WO2019225633A1 - Biological sample microchip, cover, biological sample sealing kit and method - Google Patents

Abstract

Provided is a biological sample microchip suitable for being locally irradiated with a microbeam or for observing cells, tissues, etc. in a state where a biological sample is cultured or sealed therein. In the biological sample microchip, at least one recessed section for culturing or sealing a biological sample is formed in a surface thereof, and the overall thickness of the microchip is formed to be 800 µm or less. Further, in the biological sample microchip, hydrophilicity is imparted to the surface of the microchip in which the recessed section for culturing or sealing the biological sample is formed.

Description

Biological sample microchip, cover, biological sample enclosing kit and method

The present invention relates to a microchip for a biological sample that is used when locally irradiating or observing a microbeam (eg, fluorescence imaging observation) in a state where a biological sample such as an animal or plant cell or a micro organism is cultured or encapsulated. The present invention relates to a technique for using the microchip for biological samples.

Caenorhabditis elegans (hereinafter sometimes abbreviated as “C. elegans”) is a model organism established for research on neurobiology, developmental biology, and biochemistry. is there. Studies have been conducted on the effects of radiation on animal motility using this nematode. For example, it is known that when a whole body is irradiated with a quantum beam such as a gamma ray or an ion beam, its motility decreases according to the dose. However, it remains unclear whether the reduction in nematode motility was induced by irradiation of specific tissues or regions.

Therefore, in recent years, by irradiating only a specific tissue or region such as the head with the central nervous system of the nematode, the abdomen and tail with the intestine or genital organs, the motility is reduced. Research has been conducted to determine whether or not it was induced by irradiation of the area. A microbeam is a beam in which a quantum beam is reduced to a micrometer size so that a specific region can be irradiated locally. As such a quantum beam, for example, an X-ray, an electron beam, an ion beam, a laser, or the like can be used. In this type of conventional research, anesthesia is performed on the nematode so that the nematode does not move, thereby enabling microbeam irradiation targeting a specific tissue or region. However, in this case, since the nerve calming action by anesthesia affects, it is not possible to immediately observe how the microbeam irradiation to a specific tissue or region affects the mobility of the nematode.

In order to improve this, a method has been proposed in which a nematode is retained without anesthesia using a PDMS (polydimethylsiloxane) microchip, and a microbeam is irradiated aiming at a specific tissue or region of the retained nematode. (For example, Non-Patent Document 1). The conventional PDMS microchip was developed to encapsulate and maintain nematodes for the purpose of imaging neural activity of nematodes, and is formed on the chip surface with a width of 10 to 80 μm, a depth of 40 μm, and a length. A nematode is enclosed in a linear channel of about 1 mm and used. By using such a PDMS microchip, nematodes can be encapsulated and retained in a channel without anesthesia, so that a microbeam can be used to attach a specific tissue or region of a nematode or a specific cell. You can aim and irradiate.

However, since the conventional PDMS microchip has a thickness t of about 2.5 mm, for example, an ion microbeam with an underwater range of 1 mm does not penetrate to the lower surface of the conventional PDMS microchip. Therefore, the ion detection system (scintillator-photomultiplier tube assembly, etc.) of the ion microbeam irradiation device installed on the lower surface side of the PDMS microchip that converts the beam into an electrical signal is irradiated with the nematode. There is a problem that the beam cannot be detected accurately. In particular, it is important to investigate the effects of carbon ion beams used in heavy ion beam therapy and proton beams used in proton beam therapy (also called proton ion beams). It is desirable to be able to detect the result of irradiation with a carbon ion beam or a proton beam. However, in general, the underwater range of the carbon ion beam or proton beam is not so long that it can pass through the conventional PDMS microchip, so that the conventional PDMS microchip cannot be used.

In addition, it is also known that conventional PDMS microchips cause dehydration in nematodes encapsulated and retained in channels. Therefore, even if the nematode encapsulated and retained in the channel can be irradiated with the microbeam, the decrease in mobility observed after irradiation is due to the local irradiation of the microbeam or due to dehydration. There is also a problem that it is difficult to discriminate. Furthermore, the decline in motility due to dehydration affects nematode imaging.

Therefore, the present invention is a microchip suitable for locally irradiating a microbeam or observing (for example, fluorescence imaging observation) in a state where a biological sample such as an animal or plant cell or a micro organism is cultured or encapsulated. An object of the present invention is to provide a biological sample microchip capable of solving the above-mentioned conventional problems, and a cover suitable for use with the biological sample microchip, and a biological sample An object of the present invention is to provide a biological sample enclosure kit in which a microchip and a cover are combined. Another object of the present invention is to provide various methods using a microchip for biological samples.

In order to achieve the above object, the present invention provides a microchip for a biological sample in which at least one recess for culturing or enclosing a biological sample is formed on the surface, and the thickness t of the entire microchip is 800 μm or less. It is formed in this.

The thickness t of the entire microchip in the present invention is preferably 300 μm or less. Further, the thickness t of the entire microchip in the present invention is still more preferably 100 μm or less.

In the present invention, it is preferable that hydrophilicity is imparted to at least the microchip surface. In this specification, “surface” means “exposed surface”. That is, the hydrophilicity is given to the surface as long as the outermost surface is made hydrophilic. Furthermore, in the present invention, it is more preferable that hydrophilicity is imparted to the entire microchip.

Further, the present invention is a microchip for a biological sample in which at least one recess for culturing or encapsulating a biological sample is formed on the surface, wherein hydrophilicity is imparted to at least the microchip surface. To do.

In the present invention, the microchip surface may be subjected to plasma treatment.

Also, the present invention is directed to a cover used for enclosing and holding a biological sample in the concave portion of the biological sample microchip. That is, the cover in the present invention is transparent and has a surface roughness Ra of 0.02 μm or less.

The cover thickness t2 is preferably 50 μm or more and 300 μm or less. Moreover, it is preferable that the water contact angle (25 degreeC, 30% RH) of a cover is 100 degrees or less. Further, the oxygen permeability (20 ° C., 50% RH) of the cover is preferably 30 mL / (m ² · 24 h · atm) or more. Further, the carbon dioxide permeability (20 ° C., 50% RH) of the cover is preferably 30 mL / (m ² · 24 h · atm) or more. The cover is preferably made of polystyrene.

The present invention is also directed to a biological sample enclosing kit. That is, the biological sample enclosure kit according to the present invention is configured to include the biological sample microchip and the cover.

The present invention is also directed to a biological sample retention method in which a biological sample is enclosed and retained. That is, the biological sample retention method in the present invention is a method including enclosing a biological sample in the concave portion of the biological sample microchip.

The present invention is also directed to a microbeam irradiation method for irradiating a biological sample with a microbeam. That is, the microbeam irradiation method in the present invention is a method including enclosing a biological sample in the concave portion of the biological sample microchip and irradiating the biological sample with a microbeam.

The present invention is also directed to a method for culturing or breeding a biological sample. That is, in the method of the present invention, the biological sample is sealed in the recess of the biological sample microchip, and the biological sample microchip in which the biological sample is sealed is placed in a culture environment or a breeding environment of the biological sample. Standing still.

According to the invention, a biological sample can be cultured or encapsulated in a good state, and microbeam irradiation and observation of cells and tissues (for example, fluorescence imaging observation) can be performed well.

It is a figure which shows an example of the microchip for micro organism individuals among the microchips for biological samples. It is a figure which shows an example of the microchip for animal and plant cells among the microchips for biological samples. It is a figure which shows an example of the cover used for the microchip for biological samples. It is a figure which shows the outline | summary of the apparatus of the ion microbeam local irradiation experiment using a microchip. It is a figure which shows the irradiation trace of the ion micro beam which permeate | transmitted the microchip and reached | attained the detection system. It is a figure which shows the outline | summary of the apparatus of the ion microbeam local irradiation experiment using the conventional PDMS microchip. It is a figure which shows the frequency | count of head flexion of a nematode at the time of making it move freely on an agar plate for 1 hour in Experiment 2. FIG. It is a figure which shows the relative value of the frequency | count of a head bend of a nematode at the time of using a pure water in the experiment 2. FIG. It is a figure which shows the relative value of the frequency | count of head bending of a nematode at the time of using the 1st buffer solution in the experiment 2. FIG. It is a figure which shows the relative value of the frequency | count of head bending of a nematode at the time of using the 2nd buffer solution in the experiment 2. FIG. It is a figure which shows the frequency | count of head bending of a nematode at the time of using the 1st buffer solution in the experiment 3. FIG. It is a figure which shows the frequency | count of head bending of a nematode at the time of using the 1st buffer solution in the experiment 4. FIG. It is the bright field image which image | photographed the state which enclosed and maintained the nematode in the channel of the microchip in the experiment 5. FIG. It is the fluorescence image which image | photographed the state which enclosed and maintained the nematode in the channel of the microchip in the experiment 5. FIG. It is the bright field image which image | photographed the result of having cultured the human cell using the commercially available dish for cell cultures in the experiment 6. FIG. It is the bright-field image which image | photographed the result of having cultured the human cell using the microchip which has not performed the plasma process with respect to the microchip surface in the experiment 6. FIG. It is the bright field image which image | photographed the result of having cultured the human cell using the microchip which performed the plasma process with respect to the microchip surface in the experiment 6. FIG. In Experiment 6, it is the bright field image which image | photographed the result of having cultured the human cell using the microchip which apply | coated the coating agent which improves cell adhesiveness to the well surface of a microchip. It is the fluorescence image which image | photographed the result of having cultured the human cell on the well surface of the microchip in Experiment 6. FIG. It is a figure which shows the frequency | count of head flexion of the nematode which carried out the entrapment for 3 hours using the different cover for the nematode which moved freely on the agar plate for 3 hours in experiment 7, or a microchip.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings. In the embodiments described below, members that are common to each other are denoted by the same reference numerals, and redundant descriptions thereof are omitted.

