WO2014030418A1

WO2014030418A1 - Vascular tissue and method for producing same

Info

Publication number: WO2014030418A1
Application number: PCT/JP2013/066993
Authority: WO
Inventors: 実関; 真澄山田; 優輝岩瀬
Original assignee: 国立大学法人千葉大学
Priority date: 2012-08-18
Filing date: 2013-06-20
Publication date: 2014-02-27
Also published as: JPWO2014030418A1; JP6241890B2

Abstract

Provided is a novel technique for producing functional vascular tissue having a layered structure, a hollow structure, and a branched structure combined by a simple operation, which requires neither the complex position control devices or operations that are essential to existing techniques for the ex vivo production of vascular tissue at least partially comprising cells, nor a convoluted multiple-stage process. The vascular tissue obtained by said novel technique is also provided. A method for producing the vascular tissue is used by which a first aqueous sodium alginate solution, in which cells have been suspended, is introduced into a channel structure comprising a material that contains polyvalent metal cations or that contains an inorganic salt containing the polyvalent metal cations, a first alginate hydrogel layer containing the cells is formed on the inner walls of the channel structure, and thereafter the first aqueous sodium alginate solution which remains in the channel structure and has not gelated is removed.

Description

Vascular tissue and method for producing the same

The present invention relates to a vascular tissue and a production method thereof.

An artificially constructed blood vessel-like tissue or material is essential as an artificial blood vessel graft for transplantation in the surgical field. In addition, for the purpose of regenerative medicine, in order to form a relatively large tissue body in vitro, it is necessary to efficiently supply oxygen and nutrients to internal cells, and embedding a luminal tissue. Is desirable. Furthermore, when performing physiological evaluation of cells in an environment that mimics the structure of a living body, it is necessary to create a tissue having a vascular network. In order to meet these demands, technological development for artificially producing functional vascular tissue in vitro is currently under extremely intense competition worldwide.

Vascular tissue prepared for such applications is capable of delivering liquid inside, has strength to withstand transplantation operations, etc., and is relatively close to actual living tissue It is necessary to satisfy the conditions such as

For example, as an ordinary artificial blood vessel, an artificial material such as polyester or PTFE or a biomaterial such as collagen or gelatin is generally used, but those having a diameter of about 5 mm or less are blocked by platelets or the like. Since it is easy, the diameter of the artificial blood vessel put into practical use in the medical field is usually 5 mm or more. As an artificial blood vessel having higher biocompatibility and capable of preventing thrombus formation, it is desirable to use a vascular tissue composed at least partially of cells.

The vascular tissue in the living body has a multi-layered structure in which vascular endothelial cells are organized in a single layer inside vascular smooth muscle cells, and has a hollow structure for circulating blood inside. In order to efficiently circulate nutrients throughout the body tissue, it is a relatively complex tissue that branches and merges as necessary. Therefore, in order to produce a functional vascular tissue in vitro, it is necessary to develop a technique for reproducing these structures simply and accurately, and a technique for arranging cells three-dimensionally is required.

Various techniques have been developed so far to construct a vascular tissue composed of cells in vitro. As an example, formation and winding of a cell sheet using a temperature-responsive culture substrate as shown in Non-Patent Document 1, embedding of cells in a fibrous hydrogel material as shown in Non-Patent Document 2, Seeding of cells into a hollow polymer scaffold (substrate used as a scaffold for cell culture) as shown in Non-Patent Document 3, and a cell cluster as shown in Non-Patent Document 4 as a unit structure Application of inkjet printing used.

However, a tissue structure having a branched structure is produced by a method of winding a cell sheet or a method of embedding cells in a fiber-like hydrogel material, which has been developed so far. It is difficult.

The technique using inkjet printing as reported in Non-Patent Document 4 is almost the only existing technique that enables the production of a vascular tissue having all of a layered structure, a hollow structure, and a branched structure. In this technique, spherical cell clusters called spheroids and rod-shaped agarose gels that do not contain cells are accurately placed and cultured by inkjet technology. Since the cells cannot adhere to the agarose gel, the spheroids adhere to each other, and a three-dimensional arbitrary shape tissue can be produced.

However, the method described in Non-Patent Document 4 has a problem that a very complicated and special device for controlling the position of the cell three-dimensionally is required. In addition, creating a hollow structure requires a multi-step process in which agarose gel guides and spheroids are stacked step by step, and a complex drive mechanism is required to accurately and individually control the position of the object. It is. Further, since the cell mass is ejected by the ink jet mechanism, there is a possibility of damaging the cells.

Furthermore, in order for a tissue prepared in vitro to function as a vascular tissue, it is essential that a liquid can be introduced into the inside thereof. However, a vascular tissue prepared by the technique described in Non-Patent Document 4 Since the inside of the agarose gel used as a guide is filled, a process for removing the agarose gel by heat treatment or enzyme treatment is required. On the other hand, the agarose gel present in the tissue is very difficult to remove because almost the entire surface is covered with cell clumps.

Furthermore, when a multilayered blood vessel structure composed of different cells is produced using the method described in Non-Patent Document 4, three types of objects, an inner cell, an outer cell, and an agarose gel, are sequentially used. Since it is necessary to arrange and accumulate, there is a problem that the operation process becomes more complicated, and the time required for tissue construction increases and cell function may be lowered.

The present invention has been made in view of the above-described problems of the prior art, and an object of the present invention is to produce a vascular tissue composed of cells at least partially in vitro. It does not require complex position control devices and operations and multi-step complicated processes that are essential in existing methods, and combines all the structures of layered structures, hollow structures, and branched structures with simple operations. It is intended to provide a new method for producing a functional vascular tissue and to provide a vascular tissue obtained by the new method.

