JP2008206477A - Cell seeding method using needleless syringe - Google Patents

Abstract

An object of the present invention is to provide a very simple method that allows cells to be embedded even within the scaffold without causing significant damage to the scaffold itself, and to disperse the cells over a wide area within the scaffold. A cell seeding method is provided. Furthermore, even in the case of a scaffold having a dense structure, a cell seeding method capable of dispersing cells over a wide area of the scaffold by a very simple method is provided. A method of seeding cells into a fold, wherein the cells to be seeded are injected into the scaffold by using a needleless syringe to embed the cells inside the scaffold.
[Selection] Figure 12

Description

  The present invention relates to the field of regenerative medicine. More specifically, the present invention relates to a cell seeding method useful in regenerative medicine.

  In recent years, in the field of regenerative medicine, cells are incorporated into a scaffold for cell transplantation (scaffold; carrier), and the incorporated cells are cultured and then transplanted to a patient. Attention has been focused on the development of methods of treatment by injection.

  In the field of using scaffolds for cell transplantation, in order to find materials suitable as scaffolds, research has been conducted on both synthetic and biological materials. Developments have been made that have a high porosity (gap into which cells enter) and mechanical properties.

  In the field of using a biological material as a scaffold, a scaffold obtained by decellularizing a tissue collected from a living body has been reported. Specific methods of decellularization include a method of applying an ultrahigh hydrostatic pressure (Japanese Patent Laid-Open No. 2004-97552), a method of irradiating microwaves at a low temperature (Japanese Patent Laid-Open No. 2004-187952), and the like. It is done.

As a method for seeding cells in a scaffold, several methods have been examined as general methods.
For example, the method of dripping a cell suspension to a scaffold is mentioned. This method is used as the simplest method. However, this method does not allow cells to be seeded inside the scaffold.
Another example of the cell seeding method is a method of injecting a cell suspension into the scaffold by a needled syringe. This method is a method in which seeding can be performed inside the scaffold.
Furthermore, a cell seeding method using centrifugal force can be mentioned. According to this method, the cell can be adhered to the surface of the scaffold, and in the case of a scaffold with a large gap, the cells can be seeded inside the scaffold.

  In addition, as a method for seeding cells in a scaffold, a biodegradable hollow fiber filled with lumens to be seeded is embedded in a decellularized biological tissue piece (scaffold), and inside the tissue piece, In addition, a method for releasing cells filled in hollow fibers has been reported (Japanese Patent Laid-Open No. 2007-168).

JP 2004-97552 A Japanese Patent Laid-Open No. 2004-187952 JP 2007-168 A

Transplantation using a method of seeding cells on a scaffold is very useful in the following points 1) and 2).
1) Such a transplantation method involves culturing cells to some extent in vitro and then transplanting them into the body. That is, after a certain amount of tissue is constructed under culture conditions, transplantation to a patient is performed. For this reason, fusion with a graft and the living tissue of a transplant destination is quick, and healing of an affected part is accelerated | stimulated.

2) By culturing in advance, cells attached to the scaffold are less likely to flow out after transplantation. For this reason, more cells can be held in the affected area.
On the other hand, in a general cell transplantation method, a cell suspension is injected in the usual injection manner. That is, the cells are transplanted into the patient by injecting the cell suspension directly into the patient. For this reason, after transplantation, the outflow of transplanted cells from the patient's tissue is remarkable, and it has already been reported that the transplantation efficiency is poor, which is a problem.

When cells are seeded and transplanted in a scaffold having a certain size and thickness, the cells should be injected into the inside of the scaffold.
It will be described with reference to FIG. 10 that it is preferable to seed cells to the inside of the scaffold. When transplanting to a myocardial infarction site due to myocardial infarction, a case using a scaffold without seeding cells as a graft (A), a case using a scaffold with cells seeded only on the surface as a graft (B) Consider a case (C) in which a scaffold in which cells are seeded is used as a graft. In case (A), since there are no cells in the scaffold, there is a possibility of cells entering the graft from the periphery of the graft after transplantation, but it takes time to regenerate the tissue. In case (B), cells adhered to the surface of the scaffold may gradually enter the scaffold after transplantation, but it still takes time to regenerate the tissue. On the other hand, in the case (C), since the cells are originally contained in the graft, invasion of the capillary into the graft is promoted by inducing the capillary of the patient around the graft. As a result, significant tissue regeneration can be promoted. For this reason, early healing of the myocardial injury site becomes possible.

  However, in a normal cell seeding method, it is difficult to seed cells in a deep part or a wide area. For example, in order to seed cells deeply, it is known to use a needled syringe as described above.

Cell seeding into a scaffold using a needled syringe and direct cell transplantation to an affected area are usually performed. However, this method is not effective in the following points 1) and 2).
1) Since the liquid suspension can be stored in the scaffold or the affected tissue, it is impossible to disperse the cells extensively (see FIG. 11A). When a bioscaffold is used, it is more difficult to disperse cells widely because the scaffold structure is dense.
2) The scaffold and the affected tissue are greatly damaged by the penetration of the needle (see FIG. 11B).

