JP5625269B2 - Cell observation sample stand kit, cell observation sample stand, and method for manufacturing cell observation sample stand - Google Patents

Description

  The present invention relates to a sample stage used for cell inspection.

  2. Description of the Related Art A cell observation sample stage having a plurality of compartments is known as an instrument for performing pathological examination of cells using a microscope. A plurality of holes isolated from each other are formed in the observation container, and a liquid containing cells to be observed is dropped into the holes. In observation using a microscope, antibody inspection using a fluorescent antibody method, enzyme antibody method, etc., infectious disease inspection, cancer tissue diagnosis, and the like are performed.

  As a sample stage for cell observation used in such applications, Patent Document 1 has a slide plate that is observed with a microscope and an upper member that is detachable on the slide plate for the purpose of efficient inspection. The upper member is provided with a plurality of through-holes, and a lower surface of the upper member is formed with a protrusion made of a seal material around the through-hole. When the upper member is mounted on the slide plate, There is disclosed a sample stage characterized in that the provided through holes form independent sections.

  Patent Document 2 also discloses a bottom surface of a culture container formed of a non-cell-adhesive substance in a culture container that is cultured in a state in which cells are separated into at least five cell groups for the purpose of improving working efficiency. A plurality of cell-adhesive regions formed on the upper surface of the cell, the cell-adhesive regions having a size sufficient for adhering only one of the cell groups, There is disclosed a sample stage in which adhesive regions are arranged at a predetermined interval that does not contact.

By the way, the cell observation sample stage is required to have various characteristics such as liquid leakage resistance, hermeticity, productivity, and manufacturing cost.
The liquid leakage resistance means the performance of preventing the liquid dropped into the holes from leaking into the adjacent holes and mixing the samples. This is an important characteristic because the occurrence of liquid leakage leads to shaking the reliability of analysis. In order to prevent liquid leakage, it is desirable that the member in which the hole is formed and the base portion supporting the member are firmly bonded. Liquid leakage can be prevented by integrally molding a member in which holes are formed in advance and a base. However, since a mold is required for integral molding, it is not suitable for production of small lots or customization of the hole diameter and hole position according to the intended use. In addition, when the member in which the hole is formed and the base are separately prepared and bonded with an adhesive, even if the adhesiveness can be secured, the adhesive protrudes into the hole and the adhesive component and the cell are mixed. If it does, it will cause a decrease in inspection / diagnosis accuracy.

  Hermeticity is a characteristic associated with a lid placed on top of a member in which a hole is formed. When observing the progress of cells, it is necessary to leave the sample stationary, but in order to ensure the reliability of the analysis, it is desirable that the sample retentate be sealed so that it does not volatilize. On the other hand, it is desirable that sealing is easy from the viewpoint of troublesome observation of the sample.

Patent No. 3627360 Patent No. 2965151

  An object of the present invention is to provide a sample table for cell observation that is excellent in characteristics such as liquid leakage resistance and hermeticity.

The present inventors have found that a sample stage for cell observation having excellent characteristics such as liquid leakage resistance and hermeticity can be produced at low cost by joining the sample liquid holding part and the base with a double-sided tape. completed. That is, the present invention includes the following.
(1) A sample liquid holding part in which a plurality of through holes are formed, and here, the sample liquid holding part is affixed with a double-sided tape in which a through hole is formed at a site corresponding to the through hole,
A base of a sample table joined to the sample solution holding part via the double-sided tape;
A lid placed on the sample liquid holding part and sealing the sample liquid holding part;
A sample stage kit for cell observation.
(2) The cell observation sample stage kit according to (1), wherein the sample solution holding part is made of rubber.
(3) The sample observing kit for cell observation according to (1) or (2), wherein the hardness of a portion in contact with the lid of the sample solution holding unit is lower than an average hardness of the sample solution holding unit.
(4) The sample table kit for cell observation according to any one of (1) to (3), wherein the double-sided tape includes a support layer and an adhesive layer disposed on both sides of the support layer.
(5) The sample stage kit for cell observation according to any one of (1) to (4), wherein the through holes are arranged concentrically.
(6) The sample base kit for cell observation according to any one of (1) to (5), wherein the base and the lid are made of glass.
(7) a sample solution holding part in which a plurality of through holes are formed;
A double-sided tape that is affixed to the surface of the sample solution holding part where a through hole is formed, and has a through hole formed in a portion corresponding to the through hole;
A base of a sample table joined to the sample solution holding part via the double-sided tape;
A lid placed on the sample solution holding unit and sealing the sample solution holding unit;
A sample stage for cell observation.
(8) The sample stage for cell observation according to (7), wherein the sample solution holding unit is made of rubber, the lid is made of glass, and the lid is in close contact with the sample solution holding unit by its own weight.
(9) a step of applying a double-sided tape to one surface of the sample solution holding unit;
Forming a plurality of through holes penetrating the sample solution holding part and the double-sided tape;
Bonding a base of a sample stage to the sample solution holding part via the double-sided tape;
Placing a lid for sealing the sample solution holding unit on the sample solution holding unit;
The manufacturing method of the sample stand for cell observation containing this.

