Apparatus and method for organ preservation

Abstract

An oxygenation apparatus (10) for producing an organ preservation solution (56') comprising nanobubbles of gas including oxygen, includes a flow path for passage of a precursor organ preservation solution and a nanoporous gas diffuser (12). The flow path for passage of a precursor organ preservation solution (56) comprises at least one constricted portion followed by one or more expanded portion. The nanoporous gas diffuser (12) is adapted for connection to a source of gas including oxygen (32) and disposed for fluid contact with the precursor organ preservation solution (56) passing through the flow path. An organ preservation apparatus comprising an oxygenation apparatus is also provided. Methods of use of said apparatus in organ preservation are also provided.

Description

APPARATUS AND METHOD FOR ORGAN PRESERVATION TECHNICAL FIELD

[0001] The present disclosure relates generally to organ transplantation. More particularly, the present disclosure relates to apparatus and methods for transplant organ preservation. BACKGROUND

[0002] The ability to carry out successful organ transplants has made transplantation a feasible solution for thousands of transplant candidates. However, several hurdles have presented themselves in limiting organ transplants. A major constraint is the length of time that a donor organ will remain viable after it is retrieved as well as a need to be able to bench test and assess the viability and quality of extended criteria and marginal deceased donors. For example, current technology used with heart transplants is viable only if the donor heart is transplanted rapidly.

[0003] Considerable efforts have been made to improve organ preservation and extend the viability time of an organ. Current methods and devices typically involve hypothermic storage of the donor organ to cool the organ rapidly to slow down metabolism and minimise the deleterious effects of anaerobic metabolism secondary to ischemia of the organ. The common steps in organ procurement in deceased organ donation include circulatory arrest; rapid flush with hypothermic (4–7 degrees C) preservation solution that allows for rapid cooling of the organ and removal of red blood cells from the microcirculation, followed cold storage of the organ in a hyperosmolar solution while it is maintained in a hypothermic, non-functioning state. This is otherwise referred to as static cold storage.

[0004] The limitations of static cold storage in preserving organs for extended periods or marginal or extended criteria organs; in conjunction with the development of ECMO (Extracorporeal membrane oxygenation) technologies has created renewed interest in preservation with machine perfusion where a solution is actively pumped into an organ during the storage phase with a perfusate. This can be with blood when the perfusate is normothermic (eg body temperature) and oxygenated or with a hyperosmolar perfusate (eg Belzer machine perfusion solution) when hypothermic with or without oxygenation. 1005814927

[0005] At present in the USA, UNOS data for 2023 shows that there were 103,653 patients in need of a transplant and from January to November 2023, 21,201 donors resulted in 42,601 transplants. With increasing longevity as the demand and supply gap increases, it has become increasingly imperative to explore every potential donor and ideally bench assess and even resuscitate organs prior to transplant to increase the available donor pool.

[0006] There is a need for improved apparatus and methods for the preservation of an extracted organ enabling the preservation of an organ such as heart, kidney and cornea. SUMMARY

[0007] In accordance with a first aspect of the present invention, there is provided an oxygenation apparatus for producing an organ preservation solution comprising nanobubbles of gas including oxygen, the apparatus including: a flow path for passage of a precursor organ preservation solution, wherein the flow path comprises at least one constricted portion followed by at least one or more expanded portion; a nanoporous gas diffuser adapted for connection to a source of gas including oxygen, and disposed for fluid contact with the precursor organ preservation solution passing through the flow path.

[0008] The nanoporous gas diffuser is preferably a diffuser having open pores of a nanoscale dimension. The nanoporous gas diffuser may be in the form of a nanoporous membrane. Alternatively, the nanoporous gas diffuser may be in the form of a nanoporous “stone”. Typically, the nanoporous gas diffuser will be a nanoporous metal, preferably stainless steel. The nanoporous gas diffuser should be sterilisable. The range of sizes of bubbles being produced by the nanoporous gas diffuser is preferably within the range of approximately 500 nm in diameter or smaller. It is thought that the range of sizes of bubbles includes bubbles as small as about 2-3nm. In embodiments described below using modification of a dialysis machine, the dialysis cartridge has a membrane with a pore size < 2-3nm and results suggest that the nanobubbles are diffusing across the membrane, leading to the conclusion that the nanobubbles include nanobubbles having a size of less than about 2-3nm. 1005814927

[0009] Preferably, the effect of the flow through the flow path having one or more constricted portions, followed by at least one expanded portion is such that hydrodynamic cavitation occurs. It is thought that the shockwave resulting from the hydrodynamic cavitation leads to breaking up any bubbles into smaller bubbles. Given that nanobubbles are desired and anything larger than nanobubble size is undesirable, this effect is considered desirable. The one or more constricted portions followed by the at least one expanded portion may be in the form of a Venturi.

[0010] There may be a plurality of constricted portions including a first constricted portion and a second constricted portion, the second constricted portion being downstream of the first constricted portion. The second constricted portion may be spaced from the first constricted portion. Each constricted portion may have a progressively decreasing internal diameter / dimension / cross-sectional area. For instance, the rate of decrease of the internal diameter / dimension / cross-sectional area of the first constricted portion may be different to the rate of decrease of the second constricted portion.

[0011] Alternatively, the first constricted portion may be of a uniform inside diameter / dimension and the second constricted portion may be of a uniform inside diameter / dimension. In this form of the invention, the inside diameter / dimension of the second constricted portion may be less than the inside diameter / dimension of the first constricted portion. For instance, the constriction may take a stepped form.

[0012] Alternatively, the first constricted portion may have a variable internal diameter / dimension and the second constricted portion may have a uniform inside diameter / dimension. Vice versa is also possible.

[0013] The nanoporous gas diffuser may be disposed in the flow path. The nanoporous gas diffuser may be disposed upstream of the one or more constricted portions. Alternatively, the nanoporous gas diffuser may be disposed at the one or more constricted portions.

