JP7373833B2 - Three-dimensional biological tissue culturing method, three-dimensional biological tissue culturing device and system - Google Patents

Description

The present invention relates to a method for culturing three-dimensional biological tissue. The present invention also relates to a three-dimensional biological tissue culture device and system method.

In recent years, research has been actively conducted on constructing organs or organ-like tissues called organoids, which are artificially created outside the living body. Organoids are generally created from aggregates of progenitor cells and the like that contribute to organ formation by imitating in vivo development and regeneration processes. Human tissue organoids are not only used to understand the developmental process of organs and organs, but are also expected to be applied to drug discovery research, and organoid induction research for cancer, intestine, liver, kidney, etc. has already been conducted. ing.

Various methods have been developed for culturing organoids, and for example, a method for culturing three-dimensional organoids on a porous membrane of a cell culture insert is known (for example, Non-Patent Document 1). In addition, methods for culturing organoids on microfluidic devices constructed using microfabrication technology have been developed to mimic the vascular network that supplies oxygen and nutrients to the interior of organoids (e.g., Patent Document 2).

Takasato M. , et al. , Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis, Nature. 2015 Oct. 22;526(7574):pp. 564-568. Nashimoto Y. , et al. , Integrating perfusable vascular networks with a three-dimensional tissue in a microfluidic device, Integr. Biol. (Camb). 2017 Jun. 19;9(6):506-518.

Conventional organ culture methods have had the problem that when cultured for a long period of time, the structure of three-dimensional biological tissues (eg, organoids) cannot be maintained, and organoids having a desired shape cannot be obtained. That is, the present invention provides a culture method, a culture device, and a culture system for producing organoids having a desired shape by maintaining the structure of the organoids while improving oxygen permeability.

The present inventors have conducted research and development by considering from various angles. As a result, by a simple method of providing a channel for perfusing the medium at the bottom of the porous membrane and culturing the three-dimensional biological tissue while maintaining the gas-liquid interface, three-dimensional biological tissue (e.g. We have discovered that it is possible to culture the organoids for a long period of time without their shape collapsing. The present invention was completed based on the above findings. That is, the present invention includes the following.

[1] A method for culturing three-dimensional biological tissue, comprising:
A three-dimensional biological tissue placed on one side of a porous membrane through which the components of the medium permeate is exposed to a gas phase, and the medium in contact with the other side of the porous membrane is placed parallel to the porous membrane. culturing the three-dimensional biological tissue while perfusing it with a liquid flow;
including methods.
[2] The method according to [1], wherein the porous membrane has pores that penetrate in the thickness direction of the porous membrane.
[3] The method according to [1] or [2], wherein the porous membrane is a porous membrane of a cell culture insert.
[4] The method according to any one of [1] to [3], wherein the three-dimensional biological tissue is a kidney organoid, a lung organoid, a thymus organoid, or a testis organoid.
[5] The method according to any one of [1] to [4], wherein the three-dimensional biological tissue is a renal organoid.
[6] The method according to any one of [1] to [5], wherein the medium is perfused at a flow rate of 0.01 μL/min or more.

[7] Cell culture insert holding part;
at least one channel provided in a part of the bottom of the cell culture insert holding part;
a culture medium supply pipe for supplying a culture medium to the flow path;
a culture medium discharge pipe for discharging the culture medium from the flow path;
Equipped with
Here, a part of the upper surface of the flow path has a flow top opening, and when the cell culture insert is placed on the cell culture insert holding part, the flow top opening opens the cell culture insert. A three-dimensional biological tissue culture device characterized by being covered with a lower surface of a porous membrane.
[8] The three-dimensional biological tissue culture device according to [7], wherein the cell culture insert holding section has a fitting means for making the cell culture insert adhere to the cell culture insert.
[9] The three-dimensional living tissue culture device according to [8], wherein the fitting means is one or more fitting means selected from the group consisting of a tapered structure and a sealing member.
[10] The three-dimensional biological tissue culture device according to any one of [7] to [9], wherein the flow path is formed so that a laminar flow occurs when the medium is flowed.
[11] The three-dimensional biological tissue culture device according to any one of [7] to [10], wherein the width/height of the channel is 2.0 or more.
[12] The three-dimensional living tissue culture device according to any one of [7] to [11], wherein the height of the flow path is about 0.1 mm to about 5 mm.
[13] The three-dimensional living tissue culture device according to any one of [7] to [12], further comprising a cell culture insert placed on the cell culture insert holding section.

[14] The three-dimensional biological tissue culture device according to [13],
a medium supply line connected to the medium supply pipe;
a medium supply tank that supplies a medium to the medium supply line;
a liquid pump that sends the medium to the medium supply line;
a medium discharge line connected to the medium discharge pipe;
a medium discharge tank that stores the medium discharged from the medium discharge line;
A three-dimensional biological tissue culture system.
[15] The three-dimensional biological tissue culture system according to [14], wherein the medium supply tank and the medium discharge tank are the same, and the medium circulates.

A feature of the present invention is that by using a simple device, three-dimensional living tissue can be cultured while maintaining the gas-liquid interface. Conventionally, three-dimensional biological tissues, such as kidney organoids, have been cultured in the presence of a medium or gel on top, which makes it difficult to maintain the internal epithelial cell structure due to passive penetration of the medium between cells. It was collapsing. According to the present invention, the upper part of the three-dimensional living tissue can be cultured in the gas phase, and oxygen can be sufficiently supplied without random invasion of the medium, and the tissue structure of the three-dimensional living tissue can be maintained during the culture period. becomes possible. Furthermore, the lower part of the three-dimensional biological tissue is in contact with the liquid phase through a porous membrane, and a culture medium is supplied by the reflux of the liquid phase, making it possible to supply nutrients and oxygen and remove waste products. In addition, because the fluid flow stimulation at the bottom of the three-dimensional living tissue gradually attenuates as it moves toward the upper layer of the three-dimensional living tissue, it is similar to the blood flow in the living body, for example, the blood flow supplied to the fetal kidney in the living body. It mimics the conditions inside the womb. This promotes the maturation of the three-dimensional biological tissue, making it possible to obtain a three-dimensional biological tissue that is more highly differentiated than conventional three-dimensional biological tissues.

Moreover, the three-dimensional biological tissue culture device of the present invention can also be used in combination with a commercially available cell culture insert. By appropriately selecting the pore size of the porous membrane of the cell culture insert, it is also possible to control the amount of medium refluxed to the upper perfusion tissue. Furthermore, since perfusion culture is possible using a normal culture dish, it is possible to observe the three-dimensional biological tissue during culture with a commercially available microscope, and observation over time is also possible.

