Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
  • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
  • C12M29/10—Perfusion
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
  • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
  • C12M41/34—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of gas

Definitions

• Tissue oxygenation is a crucial parameter for monitoring perfused organs ex-vivo. Under-oxygenation can be considered a partial failure of the perfusion, whereas uncontrolled overexposure to oxygen can lead to damage caused by free reactive oxygen species [Ref.17]. [005] Therefore, there is a need for improved systems and methods for determining viability and quality of a perfused tissue sample.
• the present disclosure provides systems and methods that overcome the aforementioned drawbacks by providing a system and method for tissue sample perfusion including gas inflow and sample sensors to continuously monitor perfusion.
• the present disclosure provides a perfusion system for a tissue sample, wherein the system comprises: a perfusion fluid source; an inflow conduit in fluid communication with the perfusion fluid source and the sample, the inflow conduit being configured to deliver perfusion fluid to the sample; an outflow conduit in fluid communication with the sample, the outflow conduit being configured to carry perfusion fluid away from the sample; a first gas sensor in fluid communication with the inflow conduit, the first gas sensor measuring a concentration of a gas in - 2 - ⁇ the perfusion fluid in the inflow conduit; and a second gas sensor contacting the sample, the second gas sensor measuring a concentration of a gas in the sample.
• the second gas sensor comprises: a photoluminescent oxygen-sensitive probe in contact with the sample; a photon source configured to direct photons at the photoluminescent oxygen-sensitive probe; a photodetector configured to detect light emitted from the photoluminescent oxygen-sensitive probe when the photon source directs photons at the photoluminescent oxygen-sensitive probe; and a controller in electrical communication with the photon source and the photodetector, the controller being configured to execute a program stored in the controller to calculate a concentration of oxygen adjacent to the photoluminescent oxygen-sensitive probe from an electrical signal received from the photodetector.
• the photoluminescent oxygen- sensitive probe is a formulation having an emission that provides tissue oxygen partial pressure (pO 2 ).
• the photoluminescent oxygen-sensitive probe comprises a polymeric material impregnated with a porphyrin.
• the photoluminescent oxygen-sensitive probe comprises a polymer impregnated with a phosphorescent meso-unsubstituted porphyrin.
• the second gas sensor comprises: an oxygen- sensitive probe configured for insertion into the sample; and a controller in electrical communication with the oxygen-sensitive probe, the controller being configured to execute a program stored in the controller to calculate a concentration of oxygen adjacent the oxygen- sensitive probe from an electrical signal received from the oxygen-sensitive probe.
• the second gas sensor provides tissue oxygen partial pressure (pO2).
• the perfusion system further comprises: a controller in electrical communication with the first gas sensor and the second gas sensor, the controller being 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 calculate a concentration of oxygen adjacent the first gas sensor and/or the second gas sensor.
• the first gas sensor comprises: a material configured to undergo a change in response to oxygen partial pressure (pO2); a sensor head contacting the material and configured to detect the change of the material; and a controller in electrical communication with the sensor head, the controller being configured to execute a program stored in the controller to calculate the oxygen partial pressure adjacent the material.
• pO2 oxygen partial pressure
• the perfusion system further comprises: a transparent membrane forming an outer layer of the material; a layer of a polymeric material embedded with a metalloporphyrin adjacent to the transparent membrane; and a scattering layer in contact with the layer of the polymeric material and a surface of the sample.
• the first gas sensor comprises a flow cell containing the material.
• the change in the material includes a change in phosphorescence.
• the sensor head includes a plurality of light emitting diodes (LEDs) and a photodiode.
• the sensor head further includes a temperature sensor.
• the second gas sensor comprises: a material configured to undergo a change in response to oxygen pressure; and a sensor head contacting the material and configured to detect the change of the material; and a controller in electrical communication with the sensor head, the controller being configured to execute a program stored - 4 - ⁇ in the controller to calculate the oxygen partial pressure adjacent the material.
• the material in the second oxygen sensor, is placed on an outer surface of the sample.
• the change in the material includes a change in phosphorescence intensity or phosphorescence lifetime.
• the sensor head includes a plurality of light emitting diodes (LEDs) and a photodiode.
• the sensor head further includes a temperature sensor.
• the perfusion system further comprises: a third gas sensor in fluid communication with the outflow conduit, the third gas sensor measuring a concentration of a gas in perfusion fluid in the outflow conduit.
• the third gas sensor comprises: a material configured to undergo a change in response to oxygen partial pressure (pO 2 ); a sensor head contacting the material and configured to detect the change of the material; and a controller in electrical communication with the sensor head, the controller being configured to execute a program stored in the controller to calculate the oxygen partial pressure adjacent to the material.
• the perfusion system further comprises: a transparent membrane forming an outer layer of the material; a layer of a polymeric material embedded with a metalloporphyrin adjacent to the transparent membrane; and a scattering layer in contact with the layer of the polymeric material and a surface of the sample.
• the third gas sensor includes a flow cell containing the material.
• the change in the material includes a change in phosphorescence intensity or phosphorescence lifetime.
• the sensor head includes - 5 - ⁇ a plurality of light emitting diodes (LEDs) and a photodiode.
• the sensor head further includes a temperature sensor.
• 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 inflow conduit, and the outflow conduit, wherein the oxygenator controls oxygen level in perfusion fluid flowing towards the sample.
• the perfusion system further comprises: a heat exchanger for adjusting a temperature of the perfusion fluid.
• the perfusion system further comprises: a controller in electrical communication with the oxygenator, the first gas sensor, and the second gas sensor, the controller being configured to execute a program stored in the controller to control oxygen level in perfusion fluid flowing towards the sample based on electrical signals received from the first gas sensor and the second gas sensor.
• the perfusion system further comprises: a controller in electrical communication with the pump, the first gas sensor, and the second gas sensor, the controller being configured to execute a program stored in the controller to control oxygen level in perfusion fluid flowing towards the sample based on electrical signals received from the first gas sensor and the second gas sensor.
• the perfusion system further comprises: a controller in electrical communication with the first gas sensor and the second gas sensor, the controller being 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 - 6 - ⁇ calculated based on the first concentration of the first gas adjacent the first gas sensor and the second concentration of the second gas adjacent the second gas sensor.
• the perfusion system further comprises: a controller in electrical communication with the first gas sensor and the second gas sensor, the controller being 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 when a calculated viability based on the first concentration of the first gas adjacent the first gas sensor and the second concentration of the second gas adjacent the second gas sensor exceeds a threshold viability value.
• the perfusion system further comprises: a controller in electrical communication with the first gas sensor and the second gas sensor, the controller being 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 the first concentration of the first gas adjacent the first gas sensor and the second concentration of the second gas adjacent the second gas sensor.
• the perfusion system further comprises: a controller in electrical communication with the first gas sensor and the second gas sensor, the controller being configured to execute a program stored in the controller to activate an alarm when a gas level drops below a defined threshold based on electrical signals received from the first gas sensor and the second gas sensor.
• the perfusion fluid is an acellular perfusate solution.
• the first gas sensor provides continuous circulating oxygen values delivered to the perfused sample.
• the second gas sensor provides continuous tissue oxygenation values of the perfused sample.
• the present disclosure provides a method for machine perfusion of a tissue sample. The method comprises: (a) providing a tissue sample; (b) delivering a perfusion fluid to the sample via an inflow conduit; (c) measuring a concentration of a gas in the perfusion fluid in the inflow conduit; and (d) measuring a concentration of a gas in the sample.
• the method can further comprise: (e) measuring a concentration of a gas in the perfusion fluid in an outflow conduit configured to carry perfusion fluid away from the sample.
• step (c) comprises measuring the concentration of the gas in the perfusion fluid in the inflow conduit with a first gas sensor
• step (d) comprises measuring the concentration of the gas in the sample with a second gas sensor
• step (e) comprises measuring the concentration of the gas in the perfusion fluid in the outflow conduit with 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-sensitive probe.