[0014] Preferably, the nanoporous gas diffuser is disposed in either of the first constricted portion or the second constricted portion. Alternatively, the nanoporous gas diffuser may be disposed in both of the first constricted portion and the second 1005814927constricted portion. For instance, the nanoporous gas diffuser may create either or both of the first constricted portion or the second constricted portion.

[0015] In a more preferred form of the invention, the nanoporous gas diffuser is positioned within the flow path to create the first constricted portion and the second constricted portion. For instance, the flow path may in part be defined by a tube in which the nanoporous gas diffuser is received. In a most preferred form of the invention, the tube may be closed at one end with an aperture approximately commensurate in size with the nanoporous gas diffuser, thereby creating a first constricted portion as a result of the nanoporous gas diffuser occluding part of the tube and a second constricted portion as a result of the exit to the tube i.e. the aperture, being substantially blocked by the nanoporous gas diffuser. For example, the aperture may be slightly oversized compared to the size of the nanoporous gas diffuser, creating a gap between the aperture and the nanoporous gas diffuser. For example, where the aperture is circular with an inside diameter, the nanoporous gas diffuser is preferably cylindrical with an outside diameter which is less than the inside diameter of the aperture. The nanoporous gas diffuser may comprise a diffusion “stone” which is cylindrical. The diffusion stone may be received in the tube and / or within the aperture(s). The diffuser may be supported to position it coaxially within the aperture, to create a uniform gap.

[0016] There may be aligned apertures in the tube to receive the nanoporous gas diffuser or merely a singular aperture. The aligned apertures may align with their central longitudinal axes extending substantially transversely to or aligned with the central longitudinal axis of the tube.

[0017] Alternatively, the transverse aperture(s) may be omitted from the tube and the nanoporous gas diffuser simply located within the tube. Preferably, the central longitudinal axis of the nanoporous gas diffuser is aligned with the central longitudinal axis of the tube and the outside diameter / dimension of the nanoporous gas diffuser is less than the inside diameter / dimension of the tube, thereby creating a gap therebetween. The diffuser may be supported to position it coaxially within the tube, to create a uniform gap.

[0018] The oxygenation apparatus may also include a bubble trap for escape of undesirable bubbles such as bubbles larger than nanobubble in size, e.g. microbubbles. The bubble trap may otherwise be referred to as a partial degasser. The bubble trap 1005814927may be in the form of a spillway or an overflow. For example, the tube may be housed within a container provided with a spillway or an overflow. In use, the tube may be suitably submerged within the container.

[0019] Accordingly, the organ preservation solution downstream of the diffuser and / or the constricted portion(s) may be permitted to overflow or spill over from the container in order to release the undesirable bubbles. In one preferred form of the invention, the container may be an inner container housed within an outer container, wherein the height of the wall(s) of the inner container is less than the height of the wall(s) of the outer container. Preferably, the outer container includes a fluid outlet positioned within a base of the container. The inner container also serves as the expanded portion of the flow path, downstream from the constricted portion at the exit from the tube.

[0020] Another form of bubble trap is a p trap by which the undesirable bubbles may escape. In the spillway and the p-trap, the fluid is at ambient room pressure i.e. equilibriated.

[0021] Another form of bubble trap could be a microfluidic bubble trap membrane. Alternative devices may rely upon suction to remove undesirable bubbles or activated charcoal to absorb undesirable bubbles.

[0022] Another form of bubble trap could be achieved through a liquid seal. For example, the organ preservation solution may enter a container or vessel through an inlet and be maintained at a level above an outlet to the container / vessel. Preferably, the outlet to the container is below the inlet. (For example, IV drops operate on a similar principle). A control system may be provided to maintain the level in the container / vessel.

[0023] The oxygenation apparatus may be further provided with a control system. The control system may be programmed or operable to control the flow characteristics of the precursor organ preservation solution and / or the organ preservation solution comprising nanobubbles of gas including oxygen. Preferably, the precursor organ preservation solution delivered to the organ is at pressure of 20-30 mmHg for acellular perfusate and 60-70 mmHg for normothermic blood based solution. This may result in a flow rate of approximately 10mL / min at the commencement of treatment, increasing to approximately 40-50mL / min, as the organ recovers. Accordingly, the flow rate of the 1005814927organ preservation solution may be ramped. Accordingly, the control system operates to maintain a desired pressure to the organ, while the flow rates can vary according to the organ’s ability to receive the organ preservation solution.

[0024] Additionally or alternatively, the control system may be programmed or operable to control the flow characteristics of the gas into the diffuser. For example, the pressure of gas into the apparatus may be in the range of approximately 1-14 psi, preferably approximately 2-8 psi, most preferably approximately 6psi. Preferably the gas flow rate is delivered at a rate between approximately 2-3.5 L / min.

[0025] Preferably, the oxygenation apparatus is comprised of components, the bulk of which are able to be sterilised. The components may be separable to facilitate sterilisation. For example, the inner wall(s) may be separable from the outer wall(s). Remaining components which are not sterilisable may be single use components.

[0026] In accordance with a second aspect of the present invention, there is provided an oxygenation apparatus for producing an organ preservation solution comprising nanobubbles of gas including oxygen, the apparatus including: a flow path for passage of a precursor organ preservation solution; a nanobubble generator for producing nanobubbles of gas including oxygen in the organ preservation solution, the nanobubble generator comprising at least one of: a nanoporous gas diffuser adapted for connection to a source of gas including oxygen, and disposed for fluid contact with the precursor organ preservation solution; and the flow path comprising at least one constricted portion followed by at least one or more expanded portion; and the apparatus further including a bubble trap downstream of the nanobubble generator.

[0027] Preferably, the bubble trap has any of the features described above in connection with the first aspect. The nanobubble generator may comprise either or both of the modes listed above, and optionally also any of the other modes of nanobubble generation described in the specification.