FIG. 1-1 is a diagram illustrating a three-dimensional biological tissue culture device in one embodiment. (A) Top view. (B) Bottom view. FIGS. 1-2 are diagrams illustrating a three-dimensional biological tissue culture device in one embodiment. (A) It is a diagram showing the AA cross section in FIG. 1-1(A). (B) A diagram showing a cross section taken along the line BB in FIG. 1-1(A). FIGS. 1-3 are diagrams illustrating a three-dimensional biological tissue culture device in one embodiment. (A) It is a perspective view (top side) of a three-dimensional biological tissue culture device. (B) It is a perspective view (bottom side) of a three-dimensional biological tissue culture device. FIG. 2-1 is a diagram illustrating a culture device assembly in one embodiment. FIG. 2 is a perspective view showing a culture device assembly that combines a cell culture insert, a three-dimensional biological tissue culture device, and a culture container. FIG. 2-2 is a diagram illustrating a culture device assembly in one embodiment. (A) FIG. 1-2 (A) shows a cross-sectional view of a culture device assembly that combines a cell culture insert and a culture container. (B) FIG. 1-2 (B) shows a cross-sectional view of a culture device assembly that combines a cell culture insert and a culture container. FIG. 3 is a schematic diagram showing how a part of the three-dimensional biological tissue culture system (culture device assembly) is used in one embodiment. FIG. 4-1 is a diagram illustrating a three-dimensional biological tissue culture device in one embodiment. (A) Top view. (B) Bottom view. FIG. 4-2 is a diagram illustrating a three-dimensional biological tissue culture device in one embodiment. (A) A diagram showing a cross section taken along the line CC in FIG. 4-1(A). (B) It is a diagram showing the DD cross section of FIG. 4-1(A). FIG. 5 is a diagram showing a three-dimensional biological tissue culture system in one embodiment. The culture device assembly is shown in cross-section. FIG. 6 is a diagram illustrating a three-dimensional biological tissue culture system in one embodiment. The culture device assembly is shown in cross-section. FIG. 7 is a diagram showing a three-dimensional biological tissue culture system and perfusion conditions in one embodiment. (A) It is a schematic diagram which shows the usage aspect of a part (culture device assembly) of a three-dimensional biological tissue culture system. (B) is a graph showing the results of a perfusion test using fluorescent beads. The relationship between the mobility of fluorescent beads in the channel (F1 in (A)) and the pump flow rate is shown. (C) A graph showing the results of a perfusion test using fluorescent beads. The relationship between the fluorescent bead movement speed within the cell culture insert (F2 in (A)) and the pump flow rate (μL/sec) is shown. FIG. 8 is a diagram showing the induction of renal organoids from iPS cells. (A) Shows the induction conditions for kidney organoids. (B) Macroscopic image of kidney organoids at day 18 of induction (12 days after 3D) in a 12-well cell culture insert. (C) Shows the expression of the NPHS1 gene (nephrin expression marker) over time in renal organoids. Figure 9 shows the results of perfusion culture of renal organoids. The dynamics of fluorescent lectin (red: Alexa 649 Tomato lectin) when perfused at a pump flow rate of 10 μL/min is shown. FIG. 10 is a diagram showing changes in ureteric bud tissue and induced tubule structure within renal organoids due to perfusion. FIG. 2 shows frozen section stained images of kidney organoids cultured for 18 to 21 days after induction. (A) and (B): static culture group (control), (C) and (D): 2.5 μL/min perfusion culture group, (E) and (F): 10 μL/min perfusion culture group. (A), (C) and (E) show red: ureteroblasts (CK8), green: developing tubules (Podocalyxin). (B), (D), and (F) are images obtained by superimposing cell nucleus staining images on the images of (A), (C), and (E). Figure 11 shows changes in gene expression within renal organoids due to perfusion. It is a graph showing changes in PAX8 gene expression in kidney organoids after 3 days of culture. Figure 12 shows luminal expansion of ureteroblasts upon perfusion. Showing ureteroblast structures within renal organoids after 3 days of culture at (A) static culture, (B) 2.5 μL/min, (C) 10 μL/min (red: ureteroblasts (CK8), green : Developmental tubule (podocalyxin), blue: cell nucleus). Figure 13 shows kidney organoids cultured in immersion. Upper row: Schematic diagram of the culture method, middle row: organoids after culture (arrowhead), lower row: frozen section stained image after culture (blue: cell nucleus, red: ureteroblast, green: proximal nephron). FIG. 14 shows a schematic diagram of the culture environment in the three-dimensional biological tissue culture system of one embodiment. FIG. 15 shows the analysis results of cancer cell invasion and metastasis using kidney organoids transplanted with cancer cells (G401 cells). HE-stained images of tissue sections of renal organoids transplanted with collagen gel-embedded cancer cells (G401 cells) after (A) 7 days of static culture or (B) 7 days of perfusion culture are shown. FIG. 16 shows the analysis results of cancer cell invasion and metastasis using kidney organoids transplanted with cancer cells (G401 cells). A suspension containing cancer cells (G401 cells) was transplanted dropwise into kidney organoids, and then cultured under perfusion. The cells contained in the medium after perfusion were collected and cultured separately, and the amount of MMP in the supernatant was measured by ELISA.

Embodiments of the present invention will be described below with reference to the drawings as necessary. The configuration of the embodiment is an example, and the configuration of the present invention is not limited to the specific configuration of the embodiment.

I. Three-dimensional biological tissue As used herein, "three-dimensional biological tissue" refers to a three-dimensional structure containing cells, such as a biological tissue isolated from a living body (for example, an organ and tissue or a part thereof). For example, skin tissue (e.g. skin tissue associated with hair roots, etc.), cardiac muscle tissue, skeletal muscle tissue, smooth muscle tissue, liver tissue, kidney tissue, gastrointestinal tissue, eye tissue (e.g. corneal tissue), brain tissue, thymus tissue, testis. Three-dimensional cell structures, such as organoids, reconstructed using cells constituting tissue, pancreatic tissue, thyroid tissue, mammary gland tissue, salivary gland tissue, lung tissue, etc.) or biological tissue can be applied to the present invention. The biological tissue isolated from a living body that can be applied to the present invention may be the living tissue itself collected from the living body, or a piece of tissue obtained by processing the living tissue collected from the living body into an arbitrary shape. Furthermore, the three-dimensional cell structure applicable to the present invention may be a three-dimensional cell structure formed by mixing a suspension containing cells with a gel solution or a gelling agent. The three-dimensional cell structure that can be applied to the present invention may be a three-dimensional cell structure formed by stacking a plurality of cell sheets. It may also be a three-dimensional cell structure.

As used herein, the term "organoid" refers to a tissue that is three-dimensionally constructed in vitro from a collection of organs or progenitor cells that contribute to the formation of organs. It refers to something synonymous with "organoid," which is interpreted in the broadest sense by manufacturers. Organoids are generally smaller and have a simpler structure than actual organs or organs, but exhibit characteristics anatomically and functionally similar to organs or organs existing in living bodies. Examples of organoids include kidney organoids, liver organoids, gastrointestinal organoids (e.g., intestinal organoids, oral organoids, stomach organoids, etc.), optic cup organoids, brain organoids, thymus organoids, testicular organoids, pancreatic organoids, epithelial organoids, and thyroid organoids. , mammary gland organoids, salivary gland organoids, lung organoids, etc. These organoids also include "cancer organoids" containing cancer cells. Furthermore, in this specification, organoids may include organs and tissues collected from living bodies, or parts thereof (for example, tissue pieces) (for example, see Shamir ER., Nat. Rev. Mol. Cell Biol. 2014 Oct; 15(10):647-64).

