Patent Description:
The following techniques are known as a method for observing cells using a microfluidic device. For example, <CIT> discloses a device for cell observation, which includes a cell culture chamber and a chemical liquid chamber adjacent to each other through a porous membrane, an introduction channel and a discharge channel for discharging a cell-containing solution after introduction into a cell culture chamber, an introduction channel and a discharge channel for discharging a chemical liquid after introduction into the chemical liquid chamber, and an observation window provided on a side of the chemical liquid chamber opposite to the porous membrane. Further, the reference document <NUM> discloses a cell observation method in which a cell-containing solution is introduced into a cell culture chamber and a chemical liquid is introduced into a chemical liquid chamber, and luminescence light based on cells or a product from the cells is observed through an observation window.

In addition, <CIT> (<CIT>) describes that in a filtration cell having a sample liquid passage and a carrier liquid passage, which are in contact with each other through a porous membrane, a carrier obtained by moving at least a part of a sample in a liquid to be measured into a carrier through the porous membrane is injected into a detector by an injector.

Non patent literature publication "<NPL> discloses that bovine serum albumin conjugated with fluorescein isothiocyanate is delivered to an upper microchannel by pressure-driven flow and is forced to permeate a poly(ethylene terephthalate) membrane into a lower microchannel, where it is detected by laser-induced fluorescence; the concentration of the permeate at the point of detection varies with lower channel flow rates, a sequential flow rate stepping scheme is developed and applied to obtain the permeability of cell-free and cell-bound membrane layers.

Document <CIT> discloses the evaluation of the permeability of porous membranes to red blood cells, a blood dilution is run through an upper microchannel of a blood vessel models and a physiological saline solution is run through the lower microchannels thereof; the rate of fluid delivery of the blood dilution and the physiological saline solution is set to <NUM>µL/min, the internal pressure of the upper microchannel is set to approximately <NUM> kPa, and the internal pressure of the lower microchannel is set to approximately <NUM> kPa so as to establish parameters close to the blood flow and blood pressure conditions inside actual blood vessels.

In a microfluidic device having a first flow channel, a second flow channel, and a porous membrane that separates these flow channels, the following method can be considered as an evaluation method for permeability of a porous membrane. For example, a method of monitoring an amount of light radiated from a phosphor that leaks into the second flow channel can be considered, where a liquid containing a phosphor is accommodated in a first flow channel, a liquid containing no phosphor is accommodated in a second flow channel, and the phosphor diffuses in a liquid and permeates through a porous membrane to leak to the second flow channel. However, according to this method, since the diffusion rate of the phosphor is low, it takes a lot of time (for example, about <NUM> minutes) to carry out an evaluation.

The present disclosed technology has been made in consideration of the above point, and one aspect of the technique is to evaluate the permeability of a porous membrane in a short time.

An evaluation method according to the present disclosed technology is an evaluation method for permeability of a porous membrane that separates a first flow channel and a second flow channel according to appended claim <NUM>.

According to the evaluation method according to the embodiment of the present disclosed technology, it is possible to evaluate the permeability of the porous membrane in a short time.

A chronological change of a flow rate of the liquid that passes through the second flow channel may be acquired as the evaluation indicator. In addition, a phosphor is contained in the liquid that is supplied to the first flow channel, and a chronological change in an amount of light radiated from the phosphor contained in the liquid that flows through the second flow channel may be acquired as the evaluation indicator. Further, a specific component is contained in the liquid that is supplied to the first flow channel, and a chronological change in a concentration of the specific component contained in the liquid that flows through the second flow channel may be acquired as the evaluation indicator.

The cell evaluation method according to the present disclosed technology is a cell evaluation method using the above-described evaluation method for permeability of a porous membrane, the cell evaluation method comprising acquiring the evaluation indicator acquired in a state where cells to be evaluated are cultured on a surface of the porous membrane as an indicator of performance of the cells to be evaluated, the performance being blocking of leakage of a liquid that is supplied to the first flow channel to the second flow channel.

The drug evaluation method according to the present disclosed technology is a drug evaluation method using the above-described evaluation method for permeability of a porous membrane, the cell evaluation method comprising culturing cells on a surface of the porous membrane and acquiring the evaluation indicator acquired after exposing the cells to a drug to be evaluated as an indicator of toxicity of the drug to be evaluated to the cells.

In the evaluation method according to the present disclosed technology, a microfluidic device having the first flow channel and the second flow channel may be used.

According to the present disclosed technology, it is possible to evaluate the permeability of the porous membrane in a short time.

In each of the drawings, substantially the same or equivalent configuration elements or parts are designated by the same reference numeral.

