WO2021192083A1 - Observation sample, method for producing observation sample, and sample production system - Google Patents

Abstract

An observation sample 11 is provided with a gelatinous medium M that encapsulates a spheroid SS, and a support 7 that holds the gelatinous medium M on a tip 7b, wherein the medium M is a gel that can be permeated by an adjustment solution, and the adjustment solution is an immobilizing solution that immobilizes the spheroid SS by modifying a protein of the spheroid SS, a dyeing solution that dyes the spheroid SS, a cleaning solution that cleans the spheroid SS and the medium M, or a transparentizing solution that transparentizes the spheroid SS.

Description

Observation sample, observation sample preparation method and sample preparation system

The present invention relates to an observation sample, a method for producing an observation sample, and a sample preparation system.

Conventionally, a method of microscopically observing a cell aggregate cultured three-dimensionally such as a spheroid or an organoid is known (see, for example, Patent Documents 1 and 2). In the observation method described in Patent Document 1, a gel solution such as agarose containing an observation object is gelled in a syringe. Then, while holding the gelled sample in a state of being extruded from the syringe, the observation object in the sample is observed with a light sheet microscope.

In the observation method described in Patent Document 2, a sample such as a minute object is transferred in the longitudinal direction of the measuring tube in a measuring tube filled with an aqueous sample medium such as water. Then, the sample passing through the measurement area in the measuring tube is observed with a light sheet microscope.

U.S. Pat. No. 9,733,160 U.S. Patent Application Publication No. 2017/160529

However, the observation methods described in Patent Documents 1 and 2 have the disadvantage that it is not possible to simultaneously perform adjustments such as staining and transparency for a large number of samples. Further, the observation method described in Patent Document 1 has an inconvenience that the observation object needs to be sucked into a syringe together with a gel solution such as agarose, which complicates the work. Further, the observation method described in Patent Document 2 has an inconvenience that the staining reagent cannot be changed for each sample.

The present invention has been made in view of the above circumstances, and a sample for microscopic observation capable of easily performing adjustments such as staining, washing, and transparency suitable for microscopic observation, and a sample for microscopic observation thereof are simplified. It is an object of the present invention to provide a production method and a sample preparation system that can be produced in Japan.

In order to achieve the above object, the present invention provides the following means.
The first aspect of the present invention includes a medium containing a biological sample and a support for holding the medium at the tip, the medium is a gel through which a adjusting solution can permeate, and the adjusting solution is the living body. An observational sample that is a solution that immobilizes, stains, cleans, or clears the sample.

According to this aspect, since the biological sample is contained in the gel-like medium through which the adjusting solution can permeate, the adjusting process for allowing the adjusting solution to act on the biological sample can be performed via the medium. In addition, since the gel-like medium is held at the tip of the support, the medium can be handled when adjusting the biological sample, such as moving the medium or processing a plurality of media together. It will be easier.

This eliminates the need to replace the medium before making the biological sample transparent, unlike the case where the adjustment process is performed while the biological sample is housed in a container together with the liquid medium. Therefore, there is no inconvenience that the biological sample is erroneously sucked together with the medium, or the medium is left unsucked. Further, the adjustment process is not limited to the work in the container containing the liquid medium. Therefore, adjustment suitable for microscopic observation can be easily performed.

In the above aspect, the support has a through hole penetrating in the direction intersecting the longitudinal direction of the support, a recess recessed in the direction intersecting the longitudinal direction, and an overhanging portion protruding in the direction intersecting the longitudinal direction. It may have at least one of.

The medium can be more firmly attached to the support by the gel-like medium entering the through hole or the recess or the gel-like medium being caught in the overhanging portion. Therefore, it is possible to suppress the gel-like medium from falling from the support with a simple structure.

In the above aspect, the support may include a non-circular portion having a non-circular cross section intersecting the longitudinal direction of the support.
The non-circular portion can prevent the gel-like medium from rotating around the longitudinal axis of the support. This makes it easier to observe the biological sample contained in the medium from the same direction with a microscope.

In the above aspect, the tip portion may be formed in a hook shape having the tip diameter reduced so as to be able to puncture the medium and being folded back.
With this configuration, by piercing the tip portion into the gel-like medium, the medium can be held in a state of being hooked on the tip portion.

In the above aspect, the support may be made of a material that does not contain a fluorescent substance. With this configuration, the fluorescence emitted from the biological sample can be accurately observed without being affected by the support.

In the above aspect, the plurality of the supports may be connected and the plurality of the media may be held by the respective supports.
With this configuration, it is possible to collectively adjust the biological sample contained in each medium in a state where a plurality of media are supported by each support. This makes it possible to prepare a large amount of biological samples.

