WO2014207857A1

WO2014207857A1 - Cytotoxicity testing device and cytotoxicity testing method

Info

Publication number: WO2014207857A1
Application number: PCT/JP2013/067577
Authority: WO
Inventors: 由美子五十嵐
Original assignee: 株式会社日立製作所
Priority date: 2013-06-26
Filing date: 2013-06-26
Publication date: 2014-12-31

Abstract

Provided are a cytotoxicity testing device, which makes on-site cell cultivation and toxicity testing possible and which is user-friendly, and a testing method using same. The device comprises a fluid delivery hole where a culture medium containing cells to be tested is injected, a culturing vessel into which culture medium is introduced and cells are sown and cultured, and a waste fluid vessel into which culture medium from the culturing vessel is discarded. In addition to cells being cultured using the culture medium in the culturing vessel, toxicity-testing test solution, which has been injected from the fluid delivery hole, is delivered into the culturing vessel from which the culture medium has been discharged to expose the cultured cells to the test solution, and cell toxicity is evaluated on the basis of the survival rate of the cells after the exposure.

Description

Cytotoxicity test apparatus and cytotoxicity test method

The present invention relates to an easy-to-handle cytotoxicity test apparatus and a test method using the same, which can be cultured while being transported after seeding of cells and can be easily exchanged a plurality of times.

Toxicity evaluation (bioassay) of chemical substances and the like using cells is widely used as a representative, such as the neutral red method and MTT (evaluation using Methylthiazol reagent) method, and is performed in a laboratory. The first reason that is limited to the laboratory is that it is difficult to bring out the cells as the test material outside the laboratory in terms of maintaining the culture conditions. Usually, cell culture uses a gas-exchangeable container, and is cultured in an environment of 37 ° C., saturated water vapor, and 5% CO ₂ addition, so a dedicated incubator is required. In recent years, a cell transport container for transporting cultured cells while maintaining these conditions has been developed (see Patent Document 1), but at this stage, it is limited to use for medical purposes, and a commercial product has been released. Absent. In addition, physics and chemistry products that can be transported by putting a reagent for CO ₂ addition into a sealed container are sold for research purposes, but the temperature and humidity cannot be adjusted (cell culture gas concentration regulator, Culturepal, Mitsubishi Gas) Chemical Co., Ltd.).

The second reason is that the cytotoxicity test is difficult to perform except in a laboratory equipped with facilities. The procedure of the cytotoxicity test is very complicated as cell maintenance, cell seeding and culture, medium exchange and exposure to a test substance, replacement with a detection reagent consisting of several steps, and measurement with an instrument. Furthermore, cell culture facilities (sterile operation benches, CO ₂ incubators, cell storage facilities, microscopes, centrifuges), and measuring devices suitable for detection items (spectrometers, fluorescence measuring devices, image analysis software, etc.) are required.

In recent years, chemical substances have made many contributions to improving human life, but they pollute the environment and act on living organisms to have various physiological effects and affect the structure of ecosystems. Yes. In particular, there are concerns about the risks to human health caused by nanomaterials (vertical, horizontal, and at least one side of the height is 100 millionth of a meter-nano-size chemicals). In the prior art, bioassays are carried out using aquatic organisms (algae, daphnia, fish), but they only measure the ecological effects and have not been able to sufficiently detect human health risks ( (Refer nonpatent literature 2).

Under such circumstances, cytotoxicity tests for human cells and the like in the environmental field are expected. However, in the case of surveys of environmental pollution such as rivers and lakes (including raw water from water treatment plants), factory effluents, well water, etc., samples are taken home after sampling in field surveys, so the time until analysis results are obtained Sample denaturation due to loss and storage conditions becomes a problem. It is desirable to develop a test method that is simpler and does not use an apparatus even in a laboratory.