<FIG> is a perspective view illustrating an example of a configuration of a microfluidic device <NUM> according to an embodiment of the present disclosed technology, where the microfluidic device <NUM> is used for the evaluation of the permeability of the porous membrane, and <FIG> is an exploded perspective view of the microfluidic device <NUM>. <FIG> is a schematic view illustrating a part of a cross section taken along a line <NUM>-<NUM> in <FIG>. The microfluidic device <NUM> has a cavity unit <NUM> composed of an upper cavity member <NUM> and a lower cavity member <NUM>, which are opposite to each other, as a pair of cavity members laminated in the thickness direction. The upper cavity member <NUM> and the lower cavity member <NUM> are made of a material having flexibility, such as polydimethylsiloxane (PDMS) as an example. It is noted that as the material that constitutes the upper cavity member <NUM> and the lower cavity member <NUM>, in addition to PDMS, an epoxy resin, a urethane resin, a styrenic thermoplastic elastomer, an olefinic thermoplastic elastomer, an acrylic thermoplastic elastomer, or a polyvinyl alcohol, can be used.

As illustrated in <FIG>, a recessed part <NUM> that defines a lower micro flow channel <NUM> is formed on the upper surface of the lower cavity member <NUM>, that is, on an opposite surface 14A opposite to the upper cavity member <NUM>. The lower micro flow channel <NUM> is an example of a second flow channel in the present disclosed technology. The recessed part <NUM> has an inflow port 26A, an outflow port 26B, and a flow channel part 26C that makes the inflow port 26A and the outflow port 26B communicate with each other.

Similarly, a recessed part <NUM> that defines an upper micro flow channel <NUM> is formed on the lower surface of the upper cavity member <NUM>, that is, on an opposite surface 12A opposite to the lower cavity member <NUM>. The upper micro flow channel <NUM> is an example of a first flow channel in the present disclosed technology. The recessed part <NUM> has an inflow port 20A, an outflow port 20B, and a flow channel part 20C that makes the inflow port 20A and the outflow port 20B communicate with each other. In addition, through-holes 22A and 22B that penetrate the upper cavity member <NUM> in the thickness direction are provided in the upper cavity member <NUM>. The lower ends of the through-holes 22A and 22B respectively communicate with the inflow port 20A and the outflow port 20B.

The inflow port 26A and the outflow port 26B of the lower cavity member <NUM> are provided at positions where they do not overlap with the inflow port 20A and the outflow port 20B of the upper cavity member <NUM> in a case of being viewed in a plan view. On the other hand, the flow channel part 26C of the lower cavity member <NUM> is provided at a position where it overlaps with the flow channel part 20C of the upper cavity member <NUM> in a case of being viewed in a plan view.

Through-holes 28A and 28B, which penetrate the upper cavity member <NUM> in the thickness direction and of which lower ends respectively communicate with the inflow port 26A and the outflow port 26B of the lower cavity member <NUM>, are provided in the upper cavity member <NUM>. On the outer peripheral surface of the cavity unit <NUM>, recessed parts <NUM> are provided at positions where spacers <NUM> are arranged.

A porous membrane <NUM> is arranged between the opposite surfaces 12A and 14A of the upper cavity member <NUM> and lower cavity member <NUM>. An upper surface 30A and a lower surface 30B of the porous membrane <NUM> cover the flow channel parts 20C and 26C of the upper micro flow channel <NUM> and lower micro flow channel <NUM>, and they separate the upper micro flow channel <NUM> and the lower micro flow channel <NUM>. That is, the lower micro flow channel <NUM> and the upper micro flow channel <NUM> are adjacent to each other with the porous membrane <NUM> being interposed therebetween. Specifically, the upper surface 30A of the porous membrane <NUM> defines the upper micro flow channel <NUM> together with the recessed part <NUM> of the upper cavity member <NUM>, and the lower surface 30B of the porous membrane <NUM> defines the lower micro flow channel <NUM> together with the recessed part <NUM> of the lower cavity member <NUM>.

The porous membrane <NUM> is constituted to include, for example, a hydrophobic polymer that can be dissolved in a hydrophobic organic solvent. It is noted that the hydrophobic organic solvent is liquid of which the solubility in water at <NUM> is <NUM> (g/<NUM> water) or less. Examples of the hydrophobic polymer include polystyrene, polyacrylate, and polymethacrylate.

<FIG> is a plan view illustrating an example of the configuration of the porous membrane <NUM>. <FIG> is a cross-sectional view taken along a line 4B-4B in <FIG>. A plurality of intramembrane spaces <NUM> that penetrate the porous membrane <NUM> in the thickness direction are formed in the porous membrane <NUM>, and openings 32A of the intramembrane space <NUM> are provided on both surfaces of the upper surface 30A and the lower surface 30B of the porous membrane <NUM>. In addition, the opening 32A has a circular shape in a case of being viewed in a plan view. The openings 32A are provided to be spaced from each other, and a flat portion <NUM> extends between the openings 32A adjacent to each other. The shape of the opening 32A is not limited to a circular shape, and it may be a polygonal shape or an elliptical shape.

As illustrated in <FIG>, the plurality of openings 32A are arranged in a honeycomb shape. Here, the honeycomb-shaped arrangement refers to an arrangement in which six openings 32A are equally arranged around any opening 32A (excluding the opening 32A at the edge of the membrane), centers of the six openings 32A are located at the apexes of a regular hexagon, and the center of the opening 32A located at the centers of the six openings 32A corresponds to the center of the regular hexagon. The description "equally arranged" referred to herein does not necessarily mean that the openings 32A are arranged accurately at a central angle of <NUM>°, and it suffices that the surrounding six openings 32A are arranged at substantially equal spacings with respect to the opening 32A located at the center. It is noted that "the center(s) of the opening(s) 32A" means the center(s) of the opening(s) 32A in a case of being viewed in a plan view.