In the above aspect, the supports may be configured to be separable from each other.
With this configuration, a plurality of biological samples can be grouped, and the biological samples can be individually adjusted for each group.
In the above aspect, the biological sample may be a cell mass.

In the second aspect of the present invention, the biological sample is fixed and stained by immersing the tip of the support in a liquid medium contained in the container together with the biological sample and adding a fixing agent to the medium. The medium is changed into a gel-like substance that allows the adjusting solution to be washed or made transparent to permeate, and the tip portion is held in the tip portion while the gel-like medium containing the biological sample is held in the tip portion. It is a method of preparing an observation sample taken out from.

According to this aspect, an observation sample in which a biological sample is encapsulated in a gel-like medium is prepared. Since the gel-like medium allows the adjusting solution to permeate, the adjusting process for allowing the adjusting solution to act on the biological sample can be performed via the medium. Further, since the gel-like medium is held at the tip of the support, it becomes easy to handle the medium when adjusting the biological sample. Therefore, it is possible to easily prepare an observation sample that can be easily adjusted for microscopic observation.

In the above embodiment, the gelled medium is held by the support, and at least one treatment of fixation, staining, washing, and clearing is performed on the biological sample contained in the medium. May be good.
With this configuration, it is possible to prepare an observation sample that has been adjusted to be suitable for microscopic observation.
In the above aspect, the biological sample may be treated in the order of fixation, staining, washing and clearing.

In the above aspect, the medium held by the support may be immersed in the adjusting solution.
With this configuration, an observation sample adjusted for microscopic observation can be produced by a simple operation.

In the above aspect, the medium held by the support may be sequentially immersed in various adjusting solutions according to the treatment of fixation, staining, washing or clearing for a predetermined time.
With this configuration, a large amount of biological samples can be easily prepared according to the preparation procedure, and observation samples can be efficiently prepared.

In the above embodiment, each of the tips of the plurality of supports connected to each other is immersed in each of the plurality of liquid media in the container, the fixing agent is added to each of the media, and a gel is added. The plurality of the media may be collectively immersed in the adjusting solution in a state where the plurality of the transformed media are held by the respective supports.
With this configuration, a large amount of biological samples can be collected and easily adjusted.

A third aspect of the present invention comprises an adjusting solution tank capable of containing an adjusting solution for fixing, staining, washing or clarifying a biological sample, and a gel-like medium containing the biological sample and allowing the adjusting solution to permeate. , A plurality of supports that can be held at the tip portion, the plurality of the supports are connected to each other, and the adjusting solution tank simultaneously converts the plurality of the media held by the respective supports into the adjusting solution. It is a sample preparation system having a size that can be immersed.

According to this aspect, each of the supports connected to each other can hold a plurality of gel-like media in which the adjusting solution can permeate while encapsulating the biological sample at the tip of the support. Then, a large number of observation samples adjusted for microscopic observation can be produced by a simple operation of immersing a plurality of media held by each support in the adjustment solution of the adjustment solution tank. Can be done.

The sample preparation system according to the above aspect includes a container in which a plurality of wells capable of accommodating the liquid medium together with the biological sample are arranged at a predetermined pitch, and the plurality of the supports are each of the above-mentioned containers. Each of the tips may be connected to the wells at a pitch that allows them to be inserted.

With this configuration, each tip of the plurality of supports can be immersed in a plurality of liquid media contained in each well of the container together with the biological sample. In this state, by changing each medium into a gel-like form in which the adjusting solution can permeate, a plurality of gel-like media containing a biological sample are held at each tip of a plurality of supports connected to each other. An observation sample can be easily prepared.

In the above aspect, a plurality of the adjusting solution tanks capable of separately accommodating various adjusting solutions according to the treatment of fixing, staining, washing or clearing may be provided.
With this configuration, various adjustment solutions can be separately prepared in advance in a plurality of adjustment solution tanks, and various adjustments of a large amount of biological samples become easy.

According to the sample for microscopic observation of the present invention, it is possible to easily perform adjustments such as staining, cleaning, and transparency suitable for microscopic observation. Further, according to the method for producing a sample for microscopic observation and the sample preparation system of the present invention, there is an effect that the sample for microscopic observation can be easily produced.