JP 2013-39102 A

There is a great need to expand the application field of cytotoxicity tests. However, in the prior art, it is difficult to take cells outside the laboratory, and the operation is complicated and an expensive apparatus is required. That is, there are problems such as taking out and transporting cells in a state where stress is reduced, enabling toxicology measurement that is easy to handle, and eliminating the need for expensive dedicated equipment and enabling measurement with a general-purpose instrument.

Therefore, an object of the present invention is to provide a cell toxicity test apparatus that enables cell culture and toxicity tests on-site and is easy to handle, and a test method using the apparatus.

In order to solve the above problems, main characteristics of the cytotoxicity test apparatus according to the present invention are as follows.
(1) It has a liquid feeding hole for injecting a medium containing cells to be tested, a culture tank for introducing the medium and seeding and culturing the cells, and a waste liquid tank for discarding the culture medium in the culture tank. The cells are cultured using the medium, and a test solution for testing the toxicity injected from the solution feeding hole is fed into the culture tank in which the medium is discarded, and the test solution is exposed to the cultured cells, It is characterized by assessing cell toxicity based on cell viability.

The main features of the cytotoxicity test method according to the present invention are as follows.
(2) A step of injecting a medium containing cells to be tested from a liquid supply hole, a step of supplying the injected medium to a culture tank and seeding the cells, and a step of culturing the cells seeded in the culture tank And a step of discarding the culture medium in the culture tank to the waste liquid tank, a step of feeding a test liquid for testing the toxicity injected from the liquid feed hole into the culture tank in which the medium has been drained after culturing the cells, and culturing the test liquid Exposing the exposed cells, determining the viability of the cells after the exposure, and evaluating the toxicity to the cells.

According to the present invention, cell culture and toxicity tests can be performed on-site, and a cytotoxicity test apparatus that is easy to handle and a test method using the same can be provided.

That is, the cytotoxicity test can be carried out outside the laboratory, the time until the result can be obtained can be shortened, and the problem of sample denaturation due to storage time and condition can be solved.

In addition, the operation is simple, and toxicity tests can be performed with only a micropipette and a simple temperature controller, and even those who have little experience in bio-experiments can easily perform tests.

Furthermore, the cytotoxicity test apparatus of the present invention is small in size and can save space when carried out in a laboratory.

It is a top view explaining an example of the cytotoxicity test container which can implement a cell culture and a toxicity test by an integrated type. It is sectional drawing which showed three examples of the backflow prevention structure of the waste liquid to the culture tank shown in FIG. It is sectional drawing which showed the position of the flow path attached to the culture tank shown in FIG. It is sectional drawing which showed the structure which makes an observation with a microscope easy. It is a top view which shows the solution position sent to the culture tank with the general purpose micropipette. It is a figure which shows the result of having confirmed the position holding effect in the case of horizontal placement of a solution. It is a figure which shows the result of having confirmed the position holding effect in the case of placing the solution vertically. It is a graph which shows the result of having performed cell culture with the cytotoxicity test container. It is a figure which shows the result of having carried out liquid supply and air supply with the general purpose micropipette, and implemented solution exchange. It is a figure which shows the result of having detected the cytotoxicity by chromaticity.

Embodiments of the present invention will be described below with reference to the drawings.
<First Embodiment>
A configuration example of a cytotoxicity test container according to the present invention will be described with reference to FIG. In addition, this embodiment is only an example and does not limit this invention.

In FIG. 1, 1 is a culture tank, 2 is a waste liquid tank, 3 is a liquid feed hole, 4 is a waste liquid hole, 5 is a flow path connecting the liquid feed hole to the culture tank, and 6 is a flow connecting the culture tank to the waste liquid tank. A path 7 is a flow path connecting the waste liquid tank to the waste liquid hole. The cytotoxicity test container according to the present invention is a container capable of performing cell culture and toxicity test in an integrated manner, and includes a culture tank and a waste liquid tank, from a liquid feed hole to a culture tank, from a culture tank to a waste liquid tank, and from a waste liquid tank to a waste liquid. It consists of a flow path connecting each of the holes. Cells and culture medium are injected into the culture tank 1 from the feeding hole 3 and cultured. After discarding the culture medium into the waste tank 2, the test solution / detection reagent is injected from the feeding hole 3 to treat the cells, and the toxicity test is performed. It can be performed.