  Cells can also be seeded inside the scaffold by a cell seeding method using centrifugal force, but this method can be used effectively only when a scaffold with a large gap is used. Can not. That is, cell seeding cannot be performed inside a scaffold having a dense structure.

  Therefore, an object of the present invention is a very simple method, in which cells can be embedded even inside the scaffold without causing major damage to the scaffold itself, and in a wide area within the scaffold. The object is to provide a cell seeding method capable of dispersing cells. Furthermore, the object of the present invention is to provide a cell seeding method that can disperse cells over a wide area of the scaffold by a very simple method even in the case of a scaffold having a dense structure. There is to do.

As a result of intensive studies, the present inventors have found that the object of the present invention can be achieved by seeding cells using a needleless syringe, and have completed the present invention.
The present invention includes the following inventions.
The following [1] to [7] are directed to the cell seeding method.

[1]
A method of seeding cells in a scaffold, wherein the cells to be seeded are injected into the scaffold by using a needleless syringe to embed the cells inside the scaffold.
That is, the present invention is characterized in that a needleless syringe is used as a means for seeding cells on a scaffold. In the present invention, the cells reach the inside of the scaffold by a needleless syringe. Therefore, in the present specification, “seeding” a cell into the scaffold may be described as “embedding” or “embedding” the cell inside the scaffold. In the present invention, cells are seeded in a relatively wide area of the scaffold by a needleless syringe. Thus, herein, “seeding” a cell into the scaffold may be described as “dispersing” or “spreading” the cell into the scaffold.

[2]
The cell seeding method according to [1], wherein the scaffold is made of a biological material or a synthetic material.
In the present invention, a scaffold made of a biological material may be referred to as a bioscaffold. In addition, a scaffold made of a synthetic material may be described as an artificial scaffold.

[3]
The cell seeding method according to [1] or [2], wherein the scaffold is a decellularized biological tissue piece.
The treatment of decellularization includes removal of cellular components from biological tissue pieces and removal and / or inactivation of bacteria and viruses. As treatment for decellularization, treatment by application of ultrahigh hydrostatic pressure, treatment by microwave irradiation, and the like are preferably performed.

[4]
The cell seeding method according to [3], wherein the biological tissue piece is a biological tissue piece collected from a human or a biological tissue piece collected from a mammal other than a human.

[5]
The cells to be seeded are accommodated in the needleless syringe in the form of a suspension dispersed in a liquid selected from a cell culture solution, a biocompatible sol, and a biocompatible gel solution [1] The cell seeding method according to any one of to [4].

[6]
Any of [1] to [5], wherein the cells to be seeded are selected from the group consisting of human cells and patient autologous cells to which the scaffold in which the cells to be seeded are embedded may be transplanted The cell seeding method according to claim 1.

[7]
The needleless syringe has a pressing mechanism using a method selected from the group consisting of a spring method, a gas pressure method, an explosive method, an electromagnetic force method, and a piezoelectric method, according to any one of [1] to [6]. Cell seeding method.

[8] below is directed to a needleless syringe that can be used to seed cells. The needleless syringe of the following [8] is usefully used in the methods [1] to [7].
[8]
Needleless syringe containing cells to be seeded.

According to the present invention, it is a very simple method that allows cells to be embedded inside the scaffold without causing major damage to the scaffold itself, and also distributes the cells over a wide area within the scaffold. It is possible to provide a cell seeding method that can be performed. Furthermore, according to the present invention, there is provided a cell seeding method capable of dispersing cells over a wide area of the scaffold by a very simple method even in the case of a scaffold having a dense structure. Can do.
Therefore, according to the present invention, a cell seeding method having high utility in regenerative medicine can be provided.

[1. Scaffold]
The material of the scaffold may be a biological material or a synthetic material.
[1-1. Scaffolds made of biological materials]
A scaffold made of a living body-derived material (bioscaffold) is produced by decellularizing a tissue piece collected from a living body.

[1-1-1. Preparation of bioscaffold]
The decellularization treatment includes removal of cellular components, removal of bacteria / viruses and / or inactivation. The decellularization treatment is not particularly limited. A person skilled in the art can appropriately select from chemical treatment, biochemical treatment, and physical treatment.
For example, chemical treatment includes treatment using glutaraldehyde, a surfactant, and other chemicals. Specific examples of the surfactant include Triton X-100. Specific examples of the chemical solution include SynerGraph manufactured by CryoLife.
Biochemical treatment includes treatment using a proteolytic enzyme such as trypsin.

Examples of the physical treatment include treatment by applying an ultrahigh hydrostatic pressure, treatment by microwave irradiation, and the like.
In processing by applying an ultrahigh hydrostatic pressure, an ultrahigh hydrostatic pressure is applied to a living tissue in a medium (liquid). Thereby, the connective tissue in the living tissue is separated from the cellular components and the like while maintaining the structure and biomechanical characteristics intact. In this treatment method, the ultrahigh hydrostatic pressure refers to about 5000 to 15000 atm. And as temperature, the range of 0 degreeC-40 degreeC is maintained. As an apparatus capable of performing such a treatment, an apparatus such as a doctor chef manufactured by Kobe Steel, an ultrahigh pressure experimental apparatus manufactured by Kogaku Engineering Co., Ltd., or the like can be given. Further details of the treatment by applying an ultrahigh hydrostatic pressure are described in JP-A-2004-97552.