It is an overall block diagram which shows the sample preparation system which concerns on one Embodiment of this invention. It is a perspective view explaining the tip part of the support of FIG. It is a schematic block diagram which shows the observation sample which concerns on one Embodiment of this invention. It is a flowchart explaining the manufacturing method of the observation sample which concerns on one Embodiment of this invention. It is a figure which shows the state of injecting the liquid medium in which cells are suspended into each well. It is a figure which shows the state that the cell was seeded. It is a figure which shows the appearance that the spheroid was formed. It is a figure which shows the state of exchange and cleaning of a liquid medium. It is a figure which shows the state of drug screening. It is a figure which shows the state of immersing the tip part of the support in the liquid medium in a well. It is a figure which shows the state of adding acrylamide to a medium. It is a figure which shows the state of taking out a gel-like medium from a container. It is a figure which shows the mode that each gel-like medium held by each support is simultaneously immersed in the immobilization solution in the adjustment solution tank. It is a figure which shows the state of immersing each gel-like medium held by each support at the same time in the dyeing solution in the adjustment solution tank. It is a figure which shows the state of immersing each gel-like medium held by each support at the same time in the cleaning liquid in the adjustment solution tank. It is a figure which shows the mode that each gel-like medium held by each support is simultaneously immersed in the clearing solution in the adjustment solution tank. It is a figure which shows an example of the spheroid which was immersed in the clearing solution of 37 degreeC for 12 hours. It is a figure which shows an example of the spheroid before performing a transparent treatment. It is a figure which shows an example of the observation sample which performed various adjustment processing. It is a figure which shows the state which the observation sample of FIG. 19 was immersed in the liquid in the container for observation. It is an overall block diagram which shows the sample preparation system which includes a plurality of adjustment solution tanks. It is a figure which shows an example of the spheroid heated at 500W for 30 seconds by a microwave oven. It is a figure which shows an example of the support which has a through hole. It is a figure which shows an example of the support which has a groove. It is a figure which shows an example of the support bent in the direction which the tip part intersects with the longitudinal direction of a main body part. It is a figure which shows the state which closed the opening corresponding to the bottom part of a well by a seal member. It is a figure which shows the state which opened the opening which corresponds to the bottom part of a well by peeling off a seal member.

An observation sample, a method for producing an observation sample, and a sample preparation system according to an embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, the sample preparation system 1 according to the present embodiment fixes a container 3 having a plurality of wells 3a capable of accommodating a liquid medium M together with a spheroid (biological sample) SS, and a spheroid SS. It includes an adjusting solution tank 5 capable of accommodating an adjusting solution C for dyeing, washing or clearing, and a support connecting body 9 having a plurality of supports 7 capable of holding a gel-like medium M.

Container 3 is, for example, a microplate. In the container 3, for example, a plurality of bottomed cylindrical wells 3a such as 6, 24, 96, 384, or 1536 are arranged at a predetermined pitch.

The support connecting body 9 includes a plurality of elongated supports 7 and a connecting portion 8 for connecting each of the supports 7. Each support 7 is connected by a connecting portion 8 to each well 3a of the container 3 at a pitch that can be inserted into each well. Each support 7 may be individually and separably connected, or each of the plurality of supports 7 may be separably connected.

The support 7 includes a columnar main body portion 7a and a tip portion (overhanging portion, non-circular portion) 7b provided at one end of the main body portion 7a. As shown in FIG. 2, for example, the tip portion 7b is formed in the shape of a triangular flat plate that intersects the main body portion 7a in the longitudinal direction and extends outward in the radial direction. That is, the tip portion 7b has a non-circular cross section that projects in a direction intersecting the longitudinal direction of the main body portion 7a and intersects the longitudinal direction of the main body portion 7a. Further, the support 7 is made of a material that does not contain a fluorescent substance. Here, the main body 7a and the tip 7b of the support 7 may be separably connected.

In this support 7, the gel-like medium M containing the spheroid SS is caught by the tip portion 7b, so that the medium M is held by the support 7 so as to be liftable. Further, when the medium M gels in the presence of the support 7, the medium M is more firmly attached to the support 7 due to the adhesive force of the medium M.

The adjusting solution tank 5 has a size that allows a plurality of gel-like media M held by each support 7 of the support connecting body 9 to be immersed in the adjusting solution C at the same time. The adjusting solution tank 5 may contain one type of adjusting solution, or may be partitioned so that a plurality of types of adjusting solutions C can be separately accommodated.

Examples of the adjusting solution C include an immobilized solution C1 (see FIG. 13) such as PFA (Paraformaldehyde) that denatures the protein of spheroid SS, and a staining solution C2 (FIG. 14) such as a fluorescent reagent necessary for evaluating cell S. (See), a cleaning solution C3 for cleaning the spheroid SS and the medium M (see FIG. 15), a clearing solution C4 for clearing the spheroid SS (see FIG. 16), and the like.

In the observation sample 11 according to the present embodiment, for example, as shown in FIG. 3, the gel-like medium M containing the spheroid SS is caught by the overhanging portion of the tip portion 7a of the support 7 to support the support 7. Holds the medium M attached to the tip portion 7a. Further, the support 7 and the medium M are adhered to each other by the adhesive force of the medium M, and the support 7 and the medium M are more firmly connected in an integrated state.

