System and method for determining the viability of perfused tissue - Patents.com

Abstract

The present disclosure provides a perfusion system and method for continuous or intermittent perfusion to monitor and extend the viability of tissue for transplantation. Intermittent perfusion generates perfusion cycles that alternate between baseline and elevated oxygen gas partial pressures in the circulating perfusate. The system includes a perfusate source and an inflow tube in fluid communication with the perfusate source and a sample, configured to deliver perfusate to the sample. The system further includes an outflow tube in fluid communication with the tissue sample and configured to transport perfusate from the tissue sample. A first gas sensor is in fluid communication with the outflow tube and measures the concentration of gas in the perfusate in the inflow tube, while a second gas sensor is in contact with the tissue sample and measures the concentration of gas in the sample.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is based on and claims priority to U.S. Patent Application No. 63 / 377,519 (filed September 28, 2022), the entire contents of which are incorporated herein by reference.

[0002] <Statement Regarding Federally Sponsored Research> This invention was made with government support from the Air Force Office of Scientific Research (Grant No. FA9550-20-1-0063), the National Science Foundation (Grant No. NSF1941543), and the Department of Defense (Grant No. W81XWH1910440). The government has certain rights in this invention. [Background technology]

[0003] In recent years, perfusion machine technology has experienced a remarkable resurgence, with ex vivo perfusion beginning to be routinely used as an alternative to sustained preservation of solid organs such as kidneys, livers, and hearts [References 1-4]. In the United States, normothermic machine perfusion has become standard for expanded donor livers, allowing for organ quality assessment before transplantation [Reference 5]. Hypothermic machine perfusion is also gradually replacing sustained hypothermic preservation for kidney preservation before transplantation [Reference 6]. Meanwhile, complex reconstructive procedures, such as vascularized composite allotransplantation (VCA), have attracted increasing attention from the transplant community [References 7-9]. While recent results of VCA have been positive from the functional, cosmetic, and social perspectives, long-term outcomes remain unsatisfactory, primarily due to chronic rejection [References 10, 11]. The muscle components of such allografts are prone to ischemic symptoms, whereas the skin and bone marrow components present challenges in eliciting immune responses. One important direction for mitigating VCA immune rejection is to reduce the incidence of ischemia-reperfusion injury [12,13]. The use of machine perfusion is particularly valuable for preserving delicate allografts, such as those found in solid organs

[14] .

[0004] Monitoring of perfused organs typically involves biochemical measurements, vascular pressure, and weight, which reflects edema [15,16]. However, this monitoring is not continuous, and worsening biochemical outcomes (e.g., increased lactate and potassium levels or decreased partial pressure of oxygen (pO2)) may indicate cellular distress and suboptimal preservation

[14] . Tissue oxygenation is an important parameter for monitoring ex vivo perfused organs. Inadequate oxygenation may be considered a partial failure of perfusion, and uncontrolled overexposure to oxygen may cause damage due to reactive oxygen species

[14] .

[0005] Thus, there is a need for improved systems and methods for determining the viability and quality of a tissue sample to be perfused. Summary of the Invention

[0006] The present disclosure provides a system and method that overcomes the above-mentioned drawbacks by providing a system and method for tissue sample perfusion with gas inflow and a sample sensor for continuous monitoring of perfusion.

[0007] In one aspect, the present disclosure provides a perfusion system for a tissue sample, the perfusion system including: a perfusion fluid source; an inlet tube in fluid communication with the perfusion fluid source and the sample and configured to deliver perfusion fluid to the sample; an outlet tube in fluid communication with the sample and configured to carry perfusion fluid away from the sample; a first gas sensor in fluid communication with the outlet tube and configured to measure a concentration of gas in the perfusion fluid in the inlet tube; and a second gas sensor in contact with the sample and configured to measure a concentration of gas in the sample.

[0008] In one embodiment of the perfusion system, the second gas sensor comprises a photoluminescent oxygen sensing probe in contact with the sample, a photon source for transmitting photons at the photoluminescent oxygen sensing probe, a light receiving element configured to detect light emitted from the photoluminescent oxygen sensing probe when the photon source transmits photons at the photoluminescent oxygen sensing probe, and a controller in electrical communication with the photon source and the light receiving element, the controller configured to execute a program stored in the controller to calculate the concentration of oxygen adjacent to the photoluminescent oxygen sensing probe from an electrical signal received from the light receiving element. In one embodiment of the perfusion system, the photoluminescent oxygen sensing probe is a compound having an emission that provides tissue oxygen partial pressure (pO2). In one embodiment of the perfusion system, the photoluminescent oxygen sensing probe comprises a polymer material impregnated with a porphyrin. In one embodiment of the perfusion system, the photoluminescent oxygen sensing probe comprises a polymer impregnated with a phosphorescent meso-unsubstituted porphyrin.

[0009] In one embodiment of the perfusion system, the second gas sensor comprises an oxygen sensing probe configured to be inserted into the sample, and a controller in electrical communication with the oxygen sensing probe, the controller configured to execute a program stored in the controller to calculate the concentration of oxygen adjacent to the oxygen sensing probe from an electrical signal received from the oxygen sensing probe. In one embodiment of the perfusion system, the second gas sensor provides tissue oxygen partial pressure (pO2).

[0010] In one embodiment, the perfusion system further comprises a controller in electrical communication with the first gas sensor and the second gas sensor, the controller configured to execute a program stored in the controller to receive electrical signals from the first gas sensor and the second gas sensor and calculate a concentration of oxygen adjacent to the first gas sensor and / or the second gas sensor. In one embodiment of the perfusion system, the first gas sensor comprises a material that undergoes a change in response to oxygen partial pressure (pO2), a sensor head in contact with the material and detecting the change in the material, and a controller in electrical communication with the sensor head that executes a program stored in the controller to calculate the oxygen partial pressure adjacent to the material.

[0011] In one embodiment, the perfusion system further comprises a transparent membrane forming an outer layer of the material, a layer of polymeric material having metalloporphyrins embedded therein adjacent to the transparent membrane, and a scattering layer in contact with the layer of polymeric material and the surface of the sample. In one embodiment of the perfusion system, the first gas sensor comprises a flow cell containing the material. In one embodiment of the perfusion system, the change in the material comprises a change in phosphorescence. In one embodiment of the perfusion system, the sensor head comprises a plurality of light-emitting diodes (LEDs) and a photodiode. In one embodiment of the perfusion system, the sensor head further comprises a temperature sensor.

[0012] In one embodiment of the perfusion system, the second gas sensor comprises a material that undergoes a change in response to oxygen pressure, a sensor head in contact with the material and detecting the change in the material, and a controller in electrical communication with the sensor head, the controller executing a program stored in the controller to calculate the oxygen partial pressure adjacent to the material. In one embodiment of the perfusion system, in the second oxygen sensor, the material is disposed on an outer surface of the sample. In one embodiment of the perfusion system, the change in the material comprises a change in phosphorescence intensity or phosphorescence lifetime. In one embodiment of the perfusion system, the sensor head comprises a plurality of light-emitting diodes (LEDs) and a photodiode. In one embodiment of the perfusion system, the sensor head further comprises a temperature sensor.

[0013] In one embodiment, the perfusion system further includes a third gas sensor in fluid communication with the outflow tube, the third gas sensor measuring the concentration of gas in the perfusate in the outflow tube. In one embodiment of the perfusion system, the third gas sensor includes a material that undergoes a change in response to oxygen partial pressure (pO2), a sensor head in contact with the material and detecting the change in the material, and a controller in electrical communication with the sensor head that executes a program stored in the controller to calculate the oxygen partial pressure adjacent to the material.

[0014] In one embodiment, the perfusion system further comprises a transparent membrane forming an outer layer of the material, a layer of polymeric material having metalloporphyrins embedded therein adjacent to the transparent membrane, and a scattering layer in contact with the layer of polymeric material and the surface of the sample. In one embodiment of the perfusion system, the third gas sensor comprises a flow cell containing the material. In one embodiment of the perfusion system, the change in the material comprises a change in phosphorescence intensity or phosphorescence lifetime. In one embodiment of the perfusion system, the sensor head comprises a plurality of light-emitting diodes (LEDs) and a photodiode. In one embodiment of the perfusion system, the sensor head further comprises a temperature sensor.

[0015] In one embodiment, the perfusion system further comprises an oxygenator in fluid communication with the perfusion fluid source, and a pump for circulating the perfusion fluid through the oxygenator, the inlet line, and the outlet line, the oxygenator controlling the oxygen level in the perfusion fluid flowing toward the sample.

[0016] In one embodiment, the perfusion system further comprises a heat exchanger for regulating the temperature of the perfusion solution.

[0017] In one embodiment, the perfusion system further comprises a controller in electrical communication with the oxygenator, the first gas sensor, and the second gas sensor, the controller configured to execute a program stored in the controller to control the oxygen level in the perfusion fluid flowing toward the sample based on electrical signals received from the first gas sensor and the second gas sensor.

[0018] In one embodiment, the perfusion system further comprises a controller in electrical communication with the pump, the first gas sensor, and the second gas sensor, the controller configured to execute a program stored in the controller to control the oxygen level in the perfusion fluid flowing toward the sample based on electrical signals received from the first gas sensor and the second gas sensor.

[0019] In one embodiment, the perfusion system further comprises a controller in electrical communication with the first gas sensor and the second gas sensor, the controller configured to execute a program stored in the controller to receive electrical signals from the first gas sensor and the second gas sensor and to initiate delivery of the perfusion fluid when a loss of viability is calculated based on a first concentration of a first gas adjacent to the first gas sensor and a second concentration of a second gas adjacent to the second gas sensor.

[0020] In one embodiment, the perfusion system further includes a controller in electrical communication with the first gas sensor and the second gas sensor, the controller configured to execute a program stored in the controller to receive electrical signals from the first gas sensor and the second gas sensor, and to deactivate delivery of the perfusion fluid if a viability calculated based on a first concentration of a first gas adjacent to the first gas sensor and a second concentration of a second gas adjacent to the second gas sensor exceeds a viability threshold.

[0021] In one embodiment, the perfusion system further comprises a controller in electrical communication with the first gas sensor and the second gas sensor, the controller configured to execute a program stored in the controller to receive electrical signals from the first gas sensor and the second gas sensor and to intermittently activate or deactivate delivery of the perfusion fluid based on a first concentration of a first gas adjacent to the first gas sensor and a second concentration of a second gas adjacent to the second gas sensor.

[0022] In one embodiment, the perfusion system further comprises a controller in electrical communication with the first gas sensor and the second gas sensor, the controller configured to execute a program stored therein to initiate an alarm when a gas level drops below a predetermined threshold based on the electrical signals received from the first gas sensor and the second gas sensor.

[0023] In one embodiment of the perfusion system, the perfusion solution is a cell-free perfusion solution.

[0024] In one embodiment of the perfusion system, the first gas sensor provides a continuous circulating oxygen value delivered to the sample of a perfused subject. In one embodiment of the perfusion system, the second gas sensor provides a continuous tissue oxygenation value of the sample of a perfused subject.

[0025] In another aspect, the present disclosure provides a method for machine perfusion of a tissue sample, the method including: (a) providing a tissue sample; (b) delivering perfusion fluid to the sample via an inflow conduit; (c) measuring a concentration of gas in the perfusion fluid in the inflow conduit; and (d) measuring the concentration of gas in the sample. The method may further include (e) measuring the concentration of gas in the perfusion fluid in an outflow conduit configured to carry perfusion fluid away from the sample.

[0026] In one embodiment of the method, step (c) includes measuring the concentration of the gas in the perfusion fluid in the inflow tubing using a first gas sensor, step (d) includes measuring the concentration of the gas in the sample using a second gas sensor, and step (e) includes measuring the concentration of the gas in the perfusion fluid in the outflow tubing using a third gas sensor, wherein at least one of the first gas sensor, the second gas sensor, and the third gas sensor comprises an oxygen sensing probe.

[0027] In one embodiment of the method, step (c) includes measuring the concentration of the gas in the perfusate in the inflow tubing using a first gas sensor, step (d) includes measuring the concentration of the gas in the sample using a second gas sensor, and step (e) includes measuring the concentration of the gas in the perfusate in the outflow tubing using a third gas sensor, wherein at least one of the first gas sensor, the second gas sensor, and the third gas sensor comprises a material that undergoes a change in response to oxygen partial pressure (pO2).

[0028] In one embodiment of the method, step (c) includes detecting the concentration of the gas in the perfusion fluid in the inflow tube using the first gas sensor and calculating, in the controller, the concentration of oxygen adjacent to the first gas sensor from an electrical signal sent from the first gas sensor to the controller; step (d) includes measuring the concentration of the gas in the sample using the second gas sensor and calculating, in the controller, the concentration of oxygen adjacent to the second gas sensor from an electrical signal sent from the second gas sensor to the controller; and step (e) includes detecting the concentration of the gas in the perfusion fluid in the outflow tube using the third gas sensor and calculating, in the controller, the concentration of oxygen adjacent to the third gas sensor from an electrical signal sent from the third gas sensor to the controller.

[0029] In one embodiment, the method further includes monitoring changes in the concentration from the first gas sensor and at least one of the second gas sensor and the third gas sensor, measuring variations in the oxygen concentration over a period of time, analyzing the variations in the oxygen concentration over the period of time, and estimating viability and perfusion quality of the tissue sample.