  In the treatment by microwave irradiation, microwaves are irradiated to the living tissue immersed in the treatment liquid while maintaining a temperature of 0 ° C. to 40 ° C. The frequency of the microwave can be 2450 MHz. Such a process can be performed by using a commercially available microwave rapid sample processing apparatus. Further details of the processing by microwave irradiation are described in Japanese Patent Application Laid-Open No. 2004-188792.

  Such a physical treatment method is a preferable method from the following points. It is possible to achieve removal of cellular components, bacteria, and viruses inside relatively large tissues that cannot be achieved by chemical treatment or biochemical treatment; treatment can be performed without impairing biomechanical properties. The point which is possible; and the point which can process simply and in a short time.

[1-1-2. Biology and tissue from which the bioscaffold originated]
The tissue piece serving as the biological material may be derived from a human or a non-human mammal. In the case of humans, tissues removed from brain death or heart death donors are included. In the case of non-humans, tissues extracted from mammals including pigs, cows, monkeys and the like are included.
Obtaining a safe biological material from a non-human animal by rigorous decellularization has great significance in that it enables the supply of inexpensive and useful materials in the current situation of donor shortages.

  A tissue piece serving as a biological material can be obtained from organs such as the heart, kidney, liver, pancreas, brain, and lung. Therefore, the tissue piece may be soft tissue or hard tissue. Examples of soft tissues include blood vessels, heart valve, pericardium, cornea, amniotic membrane, dura mater, and trachea. Examples of the hard tissue include bone, cartilage, and teeth.

Since the bioscaffold produced from such a tissue originally maintains the structure possessed by the living tissue, it is possible to prepare a graft having the necessary formability and mechanical properties. For this reason, the bioscaffold can be transplanted even to a difficult site in the case of a scaffold made of a synthetic material. In particular, the bioscaffold is useful when transplanting into a trachea, pericardium, cartilage, or the like.
In addition, the bioscaffold originally maintains the dense structure of living tissue. For this reason, unlike an artificial scaffold having a porous structure, the seeded cells are unlikely to flow out, and thus there is an advantage that the cell fixing property is good.

[1-2. Scaffold made of synthetic material]
A scaffold made of a synthetic material (artificial scaffold) is a porous material made of a biocompatible polymer in any appropriate shape. The shape is not particularly limited, and examples thereof include flat structures such as disks, sheets, patches, films, sponges, and meshes. Furthermore, it may be a three-dimensional structure such as a sphere, an ellipsoid, or a tube. These three-dimensional structures may be hollow bodies.

  Biocompatible polymers include those that are biodegradable or bioabsorbable. The biodegradable polymer is preferably a rapid biodegradable polymer. Rapid biodegradability refers to the property of degrading within a few days at the latest. This property can be realized by using a polymer having a relatively low molecular weight, thinning the material, impregnating the esterase enzyme into the material, and the like.

As the biocompatible polymer, a generally known polymer can be used without any particular limitation.
For example, examples of the biocompatible polymer include polymer materials such as polyesters, polyorthoesters, and polyanhydrides. Specific examples of such a polymer material include polymers of lactic acid, lactide (a cyclic dimer of lactic acid), glycolic acid, or sebacic acid, and copolymers thereof.

Biocompatible polymers also include caprolactones, amides, carbonates, amino acids, acetals, orthoesters, cyanoacrylates, urethane polymers and copolymers thereof.
Furthermore, examples of the biocompatible polymer include polysaccharides such as starch and a partial hydrolyzate thereof, and protein polymers such as collagen.

[2. Cells to be seeded]
[2-1. Preparation of cells to be seeded]
The cells to be seeded are prepared in the form of a cell suspension dispersed in a physiologically acceptable liquid. The physiologically acceptable liquid can be selected from cell culture fluids, biocompatible sols, and biocompatible gel solutions. When the cells to be seeded are used in the form of a cell suspension dispersed in a liquid containing a biocompatible sol or gel, they are seeded by hardening the gel or sol in the scaffold after seeding. Cells are included. Inclusion of the cells in the scaffold can confine moisture and prevent the cells from flowing out and leaking together with the moisture.

  The biocompatible sol and biocompatible gel in the present invention are water-soluble polymers, preferably biodegradable water-soluble polymers. As such a water-soluble polymer, those described in US Pat. No. 5,709,854 can be used.

[2-2. Cell types and origins to be seeded]
The type of cells to be seeded can be appropriately determined by those skilled in the art according to the site to be transplanted and the purpose of recellularization. For example, endothelial cells, endothelial progenitor cells, bone marrow cells, proosteoblasts, chondrocytes, fibroblasts, skin cells, muscle cells, liver cells, kidney cells, intestinal cells, stem cells, and other regenerative medicine fields Any cell can be mentioned.

  The origin of such cells is usually human. Preferred are autologous cells collected from a human to be transplanted, but cord blood-derived cells, commercially available cells, and the like are also acceptable. Specific examples of commercially available cells include HUVEC (human unbilical vein endothelial cells), PBMC (peripheral blood monoclonal cells), human skin cells, and the like. Further specific examples of commercially available human skin cells include those sold under the trade name of apligraf; Apligraph (manufactured by Organogenesis).

