JP2004201695A - Incubator container - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an incubator container not only easily positioning a specific part of a specimen but also high sensitively observing live culture state at a high magnification in cultivating a cell or an implant as the specimen. <P>SOLUTION: The incubator container 11 comprises a container main body 22 formed to freely contain specimens comprising cells or implants and a cover formed to freely cover the container main body 22. The container main body 22 has, at least on its bottom 25, a platy glass part 27 for adhering the specimen thereon. The container body 22 is formed to have plural compartments 23 separated by standing enclosure walls 24. Grid coordinates 19, comprising plural number of latitude lines and longitude lines, are formed, so as to enable specifying given positions of the adhered specimens, at least on the surface or the rear face of the platy glass 27 on which each compartment 23 is set. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

  The present invention relates to a culture vessel that can be suitably used when culturing cells, tissue pieces, and the like as a culture object for experiments and research.

  Culture vessels for experimental research widely used in laboratories for various purposes include flasks and petri dishes, as well as various types such as multiwell plates and roller bottles. And required liquids, and using these as a culture medium, it is possible to culture various cultures including cells, tissue fragments, bacteria and the like.

  Moreover, the culture vessel is generally formed of a synthetic resin material. In particular, polystyrene, which is a transparent synthetic resin material, is not only inexpensive but also adheres to cells and tissue fragments of a living body. The surface treatment work for facilitating the smoothness can be performed smoothly, and the flatness suitable for microscopic observation can be easily ensured.

  On the other hand, cells and tissue fragments in a culture vessel may need to be observed under a higher magnification under a microscope, or fluorescence observation may require a microscope under a higher sensitivity. . However, even if the above-mentioned requirement is met by using the above-mentioned culture vessel made of a synthetic resin material, it is technically necessary to mold the resin so that the thickness of the culture surface (bottom portion) is 1 mm or less while ensuring flatness. In addition to the difficulty in obtaining the depth of focus of the microscope due to the relatively large wall thickness, fluorescence (auto-fluorescence) originating from the material appears as a background, resulting in the desired high magnification and high sensitivity. There is a disadvantage that observation with a microscope becomes impossible.

  By the way, as a technique for solving the above-mentioned inconvenience, there is a method of using a glass material having high transparency and low fluorescence as well as being able to be molded to a thinner thickness while ensuring planarity as compared with a synthetic resin material. Specifically, for example, a method of using a plate glass called a slide glass, a cover glass, or a cover slip and culturing cells, tissue pieces, and the like on the surface of the plate glass.

  On the other hand, a side wall portion of the container body and a lid portion covered by the opening of the container body are formed of a synthetic resin material, and only a bottom portion of the container body is called a chamber slide formed of a slide glass. There are also culture vessels.

  Further, a glass base dish or a glass bottom in which one through-hole is provided in the bottom of the above-mentioned culture vessel made of polystyrene, particularly a petri dish, and a cover glass adhered in a state where the through-hole is closed from the lower surface side is arranged. A glass bottom type culture vessel called a culture dish is also commercially available. By using such a glass bottom type culture vessel, it is possible to solve the problem of the conventional method that "a desired high magnification and high sensitivity cannot be obtained" which is observed in a culture vessel made of a synthetic resin material. .

  In addition, when observing cells or tissue fragments cultured in a culture vessel with a microscope, for example, microscopic injection to observe the morphological changes after a minute injection of a gene or drug into a specific cell, After observing the chemotaxis of the cells, observing the population of cells in individual subpopulations in the same container, and observing the position of individual cells and the position of the cell population as a group, It may be necessary to observe repeatedly.

In response to such a request for repeated observation, a culture vessel (flask) made of a synthetic resin in which the position can be specified and alphanumeric coordinates are displayed has already been proposed (for example, see Patent Document 1). By using the culture vessel (flask), it is possible to repeatedly observe a specific position of an observation target under a culture state with a microscope.
Japanese Patent No. 2683732

  However, in the case of using the culture vessel displaying the alphanumeric coordinates, since the material is a synthetic resin material, the depth of focus cannot be obtained and observation at a high magnification cannot be performed, and the problem of autofluorescence still remains. In addition, there is also a problem in that it is not possible to display a character-numerical coordinate system that is fine enough to specify the position of each cell due to a problem in molding technology.

  On the other hand, among the circular cover glasses, those engraved with fine coordinates called a grid are already commercially available. This is because, because the material of the circular cover glass is glass, not only can the above-mentioned disadvantages in the case of a synthetic resin material be eliminated, but also a fine grid can be formed by applying a precise processing technique such as laser or etching. This is because it can be easily formed.

