Ex-vivo test system and test method using same

Abstract

An ex vivo test system 10 is constituted of a fluid circuit that circulates a prescribed circulation liquid by simulating the blood circulation state of a human body, and comprises a circulation liquid maintenance unit 17 that maintains, in the same manner as the blood environment in vivo, chemical factors in the circulation liquid, which affect the performance evaluation of a test object S. The circulation liquid maintenance unit 17 comprises: a replenishing liquid unit 23 that maintains the chloride ion concentration and protein concentration of the circulation liquid at desired values by injecting a replenishing liquid composed of an electrolyte-free isotonic solution into the fluid circuit; and a carbon dioxide supply unit 24 that maintains the pH of the circulation liquid at a desired value by supplying carbon dioxide into the fluid circuit, the state of the circulation liquid being maintained while compensating for the effect of moisture loss during circulation of the circulation liquid.

Description

In vitro testing system and testing method using the same

[0001] The present invention relates to an in vitro testing system for evaluating the performance of a test object, such as a medical material or device, in vitro, and a testing method using the same.

[0002] One treatment for cerebral aneurysms is the flow diverter placement technique, in which a cylindrical, mesh-like metallic flow diverter stent is placed in the aneurysm-affected artery to reduce blood flow into the aneurysm and promote thrombosis within the aneurysm. However, this technique poses challenges, such as the need for long-term antiplatelet medication because the flow diverter stent remains in the patient's artery, and limited retreatment options in the event of incomplete occlusion of the aneurysm. To address these challenges, research and development is underway to develop bioabsorbable flow diverter stents that degrade and are absorbed intravascularly after thrombosis within the aneurysm. Examples of bioabsorbable materials include magnesium alloys and polymers such as polylactic acid. When using such bioabsorbable materials in flow diverter stents, it is important to evaluate their corrosion resistance based on biological factors, including mechanical and chemical factors. Therefore, the present inventors are conducting research and development of a test system that can simulate these biological factors in vitro and elucidate the time-dependent degradation behavior of bioabsorbable flow diverter stents during vascular placement. During this research and development, we discovered that hemodynamic factors such as the reaction force from the blood vessel wall due to the shape of the blood vessel, pressure, and flow rate, as well as biochemical factors in the blood such as chloride ion concentration, protein concentration, and pH, affect the decomposition of magnesium alloys.

[0003] The present inventors have already developed a test device for simulating the state of blood circulation in the coronary artery and evaluating medical devices such as coronary artery stents in vitro (see Patent Documents 1 and 2).

[0004] JP 2016-126258 A JP 2017-142470 A

[0005] However, the above-mentioned test devices are specialized for simulating the blood circulation state in the coronary arteries and are not intended to simulate the blood circulation state around the cerebral arteries, which are different from the coronary arteries.Furthermore, the above-mentioned test devices are not designed to simulate chemical factors that affect the performance evaluation of bioabsorbable medical materials and devices, and are not necessarily sufficient for accurate evaluation of medical materials and devices that take chemical factors into consideration.

[0006] The present invention was devised with a focus on these problems, and its purpose is to provide an in vitro testing system and a testing method using the same that contribute to the time-dependent ex vivo evaluation of the performance of various medical materials and devices that are placed in blood vessels.

[0007] In order to achieve the above-mentioned object, the present invention provides an in vitro testing system that is primarily composed of a fluid circuit that circulates a specified circulating fluid by simulating the blood circulatory state of the human body, and that evaluates the performance of a test object, which is a medical material or device, outside the body by placing the test object in the middle of the fluid circuit.The system is configured to include a circulating fluid maintenance unit that maintains chemical factors in the circulating fluid that affect the performance evaluation of the test object at a level equivalent to the blood environment in a living body.

[0008] The present invention also provides a test method using the in vitro test system, which mainly employs the following techniques: maintaining the chloride ion concentration of the circulating fluid within a range of 96 mmol / L to 107 mmol / L; maintaining the protein concentration of the circulating fluid within a range of 55 g / L to 88 g / L; and maintaining the pH of the circulating fluid within a range of 7.3 to 7.45.

