JP2009296897A - Seal for forming observation portion, culture container, and position identification method in cell operation/observation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To enable to identify a position at a low cost without deteriorating observation performance. <P>SOLUTION: Provided is a position identification method in cell operation/observation, including the steps of: adhering a seal 10 used for forming an observation portion and having a cover portion 12 having at least one opening 11 disposed therein for forming the observation portion, to the non-culture side of a plate-like glass portion 22 constituting the bottom portion of a glass base dish 20 in which cells are cultured; putting the glass dish 20 on the stage of a microscope; selecting an observation portion for performing a cell operation among the observation portions formed with the openings 11; performing a cell operation for cells in the selected observation portion with a cell operation device; removing the glass base dish 20 from the upper side of the stage of the microscope; further after performing the culture of the cells, again putting the glass base dish 20 on the stage of the microscope; specifying the cell-operated observation portion; identifying the cell-operated cell in the specified observation portion; and then observing the cell with the microscope. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a seal for forming an observation portion to be attached to a culture vessel for culturing cells, a culture vessel, and a position identification method in cell manipulation / observation.

  When observing cells in a culture vessel, for example, observe specific cells that have been manipulated by microinjection, for example, or place the same cells in the culture vessel several times on the observation device (microscope). In some cases, it is necessary to specify the observation position in the culture container.

  Patent Document 1 discloses a culture vessel in which grid coordinates are formed on a glass portion of a glass base dish by laser or etching. By using this culture vessel, it is possible to specify which cell in the grid is to be observed.

Moreover, patent document 2 is disclosing the culture container which formed the grid coordinate in the slide glass chamber.
Japanese Patent No. 3613512 Japanese Patent No. 3942183

  In the culture vessels disclosed in Patent Documents 1 and 2, it is necessary to scan the microscope stage under a microscope and check the address information and read the position of the grid. Takes a long time to fit within the field of view of the microscope. In addition, cells to be observed may overlap on the grid, and the grid itself may interfere with the observation. In addition, since it takes a cost to form a grid in the glass part by laser and etching, a glass base dish with a grid is sold at an expensive price several times that of a general glass base dish.

  On the other hand, as a means for specifying the observation position in the culture vessel at a low cost, a grid seal provided with a grid used by being attached to a culture vessel such as a glass base dish is commercially available. Although the position can be specified with this grid seal, the observation performance deteriorates in high-magnification observation, fluorescence observation, and the like.

  The present invention has been made in view of the above points, and provides a seal for forming an observation part, a culture container, and a position identification method in cell manipulation / observation that can identify the position at low cost without degrading the observation performance. The purpose is to do.

One aspect of the observation portion forming seal of the present invention is
It is characterized by comprising a covering part provided with at least one opening part for forming an observation part when attached to the bottom part of the culture vessel.

Also, one aspect of the culture container of the present invention is
The glass part constituting the bottom part is covered with a covering part, leaving at least one observation part.

In addition, one aspect of the position identification method in the cell manipulation / observation of the present invention,
Attaching an observation part forming seal provided with a covering part provided with at least one opening for forming an observation part on the non-culture surface of the glass part constituting the bottom part of the culture vessel in which the cells are cultured; ,
The step of placing the culture vessel on a microscope stage and selecting an observation unit for performing cell operation among the observation units formed by the opening,
For the cells in the selected observation section, a step of performing cell manipulation with a cell manipulation device,
Removing the culture vessel from the microscope stage and further culturing;
Placing the culture vessel on which the culture has been further performed on the stage of the microscope, and identifying the observation unit operated with the cells;
Identifying the cell-manipulated cell in the specified observation section, and observing with the microscope,
It is characterized by having.

  ADVANTAGE OF THE INVENTION According to this invention, the seal | sticker for observation part formation which can identify a position cheaply without degrading observation performance, a culture container, and the position identification method in cell operation and observation can be provided.

  The best mode for carrying out the present invention will be described below with reference to the drawings.