[Example]
(Overall configuration of microchip)
FIG. 1 is a diagram showing an example of a microchip for microbiology among microchips 1 for biological samples (hereinafter simply referred to as “microchip 1”) in the present invention, and FIG. FIG. 1B is an enlarged sectional view of the recess 3 (channel 3a). The microchip 1 is a thin chip formed using PDMS (polydimethylsiloxane) as a base material. For example, it is used when irradiating a specific tissue or region or one specific cell with a microbeam while enclosing and holding a biological sample such as a nematode. The microchip 1 can also be suitably used when observing a biological sample (for example, fluorescence imaging observation). The microchip 1 can be rectangular, for example. For example, it is formed in a square shape in plan view having the same length in the X direction and the Y direction. For example, the size in the X direction and the Y direction is 15 mm. However, the microchip 1 is not necessarily limited to a square in plan view, and may be a rectangle, and may have a shape that matches the size of a sample stage such as an integrated microscope having an automatic photographing function.

At least one recess 3 for culturing or enclosing a biological sample is formed on the surface 2 of the microchip 1. In the microchip 1 shown in FIG. 1, the recess 3 is formed with a plurality of channels 3 a. Each channel 3 a is formed to extend in the X direction on the surface 2 of the microchip 1. Further, the plurality of channels 3a are formed at substantially equal intervals in the Y direction on the surface 2 of the microchip 1. Such a microchip 1 is in a form that can be suitably used when nematodes are used as biological samples. That is, the channel 3a is formed as a groove having a width W that can enclose the nematode. The nematode moves (crawls) by shaking its head from side to side. For this reason, the width W is set to be equal to or less than the width (thickness) B (size in the direction orthogonal to the head-to-tail direction) of the body of the nematode (typically the thickest part, for example, the abdomen). . Since the nematode has a cylindrical elongated body and is elastic, it can be enclosed in the channel 3a even when the width W of the channel 3a is narrower than the width B of the body, and the fit in the channel is good. The movement can be reliably suppressed. For example, among various nematodes, C.I. In the case of the elegance adult microchip 1, the width W is preferably set so that the difference between the width W of the channel 3a and the width B of the nematode body is approximately 20 μm or less. However, C.I. In the case of the elegance larva microchip 1, the width W of the channel 3a is preferably equal to or less than the width B of the nematode body. By setting the width W of the channel 3a to be in the above range, the movement of the nematode can be suppressed and preferably maintained. If the nematode is encapsulated with the buffer solution in the channel 3a and then a cover described later is arranged in the opening, the depth D of the channel 3a is larger than the width B of the nematode body. It is preferable to set it deeply. However, if the depth D is too deeper than the width B of the nematode body, the nematode may rotate vertically in the channel 3a or bend in two. Thus, for example, C. elegans C.I. When encapsulating the wild type of elegance, the depth D of the channel 3a is C.I. What is necessary is just to set so that it may be as large as the width | variety B of an elegance body, or about 10 micrometers. For example, the width W of the channel 3a is about 10 to 90 μm, and the depth D is about 20 to 100 μm. C. Elegance has stages of L1 to L4 larvae (approximately 2.5 days of age immediately after hatching), and the width B of the body is about 10 to 40 μm. Therefore, in the case of the microchip 1 for larvae at each stage, it is preferable that the width W of the channel 3a is 5 to 40 μm and the depth D is 10 to 50 μm. The width B of the body of a relatively young adult (about 2.5 to 4.5 days of age) is about 50 to 70 μm. Therefore, in the case of a relatively young adult microchip 1, it is preferable that the width W of the channel 3a is 40 to 60 μm and the depth D is 50 to 70 μm. Nematodes grow even after they become adults. In elegance, the width B of the body is finally about 90 μm. For this reason, in the case of the adult microchip 1 of about 4.5 days or older, the width W of the channel 3a is preferably 60 to 90 μm and the depth D is preferably 70 to 100 μm. As described above, the microchip 1 can be formed with channels 3a of various sizes according to the nematode generation stage. Further, for example, a substantially rectangular channel having a function of holding a buffer solution or bait (for example, E. coli) is formed at the end of the plurality of linear channels 3a of the microchip 1 shown in FIG. Can be reared and observed.

Alternatively, the width W of the channel 3a can be made smaller than the width B of the nematode body, and the depth D of the channel 3a can be made smaller than the width B of the nematode body. In the channel 3a having a width W and a depth D smaller than the width B of the nematode body, the nematode is stretched in the long axis direction so as to be closely attached to the channel 3a. It can be suppressed more strongly. Such a channel 3a can be suitably used for fluorescence imaging observation of an arbitrary cell or tissue. Further, if the depth D of the channel 3a is about 2/3 of the width B of the nematode body and the width W is about 1.2 to 1.5 times the width B of the nematode body, The body can be held in the channel 3a in a state of being expanded in the width direction of the channel 3a, and can be suitably used for morphological observation of cells and tissues. Here, since the microchip 1 having a small width W or depth D of the channel 3a has a small amount of buffer solution that can be held in the channel 3a, the amount of the buffer solution may be insufficient during long-term breeding and observation. is there. For this reason, it can be suitably used for morphological observation in a relatively short time. Also, if the nematode is pushed into the channel 3a where the width W or the depth D of the channel 3a is small, the nematode may be stressed. Therefore, the channel size should be selected in consideration of the influence of stress on the observed response. Is preferred.

When the width W or depth D of the channel 3a is made smaller than the width B of the nematode body, for example, the nematode C.I. For elegance wild-type L1-L2 larvae, the width W of the channel 3a is preferably 5-15 μm and the depth D is preferably 10-15 μm. For L3 to L4 larvae, the width W of the channel 3a is preferably 15 to 30 μm and the depth D is preferably 15 to 30 μm. For adults, the width W and depth D of the channel 3a are preferably about 10 μm smaller than the width B of the nematode body.

Alternatively, if the width W or depth D of the channel 3a is made larger than the width B of the nematode body, it can be kept in the observation field such as a microscope without suppressing the movement of the enclosed nematode body, The movement of the nematode can be observed. For example, C. elegans C.I. In order to enclose the wild type of elegance, the width W and depth D of the channel 3a may be about 10 to 20 μm larger than the width B of the nematode body. Such a channel 3a can be suitably used for comparatively macroscopic fluorescence imaging observation or the like targeting a group of neural cells or muscle cells of a moving nematode.

Furthermore, the microchip 1 can be suitably used not only for various nematode individuals (eggs, larvae, adults) but also for holding or observing biological samples that can be enclosed in the recesses 3. Such a biological sample may be either an animal or a plant, and the presence or absence of motor ability is not limited. For example, microscopic organisms whose body width B or diameter F is approximately equal to or less than the depth D of the recess 3, that is, approximately 2/3 or less of the thickness t of the microchip 1, animals, plants, algae cells, seeds, pollen, eggs, Sperm etc. are targeted. Specifically, pollen of various plants, cultured cells and blood cells of animals and plants, Paramecium, Tetrahymena, Euglena, Paramecium, Mikazuki, etc., microorganisms such as fertilized eggs of nematodes, fertilized eggs of Drosophila and zebrafish, It can be suitably used for animal individuals such as rotifers (for example, stink bugs and L-type rotifers), and beetles (for example, Dojarjaran rotifers). However, as the shape of the recess 3 formed on the surface 2 of the microchip 1, it is preferable to adopt a shape suitable for a biological sample. When enclosing a biological sample having motility, the depth D of the channel 3 is approximately equal to the body thickness (typically the length in the short axis direction of the thinnest surface) T or the diameter F of the biological sample. It is desirable that the width W of the channel 3 is equal to or less than the width B or the diameter F of the organism body, and the length L of the channel 3 is sufficiently longer than the body length H of the organism. However, the size of the channel 3 is not limited to the above, and may be set according to the elasticity of the biological sample to be sealed, the speed of movement, and the like.

In the microchip 1 shown in FIG. 1, the case where the width W and the depth D of the channel 3a are constant has been described as an example, but the present invention is not limited to this. Nematodes generally have a narrower head and tail than the abdomen. Therefore, a shape in which the width W or depth D of the channel 3a is matched to the width of each nematode site, for example, a narrow portion having a small width W or depth D may be provided in a part of the channel 3a. By enclosing the head or tail of the nematode in such a narrowed portion, the nematode can be suitably retained. At this time, it is preferable that the non-constricted portion is equivalent to any of the conditions described as the width W and the depth D of the channel 3a. For example, the shape of the channel 3a can be a constricted repeating pattern that repeats a wide portion and a small portion.

The shape of the channel 3a may be an arbitrary shape such as a sine wave shape that matches the trajectory of the nematode's lameness movement or an oval shape that can enclose an egg. The number of channels 3a may be freely selected according to the purpose. For example, it may be 1 or more, 2 or more, 3 or more, and 5 or more are preferable. More preferably, it is 10 or more, more preferably 15 or more. The upper limit of the number of channels 3a is not limited, and may be set within a range that can be formed on a chip. For example, it may be 100 or less and 50 or less. One or more of the channels 3a may be different from the shape and size of the other channels 3a.

FIG. 2 is a diagram showing an example of a microchip for animal and plant cells in the microchip 1. The surface 2 of the microchip 1 shown in FIG. 2 shows a structure in which a plurality of rectangular wells 3b for cultivating animal and plant cells are formed as recesses 3 for enclosing biological samples (FIG. 2 ( a)). However, the shape of the well 3b is not limited to a rectangle, and may be a circle or an ellipse. Since the cells (not moving violently) can be irradiated with microbeams or observed without being held by one cell, the size of the recess 3 may be set to be considerably larger than that of the cells. For example, as shown in FIG. 2B, the well 3b is formed in a rectangular shape having a width L in the X direction and a width W in the Y direction. For example, if the ratio of the width L in the X direction to the width W in the Y direction is set to have the same aspect ratio as the angle of view of the camera that captures the whole cell before and after the microbeam irradiation, the entire region of one well 3b Is suitable for observation for the purpose of tracking the cell dynamics after irradiation.

