JP2017205060A - Method and apparatus for creating heterogeneous tissue - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method and an apparatus, which can create a heterozygous tissue by locally exposing various tissues to a drug or locally introducing a gene into various tissues.SOLUTION: According to the present invention, a method comprises the steps of: accommodating a target tissue and a first culture solution in an upper accommodating chamber; accommodating a second culture liquid in a lower accommodating chamber positioned below the upper accommodating chamber; and a portion of the target tissue is exposed to the second culture solution from a communicating hole 27 formed in a boundary plate 26 partitioning the upper accommodating chamber and the lower accommodating chamber.SELECTED DRAWING: Figure 1

Description

  The present disclosure relates to a method and apparatus for creating a heterogeneous tissue.

  In recent years, various studies using ES cells and iPS cells have been conducted, and in previous studies, an example in which cells spontaneously form a complex structure has been reported (for example, see Non-Patent Document 1). . In addition, methods and apparatuses for culturing spheroids, which are a kind of massive cells, have also been proposed (see, for example, Patent Documents 1 and 2).

JP 2014-226046 A Japanese Patent Application No. 2014-234097

Eiraku, M .; Takata, N .; Ishibashi, H .; , Kawada, M .; Sakakura, E .; Okuda, S .; , Sekiguchi, K .; , Adachi, T .; , And Sasai, Y. et al. , “Self-organizing optical-cup morphology in three-dimensional culture” Nature 472, pp. 51-56, 2011

  However, in the conventional techniques, it has not been assumed that various cell tissues are exposed to various drugs or genes are introduced into various cell tissues from the outside.

  Here, by solving the problems of the conventional technology, various tissues are exposed to drugs locally, or genes are introduced locally into various tissues, so that heterogeneous tissues can be formed. It is an object to provide a method and apparatus that can be created.

  Therefore, in the method for creating a heterogeneous tissue, the step of storing the target tissue and the first culture solution in the upper storage chamber, and the second storage chamber located below the upper storage chamber, And a step of exposing a part of the target tissue to the second culture medium from a communication hole formed in a boundary plate that partitions the upper storage chamber and the lower storage chamber. ,including.

  In another method for creating a heterogeneous tissue, the second culture solution further contains a drug.

  In yet another method of creating a heterogeneous tissue, the second culture solution is replaced with another culture solution containing another drug.

  In still another heterogeneous tissue preparation method, the second culture solution further contains a gene.

  In another heterogeneous tissue preparation method, the method further includes the step of removing the target tissue from the upper storage chamber after exposing a part of the target tissue to the second culture solution.

  In the heterogeneous tissue creation apparatus, the upper layer portion including the upper storage chamber, the lower layer portion positioned below the upper layer portion and including the lower storage chamber, the upper storage chamber and the lower storage chamber A boundary plate partitioning the chamber, the target tissue and the first culture solution are stored in the upper storage chamber, the second culture solution is stored in the lower storage chamber, and the boundary A communication hole through which the target tissue cannot pass is formed in the plate, and a part of the target tissue is exposed from the communication hole to the lower storage chamber and exposed to the second culture solution.

  In another heterogeneous tissue creation apparatus, the lower storage chamber further includes a first side portion, a central portion, and a second side portion that communicate with each other, and an upper surface of the central portion is the boundary. Covered by a plate, a portion of the target tissue is exposed in the central portion.

  In still another heterogeneous tissue creation apparatus, the upper surfaces of the first side portion and the second side portion are at least partially open, and liquid is applied to one of the first side portion and the second side portion. The culture solution is supplied from the supply device, and the culture solution is discharged from the other side by the solution discharge device.

  In still another heterogeneous tissue creation apparatus, a pair of electrodes for electroporation are further disposed in the first side portion and the second side portion.

  According to the present disclosure, it is possible to create a heterogeneous tissue by locally exposing various tissues to a drug or introducing a gene locally into various tissues.

It is a perspective view which shows the structure of the production apparatus in 1st Embodiment. It is a figure which shows the structure of the lower layer part of the production apparatus in 1st Embodiment. It is a figure which shows the structure of the upper layer part of the production apparatus in 1st Embodiment. It is a photograph which shows the production apparatus actually produced in 1st Embodiment. It is a figure which shows the usage method of the production apparatus in 1st Embodiment. It is sectional drawing explaining the method to make the object structure | tissue in a hetero state in 1st Embodiment. It is the figure and photograph explaining the mode of the object organization fixed on the boundary board in a 1st embodiment. It is a figure explaining deformation | transformation of the object structure | tissue fixed on the boundary board in 1st Embodiment. It is the figure and photograph explaining the structure | tissue of the hetero state produced using the production apparatus in 1st Embodiment. It is a perspective view which shows the usage method of the preparation apparatus in 2nd Embodiment. It is a photograph which shows the structure | tissue of the hetero state produced using the production apparatus in 2nd Embodiment.

