WO2016035777A1

WO2016035777A1 - METHOD FOR FORMING ORGAN HAVING THREE-DIMENSIONAL VASCULAR NETWORK in vivo, AND COMPOSITION FOR FORMING ORGAN HAVING THREE-DIMENSIONAL VASCULAR NETWORK

Info

Publication number: WO2016035777A1
Application number: PCT/JP2015/074782
Authority: WO
Inventors: 孝辻; 美帆小川; 克成手塚
Original assignee: 株式会社オーガンテクノロジーズ
Priority date: 2014-09-01
Filing date: 2015-08-31
Publication date: 2016-03-10
Also published as: JP2017195778A

Abstract

　[Problem] To provide a technique for forming an organ having a three-dimensional vascular network. [Solution] A method for forming an organ having a three-dimensional vascular network in a nonhuman animal in vivo, the method including the following steps: (a) a step for arranging, in a nonhuman animal, one or a plurality of blood vessels so as to pass through a structure, the blood vessels remaining connected in a living body, or a step for arranging one or a plurality of isolated blood vessels so as to pass through a structure outside the living body, then connecting the "one or a plurality of isolated blood vessels" to a blood vessel in the living body, the structure being configured so that a hollow section is provided in the inside thereof, and an organ primordium being included inside the structure; and (b) a step for culturing the blood vessels and the organ primordium included inside the structure in vivo inside the structure, and thereby forming an organ having a three-dimensional vascular network in the hollow section inside the structure.

Description

Method for forming an organ having a three-dimensional vascular network in vivo, and a composition for forming an organ having a three-dimensional vascular network

The present invention relates to a method for forming an organ having a three-dimensional vascular network in vivo. The present invention also relates to a composition for forming an organ having a three-dimensional vascular network.

In current regenerative medicine, “cell transfer therapy” in which stem cells are transplanted and the physiological function of the organ or tissue of the transplant destination is restored has been put into practical use. However, as a next-generation regenerative medicine, the realization of so-called "organ replacement regenerative medicine" that restores physiological functions by replacing dysfunctional organs with regenerative organs artificially constructed using regenerative medicine. Is desired.

An organ in a living body is a three-dimensional collection of high-density cells. Inside, a three-dimensional (capillary) vascular network is stretched, and the organ is a high-density cell population through gas exchange, nutrient supply, waste metabolism, etc. through this vascular network. Despite being maintained and functioning.

Therefore, when artificially constructing a transplantable regenerative organ, a three-dimensional vascular network similar to the three-dimensional vascular network of an organ in a living body is indispensable.

So far, a technique has been reported in which cell sheets are laminated (multi-layered) and transplanted in order to regenerate a damaged part in a living body. Non-Patent Document 1 discloses that the thickness of the regenerated myocardial tissue changes even when three or more layers of myocardial cell sheets are laminated in the vicinity of a blood vessel that can be accessed surgically in the treatment of a damaged part of the myocardial tissue. On the other hand, a period in which a first layer of cardiomyocyte sheet is transplanted near the blood vessel and the blood vessel enters the cell sheet to form a sufficient vasculature. After waiting, the second one-layer cell sheet is transplanted onto the first cell sheet, and the cell sheets are stacked several times while angiogenesis period is provided. It reports that it was able to build an organization. However, the technique of the non-patent literature requires multiple operations to recover damaged tissue, and accelerates the speed of construction of a three-dimensional vascular network and the speed of regeneration of damaged tissue, which are indispensable for the construction of a regenerated organ. I can't.

In Non-Patent Document 2, when a cardiomyocyte sheet mixed with endothelial cells was prepared to promote angiogenesis in a layered sheet of transplanted cardiomyocytes, formation of a network of endothelial cells was observed in the cell sheet. It has been reported that the transplantation of the cell sheet promoted the regeneration of myocardial tissue as compared to the cell sheet containing only cardiomyocytes. However, in the technology of the non-patent document, the three-dimensional vascular network is indispensable for constructing a regenerative organ, not only in the construction of a two-dimensional vascular network using a sheet (planar (two-dimensional)) sheet called a cell sheet. Has not yet been provided efficiently.

Non-Patent Document 3 discloses that a three-dimensional tubular myocardial tissue is constructed in vitro by wrapping a cardiomyocyte sheet around an aorta outer periphery excised from a rat, and then replacing the aorta of a different rat individual with the aorta. After transplantation, we reported that the transplanted myocardial tissue had its own autonomous pulsation that was different from the autonomous pulsation of the host heart (i.e., the formation of myocardial tubes (pulsatile myocardial tubes) that autonomously pulsated). ing. This report shows the applicability of an autonomously pulsating myocardial tube as an auxiliary heart, and shows the applicability as an auxiliary organ that can replace part of the organ function. However, the technique of the non-patent document is simply intended to give a part of organ function to an existing biological blood vessel, and is a technique for actively constructing a vascularized three-dimensional blood vessel network. Completely different.

Non-Patent Document 4 describes that human mesenchymal stem cells (hMSC) and human umbilical cord upper vein endothelial cells (HUVEC) are mixed in a collagen gel, and then hMSC-HUVEC-mixed collagen gel is mixed with severe combined immunodeficiency (SCID). It has been reported that blood vessels derived from hMSC and HUVEC were stable and functional in vivo for a long time when transplanted into the calvaria of mice. However, in the technique of the non-patent document, various cells (for example, mesenchymal cells and the like) derived from the recipient (target to be transplanted) are mixed from the surrounding tissue of the transplanted part. In addition, since blood vessel induction from surrounding tissues is not controlled, new blood vessels enter the transplanted cells from any place. Therefore, even if the technique of the said nonpatent literature is used, the formed three-dimensional vascular network cannot be extracted and transplanted.

Thus, in the conventional technique, the construction of the three-dimensional vascular network necessary for constructing a transplantable regenerative organ is not sufficient. From such a background, establishment of a three-dimensional vascular network construction technique is desired for maintaining and functioning organ parenchymal cells (cells responsible for major functions) for the construction of regenerative organs. .