[0009] According to the present invention, the mechanical and chemical factors that are factors in the corrosion and dissolution of bioabsorbable medical materials and devices can be taken into consideration, and the blood circulation conditions of the human cerebral arteries, coronary arteries, etc. can be simulated in an ex vivo non-clinical environment, which can contribute to the evaluation of the degradation performance, etc. of test objects over time.

[0010] Fig. 2 is a conceptual diagram showing the configuration of an in vitro testing system according to the present embodiment. Fig. 3 is a partial cross-sectional view showing a state in which a stopper mechanism is disposed in an indwelling section. Fig. 4 is an exploded cross-sectional view of Fig. 2.

[0011] Hereinafter, an embodiment of the present invention will be described with reference to the drawings.

[0012] FIG. 1 shows a conceptual diagram illustrating the configuration of an in vitro testing system according to the present embodiment. In this figure, the in vitro testing system 10 is configured with an air-free fluid circuit simulating the blood circulation conditions in the human body. A test subject S, which is a medical material or device, is placed in a portion of the fluid circuit, and a predetermined circulating fluid is circulated through the fluid circuit to perform a time-dependent performance evaluation of the test subject S in vitro. In this embodiment, the test subject S is a bioabsorbable flow diverter stent made of a magnesium alloy or other material. The system is configured to simulate the blood environment of the human vascular region (from the petrous portion to the subfloor of the human internal carotid artery) in which the stent is placed. The circulating fluid used here is animal or human serum with chloride ion concentrations and total protein concentrations equivalent to those of human plasma. Examples of animal serum include serum from bovine, porcine, canine, and simian blood.

[0013] The in vitro testing system 10 comprises an annular flow path 11 through which a circulating fluid circulates, a storage section 12 for a test subject S provided midway through the flow path 11, a pulsating pump 13 that generates a flow of the circulating fluid within the flow path 11, a pressure and flow rate adjustment section 14 that adjusts the pressure and flow rate of the circulating fluid within the flow path 11, a pressure meter 15 that measures the inlet pressure of the circulating fluid on the inlet side of the storage section 12, a flow meter 16 that measures the flow rate of the circulating fluid on the inlet side of the storage section 12, and a circulating fluid maintenance section 17 that maintains the state of the circulating fluid circulating within the flow path 11.

[0014] The flow path 11 is not particularly limited, but is made of a tube made of a resin such as polyvinyl chloride, and is configured to circulate the circulating fluid in one direction, i.e., clockwise in FIG. 1, by check valves 18 arranged on the inlet and outlet sides of the pulsation pump 13.

[0015] The retention section 12 is detachably attached to the flow path 11, and is made of a cerebral aneurysm model that simulates the shape of a cerebral aneurysm and the blood vessels surrounding it, and is configured so that a test subject S can be retained in a space within the model that is connected to the flow path 11 and through which circulating fluid passes. This model is made of a translucent resin material so that the stent wall surface in contact with the aneurysm entrance (neck) can be observed with a microscope from above the aneurysm.

[0016] The pulsating pump 13 is a vortex pump including a main space 13A containing circulating fluid, an air chamber 13B containing air, and a diaphragm 13C separating the two. The diaphragm 13C is displaced by the pressure of air supplied to the air chamber 13B by a pneumatic drive unit (not shown). This displacement changes the volume of the main space 13A, thereby discharging and suctioning fluid into and from the main space 13A. In the pulsating pump 13, the reciprocating motion of the diaphragm 13C generates a pulsatile flow simulating the two time phases of the human heart, systole and diastole. The pressure of the circulating fluid pumped from the pulsating pump 13 can be changed by adjusting the air pressure that displaces the diaphragm 13C and the fluid resistance within the fluid circuit. The pulsating pump 13 is not limited to the structure described above; any structure that can impart a pulsatile flow to a fluid, like a heart, may be used. The check valve 18 is made of a polyurethane valve that allows flow in only one direction, and functions as an outflow valve and an inflow valve for the pulsation pump 13 by being attached to the outflow side and the inflow side of the pulsation pump 13 .