The size of the culture tank 1 is preferably 5 mm square or more and 1 mm to 2 mm high, which can easily determine toxicity with the naked eye. By making the size of the waste liquid tank 2 larger than the size of the culture tank and making the volume ratio 20 times or more, all of the waste liquid from one cell culture to acquisition of the toxicity test result can be accumulated in the waste liquid tank.

Also, in order to facilitate liquid feeding from the liquid feeding hole 3, a circular spot facing is inserted under the liquid feeding hole 3. It is desirable that the spot facings have the same height as the flow path and have a diameter that is at least twice the diameter of the flow path.

Further, by putting a turn back into the flow path 5 connecting the liquid feeding hole 3 to the culture tank 1 and the flow path 6 connecting the culture tank 1 to the waste liquid tank 2, the position of the solution is maintained and the reverse flow is achieved by balance with the air pressure. Can be prevented. At this time, it is desirable that the turn of the flow path 5 connecting the liquid feeding hole 3 to the culture tank 1 is one time or a plurality of times. It is desirable to put the turn of the flow path 6 connecting the culture tank 1 to the waste liquid tank 2 at least twice. Further, the shape of the turn is preferably a right angle or an acute angle so as to prevent the flow of the solution. This turn makes it possible to prevent the solution position from being retained and the waste liquid from flowing backward.

FIG. 2 is a cross-sectional view showing an example of a structure for preventing the backflow of waste liquid into the culture tank 1. This backflow prevention structure is installed on the outlet side of the culture tank 1 where the flow path 6 is connected to the culture tank 1.
As shown in FIG. 2 (a), the structure 11 in which the outlet channel of the culture tank 1 is tapered, the structure 12 in which a part of the outlet channel is slightly narrowed as shown in FIG. 2 (b), FIG. As shown in c), the structure 13 in which the height of the outlet channel is set at a position lower than the outlet can enhance the effect of preventing the backflow of waste liquid.

Due to the configuration of the cytotoxicity test container according to the present invention, the position of the medium can be maintained even if the cell is tilted or falls during transport, and the flow of the solution can be prevented, thereby reducing cell damage during transport. Can do.

FIG. 2 shows an example of a structure for preventing the backflow of waste liquid to the culture tank 1, but the same structure for preventing the backflow of waste liquid can be applied to the waste liquid tank 2. That is, any one of the

structures

11, 12, and 13 may be installed on the inlet side of the waste liquid tank 2 connected to the waste liquid tank 2 and the flow path 6. In addition, you may provide this backflow prevention structure in either the culture tank 1 side or the waste liquid tank 2 side, or both.

FIG. 3 shows the position of the flow path attached to the culture tank 1. Here, the inlet channel 21 and the outlet channel 22. As shown in this figure, both the inlet channel 21 and the outlet channel 22 are provided on the upper side of the culture tank 1. With this configuration, liquid is fed from a high position away from the bottom surface of the culture tank 1, and damage to cells attached to the bottom surface during solution exchange can be reduced. At the same time, dead cells generated during the culture can be easily discarded at the time of solution replacement, and the remaining in the culture tank can be reduced.

FIG. 4 is a side view of the test container, showing a structure that facilitates observation with a microscope. By digging 31 into the bottom surface of the container substrate in the region where the culture tank 1 is provided and reducing the thickness of the container substrate, the working distance of the microscope can be secured and observation can be facilitated.

In addition, in this figure, although the upper surface of the culture tank 1 or the waste liquid tank 2 has shown the structure open | released, it is preferable to use the airtight cover 30 shown with a dotted line.
<Second Embodiment>
This embodiment demonstrates the usage method at the time of performing a toxicity evaluation test using the cytotoxicity test container in this invention using FIG.