[0030] In one embodiment, the method further includes (e) generating a report of oxygen perfusion in the sample using the controller. In one embodiment, the method further includes (e) controlling the oxygen level in the perfusate flowing toward the sample based on the electrical signals received from the first gas sensor and the second gas sensor.

[0031] In one embodiment, the method further includes (e) activating an alarm if the gas level drops below a specified threshold based on the electrical signals received from the first gas sensor and the second gas sensor.

[0032] In one embodiment of this method, the tissue sample is a vascularized composite tissue allograft. In one embodiment of this method, the vascularized composite tissue allograft is at least a portion of a limb, face, pharynx, trachea, abdominal wall, genitourinary system tissue, uterine tissue, or solid organ, or any combination thereof. In one embodiment of this method, the tissue sample is a donor vascularized composite tissue allograft for vascularized composite tissue allotransplantation. In one embodiment of this method, the tissue sample is obtained from a human, a primate, or a pig. In one embodiment of this method, the tissue sample is a fasciocutaneous flap.

[0033] In another aspect, the present disclosure provides a perfusion system for a tissue sample, the perfusion system including a perfusion fluid source, an inlet conduit in fluid communication with the perfusion fluid source and the sample and configured to deliver perfusion fluid to the sample, an outlet conduit in fluid communication with the sample and configured to remove perfusion fluid from the sample, a first gas sensor in fluid communication with the outlet conduit and configured to measure a concentration of a first gas in the perfusion fluid in the inlet conduit, a second gas sensor in contact with the sample and configured to measure a concentration of a second gas in the sample, and a controller in electrical communication with the first gas sensor and the second gas sensor. and a controller configured to execute a program stored in the controller to (i) receive electrical signals from the first gas sensor and the second gas sensor and calculate a concentration of the first gas adjacent to the first gas sensor and a concentration of the second gas adjacent to the second gas sensor, and (ii) activate or deactivate delivery of the perfusion fluid toward the sample and / or adjust perfusion parameters based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor.

[0034] In one embodiment of the perfusion system, the controller is configured to execute the program stored in the controller to initiate delivery of the perfusion fluid when a loss of viability is calculated based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor.

[0035] In one embodiment of the perfusion system, the controller is configured to execute the program stored in the controller to deactivate delivery of the perfusion fluid if a viability calculated based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor exceeds a viability threshold.

[0036] In one embodiment of the perfusion system, the controller is configured to execute the program stored in the controller to intermittently activate or deactivate delivery of the perfusion fluid based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor.

[0037] In one embodiment, the perfusion system further includes a pump in electrical communication with the controller, the controller configured to execute the program stored in the controller to adjust the flow rate of perfusion fluid flowing toward the sample based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor.

[0038] In one embodiment, the perfusion system further includes an oxygenator in electrical communication with the controller, the controller configured to execute the program stored in the controller to control the oxygen level in the perfusion fluid flowing toward the sample based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor.

[0039] In one embodiment, the perfusion system further includes a third gas sensor in fluid communication with the outflow tube, the third gas sensor measuring a concentration of a third gas in the perfusion fluid in the outflow tube, and the controller is configured to execute a program stored in the controller to activate or deactivate delivery of the perfusion fluid flowing toward the sample and / or adjust perfusion parameters based on the first concentration of the first gas adjacent to the first gas sensor, the second concentration of the second gas adjacent to the second gas sensor, and the third concentration of the third gas adjacent to the third gas sensor.

[0040] In one embodiment of the perfusion system, the controller is configured to execute the program stored in the controller to initiate delivery of the perfusion fluid when a loss of viability is calculated based on the first concentration of the first gas adjacent to the first gas sensor, the second concentration of the second gas adjacent to the second gas sensor, and the third concentration of the third gas adjacent to the third gas sensor.

[0041] In one embodiment of the perfusion system, the controller is configured to execute the program stored in the controller to deactivate delivery of the perfusion fluid if a viability calculated based on the first concentration of the first gas adjacent to the first gas sensor, the second concentration of the second gas adjacent to the second gas sensor, and the third concentration of the third gas adjacent to the third gas sensor exceeds a viability threshold.

[0042] In one embodiment of the perfusion system, the controller is configured to execute the program stored in the controller to intermittently activate or deactivate delivery of the perfusion fluid based on the first concentration of the first gas adjacent to the first gas sensor, the second concentration of the second gas adjacent to the second gas sensor, and the third concentration of the third gas adjacent to the third gas sensor.

[0043] In one embodiment, the perfusion system further includes a pump in electrical communication with the controller, the controller configured to execute the program stored in the controller to adjust the flow rate of perfusion fluid flowing toward the sample based on the first concentration of the first gas adjacent to the first gas sensor, the second concentration of the second gas adjacent to the second gas sensor, and the third concentration of the third gas adjacent to the third gas sensor.

[0044] In one embodiment, the perfusion system further comprises an oxygenator in electrical communication with the controller, the controller configured to execute the program stored in the controller to control the oxygen level in the perfusion fluid flowing toward the sample based on the electrical signals received from the first gas sensor and the second gas sensor and the third concentration of the third gas adjacent to the third gas sensor.

[0045] In one embodiment of the perfusion system, the controller is in electrical communication with a controllable valve in the inlet line, and the controller is configured to execute a program stored in the controller to activate or deactivate delivery of the perfusion fluid by moving the controllable valve to an open position in which the perfusion fluid is delivered to the sample, or to a closed position in which the perfusion fluid is not delivered to the sample.

[0046] In one embodiment of the perfusion system, the controller is in electrical communication with a controllable valve provided in the inlet tube, and the controller is configured to execute a program stored in the controller to control the delivery of the perfusion fluid by moving the controllable valve to a fully open position in which a first amount of the perfusion fluid is delivered to the sample, an intermediate position in which a second amount of the perfusion fluid less than the first amount of the perfusion fluid is delivered to the sample, or a closed position in which no perfusion fluid is delivered to the sample.

[0047] In another aspect, the present disclosure provides a method for machine perfusion of a tissue sample, the method comprising: (a) preparing a tissue sample, (b) delivering perfusion fluid to the sample via an inflow conduit, (c) measuring a first concentration of a first gas in the perfusion fluid in the inflow conduit, (d) measuring a second concentration of a second gas in the sample, and (e) activating or deactivating delivery of the perfusion fluid to the sample and / or adjusting perfusion parameters based on the first concentration of the first gas and the second concentration of the second gas.

[0048] In one embodiment of the method, step (e) includes activating delivery of the perfusion fluid if a loss of viability is calculated based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor.

[0049] In one embodiment of the method, step (e) includes deactivating delivery of the perfusion fluid if the viability calculated based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor exceeds a viability threshold.

[0050] In one embodiment of the method, step (e) includes intermittently activating or deactivating delivery of the perfusion fluid based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor.

[0051] In one embodiment of the method, step (e) includes adjusting the flow rate of perfusion fluid flowing toward the sample based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor.

[0052] In one embodiment of the method, step (e) includes controlling the oxygen level in the perfusion fluid flowing toward the sample based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor.

[0053] In another aspect, the present disclosure provides a perfusion system for a tissue sample, the system including a perfusion fluid source, an inlet tube in fluid communication with the perfusion fluid source and the sample and configured to deliver perfusion fluid to the sample, an outlet tube in fluid communication with the sample and configured to carry perfusion fluid away from the sample, a first gas sensor in fluid communication with the outlet tube and configured to measure a concentration of a first gas in the perfusion fluid in the inlet tube, a second gas sensor in fluid communication with the outlet tube and configured to measure a concentration of a second gas in the perfusion fluid in the outlet tube, and a computer in electrical communication with the first gas sensor and the second gas sensor. and a controller configured to execute a program stored therein to (i) receive electrical signals from the first gas sensor and the second gas sensor and calculate a concentration of the first gas adjacent to the first gas sensor and a concentration of the second gas adjacent to the second gas sensor, and (ii) activate or deactivate delivery of the perfusion fluid toward the sample and / or adjust perfusion parameters based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor.

[0054] In one embodiment of the perfusion system, the controller is configured to execute the program stored in the controller to initiate delivery of the perfusion fluid when a loss of viability is calculated based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor.

[0055] In one embodiment of the perfusion system, the controller is configured to execute the program stored in the controller to deactivate delivery of the perfusion fluid if a viability calculated based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor exceeds a viability threshold.

[0056] In one embodiment of the perfusion system, the controller is configured to execute the program stored in the controller to intermittently activate or deactivate delivery of the perfusion fluid based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor.

[0057] In one embodiment, the perfusion system further includes a pump in electrical communication with the controller, the controller configured to execute the program stored in the controller to adjust the flow rate of perfusion fluid flowing toward the sample based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor.

[0058] In one embodiment, the perfusion system further comprises an oxygenator in electrical communication with the controller, the controller configured to execute the program stored in the controller to control the oxygen level in the perfusion fluid flowing toward the sample based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor.

[0059] In one embodiment, the perfusion system further comprises a third gas sensor in contact with the sample, the third gas sensor measuring a concentration of a third gas in the sample, and the controller is configured to execute a program stored in the controller to activate or deactivate delivery of the perfusion fluid toward the sample and / or adjust perfusion parameters based on the first concentration of the first gas adjacent to the first gas sensor, the second concentration of the second gas adjacent to the second gas sensor, and the third concentration of the third gas adjacent to the third gas sensor.

[0060] In one embodiment of the perfusion system, the controller is configured to execute the program stored in the controller to initiate delivery of the perfusion fluid when a loss of viability is calculated based on the first concentration of the first gas adjacent to the first gas sensor, the second concentration of the second gas adjacent to the second gas sensor, and the third concentration of the third gas adjacent to the third gas sensor.

[0061] In one embodiment of the perfusion system, the controller is configured to execute the program stored in the controller to deactivate delivery of the perfusion fluid if a viability calculated based on the first concentration of the first gas adjacent to the first gas sensor, the second concentration of the second gas adjacent to the second gas sensor, and the third concentration of the third gas adjacent to the third gas sensor exceeds a viability threshold.

[0062] In one embodiment of the perfusion system, the controller is configured to execute the program stored in the controller to intermittently activate or deactivate delivery of the perfusion fluid based on the first concentration of the first gas adjacent to the first gas sensor, the second concentration of the second gas adjacent to the second gas sensor, and the third concentration of the third gas adjacent to the third gas sensor.

[0063] In one embodiment, the perfusion system further includes a pump in electrical communication with the controller, the controller configured to execute the program stored in the controller to adjust the flow rate of perfusion fluid flowing toward the sample based on the first concentration of the first gas adjacent to the first gas sensor, the second concentration of the second gas adjacent to the second gas sensor, and the third concentration of the third gas adjacent to the third gas sensor.

[0064] In one embodiment, the perfusion system further comprises an oxygenator in electrical communication with the controller, the controller configured to execute the program stored in the controller to control the oxygen level in the perfusion fluid flowing toward the sample based on the electrical signals received from the first gas sensor and the second gas sensor and the third concentration of the third gas adjacent to the third gas sensor.

[0065] In one embodiment of the perfusion system, the controller is in electrical communication with a controllable valve in the inlet line, and the controller is configured to execute a program stored in the controller to activate or deactivate delivery of the perfusion fluid by moving the controllable valve to an open position in which the perfusion fluid is delivered to the sample or a closed position in which the perfusion fluid is not delivered to the sample. In one embodiment of the perfusion system, the controller is in electrical communication with a controllable valve in the inlet line, and the controller is configured to execute a program stored in the controller to control delivery of the perfusion fluid by moving the controllable valve to a fully open position in which a first amount of the perfusion fluid is delivered to the sample, an intermediate position in which a second amount of the perfusion fluid less than the first amount of the perfusion fluid is delivered to the sample, or a closed position in which the perfusion fluid is not delivered to the sample.

[0066] In another aspect, the present disclosure provides a method for machine perfusion of a tissue sample, the method including: (a) preparing a tissue sample, (b) delivering perfusion fluid to the sample via an inflow conduit, (c) measuring a first concentration of a first gas in the perfusion fluid in the inflow conduit, (d) measuring a second concentration of a second gas in an outflow conduit, and (e) activating or deactivating delivery of the perfusion fluid to the sample and / or adjusting perfusion parameters based on the first concentration of the first gas and the second concentration of the second gas.

[0067] In one embodiment of the method, step (e) includes activating delivery of the perfusion fluid if a loss of viability is calculated based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor.

[0068] In one embodiment of the method, step (e) includes deactivating delivery of the perfusion fluid if the viability calculated based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor exceeds a viability threshold.

[0069] In one embodiment of the method, step (e) includes intermittently activating or deactivating delivery of the perfusion fluid based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor.

[0070] In one embodiment of the method, step (e) includes adjusting the flow rate of perfusion fluid flowing toward the sample based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor.

[0071] In one embodiment of the method, step (e) includes controlling the oxygen level in the perfusion fluid flowing toward the sample based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor.

[0072] The above aspects are not intended to limit the present invention, and other aspects and configurations of the above-described systems and methods are presented below.