[0017] The pressure and flow rate adjusting section 14 comprises a compliance tube 19 arranged on the upstream side of the circuit between the retention section 12 and the outflow side of the pulsatile pump 13, a reservoir tube 20 arranged on the downstream side of the circuit between the retention section 12 and the inflow side of the pulsatile pump 13, and a resistance applying section 21 arranged between the retention section 12 and the reservoir tube 20 on the downstream side of the retention section 12.

[0018] The compliance tube 19 is formed from an elastic tube that elastically deforms so that its inner diameter can expand and contract in response to pressure changes in the circulating fluid passing through it, and the pressure and flow rate within the fluid circuit can be adjusted depending on the state of this elastic deformation. Although not particularly limited, the compliance tube 19 in this embodiment is formed from polyurethane.

[0019] The reservoir tube 20 functions as a venous reservoir element that temporarily stores the circulating fluid flowing through the flow path 11, and is made of, but not limited to, silicone.

[0020] The resistance applying section 21 is composed of a clamp or the like that applies peripheral resistance to the flow path 11, but the present invention is not limited to this, and various devices such as valves can be used instead as long as they have a similar effect.

[0021] In the pulsatile pump 13 and pressure / flow rate adjusting unit 14 configured as described above, the flow of circulating fluid in the flow path 11 near the retention portion 12 is set to simulate the blood flow and blood pressure of a human cerebral artery. That is, various conditions are set here so that the inlet pressure of the retention portion 12 measured by the pressure meter 15 will have a pressure waveform and maximum and minimum pressure magnitudes that approximate the cerebral artery flow of a human body, and so that the inlet side of the retention portion 12 measured by the flow meter 16 will have a desired blood flow state that approximates the cerebral artery flow of a human body. Specifically, the lengths, thicknesses, and inner diameters of the compliance tube 19 and reservoir tube 20 are set in accordance with the driving conditions of the pulsatile pump 13, and a predetermined peripheral resistance is applied to the flow path 11 by the resistance applying unit 21.

[0022] The circulating fluid maintenance unit 17 maintains the state of the circulating fluid while compensating for the effects of water loss due to evaporation from specific parts of the fluid circuit, and is configured to maintain the chloride ions, protein concentration, and / or pH in the circulating fluid, which are chemical factors that affect the decomposition performance evaluation of the test subject S, at levels equivalent to the blood environment in a living body.

[0023] This circulating fluid maintenance unit 17 includes a fluid replacement unit 23 that maintains the chloride ion and protein concentrations of the circulating fluid at desired values ​​by injecting a replacement fluid different from the serum that is the circulating fluid into the fluid circuit, and a carbon dioxide supply unit 24 that maintains the pH of the circulating fluid at a desired value by supplying carbon dioxide into the fluid circuit.

[0024] In the in vitro testing system 10 of this embodiment, a small amount of circulating fluid evaporates from the silicone membrane that forms the circumferential surface of the reservoir tube 20. Therefore, the fluid replacement unit 23 injects a replacement fluid consisting of an electrolyte-free isotonic solution into the fluid circuit, thereby suppressing the decrease in pressure and flow rate of the circulating fluid due to evaporation from the silicone membrane and the changes in chloride ion and protein concentrations due to concentration of the circulating fluid. This maintains the initial state of the circulating fluid, which is set to be equivalent to the blood environment in a living body. Furthermore, the carbon dioxide supply unit 24 supplies carbon dioxide gas from the outside through the silicone membrane, thereby maintaining the pH of the human blood.