FIG. 5 is a plan view showing the position of the solution fed to the culture tank by a general-purpose micropipette.
The tester performs the cytotoxicity test by injecting and discharging the solution in a single flow path by performing liquid feeding and air feeding with a general-purpose micropipette from the liquid feeding hole 3 of the cytotoxicity test container.
First, a number of cells suitable for various toxicity tests are seeded from the liquid feeding hole 3. At this time, it is desirable that the culture medium is contained up to the flow path position indicated by the black dots in FIG. In this state, the liquid feeding hole 3 and the waste liquid hole 4 are sealed and culture is performed. After stationary culture for 2 hours or more and confirmation of cell attachment, transportation is possible. It is desirable to use a simple temperature control device and carry the culture under conditions of 30 to 37 ° C. while continuing the culture.

Next, the culture solution in the culture tank is replaced with a test solution. First, the liquid feeding hole 3 and the waste liquid hole 4 are unsealed, and air is fed with a micropipette to send the medium and dead cells to the waste liquid tank. Next, the test solution is injected, and air is introduced to stop at the position shown in FIG. After sealing the liquid feeding hole 3 and the waste liquid hole 4 and exposing the cells to the test liquid for a necessary time, the test liquid is again sent to the waste liquid tank 2 by air feeding. Inject a buffer solution such as PBS buffer, discard it by air supply, and wash away harmful substances in the test solution.

For the toxicity test, a conventional method for measuring cell activity / viability can be used. Cell viability is measured by using a dye method such as neutral red, which examines cell membrane permeability as an indicator, and the phenomenon that MTT (methylthiazoletetrazolium) is reduced and decomposed along with the mitochondrial oxidative phosphorylation ability. The MTT method for obtaining the survival rate of can be used.

For example, in the case of the neutral red method, the medium is exchanged with a culture solution containing neutral red, and further cultured for 2 to 3 hours, so that neutral red is incorporated into the cells and the amount of incorporation is measured. In order to measure the amount of uptake, the following post-treatment is usually performed. That is, after removing the culture solution containing neutral red, a cultured cell is fixed by adding a calcium chloride-containing formalin solution (for example, 1% calcium chloride-containing 1% formalin solution), and the supernatant is further removed to remove cells. Neutral red that is not taken up into the cells is removed, and then neutral red taken up into the living cells is extracted by adding ethanol containing acetic acid (for example, 50% ethanol containing 1% acetic acid). As for the amount of neutral red contained in the extract, a method of measuring the absorbance at 540 nm using a spectrophotometer is simple.

Neutral red is a soluble pigment that is a substance that allows only living cells to be taken into cells. Neutral red uptake is reduced when cell growth is inhibited by toxicity, or when cell death is induced and the number of cells is reduced. Therefore, by measuring neutral red taken up into cells after washing, the number of viable cells relative to the control group can be estimated, and toxicity can be determined from the relative survival rate.

When carrying out the neutral red method with this cytotoxicity test container, it is carried out as follows. A culture solution containing neutral red is injected, similarly stopped at the position shown in FIG. 5, and the solution feeding hole 3 and the waste solution hole 4 are sealed and processed for a necessary time. Two to three hours are generally used. Next, the liquid feeding hole and the waste liquid hole are removed, and air is fed with a micropipette to send the culture solution to the waste tank. A calcium chloride-containing formalin solution (for example, 1% calcium chloride-containing 1% formalin solution) is injected and similarly stopped at the position of FIG. 5 and fixed for 1 minute or longer. Air is again supplied with a micropipette, and the calcium chloride-containing formalin solution is sent to the waste tank. Acetic acid-containing ethanol (for example, 50% ethanol containing 1% acetic acid) is injected and similarly stopped at the position of FIG. 5 and held for 10 minutes or longer.

As described above, using the cytotoxicity test container provided by the present invention, it is possible to perform a toxicity test by exchanging the solution in a single flow path by performing liquid feeding and air feeding with a micropipette. .