Although the thickness of the cultured cells is approximately 10 μm or less, the depth D of the well 3b is preferably about 50 to 100 μm in order to secure a space for injecting a culture solution or the like into the well 3b. Further, in order to maintain the shape of the formed well 3b, it is preferable that the depth D of the well 3b has an upper limit of about 2/3 of the thickness t of the microchip 1. Therefore, for example, if the thickness t of the microchip 1 is 300 μm, the depth D of the well 3b is preferably 50 to 200 μm. Here, the case where the microchip 1 in which the well 3b illustrated in FIG. 2 is used is used as an animal or plant cell has been described as an example. May be used.

(Thickness of microchip)
The microchip 1 as described above is formed in a thin sheet shape. If the thickness t is 800 μm or less, a microbeam having an underwater range of about 1000 μm can pass through the microchip 1 and reach a detection system provided on the lower surface side of the microchip 1. Such a microchip 1 having a thickness t of 800 μm or less can transmit a microbeam having an underwater range of about 1000 μm even when the cover described later is provided on both the opening side and the lower surface side of the recess. Particularly preferred. As the thickness t of the microchip 1 is thinner than 800 μm, the types of microbeams that can be detected by the ion detection system of the ion microbeam irradiation apparatus can be increased. On the other hand, the thicker the thickness t of the microchip 1, the more kinds of biological samples that can be cultured or sealed in the recess 3.

FIG. 3 is a diagram showing an example of a cover that covers the surface 2 of the microchip 1. The shape of the surface that contacts the surface of the microchip 1 is not particularly limited, and examples thereof include a rectangle, a circle, and an ellipse. The shape which rounded one or more corners of the rectangle may be sufficient. A rectangle as shown in FIG. 3 is preferable because it can be easily manufactured and manufacturing loss hardly occurs. The cover 4 may have a grip part or may have a hole. The cover 4 having a gripping portion is preferable because it can be easily arranged on the surface of the microchip 1 or peeled off. The cover 4 in which the holes are formed is preferable because air bubbles can be easily discharged through the holes when the cover 4 is brought into close contact with the surface of the microchip 1.

The underwater range of the ion beam is determined by the ion species and energy. The following Table 1 lists the major ions and energies that can be used at the Takasaki Quantum Science and Technology Research Institute of Quantum Science and Technology, and shows their relationship with the calculated values of the underwater range.

When the microchip 1 is irradiated with an ion beam having an underwater range longer than 800 μm, the ion beam passes through the microchip 1. For example, in the case of 190 MeV carbon ions having an underwater range of 930 μm exemplified in Table 1, the ion beam can pass through the microchip 1. Therefore, by setting the thickness t of the microchip 1 to 800 μm or less, it becomes possible to perform an experiment using an ion beam having an underwater range longer than 800 μm. Will also increase significantly. In particular, in the case of carbon ions used in heavy ion radiotherapy, at least 3 different carbon ions of 320 MeV with an underwater range of 2370 μm, 220 MeV of 1210 μm with an underwater range of 190 MeV with an underwater range of 930 μm. There is an advantage that various kinds of carbon ions can be used. Further, in the case of protons used for proton therapy, at least two types of protons having different energies can be used: a 15 MeV proton with an underwater range of 2440 μm and a 10 MeV proton with an underwater range of 1180 μm. This makes it possible to investigate differences in biological effects due to energy differences in the same experimental system for quantum beams used in treatment sites such as heavy ion beam therapy and proton beam therapy. However, when the thickness t of the microchip 1 is 800 μm or less, the ion beam such as neon exemplified in Table 1 does not pass through the microchip 1.

Therefore, the thickness t of the microchip 1 is preferably 300 μm or less. By setting the thickness t to 300 μm or less, the microbeam of 350 MeV neon ions having an underwater range of 700 μm and 260 MeV neon ions having an underwater range of 420 μm shown in Table 1 is also installed on the lower surface side of the microchip 1. It becomes possible to detect accurately by the ion detection system of the ion microbeam irradiation apparatus. This makes it possible to investigate the biological effects of at least four different types of ions such as helium ions, protons, carbon ions, and neon ions in the same experimental system.

More preferably, the thickness t of the microchip 1 is 100 μm or less. By setting the thickness t to 100 μm or less, microbeams of all ion species exemplified in Table 1 can pass through the microchip 1. In other words, the ion detection system of the ion microbeam irradiation apparatus installed on the lower surface side of the microchip 1 can accurately detect the microbeam transmitted through the biological sample. Therefore, if the thickness t is set to 100 μm or less, the experiment is performed using all the ion species illustrated in Table 1, that is, at least five different microbeams such as helium ions, protons, carbon ions, neon ions, and argon ions. The versatility of the microchip 1 becomes higher.

When the thickness t of the microchip 1 is set to 800 μm, the biological sample that can be cultured or sealed in the recesses 3 such as the channel 3a and the well 3b is not more than the depth D of the recesses 3, that is, approximately 530 μm or less. Become. For example, moving biological samples include Paramecium, Tetrahymena, Euglena, Paramecium, Daphnia, C. elegans. There are elegance, rotifer, caterpillar, leopard mite (dust mite), artemia and so on. Since these are preferably observed in a state where movement is suppressed, the microchip 1 shown in FIG. 1 may be used to enclose and hold the channel 3a. In addition, biological samples that do not move, or do not move so much as to interfere with microbeam irradiation and observation include pollen, mika mochi, moss, volbox, cultured cells, nematode eggs (eg, fertilized eggs), blood cells, Examples include human eggs, rotifer eggs, Drosophila eggs, and zebrafish eggs (eg, fertilized eggs). Since these do not move, or do not move so much as to interfere with microbeam irradiation and observation, the microchip 1 shown in FIG. 2 may be used to culture or enclose and hold in the well 3b. However, the microchip 1 shown in FIG. 1 may be used. In the present specification, “egg” includes both unfertilized eggs and fertilized eggs.

When the thickness t of the microchip 1 is 300 μm, the biological sample that can be cultured or sealed in the recess 3 such as the channel 3a or the well 3b has a depth D of the recess 3 or less, that is, approximately 200 μm or less. It becomes a target. For example, moving biological samples include Paramecium, Tetrahymena, Euglena, Euglena, Nematode C. There are elegance, rotifer, caterpillar, and leopard mite. Since these are preferably observed in a state where movement is suppressed, the microchip 1 shown in FIG. 1 may be used to enclose and hold the channel 3a. Biological samples that do not move, or do not move so much as to interfere with microbeam irradiation and observation include pollen, mika mochi, moss, cultured cells, nematode eggs (eg, fertilized eggs), blood cells, and human eggs. , Rotifer eggs. Since these do not move, or do not move so much as to interfere with microbeam irradiation and observation, the microchip 1 shown in FIG. 2 may be used to culture or enclose and hold in the well 3b. However, the microchip 1 shown in FIG. 1 may be used.

Further, when the thickness t of the microchip 1 is set to 100 μm, the biological sample that can be enclosed in the recesses 3 such as the channel 3a and the well 3b is intended to have a depth D of the recess 3 or less, that is, approximately 65 μm or less. Become. For example, moving biological samples include Paramecium, Tetrahymena, Euglena, Euglena, Nematode C. There are elegance and rotifer. Since these are preferably observed in a state where movement is suppressed, the microchip 1 shown in FIG. 1 may be used to enclose and hold the channel 3a. Examples of biological samples that do not move, or that do not move so much as to interfere with microbeam irradiation and observation include pollen, mika mochi, moss, cultured cells, nematode eggs (eg, fertilized eggs), and blood cells. Since these do not move, or do not move so much as to interfere with microbeam irradiation and observation, the microchip 1 shown in FIG. 2 may be used to culture or enclose and hold in the well 3b. However, the microchip 1 shown in FIG. 1 may be used.

Here, the case where the thickness t of the microchip 1 is 800 μm, 300 μm, and 100 μm has been described as an example, but the present invention is not limited to this. It can be set as appropriate in consideration of the underwater range of the ion beam to be used and the size of the biological sample to be cultured or sealed in the recess 3. For example, an arbitrary thickness such as 600 μm, 500 μm, or 200 μm may be used. If the thickness t is 600 μm, the depth D of the concave portion 3 formed on the surface 2 of the microchip 1 can be set to 400 μm, so that daphnia and the like can be suitably enclosed and retained. 700 μm 350 MeV neon ions or the like may be irradiated. Further, if the thickness t is 200 μm, the depth D of the recess 3 is set to 140 μm, so that L-type rotifer larvae, eggs, human eggs, etc. can be suitably enclosed and retained. 200 MeV neon ions having an underwater range of 270 μm and 460 MeV argon ions having an underwater range of 250 μm may be irradiated.

Furthermore, it is naturally possible to set the thickness t of the microchip 1 to be larger than 800 μm, for example, to an arbitrary thickness such as 1000 μm, 1500 μm, 2000 μm. If the thickness t is 1000 μm, the depth D of the recess 3 formed on the surface 2 of the microchip 1 can be set to 650 μm, so that fertilized eggs of silkworms can be suitably enclosed and retained. 10 MeV protons having an underwater range of 1180 μm and 220 MeV carbon ions having an underwater range of 1210 μm may be irradiated. For example, when investigating the influence of protons used in proton beam therapy, if the thickness t of the microchip 1 is 1000 μm or less, at least 2 of 10 MeV protons in the underwater range 1180 and 15 MeV protons in the underwater range 2440 μm. Different types of protons can be used. Further, if the thickness t is 1500 μm, the depth D of the recess 3 can be set to 1000 μm, so that fertilized eggs of medaka can be suitably enclosed and retained, and the ion beam is 50 MeV with an underwater range of 1770 μm. Helium ions, 320 MeV carbon ions having an underwater range of 2370 μm, and the like can be irradiated. Furthermore, if the thickness t is 2000 μm, the depth D of the recess 3 is set to 1300 μm, so that the types of micro-organisms, eggs, and plant seeds that can be enclosed can be increased. In this case, the ion beam that can be transmitted through the microchip 1 is limited to 50 MeV helium ions with an underwater range of 1770 μm, 320 MeV carbon ions with an underwater range of 2370 μm, 63 MeV helium ions with an underwater range of 2680 μm, and the like. It can be suitably used for culture or entrapment retention for fluorescence imaging observations and the like that are not limited to irradiation.