[3. Needleless syringe]
The needleless syringe used in the present invention is not particularly limited.
Specifically, it is possible to use a nozzle having a nozzle hole and having a chamber for containing a liquid (cell suspension) containing cells to be seeded and a syringe body provided with pressing means. The pressing means presses the cell suspension accommodated in the chamber in the direction of the nozzle hole and ejects the cell suspension from the nozzle hole.

  Pressing means include springs (coil springs, leaf springs, disc springs, springs, bamboo springs, and other springs), compressors (use of air pressure), cylinders (e.g., carbon dioxide cylinders), piezoelectric elements, explosives (chemical Reaction, blasting = air pressure), electromagnetic force, hydraulic pressure, water pressure / steam, electric motor (motor), prime mover (engine), magnet (permanent magnet, various magnets such as superconducting magnet), shape memory alloy and the like.

  The needleless syringe used in the present invention will be further described. The needleless syringe has a nozzle hole, and has a cell suspension containing chamber provided with a piston, and a syringe body provided with a pressing mechanism. The piston is provided so as to be located on the opposite side of the nozzle hole with the cell suspension interposed therebetween. The pressing mechanism is for pressing the piston in the direction of the nozzle hole and ejecting the cell suspension from the nozzle hole.

  As the pressing mechanism, any of a spring method (compression spring method or tension spring method), a gas pressure (carbon dioxide gas, nitrogen gas) method, an explosive method, an electromagnetic force method, a piezoelectric method, or the like may be used. As for the gas pressure system, reference can be made to JP-A-61-265147, JP-T-3-503374 (WO89 / 08469), JP-T 8-509604 (WO94 / 24263), and the like. For example, JP 2000-126293 A, JP 2001-187139 A, JP 2001-61964 A, and the like can be referred to. Of these, a compression spring method that is easy to handle and safe is preferable.

  In the present invention, as the needleless syringe, either one having one nozzle hole or one having a plurality of nozzle holes can be used. From the viewpoint that cells can be seeded in a wider range, those having a plurality of nozzle holes can be preferably used.

  In the present invention, as the needleless syringe, any one that can be used only once and one that can be used repeatedly can be used. From the viewpoint that accidents such as infection due to reuse of a syringe can be prevented, those that can be used only once are preferable.

[3-1. Specific example of needleless syringe]
Examples of the needleless syringe that can be used in the present invention include Shimadzu Corporation ShimaJET.
In the present invention, the needleless syringe is disclosed in JP 2004-329635 A, JP 2004-358234 A, and International Publication No. 2004/110533 pamphlet as described with reference to the following drawings. Can be used.

  FIG. 1 is a longitudinal sectional view of an example of a syringe that may be used in the present invention. FIG. 2 is a longitudinal sectional view of the syringe during storage. FIG. 3 is a cross-sectional view taken along line III-III in FIG. 4 is a cross-sectional view taken along the line IV-IV in FIG. FIG. 5 is an enlarged perspective view of a holding member of the syringe. In the following description, the top and bottom are referred to FIG.

  Referring to FIG. 1, the needleless syringe includes a nozzle ampule 11 and a syringe body 12 to which the nozzle ampule 11 is attached. Referring to FIG. 2, the syringe body 12 includes a safety cap 13 attached to the syringe body 12 in place of the nozzle ampoule 11.

  The nozzle ampoule 11 is composed of a bottomed cylinder having an ampoule opening 21 at the upper end and a cell suspension chamber 22 inside. The bottom surface of the cell suspension chamber 22 of the nozzle ampoule 11 is formed as an upward concave conical surface. The cell suspension chamber 22 contains a cell suspension L and a piston 23 from above. The nozzle ampoule 11 is preferably made of a transparent material suitable for the cell suspension L in order to visually recognize the cell suspension L, for example, a transparent hard plastic such as polycarbonate or glass.

  At the edge of the ampoule opening 21, two convex fitting portions 24 having an annular shape are provided so as to protrude in opposite directions. The sandwiching angle of each convex fitting part 24 is a little less than 90 degrees (see FIG. 3). A nozzle hole 25 is formed at the center of the lower surface of the nozzle ampoule 11 so as to communicate with the cell suspension chamber 22. A nozzle protection cap 26 is provided below the nozzle ampoule 11. The protective cap 26 is usually made of plastic, but it is preferable that a thin rubber material for sealing (not shown), for example, silicone rubber, is attached to the central portion in contact with the nozzle hole 25. The rubber material can prevent leakage of the cell suspension.

  The piston 23 is usually made of plastic, and has a horizontal disk shape fitted to the peripheral wall of the nozzle ampoule 11 so as to be slidable in the vertical direction. The lower surface of the piston 23 is formed in a downward convex conical surface that matches the bottom surface of the cell suspension chamber 22. An O-ring 27 is attached to the outer peripheral surface of the piston 23. The upper surface of the piston 23 is flush with the upper end surface of the nozzle ampoule 11.

  The syringe body 12 includes a vertical cylindrical housing 31, and a pressing mechanism 32 that is accommodated in the housing 31 and presses the piston 23 downward. The housing 31 is preferably made of metal in order to maintain strength, but may be made of plastic.