As illustrated in <FIG>, the intramembrane space <NUM> of the porous membrane <NUM> has a shape of spherical segment obtained by cutting an upper end and a lower end of a sphere. The sphere referred to herein does not have to be a true sphere, and it has a degree of distortion that is generally allowed to be recognized as a sphere. In addition, the intramembrane spaces <NUM> adjacent to each other have a lateral communication structure in which communication holes <NUM> communicate with each other in the inside of the porous membrane <NUM>. It is noted that the lateral communication structure refers to a space structure in which the adjacent intramembrane spaces <NUM> communicate with each other in the inside of the porous membrane <NUM>. The description "lateral" referred to herein means a plane direction orthogonal to the vertical direction in a case where the thickness direction of the porous membrane <NUM> is vertical. In the porous membrane <NUM>, since the openings 32A are arranged in a honeycomb shape, any intramembrane space <NUM> communicates with all of the six intramembrane spaces <NUM> that are equally arranged around the porous membrane <NUM>. It is noted that the intramembrane space <NUM> may have a barrel shape, a circular columnar shape, a polygonal columnar shape, or the like, and the communication hole <NUM> may be a tubular void that connects the adjacent intramembrane spaces <NUM> to each other.

The average opening diameter of the opening 32A is preferably <NUM> or more and <NUM> or less. In a case where the average opening diameter of the openings 32A is set to <NUM> or more, it is easy to form the lateral communication structure of the intramembrane space <NUM>. In addition, in a case where the average opening diameter of the openings 32A is et to <NUM> or less, it is easy to maintain a honeycomb-shaped arrangement without the adjacent openings 32A being fused with each other. It is noted that the average opening diameter means the average value of the diameters of the plurality of openings 32A on the surface of the porous membrane <NUM>. The average opening diameter can be, for example, an average value obtained by observing the surface of the porous membrane <NUM> under a microscope and measuring the diameters of a considerable number of openings 32A.

The void ratio of the porous membrane <NUM> is preferably <NUM>% or more and <NUM>% or less. In a case where the void ratio of the porous membrane <NUM> is set to <NUM>% or more, it is easy to form the lateral communication structure of the intramembrane space <NUM>. In a case where the void ratio of the porous membrane is set to <NUM>% to <NUM>% or less, it becomes easy to maintain the shape of the porous membrane <NUM>, and thus the strength does not decrease and the porous membrane <NUM> becomes difficult to be torn. It is noted that the void ratio refers to the ratio of the volume of the intramembrane space <NUM> with respect to the volume of the porous membrane <NUM>. This void ratio can be determined as a percentage which is obtained by, for example, observing the cross section of the porous membrane <NUM> under a microscope, and dividing the volume of the plurality of intramembrane spaces <NUM> by the volume of the porous membrane <NUM> in which the intramembrane spaces <NUM> are present, where the volume thereof has been determined by estimating that the observed intramembrane spaces <NUM> have a shape of spherical segment obtained by cutting two upper and lower sides and six lateral sides of a sphere.

The membrane thickness of the porous membrane <NUM> is preferably <NUM> or more and <NUM> or less. Here, the numerical value of this membrane thickness is a numerical value derived from the fact that practically, the aspect ratio of the opening diameter of the opening 32A to the height of the intramembrane space <NUM> (that is, the value obtained by dividing the opening diameter of the opening 32A by the height of the intramembrane space <NUM>) cannot exceed <NUM>. It is noted that in a case where a single-layer porous membrane <NUM> is used, the membrane thickness is preferably <NUM> to <NUM>. Further, in a case where a plurality of porous membranes <NUM> are laminated and used, the total membrane thickness of the porous membranes <NUM> is desirably <NUM> to <NUM>.

A microfluidic device <NUM> has a pair of holding plates <NUM> as holding members that hold the cavity unit <NUM> in a state of being compressed in the thickness direction. The pair of holding plates <NUM> are provided separately from the cavity unit <NUM> at both ends of the cavity unit <NUM> in the thickness direction, that is, on the upper side of the upper cavity member <NUM> and on the lower side of the lower cavity member <NUM>, and the sizes thereof are set to respectively cover the entire upper surface of the upper cavity member <NUM> and the entire lower surface of the lower cavity member <NUM>.

As illustrated in <FIG>, a plurality (eight in present embodiment) of bolt holes <NUM> are respectively formed at corresponding positions in the pair of holding plate <NUM>, where bolt holes <NUM> penetrate the holding plates <NUM> in the thickness direction. The holding plate <NUM> provided on the upper side of the upper cavity member <NUM> has the through-holes 22A, 22B, 28A, and 28B that respectively communicate with the through-holes 42A, 42B, 44A, and 44B of the upper cavity member <NUM>.

