CELL EVALUATION DEVICE

The cell evaluation device includes a cell housing unit 111, and an adapter 120 configured to be elastically deformed by being fitted into the cell housing unit to close a top opening of the cell housing unit, and the adapter includes a body 121 to be inserted into the cell housing unit, a positioning unit 122 configured to position the body in a vertical direction with respect to the cell housing unit, a culture medium inlet port 124 and a culture medium outlet port 125 penetrating the top end and the lower end of the body, and a flexible sheet member 123 which is provided on a lower end of the body and extends radially outward from an outer edge of the lower end. This makes it possible to prevent leakage of the culture medium in the cell evaluation device.

TECHNICAL FIELD

The present invention relates to a cell evaluation device.

BACKGROUND ART

Conventionally, a method for noninvasively evaluating cultured cells has been known in which a culture supernatant of cultured cells is collected and the components of the culture supernatant are analyzed. For example, the differentiation state of cultured cells can be evaluated by adding a growth factor to a culture medium of the cultured cells, which are stem cells, to induce differentiation, and then measuring the amount of a predetermined compound (specifically, a compound whose amount in the culture supernatant is known to change between the differentiated state and the undifferentiated state) in the culture supernatant. Moreover, the resistance, sensitivity, metabolic capacity, or the like of cultured cells to a drug can be evaluated by adding a predetermined drug to a culture medium of a predetermined cultured cell and then quantitatively analyzing components contained in the culture supernatant at a predetermined timing. Hereinafter, a structure in which cells to be evaluated in the evaluation of such cultured cells are housed will be referred to as a cell evaluation device.

When such cultured cells are evaluated, it is necessary to inoculate target cells in the cell evaluation device and then culture the cells in the cell evaluation device over a predetermined period. During culture, waste products accumulate in the culture medium (culture solution) in the cell evaluation device over time, and thus it is necessary to periodically refresh the culture medium. When the culture medium is refreshed, it is necessary to move the cell evaluation device to a sterile environment, remove the old culture medium from the cell evaluation device under the sterile environment, and add a new culture medium into the cell evaluation device.

Patent Literature 1 describes an adapter for enabling automatic refreshment of a culture medium by attaching the adapter to a culture vessel (such as a petri dish or a well of a multi-hole plate) in order to save time and effort for refreshment of the culture medium in cell culture. This adapter is used after being fitted into a top opening of a culture vessel, and includes a culture medium inlet port through which a culture medium is put into the culture vessel, and a culture medium outlet port through which a culture medium in the culture vessel is put out. A tapered face is formed on at least a part of an outer periphery of the adapter, so that the adapter can be easily fitted into the culture vessel by causing the tapered face to slide along the inner face of the culture vessel, and the elastically deformed adapter can be easily held in the culture vessel. Furthermore, by setting the angle of the tapered face with respect to the axis of the adapter to an appropriate value, a gap is less likely to be generated between the tapered face and the inner face of the culture vessel, and it becomes possible to suppress entry of the culture medium into the gap.

The culture medium inlet port of the adapter is connected with a first pipe connected to a culture medium supply unit, and a new culture medium is automatically supplied into the culture vessel through the first pipe. On the other hand, the culture medium outlet port of the adapter is connected with a second pipe connected to a waste liquid reservoir, and a culture medium discharged from the culture vessel with the supply of the new culture medium is discharged to the waste liquid reservoir through the second pipe.

Accordingly, it is possible to save time and effort for culture medium refreshment and reduce a burden on an operator for evaluating the cultured cells by applying such an adapter to the cell evaluation device described above.

CITATION LIST

Patent Literature

Patent Literature 1: JP 3196673 U

Technical Problem

However, in the adapter described in Patent Literature 1, particularly in a case where the size of the culture vessel is small, there is a possibility that sealing of a space between the inner peripheral face of the culture vessel and the outer peripheral face of the adapter becomes insufficient due to dimensional errors of the culture vessel and the adapter, and leakage of the culture medium may occur.

The present invention has been made in view of the above problems, and an object of the invention is to reliably prevent leakage of a culture medium in a cell evaluation device including an adapter having a configuration described above.