An object of the present invention is to provide a technique for forming an organ having a three-dimensional vascular network, which is necessary for constructing a transplantable regenerative organ.

Angiogenesis in a living body is roughly classified into angiogenesis in which fetal blood vessel generation and new blood vessels branch from mature blood vessels.
The development of blood vessels in the fetal period is the accumulation of blood vessel hemangioblasts and vascular endothelial progenitor cells such as hemangioblasts to form a luminal structure, and the vascular plexus is a collection of blood vessels of uniform vascular diameter. Begins with formation (angiogenesis). A functional vascular network is then formed by vascular remodeling, such as angiogenesis, arteriovenous differentiation, and vascular maturation.
On the other hand, angiogenesis from mature blood vessels occurs only in limited cases such as tumor cells and wound healing. Specifically, existing vascular endothelial cells respond to angiogenic factors such as VEGF released from cells that have fallen into hypoxia or undernutrition, resulting in new branching and elongation of blood vessels. The surrounding area is covered with vascular pericytes (pericytes) to form a newly matured vascular network.

In addition, organs (eg, liver and kidney) have one artery transferred into the organ, three-dimensional (capillary) blood vessels spread inside the organ, converged again into one vein, and moved out of the organ. It is known that it communicates with a blood vessel of a living body by a vascular route to be performed. In other words, for the artificial production of transplantable organs, it is not sufficient to produce only the cells and tissues that make up the organ parenchyma, and it is necessary to form a three-dimensional vascular network with the organ parenchyma in a transplantable manner. It is.

As a result of repeated studies to solve the above problems, the present inventors have not only the feature that the fetal organ primordium develops into cells / tissues constituting the organ parenchyma through normal development, Focusing on the fact that angiogenesis factors are produced during development and promotes angiogenesis inside and around the organ, and by utilizing these features, the three-dimensional blood vessels of organs in the body It has been found that an organ having a three-dimensional vascular network similar to a network can be artificially guided and constructed. That is, the present inventors create a closed space (sometimes referred to as “chamber” in this specification) inside the structure so as to wrap blood vessels in the living body with the structure, and within the closed space. In addition, it is found that organs having an organ-type three-dimensional vascular network can be formed (manufactured) by connecting the blood vessel including the organ primordia to a living body and culturing in vivo. Completed.

That is, the present invention, in one embodiment,
A method of forming an organ having a three-dimensional vascular network in vivo in a non-human animal, comprising:
The following steps:
(A) in the non-human animal,
Arranging one or more blood vessels to penetrate the structure while connected to the living body, or to pass the isolated blood vessel or blood vessels through the structure in vitro. Connecting the “isolated one or more blood vessels” to a blood vessel in a living body after placement, wherein the structure is configured to have a hollow portion therein, and the structure Contains an organ primordium;
(B) An organ having a three-dimensional vascular network in a hollow portion inside the structure by culturing the blood vessel and the organ primordium contained in the structure in vivo inside the structure. A method comprising the steps of:

In one embodiment of the method of the present invention,
The step (a)
(I) before placing the blood vessel so as to penetrate the structure, the organ primordium is included in the structure, or
(Ii) After arranging the blood vessel so as to penetrate the structure, the organ primordium is included in the structure.

In one embodiment of the method of the present invention,
In the step (a), the structure further includes vascular constituent cells in addition to the organ primordia.

In one embodiment of the method of the present invention,
The vascular constituent cells include vascular endothelial cells and / or mesenchymal stem cells.

In one embodiment of the method of the present invention,
In the step (a), an angiogenic factor is further included in the structure.

In one embodiment of the method of the present invention,
The angiogenic factor is selected from the group consisting of VEGF, FGF, EGF, PDGF, and TGF-β.

In one embodiment of the method of the present invention,
The organ primordium is selected from the group consisting of hair follicle primordia, tooth germ primordium, liver primordium, kidney primordium, ratchet sac, and endocrine organ primordium.

In one embodiment of the method of the present invention,
The organ primordium is a regenerated organ primordia prepared from ES cells, iPS cells, pluripotent progenitor cells, adult stem cells, or pluripotent cells genetically modified from these.

In one embodiment of the method of the present invention, in the method,
The following steps:
(C) After the step (b), the method further includes a step of orthotopically or ectopically transplanting the organ having the formed three-dimensional vascular network.

In one embodiment of the method of the present invention,
The structure is characterized by comprising a dialysis membrane that does not allow passage of cells but allows passage of humoral factors.

In one embodiment of the method of the present invention,
A human is used instead of the non-human animal.

Other embodiments of the invention include:
A composition for forming an organ having a three-dimensional vascular network,
The composition is a substantially sheet-like composition, and can be configured as a structure including one or a plurality of openings and a hollow portion inside.
here,
One or more blood vessels are disposed through the opening and through the structure;
The structure is substantially closed except for the opening through which the blood vessel passes;
In the structure, an organ primordium is included, and an organ having a three-dimensional vascular network is formed in a hollow portion in the structure.
It is related with the composition characterized by the above-mentioned.

Other embodiments of the present invention relate to organs having a three-dimensional vascular network that can be obtained by any of the methods of the present invention described above.

Other embodiments of the present invention relate to organs having a three-dimensional vascular network obtained by any of the methods of the present invention described above.

Other embodiments of the present invention relate to regenerative organs, including organs having a three-dimensional vascular network that can be obtained or obtained by any of the methods of the present invention described above.

Needless to say, any combination of one or more features of the present invention described above is included in the present invention.