Although the thickness t of the microchip 1 has been described by paying attention to the relationship between the underwater range of the microbeam and the object to be sealed in the recess, the present invention is not limited to this. By using the thin microchip 1 for microscopic observation of the object enclosed in the concave portion 3 of the microchip 1, observation that has been difficult in the past can be made possible. For example, in the past, observation using a water immersion lens has been performed when fluorescent imaging observation is performed while an object is alive. On the other hand, by holding the object in the concave portion 3 of the microchip 1 having a thickness t, it is possible to realize microscopic observation including live fluorescence imaging observation using an upright fluorescent microscope. From this point of view, the thickness of the microchip 1 is preferably 400 μm or less, more preferably 300 μm or less, and even more preferably 200 μm or less.

(Hydrophilicity of microchip)
Further, in the microchip 1, the microchip surface 2 on which at least the concave portion 3 is formed has hydrophilicity. PDMS is usually hydrophobic. When the microchip 1 is formed using only PDMS as a base material, the surface 2 of the microchip exhibits hydrophobicity. If the microchip surface 2 is hydrophobic, dehydration occurs in a biological sample such as a nematode enclosed in the channel 3a. In order to prevent this, the microchip 1 imparts hydrophilicity to the microchip surface 2. The hydrophilicity may be imparted only to the microchip surface 2 or may be imparted to the entire microchip. In this specification, having hydrophilicity means a state in which a water contact angle (25 ° C., 30% RH) is 100 degrees or less. Preferably, the water contact angle is 90 degrees or less, more preferably 60 degrees or less, and still more preferably 40 degrees or less.

For example, when hydrophilicity is imparted only to the microchip surface 2, a hydrophilicity imparting agent such as MPC (2-Methacryloyloxyethyl Phosphorylcholine) polymer is applied to the surface 2 of the microchip 1. That is, the thickness of the hydrophilic portion can be adjusted by the amount (thickness) of the hydrophilicity imparting agent applied. Typically, the microchip 1 subjected to such surface hydrophilic treatment includes a base material layer using PDMS as a base material and a hydrophilic layer containing a hydrophilicity-imparting agent. Thereby, hydrophilicity can be imparted to the surface 2 of the microchip 1. However, in this case, since the hydrophilicity imparting agent applied to the surface 2 of the microchip 1 reacts with air, the hydrophilicity of the microchip surface 2 is lost over time. Therefore, the microchip 1 is not suitable for long-time use or repeated use.

Further, by performing plasma treatment on the surface 2 of the microchip 1, hydrophilicity can be imparted only to the microchip surface 2. That is, the thickness of the hydrophilic portion can be adjusted by plasma irradiation conditions (irradiation intensity, irradiation time, etc.). Typically, the microchip 1 subjected to such surface hydrophilic treatment includes a plasma treated portion (portion having hydrophilicity) and a plasma untreated portion. Moreover, polarity can be provided to the outermost surface of a microchip (typically the recessed part 3) by performing a plasma process. For example, when cells are cultured using the microchip 1 for animal and plant cells shown in FIG. 2, it is preferable that the cultured cells adhere to the well 3b. Adhesive cells are known to grow well by adhering to a polar scaffold. For this reason, especially when using the microchip 1 disclosed here for the microbeam irradiation or observation for the adhesive cells, the plasma-treated microchip 1 can be preferably used. However, in the hydrophilization by plasma treatment, the hydrophilicity of the microchip surface 2 may be lost over time, as in the case of applying the hydrophilicity imparting agent such as the MPC polymer described above.

On the other hand, when hydrophilicity is imparted to the entire microchip 1, for example, when the microchip 1 is manufactured, the microchip 1 is poured into a mold and solidified by adding a hydrophilicity imparting agent to PDMS as a base material. It ’s fine. Thereby, hydrophilicity can be imparted to the entire microchip 1. As the hydrophilicity imparting agent in this case, for example, nonionic surfactants such as Tween 20, Brij 35, Triton X-100 can be used. If the nonionic surfactant is up to about 10 wt% with respect to the base material PDMS, hydrophilicity can be imparted to the PDMS without hindering the formation of the microchip 1. However, even if the addition amount of the nonionic surfactant is 10 wt% or less, if the addition amount of the nonionic surfactant increases, the transparency of the PDMS may be impaired. Therefore, it is preferable that the addition amount of the nonionic surfactant is up to about 3 wt%. If the addition amount is about 3 wt% or less, effective hydrophilicity can be imparted to PDMS without impairing the transparency of PDMS. In addition, conventionally known hydrophilicity imparting agents that are known to impart hydrophilicity to PDMS can be used without any particular limitation. By imparting hydrophilicity to the entire microchip 1, hydrophilic deterioration can be suppressed, and thus there is an advantage that hydrophilicity can be maintained even when the microchip 1 is repeatedly used. .

By imparting hydrophilicity to the microchip 1, hydrophilicity is also imparted to the recess 3 for enclosing the biological sample. Such hydrophilicity makes it easy to draw water into the recess 3. Thereby, the biological sample can be smoothly sealed in the recess 3. Moreover, the water retention of the recessed part 3 can be improved. In other words, evaporation of water from the recess 3 can be suppressed. For this reason, when a biological sample requiring a wet state is cultured or sealed, and microbeam irradiation or observation is performed, dehydration and drying of the biological sample can be reduced. For example, C. elegans C. When irradiating a microbeam while enclosing and holding a biological sample such as elegance, it is possible to suppress, for example, dehydration of the biological sample and a decrease in mobility considered to be caused by it. As a result, C.I. If the mobility of the nematode is reduced after irradiation with microbeams with elegance encapsulated and retained, it will be possible to easily determine that the cause of the decrease is due to microbeam irradiation. . Moreover, since it can hold | maintain, without producing the dehydration symptom in a biological sample, it is suitable also for observation (for example, fluorescence imaging observation) of a biological sample.

In addition, when the microchip 1 for animal and plant cells shown in FIG. 2 is produced, after imparting hydrophilicity to the entire microchip 1 as described above, plasma treatment is further performed on the microchip surface 2. You may do it. By this plasma treatment, the adhesion of cells to the microchip surface 2, in particular, the surface 6 of the well 3 b can be improved, so that the microchip 1 suitable for cultured cells can be realized. However, when the microchip 1 is used for floating cells or blood cells, cell adhesion may not be required.

When using adhesive cells as the target, it is preferable to perform a treatment for improving the cell adhesiveness of the surface 2 of the microchip 1. For example, the plasma treatment described above can be given. Moreover, cell adhesion can also be improved using methods other than plasma treatment. For example, as a process for improving the cell adhesiveness, there is a process for applying a surface coating agent for improving the cell adhesiveness to the surface 6 of the well 3b. In this case, the surface 6 of the well 3b only needs to include at least the bottom surface of the well 3b. The surface coating agent for improving cell adhesion may be appropriately selected from conventionally known cell adhesion polymers. For example, polylysine, fibronectin, Corning (registered trademark) Inc. CellTak (registered trademark) (manufactured by mussel-derived polyphenol protein) and the like can be mentioned. Such treatment for improving cell adhesion may be performed by one method alone or in combination of two or more methods. Also, a process for imparting hydrophilicity to the microchip 1, for example, a process for imparting hydrophilicity to the entire microchip 1, a process for imparting hydrophilicity to at least the surface 2 (application of a hydrophilicity imparting agent, plasma treatment, etc.) And treatment for improving cell adhesion may be performed in combination.

(cover)
The microchip 1 configured as described above can be used in combination with a cover when used. The cover is typically arranged to cover the channel 3. For example, the cover 4 shown in FIG. 3 is used. The cover 4 is a transparent sheet-like cover having a thickness of about 100 μm, for example. The cover 4 is covered so as to be in close contact with the surface 2 of the microchip 1, for example. Therefore, the biological sample enclosed in the channel 3a by the cover 4 can be prevented from being detached from the inside of the channel 3a, and can be retained in the channel 3a. Further, by covering the surface 2 of the microchip 1 with the cover 4, evaporation of the liquid (typically moisture) from the recess 3 can be suppressed. From the viewpoint of culturing or enclosing a biological sample and suppressing moisture evaporation from the recess 3, the microchip 1 and the cover 4 are preferably disposed so as to be in close contact. Here, “adhesion” refers to a state in which the cover surface 5 is in contact with the microchip surface 2 without a gap due to the self-adsorption property of PDMS. Particularly preferably, it is arranged so as to be watertight. In order to arrange the microchip 1 and the cover 4 in close contact by the self-adsorption property of PDMS, it is preferable that the contact surfaces of the microchip 1 and the cover 4, that is, the microchip surface 2 and the cover surface 5 are smooth planes. The smoothness of the surface 2 of the microchip 1 is more preferably 0.007 to 0.012 μm in surface roughness Ra (JIS 2001 standard). As for the smoothness of the surface 5 of the cover 4, the surface roughness Ra (JIS 2001 standard) is preferably 0.03 μm or less, more preferably 0.02 μm or less, and still more preferably 0.01 μm or less. Although the minimum of the smoothness of the surface 5 of a cover is not specifically limited, Preferably it is 0.002 micrometer or more, More preferably, it is 0.005 micrometer or more. Note that the cover 4 provided on the surface 2 of the microchip 1 is preferably formed of a transparent material having a smooth surface 5 (at least a surface in contact with the microchip 1) and hardly cracking. For example, a polystyrene film having a thickness of 100 μm and an Ra of about 0.008 μm is suitable because it is transparent and difficult to break, has good adhesion to PDMS, and can be easily peeled off. A PDMS cover film can also be used.

