JP3343584B2 - Bioassay device using human cells - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an apparatus for performing a bioassay using human cells.

[0002] The present invention also relates to a carrier on which human cells are immobilized for use in the device.

[0003]

2. Description of the Related Art In recent years, it has become necessary to evaluate the toxicity (environmental risk of human health) of environmental water with the aggravation of water environmental pollution. It merely specifies the substance concentration individually, and it is difficult to grasp the overall effect on the human body.

On the other hand, in a bioassay utilizing the response of an organism to a test water, the toxicity of the test water can be comprehensively evaluated without performing complicated chemical analysis. It is extremely useful as an evaluation method, and to date, toxicity evaluation devices and kits using bioassays have been developed.

[0005] Examples of such a device include a device utilizing the respiratory activity of immobilized nitrifying bacteria, and a device using luminescent bacteria.

[0006] However, the devices developed so far have been developed for the purpose of evaluating the effects on the ecosystem, and therefore have the disadvantage that the effects on the human body cannot be accurately evaluated.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to provide an apparatus capable of accurately evaluating the influence of a desired sample on a human individual by a bioassay using human cells.

[0008]

Means for Solving the Problems To solve the above problems, the present invention provides a bioassay device comprising a container containing a carrier on which human cells are immobilized.

[0009]

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to water collected from the environment,
Provided is an apparatus capable of evaluating the influence of a desired sample including soil on a human at an on-site sampling site.

[0010] Since the device contains human cells immobilized on a carrier, the sample is introduced into the device,
By examining the effect of the sample on human cells, the effect of the sample on a human individual can be easily and accurately estimated.

[0011] The present invention also provides said carrier for use in said device. When human cells are immobilized on the carrier at a high density, it becomes possible to detect substances on the order of nM. If such a carrier is used, the inspection can be completed in a short time, so that the inspection at the sample collection site can be performed.

Hereinafter, the present invention will be described in detail with reference to Examples, but is not intended to limit the scope of the present invention described in the claims in any way.

Example 1 In this example, a cell-filled chip that can easily assay the effect of a sample collected from the environment on humans will be described with reference to FIG.

The cell-filled chip 1 of the present embodiment is used by sucking a sample from a sample suction port 3 provided at the tip of the chip 1, which is mounted on a micropipette 2, like a general micropipette chip. I do.

The inside of the chip 1 is filled with a filter 4, and the inside of the chip 1 is
It is divided into two.

The sample sucked through the sample inlet 3 passes through the filter 4 and is connected to the filter 4 and the micropipette 2.
Reaches the carrier 5 which is dispersed in the compartment formed between them.

A human cell 6 is immobilized on the carrier 5. Therefore, by examining the effect of the sample on human cells 6, the effect of the sample on a human individual can be estimated.

The cell-filled chip of this embodiment can be prepared by containing a carrier on which human cells are immobilized in a commercially available filter chip filled with a filter.
The filter needs to be a hydrophobic filter because the liquid only needs to be permeable when pressure is applied with a micropipette.

A method for immobilizing human cells on a carrier is known, and a carrier binding method, an inclusive method, and the like can be applied. However, since the cells die, the cross-linking method cannot be used. Specifically, a method can be used in which human cells are added to a carrier-containing medium, and then seeded on a suspension culture plate, swirled for 5 to 10 days, and grown inside the carrier.

The shape of the carrier may be any shape such as a sphere, a plate, a string, and a hollow lattice.

The human cells to be immobilized on the carrier can be arbitrarily selected according to the type of the bioassay, the type of the carrier, the method of the immobilization, and the like. Preferably, it is used.

In the apparatus of the present invention, low-density lipoprotein (LDL;
(ow-density lipoprotein) uptake assay is preferably used as a bioassay,
It is preferable to immobilize tumor cells derived from hepatocytes in which the LDL receptor is expressed in large amounts. Such hepatocyte-derived tumor cells include Hep G2 cells (available from Japan Cancer Research Bank).

The LDL uptake assay using Hep G2 cells itself is known, and is an assay for measuring the amount of LDL labeled with a fluorescent substance, a radioactive substance, a luminescent substance, or the like, incorporated into Hep G2 cells. . LDL is a substance having the second highest metabolic rate after oxygen and can be easily labeled in various ways. Therefore, it is suitable for high-sensitivity rapid evaluation, but there is no example used for toxicity evaluation.