According to the method of the present invention, an organ having a three-dimensional vascular network in which one or a plurality of blood vessels are transferred to the inside of a structure and the hollow portion (preferably a substantially closed space) of the structure is inside. Can be formed, and the blood vessel that has converged again into one or a plurality of blood vessels is exported from the inside of the structure, and an organ that mimics the blood vessel structure of a living organ can be formed.
In a preferred embodiment, blood vessels other than one or more initially encapsulated blood vessels cannot penetrate from the surroundings into the structure where a three-dimensional vascular network is formed. Therefore, the number of blood vessels that move into and out of the structure can be controlled.
The organ having the formed three-dimensional vascular network can be used in vitro while being included in the structure or removed from the structure. Since the blood vessels transferred into and out of the structure are maintained, these blood vessels can be connected to the blood vessels of the same or different species by vascular sutures or the like. The formed three-dimensional vascular network can be easily transplanted. In addition, an organ having a three-dimensional vascular network produced by the method of the present invention is connected to an organ or tissue perfusion culture method and perfusion culture apparatus as described in WO2011 / 093268, for example, before transplantation. You may perform culture | cultivation in.
In another preferred embodiment, an organ having a three-dimensional vascular network of a desired size can be formed by changing the size of the hollow part in which the three-dimensional vascular network is formed as desired.
In still another preferred embodiment, the shape of the hollow part in which the three-dimensional vascular network is formed is changed as desired, for example, substantially the same or similar to the form of the three-dimensional vascular network possessed by the desired organ or tissue An organ having a three-dimensional vascular network having a form can be formed.

FIG. 1 is a schematic view of a method for producing an organ having a three-dimensional vascular network, which is an embodiment of the present invention. The left lobe of the liver primordium obtained from a fetus at 12 days of gestation and a gel containing vascular component cells (HUVEC and hMSC) (upper figure 2), or the left of the liver primordium obtained from a fetus at 12 days of fetus A chamber obtained after 7 days or 14 days after transplanting a gel containing leaves and vascular constituent cells (HUVEC and hMSC) and VEGF (bottom of FIG. 2) into the back of a Balb / c slnu / nu mouse. It is a figure which shows the analysis result of the structure | tissue structure by HE dyeing | staining of an inner formation. A gel containing tooth germ and VEGF obtained from a 14-day-old mouse fetus was transplanted into the back of a Balb / c slnu / nu mouse in the back of the chamber, and was obtained after 3 or 7 days. It is a figure which shows the analysis result of the structure | tissue structure by HE dyeing | staining. Latoke sac obtained from a fetus at 18 days of age and gel containing VEGF (upper part of FIG. 4), or Ratoke sac obtained from a fetus at the age of fetus 18 days and blood vessel constituent cells (HUVEC and hMSC) and VEGF (lower part of FIG. 4) ) Is a graph showing the analysis results of the tissue structure by HE staining of the formation in the chamber obtained 7 days or 14 days after transplanting the gel containing) into the back of the Balb / c slnu / nu mouse. is there.

Hereinafter, embodiments of the present invention will be described.

In the present specification, the “three-dimensional vascular network” is not two-dimensional (planar) as observed in a three-dimensional cell mass (for example, an organ or tissue in a living body), It means a network structure with three-dimensionally expanded blood vessel pathways.

In this specification, “organ-type three-dimensional vascular network” means a blood vessel of an organ in a living body in which a three-dimensional (capillary) blood vessel spreads from one arterial route and converges again into one venous route. Similar to the network structure, it means a three-dimensional blood vessel network in which blood vessels expand three-dimensionally (capillary) from one blood vessel route and converge again into one blood vessel route. In the present invention, an organ-type three-dimensional vascular network is a structure of the present invention, as in the case of a three-dimensional vascular network including a specific blood vessel serving as an entrance / exit of blood flow that passes through an organ in a living body. It may mean a three-dimensional vascular network including specific blood vessels that serve as entrances and exits for blood flow through the interior.

In the present invention, “organ or tissue” is not particularly limited as long as it is an organ or tissue intended for orthotopic transplantation or ectopic transplantation. For example, liver, heart, brain, lung, kidney, stomach, intestine , Pancreas, testis, ovary, eyeball, bone, teeth, and their constituent organs and surrounding tissues.

In the present invention, “transplantation” refers to transplanting an organ, tissue, cell, or the like from a donor (providing an organ, tissue, cell, or the like to be transplanted) to a recipient (subject to be transplanted). The type is not particularly limited. Examples of the type of transplantation include autotransplantation, syngeneic transplantation, allogeneic transplantation, xenotransplantation and the like in classification based on the relationship between the donor and the recipient. In addition, for example, classification according to the state of the donor includes living transplantation, brain death transplantation, cardiac arrest transplantation, and the like.
In one embodiment, in the present invention, the donor that provides the blood vessel used for the chamber transplant is an organ having a three-dimensional vascular network formed in the living body of the recipient who has undergone the chamber transplant, or a regenerative organ, or a structure including the same. It is understood that the recipient can be provided. It is also understood that a recipient undergoing chamber implantation can also be a donor that provides an organ or regenerative organ having a three-dimensional vascular network formed in vivo or a structure containing the same.

In this specification, the regenerative organ is an organ that is manufactured using biotechnology or regenerative medicine technology, and is an organ that imitates part or all of the function or structure of an organ in a living body, or It may mean a tissue having a function of part or all of an organ. A regenerating organ can substitute for an in vivo organ, or assist or restore the function of the in vivo organ.

<Method of forming an organ having a three-dimensional vascular network in vivo in an animal>
One embodiment of the present invention is a method of forming an organ having a three-dimensional vascular network in vivo in an animal, comprising the following steps:
(A) In the animal, the step of arranging one or more blood vessels so as to penetrate the structure while being connected to the living body, or one or more blood vessels isolated outside the body, Connecting the “isolated one or more blood vessels” to a blood vessel in a living body after being disposed so as to penetrate the structure, wherein the structure is configured to have a hollow portion therein And the structure includes an organ primordium;
(B) Inside the structure, the organ having a three-dimensional vascular network in the hollow portion of the structure is obtained by in vivo culturing the blood vessels and the organ primordium contained in the structure. It is a manufacturing method including the step to form. As long as there is no technical contradiction, the present invention may be implemented by appropriately combining any one or more of all aspects described in this specification.