In order to achieve the above object, the invention according to one aspect of the present invention is directed to a channel structure constituted by a material containing an inorganic salt containing at least one of a polyvalent metal cation or a polyvalent metal cation. The first sodium alginate aqueous solution in which cells are suspended is introduced to form a first alginate hydrogel layer containing cells on the inner wall of the channel structure, and then the gel remaining in the channel structure is not gelled. In this method, the first sodium alginate aqueous solution is removed. By using such a method, alginic acid is cross-linked by polyvalent metal cations supplied from the material constituting the flow path structure simply by introducing a sodium alginate aqueous solution in which cells are suspended in the flow path structure. Since an alginate hydrogel layer containing cells is formed on the inner wall of the flow channel structure, it is possible to produce a hollow vascular tissue in any form depending on the shape of the flow channel structure by a very simple operation. It becomes possible.

Further, in the invention according to this aspect, the second sodium alginate in which cells are suspended in the flow channel structure after removing the first sodium alginate aqueous solution that is not gelled is not necessarily limited. After introducing an aqueous solution and forming a second alginate hydrogel layer containing cells on the inner wall of the first alginate hydrogel layer, the second sodium alginate remaining in the channel structure and not gelled remains. It is preferable to remove the aqueous solution. By doing so, it becomes possible to produce a multi-layered vascular tissue structure consisting of an inner layer and an outer layer very easily, and it is possible to obtain a three-dimensional tissue body that more closely mimics the actual vascular structure. It becomes possible.

In the invention according to this aspect, although not limited, a cell suspension is introduced into the channel structure after the first sodium alginate aqueous solution or the second sodium alginate aqueous solution is removed. It is also possible to do. In this way, since it becomes possible to further adhere cells to the inner wall surface of the alginate hydrogel layer containing cells, the cells are organized into a single layer on the inner wall surface in the same manner as the actual vascular tissue. It is possible to produce a vascular tissue.

Further, in the invention according to this aspect, although not limited, immediately before the introduction of the first aqueous sodium alginate solution, the flow channel structure does not contain a polyvalent cation or a polyvalent cation. It is preferable to introduce an aqueous buffer solution having a sufficiently low cation concentration. By doing so, it becomes possible to moderately remove the polyvalent metal cations present in the vicinity of the flow channel structure, so that the gelation rate of alginic acid can be adjusted, and the blood vessel to be produced The thickness of the tissue can be easily and accurately controlled.

In the invention according to this aspect, the buffer aqueous solution may contain at least one of citric acid and ethylenediaminetetraacetic acid, although not limited thereto. By doing so, it becomes possible to more efficiently remove the polyvalent metal cations present in the material constituting the flow channel structure, so that the gelation rate of alginic acid can be adjusted more accurately. Is possible.

Further, in the invention according to this aspect, although not limited, after removing the first sodium alginate aqueous solution or after removing the second sodium alginate aqueous solution, It is preferable to introduce an aqueous solution containing a polyvalent metal cation. By doing so, it becomes possible to completely gel the alginate hydrogel layer formed on the inner wall of the flow channel structure from the inside, and it is possible to produce a vascular tissue with higher strength.

Moreover, in the invention according to this aspect, the inner wall of the flow path structure can be obtained by subjecting the material constituting the flow path structure to at least one of physical operation and chemical treatment. It is also possible to separate and recover the alginic acid hydrogel layer containing the cells formed in the above from the flow channel structure. By doing so, it is possible to collect the vascular tissue formed in the flow path, and thus, for example, it is possible to provide a vascular tissue that can be used for vascular transplantation treatment. It is possible to provide a vascular tissue as a unit structure useful for constructing a large cell tissue.

In the invention according to this aspect, the material constituting the flow channel structure is preferably at least partially a hydrogel material, although not limited thereto. By doing so, it becomes possible to efficiently supply the polyvalent metal cation necessary for gelling alginic acid to the aqueous sodium alginate solution existing inside the channel structure, and to culture cells. In some cases, oxygen and nutrients can be supplied from the material constituting the channel structure.

Further, in the invention according to this aspect, although not limited, the hydrogel material includes at least one of alginic acid, agarose, gelatin, collagen, and polyethylene glycol diacrylate. Is preferred. In this way, it is biocompatible, has low toxicity to cells, has a relatively high physical strength, has a high water retention capacity, and enables efficient supply of nutrients and oxygen to cellular tissues. It is possible to produce a channel structure.

In the invention according to this aspect, the material constituting the flow path structure may be at least partially polydimethylsiloxane, although not limited thereto. By doing in this way, since it becomes possible to produce a highly precise and simple flow channel structure with high biocompatibility, it is possible to produce a vascular tissue more easily.

In the invention according to this aspect, the polyvalent metal cation is preferably at least one of calcium ion, strontium ion, and barium ion, although not limited thereto. In this way, when alginic acid is gelled, it becomes possible to reduce the toxicity to cells and to produce a high-strength vascular tissue.

In the invention according to this aspect, the concentration of sodium alginate contained in the first aqueous sodium alginate solution and the second aqueous sodium alginate solution is 0.1% (w / v), although not limited thereto. ) Or more and 10% (w / v) or less. By doing so, it becomes possible to form an alginic acid hydrogel layer having a relatively high strength on the inner wall of the flow channel structure.

Further, in the invention according to this aspect, although not limited, it is preferable that the inner wall surface of the flow path structure is previously surface-treated with a polycation. By doing in this way, since it becomes possible to adhere | attach the alginic acid hydrogel which has a negative charge more firmly to the inner wall surface of a flow-path structure, it becomes possible to form an alginate hydrogel layer more stably.

In the invention according to this aspect, although not limited, the polycation is preferably at least one of poly-L-lysine, chitosan, and polyethyleneimine. By doing in this way, it becomes possible to adhere | attach an alginate hydrogel layer firmly on the inner wall face of a flow-path structure.

In the invention according to this aspect, although not limited, at least one of collagen and Matrigel is added to at least one of the first aqueous sodium alginate solution and the second aqueous sodium alginate solution. May be. By doing so, it becomes possible to maintain the function of the cells embedded in the first alginate hydrogel layer or the second alginate hydrogel layer, and the inside of the formed alginate hydrogel layer. Since it is possible to promote the adhesion and proliferation of the introduced cells, it is possible to more efficiently form a single cell inner layer.