Solution to Problem

A cell evaluation device according to the present invention, which has been made to solve one or more of the above problems, includes:

a concave cell housing unit having a circular bottom face and a side face defining a circular top opening; and

an adapter configured to be elastically deformed by being fitted into the cell housing unit to close the top opening, and

the adapter includes:

a body which has a circular lower end and is configured to be inserted into the cell housing unit;

a positioning unit configured to position the body in a vertical direction with respect to the cell housing unit;

a culture medium inlet port which has a through hole penetrating a top end and the lower end of the body;

a culture medium outlet port which has a through hole penetrating a top end and the lower end of the body; and

a flexible sheet member which is provided on the lower end of the body and extends radially outward from an outer edge of the lower end.

Advantageous Effects of Invention

According to a cell evaluation device according to the present invention, when the adapter is fitted into the cell housing unit, the sheet member provided on the lower end of the body is to abut on the inner peripheral face of the cell housing unit and is bent upward, and the outer peripheral part of the sheet member is brought into close contact with the inner peripheral face of the cell housing unit. As a result, the space between the adapter and the cell housing unit is sealed, and it becomes possible to prevent the culture medium in the cell housing unit from leaking to the outside.

DESCRIPTION OF EMBODIMENTS

First Embodiment

The following description will explain a cell evaluation device according to a first embodiment of the present invention with reference toFIGS. 1 to 5.

FIG. 1is a schematic configuration diagram of a cell culture system including a cell evaluation device100according to the present embodiment. In this cell culture system, a liquid feeding pump133supplies a liquid medium (which will be hereinafter simply referred to as a culture medium) in a culture medium storage container131into the cell evaluation device100through first pipe132, so that a culture medium pushed out of the cell evaluation device100accordingly is housed in a culture medium collecting unit135through second pipe134. The operation of the liquid feeding pump133is controlled by a control unit136including, for example, a personal computer, a programmable logic controller (PLC), or the like.

The cell evaluation device100includes a multi-hole plate110, which is also an example of a cell housing plate according to an embodiment of the present invention, and an adapter120to be attached to one well111(which is an example of a cell housing unit according to an embodiment of the present invention) among a plurality of wells provided in the multi-hole plate110.FIGS. 2 and 3illustrate cross-sectional views of the multi-hole plate110and the adapter120.FIG. 2illustrates a state before the adapter120is attached to the well111, andFIG. 3illustrates a state after the adapter120is attached to the well111. The well111is a recess having a circular bottom face112and a side face113defining a circular top opening, and the adapter120is attached to the well111by being fitted into the top opening of this well111.

FIG. 4illustrates a plan view of the adapter120, andFIG. 5illustrates a front view of the adapter120. The adapter120includes a truncated cone-shaped body121having a diameter decreasing downward, three retaining units122protruding from an upper outer peripheral face of the body121at equal angular intervals in a radial direction (horizontal direction), and a circular sheet member123attached to a lower end of the body121. In the sheet member123, a part in contact with the lower end of the body121is an example of a central portion according to an embodiment of the present invention, and a part located at an outer position than the outer edge of the lower end of the body121is an example of a flange portion according to an embodiment of the present invention.

In the embodiment, the number of the retaining units122is not limited to three, and may be two, or four or more. Alternatively, a single retaining unit122formed in a flange shape may also be provided.

In the adapter120, at least the body121and the sheet member123are made of a material (elastic body) having elasticity. Although the retaining unit122is not necessarily made of an elastic body, it is desirable that the entire adapter120is made of the same material (accordingly, an elastic body) so as to facilitate manufacturing. For example, polydimethylsiloxane (PDMS) can be used as the elastic body. Since PDMS is a material having gas permeability, when at least the body121and the sheet member123are made of PDMS, gas exchange can be performed between the inside and the outside of the well111even in a state where the body121and the sheet member123are fitted into the well111. In a case where the entire adapter120is made of PDMS, the adapter120can be sterilized by the autoclave and used repeatedly, so that the running cost can be suppressed. The elastic body is not limited to PDMS, and may be any elastomer such as silicone rubber, for example.