In a preferred embodiment, the biological sample placed on the microchip 1 and a liquid (typically water or an aqueous solution) are pressed with a cover, whereby the biological sample can be guided into the channel 3a. In addition, by pressing the cover 4 on the microchip 1 in this way, the adhesion between the microchip 1 and the cover 4 can be improved.

It is preferable that the biological sample held in the recess 3 of the microchip 1 using the cover 4 is easy to observe visually or using a magnifier such as a microscope. For this reason, it is preferable that the cover disposed on the microchip 1 is transparent. That is, the cover 4 that covers the surface 2 of the microchip 1 is preferably transparent. Or when arrange | positioning a cover other than the surface 2 of the microchip 1, for example, a back surface, it is preferable that the said cover is also transparent. The cover 4 that covers the front surface 2 of the microchip 1 and the cover that is disposed on the back surface of the microchip 1 may be the same or may have different properties. The cover's parallel light transmittance (according to the optical characteristic test method of JISK7105 plastic) is preferably 80% or more. Preferably it is 85% or more, more preferably 90% or more. The haze of the cover (according to the JISK7105 plastic optical property test method) is preferably 4.0% or less. Preferably it is 2.5% or less, More preferably, it is 0%. From the viewpoint of ease of observation or ease of experimentation (ease of microbeam irradiation), the refractive index of the cover disposed on the microchip 1 is preferably small. For example, the refractive index of the cover is preferably 1.0 or less, and more preferably 0.8 or less. The lower limit of the refractive index is not particularly limited, but may be, for example, 0.3 or more. In addition, a cover having no or little autofluorescence in the wavelength region of light used for observation or excitation is suitable for fluorescence observation. Further, the light transmittance of the cover disposed on the microchip 1 (by the linear transmission method using an ultraviolet-visible spectrophotometer) is preferably 70% or more, and 80% or more in the wavelength region of the light used. Is more preferable, and it is still more preferable that it is 90% or more. For example, when visible light observation is performed, the light transmittance of the cover is desirably 80% or more at a wavelength of 360 to 860 nm, more preferably 85% or more, and still more preferably 90% or more. When using light in the ultraviolet region for observation (for example, when performing multiphoton observation), the light transmittance at a wavelength of 860 to 1500 nm is preferably 80% or more, more preferably 90% or more. .

From the viewpoint of operability for attaching / detaching the cover 4 to / from the microchip 1, the cover 4 is preferably flexible. By using the supple cover 4, it is possible to prevent the cover from being damaged when the cover 4 adhered to the surface 2 of the microchip 1 is removed.

The thickness t2 of the cover is not particularly limited. However, from the viewpoint of operability when attaching / detaching the cover 4 to / from the surface 2 of the microchip 1, for example, the cover 4 is preferably 50 μm or more, and more preferably 80 μm or more. By using the cover 4 having such a thickness, the cover 4 can be prevented from being damaged during the attachment / detachment operation. On the other hand, from the viewpoint of microbeam transparency or visibility (observability), the thickness t2 of the cover 4 is preferably 300 μm or less, and more preferably 200 μm or less. For example, it is preferable to use the cover 4 having a thickness of 100 μm or more and 150 μm or less. The cover disposed on the back surface of the microchip 1 is also preferably a cover according to this.

Moreover, it is preferable that the cover 4 has hydrophilicity like the microchip 1. By using the cover 4 having hydrophilicity, evaporation of the liquid (typically moisture) from the recess 3 can be further suppressed. Thereby, drying of a biological sample can be prevented. In this case, for example, it is preferable to use the cover 4 having a water contact angle (25 ° C., 30% RH) of 100 degrees or less. For example, the cover 4 of 90 degrees or less is more preferable. In addition, although the minimum of the water contact angle of the cover 4 is not specifically limited, For example, what is necessary is just 40 degree | times or more, and may be 60 degree | times or more. From the viewpoint of suppressing the evaporation of liquid (water) from the recess 3 of the microchip 1, it is preferable that both the microchip 1 and the surface 5 of the cover 4 have hydrophilicity, for example, the water contact angle is 100 degrees or less. The microchip 1 and the cover 4 having a water contact angle of the surface 5 of 100 degrees or less are preferably used in combination. The cover 4 having a water contact angle of the surface 5 of 60 degrees or more and 100 degrees or less is exemplified as one condition of a suitable combination.

Moreover, the cover 4 which can permeate | transmit oxygen is preferable at the point which can supply oxygen to the biological sample culture | cultivated or enclosed in the recessed part 3. FIG. When a biological sample is cultured or sealed in the recess 3 for a certain time or more, or when a biological sample with high oxygen demand is used, oxygen tends to be insufficient in the space in the recess 3 of the microchip 1. Therefore, the oxygen permeability of the cover 4 is important in order to prevent the lack of oxygen in the biological sample. Therefore, for example, the cover 4 having an oxygen permeability (20 ° C., 50% RH) of 30 mL / (m ² · 24 h · atm) or more when measured by a gas chromatographic method is desirable. In consideration of long-term culture or encapsulation exceeding 1 hour, the oxygen permeability is preferably 100 mL / (m ² · 24 h · atm) or more, more preferably 1000 to 1500 mL / (m ² · 24 h. Atm).

Further, the cover 4 capable of transmitting carbon dioxide is preferable in that carbon dioxide can be supplied to a biological sample such as a plant cultured or enclosed in the recess 3 or carbon dioxide can be discharged from the recess 3. When a biological sample is cultured or enclosed in the recess 3 for a certain time or more, or when a biological sample having a high carbon dioxide requirement is used, carbon dioxide is insufficient in the space in the recess 3 of the microchip 1. The carbon dioxide permeability of the cover 4 is important in order to control the influence of carbon dioxide on a biological sample because the carbon dioxide tends to be excessive. Therefore, for example, the cover 4 having a carbon dioxide permeability (20 ° C., 50% RH) of 30 mL / (m ² · 24 h · atm) or more when measured by a gas chromatographic method is desirable. When considering the long culture or encapsulating more than 1 hour, preferably carbon dioxide transmittance of ^{300mL / (m 2 · 24h ·} atm) or more, more preferably 3000mL ^/ (m 2 · 24h · atm) or more. Here, considering the balance of oxygen permeability and carbon dioxide permeability, e.g. in the cover 4 of the oxygen permeability ^{1000mL / (m 2 · 24h ·} atm), the carbon dioxide permeability 3000mL ^/ (m 2 · 24h · atm) or more. In other words, a carbon dioxide permeability of 3 times or more than the oxygen permeability can be selected, and may be about 10 times.

The material of the cover 4 is not particularly limited, and examples thereof include polystyrene, polypropylene, polyethylene, polyester, polyethylene terephthalate, polyethylene naphthalate, glass, and polyimide. Among these, polystyrene and polypropylene are more preferable as the material of the cover 4 because they are excellent in the oxygen permeability described above. In addition, conventionally well-known various additives (a plasticizer, a crosslinking agent, a stabilizer, etc.) may be added.

Also, since the microchip 1 formed using PDMS as a base material has high self-adsorption property (adhesion), the back surface may be adsorbed on a sample stage, for example, a stereomicroscope stage, which may be difficult to operate. In order to prevent this, a cover having a thickness of about 100 μm may be laid on the back surface of the microchip 1 and placed on the sample table. The cover provided on the back surface of the microchip 1 only needs to be formed of a material having a smooth and transparent surface. For example, the above polystyrene film is suitable. For example, a cover glass or a slide glass having a thickness of 100 μm and a surface roughness Ra (JIS 2001 standard) of about 0.001 μm is preferable because it is transparent, has high adhesion to PDMS, and can be easily peeled off. It is. When irradiating an ion microbeam, the microbeam needs to pass through the cover disposed on the front surface 2 and the back surface of the microchip in addition to the microchip 1. For this reason, when ion microbeam irradiation is performed using a cover, ion species having an underwater range longer than the sum of the thicknesses of the cover 2 on the front surface 2 and the back surface of the microchip 1 and the thickness t of the microchip 1 are selected. It is preferable to select and use. When performing microbeam irradiation of ion species having a relatively short underwater range, for example, a polyimide film (Kapton (registered trademark)) manufactured by Toray DuPont Co., Ltd. having a thickness t2 of about 25 μm is used as the cover 4. May be used. Further, in the ion microbeam irradiation, in order to visually confirm ions that have passed through the biological sample, a cover provided on the back surface of the microchip 1 is provided with a plastic film for ion track detection, for example, manufactured by Fukubi Chemical Co., Ltd. Use CR-39 (Solid State Nuclear Track Detector, TNF-1). The thickness of CR-39 should be selected as appropriate such as 100 μm or 450 μm in consideration of the underwater range and the conditions of alkaline etching for detecting ion tracks depending on the ion species used. good. Furthermore, in the case of performing fluorescence imaging observation of a biological sample cultured or encapsulated and retained in the microchip 1, the covers provided on the front surface 2 and the back surface of the microchip are made of a material having no or little autofluorescence in addition to the above features. It is good to choose what. For example, a polystyrene film can be suitably used.