In the invention according to this aspect, although not limited, cell-adhesive sodium alginate is added to at least one of the first aqueous sodium alginate solution and the second aqueous sodium alginate solution. Also good. By doing so, it becomes possible to promote the adhesion and proliferation of cells introduced into the formed alginate hydrogel layer, so that it is possible to form a single cell inner layer more efficiently. Become.

In the invention according to this aspect, the cell is preferably a mammal-derived cell, although not limited thereto. By doing so, it is possible to construct a vascular tissue containing cells derived from mammals, and it is possible to provide materials useful in transplantation medicine and tissue engineering.

In the invention according to this aspect, the cells are preferably vascular smooth muscle cells, vascular endothelial cells, vascular epithelial cells, or a mixture thereof, although not limited thereto. By doing in this way, it becomes possible to produce a vascular tissue closer to a living tissue.

In the invention according to this aspect, the density of cells contained in the first sodium alginate aqueous solution and the second sodium alginate aqueous solution is 10 million or more per mL, respectively, although not limited thereto. Is preferred. By doing so, it becomes possible to embed cells densely inside the alginate hydrogel layer to be formed, adhesion between cells is promoted, and functional vascular tissue can be efficiently formed In addition, the physiological function of the cell can be maintained high.

Moreover, in the invention according to this aspect, although not limited, the cells contained in the first sodium alginate aqueous solution and the second sodium alginate aqueous solution are the type, density, and mixing ratio, respectively. It is preferable that at least one of them is different. By doing in this way, it is possible to produce a tissue closer to the vascular tissue of an actual living body constituted by a multilayer structure having different cell compositions.

Further, in the invention according to this aspect, although not limited, it is preferable that the first alginic acid hydrogel layer and the second alginic acid hydrogel layer each have a thickness of 10 micrometers or more. By doing so, it becomes possible to efficiently embed cells in the alginate hydrogel layer and to produce a high strength vascular tissue.

In the invention according to this aspect, the vascular tissue preferably has a multilayer structure, a hollow structure, and a branch structure at least partially. By doing in this way, it becomes possible to provide a vascular tissue closer to a living tissue.

The invention according to another aspect of the present invention is a vascular tissue produced using the vascular tissue production method according to any one of claims 1 to 21. Such vascular tissue can be used as a transplant material, used as a luminal structure in tissue engineering, and can be used to evaluate cell functions in a structure that highly mimics the anatomy. Alternatively, it is possible to provide materials that are very useful for academic applications.

Since the present invention is configured as described above, it does not require a special device required in the conventional method for arranging cells three-dimensionally, and the cells are suspended in the channel structure. It exhibits an excellent effect that a vascular tissue can be produced efficiently, simply and accurately by a very simple operation of introducing a turbid alginate aqueous solution.

In addition, since the present invention is configured as described above, there are only a few reported examples so far, and a functional blood vessel having all of a multilayer structure, a hollow structure, and a branched structure at the same time. Since the tissue can be easily produced, it is possible to provide a large amount of a material that highly mimics the vascular tissue in the living body.

Furthermore, since the present invention is configured as described above, the vascular tissue obtained using the present invention has excellent mechanical strength and high operability. Therefore, a material that is very useful in applications such as tissue engineering and regenerative medicine, such as creating a three-dimensional tissue incorporating a luminal structure by embedding it in another relatively large tissue, for example. It becomes possible to provide.

Furthermore, since the present invention is configured as described above, it is possible to produce a vascular tissue having an arbitrary shape by arbitrarily designing the shape of the flow path, and further, sodium alginate. By controlling the composition of the aqueous solution and the liquid feeding time, the thickness of the vascular tissue and the width of the hollow portion can be arbitrarily controlled. Therefore, it is possible to produce vascular tissue having various forms depending on the application.

Furthermore, since the present invention is configured as described above, for example, thrombus formation behavior or angiogenesis behavior in multilayered vascular tissue having various shapes, metastasis or invasion of cancer cells in blood vessels. It becomes possible to provide a very useful material for analyzing, analyzing, and evaluating various biological phenomena that occur in vascular tissue such as behavior.

It is the schematic which showed the formation process of the alginate hydrogel layer containing the most basic flow-path structure for vascular tissue preparation based on embodiment, and a cell. 1 (a) and 1 (b) are diagrams showing the structure of a device having a flow channel structure, FIG. 1 (a) is a view taken in the direction of arrow B in FIG. 1 (b), and FIG. FIG. 2B is a cross-sectional view of the flow path structure along the line A0-A1 in FIG. Fig. (C) is an enlarged view of a portion C in Fig. 1 (a) and a schematic diagram showing a process of forming an alginate hydrogel layer containing cells. It is the schematic which showed the process which forms the multilayered alginate hydrogel layer containing two types of cells based on embodiment. It is the schematic which showed a mode that the cell was organized into the monolayer inside the multilayered alginate hydrogel layer containing a different kind of cell based on embodiment. It is the schematic of the flow-path structure for producing the vascular tissue which has a branch based on embodiment. 4A is a view taken in the direction of arrow B in FIGS. 4B to 4D, and FIGS. 4B to 4D are lines A0-A1 and A2-A3 in FIG. 4A, respectively. A cross-sectional view of the flow path structure along line A4-A5 is shown. FIG. 5A is a schematic diagram of a flow path structure according to an example and a micrograph of a multilayered alginate hydrogel layer formed on the inner wall of the flow path structure, and FIG. 5A shows an overall view of the flow path structure. FIGS. 5B to 5E are photomicrographs at a position corresponding to the portion B in FIG. FIG. 5 (b) is a fluorescence micrograph showing the formation of the first alginate hydrogel layer containing fluorescent particles, and FIG. 5 (c) is just after the second sodium alginate aqueous solution is introduced. FIG. 5D shows a state in which a buffer aqueous solution containing green fluorescent particles flows inside the alginate hydrogel layer after the second alginate hydrogel layer is formed. FIG. 5E is a fluorescence micrograph, and FIG. 5E is a phase contrast micrograph of the formed alginate hydrogel layer. Example of other schematic diagram of channel structure according to Example, and multilayer alginate hydrogel layer embedded in two types of cells stained with different fluorescent dyes formed on the inner wall of channel structure FIG. 6A is a micrograph showing an overall view of the flow channel structure, and FIGS. 6B and 6C are phase contrast microscopes at positions corresponding to portion B in FIG. 6A, respectively. It is a photograph and a fluorescence micrograph. FIG. 7 is a micrograph of a multilayer alginate hydrogel layer containing different cells, separated by subjecting a material constituting a flow channel structure to chemical treatment, according to an example. ) Are a phase contrast micrograph and a fluorescence micrograph, respectively.