  Hereinafter, embodiments will be described in detail with reference to the drawings.

  FIG. 1 is a perspective view showing a configuration of a creation apparatus according to the first embodiment, FIG. 2 is a diagram showing a configuration of a lower layer of the creation apparatus according to the first embodiment, and FIG. 3 is a diagram according to the first embodiment. The figure which shows the structure of the upper layer part of a production apparatus, FIG. 4 is a photograph which shows the production apparatus actually produced in 1st Embodiment. 2 and 3, (a) is a top view, (b) is a perspective view, and in FIG. 4, (a) is a photograph showing a side surface, (b) is a photograph showing a top surface, and (c) is a photograph. It is a photograph which shows the state which arranged the some preparation apparatus in parallel.

  In the figure, reference numeral 1 denotes a creation apparatus according to the present embodiment, which is an apparatus for creating a heterogeneous tissue. Here, the tissue refers to a cellular tissue, such as a spheroid that is a kind of massive cell, a tissue piece that is a part of an animal organ, and the like. In addition, the heterogeneous state is not a homogenous but a heterogeneous state. For example, only a part of the tissue is exposed to various drugs, or various genes are introduced into only a part of the tissue. As a result, the property or character of the part or the part and the vicinity thereof is locally different from the property or character of the other part of the tissue. Examples of the spheroids include spheroids of various cancer cells, spheroids of stem cells such as iPS cells such as bovine iPS cells, human ES cells, human iPS cells, mouse ES cells, and mouse iPS cells, and ES cells. included. The tissue pieces include all tissue pieces such as human liver tissue pieces and mouse brain tissue pieces.

  In the present embodiment, the expressions indicating directions such as upper, lower, left, right, front, rear, etc. used for explaining the configuration and operation of each part of the creating apparatus 1 are not absolute. Relative and appropriate when each part of the creation device 1 has the posture shown in the figure, but when that posture changes, it should be interpreted according to the change in posture It is.

  In the example shown in the figure, the creating apparatus 1 includes a lower layer part 10 and an upper layer part 20 placed on the lower layer part 10. The lower layer portion 10 includes, for example, a lower main body 11 having a substantially rectangular parallelepiped shape, and a lower well 12 as a lower accommodation chamber formed in the lower main body 11. The upper layer portion 20 includes, for example, an upper body 21 having a substantially cylindrical shape and an upper well 22 as an upper storage chamber formed in the upper body 21.

  The lower body 11 is, for example, a member made of polydimethylsiloxane (PDMS) which is a kind of silicone rubber, but may be made of any kind of material, and the shape thereof is also a substantially rectangular parallelepiped shape. It is not limited to, and any shape may be used. In the example shown in the figure, the lower well 12 includes a first side portion 12a, a central portion 12b, and a second side portion 12c that are adjacent to each other. The first side portion 12a, the central portion 12b, and the second side portion 12c are respectively substantially cylindrical cavities extending in the thickness direction of the lower main body 11 (vertical direction in FIG. 1). As shown in 2 (a), the cavities adjacent to each other are formed so as to overlap each other and communicate with each other. The lower surface of the lower well 12 is closed by a bottom plate (not shown), and the upper surface of the lower well 12 is open.

  For example, the upper body 21 is a member made of polydimethylsiloxane (PDMS), which is a kind of silicone rubber, but may be made of any kind of material, and the shape thereof is also substantially cylindrical. It is not limited and any shape may be used. In the example shown in the figure, the upper well 22 is a substantially cylindrical cavity extending in the thickness direction of the upper main body 21 (vertical direction in FIG. 1). The lower surface of the upper well 22 is closed by a boundary plate 26, and the upper surface of the upper well 22 is open. The boundary plate 26 is, for example, a thin film member made of PDMS and having a thickness of about 20 [μm]. However, the boundary plate 26 may be made of any kind of material, and the thickness thereof can be appropriately changed. In the present embodiment, a communication hole 27 penetrating the boundary plate 26 in the thickness direction (vertical direction in FIG. 1) is formed at the center of the boundary plate 26. The diameter of the communication hole 27 is, for example, 100 [μm] or more, but is not limited to this and can be arbitrarily set. The diameter of the communication hole 27 is set such that the culture solution can pass therethrough and the target tissue 31 described later cannot pass through, preferably 100 [μm] to 3 [mm]. More preferably, the thickness is 100 [μm] to 500 [μm].