  Since the sample stage for cell observation of the present invention uses a double-sided tape to join the sample solution holding part and the base, the adhesive component oozes out to the site where the sample is dropped compared to the case where an adhesive is used. There are very few. For this reason, the reliability of inspection and diagnosis due to leakage of the sample liquid is prevented. Further, when the adhesive component oozes out, the bottom area of the through hole changes, which causes a decrease in analysis accuracy, but this can also be prevented.

  Since the sample solution holding part and the base are formed separately and joined by double-sided tape, there is no need to prepare a special mold required when integrally forming the sample solution holding part and the base. . For this reason, it is possible to deal with the production of small lots and it is easy to customize the sample stage according to customer needs.

FIG. 1 is a schematic cross-sectional view of an embodiment of the cell observation sample stage of the present invention. The cell observation sample stage 1 includes a sample solution holding unit 10, a double-sided tape 20, a sample stage base 30, and a lid 40. FIG. 2 is a schematic view of a mode in which the through holes are arranged concentrically with the point A as the center.

Hereinafter, the present invention will be described with reference to the drawings.
FIG. 1 is a schematic cross-sectional view of an embodiment of the cell observation sample stage of the present invention. A plurality of through holes 50 are formed in the sample liquid holding unit 10. The through hole 50 is a portion where the analysis sample is dropped when the sample stage is completed. The sample solution holding unit 10 and the base 30 are joined with a double-sided tape 20. The double-sided tape 20 has a through-hole formed at a site corresponding to the through-hole 50 formed in the sample solution holding part. Thus, when the through-hole is formed also in the double-sided tape 20, the contact area of an adhesive component and a sample becomes very small. If the adhesive component mixes with the sample dropped into the through-hole, the observed cell may be adversely affected and the accuracy of inspection / diagnosis may be reduced. Reduce the contact area between the adhesive component and the sample. This suppresses this harmful effect. Theoretically, a contact portion corresponding to the product of the outer peripheral length of the through hole and the thickness of the double-sided tape is generated. However, since the double-sided tape is usually very thin, the contact portion is extremely small.

  The base 30 is a member that supports the sample solution holding unit. Since the sample solution holding unit 10 and the base 30 are in close contact with each other by the double-sided tape 20, the sample dropped into the through hole is prevented from oozing out to the adjacent sample. If the sample liquid holding unit 10 is only in close contact with the base 30 without using an adhesive component, there is a possibility that the sample liquid holding part may be laterally displaced due to vibration, or the sample may leak out through a slight gap.

  The lid 40 seals the through hole 50 formed in the sample solution holding unit 10. When the lid 40 is not used, for example, contamination via air or sample volatilization may occur. These cause a decrease in inspection / diagnosis accuracy of the sample. After the sample is dropped into the through hole 50, these problems are prevented by sealing the through hole 50 with the lid 40. The lid 40 may be bonded to the sample liquid holding unit 10 using an adhesive component such as an adhesive or a tape, but preferably is in close contact with the sample liquid holding unit 10 by its own weight. When using an adhesive component, it takes time to open and close the lid, but when using its own weight, it is easy to open and close the lid. For this reason, when observing a sample multiple times over a certain period of time, or when adding a reagent such as a cell staining solution during observation, the lid and the sample solution holder are brought into close contact with each other using the weight of the lid. The technique is preferred.

