Cell injection device and method of operating the same

Abstract

To provide a device capable of preferably injecting a cell to be implanted to a biological tissue, and an operation method thereof.SOLUTION: A cell injection device comprises at least: suspension fluid 30 stored in a storage part 13; an injection needle 20 coupled to the storage part; and extrusion mechanisms (10, 50). The extrusion mechanisms are configured to extrude the suspension fluid, from the storage part to an external part, through the injection needle, so that a flow rate when the suspension fluid flows out from a tip of the injection needle is equal to or less than 1 μL / second. By injection at the low flow rate, damage on a biological cell can be further reduced and a cell to be implanted can be injected.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a cell injection device for injecting cells into a living tissue, and a method of operating the same.

Background Art

[0002] As one method of transplanting cells into a living tissue (hereinafter, also simply referred to as a tissue), a method of injecting cells to be transplanted into the tissue together with a liquid for conveyance is known (for example, Non-Patent Document 1 and the like). Hereinafter, the cells injected into the tissue for transplantation are also referred to as "transplanted cells", the living tissue at the injection site (transplantation site) of the transplanted cells is also referred to as a "host tissue", and the cells constituting the host tissue are also referred to as "host cells".

[0003] Conventionally, as a device for injecting transplanted cells into a tissue, various drug injection devices such as a syringe and an injector have been diverted (Patent Documents 1, 2, etc.). For example, in cell injection using a syringe, a suspension fluid in which transplanted cells are dispersed in a liquid for cell conveyance is sucked into a syringe with a hypodermic needle, the tip of the hypodermic needle is inserted into a host tissue, and the plunger of the syringe is manually pushed to inject the suspension fluid. The injection rate (flow rate) of the suspension fluid at this time is about several tens to several hundreds [μL / sec]. In this specification, "L" in the unit represents a liter.

[0004] However, it is known that the following problems occur in the method of injecting transplanted cells into a host tissue. For example, FIG. 19 is a diagram schematically showing the state of a treatment in which cardiomyocytes are transplanted into the heart wall by injection. In this treatment, as shown in FIG. 19(a), cardiomyocytes 210, which are the transplanted cells, are injected into the heart wall, which is the host tissue 100, through a thin tube 300 such as an injection needle in the form of a suspension 200. The suspension 200 is a dispersion of cardiomyocytes 210 in the injection solution 220. When the suspension is injected into the host tissue, as shown in FIG. 19(b), the portion of the injection solution 220 surrounding the transplanted cells 210 becomes fibrotic and forms a partition surrounding the entire transplanted cells, thereby possibly preventing direct binding between healthy host cells and transplanted cells. FIG. 20 is a microscopic photograph showing the state in which the portion surrounding the transplanted cells 210 has become a fibrotic portion 230, indicating a state where the fibrotic portion 230 serves as an insulating wall and the transplanted cells 210 cannot contact the cells of the host tissue 100.

[0005] In addition, in a treatment in which transplanted cells are injected into the dermis of the skin in the form of a suspension, it is known that when the suspension is injected, inflammation and fibrosis are induced in the dermis, which is the host tissue, and the dermis may be partially scarred. Therefore, it has been reported that in cell injection treatment for the skin, scar formation in the dermis should be suppressed.

[0006] In addition, in cell injection treatment for brain injury and spinal cord injury, it is known that when a suspension containing transplanted cells is injected, the brain and spinal cord may be damaged, glial cells may be induced and activated, resulting in scarring called glial scar. Therefore, it has been reported that in cell injection treatment for brain injury and spinal cord injury, the effectiveness may be further enhanced by suppressing the activation of glial cells induced by the injection operation itself.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Non-Patent Literature

[0008]

Non-Patent Literature 1

Summary of the Invention

Problems to be Solved by the Invention

[0009] In order to solve the above problems (the first problem), the present inventor has examined in more detail the mechanism of occurrence of the above-mentioned alterations (fibrosis, scarring, glial scar, etc.) caused by the injection of transplanted cells, and found that the host tissue or host cells at the injection site are damaged by shear, breakage, compression, etc. due to the sudden inflow of the suspension into the host tissue, thereby causing the above-mentioned alterations.

[0010] An object of the present invention is to provide an apparatus capable of reducing damage to host tissue at the injection site and preferably injecting transplanted cells into living tissue, and to provide a method of operating the apparatus.

Means for Solving the Problems

[0011] The main configuration of the present invention is as follows. 〔1〕A cell injection device for injecting cells into living tissue, the cell injection device comprising: having a housing, having a suspension fluid accommodated in the housing, the suspension fluid being one in which the cells are dispersed in a medium fluid, having an injection needle directly or connected to the housing via a pipeline, An extrusion mechanism is configured to perform an operation of extruding the suspension fluid from the storage unit through the injection needle so that the flow rate when the suspension fluid flows out from the tip of the injection needle is 1 [μL / sec] or less. The cell injection device. 〔2〕The medium fluid has a first viscosity α1 in a high-temperature-side temperature range T1 including the temperature of the living tissue, and a second viscosity α2 in a low-temperature-side temperature range T2 lower than the high-temperature-side temperature range T1. The first viscosity α1 is 1000 [mPa·s] or less, and the second viscosity α2 is 10000 [mPa·s] or more. The cell injection device has a cooler or a heat insulating material for maintaining the suspension fluid in the storage unit at the low-temperature-side temperature range T2. In use, the suspension fluid maintained at the low-temperature-side temperature range T2 in the storage unit is heated to the high-temperature-side temperature range T1 at the tip of the injection needle to become the first viscosity α1 and flows out from the tip of the injection needle. The cell injection device according to the above 〔1〕. 〔3〕The high-temperature-side temperature range T1 is 21 to 42 [°C]. The first viscosity α1 is 0.5 to 1000 [mPa·s]. The low-temperature-side temperature range T2 is 0 to 20 [°C]. The second viscosity α2 is a value greater than 5000 [mPa·s]. The cell injection device according to the above 〔2〕. 〔4〕The cell injection device according to the above 〔2〕 or 〔3〕, wherein the medium fluid becomes a sol having a first viscosity α1 in the high-temperature-side temperature range T1 and a gel having a second viscosity α2 in the low-temperature-side temperature range T2. 〔5〕The cell injection device has a syringe having a cylinder and a plunger as the extrusion mechanism. The storage unit is formed on the tip side in the cylinder by the cylinder and the plunger, and the injection needle is connected to the tip of the cylinder. The extrusion mechanism further has an actuator configured to move the plunger toward the tip of the syringe at a speed at which the above flow rate is achieved as a drive source. The cell injection device according to any one of the above [1] to [4]. [6] The actuator is a hydraulic cylinder in which a piston moves linearly by the supply of an operating fluid, and a piston rod connected to the piston is connected to the plunger of the syringe so as to push the plunger. The cell injection device according to the above [5]. [7] The actuator has an elastic body and a damper, the elastic body is arranged in a deformed state from its original shape, and a restoring force to the original shape is arranged to move the plunger, the damper is arranged to resist the movement of the plunger and limit the speed of the shape displacement of the elastic body, the restoring force of the elastic body and the resistance of the damper are selected to move the plunger toward the tip of the syringe at a speed at which the above flow rate is achieved. The cell injection device according to the above [5]. [8] On the outer front end surface of the cylinder of the syringe, a first support member is provided for intervening between the surface of the living tissue and the cylinder when the injection needle punctures the living tissue. The first support member has a counter surface for directly contacting or contacting via an adhesive with the surface of the living tissue, and the injection needle penetrates the first support member from the cylinder and protrudes from the counter surface. The cell injection device according to any one of the above [5] to [7]. [9] The cell injection device according to the above [8], wherein an adhesive layer for fixing the first support member to the surface of the living tissue is provided on the counter surface of the first support member.

[10] The injection needle has a needle tube portion that is curved in an arc, and the central angle of the arc is 45 to 90 degrees. The first support member further has an edge portion extending along and coinciding with a central axis passing through the center point of the arc and perpendicular to the plane including the arc, or including the central axis therein, extending along the central axis, and having an edge portion that is rounded concentrically with the central axis The cell injection device according to the above [8] or [9].

[11] The cell injection device according to any one of the above [8] to

[10] , wherein the angle formed by the object surface of the first support member and the needle tube portion of the injection needle protruding from the object surface is not 90 degrees.

[12] The injection needle has a needle tube portion that is curved in an arc, and the central angle of the arc is 45 to 90 degrees. A second support member is provided on the cylinder of the syringe, and the second support member has an edge portion that passes through the center point of the arc and extends in coincidence with the central axis orthogonal to the plane including the arc, or including the central axis therein, extending along the central axis, and having an edge portion that is rounded concentrically with the central axis The cell injection device according to the above [8] or [9].

[13] The cell injection device has an osmotic pump as the extrusion mechanism The osmotic pump has a first chamber and a second chamber adjacent to each other through a liquid permeable membrane The liquid disposed in the first chamber moves through the liquid permeable membrane due to an osmotic pressure difference into the osmotic pressure generating material disposed in the second chamber, whereby the second chamber expands, and the expansion of the second chamber presses the accommodating portion, and the suspension fluid is configured to be extruded from the accommodating portion through the injection needle to the outside at the above flow rate The cell injection device according to any one of the above [1] to [4].

[13] A method of operating the cell injection device according to any one of the above [1] to

[12] , comprising a step of flowing out the suspension fluid accommodated in the accommodating portion of the cell injection device from the tip of the injection needle of the cell injection device at a flow rate of 1 [μL / sec] or less by operating the extrusion mechanism of the cell injection device The above operating method. [Advantages of the Invention]

[0012] In conventional cell transplantation, a suspension containing cells was aspirated into a syringe with a hypodermic needle. After inserting the hypodermic needle into the tissue, the plunger of the syringe was manually pushed in, and the suspension was injected into the tissue at a flow rate of several tens to several hundreds [μL / sec]. Such injection was conventionally considered to be a slow injection, but according to the present invention, it is a high-speed injection that causes damage to the tissue. When a predetermined amount of the suspension flows in manually at high speed, the tissue (or cells) at the injection site is torn, compressed, and damaged. This damage triggers the infiltration of stromal cells and fibroblasts, as shown in FIG. 19(b), and a fibrotic tissue (so-called scar) having a random mesh structure of the extracellular matrix is formed. The injected transplanted cells are surrounded and insulated by this fibrotic tissue and cannot contact the surrounding host tissue, and thus the original therapeutic purpose cannot be achieved.

[0013] In contrast, in the present invention, the suspension fluid flows into the host tissue at a flow rate of 1 [μL / sec] or less when flowing out from the tip of the hypodermic needle. In a normal syringe, since the inner diameter of the cylinder is larger than the inner diameter of the hypodermic needle, the moving speed of the plunger must be made even smaller than the flow rate of the suspension fluid. Since it is substantially difficult to manually move the plunger at a speed even smaller than the flow rate of the suspension fluid sufficiently slowed down in the present invention, the extremely slow movement of the plunger is achieved by an extrusion mechanism newly developed in the present invention.

[0014] On one hand, in the host tissue, the tissue fluid exuded from the capillaries slowly flows towards the lymphatic vessels. The extremely small flow rate (1 [μL / second] or less) as described above implemented in the present invention attempts to approximate the flow of the tissue fluid in the host tissue. That is, when the suspension fluid flows into the host tissue at such an extremely small flow rate, the medium fluid constituting the suspension fluid successively flows away from the injection site into the host tissue by the flow of the tissue fluid, and only the transplanted cells remain at the injection site. As a result, the damage received by the host tissue at the injection site is minimized and is only due to the volume of the injected transplanted cells. Therefore, the generation of fibrotic tissue around the transplanted cells is suppressed, and the transplanted cells can contact and bind to the host tissue (or host cells).

[0015] (Further problems and solutions) The inventor developed an injection device in which the plunger of the syringe moves precisely at a minute speed, and newly attempted to cause the suspension to flow out from the tip of the injection needle into the host tissue at a flow rate sufficiently smaller than before. However, when the moving speed of the plunger is made extremely slow, the speed of the flow of the suspension in the cylinder towards the injection needle becomes extremely slower compared to the speed of the flow in the injection needle. In such a case, it was found that a new problem occurs, that is, only the medium liquid in the suspension in the cylinder slowly exits from the cylinder into the injection needle, and the transplanted cells often remain in the cylinder without moving. Hereinafter, such a further problem newly caused by reducing the flow rate of the suspension is also referred to as the "second problem".

[0016] In the present invention, in order to solve the above-mentioned second problem, it further has the following characteristic configurations (I) and (II). (I) The medium fluid that constitutes the suspension fluid accommodated in the accommodation part of the cell injection device has a first viscosity α1 in a high-temperature-side temperature range T1 including the temperature of the living tissue, and a second viscosity α2 in a low-temperature-side temperature range T2 lower than the high-temperature-side temperature range T1. The first viscosity α1 is 1000 [mPa·s] or less, and the second viscosity α2 is 10000 [mPa·s] or more. Hereinafter, the "high-temperature-side temperature range T1" may be abbreviated as "high temperature T1", and the "low-temperature-side temperature range T2" may be abbreviated as "low temperature T2". (II) The cell injection device has a cooler or a heat insulator, whereby the temperature of the suspension fluid in the accommodation part is maintained in the low-temperature-side temperature range T2.