Known methods may be used to obtain three-dimensional biological tissue, such as organoids, which can be applied to the present invention, and are not limited to the following methods. For example, kidney organoids can be obtained by Takasato M. , et al. , Nature. 2015, Oct. 22,526(7574), pp. 564-568; lung organoids were prepared by Unbekandt M. et al. , Kidney Int. , 2010, Mar. , 77(5), pp. 407-416; thymus organoids were prepared by Sheridan JM. , et al. , Genesis, 2009 May, 47(5), pp. 346-351; testicular organoids were obtained by Sanjo H. , et al. , PLoS One. 2018, Feb 12, 13(2), e0192884, etc. may be referred to. In addition, any organoid obtained by a method for forming organoids can also be used. The three-dimensional biological tissue applicable to the present invention may be any three-dimensional biological tissue that can be cultured on a porous membrane, and is preferably applicable to, for example, kidney organoids, lung organoids, thymus organoids, and testis organoids. is a renal organoid.

Three-dimensional biological tissues applicable to the present invention include, for example, those of mammals, birds, amphibians, reptiles, and fish. Preferably, it is a three-dimensional biological tissue of a mammal, and contains cells derived from a mammal such as a mouse, rat, human, monkey, pig, dog, sheep, cat, or goat.

The cells contained in the three-dimensional biological tissue applicable to the present invention may be primary cells collected from the biological tissue, established cell lines, pluripotent stem cells or tissue stem cells (e.g. , mesenchymal stem cells).

As used herein, "pluripotent stem cells" is intended to collectively refer to stem cells that have the ability to differentiate into cells of any tissue (pluripotency). Pluripotent stem cells include, but are not limited to, embryonic stem cells (ES cells), embryonic carcinoma cells (EC cells), trophoblast stem cells (TS cells), epiblast stem cell (EpiS cell), embryonic germ cell (EG cell), multipotent germline stem cell (mGS cell), induced pluripotent stem cell (induced p luripotent stem cell : iPS cells), Muse cells, etc. Any known pluripotent stem cells can be used, and for example, pluripotent stem cells described in International Publication No. 2009/123349 can be used.

Known methods can be used to differentiate tissue stem cells and pluripotent cells into cells that construct arbitrary three-dimensional biological tissues (eg, organoids). For example, but not limited to, cells that construct kidney organoids are described by Takasato M. , et al. , Nature. 2015, Oct. 22,526(7574), pp. 564-568; Cells that construct liver organoids were described by Takebe T. , et al. , Nature, 2013, Jul, 25, 499 (7459), pp. 481-484; Cells constructing gastrointestinal organoids were developed by Sato T. , et al. , Nature, 2009, May 14, 459 (7244), pp. 262-265; Cells that construct optic cup organoids are described by Eiraku M. , et al. , Nature, 2011, April 7, 472 (7341) pp. 51-56; Cells that construct brain organoids are described by Eiraku M. , et al. , Cell Stem Cell, 2008, Nov 6, 3(5), pp. 519-532 and Lancaster MA. , et al. , Nature, 2013, Sep 19, 501 (7467), pp. 373-379, cells constructing lung organoids were developed by Yamamoto Y. , et al. , Nat Methods. 2017 Nov, 14(11), pp. 1097-1106; Cells for constructing pancreatic organoids were obtained from Raikwar SP. , et al. , PLoS One. 2015 Jan 28, 10(1), e0116582; Cells that construct thyroid organoids were developed by Ma R. , et al. , Front Endocrinol (Lausanne). 2015 Apr 22, 6, 56. etc. can be referred to. In addition, any cells obtained by a method of differentiating into cells that construct three-dimensional biological tissues can also be used.

II. Method for culturing three-dimensional biological tissue In one embodiment, the method of the invention includes the following steps:
A three-dimensional biological tissue placed on one side of a porous membrane through which the components of the medium permeate is exposed to a gas phase, and the medium in contact with the other side of the porous membrane is placed parallel to the porous membrane. A step of culturing the three-dimensional living tissue while perfusing it with a liquid flow. In this specification, "a porous membrane through which components of a medium permeate" refers to pores through which components necessary for cell culture, such as components constituting a medium, such as water, components in a buffer solution, and proteins, can pass through. A porous membrane with

By implementing the method of the present invention, sufficient oxygen is supplied from the gas phase to the three-dimensional living tissue placed on the porous membrane, thereby preventing ischemia. Furthermore, a liquid flow is generated on the surface that contacts the three-dimensional biological tissue due to the medium that permeates from the surface opposite to the surface that contacts the three-dimensional biological tissue. The liquid flow generated on the surface that comes into contact with the 3D living tissue is an extremely slow flow, so the factors necessary for differentiation secreted by the cells that make up the 3D living tissue are not washed away, and the liquid flows around the 3D living tissue. It will remain in the Thereby, the medium containing nutrients is sufficiently supplied to the three-dimensional biological tissue. For example, when the present invention is applied to kidney organoids, the structure of the organoids is prevented from collapsing, and the maturation of the kidney organoids is also promoted. In one embodiment, the flow of medium is any unidirectional flow, for example parallel to the porous membrane. Any unidirectional flow parallel to the porous membrane may change the direction of liquid flow over time. Thereby, the direction of the liquid flow generated on the surface of the porous membrane on which the three-dimensional biological tissue is placed can also be changed.

In one embodiment, the porous membrane used in the present invention preferably has pores penetrating in the thickness direction of the porous membrane, and more preferably is a porous membrane of a cell culture insert. A commercially available cell culture insert can be used as the cell culture insert. The average pore diameter of the pores of the porous membrane used in the present invention can be appropriately selected depending on the type, size, purpose, etc. of the three-dimensional biological tissue to be cultured, and is, for example, 0.01 μm to 100 μm. It has an average pore size (eg, 0.01 μm to 50 μm, 0.01 μm to 10 μm, 0.1 μm to 50 μm, preferably 0.1 μm to 10 μm). If the average pore diameter of the porous membrane is 0.1 μm to 10 μm, cells contained in the three-dimensional biological tissue will migrate from one surface of the porous membrane with which the three-dimensional biological tissue is in contact to the other surface of the porous membrane. is prevented, which is preferable. Furthermore, the pore density of the porous membrane used in the present invention can be appropriately selected depending on the type, size, purpose, etc. of the three-dimensional biological tissue to be cultured, and is, for example, 1×10 ⁵ /cm. ² or more, preferably 5×10 ⁵ /cm ² or more, more preferably 10×10 ⁵ /cm ² or more, even more preferably 50×10 ⁵ /cm ² or more, most preferably 100×10 ⁵ /cm ² or more. Porous membranes with a density of pores can be used. The average thickness of the porous membrane used in the present invention is, for example, 5 μm to 500 μm, 5 μm to 100 μm, or 5 μm to 100 μm. Examples of the material for the porous membrane used in the present invention include, but are not limited to, polycarbonate, polystyrene, polydimethylsiloxane, silicone polycarbonate, and polyethylene terephthalate.