As illustrated in <FIG>, inflow tubes 62A and 64A are respectively connected to the through-holes 42A and 44A, and outflow tubes 62B and 64B are respectively connected to the through-holes 42B and 44B. Various treatment liquids and cell suspensions flow into the upper micro flow channel <NUM> and the lower micro flow channel <NUM> through the inflow tubes 62A and 64A. The various solutions and cell suspensions that have passed through the upper micro flow channel <NUM> and the lower micro flow channel <NUM> flow out from the outflow tubes 62B and 64B.

A plurality (eight in present embodiment) of the spacers <NUM> that define spacings between the holding plates <NUM> are provided outside the recessed part <NUM> of the cavity unit <NUM> between the pair of the holding plates <NUM>. The spacers <NUM> are cylindrical members having an inner diameter substantially the same as an inner diameter of the bolt hole <NUM> and are disposed at positions corresponding to the bolt holes <NUM>, respectively.

The pair of holding plates <NUM> are joined to each other by a plurality of bolts <NUM> that are inserted into the bolt holes <NUM> and the spacers <NUM> and fixed by nuts <NUM>. At this time, the upper cavity member <NUM> and the lower cavity member <NUM> are compressed and held in a state where the porous membrane <NUM> are sandwiched therebetween, by the pair of holding plates <NUM>.

<FIG> is a view illustrating an example of a configuration of an evaluation system <NUM> that is used for evaluating the permeability of the porous membrane according to the embodiment of the present disclosed technology. The evaluation system <NUM> is constituted to include a flow rate control device <NUM>, a storage unit <NUM>, and a flow rate sensor <NUM> in addition to the microfluidic device <NUM>.

The storage unit <NUM> stores a liquid <NUM> that is supplied to the upper micro flow channel <NUM> of the microfluidic device <NUM>. The tip part of the inflow tube 62A connected to the upper micro flow channel <NUM> is inserted into the liquid <NUM> stored in the storage unit <NUM>.

The flow rate control device <NUM> has a function of controlling the flow rate (the volume per unit time) of the liquid <NUM> that is supplied to the upper micro flow channel <NUM> of the microfluidic device <NUM>. One end of an air supply tube <NUM> is connected to an exhaust port <NUM> of the flow rate control device <NUM>, and the other end of the air supply tube <NUM> is connected to a gas introduction port <NUM> of the storage unit <NUM>. In a case where the gas is discharged from the exhaust port <NUM> of the flow rate control device <NUM>, the pressure inside the storage unit <NUM> rises, and thus the liquid <NUM> stored in the storage unit <NUM> is supplied to the upper micro flow channel <NUM>. The flow rate control device <NUM> controls the flow rate of the liquid <NUM> that is supplied to the upper micro flow channel <NUM> by controlling the pressure (hereinafter, referred to as the supply pressure) of the gas that is discharged from the exhaust port <NUM>. The supply pressure is a pressure against the liquid surface of the liquid <NUM> stored in the storage unit <NUM>. The supply pressure can be freely set by a user, and the supply pressure is continuously changed. As the flow rate control device <NUM>, for example, ELVEFLOW (registered trade name) manufactured by ELVESYS can be used.

The flow rate sensor <NUM> is connected to the outflow tube 64B connected to the lower micro flow channel <NUM>. The flow rate sensor <NUM> detects the flow rate of the liquid that flows through the lower micro flow channel <NUM> and outputs the detected flow rate.

The outflow tube 62B connected to the upper micro flow channel <NUM> and the inflow tube 64A connected to the lower micro flow channel <NUM> are each in a closed state.

Hereinafter, an evaluation method for the permeability of the porous membrane <NUM> according to the embodiment of the present disclosed technology, using the evaluation system <NUM>, will be described. <FIG> is a view schematically illustrating a flow channel configuration of the evaluation system <NUM>. <FIG> is a flowchart illustrating an example of an evaluation method for permeability of the porous membrane <NUM> according to an embodiment of the present disclosed technology.

First, the upper micro flow channel <NUM> and the lower micro flow channel <NUM> are each filled with a liquid (a step S1). Then, the flow rate control device <NUM> is operated. The supply pressure of the flow rate control device <NUM> is set so that it changes chronologically. That is, the liquid <NUM> is supplied to the upper micro flow channel <NUM> while the supply pressure is being changed (a step S2). As the supply pressure changes chronologically, the flow rate of the liquid <NUM> that is supplied from the storage unit <NUM> to the upper micro flow channel <NUM> changes. As the liquid <NUM> is supplied to the upper micro flow channel <NUM> filled with the liquid in advance, the liquid accommodated in the upper micro flow channel <NUM> permeates through the porous membrane <NUM> and flows out to the lower micro flow channel <NUM>. As a result, a liquid flow is generated in the lower micro flow channel <NUM>. The flow rate of the liquid that flows through the lower micro flow channel <NUM> changes in response to the supply pressure in the flow rate control device <NUM> and also becomes dependent on the permeability of the porous membrane <NUM>. Next, the flow rate sensor <NUM> acquires, as an evaluation indicator, a chronological change in the flow rate of the liquid that flows through the lower micro flow channel <NUM> (a step S3).