The medium M is gelled to the extent that the adjusting solution C can permeate. The medium M is, for example, a substance that forms a three-dimensional network structure with a non-interfering solution such as a physiological saline solution, a PH buffer solution, or a dextrin solution that does not react with various adjustment solutions C, and a cross-linking agent or the like, for example. Examples thereof include a mixture obtained by mixing a cross-linking solution containing acrylamide as a main component. Here, in the medium M, the porous structure formed by gelation has sufficient permeability for any liquid to reach the contained spheroid SS from the outside, and the curing state brought about by gelation. However, a component and a concentration having sufficient rigidity to lift the medium M above the container 3 while containing the spheroid SS are selected. When a known cross-linking agent such as acrylamide is used, for example, an appropriate curing state can be obtained by containing acrylamide at a concentration of 1% to 20%, preferably 3% to 10% in the medium M.

Next, a method for producing an observation sample according to the present embodiment will be described.
In the method for producing the observation sample 11 according to the present embodiment, as shown in the flowchart of FIG. 4, the tip portion 7b of the support 7 is immersed in the liquid medium M contained in the container 3 together with the spheroid SS. Step S1, step S2 for changing the liquid medium M into a gel form through which the adjusting solution C can permeate, and the tip portion of the gelled medium M containing the spheroid SS while being held by the support 7. Steps S3 of taking out 7b from the container 3 and steps S5, S6, S7 of allowing various adjusting solutions C to act on the spheroid SS contained in the medium M while the gel-like medium M is held by the support 7. Includes.

Hereinafter, a method for producing an observation sample will be specifically described.
The process from the preparation of the spheroid SS to the observation is divided into a first scheme in which the cells S are cultured and drug screening is performed, and a second scheme in which the cells S are observed and evaluated.

In the first scheme, first, as shown in FIG. 5, cells are injected as shown in FIG. 6 by injecting a liquid medium M in which a plurality of cells S are suspended into each well 3a of the container 3. Sow S. In the figure, reference numeral 13 indicates a pipette. Aggregation of a plurality of cells S in the well 3a forms a spheroid SS as shown in FIG.

Next, as shown in FIG. 8, the medium M in the well 3a is replaced, or the medium M is washed by charging the cleaning liquid C3 into the well 3a. Then, as shown in FIG. 9, drug screening is performed by administering drug P for observing the dynamics of cells S to spheroid SS.

In the second scheme, the observation sample 11 is prepared by the method for preparing the observation sample according to the present embodiment.
Specifically, as shown in FIG. 10, each support 7 of the support connector 9 is placed in each optically transparent liquid medium M housed together with the spheroid SS in each well 3a of the container 3. Immerse each of the tip portions 7b of the above (step S1). At this time, as shown in the drawing, it is preferable that the position of the tip portion 7b is arranged higher than that of the spheroid SS and lower than that of the liquid surface of the medium M.

Then, as shown in FIG. 11, by adding, for example, 5% concentration of acrylamide (fixing agent) A to each of the liquid media M, the plurality of media M are each gelled to such an extent that the adjusting solution C can permeate. (Step S2). Here, acrylamide A is, for example, N, N'-methylenebisacrylamide, which is a cross-linking agent for preparing a polyacrylamide gel. Further, as shown in the figure, the position of the tip portion 7b becomes a position lower than that in step S1 due to the addition of acrylamide A.

The order of step S1 for immersing the tip portion 7b of the support 7 in the medium M and step S2 for adding acrylamide A to the medium M may be reversed. That is, after adding acrylamide A to the liquid medium M, the tip portion 7b of the support 7 may be immersed in the medium M before the medium M gels.

Next, as shown in FIG. 12, each medium M gelled with the spheroid SS contained therein is held in a state of being hooked on the tip portion 7b of each support 7. As a result, the plurality of gel-like media M are taken out from the container 3 together with the tip portion 7b of each support 7 (step S3).

In this case, the medium M may be taken out from the container 3 by moving the support 7 in the direction of separating the support 7 from the container 3, or the medium M may be moved in the direction of separating the container 3 from the container 7. The medium M may be taken out from the container 3 according to the above. Further, as described above, when the tip portion 7b of the support 7 is separably connected, when only the tip portion 7b is placed in a state of being integrated with the medium M and then step S3 is executed. The main body 9a and the tip 7b of the support 7 may be connected to each other.

By separating the container 3 and the support connecting body 9 from each other, each gel-like medium M together with each support 7 may be extracted from each well 3a. When it is difficult to extract the gel-like medium M from the well 3a, the medium M may be dug out from the well 3a or the medium M may be extruded from the well 3a. Further, air may be introduced into the gap between the medium M and the inner surface of the well 3a to facilitate the extraction of the medium M.