• step (c) comprises measuring the concentration of the gas in the perfusion fluid in the inflow conduit with a first gas sensor
• step (d) comprises measuring the concentration of the gas in the sample with a second gas sensor
• step (e) comprises measuring the concentration of the gas in the perfusion fluid in the outflow conduit with 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 configured to undergo a change in response to oxygen partial pressure (pO2).
• pO2 oxygen partial pressure
• step (c) comprises sensing the concentration of the gas in the perfusion fluid in the inflow conduit with the first gas sensor and calculating, in a controller, - 8 - ⁇ a concentration of oxygen adjacent to the first gas sensor from an electrical signal transmitted to the controller from the first gas sensor
• step (d) comprises measuring the concentration of the gas in the sample with the second gas sensor, and calculating, in the controller, a concentration of oxygen adjacent the second gas sensor from an electrical signal transmitted to the controller from the second gas sensor
• step (e) comprises sensing the concentration of the gas in the perfusion fluid in the outflow conduit with the third gas sensor and calculating, in a controller, a concentration of oxygen adjacent to the third gas sensor from an electrical signal transmitted to the controller from the third gas sensor.
• the method further comprises: monitoring a change in the concentration from the first gas sensor, and at least one of the second gas sensor and third gas sensor; measuring a variation in the oxygen concentration over a period of time; analyzing the variation in the oxygen concentration over the period of time; and estimating a viability of the tissue sample and a perfusion quality.
• the method further comprises: (e) generating a report, using the controller, of oxygen perfusion in the sample.
• the method further comprises: (e) controlling an oxygen level in the perfusion fluid flowing towards the sample based on electrical signals received from the first gas sensor and the second gas sensor.
• the method further comprises: (e) activating an alarm when a gas level drops below a defined threshold based on electrical signals received from the first gas sensor and the second gas sensor.
• the tissue sample is a vascular composite allograft.
• the vascular composite allograft is at least a portion of a limb, face, larynx, trachea, abdominal wall, genitourinary tissue, uterine tissue, or solid organ, or any - 9 - ⁇ combination thereof.
• the tissue sample is a donor vascular composite allograft for vascular composite allograft transplantation.
• the tissue sample is obtained from a human, a primate, or a pig. In one embodiment of the method, the tissue sample is a fasciocutaneous flap.
• the present disclosure provides a perfusion system for a tissue sample, wherein the system comprises: a perfusion fluid source; an inflow conduit in fluid communication with the perfusion fluid source and the sample, the inflow conduit being configured to deliver perfusion fluid to the sample; an outflow conduit in fluid communication with the sample, the outflow conduit being configured to carry perfusion fluid away from the sample; a first gas sensor in fluid communication with the inflow conduit, the first gas sensor measuring a concentration of a first gas in the perfusion fluid in the inflow conduit; a second gas sensor contacting the sample, the second gas sensor measuring 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, the controller being configured to execute a program stored in the controller to: (i) receive electrical signals from the first gas sensor and the second gas sensor
• 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 the first gas sensor and the second concentration of the second gas adjacent the second gas sensor. - 10 - ⁇ [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 when a calculated viability based on the first concentration of the first gas adjacent the first gas sensor and the second concentration of the second gas adjacent the second gas sensor exceeds a threshold viability value.
• 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 the first gas sensor and the second concentration of the second gas adjacent the second gas sensor.
• the perfusion system further comprises: a pump in electrical communication with the controller, wherein the controller is configured to execute the program stored in the controller to adjust a flow rate of perfusion fluid flowing towards the sample based on the first concentration of the first gas adjacent the first gas sensor and the second concentration of the second gas adjacent the second gas sensor.
• the perfusion system further comprises: an oxygenator in electrical communication with the controller, wherein the controller is configured to execute the program stored in the controller to control oxygen level in perfusion fluid flowing towards the sample based on the first concentration of the first gas adjacent the first gas sensor and the second concentration of the second gas adjacent the second gas sensor.
• the perfusion system further comprises: a third gas sensor in fluid communication with the outflow conduit, the third gas sensor measuring a concentration of a third gas in perfusion fluid in the outflow conduit, wherein the controller is configured to execute the program stored in the controller to activate or deactivate delivery of the perfusion fluid flowing towards the sample and/or adapt perfusion parameters based on the first concentration of the first - 11 - ⁇ gas adjacent the first gas sensor and the second concentration of the second gas adjacent the second gas sensor, and the third concentration of the third gas adjacent the third gas sensor.
• 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 the first gas sensor and the second concentration of the second gas adjacent the second gas sensor, and the third concentration of the third gas adjacent the third gas sensor.
• the controller is configured to execute the program stored in the controller to deactivate delivery of the perfusion fluid when a calculated viability based on the first concentration of the first gas adjacent the first gas sensor and the second concentration of the second gas adjacent the second gas sensor , and the third concentration of the third gas adjacent the third gas sensor exceeds a threshold viability value.
• 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 the first gas sensor and the second concentration of the second gas adjacent the second gas sensor, and the third concentration of the third gas adjacent the third gas sensor.
• the perfusion system further comprises: a pump in electrical communication with the controller, wherein the controller is configured to execute the program stored in the controller to adjust a flow rate of perfusion fluid flowing towards the sample based on the first concentration of the first gas adjacent the first gas sensor and the second concentration of the second gas adjacent the second gas sensor, and the third concentration of the third gas adjacent the third gas sensor.
• the perfusion system further comprises: an oxygenator in electrical communication with the controller, wherein the controller is configured to execute the program stored in the controller to control oxygen level in perfusion fluid flowing towards the sample based on electrical signals received from the first gas sensor and the second gas sensor, and the third concentration of the third gas adjacent the third gas sensor.
• the controller is in electrical communication with a controllable valve in the inflow conduit, and the controller is configured to execute the 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.
• the controller is in electrical communication with a controllable valve in the inflow conduit, and the controller is configured to execute the 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, or 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.
• the present disclosure provides a method for machine perfusion of a tissue sample.
• the method comprises: (a) providing a tissue sample; (b) delivering a 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 adapting - 13 - ⁇ perfusion parameters based on the first concentration of the first gas and the second concentration of the second gas.
• step (e) comprises activating delivery of the perfusion fluid when a loss of viability is calculated based on the first concentration of the first gas adjacent the first gas sensor and the second concentration of the second gas adjacent the second gas sensor.
• step (e) comprises deactivating delivery of the perfusion fluid when a calculated viability based on the first concentration of the first gas adjacent the first gas sensor and the second concentration of the second gas adjacent the second gas sensor exceeds a threshold viability value.
• step (e) comprises intermittently activating or deactivating delivery of the perfusion fluid based on the first concentration of the first gas adjacent the first gas sensor and the second concentration of the second gas adjacent the second gas sensor.
• step (e) comprises adjusting a flow rate of perfusion fluid flowing towards the sample based on the first concentration of the first gas adjacent the first gas sensor and the second concentration of the second gas adjacent the second gas sensor.
• step (e) comprises controlling oxygen level in perfusion fluid flowing towards the sample based on the first concentration of the first gas adjacent the first gas sensor and the second concentration of the second gas adjacent the second gas sensor.
• the present disclosure provides a perfusion system for a tissue sample, wherein the system comprises: a perfusion fluid source; an inflow conduit in fluid communication with the perfusion fluid source and the sample, the inflow conduit being configured to deliver perfusion fluid to the sample; an outflow conduit in fluid communication with the sample, the outflow conduit being configured to carry perfusion fluid away from the sample; a first gas sensor - 14 - ⁇ in fluid communication with the inflow conduit, the first gas sensor 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 conduit, the second gas sensor measuring a concentration of a second gas in perfusion fluid in the outflow conduit; and a controller in electrical communication with the first gas sensor and the second gas sensor, the controller being configured
• 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 the first gas sensor and the second concentration of the second gas adjacent the second gas sensor.