[0017] By providing the above-described characteristic configurations of (I) and (II), the medium fluid of the suspension fluid has a higher viscosity (second viscosity α2) at the low temperature T2 in the accommodation part. Therefore, even if the extrusion mechanism operates extremely slowly to achieve an extremely small flow rate of 1 [μL / second] or less, the medium fluid, due to its high viscosity α2, is more likely to enter from the accommodation part into the injection needle together with the transplanted cells. On the other hand, the tip side of the injection needle is inserted into the tissue and heated to the temperature of the tissue. Also, the middle part of the injection needle may be heated by contacting the outside air. Further, the suspension fluid about to come out from the tip of the injection needle is directly heated by the tissue. Therefore, the medium fluid of the suspension fluid becomes the high temperature T1 while moving through the injection needle, and thus has a lower viscosity (first viscosity α1), and flows into the host tissue from the tip of the injection needle. Therefore, the second problem is solved.

Brief Description of the Drawings

[0018]

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DETAILED DESCRIPTION OF THE INVENTION

[0019] First, the cell injection device of the present invention will be described in detail below. As schematically shown in FIG. 1 as an example of the configuration, the cell injection device is a device for injecting transplanted cells into a living tissue 100, and includes at least a storage unit 13 that stores a suspension fluid 30, an injection needle 20, and an extrusion mechanism (10, 50). The suspension fluid 30 is formed by dispersing transplanted cells 31 in a medium fluid 32. The extrusion mechanism can cause the suspension fluid 30 to flow out from the tip of the injection needle 20 at an extremely small flow rate Q1 of 1 [μL / second] or less and at a slow flow velocity.

[0020] As a result, when the suspension fluid 30 flows into the host tissue, the medium fluid 32 flows away to the surrounding tissue along with the flow of the tissue fluid, and only the transplanted cells remain at the injection site. As a result, the damage received by the host tissue at the injection site is minimized according to the volume of only the injected transplanted cells. Therefore, the generation of fibrotic tissue around the transplanted cells is suppressed, and the transplanted cells come into contact with and bind to the host tissue (or host cells), and the desired therapeutic effect can be obtained.

[0021] (Living body) The living body into which the transplanted cells are injected according to the present invention is not particularly limited and may be a plant or an animal. In order to solve the above-described second problem, when using a medium fluid whose viscosity changes according to temperature, the action and effect of the present invention are more significantly shown in a living body with a higher tissue temperature. Such living bodies include various animals, especially mammals which are warm-blooded animals (humans, companion animals (dogs, cats, rabbits, rodents, etc.), industrial animals (cows, horses, pigs, sheep, etc.), laboratory animals (rodents, non-human primates), etc.).

[0022] (Suspension fluid) The suspension fluid 30 has a medium fluid 32 and transplanted cells 31 dispersed therein. As the medium fluid 32, a fluid or liquid that can be injected into the tissue is available. Details of the medium fluid will be described later. The transplanted cells 31 are preferably uniformly dispersed in the medium fluid, but may be partially biased in the medium fluid due to sedimentation or the like. The volume of the suspension fluid injected into the tissue varies depending on the type of living body. For example, when injecting cardiomyocytes into the heart wall for the purpose of treating the human heart, the injection volume per time is about 10 μL to 2 mL.

[0023] (Diseases to be treated) The diseases to be treated by cell injection are not particularly limited, and examples include diseases in the fields of nerves, eyes, circulatory organs, kidneys, digestive organs, muscles and skeletons, skin, etc., such as severe heart failure, spinal cord injury, Parkinson's disease, renal failure, retinitis pigmentosa, myasthenia gravis, etc.

[0024] (Transplanted cells) The transplanted cells are not particularly limited, and examples include cardiomyocytes, nerve cells, astrocytes, oligodendrocytes, glial cells, octopus cells, mesangial cells, epithelial cells, endothelial cells, stromal cells, retinal pigment cells, photoreceptor cells, hepatocytes, dermal cells, tissue stem cells, etc.

[0025] (Ratio of cells in the suspension fluid) The volume ratio of the medium fluid to the transplanted cells in the suspension fluid is not particularly limited, but from the viewpoint of shortening the transplantation time (the outflow time from the cell injection device), the volume ratio (the volume of the medium fluid / the volume of the transplanted cells) is preferably about 2 to 10.

[0026] (Flow rate Q1 when the suspension fluid flows out from the tip of the injection needle) The flow rate Q1 when the suspension fluid flows out from the tip of the injection needle is preferably 1 [μL / sec] or less from the viewpoint that the medium fluid in the suspension fluid flowing into the injection site preferably flows away into the host tissue. At that time, the flow velocity is preferably, for example, 91 [mm / sec] or less. This flow velocity of 91 [mm / sec] is the value in an example of the combination where the flow velocity is the highest, calculated from the inner diameter of 0.12 [mm] of the thinnest commercially available general-purpose injection needle 30G and the upper limit value of the above flow rate of 1 [μL / sec]. Since the flow rate in conventional cell injection was about several tens to several hundreds [μL / sec] at a high speed, the upper limit of the flow rate proposed in the present invention may exceed 1 [μL / sec], but considering the suppression of fibrosis, the upper limit of the flow rate is set to 1 [μL / sec].

[0027] The lower limit of the flow rate Q1 is not particularly limited, but if the flow rate is excessively small, the time required to inject all the transplanted cells necessary for treatment becomes unnecessarily long, which also burdens the subject. Also, from the viewpoint of the configuration of the extrusion mechanism and / or the configuration of the control, it becomes difficult to achieve an excessively small feed amount (flow rate) because the configuration becomes complicated. Therefore, in view of these incidental reasons, in the present invention, 0.01 [μL / sec] is proposed as an example of an appropriate lower limit of the flow rate. The lower limit of the flow velocity at this time is, for example, 0.015 [mm / sec]. This flow velocity of 0.015 [mm / sec] is the value in an example of the combination where the flow velocity is the lowest, calculated from the inner diameter of 1.65 [mm] of the thickest commercially available general-purpose injection needle 16G and the lower limit value of the above flow rate of 0.01 [μL / sec].

[0028] From the above points, the preferable range of the flow rate Q1 is exemplified as 0.01 to 1.0 [μL / sec]. Also, from the viewpoint of optimizing the injection time, 0.02 to 0.5 [μL / sec], further 0.05 to 0.5 [μL / sec], and further 0.05 to 0.1 [μL / sec] are exemplified as more preferable ranges. The flow velocity can be calculated from the inner diameter of the injection needle used and the flow rate as described above. If the amount of transplant cells to be injected and the amount of the suspension fluid are extremely small and a more highly precise extrusion mechanism can be used, a flow rate smaller than 0.01 [μL / sec], such as 0.005 [μL / sec], can be set as the lower limit, and within the range where the suspension fluid can be injected while achieving the object of the present invention of reducing the damage to the host tissue at the injection site, the flow rate may be appropriately determined.

[0029] (Containing portion) The mode and shape of the containing portion may be related to the configuration of the extrusion mechanism and the principle of extrusion described later, and the containing portion can also be understood as a part of the extrusion mechanism. In the preferable example shown in FIG. 1, the containing portion 13 is provided as a space in the syringe 10 which is one of the components of the extrusion mechanism. More specifically, the syringe 10 has a cylinder 11 and a plunger 12, the plunger 12 has a gasket portion 12a and a plunger rod 12b, and the containing portion 13 is provided as a space on the tip side in the cylinder 11 formed by the cylinder 11 and the gasket portion 12a. As the material of each part of the syringe, those that do not adversely affect the suspension fluid can be used, and materials of conventionally known medical syringes such as glass, plastic, and metal may be used.

[0030] (Volume of the containing portion) The volume of the accommodating part may be the volume of the suspension fluid to be injected (injection volume), or may be a volume obtained by appropriately adding losses due to dead volume to the injection volume. For example, as a general injection volume of transplanted cells for the human heart wall, dermis of the skin, brain or spinal cord, etc., about 10 [μL] to 3 [mL] is exemplified. The volume of the accommodating part may be a volume for a single injection, or may be a larger volume such that multiple injections can be performed at the same site over time or successively at different sites. Also, the volume of the accommodating part may simply be larger than the injection volume. The injection volume is determined by the extrusion mechanism, and after injection, the suspension fluid may remain in the accommodating part.

[0031] (Inner diameter of the cylinder) When the accommodating part is provided as the space inside the syringe, the inner diameter d1 of the cylinder constituting the syringe is not particularly limited. However, if the inner diameter d1 is excessively large, a large amount of suspension fluid will be sent out just by moving the plunger by a small amount, and it may be difficult to accurately control a minute flow rate. On the other hand, if the inner diameter d1 is excessively small, the syringe will become excessively long to secure a predetermined volume of the accommodating part, and the total movement distance of the plunger will also become excessively large, which may not be preferable. From these points, when performing a single normal injection of transplanted cells for the human heart wall, dermis of the skin, brain or spinal cord, etc., the inner diameter d1 of the cylinder is preferably about 3 mm to 15 mm, and more preferably about 4 mm to 10 mm.

[0032] (Injection needle) As used in the present invention, the "injection needle" includes not only conventionally known medical injection needles but also a thin tube capable of discharging a suspension fluid at an extremely low flow rate Q1 of 1 [μL / sec] or less as described above. That is, the "injection needle" as used in the present invention does not necessarily have to have a sharp tip for puncturing the skin. In terms of being able to penetrate the skin from the outside of the living body and inject the suspension fluid into the host tissue, a medical injection needle (for example, one defined in JIS T3101-1979) attached to the tip of a syringe barrel is a preferred embodiment. The form of the injection needle is not particularly limited. As shown in FIG. 1, a commercially available standard medical injection needle 20 has a needle tube portion 21 and a needle base portion 22 and can be directly attached to the cylinder 11. Examples of the material of the needle tube portion include metals such as stainless steel and plastics. A conventionally known detachable structure (such as a luer taper or a luer lock) between the needle base and the cylinder can also be used. Further, the injection needle may be connected to the cylinder via a soft tube or the like. In that case, the dead volume in the tube is appropriately considered so that a necessary amount of the suspension fluid is delivered into the tissue.

[0033] The inner diameter of the needle tube portion of the injection needle (when the injection needle is simply a thin tube, the inner diameter of the tube) is not particularly limited. However, from the point of reducing tissue damage more, the inner diameter of the needle tube portion may preferably be smaller. On the other hand, if the inner diameter of the needle tube portion is excessively small, transplanted cells (such as cell masses) may not be able to pass through and may be damaged. Also, there may be a case where the medium fluid having a high viscosity in the storage portion does not smoothly enter the needle tube portion. From these points, the inner diameter of the needle tube portion is preferably about 0.07 mm to 1.65 mm, and more preferably about 0.12 mm to 0.51 mm.

[0034] (Extrusion mechanism) As shown in FIG. 1, the extrusion mechanism is configured to perform an operation of extruding the suspension fluid 30 in the accommodating portion 13 from the accommodating portion to the outside (i.e., into the tissue into which the tip of the injection needle is inserted) through the injection needle 20, and is configured or controlled to cause the suspension fluid 30 to flow out from the tip of the injection needle 20 at a flow rate of 1 [μL / second] or less. In the example of FIG. 1, the extrusion mechanism has a syringe 10 and a drive source 50 that operates to push and move its plunger 12 into the cylinder 11 at a predetermined speed v1 [mm / second]. The predetermined speed v1 [mm / second] may be set according to the inner diameter d1 [mm] of the cylinder so that the suspension fluid flows out from the tip of the injection needle at the above-described specified flow rate Q1 (1 [μL / second] or less). For example, when the flow rate Q1 is 1 [μL / second], the moving speed v1 [mm / second] of the plunger can be obtained from the following formula. Q1 = 1 [μL / second] = {(d1) 2 π / 4} [mm 2 × v1 [mm / second] The preferred moving speed v1 [mm / second] of the plunger at the above-described preferred inner diameter d1 of the cylinder is 0.14 [mm / second] or less, and preferably, about 0.0032 to 0.02 [mm / second] is exemplified.

[0035] (Drive source of the extrusion mechanism) When the extrusion mechanism is a mechanism (10, 50) for sending out the suspension fluid 30 in the syringe 10 as shown in FIG. 1, examples of the drive source 50 include an actuator configured to push (or pull) the plunger of the syringe from the rear side to the tip side (or from the tip side) at the above-described moving speed v1.

[0036] (Example 1 of the drive source of the extrusion mechanism: hydraulic cylinder) As the actuator, as exemplified in FIG. 2 described later, a hydraulic cylinder (such as a water hydraulic cylinder or an oil hydraulic cylinder) in which a piston linearly moves by the supply of hydraulic fluid (such as water or hydraulic oil) from a hydraulic fluid supply source is exemplified. The "cylinder" in the hydraulic cylinder means an actuator device having at least a cylinder tube, a piston that linearly moves inside the cylinder tube, and a piston rod connected to the piston. The hydraulic cylinder is connected to the syringe 10 such that the tip of the piston rod pushes and moves the plunger rod of the syringe 10. In the example of FIG. 2(b), a medical syringe 50a is diverted as a hydraulic cylinder, and the hydraulic fluid is supplied into the syringe through an injection needle connected to the hydraulic fluid supply source by a pipe (tube) 55, whereby the plunger rod (piston rod) of the syringe is configured to push the plunger 12 of the syringe 10. The principle of sending the hydraulic fluid of the pump of the hydraulic fluid supply source is not particularly limited, and examples include an electric syringe pump and a peristaltic pump (tube pump).