Next, as shown in FIG. 13, each gel-like medium M is moved to the adjusting solution tank 5 while being held by each support 7, and is contained in the adjusting solution tank 5, for example, immobilization having a concentration of 4%. Each medium M is immersed in the solution C1 together. The permeation of the gel-like medium M by PFA denatures the protein of spheroid SS. As a result, the spheroid SS dies without changing its morphology, and the position of the spheroid SS is fixed in the gel-like medium M (step S4).

After the spheroid SS is fixed, as shown in FIG. 14, the gel-like medium M is moved to the next adjusting solution tank 5 while being held by each support 7, and is housed in the adjusting solution tank 5. Each medium M is immersed together in the dyeing solution C2. When the staining solution C2 permeates the gel-like medium M, desired sites such as nuclei, mitochondria, and cell membranes of the spheroid SS in the medium M are stained (step S5).

After the spheroid SS is stained, as shown in FIG. 15, each gel-like medium M is moved to the next adjusting solution tank 5 while being held by each support 7, and is housed in the adjusting solution tank 5. Each medium M is immersed together in the cleaning solution C3. The medium M and the spheroid SS are washed by the washing liquid C3 penetrating the gel-like medium M (step S6). In the cleaning treatment, for example, the stain solution C2 that has permeated the medium M is removed.

After the medium M and the spheroid SS have been washed, as shown in FIG. 16, the gel-like medium M is moved to the next adjusting solution tank 5 while being held by the support 7, and is housed in the adjusting solution tank 5. Each medium M is immersed together in the clearing solution C4. In the clearing treatment, for example, the medium M is immersed in the clearing solution C4 for several hours.

When the clearing solution C4 permeates the gel-like medium M, for example, as shown in FIG. 17, the spheroid SS contained in the medium M becomes transparent (step S7). Since the gel-like medium M is optically transparent, the transparency treatment reduces the difference in the refractive index between the gel-like medium M and the spheroid SS and suppresses the scattering of light at the boundary between the medium M and the spheroid SS. can do.

FIG. 17 shows an example of Spheroid SS immersed in a clearing solution C4 at 37 ° C. for 12 hours. FIG. 18 shows an example of spheroid SS before transparency for comparison. When the spheroid SS is subjected to the clearing treatment, as shown in FIG. 19, an observation sample 11 having been adjusted suitable for microscopic observation is prepared. Here, in the observation sample 11 of FIG. 19, when the shape of the tip portion (overhanging portion) 7b of the main body portion 7a is a perfect circle (disk shape), the gelled medium M rotates and the orientation of the spheroid SS. Sometimes changed. From this, it was found that the shape of the tip portion (overhanging portion) 7b of the main body portion 7a is preferably non-circular as described above.

The prepared observation sample 11 is, for example, as shown in FIG. 20, a gel-like medium held by each support 7 in a liquid W such as water contained in the observation container 15. Immerse M in an upside-down position, i.e., with the support 7 down and the medium M up. In this state, the spheroid SS contained in the medium M is observed using a microscope (not shown).

For example, an upright light sheet microscope or the like irradiates the spheroid SS by incidenting the excitation light focused in a plane along the horizontal direction onto the gel-like medium M from the side. Then, among the fluorescence generated in the spheroid SS, the fluorescence radiated vertically above the medium M is focused by the objective lens, and then the fluorescence is photographed by an imaging element such as a CCD.

Since the tip portion 7b of the support 7 is formed in a triangular shape, it is possible to prevent the gel-like medium M from rotating around the longitudinal axis of the support 7, and the spheroid SS can be viewed from the same direction with a microscope. It can be made easier to observe. Further, since the support 7 is made of a material that does not contain a fluorescent substance, the fluorescence emitted from the spheroid SS can be accurately observed without being affected by the support 7. The observed support 7 may be withdrawn from the gelled medium M and discarded, or may be withdrawn and then reused for a medium M containing another spheroid.

As described above, according to the observation sample 11 according to the present embodiment, adjustment suitable for microscopic observation can be easily performed. That is, since the spheroid SS is contained in the gel-like medium M through which the adjusting solution can permeate, the adjusting process for causing the adjusting solution C to act on the spheroid SS can be performed via the medium M.

As a result, unlike the case where the spheroid SS is housed in the well 3a together with the liquid medium M and the adjustment process is performed, it is not necessary to replace the medium M before making the spheroid SS transparent. Therefore, there is no inconvenience that the spheroid SS is erroneously sucked together with the liquid medium M, or the medium M is left unsucked in the well 3a.

