the fluorescence FL2 and the fluorescence FL3 are separated by the dichroic mirror DM3.

[0087] The fluorescence FL3 transmitted through the dichroic mirror DM3 is collected by the collecting lens 59. The collected light beam enters the light receiving section 53A in the third detector 53.

[0088] In this way, the dichroic mirrors DM2 and DM3 function as optical filters for separating the three types of fluorescence FL1 to FL3. The light receiving sections 51A to 53A of the first detector 51 to the third detector 53 are provided in three, one for each of the fluorescent tracers TR1 to TR3 and the corresponding fluorescence FL1 to FL3. The light receiving sections 51A to 53A of the first detector 51 to the third detector 53 receive the three types of fluorescence FL1 to FL3 separated by the dichroic mirrors DM2 and DM3.

[0089] Photomultipliers are used as an example of the light receiving sections 51A to 53A of the first detector 51 to the third detector 53. Photomultipliers have a sensitivity approximately 100 times higher than that of a fluorescent microscope, and therefore can obtain detection signals with a high S / N (Signal / Noise) ratio. Each of the first detector 51 to the third detector 53 outputs a voltage signal (voltage [V: Volt]) according to the light intensity of each of the fluorescence FL1 to FL3 as a detection signal.

[0090] Fig. 16 is an example of the detection signals of the respective fluorescence FL1 to FL3 output from each of the detectors 51 to 53. In Fig. 16, the horizontal axis is time, and the graph shown in Fig. 16 shows the change over time of each detection signal of the respective fluorescence FL1 to FL3 output from each of the detectors 51 to 53. The detection signals are signals that represent the light intensity of each of the fluorescence FL1 to FL3. Since the light intensity of the fluorescence FL represents the amount of leakage of the fluorescent tracer TR, the change over time of the detection signals represents the change over time of the amount of leakage of the fluorescent tracer TR. The amount of leakage of the fluorescent tracer TR is an example of the leakage state of each fluorescent tracer TR.

[0091] Furthermore, each of the detectors 51 to 53 can set the output interval of the detection signal to a short interval of, for example, about 1 ms at most. The output interval is appropriately set depending on the purpose.

[0092] 17, the computer constituting the control device 13 includes a storage device 60, a memory 61, a CPU (Central Processing Unit) 62, a communication unit 63, a display 64, and an input device 65. These are interconnected via a data bus 66.

[0093] The storage device 60 is a hard disk drive built into the computer constituting the control device 13, or connected via a cable, a network, or the like. Alternatively, the storage device 60 is a disk array in which a plurality of hard disk drives are connected in series. The storage device 60 may also be disposed outside the computer and connected to the control device 13 via a network. The storage device 60 stores control programs such as an operating system, various application programs (AP: Application Program) 60A, and various data associated with these programs.

[0094] The memory 61 is a work memory for executing processes by the CPU 62. The CPU 62 loads programs stored in the storage device 60 into the memory 61 and executes processes according to the programs, thereby providing overall control over each part of the computer.

[0095] The communication unit 63 is a network interface that controls the transmission of various information via a network such as a LAN (Local Area Network), the Internet, or a WAN (Wide Area Network) such as a public communication network. The display 64 displays various screens. The various screens are provided with an operation function using a GUI (Graphical User Interface). The computer constituting the control device 13 accepts input of operation instructions from an input device 65 via the various screens. The input device 65 is a keyboard, a mouse, a touch panel, etc.

[0096] The AP 60A includes an operation program for the measuring device 10. The operation program is a program for causing a computer to function as the control device 13 of the measuring device 10. In addition to the operation program, the storage device 60 stores various types of data required for executing the operation program.

[0097] 18, when the operating program is started, the CPU 62 of the computer constituting the control device 13, in cooperation with the memory 61 etc., functions as a detection control unit 621, a data processing unit 622, and an output control unit 623. In addition, the data processing unit 622 functions as a signal processing unit 622A, a concentration derivation unit 622B, and a defect state derivation unit 622C.

[0098] The detection control unit 621 transmits a control signal to the detection unit 17 to thereby control the operation of each unit of the detection unit 17, specifically, the excitation light source 56, the shutter unit 57, and the first detector 51 to the third detector 53. The detection control unit 621 also acquires detection signals from the detectors 51 to 53, and outputs the acquired detection signals to the data processing unit 622. The detection control unit 621 functions as an acquisition unit that acquires detection signals from the detection unit 17.

[0099] Data processing section 622 performs data processing on the detection signals output from each detection section 17. In data processing section 622, the detection signals output from detection control section 621 are input to signal processing section 622A.

[0100] The signal processing unit 622A performs signal processing on the detection signal. The signal processing includes a correction process, a removal process for removing overlapping parts, a conversion process, etc. The correction process includes a correction process for the dark currents of the light receiving units 51A to 53A, etc.

[0101] The overlapping portion removal process is a process of removing the overlapping portion of the wavelength ranges of multiple types of light from the detection signals corresponding to each type of light. In this example, the overlapping portion of the wavelength ranges of multiple types of fluorescence FL1 to FL3 is removed from the detection signals corresponding to each of the fluorescence FL1 to FL3. As shown by hatching in FIG. 15, a part of the wavelength range of the fluorescence FL1 partially overlaps with the wavelength ranges of the fluorescence FL2 and FL3. Therefore, the overlapping portion of the fluorescence FL1 is included in each of the detection signals corresponding to the fluorescence FL2 and FL3. In the removal process, the overlapping portion of the fluorescence FL1 is removed from the detection signals of the fluorescence FL2 and FL3.