[0028] This aspect of the invention may have any of the features described above or below in connection with other aspects of the invention. 1005814927

[0029] In accordance with the third aspect of the present invention, there is provided an organ preservation apparatus comprising: a receptacle for receiving one or more organs ex vivo; a delivery apparatus configured to deliver an organ preservation solution to at least one major vessel of the organ; an oxygenation apparatus for oxygenating at least a part of the organ preservation solution with nanobubbles of gas including oxygen. Preferably the oxygenation apparatus is according to the first or second aspects of the invention.

[0030] Suitably, the organ preservation apparatus is configured to simulate the donor physiologic system for maintaining the organ in a functioning and viable state at temperatures between 2 to 40C° or between 9 to 37.5C°, preferably 15 to 35°C, or 20°C to 30°C, or 15°C to 25°C or between 37.5C° to 39C°.

[0031] The organ preservation apparatus may include a controller for controlling the flow characteristics of at least a part of the organ preservation solution. The control system described above in connection with the first aspect may be incorporated into this aspect.

[0032] The organ preservation apparatus preferably includes an additive supply apparatus for supplying chemical substances to the organ preservation solution.

[0033] The organ preservation apparatus may further include a fluid removal apparatus configured to carry fluid media away from the organ.

[0034] The organ preservation apparatus is suitably configured to be connected to a source of oxygen or oxygen-enriched gas. For example, the organ preservation apparatus may be adapted to be connected to an oxygen concentrator. The benefit of an oxygen concentrator is firstly its portability in terms of being able to be carried, and secondly it is possible to use an oxygen concentrator on aircraft. The control system described above in connection with the first aspect may be deployed to control the flow characteristics of the gas. An oxygen cylinder may alternatively constitute the source of oxygen. Alternatively, an air compressor may be deployed. 1005814927

[0035] Preferably, the organ preservation apparatus is comprised of components, the bulk of which are able to be sterilised. The components may be separable to facilitate sterilisation.

[0036] Preferably, the organ preservation apparatus is portable, in order to transport the organ from the donor site (where the organ donor is located) to the donee site (where the organ recipient / donee is located). The size of the organ preservation apparatus may depend upon the size of the organ itself. For instance, for a liver which is approximately 1.5 – 2 kg, the organ preservation apparatus may be the size of an esky, e.g. in the range of 25L – 70L insulated box. The overall weight may be in the range of approximately 8 – 9 kg. For a kidney, typically weighing about 200g, the organ preservation apparatus may be of a size suitable to fit into cabin baggage, e.g. about 7- 8kg.

[0037] This aspect of the invention may have any of the features described above or below in connection with other aspects of the invention.

[0038] In accordance with a fourth aspect of the present invention, there is provided, a method of producing an organ preservation solution comprising nanobubbles including oxygen gas, the method including: directing a precursor organ preservation solution along a flow path which has one or more constricted portions followed by at least one expanded portion; and directing gas including oxygen through a nanoporous gas diffuser, the nanoporous gas diffuser being in fluid contact with the precursor organ preservation solution in the flow path to introduce nanobubbles of gas into the precursor organ preservation solution; wherein the flow rate through, and the configuration of the flow path induces hydrodynamic cavitation.

[0039] As a result of the nanoporous gas diffuser and the hydrodynamic cavitation, the method produces an organ preservation solution comprising oxygen nanobubbles.

[0040] As described in elsewhere in this document, the organ preservation solution may comprise an electrolyte solution. Preferably, the organ preservation solution further comprises a nutrition component. The organ preservation solution may be blood-like, 1005814927CSF-like, or plasma-like. The organ preservation solution may comprise one or more components selected from the group consisting of albumin, nutrient(s), and creatinine.

[0041] This aspect of the invention may have any of the features described above or below in connection with other aspects of the invention.

[0042] In accordance with a fifth aspect of the present invention, there is provided a method of converting hemodialysis circuits or continuous veno-venous hemofiltration circuits for preservation of one or more organs ex vivo, wherein the circuit comprises a dialyser pathway for dialysate and an extracorporeal circuit for extracorporeal fluid, the method comprising: introducing an oxygenation apparatus into the dialyser pathway for oxygenating the dialysate with nanobubbles of gas including oxygen; and adapting the extracorporeal circuit for receipt of one or more organs for preservation.

[0043] Preferably the method includes inserting the one or more organs for preservation into the extracorporeal circuit such that the extracorporeal fluid flows through the one or more organs. As converted, it is understood that the dialysate interacts with the extracorporeal fluid in the hemodialysis circuits or continuous veno- venous hemofiltration circuits, e.g. in the dialysis cartridge, such that the extracorporeal fluid is oxygenated with nanobubbles of gas including oxygen. In traditional dialysis, the extracorporeal fluid is whole blood and the dialysate is a known acellular fluid. In this method for organ preservation, the extracorporeal fluid does not need to be whole blood, it could be a blood product (suitable examples are listed in the options for a organ preservation solution) or an acellular solution (again suitable examples are listed in the options for an organ preservation solution). The extracorporeal fluid may have enriched nutrition to assist with organ metabolism as discussed elsewhere in relation to “like” fluids. The extracorporeal fluid becomes oxygen enriched following contact with the dialysate. The extracorporeal fluid may not include nanobubbles prior to contact with the dialysate. The dialysate is also optionally an organ preservation solution as described elsewhere. Preferably the dialysate is acellular. In particular, traditional dialysate (with optional added nutrition), a total parenteral nutrition solution or a solution as described in the paragraph including Table 3). The dialysate is oxygenated to become a perfusate or dialysate perfusate. 1005814927

[0044] Preferably, the oxygenation apparatus is according to any of the aspects described above. This aspect of the invention may have any of the features described above or below in connection with other aspects of the invention.

[0045] In a sixth aspect, provided herein is a method for normothermic, hypothermic, subnormothermic, or isothermic organ preservation ex vivo comprising flowing an organ preservation solution comprising nanobubbles including oxygen gas through one or more organs.

