JP2022031585A - Three-dimensional cell structure having valve function, manufacturing method thereof, and support body - Google Patents

Abstract

To provide a method of manufacturing a three-dimensional cell structure having a valve function.SOLUTION: A representative configuration of a three-dimensional cell structure having a valve function includes the steps of: preparing a tubular three-dimensional cell structure formed with a through hole in the center; and applying pressure to an end part of the three-dimensional cell structure and giving the end part a valve function that can open and close the end part. By applying pressure to the end part of the tubular three-dimensional cell structure made only of cells in a culture solution, and opening and closing the end part repeatedly, an opening/closing motion of a valve shape is generated so that the tubular three-dimensional cell structure is cultivated to a shape having a valve function. The three-dimensional cell structure having a valve function made by the present invention can be made only of human cells, for example, and enables manufacturing of an artificial heart valve having characteristics such as elimination of risks of inflammation and immune-rejection, no need to inoculate an anticoagulant, and use for an extended period.SELECTED DRAWING: Figure 7

Description

The present invention relates to a three-dimensional cell structure having a valve function, a method for producing the same, and a support thereof.

Some of the artificial heart valves that are currently mainstream are made of two types of materials. There are two types, a "biological valve" made of a heart valve of a living body such as a cow or a pig, and a "mechanical valve" made of a metal.
Since blood coagulation is unlikely to occur in "biological valves," it is not necessary to take anticoagulants, but the service life is as short as 10 to 20 years, and replacement surgery may be required again after transplantation to young people. There is.
"Mechanical valves" are prone to blood coagulation, so it is necessary to take a lifelong anticoagulant. It has been pointed out that anticoagulants have side effects such as difficulty in stopping bleeding at the time of trauma and susceptibility to other diseases.
In addition, since the shape of both is fixed, the transplanted valve does not grow as the body grows, and infants and the like need to undergo reoperation according to their growth.
If a heart valve can be formed from human cells, the above problem can be solved.

Patent Document 1 (Patent No. 6439223) shows a method for producing a tubular cell structure using only cells. According to this, it is possible to obtain a tubular cell structure made only of cells. This is a method for producing a simple tubular cell structure, and does not indicate the shape of a three-dimensional cell structure having a valve function.

Patent No. 6439223

As described above, the artificial heart valve currently used has two problems. Taking anticoagulant and useful life.
An object of the present invention is to provide a method for solving this problem by producing a three-dimensional cell structure having a valve function using cells.

In order to solve the above problems, a typical configuration of a three-dimensional cell structure having a valve function is a three-dimensional cell structure composed only of cells or mainly composed of cells, and at least a part of the three-dimensional cell structure is flexible. It is characterized by having a valve function of blocking the flow path of a fluid such as blood.
In order to prepare a three-dimensional cell structure having a valve function, a valve shape is prepared by imparting a flow of an arbitrary medium to a specific part of the three-dimensional cell structure when the tubular cell structure is cultured in a culture medium. I will do it. This is called open / closed culture.
By performing open / close culture, the valve site of the three-dimensional cell structure opens and closes, self-molding into the shape required for the valve function, and obtaining the strength required for the valve function.

According to the present invention, it is possible to produce a three-dimensional cell structure having a valve function only with cells, a valve having a long durability, no need to take an anticoagulant, and a valve that grows as the body grows. It will be possible to provide.

a: It is a photograph of an example of a tubular three-dimensional cell structure before imparting a valve shape. Below the tubular cell structure 100 is a support 110 fused to the structure. The support 110 is made of a material to which cells can adhere, and when the tubular cell structure 100 is prepared, the cells are adhered to the support 110 by being cultured while in contact with the cells. The support 110 may have a shape having many gaps such as a non-woven fabric, an individual shape having irregularities, or a flat surface. b: It is a schematic of a cross section. a: It is sectional drawing of the three-dimensional cell structure 102 which has a valve function at the time of valve opening. The flow path 150 is open at the valve site. b: FIG. 6 is a schematic cross-sectional view of a three-dimensional cell structure 103 having a valve function when the valve is closed. The valve site is closed. It is a schematic diagram which showed the movement of a valve at the time of open-and-open culture. a: The valve is opened by the culture medium extruded from the cylinder 200. b: The valve is closed by the culture medium sucked by the cylinder 200. It is a photograph of an example of a support 120 made of a rigid material assembled to a tubular cell structure. An example of a support 120 made of a hard material is attached to a tubular cell structure 100, and is a photograph when the support is opened and closed. a: When the valve is open, b: When the valve is closed (a) FIG. 3 is a schematic cross-sectional view in which an example of a support 130 made of a soft material is assembled to a tubular cell structure. Both ends of the flexible support 130 are pulled and fixed. (b) It is a photograph of an example of a support 130 made of a soft material assembled to a tubular cell structure. It is a whole photograph of an example of a three-dimensional cell structure having a valve function after culturing. It maintains its self-sustaining shape even outside the liquid, and maintains the shape of the tricuspid valve when it is open. It is a stained image of an example of a three-dimensional cell structure having a valve function after culturing. Cells are present up to the center of the structure. It can be seen that the tip of the tip has a clean curve at the part in contact with other leaflets.