[0037] (Example 2 of the drive source of the extrusion mechanism: elastic body and damper) As the actuator, as exemplified in FIG. 3 described later, there is a mechanism that combines an elastic body (in the example of FIG. 3, a tension spring) 58 and a damper (such as a viscous damper filled with fluid) 56. The elastic body 58 is arranged in a deformed state from its original shape and is maintained in the deformed state by a stopper 59 or the like. The elastic body 58 is arranged such that the restoring force to its original shape pushes and moves the plunger. On the other hand, the damper 56 resists the movement of the plunger by its viscous resistance and is arranged to limit the speed of the movement to the above movement speed v1. The restoring force of the elastic body and the resistance of the damper are selected to push and move the plunger to the tip side of the syringe at a speed at which the above-specified flow rate Q1 is achieved. The elastic body may be a compression spring. In that case, the syringe is set, for example, in an appropriate housing, and the compression spring is arranged in a compressed and deformed state behind the plunger rod. One end of the compression spring contacts the housing, and the other end contacts the plunger. A stopper for preventing the movement of the plunger is provided as appropriate. The configuration of FIG. 3 will be described in detail below.

[0038] As long as the actuator can move the plunger rod of the syringe at the above movement speed v1, various well-known hydraulic precision linear actuators can be used. For example, an electric spindle device or a ball screw actuator can convert the rotational movement of a controlled motor into a precise linear movement.

[0039] If the drive source has an electrical or electronic control device, the above-specified speed v1 may be stored or set in the control device, or may be set according to the values of elements that determine the rotational speed of the motor, or the characteristics of elements themselves that determine the operation, such as the elastic constant and the damping characteristics of the damper.

[0040] (Other aspects of the housing part and the extrusion mechanism) The accommodating part may be, for example, the space inside a thin tube. In this case, the tube is filled with a suspension fluid, and an injection needle is connected to one end side of the tube. Then, from the other end side (the rear side of the suspension fluid), a working fluid for extrusion is fed into the tube at the extremely small flow rate Q1 by a pump. As a result, the suspension fluid is pushed by the working fluid and flows out from the injection needle at the flow rate Q1. The working fluid for extrusion is preferably a highly viscous fluid that does not easily mix with the suspension fluid, and the medium fluid of the suspension fluid may be cooled and used. Also, as the pump used in such a manner, a syringe pump, a tube pump (peristaltic pump), etc., which can accurately control the flow rate of the working fluid for extrusion, can be used.

[0041] The cell injection device is preferably easy to use clinically, and at least the part that comes into contact with the suspension fluid is disposable. From this point of view, an extrusion mechanism detachably connected to a driving device or a pump, such that a syringe connected with an injection needle or a tube connected with an injection needle is disposable, is a preferred embodiment.

[0042] (The medium fluid used for the suspension fluid) Examples of the medium fluid include collagen, gelatin, carrageenan, artificial peptides, polymer polymers, polymer composites, etc., and it may be a liquid that has been conventionally used for injecting transplanted cells. When using gelatin or the like, it is preferable to use one that is compatible with the biological species of the living body into which the transplanted cells are to be injected. In particular, when the living body into which the transplanted cells are to be injected is a human, it is preferable to use artificially synthesized human gelatin.

[0043] (The medium fluid for solving the second problem) In order to solve the above-described second problem, it is preferable that the medium fluid satisfies the following conditions (a1) and (a2). (a1) In the high temperature T1 within the high temperature range including the temperature of the tissue, the medium fluid has a first viscosity α1. The first viscosity α1 is preferably equal to or lower than the viscosity of the tissue fluid in that it can flow away rapidly with the tissue fluid in the host tissue. (a2) In the low temperature T2 within the low temperature range lower than the high temperature T1, the medium fluid has a second viscosity α2. The second viscosity α2 is greater than the first viscosity α1, and in the accommodating portion, it is preferably a high viscosity that can direct the transplanted cells toward the injection needle along with the transplanted cells without leaving the transplanted cells even with an extremely slow flow.

[0044] (Temperature of living tissue) The temperature of the tissue (host tissue) varies depending on the type and site of the living body. For example, the temperature of human tissue is about 20 - 42°C, the temperature of tissue such as that of a mouse is about 20 - 42°C, and the temperature of tissue such as that of a monkey is about 36 - 40°C. In the case where the temperature of the host tissue is low because the body temperature of the living body is low in the first place, or in the case where the temperature of the host tissue is lowered due to the influence of the air temperature, etc., when injecting the transplanted cells, the whole body of the living body or the host tissue may be warmed within a range that does not have an adverse effect on the living body, thereby increasing the temperature of the host tissue.

[0045] (High temperature range T1 on the high temperature side) The high temperature T1 is the high temperature range including the temperature of the living tissue described above and is determined according to the temperature of the tissue and the outside air. The high temperature T1 also varies depending on the type and site of the living body, but for mammals, it is preferably about 20°C or higher, and more preferably about 30°C or higher. The upper limit range of the high temperature T1 is not particularly limited and is a temperature not particularly required for injecting transplanted cells, but from the viewpoint of maintaining biological functions, etc., it is preferably about 42°C or lower, and more preferably about 40°C or lower. The range of the high temperature T1 combining the above-mentioned lower limit and upper limit can be freely combined, such as 20 - 42°C, 30 - 42°C, 20 - 40°C, or 30 - 40°C.

[0046] (Temperature range T2 on the low-temperature side) The low temperature T2 can be determined as a temperature lower than the temperature of the tissue and even lower than the temperature of the outside air. From the viewpoints of the increase in viscosity and gelation due to the composition of the suspension, a temperature of about 10°C or lower is preferable, and a temperature of about 5°C or lower is more preferable. The temperature on the lower limit side of the low temperature T2 is not particularly limited, but a temperature of about 0°C or higher is mentioned from the viewpoint that the medium fluid does not freeze and the protection of the transplanted cells, and more preferably about 1°C or higher. The above-mentioned lower limit range and upper limit range can be freely combined, such as 0 to 10°C, 1 to 10°C, 0 to 5°C, 1 to 5°C, etc.

[0047] (Viscosity α1 of the medium fluid) The first viscosity α1 at the high temperature T1 is such that in the host tissue, the medium fluid does not stay at the injection site (the periphery immediately outside the tip opening of the injection needle) and can quickly flow away into the host tissue. As such a first viscosity α1, for example, when the living body is a human, at the high temperature T1 (about 21 to 42°C or higher), 1000 [mPa·s] or less is mentioned, and preferably about 0.5 to 1000 [mPa·s]. For other living bodies, the first viscosity α1 is generally the same.

[0048] (Viscosity α2 of the medium fluid) The second viscosity α2 at the low temperature T2 is greater than the first viscosity α1, and in the accommodation part, the medium fluid can be extruded into the injection needle, and without leaving the transplanted cells in the accommodation part, it can flow into the injection needle together with the transplanted cells. As such a second viscosity α2, for example, when the living body is a human, at the low temperature T2 (about 0 to 20°C or lower), 5000 [mPa·s] or more is mentioned, and preferably about 10000 to 1000000 [mPa·s].

[0049] The lower limit temperature of the high temperature T1 may be the boundary temperature (transition temperature) at which the first viscosity α1 rapidly converts to the second viscosity α2. Also, the upper limit temperature of the low temperature T2 may be lower than the lower limit temperature of the high temperature T1, and the transition temperature may be between the lower limit temperature of the high temperature T1 and the upper limit temperature of the low temperature T2. The first viscosity α1 and the second viscosity α2 may each be constant. In that case, the medium fluid exhibits two types of viscosities with the transition temperature as the boundary. The first viscosity α1 may decrease as the temperature rises. The second viscosity α2 may increase as the temperature drops. The first viscosity α1 and the second viscosity α2 may be values that achieve the object of the present invention. The medium fluid may have the first viscosity α1 at a temperature higher than the high temperature T1, and may have the second viscosity α2 at a temperature lower than the low temperature T2.

[0050] (Viscosity measurement method) The viscosity in the present invention is measured based on the "Viscosity measurement method using a cone - plate type rotational viscometer" defined in JIS Z8803. The above "cone - plate type rotational viscometer" is a device also called a cone - plate viscometer. The viscosity values described in this specification are the values measured using a TV25 type viscometer (model number: TVE - 25L) manufactured by Toki Sangyo Co., Ltd. as a cone - plate type rotational viscometer.

[0051] (Preferred embodiment of the medium fluid for solving the second problem) As a preferable medium fluid having the first viscosity α1 at the above-described high temperature T1 and the second viscosity α2 at the low temperature T2, there is a fluid which becomes a gel at the low temperature T2 in the accommodating portion and exhibits the second viscosity α2, and becomes a sol at the high temperature T1 at the tip of the injection needle and exhibits the first viscosity α1 (hereinafter, also referred to as a gel-sol change fluid). When the medium fluid is a gel-sol change fluid, the medium fluid is cooled to the low temperature T2 in the accommodating portion and gelled, and becomes not a fluid, but in this specification, it is called a gelled medium fluid. The gelled medium fluid has a property that, for example, when it is pushed by a plunger in a state of being accommodated in a syringe, a portion near the base opening of the injection needle is easily deformed, the integrity collapses, and it can flow out into the injection needle.

[0052] (gel-sol change fluid) The gel-sol change fluid that can be used in the present invention is not particularly limited, but from the viewpoint of not adversely affecting transplanted cells and living tissues, a single gelling agent selected from an aqueous gelatin solution, an aqueous κ-carrageenan solution, an aqueous ι-carrageenan solution, a polysaccharide, an artificial peptide, a polymer, a polymer complex, etc., or a mixture of the above gelling agents, and further, a substance selected from gelling aids such as an aqueous glucomannan solution, an aqueous galactomannan solution, xanthan gum, an aqueous carboxymethylcellulose solution, a polysaccharide, an artificial peptide, a polymer, a polymer complex, etc., and a mixed aqueous solution of a gelling agent are preferably exemplified.

[0053] Preferable concentrations and additives of the above-mentioned aqueous gelatin solution, aqueous carrageenan solution, and aqueous carboxymethylcellulose solution are exemplified as follows. Gelatin 0.1 to 10% (weight / volume), carrageenan (ι, κ type) 0.01 to 0.5% (weight / volume), aqueous glucomannan solution or aqueous galactomannan solution 0.01 to 0.5% (weight / volume), xanthan gum 0.01 to 0.5% (weight / volume), carboxymethylcellulose 0.5 to 12% (weight / volume).

[0054] For example, an aqueous gelatin solution with a concentration of 5% (weight / volume) becomes a sol with a viscosity of 1000 [mPa·s] or less at 30°C or higher, and becomes a gel with a viscosity of 10000 [mPa·s] or more at less than 10°C.

[0055] (Cooler) As shown in FIG. 1, in a preferred embodiment of the present invention, the cell injection device is provided with a cooler or heat insulating material (hereinafter also abbreviated as cooler) 40 configured to maintain the suspension fluid in the storage portion at a low temperature T2. As will be described later, the cooler 40 may be merely a cold insulating agent or a heat insulating material, or may be a combination of a cooling device having a function of actively cooling the storage portion and a cold insulating agent / heat insulating material. The arrangement position of the cooler 40 is not particularly limited, but an arrangement mode of winding around the storage portion is simple and preferable.

[0056] By using the above-described preferred medium fluid as the suspension fluid and providing a cooler, the suspension fluid is maintained at a low temperature and exhibits a high viscosity α2 when stored in the storage portion 13, and when flowing out from the tip of the injection needle 20 at the extremely small flow rate Q1, it is warmed by the tissue and exhibits a low viscosity α1. Therefore, as described above, the suspension fluid 30 stored in the storage portion 13 can flow into the injection needle 20 while containing the transplanted cells 31, and when the suspension fluid 30 enters the tissue 100 from the injection needle 20, the medium fluid 32 flows away to the surrounding tissue along with the flow of the tissue fluid, and only the transplanted cells remain at the injection site.

[0057] (Active cooling function) The cooling principle and configuration of the cooler are not particularly limited as long as they can keep the accommodation part at a low temperature, at least during injection. Conventional cooling devices, cold insulation agents, and heat insulating materials described later can be used. For example, as a configuration of a cooler that can actively lower the temperature of the accommodation part and the suspension fluid to a low temperature T2, a cooling pipe through which a fluid at a low temperature (for example, in the temperature range T2) supplied from a low-temperature fluid supply source flows inside (preferably, a pipe made of copper or aluminum with high thermal conductivity) is wound around the accommodation part in a coil shape or a meandering shape, a configuration using a Peltier element (cooling side), a configuration using a cooled cold insulation agent, etc. can be exemplified. When these active coolers are provided around the accommodation part, in order to further enhance the cooling efficiency, the periphery of the cooler may be further surrounded by a heat insulating material.

[0058] (Heat preservation function) The cooler may have only heat insulation properties without having a function of actively cooling the accommodation part. For example, in the case where a syringe containing a suspension fluid or the accommodation part is directly cooled to a low temperature T2 in an external refrigerator and the syringe is taken out of the refrigerator when injecting the suspension fluid, the cooler may be a heat insulating material that surrounds the periphery of the accommodation part (for example, a cylinder). The heat insulating material may be an independent component or may be integrally incorporated into the accommodation part, such as a wall material constituting the accommodation part.