  When the upper layer portion 20 is placed on the lower layer portion 10, the upper layer portion 20 is positioned immediately above the central portion 12 b of the lower well 12, and at least the upper surface of the central portion 12 b is the lower surface of the upper layer portion 20. Covered by. The upper well 22 and the lower well 12 communicate with each other through the communication hole 27. For example, the outer diameter of the upper layer portion 20 is set larger than the diameter of the central portion 12b, and the diameter of the upper well 22 is set to be approximately equal to the diameter of the central portion 12b.

  4A and 4B are photographs of the production apparatus 1 made of PDMS actually produced. FIG. 4A is a photograph of the side of the production apparatus 1 and FIG. 4B is a photograph of the top surface of the production apparatus 1. When actually creating a heterogeneous tissue, it is desirable to use a plurality of creation devices 1 arranged in parallel as shown in FIG.

  Next, a method for creating a heterogeneous tissue using the creation apparatus 1 will be described. First, an outline of a method for using the creating apparatus 1 will be described.

  FIG. 5 is a diagram illustrating a method of using the creation apparatus according to the first embodiment. In addition, in the figure, (a) is a perspective view, (b) is an AA arrow sectional drawing in (a).

  As shown in the figure, a target tissue 31 as a tissue to be created is accommodated in the upper well 22. As described above, the size of the communication hole 27 formed in the boundary plate 26 is set smaller than the outer dimension of the target tissue 31 so that the target tissue 31 cannot pass through. In the example shown in the figure, the shape of the target tissue 31 is spherical for convenience of drawing, but it should be understood that the actual shape of the target tissue 31 is not necessarily spherical.

  The first culture solution 35A is supplied into the upper well 22, and the second culture solution 35B is supplied into the lower well 12. The first culture solution 35A and the second culture solution 35B do not necessarily have to be different, and may be the same. In addition, when demonstrating the said 1st culture solution 35A and the 2nd culture solution 35B integrally, it demonstrates as the culture solution 35. FIG.

  The first culture solution 35A does not necessarily need to fill the entire upper well 22, and may be an amount that covers the entire target tissue 31. On the other hand, it is desirable that the second culture solution 35B fills the entire lower well 12 and its surface (interface) reaches the boundary plate 26 that closes the lower surface of the upper well 22. Thereby, the part exposed downward from the communicating hole 27 in the target tissue 31 is exposed to the second culture solution 35B.

  Next, a method for using the creating apparatus 1 will be described in detail.

  FIG. 6 is a cross-sectional view for explaining a method for making the target tissue in a heterogeneous state in the first embodiment, and FIG. 7 is a diagram for explaining the state of the target tissue fixed on the boundary plate in the first embodiment. FIG. 8 is a diagram for explaining deformation of the target tissue fixed on the boundary plate in the first embodiment, and FIG. 9 is a heterogeneous state created by using the creation apparatus in the first embodiment. It is the figure and photograph explaining an organization of. 6, (a) to (e) are diagrams showing each step, (f) and (g) are enlarged views of the main part in (e), and in FIG. 7, (a) is the target tissue. FIG. 9B is a photograph showing the upper surface of the target tissue. In FIG. 9, (a) is a sectional view of the tissue, (b) is a photograph of the tissue, and (c) and (d) are fluorescence of the tissue. It is a photograph.

  First, as shown in FIG. 6A, an empty preparation device 1 in which neither the culture solution 35 nor the target tissue 31 is accommodated is prepared.

  Subsequently, the first culture solution 35A is supplied into the upper well 22 of the production apparatus 1. Then, the first culture solution 35A passes through the communication hole 27 formed in the boundary plate 26 and flows into the lower well 12, and as shown in FIG. 6 (b), the lower well 12 To charge. At this time, the upper well 22 contains a certain amount of the first culture solution 35A, but is not filled with the first culture solution 35A.