[0102] For example, for the second detector 52 and the third detector 53 corresponding to the fluorescence FL2 and FL3, a correspondence relationship between the amount of incident light of the fluorescence FL1 and the amount of incident light of the fluorescence FL1 entering the first detector 51 is measured in advance. Based on this correspondence relationship, the signal processing unit 622A calculates the overlap portion in accordance with the value of the detection signal output by the first detector 51. Then, the signal processing unit 622A removes the calculated overlap portion from the detection signals of the second detector 52 and the third detector 53.

[0103] The conversion process is a process for converting a detection signal representing a voltage value into light intensity data, which is a detection signal representing light intensity.

[0104] An overview of the processing of the signal processing unit 622A is shown in Fig. 19. As described above, the signal processing unit 622A executes each of the processes of correction processing (correction) such as dark current correction, removal processing of overlapping parts (overlapped part removal), and conversion processing (conversion). The conversion processing converts the time change of the voltage value into the time change of the light intensity. The signal processing unit 622A outputs the light intensity data to the concentration derivation unit 622B.

[0105] 20, the concentration derivation unit 622B acquires light intensity data from the signal processing unit 622A. The concentration derivation unit 622B derives the respective concentrations of the multiple types of fluorescent tracers TR1 to TR3 leaked from the first flow path 25 to the second flow path 26 based on the light intensity data as a detection signal.

[0106] A calibration curve CR is used to derive the concentration. The calibration curve CR is stored in the storage device 60 as various data. The calibration curve CR indicates the relationship between the concentration of each of the fluorescent tracers TR1 to TR3 contained in the evaluation liquid 21 and the light intensity of each of the fluorescence FL1 to FL3. The concentration is a concentration when the concentration of each of the fluorescent tracers TR1 to TR3 contained in the evaluation liquid 21 supplied to the first flow path 25 is set to 100. The more leakage of each of the fluorescent tracers TR1 to TR3 from the first flow path 25 to the second flow path 26, the higher the concentration. Such a calibration curve CR is measured in advance, and the measured data is stored in the storage device 60.

[0107] The concentration derivation unit 622B uses the light intensity data and the calibration curve CR to derive concentrations such as 15% for the fluorescent tracer TR1, 10% for the fluorescent tracer TR2, and 5% for the fluorescent tracer TR3, as shown in Fig. 20 as an example. The calibration curve CR may be stored as a table or as a function. The concentration derivation unit 622B outputs the derived concentrations to the defect state derivation unit 622C.

[0108] In FIG. 18, the defect state deriving section 622C derives the state of a defect occurring in the cell sheet 20 based on the concentrations of the fluorescent tracers TR1 to TR3 derived by the concentration deriving section 622B.

[0109] Figure 21 shows the relationship between the size of fluorescent tracers TR1 to TR3 and whether they can penetrate each size of defect DF that occurs in the cell sheet 20. The small-sized fluorescent tracer TR1 can penetrate all of the defects DF shown in Figure 9, namely, the small-sized defect DF1, the medium-sized defect DF2, and the large-sized defect DF3. The medium-sized fluorescent tracer TR2 cannot penetrate the small-sized defect DF1, but can penetrate the medium-sized defect DF2 and the large-sized defect DF3. The large-sized fluorescent tracer TR3 cannot penetrate the small-sized defect DF1 or the medium-sized defect DF2, but can penetrate only the large-sized defect DF3.

[0110] In this way, the small-sized fluorescent tracer TR1 can pass through all sizes of defects DF1 to DF3, including small, medium, and large sizes, and the medium-sized fluorescent tracer TR2 can pass through medium and large defects DF2 and DF3. Therefore, the concentration of the fluorescent tracers TR1 to TR3 alone cannot be used to grasp the defect state occurring in the cell sheet 20, for example, the approximate proportion of multiple types of defects DF1 to DF3 of different sizes. In other words, the small-sized fluorescent tracer TR1 passes through not only the small-sized defect DF1 but also the medium-sized defect DF2 and the large-sized defect DF3, and therefore the concentration of the fluorescent tracer TR1 does not depend only on the area of ​​the small-sized defect DF1.

[0111] Therefore, the defect state derivation unit 622C derives the defect state in the following procedure. First, as shown in Fig. 22, a relationship G between the total area of ​​defects DF on the cell sheet 20 and the concentration, which is an index of the leakage amount of each of the fluorescent tracers TR1 to TR3, is measured in advance. The relationship G is measured in a state in which each of the fluorescent tracers TR1 to TR3 is contained in the evaluation liquid 21. As shown in Fig. 9, various sizes are possible for the defect DF, but the total area of ​​the defect DF in the relationship G is, for example, the total area of ​​defect DF of a size that allows each of the fluorescent tracers TR1 to TR3 to pass through.

[0112] 22 represents the extent to which each of the fluorescent tracers TR1 to TR3 leaks into the second flow path 26 depending on the total area of ​​the defects DF generated in the cell sheet 20. Data representing this relationship G is stored in the storage device 60 in the form of a function or a table.