Further, since the gel-like medium M is firmly held by the tip portion 7b of the elongated support 7, the medium M is moved or the plurality of media M are collectively processed, and the spheroid SS is adjusted. It becomes easy to handle the medium M when applying the above. Therefore, the adjustment process is not limited to the work in the container 3 containing the liquid medium M.

Further, according to the method for producing the observation sample 11 and the sample preparation system 1 according to the present embodiment, it is possible to easily prepare the observation sample 11 that can be easily adjusted for microscopic observation. Further, the simple operation of immersing the plurality of media M held by the supports 7 in the adjusting solution C of the adjusting solution tank 5 is sufficient. As a result, a large amount of observation sample 11 adjusted for microscopic observation can be easily produced.

In the present embodiment, for example, as shown in FIG. 21, the sample preparation system 1 may include a plurality of adjusting solution tanks 5A, 5B, 5C, and 5D. Then, the immobilized solution C1, the staining solution C2, the cleaning solution C3, and the clearing solution C4 may be individually housed in the plurality of adjusting solution tanks 5A, 5B, 5C, and 5D.

In this case, for example, it is held by the support 7 in the immobilized solution C1 of the adjusting solution tank 5A, the staining solution C2 of the adjusting solution tank 5B, the cleaning solution C3 of the adjusting solution tank 5C, and the clearing solution C4 of the adjusting solution tank 5D. The gel-like medium M that has been formed may be immersed in order for a predetermined time.

With this configuration, each solution C1, C2, C3, C4 can be prepared in advance in a plurality of adjusting solution tanks 5, and a large amount of spheroid SS can be easily adjusted according to the adjusting procedure. Therefore, the observation sample 11 can be efficiently prepared, and for example, the screening of the sample such as the effectiveness of the drug can be efficiently performed.

This embodiment can be transformed into the following configuration.
In the present embodiment, in the clearing treatment, the gel-like medium M is immersed in the clearing solution C4. Instead of this, for example, the observation sample 11 before the transparency may be made transparent by heating or applying an electric field to the observation sample 11.

Thereby, for example, as shown in FIG. 22, the spheroid SS is made transparent in a short time until the transparency is almost the same as that in the state where the medium M is immersed in the clearing solution C4 for several hours (see FIG. 17). Can be done. FIG. 22 shows an example of the observation sample 11 heated at 500 W for 30 seconds by a microwave oven. When the observation sample 11 is heated by a microwave oven, it is desirable that the support 7 is made of a material that does not excessively generate heat or change its shape or the like due to microwaves.

Further, in the present embodiment, the tip portion 7b of the support 7 functions as an overhanging portion. The support 7 may have any shape as long as the gel-cured medium M can be held in a state of being caught by the tip portion 7b.

The support 7 may have a through hole 7c penetrating in a direction intersecting the longitudinal direction of the support 7, for example, as shown in FIG. 23, instead of the overhanging portion. Further, the support 7 may have a groove (recess) 7d or a through hole that is recessed in a direction intersecting in the longitudinal direction, as shown in FIG. 24, for example. When the tip portion 7a of the support 7 has one or more through holes or ring-shaped loop portions penetrating in a direction intersecting the longitudinal axis of the support 7, the gelled medium M is used. Since it is connected to the support 7 in an annularly closed state through a through hole or a loop portion, there is an advantage that it can be realized with a simple configuration.

According to this modification, the gel-like medium M can be more firmly attached to the support 7 by allowing the gel-like medium M to enter the through hole 7c or the groove 7d, and the gel-like medium M falls from the support 7. It can be suppressed by a simple structure.

Further, in the present embodiment, the support 7 has a triangular tip portion 7b as a non-circular portion. Instead of the triangle, the tip portion 7b may be formed in the shape of a polygon, a star, or an oval flat plate having a quadrangle or more. Further, the tip portion 7b may have a shape that projects in a state of being tapered toward the tip.

Further, the main body portion 7a of the support 7 may be, for example, a plate-shaped non-circular shape shown in FIG. 23 instead of the columnar shape. In this case, the overhanging portion of the tip portion 7b is a perfect circle. There may be. Further, a part or the whole of the support 7 may have a hollow portion. Further, the tip portion 7b of the support 7 may be bent in a direction intersecting the longitudinal direction of the main body portion 7a, for example, as shown in FIG. 25.

Further, the tip portion 7b may be in the shape of a hook (hook shape) or a crane shape formed of an overhanging portion having a small diameter portion that can be punctured. In this case, after the medium M has gelled, the hook-shaped or crane-shaped tip portion 7b is pierced into the gel-shaped medium M from the lateral or oblique direction to hold the medium M in a state of being hooked on the tip portion 7b. It may be that. This makes it possible to move the medium M. In the hook-shaped or crane-shaped tip portion 7b, for example, the tip of the tip portion 7b shown in FIG. 25 can be reduced in diameter so as to be punctured by the gel-like medium M, and can be held in a state where the medium M is hooked. It may have a shape that is further folded back.