  The inside of the housing 31 is partitioned by a horizontal partition wall 33 into a lower spring chamber 34 and an upper release chamber 35. A communication hole 36 is formed in the central portion of the partition wall 33. A housing opening 37 that is matched with the ampoule opening 21 is formed at the lower end of the spring chamber 34. An end wall 38 is provided at the upper end of the release chamber 35. A holding hole 39 is formed at the center of the end wall 38.

  Two concave fitting portions 41 into which the respective convex fitting portions 24 are fitted are provided at the edge of the housing opening 37. The sandwiching angle of each concave fitting portion 41 is substantially equal to that of the convex fitting portion 24.

  The pressing mechanism 32 includes a pressing member 42 that is held up and down in the housing 31, a compression coil spring 43 that urges the pressing member 42 downward, and a spring of the pressing member 42 in a state in which the spring 43 is compressed. A holding member 44 that releasably holds the movement by 43 and a release member 45 that releases the holding by the holding member 44 are provided.

  The pressing member 42 is in contact with the upper surface of the piston 23 and is in contact with the inner surface of the peripheral wall of the spring chamber 34. 52 is provided with a vertical rod 53 that is provided upright in the center and has an upper end projecting into the release chamber 35 through the communication hole 36. An annular outwardly engaging projection 54 is provided at the upper end of the rod 53. The engagement protrusion 54 is formed in such a size that it cannot pass through the communication hole 36 of the partition wall 33. The lower surface of the protrusion 54 is formed in an upwardly expanding taper shape. The pressing member 42 is preferably made of metal.

  The spring 43 is accommodated in the spring chamber 34 so as to surround the rod 53 and to be interposed between the partition wall 33 and the spring receiver 52.

  The holding member 44 is integrally formed of, for example, hard plastic. As shown in detail in FIGS. 4 and 5, the holding member 44 surrounds the rod 53 and is received by the partition wall 33, and is concentric with the rod 53. The vertical cylindrical portion 62 is provided upright on the upper surface of the ring portion 61. An annular inward engagement protrusion 63 is provided on the upper end of the inner surface of the cylindrical portion 62. On the upper side surface of the inward engagement protrusion 63, an upwardly expanding tapered concave stopper surface 64 is formed. The inner edge portion of the stopper surface 64 is engaged with the lower surface of the outward engagement protrusion 54 of the rod 53 from below. A slit 65 is formed at a position that divides the cylindrical portion 62 into four equal parts in the circumferential direction so as to be longitudinally cut including the inward engagement protrusion 63. In the middle part of the height of both edges of each slit 65, the slits 65 are partially connected by an easily breakable breakable part 66. That is, the cylindrical portion 62 is composed of four pieces, and the pieces are partially connected to each other by the breakable portion 66. As the plastic material for the holding member 44, an elastic material is selected. In the illustrated example, the cylindrical portion 62 is divided into four equal parts in the circumferential direction by the slit 65, but it is only necessary to be divided into a plurality of parts, and it is not always necessary to be equally divided.

  The release member 45 is formed of a plastic both-end closed vertical cylinder that is fitted in the holding hole 39 so as to be movable up and down. On the part from the outer periphery of the bottom surface of the release member 45, a lower thin taper convex spreading surface 67 that is matched with the stopper surface 64 is formed. A protective cap 68 is attached to the upper end of the housing 31 so as to wrap the release member 45.

  A slight fracture gap C1 is formed between the upper end surface of the rod 53 and the center of the bottom surface of the release member 45 in a state where the spreading surface 67 is brought into contact with the stopper surface 64.

  The safety cap 13 is made of a plastic disc-like body integrally formed with a hemispherical gripping portion 71 at the center of the lower surface. Two convex fitting portions 72 having the same shape as the convex fitting portion 24 of the nozzle ampoule 11 are provided on the outer peripheral edge portion of the safety cap 13 in the same manner. At the center of the upper surface of the safety cap 13, there is provided a circular raised push-up portion 73 that is fitted into the ampoule opening 21 and has a height corresponding to the breaking gap C1. The safety cap 13 is attached to the housing 31 by fitting the convex fitting portion 72 of the safety cap 13 into the concave fitting portion 41 of the housing 31. In this state, as compared with the state in which the nozzle ampoule 11 is attached to the housing 31, the pressing member 42 is lifted by a height corresponding to the height of the push-up portion 73. As a result, a safety gap C2 is formed between the outward engagement protrusion 54 of the rod 53 and the inward engagement protrusion 63 of the holding member 44 instead of the fracture gap C1. By forming the safety gap C2, the spring force of the spring 43 does not act on the holding member 44 at all. A higher level of safety is ensured when storing the syringe.

  A safety cap 13 is attached to the syringe body 12 when the syringe is not used, that is, usually during the period from manufacture of the syringe to immediately before use of the syringe. The nozzle ampoule 11 enclosing the cell suspension may be stored separately in a refrigerator or the like.

  When using the syringe, the housing 31 and the safety cap 13 are rotated about 90 degrees in opposite directions. Then, the convex fitting portion 72 of the safety cap 13 comes out of the concave fitting portion 41 of the housing 31 and the safety cap 13 is removed from the housing 31. As a result, a fracture gap C1 is formed instead of the safety gap C2. In this state, the lower surface of the spring receiver 52 is flush with the lower end surface of the housing 31.