(kit)
As another embodiment, a biological sample culture kit or a biological sample enclosing kit combined with the microchip 1 and the cover 4 can be provided. Such a biological sample culture kit or biological sample enclosure kit cultivates or encloses a biological sample in a gap formed between the recess 3 of the microchip 1 and the cover 4. The biological sample culture kit or the biological sample enclosure kit can be used for the purpose of keeping the biological sample cultured or enclosed in the kit in a wet state. In this case, it is preferable to combine the microchip 1 having a surface roughness Ra of about 0.007 to 0.012 μm and the cover 4 having an Ra of 0.03 μm or less. As the cover 4 that covers the surface 2 of the microchip 1, for example, a polystyrene film having a thickness t2 of 100 to 130 μm and an Ra of about 0.008 μm can be used. Moreover, as a cover arrange | positioned on the back surface of the microchip 1, the cover glass and slide glass whose Ra is about 0.002 micrometer other than the said polystyrene film can be used conveniently. The biological sample culture kit or the biological sample enclosing kit can be used for the purpose of irradiating the biological sample cultured or enclosed in the kit with a microbeam. In this case, it is preferable that the total sum of the thickness t of the microchip 1 and the thickness of the cover provided on the front surface 2 and the back surface thereof is not more than the underwater range of the ionic species used. The biological sample culture kit or the biological sample enclosing kit can be used for the purpose of fluorescence imaging observation of the biological sample cultured or enclosed in the kit. In this case, it is preferable to use the microchip 1 made of a material having no or little autofluorescence and the cover in combination.

(How to use microchip)
The above-described microchip 1 can be suitably used for retaining the biological sample in the recess 3. For example, when the biological sample is a nematode, the microchip 1 shown in FIG. 1 is used.

The method of enclosing the biological sample in the microchip 1 includes an operation of guiding the biological sample into the recess 3. That is, a biological sample is disposed on the surface 2 (surface where the recess 3 is formed) of the microchip 1 and the disposed biological sample is guided to the recess 3. If nematodes are used as the biological sample, for example, a predetermined amount of liquid (typically water or an aqueous solution) is dropped on the surface 2 of the microchip 1 and the nematodes are transferred into the droplets. The nematode can be placed on the surface 2 of the microchip 1. Alternatively, the nematodes may be arranged on the surface 2 of the microchip 1 by dispersing the nematodes in a liquid and dropping a predetermined amount of the nematode dispersion on the surface 2 of the microchip 1.

The method for guiding the biological sample formed on the surface 2 of the microchip 1 to the recess 3 is not particularly limited. For example, the biological sample may be appropriately selected according to the characteristics of the biological sample, such as waiting for the biological sample to fit in the recess 3, vibrating and dropping, pushing using a platinum wire, or pushing with pressure from above. If a nematode is used as the biological sample and the nematode is placed in a droplet dropped on the surface 2 of the microchip 1 as described above, the droplet is placed on the surface of the microchip 1. The nematode in the droplet can be guided to the recess 3 by crushing toward 2 to spread the liquid (water or aqueous solution) in the recess 3.

Furthermore, the cover 4 may be used in combination in order to retain the biological sample in the recess 3 of the microchip 1. That is, the method of enclosing the biological sample in the recess 3 of the microchip 1 may further include an operation of placing the cover 4 on the surface 2 (surface where the recess 3 is formed) of the microchip 1 so as to cover the recess 3. From the viewpoint of bringing the cover 4 into close contact with the surface 2 of the microchip 1, it is preferable to press the microchip 1 and the cover 4. When the cover 4 is used in this way, it is preferable to use the cover 4 for the operation of crushing the droplets because the procedure can be simplified.

The same applies when the microchip 1 shown in FIG. 2 is used. The biological sample is sealed in the well 3b by enclosing the biological sample in the well 3b and covering the surface 2 of the microchip 1 with the cover 4. can do.

Further, the microchip 1 is suitably used when the biological sample is irradiated with the microbeam in a state where the biological sample is enclosed in the recess 3 as described above. For example, using the microchip 1 of FIG. If the elegance is held in the channel 3a, the movement of the nematode can be suppressed to the minimum at the time of the microbeam irradiation, so that the target is targeted at a specific tissue or region of the nematode or one specific cell. Can be irradiated with a microbeam.

The microchip 1 is also preferably used when culturing or raising a biological sample with the biological sample enclosed in the recess 3 as described above. For example, if the biological sample is sealed in the well 3b using the microchip 1 of FIG. 2, the microchip 1 in which the biological sample is sealed is allowed to stand for a predetermined time in a predetermined culture environment or breeding environment. Biological samples can be cultured or bred.

The microchip 1 is preferably used when observing a biological sample in a state where the biological sample is enclosed in the recess 3 as described above. For example, if the biological sample is sealed and held using the microchip 1, the movement of the biological sample can be minimized, so that it is easy to observe the biological sample having motility. In addition, by enclosing and retaining the recess 3 of the microchip 1, the encapsulated and retained biological sample can be easily distinguished. For this reason, it is easy to observe a specific cell, a specific cell population, or a specific micro organism over time. The observation method is not particularly limited, and may be appropriately selected according to the purpose such as observation with a microscope in the visible light region, observation with a fluorescence microscope.

In addition, the ion microbeam irradiation to the biological sample enclosed in the microchip 1, culture or breeding of the biological sample in the state enclosed in the microchip 1, or observation of the biological sample enclosed in the microchip 1 It may be performed alone or in combination of two or more.

[Experiment 1]
C. elegans C.I. An experiment was conducted to verify whether the microchip 1 is suitable for microbeam irradiation, with the wild type of elegance (hereinafter sometimes simply referred to as “nematode”) being the target. FIG. 4 is a diagram showing an outline of an apparatus for an ion microbeam local irradiation experiment using the microchip 1. The microchip 1 used in the experiment is the one shown in FIG. 1 and has a thickness t of 300 μm.

First, one drop (about 1 μL) of a buffer solution such as a Wash buffer is dropped between two parallel channels 3 a formed on the surface 2 of the microchip 1. Next, while observing with a stereomicroscope, one or more adult nematodes are captured from the culture plate using a platinum wire, washed, and then placed into a droplet of the solution dropped on the microchip 1. Repeat this procedure depending on the number of nematodes needed for the experiment. Thereafter, the cover 4 having a thickness of 100 μm is placed on the microchip 1, and the nematode is guided to the channel 3 a while rolling the droplet with the cover, and the cover 4 is brought into close contact with the surface 2 of the microchip 1. By covering the channel 3a with the cover 4, the nematode can be contained within the channel 3a. Further, the cover 4 can prevent the nematode from being detached from the inside of the channel 3a, and can be retained in the channel 3a. Further, the ion track detecting plastic film having a thickness of 100 μm is placed on the back surface of the microchip 1 as a cover and placed on the sample table.

Therefore, as shown in FIG. 2, the thickness of the sample placed on the sample stage of the ion microbeam irradiation apparatus is the thickness t of the microchip 1 in which nematodes are encapsulated and retained, the surface 2 and the back surface 2. The total thickness of the cover sheets is 500 μm. In this state, when a biological sample (nematode) encapsulated and retained in the channel 3a is irradiated with a 220 MeV carbon ion microbeam having an underwater range of 1210 μm from the micro aperture of the ion micro beam irradiation apparatus, the micro beam is ion micro beam. It reached the ion detection system of the irradiation device.

FIG. 5 is a diagram showing a trace (irradiation trace) of the microbeam that has reached the ion detection system of the ion microbeam irradiation apparatus. This is a visualization of the irradiation traces (etch pits) of the microbeam transmitted through the ion track detection plastic film CR-39 provided on the back surface of the microchip 1 by an alkali etching process. At the same time, in an ion detection system (such as a scintillator-photomultiplier tube assembly) arranged at the lower part of the sample stage for converting the beam into an electric signal, the reached ion beam was counted. From this, it is clear that the microbeam has passed through the microchip 1.

Next, as a comparative example, an experiment similar to the above was performed using a conventional PDMS microchip (Non-Patent Document 1). FIG. 6 is a diagram showing an outline of an experimental apparatus using a conventional PDMS microchip. The thickness t of the PDMS microchip used in the experiment is about 2.5 mm. As shown in FIG. 6, the total thickness of the microchips encapsulating and retaining nematodes and the covers disposed on the front and back surfaces of the microchips placed on the sample stage of the ion microbeam irradiation apparatus is about 2.7 mm. . In this state, when a nematode encapsulated and retained in the channel is irradiated with a 220 MeV carbon ion microbeam having an underwater range of 1210 μm from the micro aperture of the ion micro beam irradiation apparatus, the micro beam is detected by the ion micro beam irradiation apparatus. The microbeam irradiated to the nematode could not be measured without reaching the system.

From the above, it is apparent that the microchip 1 according to the present invention is suitable for locally irradiating various ion microbeams in a state in which a biological sample is encapsulated and retained. Moreover, it can be suitably used for microbeam irradiation such as X-rays, electron beams, and lasers.

[Experiment 2]
Next, an experiment was conducted to verify whether the microchip 1 imparted with hydrophilicity as described above can alleviate the dehydration of the biological sample. In this experiment, C. elegans C.I. For the wild type of elegance, as a solution used for nematode washing and encapsulation in the channel 3a, pure water (Milli Q water), a first buffer solution (Sbasal buffer), and a second buffer solution ( Three types of solutions with M9 buffer) were prepared, and the effects of these solutions on nematodes were examined. The compositions of the first buffer solution and the second buffer solution are as shown in Table 2 below.

First, in order to obtain reference data, five or more adult nematodes are captured from a plate in which nematodes are cultured using a platinum wire and washed in a droplet of one of the three solutions. Five or more washed nematodes are transferred onto an agar plate (hereinafter referred to as “NGM”) using a platinum wire and allowed to move freely for 1 hour. Thereafter, the nematode is transferred onto a new NGM using a platinum wire, and the number of flexion of the head of the nematode is counted. The number of flexion of the head is counted for 20 seconds per animal, and an average value of the number of flexion of the head of a total of 5 nematodes is obtained. FIG. 7 shows the results of an experiment in which this was independently performed 6 times for each of the three types of solutions. Note that error bars in FIG. 7 indicate standard errors.

As shown in FIG. 7, when the NGM was allowed to move freely for 1 hour, the average number of flexion of the head of the nematode was about 25, regardless of which of the three types of solutions was used for washing. No significant difference at times / 20 seconds, that is, no significant effect on the worm motility of each solution is observed (when the significance level is P <0.05). This average head flexion number is used as reference data for the next experiment.