A bioassay having all of generality, rapidity and high sensitivity seems to be difficult to achieve except for the combination of LDL and hepatocytes. Therefore, in the present invention, it is extremely preferable to use LDL and hepatocytes.

Since the number of cells immobilized on the carrier affects the measurement sensitivity, the practicality of the measuring device of the present invention largely depends on the number of cells immobilized on the carrier. Therefore, the number of cells to be immobilized on the carrier must be selected so as to be most suitable for the cells used.

Specifically, when the number of cells is excessively increased, the amount of oxygen supplied to the cells becomes insufficient in a closed space having a small volume such as a micropipette tip. For this reason, it is necessary to select a cell number that does not cause an excessive shortage of oxygen supply and that provides sufficient measurement sensitivity.

As shown in FIG. 3, in the case of the Hep G2 cells, the cell density is 1.91 × 10 ⁷ cells / mL, the cell density is too high and the cell number decreases. On the other hand, 3.21 × 10 ⁵ and 3.5
At a density of 4 × 10 ⁶ cells / mL, the number of cells is particularly small in the early stage of culture, and the number of cells is not constant. On the contrary,
At a density of 1.13 × 10 ⁷ cells / mL, the number of cells is constant and a sufficient number of cells can be obtained. Therefore, when Hep G2 cells are used, the cell density in the solution is 1.0 to 1.2 × 10 ⁷ cells / mL, more preferably 1.10 to 1.15 × 10 ⁷ cells / mL. First, the cells should be immobilized on the carrier.

Thus, when human cells are used,
It was first discovered by the present inventors that there is a cell density that can provide sufficient measurement sensitivity and stability without causing a decrease in cell activity due to excessive lack of oxygen supply in a small enclosed space. It is knowledge.

Based on the findings found by the present inventors, it will be possible to select an optimal cell density even when using cells other than Hep G2 cells.

As a carrier for immobilizing human cells, it is preferable to use a microcarrier capable of achieving the above-mentioned appropriate cell density. Specifically, the pore size is 10 ~ 100μm
It is preferable, but not limited, to use a certain amount of porous carrier. The particle size of the carrier itself is generally 100 to 1000 μm, but is not limited thereto. As the carrier, collagen, cellulose and the like can be used, but the most appropriate carrier must be selected according to the type of cells to be immobilized.

As shown in FIG. 4, the number of cells immobilized is significantly different depending on the type and size of the carrier.
When Hep G2 cells are immobilized, spherical porous collagen having a pore size of 10 to 40 μm, more preferably 20 to 40 μm should be used as a carrier.

After preparing the carrier on which the cells are immobilized, the carrier is stored in an appropriate volume of a filter chip,
Alternatively, it is stored at a low temperature, more preferably at 4 ° C. or lower, in a state where it is not contained. If the immobilized cells have been organized, they can be stored frozen. The carrier is
Preferably, it is stored in a suitable storage medium.

When the carrier is accommodated in a filter chip, it is preferable to uniformly fill the carrier in order to improve the measurement accuracy. In order to uniformly fill the carrier, it is preferable to prepare a micropipette tip having an opening that is sufficiently larger than the diameter of the carrier, and to fill it after sufficient pipetting using the tip.

In this embodiment, the carrier is accommodated in the filter chip, but the container for accommodating the carrier is:
The container is not limited to the filter chip, and a container having an arbitrary size and material can be used. For example, the carrier may be housed in microtiter wells, plastic sample tubes, or glass labware.

[Embodiment 2] In this embodiment, the result of detecting a toxic substance using the cell-filled chip described in Embodiment 1 will be described.

The substances measured in this example are the 30 kinds of substances listed in Table 1 below. First, for these substance groups, the results of detection of toxic substances using a cell-filled chip and the conventional method The results were compared.

[0037]

[Table 1]

The operation of detecting a toxic substance using a cell-filled chip was performed as follows.