[0073] The above-described configuration of the embodiment will be more easily understood by referring to the following detailed description with reference to the accompanying drawings. [Brief explanation of the drawings]

[0074] [Figure 1] 1 is a schematic diagram illustrating multiple aspects of the perfusion system of the present disclosure with a fascia flap of an oxygen monitoring device. [Figure 2A-2B] Figure 2A is a side view of the film of the perfusion system of Figure 1 positioned over a source of perfusion fluid, and Figure 2B is a side view of the film of the perfusion system of Figure 1 positioned over a tissue sample. [Figure 3] FIG. 2 is a perspective view of a sensor head of the perfusion system of FIG. 1. [Figure 4] 1 is a flowchart of a method for machine perfusion of a tissue sample in accordance with aspects of the present disclosure. [Figure 5] This figure shows the monitoring of transcutaneous oxygen (TcPO2, orange curve, right Y-axis) and circulating inflow oxygen (PO2, blue curve, left Y-axis) during extended intermittent perfusion of a fasciocutaneous flap. The inflow values ​​were compared with analysis and intermittent sampling of gases in the perfusate. The insert shows the time delay between the rise in oxygen in the perfusate and the rise in oxygen in the skin. [Figure 6] This figure plots the measurement results of a three-device system providing transcutaneous oxygen values ​​(orange curve, right Y-axis) and circulating inflow oxygen values ​​(dark blue, left Y-axis) and outflow oxygen values ​​(light blue, left Y-axis). [Figure 7] Figure 10 plots the statistically significant correlation between continuous PO2 values ​​provided by a flow cell device and a Siemens Rapidpoint® 500 blood gas analyzer, with the 95% confidence interval indicated by the dashed line. [Figures 8A-8D] Figure 8A shows the maximum P02 and TcP02 values ​​during ex vivo perfusion of the fascial flap. Figure 8B shows the minimum P02 and TcP02 values ​​during ex vivo perfusion of the fascial flap. Figure 8C shows the change in P02 and TcP02 values ​​(Δ=Max-Min) during ex vivo perfusion of the fascial flap. Figure 8D shows the delay between inflow P02 in the circulation and skin TcP02, which shows a similar trend of deviation between inflow and skin values. Vascular resistance is also shown, which shows a strong correlation with the delay. DETAILED DESCRIPTION OF THE INVENTION

[0075] In the following description, like parts in each drawing are given like reference numerals.

[0076] Described herein is a method for assessing the viability of perfused tissue, which involves continuous measurement of oxygen partial pressure (pO2) at multiple locations, including, but not limited to, oxygen-rich (inflow) and oxygen-depleted (outflow) perfusates, tissue depth, and the surface of the perfused tissue. In one embodiment, the following is used: (a) a tissue perfusion system that circulates oxygenated perfusate within the tissue via vascular connections and drains; (b) an oxygen-sensing flow cell within or part of the tubing of the system and a needle, microneedle, or skin patch type material that is placed within or on the tissue to be perfused, respectively; (c) a programmable electronic readout sensor of the oxygen-sensing material for signal collection; (d) an algorithm loaded onto a computer or mobile device that controls the reading device, which is capable of recording, processing, and reporting the signals output from the multiple devices in the form of pO2 values ​​in real time; (e) An algorithm loaded onto the computer or mobile device, the computer or mobile device controlling the perfusion system pumps and pressure sensors to adjust perfusion parameters in response to the oxygen measurement level.

[0077] To provide a thorough understanding of the structure, function, manufacture, and use of the devices and methods disclosed herein, specific example embodiments will be described. One or more examples of these embodiments are illustrated in the accompanying drawings. Those skilled in the art will appreciate that the devices and methods specifically described herein and illustrated in the accompanying drawings are exemplary embodiments, not limiting the scope of the invention, the scope of which is defined by the claims. Features shown or described with respect to one example embodiment may be combined with features of other embodiments. Such modifications and variations are intended to be within the scope of the present invention.

[0078] As used in this specification and claims, the singular forms "a," "an," and "the" include the plural forms as well, unless the context clearly dictates otherwise.

[0079] As used herein, the terms "about," "approximately," "substantially," and "significantly" will be clear to those of ordinary skill in the art and may vary depending on the context. When terms are used that are not clear to those of ordinary skill in the art in the context, "about" or "approximately" will mean a range of up to ±10% of the term, and "substantially" and "significantly" will mean a range of more than ±10% of the term.

[0080] Here, "include" and "including" are synonymous with "comprise" and "comprising," respectively, and the term "comprising" should be interpreted as an "open" term that allows for the inclusion of additional elements in addition to those recited in the claims. "Consist" and "consisting of" should be interpreted as a "closed" term that does not allow for the inclusion of additional elements other than those recited in the claims. "Consisting essentially of" is partially closed and should be interpreted as allowing for the inclusion of only additional elements that do not fundamentally change the nature of the claimed invention.

[0081] The phrase "such as" should be interpreted as "including, for example." Furthermore, the use of all exemplary language, including but not limited to "such as," is intended to facilitate understanding of the invention and does not limit the scope of the invention unless otherwise stated in the claims.

[0082] Furthermore, when idiomatic expressions such as "at least one of A, B, C, etc." are used, such structures are generally intended to be understood in the sense that one of ordinary skill in the art would understand them (e.g., "a system comprising at least one of A, B, and C" includes, but is not limited to, a system including only A, only B, only C, A and B, A and C, B and C, and / or A, B, and C). Those of ordinary skill in the art should understand that any disjunctive term and / or phrase indicating two or more alternative terms, whether in the specification or drawings, is intended to include in effect one of the terms, only one of the terms, or both terms. For example, the phrase "A or B" is understood to include the possibilities of "A" or "B" or "A and B."

[0083] All terms such as "up to," "at least," "greater than," "less than," etc., refer to ranges that are inclusive of the numerical values ​​recited therein and that can be subsequently broken down into multiple ranges or subranges. A range includes each individual member. Thus, for example, a group having 1 to 3 members means groups having 1, 2, or 3 members. Similarly, a group having 6 members means groups having 1, 2, 3, 4, or 6 members.

[0084] The modal verb "may" refers to the preferred use or selection of one or more options or alternatives from among multiple described embodiments or configurations contained within those embodiments. When no options or alternatives are disclosed with respect to a particular embodiment or configuration contained within a particular embodiment, the modal verb "may" refers to a positive action regarding how to produce or use a particular aspect of the described embodiment or configuration contained within that embodiment, or a definitive decision to exercise special abilities with respect to the described embodiment or configuration contained therein. In this latter context, the modal verb "may" is synonymous with and implies the auxiliary verb "can."

[0085] FIG. 1 illustrates an exemplary embodiment of a perfusion system 100 of the present disclosure. The perfusion system 100 includes a perfusion fluid source 102. In one non-limiting example, the perfusion fluid 102 is an acellular crystal solution. The system 100 includes a membrane oxygenator 104 capable of oxygenating the circulating perfusion fluid 102. The membrane oxygenator can receive an oxygen supply 150 via tubing 151. The system 100 further includes an inlet tube 106 in fluid communication with the perfusion fluid source 102 and a tissue sample 108 via an artery. In one non-limiting example, the tissue sample 108 is a fascia flap. In one non-limiting example, the inlet tube 106 is tubing. For example, the tubing can be, but is not limited to, silicone tubing or polyvinyl chloride (PVC) tubing. The system 100 further includes an outlet tube 110 in fluid communication with the sample, the outlet tube 110 configured to carry the perfusion fluid away from the sample. The outflow tube 110 can be made of the same material as the inflow tube 106. The perfusate is venously carried away from the tissue sample 108 through the outflow tube 110 after perfusion of the sample 108. The system 100 further includes a first gas sensor 112 in fluid communication with the inflow tube 106 and a second gas sensor 114 in contact with the sample 108. The first gas sensor 112 measures the concentration of gas in the perfusate 102, and the second gas sensor 114 measures the concentration of gas in the sample 108.

[0086] 1, the perfusion system 100 includes a third gas sensor 116. The third gas sensor 116 may be identical to the first gas sensor 112 described above. In one non-limiting example, the third gas sensor is in fluid communication with the outflow conduit 110 and is configured to measure the concentration of gas in the perfusate in the outflow conduit 110.

[0087] In one non-limiting example, the perfusion solution can be a composition of Steen+ solution (Department of Surgery, Center for Engineering in Medicine and Surgery, Harvard Medical School, Massachusetts General Hospital, Boston, MA 02114, USA). Steen+ solution is based on deionized water and contains: NaCl: 86 mmol / L KCl: 4.6 mmol / L CaCl2.2H20: 1.5 mmol / L NaH2PO4: 1.2 mmol / L NaHCO3: 16 mmol / L MgCl2.6H2O: 1.2 mmol / L D-glucose: 22 mmol / L Polyethylene glycol (PEG) 35Kda: 5g / L Bovine serum albumin: 15g / L Insulin: 200UI / L Hydrocortisone: 10 mg / L Heparin: 200UI / L Piperacillin-tazobactam: 2.25 g / L Vancomycin: 1.5 g / L

[0088] In one non-limiting example, the first gas sensor 112 and, optionally, the third gas sensor 116 each include a sensor head 118 and a flow cell 120 with a film 122. In one embodiment, the flow cell 120 of the first gas sensor communicates with the inlet tube 106, the flow cell 120 of the third gas sensor communicates with the outlet tube 110, and the film 122 disposed within the flow cell 120 is in contact with the perfusion fluid 102. In one non-limiting example, the flow cell can be incorporated into the perfusion system by cutting the inlet tube and inserting the flow cell into the tubing, thereby restoring tubing continuity. The flow cell is designed to have an inner diameter similar to that of a single silicone tube. The outlet sensor can be positioned similarly. In one non-limiting embodiment, the flow cell 120 has a first opening 124 through which the perfusion fluid 102 flows into the flow cell 120 and a second opening 126 through which the perfusion fluid 102 flows out of the flow cell 120.

[0089] The film 122 or 123 is configured to change in response to pO2. The film 122 or 123 can have a light-based, photoluminescent oxygen-sensing formulation and have emission properties capable of providing tissue pO2. The film 122 or 123 can include an oxygen-sensing polymer or a polymer containing oxygen-sensing molecules. As shown in FIG. 2A, the film 122 or 123 can include three layers: (i) a transparent membrane 202 forming the outer layer of the film; (ii) a polymer material layer 204 having embedded therein an oxygen-sensing lumiphore 206 adjacent to the transparent membrane 202; and (iii) a scattering layer 208 (e.g., a polymer film pigmented with white particles) in contact with the polymer material layer 204 and the irrigation fluid 102.

[0090] In one non-limiting example, the oxygen-sensing lumiphore 206 is a metalloporphyrin that can emit red phosphorescence when excited by blue light, and the intensity and lifetime of the phosphorescence can be inversely proportional to pO2. A reference sensor in the form of a green-emitting dye can also be incorporated into the sensor film 122 or 123 as a reference standard for accurate pO2 measurements.

[0091] In one non-limiting example, the polymer can be, but is not limited to, poly(propyl methacrylate) (PMMA) or polydimethylsiloxane (PDMS). For example, silicones such as PDMS have extremely high gas permeability, which can allow for rapid readings of tissue oxygen dynamics.

[0092] In some embodiments, porphyrin-based oxygen sensing molecules 206 embedded in polymeric material 204 are designed to provide exceptional sensitivity and accuracy for measuring tissue oxygenation. Porphyrin-based oxygen sensing molecules 206 can be formed through a modular synthetic route that allows for tuning both the oxygen sensitivity range of the oxygen sensing molecule and its compatibility with the matrix material in which it is embedded. The matrix material can be further configured to tune the oxygen sensitivity range of the oxygen sensing molecule. In one embodiment, the change in film 122 or 123 includes a change in phosphorescence. For example, oxygen sensing molecules 206 can be specifically designed to feature bright red phosphorescent emissions, providing a visual response to changes in oxygenation levels that are visible under ambient light. These properties simplify the collection and interpretation of oxygen-dependent emissions, allowing analysis to be performed using simple and inexpensive equipment.

[0093] In one embodiment, the oxygen sensing lumiphore 206 comprises a phosphorescent meso-unsubstituted porphyrin having the chemical formula (I): [ka] wherein M is a metal, each R is independently an atom or group of atoms, and at least one R is -OR 1 and R 1 is an atom or group of atoms.

[0094] In the porphyrin of formula (I), R 1can be selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkylcarbonyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, heteroaryl, halo, cyano, and nitro. In one example of a porphyrin of formula (I), R 1 is hydrogen. In another example of a porphyrin of formula (I), R 1 is alkynyl, for example 2-propynyl (propargyl). In yet another example of a porphyrin of formula (I), R 1 is alkylcarbonyl, such as 2,2-dimethylpropanoyl (also called trimethylacetyl or pivaloyl). In the porphyrin of formula (I), multiple R's are -OR 1 and optionally, all R's can be -OR'd 1 It can be said that:

[0095] In one example of a porphyrin of formula (I), R 1 contains a triazolyl group. The triazolyl group can be attached to O through an alkyl chain. In one example of a porphyrin of formula (I), R 1 contains an alkyl glutamate group. 1 can be terminated with a pair of alkyl glutamate groups. In another example of a porphyrin of formula (I), R 1 contains a triazolyl group, and R 1 is a pair of ethyl glutamate groups at the end of the chain, and all R are -OR 1 In one example of a porphyrin of formula (I), the metal is platinum or palladium.