Then, each member which comprises the sample stand for cell observation is demonstrated.
The material for the sample solution holding part may be any material such as plastic, rubber, organic materials such as cardboard, inorganic materials such as metal, glass, etc., but is preferably rubber. Where a through hole through which a sample is dropped is formed in the sample liquid holding part, the rubber member is easy to drill. The base is preferably made of a non-deformable material such as glass from the viewpoint of firmly holding the cell observation sample stage, and rubber has excellent adhesion to glass. The types of rubber include natural rubber, synthetic rubber (silicone rubber, acrylic rubber, acrylonitrile butadiene rubber, isoprene rubber, urethane rubber, ethylene propylene rubber, epichlorohydrin rubber, chloroprene rubber, styrene butadiene rubber, butadiene rubber, fluorine rubber, polyisobutylene. ). In these, the silicone rubber which is excellent in chemical resistance is preferable. As the thick paper, YUPO paper or water-repellent paper can be used.

  The size of the sample solution holding part is not particularly limited, but considering the ease of use as a sample stage, preferably the height (T), width (L), and depth (W) are 0.1 to 100 mm, 1 to 1, respectively. 500 mm and 0.1 to 500 mm. Especially preferably, they are 1.0-10 mm, 10-50 mm, and 1.0-50 mm, respectively. As for the width and depth, the size of the other stacked members is determined based on the size of the sample liquid holding portion. For example, the size of the double-sided tape is made equal to that of the sample solution holding unit, and the size of the base and the lid is made slightly larger than that of the sample solution holding unit.

  The sample solution holding part may be formed of a single material, but may be a composite material made of a plurality of materials. For example, when the lid and the sample solution holding unit are brought into close contact with each other due to the weight of the lid, the compatibility between the lid and the sample solution holding unit is important. Specifically, it is preferable to deform following the shape of the contacting member. On the other hand, considering the drilling of the sample solution holding part, it is preferable that the sample solution holding part has a certain degree of hardness. In order to satisfy both of these requirements, a sample solution holding unit in which the hardness of the portion in contact with the lid is lower than the average hardness of the sample solution holding unit can be considered. In other words, a soft material is used for the portion in contact with the lid. For example, the adhesiveness between the sample liquid holding part and the lid can be secured by bonding a silicone film having a high content of low molecular weight siloxane to a portion in contact with the lid.

  Specifically, the hardness of the portion in contact with the lid is preferably 45 ° or less. The hardness can be determined, for example, according to JIS K6253 (vulcanized rubber and thermoplastic rubber—how to determine hardness). The average hardness of the sample solution holding part can be obtained from the volume% and the hardness of each member constituting the sample solution holding part. For example, when a sample liquid holding portion is formed by laminating rubber having a thickness of 5 mm with a hardness of a and rubber having a thickness of 10 mm with a hardness of b degrees, the average hardness is (a × 5 + b X10) / 15.

  The material of the double-sided tape is not particularly limited as long as it is a material capable of bonding the sample solution holding part and the base. A preferable material may be selected in consideration of the material of the sample solution holding part and the material of the base. In addition, since there is a possibility that the double-sided tape and the sample come into contact with each other, it is preferable to use a material that causes little elution into the sample and has low cytotoxicity. Specific examples of the adhesive component include acrylic resin, epoxy resin, siloxane, and rubber. When the sample solution holding part is rubber, it is preferable that the adhesive component on the side to which the sample solution holding part is bonded is also rubber.

  The double-sided tape may be either a single layer type or a laminated type. In the case of a single layer type, a double-sided tape having a laminated structure in which a release paper is disposed on the surface of an adhesive layer is used by peeling the release paper when used. The configuration of the sample table is sample solution holding unit / adhesive layer / base.

  The laminated type is a double-sided tape in which two adhesive layers are laminated. In the case where it is difficult to obtain a material having adhesive force on both the sample liquid holding part and the base, an adhesive component having excellent adhesion to the material of the sample liquid holding part is disposed on the joint surface with the sample liquid holding part, An adhesive component having excellent adhesion to the base is disposed on the joint surface with the base. Between the two adhesive layers, a support layer excellent in adhesiveness with each adhesive component is disposed. The configuration of the sample stage is as follows: sample solution holding part / first adhesive layer / support layer / second adhesive layer / base. Another advantage of such a support layer is that the structure of the double-sided tape is easily maintained, and the shape of the through hole is not easily distorted when the through hole is formed by punching. Examples of the material for the support layer include glass mesh and film.