[First Embodiment]
FIG. 1A is a view showing an observation portion forming seal 10 according to the first embodiment of the present invention, and FIGS. 1B and 1C are attached with the observation portion forming seal 10. FIG. 2 is a top view and a cross-sectional view of a culture vessel, in this example a glass base dish 20.

  As shown in FIG. 1 (A), the observation portion forming seal 10 has a covering portion 12 provided with at least one opening 11 for forming an observation portion when attached to the bottom of the culture vessel. I have. Although not particularly illustrated, the observation portion forming seal 10 is provided with an adhesive layer on one surface of a support made of a transparent resin film. And the adhesive layer is affixed on release paper, and a plurality of observation part forming seals 10 are provided in the form of a seal sheet 13.

  The observation part forming seals 10 are peeled from the seal sheet 13 one by one and, as shown in FIGS. 1 (B) and 1 (C), cells (FIG. 1) arranged on the bottom plate part 21 of the glass base dish 20. (Not shown) is attached to a transparent plate-like glass portion 22 for culturing with adhering to a back surface 22a which is a non-culture surface of cells, for example.

  Here, the glass base dish 20 is formed in a cylindrical shape with a bottom plate portion 21 and a peripheral side portion 23 made of a synthetic resin material such as polystyrene, and a circular transparent portion is formed at the central portion of the bottom plate portion 21. One hole 24 is formed. A plate-like glass portion 22 that is sufficient to completely cover the opening surface 24a of the through hole 24 from the lower surface 21a side is joined to the bottom plate portion 21 via an adhesive S made of a silicon-based adhesive or an acrylate-based adhesive. Thus, the entire opening surface 24 a of the through hole 24 is shielded by the plate-like glass portion 22. Thus, the bottom portion of the glass base dish 20 is constituted by the bottom plate portion 21 and the plate-like glass portion 22.

  The thickness of the observation portion forming seal 10 is preferably 100 μm or less. This is because when the thickness is 100 μm or more, there is a possibility that bubbles may enter and cannot be observed with a microscope using an immersion objective lens.

  Moreover, it is preferable that the size of the opening part 11 which forms an observation part is equivalent to a visual field size. For example, the diameter is 500 μm for a 40 × objective, the diameter is 1 mm for a 20 × objective, and the diameter is 2 mm for a 10 × objective. In addition, in order to make each opening part 11 easy to distinguish, it is preferable to arrange | position the space | interval more than the size of the opening part 11 between the adjacent opening parts 11. FIG.

  Here, with reference to FIG. 2, the cell operation apparatus and the microscope for operating and observing the cells cultured on the glass base dish 20 to which the observation unit forming seal 10 according to the first embodiment is attached will be described. I will explain.

  The cell manipulation device 30 according to the present embodiment is provided in an inverted microscope 40 for observing cells.

  The inverted microscope 40 includes a culture container holder 41 on which the glass base dish 20 and the like containing the cells are placed, an illumination device 42 that illuminates the cells on the culture container holder 41, light reflected or transmitted by the cells, Alternatively, an observation device 43 that observes fluorescence generated from the cells, a microscope XY stage 44 that moves the culture vessel holder 41 in the X direction and the Y direction, and a microscope XY stage controller 45 that drives and controls the microscope XY stage 44, A microscope controller 46 that controls the illumination device 42 and the observation device 43, an image processing device 47 that processes an image obtained by the observation device 43, and a monitor 48 that displays an image processed by the image processing device 47. A control computer for controlling the entire cell manipulation device 30 and the inverted microscope 40 It is provided with a 9, a.

  Further, the illumination device 42 collects the transmitted illumination light source 42A that irradiates the cells with illumination light from the side opposite to the observation device 43, and the illumination light emitted from the transmitted illumination light source 42A on the cells. And a condenser lens 42B for irradiating the cells with illumination light from the same direction as the observation device 43.

  On the other hand, the observation apparatus 43 includes an observation optical system including an objective lens (not shown), a CCD camera 43A that captures light from the cell via the observation optical system and acquires an image, and direct light from the cell. An eyepiece 43B for observation is provided.