According to this modification, in each case, the cross section intersecting the longitudinal direction of the support 7 has a non-circular shape. As a result, the gel-like medium M can be prevented from rotating around the longitudinal axis of the support 7, and the spheroid SS contained in the medium M can be easily observed from the same direction.

Further, in the present embodiment, as the support, the support 7 including the columnar main body 7a and the tip 7b has been illustrated and described. Alternatively, the support may be formed, for example, in a cylindrical shape. When the medium M is gelled while the cylindrical support is immersed in the medium M of the well 3a, the gel-like medium M is held inside the cylindrical support.

In this case, the inner surface of the cylindrical support may have an overhanging portion or the like that projects inward in the radial direction. The gel-like medium M held inside the cylindrical support is caught by the overhanging portion, so that the medium M can be firmly attached to the support.

Further, the cylindrical support may have an outer diameter slightly smaller than the inner diameter of the well 3a. In this case, since most of the gel-like medium M has entered the inside of the cylindrical support, the gel-like medium M can be easily extracted from the well 3a together with the support. Further, when the support has a cylindrical shape, the spheroid SS in the medium M can be observed via the inside of the support.

Further, in the present embodiment, the container 3 is provided with a bottomed cylindrical well 3a. Instead, for example, as shown in FIG. 26, the well 3a may not have a bottom, and the opening 3b corresponding to the bottom portion of the well 3a may be closed by a transparent sealing member 17 or the like. Further, the shape of the well 3a is not limited to a cylindrical shape, and the shape of the bottom and / or the side surface may be appropriately changed.

According to this modification, as shown in FIG. 27, by peeling off the seal member 17, air enters between the well 3a and the medium M from the opening 3b corresponding to the bottom portion of the well 3a, so that the medium M is introduced from the well 3a. Can be easily taken out. In this modification, the well 3a has no bottom, but it is sufficient if air can be introduced from the bottom of the well 3a. For example, a through hole is provided in the bottom of the well 3a, and the through hole is provided by the seal member 17. It may be closed so that it can be opened.

Further, in the present embodiment, the method of preparing the observation sample may be manually performed, or may be automatically performed by a control device (not shown). In the case of automatic operation, for example, the tip portion of the support 7 is moved to the liquid medium M in the well 3a by relatively moving the container 3 and the support 7 in the proximity direction by a drive mechanism (not shown). 7b may be immersed. Further, acrylamide A may be added to the liquid medium M by a supply mechanism (not shown). Further, the gel-like medium M may be taken out from the container 3 by relatively moving the container 3 and the support 7 in the direction of separating them by a drive mechanism (not shown). The above processing may be executed by at least one processor including hardware.

Although the embodiments of the present invention have been described in detail with reference to the drawings, the specific configuration is not limited to this embodiment, and design changes and the like within a range not deviating from the gist of the present invention are also included.
For example, in the above embodiment, the step S1 of immersing the tip portion 7b of the support 7 in the medium M and the step S2 of gelling the medium M are performed before the second scheme for observing and evaluating the cells S. Although it is carried out, the invention may be applied to all schemes by carrying out before the first scheme in which cells S are cultured or drug screening is performed.

Further, in the above embodiment, a mixture containing acrylamide as a main component was exemplified and described as the medium M. Instead of this, sodium alginate may be adopted as the medium M. When sodium alginate is used as the medium M, for example, a calcium chloride aqueous solution or the like, which is an aqueous solution containing divalent metal ions (calcium, magnesium, strontium, etc.), may be used as the immobilizing agent. Further, as the medium M, for example, a substance that forms a three-dimensional network structure depending on physical conditions such as temperature and pH, for example, a mixture such as agarose containing a polysaccharide component as a main component may be adopted.

Further, in the present embodiment, spheroid SS has been described as an example as a biological sample, but it can be applied to all cell masses having a three-dimensional shape such as organoids, colonies, and cell tissues. Alternatively, it may be, for example, one or more isolated cells.

According to the present invention, it is facilitated to move back and forth between both gas and liquid environments using the support 7 while maintaining the three-dimensional shape and / or orientation of the biological sample in the gel. In addition, the biological sample encapsulated in the permeable gel comes into contact with a different liquid in order to interact with the biological sample without being exposed to the gas. In general, when a biological sample is exposed to a gas, the surface of the biological sample becomes dry, deformed by atmospheric pressure, the pH changes to the alkaline side, and the orientation of the biological sample tends to rotate. In addition, if the biological sample is coated or encapsulated with a coating agent to increase the thickness so that it is not exposed to the outside air, careful handling must be performed to prevent the coating agent from peeling off, and the orientation of the biological sample changes. It was difficult to prevent.