In one embodiment, in the method of the present invention, the medium brought into contact with the porous membrane is perfused at a flow rate of 0.01 μL/min or more. For example, the flow rate of the medium is 0.01 μL/min or more, 0.05 μL/min or more, 0.1 μL/min or more, 0.5 μL/min or more, 1.0 μL/min or more, or 2.0 μL/min or more. There may be. Further, the flow rate is, for example, 0.01 μL/min to 10 mL/min, 0.1 μL/min to 10 mL/min, 1.0 μL/min to 10 mL/min, 2.0 μL/min to 10 mL/min, 0.01 μL /min~2mL/min, 0.1μL/min~2mL/min, 1.0μL/min~2mL/min, 2.0μL/min~2mL/min, 0.01μL/min~500μL/min, 0.1μL /min~500μL/min, 1.0μL/min~500μL/min, 2.0μL/min~500μL/min, 0.01μL/min~100μL/min, 0.1μL/min~100μL/min, 1.0μL/min /min~100μL/min, 2.0μL/min~100μL/min, 0.01μL/min~20μL/min, 0.1μL/min~20μL/min, 1.0μL/min~20μL/min, 2.0μL/min /min~20μL/min, 0.01μL/min~12.5μL/min, 0.1μL/min~12.5μL/min, 1.0μL/min~12.5μL/min, 2.0μL/min~12 .5 μL/min, 0.01 μL/min to 8.0 μL/min, 0.1 μL/min to 8.0 μL/min, 1.0 μL/min to 8.0 μL/min, 2.0 μL/min to 8.0 μL /minute may be sufficient. The medium brought into contact with the porous membrane is preferably refluxed to form a laminar flow.

In one embodiment, the method of the invention can be applied to lung organoids, thymic organoids or testicular organoids, preferably renal organoids.

III. Three-Dimensional Biological Tissue Culture Device FIG. 1 (FIGS. 1-1-3) shows a three-dimensional biological tissue culture device 10 in one embodiment. The three-dimensional living tissue culture device 10 includes a cell culture insert holding section 11, at least one channel 12 provided in a part of the bottom 110 of the cell culture insert holding section 11, and a structure for supplying a medium to the channel 12. A culture medium supply pipe 13 and a culture medium discharge pipe 14 for discharging the culture medium from the flow path 12 are provided.

As shown in FIG. 2, the cell culture insert holding part 11 is designed to hold the cell culture insert 20, and is defined by an inner wall 111, which is a part of the main body part 15, and a bottom part 110. Cell culture insert 20 is placed on bottom 110. The shape and size of the cell culture insert holding section 11 are selected depending on the shape of the cell culture insert 20 to be placed. For example, as shown in FIG. 2-1(A), when the cell culture insert 20 has a truncated conical shape, the inner wall 111 of the main body 15 is fitted so that the cell culture insert main body 21 is fitted tightly. As a coupling means, a tapered structure may be provided. Thereby, the cell culture insert 20 can be brought into close contact with the cell culture insert holding part 11. Moreover, the cell culture insert holding part 11 can also bring the cell culture insert 20 into close contact with the cell culture insert 20 by having a sealing member, for example, an O-ring, as a fitting means. When the cell culture insert 20 is, for example, cylindrical or prismatic, the inner wall 111 of the cell culture insert holding part 11 also has a cylindrical or prismatic shape so that they fit tightly together.

At least one channel 12 is formed in a portion of the bottom portion 110 . A culture medium supply pipe 13 and a culture medium discharge pipe 14 provided in the main body 15 are in fluid communication with the flow path 12 . In one embodiment, the medium supply pipe 13 and the medium discharge pipe 14 are provided approximately perpendicularly to the main body upper surface 150 of the main body 15, as shown in FIG. 2-1(A). There is. Thereby, when the three-dimensional biological tissue culture device 10 is installed in the culture container 30, the culture medium can be supplied and discharged from the top of the culture container 30. However, the positions and shapes of the culture medium supply pipe 13 and the culture medium discharge pipe 14 are not particularly limited because they can be changed as appropriate depending on the shape of the culture container 30 to which they are applied, the place where they are installed, etc.

As shown in FIGS. 1 and 2, a portion of the upper surface of the flow path 12 has a flow upper opening 121. As shown in FIGS. The upper flow opening 121 is formed in a part of the bottom 110 of the cell culture insert holding part 11. When the cell culture insert 20 is placed on the cell culture insert holding part 11 , the upper flow opening 121 is covered by the lower surface of the porous membrane 22 of the cell culture insert 20 . Thereby, the culture medium flowing through the channel 12 passes directly under the porous membrane 22.

In one embodiment, the channel 12 has a channel bottom opening 122 (see FIGS. 1-1(B) and 1-2). When the main body bottom surface 151 of the main body section 15 comes into close contact with the culture container bottom surface 31 of the culture container 30, the culture container bottom surface 31 becomes the bottom surface of the flow path 12. Since the three-dimensional biological tissue culturing device 10 has the channel bottom opening 122, it becomes possible to observe the three-dimensional biological tissue using, for example, an inverted microscope while culturing the three-dimensional biological tissue. Furthermore, when manufacturing the three-dimensional biological tissue culture device 10, it is also possible to manufacture it as one part using, for example, a 3D printer or injection molding, which simplifies the manufacturing process.

In one embodiment, when the culture container 30 has a truncated cone shape, the main body portion 15 may have, for example, a tapered structure as a fitting means so as to closely fit into the culture container 30. Thereby, the three-dimensional biological tissue culture device 10 can be brought into close contact with the culture container 30. Moreover, the main body part 15 can also be brought into close contact with the inner wall of the culture container 30 by having a sealing member, for example, an O-ring, as a fitting means. When the inner wall of the culture container 30 is, for example, cylindrical or prismatic, the main body 15 also has a cylindrical or prismatic shape so that they can fit tightly together.

ここで、Ｕ：代表流速（円管の場合、平均流速）、Ｌ：代表長さ（円管の場合、内径）、ν：動粘性係数、を表す。 The flow path 12 is designed so that a laminar flow is expected when the culture medium flows. Generally, whether a fluid will be laminar or turbulent can be predicted by calculating the Reynolds number. The Reynolds number can be determined by the following formula.

Here, U: representative flow velocity (in the case of a circular pipe, average flow velocity), L: representative length (inner diameter, in the case of a circular pipe), and ν: kinematic viscosity coefficient.

Generally, when the flow path is a circular pipe, if the Reynolds number (Re) is less than 2300, it is determined that the flow is laminar. As in one embodiment, when the cross section of the flow path 12 (the cross section perpendicular to the flow direction of the medium) is rectangular, the fluid average depth m=A/S (A: cross-sectional area, S: perimeter of the cross-section) For approximation, the Reynolds number can be calculated by substituting the representative length (L)=4 m, that is, "L=4×height of the flow path" into the above equation. By calculating the Reynolds number, it becomes possible to appropriately design the flow path 12 that is expected to provide laminar flow.

Therefore, the height of the flow path 12 for laminar flow is not limited because it varies depending on the flow rate and kinematic viscosity coefficient of the medium used, but for example, when perfusing a commonly used medium, the height of the flow path 12 is Laminar flow is expected if the height is 0.1 mm to 5.0 mm, 0.5 mm to 3.0 mm, 0.5 mm to 2.0 mm, or 1.0 mm to 2.0 mm. When used for observing three-dimensional living tissues during culture, the height of the channel 12 is preferably 2.0 mm or less, for example, 0.1 mm to 2.0 mm.