Hereinafter, the method for producing a vascular tissue and the best mode of the vascular tissue according to the present invention will be described in detail. However, the present invention can be implemented in many different forms, and is not limited only to the embodiments and examples described below.

FIG. 1 is a schematic view showing the most basic flow channel structure for producing a vascular tissue and the formation process of an alginate hydrogel layer containing cells. 1 (a) and 1 (b) are diagrams showing the structure of a device having a flow channel structure, FIG. 1 (a) is a view taken in the direction of arrow B in FIG. 1 (b), and FIG. FIG. 2B is a cross-sectional view of the flow path structure along the line A0-A1 in FIG. Fig. (C) is an enlarged view of a portion C in Fig. 1 (a) and a schematic diagram showing a process of forming an alginate hydrogel layer containing cells.

The material constituting the flow channel structure has physical strength that can maintain the formed flow channel structure, has low toxicity to cells, and alginic acid in which polyvalent metal cations contained in the material are introduced into the flow channel structure Any material can be used as long as it diffuses into the aqueous sodium solution, and various hydrogel materials and polymer materials can be used. However, from the viewpoint of operability, it is preferable that the medium component has a certain degree of strength and can be sterilized in advance, and the medium component penetrates into the material in order to assist the maintenance of cell functions. Therefore, a material having high water retention such as hydrogel is suitable. Typical hydrogel materials for constituting the channel structure include alginic acid, agarose, gelatin, collagen, polyethylene glycol diacrylate and the like.

On the other hand, a silicone polymer material such as polydimethylsiloxane has an advantage that the flow channel structure is easy to process, and can be premixed with an inorganic salt composed of polyvalent cations, and further an aqueous sodium alginate solution. When introduced into the inside of the channel structure, the alginate hydrogel layer is formed by elution of polyvalent cations from their salts, etc., so that the channel formed by such a polymer material. It is also possible to use a structure.

The flow channel structure shown in FIG. 1 is formed by laminating a flat substrate having a groove structure and a flat substrate having no groove structure on each other. A flat substrate having a groove structure can be formed, for example, by molding using a mold.

As shown in FIG.1 (c), in this invention, the alginic acid which suspended the cell with respect to the flow-path structure comprised with the raw material containing the polyvalent metal cation or the inorganic salt containing a polyvalent metal cation was shown. When an aqueous sodium solution is introduced, alginic acid is cross-linked by the polyvalent metal cations supplied from the material constituting the flow path structure, and an alginate hydrogel layer containing cells is formed on the inner wall of the flow path. It is possible to produce a vascular tissue having a hollow structure and a layered structure depending on the shape.

In order to include a polyvalent metal cation in the material constituting the flow path, when the material constituting the flow path is a hydrogel material, the polyvalent metal cation is previously added to the sol solution before forming the hydrogel. An aqueous solution containing a cation or an inorganic salt containing a polyvalent metal cation can be mixed and then gelled to produce a flow channel structure. A valent metal cation can be included. Moreover, it is also possible to include polyvalent metal cations in the material constituting the channel structure by immersing the entire channel structure thus prepared in an aqueous solution containing polyvalent metal cations for a certain period of time. On the other hand, when polydimethylsiloxane is used as a material constituting the flow channel structure, an inorganic salt containing a polyvalent metal cation is mixed in advance with the polydimethylsiloxane prepolymer, and then the flow channel structure is prepared by molding. Is preferred.

The size of the flow channel structure can be set so that the depth and the width are arbitrary values of 50 micrometers or more and 10 millimeters or less, respectively, according to the structure of the vascular tissue to be manufactured.

The cross section of the channel structure can be rectangular, circular, or any other shape. However, the rectangular cross section has the advantage that it can be easily formed by a molding process, and the circular section has the advantage that a vascular tissue closer to the anatomy can be easily formed.

As the polyvalent metal cation contained in the material constituting the channel structure, any polyvalent metal ion capable of gelling alginic acid can be used. However, at least one of calcium ion, strontium ion, and barium ion is preferable from the viewpoint of low toxicity to cells and cost. More specifically, when the material constituting the flow channel structure is a hydrogel material, it is preferable to dissolve these chlorides in a sol solution in advance, and when polydimethylsiloxane is used as the material, these chlorides are used. Products, sulfates and carbonates are preferably used.

The concentration of the polyvalent metal cation contained in the material constituting the flow channel structure may be any value as long as it allows gelation of alginic acid and is not toxic to cells. It is preferably 100 mM or less. In addition, since the time required for gelatinization of alginic acid becomes shorter as the concentration of the polyvalent metal cation is higher, it is preferable to optimize this concentration according to the size of the flow channel structure to be used.

It is necessary to suspend cells in the sodium alginate aqueous solution. Therefore, the ionic strength of the aqueous sodium alginate solution needs to be appropriately adjusted in advance, and the aqueous sodium alginate solution is preferably sterilized before suspending the cells.

By using vascular smooth muscle cells, vascular endothelial cells, vascular epithelial cells, etc. as cells suspended in an aqueous sodium alginate solution, a multilayered vascular tissue close to the actual vascular tissue can be prepared. However, if necessary, construct a complex vascular structure in individual tissues or organs by using cells such as fibroblasts, myoblasts, hepatocytes, nerve cells, cardiomyocytes, etc. Is also possible.