<FIG> is a graph illustrating an example of a time course of supply pressure in the flow rate control device <NUM>. As illustrated in <FIG>, the supply pressure may be set to change linearly in time. <FIG> is a graph illustrating an example of a time course of the flow rate of the liquid that flows through the lower micro flow channel <NUM> in a case where the supply pressure is linearly changed in time. In <FIG>, the solid line corresponds to a case where the permeability of the porous membrane <NUM> is relatively high, and the dotted line corresponds to a case where the permeability of the porous membrane <NUM> is relatively low. In a case where the permeability of the porous membrane <NUM> is relatively high, the rate of change (the slope) of the flow rate of the liquid that flows through the lower micro flow channel <NUM> becomes higher than that in a case where the permeability of the porous membrane <NUM> is relatively low. As a result, it is possible to evaluate the permeability of the porous membrane <NUM> by monitoring the flow rate of the liquid that flows through the lower micro flow channel <NUM>.

A graph showing the time course of the flow rate, as illustrated in <FIG>, may be acquired as the chronological change of the flow rate of the liquid that flows through the lower micro flow channel <NUM>, which is acquired in the step S3. In addition, a rate of change (the slope) of the flow rate of the liquid that flows through the lower micro flow channel <NUM> may be acquired as the chronological change of the flow rate of the liquid that flows through the lower micro flow channel <NUM>. Specifically, in a case where the amount of change in the flow rate of the liquid that flows through the lower micro flow channel <NUM> in the period ΔT is denoted by ΔQ, ΔQ/Δt may be acquired as the above rate of change (the slope). Further, in a case where the flow rate of the liquid that flows through the lower micro flow channel <NUM> is denoted by Q<NUM> in a case where the supply pressure is P<NUM>, Q<NUM>/P<NUM> may be acquired as a chronological change in the flow rate of the liquid that flows through the lower micro flow channel <NUM>. In addition, in a case where the flow rates of the liquids that flow through the lower micro flow channel <NUM> at different supply pressures P<NUM>, P<NUM>,. , Pn are respectively denoted by Q<NUM>, Q<NUM>,. , Qn, the average value of Q<NUM>/P<NUM>, Q<NUM>/P<NUM>,. , Qn/Pn may be acquired as a chronological change of the flow rate of the liquid that flows through the lower micro flow channel <NUM>.

<FIG> is a graph showing an example of results of evaluating the permeability of a plurality of kinds of porous membranes having different pore diameters or opening ratios by using the evaluation method according to the embodiment of the present disclosed technology. That is, <FIG> a graph showing, regarding each of the plurality of kinds of porous membranes A to I, the time course of the flow rate of the liquid that flows through the lower micro flow channel <NUM> in a case where the supply pressure is linearly changed in time. The outlines of the porous membranes A to I are summarized in Table <NUM> below.

<FIG> shows results that the larger the pore diameter of the porous membrane and the larger the opening ratio of the porous membrane, the larger the rate of change (the slope) of the flow rate of the liquid that flows through the lower micro flow channel <NUM>. It is noted that in the porous membrane F and the porous membrane G, the change in the flow rate with respect to the time change is non-linear since an unintended leak has occurred in the flow channel.

As described above, in the evaluation method for permeability of a porous membrane according to the embodiment of the present disclosed technology, the change that occurs in the liquid accommodated in the lower micro flow channel <NUM> is acquired as an evaluation indicator of the permeability of the porous membrane in a case where the liquid is supplied to the upper micro flow channel <NUM> while the supply pressure is being changed. In the present embodiment, a chronological change in the flow rate of the liquid that flows through the lower micro flow channel <NUM> is acquired as "the change that occurs in the liquid accommodated in the lower micro flow channel <NUM>".

According to the evaluation method according to the embodiment of the present disclosed technology, it is possible to evaluate the permeability of the porous membrane in a short time as compared with, for example, a method of monitoring an amount of light radiated from a phosphor that leaks into the second flow channel, where a liquid containing a phosphor is accommodated in the upper micro flow channel <NUM>, a liquid containing no phosphor is accommodated in the lower micro flow channel <NUM>, and the phosphor diffuses in a liquid and permeates through a porous membrane to leak to the lower micro flow channel <NUM>.

<FIG> is a view illustrating an example of an evaluation system 200A according to the second embodiment of the present disclosed technology. <FIG> is a view schematically illustrating a flow channel configuration of the evaluation system 200A. The evaluation system 200A includes a light amount sensor <NUM> instead of the flow rate sensor <NUM> in the evaluation system <NUM> according to the first embodiment.

Hereinafter, an evaluation method for the permeability of the porous membrane <NUM> according to the second embodiment of the present disclosed technology, using the evaluation system 200A, will be described. <FIG> is a flowchart illustrating an example of an evaluation method for permeability of the porous membrane <NUM> according to the second embodiment of the present disclosed technology.