FIG. 3 is a schematic diagram showing a state in which a culture medium is perfused through the channel 12 in the culture device assembly 4 which is a combination of the three-dimensional biological tissue culture device 10, the cell culture insert 20, and the culture container 30. As described above, since the medium flows in the flow path 12 in a laminar flow, it can be assumed that the amount of the medium passing through the porous membrane 22 is sufficiently small. When the width of the flow path 12 is wider than the height of the flow path 12, for example, the "width/height" (i.e., aspect ratio) of the flow path 12 is 2.0 or more, preferably 3.0 or more. , more preferably 3.5 or more, still more preferably 4.0 or more (for example, 4.5 or more, 5.0 or more, 10.0 or more), the flow of the medium is Approximately to a plane Poiseuille flow, a slight pressure gradient occurs from the channel inlet 123 to the channel outlet 124. That is, in the vicinity of the channel inlet 123, a microcurrent flows through the porous membrane into the cell culture insert 20, and in the vicinity of the channel outlet 124, a microcurrent flows in the direction of passing through the porous membrane and being discharged from the cell culture insert 20. A flow occurs. This causes an extremely gentle flow of the medium around the three-dimensional biological tissue OG placed on the porous membrane (see FIG. 3). As a result, the three-dimensional biological tissue OG is supplied with a medium containing nutrients in which the factors necessary for differentiation secreted by the cells constituting the three-dimensional biological tissue remain without being washed away.

FIG. 4 (FIGS. 4-1 and 4-2) shows a three-dimensional biological tissue culture device 10a that is another embodiment. The description of each member having the same code number as each member provided in the three-dimensional biological tissue culture device 10, except that "a" is added to the code number of each member, is as follows. The above description for each corresponding member of the biological tissue culture device 10 applies. Here, only the members to which the description of each member of the three-dimensional biological tissue culture device 10 does not apply will be described.

The three-dimensional living tissue culture device 10a is provided with a culture medium supply pipe 13a and a culture medium discharge pipe 14a on the side surface of the main body 15a. The culture medium supply pipe 13a and the culture medium discharge pipe 14a are each in fluid communication with the flow path 12a. Unlike the three-dimensional living tissue culture device 10, the channel 12a does not have a channel bottom opening 122 (see FIGS. 1-1(B) and 1-2). Therefore, the three-dimensional biological tissue culture device 10a does not need to be used together with the culture container 30. Since the main body portion 15a is not used together with the culture container 30, it may have any shape.

In other embodiments, the three-dimensional biological tissue culture device (10, 10a) may be provided with a cell culture insert 20 placed on the cell culture insert holding section (11, 11a). .

IV. Three-dimensional biological tissue culture system FIG. 5 shows a three-dimensional biological tissue culture system 1 in one embodiment. The three-dimensional biological tissue culture system 1 is connected to a three-dimensional biological tissue culture device 10, a cell culture insert 20 placed on a cell culture insert holding part 11 of the three-dimensional biological tissue culture device 10, and a medium supply pipe 13. a culture medium supply line 17 that supplies the culture medium to the culture medium supply line 17 , a liquid supply pump 19 that supplies the culture medium to the culture medium supply line 17 , a culture medium discharge line 18 connected to the culture medium discharge pipe 14 , A culture medium discharge tank (corresponding to the culture medium supply tank 5 in FIG. 5) that stores the culture medium discharged from the culture medium discharge line 18 is provided. The medium supply line 17 and the medium supply pipe 13 and the medium discharge line 18 and the medium discharge pipe 14 are connected by an adapter 16.

As shown in FIG. 5, in the three-dimensional biological tissue culture system 1, the medium supply tank 5 and the medium discharge tank may be the same, and the medium may be circulated. may be provided separately and always provided with fresh medium. The liquid feeding pump 19 can be any known pump, and may be a tube pump (peristaltic pump) or a piezo pump, and any pump that can pump fluid can be used. can. In another embodiment, a syringe pump in which the liquid feeding pump 19 and the culture medium supply tank 5 are integrated may be used.

As shown in FIG. 6, in another embodiment, the three-dimensional biological tissue culture system 1a includes two or more three-dimensional biological tissue culture devices 10 (corresponding to the culture device assembly 4 in FIG. 6) connected by a connection line 180. They may be connected. By connecting two or more three-dimensional biological tissue culture devices 10, for example, it becomes possible to apply the same or different three-dimensional biological tissues to each cell culture insert 20 and perform perfusion culture in which the culture medium is shared. . This makes it possible to observe phenomena that occur when three-dimensional biological tissues cultured at separate locations share a medium. For example, arbitrary cells (e.g., cancer cells, mesenchymal stem cells, bone marrow cells, etc.) are transplanted into a three-dimensional biological tissue on a cell culture insert 20 installed in one three-dimensional biological tissue culture device 10, Thereafter, by performing perfusion culture, it is possible to analyze the phenomenon in which the transplanted cells infiltrate and metastasize to the three-dimensional biological tissue on the cell culture insert 20 installed in another three-dimensional biological tissue culture device 10.

The three-dimensional biological tissue culture device (10, 10a) used in the three-dimensional biological tissue culture system (1, 1a) may be provided as a kit, for example, the three-dimensional biological tissue culture device (10, 10a) and , the cell culture insert 20 may be included in the kit, or the three-dimensional biological tissue culture device (10, 10a) and the cell culture insert 20 may be provided separately. A commercially available cell culture insert 20 can be used, and the average pore diameter of the porous membrane 22 of the cell culture insert 20 used varies depending on the type, size, purpose, etc. of the three-dimensional biological tissue to be cultured. , can be selected as appropriate, for example, porous having an average pore diameter of 0.01 μm to 100 μm (for example, 0.01 μm to 50 μm, 0.01 μm to 10 μm, 0.1 μm to 50 μm, preferably 0.1 μm to 10 μm). A cell culture insert 20 with a membrane can be used. If the average pore diameter of the porous membrane 22 is 0.1 μm to 10 μm, cells contained in the three-dimensional living tissue are prevented from moving to the lower surface of the porous membrane 22, which is preferable. Further, the density of pores in the porous membrane 22 of the cell culture insert 20 used can be appropriately selected depending on the type, size, purpose, etc. of the three-dimensional biological tissue to be cultured. From the viewpoint of medium exchange efficiency inside and outside the porous membrane 22, the density is 1×10 ⁵ /cm ² or more, preferably 5×10 ⁵ /cm ² or more, more preferably 10×10 ⁵ /cm ² or more, and even more preferably 50× A cell culture insert 20 comprising a porous membrane 22 having a pore density of 10 ⁵ /cm ² or more, most preferably 100×10 ⁵ /cm ² or more can be used. Further, the average thickness of the porous membrane 22 of the cell culture insert 20 is, for example, 5 μm to 500 μm, 5 μm to 100 μm, or 5 μm to 50 μm. Furthermore, examples of the material for the porous membrane 22 of the cell culture insert 20 include, but are not limited to, polycarbonate, polystyrene, polydimethylsiloxane, silicone polycarbonate, and polyethylene terephthalate.