[0025] Specifically, the fluid replacement unit 23 is disposed so as to be able to inject replacement fluid into the flow path 11 downstream of the reservoir tube 20, and includes a connector 26 connected to the flow path 11, and a low flow rate pump 27 connected to the connector 26 to supply replacement fluid to the connected portion. In this embodiment, a glucose solution is used as the replacement fluid, but the present invention is not limited to this, and various liquids can be used as the replacement fluid as long as they are isotonic solutions that do not contain electrolytes.

[0026] The connector 26 is composed of two three-way stopcocks connected in series within the flow path 11. A line from a low-flow pump 27 is connected to the downstream three-way stopcock, and by opening it as needed, it is possible to inject replenishment fluid into the circulating fluid. On the other hand, the upstream three-way stopcock is a sampling port connected to a sampling tube 29 for preventing contamination of the fluid circuit. By opening it as needed, it is possible to sample the circulating fluid upstream of the injection point for replenishment fluid. Therefore, the connector 26 and the tube 29 function as a sampling part that allows a portion of the circulating fluid to be extracted to the outside while keeping the circulating fluid in the fluid circuit clean.

[0027] The low flow rate pump 27 is a syringe pump that operates to continuously supply replenishment fluid into the flow path 11 at a predetermined flow rate, and is designed to suppress the pressure and flow rate reduction caused by water loss in the circulating fluid within the flow path 11, as well as the increase in chloride ion and protein concentrations in the circulating fluid, thereby maintaining them within human standard values.

[0028] The carbon dioxide supply unit 24 is composed of a storage box 31 that stores the reservoir tube 20 in a sealed state, and a gas supply unit 32 that supplies carbon dioxide gas to the internal space of the storage box 31. In the carbon dioxide supply unit 24, the carbon dioxide gas from the gas supply unit 32 permeates the silicone membrane of the reservoir tube 20 and is supplied into the flow path 11, thereby making it possible to adjust the pH of the circulating fluid in the flow path 11.

[0029] The in vitro testing system 10 according to the above embodiment can simulate the blood environment of the human body with respect to mechanical and chemical factors that affect the decomposition performance of a bioabsorbable test object S for treating cerebral aneurysms. That is, the retention section 12, the pulsatile pump 13, and the pressure and flow rate adjusting section 14 can simulate mechanical factors, and the circulating fluid maintaining section 17 can simulate chemical factors. This makes it possible to grasp the changes in the external shape of the test object S over time, which are visible in the retention section 12, and the changes in the properties of the test object S after a predetermined time, just as if the test object S had been used inside the human body, thereby contributing to the evaluation of the decomposition performance of the test object S over time.

[0030] Although not particularly limited, when a bioabsorbable flow diverter stent is used as the test subject S, in one embodiment, the in vitro testing system 10 is designed and configured in the following manner.

[0031] In this embodiment, bovine serum is used as the circulating fluid. The flow path 11 is formed of a polyvinyl chloride tube, with an inner diameter of 7 mm and a length of 220 mm between the compliance tube 19 and the indwelling portion 12, an inner diameter of 7 mm and a length of 100 mm between the indwelling portion 12 and the reservoir tube 20, and an inner diameter of 7 mm and a length of 50 mm between the reservoir tube 20 and the connector 26. The pulsatile pump 13 has a pump capacity of 15 mL. The compliance tube 19 is made of polyurethane and has an inner diameter of 15 mm and a length of 180 mm. The reservoir tube 20 is made of silicone and has an outer diameter of 20 mm, a thickness of 1.5 mm, and a length of 210 mm. The pulsatile pump 13 and pressure / flow rate adjuster 14 adjust the blood flow at the inlet side of the indwelling portion 12 to a maximum flow rate of approximately 400 mL / min, an average flow rate of approximately 275 mL / min, and a minimum flow rate of approximately 130 mL / min. The blood pressure is set to a maximum pressure of approximately 120 mmHg and a minimum pressure of approximately 50 mmHg. A 5% glucose solution is continuously injected into the flow path 11 from the low-flow pump 27 at a flow rate of 0.25 mL / h. Carbon dioxide gas with a partial pressure of 10% is continuously injected into the storage box 31 at a flow rate of 0.3 L / min throughout the test. The in vitro testing system 10 is placed in an insulated box including a heater (not shown), and the temperature environment is maintained at approximately human body temperature, i.e., between 35°C and 37°C. With these settings, the chloride ion concentration of the circulating fluid is maintained within a range of 96 mmol / L to 107 mmol / L. The protein concentration of the circulating fluid is maintained within a range of 55 g / L to 88 g / L, preferably between 60 g / L and 83 g / L. Furthermore, the pH of the circulating fluid is maintained within a range of 7.3 to 7.45.