First, the upper micro flow channel <NUM> is filled with a liquid containing a phosphor (a step S11). Next, the lower micro flow channel <NUM> is filled with a liquid containing no phosphor (a step S12). Then, the flow rate control device <NUM> is operated. The supply pressure of the flow rate control device <NUM> is set so that it changes chronologically. The liquid <NUM> containing a phosphor is accommodated in the storage unit <NUM>. That is, the liquid <NUM> that contains a phosphor is supplied to the upper micro flow channel <NUM> while the supply pressure is being changed (a step S13). As the supply pressure changes, the flow rate of the liquid <NUM> that is supplied from the storage unit <NUM> to the upper micro flow channel <NUM> changes. As the liquid <NUM> that contains a phosphor is supplied to the upper micro flow channel <NUM> filled with the liquid that contains a phosphor in advance, the liquid that contains a phosphor, which is accommodated in the upper micro flow channel <NUM>, permeates through the porous membrane <NUM> and flows out to the lower micro flow channel <NUM>. As a result, a liquid flow due to the liquid that contains a phosphor is generated in the lower micro flow channel <NUM>. The liquid that flows through the lower micro flow channel <NUM> is irradiated with excitation light from a light source, which is not illustrated in the drawing. As a result, light is radiated from the phosphor contained in the liquid that flows through the lower micro flow channel <NUM>. The rate of change in the amount of light (hereinafter, referred to as the fluorescent light amount) radiated from the phosphor contained in the liquid that flows through the lower micro flow channel <NUM> changes in response to the supply pressure in the flow rate control device <NUM> and also becomes dependent on the permeability of the porous membrane <NUM>. Next, the light amount sensor <NUM> acquires the chronological change of the fluorescent light amount as an evaluation indicator (a step S14).

<FIG> is a graph illustrating an example of a time course of a fluorescent light amount in a case where the supply pressure is linearly changed in time. In <FIG>, the solid line corresponds to a case where the permeability of the porous membrane <NUM> is relatively high, and the dotted line corresponds to a case where the permeability of the porous membrane <NUM> is relatively low. In a case where the permeability of the porous membrane <NUM> is relatively high, the rate of change (the slope) of the fluorescent light amount becomes higher than that in a case where the permeability of the porous membrane <NUM> is relatively low. As a result, it is possible to evaluate the permeability of the porous membrane <NUM> by monitoring the fluorescent light amount.

A graph showing the time course of the fluorescent light amount, as illustrated in <FIG>, may be acquired as the chronological change of the fluorescent light amount, which is acquired in the step S14. In addition, the rate of change (the slope) of the fluorescent light amount may be acquired as the chronological change of the fluorescent light amount. Specifically, in a case where the amount of change of the fluorescent light amount in the period ΔT is denoted by ΔL, ΔL/Δt may be acquired as the above rate of change (the slope). In addition, in a case where the fluorescent light amount is denoted by Li in a case where the supply pressure is denoted by P<NUM>, L<NUM>/P<NUM> may be acquired as a chronological change of the fluorescent light amount. In a case where the fluorescent light amounts at different supply pressures P<NUM>, P<NUM>, ···, Pn are respectively denoted by L<NUM>, L<NUM>, ···, Ln, the average value of L<NUM>/P<NUM>, L<NUM>/P<NUM>, ···, Ln/Pn may be acquired as a chronological change in the fluorescent light amount.

As described above, in the present embodiment, a chronological change in the amount of light radiated from the phosphor contained in the liquid that flows through the lower micro flow channel <NUM> is acquired as "the change that occurs in the liquid accommodated in the lower micro flow channel <NUM>". According to the evaluation method according to the present embodiment, it is possible to evaluate the permeability of the porous membrane in a short time as in the evaluation method according to the first embodiment.

<FIG> is a view illustrating an example of an evaluation system 200B according to the third embodiment of the present disclosed technology. <FIG> is a view schematically illustrating a flow channel configuration of the evaluation system 200B. The evaluation system 200B includes a concentration sensor <NUM> instead of the flow rate sensor <NUM> in the evaluation system <NUM> according to the first embodiment.

Hereinafter, an evaluation method for the permeability of the porous membrane <NUM> according to the third embodiment of the present disclosed technology, using the evaluation system 200B, will be described. <FIG> is a flowchart illustrating an example of an evaluation method for permeability of the porous membrane <NUM> according to the third embodiment of the present disclosed technology.

First, the upper micro flow channel <NUM> is filled with a liquid containing a specific component (a step S21). Next, the lower micro flow channel <NUM> is filled with a liquid containing no specific component (a step S22). Then, the flow rate control device <NUM> is operated. The supply pressure of the flow rate control device <NUM> is set so that it changes chronologically. The liquid <NUM> containing a specific component is accommodated in the storage unit <NUM>. That is, the liquid <NUM> that contains a specific component is supplied to the upper micro flow channel <NUM> while the supply pressure is being changed (a step S23). As the supply pressure changes, the flow rate of the liquid <NUM> that is supplied from the storage unit <NUM> to the upper micro flow channel <NUM> changes. As the liquid <NUM> that contains a specific component is supplied to the upper micro flow channel <NUM> filled with the liquid that contains a specific component in advance, the liquid that contains a specific component, which is accommodated in the upper micro flow channel <NUM>, permeates through the porous membrane <NUM> and flows out to the lower micro flow channel <NUM>. As a result, a liquid flow due to the liquid that contains a specific component is generated in the lower micro flow channel <NUM>. The rate of change in the concentration of the specific component in the liquid that flows through the lower micro flow channel <NUM> (hereinafter, referred to as the specific component concentration) changes in response to the supply pressure in the flow rate control device <NUM> and also becomes dependent on the permeability of the porous membrane <NUM>. Next, the concentration sensor <NUM> acquires the chronological change of the specific component concentration as an evaluation indicator (a step S24). It is noted that the specific component may be any substance that can be quantified, and examples thereof include a dye, a conductive substance, an enzyme, a nanoparticle, a substance containing a radioisotope, a nucleic acid, and a sugar chain. Most substances can be quantified by using techniques such as liquid chromatography.