[0113] As shown in FIGS. 23 and 24, the defect state deriving section 622C uses the measured value of the concentration derived by the concentration deriving section 622B and the relationship G to execute a defect state deriving process.

[0114] As shown in Fig. 24, in the defect state derivation process, the defect state derivation unit 622C first executes a first process. The first process is a process for individually deriving a provisional total area of ​​defects DF from the respective concentrations of the fluorescent tracers TR1 to TR3. In the first process, the defect state derivation unit 622C refers to the relationship G based on the respective concentrations of the fluorescent tracers TR1 to TR3 to determine the total area corresponding to the concentration. This total area is set as the provisional total area.

[0115] As shown as an example in Figure 24, when the concentration of small-sized fluorescent tracer TR1 is 15%, the provisional total area of ​​defect DF in the cell sheet 20 is 1000. When the concentration of medium-sized fluorescent tracer TR2 is 10%, the provisional total area of ​​defect DF is 700, and when the concentration of large-sized fluorescent tracer TR3 is 5%, the provisional total area of ​​defect DF is 200. Note that in Figure 24, the provisional total area is a standardized value as an example and has no unit.

[0116] After the first process, the defect state derivation unit 622C executes the second process. In the second process, the area of ​​the defects DF1 to DF3 for each size is derived from the provisional total area. The defect state derivation unit 622C calculates the area of ​​the defects DF1 to DF3 of different sizes in order from the largest. First, as shown in FIG. 21, the large-sized fluorescent tracer TR3 only transmits the large-sized defect DF3. Therefore, the provisional total area of ​​the defects DF, 200, calculated based on the large-sized fluorescent tracer TR3 is considered to be the total area of ​​only the large-sized defect DF3. Therefore, the provisional total area of ​​200 calculated based on the large-sized fluorescent tracer TR3 is calculated as the area of ​​the large-sized defect DF3 as it is.

[0117] Next, the defect state derivation unit 622C calculates the area of ​​the medium-sized defect DF2. The medium-sized fluorescent tracer TR2 transmits through the large-sized defect DF3 in addition to the medium-sized defect DF2. Therefore, it is considered that the provisional total area of ​​defects DF, 700, calculated based on the medium-sized fluorescent tracer TR2, includes the area of ​​the large-sized defect DF3. Therefore, the defect state derivation unit 622C subtracts 200, which is the area of ​​the large-sized defect DF3, from the provisional total area, 700, calculated based on the medium-sized fluorescent tracer TR2. The defect state derivation unit 622C sets the value of 500 calculated in this way as the area of ​​the medium-sized defect DF2.

[0118] Finally, the defect state derivation unit 622C calculates the area of ​​the small defect DF1. The small fluorescent tracer TR1 transmits not only the small defect DF1, but also the medium and large defects DF2 and DF3. Therefore, the provisional total area of ​​defects DF, 1000, calculated based on the small fluorescent tracer TR1, is considered to include the area of ​​the medium defect DF2 and the area of ​​the large defect DF3. Therefore, the area of ​​the medium defect DF2, 500, and the area of ​​the large defect DF3, 200, are subtracted from the provisional total area, 1000, calculated based on the small fluorescent tracer TR1. The defect state derivation unit 622C determines the value of 300 calculated in this way as the area of ​​the small defect DF1.

[0119] The defect state derivation unit 622C thus derives, based on the respective concentrations of the different sized fluorescent tracers TR1 to TR3 and the relationship G, the approximate proportion of the different sized defects DF1 to DF3 that have occurred in the cell sheet 20 as the defect state.

[0120] A measurement result display screen 69 shown in Fig. 25 is an example of a screen displayed on the display 64. The output control unit 623 shown in Fig. 18 generates such a measurement result display screen 69 and updates the contents of the measurement result display screen 69 in real time. As shown in Fig. 18, measurement data such as light intensity data processed by the signal processing unit 622A, the concentration derived by the concentration derivation unit 622B, and the defect state derived by the defect state derivation unit 622C are input to the output control unit 623. Such measurement data indicates the leakage status of the fluorescent tracers TR1 to TR3, and serves as an index for evaluating the barrier property of the cell sheet 20.

[0121] The output control unit 623 displays such measurement data as measurement results on the measurement result display screen 69. Measurement data such as light intensity data, concentration, and defect state are updated based on detection signals acquired in real time. The output control unit 623 displays the measurement data in real time. As a result, the leakage status of the fluorescent tracers TR1 to TR3 is output to the display 64 in real time. The output control unit 623 functions as a processing unit that executes output control processing to output the leakage status.

[0122] The operation of the above configuration will be described below with reference to the flowchart shown in Fig. 26. In this example, the user of the measuring device 10 is a manufacturer of the flow channel device 11. The user uses the measuring device 10 when evaluating the barrier property of the cell sheet 20 of the manufactured flow channel device 11.

[0123] First, in step S1100, the flow path device 11 is set in the measurement apparatus 10. Specifically, the flow path device 11 is mounted on the mounting portion 16. Then, as shown in Fig. 1, a supply tube 22A and a discharge tube 22B are attached to the flow path device 11. Furthermore, an evaluation liquid 21 containing a mixture of three types of fluorescent tracers TR1 to TR3 is prepared in advance, and the prepared evaluation liquid 21 is set in a tank of a pump 22C.