  Subsequently, as illustrated in FIG. 6C, the target tissue 31 is introduced into the upper well 22 from the upper surface of the upper well 22 using the introduction device 40. In this case, it is desirable to introduce the first culture solution 35 </ b> A together with the target tissue 31 into the upper well 22. Thereby, the first culture solution 35A is replenished into the upper well 22. The introduction device 40 is, for example, a pipette having a diameter large enough to introduce the target tissue 31 immersed in the first culture solution 35A. Any kind of tool can be used.

  Since the target tissue 31 is introduced into the upper well 22 and the first culture solution 35A is replenished, the upper well 22 is also filled with the first culture solution 35A, as shown in FIG. 6 (d). The Then, the liquid level of the first culture solution 35A in the upper well 22 becomes higher than the liquid level of the first culture solution 35A in the lower well 12 as indicated by the arrow H. As indicated by the arrow P, hydraulic pressure acts. As a result, the target tissue 31 is pressed onto the communication hole 27 in the boundary plate 26, and a part thereof is exposed from the communication hole 27 to the central portion 12 b of the lower well 12.

  FIG. 7A shows a case where the target tissue 31 that is pressed onto the communication hole 27 in the boundary plate 26 is a cell spheroid. Most of the spheroid is located in the upper well 22, but a part of the lower part is exposed to the lower well 12 from the communication hole 27. The spheroids cultured in such a state change to a segmental shape as shown in FIG.

  FIG. 7B shows, as an experimental example, a photograph taken from above of a state in which a spheroid as the target tissue 31 is pressed onto the communication hole 27 in the boundary plate 26 of the production device 1 actually produced. It is shown. In the example shown in FIG. 7B, the target tissue 31 is an EB (Embryoid Body) of mouse ES cells, and the diameter of the communication hole 27 is 200 [μm].

  When the culture medium 35 in the lower well 12 is exchanged from the first culture liquid 35A to the second culture liquid 35B, as shown in FIG. 6 (e), the first well 12 in the lower well 12 is replaced. From the upper surface of the side part 12a, the liquid discharger 42 sucks and discharges the first culture solution 35A in the lower well 12, and from the upper surface of the second side part 12c of the lower well 12, the liquid supply device 41 supplies the second culture solution 35B into the lower well 12. Thus, by discharging the first culture solution 35A from one end of the upper surface of the lower well 12 and supplying the second culture solution 35B from the other end of the upper surface of the lower well 12, the lower well 12 is supplied. The inside culture solution 35 can be exchanged from the first culture solution 35A to the second culture solution 35B. The liquid discharge device 42 is, for example, an aspirator, but may be any type of device or tool that can suck and discharge a liquid such as the culture solution 35. The liquid supply device 41 is, for example, a pipette, but may be of any type as long as it is a device or tool capable of supplying a liquid such as the culture solution 35.

  When the culture solution 35 is supplied from the upper surface of the second side portion 12c of the lower well 12 by the liquid supply device 41, as shown in FIG. 6 (f), on the upper surface of the first side portion 12a of the lower well 12 The liquid surface 35s of the culture solution 35 rises like a spherical surface due to surface tension. Therefore, it is desirable that the culture medium 35 is sucked and discharged from the spherical portion by the liquid discharge device 42. Thereby, the liquid level 35s of the culture solution 35 on the upper surface of the first side portion 12a of the lower well 12 becomes substantially flat as shown in FIG. 6 (g).

  In this way, the culture medium 35 in the lower well 12 can be exchanged from the first culture liquid 35A to the second culture liquid 35B without transferring the target tissue 31 into the upper well 22, so that the target The tissue of the tissue 31 is not damaged and there is no risk of contamination.

  Here, if the second culture solution 35B contains, for example, a predetermined drug, a portion exposed downward from the communication hole 27 in the target tissue 31 is locally exposed to the predetermined drug. The Rukoto. As a result, only a part of the target tissue 31 is exposed to a predetermined drug, so that the nature or character of the part or the part and the vicinity thereof is different from other parts. Then, by taking out the target tissue 31 that has become a heterogeneous tissue in this way from the upper well 22 of the creation apparatus 1, the heterogeneous tissue can be easily and accurately observed or inspected. Can do.

