WO2004100832A1

WO2004100832A1 - Living organism tissue retaining unit and living organism tissue treating apparatus including the same

Info

Publication number: WO2004100832A1
Application number: PCT/JP2004/006665
Authority: WO
Inventors: Mitsuo Umezu; Kiyotaka Iwasaki; Shigeyuki Ozaki; Yuji Morimoto; Osamu Endo
Original assignee: Waseda University; Aubex Corporation
Priority date: 2003-05-15
Filing date: 2004-05-12
Publication date: 2004-11-25
Also published as: JP2006223101A

Abstract

Living organism tissue treating apparatus (10) comprises circulation unit (10A) capable of circulating a cell removing solution or cell-containing solution through circulation channel (13) under conditions similar to those of blood circulation of a living body and retention unit (10B) retaining a different tissue valve, disposed along the circulation channel (13). The retention unit (10B) is fitted with retention member (39) capable of retaining a living organism tissue in the state of being immersed in a solution and irradiation unit (42) capable of irradiating the living organism tissue retained by the retention member (39) with microwave. The retention member (39) includes inflow part (62) and outflow part (63) for the solution between which space (64) for accommodating the living organism tissue is provided, so that cell removal or cell sowing treatment can be performed while the living organism tissue is arranged in the solution flow from the inflow part (62) to the outflow part (63).

Description

Specification

Living tissue holding device and living tissue processing device using the same

Technical field

The present invention relates to a biological tissue holding device and a biological tissue processing device using the same, and is particularly suitable for effectively performing a decellularization process and / or a cell seeding process when transplanting a predetermined biological tissue. The present invention relates to a living tissue holding device and a living tissue processing device using the same. Background art

If the heart valve of the human body does not work properly and dysfunction occurs, such as stenosis of the opening of the valve or regurgitation of blood, it is necessary to replace the heart valve with a predetermined substitute valve. At present, there are a mechanical valve formed of a predetermined artificial material, a heterogeneous biological valve collected from an animal such as a pig, and a similar biological valve provided by another human body. Here, although the mechanical valve is durable, there is a problem that the anticoagulant must be continuously consumed for a lifetime. On the other hand, the above-mentioned xenogeneic biological valve does not need to keep taking the anticoagulant for a lifetime.However, valve malfunction occurs due to long-term deposition of calcium, etc., and it is necessary to replace it with a new replacement valve in about 15 years. There is a problem that occurs. Further, there is a problem that it is difficult to secure a large amount of the same type of biological valve due to a shortage of donors.

By the way, since a sufficient number of the heterogeneous biological valves can be supplied, and the patient does not need to keep taking the anticoagulant for a lifetime after transplantation, if the durability which is regarded as a disadvantage is improved, other It can be expected to be more useful than alternative valves.

Therefore, the following treatment methods are known for suppressing the immune rejection reaction after transplantation and improving the durability of xenogeneic biological valves collected from animals such as pigs (for example, Japanese Patent Laid-Open Publication No. Hei 6-26). No. 193,3). That is, first, a foreign body valve is immersed in a cell removing solution such as bile acid or a surfactant to remove animal original cells such as animal endothelial cells and fibroblasts (decellularization treatment). Thereafter, the xenogeneic biological valve from which the original cells have been removed is immersed in a cell-containing solution containing autologous cells such as endothelial cells and fibroblasts of the transplanted human body, so that the autologous cells are seeded on the xenogeneic biological valve. (Cell seeding treatment). However, according to the above-mentioned treatment methods, the decellularization treatment and the cell seeding treatment for the xenobiotic valve collected from the animal cannot be performed effectively, and the xenobiotics after the respective treatments have sufficient biocompatibility. Cannot be provided. That is, in the above-described decellularization treatment, the original cells remain to some extent, and the biocompatibility of the heterogeneous biological valve after the treatment is reduced due to the presence of the original cells. In addition, in the above-described cell seeding treatment, autologous cells do not uniformly adhere to the entire area of the xenogeneic biological valve, and the number of autologous cells seeded on the xenogeneic biological valve is not sufficient.

Therefore, the present inventors have conducted intensive experimental research to solve the above-mentioned problem. As a result, during the decellularization treatment, the blood flow of the human body was introduced into the cell removal solution in which a foreign biological valve was immersed. And / or applying microwaves to a heterogeneous biological valve immersed in the cell-removing solution, the number of remaining progenitor cells is significantly reduced as compared to the above-described treatment method. I learned. Further, at the time of the cell seeding treatment, the heterogeneous biological valve after the decellularization treatment is rotated up and down in the cell-containing solution, and further, a flow substantially corresponding to the blood flow of the human body flows through the cell-containing solution. As a result, it was found that the autologous cells adhered more uniformly and the number of the adhered cells increased as compared with the above-mentioned treatment method. Disclosure of the invention

The present invention has been devised based on such knowledge, and has as its object the purpose of a biological tissue that can contribute to the effective decellularization and cell seeding of a biological tissue such as a heterogeneous biological valve. And a biological tissue processing apparatus using the same.

In order to achieve the above object, a holding device according to the present invention includes a holding body capable of holding a predetermined living tissue in a solution for decellularization treatment or cell seeding treatment,

The holding body includes an inflow portion and an outflow portion of the solution, and an installation space for the living tissue located between the inflow portion and the outflow portion. In a state where the living tissue is arranged in the flow, a decellularization treatment and / or a cell seeding treatment is provided so as to be possible. According to such a configuration, the living tissue can be immersed in a solution having a flow substantially corresponding to the blood flow of the living body to be transplanted. According to the above findings of the present inventors, the number of progenitor cells remaining during the decellularization treatment can be significantly reduced, and the number of living cells to be seeded during the cell seeding treatment can be reduced. Can be increased. Therefore, by using the device of the present invention, it is possible to effectively perform decellularization treatment and cell seeding treatment when a living tissue such as a porcine xenobiopsy valve is implanted into a different kind or the same kind of living body (human body). Can be. In the present invention, it is preferable to adopt a configuration further comprising an irradiating means for irradiating the living tissue held by the holder with microwaves during the decellularization treatment. With such a configuration, it is possible to impart a higher decellularization effect to the living tissue to be subjected to the decellularization treatment.

Further, a configuration in which the holder and the irradiation unit are provided so as to be relatively rotatable can also be adopted. This makes it possible to irradiate the microwave from the irradiating means to substantially the entire region in the rotational direction of the biological tissue, and it is possible to uniformly remove cells in substantially the entire region in the rotational direction of the biological tissue.

Further, the irradiating means stops irradiation of the microphone mouth wave when the solution has reached a predetermined temperature or higher, and starts irradiation of the microwave when the solution has reached a predetermined temperature or lower. Then, According to such a configuration, excessive temperature rise due to microwave irradiation can be suppressed, and by appropriately maintaining the temperature of the solution, tissue damage to living tissue due to temperature rise or the like is prevented. be able to.