[0124] In step S1200, when a measurement start instruction is input by operating a measurement start button or the like, the measurement device 10 starts measurement. Specifically, the detection control unit 621 starts the operation of each unit of the detection unit 17, such as the excitation light source 56 and the first detector 51 to the third detector 53. As a result, the first detector 51 to the third detector 53 start outputting detection signals.

[0125] In step S1300, when the operation of the pump 22C is started, the evaluation liquid 21 containing the three types of fluorescent tracers TR1 to TR3 as the multiple types of tracers is supplied to the flow path device 11. As a result, the transfer of the evaluation liquid 21 to the first flow path 25 is started.

[0126] In step S1400, the first detector 51 to the third detector 53 of the detection unit 17 each output a detection signal. If no defect DF occurs in the cell sheet 20, there is no leakage of the fluorescent tracer TR into the second flow path 26. In this case, the first detector 51 to the third detector 53 output a detection signal indicating a baseline.

[0127] On the other hand, when a defect DF occurs in the cell sheet 20, at least one of the three types of fluorescent tracers TR1 to TR3 mixed in the evaluation liquid 21 passes through the defect DF and leaks from the first flow path 25 to the second flow path 26. The detection unit 17 irradiates the detection position 46 of the second flow path 26 with excitation light LB. When the fluorescent tracers TR1 to TR3 leak into the second flow path 26, the excitation light LB is irradiated onto the fluorescent tracers TR1 to TR3, causing each of the fluorescent tracers TR1 to TR3 to emit fluorescence FL1 to FL3. The dichroic mirrors DM1 to DM3 in the first mirror barrel 54 of the detection unit 17 optically separate each of the fluorescence FL1 to FL3 and enter the first detector 51 to the third detector 53, respectively.

[0128] The first detector 51 to the third detector 53 output a detection signal for each leaked fluorescent tracer TR according to the light intensity of each of the fluorescence FL1 to FL3 corresponding to each of the three types of fluorescent tracers TR1 to TR3. That is, the light intensity of each of the fluorescence FL1 to FL3 represents the leakage status of each of the fluorescent tracers TR1 to TR3. That is, the detection unit 17 optically distinguishes and detects the leakage status of each of the fluorescent tracers TR1 to TR3 by distinguishing and detecting the light intensity of each of the fluorescence FL1 to FL3. The detection control unit 621 acquires the detection signal from the detection unit 17 and outputs it to the data processing unit 622.

[0129] In step S1500, the data processing unit 622 performs data processing on the detection signal. As shown in Fig. 19, the signal processing unit 622A performs correction processing, overlap removal processing, conversion processing, and the like on the detection signal. The concentration derivation unit 622B performs concentration derivation processing shown in Fig. 20 on the light intensity data that has been subjected to the signal processing to derive the concentration. The defect state derivation unit 622C performs defect state derivation processing shown in Figs. 23 and 24 based on the concentration derived by the concentration derivation unit 622B to derive the defect state.

[0130] In step S1600, the output control unit 623 generates a measurement result display screen 69 using the measurement data processed by the data processing unit 622 as the measurement results, and displays the measurement results in real time on the display 64. Based on the measurement results on the measurement result display screen 69, the user can check the leakage status including the presence or absence of leakage of each of the fluorescent tracers TR1 to TR3, the amount of leakage and the change in the amount of leakage over time, and further the defective state of the cell sheet 20. Since these measurement results are indices for evaluating the barrier property of the cell sheet 20, the user evaluates the barrier property of the cell sheet 20 based on these measurement results.

[0131] In step S1700, if the preset time has not elapsed (N in S1700), the measurement process from steps S1400 to S1600 continues. In step S1700, if the preset time has elapsed (Y in S1700), the process proceeds to step S1800. In step S1800, the delivery of the evaluation liquid and the operation of detection unit 17 are stopped, thereby ending the measurement.

[0132] The measuring device 10 according to the technology of the present disclosure has a detection unit 17 that optically distinguishes and detects the leakage states of the multiple fluorescent tracers TR1 to TR3 leaking from the first flow path 25 to the second flow path 26 through defects DF generated in the cell sheet 20 when an evaluation liquid 21 containing three types of fluorescent tracers TR1 to TR3 as multiple types of tracers with different sizes is sent to the first flow path 25. Therefore, multiple types of defects DF with different sizes generated in the cell sheet 20 can be detected without spending time and effort compared to the conventional method.

[0133] The effect of the measuring device 10 according to the technique of the present disclosure will be described in more detail using a comparative example shown in Fig. 27. The comparative example shown in Fig. 27 is similar to the measuring device 10 in that it uses three types of fluorescent tracers TR1 to TR3. The difference is that it does not use an evaluation liquid 21 in which the fluorescent tracers TR1 to TR3 are mixed, but rather uses each of the fluorescent tracers TR1 to TR3 one by one for measurement.

[0134] More specifically, the comparative example uses three types of evaluation liquids: a first evaluation liquid containing only small-sized fluorescent tracer TR1, a second evaluation liquid containing only medium-sized fluorescent tracer TR2, and a third evaluation liquid containing only large-sized fluorescent tracer TR3. In the comparative example, the first evaluation liquid is replaced with the third evaluation liquid, and measurements are performed three times.