  The thickness of the double-sided tape is preferably 0.01 to 5.0 mm. If it is too thin, sufficient adhesive force cannot be ensured. On the other hand, if it is too thick, there is a higher possibility that the observed cells will be adversely affected by the seepage of the adhesive component into the through-hole.

  The shape and arrangement of the through holes formed in the sample solution holding part and the double-sided tape are not particularly limited. The shape of the through hole is preferably designed according to the amount of the sample to be dropped. In the present invention, the through hole of the sample liquid holding part holds the sample liquid containing cells, and therefore the shape of the through hole is preferably determined according to the type and amount of cells to be observed and the observation means. The Specifically, the pore diameter is reduced if the amount of sample dripping is small, and the hole diameter is increased if the amount of sample dripping is large. Since the present invention is a form in which the sample solution holding part and the base are separately formed and joined by a double-sided tape, a special mold required for integrally forming the sample solution holding part and the base is prepared. There is no need to do. For this reason, customization according to the intended use is easy. For example, it is possible to adopt a supply system in which the base is made common, a plurality of sample liquid holding portions having different hole diameters and through hole arrangements are prepared, and an appropriate combination is provided according to the customer's request.

  Considering the ease of formation of the through hole, the shape of the through hole is preferably a cylindrical shape. The size of the through hole is usually about 0.1 to 499 mm in diameter.

  When analysis using electromagnetic waves such as MRI analysis is performed, the through holes are preferably arranged concentrically around one point. FIG. 2 is a schematic view of a mode in which the through holes are arranged concentrically with the point A as the center. In this way, the through holes 50 are arranged concentrically around the point A, and the analysis is performed so that the applied magnetic field is symmetric with respect to the point A, thereby preventing uneven influence of the magnetic field during measurement. Thus, the variation factor for the sample can be reduced. Further, when the through holes are concentrically arranged, all the through holes can be put in the same visual field at the time of microscopic observation, and the convenience of sample observation is enhanced.

  The through holes may be arranged more than double around a certain point. At this time, the magnetic field applied to the through hole arranged on the inner concentric circle and the magnetic field applied to the through hole arranged on the outer concentric circle may be different. However, by arranging one sample group on the inner concentric circle and arranging the other sample groups on the outer concentric circle, a plurality of sample groups can be analyzed simultaneously by one MRI.

  Examples of the material for the base of the sample table include glass, plastic, and metal. It is also possible to use existing laboratory instruments such as slide glass and cover glass. Those using metal are suitable for observing surface reflections such as SRR / surface plasmon resonance and ellipsometer. Preferably, the foundation is made of glass. By using glass that does not substantially deform as a base, the base is stabilized.

  The thickness of the base is preferably 0.01 to 50 mm. If it is too thin, the strength as a base is insufficient, and there is a risk of cracking. On the other hand, if it is too thick, there is a possibility that it will be an obstacle to microscopic observation.

  The foundation may be coated with a functional coat layer. For example, the value of the sample stage can be increased by forming a functional coat layer such as a water repellent ink, a cell adhesive ink, a cell non-adhesive ink, or a tissue culture coat.

  Examples of the material of the lid include glass, plastic, and metal. It is also possible to use existing laboratory instruments such as slide glass and cover glass. Preferably, the lid is made of glass. For the sample holding part, it is desirable to use a material that is easy to process, such as rubber, rather than a material that is difficult to process, such as glass or metal, in order to form a through hole. Therefore, by using glass having excellent adhesion to rubber, contamination via air and sample volatilization can be prevented.

  The thickness of the lid is preferably 0.01 to 50 mm. If it is too thin, there is a possibility that the adhesion is insufficient due to the lack of its own weight when securing the adhesion between the lid and the sample holder due to its own weight. On the other hand, if it is too thick, the sample holder may be damaged or deformed by a load on the sample holder.