A method for creating a three-dimensional cell structure having a valve function will be described with reference to the drawings.

First, a tubular three-dimensional cell structure 100 prepared using only cells or mainly using cells is prepared. This is produced by using the method shown in Japanese Patent No. 6439223 and the like. As shown in FIG. 1, the tubular three-dimensional cell structure 100 is composed of a support 110 made of a material to which cells can adhere. The support 110 may be made of a highly flexible material such as a non-woven fabric, or may be a plate-shaped material. As shown in FIG. 1-b, the cell portion (before open / closed culture) 101 and the support 110 are fused. It does not have a valve function at this stage.

FIG. 2 is a cross-sectional view after the three-dimensional cell structure is formed into a valve shape. a: At the time of opening, the tips of the valve sites of the three-dimensional cell structure (disclosure) 102 having a valve function are separated from each other, and the flow path 150 appears. b: When closed, the three-dimensional cell structures having a valve function (when closed) 103 are in contact with each other and the flow path 150 disappears, resulting in a valve function.

FIG. 3 is an overall view of the open / closed culture system.
a: When the valve is opened, the stepping motor 400 is rotated at a speed and angle programmed in advance in the controller 500, the piston 310 is pushed into the cylinder 210, and at the same time, the culture medium is discharged, and the three-dimensional cell structure having a valve function ( Disclosure) 102 is pushed open and the valve is opened.
b: When the valve is closed, the movement is reversed, the culture medium is sucked, and the three-dimensional cell structure (when closed) 103 having a valve function is closed, and the valve is closed.
By repeating the above steps a and b, the three-dimensional cell structure 100 is shaped to match the valve function, and the strength required for the valve function is obtained to obtain the three-dimensional cell structure 105 having the valve function.

A form of mammalian heart valve is called the tricuspid valve, which has three leaflets. In order to create a shape similar to this tricuspid valve, a support that restricts movement is inserted into the lumen of the tubular cell structure and cultured. FIG. 4 is a photograph in which a rigid support is fixed in a lumen.
FIG. 5 is a photograph of a: when the valve is opened and b: when the valve is closed during the open / closed culture shown in FIG.
The material of the support 120 is, of course, limited to those that are not cytotoxic. Further, a material having biocompatibility is desirable. Further, it may be a material to which cells can adhere or a material to which cells cannot adhere. When cells adhere, a material with a large space such as a non-woven fabric is desirable, but it is also possible to provide irregularities on the surface of the plate material to facilitate adhesion. In addition, it suffices if the treatment is performed so that the cells can adhere even on a smooth surface. When a non-cellular material is used for the support 120, the support 120 can be separated from the three-dimensional cell structure 105 and the three-dimensional cell structure 105 can be transplanted into the heart. When a cell-adhesive material is used for the support 120, the support 120 can be transplanted into the heart while retaining the function of shape retention.

FIG. 6a is an image diagram of a soft support made of a soft material placed in the lumen of a tubular cell structure. The lower part of the flexible support 130 is fixed to the support 110 or the like, and the upper part is fixed to some kind of holding portion so that the flexible support 130 is held while maintaining a substantially linear shape. The flexible support 130 is cultured in contact with the tubular cell structure 100, and in the case of a cell-adhesive material, it fuses and adheres to cells as the culture progresses.
For the flexible support 130, for example, when making a tricuspid valve, the supports are installed at three places on the circumference. The upper and lower ends of the support are fixed to a container, a base, or the like. By repeating opening and closing by the above-mentioned method, the three-dimensional cell structure 100 is shaped into a shape suitable for the valve function, and at the same time, the strength required for the valve function is obtained to become the three-dimensional cell structure 105 having the valve function.
When a non-cellular material is used for the support 130, the support 130 can be separated from the three-dimensional cell structure 105 and the three-dimensional cell structure 105 can be transplanted into the heart. When a cell-adhesive material is used for the support 130, the support 130 can be transplanted into the heart while retaining the function of maintaining strength. Further, the support 130 may be made of a material that is absorbed by a living body.
b is an example photograph. Flexible supports 130 are placed on three sides and fused with tubular cell structure 100.

FIG. 7 is an overall photograph of a three-dimensional cell structure having a valve function after opening and closing culture and removing the rigid support 120. It has a shape similar to that of a tricuspid valve and has a valve function.