  The cell manipulation device 30 is disposed in the vicinity of the culture container holder 41 of the inverted microscope 40, and the introduction needle 31 inserted into the cell, the introduction needle holder 32 for holding the introduction needle 31, A drive unit 33 for moving the introduction needle 31 by moving the introduction needle holder 32 and a control box 34 for controlling the cell manipulation device 30 are provided. Here, the drive unit 33 includes, for example, a triaxial linear movement mechanism that moves the introduction needle 31 in the X, Y, and Z directions.

  As shown in FIG. 3A, the introduction needle 31 is provided on the free end side of the cantilever 35 so as to extend in a direction intersecting the longitudinal direction of the cantilever 35. The cantilever 35 is fixed to an attachment member 36, and the attachment member 36 is fixed to the arm 32 </ b> A of the introduction needle holder 32 via a Z-axis actuator 37. As a result, the cantilever 35 is held obliquely downward, and the introduction needle 31 is held at the free end of the cantilever 35 with its tip directed vertically downward.

  As shown in FIG. 3B, the cantilever 35 includes a flexible silicon base portion 35A and a lever portion 35B in which a fine introduction needle 31 is formed at the tip. It is fixed to be exchangeable. Therefore, by exchanging the cantilever 35, the cell manipulation device 30 can be used repeatedly without fear of contamination.

  The Z-axis actuator 37 disposed between the mounting member 36 and the introduction needle holder 32 is composed of, for example, stacked piezoelectric elements, and expands and contracts according to the applied voltage. As shown in FIG. 3A, since the Z-axis actuator 37 is disposed vertically between the mounting member 36 and the introduction needle holder 32, the applied voltage is adjusted to adjust the applied needle holder 32. Thus, the attachment member 36 is slightly displaced in the vertical direction, and the tip of the introduction needle 31 attached vertically downward to the attachment member 36 can be moved by a minute distance in the vertical direction.

  The control box 34 includes a drive unit control circuit 34A for controlling the drive unit 33 and an actuator control circuit 34B for controlling the Z-axis actuator 37.

  Hereinafter, the position identification method in the cell manipulation / observation according to the first embodiment of the present invention using the observation part forming seal 10, the glass base dish 20, the cell manipulation device 30, and the inverted microscope 40 will be described. This will be described with reference to the flowchart of FIG. Here, as an example of a cell operation using the cell operation device 30, an operation for introducing a gene into cells cultured in a culture solution in the glass base dish 20 will be described as an example.

  First, one observation portion forming seal 10 is peeled off from the seal sheet 13 and is pasted on the rear surface 22a of the plate-like glass portion 22 of the glass base dish 20 in which the gene introduction material is dispersed in the cell culture solution (step). S1).

  Thereafter, the glass base dish 20 to which the observation part forming seal 10 is attached is placed on the culture container holder 41 of the inverted microscope 40 and formed by the opening 11 of the observation part forming seal 10. Among the observation units, an observation unit for performing cell manipulation is selected (step S2). Here, although the said observation part formation seal | sticker 10 is comprised with the support body which consists of a transparent resin film, resolution | decomposability is impaired by an adhesive layer. Therefore, although the illumination light from the illumination device 42 is transmitted at a location corresponding to the covering portion 12, it is not suitable for cell observation. On the other hand, since the observation part is formed by the opening part 11, it is possible to observe the cell without impairing the resolution. In addition, as described above, the size of the opening 11 forming the observation portion is equivalent to the visual field size based on the magnification of the inverted microscope 40. Accordingly, while observing with the eyepiece 43B or the monitor 48, the microscope XY stage controller 45 and the microscope controller 46 are operated to operate the microscope XY stage 44, and the observation unit having the cell into which the gene is to be introduced is placed under the microscope observation. .

  Thus, if a desired cell in the selected observation part is placed under the microscope observation, a gene introduction operation is performed by the cell operation device 30 as a cell operation on the cell (step S3). This is performed, for example, as follows.