The inflow portion and the outflow portion have first and second flow paths, respectively. Each first flow path is provided so as to be able to communicate through the inside of the tubular biological tissue, while each second flow path is provided with a second flow path. The flow path may be provided so as to be able to communicate through the outside of the living tissue. By doing so, the solution flows inside and outside the living tissue from the inflow portion to the outflow portion, and the temperatures inside and outside the living tissue can be kept substantially the same. Therefore, even when the living tissue is irradiated with microwaves, a temperature difference between the inside and the outside of the living tissue hardly occurs, and the tissue failure due to the temperature difference can be prevented.

Here, a configuration may be adopted in which a guide path for guiding a temperature sensor is provided in the first flow path and / or the second flow path in the outflow portion. According to such a configuration, the temperature sensor can be easily inserted into the first flow path and the Z flow path or the second flow path. Thus, the temperature in these flow paths can be easily monitored. In particular, since the guideway is provided on the outflow side, the flow of the solution before passing through the living tissue is not obstructed by the temperature sensor or the like, and the living tissue is placed in an appropriate flow of the solution. Thus, the processing can be appropriately performed.

In addition, it is preferable to adopt a configuration in which the holding member is provided so as to be rotatable in a direction of inverting the upper and lower sides of the living tissue during the cell seeding process. With this configuration, since the living tissue is seeded while rotating in the upside down direction, the influence of gravity when the cells adhere to the living tissue is suppressed, and the cells are attached by the influence of the gravity. The cells can be attached to almost the entire area of the living tissue without unevenness.

Further, the biological tissue processing device according to the present invention is a biological tissue processing device that includes the holding device, and performs a decellularization process and / or a cell seeding process on the biological tissue. A circulation device for circulating the solution in a manner simulating the circulation of blood in a living body,

The circulation device is connected to the inflow portion and the outflow portion, and applies a flow substantially corresponding to the blood flow in the living body to the solution flowing from the inflow portion to the outflow portion. Even with such a configuration, the above-described object can be achieved. In addition, when the decellularization treatment and the cell seeding treatment are performed by one device, a series of processing operations before transplantation of the living body and the tissue can be performed by one closed circuit, and the contamination of the living tissue can be prevented. The cleanliness can be kept.

Here, the circulation device includes a drive pump that generates a pulsating flow, and a circulation path that is disposed between the inflow port and the outflow port of the drive pump and that includes the holding device in the middle of the drive pump.

The circulation path includes a resistance applying unit that applies flow resistance to the solution, and a hydraulic pressure attenuating unit that is disposed downstream of the resistance applying unit and attenuates the liquid pressure of the solution. Can be. According to such a configuration, the solution can be circulated in a blood circulation state simulating the human body circulation. Therefore, by changing the installation location of the holding device in the circulation, different blood flow conditions in the body such as arteries and veins The flow conditions of the solution can be selected according to the conditions, and the solution can be immersed in various biological tissues with different conditions of the blood that actually flows, with a flow substantially equivalent to them, and the various biological tissues have high A versatile device that provides a decellularization effect and a cell seeding effect can be provided.

Further, it is preferable that the circulation path includes an amplitude adjusting unit that adjusts the amplitude of the hydraulic pressure upstream of the resistance applying unit. With such a configuration, it is possible to perform the decellularization treatment and the cell seeding treatment under various conditions in which the solution pressure of the solution is arbitrarily changed. Here, the amplitude adjusting means may be constituted by an adjusting tube capable of adjusting the amplitude by changing the wall thickness, or may be constituted by a plurality of tubes made of different materials. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic configuration diagram of a biological tissue processing apparatus applied to the present invention, FIG. 2 is a schematic longitudinal sectional view of a driving pump, FIG. 3 is a schematic front view of a holding device constituting the biological tissue processing apparatus, and FIG. 5 is an exploded perspective view of the cover of the holding device, FIG. 5 is a schematic cross-sectional view of the holding body, FIG. 6 is an exploded cross-sectional view of the right side in FIG. 5, FIG. 8 is an exploded cross-sectional view on the left side in FIG. 5, FIG. 9 is a schematic exploded perspective view of a flow path forming member at an outflow portion, and FIG. 10 (A) is a partial extract of FIG. (B) is a view showing a state where the whole is rotated by 180 degrees around the support axis from the state of (A), and FIG. 11 is a diagram showing remaining original cells per unit area in Examples 1, 2 and Comparative Example 1. FIG. 12 is a diagram showing an enlarged micrograph of the living tissue obtained in Example 3, and FIG. 13 is an enlarged view of the living tissue obtained in Example 4. Is a diagram representing the micrograph. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments of the present invention will be described with reference to the drawings.

FIG. 1 shows a schematic configuration diagram of a biological tissue processing apparatus according to the present embodiment. In this figure, a living body / tissue processing apparatus 10 is an apparatus used when performing decellularization processing or cell seeding processing of a heterogeneous biological valve, which is a biological tissue. Here, the decellularization treatment is performed before transplanting a xenobiotic valve collected from an animal such as a pig into a human body. The biological valve is immersed in a cell-removing solution such as bile acid to remove the animal cells (hereinafter referred to as “progenitor cells”) so that only a substrate composed of collagen or the like is obtained. On the other hand, in the cell seeding treatment, the autologous biological valve after decellularization is immersed in a cell-containing solution containing cells of a human body to be transplanted (hereinafter, referred to as “autologous cells”). This is a process for causing the particles to adhere.

The biological tissue processing apparatus 10 is provided with a circulating apparatus 1 OA for circulating the cell removing solution or the cell-containing solution through a predetermined circuit, and is provided in association with the circulating apparatus 1 OA and holds the heterogeneous biological valve. And a holding device 10B. In the following, the cell removing solution and the cell-containing solution are collectively referred to as each solution unless otherwise specified.

The circulating device 1 OA includes a known suction / inlet device 11, a polyurethane drive pump 12 connected to the suction / inlet device 11, and each solution discharged from the drive pump 12. The suction / inhalation device 11 configured to include a circulation path 13 disposed so as to return to the position 2 includes a known structure that is capable of inhalation and inhalation with respect to the drive pump 12. Here, detailed description is omitted here.

The drive pump 12 is a pulsating pump capable of generating a swirling vortex therein and generating a pulsating flow at the time of discharge. That is, as shown in FIG. 2, the drive pump 12 adopts a structure already proposed by the present applicant (see Japanese Patent Application No. 200202_1667836). A hollow upper structure 17 having a substantially conical outer shape formed with an inflow port 15 and an outflow port 16, and a lower dome-shaped outer structure located below the upper structure 17. It comprises a hollow lower component 18 and a flexible diaphragm 20 that partitions the internal space 31, S 2 of each of these components 17, 18. Here, the inflow port 15 is provided at a position on the right end side in FIG. 2 that is continuous with the peripheral wall of the upper structure 17, and the outflow port 16 is an upper end in FIG. 2 that is on the top side of the upper structure 17. It is provided at the side position.