[0096] Porphyrins of formula (I) can be oxygen-sensing phosphors with emission intensities that depend on oxygen partial pressure. In one example of a porphyrin of formula (I), the porphyrin can be excited when irradiated with a first wavelength in the range of 350 to 600 nanometers and then emit phosphorescence at a second wavelength in the range of 600 to 700 nanometers. The first wavelength can be 532 nanometers and the second wavelength can be 644 nanometers. The first wavelength can also be 546 nanometers and the second wavelength can be 674 nanometers.

[0097] In another embodiment, the oxygen-sensing lumiphore 206 comprises a phosphorescent meso-unsubstituted porphyrin having the chemical formula (II): [ka] wherein M is a metal, each R is independently an atom or group of atoms, and at least one R is -OR 1 and R 1 is an atom or group of atoms.

[0098] In the porphyrin of formula (II), R 1 can be selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkylcarbonyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, heteroaryl, halo, cyano, and nitro. In one example of a porphyrin of formula (II), R 1 is hydrogen. In another example of a porphyrin of formula (II), R 1 is alkynyl, for example 2-propynyl (propargyl). In yet another example of a porphyrin of formula (II), R 1 is alkylcarbonyl, such as 2,2-dimethylpropanoyl (also called trimethylacetyl or pivaloyl). In the porphyrin of formula (II), multiple R's are -OR 1 Optionally, all R's can be -OR 1It can also be done as follows.

[0099] In one example of a porphyrin of formula (II), R 1 contains a triazolyl group. The triazolyl group can be attached to O through an alkyl chain. In one example of a porphyrin of formula (II), R 1 contains an alkyl glutamate group. 1 can be terminated with a pair of alkyl glutamate groups. In another example of a porphyrin of formula (II), R 1 contains a triazolyl group, and R 1 is a pair of ethyl glutamate groups at the end of the chain, and all R are -OR 1 In one example of a porphyrin of formula (II), the metal is platinum or palladium.

[0100] Porphyrins of formula (II) can be oxygen-sensing phosphors with emission intensities that depend on oxygen partial pressure. In one example of a porphyrin of formula (II), the porphyrin can be excited when irradiated with a first wavelength in the range of 350 to 650 nanometers and subsequently emit phosphorescence at a second wavelength in the range of 700 to 800 nanometers. The first wavelength can be 594 nanometers and the second wavelength can be 740 nanometers. The first wavelength can be 605 nanometers and the second wavelength can be 770 nanometers. The first wavelength can be 600 to 615 nanometers and the second wavelength can be 760 to 800 nanometers.

[0101] In one non-limiting embodiment, the second gas sensor 114 comprises a photoluminescent oxygen sensing probe comprising a polymeric material impregnated with porphyrin as disclosed in the first and third gas sensors 112 and 116 above. The photoluminescent oxygen sensing probe is a compound having an emission that provides tissue pO2. For example, the second gas sensor 114 is a skin sensor that is placed in or on a fascial flap after harvesting and before initiating perfusion. In one example, the second gas sensor 114 is an oxygen sensing needle that can be placed in the core tissue 108. In another example, the sensor film can be covered with a transparent adhesive dressing (e.g., Tegaderm™, 3M, St. Paul, MN). In one non-limiting example, the film 123 described above is in contact with the surface of the tissue sample 108. As shown in FIG. 2B, the scattering layer 208 is in contact with the polymeric material 204 and the surface of the sample 108. The second gas sensor 114 provides tissue pO2.

[0102] In some embodiments, the sensor head 118 can be in contact with a porphyrin as described in U.S. Patent Application Publication No. 2016 / 0159842, which is incorporated herein by reference. For example, the porphyrin can be an oxygen-sensing phosphor with an emission intensity that depends on the partial pressure of oxygen.

[0103] In one non-limiting example, the first, second, and third gas sensors 112, 114, 116 can each incorporate a probe head 118 in contact with a film 122 or 123 including an optical-based oxygen sensing formulation having emissions that provide tissue pO2, an emission source, and a detector that can be attached to the gas sensor 112, 114, 116. The sensor head can be as described in WO 2017 / 197385, discussed above and incorporated herein by reference.

[0104] In one non-limiting example, the first, second, and third gas sensors 112, 114, 116 can each have a light-based, photoluminescent oxygen-sensing compound in the film 122 or 123, whose emission provides tissue pO2. The sensor head 118 can be in contact with the oxygen-sensing film 122 or 123 or polymer containing the oxygen-sensing molecules. For the first and / or third gas sensors 112, 116, the sensor head 118 can be positioned in the flow cell 120, in contact with the inner surface of the inlet tube 106 and / or outlet tube 110, and in direct contact with the perfusate. The second gas sensor 114 can be positioned in direct contact with the sample tissue 108. The probe head 118 can take the form of a circular pad in contact with the film containing the oxygen-sensing molecules. The film can also include other sensors, such as a reference sensor. The reference sensor can provide a baseline for oxygenation measurements or a reference for calibration of the acid sensor. In another embodiment, the probe head 118 may be integrated with the flow cell 120 and the sensing film 122 into a single package.

[0105] The second gas sensor 114 can have a contact mechanism that can be configured to bring the probe head 118 of the film 123 into contact with the sample tissue 108. In some embodiments, the contact mechanism can provide contact between the second gas sensor 114 and the tissue 108 using an adhesive that adheres the contact surface of the second gas sensor 114 to the tissue 108. A contact mechanism in the form of an adhesive can directly and reversibly adhere the second gas sensor 114 to the tissue 108. In other embodiments, the contact mechanism can be a strap, band, elastic element, pocket, or any suitable contact mechanism that can bring the second gas sensor 114 into contact with the patient's tissue. In one non-limiting example, a seal can be used between the film 122 and the tissue 108.

[0106] In one embodiment, the first, second, and third gas sensors 112, 114, and 116 each include a sensor head 118. The sensor head 118 can include a photon source configured to send photons to the photoluminescent oxygen sensing film 122 or 123. The sensor head 118 can include a light receiving element configured to detect light emitted from the photoluminescent oxygen sensing film 122 or 123 when the photon source sends photons to the photoluminescent oxygen sensing film 122. The sensor head 118 can include a controller in electrical communication with the photon source and the light receiving element, the controller configured to execute a program stored on the controller to calculate the concentration of oxygen adjacent to the photoluminescent oxygen sensing film 122 or 123 from the electrical signal received from the light receiving element.

[0107] 3 is a non-limiting example of a sensor head 118. The figure shows a circuit board 302 with a photon source 304 and a detector 306 that can be attached to the sensor head 300. In some embodiments, the circuit board 302 can be attached to the sensor head 300 on an opposite side of the sensor head from the photon source 304 and the detector 306. In some embodiments, the circuit board 302 can be connected to the sensor head 300 via an optical fiber (not shown). The circuit board 302 can be a flexible board and can have a substrate, and the emission source 304 and the detector 306 can be embedded in or disposed on the substrate of the circuit board 302.

[0108] The emission sources 304 can be positioned on the substrate such that each photon source 304 emits photons toward the film 122 or 123. In one non-limiting example embodiment, the photon sources 304 are positioned at four radial positions around the periphery of the circuit board 302, such that the photon sources 304 can send photons to the film 122 or 123. In some embodiments, the photon sources 304 can be blue light emitting diodes. In other embodiments, the photon sources 304 can be green, yellow, or orange light emitting diodes. In other embodiments, the photon sources 304 can be optical fibers that transmit light of a particular color.

[0109] The detectors 306 can be positioned on the substrate such that each detector 306 detects or receives photons emitted from the film 122 or 123. In one non-limiting example embodiment, the detectors 306 are positioned at four radial positions around the circuit board 302, allowing the detectors 306 to detect or receive photons emitted from the film 122. The detectors 306 on the circuit board 302 can include one or more light receiving elements. In one embodiment, the light receiving elements can be configured to be sensitive to different wavelengths of light. In some embodiments, the detectors 306 can be photodiodes. The one or more green light receiving elements can be green photodiodes, and the one or more red light receiving elements can be photodiodes. In other embodiments, the detectors 306 can be charge-coupled devices (CCDs). In other embodiments, the detectors 306 can be optical fibers that couple emitted light to the light receiving elements, and can include photodiodes and charge-coupled devices (CCDs). In other embodiments, the light receiving elements can detect both green and red emissions.

[0110] The circuit board 302 can include a controller 308 that can be in electrical communication with the photon source 304 and the detector 306. The controller 308 can be a microcontroller or a system-on-chip and can include non-transitory memory that can store an executable program. In some embodiments, the controller 308 can store an oxygen calculation program that calculates the oxygen level adjacent to the sensor head 118, 300 from one or more electrical signals received from the detector 306. The circuit board 302 can include an output 310, which can be a bundle of wires. The output 310 can be connected to the external interface 128 of FIG. 1, which can be used to display, store, or analyze the results of the executable program. As an example, the output 310 can be connected to the external interface 128 via a universal serial bus (USB) hub 130. In other embodiments, the controller 308 can be configured with a wireless output that can provide wireless communication. Non-limiting examples of wireless communication that can be incorporated include Wi-Fi, Bluetooth, near-field communication, cellular networks, radio frequency, etc. The circuit board 302 may include an on-board power source (not shown), such as a battery, to provide power to the photon source 304, the detector 306, and the controller 308. In other embodiments, the circuit board 302 may include an external power source (not shown), such as an electrical connection to grid power, to provide power to the photon source 304, the detector 306, and the controller 308.

[0111] The optical first, second, and third gas sensors 112, 114, and 116 can measure pO2 directly and do not require perfusion with an oxygen carrier such as red blood cells or whole blood. The sensing components can be safely held within the sensor head 118, 300, which can be non-invasive, thus eliminating the need for external dyes, injections, and needles. The gas sensors 112, 114, and 116 can require short setup times and provide virtually instantaneous readout.

[0112] Now that the components of the perfusion system 100 have been described in detail, their function can be understood. In some embodiments, the sensor head 118 can contact the film 122 in the flow cell 120 or the film 123 on the tissue sample 108 (FIGS. 2A-2B). In some embodiments, the films 122 and 123 can include an oxygen-sensing polymer that can be in direct contact with the perfusion fluid 102 or the patient's tissue sample 108. Direct contact of the sensor head 118 with the film 123 on the patient's tissue allows the sensor head 118 to provide tissue pO2 via the photoluminescent oxygen-sensing compound in the film 123. The photoluminescent oxygen-sensing compound in the film 123 can have an emission that can be indicative of tissue pO2.

[0113] The photoluminescent oxygen sensing formulations of films 122 and 123 can emit red phosphorescence when excited by blue light from one or more photon sources 304, and the phosphorescence intensity can be detected by one or more detectors 306. The phosphorescence intensity can be inversely proportional to the pO2 of the patient's tissue. A reference sensor, which can be in the form of a green-emitting dye, can also be attached to films 122 and 123 and serve as a reference standard for accurate pO2 measurements. In one embodiment, the phosphorescence lifetime can also be inversely proportional to the pO2 of the patient's tissue and can alternatively or additionally be measured and analyzed to provide an accurate pO2 measurement.

[0114] The circuit board 302 may be a flexible substrate and may be attached to the surface of the sensor head 118 via an oxygen-impermeable membrane. The circuit board 302 may include a photon source 304 and a detector 306. The photon source 304 may be one or more blue light emitting diodes (LEDs), and the detector 306 may include one or more light receiving elements in the form of green-sensitive photodiode detectors and one or more red light receiving elements in the form of red-sensitive photodiode detectors. In some embodiments, the detector may be a photodiode, photomultiplier tube, avalanche photodiode, charge-coupled device (CCD), complementary metal-oxide semiconductor (CMOS) device, or a combination of these or similar light receiving elements.

[0115] The oxygen-dependent change in red phosphorescence intensity can be captured by a red receiving element in the form of a red-sensitive photodiode detector and compared with the green emission captured by a green receiving element in the form of a green-sensitive photodiode detector to provide a pre-calibrated and robust transcutaneous oxygen tension measurement of the sample 108 using analog circuitry in the controller 308.

[0116] The oxygen-dependent change in phosphorescence lifetime can be captured by a light-receiving element in the form of a photodiode detector. The LED illumination can be modulated (e.g., with a sine wave) to induce a modulated (e.g., sinusoidally) phosphorescence emission from the oxygen sensor, and the time delay relative to the excitation light can be measured to calculate the phosphorescence lifetime and thereby pO2.

[0117] The analog or digital circuitry of the controller 308 can provide a pre-calibrated, robust measurement of oxygen partial pressure in the patient's tissue. In some embodiments, the oxygen partial pressure measurement can be analyzed and reported using a molecule with an emission characteristic insensitive to oxygen (e.g., a fluorophore probe) and a molecule with an emission characteristic sensitive to molecular oxygen concentration (e.g., a phosphorus probe). The molecule with an emission characteristic insensitive to oxygen can be a reference sensor, and the molecule with an emission characteristic sensitive to molecular oxygen concentration can be a fluorophore probe. Emissions from the fluorophore and fluorophore probes can be used to measure oxygen partial pressure in biological systems with high precision and reversibility. The fluorophore and fluorophore probes can be calibrated so that the spectral ratio between the fluorophore emission and the fluorophore emission correlates with the oxygen concentration in the tissue sample 108. This calibration can be used to read a map of oxygen concentration in the patient's tissue. This calibration can also be used to read the average oxygen concentration in the area covered by the first, second, or third gas sensors 112, 114, 116. Fluorophore and fluorescent probes can be calibrated so that the lifetime of the fluorophore and fluorescent emission can be analyzed to provide the oxygen concentration of the tissue sample 108. Colorimetric oxygen measurements based on light absorption can also be performed using molecules such as dyes whose light absorption properties (e.g., absorption wavelength or absorption cross section) are modulated in the presence of an analyte such as oxygen.