Examples of cells include cells derived from vertebrates such as humans, mice, rats, dogs, monkeys, rabbits, goats, cows, horses, pigs and cats, cells derived from invertebrates such as Drosophila and silkworms, and fungi such as yeast and Escherichia coli. , Pluripotent stem cells such as ES cells and iPS cells, mesenchymal stem cells, adipose stem cells, hematopoietic stem cells, nerve stem cells, hepatic stem cells, stem cells such as muscle stem cells and the like. Further, as the biological sample, an abiotic material having fluorescence, light emission, phosphorescence or dye may be adopted.

1 Sample preparation system 3 Containers 5, 5A, 5B, 5C, 5D Adjustable solution tank 7 Support 7b Tip (overhang, non-circular part)
7c through hole 7d groove (recess)
9 Support connector 11 Observation sample A Acrylamide (fixing agent)
C adjustment solution C1 immobilization solution (adjustment solution)
C2 stain solution (adjustment solution)
C3 cleaning solution (adjustment solution)
C4 clearing solution (adjusting solution)
M medium SS spheroid (biological sample)

Claims (17)

1. A medium containing a biological sample and
   A support that holds the medium at the tip is provided.
   An observation sample in which the medium is a gel through which the conditioning solution can permeate, and the conditioning solution is a solution that immobilizes, stains, cleanses or clears the biological sample.
2. At least one of a through hole through which the support intersects in the longitudinal direction, a recess that is recessed in the direction that intersects in the longitudinal direction, and an overhang that projects in the direction that intersects in the longitudinal direction. The observation sample according to claim 1.
3. The observation sample according to claim 1 or 2, wherein the support includes a non-circular portion having a non-circular cross section at which the support intersects in the longitudinal direction of the support.
4. The observation sample according to claim 1, wherein the tip portion has a small diameter so that it can be punctured by the medium and is formed in a hook shape formed by folding back.
5. The observation sample according to any one of claims 1 to 4, wherein the support is made of a material that does not contain a fluorescent substance.
6. A plurality of the supports are connected and
   The observation sample according to any one of claims 1 to 5, wherein the plurality of the media are held by the respective supports.
7. The observation sample according to claim 6, wherein the supports are configured to be separable from each other.
8. The observation sample according to any one of claims 1 to 7, wherein the biological sample is a cell mass.
9. Immerse the tip of the support in a liquid medium contained in a container together with a biological sample.
   By adding an immobilizing agent to the medium, the medium is transformed into a gel that allows the adjusting solution for immobilizing, staining, washing or clearing the biological sample to permeate.
   A method for producing an observation sample in which the tip portion is taken out from the container while the gel-like medium containing the biological sample is held at the tip portion.
10. The observation according to claim 9, wherein the biological sample contained in the medium is subjected to at least one treatment of fixation, staining, washing and clearing while the gelled medium is held by the support. How to make a sample for.
11. The method for producing an observation sample according to claim 10, wherein the biological sample is processed in the order of fixation, staining, washing, and clearing.
12. The method for preparing an observation sample according to claim 10, wherein the medium held by the support is immersed in the adjusting solution.
13. The observation sample according to claim 11, wherein the medium held by the support is sequentially immersed in various adjusting solutions according to the treatment of fixation, staining, washing or clearing for a predetermined time. Manufacturing method.
14. Each of the tips of the supports connected to each other is immersed in each of the liquid media in the container.
    The immobilizing agent is added to each of the media,
    The method for producing an observation sample according to claim 10, wherein the plurality of gelled media are held together by the respective supports, and the plurality of the media are collectively immersed in the adjusting solution.
15. An adjustment solution tank capable of containing an adjustment solution for fixing, staining, washing or clarifying a biological sample,
    A gel-like medium containing the biological sample and allowing the adjustment solution to permeate is provided with a plurality of supports capable of holding the gel-like medium at the tip.
    A plurality of the supports are connected to each other,
    A sample preparation system in which the adjusting solution tank has a size capable of simultaneously immersing a plurality of the media held by each of the supportings in the adjusting solution.
16. A container is provided in which a plurality of wells capable of accommodating the liquid medium together with the biological sample are arranged at a predetermined pitch.
    The sample preparation system according to claim 15, wherein a plurality of the supports are connected to each of the wells of the container at a pitch at which each tip thereof can be inserted.
17. The sample preparation system according to claim 16, further comprising a plurality of the adjusting solution tanks capable of separately accommodating various adjusting solutions according to the fixing, staining, washing or clearing treatment.
    

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