[0135] That is, first, in step S2100, a first measurement is performed using a first evaluation liquid containing only the small-sized fluorescent tracer TR1. By performing the first measurement, the leakage state of the small-sized fluorescent tracer TR1 is detected.

[0136] Before the next measurement, in step S2200, a buffer is caused to flow through first flow path 25 to wash first flow path 25.

[0137] In step S2300, a second measurement is performed using a second evaluation liquid containing only the medium-sized fluorescent tracer TR2. By performing the second measurement, the leakage state of the medium-sized fluorescent tracer FL2 is detected.

[0138] Before the next measurement, in step S2400, a buffer is caused to flow through first flow path 25 to wash first flow path 25.

[0139] Finally, in step S2300, a third measurement is performed using a third evaluation liquid containing only the large-sized fluorescent tracer TR3. By performing the third measurement, the leakage state of the large-sized fluorescent light FL3 is detected.

[0140] The leakage status of each of the fluorescent tracers TR1 to TR3 can also be detected by using three types of evaluation liquids in an interchangeable manner as in the comparative example.

[0141] However, in the comparative example, it is necessary to perform three measurements while replacing the three types of evaluation liquid, which requires more time and effort than the measurement device 10 and measurement method according to the technology of the present disclosure. In addition, in the comparative example, it is difficult to detect the leakage status of each of the fluorescent tracers TR1 to TR3 under the same conditions because three measurements are performed while replacing the three types of evaluation liquid. This is because the cell sheet 20 is damaged to some extent every time the evaluation liquid is delivered. This damage causes the defective state of the cell sheet 20 to change in each measurement.

[0142] Furthermore, when a different type of evaluation liquid is replaced as in the comparative example, the first flow path 25 needs to be washed every time the evaluation liquid is replaced. Since the cell sheet 20 and the buffer come into contact with each other during washing, the washing also causes damage to the cell sheet 20. Furthermore, the need for washing further increases time and labor.

[0143] The measuring device 10 according to the technology of the present disclosure detects the leakage status of each tracer in one measurement using an evaluation liquid containing a mixture of multiple types of tracers. Therefore, not only can it reduce time and labor, but it also has the effect of being able to detect the leakage status of each tracer under the same conditions compared to the comparative example.

[0144] In addition, fluorescent tracers TR1 to TR3 that emit fluorescence in different wavelength ranges are used as multiple types of tracers. Fluorescent tracers TR are available in a wide variety of sizes and fluorescent colors, making it easy to select an appropriate tracer.

[0145] Furthermore, the detection unit 17 that detects the fluorescence includes an excitation light source 56, dichroic mirrors DM1 to DM3 as optical filters for separating the multiple types of fluorescence FL1 to FL3, and light receiving units 51A to 53A that receive the fluorescence FL1 to FL3 separated by the dichroic mirrors DM1 to DM3. Such a configuration for detecting fluorescence is common, and therefore the detection unit 17 can be easily manufactured.

[0146] Furthermore, the measuring device 10 uses a laser light source as the excitation light source 56, and is therefore capable of detecting strong fluorescence FL1 to FL3. This improves the S / N ratio of the detection signals. Furthermore, photomultipliers are used as the first detector 51 to the third detector 53. This improves the S / N ratio of the detection signals compared to a fluorescence microscope.

[0147] Furthermore, the measuring device 10 has one excitation light source 56, and three light receiving units, light receiving unit 51A to light receiving unit 53A, are provided for each of the three types of fluorescence FL1 to FL3. By providing only one excitation light source 56, it is possible to simplify the configuration, reduce the size, and reduce manufacturing costs compared to a case where multiple excitation light sources 56 are provided. Furthermore, since three light receiving units are provided, it is easy to make adjustments to match the wavelength ranges of the respective fluorescence FL1 to FL3, for example.

[0148] Furthermore, the measurement apparatus 10 according to the technique of the present disclosure irradiates the excitation light LB at the detection position 46 in the flow path device 11 mounted on the mounting portion 16. The detection position 46 is downstream in the flow direction of the fluorescent tracers TR1 to TR3, which are an example of a tracer leaking from a communicating portion corresponding to the slit 34A, and is an example of a position where the fluorescent tracers are incident only on the second flow path 26 out of the first flow path 25 and the second flow path 26. Since the excitation light LB is not incident on the first flow path 25, only the tracer leaking into the second flow path 26 can be reliably detected.

[0149] The leakage status detected by the measuring device 10 according to the technique of the present disclosure includes the time change in the leakage amount of the fluorescent tracers TR1 to TR3, which are an example of a tracer, so that the time change in the defect state of the cell sheet 20 can be observed.

[0150] The measuring device 10 according to the technique of the present disclosure also includes a data processing unit 622 that performs data processing on the detection signal output by the detection unit 17. This allows the detection signal representing a voltage value to be converted into light intensity data or otherwise processed into a form suitable for observation. Furthermore, the data processing unit 622 allows various measurement results, such as concentration and defect state, to be obtained.

[0151] Furthermore, in the measuring device 10, the signal processing unit 622A performs a correction process on the detection signal and a removal process to remove from the detection signal the overlapping portions of the wavelength ranges of the fluorescence FL1 to FL3 as multiple types of light, thereby obtaining a detection signal with higher accuracy.