  Next, instead of the safety cap 13, the nozzle ampoule 11 in which the cell suspension is sealed in the housing 31 is attached. When the two convex fitting parts 24 of the nozzle ampoule 11 are positioned between the two concave fitting parts 41 of the housing 31 and the housing 31 and the safety cap 13 are rotated in the opposite directions by about 90 degrees, The fitting portions 24 and 41 are fitted to each other.

  After removing the upper and lower protective caps 26, 68, the nozzle hole 25 is pressed against the scaffold to be sprayed, and the release member 45 is pressed down. When the release member 45 is pushed down, the spreading surface 67 presses the stopper surface 64 obliquely downward, and the cylindrical portion 62 tends to be expanded upward. Thereby, first, all the breakable parts 66 or a part thereof is broken. Subsequently, when the release member 45 is pushed down, the tubular portion 62 is further expanded upward, and each piece of the tubular portion 62 is elastically deformed and falls outward. Along with this, the stopper surface 64 gradually moves outward, and eventually the engagement between the inward engagement protrusion 63 of the stopper surface 64 and the outward engagement protrusion 54 of the rod 53 is released. Then, the pressing member 42 is vigorously pushed downward by the force of the spring 43. At the same time, the piston 23 is pushed down, so that the cell suspension L is compressed at a high pressure, and is ejected from the nozzle hole 25 at a high speed and is scanned. An injection is made inside the fold. Since the engagement protrusion 54 of the rod 53 cannot pass through the communication hole 36 of the partition wall 33, after the cell suspension is injected, the pressing member 42 is stopped at a predetermined position to ensure safety.

  In the above, the needleless syringe in which the nozzle ampoule and the syringe body are detachable has been described. However, other configurations, except that the nozzle ampoule and the syringe main body are detachable, in particular, each configuration of the extrusion mechanism 32, particularly the holding member 44, the breakable portion 66, the extrusion member 42, and the release member 45 are nozzles. This also applies to a needleless syringe in which the ampule is fixed to the syringe body.

  6 and 7 show a modified example of the nozzle ampoule 11. 6 and 7, parts corresponding to those shown in FIG. 1 are denoted by the same reference numerals.

  The cell suspension chamber 22 of the nozzle ampoule 11 according to this modification includes one large-diameter chamber 81 located at the upper part and four small-diameter chambers 82 respectively connected to the lower end thereof. As shown in FIG. 7, the small diameter chambers 82 are arranged at equal intervals in the circumferential direction in a circle defined by the inner surface of the peripheral wall of the large diameter chamber 81.

  The piston 23 is slidably fitted to the large-diameter chamber 81 and has one horizontal disk-shaped large-diameter piston 92 having an air vent 91 in the center, and is provided in a suspended manner on the lower surface of the large-diameter piston 92. Each of the small diameter chambers 82 is slidably fitted with four vertical round bar-shaped small diameter pistons 93.

  The bottom surface of each small diameter chamber 82 is formed as an upward concave conical surface, and the lower surface of each small diameter piston 93 is formed as a downward convex conical surface that matches the bottom surface of the small diameter chamber 82. A nozzle hole 94 is formed at the lower end of each small-diameter chamber 82. An O-ring 95 is attached to the outer surface of each small diameter piston 93.

  When a plurality of nozzle holes 94 are formed as in the nozzle ampoule 11 according to this modification, the cell suspension L can be uniformly injected over a wide range in the scaffold tissue as compared with the case of a single nozzle hole. There is an advantage. In the needleless syringe used in the present invention, a nozzle ampoule having a plurality of nozzle holes may be used depending on the purpose of treatment and the type of cell suspension. In this modification, four nozzle holes are shown, but it goes without saying that the number of nozzle holes can be changed as appropriate. The change in the number of nozzle holes is applied to a needleless syringe in which the nozzle ampule and the syringe main body are detachable and a needleless syringe in which the nozzle ampule is fixed to the syringe main body.

  8 and 9 show modified examples of the holding member 44. FIG. In this modified example, a thick holding portion 101 is provided on a part of the peripheral wall of the release chamber 35 of the housing 31, and a guide portion 102 is provided on the opposite side of the holding portion 101 with the rod 53 interposed therebetween. ing. A rod 53 is brought into sliding contact with the guide portion 102.

  The holding portion 101 is formed with a lock hole 103 extending in a direction perpendicular to the rod 53 through the inside and outside. On the other hand, an internal enlarged guide groove 104 extending vertically is formed on the outer peripheral surface of the release chamber 35. On the bottom surface of the guide groove 104, the outer end of the lock hole 103 is opened. Further, an engaging protrusion 105 extending toward the holding portion 101 is provided on the outer surface of the upper end portion of the rod 53. The lower surface of the engaging protrusion 105 is an inclined surface. The engagement protrusion 105 is formed in such a size that it cannot pass through the communication hole 36 of the partition wall 33.

  The holding member 44 includes a stopper piece 111 that is movably inserted into the lock hole 103 and has an upward stopper surface 112 provided at the inner end thereof.