  Further, assuming that the first culture solution 35A and the second culture solution 35B contain different drugs, a portion in the upper well 22 and a portion exposed downward from the communication hole 27 in the target tissue 31; Each will be exposed locally to a different drug. Thereby, the said target tissue 31 turns into the structure | tissue of the hetero state from which the property thru | or character of the said parts or the said part and its vicinity part differ mutually by exposing a several part to a different chemical | medical agent. Then, by taking out the target tissue 31 that has become a heterogeneous tissue in this way from the upper well 22 of the creation apparatus 1, the heterogeneous tissue can be easily and accurately observed or inspected. Can do.

  In the present embodiment, as an experiment example, an experiment was performed in which upper and lower portions of a cell spheroid are stained with different fluorescent substances. In FIG. 9A, the target tissue 31 that is pressed onto the communication hole 27 in the boundary plate 26 is a cell spheroid, and the first culture solution 35 </ b> A is supplied into the upper well 22. The case where the second culture solution 35B is supplied into the lower well 12 is shown. The target tissue 31 is an EB of a mouse ES cell.

  In the production apparatus 1 actually produced, the spheroids as the target tissue 31 cultured overnight changed to a segmental shape as shown in FIG. 9A. Subsequently, the culture medium 35 containing Hoechst, which is a fluorescent dye, is used as the first culture liquid 35A in the upper well 22, and the mitochondrial fluorescent staining dye is used as the second culture liquid 35B in the lower well 12 The spheroids were stained using a culture solution 35 containing MitoTracker® Orange. Then, the target tissue 31 that is a stained spheroid was taken out from the upper well 22 of the creating apparatus 1.

  FIG. 9B shows a photograph of the extracted spheroid. It can be seen that it has a segmented shape. FIG. 9C is a fluorescent photograph of the extracted spheroid showing staining with MitoTracker® Orange, and FIG. 9D is a fluorescent photograph of the extracted spheroid showing staining with Hoechst. It can be seen from FIGS. 9B to 9D that each part of the spheroid was stained with a different color.

  Accordingly, when the first culture solution 35A supplied to the upper well 22 and the second culture solution 35B supplied to the lower well 12 contain different drugs, the upper well in the target tissue 31 22 and a portion exposed downward from the communication hole 27 are locally exposed to different drugs, and it is confirmed that the tissue is in a heterogeneous state including a plurality of portions having different properties or traits. It was done.

  Also, a plurality of types of second culture solutions 35B each containing another type of drug are prepared in advance, and the plurality of types of second culture solutions 35B supplied to the lower well 12 are sequentially replaced, whereby a plurality of types of second culture solutions 35B are prepared. The influence of the drug on the target tissue 31 can be observed and examined sequentially. That is, it is possible to efficiently perform drug screening for finding a drug that has an effect on a target tissue 31 composed of a certain type of tissue.

  Furthermore, if the second culture solution 35B contains, for example, a predetermined gene, the gene is locally introduced into a portion exposed downward from the communication hole 27 in the target tissue 31. The Rukoto. As a result, by introducing a predetermined gene into only a part, a tissue in a heterogeneous state in which the nature or character of the part or the part and its vicinity is different from other parts.

  For example, since cancer cells in a living body are generally attached to blood vessels, a portion attached to the blood vessels is first administered to a living body and becomes a drug such as an anticancer agent contained in blood. It is considered that the nature or character of the part exposed to and attached to the blood vessel or the part in the vicinity thereof and the vicinity thereof are different from those of other parts by the drug. Therefore, by taking out the target tissue 31 that has become a heterogeneous tissue as described above from the upper well 22 of the creation apparatus 1, a heterogeneous tissue similar to various tissues in the living body can be observed. Can be inspected.

  Thus, in the present embodiment, the method for creating a heterogeneous tissue is located under the upper well 22 and the step of accommodating the target tissue 31 and the first culture solution 35A in the upper well 22. The second culture solution 35B is accommodated in the lower well 12, and a part of the target tissue 31 is separated from the communication hole 27 formed in the boundary plate 26 that partitions the upper well 22 and the lower well 12. Exposing to the second culture solution 35B. Thereby, the structure | tissue of a hetero state can be created easily and reliably.

  In addition, the heterogeneous tissue creation apparatus 1 includes an upper layer portion 20 including an upper well 22, a lower layer portion 10 including a lower well 12, and an upper well 22 and a lower side. A boundary plate 26 for partitioning the well 12, the target tissue 31 and the first culture solution 35 </ b> A are accommodated in the upper well 22, and the second culture solution 35 </ b> B is contained in the lower well 12. In the boundary plate 26, a communication hole 27 through which the target tissue 31 cannot pass is formed, and a part of the target tissue 31 is exposed to the lower well 12 from the communication hole 27 and exposed to the second culture solution 35B. Is done. Thereby, it is possible to easily and reliably create a heterogeneous tissue with a simple configuration. Then, the created heterogeneous tissue can be taken out from the creation device 1 and accurately observed or inspected.