• the controller is configured to execute the program stored in the controller to deactivate delivery of the perfusion fluid when a calculated viability based on the first concentration of the first gas adjacent the first gas sensor and the second concentration of the second gas adjacent the second gas sensor exceeds a threshold viability value.
• 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 the first gas sensor and the second concentration of the second gas adjacent the second gas sensor. - 15 - ⁇ [0057]
• the perfusion system further comprises: a pump in electrical communication with the controller, wherein the controller is configured to execute the program stored in the controller to adjust a flow rate of perfusion fluid flowing towards the sample based on the first concentration of the first gas adjacent the first gas sensor and the second concentration of the second gas adjacent the second gas sensor.
• the perfusion system further comprises: an oxygenator in electrical communication with the controller, wherein the controller is configured to execute the program stored in the controller to control oxygen level in perfusion fluid flowing towards the sample based on the first concentration of the first gas adjacent the first gas sensor and the second concentration of the second gas adjacent the second gas sensor.
• the perfusion system further comprises: a third gas sensor contacting the sample, the third gas sensor measuring a concentration of a third gas in the sample, wherein the controller is configured to execute the program stored in the controller to activate or deactivate delivery of the perfusion fluid flowing towards the sample and/or adapt perfusion parameters based on the first concentration of the first gas adjacent the first gas sensor and the second concentration of the second gas adjacent the second gas sensor, and the third concentration of the third gas adjacent the third gas sensor.
• 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 the first gas sensor and the second concentration of the second gas adjacent the second gas sensor, and the third concentration of the third gas adjacent the third gas sensor. - 16 - ⁇ [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 when a calculated viability based on the first concentration of the first gas adjacent the first gas sensor and the second concentration of the second gas adjacent the second gas sensor , and the third concentration of the third gas adjacent the third gas sensor exceeds a threshold viability value.
• 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 the first gas sensor and the second concentration of the second gas adjacent the second gas sensor, and the third concentration of the third gas adjacent the third gas sensor.
• the perfusion system further comprises: a pump in electrical communication with the controller, wherein the controller is configured to execute the program stored in the controller to adjust a flow rate of perfusion fluid flowing towards the sample based on the first concentration of the first gas adjacent the first gas sensor and the second concentration of the second gas adjacent the second gas sensor, and the third concentration of the third gas adjacent the third gas sensor.
• the perfusion system further comprises: an oxygenator in electrical communication with the controller, wherein the controller is configured to execute the program stored in the controller to control oxygen level in perfusion fluid flowing towards the sample based on electrical signals received from the first gas sensor and the second gas sensor, and the third concentration of the third gas adjacent the third gas sensor.
• the controller is in electrical communication with a controllable valve in the inflow conduit, and the controller is configured to execute the - 17 - ⁇ 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.
• the controller is in electrical communication with a controllable valve in the inflow conduit, and the controller is configured to execute the 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, or 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.
• the present disclosure provides a method for machine perfusion of a tissue sample.
• the method comprises: (a) providing a tissue sample; (b) delivering a 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 adapting perfusion parameters based on the first concentration of the first gas and the second concentration of the second gas.
• step (e) comprises activating delivery of the perfusion fluid when a loss of viability is calculated based on the first concentration of the first gas adjacent the first gas sensor and the second concentration of the second gas adjacent the second gas sensor.
• step (e) comprises deactivating delivery of the perfusion fluid when a calculated viability based on the first concentration of the first gas adjacent the first - 18 - ⁇ gas sensor and the second concentration of the second gas adjacent the second gas sensor exceeds a threshold viability value.
• step (e) comprises intermittently activating or deactivating delivery of the perfusion fluid based on the first concentration of the first gas adjacent the first gas sensor and the second concentration of the second gas adjacent the second gas sensor.
• step (e) comprises adjusting a flow rate of perfusion fluid flowing towards the sample based on the first concentration of the first gas adjacent the first gas sensor and the second concentration of the second gas adjacent the second gas sensor.
• step (e) comprises controlling oxygen level in perfusion fluid flowing towards the sample based on the first concentration of the first gas adjacent the first gas sensor and the second concentration of the second gas adjacent the second gas sensor.
• FIG.1 is a schematic diagram of the perfusion system, along with the fasciocutaneous flap and the oxygen monitoring devices, according to aspects of the present disclosure.
• FIG.2A is a side view of the film of the perfusion system of FIG.1 over a perfusion fluid source.
• FIG.2B is a side view of the film of the perfusion system of FIG.1 over a tissue sample.
• FIG.3 is a perspective view of the sensor head of the perfusion system of FIG.1.
• FIG.4 is a flow chart of the method for machine perfusion of a tissue sample, according to aspects of the present disclosure.
• FIG.5 is a plot of the monitoring of the transcutaneous (TcPO 2 orange curve, right Y axis) and circulating inflow (PO2, blue curve, left Y axis) oxygen values during extended intermittent perfusion of a fasciocutaneous flap. Inflow values were compared to intermittent sampling and gas analysis in the perfusate. The inset depicts the time delay between the rise in oxygen of the perfusate and the skin.
• FIG.6 is a plot of the measurements in a 3-device system providing transcutaneous (orange curve, right Y axis), circulating inflow (dark blue, left Y axis) and outflow (light blue, left Y axis) oxygen values.
• FIG. 7 is a plot of the statistically significant correlation between continuous PO2 values provided by the flowcell device and by a Siemens Rapidpoint 500 blood gas analyzer. The 95% confidence interval is represented in dashed lines.
• FIG. 8A is the maximum PO2 and TcPO2 values during ex-vivo perfusion of a fasciocutaneous flap.
• FIG. 8B is the minimum PO2 and TcPO2 values during ex-vivo perfusion of a fasciocutaneous flap.
• FIG. 8D is the delay between circulating inflow PO 2 and skin TcPO 2 showing a similar trend towards disconnection between inflow and skin values. The vascular resistance is also plotted, showing a strong correlation with the delay. - 20 - ⁇ [0086]
• Like reference numerals will be used to refer to like parts from Figure to Figure in the following description of the drawings.
• the method of assessing perfused tissue viability described herein involves continuous measurement of partial oxygen pressure (pO 2 ) at multiple locations, including but not limited to the oxygen-rich (inflow) and oxygen-depleted (outflow) perfusate, at depths in the tissue, and the surface of the perfused tissue, and correlation of the measurement results.
• pO 2 partial oxygen pressure
• One embodiment employs: (a) a tissue perfusion system that circulates oxygenated perfusate through tissue via vascular connection and drainage; (b) oxygen- sensing flow-cell within or part of the system tubing and a needle, microneedle, or skin patch type materials placed into/onto the perfused tissue surface, respectively; (c) programmable, electronic readout sensors for the oxygen-sensing materials for collecting the signal; (d) an algorithm loaded onto a computer or mobile device that controls the readout devices that may log, process and report the signal output from multiple devices in realtime in the form of pO2 values; and (e) an algorithm loaded onto a computer or mobile device that controls the perfusion system pump and pressure sensor for adapting the perfusion parameters depending on the oxygen measurement levels.
• the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.”
• the terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims.
• the terms “consist” and “consisting of” should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims.
• the term “consisting essentially of” should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.
• the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”
• All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges.
• a range includes each individual member.
• a group having 1-3 members refers to groups having 1, 2, or 3 members.
• a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth.
• the modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same.
• FIG.1 shows an example embodiment of the perfusion system 100 of the disclosure.
• the perfusion system 100 includes a perfusion fluid source 102.
• the perfusion fluid 102 is an acellular crystalloid solution.
• the system 100 includes a membrane oxygenator 104 allowing for oxygenation of the circulating perfusion fluid 102.
• the membrane oxygen may receive its oxygen supply 150 via tube 151.