The density of cells suspended in a sodium alginate aqueous solution is preferably 10 million or more per mL. By suspending cells in high density in this way, the cells are densely embedded inside the alginate hydrogel layer that is formed, which promotes adhesion between cells and efficiently creates functional vascular tissue. In addition to being able to form, it is possible to maintain high physiological functions of cells.

Collagen, Matrigel, etc. can be added to the sodium alginate aqueous solution in advance. By doing so, it becomes possible to assist the maintenance of the function of the cells embedded in the sodium alginate aqueous solution, and the cells can adhere to the alginate hydrogel layer to be formed. By introducing cells inside, it becomes possible to form a vascular tissue in which the inner cells are organized into a single layer, and it is possible to construct a tissue in a form closer to the actual vascular tissue.

Moreover, cell-adhesive sodium alginate may be added to the sodium alginate aqueous solution. By doing so, it becomes possible to impart cell adhesion to the formed alginate hydrogel layer, and by introducing cells inside the alginate hydrogel layer, the inner cells are organized into a single layer. It becomes possible to produce a vascular tissue in a morphological form. For example, sodium alginate to which RDG peptide or collagen is covalently bonded can be used.

In order to stably form the alginate hydrogel layer on the inner wall of the channel structure, it is desirable that the inner wall surface of the channel structure is modified with a polycation in advance. For example, it is possible to modify the inner wall surface of the channel structure by introducing a polycation aqueous solution.

As the polycation contained in the polycation aqueous solution, at least one of poly-L-lysine, chitosan, and polyethyleneimine can be used. In addition, since it exists in the tendency for the adhesive strength of an alginate hydrogel layer and the inner wall surface of a flow-path structure to increase, so that the molecular weight of these polycations becomes high, it is preferable to use a thing with an average molecular weight of 100,000 or more. In addition, the concentration of the polycation aqueous solution and the amount of the introduced liquid may be any value, but it is necessary to consider that there is an amount of polycation molecules that sufficiently coats the inner wall surface of the channel structure. is there. Furthermore, since polyvalent metal cations contained in the material constituting the channel structure may be partially removed by introducing the polycation aqueous solution, the polycation aqueous solution may be used as necessary. A polyvalent metal cation may be added in advance.

In order to control the gelation rate of alginic acid, it is possible to introduce an aqueous buffer solution that does not contain a polyvalent metal cation or has a sufficiently low concentration just before introducing an aqueous sodium alginate solution, if necessary. . This operation is effective in controlling the gelation rate of alginic acid, particularly when using a thin channel structure made of hydrogel and having a diameter of several millimeters or less. Further, by dissolving a substance having a chelating effect on the metal cation such as citric acid or ethylenediaminetetraacetic acid (EDTA) in the buffer aqueous solution, the metal cation can be more efficiently removed in a short time. The higher the concentration of citric acid or EDTA in the buffer aqueous solution, or the longer the solution feeding time of the buffer aqueous solution, the slower the gelation of alginic acid. Therefore, these values tend to form an alginate hydrogel layer. It is preferable to optimize in advance.

After the alginate hydrogel layer is formed, it is preferable to introduce an aqueous solution containing a polyvalent metal cation into the flow channel structure so that the alginate hydrogel layer is completely gelled from the inside. By doing so, it is possible to prevent the alginate hydrogel layer and the cells embedded therein from being removed by the shearing force of the solution flowing inside the alginate hydrogel layer. In addition, the polyvalent metal cation to be introduced may be the same as or different from that contained in the material constituting the flow channel structure, but should be less toxic to cells. Is preferable, and is preferably at least one of calcium, strontium, and barium ions.

After forming an alginate hydrogel layer containing cells, vascular tissue can be efficiently formed by performing a culture operation as necessary. In addition, it is preferable to introduce a liquid medium into the channel structure during culture. By doing so, it is possible to efficiently supply oxygen and nutrients to the cells. In addition, when a hydrogel material is used as the material constituting the flow channel structure, cells in the flow channel can be more efficiently immersed in advance by immersing the entire device in a liquid medium and allowing the medium components to penetrate into the material. It is possible to supply oxygen and nutrients. In addition, it is possible to perform culture | cultivation operation, without impairing the mechanical strength of an alginate hydrogel layer by using the culture medium which added the polyvalent metal cation previously.

FIG. 2 is a schematic diagram showing a process for forming a multilayer alginate hydrogel layer containing two types of cells.

As shown in FIG. 2, a first sodium alginate aqueous solution containing cells A is introduced into a channel structure composed of a material containing a polyvalent metal cation, and the first alginate hydrogel layer containing cells A is introduced. Is formed, a second aqueous sodium alginate solution containing cells B is introduced. After the second alginate hydrogel layer containing cells B is formed inside the first alginate hydrogel layer containing cells A, an aqueous solution containing a polyvalent metal cation is introduced to completely gel the alginate hydrogel layer. As a result, it is possible to form a multilayer alginate hydrogel layer containing two types of cells in the outer layer and the inner layer on the inner wall of the channel structure.

In FIG. 2, a sodium alginate aqueous solution is introduced in two stages to form a two-layer alginate hydrogel layer. By repeating this introduction operation, three or more alginate hydrogel layers are produced. Is also possible. It is also possible to construct three or more layers of vascular tissue in which different cells are arranged in each layer by stepwise introducing a plurality of types of sodium alginate aqueous solutions in which different types of cells are suspended.

FIG. 3 shows a schematic diagram showing how cells are organized into a single layer inside a multilayer alginate hydrogel layer containing different types of cells.

As shown in FIG. 3, after a multilayer alginate hydrogel layer containing cells A in the outer layer and cells B in the inner layer is formed, an aqueous solution in which the cells C are suspended is introduced. In the state where it adheres to the inner wall surface of the gel layer, by culturing and growing the cells as necessary, different cells are organized into a single layer inside the multilayered alginate hydrogel layer containing different cells. It is possible to obtain an organized tissue structure. When cells that do not proliferate or have a low growth rate are used, it is preferable to embed cells A and B in high density in an alginate hydrogel layer in advance as necessary. It is also possible to introduce.