In this specification, “chamber transplantation (method)” means that a chamber (closed space) is provided, and an organ, tissue or organ to be transplanted is enclosed therein, and the chamber is penetrated by a blood vessel. A new transplantation (method) invented by the present inventors, in which an organ, tissue or organ inside the chamber is fed with nutrients by the blood vessel, and the organ, tissue or organ inside the chamber is cultured in vivo. It is.
In conventional subcutaneous transplantation, an organ, a tissue, an organ, and the like are transplanted into a transplanted portion so as to come into contact with a living tissue in a recipient (a subject to receive the transplant).
In the chamber transplantation used in the present invention, the organ primordium or the like that is the material of the organ (or organ) to be transplanted is isolated within the structure (that is, isolated in a closed space (chamber)). Provided to the recipient. The organ primordium can communicate with living tissue in the recipient only through the blood vessels used to cause the recipient to form a vascular network. Since the blood vessel is configured to penetrate the structure, it is possible to communicate / contact with the organ primordial or the like in a closed space. As a result, the organ primordium that has received nutrient supply from the blood vessels can proliferate without contact with the living tissue in the recipient other than the blood vessels. By using such a chamber transplantation, it is possible to form a blood vessel in a hollow portion provided inside the structure and form an organ having a three-dimensional vascular network. In this specification, chamber implantation (method) will be further described below, and those skilled in the art will understand that all relevant descriptions are one non-limiting aspect of chamber implantation (method).

In the present invention, an animal that can be used is not limited. For example, non-human animals (non-human mammals (mouse, rat, dog, cat, rabbit, cow, horse, sheep, goat, pig, monkey, etc.) ), Non-mammals (fish, reptiles, amphibians, birds, etc.)) and humans.

In the present invention, an organ having a three-dimensional vascular network or a regenerative organ including the three-dimensional vascular network may be formed by culturing in vivo using any animal. In addition, an organ having a three-dimensional vascular network to be formed, a regenerative organ including the three-dimensional vascular network, or a structure including the same is orthotopically or ectopically with respect to any animal. It may be used for transplantation (autologous transplant, syngeneic transplant, allogeneic transplant, xenotransplant, artificial transplant, etc.). In a preferred embodiment, the animal used in the present invention is immunodeficient in order to be compatible with ectopic transplantation, or the immune response is temporarily dysfunctional using an immunosuppressant or the like. It may be.
In vivo transplantation of an animal having a three-dimensional vascular network or a regenerative organ, an organ having a formed three-dimensional vascular network, a regenerative organ containing the three-dimensional vascular network, or a structure containing the same When the recipient animal is a heterogeneous or different individual, in an animal that forms an organ having a three-dimensional vascular network or a regenerative organ, the blood vessel used by penetrating the structure for the formation of the three-dimensional vascular network is: It is preferably a blood vessel derived from an animal that undergoes transplantation of the formed “organ having a three-dimensional vascular network, or a regenerative organ including the three-dimensional vascular network, or a structure including the same”.

In the present invention, “one or more blood vessels” used to form a three-dimensional blood vessel network may be one or more blood vessels that are non-branched or branched. The blood vessel may be derived from the same or different species of animals that form an organ having a three-dimensional vascular network, and is preferably different. For example, when an organ having a three-dimensional vascular network is formed in vivo in a rat or pig, the blood vessel may be a blood vessel extracted from a mouse or a human, respectively. The “extracted blood vessel” may be derived from a human transplant donor who died due to brain death or cardiac arrest. Alternatively, the blood vessel may be an artificial blood vessel prepared by differentiation from ES cells, iPS cells, pluripotent progenitor cells, or the like, which are pluripotent stem cells. An “iPS cell” is a pluripotent stem cell similar to an ES cell prepared by introducing, for example, four genes encoding transcription factors into an adult cell (Cell., Vol 126, Issue 4, 25 August 2006, pp. 663-676). However, iPS cells that can be used in the present invention may be iPS cells produced by any method as long as they have iPS cell properties. Since iPS cells can be prepared from somatic cells derived from patients, iPS cells are preferred as cells capable of regenerating transplantable tissues and organs with little risk of ethical problems and immune rejection. The origin of pluripotent stem cells and pluripotent progenitor cells is not limited, and for example, cells derived from humans, mice, rats, pigs, and monkeys can be used. Further, the blood vessel may contain a material not derived from a living body, for example, a synthetic polymer material such as nylon or Teflon, or may be used in combination with an artificial blood vessel made of the synthetic polymer material.

In the present invention, the “structure” that penetrates “one or more blood vessels” has a hollow portion (closed space) inside thereof, the blood vessel penetrates the hollow portion, and the organ source in the hollow portion. Any configuration may be used as long as a configuration including a group or the like can be adopted. As long as the said structure does not permeate | transmit the cell contained in the inside of a structure, and the cell derived from the tissue of the recipient which exists in the exterior of a structure (cell impermeable material, a film | membrane, a film, etc.) It may be composed of various materials. The structure is formed from, for example, the “substantially sheet-like composition” in the present invention, or formed from the substantially sheet-like composition (that is, the substantially sheet-like composition is molded in advance, For example, it may be a three-dimensional form such as a bag. The structure in the present invention is not limited, but is preferably formed from a material that can be easily molded. For example, when it is intended to remove and use an organ having a three-dimensional vascular network or a regenerated organ formed from a structure, the structure in the present invention is a blood vessel or an organ source contained in the structure. It is preferably non-adhesive with the group or the like or can be easily separated. For the structure, a material that can be expanded and contracted or deformed may be used.

By changing the size of the hollow part provided in the structure of the present invention as desired, an organ having a three-dimensional vascular network having a desired size or a regenerated organ can be formed in the hollow part. Further, by changing the shape of the hollow part provided in the structure of the present invention as desired, for example, the form of an organ or tissue intended for transplantation or the three-dimensional vascular network that they have is substantially the same or similar. A reconstructed organ having a three-dimensional vascular network having the above-described form can be formed in the hollow portion. The structure in the present invention may be disposed in the living body so that a part of the structure protrudes (protrudes) out of the living body of the animal receiving the chamber transplantation.