• the system 100 further includes an inflow conduit 106 in fluid communication with the perfusion fluid source 102 and a tissue sample 108 via an artery.
• the tissue sample 108 is a fasciocutaneous flap.
• the inflow conduit 106 is a tube.
• the tube may be, but is not limited to, silicone tubing or polyvinyl chloride (PVC) tubing.
• the system 100 further includes an outflow conduit 110 in fluid communication with the sample, the outflow conduit 110 being configured to carry perfusion fluid away from the sample.
• the outflow conduit 110 may be the same material as the inflow tubing 106.
• the perfusion fluid carried away from the tissue sample 108 by a vein via the outflow conduit 110 has perfused through the sample 108.
• the system 100 further includes a first gas sensor 112 in fluid communication with the inflow conduit 106, and a second gas sensor 114 contacting the sample 108.
• the first gas sensor 112 measures a concentration of a gas in the perfusion fluid 102, while the second gas sensor 114 measures a concentration of a gas in the sample 108.
• the perfusion system 100 includes a third gas sensor 116 as shown in FIG.1.
• the third gas sensor 116 may be identical to the first gas sensor 112 as previously described.
• the third gas sensor is in fluid communication with the outflow conduit 110, configured to measure a concentration of a gas in perfusion fluid in the outflow conduit 110.
• the perfusion fluid may be a Composition of Steen+ Solution – Massachusetts General Hospital, Center for Engineering in Medicine and Surgery, Department of Surgery, Harvard Medical School, Boston MA 02114 USA.
• the Steen+ solution contains, in a de- ionized water basis : NaCl : 86 mmol/L KCl : 4.6 mmol/L CaCl 2 .2H 2 0 : 1.5 mmol/L NaH2PO4 : 1.2 mmol/L NaHCO 3 : 16 mmol/L MgCl2.6H2O : 1.2 mmol/L D-Glucose : 22 mmol/L Polyethylene Glycol (PEG) 35Kda : 5g/L Bovine Serum Albumin : 15 g/L Insulin : 200 UI/L Hydrocortisone : 10mg/L Heparin : 200 UI/L Piperacillin-Tazobactam : 2.25G/L Vancomycin : 1.5G/L [0099]
• the first gas sensor 112 and optionally the third gas sensor 116 include a sensor head 118 and flow cell 120 containing a film 122.
• the flow cell 120 of the first gas sensor is in communication with the inflow conduit 106, while the flow cell 120 of the third gas sensor is in communication with the outflow conduit 110, and the film 122 contained within the flow cell 120 is in contact with the perfusion fluid 102.
• the flowcell can be incorporated into the perfusion system by cutting the inflow tubing and plugging the flowcell inside the tubings, restoring the continuity of the tubing.
• the flowcell is designed to have an internal diameter close to one of the silicone tubings.
• the outflow sensor can be similarly placed.
• the flow cell 120 includes a first opening 124 configured to allow the perfusion fluid 102 to enter the flow cell 120 and a second opening 126 configured to allow the perfusion fluid 102 to exit the flow cell 120.
• the film 122 or 123 is configured to undergo a change in response to pO 2 .
• the film 122 or 123 can have a light-based, photoluminescent oxygen sensing formulation that can have emission properties that can provide tissue pO2.
• the film 122 or 123 can include an oxygen- sensing polymer or polymer containing an oxygen-sensing molecule. As shown in FIG.
• the film 122 or 123 may include three layers: (i) a transparent membrane 202 forming an outer layer of the film, (ii) a layer of polymeric material 204 embedded with an oxygen-sensing lumiphore 206 adjacent to the transparent membrane 202, and (iii) a scattering layer 208 (such as a polymeric film pigmented with white particles) in contact with the layer of the polymeric material 204 and the perfusion fluid 102.
• the oxygen-sensing lumiphore 206 can be a metalloporphyrin can emit red phosphorescence when excited by blue light, and the phosphorescence intensity and lifetime 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 to serve as a reference standard for precise pO 2 measurements.
• the polymer may be, but is not limited to poly(propyl methacrylate) (PMMA) or polydimethylsiloxane (PDMS).
• PMMA poly(propyl methacrylate)
• PDMS polydimethylsiloxane
• silicones such as PDMS can be extremely gas-permeable and can allow rapid readout of tissue oxygen dynamics.
• porphyrin-based, oxygen sensing molecules 206 embedded in the polymeric material 204 are designed to provide extremely high sensitivity and accuracy for the measurement of tissue oxygenation.
• Porphyrin-based, oxygen sensing molecules 206 can be built via a modular synthetic pathway that enables the tailoring of both the oxygen sensing molecules' oxygen sensitivity range and the oxygen sensing molecules' compatibility with the matrix material the oxygen sensing molecules can be embedded in.
• the matrix material can further be configured to tailor the oxygen sensing molecules' oxygen sensitivity range.
• the change in the film 122 or 123 includes a change in the phosphorescence.
• the oxygen sensing molecules 206 can be specifically designed to feature bright, red phosphorescence emission, with a visual response to changes in oxygenation level that can be seen under ambient light. These properties simplify the collection and interpretation of their oxygen-dependent emission, enabling the analysis to be performed with simple and inexpensive equipment.
• the oxygen-sensing lumiphore 206 comprises a phosphorescent meso- unsubstituted porphyrin having the Formula (I): ⁇ wherein M is a metal, wherein each R is independently an atom or a group of atoms, and wherein at least one R is —OR 1 , wherein R 1 is an atom or a group of atoms.
• R 1 may be selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkyl carbonyl, 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.
• R 1 is hydrogen.
• R 1 is alkynyl, such as 2-propynyl (propargyl).
• R 1 is alkyl carbonyl, such as 2,2- dimethylpropanoyl (also known as trimethylacetyl or pivaloyl).
• a plurality of R can be —OR 1 , and optionally, every R can be —OR 1 .
• R 1 includes a triazolyl group. The triazolyl group may be bonded to O via an alkyl chain.
• R 1 includes an alkylglutamate group. R 1 may terminate in a pair of alkylglutamate groups. In another example of the porphyrin of Formula (I), R 1 includes a triazolyl group, and R 1 terminates in a pair of ethylglutamate groups, and every R is —OR 1 . In one example of the porphyrin of Formula (I), the metal is platinum or palladium.
• the porphyrin of Formula (I) may be an oxygen-sensitive phosphor whose emission intensity is dependent on oxygen partial pressure.
• the porphyrin in one example of the porphyrin of Formula (I), can be excited when illuminated at a first wavelength in a range of 350-600 nanometers, followed by emission of phosphorescence at a second wavelength in a range of 600- 700 nanometers.
• the first wavelength can be 532 nanometers
• the second wavelength can be - 28 - ⁇ 644 nanometers.
• the first wavelength can also be 546 nanometers and the second wavelength can be 674 nanometers.
• the oxygen-sensing lumiphore 206 comprises a phosphorescent meso-unsubstituted porphyrin having the Formula (II): wherein M is a metal, wherein of atoms, and wherein at least one R is —OR 1 , wherein R 1 is an atom or a group of atoms.
• R 1 may be selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkyl carbonyl, 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.
• R 1 is hydrogen.
• R 1 is alkynyl, such as 2- propynyl (propargyl).
• R 1 is alkyl carbonyl, such as 2,2-dimethylpropanoyl (also known as trimethylacetyl or pivaloyl).
• a plurality of R can be —OR 1 , and optionally, every R can be —OR 1 . - 29 - ⁇ [00110]
• R 1 includes a triazolyl group. The triazolyl group may be bonded to O via an alkyl chain.
• R 1 includes an alkylglutamate group. R 1 may terminate in a pair of alkylglutamate groups.
• R 1 includes a triazolyl group, and R 1 terminates in a pair of ethylglutamate groups, and every R is —OR 1 .
• the metal is platinum or palladium.
• the porphyrin of Formula (II) may be an oxygen-sensitive phosphor whose emission intensity is dependent on oxygen partial pressure.