To evaluate the toxicity, a color sample for the degree of cytotoxicity is prepared in advance, and it can be easily judged by the color density. It is also possible to photograph a culture tank using a device having a camera function such as a smartphone, perform image processing, and determine the color intensity numerically. For image processing, you can use ImageJ distributed by National Institutes of Health, which can be downloaded for free, and Photoshop from Adobe.

In this embodiment, by turning the channel 5 connecting the solution feeding hole 3 to the culture tank 1 and the channel 6 connecting the culture tank 1 to the waste solution tank 2, the position of the solution is balanced by the air pressure. This confirms the effect of the mechanism that prevents the holding and backflow, that is, the structure that suppresses the flow of the medium in the culture tank.

As shown in FIG. 1 or FIG. 5, the turn of the flow path 5 connecting the liquid feeding hole 3 to the culture tank 1 is one time, and the turn of the flow path 6 connecting the culture tank 1 to the waste liquid tank 2 is A test vessel that was 4 times was used. The shape of the turn was a right angle.

6A and 6B are diagrams showing the results of confirming the effect of maintaining the position of the solution, FIG. 6A is a case where the container is placed horizontally, and FIG. 6B is a case where the container is placed vertically. Here, “horizontal placement” refers to a state in which the long side of the container as shown in FIG. 1 is installed perpendicular to the installation surface. In addition, “vertical placement” refers to a state where the short side of the container as shown in FIG. 1 is installed perpendicular to the installation surface.

The confirmation of the effect of maintaining the position of the solution was performed as follows. Assuming the transportation state of the container, the container is vibrated 10 times, the culture tank is rotated, and then, as shown in FIG. Considered moving.

In FIG. 6B, the container in the “horizontal” state is set to “vertical”, that is, the container is stood still in the vertical direction and the movement of the liquid was examined.

FIG. 6A (a) shows a photograph of the above confirmation result, and FIG. 6 (b) is a schematic diagram for easy understanding of the state of (a). The same applies to FIGS. 6B (a) and (b).

As shown in FIG. 6A (b), it can be seen that the position of the solution after each elapsed time of 0h (hour), 3h (hour), and 24h (hour) after being placed horizontally is held substantially the same. . FIG. 6B (b) shows the position of the solution after the elapse of 24 h (hours) from the vertical placement, and it can be seen that almost no change is seen from the position of the solution at the time of 0 h (hours).

From the above, as shown in FIGS. 6A and 6B, the position retention of the solution was good.

This example is evaluated from the viewpoint of cell culture in the test container. Normally, cell culture is performed in a container capable of gas exchange by using a dedicated incubator under 37 ° C., saturated water vapor, with 5% CO ₂ addition. 2 × 10 ⁵ human liver cancer cell lines HepG2 are seeded in a cytotoxicity test container, sealed in the liquid feeding hole 3 and the waste liquid hole 4, and kept at 37 ° C., without humidity control, without CO ₂ addition. Culture was performed under conditions. This evaluation condition is hereinafter referred to as a sealed or sealed system.

For comparison, 2 × 10 ⁵ human liver cancer cell lines HepG2 were cultured under normal open culture conditions using a culture tank without a lid of the test container and a container with a flow channel exposed. This evaluation condition is hereinafter referred to as open or open system.

Cells were collected after 24 hours and 48 hours, and the number of viable cells was measured to determine the ratio to the number of seeding.

FIG. 7 is a graph showing the results of cell culture in a cytotoxicity test container. The doubling rate and viability after 24 hours and 48 hours are shown as the ratio of the number of cells after time to the initial number of viable cells.
Further, the white in the graph is the result of the evaluation in the open system shown above, and the dot shows the result of the evaluation in the closed system shown above.

As shown in FIG. 7, by using this test vessel, the doubling rate and the survival rate are equivalent in 24 hours as compared with the culture in the open system, and the survival rate is inferior in doubling rate in 48 hours. It was confirmed that an equivalent culture effect was obtained.