As shown in FIG. 3, by further organizing different cells into a single layer inside a multilayered alginate hydrogel layer containing different types of cells, the intima, media and outer layers in actual vascular tissue It is possible to produce a vascular tissue that highly mimics a multilayered form such as a membrane.

FIG. 4 shows a schematic view of a flow channel structure for producing a vascular tissue having a branch, and FIG. 4 (a) is a view as seen from the direction of arrow B in FIGS. 4 (b) to 4 (d). FIGS. 4B to 4D are cross-sectional views of the channel structure taken along lines A0-A1, A2-A3, and A4-A5 in FIG. 4A, respectively.

As shown in FIG. 4, by using a flow channel structure in which the flow channel connected to the inlet branches into a plurality of flow channels, finally converges to one flow channel and is connected to the outlet, It is possible to produce a vascular tissue that branches into and joins. A tissue having such a shape highly mimics the structure of an actual vascular tissue, so that it is useful for analyzing the physiological behavior of cells in the vascular tissue and is relatively large in vitro. It is also very useful as a material for forming tissue. In the case of producing a complex vascular tissue, a flow channel structure having a plurality of inlets and outlets may be used, or a flow channel structure that branches in the vertical direction may be used. Alternatively, different channel structures may be used in stages.

After forming an alginate hydrogel layer containing cells on the inner wall of the flow channel structure and performing a culture operation as necessary, the material constituting the flow channel structure is subjected to physical operation, chemical treatment, or both. By applying, it is possible to separate the formed vascular tissue from the channel structure.

For example, when a hydrogel material that can be decomposed by an enzyme is used as a material constituting the channel structure, the hydrogel can be chemically decomposed by immersing the entire device including the channel structure in an aqueous solution containing the enzyme. In addition, when the flow channel structure is composed of a hydrogel material that is decomposed or dissolved by temperature change, hydrogen ion concentration change, or light irradiation, the hydrogel is decomposed by performing these operations. It is possible to separate the vascular tissue from the channel structure. Further, in the case of a flow path structure constituted by two flat-plate substrates, it is possible to perform a physical operation such as physically peeling these substrates. However, regardless of which method is used, it is preferable to separate the formed vascular tissue under conditions that cause little damage to the cells.

Hereinafter, the effect of the present invention was confirmed by actually performing the method for producing a vascular tissue according to the above embodiment. This will be described below.

FIG. 5 shows a schematic diagram of the channel structure and a micrograph of a multilayer alginate hydrogel layer formed on the inner wall of the channel structure. FIG. 5 (a) shows the entire channel structure. FIGS. 5B to 5E are photomicrographs at positions corresponding to the portion B in FIG. FIG. 5 (b) is a fluorescence micrograph showing the formation of the first alginate hydrogel layer containing fluorescent particles, and FIG. 5 (c) is just after the second sodium alginate aqueous solution is introduced. FIG. 5D shows a state in which a buffer aqueous solution containing green fluorescent particles flows inside the alginate hydrogel layer after the second alginate hydrogel layer is formed. FIG. 5E is a fluorescence micrograph, and FIG. 5E is a phase contrast micrograph of the formed alginate hydrogel layer.

The flow channel structure shown in FIG. 5 (a) is composed of an agarose hydrogel prepared using an agarose aqueous solution containing calcium chloride. This flow channel structure is formed by producing an agarose hydrogel having a fine groove structure by molding using a mold, and superposing and bonding to a flat agarose hydrogel.

The agarose concentration in the agarose aqueous solution used to form the channel structure was 5% (w / v). Further, 10 mM calcium chloride was mixed in advance as a gelling agent for sodium alginate. The concentration of agarose may be any value as long as it is strong enough to perform a molding operation or the like for producing a flow channel structure during the formation of agarose hydrogel.

In this example, in order to accurately manufacture the channel structure, first, a template having a fine structure composed of SU-8 photoresist is prepared on a silicon substrate by photolithography, and molding is performed using the template. It was. Therefore, the cross-sectional shape of the flow channel structure is rectangular, and the width and depth thereof are 500 micrometers, respectively. Depending on the structure of the vascular tissue to be produced, these values can be set to any value between 50 micrometers and 10 millimeters, and are circular, elliptical, and other complicated cross sections. It is also possible to use a channel structure having a shape. In addition, the shape of the flow channel structure can be any shape depending on the structure of the vascular tissue to be produced.

In order to introduce the solution into the channel structure, silicon tubes were connected to the inlet and the outlet of the channel structure, respectively. In this embodiment, the end of the tube connected to the inlet is immersed in a container containing the aqueous solution to be introduced, and the aqueous solution is introduced into the flow path structure by suctioning from the end of the tube connected to the outlet using a syringe pump. Was introduced. In this way, liquid is delivered by suction, and different types of aqueous solutions are stepped into the channel structure by sequentially immersing them in individual containers containing aqueous solutions that introduce the ends of the silicon tubes connected to the inlet. Can be introduced efficiently and efficiently. It should be noted that any method can be used as long as the solution can be sent in order to the inside of the flow channel structure and the switching of the solution can be performed in a short time even if the solution is not sent by suction. Good. For example, the capillary tube can be prefilled with the aqueous solution to be introduced in order, then the end of the tube can be connected to the inlet of the flow channel structure, and the pressure can be applied from the end to send the liquid. is there.

Using the flow path structure and liquid feeding system configured as described above, first, it was verified whether an alginate hydrogel layer could be formed without using cells. The manufacturing method will be described.

For the channel structure constituted by the agarose hydrogel containing 10 mM calcium chloride shown in FIG. 5A, first, 0.05% (w / v) poly-L-lysine aqueous solution containing 10 mM calcium chloride is added. The solution was fed for 1 minute, and the inner wall surface of the channel structure was coated with polycation. Next, after feeding a 10 mM sodium citrate aqueous solution for 30 seconds, a first sodium alginate aqueous solution having a concentration of 0.5% (w / v) containing red fluorescent particles was fed. The flow rate of these aqueous solutions was 100 μL per minute as an example. The concentration, flow rate, and liquid feeding time of the poly-L-lysine and citric acid aqueous solution are conditions that allow the inner wall surface of the channel structure to be sufficiently coated and that calcium ions contained in the agarose hydrogel can be removed appropriately. Any value can be used.