• the porphyrin in one example of the porphyrin of Formula (II), can be excited when illuminated at a first wavelength in a range of 350-650 nanometers, followed by emission of phosphorescence at a second wavelength in a range of 700- 800 nanometers.
• the first wavelength can be 594 nanometers
• the second wavelength can be 740 nanometers.
• the first wavelength can be 605 nanometers
• the second wavelength can be 770 nanometers.
• the first wavelength can also be 600-615 nanometers and the second wavelength can be 760-800 nanometers.
• the second gas sensor 114 includes the photoluminescent oxygen-sensitive probe comprising a polymeric material impregnated with a porphyrin as disclosed for the first and third gas sensors 112, 116 above.
• the photoluminescent oxygen- sensitive probe is a formulation having an emission that provides tissue pO 2 .
• the second gas sensor 114 is a skin sensor placed within or on a fasciocutaneous flap after its harvesting, and before starting the perfusion.
• the second gas sensor 114 is an oxygen sensing needle that can be placed within the center tissue 108.
• a sensor film can be covered by a transparent adhesive dressing (for example TegadermTM, 3M, St Paul, MN).
• the film 123 as previously described above is in contact with the - 30 - ⁇ surface of the tissue sample 108.
• the scattering layer 208 is in contact with polymeric material 204 and a surface of the sample 108.
• the second gas sensor 114 provides tissue pO 2 .
• the sensor head 118 can interface with a porphyrin described in U.S. Patent Application Publication No.2016/0159842, which is incorporated herein by reference.
• the porphyrin may be an oxygen-sensitive phosphor whose emission intensity is dependent on oxygen partial pressure.
• the first, second, and third gas sensors 112, 114, 116 can each incorporate a probe head 118 placed in contact with a film 122 or 123 containing a light-based oxygen sensing formulation that can have an emission that can provide tissue pO 2 , emission sources, and detectors that can be mounted on the gas sensors 112, 114, 116.
• the sensor heads may be those previously described in PCT Patent Application Publication No. WO 2017/197385, which is herein incorporated by reference.
• the first, second, and third gas sensor 112, 114, 116 can have a light-based, photoluminescent oxygen sensing formulation in the film 122 or 123, that can have an emission that can provide tissue pO2.
• the sensor heads 118 can interface with an oxygen- sensing film 122 or 123 or polymer containing an oxygen-sensing molecule.
• the second gas sensor 114 can be positioned to be in direct contact with the sample tissue 108.
• the probe heads 118 can be in the form of a circular pad that interfaces with a film containing oxygen sensing molecules.
• the film can also contain other sensors such as - 31 - ⁇ reference sensors.
• the reference sensors can provide a baseline for oxygenation measurement or a reference for calibrating the oxygen sensor.
• the probe head 118 can be integrated in a single package with the flow cell 120 and sensing film 122.
• the second gas sensor 114 can have an interface mechanism that can be configured to place the probe head 118 in contact with the film 123 sample tissue 108.
• the interface mechanism can provide contact between the second gas sensor 114 and a tissue 108 using an adhesive that adheres an interface surface of the second gas sensor 114 to the tissue 108.
• the interface mechanism in the form of an adhesive can directly and reversibly adhere the second gas sensor 114 to the tissue 108.
• the interface mechanism can be a strap, a band, an elastic element, a pocket, or any other suitable interface mechanism capable of placing the second gas sensor 114 in contact with a tissue of a patient.
• a seal may be utilized between the film 122 and the tissue 108.
• the first, second, and third gas sensors 112, 114, 116 each include a sensor head 118.
• the sensor head 118 may include a photon source configured to direct photons at the photoluminescent oxygen-sensitive film 122 or 123.
• the sensor head 118 may include a photodetector configured to detect light emitted from the photoluminescent oxygen-sensitive film 122 or 123 when the photon source directs photons at the photoluminescent oxygen-sensitive film 122.
• the sensor head 118 can also include a controller in electrical communication with the photon source and the photodetector, the controller being configured to execute a program stored in the controller to calculate a concentration of oxygen adjacent to the photoluminescent oxygen- sensitive film 122 or 123 form an electrical signal received from the photodetector.
• FIG. 3 is a non-limiting example of a sensor head 118.
• circuit board 302 containing the photon sources 304 and the detectors 306 that can be attached to the sensor head - 32 - ⁇ 300.
• the circuit board 302 can be attached to the sensor head 300 on an opposite side of the sensor head relative to the photon sources 304 and detectors 306.
• the circuit board 302 can be linked to the sensor heads 300 via optical fibers (not shown).
• the circuit board 302 can be flexible and can have a substrate, wherein the emission sources 304 and the detectors 306 can be embedded in or on the circuit board 302 substrate.
• the emission sources 304 can be positioned in or on the substrate such that each photon source 304 is positioned to emit photons directed at the film 122 or 123.
• the photon sources 304 are positioned at four radial positions around the circuit board 302 such that the photon sources 304 can direct photons to the film 122 or 123.
• the photon sources 304 can be a blue light-emitting diode.
• the photon sources 304 can be a green, yellow, or orange light-emitting diode.
• the photon sources 304 can be optical fibers that deliver specific colors of light.
• the detectors 306 can be positioned in or on the substrate such that each of the detectors 306 is positioned to detect or receive photons emitted from the film 122 or 123.
• the detectors 306 are positioned at four radial positions around the circuit board 302 such that the detectors 306 can detect or receive photons emitted from the film 122.
• the detectors 306 on the circuit board 302 can comprise one or more photodetectors.
• the photodetectors can be configured to be sensitive to different wavelengths of light.
• the detectors 306 can be a photodiode.
• the one or more green photodetectors can be green photodiodes, and the one or more red photodetectors can be photodiodes.
• the detectors 306 can be a charge-coupled device (CCD).
• CCD charge-coupled device
• the detectors 306 can be optical fibers that couple emitted light to - 33 - ⁇ photodetectors, including photodiodes and charge-coupled devices (CCDs). In other embodiments, the photodetectors can detect both green and red emission.
• the circuit board 302 can also comprise a controller 308 that can be in electrical communication with the photon sources 304 and the detectors 306.
• the controller 308 can be a microcontroller, or system-on-a-chip, and can comprise a memory which can be a non-transitory memory that can store executable programs on the controller 308.
• the controller 308 can store an oxygen calculation program that can calculate a level of oxygen adjacent the sensor head 118, 300 from one or more electrical signals received from the detectors 306.
• the circuit board 302 can also include an output 310, which can be a wire bundle.
• the output 310 can connect to an external interface 128 in FIG. 1 which can be used for at least one of displaying, storing, and analyzing the results of the executable program.
• the output 310 can connect to the external interface 128 via a universal serial bus (USB) hub 130.
• USB universal serial bus
• the controller 308 can be configured to have a wireless output; the wireless output can perform wireless communication.
• Non-limiting examples of wireless communication that can be incorporated are Wi-Fi, Bluetooth ® , near-field communication, cellular network, radiofrequency, etc.
• the circuit board 302 can comprise an on-board power source (not shown), for example a battery, that can provide power to the circuit board 302 such that the photon sources 304, the detectors 306, and the controller 308 can be powered.
• the circuit board 302 can comprise an external power source (not shown), for example electrical connection to grid power, that can provide power to the circuit board 302 such that the photon sources 304, the detectors 306, and the controller 308 can be powered.
• optical first, second, and third gas sensors 112, 114, 116 can directly measure pO 2 , it does not require perfusion with oxygen carriers such as red blood cells or whole blood.
• the - 34 - ⁇ sensing components can be safely retained within the sensor head 118, 300, which can be non- invasive, and as such, exogenous dyes, injectable agents, and needles may not be required.
• the gas sensors 112, 114, 116 can require low preparation time, and a readout can be essentially instantaneous.