[0118] In one embodiment, the analog or digital circuitry of the controller 308 can use the Stern-Volmer relationship to provide pre-calibrated, robust transcutaneous oxygen tension measurement and analysis of the tissue sample 108. The Stern-Volmer relationship can be used to characterize the oxygenation of the fluid 102 or sample 108 based on a photoluminescent oxygen-sensing formulation of the film 122 or 123, which emits red phosphorescence when excited by blue light from at least one photon source 304. The intensity and / or lifetime of the phosphorescence is detected by at least one detector 306 and is inversely proportional to the pO2 of the patient's tissue. Dynamic (collisional) quenching by oxygen is a photophysical, rather than photochemical, process. It is fully reversible and does not alter the optical probe, thereby affecting its absorption spectrum. Rather, it causes changes in luminescence intensity and luminescence lifetime. The relationship between intensity (or decay time) and oxygen concentration ([O2]) is expressed in its simplest form by the Stern-Volmer equation: F=F0 / (1+K SV ·[O2]);τ=τ0 / (1+K SV [O2]) where F0 and F are the luminescence intensities of the probe in the absence and presence of oxygen, respectively; τ0 and τ are the luminescence lifetimes of the probe in the absence and presence of oxygen, respectively; KSV is the Stern-Volmer constant, which depends on the lifetime of the probe and its environment (polymer matrix, solvent, etc.); and [O2] is the oxygen concentration in the sample. The term [O2] (concentration) can be replaced with pO2, the partial pressure of oxygen.

[0119] In some embodiments, a linear relationship can be established between F / F (or τ / τ) and oxygen concentration. Stern-Volmer plots (SVPs) can be established by measuring either luminescence intensity or lifetime. However, luminescence intensity data can be adversely affected by poor light source stability, variations in the efficiency of the delivery optics, drift in detector sensitivity, probe leaching and photodegradation, uneven probe distribution, background emission, and stray light. To correct for these effects, a reference sensor (such as an inert reference fluorophore emitting at a different wavelength) can be used. Alternatively, phosphorescence lifetime, which is not adversely affected by such factors, can be measured to correct for the above effects.

[0120] In some embodiments, the controller 308 can analyze the lifetime and intensity of the phosphorescent emissions to determine the oxygen concentration of the sample 108. In other embodiments, the first, second, or third gas sensor 112, 114, 116 can transmit the emission results obtained from the at least one detector 306 externally to be analyzed by an external device 128.

[0121] In some embodiments, the first and second sensor heads 118 may also include a display 132. The display 132 may be configured to display the oxygenation of the patient's tissues as determined by the first, second, or third gas sensors 112, 114, 116. In some embodiments, the display may be attached to the sensor head 118, while in other embodiments, the display may be located externally, such as on the external interface 128.

[0122] In another embodiment, the second gas sensor includes an oxygen sensing probe configured to be inserted into the sample 108. By way of example, the second gas sensor may comprise a transcutaneous sensor and a separate oxygen sensing needle, as described herein. For example, the oxygen sensing probe may be an oxygen sensing needle and may be placed in the center of the tissue sample 108 to provide subcutaneous, intramuscular, or intraorgan pO2 measurements.

[0123] In another embodiment, the perfusion system 100 includes an oxygenator 104 in fluid communication with the perfusion fluid source 102. For example, the oxygenator 104 is connected to an inlet line 106. The oxygenator 104 further controls the oxygen level of the perfusion fluid flowing toward the sample 108. In one non-limiting example, a controller, such as an external interface device 128, electrically connected to the first gas sensor, the second gas sensor, and / or the third gas sensor is configured to execute a program stored in the controller to receive electrical signals from the first gas sensor, the second gas sensor, and / or the third gas sensor and calculate the oxygen concentration adjacent to the first gas sensor, the second gas sensor, and / or the third gas sensor. As another non-limiting example, a controller, such as an external interface device 128, electrically connected to the first gas sensor, the second gas sensor, and / or the third gas sensor is configured to execute a program stored in the controller to modify or adjust the perfusion of the tissue sample in response to information from the first, second, and / or third gas sensors. As another non-limiting aspect, the controller 128 is electrically connected to a valve 162 to modify or adjust the perfusion of the tissue sample.

[0124] The perfusion system 100 may further include a pump 136 for circulating the perfusion solution 102 through the oxygenator 104, the inlet line 106, the tissue sample 108, the outlet line 110, the reservoir 134, and the recirculation line 140. The perfusion system 100 may further include a bubble trap 138 disposed between the oxygenator 104 and the first gas sensor 112. The bubble trap 138 is configured to remove air bubbles in the perfusion solution 102 formed by passing through the oxygenator 104.

[0125] In another embodiment, the perfusion system 100 includes a heat exchanger (not shown) for regulating the temperature of the perfusion fluid.

[0126] The present disclosure also provides a method 400 for machine perfusion of a tissue sample, as shown in the flowchart of FIG. 4 . The method 400 includes step 402 of preparing a tissue sample. The tissue sample can be a vascularized composite tissue allograft. For example, the vascularized composite tissue allograft can be at least a portion of a limb, face, larynx, trachea, abdominal wall, genitourinary system tissue, uterine tissue, or a solid organ, or any combination thereof. In another embodiment, the tissue sample is a donor vascularized composite tissue allograft for vascularized composite tissue allograft transplantation. By way of example, the tissue sample can be obtained from a human, a primate, or a pig. In another example, the tissue sample can be a fascia flap. In step 404, perfusate is supplied to the sample via an inflow tube. In step 406, the concentration of gas in the perfusate in the inflow tube is measured. In one non-limiting example, the measurement is performed using a first gas sensor, such as the first gas sensor 112 described above. In step 408, the concentration of the gas in the sample is measured. In one non-limiting example, the measurement is made using a second gas sensor, such as the second gas sensor 114 described above. Alternatively, the measurement is made using an oxygen sensing needle, as previously described.

[0127] In one example, the first gas sensor, the second gas sensor, the third gas sensor, or all or a combination of the sensors comprises an oxygen sensing probe. In another example, the first gas sensor, the second gas sensor, the third gas sensor, or all or a combination of the sensors comprises a film configured to change in response to pO2.

[0128] As an example, step 406 further includes detecting the concentration of gas in the perfusate in the inflow line using a first gas sensor and calculating, with the controller, the oxygen concentration adjacent to the first gas sensor from an electrical signal transmitted from the first gas sensor to the controller. Similarly, step 408 further includes measuring the concentration of gas in the sample with a second gas sensor and calculating, with the controller, the oxygen concentration adjacent to the second gas sensor from an electrical signal transmitted from the second gas sensor to the controller.

[0129] Optionally, the method 400 may include step 410 of detecting the concentration of gas in the perfusate in the outflow tubing with a third gas sensor and calculating with the controller the concentration of oxygen adjacent to the third gas sensor from an electrical signal transmitted from the third gas sensor to the controller.

[0130] In another alternative embodiment, the method may include steps 402-406 and 410, and step 408 may be omitted.

[0131] In one non-limiting example, the controller is used to generate a report of oxygen perfusion in the sample based on measurements from the first, second, and / or third gas sensors.

[0132] In one non-limiting example, the method includes controlling an oxygen level in perfusate flowing toward the sample based on electrical signals received from the first, second, and / or third gas sensors.

[0133] In one non-limiting example, the method includes activating an alarm when the gas level falls below a predetermined threshold based on electrical signals received from the first, second, and / or third gas sensors or a calculation based on data collected from the sensors. In one non-limiting example, the method includes adjusting perfusion parameters in accordance with the data collected from the sensors by sending a signal to a pump of the perfusion system or changing oxygenation parameters of the perfusion system.

[0134] In one embodiment, the tissue sample is perfused intermittently, ie, producing perfusion cycles that alternate between baseline and elevated oxygen tensions in the perfusate.

[0135] In one embodiment of the present invention, three device measurements (including measurements from the first, second, and third gas sensors) connected to an intermittently perfused sample simultaneously, continuously, and in real time provide inflow and outflow pO2 values ​​in the perfusate and transcutaneous pO2 values ​​at the perfused tissue surface. Intermittent perfusion allows for the observation of tissue response to changes in oxygenation, which depend on the state of the sample's capillary bed, thereby allowing for the observation of tissue viability (e.g., correlation or non-correlation between sensor measurements). When the tissue is viable, the transcutaneous pO2 follows trends in the inflow measurement consistent with increases and decreases in pO2 following operator-induced perfusate oxygenation cycling (e.g., turning the oxygenator on and off, cyclical fluctuations in oxygen levels, or fluctuations in oxygen levels via programmable means). The outflow measurement may indicate pO2 deficiencies and oxygenation cycle-dependent trends. If the tissue is not viable, there will be no change in tissue pO2 over time (decorrelated), and outflow measurements will show an increase in pO2 as the tissue no longer consumes oxygen. Other quantities that reflect tissue non-viability can be the difference in oxygenation between inflow and sample, or between inflow and outflow. Alternatively, a decrease in the time difference between the increase or decrease in oxygenation of the sample relative to the perfusate at inflow, the increase or decrease in oxygenation of the sample relative to outflow, or the increase or decrease in oxygenation of the outflow relative to inflow indicates tissue non-viability.

[0136] Example Examples are provided herein to illustrate and further explain certain embodiments and aspects of the combination of intermittent perfusion with continuous measurement of pO2 of the perfusate entering and leaving the tissue and the tissue's own pO2 (e.g., transcutaneous, intramuscular), and should not be construed as limiting the scope of the invention.

[0137] <Outline of the Example> The use of perfusion machines is rapidly increasing in the field of transplantation. Vascularized composite tissue allotransplantation and reconstructive surgery are comparable to the first described machine perfusion (MP) applications. Its purpose is to improve preservation by continuously delivering oxygen and nutrients essential for cellular activity while removing metabolic waste products, significantly reducing ischemic complications during preservation. By reducing ischemia-reperfusion injury, MP can result in improved short-term success and long-term outcomes. Oxygenation is a critical parameter for ensuring tissue viability during ex vivo MP. This study introduces an innovative technology based on oxygen-sensing phosphorescent metalloporphyrins that can continuously and noninvasively monitor the oxygen of vascularized fasciocutaneous flaps. The device comprises a transparent oxygen-sensing film applied to the flap's skin paddle, probed via a compact, energy-saving electronic device, providing continuous monitoring of oxygen concentration. Furthermore, this oxygen-sensing technology was also used to monitor the oxygen level of the perfusate inflow in parallel with the oxygenation of the skin paddle. To demonstrate the responsiveness and accuracy of this technology, we chose to test it using intermittent ex vivo perfusion of porcine fasciocutaneous flaps. We present the first proof-of-concept results of ex vivo machine perfusion using this technique, providing evidence of the technique's high accuracy, responsiveness, reliability, and ease of use.

[0138] This example describes the use of a novel device that can noninvasively, continuously, and responsively measure tissue oxygen levels in porcine vascularized fasciocutaneous flaps during ex vivo perfusion. This example serves as a proof-of-concept and demonstrates the feasibility of continuous and reliable monitoring of the VCA and other organs during ex vivo perfusion. This technology is believed to be a viable solution for clinical routine use in current reconstructive surgery techniques, maximizing monitoring of microsurgical flaps.

[0139] <Materials and Methods> (Animals and surgical procedures) All animal care and procedures were approved by the local Institutional Animal Care and Use Committee (IACUC). This example study included four female Yorkshire pigs weighing 30–35 kg. The animals were housed by the local Center for Comparative Medicine (CCM) and provided with food and water in accordance with IACUC guidelines. Surgery was performed after at least 24 h of acclimatization. The animals were sedated, intubated, and maintained under general anesthesia throughout the procedure by a veterinarian. Continuous monitoring included heart rate, electrocardiogram, blood pressure, core temperature, respiratory rate, and oxygen saturation. After a single systemic heparin injection (100 UI / kg), a saphenous fascial flap was harvested from the animal's right groin. The flap boundaries and anatomical markings were delineated prior to incision to allow for the creation of an elliptical skin paddle with a short axis of 5.5 cm and a long axis of 9 cm, respectively. The surgical technique was previously described by our team

[21] . The saphenous flap was harvested on the femoral vessels, allowing for smooth cannulation of the artery and easy access to the venous outflow from the two accompanying veins. After meticulous dissection, the distal femoral vessels were ligated, and the valve was released after division of the proximal portion. The vascularized flap was then flushed with 4°C heparinized saline until clear outflow was achieved. With permission from the IACUC, movement on the table was permitted, allowing other groups to harvest organs and tissues to optimize the number of animals used at the facility. The animals were euthanized at the end of surgery.