In one embodiment, the “structure” may include an organ primordium or the like in advance inside the structure before penetrating the blood vessel or before being placed in a living body. . Alternatively, in another embodiment, the “structure” may include an organ primordia or the like inside the structure after penetrating the blood vessel or after being placed in a living body.

In the present invention, the angiogenic factor is not particularly limited as long as it is a factor involved in angiogenesis or angiogenesis. Examples of the angiogenic factor include, but are not limited to, fibroblast growth factor (FGF) (eg, b-FGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), angiopoietin, platelets Derived growth factor (PDGF), transforming growth factor-β (TGF-β), matrix metalloprotease (MMP), VE-cadherin (CD144), CD31, ephrin, plasminogen activator, inducible nitric oxide synthase (INOS), cyclooxygenase-2 (COX-2), prostaglandins, placental growth factor (PIGF).

In the present invention, the organ primordium refers to a region of the embryo or the structure of the embryo that is determined to occur in a specific organ as the developmental stage proceeds in the living body, and may be simply referred to as “primordium”. . Almost all organs in a living body are generated from organ primordia derived from epithelial stem cells and mesenchymal stem cells by a fetal development program, and develop into a predetermined position and a predetermined number. The organ primordium produces a blood vessel producing factor in the course of development, and induces blood vessels inside or around the organ primordium. By culturing the organ primordia in the structure of the present invention, An organ having a three-dimensional vascular structure can be produced. The organ primordium used in the present invention may be fetal or may be an organ prima artificially derived from ES cells or iPS cells (may be called regenerative organ primordia).

The type of organ primordium used in the method of the present invention is not particularly limited. For example, hair follicle primordium, tooth germ primordium, liver primordium, kidney primordium, rat sac, or endocrine organ (eg, adrenal gland, pancreas) , Thyroid, parathyroid)).

In addition, an organ having a three-dimensional vascular network formed by the method of the present invention may be an organ having a complete structure and function similar to an organ in a living body, and a part of the structure and function of the organ in the living body. It may be an organ. For example, when the method of the present invention is performed using an organ primordium of an endocrine organ, the formed organ has at least an endocrine function even if it does not have a structure and function that can completely replace the endocrine organ in the living body. If it has, it can be suitably used for the treatment of endocrine diseases, for example. In addition, the kidney functions not only as a urinary organ but also as an endocrine organ that secretes erythropoietin. When the method of the present invention is performed using a renal primordium, the formed organ may have at least an endocrine function (for example, an erythropoietin secretion function) even if it does not have a structure and function that can completely replace the kidney in the living body. Can be suitably used for the treatment of endocrine diseases, for example.

Further, in the present invention, the type, shape, size, number of cells of vascular constituent cells contained in the structure, the size of the organ having the desired three-dimensional vascular network, the size of the regenerated organ, etc. Depending on the situation, those skilled in the art can appropriately determine.

In the present invention, “place one or more blood vessels so as to penetrate the structure while being connected to the living body” means that when the blood vessel is placed in the structure, the blood vessel is the recipient ( It may mean that the structure is arranged while being physically connected to the living body without being separated (isolated) from the living body. It is preferable that the blood vessel is enclosed with the structure in such a manner that the blood vessel is entirely or partially detached from the organ or tissue of the living body while being connected to the living body and penetrates the structure. The blood vessel to be exfoliated is preferably a blood vessel or an artificial blood vessel derived from the same or different animal previously transplanted to the recipient, and more preferably a blood vessel derived from a different animal. When a blood vessel or an artificial blood vessel derived from the same or different animal is previously transplanted to the recipient, it is preferable to replace the previously prepared blood vessel with the recipient's existing blood vessel in the recipient who is preferably an immunodeficient animal. Alternatively, it is preferably transplanted as a shunt (to create a state in which blood flows through a different route from the blood vessel through which blood originally passes), and the structure is penetrated through the transplanted blood vessel. When the transplanted blood vessel is a blood vessel derived from a heterogeneous animal, an organ having a three-dimensional blood vessel network to be formed, a regenerative organ containing the three-dimensional blood vessel network, or a structure containing the same is referred to as the “heterologous animal”. It is preferable to transplant (in such a mode that the transplanted blood vessel is returned to the living body again).

In the process of penetrating the blood vessel into the structure, for example, the blood vessel is arranged so as to penetrate the structure in vitro, and then the blood vessel penetrating the structure is intended for transplantation. It may be connected to a blood vessel in a living body by vascular suture or the like at a place. In such a case, organ primordia and vascular constituent cells may be included in the structure before the blood vessel penetrating the structure is connected to the blood vessel in the living body. Or after connecting the said blood vessel which penetrated the said structure to the blood vessel in a biological body, you may include an organ primordia and a blood vessel constituent cell.

The organ primordium and blood vessel constituent cells contained in the structure and the blood vessel penetrating the structure do not contact each other as long as nutrient supply or the like is performed from the blood vessels to the organ primordia or blood vessel constituent cells. It may be, but it is preferable to touch.

In the present invention, the phrase “one or more blood vessels penetrate the structure” may mean that the hollow portion provided in the structure is moved in and out so that the blood vessel penetrates. There may be one or a plurality of portions where the blood vessel enters and exits the structure, that is, the opening provided in the structure. For example, a plurality of (for example, two) branched blood vessels are transferred to the hollow portion of the structure through a number of openings corresponding to the number of the branched blood vessels provided in the structure, and the transferred blood vessels The blood vessels branched into a plurality, which may be the same as or different from each other, are transferred from the hollow portion of the structure through an opening (export opening) different from the transferred opening (transfer opening). May be. For example, when there is one opening, one or a plurality of blood vessels can be configured to move in and out of the hollow portion provided in the structure through the same opening.
In a preferred embodiment, one unbranched blood vessel may be configured to import into one opening, penetrate the hollow portion of the structure, and exit from another opening. As a result, an organ having an organ-type three-dimensional vascular network can be constructed in the hollow portion of the structure.