By the way, the cytotoxicity test cell is a short-term test in which results are obtained within approximately 48 hours, and the culture time before exposure to the test solution is 24 hours at the maximum. Therefore, it can be said that the effect of the present invention can be sufficiently confirmed if the culture effect of the doubling rate and the survival rate in 24 hours can be confirmed.

Therefore, the results of this implementation showed that test cells equivalent to normal culture can be donated to the toxicity test by simple culture using the cytotoxicity test vessel of the present invention.

This example is the result of carrying out solution replacement of the culture tank by air supply and liquid supply.
FIG. 8 is a diagram showing a result of solution exchange performed by feeding and feeding with a general-purpose micropipette.
FIG. 8A is a photograph showing the results of the implementation, and FIG. 8B is a schematic diagram for making the results easier to see.

Next, the drawings (1) to (6) will be described.
(1) First, 160 μl of the solution A was injected into the culture tank 1 from the liquid feeding hole 3 using a micropipette. A little air was sent to the liquid feeding side, and the solution was stopped in the middle of the flow path.
(2) Next, 200 μl of air was supplied three times to completely remove the solution A from the culture tank.
(3) Assuming washing, 200 μl of solution B was injected, and this was repeated three times.
(4) Next, 200 μl of air is supplied twice again to remove the solution B from the culture tank,
(5) 200 μl of solution C was injected.
(6) Furthermore, 200 μl of air was supplied twice, and the solution C was completely moved to the waste liquid tank.

As can be seen from the schematic diagrams (b) of (1) to (6) above, the solution exchange in the culture tank can be easily performed by combining air feeding and liquid feeding. All the waste liquid could be stored in the waste liquid tank.

In this example, a neutral red assay was performed using this test container. In order to evaluate toxicity using the red density of neutral red, seeding was performed in advance by changing the number of seeded cells, and the result of performing only the neutral red assay without performing toxicity tests was obtained. The difference in the number of cells can be evaluated by the color shading.

FIG. 9 is a diagram showing the result of detecting the number of viable cells, that is, the survival rate in the toxicity test by chromaticity.
FIG. 9A is a photograph of the detection result, and FIG. 9B is a schematic diagram for easily viewing the result. This evaluation actually uses color shading, but in this figure, the shading is represented by dot density instead of color display.

After taking a picture of the part of the culture tank and processing it in black and white using image analysis software Photoshop (Adobe), the color density in the culture tank was measured. When the chromaticity ratio at a survival rate of 0% was 1, the survival rate was 30%, the chromaticity ratio was 15, and the survival rate was 100%, and the chromaticity ratio was 92. Here, the standard of the chromaticity ratio is the darkness of black. A reference sample is determined and expressed as a relative value. In this embodiment, the standard is one having a small number of cells.

As a result of detection, there was no toxicity as shown in FIG. 9 (1) (number of seeded cells 2 × 10 ⁵ ), and there was toxicity as shown in (2) I (survival rate 30%, number of seeded cells 6 × 10 ⁴ ). ) And (3) showed toxicity II (survival rate 0%, seeded cell number 0) color shading (dot shading in this figure (b)).

Toxicity can be evaluated by either comparing these color samples with actual test results with the naked eye, or taking a picture with a smartphone and comparing the color density numerically with image analysis software. .

1: culture tank,
2: Waste tank
3: Liquid feed hole,
4: Waste liquid hole,
5: A flow path connecting the liquid feeding hole to the culture tank,
6: A flow path connecting the culture tank to the waste liquid tank,
7: Flow path connecting the waste liquid tank to the waste liquid hole,
11: Tapered structure installed in the outlet channel of the culture tank,
12: Structure in which the outlet channel of the culture tank is partially narrowed,
13: Structure in which the outlet channel of the culture tank is installed at a position lower than the outlet,
21: Inlet channel of the culture tank,
22: outlet channel of the culture tank,
30: sealing lid,
31: Excavation of the bottom of the culture tank placed for microscopic observation.