  That is, the drive unit 33 is operated by the operation of the drive unit control circuit 34A, and the introduction needle 31 of the cantilever 35 is moved close to the cell from above the desired cell. Since the cells seeded on the plate-like glass portion 22 of the glass base dish 20 have only a thickness of about 2 μm to 10 μm, the minute descent from this close position is caused by the operation of the actuator control circuit 34B by the Z-axis actuator 37. This is done by operating.

  By operating the Z-axis actuator 37, the introduction needle 31 is lowered and brought closer to the plate-like glass portion 22 as shown in FIG. As a result, while the tip of the introduction needle 31 is descending, it enters into the culture solution in which the genes in the glass base dish 20 are dispersed (hereinafter referred to as DNA dispersion solution 50), and the glass plate of the glass base dish 20 is introduced. The cell 60 seeded on the part 22 is contacted. Here, the introduction needle 31 is further lowered to perforate the cell membrane 61 and the nucleus 62.

  Then, as shown in FIG. 5B, if the introduction needle 31 is lowered to a position where the tip of the introduction needle 31 comes into contact with the surface of the plate-like glass portion 22, the contact position with the plate-like glass portion 22 surface. Furthermore, the introduction needle 31 is lowered. As a result, as shown in FIG. 5C, the tip of the introduction needle 31 slides with respect to the surface of the plate-like glass portion 22, and by this sliding, the bottom surface side is also broken, and the top surface side and bottom surface of the cell membrane 61 A hole penetrating between the two fractured portions 70 on the side is opened.

  When the DNA dispersion solution 50 flows through the thus formed through-holes, the genes dispersed in the DNA dispersion solution 50 flow into the cells 60.

  Note that after the introduction needle 31 is lifted and the introduction needle 31 is pulled out of the cell 60, the cell membrane 61 is recovered by self-repair and a gene is taken into the cell 60 after a certain period of time.

  In this way, it is possible to introduce the gene into the cell 60 reliably and with high introduction efficiency while maintaining a low survival rate and high survival rate as in the prior art.

  The sliding method of the introduction needle 31 is not limited to the method performed by lowering the introduction needle 31 further than the contact position with the surface of the plate-like glass portion 22 as described above. Similarly to the Z-axis actuator 37, the arm 32A of the holder 32 is also composed of stacked piezoelectric elements, and the arm 32A is expanded and contracted to introduce the tip of the introduction needle 31 in contact with the plate-like glass portion 22. It can also be performed by moving the needle 31 horizontally.

  Alternatively, when the introduction needle 31 is lowered and the tip thereof enters the DNA dispersion solution 50 in the glass base dish 20, the introduction of the introduction needle 31 is performed while the tip of the introduction needle 31 is vibrated in the horizontal direction by operating the arm 32A. The needle 31 may be lowered. As a result, when the tip of the introduction needle 31 is lowered, when the cell 60 is contacted, a cut is formed in the cell membrane 61 by the vibration of the introduction needle 31. That is, the cell membrane 61 has fluidity, and when the introduction needle 31 is simply moved in the length direction and stabbed, the cell membrane 61 is deformed following the introduction needle 31 and cannot penetrate the cell membrane 61. By moving the introduction needle 31 in the length direction while vibrating in a direction intersecting the longitudinal direction, a cut is formed in the cell membrane 61, and the cell membrane 61 can be easily penetrated.

  As a result, the introduction needle 31 can be reliably inserted into the nucleus 62 in the cell membrane 61. The introduction needle 31 is pulled out from the cell 60 and cultured for a predetermined time, so that the gene dispersed in the DNA dispersion solution 50 is more reliably introduced into the hole formed up to the inside of the nucleus 62.

  Of course, when the drive unit 33 can finely adjust in the XYZ directions, the Z-axis actuator 37 and the arm 32A need not be formed of piezoelectric elements.

  If the gene introduction operation is performed as described above, the glass base dish 20 is removed from the culture vessel holder 41 of the inverted microscope 40 and further culturing is performed (step S4).