The culture container 30 applied to the three-dimensional biological tissue culture device 10 can be appropriately selected depending on the shape of the main body 15, and can be selected from commercially available culture containers (for example, 6-well culture dish, 12-well culture dish, 35 mm culture dish). dish, 100 mm culture dish, etc.) may be used.

By using the method for culturing three-dimensional biological tissue and the three-dimensional biological tissue culture device and system of the present invention, it becomes possible to maintain and culture three-dimensional biological tissue, which has been difficult to maintain and culture in the past. Furthermore, by using the three-dimensional biological tissue culture device and system of the present invention, it is also possible to screen new therapeutic agents, perform toxicity tests, and the like.

EXAMPLES The present invention will be explained in more detail below based on examples, but these are not intended to limit the invention in any way.

(Example 1)
1. Materials and experimental methods 1-1. Fabrication of a three-dimensional biological tissue culture device The three-dimensional biological tissue culture device was manufactured using a 3D printer (Stratasys object Eden series, resin MED610) based on 3D CAD design data, and the support material was dissolved in sodium hydroxide. It was made by

Specifically, the cell culture insert holding part of the three-dimensional biological tissue culture device was designed to have a diameter of 14.2 mm so that a 12-well cell culture insert (manufactured by Falcon) could be placed there, and the width of the flow path was 5 mm. The height of the channel was designed to be 1 mm (see Figure 1-3). Furthermore, the main body of the three-dimensional biological tissue culture device was designed to be able to be installed in a 6-well plate. The Reynolds number was calculated for the medium flowing through the flow path of the three-dimensional biological tissue culture device thus designed.

Assuming that the viscosity of the medium is 0.6 mPa·S and the density of the medium is 1000 kg/m ³ , the kinematic viscosity coefficient ν=0.6 mm ² /s. Since the width of the flow path is 5 mm and the height of the flow path is 1 mm, flow rate Q = 5 mm x 1 mm x representative flow velocity U, so U = Q/5 mm ² . Since the representative length (L)=4×fluid average depth, L=4×1 mm=4 mm. From this, the Reynolds number can be calculated as follows. Re = UL/ν=(Q/5mm ² )×(4mm)/(0.6mm ² /s) ≒ Q×1.33s/μL

Assuming that the flow rate Q=25 μL/m (ie, Q≈0.42 μL/s), Re≈(0.42 μL/s)×(1.33 s/μL) ≈0.56. That is, since the Reynolds number is much smaller than 2300, the liquid flow of the medium can be expected to be laminar.

式中、ｘ：流路長手方向座標、ｙ：高さ方向座標、ｕ：流速、Ｕ_ｍａｘ：最大流速、Ｕ_ｍｅａｎ：平均流速、Ｑ：単位奥行き当たりの流量、ｐ：圧力、ρ：密度、ｈ：流路の高さ、ν：動粘性係数、を示す。 The flow channel of the three-dimensional biological tissue culture device is a rectangular channel that is sufficiently wide relative to its height (width: 5 mm, height: 1 mm), so the flow of viscous fluid at the center of the channel is , which approximates a planar Poiseuille flow. As mentioned above, the flow of the medium is a laminar flow, and assuming that the amount of liquid passing through the porous membrane covering the opening of the channel is sufficiently small, the flow velocity will have a distribution approximated by the following formula: .

In the formula, x: channel longitudinal coordinate, y: height direction coordinate, u: flow velocity, U _max : maximum flow velocity, U _mean : average flow velocity, Q: flow rate per unit depth, p: pressure, ρ: density, h: Height of the flow path, ν: Kinematic viscosity coefficient.

Transforming the above formula (3), we get
dp/dx = -U _max × (8ρν)/h ² (...(6))
becomes.
Furthermore, from equation (5), equation (6) becomes
dp/dx = -3/2U _mean × (8ρν)/h ² (...(7))
It can be transformed into Since ρν is the viscosity, ρν = 0.6 mPa・S, and by substituting U _mean = Q/5mm ² = (0.42 μL/s)/5mm ² and h = 1 mm into equation (7), The following values are calculated.
dp/dx = -3/2U _mean × (8ρν)/h ² = -3/2 ((0.42μL/s)/5mm ² ) × (8×0.6mPa・S)/(1mm) ² = - 0.6mPa/mm

Therefore, a minute pressure gradient of -0.6 mPa/mm is generated from the channel inlet to the channel outlet with a channel length of 14.2 mm (corresponding to the diameter of the cell culture insert holding part) of the three-dimensional biological tissue culture device. As a result, near the flow path inlet, a microflow is created in the direction of flowing through the porous membrane to the cell culture insert, and near the flow path exit, a microflow is created in the direction of exiting from the cell culture insert through the porous membrane. , a gentle flow of medium occurs in the three-dimensional biological tissue placed on the porous membrane.

1-2. Assembly of Three-Dimensional Biological Tissue Culture System For the three-dimensional biological tissue culture device, holes for inserting a medium supply pipe and a medium discharge pipe were prepared in the lid of a 6-well insert plate (Falcon), and the device was sterilized. The three-dimensional biological tissue culture device was placed in a 6-well plate, and a 0.4 μm pore or 3.0 μm, 12-well cell culture insert (Corning, Falcon Cell Culture insert, Cat#) was placed on which the three-dimensional biological tissue was placed. 353494 or #353292) was placed on a three-dimensional biological tissue culture device. It was covered with a lid, and silicone tubes were connected to the medium supply tube and medium discharge tube, and the tubes were connected to a microperistaltic pump (Icams Lab). Silicon tubes serving as a medium supply line and a medium discharge line were connected to the medium supply tank.

1-3. Perfusion Test To examine the kinetics of perfusion, 200 μL of PBS (0.0 μm) containing fluorescent beads (Cat. #17149, Lot. 536309, average particle size 0.0394 μm, 2.5% (w/v), Polyscience Inc.) was added. 05% solution) was introduced from a three-way stopcock provided in the medium supply line. The beads were photographed for 1 minute using a camera connected to a Nikon microscope, and the distance traveled by the beads was measured using Aqua Cosmos (Hamamatsu Photonics) to analyze the mobility (F1 in FIG. 7(A) and FIG. 7(B)). In addition, the dynamics of the liquid inside the cell culture insert was determined by adding a PBS solution containing about 6% of fluorescent beads (3.6 × 10 ⁶ beads/mL, FluoSpheres (trademark), Thermo Fisher Scientific) with a diameter of 10 μm only inside the cell culture insert. The bead dynamics were recorded every 2 seconds for 1 to 1.5 minutes, and the mobility was analyzed (F2 in FIG. 7(A) and FIG. 7(C)).

2. Results We confirmed that fluorescent microbeads on the porous membrane moved by flowing liquid through the channel. Therefore, it was confirmed that a gentle liquid flow was generated on the porous membrane as theoretically expected (FIG. 7(C)).