First, as shown in FIG. 1, a tip 1 (200 μL) is mounted on a micropipette 2 for 200 μL. Press the head 7 of the micropipette 2 and press the human cell 6
The medium added in the chip 1 for preserving (in this example, Hep G2 cells) is discharged from the chip 1.

Next, the tip of the chip 1 is immersed in the sample to be measured, the head is rotated to adjust the scale to 10 μL, and 10 μL of the concentrated medium is sucked through the sample inlet 3. The concentrated medium may be a concentrated storage medium (eg, a DME medium). The concentrated medium used in this example was LDL labeled with the fluorescent substance fluorescein isothiocyanate.
When a concentrated medium containing (hereinafter, FITC-LDL) but not containing FITC-LDL is used, FITC-LDL may be added to the chip 1 after the sample is introduced.

Subsequently, the head 7 is rotated to set the scale to 90 μL.
90 μL of the sample is sucked from the sample inlet 3 in accordance with the time. The volume of the sample to be inhaled is preferably such that the volume of the chip 1 is such that the human cell 6 is completely immersed in the sample.
It is sufficient to inhale a sample equivalent to half of the chip volume.

When the sample is introduced into the chip 1, the sample and the human cells 6 come into contact. Since the LDL receptor is expressed on the membrane of the Hep G2 cells used in this example, when the sample comes into contact with human cells, the FITC-LDL in the storage medium is taken into the human cells 6. It is.

Two hours after the sample is introduced into the chip 1, the sample is discarded from the chip 1 and a washing solution (eg, PBS)
Is introduced into the chip 1 by 100 μL. After several pipetting steps, discard the washing PBS.

100 μL of an alkaline solution such as 1N-NaOH is added to the chip 1. After pipetting several times, the solution is directly put into the cell of the fluorescence intensity meter. The fluorescence intensity meter
In general, it is preferable to use a portable small fluorescent intensity meter that is commercially available. In addition, when the sample volume is 100 μL,
Because high-sensitivity evaluation is required, cells for small volumes (about 50
200200 μL) should be used.

The storage medium, the concentrated medium, the washing solution, and the alkaline solution used for such an operation are the same as the chip 1
It may be provided together with the kit.

Since the fluorescence intensity is proportional to the amount of LDL taken in, the fluorescence intensity (Ex = 490 nm, Em = 520 nm) was measured.
Compared to control, relative LDL uptake activity can be measured. As a control, ultrapure water is preferably used, but distilled water or ion-exchanged water may be used. Tap water is not preferred as a control because it may itself contain a trace amount of toxic substances and may not be sufficiently degassed with chlorine for disinfection.

If the sample contains a toxic substance that damages human cells, the activity of LDL uptake by human cells 6 is very quickly affected by the toxic substance. Therefore, if the activity of LDL uptake by the human cells 6 is measured, it is possible to estimate the toxicity of the toxic substance in the sample to humans.

The above detection method using a cell-filled chip (this detection method) is the following conventional method (using non-fixed cultured cells in a 96-well plate): 48-hour cell viability test 4-hour LDL Assay Compared to the 48 hour LDL assay.

FIG. 5 shows the results.

FIGS. 5A, 5B, and 5C respectively show:
It shows the results of comparing the ED ₅₀ (horizontal axis) of each substance measured by the present detection method with the ED ₅₀ (vertical axis) measured by the above method and the conventional method.

Here, “ED ₅₀ ” means the concentration of a substance that reduces cell activity by 50% when a control is used.

In the drawing, the numbers assigned to the points are as follows:
It corresponds to each substance described in Table 1.

[0053] ED ₅₀ measured by the detection method, and the ED ₅₀ measured by the above method, but were differed in more than one order of magnitude depending on materials, the highest correlation with ED ₅₀ measured by the above method (See FIG. 5 (b)). Except for 4-nitroquinoline-N-oxide (20) and tributyltin chloride (27), a good correlation with the 48-hour cell viability test (the method described above) was obtained. Indicated.

From the above results, it is clear that the present detection method can detect a toxic substance in a short time of 2 hours, almost in the same manner as the conventional method.

Next, six substances (acetaldehyde (1), paraquat (30), pentachlorophenol (22), phenol (23), tributyltin chloride (27),
2,4,5-trichlorophenol (28) and sodium lauryl sulfate (31)) were selected, and a dose-response curve was prepared using this detection method and the above-mentioned methods.