  And after lapse of a predetermined period, in order to observe the time-dependent change of the cell 60 into which the gene has been introduced, the glass-based dish 20 which has been further cultured is placed on the culture container holder 41 of the inverted microscope 40, The observation part where the gene introduction operation has been performed is placed in the field of view (step S5). At this time, the glass base dish 20 has only four observation parts in this example, and the observation part where the gene introduction operation was easily performed can be put in the field of view, and the periphery of the observation part is covered. Therefore, the position of the observation part can be easily specified even under microscopic observation. Therefore, it becomes possible to easily identify the cell that has undergone the gene transfer operation.

  Then, the cell-operated cell in the identified observation part is observed with the inverted microscope 40 (step S6).

  As described above, according to the first embodiment, it is possible to provide a seal for forming an observation part that can identify the position at low cost without degrading the observation performance, thereby preventing the observation performance from degrading. It is possible to provide a position identification method for cell manipulation / observation that can identify the position at low cost.

[Modification 1]
In addition, the shape of the opening 11 for forming the observation part in the observation part forming seal 10 is not a circle as shown in FIG. 1A, but as shown in FIG. 6A or 6B. Further, it may have one or more sides of a straight line. In this case, as shown in those figures, it is not necessary for all the openings 11 to be, and it is sufficient that at least one has at least one side of a straight line.

  Thereby, since it becomes easy to reproduce the arrangement angle of the glass base dish 20, it is possible to more easily identify cells that have undergone cell manipulation (gene introduction manipulation) in the observation section.

  Furthermore, as shown in FIG. 6 (C), an identification symbol 13 such as a number for identifying each opening 11 is described on the covering part in the vicinity of each opening 11 forming each observation part. The position of the observation part where the cell operation (gene introduction operation) has been performed can be easily identified.

  Furthermore, as shown in FIG. 6D, the arrangement of the openings 11 forming the observation units may be arranged with directionality.

[Modification 2]
Further, as shown in FIG. 7, the covering portion of the observation portion forming seal 10 can be made black, for example, to form the light-shielding covering portion 14 having light shielding properties.

  Thereby, since the illumination light by the illuminating device 42 is transmitted only to the observation part being observed, it is easy to determine which observation part is being viewed.

[Modification 3]
Note that the number of observation portions, that is, the openings 11 in the observation portion forming seal 10 is not limited to the above four, and may be larger as shown in FIG. Conversely, the number may be less than four. In other words, the observation portion forming seal 10 is provided with at least one opening 11 for forming an observation portion when being attached to the plate-like glass portion 22 constituting the bottom portion of the glass base dish 20. good.

[Modification 4]
Moreover, although the observation part formation seal | sticker 10 shall be affixed on the plate glass part 22 back surface 22a of the glass base dish 20, it is also possible to affix on the surface side which is a culture surface of a cell.

  In that case, the observation part forming seal 10 is first attached to the surface of the plate-like glass part 22 of the glass base dish 20, and then the plate-like glass part 22 on which the observation part forming seal 10 is attached. Cells are seeded on the opening surface 24a, and a culture solution is added to culture the cells.

[Second Embodiment]
9A and 9B are a top view and a cross-sectional view of a glass base dish 80 as a culture container according to the second embodiment of the present invention. Here, about the structure similar to the glass base dish 20 in the said 1st Embodiment, the description is abbreviate | omitted by attaching | subjecting the same referential mark as FIG. 1 (B) and (C).

  In the glass base dish 80 in the present embodiment, at least one plate glass portion 22 constituting the bottom portion is covered with a coating film 82 that becomes a covering portion, leaving four observation portions 81 in the drawing. That is, in the first embodiment, the observation part is formed by attaching the observation part forming seal 10 to the plate-like glass part 22, but in this embodiment, the observation part is directly attached to the plate-like glass part 22. 81 is provided.