[0046] In a seventh aspect, provided herein is an ex vivo method for organ preservation using oxygenated perfusion, the method comprising: - flowing an organ preservation solution comprising nanobubbles including oxygen gas through one or more organs, preferably the nanobubbles are bulk nanobubbles.

[0047] Optionally, the one or more organs is preserved in hypothermic, subnormothermic, normothermic, or isothermic conditions, or a combination thereof, preferably subnormothermic or isothermic conditions.

[0048] The following are embodiments of the sixth or seventh aspects of the invention.

[0049] In some embodiments, the organ is preserved at a temperature from 2 to 40 °C, preferably from 4 to 40 °C, preferably from 9 to 37.5°C, most preferably from 15 to 35 °C or 20 to 30 °C or 15 to 25 °C. Alternatively, the temperature is from 35.5 to 39°C.

[0050] The organ may be preserved ex vivo for a preservation period of at least at least about 30 minutes, at least about an hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, or at least about 14 hours, preferably at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours or longer, such as up to about 2 days, 3 days or 4 days. Optionally, the one or more organs is / are preserved ex vivo for at least 2, 3, 4, 5 or 6 hours. 1005814927

[0051] Optionally, the method is conducted under subnormothermic conditions such as between 5 to 25 °C, between 9 to 25°C, between 15 to 25 °C; optionally 15 to 20 °C, preferably the conditions are subnormothermic and isothermic conditions.

[0052] Preferably, the organ preservation solution is provided to the one or more organs via oxygenated machine perfusion, preferably subnormothermic machine perfusion.

[0053] Preferably, the one or more organs conducts aerobic respiration during a preservation period.

[0054] Optionally, the one or more organs is selected from: a kidney, a small bowel, a pancreas, a liver, a heart, a cornea, a lung or combinations thereof such as an organ block. Optionally, the organ block is an abdominal organ block. An abdominal block can be the kidneys, liver and pancreas or the liver, small bowel, pancreas, and kidneys. Other organ blocks will be known to the skilled person.

[0055] In some embodiments, the organ preservation solution is supplied to one or more vessels of the one or more organs, preferably the one or more vessels are one or more arteries. Optionally, the organ preservation solution is directly supplied to the one or more organs via one or more catheters or cannulas.

[0056] Optionally, after the organ preservation solution flows through one or more organs, the organ preservation solution is dialyzed before returning to the one or more organs.

[0057] Optionally, the method further comprises one or more of: - determining the viability of the one or more organs; and / or - determining the one or more organs is free from a pathogen or pathogens; - obtaining the one or more organs to be preserved by harvesting the one or more organs.

[0058] In some embodiments, the method results in resuscitation or partial resuscitation of the organ. 1005814927

[0059] In some embodiments of the sixth and seventh aspects, the organ preservation solution is produced in accordance with the method of the fourth aspect of the invention.

[0060] In some embodiments of the sixth and seventh aspects, the method is performed using the oxygenation apparatus of the first, second, third or fifth aspect.

[0061] The following embodiments apply to all aspects of the invention including an organ preservation solution. Optionally, the organ preservation solution is whole blood or a fractionated blood product comprising nanobubbles including oxygen; preferably wherein the method is conducted in normothermic conditions such as between 35.5 to 37.5 degrees. Organ preservation solutions that are blood or fractionated blood product may not require nanobubbles. Alternatively, the organ preservation solution is an acellular perfusate comprising nanobubbles including oxygen; optionally wherein the method is conducted in subnormothermic or normothermic conditions.

[0062] The organ preservation solution is oxygenated either directly with nanobubbles or indirectly via contact with an oxygen nanobubble enriched solution across a membrane (ie when prepared using a dialysis system).

[0063] Preferably, the acellular perfusate comprises electrolytes. Preferably, the organ preservation solution comprises one or more components selected from the group consisting of albumin, nutrient(s), and creatinine. Preferably, the organ preservation solution includes a total parenteral nutrition formula, optionally at 20-30 ml / kg, preferably 25 mg / kg.

[0064] The albumin may be present at from 3g / dl to 5 g / dl, preferably 4 g / dl.

[0065] The creatinine may be present at from 5 to 15 mg / dL, preferably 10 mg / dL.

[0066] Preferably, the organ preservation solution has a partial oxygen pressure of 275 to 375 mmHg, 300 to 350 mmHg, or 315 to 335 mmHg.

[0067] Preferably, the electrolytes include one or more of sodium (such as 130-140 mmol / L), calcium (such as 1-2 mmol / l), magnesium (such as 0.8-1 mmol / l), potassium (such as 3.5-3.5 mmol / l), acetate (such as 0.2-0.6 mmol / l), and bicarbonate (such as 22-29 mmol / l). 1005814927

[0068] Preferably, the osmolarity of the organ preservation solution is 200 to 400 mosml / l, 250 to 350 mosml / l, 275 to 325 mosml / l, or about 300 mosml / l (ie 290-310 mosml / l).

[0069] Preferably, the organ preservation solution comprises glucose, preferably at 5- 10 mmol / l or 0.5-10, 0.5-1.5, 4-7, 5-6, about 1 or about 5.5 g / dl.

[0070] Preferably, the organ preservation solution is buffered to pH 7 to 8, 7.3 to 7.7, or about 7.5.

[0071] Preferably, the organ preservation solution flows at a flow rate from 15 to 450 ml / min, preferably 15 to 30 ml / min or about 25 ml / min for an acellular organ preservation solution or 350 to 450 ml / min or 400 ml / min for a blood organ preservation solution.

[0072] Preferably, the organ preservation solution is at a pressure of 20 to 70 mmHg, preferably 30 to 40 mmHg for an acellular organ preservation solution or 55 to 65 mmHg for a blood organ preservation solution.

[0073] Preferably, the partial oxygen pressure of the organ preservation solution is 250 to 280 mmHg.