  However, the circular cover glass is used for culturing cells and the like on its surface while immersing it separately in a culture vessel made of a synthetic resin containing a liquid medium. Therefore, when observing the cells and the like on the circular cover glass with a microscope, the cells can be observed for a time in a state of being immersed in a liquid medium in a culture vessel made of synthetic resin, but the synthetic resin material is used. The above-mentioned drawbacks derived from the above have not been solved yet. In addition, when it is necessary to observe with higher magnification and high sensitivity, it is necessary to take out the circular cover glass from the culture vessel, place it on a slide glass, and observe with a microscope, Not only is it complicated, but there is also a problem that removing it from the liquid culture medium makes it impossible to perform microscopic observation in a living culture state.

  The present invention has been made in view of the above-mentioned problems in the prior art, and when culturing cells and tissue pieces as a culture, not only can a predetermined site of the culture be easily located, but also culture while living It is an object of the present invention to provide a culture vessel capable of observing a state under a microscope with high magnification and high sensitivity.

  SUMMARY OF THE INVENTION The present invention has been made to achieve the above-mentioned object, and its structural features are that a container body formed freely to accommodate a culture object composed of cells, tissue fragments, and the like, and that the container body is applied to the container body. In a culture container comprising a lid having a lid freely formed, the container main body is provided with a plate-like glass portion for attaching the culture object to at least a bottom portion thereof, and a surrounding partition is provided on the plate-like glass portion. It is formed having a plurality of compartments that are set up and partitioned, and either one of the front and back surfaces of the plate-like glass portion where the individual compartments are located, That is, grid coordinates composed of a plurality of latitude lines and meridians formed so as to enable a specified position to be specified are provided.

  In this case, the thickness of the plate-shaped glass portion is preferably 0.04 to 1.50 mm because the thickness is easily broken when the thickness is too thin, and the depth of focus cannot be obtained by microscopic observation when the thickness is too large. Further, it is desirable to coat an extracellular matrix or polycations on the surface of the plate-like glass part in order to enhance the adhesiveness when culturing cells or tissue fragments.

  According to the present invention, when a culture object such as a cell or a tissue piece attached and cultured on a plate-shaped glass part of a container body needs to be observed under a microscope with higher magnification and high sensitivity. Even in this case, the depth of focus of the microscope can be taken, and further, the transmittance is high and the characteristics of low fluorescence can be obtained.

  In addition, since the plate-shaped glass portion is provided with grid coordinates composed of a plurality of latitude lines and meridians, even if it becomes necessary to repeatedly observe a cultured object such as cells or tissue fragments in culture, After specifying the position of each cell or the position of a group of cells as a group, the microscope observation can be performed accurately each time, which can greatly contribute to the improvement of the experiment and research accuracy. In addition, cells to be cultured, such as cells and tissue fragments, can be microscopically observed in a living culture state without requiring complicated preparation work.

  Further, when an extracellular matrix or polycations is applied to the plate-like glass portion, the adherence of a culture object such as a cell or a tissue piece to the culture surface can be increased.

  FIG. 1 is a plan view showing an example of a culture vessel according to the present invention in an open state, and FIG. 2 is a longitudinal sectional view taken along line AA in FIG.

  According to these figures, the culture vessel 11 has a transparent plate-like glass portion 27 for attaching an appropriate culture object (not shown) composed of cells, tissue pieces, and the like to the bottom 25 thereof. It comprises a container body 22 and a lid (not shown) formed freely to cover the container body 22.

  Among them, the container body 22 has a plurality of compartments 23 which are partitioned by arranging a surrounding partition wall 24 made of a synthetic resin material such as polystyrene on a bottom 25 made of one transparent glass plate 28. It is formed.

  In this case, the plate-like glass portion 27 is formed by the individual partition portions 29 of the glass plate 28 located at least on the bottom 25 side separately from each compartment 24. Reference numeral 30 in the drawing indicates a reinforcing rib interposed between adjacent surrounding partitions 24, 24.

  The surface of the plate-shaped glass portion 27 was formed by an appropriate method such as engraving by laser or etching so as to be able to specify the position of a predetermined portion of the culture object such as attached cells. A grid coordinate 19 composed of a plurality of fine latitude lines 19a and meridians 19b is provided. Note that the grid coordinates 19 can be formed on the back surface of the plate-like glass portion 27 as necessary.