[0059] (Example aspect 1 of the cell injection device) FIG. 2(a) is a cross-sectional view schematically showing the configuration of the cell injection device manufactured in the following example as an example of a preferred embodiment of the cell injection device. FIG. 2(b) is a photographic view showing the appearance of a device obtained by modifying the details of the device of FIG. 2(a) (the operating principle is the same as that of FIG. 2(b)). In the embodiment of FIG. 2, a syringe 10 containing a suspension fluid 30 and a hydraulic cylinder 50a for moving its plunger 12 are fixed to a support frame S1, and the two are connected. An operating fluid (water in the example) is supplied from an operating fluid supply source 54 to the hydraulic cylinder 50a through a pipe 55, the piston 52 moves linearly, the tip of the piston rod 53 pushes the plunger 12 of the syringe 10 to move it at a moving speed v1, and the suspension fluid 30 flows out from the tip of the injection needle 20 at the above flow rate Q1. Around the syringe 10, as a cooler 40, a copper cooling pipe is densely wound, and the accommodating part is cooled to a low temperature T2. A fluid at a low temperature T2 is supplied to the cooling pipe from a low-temperature fluid supply source (not shown). The operating fluid supply source 54 has a syringe as a pump inside itself and a device for accurately moving its plunger, and can accurately send out the operating fluid. In the embodiment shown in FIG. 2(a), the entire cylinder 11 and the cooler 40 are accommodated in the support frame S1, whereas in the embodiment shown in FIG. 2(b), about 3 / 4 of the total length of the cylinder 11 protrudes outside the support frame S1, and a copper cooling pipe 40 is densely wound around the protruding part. Further, in the embodiment shown in FIG. 2(b), a connecting fixture S2 is fixed to the support frame S1. The connecting fixture S2 is configured to connect the cell injection device to the arm of a fixing stand and to be able to finely adjust the angle and position. With the above configuration, it is preferable that the large and heavy operating fluid supply source 54 is separated from the syringe and does not interfere with the operation of puncturing the living body with the injection needle.

[0060] (Example 2 of the embodiment of the cell injection device) FIG. 3 is a diagram showing another example of a preferred embodiment of the cell injection device. In the example of FIG. 3, the extrusion mechanism is an exchangeable syringe 10. The actuator 50 that advances the plunger 12 of the syringe 10 has a configuration in which a tension spring 58 serving as a driving force source and a viscous damper 56 for limiting the moving speed are combined so that the moving speed v1 is created.

[0061] In the example of FIG. 3, the viscous damper 56 and the syringe 10 are coaxially and serially accommodated in the housing 60. The drive rod 57 penetrates the viscous damper 56, and its tip 57a is in contact with the rear end 12a of the plunger 12 of the syringe 10. The tip 57a of the drive rod 57 and the rear end 12a of the plunger 12 of the syringe 10 may be in a relationship of merely contacting each other, or may be in a relationship of being detachably fixed to each other via a one-touch coupling or the like. The drive rod 57 protrudes from the upper surface of the housing 60 (in the example of the figure, the upper surface of the lid 63), and a flange 57b is provided at its end (rear end). A tension spring 58 for generating a force to advance the drive rod 57 downward in the figure is provided between the flange 57b and the housing 60, and both ends of the tension spring 58 are fixed to the flange 57b and the lid 63, respectively. Thus, when the drive rod 57 is pulled upward in the figure by an external force or the like, the tension spring 58 is stretched to accumulate elastic energy, and the return force tries to advance the drive rod 57.

[0062] In the example of FIG. 3, the drive rod 57 is pulled and the tension spring 58 is in a stretched state, and a stopper 59 is inserted between the flange 57b and the lid 63 to prevent the return (contraction) of the tension spring 58. On the other hand, the syringe 10 is set in the housing 60 while containing the suspension fluid 30. When the stopper 59 is removed, the cell injection device starts to operate, the drive rod 57 advances at the moving speed v1 to push the plunger 12, and the suspension fluid 30 flows out from the tip of the injection needle 20 at a flow rate of 1 [μL / second] or less.

[0063] (Syringe) The syringe 10 is set in the housing in an exchangeable manner while containing a suspension therein. The syringe 10 may be a generally known conventional medical syringe, but in the example of FIG. 3, a preferable configuration considering the cell discharge property is provided inside (described later).

[0064] (Viscous damper) As for the mechanism of the viscous damper 56 itself, a conventionally known mechanism of a viscous damper in which a viscous fluid resists the movement of the piston can be referred to. In the example of FIG. 3, the cylinder of the viscous damper 56 is composed of a cylindrical body 56a and a sealing cap 56b. The cylinder is filled and sealed with a viscous fluid 56m having an appropriately selected viscosity. The drive rod 57 passes through the cylinder and also functions as the piston rod of the viscous damper 56. Inside the cylinder of the viscous damper 56, a piston 56p for the damper is fixed to the drive rod 57 and slides inside the chamber like a gasket in the syringe. In the figure, for simplicity of explanation, the drive rod 57 and the piston 56p are drawn as being integrated, but it may be a structure in which the shaft member (drive rod 57) passes through the central hole of the piston member (piston 56p) and the two are fixed. The piston 56p is provided with minute through-holes 56h. Thereby, the viscous fluid 56m can pass through the through-holes 56h, and thus the piston 56p can reciprocate inside the chamber. Then, due to the resistance force when the viscous fluid 56m passes through the through-holes 56h, the moving speed of the piston 56p is limited to the above-mentioned moving speed v1. The diameter of the through-holes 56h, the restoring force of the tension spring 58, and the viscosity of the viscous fluid 56m can be appropriately selected so that the above-mentioned moving speed v1 is achieved. Incidentally, as the tension spring 58 returns, the restoring force decreases, and accordingly the moving speed of the drive rod 57 also decreases, but it suffices as long as it is within the range of the above-mentioned moving speed v1. In relation to this, by making the total moving distance of the plunger smaller and the natural length of the tension spring larger, the rate of decrease in the restoring force of the tension spring can be reduced.

[0065] (Viscous fluid) The viscous fluid 56m in the viscous damper 56 may be an oil, water, air, various working fluids, etc. for a general viscous damper. Further, the viscous fluid 56m preferably has heat resistance so that the entire cell injection device can be sterilized at a high temperature.

[0066] (Housing) The housing 60 is a kind of holder that holds the viscous damper 56 and the drive rod 57 in a coaxially aligned state with each other, and is also a support that enables the cell injection device to be disposed on the surface of a living tissue (such as the skin surface or the surface of an organ, the outer surface of a host tissue) in a stable state and at a predetermined puncture angle. In the example of FIG. 3, the housing 60 has a lower surface (contact surface) 60b that is wider than the upper surface 60a so as to be able to stably contact the surface of the living tissue, and thus has an overall shape of a frustum of a cone or a frustum of a pyramid. A syringe holding hole 61 for holding the syringe 10 in an exchangeable manner is provided in the lower surface 60b of the housing 60. Auxiliary structures such as fixing members, positioning protrusions, and steps for fixing the syringe 10 at a predetermined position in the syringe holding hole 61 may be appropriately provided (not shown in the figure). On the other hand, a damper accommodation hole 62 for accommodating the viscous damper 56 is provided in the upper surface 60a of the housing 60. The viscous damper 56 is fixed in the damper accommodation hole 62. The lid 63 can be appropriately fixed to the housing 60 by bolts, screwing, or the like.

[0067] The structure and shape of the housing shown in FIG. 3 are merely examples. For example, a structure that can be appropriately disassembled so that necessary devices such as a viscous damper can be incorporated therein, such as a structure that is split into two vertically, a structure provided with an internal space and a lid for closing it at necessary positions, a structure having only a skeleton without a wall portion, etc. can be adopted. The support frame S1 shown in FIG. 2(a) is also an aspect of the housing. Further, the shape of the housing may be a straight shape such as a cylindrical shape or a polygonal column shape (including a triangular column shape and a quadrangular column shape), and a flange portion or an auxiliary leg portion protruding laterally may be provided at the lower end surface of the body thereof to stabilize the contact with the surface of the living tissue.

[0068] In a preferred embodiment of the present invention, the syringe is cooled to a low temperature. On the other hand, due to the ultra-low speed injection peculiar to the present invention, the housing is in contact with the surface of the living tissue for a long time. Therefore, the surface layer of the lower surface of the housing (the surface in contact with the surface of the living tissue) may be made of a heat-insulating material so that the housing does not cool the surface of the living tissue or the surface of the living tissue does not warm the housing.

[0069] (Cooler) In the example of FIG. 3, a cooler 64 is disposed inside the housing 60. The cooler 64 may be directly filled with a coolant in a space provided inside the housing 60, or the coolant may be housed in a flexible bag or a rigid case. In the former case, the entire housing 60 may be cooled in a refrigerator. In the latter case, only the bag containing the coolant may be cooled and disposed in the space. Also, various cooling devices may be disposed as the cooler. The cooler 64 may be in direct contact with the syringe 10, or the material between the cooler 64 and the syringe 10 may be a metal material with high thermal conductivity. Also, the material and members of the housing surrounding the outside of the cooler 64 may be made of a material with high heat insulation (for example, plastic, foam, the vacuum double structure used in a heat-insulating container, etc.).

[0070] In the example of FIG. 3, the stopper 59 is depicted as a simple block piece, but it may be a collar or the like having a U-shaped cross-sectional shape in the horizontal direction in the figure so as to support the flange 57b in a balanced manner and be detachable. Also, a release mechanism may be provided in which the stopper member moves to release the prevention of the forward movement of the drive rod 57 when the user presses a start button or the like.

[0071] In the example of Fig. 2(a), the syringe 10 incorporated in the support frame S1 and the actuator (hydraulic cylinder 50a) can be incorporated in the housing 60 shown in Figs. 3 and 4. In other words, the actuator (a device combining a viscous damper and a tension spring) in the cell injection device shown in Figs. 3 and 4 can be replaced with the actuator shown in Fig. 2. In that case, in Fig. 2, the hydraulic cylinder can be set in place of the viscous damper so as to push the plunger of the syringe 10 in Fig. 3, and the hydraulic fluid supply source in Fig. 2(a) may be arranged outside the housing 60.

[0072] The materials of the respective elements constituting the cell injection device shown in Fig. 3 are not particularly limited, and materials used for medical devices such as metals, polymers, glasses, and ceramics can be appropriately used.

[0073] (Preferred embodiment 1 of each part: gasket) In a conventional syringe, the inner front end surface of the cylinder (where a pipe leading to the injection needle opens) is a funnel-shaped surface, and the front end surface of the gasket portion (made of hard rubber) of the plunger may be a convex surface that fits the funnel-shaped surface. However, when injecting the suspension fluid to the end in such a conventional syringe, the entire front end surface of the gasket portion comes into close contact with the entire inner front end surface of the cylinder at once. When the front end surface of the gasket portion and the inner front end surface of the cylinder come into contact with each other entirely at once, at the moment of contact, many transplanted cells are left between these two surfaces and are pinched and destroyed.

[0074] Therefore, in the present invention, as shown in FIG. 3, the gasket portion is provided with a two-layer structure (12a1, 12a2). This two-layer structure has a hard rubber portion 12a1 having sealing properties and slidability as a plunger, and a tip member 12a2 joined to the tip side thereof. This tip member 12a2 is made of a material that is softer and more flexible than the hard rubber portion 12a1. The tip surface of the tip member 12a2 is a flat surface (a surface perpendicular to the central axis of the plunger), or a gentle convex shape that does not fit the funnel-shaped surface of the cylinder. Here, for the sake of explanation, it is described as a simple two-layer structure, but a multilayer structure with a functional layer such as an adhesive layer may be further added.

[0075] With the above configuration, when the plunger moves in the tip direction and the gasket portion reaches the inner tip surface (funnel-shaped surface) of the cylinder, first, a conical space is left by the funnel-shaped surface of the cylinder and the flat surface of the gasket portion, and the transplanted cells are not crushed. Next, when the plunger moves further in the tip direction, the soft and flexible tip member 12a2 deforms into a convex shape according to the funnel-shaped surface 11a, and sequentially adheres to the funnel-shaped surface from the outer peripheral edge side to the central side, and finally deforms until it fits the funnel-shaped surface entirely. Therefore, the transplanted cells in the suspension fluid remaining in the conical space are also sequentially moved from the outer peripheral edge side to the central side, and thus it becomes possible to move all the transplanted cells from inside the cylinder into the injection needle. Further, such a two-layer structure of the gasket portion may be applied not only to the cell injection device of the present invention but also to the gasket portion of a conventionally known plunger for cell injection.

[0076] The thickness of the tip member 12a2 of the above gasket (dimension in the central axis direction of the plunger) is preferably at least the depth of the funnel-shaped surface of the cylinder (dimension in the central axis direction of the plunger) or more in terms of fitting the concave surface of the cylinder.

[0077] In addition, the material of the tip member 12a2 of the gasket is not particularly limited, and materials (such as elastomers) of gaskets conventionally known to be provided at the tip of a syringe plunger can be used. Also, its softness and flexibility may be appropriately determined according to the pressing force of the plunger. For example, the hardness measured with a Type C durometer (SRIS 0101 / ASKER C) may be 5 to 25.

[0078] (Preferred embodiment 2 of each part: The first support member interposed between the device and the living body surface) Even if the injection needle is pushed into the heart wall or the like, the injection needle is pushed back by the contraction force of the muscle. Conventionally, when injecting a drug, transplanted cells, etc. into the heart wall or the like, an operation such as manually holding the syringe at a predetermined position (i.e., with one hand) and manually pushing down the piston (i.e., with the other hand) was performed. However, with such an operation, it was difficult to stably hold the syringe, and it was also difficult to inject drugs, transplanted cells, etc.