  Furthermore, the 2nd culture solution 35B contains a chemical | medical agent. Therefore, it is possible to create a tissue locally affected by the drug.

  Furthermore, the second culture solution 35B is replaced with another culture solution containing another drug. Thereby, drug screening can be performed efficiently.

  Further, the lower well 12 includes a first side portion 12a, a central portion 12b, and a second side portion 12c that are in communication with each other. The upper surface of the central portion 12b is covered with a boundary plate 26, and a part of the target tissue 31 is formed. It is exposed at the central portion 12b. Furthermore, at least a part of the upper surfaces of the first side portion 12a and the second side portion 12c is opened, and the culture solution is supplied from the liquid supply device 41 to one of the first side portion 12a and the second side portion 12c. The culture medium is discharged by the liquid discharge device 42. Thereby, the 2nd culture solution 35B can be replaced easily, the structure | tissue of the object structure | tissue 31 is not damaged, and there is no danger of contamination.

  Next, a second embodiment will be described. In addition, about the thing which has the same structure as 1st Embodiment, the description is abbreviate | omitted by providing the same code | symbol. The description of the same operation and the same effect as those of the first embodiment is also omitted.

  FIG. 10 is a perspective view showing a method of using the creating apparatus according to the second embodiment, and FIG. 11 is a photograph showing a heterogeneous tissue created using the creating apparatus according to the second embodiment. In FIG. 11, (a) is a photograph of the tissue, and (b) is a fluorescence photograph of the tissue.

  As shown in FIG. 10, in the present embodiment, the creation apparatus 1 further includes a pair of electrodes 45. Each of the electrodes 45 has a distal end disposed in the first side portion 12a and the second side portion 12c in the lower well 12, and a proximal end thereof is connected to a power source (not shown). Thereby, an electric pulse can be applied to the culture solution 35 in the lower well 12.

  In the present embodiment, as an experimental example, an experiment was performed in which a gene for green fluorescent protein (GFP) was introduced into spheroids of human iPS cells.

  Specifically, a spheroid prepared by culturing human iPS cells (409B2 strain) using an Essential 8 culture solution was used as the target tissue 31. The created target tissue 31 that is a spheroid was transferred to the creating apparatus 1 and housed in the upper well 22 as in the first embodiment. In addition, the Essential 8 culture solution was supplied into the upper well 22 as the first culture solution 35A. Further, in the lower well 12, as the second culture solution 35B, a cell suspension in which the GFP gene is suspended, specifically, a solution in which the cag-GFP gene is added to the Opti-MEM culture solution Was supplied.

  Subsequently, by the pair of electrodes 45, a poreing pulse that pierces the micropores in the cell membrane and a transfer pulse that sends a gene or drug into the cell as an electric pulse to the second culture solution 35 B in the lower well 12. Applied and electroporation, ie electroporation, was performed. The poreing pulse had a voltage of 200 [V], a pulse length of 5 [msec], a pulse interval of 50 [msec], a number of pulses of 2, and an attenuation factor of 10. The transfer pulse had a voltage of 30 [V], a pulse length of 50 [msec], a pulse interval of 50 [msec], a number of pulses of 5, and an attenuation factor of 10. In addition, since electroporation is a well-known method, the description is abbreviate | omitted. As a result, the GFP gene was introduced into a portion of the target tissue 31 exposed downward from the communication hole 27.

  Subsequently, the target tissue 31 into which the GFP gene has been introduced is taken out from the upper well 22 of the production apparatus 1 and transferred to a dish-shaped culture container, and immersed in a medium accommodated in the culture container. Incubated.

  FIG. 11 shows a photograph of a spheroid of human iPS cells as the target tissue 31 that has passed overnight since the start of culture in the culture vessel. FIG. 11A is a photograph of the spheroid of the human iPS cell, and FIG. 11B is a fluorescence photograph of the spheroid of the human iPS cell for observing fluorescence due to the GFP gene.