  A lock member 121 is fitted in the guide groove 104 so as to be slidable up and down. A recess 122 that allows insertion of the outer end of the stopper piece 111 is formed in the inner surface of the lock member 121.

  In a state where the lock member 121 is brought into contact with the upper end surface of the guide groove 104, the recess 122 is closed by the bottom surface of the guide groove 104. Further, in this state, a gap is formed between the lower end surface of the guide groove 104 and the lock member 121, and the safety pin 131 is inserted into the gap so as to be freely inserted and removed.

  The downward movement of the lock member 121 is restricted by the safety pin 131, and at the same time, the outward movement of the stopper piece 111 is restricted by the lock member 121. When the safety pin 131 is removed, the movement of the lock member 121 becomes free. When the lock member 121 is moved downward and the heights of the stopper piece 111 and the recess 122 are made coincident, the outward movement of the stopper piece 111 becomes free. Since the rod 53 is biased downward by the force of the spring 43, the stopper piece 111 is pushed outward by the engagement protrusion 105 of the rod 53. Then, the engagement protrusion 105 and the stopper piece 111 are disengaged, and the rod 53 is pushed down by the force of the spring 43. At the same time, the piston 23 is pushed down, so that the cell suspension L is at a high pressure. It is compressed and injected from the nozzle hole 25 at a high speed, and injection is performed inside the scaffold. Since the engagement protrusion 105 of the rod 53 cannot pass through the communication hole 36 of the partition wall 33, after the cell suspension is injected, the pressing member 42 is stopped at a predetermined position to ensure safety.

  The modified example of the holding member 44 shown in FIGS. 8 and 9 is applicable to a needleless syringe in which the nozzle ampule and the syringe main body are detachable and a needleless syringe in which the nozzle ampule is fixed to the syringe main body. Is done.

  In the case of a needleless syringe in which the nozzle ampoule and the syringe main body are detachable, the modified example of FIG. 8 represents a state when the syringe is in use, and corresponds to FIG. When the syringe shown in the modification of FIG. 8 is stored, a safety cap 13 is attached to the syringe body 12 in the same manner as shown in FIG. By attaching the safety cap 13, the rod 53 is lifted by a height corresponding to the height of the push-up portion 73 of the cap 13, and a safety gap (see FIG. 5) is formed between the engagement protrusion 105 of the rod 53 and the stopper piece 111. 2) corresponding to the safety gap C2 in FIG. Due to the safety gap, the spring force of the spring 43 does not act on the stopper piece 111 at all, so that higher safety of the syringe during storage is ensured.

  As described above, the modified example of the nozzle ampoule having a plurality of nozzle holes and the modified example of the holding member have been shown, but the needleless syringe that can be used in the present invention is not limited to this, Forms can be used. Therefore, the above-described embodiment is merely an example and should not be interpreted in a limited manner.

[3-2. Injection method]
The injection method of the needleless syringe is not particularly limited. Basically, the nozzle hole of a needleless syringe containing the cell suspension is pressed against the scaffold, or the cell suspension is suspended from the nozzle hole with a certain distance between the nozzle hole and the scaffold. Spray liquid into the scaffold.

  In a needleless syringe, the spring force and the jetting force of the cell suspension are generally correlated. For this reason, the spring force is appropriately adjusted by those skilled in the art so as not to damage the scaffold according to the material composition of the scaffold, the shape such as thickness, and the mechanical characteristics.

  In addition, when making such adjustments, a cushioning material is interposed between the scaffold and the nozzle hole of the needleless syringe, and the jetting force of the cell suspension directly applied to the scaffold can be reduced by the cushioning material. good. In this case, it is possible to make finer adjustments by further considering the material composition, thickness, mechanical characteristics, etc. of the cushion material. As long as the cushioning material is sandwiched between the scaffold and the nozzle hole of the syringe, the cell suspension sprayed from the nozzle hole can reach the inside of the scaffold through itself. Well, not particularly limited. For example, agar can be used.

[3-3. Advantages of using a needleless syringe]
Among the conventional cell seeding methods, methods other than the method using a needle syringe do not incorporate cells into the scaffold, so that only the surface of the scaffold is transformed into cells even after culturing. Even when a scaffold having a cell surface only is transplanted, cells that adhere to the surface of the scaffold may gradually enter the scaffold at the transplant destination, but it takes time to regenerate the tissue ( (See case (B) in FIG. 10).

  On the other hand, in the cell seeding method of the present invention using a needleless syringe, since cells can be embedded even inside the scaffold, the cells are transformed into the scaffold by culturing. When a scaffold that has been cellized to the inside is transplanted, since the cells are originally inside the scaffold, surrounding capillaries are induced at the transplant destination and tissue regeneration is promoted. For this reason, early healing of a transplant site | part is attained (refer FIG. 10 case (C)).

  Of the conventional cell seeding methods, the method using a needle syringe can inject cells into the scaffold. However, as shown in FIG. 11 (A), after the cell seeding using the needle injector, the horizontal cross section (cross section by plane (a)) of the scaffold is observed. A liquid pool (see white arrow in FIG. 11A) can be formed. As described above, the method using the needle injector can only perform local cell seeding and cannot seed cells over a wide range. In addition, as shown in FIG. 11B, when the vertical longitudinal section (cross section by plane (b)) of the scaffold is observed, the scaffold tissue itself is greatly damaged by the passage of the needle (FIG. 11 (B). ) Receive white arrow).