[0079] Therefore, in the present invention, as shown in FIG. 4, a first support member 70 is provided in front of the outer tip of the cylinder 11 of the syringe of the cell injection device 1. The cell injection device 1 shown in FIG. 4 has the same configuration as the cell injection device shown in FIG. 3 except that the shape of the housing 60 is cylindrical or prismatic. The first support member 70 is interposed between the surface 100a of the living tissue and the housing 60 (or the cylinder 11) when the injection needle (the needle tube portion 21) punctures the living tissue 100. The first support member 70 has a mating surface 70a for contacting the surface 100a of the living tissue directly or via an adhesive layer (not shown). The mating surface 70a may be a flat surface, a curved surface according to the shape of the surface of the living tissue, or may have a flexible surface layer that can be curved according to the shape of the surface of the living tissue. The injection needle passes through the first support member 70 and protrudes from the mating surface 70a.

[0080] By providing the first support member 70, the contact surface of the cell injection device 1 with respect to the surface 100a of the biological tissue becomes wider and flatter, and the cell injection device 1 can be held more stably. The first support member 70 can also exhibit the effect of preventing the blood that has flowed out to the outside from diffusing around by surrounding the portion where the injection needle punctures the surface of the biological tissue. The configuration of providing the first support member may be provided not only in the cell injection device of the present invention but also in a conventionally known syringe for cell injection.

[0081] The material of the first support member 70 is not particularly limited, but examples include those having rigidity, hardness, and mechanical strength that can function as a support member, such as metal materials such as stainless steel and plastics.

[0082] In a preferred embodiment of the present invention, an adhesive layer (not shown) is provided on the opposing surface 70a of the first support member 70 shown in FIG. 4. Thereby, the first support member 70 is fixed to the surface 100a of the biological tissue, and there is no need to apply manual force to hold the syringe in a predetermined position. As the material of the adhesive layer (adhesive, tackifier), a biocompatible material is preferable, and examples include collagen sponge.

[0083] The adhesive layer may be configured to peel off from the support member 70 and remain on the surface 100a of the biological tissue after the injection operation of the transplanted cells. Thereby, it can also exhibit the effect of preventing blood from flowing out from the hole on the surface of the biological tissue after the needle is withdrawn (hemostatic effect) and the effect of preventing blood from blowing out from around the needle during puncture (backflow prevention effect). Such a configuration may be provided not only in the cell injection device of the present invention but also in a conventionally known syringe for cell injection.

[0084] (Tilt of the injection needle by the first support member) In a preferred embodiment of the present invention, as shown in FIG. 4, the angle θ1 formed between the objective surface 70a of the first support member 70 and the syringe tube portion 21 protruding from the objective surface is not 90 degrees but an inclined angle. In the example of FIG. 4, the upper surface 70b and the objective surface 70a of the first support member 70 are not parallel to each other, and the overall shape of the first support member 70 is in the shape of a wedge. The angle θ1 is not particularly limited, but on the acute angle side, 20 to 70 degrees is preferable. By inserting the needle along the muscle fiber direction, invasion can be avoided, needle detachment can be prevented, and a long insertion length can be achieved, so that the effect of preventing the backflow of body fluid into the syringe tube can be obtained. When the angle θ1 exceeds the above upper limit and approaches a right angle, the needle is likely to come off, and after the needle is removed, the transplanted cells are likely to leak from the punctured hole. On the other hand, when the angle θ1 becomes excessively small beyond the above lower limit, when the site where the cells should be injected is at a deep position from the surface of the living tissue, it becomes difficult to reach the site or a long needle is required, which is not preferable.

[0085] (Curvature of the injection needle) Further, in a preferred embodiment of the present invention, as shown in FIG. 5(a), the syringe tube portion 21 of the injection needle is curved in an arc, and the central angle of the arc is preferably 45 to 90 degrees, more preferably 60 to 85 degrees. In the example of FIG. 5(a), the first support member 70 is a simple plate shape, and at the end of the objective surface 70a, there is an edge portion 70d that functions as a rotation center axis portion for preferably inserting the curved syringe tube portion 21 into the tissue 100. The form of this edge portion 70d is not particularly limited, but for example, the following (i) and (ii) are exemplified. (i) As shown in FIG. 5(a), a sharp-edged ridge line that passes through the center point of the arc of the syringe tube portion 21 (the point that coincides with the point 70d indicating the edge portion in the figure) and extends in coincidence with the central axis line (that is, a straight line perpendicular to the paper surface of the figure) perpendicular to the plane including the arc (the paper surface in the figure). (ii) As shown in FIG. 5(b), a ridge line 70d that passes through the center point 70c of the arc, includes the central axis line perpendicular to the plane including the arc inside, extends along the central axis line, and has a rounded shape concentric with the central axis line.

[0086] Due to the configuration in which the syringe portion 21 is curved in an arc and the first support member 70 has an edge portion 70d, as shown in FIGS. 5(c) and 5(d), by a rotation operation with the edge portion 70d as the center (fulcrum), the syringe portion can be inserted into the tissue along an arc trajectory, and it is also possible to pull out the syringe portion from the tissue along an arc trajectory. More specifically, this operation is performed as follows. As shown in FIG. 5(c), with the cell injection device 1 placed on the surface 100a of the tissue, the edge portion 70d of the first support member 70 is brought into contact with the surface 100a. The first support member 70 in FIGS. 5(c) and 5(d) is in a wedge shape, similar to the example in FIG. 4. Next, as shown in FIG. 5(d), while rotating the cell injection device 1 so that the edge portion 70d becomes the fulcrum (rotation center axis). Thereby, the syringe portion 21 can enter the tissue 100 along an arc trajectory. As shown in FIG. 5(d), even if the tissue 100 contracts, the arc-shaped syringe portion 21 that has entered the tissue 100 is unlikely to exert a force to push the syringe portion 21 back along the arc trajectory. Therefore, the syringe portion 21 is suppressed from coming out of the tissue.

[0087] The edge portion in the above (i) may be a sharp edge (ridge line shape) such as a right-angled corner portion, or may be a rounded edge portion (with a central axis on its surface). In the case of a rounded edge portion, when the cell injection device is lifted while rotating, strictly speaking, the central axis deviates from the center of the rotational movement. However, by appropriately reducing the radius of curvature of the roundness, such deviation from the center of the rotational movement can be ignored. The same applies to the radius of curvature of the roundness of the edge portion in the above (ii). Also, a rotatable roller (not shown) having a radius approximately the same as the radius of curvature of the roundness may be provided on the edge portions in the above (i) and (ii), or a hinge (not shown) may be provided to make the position of the rotation center axis portion more immovable.

[0088] As shown in FIGS. 5(c) and 5(d), the configuration in which the first support member 70 is provided with the edge portion 70d may be combined with the configuration shown in FIG. 4.

[0089] The radius of the arc of the syringe portion 21 may be appropriately determined according to the application site. For example, about 4 to 10 [mm] is useful, and about 5 to 8 [mm] is particularly useful. The total length of the edge portion provided on the first support member 70 is not particularly limited, but from the viewpoint of functioning as a stable rotation center axis portion, about 4 to 15 [mm] is preferable.

[0090] In the example of FIG. 5, the edge portion 70d is provided on the first support member 70 located in front of the syringe. However, as the second support member (not shown), any member extending laterally from the cylinder may be provided, and the edge portion of the above (i) or (ii) may be provided at the tip thereof.

[0091] (Embodiment Example 3 of the Cell Injection Device) FIG. 6 is a cross-sectional view showing another example of a preferred embodiment of the cell injection device. In the example of FIG. 6, an osmotic pump is used as the extrusion mechanism. The osmotic pump according to the present invention has a first chamber 81 and a second chamber 82 adjacent to each other via a liquid permeable membrane 83 such as a semipermeable membrane or a porous membrane. Using the osmotic pressure (or osmotic pressure difference) as the driving source for liquid movement, the liquid (first liquid) in the first chamber 81 moves into the osmotic pressure generating material (liquid or solid) in the second chamber 82, whereby the osmotic pressure generating material and the second chamber 82 expand to press an object. (Note that the first chamber 81 may be the outside such as a living tissue or a body cavity as described later.) Here, the osmotic pressure (or osmotic pressure difference) may be the difference in osmotic pressure generated between two liquids (the first liquid and the liquid osmotic pressure generating material) sandwiching the semipermeable membrane as the liquid permeable membrane, or the osmotic pressure acting when the first liquid that has freely passed through the porous membrane as the liquid permeable membrane penetrates into the osmotic pressure generating material (such as a gel-like or solid water-absorbing polymer) in the second chamber. In other words, the driving source for the first liquid to pass through the semipermeable membrane may be the osmotic pressure, or after the first liquid passes through the porous membrane regardless of the osmotic pressure and then contacts the osmotic pressure generating material and penetrates into the osmotic pressure generating material, the driving source may be the osmotic pressure. The osmotic pressure generating material may be a liquid or a solid (such as a porous mass, a fiber aggregate, or particles), and any material that absorbs the first liquid and expands may be used.

[0092] In FIG. 6, reference numeral 81 represents the first chamber by indicating the inner surface of the wall portion that defines the outer shape of the first chamber, and reference numeral 82 represents the second chamber by indicating the inner surface of the liquid permeable membrane 83 that defines the outer shape of the second chamber. The first chamber 81 and the second chamber 82 are isolated from each other by the liquid permeable membrane 83, and the substance filled in the first chamber 81 and the substance filled in the second chamber 82 will be adjacent to each other through the liquid permeable membrane 83. During use, a liquid (first liquid) 81m is disposed in the first chamber 81 of the osmotic pump. In the example of FIG. 6, the first liquid 81m is injected into the first chamber 81 through the liquid injection tube 88 during use. On the other hand, an osmotic pressure generating material 82m is disposed in the second chamber 82. The osmotic pressure generating material 82m may be disposed in the second chamber 82 in advance, or an injection tube may be provided and the osmotic pressure generating material may be injected into the second chamber 82 during use (when the osmotic pressure generating material is injected into the second chamber 82 during use, the first liquid 81m may be disposed in the first chamber 81 from the beginning). The first liquid 81m disposed in the first chamber 81 permeates through the liquid permeable membrane 83 due to the osmotic pressure (osmotic pressure difference) generated between the first liquid 81m and the osmotic pressure generating material 82m in the second chamber 82, and moves into the osmotic pressure generating material 82m in the second chamber 82. As a result, the osmotic pressure generating material 82m absorbs the first liquid 81m and expands, and thus the second chamber 82 also expands. When the osmotic pressure generating material 82m and the second chamber 82 expand, the accommodating portion (accommodating chamber) 85 disposed adjacent to the second chamber 82 is pressed, and the suspension fluid 30 (in which the transplanted cells 31 are dispersed in the medium fluid 32) is pushed out from the accommodating portion 85 to the outside (i.e., into the tissue into which the tip of the injection needle is inserted) through the injection needle 20 at the specified flow rate Q1 described above.

[0093] In the example of Fig. 6, from the perspective of cooling, the first chamber 81 is structurally provided as one chamber of the osmotic pump and functions as a chamber for supplying the first liquid 81m. However, the first chamber 81 does not necessarily have to be provided as a part of the device. For example, when cooling is not required, an external space such as a biological tissue or a body cavity may be used as the first chamber. Commercially available osmotic pumps such as the Alzet osmotic pump (manufactured by DURECT Corporation) are devices premised on being implanted in subcutaneous biological tissues or body cavities (i.e., devices premised on being supplied with body fluid (water) from the surrounding biological tissues or the space within the body cavity), and have a configuration that structurally includes only the second chamber 82 (filled with the osmotic pressure generating material 82m) and the accommodating portion 85 in Fig. 6. In the case of such a commercially available osmotic pump, during use, the external space during use such as a biological tissue or a body cavity functions as the first chamber.

[0094] At least a part of the wall portion constituting the accommodating portion 85 is made of a flexible liquid-impermeable film so that it can be pressed and contracted by the second chamber 82 or the osmotic pressure generating material 82m. In the example of Fig. 6, the accommodating portion 85 is a contractable bag entirely constituted by a flexible liquid-impermeable film 84, to which the needle base portion of the injection needle 20 is connected. The injection needle may be directly connected to the accommodating portion 85 without the needle base portion. When the film 84 is pressed from the outside by the expansion of the second chamber 82 (expansion of the osmotic pressure generating material 82m) of the osmotic pump, the volume inside the accommodating portion 85 decreases, and the suspension fluid 30 is extruded from the injection needle 20. In the example of Fig. 6, the entire outside of the accommodating portion 85 is surrounded by the space of the second chamber 82. The second chamber 82 is a bag entirely constituted by a flexible semipermeable membrane 83. The entire outside of the second chamber 82 is surrounded by the first chamber 81, and the first chamber 81 is a room provided within the housing 80.

[0095] In the example of FIG. 6, a suspension fluid injection tube 87 for injecting a suspension fluid into the housing portion from the outside is inserted. A check valve 87a is provided at the tip port of the suspension fluid injection tube 87, whereby the suspension fluid 30 is prevented from flowing out from the suspension fluid injection tube 87 to the outside. The suspension fluid injection tube 87 is not essential, and the suspension fluid 30 may be injected into the housing portion from the tip port of the injection needle 20. Further, in the example of FIG. 6, a liquid injection tube 88 for injecting a first liquid into the first chamber from the outside of the housing 60 is inserted. A check valve 88a is provided at the tip port of the liquid injection tube 88, so that the first liquid 81m in the first chamber 81 does not flow out from the liquid injection tube 88 to the outside. Each of the check valves 87a and 88a is a preferred embodiment but not essential, and on-off valves may be provided on the inlet sides (the sides located outside the housing 80) of the suspension fluid injection tube 87 and the liquid injection tube 88, respectively.