  FIGS. 3 and 4 illustrate the grid coordinates 19 provided on the surface of the plate-shaped glass portion 27 in an enlarged manner and exemplify each of the patterns. Among them, FIG. 3 shows an example of a pattern of grid coordinates 19 formed by engraving a latitude line 19a and a meridian line 19b suitable for observing a population, which is a group of cell populations, as individual sub-populations. FIG. 4 shows a pattern example of grid coordinates 19 formed by engraving a latitude line 19a and a meridian line 19b suitable for specifying the position of an individual cell or the position of a cell group, for example. Note that the above example of the pattern of the grid coordinates 19 is a representative example to the last, as long as the position of a predetermined portion of the culture can be specified. Can be appropriately adopted as desired.

  In the present invention, the transparent plate-shaped glass portion is fragile if it is too thin, and has a problem in processing. If it is too thick, the depth of focus cannot be obtained in microscopic observation, and the advantage of glass is lost. It is desirable to adopt a thickness of 0.04 to 1.50 mm. Further, from the viewpoint of enabling microscopic observation with higher magnification and higher sensitivity, it is desirable to use a plate-like glass portion having higher transmittance and lower fluorescence characteristics.

  Furthermore, when culturing a culture such as cells or tissue fragments, from the viewpoint of enhancing the adherence of the culture to the culture surface, for example, extracellular matrix such as collagen, laminin, fibronectin, and polylysine, polyethyleneimine It is preferable to apply a polycation such as polyortinin on the surface of the sheet glass part in advance.

  Next, the operation of the culture vessel 11 according to the present invention will be described with reference to the examples shown in FIGS. 1 and 2. The cells and tissue fragments are placed on the surface of the plate-like glass portion 27 of the vessel body 22 in the open state. After attaching a culture medium such as the above, the culture medium can be cultured by covering with a lid (not shown) and placing it in an appropriate culture environment.

  In addition, it is necessary to observe a culture object such as a cell or a tissue piece in the culture vessel 11 under a higher magnification with a microscope, or to observe a fluorescence with a higher sensitivity when observing a fluorescence. Even in this case, if the thickness of the plate-like glass portion 17 itself is 0.04 to 1.50 mm, not only the depth of focus of the microscope can be obtained, but also the transmittance is high and the characteristics of low fluorescence are obtained. Therefore, it is possible to sufficiently cope with the situation.

  In addition, since a grid coordinate 19 composed of a plurality of latitude lines 19a and meridians 19b is provided on the front surface or the back surface of the plate-like glass portion 27, a small amount of a gene or a drug is injected into specific cells by, for example, microinjection. There is a need for repeated observations such as observing later morphological changes with time, observing cell chemotaxis, and observing the cell population in individual subpopulations in the same container. However, it is possible to accurately observe the microscope each time after specifying the position of each cell or the position of a cell group as a group. In addition, cells to be cultured, such as cells and tissue fragments, can be microscopically observed in a living culture state without a complicated preparation operation such as removal from the culture medium. When the grid coordinates 19 are provided on the back surface of the plate-shaped glass part 27, the grid coordinates 19 are not visible after confirming the position of the grid coordinates 19 in advance, for example, when taking a microscopic photograph. It will be possible to shoot in the state.

  Further, according to the present invention, a culture target such as a cell or a tissue piece can be cultured at a plurality of locations in the same culture vessel 11, and a plate-like glass portion formed of a partition portion 29 of a glass plate 28 in the container body 22. 27, high-magnification and high-sensitivity microscopic observation can be repeatedly performed under the same environment.

The top view which shows only the container main body side about an example of this invention. FIG. 2 is a longitudinal sectional view in the direction of arrows AA in FIG. 1. The top view which expands and shows an example of a grid coordinate. The top view which expands and shows another example of a grid coordinate.

Explanation of reference numerals

DESCRIPTION OF SYMBOLS 11 Culture container 19 Grid coordinate 19a Latitude 19b Meridian 22 Container main body 23 Compartment 24 Surrounding partition wall 25 Bottom 27 Plate glass part 28 Glass plate 29 Partition part 30 Reinforcement rib

Claims (3)

In a culture container comprising a container body freely formed to accommodate a culture medium such as cells and tissue pieces, and a lid formed freely to cover the container body,
The container main body includes a plate-shaped glass portion for attaching the culture object to at least a bottom portion thereof, and has a plurality of compartments partitioned by erecting a surrounding partition on the plate-shaped glass portion. Formed
A plurality of latitude lines and meridians formed to enable identification of a predetermined position of the attached culture object on either the front or back surface of the plate-shaped glass portion where the individual compartments are located. A culture vessel provided with grid coordinates consisting of: The culture vessel according to claim 1, wherein the thickness of the plate-shaped glass part is 0.04 to 1.50 mm. The culture vessel according to claim 1, wherein an extracellular matrix or polycations is applied to a surface of the plate-shaped glass portion.

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