After the blood vessel has penetrated the structure and the internal structure of the structure contains the organ primordium and blood vessel constituent cells, the structure is substantially removed except for the opening through which the blood vessel passes. It is desirable to close it. Here, “substantially close” means that the structure is structured such that cells and blood vessels do not invade from the outside, and the organ primordia and blood vessel constituent cells are formed from the inside to the outside of the structure. This means that the neovascularization formed in the hollow portion of the structure does not leak out and does not leak out. In one embodiment, the organ primordium, blood vessel constituent cells, and the like contained in the structure may be included in a gel substance or the like so as not to leak from the structure to the outside. Alternatively, it may be embedded or adhered inside the structure.

In the present invention, “culturing in vivo” is not limited, but may mean growing a recipient who has undergone chamber transplantation. The in vivo culture period is the size of the structure used for chamber transplantation; the type of cells contained in the structure, the number of cells and the cell growth rate; an organ having a three-dimensional vascular network to be formed or a regenerative organ Those skilled in the art can appropriately determine the size according to the size.

In the present invention, the blood vessel constituent cell is not particularly limited as long as it is a cell constituting a blood vessel. Angiogenesis can be stabilized by combining vascular constituent cells with the organ primordia and including them in the structure, followed by in vivo culture. Vascular component cells include, but are not limited to, vascular endothelial progenitor cells such as blood cell hemangioblasts and hemangioblasts; vascular endothelial cells (eg, venous endothelial cells and arterial endothelial cells); vascular stem cells; vascular smooth muscle cells and blood vessels Vascular wall cells such as pericytes (pericytes); mesenchymal stem cells; fibroblasts; blood cells; and other cells that assist in angiogenesis. The mesenchymal stem cells can efficiently stabilize blood vessels as progenitor cells of vascularized blood vessels and mural cells. In one embodiment, the vascular component cells included with the organ primordium preferably comprise vascular endothelial cells and / or mesenchymal stem cells. The vascular constituent cells used in the present invention may be derived from living organisms, and are vascular constituent cells produced by differentiation from ES cells, iPS cells, pluripotent progenitor cells, etc., which are pluripotent stem cells. Also good.

Organ primordia and vascular constituent cells are the nature, type, and growth stability of organ primordia; the efficiency of angiogenesis; the mode of the three-dimensional vascular network that is formed; the function of the cells that form the network with the three-dimensional vascular network Depending on the properties and the like, those skilled in the art can appropriately include them in the structure at a predetermined mixing ratio.

In one embodiment, the structure may further contain an angiogenic factor. That is, in addition to the angiogenic factor produced from the organ primordia, an angiogenic factor may be additionally injected into the hollow part of the structure.

It is understood that the organ having the three-dimensional vascular network formed by the step (b) in the present invention can function as a regenerative organ having or imitating all or part of the function of the organ or tissue intended for transplantation or the like. Let's be done. The formed regenerative organ may be used after being removed from the living body and taken out (removed) from the inside of the structure or included in the structure. In addition, a regenerated organ removed from a living body may be used for orthotopic or ectopic transplantation. The regenerative organ moves into and out of the structure that was originally used to form the three-dimensional vascular network to replace, reinforce, or restore the target organ or tissue or part thereof. The vascularized blood vessel and the blood vessel of the living body to be transplanted may be connected to the living body by, for example, vascular stitching.

A person skilled in the art will include organ primordia, vascular constituent cells, and optionally, factors such as angiogenic factors and drugs, in the desired order, as appropriate, within the structure, chamber transplantation, three-dimensional vessel It will be understood that formation of a net or formation of a regenerative organ can be performed. For example, the vascular constituent cells may be included in the structure simultaneously with the organ primordium, or may be included in the structure before or after the organ primordium. In addition to the angiogenic factor produced from the organ primordia, when an additional angiogenic factor is injected into the structure, the additional angiogenic factor is included when the organ primordia or vascular constituent cells are included in the structure. May be injected at the same time, or an additional angiogenic factor may be injected at a different time from when organ primordia or vascular constituent cells are included in the structure.

In the present invention, after removing an organ having a three-dimensional vascular network formed in vivo or a reconstructed organ or a structure containing the same, a living body that has been excised using, for example, an artificial blood vessel from the viewpoint of life maintenance. It is preferable to connect blood vessels in the body.

In another embodiment, the present invention can be formed (obtainable) by the “method of forming an organ having a three-dimensional vascular network in vivo in an animal” of the present invention. Or a regenerative organ or a structure containing the same. Here, the organ or regenerative organ having a three-dimensional vascular network that can be formed (obtainable) or a structure including the same is limited to that manufactured by the method specifically exemplified in this specification. Of course, those manufactured by any forming method included in the concept of the present invention are also targeted.

Furthermore, in another embodiment, the present invention is a three-dimensional shape formed (obtained) by the “method for forming an organ having a three-dimensional vascular network in vivo in an animal” of the present invention. Provided is an organ having a vascular network or a regenerative organ or a structure including the same. The formed organ or regenerative organ having a three-dimensional vascular network or a structure containing the same may be used for transplantation after being removed from a living body. In addition, an organ having a three-dimensional vascular network produced by the method of the present invention is connected to an organ or tissue perfusion culture method and perfusion culture apparatus as described in WO2011 / 093268, for example, before transplantation. You may perform culture | cultivation in.

In the present invention, the transplantation of an organ having a three-dimensional vascular network to be formed or a regenerative organ or a structure including the same is performed by setting the temperature of the organ having a three-dimensional vascular network to be formed or a structure including the organ to 4. It can be carried out while maintaining a state of from 35 ° C. to 37 ° C., preferably carried out while maintaining a state of from 15 ° C. to 33 ° C., and most preferably carried out while maintaining a state of from 20 ° C. to 25 ° C.

<Composition for forming a three-dimensional vascular network>
Another embodiment of the present invention is a composition for forming an organ having a three-dimensional vascular network, comprising:
The composition is a substantially sheet-like composition, and can be configured as a structure including one or a plurality of openings and a hollow portion inside.
here,
One or more blood vessels are disposed through the opening and through the structure;
The structure is substantially closed except for the opening through which the blood vessel passes;
In the structure, an organ primordium is included, and an organ having a three-dimensional vascular network is formed in a hollow portion in the structure.
It is characterized by that.
As long as there is no technical contradiction, the present invention may be implemented by appropriately combining any one or more of all aspects described in this specification.