[0032] 2 and 3, it is preferable to provide a stopper mechanism 35 for restricting movement of the test subject S downstream in the flow direction of the circulating fluid (arrows in FIGS. 1 and 2) in the in-vitro testing system 10 having the above configuration. This stopper mechanism 35 is structured to restrict the test subject S from being carried downstream by the flow of the circulating fluid due to a decrease in the expansive force of the test subject S in the in-dwelling portion 12 as the test subject S decomposes over time.

[0033] The stopper mechanism 35 is provided up to the resistance applying section 21 (see FIG. 1) that applies peripheral resistance, and is composed of a downstream portion of the retention section 12 and a connector 36 connected to the downstream portion. The retention section 12 here includes a retention flow path 12A that connects to the flow path 11 (see FIG. 1) and serves as an area where the test subject S is retained, and an outlet flow path 12B that connects to the downstream side of the retention flow path 12A. The inner diameter of the outlet flow path 12B is designed to be larger than that of the retention flow path 12A, as shown in FIG. 3.

[0034] The connector 36 comprises a tip portion 36A that is disposed inside the retention flow path 12A when connected to the retention section 12, an intermediate portion 36B that is connected to the tip portion 36 and is disposed inside the outlet-side flow path 12B when connected, and a stepped rear end portion 36C that is connected to the intermediate portion 36B and is disposed outside the retention section 12 when connected. An internal connector flow path 36D that is connected to the retention flow path 12A and the flow path 11 (see FIG. 1 ) when connected to the retention section 12 is formed inside the tip portion 36A, the intermediate portion 36B, and the rear end portion 36C, and that gradually expands in diameter according to the outer diameter of each portion.

[0035] The intermediate section 36B has an outer diameter approximately equal to the inner diameter of the outlet-side flow path 12B, and is approximately equal in length to the outlet-side flow path 12B, and is designed to be accommodated almost snugly within the outlet-side flow path 12B when the detention section 12 and the connector 36 shown in Fig. 2 are connected. Therefore, when connected, the boundary wall 36E, which forms the boundary between the tip section 36A and the intermediate section 36B, comes into contact with the test object S as it moves downstream, and serves as a restricting portion that restricts further movement of the test object S.

[0036] Furthermore, although not shown, hole-like outer communication passages that communicate between the outside of the connector 36 and the internal connector flow path 36D in the intermediate portion 36B can be formed at multiple locations in the circumferential direction of the boundary wall 36E that serves as the restriction portion. The outer communication passages can make it difficult for a partial difference in concentration of the circulating fluid due to a process such as decomposition of the test subject S to occur when the retention portion 12 and the connector 36 are connected as shown in Figure 2, i.e., a difference in concentration between the circulating fluid in the retention flow path 12A located on the outer periphery of the tip portion 36A and the circulating fluid passing through the internal connector flow path 36D of the tip portion 36A.

[0037] Although the in vitro testing system 10 uses a bioabsorbable flow diverter stent as the test subject S, the present invention is not limited to this, and other medical materials or medical devices, such as bioabsorbable materials or devices, can also be used as the test subject S. In this case, not only can the blood circulation conditions of the cerebral artery be simulated, but also the blood circulation conditions of the coronary artery or other parts of the human body where the test subject S is placed can be simulated by changing the configuration, such as the shape, of the indwelling portion 12 and adjusting the pulsation pump 13 and pressure / flow rate adjusting portion 14.