• the sensor head 118 can be placed in contact with the film 122 in the flow cell 120 or with the film 123 on the tissue sample 108 (FIGS. 2A-2B).
• films 122 and 123 can include an oxygen-sensing polymer that can be in direct contact with the perfusion fluid 102 or tissue sample 108 of the patient. Direct contact between the sensor head 118 and the film 123 on the tissue of the patient can allow for the sensor head 118 to provide tissue pO2 via the photoluminescent oxygen sensing formulation of the film 123.
• the photoluminescent oxygen sensing formulation of the film 123 can have an emission that can be indicative of the tissue pO 2 .
• the photoluminescent oxygen sensing formulation of the films 122 and 123 can emit red phosphorescence when excited by blue light from one or more of the photon sources 304, with a phosphorescence intensity detected by one or more of the detectors 306.
• the phosphorescence intensity can be inversely proportional to pO 2 of the tissue of the patient.
• a reference sensor which can be in the form of a green-emitting dye, can also be loaded into the films 122 and 123 and can serve as a reference standard for precise pO2 measurements.
• the phosphorescence lifetime can be also inversely proportional to pO 2 of the tissue of the patient, and alternatively or additionally measured and analyzed to provide precise pO2 measurements.
• the circuit board 302 can be flexible and can be attached to a surface of the sensor heads 118 via an oxygen-impermeable membrane.
• the circuit board 302 can contain photon sources 304 and detectors 306.
• the photon sources 304 can be one or more blue light emitting diodes (LEDs), and the detectors 306 include one or more photodetectors in the form of green sensitive photodiode detectors and one or more red photodetectors in the form of red sensitive photodiode detectors.
• the detectors can be a photodiode, photomultiplier tube, avalanche photodiode, charge-coupled device (CCD), complementary metal-oxide semiconductor (CMOS) device, or combination of similar photodetectors.
• CCD charge-coupled device
• CMOS complementary metal-oxide semiconductor
• the oxygen-dependent change in red phosphorescence intensity can be captured by the red photodetectors in the form of red sensitive photodiode detectors and referenced against the green emission captured by the green photodetectors in the form of green sensitive photodiode detectors using analog circuitry of the controller 308 to provide pre-calibrated, robust transcutaneous oxygen tension measurements of the sample 108.
• the oxygen-dependent change in phosphorescence lifetime can be captured by the photodetectors in the form of photodiode detectors.
• LED illumination can be modulated (e.g., by a sinusoidal wave), causing a (e.g., sinusoidally) modulated phosphorescence emission from the oxygen sensors whose time delay with respect to the excitation light can be measured to calculate phosphorescence lifetime, and thus pO2.
• the analog or digital circuitry of the controller 308 can provide pre-calibrated, robust oxygen tension measurements of the tissue of the patient.
• the oxygen tension measurements can be analyzed and reported by utilizing molecules (e.g., fluorophore probes) whose emission properties are insensitive to oxygen along with molecules (e.g., phosphor probes) whose emission properties are influenced by molecular oxygen concentration.
• molecules e.g., fluorophore probes
• phosphor probes molecules whose emission properties are insensitive to oxygen
• the - 36 - ⁇ molecules whose emission properties are insensitive to oxygen can be the reference sensor, and the molecules whose emission properties are influenced by molecular oxygen concentration can be the phosphor probes. Emission from the fluorophore probes and phosphor probes can be used to measure oxygen tension in biological systems reversibly with high fidelity.
• the fluorophore probes and phosphor probes can be calibrated so that a spectral ratio between fluorophore and phosphor emission correlates with oxygen concentration of the tissue sample 108.
• the calibration can be used to read out a map of oxygen concentration in the tissue of the patient.
• the calibration can also be used to read out an average oxygen concentration of the area covered by the first, second, or third gas sensor 112, 114, 116.
• the fluorophore probes and phosphor probes can be calibrated so that the lifetime of the fluorophore and phosphor emission can be analyzed to provide the oxygen concentration of the tissue sample 108.
• the analog or digital circuitry of the controller 308 can provide pre- calibrated, robust transcutaneous oxygen tension measurements and analysis of the tissue sample 108 using the Stern-Volmer relationship.
• the Stern-Volmer relationship can be used to characterize the oxygenation of the fluid 102 or sample 108 based on the photoluminescent oxygen sensing formulation of the film 122 or 123, respectively, that can emit red phosphorescence when excited by blue light from the at least one photon source 304, with a phosphorescence intensity and/or lifetime detected by the at least one detector 306 can be inversely proportional to pO2 of the tissue of the patient.
• a dynamic (collisional) quenching by oxygen is a photophysical (rather than a photochemical) process. It is fully reversible, does not alter the optical probe, and thus has - 37 - ⁇ no effect on its absorption spectrum.
• [O 2 ] (a concentration) may be replaced by pO 2 , the partial pressure of oxygen.
• [00130] there can be a linear relationship between F0/F (or IJ0/IJ) and oxygen concentration.
• Stern-Volmer plots (SVPs) can be established by measurement of either luminescence intensity or lifetime.
• luminescence intensity data can be adversely affected by poor stability of the light source, variations in the efficiency of the transmission optics, drifts in detector sensitivity, leaching and photodecomposition of probes, inhomogeneous probe distribution, background luminescence and stray light.
• a reference sensor e.g., an inert reference fluorophore emitting at a different wavelength
• the controller 308 can analyze the lifetime and intensity of the phosphorescence emission to determine the oxygen concentration of the sample 108.
• the first, second, or third gas sensor 112, 114, 116 can communicate emission results acquired from the at least one detector 306 externally to be analyzed by an external device 128.
• the first and second sensor head 118 can also comprise a display 132. The display 132 can be configured to indicate the oxygenation of the tissue of the patient as determined by the first, second, or third gas sensors 112, 114, 116.
• the display can be attached to the sensor head 118, while in other embodiments, the display can be positioned externally, such as in the external interface 128.
• the second gas sensor comprises an oxygen-sensitive probe configured for insertion into the sample 108.
• the second gas sensor may include a transcutaneous sensor and a separate oxygen-sensing needle, as described herein.
• the oxygen-sensitive probe may be an oxygen-sensing needle and placed in the center of the tissue sample 108 to perform subdermal, intramuscular, or intra organ pO 2 measurements.
• the perfusion system 100 comprises an oxygenator 104 in fluid communication with the perfusion fluid source 102.
• the oxygenator 104 is connected to the inflow conduit 106. Further, the oxygenator 104 controls oxygen level in perfusion fluid flowing towards the sample 108.
• a controller such as the external interface device 128 in electrical communication with the first gas sensor, the second gas sensor, and/or 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 to calculate a concentration of oxygen adjacent the first gas sensor, the second gas sensor, and/or the third gas sensor.
• the controller such as the external interface device 128 in electrical communication with the first gas sensor, the second gas sensor, and/or third gas sensor is configured to execute a program stored in the controller in response to the - 39 - ⁇ information from the first, second, and/or third gas sensors to change or modulate the perfusion of the tissue sample.
• the controller 128 is in electrical communication 160 with a valve 162 to change or modulate the perfusion of the tissue sample.
• the perfusion system 100 may further include a pump 136 for circulating the perfusion fluid 102 through the oxygenator 104, inflow conduit 106, tissue sample 108, the outflow conduit 110, a reservoir 134, and a recirculating conduit 140.
• the perfusion system 100 may further include a bubble trap 138 positioned in line between the oxygenator 104 and the first gas sensor 112.
• the bubble trap 138 is configured to remove bubbles in the perfusion fluid 102 formed as a result of flowing through the oxygenator 104.
• the perfusion system 100 comprises a heat exchanger (not shown) for adjusting the temperature of the perfusion fluid.
• 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 providing a tissue sample.
• the tissue sample may be a vascular composite allograft.
• the vascular composite allograft is at least a portion of a limb, face, larynx, trachea, abdominal wall, genitourinary tissue, uterine tissue, or solid organ, or any combination thereof.