The lower structure 18 is provided with a vent 22 connected to the suction / intake air device 11, and compressed air is sucked and sucked alternately at a predetermined timing in the lower internal space S 2. It is becoming noticeable. Thus, the compressed air is sucked and sucked into the internal space S 2 Due to the deformation of the diaphragm 20, the volume in the upper internal space S1 increases and decreases, thereby generating a pulsating flow in each solution discharged from the outflow port 16. At this time, in the upper internal space S1, as shown by the broken line in FIG. 2, a swirling vortex is generated in which a flow stagnation region is hardly generated. Although not particularly limited, in this embodiment, the pressure (positive pressure) of the air supplied into the internal space S2 is set to about 14 OmmHg to about 26 OmmHg. On the other hand, the pressure (negative pressure) of the air sucked from the interior space S2 is set to about 13 OmmHg to about 15 OmmHg.

As shown in FIG. 1, the circulation path 13 has a closed loop shape in which each solution discharged from the outlet port 16 of the drive pump 12 flows into the inlet port 15 in a state of not contacting the outside air. Is set. That is, the circulation path 13 includes resistance applying means 23 for applying flow resistance to each solution discharged from the outflow port 16, an upstream tube 24 connected to the outflow port 16, and an upstream side An adjusting tube 25 as an amplitude adjusting means connected to the downstream end of the tube 24; a connecting tube 26 connected to the downstream end of the adjusting tube 25; and the connecting tube 26 The downstream tube 27 connected to the inflow port 15 of the drive pump 12 and the connection pump 28 provided between the connection tube 26 and the downstream tube 27 are also disposed on the downstream side. It is comprised including. A well-known connector 29 is used to connect the members 24 to 28 here. In the following, for convenience of explanation, the upstream side of the circulation path 13 across the resistance applying means 23 is referred to as an upstream line L1, and the downstream side is referred to as a downstream line L2.

The resistance applying means 23 is provided at one place in the connecting tube 26 assuming the peripheral resistance of the human body, and is not shown, but is a pinch-shaped for tightening the connecting tube 26. It is composed of members. That is, due to the tightening of the connection tube 26 by the resistance applying means 23, even if the drive pump 12 pulsates, the diastolic blood pressure in the upstream line L1 does not become O mmHg, and the blood flow in the arteries of the human body To be simulated. Here, the average pulse pressure in the upstream line L1 can be adjusted to a predetermined value by tightening the connection tube 26. In the present embodiment, the average pulse pressure is substantially equivalent to the average pressure of the human body. To be adjusted to about 10 O mmH g . In addition, as the resistance applying means 23, other members such as a variable stop can be adopted as long as the above-described operation is achieved, in addition to the above-described pinch-shaped member.

The upstream tube 24, the connection tube 26, and the downstream tube 27 are not particularly limited, but are formed of Shiridani vinyl. As described later, the holding device 10B is provided in the middle of the upstream tube 24, and the heterogeneous biological valve held in the holding device 10B and the downstream tube 27 are provided. The check valve 30 provided at the bottom of the housing ensures that the solutions can be circulated in the direction of the arrow in FIG. 1 without backflow.

The adjusting tube 25 is arranged upstream of the resistance applying means 23 and adjusts the amplitude of the pulse pressure of the upstream line L1. That is, the adjustment tube 25 is formed of a soft material that can adjust the amplitude of the pulse pressure in the upstream line L1 by changing its thickness, and is formed of, for example, segmented polyurethane or silicon. I have. In the present embodiment, the amplitude of the pulse pressure in the upstream line L1 is set so as to approximate the human body, for example, ± 20 mmHg of the average pulse pressure (100 mmHg). .

The connection pump 28 uses a pulsating flow pump having the same configuration as the drive pump 12, and the same reference numerals are used for the same or equivalent components to the drive pump 12. The description is omitted. The connection pump 28 is also attached so that each solution flows in from the inlet port 15 and the solution is discharged from the outlet port 16. Further, each ventilation port 22 of the connection pump 28 is open to the outside, and the diaphragm 20 is displaced in accordance with the flow of each solution. Also, the amount of the solution filled in the circulation device 1OA is slightly smaller than the maximum filling amount allowed by the device 1OA, and the displacement amount of the diaphragm 20 of the connection pump 28 is Slightly smaller than the maximum allowable displacement. Thus, when blood passes through the inside of the connection pump 28, a damper effect that allows displacement of the diaphragm 20 and causes a pressure loss is provided. Therefore, the connection pump 28 is arranged downstream of the resistance applying means 23 and constitutes a pulse pressure attenuating means for attenuating the liquid pressure of the solution in the downstream line L2. In the present embodiment, the blood pressure passing through the connection pump 28 becomes approximately 1 OmmHg, which is equivalent to the left atrial pressure of the human body. Is set to

The connection pump 28 may be provided between the adjustment tube 25 and the connection tube 26.

The holding device 10B is a device that guides a solution flowing in the circulation device 10A and holds a heterogeneous biological valve that is immersed in the solution. In the present embodiment, a porcine aortic valve is applied as a heterogeneous biological valve for performing the decellularization process and the cell seeding process. For this reason, the holding device 10B is connected to a portion of the circulation path 13 near the blood flow state of the aortic valve portion of the human body, that is, in the middle of the upstream tube 24. In the following, for convenience of explanation, the upstream side of the upstream side tube 24 will be referred to as the inlet side tube 24A and the downstream side thereof will be referred to as the outlet side tube 24B with the holding device 10B as a boundary. The holding device 10B can be freely connected to each part of the living tissue to be processed by appropriately selecting a part or the like in the circulation path 13 that is close to the state (pressure or the like) of the blood flowing through the living tissue. can do.

As shown in FIG. 3, the holding device 10 B includes a flat base 33 that can be installed on a predetermined surface F, a device body 35 located above the base 33, and a base 33. Support members 36, 36 erected on the left and right sides to support the apparatus main body 35 are provided.

The device main body 35 includes a box-shaped cover 38 having a hexagonal shape in a front view, a holder 39 provided inside the cover 38, and holding the heterogeneous biological valve V, and a holder

An octagonal frame member 40 in a plan view, which is arranged along the inner wall of the cover 38, and a heterogeneous biological valve provided on the back side of the cover 38 and held by the holder 39. Irradiation device 4 2 (irradiation means) for irradiating microwave toward V, and frame member

A support shaft 44, 44 extending in the direction of each support member 36, 36 penetrating the cover 38 from the left and right sides of 40, two introduction tubes 46 connected to the holder 39, and a discharge tube. 4 and 7 are provided.