A substantially sheet-like composition (that is, a substantially sheet-like composition) that can be used to form (manufacture) an organ having a three-dimensional vascular network of the present invention finally encloses (encloses) the blood vessel. Any shape is possible as long as it is possible. The substantially sheet-like composition may be molded in advance so as to have a hollow portion inside, or when arranged so as to enclose the blood vessel, it is molded so as to have a hollow portion inside. Also good. For example, a structure in which the inner peripheries of two substantially sheet-shaped compositions that are overlapped are finally fixed over one circumference so as to form a bag (closed space), and a hollow portion is provided inside. May be provided. Alternatively, one substantially sheet-shaped composition is folded in half, and the inner peripheries that come into contact with each other, for example, are wrapped in the skin of dumplings (a kind of Chinese or Japanese food), and there is a space inside It may be fixed as possible. Alternatively, one substantially sheet-shaped composition is folded, or a plurality of substantially sheet-shaped compositions are joined together to have a hollow portion inside, for example, a substantially columnar body (including a cylinder and a prism (including a rectangular parallelepiped and a cube). Etc.), a substantially conical shape (such as a cone or a pyramid), or a substantially spherical shape.

The substantially sheet-like composition does not permeate cells contained in the structure and cells derived from the recipient tissue existing outside the structure (cell-impermeable material, membrane, film, etc.) Any material may be used as long as it is. Moreover, it is preferable that the said substantially sheet-like composition is comprised with a biocompatible material. The substantially sheet-like composition may be made of a material that can be stretched or deformed. In a preferred embodiment, the substantially sheet-shaped composition does not pass cells contained in the structure and cells derived from the recipient tissue existing outside the structure, but humoral factors (water, cytokines, etc.) ) May be (freely) allowed to pass through (eg, a semipermeable membrane / porous membrane such as a dialysis membrane). The material of the substantially sheet-shaped composition is not limited, but may be, for example, regenerated cellulose (cellophane), acetylcellulose, polyacrylonitrile, Teflon, polyester polymer alloy, or polysulfone.

In addition, for example, when it is intended to use an organ having a three-dimensional vascular network formed or a regenerative organ by removing it from the structure, the substantially sheet-like composition contains blood vessels and cells contained in the structure. It is preferable that it is non-adhesive or can be easily separated. Moreover, it is preferable that the said substantially sheet-like composition is transparent or semi-transparent so that the inside of a structure can be confirmed easily.

The terms used in the present specification are used to describe a specific embodiment, and are not intended to limit the invention.

As used herein, the terms “comprising” or “including” include the items (members, steps, elements, numbers, etc.) described, unless the context clearly requires different understanding. It does not exclude the presence of other matters (members, steps, elements, numbers, etc.). When it is possible to exclude the presence of other items (members, steps, elements, numbers, etc.), the term “consist of” may be used. The term “contains” or “includes” encompasses the concept of the term “consisting of”.

Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms used herein should be interpreted as having a meaning consistent with the meaning in this specification and the related technical field, unless otherwise defined, idealized, or overly formal. It should not be interpreted in a general sense.

The embodiment of the present invention may be described with reference to a schematic diagram, but in the case of a schematic diagram, it may be exaggerated for clarity of explanation.

Although terms such as first, second, etc. are used to represent various elements, it is understood that these elements should not be limited by those terms. These terms are only used to distinguish one element from another, for example, the first element is referred to as the second element, and similarly, the second element is the first element. Can be made without departing from the scope of the present invention.

In the present specification, any numerical value used to indicate the component content, numerical value range, etc. is interpreted as including the meaning of the term “about” unless otherwise specified.

References cited in this specification are to be regarded as the entire disclosure of which is hereby incorporated by reference, and those skilled in the art will recognize the spirit of the invention in accordance with the context of this specification. And without departing from the scope, the relevant disclosures in those prior art documents are hereby incorporated by reference.

In the following, the present invention will be described in more detail with reference to examples. However, the invention can be embodied in various ways and should not be construed as limited to the embodiments set forth herein.

It is known that immature cells obtained from fetuses in the process of organ transplantation of organ primordia (and vascular constituent cells) release abundant angiogenic factors. In this example, chamber transplantation using a plurality of types of fetal organ primordia was performed.
The organ primordium immediately after transplantation is maintained only by substance supply by natural diffusion. Accordingly, the present inventors have found that it is preferable to place the organ primordium close to the blood vessel and reduce the volume of the suspension of vascular constituent cells relative to the organ primordia. In addition, to reduce the amount of gel and the number of cells that can be placed in close proximity to blood vessels and to minimize the size of the implant, a wide variety of tissues can be used for chamber transplantation. The present inventors have found that can be used.

<Chamber transplantation of organ primordia>
As vascular constituent cells, HUVEC (Lonza, Basel, Switzerland) and hMSC (Lonza, Basel, Switzerland) were each collected after culture and subjected to cell count. HUVEC (8 × 10 ⁵ cells) and hMSC (2 × 10 ⁵ cells) were mixed in a 50 ml tube (BD Falcon, NJ, USA). In order to remove the influence of cytokines contained in the culture medium, an additive-free DMEM medium was added and centrifugation was repeated twice to wash the cells. The cell suspension was transferred to a 1.5 ml tube (eppendorf, Tokyo, Japan), centrifuged again, and all the supernatant was removed and tapped. Then, it was suspended in collagen gel type-IA (Nitta Gelatin, Osaka, Japan) and used for chamber transplantation.