Next, a conventional PDMS microchip (hereinafter sometimes referred to as “chip L”) and a microchip that has the same shape as the above-described microchip 1 (see FIG. 1) and has no hydrophilicity ( Hereinafter, three microchips are prepared, which may be referred to as “chip B1”) and microchip 1 (hereinafter also referred to as “chip B2”) in which hydrophilicity is imparted to the entire microchip. The same experiment as described above was performed using each microchip. That is, 5 or more adult nematodes are captured from a plate in which nematodes are cultured using a platinum wire and washed in a droplet of one of the three types of solutions. Five or more washed nematodes are transferred into droplets of the same solution on a microchip using a platinum wire, all nematodes are placed in a channel using a cover, and the cover is adhered to the microchip. That is, the nematode is encapsulated and retained inside the channel. After standing for 1 hour in that state, the cover is removed from the surface of the microchip, and the same solution is dropped into the channel containing the nematode. The nematode in the droplet is transferred onto the NGM using a platinum wire, and the number of head flexion of the nematode is counted. Similarly to the above, the number of head flexions is counted for 20 seconds per animal, and the average value of the number of head flexions of a total of 5 nematodes is obtained.

FIG. 8 is a diagram showing the relative value of the number of flexion times of nematode heads when pure water is used as a solution, and the average head when using pure water of nematodes freely moved as shown in FIG. The number of bends is normalized and represented as 1. As shown in FIG. 8, when pure water was used as the solution, the motility after the nematode was encapsulated and retained in the channel for 1 hour was significantly reduced only in the conventional PDMS microchip (chip L) (* P <0.05). * In FIG. 8 indicates that there is a significant difference from the control group at the significance level P <0.05. This point is the same in other drawings below.

FIG. 9 is a diagram showing a relative value of the number of times the head of the nematode bends when the first buffer is used as a solution. The average head of the nematode when the first buffer shown in FIG. 7 is used. The number of times of partial bending is normalized and expressed as 1. ** in FIG. 9 indicates that there is a significant difference from the control group at the significance level P <0.01. This point is the same in other drawings below. As shown in FIG. 9, when a first buffer solution having a high salt concentration is used as a solution, a conventional PDMS microchip (chip L) and a microchip (chip B1) not imparting hydrophilicity are used. In one microchip, motility was significantly reduced after nematodes were encapsulated in channels for 1 hour (* P <0.05, ** P <0.01).

FIG. 10 is a diagram showing a relative value of the number of times the nematode head bends when the second buffer solution is used as a solution, and the nematode head portion when the second buffer solution shown in FIG. 7 is used. The number of bends is normalized and represented as 1. As shown in FIG. 10, when the second buffer solution having a high phosphate and salt concentration was used as the solution, the nematode was sealed and retained in the channel for 1 hour only with the conventional PDMS microchip (chip L). Motility was significantly reduced (** P <0.01).

From the above, as in the present invention, hydrophilic microchip 1 (chip B2) is encapsulated and retained in channel 3a for 1 hour regardless of the type of solution used when nematodes are washed and encapsulated. Even after doing so, no effect on the worm's motility is observed. That is, it is clear that the use of the microchip 1 having hydrophilicity throughout the microchip can suppress dehydration of micro-organisms such as nematodes.

[Experiment 3]
Next, an experiment was conducted comparing the case where only the microchip surface was hydrophilized and the case where the entire microchip was hydrophilized. A microchip that has the same shape as the above-described microchip 1 and has no hydrophilicity (hereinafter, may be referred to as “chip B1”), and that has the same shape as the above-described microchip 1 and that is subjected to plasma treatment. A microchip having hydrophilicity only on the surface (hereinafter sometimes referred to as “chip B1”) and a microchip 1 having hydrophilicity imparted to the entire microchip (hereinafter referred to as “chip B2”). .) And three microchips were prepared, and an experiment similar to the above was performed using each microchip. In this experiment, C. elegans C.I. For the elegance wild type, the first buffer solution (S basal buffer) having a high salt concentration, in which the motility of the nematode is reduced most in Experiment 2, is used for washing and encapsulating the nematode. Assuming long-term use in fluorescence imaging observation, etc., the nematode channel 3a was sealed for 3 hours, and the number of head flexions of 5 nematodes transferred to NGM after 3 hours was counted. Find the average value. As a comparison object, the number of flexion of the heads of five nematodes that were moved freely on the NGM for 3 hours and then transferred to a new NGM was also counted to obtain an average value. FIG. 11 shows the result of an experiment in which this was independently performed three times for each of the three types of microchips and NGM.

As shown in FIG. 11, a microchip (chip B1) that is not hydrophilic and has a hydrophobic property and a microchip (chip B1) that is hydrophilic only on the surface by plasma treatment are encapsulated and held for 3 hours. In all insects, motility was significantly reduced (* P <0.05). On the other hand, the motility of the nematode encapsulated and retained for 3 hours in the microchip (chip B2) provided with hydrophilicity throughout the microchip is significantly different from that of the nematode freely moved on NGM. It was revealed that there was no decrease in motility due to dehydration.

Therefore, in order to suppress dehydration of the biological sample, it is particularly preferable to impart hydrophilicity to the entire microchip rather than to impart hydrophilicity only to the channel 3a on the surface 2 of the microchip 1. It has been found that a remarkable effect can be obtained when used for a long time.

[Experiment 4]
Next, an experiment was conducted to verify whether the hydrophilicity does not deteriorate due to repeated use of the microchip 1. In this experiment, C. elegans C.I. For the wild type of elegance, the first buffer solution used in Experiment 3 was used for washing and encapsulating nematodes, and the microchip 1 (hereinafter referred to as “chip B2”) was given hydrophilicity to the entire microchip. The nematode is encapsulated and held in the channel 3a for 3 hours, and then the nematode is taken out and washed with pure water (Milli Q water) and sterilized with 70% ethanol. And then dried. A series of operations of entrapment and retention of nematodes, microchip cleaning and sterilization are repeated 9 times. At the time of use for the tenth time, the nematode is encapsulated and held in the channel 3a of the microchip 1 for 3 hours, and then the nematode is transferred onto the NGM, and the number of flexion of five heads is counted to obtain an average value. As a comparison object, the number of flexion of the heads of five nematodes that were moved freely on the NGM for 3 hours and then transferred to a new NGM was also counted to obtain an average value. FIG. 12 shows the results of an experiment in which this was independently performed three times for chips B2 and NGM.

As shown in FIG. 12, even when the microchip 1 imparted with hydrophilicity to the entire microchip is used ten times, the mobility of the nematode sealed and held in the channel 3a of the microchip 1 for 3 hours is There was no significant difference from nematodes that were allowed to move freely on NGM for 3 hours. Therefore, the microchip 1 imparted with hydrophilicity to the entire microchip retains hydrophilicity even when used repeatedly for a long time. It is clear that dehydration of micro-organisms such as elegance can be suppressed.

[Experiment 5]
Next, an experiment was conducted to verify whether the microchip 1 is suitable for observation, particularly for fluorescence imaging observation. FIG. 13 is a bright-field image obtained by photographing a state in which nematodes are encapsulated and retained in the channel 3 a of the microchip 1. The nematodes encapsulated and retained in the channel 3a are genetically modified nematodes in which only body wall myocytes extending from the head to the tail are labeled with a fluorescent probe. In the bright field image of FIG. 13 where the fluorescence is not known, it is not different from the wild type nematode. On the other hand, FIG. 14 is a fluorescent image of the same nematode. The fluorescence image is an image having a fluorescence intensity corresponding to the contraction intensity of the cells. The microchip 1 suppresses the movement of the nematode without using an anesthetic, and enables observation in a living state. Moreover, the nematode dehydration can be suppressed, and it is also suitable for long-term observation. Further, the above-described microchip 1 has no autofluorescence. Therefore, it can be suitably used for fluorescence imaging observation for the purpose of capturing activities of nematode muscle cells, nerve cells, and the like.

[Experiment 6]
Next, an experiment was conducted to verify whether or not the microchip 1 shown in FIG. 2 is suitable for cell culture. FIG. 15 is a bright field image obtained by photographing the results of culturing human cells using a commercially available dish for cell culture. That is, a human cell suspension was spotted (seeded) on a commercially available dish for cell culture, covered with a lid to prevent dehydration, cultured at 37 ° C., and the inside of the dish was photographed after 6 hours. As shown in FIG. 15, when a commercially available dish for cell culture is used, it can be seen that the cells adhere to the dish and start to grow.

FIG. 16 shows the result of culturing human cells using the microchip 1 shown in FIG. 2 that imparts hydrophilicity to the entire microchip and the microchip surface 2 is not subjected to plasma treatment. A bright field image taken. That is, after sterilizing the microchip 1 of FIG. 2, it is placed in an aseptic processing dish, seeded with a human cell suspension, covered with a lid to prevent dehydration, and cultured at 37 ° C. 3b is taken. As shown in FIG. 16, it can be seen that in the microchip 1 in which hydrophilicity is imparted to the entire microchip, cell adhesion and proliferation after 6 hours are insufficient.

Next, FIG. 17 shows the microchip 1 shown in FIG. 2, in which human cells are cultured using the microchip in which hydrophilicity is imparted to the entire microchip and the surface 2 is subjected to plasma treatment. It is a bright field image which image | photographed the result. That is, after sterilizing the microchip 1 subjected to plasma treatment, it is placed in an aseptic treatment dish, seeded with a human cell suspension, covered with a lid to prevent dehydration, and cultured at 37 ° C. for 6 hours. The inside of the well 3b was taken later. As shown in FIG. 17, in the microchip 1 in which the microchip surface 2 is subjected to the plasma treatment, the cells start to adhere and proliferate in the same manner as in the case of using the commercially available cell culture dish shown in FIG. Can be confirmed. Therefore, the above-described microchip 1 can be suitably used for cultured cells if the microchip surface 2 is subjected to plasma treatment.