[0140] <Ex vivo fasciocutaneous flap perfusion> Immediately after cleaning, the valve was transported under a class II biosafety hood. The 18G arterial cannula was connected to a customized machine perfusion system (Figure 1: Machine Perfusion Setup). A hollow-fiber oxygenator (Affinity Pixie, Medtronic, Dublin, Ireland) was connected to an oxygen tank (95% O2, 5% CO2) to enable oxygenation of the perfusate during circulation. A pressure transducer was incorporated into the closed system to enable continuous pressure monitoring. Modified Steen's solution was used as the perfusate. The perfusate circulated through the valve's vascular tree, and the outflow was sampled through a cannulated femoral vein. The perfusate was recirculated, with complete perfusate changes every 24 hours as needed. A total volume of 350 ml was circulated. Sodium bicarbonate can be used to correct pH. The initial flow rate was based on our experience and perioperative color Doppler velocimetry performed intraoperatively

[22] . The perfusion rhythm was intermittent: the valve was perfused for 30–45 min, followed by a 75–90 min ischemic period. The roller pump (DRIVE MASTERFLEX L / S, Cole-Parmer, Vernon Hills, IL, USA) used in the perfusion system allowed for programmable cycles. The delivered flow was nonpulsatile. Perfusate samples were obtained frequently, including blood gas analysis (BGA), which was performed using a clinical-grade measurement system (Rapidpoint® 500, Siemens, Munich, Germany). Perfusion was terminated if weight gain reached 50% of the initial weight or if vascular resistance became too high to allow the flow rate F to exceed 50% of the initial value.

[0141] (Oxygen monitoring) The technique used in this example is based on detecting changes in the phosphorescence lifetime and intensity of ultrabright metalloporphyrin molecules that exhibit oxygen quenching of phosphorescence [Ref. 23]. The changes in the phosphor's lifetime (τ) and intensity (I) depend on oxygen according to the Stern-Volmer relationship [Ref. 24]. This phosphor can be easily embedded within polymer-based films, resulting in ultrathin, breathable films that exhibit bright emission throughout the oxygen partial pressure (pO2) range of 0–760 mmHg and are insensitive to changes in relative humidity [Refs. 25, 26].

[0142] In this example, the oxygen sensor was used for two purposes: to detect the transcutaneous oxygen partial pressure and to detect the oxygen partial pressure in the perfusate.

[0143] To sense pO2 through the skin, we designed an adhesive medical-grade film

[27] and applied it to the skin paddle of the valve after rigorous drying. The oxygen-sensing film consists of three layers: (i) a medical-grade semi-permeable transparent membrane (Bioclusive, McKesson), which partially blocks atmospheric oxygen from reaching the skin; (ii) a thin poly(propyl methacrylate) (PPMA) layer containing embedded metalloporphyrins; and (iii) a highly breathable white scattering layer that enhances phosphorescence collection, acts as optical insulation, improves oxygenation estimation, and prevents external light interference.

[0144] Oxygen tension in the perfusate was measured with a 3D-printed flow cell containing the same polymer membrane and a scattering layer on top. A breathable layer of medical adhesive (3M) covered the scattering layer, acting as a physical barrier between the O2-sensing membrane and the circulating perfusate. The flow cell, which monitored oxygenation of the perfusate inflow, was placed 4 cm upstream of the arterial cannula. When possible, oxygen outflow was measured with a flow cell placed 4 cm downstream of the cannulated femoral vein.

[0145] Changes in the phosphorescence of the O2-sensing layer are detected by a portable sensor, originally designed as a wearable transcutaneous oxygenation monitoring (TCOM) device

[27] . The sensor head of the prototype device is attached to a film or flow cell centrally positioned above the O2-sensing layer, exciting the phosphorescence of the porphyrin via two ultraviolet LEDs and detecting the phosphorescence with a miniature photodiode. A miniature temperature sensor (thermistor) within the sensor head accounts for the temperature-dependent effects of phosphorescence, allowing for the generation of temperature-compensated pO2 readings.

[0146] The sensor head is connected to the main control electronics, built around a commercially available microcontroller board (Particle Photon), which is then connected to a laptop via a USB serial port (Figure 1: Device Setup on the Valve + Device Configuration). The device allows for continuous monitoring of the oxygen levels of the underlying tissue (TcpO2) and perfusate during multiday experiments, with a selected sampling time of 30 seconds. Continuous oxygenation readings from all devices are stored and displayed in real time in mmHg by a Python [Reference 28] script on a PC via the USB serial port. Oxygenation readings are obtained from phosphorescence by pre-calibrating the material response and fitting the resulting dependence on pO2 using the Stern-Volmer relationship.

[0147] (result) In several experiments, the perfusate inflow oxygen level could be simultaneously monitored in parallel with the skin paddle oxygenation. Figure 5 plots the results of an experiment in which two devices were used, showing continuous readings of inflow circulating oxygen (pO2) and skin oxygen level (TcpO2, right y-axis). In experiments in which the femoral vein was also cannulated, the outflow pO2 could also be monitored from the inflow gas sensor 602, sample gas sensor 604, and outflow gas sensor 606, and the results are shown in Figure 6.

[0148] Correlation tests were performed to evaluate the circulating oxygen values ​​provided by the flow cell-based device. It should be noted that the calibrated device incorporated into the perfusion system provided continuous values ​​while conventional blood gas analysis was performed on repeated perfusate samples. To compare continuous and discrete variables, 5-minute average values ​​of flow cell readings were calculated for comparison with each BGA measurement. Figure 7 shows the correlation plot and Pearson correlation coefficient, which showed a statistically significant correlation between measurements (r = 0.982, p < 0.001).

[0149] The statistical analysis described below included four ex vivo perfused saphenous fasciocutaneous flaps. The mean warm ischemia time was 13.4 ± 2.7 minutes. The mean time to flow stabilization was 33.8 ± 21.4 minutes. After this initial phase, in which flow adapted to vascular resistance, the flaps underwent intermittent perfusion. The first two flaps underwent 30 minutes of perfusion (ON), followed by 90 minutes of ischemia (OFF). The perfusion rhythm was changed to 30 minutes ON / 75 minutes OFF. The mean total perfusion time was 50 hours (range 28-76 hours). The mean total number of perfusion cycles per flap was 19.0 ± 5.5, for a total of 76 cycles. Statistical analyses obtained from each cycle included the maximum (Max) and minimum (Min) oxygen values ​​of the skin (TcpO2) and the inflow perfusate (pO2) upstream of the arterial cannula, the difference between TcpO2 and pO2 for each cycle (Δ=Max-Min), and the time delay between the measured inflow pO2 and TcpO2. The delay was calculated by normalizing the inflow pO2 and TcpO2 between [0,1] and finding the time difference at which both curves reached a value of 0.4 (see insert in Figure 5).

[0150] The descriptive variables extracted from the measurements in Figure 6 are shown in Figures 8A-8D, and the following characteristics are observed: Both maximum and minimum TcpO2 experience a sharp decline after cycle no. 8. Furthermore, the difference between pO2 and TcpO2 increases at the end of perfusion, corresponding to perfusion failure supported by a significant increase in vascular resistance. The delay between circulatory inflow pO2 and skin TcpO2 showed a rapid increase after cycle 8, which correlated with the increase in vascular resistance (Fig. 8D).

[0151] (Consider) One of the objectives of this example was to test the responsiveness and reliability of continuous oxygen readings using a novel technology. The vascularized saphenous valve used in the included experiments allowed for testing of circulating oxygen values ​​associated with all machine perfusion-based approaches. The perfusion design, with 75-90 min of interruptions followed by 30-45 min of vigorous oxygenation, allowed for periodic assessment of responsiveness throughout the experiment, providing a total of 76 cycles to be used as replicates. The proposed system was easily implemented in a custom-made perfusion system, and its easy-to-use computer interface allowed it to be used by non-specialized surgical teams.

[0152] To control effective oxygenation during machine perfusion, a measuring flow cell was placed upstream of the arterial cannula to provide continuous circulating oxygen (pO2) values ​​delivered to the perfused organ. To our knowledge, no perfusion systems currently in clinical use utilize such monitoring. Oxygen is critical for ATP generation in mitochondria. While ischemia is nearly unavoidable during transplant surgery, recent advances in machine perfusion have significantly reduced total ischemic time. However, most damage occurs during the reperfusion process by restoring high oxygen levels after metabolic arrest. To date, transplant teams lack a target oxygen level to halt the progression of ischemia-reperfusion injury (IRI) before reactive oxygen species (ROS) are generated.

[0153] While machine perfusion appears to be a key step toward improving transplant outcomes, this limitation leaves clinicians with the challenge of mitigating its consequences. The challenges of IRI and ROS are particularly problematic for extended criteria donor organs. The molecular mechanisms occurring during machine perfusion are not yet fully understood, and even though several teams are actively researching specific areas, they are limited by the need for repeated blood gas measurements. Furthermore, machine perfusion is also used to assess organ function. Oxygen consumption is an important indicator of cellular, tissue, and organ activity and can be determined by implementing arteriovenous oxygen differentials, flow rates, and weights. The ability to continuously measure oxygen values ​​in the inflow and outflow perfusate opens the door to live O2 consumption monitoring, in addition to the level of tissue oxygenation provided by inflow and tissue oxygen readings.

[0154] A delay was observed between the measured pO2 and TcpO2 changes. This was expected due to the time required for the perfusate to reach the capillaries distal to the valve. Interestingly, this delay correlated with vascular resistance, which was continuously monitored. However, only a few time points were assessed, and further studies are needed to demonstrate reproducibility and fully explore and utilize the potential of this feature.

[0155] This example also demonstrated reliable percutaneous oxygenation readings in fasciocutaneous flaps. The saphenous flap model relates to the cutaneous component of VCA. Continuous monitoring of tissue oxygenation in these complex organs (e.g., face and hand transplants) could significantly improve the outcomes of these challenging surgeries characterized by prolonged immune rejection. IRI is known to contribute to VCA rejection by increasing antigen release in highly immunogenic tissues such as skin. Similar to its potential contributions in the field of solid organ transplantation, this technique may be valuable in these reconstructive transplants. The model used is also directly relevant to a wide range of autologous reconstructive procedures, including flap transfer and monitoring. Fasciocutaneous flaps have become the gold standard for complex reconstruction over the past decade. While the technique has been increasingly refined and mastered, failure rates for pedicled and free flaps still exist (2–10% according to the authors). Delayed surgical revision is a significant factor in cases of flap vascularization dysfunction. Early revision correlates with the rate of flap salvage. To date, flap monitoring has primarily been performed clinically by assessing color, skin capillary refill time (CRT), and overall appearance. These parameters are poorly responsive, and the delayed occurrence of paleness, coldness, and increased CRT after acute vascular complications significantly compromises flap viability and salvageability. Some authors have described oxygen monitoring using near-infrared spectroscopy (NIRS) and a LiCox probe (Integra Lifescience, Princetown, New Jersey, USA) [19,29,30]. NIRS has the disadvantage of being discontinuous and hemoglobin-dependent, while the repurposed use of LiCox is invasive, highly sensitive to motion, and requires bulky, expensive equipment. Furthermore, ease of implementation and readability of measurements must be adequate to enable efficiency in clinical routine. The technique described in this example allows for accurate and reactive live monitoring of fasciocutaneous flap oxygenation. This opens up the prospect of efficient monitoring, with the potential to activate an alarm and alert the team if oxygen levels fall below a defined threshold post-operatively, optimizing valve viability after surgical revision.However, the transcutaneous oxygenation measurements presented here are affected by atmospheric oxygen and provide readings slightly above tissue oxygen concentration. True TcpO2 can be calculated via mathematical models

[31] . It is conceivable to implement such calculations in device firmware to provide live readings of true tissue oxygenation. This noninvasive tissue monitoring technique has been separately validated for monitoring deep inferior epigastric perforator (DIEP) valves and can be used for all free or pedicled valves with a cutaneous component. Furthermore, the early application of machine perfusion in autologous procedures, as demonstrated by the technique developed by Wolff et al. [32, 33], opened new horizons in reconstructive surgery. They achieved free flap reconstruction without anastomosis by perfusing the transferred free flap with diluted compatible blood. Their innovative technique was limited by ischemic complications. No technology has been used in this field for continuous monitoring of perfused organs in clinical transplantation. In autologous reconstructive surgery, some authors have described the use of devices to measure oxygen within the flap, potentially detecting early signs of vascular compromise and leading to effective reintervention [18,19]. However, these measurements are ineffective because the techniques used are often hemoglobin-dependent, and most ex vivo perfusion work, including VCA, uses acellular perfusion solutions [1,15,20]. In this example, our team demonstrated that extensive perfusion of a fasciocutaneous flap is possible using acellular perfusion solutions. The idea of ​​a universal monitoring system that can be used regardless of perfusion and temperature conditions could enhance the safety of these new procedures and help overcome limitations currently faced by plastic surgeons.