(Example 2)
1. Materials and experimental methods 1-1. Maintenance culture of iPS cells A strain of iPS cells (201B7, Lot No. 18) distributed by RIKEN Cell bank was used, and iPS cells between passages p8 to 23 were used in the experiment. iPS cells were maintained and cultured without a feeder. Specifically, 4 to 5 × 10 iPS cells were seeded in a culture dish coated with 0.4 mg/cm ² imatrix-511 (WAKO) in advance, and 10 μM Rock inhibitor (Y27632 ^, WAKO) was added. The cells were cultured in a maintenance medium and replaced with a maintenance medium (AK02N, AJINOMOTO) within 24 hours. The medium was exchanged once every two days until the number of cells increased, and after the number of cells increased and the colony grew to around 1 mm in diameter, the medium was exchanged every day. Passage was carried out at 40-50% confluence, at which point the colonies began to fuse with each other. After washing with PBS (WAKO), 1/2 diluted TrypLET (Thermo) was added, and after incubation at 37° C. for 7 minutes, the cells were detached, the number of cells was counted, and culture was continued as appropriate. When preserving, it was frozen by slow freezing using Stem Cell Banker (Zenoac).

1-2. Induction of kidney organoids Kidney organoids were generated according to the method of Takasato et al. (Takasato M., et al., Nature. 2015 Oct. 22; 526 (7574): pp. 564-568.). Specifically, iPS cells were cultured on a 35 mm ² culture dish, and when they reached 70 to 80% confluence, they were cultured in Xeno-free medium (APEL2, STEMCELL) containing 0.8 μM CHIR99021 (WAKO) for 4 days. Cultured. Thereafter, culture was continued for 2 days in a Xeno-free medium containing 2 μg/mL FGF9 (PEPRO TECH). Then, in order to make the cells three-dimensional, they were detached from the culture dish using a passage reagent, a suspension was prepared at a concentration of 6.25 x ¹⁰ cells/100 to 200 μL/tube, and the cells were incubated at 400 x g. Centrifugation was performed for 3 minutes at room temperature. The resulting pellet was transferred to the center of a 0.4 μm pore cell culture insert (Corning, Falcon Cell Culture insert, Cat #353494) for a 12-well plate. Thereafter, cell culture inserts were placed in wells of a 12-well plate, and a medium (0.5 μM CHIR99021, 10 μM Y27632-added APEL) was added only to the wells, followed by incubation at 37° C. for 1 hour. Thereafter, the culture medium was changed to APEL medium supplemented with 2 μg/mL FGF9. The culture medium was replaced once every two days and culture was continued (see FIG. 8(A)).

1-3. Perfusion culture conditions Genetic analysis was performed to explore the timing at which kidney organ guide induces kidney cells. Organoids were perfused cultured for 3 days during the induction period of kidney cells. The total volume of medium for perfusion was 2.5 mL to 3 mL, and the perfusion rate was set between 0 and 100 μL/min. The entire amount of the medium was replaced every 1.5 days. The organoid culture system was placed in a 37°C, 5% _CO2 incubator. The pump controller was installed outside the incubator. During time-lapse observation, the chamber around the microscope was kept at 37°C, sterile water was added to unused wells of the multiwell plate, and 5% CO ₂ was supplied by bubbling for observation.

1-4. Kidney organoid perfusion culture From the results of genetic analysis and morphological analysis, perfusion culture of organoids was performed from day 18 of induction, when kidney cell induction had definitely progressed. The organoid culture device was placed in a 6-well multiwell plate, a 12-well cell culture insert with organoids placed thereon, and a lid was placed. Thereafter, a silicone perfusion tube connected to a pump with three-way activation was connected, and after filling with the culture medium and removing bubbles, perfusion was started. As a control, a 12-well cell culture insert was placed on an organoid culture device with a gap of about 2 mm, and then placed in a 5 cm culture dish containing 3 mL of medium, and the perfusion group and the amount of medium used were determined. We considered this by arranging the following.

1-5. Genetic Analysis Organoids obtained during each culture period were collected, and RNA was extracted using an RNA extraction kit (Ambion Thermo). After measuring the amount of RNA, the same amount of RNA was subjected to reverse transcription reaction using High Capacity cDNA (ABI Thermo) to produce cDNA. Real-time PCR was performed using Taqman probe (ABI Thermo), and reaction and analysis were performed using Via7. A relative standard curve was created using standard samples, and quantitative calculations were performed using it. ACTB was used as an internal standard.

1-6. Immunohistochemical staining Renal organoids obtained during each culture period were fixed with 4% paraformaldehyde (Muto Chemical) and stored at 4°C. To create cryoblocks, fixed renal organoids were treated with 15% sucrose/PBS for 30 minutes, then replaced with fresh 15% sucrose/PBS and treated again for 30 minutes. Thereafter, the solution was replaced with 30% sucrose/PBS and treated for 30 minutes, and then replaced with fresh 30% sucrose/PBS and treated again for 30 minutes. Tissue Tek O. C. T. A frozen block was prepared using Compound (Sakura Finetech Japan) and stored at -80°C. Using a cryostat (Leica), frozen sections were prepared at a thickness of 8 μm and used for staining.

1-7. Visualization of distribution of perfusion culture medium in tissues During perfusion culture, cells were perfused with a culture medium containing 0.5 mg/mL Texas Red-conjugated dextran (70,000 MW Molecular Probes, Eugene, OR, USA) for 2 days, and then, Perfusion fixation was performed with 4% paraformaldehyde. Frozen sections were prepared from the obtained organoids and observed using a confocal laser microscope. Dynamics were visualized by considering the Texas Red positive region (FIG. 9A) as the region through which the perfusion fluid passes through the tissue. The reagents used are as follows.

<Primary antibody>
・CK8 (mouse monoclonal antibody: abcam) 1/200 dilution ・Podocalyxicin (goat polyclonal antibody: R&D) 1/500 dilution ・PDGFβ receptor (rabbit monocl onal antibody: abcam) 1/200 dilution/Active-Caspase-3 (rabbit polyclonal antibody: abcam) 1/200 dilution/CK19 (rabbit monoclonal antibody: abcam)
<Secondary antibody>
・moues IgG Rhodamine (Jackson immunology)
・rabit IgG FITC (Jackson immunology)
・Goat IgG FITC (Jackson immunology)
Each was used at 1/200 dilution.
<Nuclear staining>
・Hoechxist 33528 (Dojindo) 1/500 dilution

2. Results Kidney organoids were induced from iPS cells (Fig. 8(A)). As a result, it was revealed that gene expression and morphogenesis of kidney cells began to gradually increase after 12 days of induction, and reached a peak after 18 days (FIGS. 8(B) and (C)).

Figure 9 shows the results of perfusion culture of renal organoids. It was confirmed that the medium had invaded approximately 100 μm below the kidney organoid.

Figure 10 shows the results of changes in ureteric bud tissue and induced tubular structure within renal organoids due to perfusion. A tendency for CK8-positive cells to increase with perfusion of 2.5 μL/min was observed.

Figure 11 shows changes in gene expression within renal organoids upon perfusion. The expression of PAX8, which is expressed at the ureteric bud junction, was highest when perfused at 2.5 μL/min.

FIG. 12 is a diagram showing the state of lumen expansion of ureteroblasts due to perfusion. A tendency for the ureteroblast lumen to expand was observed as the perfusion rate increased.