<FIG> is a graph illustrating an example of a time course of a specific component concentration in a case where the supply pressure is linearly changed in time. In <FIG>, the solid line corresponds to a case where the permeability of the porous membrane <NUM> is relatively high, and the dotted line corresponds to a case where the permeability of the porous membrane <NUM> is relatively low. In a case where the permeability of the porous membrane <NUM> is relatively high, the rate of change (the slope) of the specific component concentration becomes higher than that in a case where the permeability of the porous membrane <NUM> is relatively low. As a result, it is possible to evaluate the permeability of the porous membrane <NUM> by monitoring the specific component concentration.

A graph showing the time course of the specific component concentration, as illustrated in <FIG>, may be acquired as the chronological change of the specific component concentration, which is acquired in the step S24. In addition, the rate of change (the slope) of the specific component concentration may be acquired as the chronological change of the specific component concentration. Specifically, in a case where the amount of change of the specific component concentration in the period ΔT is denoted by ΔC, ΔC/Δt may be acquired as the above rate of change (the slope). In addition, in a case where the specific component concentration is denoted by Ci in a case where the supply pressure is denoted by P<NUM>, C<NUM>/P<NUM> may be acquired as a chronological change of the specific component concentration. In a case where the specific component concentration at different supply pressures P<NUM>, P<NUM>, ···, Pn are respectively denoted by C<NUM>, C<NUM>, ···, Cn, the average value of C<NUM>/P<NUM>, C<NUM>/P<NUM>, ···, Cn/Pn may be acquired as a chronological change in the specific component concentration.

According to the evaluation method according to the present embodiment, it is possible to evaluate the permeability of the porous membrane in a short time as in the evaluation method according to the first embodiment.

<FIG> are respectively a flow channel configuration view and a flowchart, illustrating an example of a cell evaluation method according to the fourth embodiment of the present disclosed technology.

The cell evaluation method according to the present embodiment includes culturing cells to be evaluated on the surface of the porous membrane <NUM> of the microfluidic device <NUM> (a step S31). For example, endothelial cells <NUM> may be cultured on the surface of the porous membrane <NUM> on the side of the upper micro flow channel <NUM>, and smooth muscle cells <NUM> may be cultured on the surface of the porous membrane <NUM> on the side of the lower micro flow channel <NUM>. This makes it possible to form a structure that simulates the blood vessel (the artery) in the microfluidic device <NUM>. The endothelial cells <NUM> and the smooth muscle cells <NUM> are cultured in a state of being immersed in the culture solution accommodated in the upper micro flow channel <NUM> and the lower micro flow channel <NUM>, respectively. It is noted that the cells to be evaluated may be cultured only on one surface of the porous membrane <NUM>.

The cell evaluation method according to the present embodiment includes acquiring the evaluation indicator of the permeability of the porous membrane <NUM> according to any one of the first to third embodiments described above, as an indicator of the barrier property of cells to be evaluated, which are cell cultured on the surface of the porous membrane <NUM>. That is, the change that occurs in the liquid accommodated in the lower micro flow channel <NUM> is acquired as an indicator of the barrier property of cells to be evaluated, in a case where the liquid is supplied to the upper micro flow channel <NUM> while the supply pressure is being changed (a step S32). Here, the barrier property of cells means the performance of cells to be evaluated, which are cultured on the surface of the porous membrane <NUM>, where the performance is the blocking of leakage of the liquid that is supplied to the upper micro flow channel <NUM> to the lower micro flow channel <NUM>.

In a case where cells cultured on the surface of the porous membrane <NUM> are healthy, the barrier property of cells suppresses the outflow of the liquid from the upper micro flow channel <NUM> to the lower micro flow channel <NUM>. On the other hand, in a case where an abnormality occurs in cells cultured on the surface of the porous membrane <NUM> and the barrier property of cells decreases, the outflow amount of the liquid from the upper micro flow channel <NUM> to the lower micro flow channel <NUM> increases. As a result, it is possible to use the evaluation indicator of the permeability of the porous membrane <NUM> according to the first to third embodiments described above, as an indicator of the barrier property of cells to be evaluated, which are cells cultured on the surface of the porous membrane <NUM>.

For example, in a case where the evaluation indicator of the permeability of the porous membrane <NUM> according to the first embodiment is used as an indicator of the barrier property of cells to be evaluated, the chronological change of the flow rate of the liquid that flows through the lower micro flow channel is used as an indicator of the barrier property of cells to be evaluated, in a case where the liquid is supplied to the upper micro flow channel <NUM> while the supply pressure is being changed.