  The lid may be coated with a functional coat layer. For example, the value of the sample stage can be increased by forming a functional coat layer such as water-repellent ink or cell non-adhesive ink.

  The sample stage for cell observation of the present invention can be used also for cell culture, and can be advantageously used also when performing cell culture and cell observation in parallel. Therefore, cell observation in the present invention includes culturing cells and assaying cells.

  Next, an embodiment of a sample stage manufacturing method will be described. In addition, since the shape, configuration, and composition of each member have already been described, description thereof will be omitted in the following description of the manufacturing method.

  First, a sample solution holding part is prepared. At this stage, a through hole has not yet been formed, and a sheet-like material having a certain thickness corresponding to the height of the sample liquid holding part can be preferably used. A double-sided tape is prepared and a double-sided tape is affixed to a sample liquid holding part using one adhesive surface. At this stage, it is preferable that a release paper is affixed to the opposite surface of the double-sided tape that is in contact with the sample solution holding portion.

  A through hole is formed in the sample solution holding part to which the double-sided tape is attached. The through hole penetrates the surface on which the double-sided tape is affixed and the surface facing the surface. By using a technique in which the sample liquid holding part and the double-sided tape are simultaneously punched with the blade type, the working efficiency can be improved. A through hole may be separately formed in the sample solution holding part and the double-sided tape, and then both may be bonded together, but it takes time to align the through hole.

  The through hole may be formed by punching a plurality of formed through holes at a time or by punching one by one. When punching a plurality of through holes at a time, productivity is high, but when the position of the blade of the blade type cannot be changed, it is difficult to cope with customization of the part of the through hole. On the other hand, when punching through holes one by one, productivity decreases, but fine adjustment of the location of the through holes is easy.

  The release paper of the double-sided tape is peeled off, and the sample solution holding part and the base of the sample table are joined. If there are no special circumstances, the sample solution holding part is joined near the center of the sample table. A certain amount of pressure may be applied between the base and the double-sided tape so that the adhesive force of the double-sided tape is sufficient.

  Thereafter, a lid for sealing the sample solution holding unit is placed on the surface opposite to the side on which the base of the sample solution holding unit is disposed. Thereby, the sample stage for cell observation is completed. Each constituent material is preferably sterilized prior to the manufacture of the sample stage.

  The present invention can be sold as a sample stage kit for cell observation including a sample solution holding part, a base of a sample stage, and a lid. In this case, it is preferable to pack the members constituting the sample stage together with instructions such as an assembly method. The sample stage kit is assembled by the user according to the instructions prior to use of the sample stage. The assembled sample stage for cell observation may be sold, and any of the embodiments can be included in the scope of the present invention.

Example 1
A silicone rubber of 5 mm (T) × 30 mm (L) × 26 mm (W) was prepared. One release paper of the double-sided tape (manufactured by Teraoka Seisakusho / No.765H) was peeled off, and the double-sided tape was attached to silicone rubber. A through-hole having a diameter of 6 mm was formed in a composite of silicone rubber and double-sided tape to form a sample liquid holding portion in which a plurality of through-holes were formed. The arrangement of the through holes was concentric.

  The other release paper of the double-sided tape was peeled off, and the sample solution holding part and the slide glass were joined to the slide glass serving as the base of the sample stage via the double-sided tape to complete the sample stage for cell observation. 100 μl of a cell suspension incorporating a metal detectable by MRI was dropped into the through hole of the sample solution holding part. Then, the sample liquid holding | maintenance part was sealed by mounting a slide glass as a lid | cover. Silicone rubber, a glass slide serving as a base, and a glass slide serving as a lid were put out before use and sterilized.

  The sample stage was fixed to the measurement stage, and MRI measurement was performed. After the MRI measurement, the slide glass lid was carefully removed, and a cell staining solution was added and allowed to stand. After washing the cell suspension, the sample was observed with a microscope. It was confirmed that cells were detected at the same position as confirmed by MRI measurement.

(Example 2)
A silicone rubber of 5 mm (T) × 30 mm (L) × 26 mm (W) was prepared. One release paper of the double-sided tape (manufactured by Teraoka Seisakusho / No.765H) was peeled off, and the double-sided tape was attached to silicone rubber. A through-hole having a diameter of 5 mm was formed in a composite of silicone rubber and double-sided tape, and a sample liquid holding portion in which a plurality of through-holes were formed was formed. The arrangement of the through holes was concentric.