[0152] For example, the upper graph in Fig. 19 is a graph showing the change over time in voltage value before the process of removing the overlapping portion is executed. The lower graph in Fig. 19 is a graph showing the change over time in light intensity after the process of removing the overlapping portion is executed. In the upper graph, all three types of fluorescence FL, fluorescence FL1 to fluorescence FL3, appear to increase monotonically.

[0153] However, in the graph shown in the lower part of Fig. 19, the light intensity of the fluorescence FL1 does not increase monotonically. Specifically, the light intensity of the fluorescence FL1 increases at first, but decreases after reaching a peak. In this way, by performing a process to remove the overlapping parts, it is possible to grasp the accurate time change of each detection signal that indicates the leakage state of the fluorescent tracer TR.

[0154] In addition, in the measurement device 10, the concentration deriving unit 622B derives the concentration of each of the fluorescent tracers TR1 to TR3, which are an example of multiple types of tracers. Therefore, compared to displaying the leakage status of the fluorescent tracer TR as a voltage value, it is easier to intuitively grasp the leakage status by displaying it as a concentration.

[0155] In the measurement device 10, the defect state derivation unit 622C derives a defect state, which is the state of the defects DF occurring in the cell sheet 20, based on the respective concentrations of the fluorescent tracers TR1 to TR3, which are an example of multiple types of tracers. The defect state includes, for example, an approximate proportion of each of multiple types of defects DF1 to DF3 of different sizes. By deriving such a defect state, the barrier property of the cell sheet 20 can be evaluated in more detail than when simply detecting the presence or absence of defects DF.

[0156] In the measuring device 10, the output control unit 623 displays in real time on the display 64 the leakage status of each of the fluorescent tracers TR1 to TR3, which are examples of tracers, detected by the detection unit 17. This makes it possible to observe the time change in the leakage status of the fluorescent tracer TR and the time change in the defective state of the cell sheet 20 in real time.

[0157] In addition, in the flow channel device 11, the permeable membrane 36 that functions as a support for the cell sheet 20 has holes with a diameter of 1 μm or more. In the conventional JP2018-522586A, a gel is used as the support for the cell sheet 20. A tracer having a large size of 1 μm or more is unlikely to permeate the gel. Therefore, by using the permeable membrane 36, it is possible to detect defects DF having a large size of 1 μm or more that occur in the cell sheet 20.

[0158] A gel may be used as a support for the cell sheet 20 instead of the permeable membrane 36. When a gel is used as a support for the cell sheet 20, the size of the tracer may be selected according to the properties of the gel. However, since the permeable membrane 36 has the above-mentioned advantages, it is preferable to use the permeable membrane 36.

[0159] Moreover, the size (particle size) of each of the fluorescent tracers TR1 to TR3, which is an example of a plurality of types of tracers, is within the range of 1 nm to 100 μm, thereby making it possible to detect defects DF of various sizes in the cell sheet 20.

[0160] Fluorescent tracers TR1 to TR3, which are examples of multiple types of tracers, include tracers with sizes (particle diameters) on the order of 1 nm to several hundred nm, and tracers with sizes (particle diameters) on the order of 1 μm to several tens of μm. When the purpose is to detect defects DF of different sizes, there is little advantage to using multiple types of tracers with similar sizes. By using tracers with large differences in size (particle diameters) on different orders of magnitude, it is possible to efficiently detect defects DF of different sizes.

[0161] The measurement device 10 includes a supply mechanism 22 that supplies the evaluation liquid 21 to the first flow path 25. This makes it easier to deliver the evaluation liquid 21 compared to a case where a separate supply mechanism is provided. The measurement device 10 does not necessarily need to include the supply mechanism 22. A syringe, a pipette, or the like can be used instead of the supply mechanism 22.

[0162] (Variation 1: Forming cell sheets on both sides of a permeable membrane) 8 and 9, in the flow path device 11 of the above example, the cell sheet 20 is formed only on one side of the permeable membrane 36, i.e., the side on the first flow path 25 side. As shown in Fig. 28 and 29, the cell sheet 80 may be formed on the other side of the permeable membrane 36, i.e., the side on the second flow path 26 side. In other words, the example shown in Fig. 28 and 29 is an example in which a cell sheet is formed on both sides of the permeable membrane 36.

[0163] The cell sheet 80 is, for example, an array of cells 80A, and the cells 80A are smooth muscle cells (SMCs). The tissue that constitutes the inner wall of a blood vessel includes endothelial cells formed on one side of a basement membrane and smooth muscle cells formed on the other side. Therefore, when simulating a blood vessel using the flow channel device 11, as shown in Figs. 28 and 29, the permeable membrane 36 is regarded as a basement membrane, and a cell sheet 20 is formed on one side of the permeable membrane 36, and a cell sheet 80 is formed on the other side, thereby making it possible to more closely resemble the structure of an actual blood vessel tissue.

[0164] Unlike the cells 20A, which are endothelial cells, the cells 80A, which are smooth muscle cells, do not have tight junctions. Therefore, as shown in Figure 29, even when a plurality of cells 80A are arranged in the cell sheet 80, a small-sized fluorescent tracer TR1 can penetrate the gaps between the cells 80A. Furthermore, the cell sheet 80 is easier to peel off than the cell sheet 20.