FIG. 8 is an HE-stained image obtained by slicing the cell portion of FIG. 7 to a thickness of 5 microns. It can be seen that the right apex is the part in contact with the other leaflets, but the curve is clean and smooth. In addition, it can be seen that the nucleus of the cell exists inside.

INDUSTRIAL APPLICABILITY According to the present invention, a three-dimensional cell structure having a valve function can be produced only by cells. The present invention is used, for example, for heart valve transplantation in humans. In this case, the three-dimensional cell structure having a valve function is made only of human cells, so that there is no risk of inflammation caused by different species, and since no metal or the like is used, no blood clots occur and no blood anticoagulant is required. In addition, the risk of immune rejection can be eliminated if the cells are prepared using the patient's own cells. Furthermore, transplantation to infants and the like is thought to cause the valve to become larger as the body grows, eliminating the need for reoperation. In addition to the heart, it can also be used for blood vessels with valve functions such as lower limbs.

100 Photograph of an example of a tubular three-dimensional cell structure (straight tube shape, no valve shape).
101 A schematic cross-sectional view of a tubular three-dimensional cell structure.
102 A schematic cross-sectional view of a three-dimensional cell structure having a valve function when it is open.
103 Schematic cross-section of a three-dimensional cell structure with a valve function when it is closed 105 Photograph of an example of a three-dimensional cell structure with a valve function 108 Example of a stained image of a three-dimensional cell structure with a valve function 109 Three-dimensional cell with a valve function An example of magnifying a stained image of a structure 110 A support connected to a three-dimensional cell structure.
120 A support made of a hard material placed inside a cell structure.
130 Support 200 made of flexible material placed inside the cell structure Cylinder holding the culture medium.
300 The piston sucks the culture solution.
310 The piston discharges the culture solution.
400 Stepping motor that operates the piston 500 Controller that controls the stepping motor

Claims (20)

It is a method for manufacturing a three-dimensional cell structure.
The process of preparing a tubular three-dimensional cell structure with a through hole formed in the center,
A step of applying pressure to the end of the three-dimensional cell structure to impart a valve function capable of opening and closing the end to the end.
Manufacturing method having. The manufacturing method according to claim 1, wherein the step of imparting the valve function repeatedly opens and closes the end portion by applying pressure. The production method according to claim 1, wherein the step of imparting the valve function includes applying a fluid into the through hole of the three-dimensional cell structure. The manufacturing method according to claim 2, wherein the step of applying the fluid includes repeatedly applying pressures in different directions into the through hole. The manufacturing method according to claim 3, wherein the step of applying the fluid includes discharging the fluid from the end portion and sucking the fluid from the end portion. The manufacturing method according to claim 1, wherein the step of imparting the valve function includes fixing a predetermined position of the end portion, and the end portion opens and closes with the fixed position as a fulcrum. The production method according to any one of claims 1 to 4, wherein the step of imparting the valve function is carried out in a container containing a culture solution. The manufacturing method according to claim 1, wherein the step of imparting the valve function is to impart three valve functions in the circumferential direction of the end portion. A device for manufacturing a three-dimensional cell structure having a valve function.
A supporting means for supporting one end of a tubular three-dimensional cell structure having a through hole in the center,
A forming means for applying a fluid into the through hole of the three-dimensional cell structure to form a valve function at the other end of the three-dimensional cell structure so that the other end can be opened and closed.
Manufacturing equipment with. The manufacturing apparatus according to claim 9, wherein the forming means repeatedly opens and closes the other end portion by applying pressure. The forming means includes a cylinder capable of reciprocating movement and a drive unit for driving the cylinder.
The manufacturing apparatus according to claim 9 or 10, wherein the forming means changes the pressure applied into the through hole in response to the reciprocating motion of the cylinder. The manufacturing apparatus according to claim 9, wherein the forming means applies the culture solution into the through-holes of the three-dimensional structure cell body in a container containing the culture solution. The manufacturing apparatus according to claim 9, wherein the forming means includes a fixing member for fixing a fixed position of the other end portion, and the other end portion opens and closes with the fixing member as a fulcrum. 13. The manufacturing apparatus according to claim 13, wherein the fixing member fixes three positions in the circumferential direction of the other end portion. The manufacturing apparatus according to claim 13, wherein the fixing member is made of a biocompatible material. The manufacturing apparatus according to claim 10, wherein the supporting means is made of a non-woven fabric. A three-dimensional cell structure having a valve function produced by the production method according to any one of claims 1 to 9. The three-dimensional cell structure according to claim 17, wherein the three-dimensional cell structure provides a valve function of the heart. A three-dimensional cell structure having a valve function manufactured by the manufacturing apparatus according to any one of claims 10 to 16. The three-dimensional cell structure according to claim 19, wherein the three-dimensional cell structure provides a valve function of the heart.

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