[0096] In the example of FIG. 6, the second chamber 82 of the osmotic pump directly presses the housing portion 85. However, an osmotic pump is used as the working fluid supply source 54 shown in FIG. 2, and the second chamber of the osmotic pump presses an adjacent chamber (not shown), and the working fluid contained in the chamber is injected into the hydraulic cylinder 50a. Such a configuration may be adopted.

[0097] The material and film thickness of the flexible liquid-impermeable film constituting the housing portion 85 are not particularly limited and may be appropriately determined so as to be well contracted when pressed by the osmotic pump. For example, flexible films made of polymers such as polypropylene and vinyl chloride having a thickness of about 1 to 100 μm can be mentioned.

[0098] The first liquid may be a liquid containing only a solvent without a solute, or may be a solution containing a solute. The solvent is not particularly limited, but water is preferably mentioned as a solvent from the viewpoint of safety.

[0099] The osmotic pressure generating material disposed in the second chamber may be solid or liquid (solution). The combination and concentration difference between the first liquid and the osmotic pressure generating material may be appropriately selected so as to satisfy the conditions of the following (I). (I) When the first liquid and the osmotic pressure generating material come into contact through the liquid permeable membrane, due to the osmotic pressure (osmotic pressure difference), the first liquid moves into the osmotic pressure generating material, thereby expanding the osmotic pressure generating material and the second chamber and pressing the accommodating portion, and the above-described flow rate Q1 is created.

[0100] In order to satisfy the conditions of (I) above, and particularly to obtain the above-described flow rate Q1, the thickness and area of the liquid permeable membrane can be adjusted as appropriate, the liquid permeability of the liquid permeable membrane can be adjusted, the concentration difference of the liquids in the first chamber and the second chamber can be adjusted (adjustment of the osmotic pressure difference), the amount of the osmotic pressure generating material can be adjusted, the expansibility of the osmotic pressure generating material can be adjusted, and the like. Examples of the liquid permeable membrane include porous films such as tetrafluoroethylene resin and polytetrafluoroethylene, dialysis membranes derived from cellulose such as regenerated cellulose and cellulose acetate, and semipermeable membranes of synthetic polymers such as polymethyl methacrylate, ethylene vinyl alcohol copolymer, polyacrylonitrile, and polysulfone. The membrane thickness can be appropriately determined according to the permeability and the overall shape.

[0101] The osmotic pressure generating material is not particularly limited, and those that can generate an osmotic pressure difference (the osmotic pressure difference that moves the first liquid to the second chamber) with respect to the first liquid, thereby moving the first liquid to the second chamber and increasing the volume of the second chamber, can be used. Such materials may include, for example, one or more materials selected from the group consisting of ionic hydrophilic materials, non-ionic hydrophilic materials, water-absorbing materials, non-volatile water-soluble materials, salts, saccharides, polysaccharides, polymers, hydrogels, osmolymers, hydrophilic polymers, and water-absorbing polymers.

[0102] The above hydrophilic material may contain non-volatile substances and water-soluble substances such as, for example, magnesium sulfate, magnesium chloride, potassium sulfate, sodium chloride, sodium sulfate, lithium sulfate, sodium phosphate, potassium phosphate, d-mannitol, sorbitol, inositol, urea, magnesium succinate, tartaric acid, raffinose, various monosaccharides, oligosaccharides, polysaccharides (such as sucrose, glucose, lactose, fructose, dextran, etc.), and mixtures thereof. The above water-absorbing material contains an osmopolymer, for example, a hydrophilic polymer (water-absorbing polymer) that swells upon contact with water.

[0103] For various configuration examples of the osmotic pump and specific osmotic pressure generating materials (ionic hydrophilic materials, non-ionic hydrophilic materials, water-absorbing polymers, etc.), reference can be made to, for example, the descriptions in Japanese Patent Application Laid-Open No. 2009-520511 (International Publication No. 2007 / 056501), U.S. Patent Application Publication No. 2004 / 0106914, U.S. Patent Application Publication No. 2004 / 0015154, etc.

[0104] The cell injection device illustrated in FIG. 6 is used, for example, as follows. With the tip of the injection needle 20 facing upward, the suspension fluid 30 warmed to a sol state is injected into the housing portion 85 from a medical syringe (not shown) through the suspension fluid injection tube 87. After confirming that the suspension fluid is overflowing from the tip of the injection needle 20 (i.e., the housing portion is filled with the suspension fluid), the medical syringe is removed. The backflow prevention valve 87a prevents the suspension fluid 30 in the housing portion 85 from flowing out from the suspension fluid injection tube 87. The medium fluid of the suspension fluid 30 in the housing portion 85 is cooled by the cooler 86 disposed inside the housing 80 and gelled. Next, the first liquid (for example, water) 81m is injected into the first chamber 81 through the liquid injection tube 88. Thereby, the operation of the osmotic pump starts. Immediately after the osmotic pump starts operating, the injection needle 20 is inserted into the living tissue, and the objective surface 80b of the housing 80 is brought into close contact with the surface of the living tissue to stabilize the cell injection device.

[0105] A heat insulating material layer 90 may be provided on the objective surface 80b of the housing 80 so that the cooled housing 80 does not cool the biological tissue and the inside of the housing 80 is not warmed by the biological tissue. Also, in the example of FIG. 6, by making the space in the second chamber in the osmotic pump exist more in the upper part of the accommodating portion, it is possible to operate the osmotic pump so as to squeeze out the accommodating portion from top to bottom.

[0106] (Operation method according to the present invention) The operation method is a method of operating the cell injection device according to the present invention described above. The operation method includes, for example, as shown in FIG. 1, a step of operating the extrusion mechanism (10, 50) of the cell injection device to cause the suspension fluid 30 accommodated in the accommodating portion 13 of the cell injection device to flow out from the tip of the injection needle 20 of the cell injection device at a flow rate of 1 [μL / second] or less.

[0107] The start of the operation of the extrusion mechanism may be, for example, the release of a stopper by a human or the input of a start signal by a human (pushing a start button). When the extrusion mechanism becomes startable, the extrusion mechanism operates by itself by its own drive source and causes the suspension fluid to flow out from the tip of the injection needle at the above flow rate Q1.

[0108] (Method for storing suspension fluid) When the cell injection device according to the present invention has a disposable extrusion mechanism including an accommodating chamber, such as a syringe to which an injection needle is connected or a tube to which an injection needle is connected, the suspension fluid is cooled to a low temperature T2 while being accommodated in the accommodating portion of the disposable extrusion mechanism, supplied to the medical site (the site where transplanted cells are injected), and may be mounted on the cell injection device there. The suspension fluid cooled to the low temperature T2 is maintained at the low temperature T2 by the cooler of the cell injection device.

[0109] In addition, the suspension fluid may be stored at a low temperature T2 in a refrigerator (including a cool box) while being contained in another container such as a vial or a culture bag until the cell injection device is used. This stored suspension fluid may be heated to a high temperature T1 once when the cell injection device is used, sucked by the cell injection device, transferred into the storage part, and then cooled to the low temperature T2 again there. Further, the suspension fluid may be stored at room temperature while being contained in another container such as a vial or a culture bag until the cell injection device is used. This stored suspension fluid may be sucked by the cell injection device and transferred into the storage part when the cell injection device is used, and then cooled to the low temperature T2 for the first time there.

Example

[0110] Hereinafter, experimental examples for confirming the usefulness of the present invention will be shown. Hereinafter, according to the present invention, an operation of injecting the suspension fluid from an injection needle into a tissue so that the flow rate when the suspension fluid flows out from the tip of the injection needle is 1 [μL / sec] or less is referred to as "slow injection". Further, as a comparative example (an injection example according to the prior art), an operation of injecting the suspension fluid from an injection needle into a tissue so that the flow rate when the suspension fluid flows out from the tip of the injection needle is a conventional flow rate (about 10 [μL / sec]) is referred to as "conventional injection".

[0111] (Example 1) Slow injection using a low-viscosity medium fluid In this example, slow injection was performed using an Ads buffer having a relatively low viscosity as the medium fluid of the suspension fluid (cell suspension), and it was examined that the collagen fibrosis around the injected transplanted cells was suppressed, and how much the cells in the suspension fluid contained in the syringe could move into the living body together with the low-viscosity medium fluid was examined.

[0112] (Preparation of transplanted cells) Newborn rats were anesthetized with air containing 5% isoflurane (Wako Pure Chemical 099-06571) to obtain a deep anesthetic state. After confirming the absence of body movement and insensitivity to pain stimuli, euthanasia was performed. The chest wall was incised widely to expose the heart by compressing the chest cavity. The ventricular tissue was cut with scissors and collected. The tissue was immersed in Ads buffer (116 mM NaCl, 20 mM HEPES, 12.5 mM NaH2PO4, 5.6 mM glucose, 5.4 mM KCl, 0.8 mM MgSO4; pH 7.35, 25 °C) containing 1% collagenase (Wako Pure Chemical 032-22364) and 0.1% trypsin (Difco 215240), stirred at 37 °C to disperse the cells constituting the tissue. The supernatant containing the dispersed cells was collected, the enzyme reaction was neutralized using fetal bovine serum (biowest S1820-500), and then the cells were washed with Ads buffer.

[0113] Cardiomyocytes were purified from the collected cells based on the methods described in known literature (Hattori et al., "Nongenetic method for purifying stem cell-derived cardiomyocytes", Nature Methods 2010 Jan; 7(1):61-6.) and International Publication WO2006 / 022377. Subsequently, based on the methods described in the above-mentioned non-patent literature and International Publication WO2009 / 017254, about 300 cells were grouped together to create as many cardiomyocyte clusters as required for this experiment. At this time, the cell surface was labeled with red fluorescence in a living state using PKH 26 red (Sigma).

[0114] (Fabrication of cell injection device) As a cell injection device of the present invention, the device shown in Fig. 2(b) was fabricated. As the syringe 10 constituting the extrusion mechanism, a syringe with an injection needle (SS-10M2913) manufactured by Terumo Corporation was used. This syringe is equipped with a 29-gauge injection needle 20. On the body of the cylinder 11 of the syringe 10, as a cooler 40, a copper pipe (Yamato Hardware Store) with an outer diameter of 1 mm, an inner diameter of 0.3 mm (wall thickness 0.35 mm) was wound around without a gap, and an external pump (manufactured by AS ONE Corporation, 1-9419-02, cassette tube pump SMP-23AS), not shown in the figure, was used to forcibly circulate cooling water in the copper pipe, so that the temperature was around 2°C. By this cooler, the suspension fluid in the cylinder was maintained at a temperature of about 2°C.

[0115] As the hydraulic cylinder 50a as a driving source for pushing the plunger of the syringe 10, a syringe manufactured by Terumo Corporation (for tuberculin, 1 mL) with an 18G needle manufactured by Terumo Corporation attached thereto was used. The 18G needle of the hydraulic cylinder 50a and the working fluid supply source (syringe pump not shown in Fig. 2(b)) are connected by a tubing 55 for piping. The tubing 55 for piping is made of polypropylene, is thin and has low expandability of the inner cavity, and does not inhibit the accurate supply of the working fluid. The working fluid is water, there are no air bubbles in the tubing for piping, and it is filled with water. As the working fluid supply source, a syringe pump (remote control type DR-10) and a controller CT-10 manufactured by AS ONE Corporation were used.

[0116] (Implementation of slow injection) The above Ads buffer was used as the medium fluid of the suspension fluid, 100 of the above myocardial cell clusters were mixed and dispersed therein to prepare a suspension fluid, and 0.02 mL of the suspension fluid was aspirated into the syringe with an injection needle of the above cell injection device. Next, the extrusion mechanism was operated to perform a slow injection at 0.02 [μL / sec], and the suspension fluid was injected into the heart of the subject (number of samples n = 3) for Example 1. The plunger of the syringe was moved until the gasket hit the tip surface of the cylinder.

[0117] (Evaluation of the effect of slow injection) When observing the heart tissue after injecting the suspension fluid, it was found that fibrosis did not occur. Thereby, the usefulness of the cell injection device according to the present invention and the slow injection thereby was revealed.

[0118] (Evaluation of the residual of myocardial cell clusters in the syringe) When observing inside the syringe after injection, it was found that among the 100 myocardial cell clusters aspirated into the syringe, 50% ± 10% (sample number n = 3) of the myocardial cell clusters remained sandwiched between the gasket and the tip surface of the cylinder.

[0119] (Example 2) Use of a medium fluid that gels at low temperature and does not gel under body temperature conditions From the results of the residual of myocardial cell clusters in Example 1 above, the present inventor considered that a high viscosity is required for the medium fluid of the suspension fluid in order to extrude more of all the cells in the syringe, and considered using a gelled medium fluid. However, if the medium fluid remains gelled in the host tissue at the injection site (transplantation site) due to injection, it may inhibit the dispersion of the injected cells in the tissue and inhibit the contact conjugation with the myocardium which is the host tissue. Therefore, in this Example 2, as the medium fluid, a substance that gels in a syringe cooled to a low temperature and does not gel under body temperature conditions after injection was used. Among the group of substance candidates showing such properties, in this Example 2, κ-carrageenan was selected. The medium fluid is an aqueous solution containing 0.2% by weight of κ-carrageenan, shows a gel state at a low temperature (0 to 30°C), and becomes liquid at a temperature exceeding 30°C.

[0120] (Implementation of slow injection) During slow injection, the syringe was cooled to set the temperature of the suspension fluid to 5°C, and the syringe was surrounded by a heat insulating material to maintain the temperature. Also, the temperature of the host tissue was 37°C. The configuration of the device and other implementation conditions other than the temperature of the medium fluid and the syringe were the same as in Example 1.