[0074] In an eighth aspect, provided herein is a method for determining the suitability of one or more organs for transplantation, comprising determining the viability of one or more organs by measuring one or more of: - aerobic respiration; - glucose utilisation; - ATP generation; - ICG clearance (where the one or more organs includes a liver); - creatinine clearance (where the one or more organs includes a kidney) - insulin and / or GLP-1 response to enteral glucose stimulation (where the one or more organs includes a pancreas), preferably measuring at least one or more of aerobic respiration, glucose utilisation, and 1005814927ATP generation. Optionally, the method further comprises transplanting into a subject in need thereof one or more organs determined as suitable for transplantation.

[0072] In a ninth aspect, provided herein is a method for treating a subject in need of organ transplantation, by transplanting into the subject one or more organs preserved by the method of the sixth or seventh aspects.

[0075] This aspect of the invention may, but does not need to, use any apparatus of the invention with any of the features described in the above aspects of the invention.

[0076] Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE FIGURES Embodiments of the present disclosure will be discussed with reference to the accompanying figures wherein: Figure 1 shows a perspective drawing of a nanoscale pore diffuser for an oxygenation apparatus illustrated in Figure 5, in accordance with an embodiment of the present disclosure; Figure 2 shows a perspective drawing of a tube component of an oxygenation apparatus in accordance with an embodiment of the present disclosure; Figure 3 shows the scale and dimensions of various bubble sizes; Figure 4 shows a photograph of a diffraction pattern generated using a green 512nm green light laser on a fluid treated in the oxygenation apparatus in accordance with an embodiment of the present disclosure; Figure 5 shows a schematic diagram of an organ preservation apparatus in accordance with an embodiment of the present disclosure; Figure 6 shows a plot of oxygen consumption over time for organs preserved using static cold storage (SCS), normothermic oxygenated perfusion (NMP), and isothermic preservation (IMP); 1005814927Figure 7 shows a plot of carbon dioxide production over time for organs preserved using static cold storage (SCS), normothermic oxygenated perfusion (NMP), and isothermic preservation (IMP); Figure 8 shows a plot of kidney ATP over time for organs preserved using static cold storage (SCS), normothermic oxygenated perfusion (NMP), and isothermic preservation (IMP); Figure 9 shows a plot of liver ATP over time for organs preserved using static cold storage (SCS), normothermic oxygenated perfusion (NMP), and isothermic preservation (IMP); Figure 10 shows plots of IMP glucose over time for organs preserved using static cold storage (SCS), normothermic oxygenated perfusion (NMP), and isothermic preservation (IMP); Figure 11 shows plots of post OGTT GLP-1 and post OGTT insulin in organs preserved using normothermic oxygenated perfusion (NMP), and isothermic preservation (IMP) over time; Figure 12 shows plots of creatine clearance over time for organs preserved using isothermic preservation (IMP; four samples – IMP1, IMP2, IMP4 and IMP5) and normothermic oxygenated perfusion (NMP; two samples – NMP2 and NMP3); Figure 13 shows plots of pancreas histology, small bowel histology, and liver histology over time for organs preserved using static cold storage (SCS), normothermic oxygenated perfusion (NMP), and isothermic preservation (IMP); Figure 14 shows plots of oxygen consumption, arterial and venous pH, and arterial and venous CO2 over time for abdominal blocks. A Baxter PrismaflexTMDialysis System was modified to use oxygenated dialysate. Whole blood was used to perfuse 12 composite porcine abdominal blocks. Serial arterial and venous blood gas samples were taken to assess oxygenation across the dialysis membrane, oxygen consumption, carbon dioxide production and pH maintenance. Oxygen transfer across the dialysis cartridge was effective, with significantly higher PaO2 (M = 318 mmHg, SD = 119) as compared to room air oxygenation (p<0.05). The dialysis function was able to maintain pH with a range of normality and the blocks were successfully perfused and preserved 1005814927for 6 hours. Oxygen consumption alongside carbon dioxide production was observed, with a baseline approximately three times that of normal resting tissue oxygen consumption. Figure 15A shows exemplary perfusion apparatus with single inflow, suitable for offsite resuscitation and transportation. Figure 15B shows exemplary perfusion apparatus with dual inflow, suitable for offsite resuscitation and transportation. Whether an organ is suitable for single or dual inflow will depend on the organ vasculature. For some organs, single inflow will sufficient because the perfusate will travel through the vasculature and sufficiently oxygenate / nourish the organ. For some organs, the vasculature attached to a single inflow is not sufficient to oxygenate / nourish the organ. In these instances dual inflow is recommended. Figure 16 shows an exemplary configuration for conversion of a hemodialysis circuit to the apparatus of the invention, comprising a nanofuser (NF) for the generation of nanobubbles including oxygen gas. Figure 17 (none). Figure 18 shows a schematic diagram of an alternative exemplary continuous flow oxygenation apparatus suitable for the organ preservation apparatus illustrated in Figure 5. Figure 19 shows an exemplary diffusion “stone” for generation of nanobubbles. Figure 20 shows the detection of nanobubbles in fluid bubbled with air, as detected by 1000x magnification with green light laser (wavelength of 512 nm). Figure 21 plots oxygenation over time, comparing perfusion of nanobubbles (top line), perfusion with macrobubbles (middle line), or normal blood (bottom line). To assess whether the nanobubbles generated remained in solution for a prolonged period, oxygenation of 1L Hartmann’s solution in a reservoir with medical air from an oxygen concentrator at 2 lpm and 6psi for 1 minute followed by serial measurements were taken for 1 hour.2 groups comprising of fluid oxygenated by simple bubbling and fluid infused by nanobubbles were tested. 1005814927Figure 22 is a perspective view from above of an organ preservation apparatus according to another preferred embodiment. Figure 23 is a side view of the organ preservation apparatus according to Figure 22. Figure 24A shows a diffuser from the organ preservation apparatus of figure 22. Figure 24B shows a T-fitting from the organ preservation apparatus of Figure 22. Figure 24C shows the T-fitting and the diffuser in assembled or partially assembled configurations. Figure 24D is a top view of the assembly shown in figure 24C. DETAILED DESCRIPTION