As described above, since the body121of the adapter120has a truncated cone shape having a diameter decreasing downward, the outer peripheral face of the body121forms a tapered face inclined with respect to a central axis X of the body121. Furthermore, the lower end of the body121has a diameter smaller than the top opening of the well111, and the upper face of the body121has a diameter larger than the top opening of the well111. Therefore, by inserting the adapter120into the well111with the outer peripheral face of the body121of the adapter120moved along the inner face of the well111, the body121of the adapter120can be easily fitted into the well111, and the body121can be elastically deformed to fix the body121to the well111by the elastic force. The distance from the central axis X of the body121of the adapter120to the tip of each retaining unit122is larger than the radius of the top opening of the well111. Therefore, when the body121of the adapter120is fitted into the well111, the lower end (lower face) of each retaining unit122abuts on the upper end of the side face113of the well111(peripheral edge of the top opening of the well111), so that the positional relationship between the bottom face of the well111and the lower face of the adapter120can be kept constant (that is, the depth of the culture medium in the well111is defined). In the present embodiment, the retaining unit122corresponds to a positioning unit in the present invention.

Furthermore, two through holes extending from the upper face to the lower face of the body121are formed in the body121of the adapter120. Hereinafter, one through hole will be referred to as a culture medium inlet port124, and the other through hole will be referred to as a culture medium outlet port125. An upper end of the culture medium inlet port124is connected with the first pipe132connected to the culture medium storage container131via the liquid feeding pump133, and an upper end of the culture medium outlet port125is connected with the second pipe134leading to the culture medium collecting unit135. In the sheet member123, an inlet hole128and an outlet hole129are formed respectively at positions corresponding to the lower end of the culture medium inlet port124and the lower end of the culture medium outlet port125.

As illustrated inFIG. 3, the diameter of the sheet member123is larger than the inner diameter Di of the well111at the height corresponding to the lower end of the body121when the body121of the adapter120is inserted into the well111until the lower end (lower face) of the retaining unit122abuts on the peripheral edge (upper end of the side face113) of the top opening of the well111. Although it is preferable that the diameter of the sheet member123is 1.2 times or less the inner diameter Di, the present invention is not limited to this. Although it is preferable that the thickness of the sheet member123is approximately 0.05 mm to 0.5 mm, the present invention is not limited to such values.

As illustrated inFIG. 3, when the body121of the adapter120is fitted into the well111, the outer peripheral part of the sheet member123abuts on the inner peripheral face of the well111, is bent upward, and is brought into close contact with the inner peripheral face. Since this outer peripheral part plays a role like an O-ring to seal the space between the adapter120and the well111, it is possible with the cell evaluation device100according to the present embodiment to prevent the culture medium housed in the well111from leaking to the outside.

Although the inner peripheral face of the well111may be a vertical face or a tapered face, in a case where the inner peripheral face is a tapered face, the inclination angle θ2of the tapered face with respect to the central axis (not shown) of the well111is set to be smaller than the inclination angle θ1of the tapered face of the body121of the adapter120with respect to the central axis X of the body121.

The adapter120described above can be produced by, for example, pouring liquid PDMS into a mold and curing the PDMS. The culture medium inlet port124and the culture medium outlet port125can be formed by previously setting tubes126and127made of silicone rubber or the like in the mold before pouring the PDMS.

A procedure for culturing and evaluating cells using the above cell culture system including such a cell evaluation device100will be described. First, an appropriate amount of culture medium is put into the well111under a sterile environment, and the target cells are inoculated in the culture medium. Then, after the adapter120is attached to the well111, the multi-hole plate110is moved to an incubator (thermostatic chamber) to start cell culture. At this time, the culture medium inlet port124and the culture medium outlet port125are respectively connected with the first pipe132and the second pipe134, and the culture medium in the culture medium storage container131is fed to the culture medium inlet port124by operating the liquid feeding pump133under the control of the control unit136. As a result, a new culture medium is supplied to the well111, and the culture medium in the well111is swept away and fed from the culture medium outlet port125to the culture medium collecting unit135through the second pipe134. As described above, according to a cell culture system including the cell evaluation device100of the present embodiment, since the culture medium in the cell evaluation device100is automatically refreshed, it is possible to save time and effort for manual refreshment of the culture medium. Such culture medium refreshment may be performed continuously by constantly driving the liquid feeding pump133, or may be performed intermittently by driving the liquid feeding pump133at regular time intervals.