FIG. 6 shows the results.

As shown in FIG. 6, the present detection method (2
The time-response curve of the time chip; open triangle) was most consistent with the method described above (4 hour LDL; closed triangle), but was not
Time cell viability test; black square) and method (48 hour LD)
L; white square).

From the above results, it has been clarified that the toxicity as an integral of various substances in a sample collected from the environment can be detected in 2 hours by using the cell-filled chip described in Example 1. In this example, the measurement time was set to 2 hours. However, when a more sensitive evaluation is required, the time may be extended as appropriate, as is clear from FIG.

Since the compounds shown in Table 1 are composed of various substances such as hormones, organic substances, metals and salts thereof, and surfactants, the bioassay device of the present invention can be used to convert any substance to human individuals. The impact can be evaluated.

Example 3 In this example, the test water collected from a river was measured using the cell-filled chip described in Example 1, and compared with the measurement by the conventional method (the above-mentioned methods).

FIG. 7 shows the results.

FIG. 7 (a) shows 16: 20-1 on January 26, 1999.
Fig. 7 (b) shows the measurement results of the test water collected at Shiotsuru Bridge on the Tsurumi River over 14:30 on March 27.
This is a measurement result of the test water collected at Araibashi in Asakawa from 12:00 to 12:00 on June 19. In each plot of FIG.
The standard deviation is described as the error range, which is within approximately ± 10% of the measured value. From this, it can be said that the measurement of this chip is excellent in stability and reproducibility.

From FIGS. 7 (a) and 7 (b), the measurement results obtained by the present detection method (two-hour chip; open triangle) are obtained by the above method (4
Time LDL; closed triangle, the best agreement was obtained with the method (48 hour cell viability test; closed square) and the method (48
Time LDL; open squares).

According to this example, it was clarified that the effect of test water in the environment on humans can be evaluated using the cell-filled chip of Example 1.

In the present embodiment, water collected from a river was used as a sample. However, the sample is not limited to environmental water, and a solution in which soil, plume, food, airborne substances, artificial substances, and the like are dissolved also serves as a sample. obtain.

[0066]

According to the apparatus of the present invention, whether or not a toxic substance for humans is contained in a sample water collected from the environment can be accurately inspected at a sampling site in a short time.

[Brief description of the drawings]

FIG. 1 is a diagram showing a cell-filled chip of Example 1.

FIG. 2 is an enlarged view of a carrier contained in the cell-filled chip of Example 1.

FIG. 3 is a diagram showing the relationship between the cell density in a container and the number of viable cells.

FIG. 4 is a diagram showing the relationship between the type and size of a carrier and the number of viable cells immobilized on the carrier.

FIG. 5 is a diagram comparing a detection method using the cell-filled chip of Example 1 with a conventional detection method.

FIG. 6 is a diagram comparing a dose response curve obtained by the detection method using the cell-filled chip of Example 1 with a dose response curve obtained by the conventional detection method.

FIG. 7 is a diagram comparing a measurement result of river water by a detection method using the cell-filled chip of Example 1 with a measurement result of river water by a conventional detection method.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-9-316428 (JP, A) JP-A-9-191896 (JP, A) JP-A-3-10685 (JP, A) Production Research Vol. 51, No. 6 (1999) p. 571-574 (58) Field surveyed (Int.Cl. ⁷ , DB name) G01N 33/48-33/98 C12M 1/34 C12Q 1/00 G01N 33/18

Claims (5)

(57) [Claims] 1. A bioassay device comprising a bioassay carrier having human cells immobilized on a pipette chip. 2. The device of claim 1, wherein said bioassay is an LDL uptake assay. 3. The device according to claim 1, wherein the human cells are Hep G2 cells. 4. The method according to claim 1, wherein the human cells are 1.10 to 1.15 × 10 ⁷ cells / cell.
The device according to any one of claims 1 to 3, wherein the device is immobilized on a carrier at a cell density of mL. 5. A bioassay comprising the device according to claim 1, and at least one reagent selected from the group consisting of a storage medium, a concentrated medium, a washing solution, and an alkaline solution. kit.