As shown in FIG. 4, the cover 38 has a configuration in which a box having a predetermined shape is vertically divided into two parts, and a front cover 51 located on the front side and a rear cover located on the rear side. 5 and 2 The abutting portion of the front cover 51 and the rear cover 52 is formed at a position intersecting the support shafts 44, 44, and the outer edge side of each abutting portion. Are formed with flange surfaces 54, 54 substantially along the outer edge. Each of these flange surfaces 54, 54 has a large number of through holes 55 for bolts and the like, not shown, and the flange surfaces 54, 54 are in contact with each other. The front cover 51 and the rear cover 52 can be integrated by using. If the bolt is removed from this state, the front cover 51 can be separated from the rear cover 52 and can be removed. Near the center of the inside of the rear cover 52, a rectangular microwave irradiation port 57 that opens forward is formed. The irradiation port 57 is formed at a position substantially opposite to the heterogeneous living body valve V held by the front holder 39 (see FIG. 1), and is provided with a micro-beam from the irradiation device 42 provided on the rear side. Waves are applied to the foreign body valve V in the holder 39 in front through the irradiation port 57. Further, the front cover 51 and the rear cover 52 have a shape and a structure such that the microwave does not leak to the outside when the microwave is irradiated from the irradiation port 57 toward the holder 39. Has become. At the front end side of the front cover 51, a door plate 59 of a known material that can see through the state of the holding body 39 and can prevent leakage of microwaves to the outside is provided. .

As shown in FIG. 5, the holding body 39 includes an acrylic cylindrical member 61 extending in the left-right direction in FIG. 5 and an inflow portion 62 located on the right end side of the cylindrical member 61 in FIG. An outflow portion 63 located on the left end side, and an installation space 64 for the heterogeneous biological valve V located between the inflow portion 62 and the outflow portion 63 are provided. In the holder 39, as described later, each of the solutions flows into the cylindrical member 61 from the inflow portion 62, and is discharged from the outflow portion 63 to the outside through the installation space 64. ing. Both ends of the cylindrical member 61 in the extending direction are open, and an inflow portion 62 and an outflow portion 63 are set in each of the open portions. Further, on the outer peripheral surface of the cylindrical member 61, an aluminum thin plate 66 is attached on the entire periphery except for a central portion substantially opposed to the installation space 64. For this reason, the microwave from the irradiation device 42 (see FIG. 3) passes only through the central region of the peripheral wall of the cylindrical member 61 substantially opposed to the installation space 64, and concentrates the microphone mouth wave in the region. In addition to the above, it is possible to suppress a rise in temperature in a portion located outside the installation space 64.

As shown in FIGS. 5 and 6, the inflow portion 62 is formed on the outer peripheral surface of the cylindrical member 61. An end member 68 to be engaged and a flow path forming member 69 located inside the end member 68 are provided.

The end member 68 is provided in the shape of a bottomed container having the left side open in FIG. 6, and a bottom wall 71 located on the right end side in FIG. A side wall 72 extends substantially perpendicularly to the bottom wall 71 (leftward in FIG. 6). The bottom wall 71 has a through hole 73 formed substantially at the center thereof. The side wall 72 has a thread groove 74 formed on an inner peripheral portion on the open side. The thread groove 74 engages with a thread groove 75 formed on the outer peripheral surface of the cylindrical member 61. It has become.

As shown in FIGS. 5 to 7, the flow path forming member 69 includes a hollow conical tube 77 having a shape similar to a cone, and an insertion tube 78 inserted into the conical tube 77. It consists of

The conical tube 77 has a cylindrical top 79 located on the installation space 64 side, and gradually expands outward from the top 79 to the right in FIG. A skirt-shaped portion 80 extending in the shape of a skirt, a skirt-shaped flange 81 connected to the right end side in FIG. 6 of the skirt-shaped portion 80, and formed inside these portions 79-81, In FIG. 6, an inner space 83 penetrating in the left-right direction is provided. In the following description of the flow path forming member 69, unless otherwise specified, the “front end side” means the top part 79 side of the conical tube 77, and the “rear end side” means Means the hem 8 1 side of tube 77.

A single stop groove 85 is formed on the outer peripheral surface of the top portion 79 along the circumferential direction. As shown in FIG. 5, the stopper groove 85 is covered with an end portion of porcine vascular tissue B including an aortic valve as a heterogeneous living body valve V, and a binding member not shown. By being clamped at, the detachment of the vascular tissue B from the top 79 can be regulated.

In the skirt-shaped portion 80, the outer diameter of the region on the hem portion 81 side is set to be substantially the same as the inner diameter of the cylindrical member 61, and the region on the hem portion 81 side is almost exactly inside the cylindrical member 61. Is to be contained.

The skirt portion 81 is provided in an annular shape, and the outer diameter thereof is set to be substantially the same as the inner diameter of the side wall 72 of the end member 68 so that the hem portion 81 is almost exactly accommodated in the end member 68. Nana I'm wearing A thread groove 87 is formed on the inner peripheral surface of the skirt portion 81.

The inner space 83 is opened at the right end side in FIG. 6, a base space 89 into which the distal end side of the insertion tube 78 is inserted from the open portion, and formed at the distal end side in communication with the base space 89. A main flow path 91 extending to the open portion 90 and sub flow paths 92 provided at four locations around the main flow path 91 and communicating with the base space 89 are provided. The base space 89 is provided in an internal shape along the distal end shape of the insertion tube 78 so that the insertion tube 78 can be received almost exactly. Although not particularly limited, the main flow passage 91 is formed in a tapered hole shape whose inner diameter gradually decreases from the top 79 side toward the inside. As shown in FIG. 7, the sub-channels 92 are opened at four positions in the circumferential direction at substantially equal intervals on the inclined surface of the skirt portion 80. In the present embodiment, the total opening area obtained by combining the opening areas of the sub flow paths 92 is set to be substantially the same as the opening area of the main flow path 91. As shown in FIG. 6, the insertion tube 78 has a spherical portion 94 on the distal end side having a spherical outer shape, and an outer diameter smaller than the spherical portion 94. A cylindrical portion 95 connected to the cylindrical portion 95 through a step, an outer cylindrical portion 96 rotatably inserted into the outer peripheral surface of the cylindrical portion 95, and a rear end side of the outer cylindrical portion 96. And an annular space formed inside the spherical portion 94 and the cylindrical portion 95 having an annular flange 97 having an outer diameter smaller than the inner diameter of the through hole 73 of the end member 68. 9 and 9 are provided. A screw groove 101 is formed on the outer peripheral surface of the outer cylindrical portion 96, and the screw groove 101 is related to a screw groove 87 formed on an inner peripheral portion of the skirt portion 81. Thus, the insertion tube 78 is attached to the conical tube 77.

The internal space 99 includes a base passage 103 through which the respective solutions pass, a tip hole 104 communicating with the base passage 103 and opening at the tip side of the spherical portion 94, It is provided at four places around the tip hole 104 and includes a side hole 105 communicating with the base passage 103. When the distal end side of the insertion tube 78 is received in the internal space 83 of the conical tube 77, the distal end hole 104 is always in communication with the main flow path 91. The side holes 105 are opened at four positions in the circumferential direction at substantially equal intervals on the surface of the spherical portion 94. These side holes 105 are completely connected to all sub-channels 92 by their relative rotation when the insertion tube 78 is received in the conical tube 77. Rotational displacement is possible from the passing position (maximum communication position) to the position (blocking position) where all sub-flow paths 92 are completely blocked. Therefore, the insertion pipes 78 move all the sub-flows from a position where the total flow rate of each solution flowing out of all the sub-flow paths 92 is almost the same as the flow rate of each solution flowing out of the main flow path 91. It has the same function as a variable throttle valve that can adjust the flow rate of each solution flowing out of the sub flow path 92 up to a position where the total flow rate of the solution flowing out of the flow path 92 becomes substantially zero.