After a predetermined period has elapsed from the start of the cell culture, a growth factor, a predetermined drug, or the like is added to the well111as necessary, and then the culture medium, that is, the culture supernatant is collected from the culture medium collecting unit135at a predetermined timing. The collected culture supernatant is subjected to analysis using a mass spectrometer, a liquid chromatograph, a gas chromatograph, or the like, and the cultured cells are evaluated on the basis of the result of the analysis. Specifically, for example, the differentiation state of stem cells can be evaluated by culturing stem cells in the well111, then adding a growth factor to the well111to induce the differentiation of the stem cells, then collecting and analyzing the culture supernatant, and finding the abundance of a predetermined compound in the culture supernatant. Alternatively, the sensitivity, resistance, metabolic capacity, or the like of a cell to a drug can be evaluated by culturing a predetermined cell in the well111, then adding a predetermined drug to the well111, and then collecting a culture supernatant and analyzing components in the culture supernatant.

Second Embodiment

The following description will explain a second embodiment of the present invention with reference toFIGS. 6 and 7. The same or corresponding components as those described in First Embodiment are denoted by the same reference numerals in the last two digits, and the description of such components is appropriately omitted.

FIG. 6is a schematic configuration diagram of a cell culture system including a cell evaluation device200according to the present embodiment. This cell culture system includes a resistance meter260(details will be described later) in addition to the same configuration as that in First Embodiment. The cell evaluation device200according to the present embodiment includes a multi-hole plate210and a cell culture insert (which will be hereinafter simply referred to as an insert240).FIG. 7illustrates a cross-sectional view of the multi-hole plate210, the insert240, and an adapter220in the present embodiment.

The insert240includes a truncated cone-shaped body portion241(which is an example of a cell housing unit according to an embodiment of the present invention) having a diameter decreasing downward, and a plurality of retaining units242which are provided at an upper portion of the body portion241and protrude radially from an outer periphery of the body portion241. The bottom face of the body portion241is made of a porous film243. This insert240is used in a state where the body portion241is positioned in a well211(which is an example of a recess according to an embodiment of the present invention) of the multi-hole plate210(which is an example of a culture vessel according to an embodiment of the present invention), and the retaining units242are retained to the upper face of the outer peripheral edge of the well211, that is, the body portion241is suspended inside the well211. Since a large number of minute through holes having an inner diameter of approximately 0.1 um to 10 um are formed in the porous film243constituting the bottom face of the insert240, a substance (a drug, a nutrient, oxygen, or the like) can move between the inside of the well211and the inside of the insert240through the porous film243.

The adapter220in the present embodiment is to be used after fitting a body221of the adapter into the top opening of the insert240, and includes a pair of electrodes (which will be referred to respectively as a first internal electrode251and a second internal electrode252) contained in the body221, in addition to a culture medium inlet port224, a culture medium outlet port225, a retaining unit222, and a sheet member223similar to those in First Embodiment.

Furthermore, the cell evaluation device200according to the present embodiment includes a pair of external electrodes (which will be referred to respectively as a first external electrode253and a second external electrode254) arranged in the well211of the multi-hole plate210(that is, between the inner peripheral face of the well211and the outer peripheral face of the insert240). The first internal electrode251and the first external electrode253are connected with current supply terminals261and264of the resistance meter260, and the second internal electrode252and the second external electrode254are connected with voltage measurement terminals262and263of the resistance meter260. Each of these electrodes251,252,253, and254corresponds to each terminal of a chopstick type electrode, and the first internal electrode251and the second internal electrode252are embedded in the adapter220with the end faces of the internal electrodes exposed to the lower face of the adapter220(that is, the lower end of the body221). The adapter220in the present embodiment is made of a material having elasticity and no conductivity. For example, PDMS can be suitably used as such a material.