  From FIG. 11, in the spheroid of the human iPS cell as the target tissue 31, only the part into which the GFP gene is introduced or the part and the vicinity thereof, the property is changed or the character is transformed, It turns out that it is emitting fluorescence.

  Thus, when the second culture solution 35B supplied into the lower well 12 contains a predetermined gene, the portion of the target tissue 31 exposed downward from the communication hole 27 is locally, Thus, the target tissue 31 is in a heterogeneous state in which the nature or character of the part or the part and the vicinity thereof is different from other parts by introducing a predetermined gene into only a part. It was confirmed that it became an organization.

  Thus, in the present embodiment, the second culture solution 35B includes a gene. Therefore, a gene can be locally introduced, and a heterogeneous tissue in which properties are locally changed or transformed can be obtained.

  A pair of electrodes 45 for electroporation are disposed in the first side portion 12a and the second side portion 12c. Therefore, the gene can be reliably introduced into only a part of the target tissue 31.

  As described above, the heterogeneous tissue creation method and heterogeneous tissue creation apparatus 1 in the first and second embodiments are used for culturing various cells, spheroids, tissue fragments, and other tissues. In addition, it is possible to easily and reliably create a heterogeneous tissue similar to various tissues in a living body. Accordingly, the disclosures in the first and second embodiments include the induction of differentiation of stem cells such as iPS cells and ES cells, confirmation of the effects of various drugs on specific sites of cancer cells and other various cells and various tissue fragments, Applicable to a wide range of fields, such as screening for drugs that are effective against specific parts of cancer cells and other various cells and various tissue fragments, and introduction of specific genes into specific regions of various cells and various tissue fragments be able to.

  It should be noted that the disclosure of the present specification describes features related to preferred and exemplary embodiments. Various other embodiments, modifications and variations within the scope and spirit of the claims attached hereto can naturally be conceived by those skilled in the art by reviewing the disclosure of this specification. is there.

  The present invention can be applied to a method and apparatus for creating a heterogeneous tissue.

DESCRIPTION OF SYMBOLS 1 Creation apparatus 10 Lower layer part 12 Lower side well 12a First side part 12b Central part 12c Second side part 20 Upper layer part 22 Upper well 26 Boundary board 27 Communication hole 31 Target tissue 35A 1st culture solution 35B 2nd culture solution 41 Liquid supply device 42 Liquid discharge device 45 Electrode

Claims (9)

Storing the target tissue and the first culture solution in the upper storage chamber;
A second culture solution is stored in a lower storage chamber located below the upper storage chamber, and a part of the target tissue is formed on a boundary plate that partitions the upper storage chamber and the lower storage chamber. Exposing to the second culture solution from the formed communication hole;
A method for creating a heterogeneous tissue, comprising:   The method for creating a heterogeneous tissue according to claim 1, wherein the second culture solution contains a drug.   The method for creating a heterogeneous tissue according to claim 2, wherein the second culture solution is replaced with another culture solution containing another drug.   The method for creating a heterogeneous tissue according to claim 1, wherein the second culture solution contains a gene.   The tissue in a heterogeneous state according to any one of claims 1 to 4, further comprising a step of taking out the target tissue from the upper storage chamber after exposing a part of the target tissue to the second culture solution. How to create An upper layer portion including the upper storage chamber, a lower layer portion including the lower storage chamber, located on the lower side of the upper layer portion, and a boundary plate that partitions the upper storage chamber and the lower storage chamber;
In the upper storage chamber, the target tissue and the first culture solution are stored,
A second culture solution is stored in the lower storage chamber,
The boundary plate is formed with a communication hole through which the target tissue cannot pass,
An apparatus for creating a heterogeneous tissue, wherein a part of the target tissue is exposed to the second culture solution through the communication hole and exposed to the lower accommodation chamber.   The lower storage chamber includes a first side portion, a central portion, and a second side portion that communicate with each other, an upper surface of the central portion is covered with the boundary plate, and a part of the target tissue is located in the central portion. The apparatus for creating a heterogeneous tissue according to claim 6 exposed.   At least a part of the upper surface of the first side part and the second side part is opened, the culture medium is supplied from one liquid supply device to one of the first side part and the second side part, and the culture is performed by the liquid discharge device from the other side. The apparatus for creating a heterogeneous tissue according to claim 7, wherein the liquid is discharged.   The apparatus for creating a heterogeneous tissue according to claim 7, wherein a pair of electrodes for electroporation are disposed in the first side portion and the second side portion.