As shown in FIGS. 5, 8, and 9, the outflow portion 63 has substantially the same components as the inflow portion 62, although the shapes are partially different from each other. It has come to play.

Therefore, in the following, for the components of the outflow portion 63 that are the same as or similar to the inflow portion 62, the description will be omitted or simplified using the same reference numerals, and only the differences from the inflow portion 62 will be supplementarily described. .

The end member 68 of the outflow portion 63 is provided in a nut shape, and a screw hole 1 • 7 penetrating in the left-right direction in FIG.

The conical tube 77 of the outflow portion 63 differs from the inflow portion 62 in the shape of the skirt portion 81 connected to the skirt portion 80. That is, the skirt portion 81 here is formed in a screw cylindrical shape having an outer diameter smaller than the maximum outer diameter of the skirt-shaped portion 80, and its outer peripheral portion is formed in the screw hole 1 of the end member 68. 07 are engaged. The conical tube 77 has a guide passage 109 through which a temperature sensor (not shown) is inserted. The guideway 109 is formed outside the internal space 83 and has a first guideway 109A that penetrates from the rear end of the skirt 81 to the slope of the skirt 80. And a second guideway 109B extending from the middle of the first guideway 109A toward the main flow passage 91. It is also possible to omit all or one of the guideways 109A and 109B.

The overall shape of the insertion tube 78 of the outflow portion 63 differs from that of the inflow portion 62. That is, the inlet pipe 8 here is provided in such a shape that a cylindrical portion 95 extends toward the distal end side instead of the spherical portion 94. Here, the tip hole 104 is provided on the tip side of the cylindrical portion 95, and the side hole 105 is provided at approximately four places on the outer peripheral surface on the tip side of the cylindrical portion 95 in the circumferential direction. It is provided at intervals. In FIGS. 5, 6, and 8, black members are O-rings for sealing. As shown in FIG. 3, the frame member 40 rotatably supports the holding body 39 inside thereof. That is, the insertion pipes 78, 78 protruding outward from the approximate centers of the end members 68, 68 are connected to the rotary pipes 112, 112 via pipe nuts 111, 111, respectively. Has become. The rotating tubes 1 12 and 112 are configured to protrude outward from the frame member 40 through bearings (not shown) attached to a part of the frame member 40. Here, the holding body 39 is attached to the inside of the frame member 40 so that the rotating pipes 1 12 and 1 12 extend obliquely at 45 degrees in FIG. Rotation (rotation) around 12 is possible. Here, on the outer surface of the frame member 40, near the portion where the rotary tube 112 protrudes on the outflow portion 63 side on the upper right in FIG. By the operation of the rotation driving means 114, the holding body 39 rotates inside the frame member 40. In addition, each of the rotary pipes 112 and 112 is connected to an inlet tube 46 and a discharge tube 47 via a rotary joint 115 fixed to the frame member 40 side. The rotary joint 115 here is a known joint that connects two tubular members (rotating pipes 112, 112 and tubes 46, 47) to be connected to each other so as to be rotatable relative to each other and to communicate the inside of the tubular member. Is used. Thus, even if each of the rotary tubes 112 and 112 is rotated by the operation of the rotation driving means 114, the rotational force is not transmitted to each of the tubes 46 and 47. The irradiating device 42 is a known device using a magnet port (not shown) as a source of the microphone port wave, and a detailed description thereof will be omitted.

The support shaft 44 has a hollow pipe shape, and accommodates the ends of an introduction tube 46 and a discharge tube 47 therein. That is, a slot hole 117 is formed in a portion of the outer peripheral surface of the support shafts 44, 44 which is located near the inside of each support member 36, 36, and each rotary pipe 117 is formed in the slot hole 117. Tubes 46, 47 extending from 112, 112 are accommodated.

The support member 36 is provided inside the first support portion 120 that rotatably supports the support shafts 44, 44, and is provided outside the first support portion 120, and has a rotation structure similar to that of the rotary joint 115 described above. And a joint support portion 122 that supports the joint 121. I have. Here, the rotary joint 1 21 supported by the support member 36 on the left side in FIG. 3 connects the discharge tube 47 inserted into the support shaft 44 on the left side and the outlet tube 24 B. They are connected so that they can rotate relative to each other. On the other hand, the rotary joint 12 1 supported by the support member 36 on the right side in FIG. 3 connects the introduction tube 46 inserted into the support shaft 44 on the right side and the inlet side tube 24 A. They are connected so that they can rotate relative to each other. Further, the driving member 124 for revolving composed of a motor, gears, etc. is provided on the support member 36 on the right side in FIG. 3. The shafts 4 4 and 4 4 can be rotated. In this way, when the support shafts 44, 44 rotate, the holder 39 rotates (revolves) together with the frame member 40 around the support shafts 44, 44. In addition, the introduction tube 46 and the discharge tube 47 accommodated in the support shaft 44 are provided in the space between the base 33 and the support shaft 44 so as not to interfere with the base 33 during the revolution. The length and arrangement are such that they can pass. As shown in FIG. 10, when the support shaft 44 rotates 180 degrees (half a rotation) from the state of (A) in FIG. 10 as shown in FIG. 10, as shown in FIG. 10 (B), The frame member 40 rotates and the upper and lower sides of the holder 39 are inverted. In other words, the holding body 39 can perform the revolving operation by the revolving driving means 124 in addition to the revolving operation by the revolving driving means 114 described above. At the time of the revolving operation, the held vascular tissue B Rotate in the direction to flip the upper and lower sides. Reference numeral 125 in FIG. 3 denotes an operation switch of the driving means 124 for revolution. Further, a cooling means for cooling the periphery of the introduction tube 46 may be provided in order to suppress the temperature rise of the solution.

Next, a procedure for setting the heterogeneous living body valve V to the holding device 10B configured as described above and an operation of the holding device 10B at the time of the setting will be described.

First, from the state shown in FIG. 3, after removing the joint between the rotary pipe 1 12 and the rotary joint 1 15, the pipe nut 111 is rotated with respect to the holder 39 so that the holder 3 is rotated. 9 is removed from the rotating tubes 1 1 2 and 1 1 2, the end members 6 8 and 6 8 are removed, and the inflow portion 6 2 and the outflow portion 6 3 are taken out from the cylindrical member 6 1. Then, as shown in FIG. 5, with the tops 79, 79 of the inflow section 62 and the outflow section 63 facing each other, the pigs including the aortic valve V at the respective tops 79, 79 are shown. Both ends of the vascular tissue B are fitted, and clamped on the stopper grooves 85, 85 using a binding member not shown. here The vascular tissue B is set by the aortic valve V, which is a one-way valve, in a direction that allows the flow of each solution from right to left in FIG. Thereafter, the inflow portion 62 and the outflow portion 63 interconnected via the vascular tissue B are returned into the cylindrical member 61 again, and the end members 68, 68 are attached to the cylindrical member 61, As shown in FIG. 3, the holder 39 is again attached to the rotating tubes 112, 112, and the setting of the vascular tissue B is completed.