The cell evaluation device200in the present embodiment can be suitably used for culturing and evaluating cells (e.g., intestinal epithelial cells) having a property of forming tight junctions between cells. When this cell evaluation device200cultures cells, a first culture solution and the target cells are housed in the insert240, and a second culture solution is housed in the well211(strictly speaking, between the well211and the insert240).

While cell culture is performed using the above cell culture system including such a cell evaluation device200, a culture medium in a culture medium storage container231sucked by a liquid feeding pump233is supplied to the inside of the insert240through first pipe232, and a culture medium swept away from the inside of the insert240accordingly is housed in a culture medium collecting unit235through second pipe234. Accordingly, also in the present embodiment, the refreshment of the culture medium in the insert240can be automatically performed.

After the start of the cell culture, the electric resistance is periodically or constantly measured using the resistance meter260to check the formation state of tight junctions between cells in the insert240. Specifically, the resistance meter260supplies a constant current across the first internal electrode251and the first external electrode253, so as to measure the voltage generated between the second internal electrode252and the second external electrode254. The resistance meter260finds a resistance value from the value of the supplied current and the value of the measured voltage, and displays the resistance value on a display unit (not shown) provided in the resistance meter260. The electric resistance found in such a manner includes the electric resistance of the porous film243constituting the bottom face of the insert240, and the electric resistance of the cells cultured on the porous film243. Among them, the electric resistance of the porous film243is constant, while the electric resistance of cells increases as the number of tight junctions formed between cells increases after the start of culture. Therefore, the value of the electric resistance measured by the resistance meter260gradually increases, and when sufficient tight junctions are formed between cells, the electric resistance does not increase any more. Accordingly, it can be determined that sufficient tight junctions have been formed between cells by checking that the measured value of the electric resistance by the resistance meter260becomes substantially constant.

After it is checked that sufficient tight junctions have been formed between cells as described above, the sensitivity, resistance, permeability, metabolic capacity, or the like of cells to the substance is evaluated by putting in a culture solution containing a predetermined substance instead of the first culture solution or the second culture solution, and observing changes in the cells, or collecting a culture supernatant in the insert240or a culture medium in the well211and analyzing components of the culture supernatant or the culture medium.

The cell evaluation device200according to the present embodiment may include terminals of sheet type electrodes instead of the chopstick type electrodes251,252,253, and254. Although the above example has illustrated a configuration in which the electric resistance is measured using a four-terminal measurement method, a cell evaluation device according to the present embodiment may measure the electric resistance using a two-terminal measurement method. In this case, a single electrode is each disposed inside the adapter220and between the well211and the insert240instead of the pair of electrodes.

Moreover, the adapter120having no internal electrode as illustrated inFIGS. 2 and 3may be applied to the combination of the multi-hole plate210(in a state where no external electrode is housed) and the insert240described above. In this case, although the electric resistance of the cells cannot be measured, a nutrient, oxygen, a drug, or the like can be moved between the inside of the insert240and the inside of the well211through the porous film243constituting the bottom face of the insert240as described above, and thus the cells can be cultured and evaluated under various conditions.

Although the retaining units122and222are provided in the upper portions of the bodies121and221of the adapters120and220in both the first and second embodiments, such a retaining unit may not necessarily be provided. Even in a case where a retaining unit is not provided, it is possible to keep the positional relationship between the bottom face of the cell housing unit and the lower end of the body of the adapter substantially constant when the body of the adapter is inserted into the cell housing unit, by defining an inclination angle (corresponding to the angle θ1inFIG. 2) of the peripheral face (tapered face) of the body with respect to the central axis X of the body of the adapter such that the lower end of the body stays at a position higher than the bottom face of the cell housing unit. That is, in this case, the peripheral face of the body of the adapter corresponds to the positioning unit in the present invention.

Although the circular sheet members123and223are attached to the lower ends of the bodies121and221of the adapters120and220in the first and second embodiments, an annular sheet member may be attached to the outer edge of the lower end of the body121or221instead.