[0038] Furthermore, the configuration of each part of the device in the present invention is not limited to the illustrated configuration example, and various modifications are possible as long as they provide substantially the same effect.

[0039] REFERENCE SIGNS LIST 10 In vitro test system 11 Flow path 12 Indwelling section 13 Pulsatile pump 17 Circulating fluid maintenance section 23 Fluid replacement section 24 Carbon dioxide supply section 26 Connector (sampling collection section) 29 Tube (sampling collection section) 35 Stopper mechanism 36 Connector 36D Flow path within connector 36E Boundary wall (restriction section) S Test object

Claims

1. An in vitro testing system that is composed of a fluid circuit that circulates a specified circulating fluid, simulating the blood circulation state of the human body, and that evaluates the performance of a test object, which is a medical material or device, outside the body by placing the test object in the middle of the fluid circuit, characterized by having a circulating fluid maintenance unit that maintains the chemical factors in the circulating fluid that affect the performance evaluation of the test object at a level equivalent to the blood environment in a living body.

2. An in vitro testing system according to claim 1, wherein said circulating fluid maintenance unit maintains the state of said circulating fluid while correcting the effect of water loss during circulation of said circulating fluid.

3. An in vitro testing system as described in claim 1 or 2, characterized in that the circulating fluid maintenance unit comprises a fluid replacement unit that maintains the chloride ion concentration and protein concentration of the circulating fluid at desired values ​​by injecting a replacement fluid consisting of an isotonic solution that does not contain electrolytes into the fluid circuit, and a carbon dioxide supply unit that maintains the pH of the circulating fluid at a desired value by supplying carbon dioxide into the fluid circuit.

4. The in vitro testing system according to claim 1 or 2, characterized in that the fluid circuit includes a circular flow path through which the circulating fluid circulates in one direction, a storage section for the test object provided midway along the flow path, a pulsating pump that generates a flow of the circulating fluid within the flow path, and a pressure and flow rate adjusting section that adjusts the pressure and flow rate of the circulating fluid within the flow path, and is configured so that the blood flow and blood pressure in cerebral arteries and coronary arteries can be simulated within the fluid circuit by setting the pulsating pump and the pressure and flow rate adjusting section.

5. The in vitro testing system according to claim 4, wherein said retention portion is translucent and is formed so that said test object can be observed from the outside.

6. An in vitro testing system according to claim 1 or 2, characterized in that it is provided with a sampling section that enables a portion of the circulating fluid in the fluid circuit to be extracted to the outside while keeping the circulating fluid in a clean state.

7. The in vitro testing system according to claim 4, further comprising a stopper mechanism provided downstream of said retention section for restricting movement of said test object.

8. The in vitro testing system described in claim 7, characterized in that the stopper mechanism includes a connector connected to the downstream portion of the retention section, the connector including an internal flow path in the connector that is connected to the flow path, and a regulating portion that is arranged outside the internal flow path in the connector and that abuts against the test subject when it moves, and an external connecting passage that connects the flow path and the internal flow path in the connector is formed in the regulating portion.

9. A testing method using the in vitro testing system according to claim 1, characterized in that the chloride ion concentration of the circulating fluid is maintained within a range of 96 mmol / L to 107 mmol / L.

10. A testing method using the in vitro testing system of claim 1, characterized in that the protein concentration of the circulating fluid is maintained within the range of 55 g / L to 88 g / L.

11. A testing method using the in vitro testing system according to claim 1, characterized in that the pH of the circulating fluid is maintained within the range of 7.3 to 7.

45.

12. The test method according to claim 9, 10 or 11, characterized in that the circulating fluid is bovine, porcine, canine, monkey or human serum.

13. The test method according to claim 9, 10 or 11, characterized in that the temperature of the in vitro test system is maintained within the range of 35°C to 39°C.

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