(Comparative example)
Immersion Culture of Kidney Organoids 300 μL of medium was added to the cell culture insert in which the kidney organoids were cultured on the 18th day of induction (12th day of three-dimensionalization), and the culture was continued for an additional 3 days (FIG. 13).

As a result, when switching from organ culture (air-liquid interface maintenance culture) to immersion culture, the three-dimensional structure and internal tissue structure of kidney organoids collapsed after 3 days (Figure 13).

(Consideration)
FIG. 14 is a diagram illustrating the similarities between the environment of kidney organoids cultured in the present invention and the fetal kidney environment. According to the present invention, organoids are supplied with oxygen from the gas phase, and at the same time, nutrients are supplied by the liquid phase (medium) flowing directly under the porous membrane. The culture environment realized by the present invention is similar to the blood flow environment of the fetal kidney, where blood flow is transmitted in layers from the bottom to the top, and is considered to be an environment that imitates this.

(Example 3)
1. Experimental Method The flow channels of the two three-dimensional biological tissue culture devices produced in Example 1 were connected using a polypropylene connector of about 2.8 cm. The kidney organoids obtained by the method of Example 2 were placed on cell culture inserts on two three-dimensional biological tissue culture devices, respectively. 10 ⁵ G401 cells (pediatric renal cancer cell line) embedded in 50 μL of type I collagen gel were transplanted into one kidney organoid, and approximately 3 mL of medium (APEL; STEMCELL's kidney organoid medium) was added to the gel. The cells were cultured for 7 days with perfusion at .5 μL/min.

As a control (static culture), a cell culture insert with renal organoids placed thereon was set in one well of a 6-well plate containing 3 mL of APEL medium, and 10 ⁵ cells were embedded in type I collagen gel. G401 cells (pediatric renal cancer cell line) were transplanted and similarly cultured for 7 days.

2. Results The type I collagen gel in which cancer cells were embedded progressed to dissolve during perfusion culture (Figure 15)

(Example 4)
1. Experimental Method The flow channels of the two three-dimensional biological tissue culture devices produced in Example 1 were connected using a polypropylene connector of about 2.8 cm. The kidney organoids obtained by the method of Example 2 were placed on cell culture inserts on two three-dimensional biological tissue culture devices, respectively. One hundred G401 cells suspended in 1 uL of medium were dropped and transplanted into one kidney organoid. The cells were cultured for 14 days while perfusing with about 3 mL of a medium (APEL; STEMCELL's renal organoid medium) at a rate of 2.5 μL/min.

After medium exchange, the cells were collected by centrifuging the collected post-perfusion medium, suspended in MaCoy's 5A medium supplemented with 10% serum for G401, added to 1 well of a 6-well plate, and grown until confluent. . Thereafter, the cells were cultured in a serum-free medium for 24 hours, and the amount of Human MMP9 contained in the culture supernatant was measured by ELISA. As a control, a culture supernatant in which 1 G401 cell was cloned in a 96-well plate and grown in the same manner was used ("1 cell/w (M)" in FIG. 16).

2. Results Cells collected from the perfusion medium after 5 days of perfusion culture were cultured in serum-containing medium for 18 days. As a result, cancer cells were detected, and it was confirmed that cancer cells had infiltrated into the perfusion medium after drop transplantation.

Cells collected from the perfusion medium after 3 and 7 days of perfusion culture were cultured in serum-containing medium for 18 days, and as a result, mesenchymal cells highly expressing MMP9 were detected (FIG. 16). Metalloprotease activity was low in G401 cells (Figure 16).

1, 1a Three-dimensional biological tissue culture system 10, 10a Three-dimensional biological tissue culture device 11, 11a Cell culture insert holding part 110, 110a Bottom 111 Inner wall 12, 12a Channel 120, 120a Channel inner wall 121, 121a Upper opening of channel Part 122 Channel bottom opening 122a Channel bottom 123 Channel inlet 124 Channel outlet 13 Medium supply pipe 130 Lower medium supply port 131 Upper medium supply port 14 Medium discharge pipe 140 Lower medium discharge port 141 Upper medium discharge port 15, 15a Main body 150, 150a Main body top 151, 151a Main body bottom 16 Adapter 17 Medium supply line 18 Medium discharge line 180 Connection line 19 Liquid pump 20 Cell culture insert 21 Cell culture insert main body 22 Porous membrane 30 Culture vessel 31 Bottom of culture vessel 4 Culture device assembly 5 Medium supply tank OG Three-dimensional biological tissue M Medium

Claims (14)

A method for culturing three-dimensional biological tissue, the method comprising:
A three-dimensional biological tissue placed on one side of a porous membrane through which the components of the medium permeate is exposed to a gas phase, and the medium in contact with the other side of the porous membrane is placed parallel to the porous membrane. a step of culturing the three-dimensional biological tissue while perfusing it with a liquid flow, the liquid flow being a laminar flow ;
including methods. The method according to claim 1, wherein the porous membrane has pores that penetrate in the thickness direction of the porous membrane. The method according to claim 1 or 2, wherein the porous membrane is a porous membrane of a cell culture insert. The method according to any one of claims 1 to 3, wherein the three-dimensional biological tissue is a kidney organoid, a lung organoid, a thymus organoid, or a testis organoid. The method according to any one of claims 1 to 4, wherein the three-dimensional biological tissue is a kidney organoid. The method according to any one of claims 1 to 5, wherein the medium is perfused at a flow rate of 0.01 μL/min or more. A cell culture insert holding part;
at least one channel provided in a part of the bottom of the cell culture insert holding part;
a culture medium supply pipe for supplying a culture medium to the flow path;
a culture medium discharge pipe for discharging the culture medium from the flow path;
Equipped with
Here, a part of the upper surface of the flow path has a flow top opening, and when the cell culture insert is placed on the cell culture insert holding part, the flow top opening opens the cell culture insert. Covered by the lower surface of the porous membrane, the shape of the flow path is formed so that when the medium flows, it becomes a laminar flow .
Three-dimensional biological tissue culture device. The three-dimensional living tissue culture device according to claim 7, wherein the cell culture insert holding section has a fitting means for bringing the cell culture insert into close contact with the cell culture insert. The three-dimensional living tissue culture device according to claim 8, wherein the fitting means is one or more fitting means selected from the group consisting of a tapered structure and a sealing member. The three-dimensional living tissue culture device according to any one of claims 7 to 9 , wherein the shape of the flow path has a width/height of 2.0 or more. The three-dimensional biological tissue culture device according to any one of claims 7 to 10 , wherein the height of the flow path is about 0.1 mm to about 5 mm. The three-dimensional biological tissue culture device according to any one of claims 7 to 11 , further comprising a cell culture insert placed on the cell culture insert holding section. The three-dimensional biological tissue culture device according to claim 12 ,
a medium supply line connected to the medium supply pipe;
a medium supply tank that supplies a medium to the medium supply line;
a liquid pump that sends the medium to the medium supply line;
a medium discharge line connected to the medium discharge pipe;
a medium discharge tank that stores the medium discharged from the medium discharge line;
A three-dimensional biological tissue culture system. The three-dimensional living tissue culture system according to claim 13 , wherein the medium supply tank and the medium discharge tank are the same, and the medium circulates.

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