The above description has explained embodiments of the present invention in detail with reference to the accompanying drawings. Finally, various aspects of the present invention will be described.

A cell evaluation device according to a first aspect of the present invention includes:

a concave cell housing unit having a circular bottom face and a side face defining a circular top opening; and

an adapter configured to be elastically deformed by being fitted into the cell housing unit to close the top opening, and

the adapter includes:

a body which has a circular lower end and is configured to be inserted into the cell housing unit;

a positioning unit configured to position the body in a vertical direction with respect to the cell housing unit;

a culture medium inlet port which has a through hole penetrating a top end and the lower end of the body;

a culture medium outlet port which has a through hole penetrating a top end and the lower end of the body; and

a flexible sheet member which is provided on the lower end of the body and extends radially outward from an outer edge of the lower end.

According to the cell evaluation device of the first aspect, when the adapter is fitted into the cell housing unit, the sheet member abuts on the inner peripheral face of the cell housing unit and is bent upward, and the outer peripheral part of the sheet member is brought into close contact with the inner peripheral face of the cell housing unit. As a result, the space between the adapter and the cell housing unit is sealed, and it becomes possible to prevent the culture medium housed in the cell housing unit from leaking to the outside.

A second aspect of the present invention is the cell evaluation device according to the first aspect,

in which an outer diameter of the sheet member is larger than an inner diameter of the cell housing unit at a height corresponding to the lower end of the body with the body of the adapter being inserted into the cell housing unit and being positioned in the vertical direction by the positioning unit.

A third aspect of the present invention is the cell evaluation device according to the first or second aspect,

in which the positioning unit is a retaining unit protruding in a horizontal direction from the adapter, and

a lower end of the retaining unit abuts on a peripheral edge of the top opening of the cell housing unit with the body being inserted into the cell housing unit.

According to the cell evaluation device of the third aspect, when the body of the adapter is inserted into the cell housing unit, the lower end of the retaining unit abuts on the peripheral edge of the top opening of the cell housing unit, so that the positional relationship between the bottom face of the cell housing unit and the lower end of the body of the adapter can be kept constant.

A fourth aspect of the present invention is the cell evaluation device according to any one of the first to third aspects, including

a cell housing plate in which a plurality of the cell housing units is formed.

According to the cell evaluation device of the fourth aspect, cell culture and evaluation of a plurality of samples can be collectively performed on one cell housing plate.

A fifth aspect of the present invention is the cell evaluation device according to any one of the first to fourth aspects, further including

a culture vessel having a recess in which a culture medium is housed,

in which the cell housing unit is configured to be detachably attached in the recess, and the bottom face of the cell housing unit is made of a porous film.

According to the cell evaluation device of the fifth aspect, since a substance (a drug, a nutrient, oxygen, or the like) can move between the recess and the cell housing unit through the porous film, cells can be cultured and evaluated under various conditions.

A sixth aspect of the present invention is the cell evaluation device according to the fifth aspect,

in which the culture vessel is a multi-hole plate.

According to the cell evaluation device of the sixth aspect, cell culture and evaluation of a plurality of samples can be collectively performed on one multi-hole plate.

A seventh aspect of the present invention is the cell evaluation device according to any one of the first to sixth aspects,

in which the sheet member includes a circular central portion attached to the lower end of the body, a flange portion protruding radially outward from an outer edge of the central portion, and an inlet hole and an outlet hole respectively formed at positions corresponding to a lower end of the culture medium inlet port and a lower end of the culture medium outlet port in the central portion.

According to the cell evaluation device of the seventh aspect, the sheet member can be easily and reliably attached to the lower end of the body of the adapter.

An eighth aspect of the present invention is the cell evaluation device according to the fifth or sixth aspect,

an internal electrode which is embedded in the adapter and has a part exposed onto a lower end of the adapter; and

an external electrode housed in the recess.

According to the cell evaluation device of the eighth aspect, the electric resistance of cells cultured on the porous film can be measured by connecting the internal electrode and the external electrode with a resistance meter.

REFERENCE SIGNS LIST