[0077] The present disclosure arises from the inventor’s hypothesis that the need for normothermia an evolutionary adaptive response in becoming terrestrial in nature and erect in posture utilizing the physiological properties of haemoglobin to optimise oxygen absorption and delivery to the brain and other organs by the cardiovascular system to ensure cerebral perfusion during erect posture. The pulsatile nature of the flow is such that at the level of the capillary, flow is continuous, and the entire heart and blood vessels act as a Windkessel to dampen the pulsatility of the flow. The evolution of the normothermic closed circulatory system also allows the body to autoregulate and allow targeted flow response to various organs and systems to maintain the organism and to prepare for fight or flight. Based on this hypothesis, at an organisational level, haemoglobin is nature’s oxygen concentrator and the heart and blood vessels akin to a pump and tubing system that can maintain continuous flow in the capillary beds.

[0078] From an evolutionary perspective, the inventor also proposes that the issue of temperature control in organs preserved with oxygenated perfusion may be irrelevant. Additionally, the single most important organ (the brain) in a human, which has a high metabolic activity and controls all other functions, has a blood-brain barrier and all metabolic processes occur against the cerebrospinal fluid (CSF) that in composition is similar to transcellular fluid. Therefore, given that from an evolutionary perspective, the only oxygenated fluid that has demonstrated the ability to support a metabolically active organ is CSF, it would seem a reasonable assumption that a perfusion fluid with a 1005814927composition similar to CSF should be able to support the metabolic needs of an organ if adequately oxygenated.

[0079] Since red cells with an average diameter of 8 microns cannot cross the capillary membranes that have 1-2 micron pores, in theory, if one could have a continuous supply of nanobubbles of oxygen in a balanced electrolyte solution with appropriate nutrition, it should be able to support the metabolic needs of an organ, without the need for normothermia – and the organ should be able to survive at isothermia – at equilibrium with its environment between 4 – 40C° rather than at a user- defined temperature in hypothermic, subnormothermic or normothermic conditions.

[0080] To test this hypothesis, the present inventor investigated the feasibility of machine preservation at environmental temperature (isothermia) using an acellular perfusate similar in composition to cerebrospinal fluid achieving oxygenation by a continuous infusion of nanobubbles. Definitions

[0081] "About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, in some instances ±5%, in some instances ±1%, and in some instances ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0082] Ranges: throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0083] As used herein, the term “nanobubbles” and related terms is a term of the art and refers to bubbles that are less than 1 micron in size (diameter). In preferred 1005814927embodiments of any aspect of the invention, nanobubbles are bubbles of less than 500 nm in diameter. Nanobubbles have interesting properties of high stability, longevity and high surface area per volume. They are classified as either surface nanobubbles or bulk nanobubbles. While larger bubbles collapse, nanobubbles randomly drift because of Brownian motion and can remain in liquids for extended periods. One of the main factors supporting the stability of bulk nanobubbles in a solution is the negative ζ- potential. “Nanobubbles of gas including oxygen” and “nanobubbles including oxygen” and “oxygen-enriched nanobubbles” “oxygen nanobubbles” are used interchangeably in the specification. Similarly, an organ preservation solution referred to as “oxygen nanobubble organ preservation solution” or “oxygen-enriched” relates to an organ preservation solution comprising nanobubbles of gas including oxygen.

[0084] ‘Organ’ or ‘organs’ – An organ refers to a collection of tissues that structurally form a functional unit specialized to perform a particular function. ‘Organs’ as referred to herein includes an organ block comprising multiple organs (such as an abdominal block). Suitable organs for transplantation include but are not limited to a liver, small bowel, a pancreas, a kidney, a heart, a cornea, and a lung, or combinations thereof. In some embodiments, the organ is one or more of: a liver, small bowel, a kidney, or a pancreas. The organ block may be an abdominal organ block optionally including a kidney or kidneys, a liver and a pancreas from a single donor. A donor organ is an ex vivo organ from a donor that is to be transplanted into a recipient or assessed for viability prior to transplant into a recipient to determine suitability of the organ for transplantation. The organ may be human. Alternatively, the organ is from a non-human animal, preferably a mammal (eg pig, sheep).

[0085] ‘Normothermia’ or ‘normothermic conditions’ – are used herein to refer to storing or maintaining an organ at normal body / physiological temperature. Normothermic conditions are often, but not limited to, 35.5°C° to 37.5 °C.

[0086] ‘Subnormothermia’ or ‘subnormothermic’ – are used herein to refer to a temperature lower than normothermic temperature. In the context of this disclosure, sub-normothermic temperatures can be, but are not limited to, between 12°C to 30°C, 9°C to 37.5°C, 10 to 25 °C, 15 to 25 °C , 20 to 25 °C ,or 15 to 20°C.

[0087] ‘Isothermia’ or ‘isothermic conditions’ – are used herein to refer to environmental conditions such as at room temperature. In isothermic conditions, the 1005814927organs are not cooled like in cold storage or hypothermic conditions and not warmed for body temperature for normothermic conditions. Isothermic conditions can be from 4 to 40 °C or 10 to 30 °C or 15 to 25 °C or 35.5°C to 37.5°C. Preferred conditions for organ preservation in accordance with this disclosure and the conditions of the isothermic experiments reported herein are between 15 to 20 degrees C.

[0088] ‘Hypothermia’ or ‘hypothermic conditions’ – are used herein to refer to storing or maintaining an organ at a lower temperature than room temperature. Hypothermic conditions can be, but are not limited to, 2 to 10 °C or 4 to 8 °C. Hypothermic conditions includes static cold storage (SCS) temperatures, which tend to be 2 to 10 °C or 4 to 8 °C.