[0121] (Evaluation of the effect of slow injection) Observation of the heart tissue after injection of the suspension fluid revealed that fibrosis did not occur. This clarified the usefulness of the cell injection device according to the present invention and the slow injection thereby.

[0122] (Evaluation of the residual of cardiomyocyte aggregates in the syringe) Observation of the inside of the syringe after injection revealed that among the 100 cardiomyocyte aggregates aspirated into the syringe, the cardiomyocyte aggregates remaining sandwiched between the gasket and the front end surface of the cylinder decreased to 5.9% (sample number n = 3).

[0123] (Comparative Example 1) Examination of other medium fluids In order to show that it is necessary for the gel-like suspension fluid to become a sol after injection and that the flow of tissue fluid is necessary to produce the effect of slow injection, the following comparative experiment was conducted.

[0124] As the medium fluid, Ads buffer containing 5 wt% hydrolyzed gelatin (Sigma G9382) at the final concentration was used, and 100 cardiomyocyte masses were mixed therein to prepare 0.02 mL of suspension fluid. This suspension fluid was aspirated into a syringe, and after cooling, slow injection was performed on the heart of a dead mouse at 0.02 [μL / sec] in the same manner as in Example 2 above.

[0125] (Evaluation of the effect of slow injection) In this Comparative Example 1, gelatin solubilization did not occur due to body temperature and there was no dilution effect by tissue fluid, so a blister-like raised structure appeared around the transplantation site.

[0126] (Example 3) In the slow injection as Example 3 and also in the conventional injection as Comparative Example 2, cardiomyocytes of neonatal rats were injected into the heart of an immunodeficient nude rat as a subject, and the amount of collagen fibrosis around each injected transplanted cell was examined and compared. In both Example 3 and Comparative Example 2, the number of subjects n was 3.

[0127] (Preparation of transplanted cells) In the same manner as in Example 1, a myocardial cell mass was obtained. Also, using PKH 26 red (Sigma), the cell surface was labeled with a red fluorescent color while alive.

[0128] (Cell injection device) The same cell injection device as in Example 1 was used. The temperature of the suspension fluid in the cylinder was maintained at about 2°C. Note that the moving speeds of the pistons of the hydraulic cylinders 50a in Example 3 and Comparative Example 2 are different from each other depending on the settings of the working fluid supply source.

[0129] (Suspension fluid) As the medium fluid, Ads buffer containing hydrolyzed gelatin (Sigma G9382) with a final concentration of 5% was used, and 100 myocardial cell masses were mixed and dispersed therein to obtain 0.1 mL of the suspension fluid of this Example 3. This medium fluid solates at 25°C or higher and exhibits a first viscosity of 50 mPa·s, and gels at less than 10°C and exhibits a second viscosity of 10,000 mPa·s or higher. Also, the same suspension fluid as this was prepared and used as the suspension fluid of Comparative Example 2. Note that the suspension fluid of Comparative Example 2 was used at room temperature without cooling the syringe so as not to gel in the syringe.

[0130] (Transplantation) As subjects for the examples and comparative examples, male immunodeficient nude rats were used, and anesthesia was induced in each subject with air containing 3% Isoflurane. A cannula was inserted into the trachea and connected to a ventilator. An Isoflurane vaporizer was connected to the ventilator to continuously maintain deep anesthesia. The chest skin and pectoral muscles were cut, the left third intercostal muscle was incised, and further the pleura was incised, and a rib retractor was inserted to maintain the open-chest state.

[0131] (Injection according to Example 3) The 0.02 mL of the suspension fluid was aspirated into the syringe with a needle of the cell injection device, cooled to 4°C to gel the medium fluid. Next, the extrusion mechanism was activated to perform a slow injection at 0.02 [μL / sec], and the suspension fluid was injected into the heart of the subject for the example (number of samples n = 3).

[0132] (Injection according to Comparative Example 2) The 0.02 mL of the suspension fluid was aspirated into the syringe with a needle of the cell injection device, and while the medium fluid remained in a sol state at room temperature, the extrusion mechanism was activated to perform a conventional injection at about 10 [μL / sec], and the suspension fluid was injected into the heart of the subject for the comparative example (number of samples n = 3).

[0133] For both the subjects of Example 3 and Comparative Example 2, sutures were placed between the second and fourth intercostal spaces, the chest opening was closed, and the muscle and skin were sutured.

[0134] (Results) After two months had passed since the injection of the cardiomyocyte mass, anesthesia was induced with Isoflurane vapor anesthesia for each subject, and deep anesthesia was performed by administering Somnopentyl 1 μL / g (Pentobarbital 65 μg / g equivalent) intraperitoneally. The chest was incised under anesthesia, and the heart was removed.

[0135] Each heart as a sample was fixed with 4% paraformaldehyde (Wako Pure Chemical 163-20145), replaced with TBS solution (Bio-rad 1706435) containing 20% Sucrose (Wako Pure Chemical 195-07925), embedded and frozen in OCT compound (Sakura Tissue-Tek), and frozen heart sections were prepared using a Leica cryostat CM3050S. The frozen sections were washed with TBS solution and the tissue images were observed under a microscope. For microscopic observation, type I collagen, which is representative of the extracellular matrix in fibrosis, was immunostained (primary antibody: Chondrex Anti-Rat Type I Collagen Antibody, Clone 1F10C2, secondary antibody: Invitrogen donkey anti-mouse IgG-Alexa fluor488) (Figs. 7(a), 8(a)). The nuclei were stained with DAPI (Molecular Probe) (Figs. 7(b), 8(b)). The cells were stained with PKH26 red (Figs. 7(c), 8(c)). These were photographed using a fluorescence microscope (Olympus IX-71, pixera pro 600es).

[0136] As a result, it was found that the number of engrafted cells in the sample according to Comparative Example 2 was extremely small compared to the number of engrafted cells in the sample according to Example 3.

[0137] Also, it was examined whether the interstitial fibrosis differed between the slow injection of Example 3 and the conventional injection of Comparative Example 2.

[0138] As a result, in the tissue injected with the slow injection of Example 3, as shown in the micrograph of Fig. 7(d), type I collagen was not seen around the injected (transplanted) myocardial cell mass and between the myocardial cell masses. In contrast, in the tissue injected with the conventional injection of Comparative Example 2, as shown in the micrograph of Fig. 8(d), it was found that type I collagen was present extensively around the grafted cells and between the myocardial cell masses.

[0139] (Example 4) Using slow injection as Example 4 and conventional injection as Comparative Example 3, cardiomyocytes of neonatal rats were injected into the hearts of immunodeficient nude rats as the subjects, the engraftment amount after transplantation (after injection) was compared, and the presence or absence of contact between the host tissue and the transplanted cells after transplantation was confirmed.

[0140] (Experimental method) Similar to Example 3 and Comparative Example 2 above, a suspension fluid containing 100 cardiomyocyte masses (300 cells per mass) of neonatal rats was prepared, and slow injection (about 0.02 [μL / sec]) as Example 4 and conventional injection (about 10 [μL / sec]) as Comparative Example 3 were performed. In both Example 4 and Comparative Example 3, the number of subjects was 3.

[0141] Two months after the injection of the cardiomyocyte masses, the hearts of each subject were excised and sections were prepared. Red-labeled cardiomyocytes were photographed at low magnification using a fluorescence microscope. Using the image analysis software ImageJ (open source of the National Institutes of Health, USA), the engraftment area of cardiomyocytes was quantified (number of pixels), and the volume was numerically determined by multiplying the distance between sections (μm). For both Example 4 and Comparative Example 3, the average value, standard deviation, and results of the significance test of the numerical values of the three subjects are shown as a bar graph in Fig. 9. The vertical axis of the bar graph is the volume of the transplanted cells that survived after injection, which is in arbitrary units. The significance test was analyzed by Student-t using EZR (open source of Jichi Medical University).

[0142] (Results) As is clear from the bar graph in Fig. 9, the volume of the cardiomyocytes engrafted after injection showed a significantly higher engraftment rate in Example 4 than in Comparative Example 3.

[0143] (Example 5) In this example, neonatal rat cardiomyocytes were injected into a left ventricular infarction model created using nude rats by slow injection (at approximately 0.02 [μL / sec]) to examine engraftment and therapeutic effects. As Comparative Example 4, phosphate buffered saline (PBS) was injected by the same slow injection for comparison with Example 5.

[0144] (Creation of left ventricular infarction model) Male nude rats were anesthetized with air containing 3% isoflurane. A cannula was inserted into the trachea and connected to a ventilator. An isoflurane vaporizer was connected to the ventilator to maintain deep anesthesia continuously. The rat's body was twisted to the right around the body axis, and with the left flank as the surgical field, the skin and pectoral muscles were incised, the left third intercostal muscle was incised, and then the pleura was incised. A rib retractor was inserted to maintain an open-chest state. The obtuse marginal branch and the first diagonal branch of the coronary artery were ligated with a single thread to construct a left ventricular infarction model. Sutures were placed between the second and fourth intercostal spaces to close the chest cavity. The muscles and skin were sutured and the rats were returned to their cages and raised for one week.

[0145] Changes in cardiac systolic pump function were analyzed by echocardiography. Due to left ventricular myocardial infarction, cardiac function was reduced. For this model in this state (4 rats), as Example 5, a suspension fluid containing cardiomyocyte aggregates prepared as in Example 1 above was injected by slow injection without thoracotomy. For a similar model (3 rats), as Comparative Example 4, an Ads buffer mixed with PBS instead of cardiomyocyte aggregates was injected by slow injection without thoracotomy. For the injection, nude rats with a left ventricular infarction model were fixed on an echocardiography table while under inhalation anesthesia, and slow injection was performed in the same manner as in Example 2 except without thoracotomy while performing echocardiographic imaging. The cell injection device used for slow injection was installed via a micromotion device that can finely adjust the three-dimensional position by smooth screwing, and the injection was performed while confirming the echocardiographic image in real time.

[0146] After two months had passed since the injection, echocardiography was performed on the models of Example 5 and Comparative Example 4, and they were euthanized by the method described in Example 1, and their hearts were collected respectively. Furthermore, frozen sections of the heart were prepared by the method described in Example 1. For the purpose of verifying the presence or absence of gap junctions, which are part of the intercalated disc junctions, Connexin 43, which is a constituent protein of gap junctions in mature cardiomyocytes, was immunostained (primary antibody: Sigma Rabbit Anti-Connexin-43 C6219, secondary antibody: Invitrogen donkey anti-rabbit IgG-Alexa fluor488).

[0147] (Results) A significance test was performed on the cardiac pump function before and after injection in Example 5 and Comparative Example 4. The results are shown in the graph of FIG. 10. As shown in the graph, in the model group injected with PBS (dashed graph), the cardiac function did not recover significantly, but in the model group injected with cardiomyocytes (thick solid line graph), a significant recovery of cardiac contraction and pump function was observed compared to before injection.

[0148] Also, in the model group injected with cardiomyocytes, as shown in the microscopic photograph of FIG. 11, and the microscopic photographs of FIGS. 12 and 13, which are local magnifications of FIG. 11, it was found that the cardiomyocytes stained with fluorescent red (PHK 26 red) were directly connected to the cardiomyocytes, which are host cells, and showed a mature cell morphology equivalent to that of the host cells. It was found that these connections include the mode of gap junctions by Connexin 43. From the above, it is considered that the transplanted cells and the host cells form an intercalated disc and achieve electrical and mechanical coupling.

[0149] (Example 6) In this example, another medium fluid that thickens (gels) at a temperature lower than body temperature and thins (solates) at body temperature was actually prepared, and it was confirmed that the viscosity changes with temperature.

[0150] An aqueous solution containing 0.2% by weight of κ-carrageenan (Wako Pure Chemical 033-09292) was prepared. This aqueous solution was gel-like at low temperature T2 (0 to 30 °C) and became a sol with low viscosity at temperatures higher than that.

[0151] 100 cardiomyocyte clusters derived from marmoset ES cells (the number of cells per cluster is 300) were mixed with the above aqueous solution (0.02 mL) to prepare a suspension fluid. This suspension fluid was injected in its entirety by slow injection into the adult marmoset heart wall using the same cell injection device as in Example 1. The number of cardiomyocyte clusters remaining in the syringe after injection was on average 6 (sample number n = 3).

[0152] In the same manner as above, an aqueous solution containing 3% by weight of gelatin (Sigma G9382) was prepared. This aqueous solution was gel-like at low temperature T2 (0 to 37 °C) and became a sol with low viscosity at temperatures higher than that. 100 cardiomyocyte clusters derived from marmoset ES cells (the number of cells per cluster is 300) were mixed with the above aqueous solution (0.02 mL) to prepare a suspension fluid. This suspension fluid was injected in its entirety by slow injection into the adult marmoset heart wall using the same cell injection device as in Example 1. The number of cardiomyocyte clusters remaining in the syringe after injection was on average 4 (sample number n = 3).

[0153] (Summary of Examples 1 to 6) In conventional injection, deposition of fibrosis (type 2 collagen) was observed around the injection site of the suspension fluid, whereas in slow injection, no fibrosis was observed around the injected transplanted cells. Also, in an injection experiment on a myocardial infarction model of 8-week-old nude rats subjected to left coronary artery ligation, in the group injected with transplanted cells, improvement in heart wall motion was observed after transplantation compared to the heart function before transplantation, whereas in the group not injected with transplanted cells (Vehicle transplantation group), no significant improvement in wall motion was observed. Furthermore, it was found that the transplanted cells (cardiomyocytes of neonatal rats stained with red pigment) clearly formed an intervening plate that was electro-mechanically coupled to the host cells. When cardiomyocytes of neonatal rats were conventionally injected, such formation of an intervening plate could not be found. Furthermore, when comparing the engraftment rates of the transplanted cells after injection, the cardiomyocytes injected by slow injection survived statistically significantly more than the cardiomyocytes injected by conventional injection. One of the reasons for the difference in cell survival rate due to the difference in the injection flow rate (or outflow rate) is considered to be due to the difference in shear stress when passing through the injection needle during injection (for example, shear stress generated by friction with the inner wall surface of the injection needle, or shear stress generated when trying to enter the host tissue from the outlet of the injection needle, etc.).