[0165] (Variation 2: Rotating Filter) In the above example, a plurality of first to third detectors 51 to 53 are provided corresponding to the respective fluorescence FL1 to FL3 of the fluorescent tracers TR1 to TR3. However, as shown in Fig. 30 and Fig. 31, by using a rotary filter 92, it is possible to detect three types of fluorescence FL1 to FL3 with one detector 91.

[0166] The rotary filter 92 is provided with a plurality of types of dichroic mirrors DM21 to DM23 as optical filters with different transmission characteristics. The rotary filter 92 is rotated, for example, by driving a motor (not shown). Due to this rotation, each of the dichroic mirrors DM21 to DM23 is selectively inserted into the optical paths of the fluorescence FL1 to FL3.

[0167] In Fig. 31, as shown in Fig. 31A, the dichroic mirror DM21 is a bandpass filter having a transmission characteristic that transmits only the wavelength range of the fluorescence FL1. As shown in Fig. 31B, the dichroic mirror DM22 is a bandpass filter having a transmission characteristic that transmits only the wavelength range of the fluorescence FL2. As shown in Fig. 31C, the dichroic mirror DM23 is a bandpass filter having a transmission characteristic that transmits only the wavelength range of the fluorescence FL3.

[0168] The combinations of the wavelength range of the fluorescence and the size of the fluorescent tracer shown in the above example are merely examples, and these combinations are selected as appropriate. For example, the size of the fluorescent tracer is selected according to the characteristics of the defect DF, such as the size of the defect DF that occurs in the cell sheet. In addition, the wavelength range of the fluorescence is also selected as appropriate according to the characteristics of the optical filter of the detection unit 17 and the light receiving unit of the detector.

[0169] Although an example has been described in which three types of fluorescent tracers TR are used as the multiple types of tracers, the number of tracers is not limited to three, and may be two, or four or more.

[0170] In the above example, a fluorescent tracer is used as an example of a tracer, and the leakage status of each tracer is optically distinguished by using the fluorescence emitted by the fluorescent tracer. However, Raman scattered light may be used instead of fluorescence. When light is irradiated onto a substance, the light interacts with the substance to produce light called Raman scattered light, which has a wavelength different from that of the incident light. Raman scattered light is light with a spectrum specific to the substance. When Raman scattered light is used, multiple types of tracers with different sizes that emit Raman scattered light with different spectra are used as multiple types of tracers. Then, the leakage status of each tracer is optically distinguished based on the Raman scattered light emitted by each of these multiple types of tracers.

[0171] In addition, such Raman scattered light is very weak, and the device configuration is more complicated than the configuration of the detection unit 17 that detects fluorescence. Therefore, it is preferable that the measurement device 10 utilizes fluorescence.

[0172] In each of the above embodiments, the following various processors can be used as the hardware structure of the processing unit that executes various processes, such as the detection control unit 621 and the data processing unit 622. The various processors include a CPU (Central Processing Unit), which is a general-purpose processor that executes software and functions as various processing units, as well as a programmable logic device (PLD), which is a processor whose circuit configuration can be changed after manufacture, such as an FPGA (Field Programmable Gate Array), and / or a dedicated electric circuit, which is a processor having a circuit configuration designed specifically for executing specific processes, such as an ASIC (Application Specific Integrated Circuit).

[0173] A single processing unit may be configured with one of these various processors, or may be configured with a combination of two or more processors of the same or different types (e.g., a combination of multiple FPGAs and / or a combination of a CPU and an FPGA). Also, multiple processing units may be configured with a single processor.

[0174] An example of configuring multiple processing units with one processor is one in which one processor is configured with a combination of one or more CPUs and software, and this processor functions as multiple processing units. Secondly, there is a form in which a processor is used that realizes the functions of the entire system including multiple processing units with one IC (Integrated Circuit) chip, as typified by System On Chip (SoC). In this way, the various processing units are configured as a hardware structure using one or more of the above various processors.

[0175] Furthermore, more specifically, the hardware structure of these various processors can be an electric circuit that combines circuit elements such as semiconductor elements.

[0176] In addition to the operating program of the measuring device, the technology disclosed herein also extends to a computer-readable storage medium (such as a Universal Serial Bus (USB) memory or a Digital Versatile Disc (DVD)-Read Only Memory (ROM)) that non-temporarily stores the operating program of the measuring device.

[0177] In this specification, "A and / or B" is synonymous with "at least one of A and B." In other words, "A and / or B" means that it may be only A, only B, or a combination of A and B. In addition, in this specification, the same idea as "A and / or B" is also applied when three or more things are expressed by connecting them with "and / or."

[0178] The above description and illustrations are detailed descriptions of the parts related to the technology of the present disclosure, and are merely an example of the technology of the present disclosure. For example, the above description of the configuration, function, action, and effect is an example of the configuration, function, action, and effect of the parts related to the technology of the present disclosure. Therefore, it goes without saying that unnecessary parts may be deleted, new elements may be added, or replaced with respect to the description and illustrations shown above, within the scope of the gist of the technology of the present disclosure. In addition, in order to avoid confusion and to facilitate understanding of the parts related to the technology of the present disclosure, the description and illustrations shown above omit explanations of technical common sense that do not require explanation in order to enable the implementation of the technology of the present disclosure.