Claims

A feeding hole for injecting a medium containing cells to be tested;
A culture vessel for introducing the medium and seeding and culturing the cells;
A waste liquid tank for discarding the culture medium of the culture tank,
Culturing the cells using the medium in the culture vessel;
Based on the survival rate of the cells after the exposure, the test solution for testing the toxicity injected from the liquid supply hole is fed into the culture tank in which the medium is discarded, and the test solution is exposed to the cultured cells. A cytotoxicity test apparatus characterized by evaluating toxicity to the cells.
A first flow path connecting the liquid feeding hole and the culture tank;
A second flow path connecting the culture tank and the waste liquid tank,
By providing a folded structure in each of the first channel and the second channel, the liquid in the first and second channels is prevented from moving, and the cells are conveyed while being cultured in the culture tank. The cytotoxicity test apparatus according to claim 1, wherein the cytotoxicity test apparatus is possible.
The cytotoxicity test apparatus according to claim 2, wherein the folding number of the folding structure of the second channel is greater than the folding number of the first channel.
The viability of the cells is determined by processing the cells after the exposure using the detection reagent injected from the liquid feeding hole, and the color density of the cells after the treatment is set based on a preset color density The cytotoxicity test apparatus according to claim 1, wherein:
A backflow prevention mechanism for preventing a backflow from the culture tank or the waste liquid tank is provided in at least one of the second flow path on the outlet side from the culture tank or the inlet side of the waste liquid tank. The cytotoxicity test apparatus according to claim 2.
The backflow prevention mechanism has a tapered structure in which the cross-sectional structure of the second flow path gradually increases from the outlet side of the culture tank or the inlet side of the waste liquid tank. Cytotoxicity test equipment.
The backflow prevention mechanism is characterized in that the second channel on the outlet side of the culture tank or the inlet side of the waste liquid tank has a region having a smaller cross-sectional area than the other part of the channel. The cytotoxicity test apparatus according to claim 5.
In the second flow path on the outlet side of the culture tank or on the inlet side of the waste liquid tank, the reverse flow prevention mechanism is configured such that the flow path gradually becomes the bottom surface of the culture tank from the surface side of the culture tank or the waste liquid tank. The cytotoxicity test apparatus according to claim 5, wherein the cytotoxicity test apparatus has a structure inclined to a side or a bottom side of the waste liquid tank.
The first and second flow paths are installed at a position where liquid is introduced or discharged from the upper part of the side surface of the culture tank, thereby reducing damage to the cells during the medium exchange. The cytotoxicity test apparatus according to claim 2.
The substrate on which the culture tank is formed is provided with a scraped structure at the top or bottom of the culture tank, so that the cell state in the culture tank can be easily observed. The cytotoxicity test apparatus as described.
The cytotoxicity test apparatus according to claim 1, wherein the waste liquid from the start to the end of the test is stored by setting a volume ratio of the culture tank and the waste liquid tank to 1:20 or more.
Injecting a medium containing cells to be tested from a liquid feeding hole;
Feeding the injected medium to a culture tank and seeding the cells;
Culturing the seeded cells in the culture vessel;
Discarding the culture medium in the culture tank to a waste liquid tank;
After culturing the cells, feeding a test solution for testing the toxicity injected from the feeding hole into the culture tank in which the medium is discarded;
Exposing the test solution to the cultured cells, determining the survival rate of the cells after the exposure, and evaluating the toxicity to the cells.
The cytotoxicity test method according to claim 12, wherein the culture medium in the culture tank removes the solution injected from the liquid supply hole from the culture tank by supplying gas from the liquid supply hole. .
The survival rate of the cells is obtained by processing the cells after the exposure using the detection reagent injected from the liquid feeding hole, and setting the color density of the cells after the treatment based on a preset reference color density. The cytotoxicity test method according to claim 12, wherein the cytotoxicity test method is determined.