  Here, the size of the observation unit 81 is preferably equivalent to the visual field size. For example, the diameter is 500 μm for a 40 × objective, the diameter is 1 mm for a 20 × objective, and the diameter is 2 mm for a 10 × objective. In order to make it easy to distinguish each observation unit 81, it is preferable that the adjacent observation units 81 are arranged with an interval larger than the size of the observation unit 81.

  According to such a configuration, it is possible to provide a culture vessel that can identify the position at low cost without degrading the observation performance.

  Further, since the covering portion is formed of a coating film 82 having a light shielding property such as black paint, the illumination light from the illumination device 42 is transmitted only to the observation portion 81 being observed. It is easy to determine whether

  As described in the first embodiment, it is sufficient that the number of observation units 81 is at least one, and at least one of the observation units 81 has one or more sides of a straight line. The identification symbol 13 for identification may be described on the coating film 82, or the observation units 81 may be arranged in a directional array.

  Moreover, although the coating film 82 which comprises a coating | coated part is formed in the surface of the plate glass part 22 which is a cell culture surface, it is needless to say that you may provide in the back surface 22a of the plate glass part 22. FIG.

[Third Embodiment]
In the first and second embodiments, the glass base dish is described as an example of the culture container. However, the present invention is not limited to such a glass base dish, and the cover glass chamber, the slide glass chamber, the well It can be applied to various culture vessels such as plates.

  For example, FIG. 10 is a cross-sectional view showing an example in which the observation unit forming seal 10 according to the first embodiment is applied to the slide glass chamber 90.

  The slide glass chamber 90 has a plurality of compartments 94 that are partitioned by standing a surrounding partition wall 93 made of a synthetic resin material such as polystyrene on a bottom 92 made of a single transparent glass plate 91. Is formed. Thereby, the transparent plate-like glass part 95 for making a cell adhere to the bottom part 92 separately of each compartment 94 will be comprised.

  And the said observation part formation seal | sticker 10 is affixed on each plate-shaped glass part 95, respectively.

  Further, instead of the observation part forming seal 10, a covering part by the coating film 82 for forming the observation part 81 as in the second embodiment may be formed on each plate-like glass part 95.

  As described above, the same effects as those of the first and second embodiments can be obtained in any culture vessel.

  Although the present invention has been described above based on the embodiments, the present invention is not limited to the above-described embodiments, and various modifications and applications are naturally possible within the scope of the gist of the present invention.

(Appendix)
The invention having the following configuration can be extracted from the specific embodiment.

  (1) An observation portion forming seal comprising a covering portion provided with at least one opening for forming an observation portion when attached to the bottom of a culture vessel.

(Corresponding embodiment)
The first and third embodiments correspond to the embodiment relating to the observation portion forming seal described in (1). In these embodiments, for example, the glass base dish 20 and the slide glass chamber 90 are in the culture vessel, the plate glass portions 22 and 95 are in the bottom portion, the opening portion 11 is in the opening portion, and the covering portion 12 is in the covering portion. The observation portion forming seal 10 corresponds to the observation portion forming seal.

(Function and effect)
According to the observation portion forming seal described in (1), it is possible to provide an observation portion forming seal that allows the position to be identified at low cost without degrading the observation performance.

  (2) The observation portion forming seal according to (1), wherein at least one of the openings for forming the observation portion has a linear portion having one or more sides.

(Corresponding embodiment)
The embodiment relating to the observation portion forming seal described in (2) corresponds to the first modification, in particular, of the first embodiment.

(Function and effect)
According to the observation part forming seal described in (2), it is possible to more easily identify the cells in which the cells are manipulated in the observation part.

  (3) The observation portion forming seal according to (1), wherein the thickness of the observation portion forming seal is 100 μm or less.

(Corresponding embodiment)
The first and third embodiments correspond to the embodiment relating to the observation portion forming seal described in (3).

(Function and effect)
According to the observation part forming seal described in (3), observation can be performed without bubbles even in a microscope using an immersion objective lens.

  (4) The observation portion forming seal according to (1), wherein the covering portion has a light shielding property.