Next, FIG. 18 shows the microchip 1 shown in FIG. 2, which is a coating agent (CellTak) that imparts hydrophilicity to the entire microchip and further improves cell adhesion on the surface 6 of the well 3 b of the microchip 1. It is the bright field image which image | photographed the result of having cultured the human cell using the microchip 1 which apply | coated (registered trademark). That is, after sterilizing the microchip 1 coated with a coating agent that improves cell adhesion on the surface 6 of the well 3b, the microchip 1 is placed in an aseptic processing dish, seeded with a human cell suspension, and covered with a lid to prevent dehydration. The well 3b was photographed after 6 hours. As shown in FIG. 18, in the microchip 1 in which a coating agent for improving cell adhesion is applied to the surface 6 of the well 3b of the microchip 1, the same as in the case of using the commercially available cell culture dish shown in FIG. It can be confirmed that the cells adhere to the cell and start to proliferate. Therefore, the microchip 1 in which the coating agent that improves cell adhesion is applied to the surface 6 of the well 3b can be suitably used for cultured cells.

Next, an experiment was conducted to verify whether the microchip 1 is suitable for cultured cell observation, particularly for fluorescence imaging observation. As the microchip 1, a microchip having a hydrophilic property throughout the microchip (a microchip made of the same material as “chip B2” in Experiment 2 above) was used. FIG. 19 shows that the entire microchip 1 is made hydrophilic, and further, a coating agent (CellTak (registered trademark)) for improving cell adhesion is applied to the surface 6 of the well 3b, and then the whole is stained with a fluorescent dye. It is the fluorescence image which image | photographed using the erecting fluorescence microscope, making the cover closely_contact | adhere to the surface 6 of the well 3b in the state which seed | inoculated and adhere | attached the obtained human cell. The microchip 1 can acquire a fluorescence image with the cells adhered, and has no autofluorescence. Since the microchip 1 has a small thickness t, it is possible to take an image using an upright microscope that cannot be realized without using a water immersion lens in normal fluorescent imaging observation of living cells. Therefore, it can also be suitably used for fluorescence imaging observation for the purpose of analyzing the dynamics of living cells of animals and plants using biological fluorescent staining.

[Experiment 7]
Next, an experiment was conducted to verify the influence of the oxygen permeability of the cover 4 placed on the microchip 1 on the biological sample. In this experiment, C. elegans C.I. Cover 4 for covering the channel 3a of the microchip 1 (chip B2) in which the entire microchip used in the experiment 2 is made hydrophilic for the elegance wild-type adult, for example, a wavelength of 360 to 1500 nm The light transmittance at 90 ° C. is 90% or more, which is suitable for observation, but does not transmit oxygen (hereinafter sometimes referred to as “glass cover”), oxygen transmittance (20 ° C., 50% RH) Is a very low polyester film (hereinafter sometimes referred to as a PET cover) of about 30 mL / (m ² · 24 h · atm), and an oxygen transmission rate of 1000 mL / (m ² · 24 h · atm) or more. (Hereinafter, it may be referred to as PS cover.) And the effect of their use on nematodes was examined. The glass cover has a thickness t of 130 to 170 μm, the PET cover has a thickness t of about 130 μm, and the PS cover has a thickness t of 125 μm. For the nematode washing and encapsulation in the channel 3a, the first buffer solution (S basal buffer) having the lowest salt motility, that is, the salt concentration that tends to cause dehydration, was used in Experiment 2. . Ten or more adult nematodes were captured from a plate in which the nematodes were cultured using a platinum wire and washed in a first buffer droplet. Ten or more washed nematodes are transferred into a buffer droplet dropped on the microchip 1 using a platinum wire, all the nematodes are placed in the channel 3a using the cover 4, and the cover 4 is micro The chip 1 was brought into close contact with the surface. The entrapment time of the nematode in the channel 3a was set to 3 hours on the assumption of long-time use in imaging or the like. That is, nematodes were sealed in the channels 3a of the three microchips 1 and the respective microchips 1 were covered with different covers 4 and allowed to stand for 3 hours. Thereafter, the cover 4 is removed from the surface 2 of each microchip 1, and the first buffer solution is dropped onto the channel 3a enclosing the nematode. The nematodes in the droplets were transferred onto NGM, and the number of head flexions per nematode was counted for 20 seconds, and the average value of the total number of head flexes of 10 nematodes was determined. In addition, as a comparison object, the number of flexion of the heads of 10 nematodes that were moved freely on NGM for 3 hours and then transferred to a new NGM was counted to obtain an average value. FIG. 20 shows the results of performing this experiment independently for each of glass cover, PET cover, PS cover and NGМ five times. The measured values of the physical properties of the three types of covers used are shown in Table 3 below.

As shown in FIG. 20, the movement of a nematode encapsulated for 3 hours by covering the channel 3a of the microchip 1 (chip B2) with hydrophilicity imparted to the entire microchip and enclosing the PET cover for 3 hours. Both sexes were significantly reduced compared to nematodes that were allowed to move freely on NGM for 3 hours (* P <0.05, ** P <0.01). On the other hand, the motility of the nematode encapsulated with the PS cover was not significantly different from that of the comparative example which was allowed to move freely on NGM for 3 hours. That is, it has been clarified that if a PS cover is used when the nematode is encapsulated in the channel 3a, motility is not reduced due to lack of oxygen or dehydration.

Considering the above results, when encapsulated and retained with a glass cover, individuals exhibiting normal motility without causing dehydration at all after 3 hours of encapsulated retention, and individuals with significantly decreased motility due to dehydration And was mixed. One reason for this is thought to be that the droplets of the buffer solution did not become sufficiently fine on the glass surface, and sufficient moisture did not reach the channel 3a. Moreover, since oxygen is not allowed to pass through at all, even when it can be sealed together with moisture, it is thought that the motility is reduced due to lack of oxygen.

On the other hand, in the case of the PS cover, a droplet dropped on the surface 2 of the microchip 1 is spread by the cover 4 to a diameter of 5 to 10 mm and spreads into a plurality of channels 3a. The nematode is in close contact with the surface 2 of 1 and sealed in the channel 3a. Therefore, the nematodes encapsulated and retained with the PS cover were maintained in a suitable state without causing dehydration in the channel 3a filled with the buffer solution, and exhibited normal motility even after the encapsulated retention for 3 hours.

When encapsulated and retained with a PET cover, decreased mobility was observed in almost all individuals after 3 hours of encapsulated retention. That is, a tendency that the motility of some individuals did not decrease, but decreased overall. As shown in Table 3, the PET cover is a cover with extremely low oxygen permeability. Therefore, it is considered that the nematode encapsulated and held for 3 hours with a PET cover was deficient in oxygen and decreased in mobility.

In contrast, the PS cover is a material having higher oxygen permeability than the PET cover. Therefore, nematodes encapsulated and retained with the PS cover did not lack oxygen, and exhibited normal motility even after 3 hours of encapsulated retention.

From the above results, it is clear that the cover 4 used when culturing or encapsulating an oxygen-requiring biological sample in the microchip 1 is preferably one having high oxygen permeability.

[Experimental result]
As described above, the microchip 1 and the cover 4 according to the present invention have characteristics suitable for locally irradiating and observing a microbeam in a state where animal and plant cells and micro organisms are cultured or encapsulated. The conventional problems can be solved.

1 Microchip (Microchip for biological samples)
2 Microchip surface 3 Recess 3a Channel 3b Well 4 Cover 5 Cover surface 6 Well surface

Claims (17)

1. A biological sample microchip having a surface on which at least one recess for culturing or enclosing a biological sample is formed, wherein the thickness of the entire microchip is 800 μm or less. Chip.
2. The microchip for biological samples according to claim 1, wherein the thickness of the entire microchip is 300 µm or less.
3. The microchip for biological samples according to claim 1, wherein the thickness of the entire microchip is 100 µm or less.
4. The microchip for biological samples according to any one of claims 1 to 3, wherein at least the surface of the microchip is hydrophilic.
5. The biochip microchip according to claim 4, wherein hydrophilicity is imparted to the entire microchip.
6. A biochip microchip having at least one concave portion for culturing or enclosing a biological sample on the surface, wherein hydrophilicity is imparted to at least the microchip surface. .
7. The microchip for biological samples according to any one of claims 1 to 6, wherein the surface of the microchip is subjected to plasma treatment.
8. A cover used for enclosing and retaining a biological sample in the recess of the microchip for biological sample according to claim 1, wherein the cover is transparent and has a surface roughness of 0.03 μm or less. Features a cover.
9. The cover according to claim 8, wherein the thickness is 50 μm or more and 300 μm or less.
10. The cover according to claim 8 or 9, wherein a water contact angle is 100 degrees or less.
11. The cover according to any one of claims 8 to 10, wherein the oxygen permeability is 30 mL / (m ² · 24 h · atm) or more.
12. The cover according to any one of claims 8 to 11, wherein the carbon dioxide permeability is 30 mL / (m ² · 24 h · atm) or more.
13. The cover according to any one of claims 8 to 12, wherein the cover is made of polystyrene.
14. A biological sample enclosing kit comprising the biological sample microchip according to any one of claims 1 to 7 and the cover according to any one of claims 8 to 13.
15. A biological sample retention method for retaining a biological sample, comprising enclosing the biological sample in a recess of the microchip for biological sample according to any one of claims 1 to 7. .
16. A microbeam irradiation method for irradiating a biological sample with a microbeam, wherein the biological sample is sealed in a concave portion of the microchip for biological sample according to any one of claims 1 to 7, and the microbeam is applied to the biological sample. Irradiating a microbeam irradiation method.
17. A method for culturing or rearing a biological sample, wherein the biological sample is enclosed in a recess of the biological sample microchip according to any one of claims 1 to 7, and the biological sample microcapsule in which the biological sample is enclosed. Leaving the chip in the culture or breeding environment of the biological sample;
    A method comprising the steps of:

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