[0156] 《References》 (1) van Beekum, C.J.; Vilz, T.O.; Glowka, T.R.; von Websky, M.W.; Kalff, J.C.; Manekeller, S., “Normothermic machine perfusion (NMP) of the liver-current status and future perspectives”, Annals of Transplantation 2021, 26, e931664-1. (2) Czigany, Z.; Pratschke, J.; Fronek, J.; Guba, M.;Schoning, W.; Raptis, D.A.; Andrassy, J.; Kramer, M.; Strnad, P.; Tolba, R.H., et al., “Hypothermic oxygenated machine perfusion reduces early allograft injury and improves post-transplant outcomes in extended criteria donation liver transplantation from donation after brain death: results from a multicenter randomized controlled trial (HOPE ECD-DBD)”, Annals of Surgery 2021, 274, 705-712. (3) MacConmara, M.; Hanish, S.I.; Hwang, C.S.; De Gregorio, L.; Desai, D.M.; Feizpour, C.A.; Tanriover, B.; Markmann, J.F.; Zeh III, H.; Vagefi, P.A., “Making every liver count: increased transplant yield of donor livers through normothermic machine perfusion”, Annals of Surgery 2020, 272, 397-401. 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(25) Roussakis, E.; Cascales, J.P.; Marks, H.L.; Li, X.; Grinstaff, M.; Evans, C.L., “Humidity-Insensitive Tissue Oxygen Tension Sensing for Wearable Devices”, Photochemistry and Photobiology2020, 96, 373-379. (26) Li, X.; Roussakis, E.; Cascales, J.P.; Marks, H.L.; Witthauer, L.; Evers, M.; Manstein, D.; Evans, C.L., “Optimization of bright, highly flexible, and humidity insensitive porphyrin-based oxygen-sensing materials”, Journal of Materials Chemistry C 2021, 9, 7555-7567. (27) Cascales, J.P.; Roussakis, E.; Witthauer, L.; Goss, A.; Li, X.; Chen, Y.; Marks, H.L.; Evans, C.L., “Wearable device for remote monitoring of transcutaneous tissue oxygenation”, Biomed. Opt. Express 2020, 11, 6989-7002. (28) Van Rossum, G.; Drake, F.L., The Python language reference manual; Network Theory Ltd., 2011. (29) Repez, A.; Oroszy, D.; Arnez, Z.M. “Continuous postoperative monitoring of cutaneous free flaps using near infrared spectroscopy”, Journal Of Plastic, Reconstructive & Aesthetic Surgery2008, 61, 71-77. (30) Newton, E.; Butskiy, O.; Shadgan, B.; Prisman, E.; Anderson, D.W., “Outcomes of free flap reconstructions with near-infrared spectroscopy (NIRS) monitoring: A systematic review”, Microsurgery 2020, 40, 268-275. (31) Cascales, JP; Draghici, AE; Keshishian, H.; Taylor, JA; Evans, CL, “Calculation of Tissue Oxygenation via an Inverse Boundary Problem for Transcutaneous Oxygenation Wearable Applications”, ACS Measurement Science Au 2023. (32) Wolff, K.-D.; Mucke, T.; von Bomhard, A.; Ritschl, LM; Schneider, J.; Humbs, M.; Fichter, AM, “Free flap transplantation using an extracorporeal perfusion device: First three cases”, Journal of Cranio-Maxillofacial Surgery 2016, 44, 148-154. (33) Wolff, K.-D., “New aspects in free flap surgery: Miniperforator flaps and extracorporeal flap perfusion”, Journal Of Stomatology, Oral And Maxillofacial Surgery 2017, 118, 238-241. The citation of any document shall not be construed as an admission that it is prior art with respect to the present invention.

[0157] As can be seen, the present invention provides an improved perfusion system for tissue samples.

[0158] Although the present invention has been described in detail with reference to specific embodiments, it will be apparent to those skilled in the art that the present invention may be practiced in other embodiments alternative to those described above, and the embodiments described above are presented by way of example only and are not a limiting list. Therefore, the scope of the appended claims should not be limited to the description of the embodiments herein.

Claims

1. 1. A perfusion system for a tissue sample, comprising: A liquid source; an inlet conduit in fluid communication with the perfusion fluid source and the sample, the inlet conduit configured to deliver perfusion fluid to the sample; an outflow conduit in fluid communication with the sample and configured to carry perfusate away from the sample; a first gas sensor in fluid communication with the outflow conduit for measuring a concentration of gas in the perfusion fluid in the inflow conduit; a second gas sensor in contact with the sample to measure the concentration of gas in the sample; A perfusion system comprising:

2. The second gas sensor comprises: a photoluminescent oxygen sensing probe in contact with the sample; a photon source for transmitting photons at said photoluminescent oxygen sensing probe; a light receiving element configured to detect light emitted from the photoluminescent oxygen sensing probe when the photon source transmits photons at the photoluminescent oxygen sensing probe; a controller in electrical communication with the photon source and the light receiving element, the controller configured to execute a program stored on the controller to calculate a concentration of oxygen adjacent to the photoluminescent oxygen sensing probe from an electrical signal received from the light receiving element; The perfusion system of claim 1 , comprising:

3. The photoluminescent oxygen sensing probe measures tissue oxygen tension (pO ２ ) a formulation having an emission that provides The perfusion system of claim 2.

4. the photoluminescent oxygen sensing probe comprises a polymeric material impregnated with a porphyrin; The perfusion system of claim 3.

5. The photoluminescent oxygen sensing probe comprises a polymer impregnated with a phosphorescent meso-unsubstituted porphyrin; The perfusion system of claim 3.

6. The second gas sensor comprises: an oxygen sensing probe configured to be inserted into the sample; a controller in electrical communication with the oxygen sensing probe, the controller configured to execute a program stored on the controller to calculate a concentration of oxygen adjacent to the oxygen sensing probe from electrical signals received from the oxygen sensing probe; The perfusion system of claim 1 , comprising:

7. The second gas sensor measures tissue oxygen partial pressure (pO ２ ) to provide The perfusion system of claim 1 .

8. a controller in electrical communication with the first gas sensor and the second gas sensor, the controller configured to execute a program stored in the controller to receive electrical signals from the first gas sensor and the second gas sensor and calculate a concentration of oxygen adjacent the first gas sensor and / or the second gas sensor. The perfusion system of claim 1 .

9. The first gas sensor comprises: Oxygen partial pressure (pO ２ a material that undergoes a change in response to the a sensor head that contacts the material and detects the change in the material; a controller in electrical communication with the sensor head, the controller executing a program stored in the controller to calculate the oxygen partial pressure adjacent the material; The perfusion system of claim 1 , comprising:

10. a transparent film that forms the outer layer of the material; a layer of polymeric material having metalloporphyrin embedded therein adjacent to said transparent film; a scattering layer in contact with the layer of polymeric material and the surface of the sample; 10. The perfusion system of claim 9, further comprising:

11. the first gas sensor comprises a flow cell containing the material; The perfusion system of claim 9.

12. the change in the material comprises a change in phosphorescence; The perfusion system of claim 9.

13. The sensor head includes a plurality of light emitting diodes (LEDs) and a photodiode. The perfusion system of claim 9.

14. The sensor head further includes a temperature sensor.

14. The perfusion system of claim 13.

15. The second gas sensor comprises: a material that undergoes a change in response to oxygen pressure; a sensor head that contacts the material and detects the change in the material; a controller in electrical communication with the sensor head, the controller executing a program stored in the controller to calculate the oxygen partial pressure adjacent the material; Equipped with The perfusion system of claim 1 .

16. In the second oxygen sensor, the material is disposed on an outer surface of the sample.

16. The perfusion system of claim 15.

17. The change in the material includes a change in phosphorescence intensity or phosphorescence lifetime.

16. The perfusion system of claim 15.

18. The sensor head includes a plurality of light emitting diodes (LEDs) and a photodiode.

16. The perfusion system of claim 15.

19. The sensor head further includes a temperature sensor.

19. The perfusion system of claim 18.

20. a third gas sensor in fluid communication with the outlet conduit; the third gas sensor measures the concentration of gas in the perfusion fluid in the outflow tube; The perfusion system of claim 1 .

21. The third gas sensor comprises: Oxygen partial pressure (pO ２ a material that undergoes a change in response to the a sensor head that contacts the material and detects the change in the material; a controller in electrical communication with the sensor head, the controller executing a program stored in the controller to calculate the oxygen partial pressure adjacent the material; 21. The perfusion system of claim 20, comprising:

22. a transparent film that forms the outer layer of the material; a layer of polymeric material having metalloporphyrin embedded therein adjacent to said transparent film; a scattering layer in contact with the layer of polymeric material and the surface of the sample; 22. The perfusion system of claim 21, further comprising:

23. the third gas sensor comprises a flow cell containing the material; 22. The perfusion system of claim 21.

24. The change in the material includes a change in phosphorescence intensity or phosphorescence lifetime.

22. The perfusion system of claim 21.

25. The sensor head includes a plurality of light emitting diodes (LEDs) and a photodiode.

25. The perfusion system of claim 24.

26. The sensor head further includes a temperature sensor.

26. The perfusion system of claim 25.

27. an oxygenator in fluid communication with the source of perfusion fluid; a pump for circulating the perfusion fluid through the oxygenator, the inflow line, and the outflow line; It also has The oxygenator controls the oxygen level in the perfusate flowing toward the sample. The perfusion system of claim 1 .

28. Further comprising a heat exchanger for adjusting the temperature of the perfusion solution.

28. The perfusion system of claim 27.

29. a controller in electrical communication with the oxygenator, the first gas sensor, and the second gas sensor; the controller is configured to execute a program stored in the controller to control the oxygen level in the perfusion fluid flowing toward the sample based on the electrical signals received from the first gas sensor and the second gas sensor.

28. The perfusion system of claim 27.

30. a controller in electrical communication with the pump, the first gas sensor, and the second gas sensor; the controller is configured to execute a program stored in the controller to control the oxygen level in the perfusion fluid flowing toward the sample based on the electrical signals received from the first gas sensor and the second gas sensor.

28. The perfusion system of claim 27.

31. a controller in electrical communication with the first gas sensor and the second gas sensor; the controller is configured to execute a program stored in the controller to receive electrical signals from the first gas sensor and the second gas sensor, and to activate delivery of the perfusion fluid when a loss of viability is calculated based on a first concentration of a first gas adjacent to the first gas sensor and a second concentration of a second gas adjacent to the second gas sensor. The perfusion system of claim 1 .

32. a controller in electrical communication with the first gas sensor and the second gas sensor; the controller is configured to execute a program stored therein to receive electrical signals from the first gas sensor and the second gas sensor, and to deactivate delivery of the perfusion fluid when a viability calculated based on a first concentration of a first gas adjacent to the first gas sensor and a second concentration of a second gas adjacent to the second gas sensor exceeds a viability threshold. The perfusion system of claim 1 .

33. a controller in electrical communication with the first gas sensor and the second gas sensor; the controller is configured to execute a program stored therein to receive electrical signals from the first gas sensor and the second gas sensor and to intermittently activate or deactivate delivery of the perfusion fluid based on a first concentration of a first gas adjacent to the first gas sensor and a second concentration of a second gas adjacent to the second gas sensor. The perfusion system of claim 1 .

34. a controller in electrical communication with the first gas sensor and the second gas sensor; the controller is configured to execute a program stored in the controller to initiate an alarm when a gas level drops below a predetermined threshold based on the electrical signals received from the first gas sensor and the second gas sensor. The perfusion system of claim 1 .

35. The perfusion solution is a cell-free perfusion solution. The perfusion system of claim 1 .

36. the first gas sensor providing a continuous circulating oxygen value delivered to the sample of the perfused subject; The perfusion system of claim 1 .

37. the second gas sensor providing a continuous tissue oxygenation value of the sample of the perfused subject. The perfusion system of claim 1 .

38. 1. A method for performing machine perfusion of a tissue sample, comprising: (a) providing a tissue sample; (b) delivering a perfusion fluid to the sample via an inlet tube; (c) measuring the concentration of gas in the perfusion fluid in the inflow line; (d) measuring the concentration of gas in the sample; A method comprising:

39. (e) measuring the concentration of gas in the perfusate in an outlet tube configured to carry the perfusate away from the sample.

39. The method of claim 38, further comprising:

40. step (c) includes measuring the concentration of the gas in the perfusion solution in the inflow line with a first gas sensor; step (d) includes measuring the concentration of the gas in the sample using a second gas sensor; step (e) includes measuring the concentration of the gas in the perfusion solution in the outflow line using a third gas sensor; at least one of the first gas sensor, the second gas sensor, and the third gas sensor comprises an oxygen sensing probe; 40. The method of claim 39.

41. step (c) includes measuring the concentration of the gas in the perfusion solution in the inflow line with a first gas sensor; step (d) includes measuring the concentration of the gas in the sample using a second gas sensor; step (e) includes measuring the concentration of the gas in the perfusion solution in the outflow line using a third gas sensor; At least one of the first gas sensor, the second gas sensor, and the third gas sensor detects an oxygen partial pressure (pO ２ ) a material that undergoes a change in response to 40. The method of claim 39.

42. step (c) includes detecting the concentration of the gas in the perfusion solution in the inflow line using the first gas sensor, and calculating, in a controller, the concentration of oxygen adjacent to the first gas sensor from an electrical signal transmitted from the first gas sensor to the controller; step (d) includes measuring the concentration of the gas in the sample using the second gas sensor and calculating, in the controller, the concentration of oxygen adjacent to the second gas sensor from an electrical signal transmitted from the second gas sensor to the controller; step (e) includes detecting the concentration of the gas in the perfusion solution in the outflow tube using the third gas sensor, and calculating, in a controller, the concentration of oxygen adjacent to the third gas sensor from an electrical signal transmitted from the third gas sensor to the controller; 41. The method of claim 40.