• the tissue sample is a donor vascular composite allograft for vascular composite allograft transplantation.
• the tissue sample is obtained from a human, a primate, or a pig.
• the tissue sample is a fasciocutaneous flap.
• a perfusion fluid is delivered to the sample via an inflow conduit.
• a concentration of a gas in the perfusion fluid in the inflow conduit is measured.
• the measurement is performed using a first gas sensor, such as the first gas sensor 112 as described above.
• a concentration of gas is measured in the sample.
• the measurement - 40 - ⁇ is performed using a second gas sensor, such as the second gas sensor 114 as described above.
• the measurement is performed using an oxygen-sensing needle as previously described.
• the first gas sensor, second gas sensor, third gas sensor, or all or a combination of sensors comprise an oxygen-sensitive probe.
• the first gas sensor, second gas sensor, third gas sensor, all or a combination of sensors comprise a film configured to undergo a change in response to pO 2 .
• step 406 further comprises sensing the concentration of gas in the perfusion fluid in the inflow conduit with a first gas sensor and calculating, in a controller, a concentration of oxygen adjacent to the first gas sensor from an electrical signal transmitted to the controller from the first gas sensor.
• step 408 further comprises measuring the concentration of the gas in the sample with the second gas sensor, and calculating, in the controller, a concentration of oxygen adjacent to the second gas sensor from an electrical signal transmitted to the controller from the second gas sensor.
• the method 400 may include step 410 comprising sensing the concentration of gas in the perfusion fluid in the outflow conduit with a third gas sensor and calculating, in a controller, a concentration of oxygen adjacent to the third gas sensor from an electrical signal transmitted to the controller from the third gas sensor.
• the method may comprise steps 402-406 and 410, omitting step 408.
• a report is generated, using the controller, of oxygen perfusion in the sample from measurements from the first, second, and/or third gas sensors.
• the method comprises controlling an oxygen level in the perfusion fluid flowing towards the sample based on electrical signals received from the first gas sensor, the second gas sensor, and/or the third gas sensor.
• the method comprises activating an alarm when a gas level drops below a defined threshold based on electrical signals received from the first gas sensor, second gas sensor, and/or third gas sensor or any calculation on the data collected from the sensors.
• the method comprises adapting the perfusion parameters to the data collected from the sensors, by sending a signal to the perfusion system’s pump or altering the perfusion system’s oxygenation parameters
• the tissue sample is perfused intermittently, i.e., generating perfusion cycles alternating between baseline and high oxygen partial pressure in the perfusate.
• a 3-device measurement (including measurements from the first, second, and third gas sensors) connected to intermittently perfused sample, provides, simultaneous, continuous, real-time inflow and outflow pO2 values in the perfusate as well as transcutaneous pO 2 values on the perfused tissue surface.
• Intermittent perfusion allows the observation of the response of the tissue to changes in oxygenation which is dependent on the state of the capillary bed of the sample, and therefore the tissue viability (e.g., correlation or decorrelation between the sensor measurements).
• tissue viability e.g., correlation or decorrelation between the sensor measurements.
• transcutaneous pO2 follows the trend of the inflow measurement matching the pO2 increase/decrease according to the operator-induced perfusate oxygenation cycles (oxygenator on/off, oxygen levels varied periodically, or oxygen levels varied through a programmable means).
• the outflow measurement may show a depletion in pO 2 as well as the same oxygenation cycle-dependent trend.
• tissue pO2 When the tissue becomes non-viable, no more change is observed in tissue pO2 over time (decorrelation) and - 42 - ⁇ the outflow measurement shows an increase in pO2 due to the tissue no longer consuming oxygen.
• Other quantities which reflect tissue non-viability can be the difference in oxygenation between inflow and sample, inflow and outflow.
• the time difference in the increase/decrease in oxygenation of the sample with respect to the perfusate at the inflow, or the sample with respect to the outflow, or the inflow with respect to the outflow shows a decrease indicative of the tissue becoming non-viable.
• Example [00147] Described herein are examples to demonstrate and further illustrate certain embodiments and aspects for the combination of intermittent perfusion and continuous measurement of pO2 of the perfusate entering/exiting the tissue and the pO 2 of the tissue itself (transcutaneous, intramuscular, etc.).
• the Example is not to be construed as limiting the scope of the invention.
• Overview of the Example [00148] The use of perfusion machines is rapidly growing in the field of transplantation. Vascularized Composite Allotransplantations and reconstructive surgery are not to be outdone with first descriptions of machine perfusion (MP) applications. The goal is to improve preservation by continuously delivering oxygen and nutrients essential for cellular activity while clearing metabolic waste, greatly decreasing ischemic complications during preservation.
• MP machine perfusion
• Oxygenation is a critical parameter to ensure tissue viability during ex-vivo MP.
• This work presents an innovative technology based on an oxygen-sensitive, phosphorescent metalloporphyrin, which allows continuous and non-invasive oxygen monitoring of vascularized fasciocutaneous flaps.
• the device comprises a transparent oxygen sensing film to be applied on the flap’s skin paddle, which is probed via a small, low-energy, electronic device that provides - 43 - ⁇ continuous monitoring of oxygen concentration. Additionally, this oxygen-sensing technology was used to monitor the oxygen levels of the perfusate inflow in parallel with the skin paddle’s oxygenation.
• the saphenous fasciocutaneous flap was harvested from the right groin of the animal.
• the flap limits - 44 - ⁇ and anatomic markings were drawn to allow an elliptical skin paddle with a short and long axis of 5.5 and 9 cm before incision, respectively.
• the surgical technique has been previously described by our team [Ref.21].
• the saphenous flap was harvested on the femoral vessels, allowing smooth cannulation of the artery and easy procurement of the outflow from the vein receiving two venae comitantes from the flap. Following meticulous dissection, the distal femoral vessels were ligated, and the flap was freed after division of the proximal portion.
• the vascularized flap was then flushed using 4°C heparinized saline until clear outflow. Permission from the IACUC could have been granted for on-table transfers, allowing other groups to harvest organs and tissues to optimize the number of animals used in the institution. Animals were euthanized at the end of the procedure. Fasciocutaneous Flap Ex-Vivo Perfusion [00151] Immediately after flushing, the flap was transported under a Class II biosafety hood. The 18G arterial cannula was linked to a customized machine perfusion system (FIG. 1: machine perfusion setup).
• a hollow fiber oxygenator (Affinity Pixie, Medtronic, Dublin, Ireland) was connected to an oxygen tank (95% O2, 5% CO2), which allowed for oxygenation of the circulating perfusate.
• a pressure transducer was incorporated into the closed system and allowed for continuous monitoring of the pressure.
• a modified Steen solution was used as a perfusate. The perfusate was circulating through the flap’s vascular tree, and the outflow was sampled through the cannulated femoral vein. The perfusate was recirculated and a full perfusate exchange was performed every 24 hours if needed. A total volume of 350 ml was circulating. Sodium bicarbonate could be used to correct the pH.
• the initial flow rate was based on our experience and perioperative color Doppler velocity measurements performed intraoperatively [Ref. 22].
• the perfusion rhythm was intermittent: The flap was perfused between 30 and 45 minutes, followed by an ischemic period of 75 to 90 minutes.
• the roller pump used in the perfusion system (DRIVE - 45 - ⁇ MASTERFLEX L/S, Cole-Parmer, Vernon Hills, Illinois, USA) allowed for programming the cycles.
• the delivered flow was nonpulsatile.
• Perfusate samples were procured frequently and included blood gas analyses (BGA) performed with a clinical-grade measurement system (Rapidpoint 500, Siemens, Kunststoff, Germany).
• the perfusion was terminated if the weight gain reached 50% of the initial weight, or if the vascular resistance was too high to allow a flow F ⁇ 50% of the initial value.