[0089] ‘Organ resuscitation’ – refers to reviving an organ that is non-functioning or functioning suboptimal (for example suffering non-lethal organ injury) to normal functional levels or improved functioning levels. Revival of the organ can be determined by confirmed by assessing organ function using of one or more measures of respiration (use of oxygen, expulsion of carbon dioxide or both), metabolic function in the organ (processing sugar or other metabolic indicator known to the skilled person), or combinations thereof. In the context of this invention, an organ may be resuscitated ex vivo in the context of the organ preservation methods of the invention including perfusion, such as machine perfusion. Alternatively, the organ is fully resuscitated upon transplant into a recipient body. Resuscitation may be following warm ischemia (deprivation of blood flow but kept at physiological temperature) or cold ischemia (blood flow deprivation and cooled temperature). Warm ischemia is the preferred option for the present disclosure.

[0090] ‘Organ preservation’ – maintaining, extending, or otherwise prolonging the viability of an organ ex vivo so that it is suitable for transplantation (ie viable for resuscitation upon transplantation into a recipient body). The ‘preservation period’ includes the period after an organ or organ block has been removed from a donor body or cadaver, during storage or transportation before transplantation surgery, to when the organ or organ blocks is received by a recipient body. Depending on the organ, the current preservation periods in clinical transplantation vary from 4 – 8 hrs for hearts to 30 hours for kidneys with static cold storage at which time points accumulation of oxygen debt in the organ and injury on reperfusion during transplantation leads to lethal 1005814927damage and non-function of the organ. An ideal preservation method should maintain the organ viable till the organ and the recipient are optimized to give the best outcome. This may be from several hours to days. In the methods herein, an organ / organs may be preserved for a preservation period of at least about 30 minutes, at least about 3 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 14 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, at least about 22 hours, at least about 24 hours, at least about 30 hours, at least about 36 hours – preservation may be up to about 2 days, 3 days or 4 days. Optionally, preservation is for 8 hours to 24 hours or 36 hours to 4 days.

[0091] The organ may be preserved for a preservation period of at least about 30 minutes, at least about an hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, or at least about 14 hours, preferably at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours or longer, such as up to about 12 hours, about 1 day, about 2 days, 3 days or 4 days. The preservation period may be between 2 hours and 4 days, 2 hours and 2 days, 4-8 hours, and 6-10 hours. There is some variability in relation to different organ types that the skilled person will understand. For example, while a kidney can be preserved up to 40 hours and while 14 hours is a good outcome for a liver.

[0092] ‘Organ preservation solution’ and ‘perfusate’ are used interchangeably to refer to the solution that is perfused into the organ or organ block during the organ preservation method. The solution is supplied to the organ or organ block under such conditions that the solution permeates the organ or organ block. Perfusion (also referred to as permeation) may occur by directly delivering the organ preservation solution to a vessel or other suitable cavity of the organ (eg using a catheter or cannular to an artery or other vessel), or passively via at least partial submersion of the organ in a reservoir of the organ preservation solution, such that the organ preservation solution perfuses / permeates the organ. The organ preservation solution may be dialysate (that is, a fluid suitable for use in kidney dialysis), cerebral spinal fluid (CSF), whole blood 1005814927(referred to herein interchangeably with the term ‘blood’), a fractionated blood product (such as plasma or serum) or a solution as defined by components herein. In all situations, the organ preservation solution includes nanobubbles (ie nanobubbles including oxygen or consisting of oxygen). Consequently, the dialysate (traditional dialysis fluid), CSF, whole blood, or fractionated blood product used may be dialysate- like, CSF-like, or plasma-like because they have been modified to include oxygen nanobubbles and are therefore oxygen-enriched fluids. As blood can deliver oxygen via red blood cells, it is not essential for a blood organ preservation solution to include oxygen nanobubbles. A further modification in a “like” product could be to add nutrition to the solution, which may improve organ metabolism.

[0093] A ‘nanoporous material’ – refers to a regular organic or inorganic structure in which a porous structure is present. Nanoporous materials exhibit pore diameters that are most appropriately quantified using units of nanometres. The diameter of pores in nanoporous materials is thus typically smaller than 1 micron, such as 100 nanometres or smaller. The pores of the nanoporous diffuser are preferably open pores.

[0094] ‘Hydrodynamic cavitation’ – refers to the cavitation phenomenon that involves the development of vapor cavities inside a liquid medium. Hydrodynamic cavitation is induced by a drop in the static pressure of the flowing fluid. Hydrodynamic cavitation involves three mechanisms: including nucleation, bubble growth, and bubble implosion. When a fluid flow passes through irregular geometries or narrow / constricted orifices, the velocity of the fluid rises. The rise in velocity decreases the static pressure. Whenever the pressure becomes less than the local saturated vapor pressure, a large number of cavities are released (nucleation). Upon the pressure drop, the generated cavities expand and break down (growth and implosion). When the cavities collapse, they release sharp shock waves of energy into the surrounding fluids. It is thought that the shock waves break down the remaining generated cavities into smaller sized bubbles, preferably nanobubbles.

[0095] ‘Acellular perfusate’ – a perfusate that is substantially cell-free. Preferably, the perfusate is free of red blood cells and provides oxygen via an alternate mechanism such as oxygen nanobubbles. Substantially cell-free can be 90%, 95%, 98%, 99%, 99.5% or 99.9% cell free by volume. An acellular perfusate may be a decellularized 1005814927blood product (eg plasma) or decellularized bodily fluid (eg CSF) . Otherwise, the acellular perfusate may be a solution that is does not comprises cells initially (eg a synthetically produced dialysate solution or an organ compatible nutritional solution, such as a total parenteral nutrition product like used in Table 3).