Next, when performing the decellularization treatment, the cell removing solution is supplied from the introduction tube 46 side to the insertion tube 78 of the inflow portion 62 through the rotary tube 112. At this time, as shown in FIG. 5, in both the inflow section 62 and the outflow section 63, the insertion pipes 78, 78 are provided with the side holes 105 and the sub-flow passages 92 of the conical pipe 77, respectively. Are in a position where they can completely communicate with each other. Then, the cell removing solution supplied to the inflow portion 62 enters the vascular tissue B through the tip hole 104 and the main flow passage 91 of the inflow portion 62, and passes through the aortic valve V therein. In addition to being sent to the main flow passage 91 of the outflow portion 63 and flowing through the side hole 105 and the sub flow passage 92 of the inflow portion 62 at almost the same flow rate, the outside of the blood vessel and the tissue B is It is sent from the internal space of the cylindrical member 61 to the sub flow path 92 of the outflow part 63 so as to bypass. Then, the cell-removing solution flowing in the main flow path 91 and the sub flow path 92 of the outflow section 63 flows from the inlet pipe 78 of the outflow section 63 through the rotary pipe 112 of FIG. 47 Discharged to the 7 side. Therefore, the main flow passage 91 and the tip hole 104 provided in the inflow portion 62 and the outflow portion 63, respectively, are connected to the first flow passage through the inside of the tubular living tissue (vascular tissue B). The sub-flow channel 92 and the side hole 105 provided in the inflow portion 62 and the outflow portion 63 respectively communicate with the second through the outside of the living tissue (vascular tissue B). This constitutes a flow path.

When performing the above-described decellularization treatment, the irradiation device 42 and the rotation driving means 114 are operated to irradiate the microwave to the vascular tissue B held by the holder 39. While rotating, the holder 39 rotates. As a result, it is possible to perform the decellularization process evenly over the entire circumferential direction of the vascular tissue B. At this time, it is preferable to insert a temperature sensor (not shown) into the first and second guide paths 109A and 109B (see FIG. 5 and the like) and measure the solution temperature inside and outside the vascular tissue B. Les ,. That is, in this case, the cell removal solution flows at substantially the same flow rate on both the inner and outer sides of the vascular tissue B so that a temperature difference does not occur on both the inner and outer sides, but the first and second guide paths 109 A, Ten By inserting a temperature sensor (not shown) into 9B, it is possible to confirm whether or not there is a temperature difference between the inside and outside. The irradiation device 42 automatically stops microwave irradiation when the temperature on the inside and outside both rises above the human body temperature (for example, 37 ° C.), and when the temperature falls below the body temperature. In addition, microwave irradiation is automatically started. The irradiation device 42 of the present embodiment uses a device capable of irradiating a microwave having a frequency of 2.45 GHz and an output of about OW to 1200 W. Note that the temperature sensor may be arranged so as to be guided into the first flow path from a hole formed in the middle of the discharge tube 47.

On the other hand, when the cell seeding process is performed, the irradiation of the microwave by the irradiation device 42 and the rotation of the holder 39 are stopped. Then, the respective insertion pipes 78 and 78 in the holding body 39 are rotated from the state shown in FIG. The hole 105 (see FIG. 5 and the like) is changed to a position where the sub flow path 92 is completely shut off. After the above, the cell-containing solution is supplied into the insertion tube 78 of the inflow portion 62, and enters the vascular tissue B through the distal end hole 104 of the inflow portion 62 and the main flow passage 91. After passing through the internal aortic valve V, it is fed into the main flow passage 91 of the outflow portion 63 and discharged from the inlet tube 78 side of the outflow portion 63. At this time, since each of the insertion tubes 78 and 78 is at the blocking position, the cell-containing solution does not flow outside the vascular tissue B at all, unlike the case of the aforementioned decellularization treatment.

In addition, when performing the above-described cell seeding treatment, the driving means for revolution 124 shown in FIG. 3 is operated, and the holder 39 rotates together with the frame member 40 around the support shafts 44, 44. As a result, the holder 39 is rotated in the direction to reverse the top and bottom, as shown in FIG. By such a revolution of the holder 39, the influence of gravity can be eliminated, and the autologous cells in the cell-containing solution can be uniformly attached to the vascular tissue B.

Next, the decellularization method and cell seeding method using the biological tissue processing apparatus 10 will be described with reference to FIG.

First, vascular tissue B including aortic valve V is collected from an animal such as a pig. Then, as described above, the vascular tissue B is set in the holding device 10B, the cell removing solution is injected into the biological tissue processing device 10, and the cell removing solution is circulated. Here, as the cell removing solution, for example, deoxycholic acid (bile acid), sodium dodecyl sulfate Surfactants such as PDS (SDS) and Triton X_100 are used. At this time, in the living tissue processing apparatus 10, the cell removing solution circulates in a state similar to the blood flow of the human body as follows.

When a predetermined switch (not shown) is turned on, the suction / inhalation device 11 shown in FIG. 1 is operated, and the cell removing solution is circulated in the circulation path 13 by the pulsation of the drive pump 12. That is, the cell removing solution discharged from the drive pump 12 flows in the upstream line L1 at a pressure substantially equivalent to the general aortic pressure of the human body, and the holding device provided in the middle of the upstream line L1 It passes through 10 B and reaches resistance applying means 23 corresponding to the peripheral resistance of the human body. After passing through the resistance applying means 23, the cell removing solution passes through the connection pump 28, and then has a pressure of about 1 OmmHg, which is substantially equivalent to the left atrial pressure of the human body, and is supplied to the drive pump 12. Inflow.

At this time, in the holding device 1 OB, the cell removing solution flowing on the inside and outside of the vascular tissue B held by the holding body 39 is given a flow substantially equivalent to the blood flow flowing in the aorta of the human body, and As described above, by the rotation of the holder 39, the microwave is irradiated while rotating the vascular tissue B. As a result, the vascular tissue B collected from the animal has various progenitor cells (endothelial cells, fibroblasts, smooth muscle cells) removed, and becomes only a substrate composed of collagen and the like. In the present embodiment, the conditions of the irradiated microwave are set to a frequency of 2.45 GHz and an output of about 100 W to 500 W, but the present invention is not limited to this. As long as tissue B does not cause degeneration affecting the human body and can obtain a predetermined decellularization effect, the output can be changed within a predetermined range, and electromagnetic waves and sound waves of other frequencies can be obtained. Irradiation is also possible. Also, the flow applied to the cell removing solution may be a pulsating flow that does not simulate the blood flow of the human body.