A subcutaneous tissue including blood vessels in the dorsal skin of a nude mouse was partially exfoliated, and then a dialysis membrane with a molecular weight of 50 kD cut-off using Aron Alpha A (Sankyo, Tokyo, Japan) on one exfoliated blood vessel part ( (Adia, Tokyo, Japan). Next, after placing the organ primordium so as to directly touch the blood vessel in the peeled subcutaneous tissue from the unencapsulated part provided in the upper part of the chamber, the already prepared HUVEC and hMSC cell suspension-containing gel was prepared. It added so that it might cover from a top with respect to a base. Next, the unencapsulated portion was sealed, and a completely closed space was created except that one peeled blood vessel penetrated the chamber.
The combinations of organ primordia and vascular constituent cells used for chamber transplantation are as follows:
(1) The left lobe of the liver primordium obtained from a fetal mouse of day 12; collagen gel type-IA supplemented with vascular constituent cells (HUVEC and hMSC);
(2) The left lobe of the liver primordium obtained from a fetus at 12 days of gestation; collagen gel type-IA supplemented with vascular constituent cells (HUVEC and hMSC) and VEGF;
(3) Collagen gel type-IA supplemented with a tooth germ obtained from a 14-day-old mouse fetus and VEGF;
(4) Collagen gel type-IA supplemented with ratoke sac obtained from a fetus at 18 days of age and VEGF;
(5) Collagen gel type-IA supplemented with ratchet sac obtained from a mouse fetus at 18 days of gestation, vascular component cells (HUVEC and hMSC) and VEGF
VEGF was used at a final concentration of 100 ng / ml.
The skin in which the chamber was installed was sutured and the progress was observed.

<Tissue analysis after organ transplantation of organ primordia (and vascular constituent cells)>
In order to analyze the tissue structure of the transplanted organ primordia, nude mice transplanted with each organ primorum excluding the above-mentioned tooth germ were bred up to 7th or 14th day after transplantation. In addition, nude mice transplanted with tooth germs were grown until the third or seventh day after transplantation. Each mouse was then sacrificed and the formation in the chamber was formalin fixed. For each formation, 5 μm tissue slices were prepared and stained with hematoxylin and eosin (HE). HE staining was performed by staining with 1% eosin solution (Mudo Chemical Co., Tokyo, Japan) after staining with Mayer's hematoxylin (Wako, Osaka, Japan) by a conventional method.
As a result, in the chamber transplantation using the liver primordium, hepatocyte engraftment was confirmed on the 7th and 14th days (FIG. 2). In the chamber transplantation using the tooth germ, the tissue structure of the tooth germ was maintained on the third day, and the invasion of blood vessels from the mesenchymal side was also confirmed. Furthermore, on the 7th day, it was confirmed that the accumulation of hard tissue progressed and the generation of tissue progressed compared to the 3rd day (FIG. 3). In the chamber transplantation using Ratoke sac, maintenance of the tissue structure was observed on the 7th day, and the tuft-like tissue structure observed in the pituitary was confirmed on the 14th day (FIG. 4).
From the above results, when chamber transplantation was performed using organ primordia, it was confirmed that a three-dimensional vascular network was constructed in the tissue along with the generation of organ primordia. It was shown that training is possible.

The method for forming an organ having a three-dimensional vascular network of the present invention in vivo can be advantageously used in the field of regenerative medicine, application to a screening system for evaluating the effects of drugs, and the like.

Claims

A method of forming an organ having a three-dimensional vascular network in vivo in a non-human animal, comprising:
The following steps:
(A) in the non-human animal,
Arranging one or more blood vessels to penetrate the structure while connected to the living body, or to pass the isolated blood vessel or blood vessels through the structure in vitro. Connecting the “isolated one or more blood vessels” to a blood vessel in a living body after placement, wherein the structure is configured to have a hollow portion therein, and the structure Contains an organ primordium;
(B) An organ having a three-dimensional vascular network in a hollow portion inside the structure by culturing the blood vessel and the organ primordium contained in the structure in vivo inside the structure. Forming the method.
The method of claim 1, comprising:
The step (a)
(I) before placing the blood vessel so as to penetrate the structure, the organ primordium is included in the structure, or
(Ii) The organ primordium is included in the structure after the blood vessel is disposed so as to penetrate the structure.
Method.
The method according to claim 1 or 2, wherein
In the step (a), in addition to the organ primordium, the structure further contains vascular constituent cells.
Method.
The method of claim 3, comprising:
The vascular constituent cells include vascular endothelial cells and / or mesenchymal stem cells,
Method.
A method according to claim 3 or 4, wherein
In the step (a), the structure further contains an angiogenic factor.
Method.
6. A method according to claim 5, wherein
The angiogenic factor is selected from the group consisting of VEGF, FGF, EGF, PDGF, TGF-β;
Method.
The method of claim 1, comprising:
The organ primordium is selected from the group consisting of hair follicle primordium, tooth germ primordium, liver primordium, kidney primordium, ratchet sac, and endocrine organ primordium,
Method.
The method of claim 1, comprising:
The organ primordium is an ES cell, iPS cell, pluripotent progenitor cell, adult stem cell, or regenerative organ primordium prepared from a pluripotent cell genetically modified from these.
Method.
A method according to any one of claims 1 to 8, comprising
The following steps:
(C) after the step (b), further comprising the step of orthotopically or ectopically transplanting the organ having the formed three-dimensional vascular network,
Method.
A method according to any one of claims 1 to 9, comprising
The structure consists of a dialysis membrane that does not allow cells to pass but allows humoral factors to pass through.
Method.
A method according to any one of claims 1 to 10, comprising
Instead of the non-human animal, a human is used.
Method.
A composition for forming an organ having a three-dimensional vascular network,
The composition is a substantially sheet-like composition, and can be configured as a structure including one or a plurality of openings and a hollow portion inside.
here,
One or more blood vessels are disposed through the opening and through the structure;
The structure is substantially closed except for the opening through which the blood vessel passes;
In the structure, an organ primordium is included, and an organ having a three-dimensional vascular network is formed in a hollow portion in the structure.
The composition characterized by the above-mentioned.
An organ having a three-dimensional vascular network, obtainable by the method according to any one of claims 1 to 11.
An organ having a three-dimensional vascular network obtained by the method according to any one of claims 1 to 11 (obtained).
A regenerative organ including the organ having the three-dimensional vascular network according to claim 13 or 14.