Under the above conditions, an alginate hydrogel layer having a thickness of about 100 micrometers was formed by feeding a sodium alginate aqueous solution for about 1 minute. The thickness of the alginate hydrogel layer can be controlled by changing the flow rate of the sodium alginate aqueous solution or the liquid feeding time. It is also possible to adjust the strength of the formed alginate hydrogel layer by changing the concentration of the aqueous sodium alginate solution.

As shown in FIGS. 5B and 5C, after the first alginic acid hydrogel layer was formed, the second alginic acid hydrogel aqueous solution containing blue fluorescent particles was introduced. It was confirmed that the second alginic acid hydrogel layer was formed inside the hydrogel layer, and a multilayered alginic acid hydrogel layer was formed. Also, as shown in FIG. 5 (d), when a 10 mM calcium chloride aqueous solution containing green fluorescent particles was introduced as a gelling agent aqueous solution inside the formed multilayer alginate hydrogel layer, the formed alginate was obtained. It was observed that green fluorescent particles flowed inside the hydrogel layer, and it was confirmed that a multilayer alginate hydrogel layer having a hollow structure depending on the shape of the channel was formed.

Next, vascular tissue was constructed using a sodium alginate aqueous solution in which cells were suspended. The manufacturing method will be described.

FIG. 6 shows another schematic example of the flow path structure and a multilayer alginate hydrogel layer embedded in two types of cells stained with different fluorescent dyes formed on the inner wall of the flow path structure. FIG. 6A is an overall view of the flow channel structure, and FIGS. 6B and 6C are phase differences at positions corresponding to the portion B in FIG. 6A, respectively. It is a micrograph and a fluorescence micrograph.

As the sodium alginate solution in which the cells are suspended, 10 mM HEPES containing 0.5% (w / v) sodium alginate, 0.9% (w / v) sodium chloride, 0.1% (w / v) collagen is used. Buffer was used.

The flow channel structure shown in FIG. 6 (a) was produced by molding using 5% (w / v) agarose containing 10 mM calcium chloride and 0.8% (w / v) sodium chloride. In addition, when producing a flow channel structure using a hydrogel in this way, the ionic strength of the solution contained in the material constituting the flow channel structure may be adjusted in advance so as not to affect the cells. preferable.

The whole hydrogel material constituting the flow channel structure was immersed in a liquid medium containing 10 mM calcium chloride for 24 hours in advance to infiltrate nutrients into the hydrogel material.

As cells, fibroblasts were used as cells A contained in the first aqueous sodium alginate solution, and myoblasts were used as cells B contained in the second aqueous sodium alginate solution. These concentrations were 4 × 10 ⁷ cells / mL and ⁷ × 10 ⁷ cells / mL, respectively.

As shown in FIGS. 6B and 6C, the first sodium alginate aqueous solution containing the cells A is introduced into the flow channel structure constituted by the agarose hydrogel containing calcium chloride, and the cells A are contained. After the first alginate hydrogel layer is formed, a second sodium alginate aqueous solution containing cells B is introduced, whereby the second alginate hydrogel layer containing cells B is contained inside the first alginate hydrogel layer containing cells A. It was confirmed that an alginate hydrogel layer was formed. Further, when a 10 mM calcium chloride aqueous solution containing green fluorescent particles is introduced as a gelling agent aqueous solution inside the formed multilayer alginate hydrogel layer, green fluorescent particles are formed inside the formed alginate hydrogel layer. It was confirmed that a multi-layered alginate hydrogel layer containing different cells and having a hollow structure was formed by such an operation.

Even when human-derived vascular smooth muscle cells and vascular endothelial cells were used as the cells A and B, it was possible to prepare vascular tissues in the same manner. Moreover, it was possible to produce a vascular tissue in the same way inside the channel structure having branches.

FIG. 7 shows a micrograph of a multi-layered alginate hydrogel layer containing different cells separated by subjecting the material constituting the channel structure to chemical treatment, and FIG. And (b) are a phase contrast micrograph and a fluorescence micrograph, respectively.

As shown in FIG. 7, after the alginate hydrogel layer containing cells is formed on the inner wall of the channel structure, the entire device constituted by the agarose hydrogel is immersed in a phosphate buffer containing agarase. By performing the operation, it was possible to dissolve the agarose hydrogel by enzymatic reaction and to separate the vascular tissue from the channel structure.

Further, as shown in FIG. 7, it was confirmed that the vascular tissue separated and recovered from the flow channel structure maintained a multilayer structure even after being separated. Furthermore, it was confirmed that the mechanical strength was high enough to enable operation with tweezers or the like. In addition, it was possible to control the mechanical strength of the structure | tissue formed by changing the density | concentration of the sodium alginate aqueous solution.

Furthermore, it has been confirmed that the collected vascular tissue can circulate liquid inside, and the vascular tissue produced by using this method can be used as an artificial vascular graft for transplantation in a surgical field or a luminal structure. It was shown that it can be used as a unit structure for the construction of a relatively large three-dimensional organization that incorporates.

Since the present invention is configured as described above, the present invention highly mimics the original vascular tissue morphology, which has all of the multilayer structure, hollow structure, and branch structure, which has never been reported so far. Therefore, it is possible to provide a useful technique in the medical field and the biochemical research field because it is possible to easily produce the tissue body without requiring a special device or a complicated process.

In addition, since the present invention is configured as described above, it is possible to produce and provide a fine artificial blood vessel graft having a diameter of about 5 mm or less, which has been practically used so far and is composed of cells. Therefore, a wide range of applications in the surgical field is expected.