Then, after the cell removing solution is discharged from the living tissue processing apparatus 10, physiological saline is injected into the living tissue processing apparatus 10, and the physiological saline is circulated to circulate the inside of the apparatus 10. After washing, the physiological saline is discharged from the living tissue processing apparatus 10. Then, a binder such as fibronectin is directly injected into the first flow path in the holder 39, and the open ends of the inflow portion 62 and the outflow portion 63 are closed, respectively, to thereby obtain the holder 39 The vascular tissue B after decellularization held in the above is immersed in a binder for a predetermined time. Then The mixture is discharged from the inside of the holder 39 to the outside, and the cell-containing solution is injected into the holder 39. Here, the cell-containing solution is obtained by collecting autologous cells (endothelial cells, fibroblasts, and no or smooth muscle cells) of a transplant recipient, culturing for a predetermined time, and adding a predetermined culture solution. Can be The culture solution may be any culture solution that can be used for the cell seeding treatment, and examples thereof include M199 (Mediuml99 medium, manufactured by Life Technology ogno ogies). Then, in the same manner as in the case of the binder, the vascular tissue B after decellularization is immersed in the cell-containing solution for a predetermined time, and then the circulating device 10A is activated, and the vascular device 10A The cell-containing solution is circulated in a flow substantially corresponding to the blood flow of the human body. As a result, the decellularized vascular tissue B is placed in a cell-containing solution having a flow substantially equivalent to blood flowing in the aorta of the human body, and the autologous cells are seeded on the vascular tissue B. . Here, the revolving drive means 124 (see FIG. 3) is activated before the pulsating flow is generated by the circulating device 10A, so that the vascular tissue B immersed in the cell-containing solution is vertically moved. As a result, the cell-containing solution removes the influence of gravity and adheres to substantially the entire vascular tissue B evenly. In addition, such a revolving motion may be continuously performed when a pulsatile flow of the cell-containing solution is generated.

Therefore, according to such an embodiment, not only the decellularization effect and the cell seeding effect can be significantly improved than in the conventional treatment method, but also the vascular tissue B is held in the holder 39 while maintaining the vascular tissue B in the holder 39. The decellularization process and the cell seeding process can be performed in a series of operations, and the effect of easily and quickly processing heterogeneous biological valves accompanying transplantation can be obtained. In addition, such a series of processing operations can be performed in one closed circuit, and the effect of preventing contamination of the heterogeneous living body valve and maintaining cleanliness can be obtained.

Next, the present inventors conducted an experiment for demonstrating the decellularizing effect and cell seeding effect based on the present invention.

(1) Decellularization effect experiment

[Example 1]

In Example 1, bile acid at 37 ° C. was used as the cell removing solution. Then, the bile acid is injected into the circulation device 1OA, and a flow in a state substantially equivalent to the blood flow (pulsating flow) in the human aorta is given to the bile acid. Valve including valve The blood vessels and tissues were left for 24 hours. The condition of the circulation device 10 A at this time is that the average flow rate is 5 liters per minute, the number of beats of the drive pump 12 is 70 times per minute, and the maximum and minimum fluid pressures of bile acids are as follows. The average fluid pressure was set to about 90 mmHg, which roughly corresponded to the general maximum and minimum pulse pressures of humans. Then, the vascular tissue after the treatment was imaged in an enlarged state with an electron microscope, and the average number of the original cells (endothelial cells and fibroblasts) per unit area (1 mm ² ) remaining in the vascular tissue was counted.

As a result, as shown in FIG. 11, about 850 original cells remained per unit area.

[Example 2]

In the second embodiment, in addition to the conditions of the first embodiment, a predetermined microwave is applied while rotating the vascular tissue using the holding device 10B. Here, a microwave with a frequency of 2.45 GHz was used, and three combinations of output and irradiation time were performed. That is, irradiation was performed for 8 hours at an output of 100 W, irradiation for 12 hours at an output of 500 ^, and irradiation for 24 hours at an output of 500 W. In each case, the rotation speed (rotation speed) of the vascular tissue was set to 4 rotations per minute. Then, as in the case of Example 1, the average number of the original cells per unit area (1 mm ² ) remaining in the vascular tissue was counted.

As a result, as shown in FIG. 11, when irradiation was performed at 100 W for 8 hours, about 380 original cells remained per unit area. On the other hand, in the case of irradiation at 500 W for 12 hours, and the case of irradiation at 500 W for 24 hours, no residual original cells were observed. In addition, proteins contained in the original tissue were substantially removed. On the other hand, in each case, almost no damage was seen in collagen and elastin.

[Comparative Example 1]

As a comparative example to the above Examples 1 and 2, the vascular tissue was immersed for 24 hours in a non-flowing bile acid contained in a predetermined container. Then, as in the case of Example 1, etc., the average number of original cells per unit area (1 mm ² ) remaining in the vascular tissue was counted.

As a result, as shown in FIG. 11, about 970 original cells remained per unit area. The vascular tissue used in Examples 1 and 2 and Comparative Example 1 had about 1880 original cells per unit area (1 mm ² ) in the initial state before the decellularization treatment. From the above results, it can be understood that in Examples 1 and 2, the original cells were significantly removed and a higher decellularization effect was obtained than in Comparative Example 1. In particular, a higher decellularization effect was obtained when microwave irradiation was performed than when only vascular tissue was immersed in bile acid under pulsatile flow (Example 1), and the output was 100 W. From 500W, the original cells will not remain. Although the explanation is omitted this time, the above-mentioned microwave can be applied to the vascular tissue immersed in bile acid with no flow, and even in this case, the decellularization is higher than that of Comparative Example 1. The effect is obtained.

(2) Experiment of cell seeding effect

[Example 3]

In Example 3, first, the vascular tissue after the decellularization treatment was washed with physiological saline for about 1 hour, and then immersed in fibronectin for 4 hours. Before or after that, autologous cells (endothelial cells) were collected from the living body to be transplanted to generate a cell-containing solution. In the cell-containing solution, the collected autologous cells are cultured on a culture dish using the aforementioned M199 for 5 days, and then the cultured autologous cells are detached from the culture dish with trypsin and contained in Ml99. Was obtained. Here, at the time of culture, FBS (Fetal Bovine Serum IWK-500, manufactured by Iwaki), antibiotics (mixed solution of penicillin nostreptomycin), and FGF-2 (Pepro Tech Ec Ltd) was added.

Thereafter, the vascular tissue was rotated vertically while the vascular tissue was immersed in the cell-containing solution for about 4 hours. Then, the blood vessel tissue after the cell seeding treatment was imaged in an enlarged state with an electron microscope, and the state of the autologous cells (endothelial cells) seeded in the blood vessel tissue was observed.

As a result, as shown in FIG. 12, autologous cells were seeded on almost the entire vascular tissue. Here, on average, about 850 autologous cells adhered per unit area (1 mm ² ) of vascular tissue. The area ratio (density) of the autologous cells to the unit area (1 mm ² ) of the vascular tissue was about 18% on average.

[Example 4] In Example 4, after the same steps as in Example 3 were performed, the cell-containing solution was circulated in the circulation device 10A under the same flow conditions as in Example 1.

Specifically, after the blood vessel tissue was immersed in the cell-containing solution in a stationary state for about 48 hours, the blood vessel and the tissue were rotated vertically for about 4 hours. Then, a flow having a state substantially equivalent to the blood flow in the human aorta was applied to the cell-containing solution, and the vascular tissue was left in the flow for 1 hour. Then, as in Example 3, the state of the autologous cells seeded in the vascular tissue was observed.