• Oxygen Monitoring [00152] The technology used in this Example is based on the detection of changes in the phosphorescence lifetime and intensity of an ultrabright metalloporphyrin molecules [Ref. 23], which exhibit oxygen quenching of phosphorescence. The change in the phosphor’s lifetime IJ and intensity I depends on oxygen following the Stern-Volmer relation [Ref.24].
• This phosphor can be readily embedded within polymer-based films, resulting in ultra-thin, breathable films that exhibit bright emission through the oxygen partial pressure (pO 2 ) range 0-760mmHg, and are impervious to changes in relative humidity [Ref.25, 26].
• pO 2 oxygen partial pressure
• an adhesive, medical grade film [Ref.27] which were applied to the skin paddle of the flap after rigorous drying.
• the oxygen sensing film is composed 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 with the embedded metalloporphyrins; (iii) a highly breathable white scattering layer which both enhances the collection of the phosphorescence and - 46 - ⁇ acts as optical insulation, improving the estimation of oxygenation and preventing external light interference.
• Oxygen partial pressure in the perfusate fluid is carried out with a 3D-printed flow cell containing the same polymer film and scattering layer in its top lining.
• a breathable layer of medical adhesive (3M) covers the scattering layer and acts as a physical barrier between the O 2 - sensing film and the circulating perfusate.
• the flow cell monitoring the perfusate inflow oxygenation was placed 4 cm upstream of the arterial cannula. Outflow oxygen was measured when possible, placing the flow cell 4 cm downstream the cannulated femoral vein.
• Changes in the phosphorescence of the O2-sensing layers are detected by a portable sensor, initially designed as a wearable transcutaneous oxygenation monitoring (TCOM) device [Ref.27].
• the prototype device’s sensor head is affixed to the films or flow cells centered over the O2-sensing layer, exciting the phosphorescence of the porphyrins via two ultraviolet LEDs, with the phosphorescence detected via a small photodiode.
• a small temperature sensor (thermistor) in the sensor head allows us to account for temperature-dependent effects in the phosphorescence and to produce a temperature compensated pO 2 reading.
• the sensor head is linked to the main control electronics, built around a commercially available microcontroller board (Particle Photon), which is then connected to a laptop through a USB serial port (FIG.1: Setup of the device on the flap + composition of the device).
• the devices allow us to continuously monitor oxygen levels of the tissue underneath (TcPO2) and the perfusate during experiments lasting multiple days, with a chosen sampling time of 30 seconds.
• the continuous oxygenation readings from all devices were saved and displayed real-time, in units of mmHg, via a Python [Ref.28] script on a PC through USB serial port.
• Oxygenation readings are - 47 - ⁇ obtained from the phosphorescence by previously calibrating the response of the materials and fitting the resulting dependence to pO 2 with the Stern-Volmer relation.
• Results [00158] We were able to simultaneously monitor the oxygen levels of the perfusate inflow in parallel with the skin paddle’s oxygenation in multiple experiments.
• FIG.5 plots the results from an experiment in which two devices were used, showing continuous readings of the circulating oxygen (PO 2 ) in the inflow and the skin oxygen levels (TcPO 2 , the right Y axis).
• PO 2 the circulating oxygen
• TcPO 2 the skin oxygen levels
• the perfusion rhythm was changed to 30 minutes ON / 75 minutes OFF.
• the mean total perfusion - 48 - ⁇ 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.
• We included the following variables in the statistical analysis obtained from each cycle: the maximum (Max) and minimum (Min) oxygen values in both skin (TcPO2) and in the inflow perfusate (PO2) upstream the arterial cannula; the difference ⁇ Max ⁇ Min for TcPO 2 and PO 2 for each cycle; the time delay between the measured inflow PO 2 and TcPO2.
• the delay was calculated by normalizing the inflow PO2 and TcPO2 between [0,1], and finding the time difference between both curves reaching a value of 0.4 (see inset of FIG.5).
• the described variables, extracted from the measurement in FIG. 6, are shown in FIGS. 8A-8D, where the following features are observed: • Both the maximum and minimum TcPO 2 experience a sharp drop following cycle #8. • Additionally, the difference between PO2 and TcPO2 increases at the end of the perfusion, corresponding with perfusion failure backed by substantial increase in vascular resistance. • The delay between circulating inflow PO 2 and skin TcPO 2 shows a sharp increase following cycle #8, which was also correlated to increasing vascular resistance (FIG 8D).
• One objective of this Example was to test the reactivity and reliability of continuous oxygen readings using a novel technology.
• the vascularized saphenous flaps used in the included experiments allowed testing circulating oxygen values, relevant to all machine-perfusion based approaches.
• the perfusion design with interruptions of 75 to 90 minutes, followed by active oxygenation of 30 to 45 minutes allowed periodically assessing the response during the experiments, providing a total of 76 cycles used as replicates.
• the proposed system was easily - 49 - ⁇ implemented in a custom-made perfusion system, and the user-friendly computer interface made it possible to be used by a non-expert surgical team.
• the saphenous flap model is relevant for the skin component of VCAs.
• tissue oxygenation of these complex organs such as face or hand transplants
• reconstructive surgeons could substantially improve the outcomes of these difficult procedures marked by long term immune rejection.
• IRI are indeed known to be implicated in VCA rejection by increasing antigen release in a highly immunogenic tissue such as skin. Similar to the potential contributions in the field of solid organ transplantation, the technology could be precious in these reconstructive transplants.
• the model used is also directly relevant for wide autologous reconstructive surgery involving flap transfer and monitoring. Fasciocutaneous flaps have become the gold standard in complex reconstructions over the last decade. Although the techniques are increasingly improved and mastered, a failure rate persists for pedicled and free flaps (2 to 10% according to authors).
• the delay of surgical revision is a significant element in the case of vascularization dysfunction of flaps.
• the precocity of revision is correlated with the flap salvage rate.
• flap monitoring is mainly clinical by assessing color, skin capillary refill time (CRT), and overall appearance. Those parameters show poor reactivity, since paleness, coldness and increase of the CRT occur late after acute vascular complications, highly compromising the flap survivability and salvage feasibility.
• Some authors described oxygen monitoring using Near- Infrared Spectroscopy (NIRS) and LiCox probes (Integra Lifescience, Princetown, New Jersey, - 51 - ⁇ USA) [Ref. 19, 29, 30].
• NIRS Near- Infrared Spectroscopy
• LiCox probes Integra Lifescience, Princetown, New Jersey, - 51 - ⁇ USA
• NIRS has the disadvantage of being discontinuous and hemoglobin dependent, while the diverted use of LiCox is invasive and highly susceptible to movement, using bulky and expensive equipment. Moreover, the simplicity of implementation and the readability of the measurements must be appropriate to allow efficiency in the clinical routine.
• the technology we describe in this Example allows live monitoring in a precise and reactive way of the oxygenation of fasciocutaneous flaps. This opens perspectives of efficient monitoring, with the possibility of activating an alarm when the oxygen level drops below a defined threshold postoperatively in order to alert the teams and optimize the chances of survival of the flap after surgical revision.
• the transcutaneous oxygenation measurement presented herein is influenced by atmospheric oxygen and provides readings which are slightly above tissue oxygen concentration.
• the true TcpO2 can be calculated via mathematical models [Ref. 31].
• This non-invasive tissue monitoring technique whose technology has been validated in another form for monitoring Deep Inferior Epigastric Perforator (DIEP) flaps, can be used for all free or pedicled flaps with a skin component.
• DIEP Deep Inferior Epigastric Perforator
• the early applications of machine perfusion in autologous procedures open new horizons in reconstructive surgery, as shown by the techniques developed by Wolff et al. [Ref. 32, 33]. They achieved free flap reconstruction with no anastomoses, by perfusing the transferred free flap with diluted compatible blood.
• Their innovative technique was limited by ischemic complications. In the field, no technology has been used to continuously monitor perfused organs in clinical transplantation.

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