In addition, since the present invention is configured as described above, by appropriately setting the shape of the flow channel structure to be used and the feeding time of the sodium alginate aqueous solution according to the structure of the vascular tissue to be produced, A vascular tissue having an arbitrary shape and thickness can be easily produced, and a vascular tissue having various forms can be constructed depending on the application. Therefore, for example, it is possible to provide a new technique for producing various types of artificial blood vessel grafts having high strength, which are essential for blood vessel transplantation.

In addition, since the present invention is configured as described above, for example, it is possible to produce a tissue body in which vascular endothelial cells are organized into a single layer on the inner wall surface of an alginate hydrogel layer containing vascular smooth muscle cells. It is possible to accurately reproduce the morphology of the actual vascular tissue. Therefore, the vascular tissue prepared using the present invention is a tool for evaluating the metastatic behavior and invasion behavior of cancer cells moving in the blood, or analyzing the physiological behavior of cells such as thrombus formation and angiogenesis. Expected to be widely used in the fields of medicine and biochemistry.

Furthermore, when the present invention is used, the produced vascular tissue can be separated from the flow channel structure and recovered, and has high operability from the viewpoint of strength. Therefore, for example, by implanting the collected vascular tissue in another cellular tissue to form a tissue body having a luminal structure, oxygen and nutrients can be efficiently supplied to cells inside the tissue. It is possible to build a large three-dimensional tissue, and a wide range of applications in the fields of tissue engineering and regenerative medicine are expected.

Claims

For a channel structure constituted by a material containing an inorganic salt containing a polyvalent metal cation or a polyvalent metal cation,
After introducing a first aqueous sodium alginate solution in which cells are suspended and forming a first alginate hydrogel layer containing cells on the inner wall of the flow channel structure,
A method for producing a vascular tissue, wherein the first sodium alginate aqueous solution that is not gelled and remains in the flow channel structure is removed.
After removing the first sodium alginate aqueous solution not gelled,
After introducing a second sodium alginate aqueous solution in which cells are suspended into the channel structure, and forming a second alginate hydrogel layer containing cells on the inner wall of the first alginate hydrogel layer,
The method for producing a vascular tissue according to claim 1, wherein the second sodium alginate aqueous solution that is not gelled and remains in the flow channel structure is removed.
3. The cell suspension is introduced into the flow channel structure after removing the first sodium alginate aqueous solution or after removing the second sodium alginate aqueous solution. 4. 2. A method for producing a vascular tissue according to 1.
The buffer aqueous solution which does not contain a polyvalent cation or has a sufficiently low polyvalent cation concentration is introduced into the flow channel structure immediately before introducing the first sodium alginate aqueous solution. The method for producing a vascular tissue according to any one of the above.
The method for producing a vascular tissue according to claim 4, wherein the aqueous buffer solution contains at least one of citric acid and ethylenediaminetetraacetic acid.
6. After removing the first sodium alginate aqueous solution or after removing the second sodium alginate aqueous solution, an aqueous solution containing a polyvalent metal cation is introduced into the flow channel structure. The method for producing a vascular tissue according to any one of the above.
By applying at least one of physical operation and chemical treatment to the material constituting the flow channel structure, an alginate hydrogel layer containing the cells formed on the inner wall of the flow channel structure is formed in the flow channel structure. The method for producing a vascular tissue according to any one of claims 1 to 6, wherein the blood vessel tissue is separated and recovered from the blood vessel.
The method for producing a vascular tissue according to any one of claims 1 to 7, wherein the material constituting the flow channel structure is at least partially a hydrogel material.
The method for producing a vascular tissue according to claim 8, wherein the hydrogel material includes at least one of alginic acid, agarose, gelatin, collagen, and polyethylene glycol diacrylate.
The method for producing a vascular tissue according to any one of claims 1 to 7, wherein the material constituting the flow channel structure is at least partially polydimethylsiloxane.
The method for producing a vascular tissue according to any one of claims 1 to 10, wherein the polyvalent metal cation is at least one of calcium ion, strontium ion, and barium ion.
The concentration of sodium alginate contained in the first aqueous sodium alginate solution and the second aqueous sodium alginate solution is 0.1% (w / v) or more and 10% (w / v) or less, respectively. 12. The method for producing a vascular tissue according to any one of 11 above.
The method for producing a vascular tissue according to any one of claims 1 to 12, wherein an inner wall surface of the channel structure is previously surface-treated with a polycation.
14. The method for producing a vascular tissue according to claim 13, wherein the polycation contains at least one of poly-L-lysine, chitosan, and polyethyleneimine.
The blood vessel according to any one of claims 1 to 14, wherein at least one of collagen and Matrigel is added to at least one of the first sodium alginate aqueous solution and the second sodium alginate aqueous solution. Tissue making method.
The vascular tissue according to any one of claims 1 to 15, wherein cell-adhesive sodium alginate is added to at least one of the first aqueous sodium alginate solution and the second aqueous sodium alginate solution. Manufacturing method.
The method for producing a vascular tissue according to any one of claims 1 to 16, wherein the cell is a cell derived from a mammal.
The method for producing vascular tissue according to any one of claims 1 to 17, wherein the cells include at least one of vascular smooth muscle cells, vascular endothelial cells, and vascular epithelial cells.
The density of the cells contained in the first aqueous sodium alginate solution and the second aqueous sodium alginate solution is 1 respectively.
The method for producing a vascular tissue according to any one of claims 1 to 18, wherein the number is 10 million or more per mL.
The cell contained in each of the first sodium alginate aqueous solution and the second sodium alginate aqueous solution is different in at least one of its kind, density, and mixing ratio. 2. A method for producing a vascular tissue according to item 1.
21. The method for producing a vascular tissue according to claim 1, wherein the first alginate hydrogel layer and the second alginate hydrogel layer each have a thickness of 10 micrometers or more.
The method for producing a vascular tissue according to any one of claims 1 to 21, wherein the vascular tissue has at least partially a multilayer structure, a hollow structure, and a branched structure.
A vascular tissue produced using the vascular tissue production method according to any one of claims 1 to 22.