As a result, as shown in FIG. 13, the autologous cells were seeded evenly over substantially the entire area of the vascular tissue, and the autologous cells were regularly arranged in the flow direction of the cell-containing solution. The autologous cells proliferated and became denser than that. Here, an average of about 217 autologous cells adhered per unit area (1 mm ² ) of the vascular tissue. The area ratio (density) of the autologous cells to the unit area (1 mm ² ) of the vascular tissue was about 63% on average.

[Example 5]

The fifth embodiment is different from the fourth embodiment in that the stationary of the vascular tissue in the cell-containing solution and the vertical rotation of the vascular tissue are processed in reverse order. That is, in this embodiment, after rotating the vascular tissue immersed in the cell-containing solution in the vertical direction for about 4 hours, the vascular tissue is immersed in the cell-containing solution in a stationary state for 48 hours, A flow having a state substantially equivalent to the blood flow in the human aorta was applied to the solution, and the vascular tissue was left in the flow for 1 hour. Other conditions were the same as in Example 4. Then, as in the case of Example 4, the state of the autologous cells seeded in the vascular tissue was observed,

As a result, the autologous cells proliferated and became denser as in the case of Example 4, and the autologous cells were seeded more evenly than in the case of Example 4.

[Example 6]

In Example 6, compared to Example 5, the time for leaving the vascular tissue in the flow of the cell-containing solution under the same conditions as Example 5 was set to 48 hours. Other conditions were the same as in Example 5.

As a result, the seeded cells proliferated and came to cover almost the entire surface of the vascular tissue. [Example 7]

Example 7 differs from Example 6 in that the flow conditions of the cell-containing solution were changed, and the other conditions were the same as Example 6. That is, in this example, the average flow rate of the cell-containing solution was 2 liters per minute, and the average liquid pressure of the cell-containing solution was about 2 OmmHg.

As a result, substantially the same effects as in Example 6 were obtained. In other words, using the cell seeding method of the present invention, seeding was performed even if the flow conditions of the cell-containing solution were changed in consideration of the state of the blood flow and the like of the recipient patient and the location of the heart valve at the recipient. Autologous cells proliferate and cover almost the entire surface of vascular tissue. In general, even in the case of adults and children with different amounts of blood circulating in the body, various heart valves with different blood pressures to be used, etc., it is possible to create an optimal biological valve according to the use condition.

[Comparative Example 2]

As a comparative example with respect to the above Examples 3 to 7, as compared with Example 3, the vascular tissue was kept stationary without rotating in the vertical direction. Then, as in Example 3 and the like, the state of the autologous cells seeded in the vascular tissue was observed.

As a result, unevenness was observed in the autologous cells seeded in the vascular tissue, whereby the number of autologous cells seeded in the vascular tissue was significantly reduced as compared with the cases of Examples 3 to 7.

As a result, in Examples 3 to 7, autologous cells adhered much more than in Comparative Example 2, and a higher cell seeding effect was obtained. In particular, when vascular tissue is immersed in a cell-containing solution under a pulsating flow, the orientation of the cells is improved, and the cell functions are more exerted by activating the cells and the like, so that more cells can be seeded.

In addition, in the present invention, in addition to the vascular tissue including the aortic valve described in the above embodiment, the present invention is also applicable to a decellularization process and a cell seeding process for other living tissues that come into contact with blood. In addition, the present invention can be applied to processing for the same type of biological valve in addition to processing for the different type of biological valve.

Further, in the above-described embodiment, the holder 39 is rotated at the time of microwave irradiation. On the contrary, the holder 39 is kept stationary, and the irradiation port 57 side rotates around the holder 39. It may be. Furthermore, in the above-described embodiment, the holding device 10B is used for both the decellularization treatment and the cell seeding treatment. However, even when only one of these treatments is performed, the holding device 10B can of course be applied. .

In addition, the configuration of each unit of the device according to the present invention is not limited to the illustrated configuration example, and various changes can be made as long as substantially the same operation is achieved. As described above, according to the present invention, it is possible to effectively perform the decellularization process and the cell seeding process on the biological tissue of a heterogeneous biological valve or a homogeneous biological valve. Industrial applicability

INDUSTRIAL APPLICABILITY The present invention can be used for processing a biological valve collected from an animal including a human so as to be transplantable into a predetermined human body.

Claims

The scope of the claims

1. With a holder capable of holding a predetermined biological tissue in a solution for decellularization treatment or cell seeding treatment,

The holding body includes an inflow portion and an outflow portion of the solution, and an installation space for the living tissue located between the inflow portion and the outflow portion, and the flow of the solution flowing from the inflow portion to the outflow portion is provided. A biological tissue holding device provided with a decellularization process and / or a cell seeding process in a state where the biological tissue is disposed therein.

2. The living tissue preservation according to claim 1, further comprising irradiation means for irradiating the living tissue held by the holder with microwaves during the decellularization treatment.

3. The biological tissue holding device according to claim 2, wherein the holder and the irradiation unit are provided so as to be relatively rotatable.

4. The irradiating means stops the irradiation of the microwave when the temperature of the solution becomes higher than a predetermined temperature, and starts the irradiation of the microwave when the temperature of the solution becomes lower than the predetermined temperature. 4. The device for holding a living tissue according to claim 2 or 3, characterized by:

5. The inflow section and the outflow section have first and second flow paths, respectively. Each first flow path is provided so as to be able to communicate via the inside of a tubular living tissue, The biological tissue holding device according to any one of claims 1 to 4, wherein the second flow path is provided so as to be able to communicate through the outside of the biological tissue.

6. The preservation of living tissue according to claim 5, wherein the outflow portion is provided with a guide path for accommodating a temperature sensor in the first flow path and the Z or the second flow path.

7. The living tissue holding device according to any one of claims 1 to 6, wherein the holding body is provided so as to be rotatable in a direction of inverting the upper and lower sides of the living tissue during the cell seeding process. .

8. A biological tissue processing device comprising the holding device according to any one of claims 1 to 7, wherein the biological tissue is subjected to a decellularization process and / or a cell seeding process.

The solution circulates in a manner simulating the circulation of blood in a living body to be transplanted with the living tissue. Equipped with a circulating device,

The circulating device is connected to the inflow portion and the outflow portion, and applies a flow substantially corresponding to the blood flow in the living body to the solution flowing from the inflow portion to the outflow portion. .

9. The circulating device includes a driving pump that generates a pulsating flow, and a circulating passage that is disposed between an inflow port and an outflow port of the driving pump and that has the holding device provided in the middle thereof.

The circulation path includes: a resistance applying unit that applies a flow resistance to the solution; and a hydraulic pressure attenuating unit that is disposed downstream of the resistance applying unit and attenuates a hydraulic pressure of the solution. The biological tissue processing apparatus according to claim 8.

10. The biological tissue processing apparatus according to claim 9, wherein the circulation path includes amplitude adjustment means for adjusting the amplitude of the hydraulic pressure upstream of the resistance applying means.