43. monitoring the change in concentration from the first gas sensor and at least one of the second gas sensor and the third gas sensor; measuring the variation in the oxygen concentration over a period of time; analyzing the variation in the oxygen concentration over the period of time; estimating the viability and perfusion quality of the tissue sample; 40. The method of claim 39, further comprising:

44. (e) generating a report of oxygen perfusion in the sample using the controller; 39. The method of claim 38, further comprising:

45. (e) controlling the oxygen level in the perfusion fluid flowing toward the sample based on the electrical signals received from the first gas sensor and the second gas sensor.

39. The method of claim 38, further comprising:

46. (e) activating an alarm when the gas level drops below a predetermined threshold based on the electrical signals received from the first gas sensor and the second gas sensor.

39. The method of claim 38, further comprising:

47. the tissue sample is a vascularized composite tissue allograft; 39. The method of claim 38.

48. The vascularized composite tissue allograft is at least a portion of a limb, face, pharynx, trachea, abdominal wall, genitourinary system tissue, uterine tissue, or solid organ, or any combination thereof.

48. The method of claim 47.

49. The tissue sample is a donor vascularized composite tissue allograft for vascularized composite tissue allograft transplantation.

39. The method of claim 38.

50. the tissue sample is obtained from a human, a primate, or a pig; 39. The method of claim 38.

51. the tissue sample is a fasciocutaneous flap; 39. The method of claim 38.

52. 1. A perfusion system for a tissue sample, comprising: a source of perfusion fluid; an inlet conduit in fluid communication with the perfusion fluid source and the sample, the inlet conduit configured to deliver perfusion fluid to the sample; an outflow conduit in fluid communication with the sample and configured to carry perfusate away from the sample; a first gas sensor in fluid communication with the outflow conduit for measuring a concentration of a first gas in the perfusion fluid in the inflow conduit; a second gas sensor in contact with the sample to measure the concentration of a second gas in the sample; a controller in electrical communication with the first gas sensor and the second gas sensor; It is equipped with The controller executes a program stored in the controller, (i) receiving electrical signals from the first gas sensor and the second gas sensor and calculating a concentration of the first gas adjacent to the first gas sensor and a concentration of the second gas adjacent to the second gas sensor; (ii) activating or deactivating delivery of the perfusion fluid toward the sample and / or adjusting perfusion parameters based on a first concentration of a first gas adjacent to the first gas sensor and a second concentration of a second gas adjacent to the second gas sensor. A perfusion system characterized by being configured as follows.

53. the controller is configured to execute the program stored in the controller to activate delivery of the perfusion fluid when a loss of viability is calculated based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor.

53. The perfusion system of claim 52.

54. the controller is configured to execute the program stored in the controller to deactivate delivery of the perfusion fluid when a viability calculated based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor exceeds a viability threshold.

53. The perfusion system of claim 52.

55. the controller is configured to execute the program stored in the controller to intermittently activate or deactivate delivery of the perfusion fluid based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor.

53. The perfusion system of claim 52.

56. further comprising a pump in electrical communication with the controller; the controller is configured to execute the program stored in the controller to adjust a flow rate of perfusion fluid flowing toward the sample based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor.

53. The perfusion system of claim 52.

57. further comprising an oxygenator in electrical communication with the controller; the controller is configured to execute the program stored in the controller to control the oxygen level in the perfusate flowing toward the sample based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor.

53. The perfusion system of claim 52.

58. a third gas sensor in fluid communication with the outlet conduit; the third gas sensor measures the concentration of a third gas in the perfusion fluid in the outflow tube; the controller is configured to execute a program stored therein to activate or deactivate delivery of the perfusion fluid toward the sample and / or adjust perfusion parameters based on the first concentration of the first gas adjacent to the first gas sensor, the second concentration of the second gas adjacent to the second gas sensor, and the third concentration of the third gas adjacent to the third gas sensor.

53. The perfusion system of claim 52.

59. the controller is configured to execute the program stored in the controller to activate delivery of the perfusion fluid when a loss of viability is calculated based on the first concentration of the first gas adjacent to the first gas sensor, the second concentration of the second gas adjacent to the second gas sensor, and the third concentration of the third gas adjacent to the third gas sensor.

59. The perfusion system of claim 58.

60. the controller is configured to execute the program stored in the controller to deactivate delivery of the perfusion solution when a viability calculated based on the first concentration of the first gas adjacent to the first gas sensor, the second concentration of the second gas adjacent to the second gas sensor, and the third concentration of the third gas adjacent to the third gas sensor exceeds a viability threshold.

59. The perfusion system of claim 58.

61. the controller is configured to execute the program stored in the controller to intermittently activate or deactivate delivery of the perfusion fluid based on the first concentration of the first gas adjacent to the first gas sensor, the second concentration of the second gas adjacent to the second gas sensor, and the third concentration of the third gas adjacent to the third gas sensor.

59. The perfusion system of claim 58.

62. further comprising a pump in electrical communication with the controller; the controller is configured to execute the program stored in the controller to adjust a flow rate of perfusion fluid flowing toward the sample based on the first concentration of the first gas adjacent to the first gas sensor, the second concentration of the second gas adjacent to the second gas sensor, and the third concentration of the third gas adjacent to the third gas sensor.

59. The perfusion system of claim 58.

63. further comprising an oxygenator in electrical communication with the controller; the controller is configured to execute the program stored in the controller to control the oxygen level in the perfusate flowing toward the sample based on the electrical signals received from the first gas sensor and the second gas sensor and the third concentration of the third gas adjacent to the third gas sensor.

59. The perfusion system of claim 58.

64. the controller is in electrical communication with a controllable valve disposed in the inlet line; the controller is configured to execute a program stored in the controller to activate or deactivate the delivery of the perfusion fluid by moving the controllable valve to an open position in which the perfusion fluid is delivered to the sample or to a closed position in which the perfusion fluid is not delivered to the sample.

53. The perfusion system of claim 52.

65. the controller is in electrical communication with a controllable valve disposed in the inlet line; the controller is configured to execute a program stored in the controller to control delivery of the perfusion fluid by moving the controllable valve to a fully open position to deliver a first amount of the perfusion fluid to the sample, an intermediate position to deliver a second amount of the perfusion fluid to the sample that is less than the first amount of the perfusion fluid, or a closed position to deliver no perfusion fluid to the sample.

53. The perfusion system of claim 52.

66. 1. A method for performing machine perfusion of a tissue sample, comprising: (a) providing a tissue sample; (b) delivering a perfusion fluid to the sample via an inlet tube; (c) measuring a first concentration of a first gas in the perfusion fluid in the inflow line; (d) measuring a second concentration of a second gas in the sample; (e) activating or deactivating delivery of the perfusion solution to the sample and / or adjusting perfusion parameters based on the first concentration of the first gas and the second concentration of the second gas; A method comprising:

67. step (e) includes activating delivery of the perfusion fluid when a loss of viability is calculated based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor.

67. The method of claim 66.

68. step (e) includes deactivating delivery of the perfusion fluid if a viability calculated based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor exceeds a viability threshold.

67. The method of claim 66.

69. step (e) includes intermittently activating or deactivating delivery of the perfusion fluid based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor.

67. The method of claim 66.

70. step (e) includes adjusting a flow rate of perfusion fluid flowing toward the sample based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor; 67. The method of claim 66.

71. step (e) includes controlling an oxygen level in the perfusate flowing toward the sample based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor; 67. The method of claim 66.

72. 1. A perfusion system for a tissue sample, comprising: a source of perfusion fluid; an inlet conduit in fluid communication with the perfusion fluid source and the sample, the inlet conduit configured to deliver perfusion fluid to the sample; an outflow conduit in fluid communication with the sample and configured to carry perfusate away from the sample; a first gas sensor in fluid communication with the outflow conduit for measuring a concentration of a first gas in the perfusion fluid in the inflow conduit; a second gas sensor in fluid communication with the outflow tube for measuring the concentration of a second gas in the perfusate in the outflow tube; a controller in electrical communication with the first gas sensor and the second gas sensor; It is equipped with The controller executes a program stored in the controller, (i) receiving electrical signals from the first gas sensor and the second gas sensor and calculating a concentration of the first gas adjacent to the first gas sensor and a concentration of the second gas adjacent to the second gas sensor; (ii) activating or deactivating delivery of the perfusion fluid toward the sample and / or adjusting perfusion parameters based on a first concentration of a first gas adjacent to the first gas sensor and a second concentration of a second gas adjacent to the second gas sensor. A perfusion system characterized by being configured as follows.

73. the controller is configured to execute the program stored in the controller to activate delivery of the perfusion fluid when a loss of viability is calculated based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor.

73. The perfusion system of claim 72.

74. the controller is configured to execute the program stored in the controller to deactivate delivery of the perfusion fluid when a viability calculated based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor exceeds a viability threshold.

73. The perfusion system of claim 72.

75. the controller is configured to execute the program stored in the controller to intermittently activate or deactivate delivery of the perfusion fluid based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor.

73. The perfusion system of claim 72.

76. further comprising a pump in electrical communication with the controller; the controller is configured to execute the program stored in the controller to adjust a flow rate of perfusion fluid flowing toward the sample based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor.

73. The perfusion system of claim 72.

77. further comprising an oxygenator in electrical communication with the controller; the controller is configured to execute the program stored in the controller to control the oxygen level in the perfusate flowing toward the sample based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor.

73. The perfusion system of claim 72.

78. a third gas sensor in contact with the sample; the third gas sensor measures the concentration of a third gas in the sample; the controller is configured to execute a program stored therein to activate or deactivate delivery of the perfusion fluid toward the sample and / or adjust perfusion parameters based on the first concentration of the first gas adjacent to the first gas sensor, the second concentration of the second gas adjacent to the second gas sensor, and the third concentration of the third gas adjacent to the third gas sensor.

73. The perfusion system of claim 72.

79. the controller is configured to execute the program stored in the controller to activate delivery of the perfusion fluid when a loss of viability is calculated based on the first concentration of the first gas adjacent to the first gas sensor, the second concentration of the second gas adjacent to the second gas sensor, and the third concentration of the third gas adjacent to the third gas sensor.

79. The perfusion system of claim 78.

80. the controller is configured to execute the program stored in the controller to deactivate delivery of the perfusion solution when a viability calculated based on the first concentration of the first gas adjacent to the first gas sensor, the second concentration of the second gas adjacent to the second gas sensor, and the third concentration of the third gas adjacent to the third gas sensor exceeds a viability threshold.

79. The perfusion system of claim 78.

81. the controller is configured to execute the program stored in the controller to intermittently activate or deactivate delivery of the perfusion fluid based on the first concentration of the first gas adjacent to the first gas sensor, the second concentration of the second gas adjacent to the second gas sensor, and the third concentration of the third gas adjacent to the third gas sensor.

79. The perfusion system of claim 78.

82. further comprising a pump in electrical communication with the controller; the controller is configured to execute the program stored in the controller to adjust a flow rate of perfusion fluid flowing toward the sample based on the first concentration of the first gas adjacent to the first gas sensor, the second concentration of the second gas adjacent to the second gas sensor, and the third concentration of the third gas adjacent to the third gas sensor.

79. The perfusion system of claim 78.

83. further comprising an oxygenator in electrical communication with the controller; the controller is configured to execute the program stored in the controller to control the oxygen level in the perfusate flowing toward the sample based on the electrical signals received from the first gas sensor and the second gas sensor and the third concentration of the third gas adjacent to the third gas sensor.

79. The perfusion system of claim 78.

84. the controller is in electrical communication with a controllable valve disposed in the inlet line; the controller is configured to execute a program stored in the controller to activate or deactivate the delivery of the perfusion fluid by moving the controllable valve to an open position in which the perfusion fluid is delivered to the sample or to a closed position in which the perfusion fluid is not delivered to the sample.

73. The perfusion system of claim 72.

85. the controller is in electrical communication with a controllable valve disposed in the inlet line; the controller is configured to execute a program stored in the controller to control delivery of the perfusion fluid by moving the controllable valve to a fully open position to deliver a first amount of the perfusion fluid to the sample, an intermediate position to deliver a second amount of the perfusion fluid to the sample that is less than the first amount of the perfusion fluid, or a closed position to deliver no perfusion fluid to the sample.

73. The perfusion system of claim 72.

86. 1. A method for performing machine perfusion of a tissue sample, comprising: (a) providing a tissue sample; (b) delivering a perfusion fluid to the sample via an inlet tube; (c) measuring a first concentration of a first gas in the perfusion fluid in the inflow line; (d) measuring a second concentration of the second gas in the outflow conduit; (e) activating or deactivating delivery of the perfusion solution to the sample and / or adjusting perfusion parameters based on the first concentration of the first gas and the second concentration of the second gas; A method comprising:

87. step (e) includes activating delivery of the perfusion fluid when a loss of viability is calculated based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor.

87. The method of claim 86.

88. step (e) includes deactivating delivery of the perfusion fluid if a viability calculated based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor exceeds a viability threshold.

87. The method of claim 86.

89. step (e) includes intermittently activating or deactivating delivery of the perfusion fluid based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor.

87. The method of claim 86.

90. step (e) includes adjusting a flow rate of perfusion fluid flowing toward the sample based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor; 87. The method of claim 86.

91. step (e) includes controlling an oxygen level in the perfusate flowing toward the sample based on the first concentration of the first gas adjacent to the first gas sensor and the second concentration of the second gas adjacent to the second gas sensor; 87. The method of claim 86.

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