Claims

1. a mounting section to which a flow path device including a first flow path, a second flow path having a communicating portion communicating with at least a part of the first flow path, and a cell sheet in which a plurality of cultured cells are arranged in a bonded state, the cell sheet separating the first flow path and the second flow path at the communicating portion is detachably mounted; a detection unit that optically distinguishes and detects the leakage status of each of a plurality of types of tracers having different sizes that leak from the first flow path to the second flow path through defects that occur in the cell sheet, the plurality of types of tracers being mixed in an evaluation liquid for evaluating the barrier property of the cell sheet that is sent to the first flow path; Equipped with Each of the plurality of types of tracers is a fluorescent tracer that emits fluorescence having a different wavelength range, The detection unit detects a plurality of types of fluorescence emitted by each of the plurality of types of tracers and outputs a detection signal; The measuring device further includes a data processing unit that derives the concentration of each of the multiple types of tracers that have leaked from the first flow path to the second flow path based on the detection signal output by the detection unit, and derives the state of defects of different sizes that have occurred in the cell sheet based on the concentration of each of the multiple types of tracers.

2. The detection unit includes an excitation light source that irradiates each of the plurality of types of tracers with excitation light that excites the fluorescence; an optical filter for separating the plurality of types of fluorescence; The measuring device according to claim 1 , further comprising a light receiving section that receives the fluorescent light separated by the optical filter.

3. The excitation light source is one, The measuring device according to claim 2 , wherein the light receiving section is provided for each of the plurality of types of fluorescence.

4. 4. The measuring device according to claim 3, wherein the excitation light source irradiates excitation light at a position downstream in a flow direction of the tracer leaking from the communicating portion in the flow path device attached to the attachment portion, the excitation light being incident only on the second flow path out of the first flow path and the second flow path.

5. 5. The measuring device according to claim 1, wherein the leakage condition includes a change in leakage amount over time.

6. 6. The measurement device according to claim 5, wherein the data processing unit has a signal processing unit that executes at least one of a correction process that corrects a dark current of the detection unit, a removal process that removes overlapping portions of the wavelength ranges of the multiple types of light from the detection signals corresponding to each type of light, and a conversion process that converts the detection signal output as a voltage signal into a light intensity.

7. The measuring device according to claim 1 , further comprising an output control unit that outputs the leakage state detected by the detection unit on a display in real time.

8. The cell sheet is cultured on a support, 8. The measuring device according to claim 1, wherein the support is a permeable membrane having pores with a diameter of 1 μm or more.

9. 9. The measuring device according to claim 8, wherein the particle diameter of each of the plurality of types of tracers is in the range of 1 nm to 100 μm.

10. 10. The measuring apparatus according to claim 9, wherein the plurality of types of tracers include tracers having particle sizes on the order of 1 nm to several hundreds of nm and tracers having particle sizes on the order of 1 μm to several tens of μm.

11. The measurement device according to claim 1 , further comprising a supply mechanism that supplies the evaluation liquid to the first flow path.

12. An operating program for a measurement device for evaluating a barrier property of a cell sheet using a flow path device including a first flow path, a second flow path having a communicating portion communicating with at least a part of the first flow path, and a cell sheet on which a plurality of cultured cells are arranged in a bound state, the cell sheet separating the first flow path and the second flow path at the communicating portion, an acquisition unit that acquires a detection signal from a detection unit that optically distinguishes and detects the leakage status of each of a plurality of types of tracers with different sizes that leak from the first flow path to the second flow path through defects occurring in the cell sheet, the plurality of types of tracers being mixed in an evaluation liquid for evaluating the barrier property of the cell sheet that is sent to the first flow path, and each of the plurality of types of tracers is a fluorescent tracer that emits fluorescence in a different wavelength range, and the detection unit detects the plurality of types of fluorescence emitted by each of the plurality of types of tracers to acquire a detection signal that distinguishes each of the plurality of types of tracers and detects the leakage status; a data processing unit that derives the respective concentrations of the plurality of types of tracers that have leaked from the first flow path to the second flow path based on the acquired detection signals, and derives the state of defects of different sizes occurring in the cell sheet based on the respective concentrations of the plurality of types of tracers; a processing unit that executes an output control process for outputting states of the defects having different sizes in accordance with the measurement results derived by the data processing unit, The operating program of the measuring device that causes the computer to function.

13. a flow path device including a first flow path, a second flow path having a communicating portion communicating with at least a part of the first flow path, and a cell sheet on which a plurality of cultured cells are arranged in a bonded state, the cell sheet separating the first flow path and the second flow path at the communicating portion; a liquid sending step of sending an evaluation liquid for evaluating the barrier property of the cell sheet, the evaluation liquid having a mixture of a plurality of types of tracers with different sizes, to the first flow path; a detection step of optically distinguishing and detecting leakage states of each of the plurality of types of tracers leaking from the first flow path to the second flow path through defects occurring in the cell sheet; Equipped with Each of the plurality of types of tracers is a fluorescent tracer that emits fluorescence having a different wavelength range, The detection step includes outputting detection signals obtained by detecting a plurality of types of fluorescence emitted by the plurality of types of tracers, The measurement method further includes a data processing step of deriving the concentration of each of the multiple types of tracers that have leaked from the first flow path to the second flow path based on the detection signal output by the detection step, and deriving the state of defects of different sizes that have occurred in the cell sheet based on the concentration of each of the multiple types of tracers.

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