  The other release paper of the double-sided tape was peeled off, and the sample solution holding part and the slide glass were joined to the slide glass serving as the base of the sample stage via the double-sided tape to complete the sample stage for cell observation. 60 μl of cell suspension was dropped into the through hole of the sample solution holding part. Then, the sample liquid holding | maintenance part was sealed by mounting a slide glass as a lid | cover. Silicone rubber, a glass slide serving as a base, and a glass slide serving as a lid were put out before use and sterilized.

The sample stage was allowed to stand for 7 days in a CO ₂ environment at 37 ° C. and 5% concentration. Cell images were acquired using a microscope. Thereafter, the lid of the slide glass was carefully removed, and a cell staining solution was added and allowed to stand. After washing the cell suspension, the samples were used for biological assays.

(Comparative Example 1)
A silicone rubber of 5 mm (T) × 30 mm (L) × 26 mm (W) was prepared. A through hole having a diameter of 6 mm was formed in the silicone rubber to form a sample solution holding part in which a plurality of through holes were formed. The arrangement of the through holes was concentric.

  A sample solution holding unit was placed on a slide glass serving as a base of the sample table, and was attached by its own weight to complete a cell observation sample table. 60 μl of cell suspension was dropped into the through hole of the sample solution holding part. Then, the sample liquid holding | maintenance part was sealed by mounting a slide glass as a lid | cover. Silicone rubber, a glass slide serving as a base, and a glass slide serving as a lid were put out before use and sterilized.

The sample stage was allowed to stand for 7 days in a CO ₂ environment at 37 ° C. and 5% concentration. Cell images were acquired using a microscope. Since the silicone rubber and the slide glass as a base were only fixed by self-adhesion, leakage of the cell suspension into the adjacent holes was confirmed.

Claims (9)

A sample solution holding part in which a plurality of through holes are formed; and here, the sample solution holding part is affixed with a double-sided tape in which a through hole is formed in a portion corresponding to the through hole,
A base of a sample table joined to the sample solution holding part via the double-sided tape;
A lid placed on the sample liquid holding part and sealing the sample liquid holding part;
Including
The sample liquid holding part has a hardness of a portion in contact with the lid that is lower than an average hardness of the sample liquid holding part,
Sample stand kit for cell observation. The sample stand kit for cell observation according to claim 1, wherein the sample solution holding part is made of rubber, and the base and the lid are made of glass.   The sample table kit for cell observation according to claim 1 or 2, wherein the double-sided tape has a support layer and an adhesive layer disposed on both sides of the support layer.   The sample stand kit for cell observation according to any one of claims 1 to 3, wherein the through hole formed in the sample solution holding part and the through hole formed in the double-sided tape are arranged concentrically. . A sample solution holding part in which a plurality of through holes are formed;
A double-sided tape that is affixed to the surface of the sample solution holding part where a through hole is formed, and has a through hole formed in a portion corresponding to the through hole;
A base of a sample table joined to the sample solution holding part via the double-sided tape;
A lid placed on the sample solution holding unit and sealing the sample solution holding unit;
Including
The sample liquid holding part has a hardness of a portion in contact with the lid that is lower than an average hardness of the sample liquid holding part,
Sample stage for cell observation. The sample stand for cell observation according to claim 5, wherein the sample solution holding part is made of rubber, and the base and the lid are made of glass.   The sample stage for cell observation according to claim 5 or 6, wherein the lid is in close contact with the sample solution holding part by its own weight. A step of applying a double-sided tape to one side of the sample liquid holding part;
Forming a plurality of through holes penetrating the sample solution holding part and the double-sided tape;
Bonding a base of a sample stage to the sample solution holding part via the double-sided tape;
Placing a lid for sealing the sample solution holding unit on the sample solution holding unit;
Including
The sample liquid holding part has a hardness of a portion in contact with the lid that is lower than an average hardness of the sample liquid holding part,
A method for producing a sample stage for cell observation. The manufacturing method of the sample stand for cell observation of Claim 8 whose said sample liquid holding | maintenance part is rubber, and the said base and the said lid | cover are glass.

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