[0154] (Example 7) (Prototype of a cell injection device using a commercially available osmotic pump) In this example, using a commercially available osmotic pump, two types of prototypes of an extrusion mechanism similar to the embodiment during cell transplantation were fabricated, one large and one small (hereinafter also referred to as prototype (large) and prototype (small)). The prototype is equipped with a cooling device and is configured to be able to perform slow injection while maintaining the gel at a low temperature. The prototype (large) and the prototype (small) only differ in the size and capacity of the parts, and the operating principle of slow injection using the osmotic pump itself is the same as the mode shown in FIG. 6.

[0155] The basic configuration common to both the prototype (large) and the prototype (small) is, as shown in Fig. 14, a commercially available osmotic pump 810, a cylindrical container (a container with a cylindrical body shape) 820 surrounding it, the tip portion 840 of a cylinder attached to the tip of the osmotic pump 810, and an injection needle 841 connected to the tip thereof. A first chamber 820s is formed around the osmotic pump 810 by the cylindrical container 820. The tubular member 830 is a packing that seals the gap between the osmotic pump 810 and the cylindrical container 820, and the tubular member 850 is a packing that seals the gap between the needle 811 at the tip of the osmotic pump 810 and the cylinder 840. The first chamber 820s is filled with pure water 820m for operating the osmotic pump 810. A cooling pipe 900 is connected to the cylindrical container 820, and the pure water 820m advances through the cooling pipe 900 by a peristaltic pump (also called a roller pump) 910, is cooled through a cooling device 920, and returns to the first chamber 820s. By this circulation device, the temperature of the pure water 820m in the first chamber 820s can be maintained at an arbitrary low temperature (about 3 to 5 °C in this embodiment).

[0156] The specifications of each part of the prototype are as follows. 〔Prototype (Large)〕 Osmotic pump 810; manufactured by DURECT Corporation, Alzet osmotic pump, model number 2ML1, reservoir capacity 2000 μL, flow rate 10.0 (μL / hr). μL represents microliter. Cylindrical container 820 for the first chamber; polypropylene round-bottom tube (Falcon (registered trademark), part number 352059, inner diameter 17 mm, length 199 mm, capacity 14 mL). mL represents milliliter. Packing 830: silicone tube (Tiger polymer corporation, part number SR1554), inner diameter 8 mm, outer diameter 11 mm, length 5 mm. Cylinder 840 and needle 841: Only the cylinder and needle of a 29G needle syringe (BD Luer Lok (trademark), 1 / 2 mL, 29G, 12.7 mm, catalog number 326666) were used, and the tip portion 5 mm of the cylinder was cut and used. Packing 850: silicone tube, inner diameter 0.2 mm, outer diameter 3.6 mm, length 4 mm. 〔Prototype (small)〕 Osmotic pump 810; manufactured by DURECT Corporation, Alzet osmotic pump, model number 2001D, reservoir volume 200 μL, flow rate 8.0 (μL / hr). Cylindrical container 820 for the first chamber; ceramic tube (Sumitomo Bakelite, part number MS-4504W, inner diameter 10.5 mm, length 60 mm, volume 4 mL) Packing 830: silicone tube, inner diameter 7 mm, outer diameter 9 mm, length 5 mm. Cylinder 840 and needle 841: same as the prototype (large) Packing 850: same as the prototype (large)

[0157] Figure 15(a) is a photographic view showing the appearances of the prototype (large); reference numeral 800A and the prototype (small); reference numeral 800B. Further, Figure 15(b) is a photographic view showing the appearances of the osmotic pump 810A used in the prototype (large) and the osmotic pump 810B used in the prototype (small).

[0158] (Operation verification) In order to confirm whether the above-configured prototype (large) and prototype (small) actually have the ability to extrude the gel containing cells, the above-mentioned commercially available large and small Alzet osmotic pumps (model numbers 2ML1 and 2001D) and the following two types of suspensions (1) and (2) with different concentrations of hydrolyzed gelatin were used, and an operation experiment was conducted as follows. Among the above-mentioned large and small Alzet osmotic pumps, the larger Alzet osmotic pump is referred to as the "(large) osmotic pump", and the smaller Alzet osmotic pump is referred to as the "(small) osmotic pump". Also, in this example, these (large) osmotic pump and (small) osmotic pump are also simply referred to as the "osmotic pump". Suspension (1): Instead of cells, hard gel beads (Thermofisher product number F8842, product name FluoSpheres® Polystyrene Microspheres, 15 μm, red fluorescent (580 / 605), for blood flow determination) having the same size and specific gravity as cells and containing a red fluorescent dye were used. 2×10 5 of these hard gel beads were dispersed in a medium fluid (2 mL of Ads buffer containing hydrolyzed gelatin (Sigma G9382) with a final concentration of 5 wt% solubilized by heating to 37°C) to prepare suspension (1). Suspension (2): 2×10 5 of the above hard gel beads were dispersed in a medium fluid (2 mL of Ads buffer containing hydrolyzed gelatin (Sigma G9382) with a final concentration of 0.3 wt% solubilized by heating to 37°C) to prepare suspension (2).

[0159] As shown in Fig. 16, one (large) osmotic pump (reference numeral 810A) and two (small) osmotic pumps (reference numerals 810B1, 810B2) were used. Suspension (1) was injected into the (large) osmotic pump 810A. Suspension (1) was injected into the first (small) osmotic pump 810B1, and suspension (2) was injected into the second (small) osmotic pump 810B2.

[0160] Silicone tube packings were attached around the body of the tip of each of the three osmotic pumps (810A, 810B1, 810B2). These osmotic pumps were held by fitting them into through-holes provided in a holding plate 950 made of a soft polymer material as shown in Figs. 16 and 17, and the body portions of each osmotic pump were immersed in pure water 940 (4°C, 500 mL) in a beaker 930 to construct a set for an operation experiment. The entire set for the operation experiment (including the beaker) was left in a refrigerator (4°C) for 24 hours. The reason for immersing the three osmotic pumps in one beaker was to supply the same pure water at the same temperature to the three osmotic pumps simultaneously with a simple configuration.

[0161] Twenty-four hours after the start of the immersion, the pure water in the beaker had permeated into each osmotic pump, and the discharge of the gel had started from the tip of each osmotic pump. Further, as shown in Fig. 17, after being placed in the refrigerator, 72 hours later, a larger amount of the gel had been discharged than at the time when 24 hours had elapsed.

[0162] When the discharged gel was collected and observed with a fluorescence microscope, as shown in the micrograph of Fig. 18, fluorescent beads were contained in the gel. Fig. 18(a) is a transmission image (the horizontal width of the entire screen is about 2 mm) showing the state in which the fluorescent beads are dispersed in the gel, and Fig. 18(b) is a micrograph (the horizontal width of the entire screen is about 2 mm) showing the state of the beads that emitted red fluorescence when irradiated with excitation light having a wavelength of 475 nm.

[0163] From the operation experiment shown in Example 7, it was shown that it is possible to discharge the medium fluid containing cells in a dispersed state and gelled using an osmotic pump by slow injection. From this, it was suggested that by appropriately changing the configuration, specifications, operating conditions, etc. of the osmotic pump, it is possible to inject the medium fluid containing cells in a dispersed state and gelled into the target site at a low speed targeted by the present invention.

Industrial Applicability

[0164] According to the present invention, the damage received by the host tissue at the site where the suspension fluid flows in is minimized and is only due to the volume of the injected transplanted cells. Therefore, the present invention can contribute to various treatments for injecting transplanted cells into living tissues. According to the present invention, a method for injecting (administering) transplanted cells into a living body by slow injection and a treatment method by injecting transplanted cells into a living body by slow injection can be implemented.

Explanation of Reference Numerals

[0165] 10 Syringe 11 Cylinder 12 Piston 13 Accommodating portion 20 Injection needle 30 Suspended fluid 31 Transplanted cells 32 Medium fluid 40 Cooler 50 Driving source 100 Biological tissue

Claims

1. A cell injection device for injecting cells into a biological tissue, the cell injection device comprising: having a housing portion; having a suspension fluid contained in the housing portion, the suspension fluid being a dispersion of the cells in a medium fluid; having an injection needle directly or indirectly connected to the housing portion via a pipeline; having an extrusion mechanism configured to perform an operation of extruding the suspension fluid from the housing portion through the injection needle to the outside such that the flow rate when the suspension fluid flows out from the tip of the injection needle is 1 [μL / sec] or less; The medium fluid has a first viscosity α1 in a high-temperature temperature range T1 including the temperature of the biological tissue, and a second viscosity α2 in a low-temperature temperature range T2 lower than the high-temperature temperature range T1. The first viscosity α1 is 1000 [mPa·s] or less, and the second viscosity α2 is 10000 [mPa·s] or more; The cell injection device has a cooler or heat insulating material for maintaining the suspension fluid in the housing portion at the low-temperature temperature range T2; During use, the suspension fluid maintained at the low-temperature temperature range T2 in the housing portion is heated to the high-temperature temperature range T1 at the tip of the injection needle to become the first viscosity α1 and flows out from the tip of the injection needle. The cell injection device.

2. The high-temperature temperature range T1 is 21 to 42 [°C]; The first viscosity α1 is 0.5 to 1000 [mPa·s]; The low-temperature temperature range T2 is 0 to 20 [°C]; The second viscosity α2 is a value greater than 5000 [mPa·s]; The cell injection device according to Claim 1.

3. The cell injection device according to Claim 1 or 2, wherein the medium fluid becomes a sol having a first viscosity α1 in a high-temperature temperature range T1 and a gel having a second viscosity α2 in a low-temperature temperature range T2.

4. The cell injection device has a syringe having a cylinder and a plunger as the extrusion mechanism. The housing portion is formed on the tip side in the cylinder by the cylinder and the plunger, and the injection needle is connected to the tip of the cylinder. The extrusion mechanism further has an actuator configured to move the plunger toward the tip of the syringe at a speed at which the above flow rate is achieved, as a drive source. The cell injection device according to any one of claims 1 to 3.

5. The actuator is a hydraulic cylinder in which a piston moves linearly by the supply of a working fluid. A piston rod connected to the piston is connected to the plunger of the syringe so as to push the plunger. The cell injection device according to claim 4.

6. The actuator has an elastic body and a damper. The elastic body is arranged in a deformed state from its original shape and is arranged so that the restoring force to the original shape moves the plunger. The damper is arranged to resist the movement of the plunger and limit the speed of the shape displacement of the elastic body. The restoring force of the elastic body and the resistance of the damper are selected so as to move the plunger toward the tip of the syringe at a speed at which the above flow rate is achieved. The cell injection device according to claim 4.

7. On the outer tip surface of the cylinder of the syringe, a first support member is provided for intervening between the surface of the living tissue and the cylinder when the injection needle punctures the living tissue. The first support member has a mating surface for directly contacting or contacting the surface of the living tissue via an adhesive. The injection needle penetrates the first support member from the cylinder and protrudes from the mating surface. The cell injection device according to any one of claims 4 to 6.

8. On the objective surface of the first support member, an adhesive layer for fixing the first support member to the surface of the living tissue is provided. The cell injection device according to claim 7.

9. The injection needle has a needle tube portion that is curved in an arc, and the central angle of the arc is 45 to 90 degrees. The first support member further has An edge portion that passes through the center point of the arc and extends along a central axis perpendicular to the plane including the arc, or An edge portion that includes the central axis therein, extends along the central axis, and has a rounded shape concentric with the central axis. The cell injection device according to claim 7 or 8.

10. The angle formed between the objective surface of the first support member and the needle tube portion of the injection needle protruding from the objective surface is not 90 degrees. The cell injection device according to any one of claims 7 to 9.

11. The injection needle has a needle tube portion that is curved in an arc, and the central angle of the arc is 45 to 90 degrees. A second support member is provided on the cylinder of the syringe, and the second support member has An edge portion that passes through the center point of the arc and extends along a central axis perpendicular to the plane including the arc, or An edge portion that includes the central axis therein, extends along the central axis, and has a rounded shape concentric with the central axis. The cell injection device according to claim 7 or 8.

12. The cell injection device has an osmotic pump as the extrusion mechanism. The osmotic pump has A first chamber and a second chamber that are adjacent to each other through a liquid permeable membrane. The liquid disposed in the first chamber permeates through the liquid permeable membrane due to an osmotic pressure difference and moves into the osmotic pressure generating material disposed in the second chamber, whereby the second chamber expands. The expansion of the second chamber presses the housing portion, and the suspension fluid is configured to be extruded from the housing portion to the outside through the injection needle at the above flow rate. The cell injection device according to any one of claims 1 to 3.

13. A method of operating the cell injection device according to any one of claims 1 to 12, comprising: a step of causing the suspension fluid accommodated in the housing portion of the cell injection device to flow out from the tip of the injection needle of the cell injection device at a flow rate of 1 [μL / sec] or less by operating the extrusion mechanism of the cell injection device. The above method of operation.

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