  In contrast, in the cell seeding method of the present invention using a needleless syringe, since the cell suspension is sprayed to the scaffold, the cells can be compared even by a single injection without any liquid pooling inside the scaffold. It becomes possible to scatter and embed over a wide area. In the cell seeding method of the present invention, since the cell suspension is ejected using pressure by a spring force rather than injection of the cell suspension using a needle, the scaffold tissue can be maintained. .

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to the following examples.

[Example 1]
1. A cell suspension was prepared as follows.
L929 cells cultured in a culture dish (diameter 100 mm) were detached with trypsin / EDTA solution and collected in a centrifuge tube. The cells are precipitated by centrifugation (1000 rpm / minute, 5 minutes), the supernatant is discarded, and the carefully prepared collagen gel (for cell culture) is adjusted to a cell density of 1 x 10 ⁶ / mL. Added and suspended.

2. The cell suspension was sealed in a nozzle ampoule attached to a needleless syringe (Shimadzu Shimajet; ShimaJET). At this time, the amount of liquid sealed in the needleless syringe was set to 1 unit amount set in the syringe.

3. Place the myocardial scaffold (decellularized myocardial tissue; thickness 3.2 mm) in a culture dish (diameter 60 mm), and the distance between the scaffold surface and the nozzle hole of the needleless syringe will be 15 cm Syringes were installed as follows.
The opening member of the syringe was pushed down to eject the cell suspension, and the cells were seeded.

4. 4 mL of the medium was added to the culture dish containing the scaffold, and the culture was allowed to stand in an atmosphere of 37 ° C, 5% CO _{2 and} 100% humidity. The medium was changed twice a week.

5. After culturing for one month, the scaffold was cut in half in the transverse direction, one was subjected to life / death discrimination staining with calcein AM / PI staining solution, and the other was stained with hematoxylin eosin and observed under a microscope.

FIG. 12 (A) is an example of a scaffold horizontal cross-section image obtained by observing a tissue stained with calcein AM / PI with a confocal laser scanning microscope. It can be confirmed that living cells are interspersed between scaffolds having an elongated shape.
In order to observe the horizontal section from the tissue stained with hematoxylin and eosin, a plurality of sections having a thickness of 4 micrometers in the horizontal section direction were prepared and observed using an optical microscope. FIG. 12 (B) is a horizontal cross-sectional photograph at a depth of 300 micrometers from the surface of the scaffold. In addition, it was confirmed that cells were fixed at other depths as well.

It is a longitudinal cross-sectional view at the time of use of an example of the syringe which can be used by this invention. It is a longitudinal cross-sectional view at the time of preservation | save of the syringe. FIG. 3 is a transverse sectional view taken along line III-III in FIG. 1. It is a cross-sectional view which follows the IV-IV line of FIG. It is an expansion perspective view of the holding member of the syringe. It is a longitudinal cross-sectional view of the nozzle ampule by the modification of the syringe which can be used by this invention. It is a cross-sectional view which follows the VII-VII line of FIG. It is a longitudinal cross-sectional view of the holding member by the modification of the syringe which can be used by this invention. It is a side view of the part shown in FIG. It is a figure for demonstrating that it is preferable to seed | inoculate a cell to the inside of a scaffold. It is the horizontal cross-sectional photograph (A) and vertical vertical cross-sectional photograph (B) of a scaffold at the time of performing cell seeding to the inside of the scaffold using a needled syringe. In Example 1, a scaffold horizontal cross-sectional photograph (A) stained with calcein AM / PI and a scaffold horizontal cross-sectional photograph stained with hematoxylin and eosin when cells are seeded inside the scaffold using a needleless syringe ( B).

Explanation of symbols

11: Nozzle ampoule
12: Syringe body
23: Piston
25: Nozzle hole
32: Pressing mechanism L: Cell suspension

Claims (8)

A method of seeding cells in a scaffold, wherein the cells to be seeded are ejected into the scaffold using a needleless syringe to embed the cells inside the scaffold. The cell seeding method according to claim 1, wherein the scaffold is made of a biological material or a synthetic material. The cell seeding method according to claim 1 or 2, wherein the scaffold is a decellularized biological tissue piece. The cell seeding method according to claim 3, wherein the biological tissue piece is a biological tissue piece collected from a human or a biological tissue piece collected from a mammal other than a human. The cells to be seeded are accommodated in the needleless syringe in the form of a suspension dispersed in a liquid selected from a cell culture solution, a biocompatible sol, and a biocompatible gel solution. The cell seed | inoculation method of any one of -4. The cell to be seeded is selected from the group consisting of a human cell and a patient's autologous cell into which a scaffold in which the cell to be seeded is embedded may be transplanted. The cell seeding method according to Item. The needleless syringe has a pressing mechanism using a method selected from the group consisting of a spring method, a gas pressure method, an explosive method, an electromagnetic force method, and a piezoelectric method. Cell seeding method. Needleless syringe containing cells to be seeded.