(Corresponding embodiment)
The embodiment relating to the observation portion forming seal described in (4) corresponds to the second modification, in particular, of the first embodiment. In the modification, for example, the light-shielding covering portion 14 corresponds to the covering portion having the light-shielding property.

(Function and effect)
According to the observation part forming sticker described in (4), since the illumination light is transmitted only through the observation part being observed, it is easy to determine which observation part is being viewed.

  (5) The seal for forming an observation part according to (1), wherein an identification symbol is written in the vicinity of the observation part.

(Corresponding embodiment)
The embodiment relating to the observation portion forming seal described in (5) corresponds to the first modification, in particular, of the first embodiment. In the modification, for example, the identification symbol 13 corresponds to the identification symbol.

(Function and effect)
According to the observation portion forming seal described in (5), it is possible to more easily identify the position of the observation portion where the cell operation has been performed.

  (6) A culture vessel characterized in that the glass part constituting the bottom part is covered with a covering part leaving at least one observation part.

(Corresponding embodiment)
The embodiment relating to the culture container described in (6) corresponds to the second and third embodiments. In these embodiments, for example, the plate-like glass portions 22 and 95 are the glass portion constituting the bottom portion, the observation portion 81 is the observation portion, the coating film 82 is the covering portion, the glass base dish 80, and the slide glass. The chamber 90 corresponds to each of the culture containers.

(Function and effect)
According to the culture container described in (6), it is possible to provide a culture container whose position can be identified inexpensively without deteriorating the observation performance.

  (7) The culture container according to (6), wherein the covering portion is a coating film.

(Corresponding embodiment)
The embodiment relating to the culture container described in (7) corresponds to the second and third embodiments. In those embodiments, the coating film 82 corresponds to the coating film.

(Function and effect)
According to the culture container described in (7), since the illumination light is transmitted only through the observation part being observed, it is easy to determine which observation part is being viewed.

  (8) The culture container according to (6), wherein at least one of the observation units has a linear portion having one or more sides.

(Corresponding embodiment)
The embodiment relating to the culture container described in (8) corresponds to the second and third embodiments.

(Function and effect)
According to the culture container described in (8), it is possible to more easily identify the cell that has been manipulated in the observation section.

  (9) The culture container according to (6), wherein the abdomen is marked with an identification symbol in the vicinity of the observation part.

(Corresponding embodiment)
The embodiment relating to the culture container described in (9) corresponds to the second and third embodiments. In these embodiments, for example, the identification symbol 13 corresponds to the identification symbol.

(Function and effect)
According to the culture container described in (9), it is possible to more easily identify the cell in which the cell is manipulated in the observation unit.

(10) An observation part forming seal provided with a covering part provided with at least one opening for forming an observation part is pasted on the non-culture surface of the glass part constituting the bottom part of the culture vessel in which the cells are cultured. Attaching process,
The step of placing the culture vessel on a microscope stage and selecting an observation unit for performing cell operation among the observation units formed by the opening,
For the cells in the selected observation section, a step of performing cell manipulation with a cell manipulation device,
Removing the culture vessel from the microscope stage and further culturing;
Placing the culture vessel on which the culture has been further performed on the stage of the microscope, and identifying the observation unit operated with the cells;
Identifying the cell-manipulated cell in the specified observation section, and observing with the microscope,
A position identification method in cell manipulation / observation characterized by comprising:

(Corresponding embodiment)
The first and third embodiments correspond to the embodiment relating to the position identifying method in the cell manipulation / observation described in (10). In these embodiments, for example, step S1 is a step for attaching the observation portion forming seal, step S2 is a step for selecting the observation portion, step S3 is a step for performing the cell operation, and step S4 is a step for the above. Further, the step of culturing corresponds to the step of specifying the observation part in step S5, and the steps of observation in steps S5 and S6, respectively.

(Function and effect)
According to the position identification method in cell manipulation / observation described in (10), it is possible to provide a position identification method in cell manipulation / observation that can identify the position at low cost without degrading the observation performance.