According to the cell evaluation method according to the present embodiment, it is possible to evaluate the barrier property of cells in a short time as compared with, for example, a method of monitoring an amount of light radiated from a phosphor that leaks into the second flow channel, where a liquid containing a phosphor is accommodated in the upper micro flow channel <NUM>, a liquid containing no phosphor is accommodated in the lower micro flow channel <NUM>, and the phosphor diffuses in a liquid and permeates through a porous membrane to leak to the lower micro flow channel <NUM>.

<FIG> is a flowchart illustrating an example of a drug evaluation method according to the fifth embodiment of the present disclosed technology.

The drug evaluation method according to the present embodiment includes culturing cells on the surface of the porous membrane <NUM> of the microfluidic device <NUM> (a step S41). For example, as illustrated in <FIG>, the endothelial cells <NUM> may be cultured on the surface of the porous membrane <NUM> on the side of the upper micro flow channel <NUM>, and the smooth muscle cells <NUM> may be cultured on the surface of the porous membrane <NUM> on the side of the lower micro flow channel <NUM>. This makes it possible to form a structure that simulates the blood vessel (the artery) in the microfluidic device <NUM>. It is noted that the cells to be evaluated may be cultured only on one surface of the porous membrane <NUM>.

The drug evaluation method according to the present embodiment includes exposing cells cultured on the surface of the porous membrane <NUM> to a drug to be evaluated (a step S42). That is, a liquid containing a drug to be evaluated is supplied to each of the upper micro flow channel <NUM> and the lower micro flow channel <NUM>.

The drug evaluation method according to the present embodiment includes acquiring the evaluation indicator of the permeability of the porous membrane <NUM> according to any one of the first to third embodiments described above as an indicator of toxicity of the drug to be evaluated to the cells. That is, the change that occurs in the liquid accommodated in the lower micro flow channel <NUM> is acquired as an indicator of toxicity of the drug to be evaluated to the cells, in a case where the liquid is supplied to the upper micro flow channel <NUM> while the supply pressure is being changed (a step S43).

In a case where cells cultured on the surface of the porous membrane <NUM> are healthy, the barrier property of these cells suppresses the outflow of the liquid from the upper micro flow channel <NUM> to the lower micro flow channel <NUM>. In a case where the drug to be evaluated has toxicity to cells cultured on the surface of the porous membrane <NUM> and thus the barrier property of the cells decreases, the outflow amount of the liquid from the upper micro flow channel <NUM> to the lower micro flow channel <NUM> increases in a case where an abnormality occurs in the cells. As a result, it is possible to use the evaluation indicator of the permeability of the porous membrane <NUM> according to the first to third embodiments described above, as an indicator of toxicity of the drug to be evaluated to the cells.

For example, in a case where the evaluation indicator of the permeability of the porous membrane <NUM> according to the first embodiment is acquired as an indicator of the toxicity of the drug to to be evaluated to the cells, the chronological change of the flow rate of the liquid that flows through the lower micro flow channel is acquired as an indicator of toxicity of the drug to be evaluated to the cells, in a case where the liquid is supplied to the upper micro flow channel <NUM> while the supply pressure is being changed.

<FIG> is a graph showing an example of results of evaluating the toxicity of drugs by using the evaluation method according to the present embodiment. That is, <FIG> a graph showing, regarding each of the liquids to be evaluated A to E, the time course of the flow rate of the liquid that flows through the lower micro flow channel <NUM> in a case where the supply pressure is linearly changed in time. The outlines of the liquids A to E are summarized in Table <NUM> below. The liquid A and the liquid B are media obtained by adding dimethyl sulfoxide (DMSO) to a basal medium as a solvent, where the media contains cytochalasin having a concentration of <NUM>µg/ml, which is a drug to be evaluated. The liquid C and the liquid D are media obtained by adding DMSO to a basal medium as a solvent, and they do not contain a drug to be evaluated. The liquid E contains a basal medium and does not contain DMSO and a drug to be evaluated. It is noted that the endothelial cells <NUM> were cultured on the surface of the porous membrane <NUM> on the side of the upper micro flow channel <NUM>, and the smooth muscle cells <NUM> were cultured on the surface of the porous membrane <NUM> on the side of the lower micro flow channel <NUM>. As the porous membrane <NUM>, Millipore <NUM> (pore diameter: <NUM>) was used.

In a case where cells were exposed to the liquid A and the liquid B, containing cytochalasin as a drug, the rate of change (the slope) of the flow rate of the liquid that flows through the lower micro flow channel <NUM> was significantly larger than those of Vehicle (the liquid C and the liquid D) and Control (the liquid E). This indicates that the cytochalasin contained in the liquid A and the liquid B has toxicity to the endothelial cells <NUM> and the smooth muscle cells <NUM>.

Claim 1:
An evaluation method for permeability of a porous membrane (<NUM>) that is inserted between a first flow channel (<NUM>) and a second flow channel (<NUM>), the evaluation method comprising:
acquiring a change that occurs inside a liquid accommodated in the second flow channel (<NUM>) as an evaluation indicator of permeability of the porous membrane (<NUM>) in a case of supplying a liquid to the first flow channel (<NUM>) while continuously changing a supply pressure.