FIG. 1 (A) is a view showing an observation part forming seal according to the first embodiment of the present invention, and FIGS. 1 (B) and (C) are cultures to which the observation part forming seal is attached. It is the upper side figure and sectional drawing of the glass base dish which is a container. FIG. 2 is a diagram showing a configuration of a cell manipulation device and an inverted microscope for performing manipulation / observation on cells cultured in a glass base dish. 3A is an enlarged view showing the structure near the cantilever, and FIG. 3B is an enlarged view showing the structure of the cantilever. FIG. 4 is a flowchart for explaining the position identification method in the cell manipulation / observation according to the first embodiment of the present invention. FIG. 5 is a diagram showing a series of states of an introduction needle provided on a cantilever for explaining a method of gene introduction as a cell manipulation. 6A and 6B are diagrams showing examples of openings having one or more sides of a straight line, and FIG. 6C is a diagram showing examples of identification symbols for identifying the openings. FIG. 6D is a diagram showing an example of the arrangement of openings having directionality. FIG. 7 is a view showing an observation portion forming seal provided with a light shielding covering portion. FIG. 8 is a view showing an observation portion forming seal having ten openings. 9A and 9B are a top view and a cross-sectional view of a glass base dish as a culture vessel according to the second embodiment of the present invention. FIG. 10 is a cross-sectional view of a slide glass chamber as a culture container in the third embodiment of the present invention.

Explanation of symbols

    DESCRIPTION OF SYMBOLS 10 ... Seal for observation part formation, 11 ... Opening part, 12 ... Covering part, 13 ... Seal sheet, 14 ... Light-shielding coating part, 20, 80 ... Glass base dish, 21 ... Bottom plate part, 22, 95 ... Sheet glass 23: Peripheral side part 24 ... Through hole 30 ... Cell manipulation device 31 ... Introducing needle 40 ... Inverted microscope 41 ... Culture vessel holder 42 ... Illumination device 43 ... Observation device 44 ... XY stage 50 ... DNA dispersion solution, 60 ... cells, 81 ... observation part, 82 ... painted film, 90 ... slide glass chamber, 91 ... glass plate, 92 ... bottom part, 93 ... going partition wall, 94 ... compartment, S ... adhesive .

Claims (10)

  An observation portion forming seal comprising a covering portion provided with at least one opening for forming an observation portion when attached to the bottom of a culture vessel.   2. The observation part forming seal according to claim 1, wherein at least one of the openings for forming the observation part has a linear portion having one or more sides.   The thickness of the said observation part formation seal | sticker is 100 micrometers or less, The observation part formation seal | sticker of Claim 1 characterized by the above-mentioned.   The observation portion forming seal according to claim 1, wherein the covering portion has a light shielding property.   2. The observation part forming seal according to claim 1, wherein an identification symbol is written in the vicinity of the observation part.   A culture vessel characterized in that a glass part constituting a bottom part is covered with a covering part leaving at least one observation part.   The culture container according to claim 6, wherein the covering portion is a coating film.   7. The culture vessel according to claim 6, wherein at least one of the observation parts has a straight part having one side or more.   The culture container according to claim 6, wherein an identification symbol is written in the vicinity of the observation part of the abdomen. Attaching an observation part forming seal provided with a covering part provided with at least one opening for forming an observation part on the non-culture surface of the glass part constituting the bottom part of the culture vessel in which the cells are cultured; ,
The step of placing the culture vessel on a microscope stage and selecting an observation unit for performing cell operation among the observation units formed by the opening,
For the cells in the selected observation section, a step of performing cell manipulation with a cell manipulation device,
Removing the culture vessel from the microscope stage and further culturing;
Placing the culture vessel on which the culture has been further performed on the stage of the microscope, and identifying the observation unit operated with the cells;
Identifying the cell-manipulated cell in the specified observation section, and observing with the microscope,
A position identification method in cell manipulation / observation characterized by comprising:

Images