[0096] ‘Vessel’ and ‘blood vessel’ are used interchangeable herein to refer to any blood vessel in an organ including arteries, veins and capillaries. Preferably, perfusate is perfused into one or more arteries of the one or more organs. Perfusate that exits the organ or organ block after flowing through the one or more organs (which may be referred to as ‘waste’ organ preservation solution and comprises metabolic waste products such as lactate, ammonia) preferably exits via a vein, and optionally is collected from the vein. Optionally, the waste organ preservation solution is recirculated or dialysed and recirculated. Optionally, the nutrition and / or nanobubbles are supplemented in the recirculated solution. “Vessel” may also be used to refer to apparatus which the context will make clear. Nanobubbles

[0097] Nanobubbles are a natural phenomenon that are seeing increasing application in medicine. The use of nanobubbles has been described in ultrasound diagnostics to assess flow, in cancer chemotherapeutics, to ameliorate the impact of hypoxia in experimental models on cells and tissues during surgery and a means to deliver pharmaceuticals. To the best of the inventor’s knowledge, the role of nanobubbles in organ preservation has not been explored. The stability of nanobubbles is shown in Table 1 (from Michailidi et al.) Table 1 – Stability of nanobubbles 1005814927

[0098] Bulk nanobubbles are able to be produced through hydrodynamic cavitation under a pressure of ~3 bar. While nanobubbles can exist as surface nanobubbles and bulk nanobubbles, the nanobubbles of interest for organ preservation are bulk nanobubbles. Oxygen containing nanobubbles

[0099] The nanobubbles of the invention are for including in an organ preservation solution to oxygenate or oxygen enrich (if some oxygen is already present) the organ preservation solution. The nanobubbles of the invention therefore need to contain oxygen (ie be ‘oxygen nanobubbles’ either consisting of oxygen or including oxygen with another gas such as nitrogen). Oxygen or oxygen-enriched gas is used to prepare the oxygen nanobubbles of this disclosure. Preferably, the oxygen or oxygen-enriched gas is medical grade (ie safe for contact with an organ). Preferably, the oxygen content of the air in the nanobubbles is greater than atmospheric levels (ie greater than 21%). Preferably, the gas is 90 to 100% oxygen, 95 to 100% oxygen, 95 to 98% oxygen ir over 99.5% oxygen (ie medical oxygen cylinder levels) v / v. As shown in Table 2 (from Wang et al.), various techniques have been described to generate nanobubbles each with their advantages and disadvantages. Table 2 – Methods for generating nanobubbles 1005814927

[0100] In the context of organ preservation, in a pressure-controlled circuit with hypothermic machine perfusion, what has been observed is a gradual increase in flow rate of perfusate and therefore scalability is an important consideration in design.

[0101] To assess whether the nanobubbles generated remained in solution for a prolonged period, oxygenation of 1L Hartmann’s solution in a reservoir with medical air from an oxygen concentrator at 2 Lpm and 6psi for 1 minute followed by serial measurements were taken for 1 hour.2 groups comprising (i) fluid oxygenated by simple bubbling, and (ii) fluid infused by nanobubbles (diffuser device only) were tested. Nanobubbles were shown to generate the greatest amount of oxygenation over time, when compared to perfusion with macrobubbles or no perfusion (normal blood) (Figure 21). The samples were tested till 4 hours and the nanobubble containing solutions showed only minor attrition during that time, whereas the oxygenation from simple bubbling resulted in a significant drop across the 4 hour time period. The perfusate of the present disclosure are viable for about 2, 3, 4 and 5 hours following oxygen- enrichment with nanobubbles. Oxygenation Apparatus

[0102] It is against this background that the present inventor developed an oxygenation apparatus for producing an organ preservation solution comprising oxygen nanobubbles. The preferred apparatus utilises a combination of a nanoscale pore 1005814927method with hydrodynamic cavitation (either mode or the combination being referred to herein as a “nanobubble generator”) to provide a continuous flow of an organ preservation solution including nanobubbles including oxygen gas (so-called oxygen nanobubbles) (ie a solution enriched with oxygen). The term “nanofuser” used herein is also adopted as a shorthand way of referring to this oxygenation apparatus.

[0103] The nanoscale pore method (for instance using a diffusion stone) typically produces surface nanobubbles. On the other hand, with the at least one constricted portion of the flow path followed by the one or more expanded portion results in hydrodynamic cavitation, producing bulk nanobubbles. This combination of modes of nanobubble creation leads to an improved organ preservation solution including oxygen nanobubbles.

[0104] As shown in Figure 5, the oxygenation apparatus 10 comprises a flow path for passage of a precursor organ preservation solution 56. A nanobubble generator 11 conveys the precursor organ preservation solution 56 along the flow path. The nanobubble generator 11 is configured such that, in operation, oxygen nanobubbles are introduced into the precursor organ preservation solution 56 to produce an organ preservation solution comprising oxygen nanobubbles 56’ suitable for normothermic, hypothermic, subnormothermic or isothermic preservation of an organ 54.

[0105] The nanobubble generator 11 combines a nanoporous gas diffuser (aka nanoscale pore diffuser) 12 disposed in the flow path designed to induce hydrodynamic cavitation to reliably and stably produce bulk nanobubbles.

[0106] Turning to Figures 1 to 5, there is shown an embodiment of a nanobubble generator 11 according to a preferred embodiment of the present invention.

[0107] Figures 1 and 2 show components of a nanobubble generator 11 comprising a nanoscale pore diffuser 12 and a tube 18. The nanoscale pore diffuser 12 comprises a cylindrical diffusion stone 14 comprising a medical grade 0.5 micron pore 316 stainless steel nanoporous diffusion stone 14. The diameter of the diffusion stone is 12.6 mm. Diffusion stones are readily available off-the-shelf and are typically used in the brewing industry to introduce carbon dioxide into beer. The diffusion stone needs to be suitable for contacting a solution that will subsequently contact one or more organs. 1005814927

[0108] As shown, the diffuser 12 has a cylindrical diffusion stone 14 having a central longitudinal axis. The diffuser 12 has a nipple 16 in the form of a cylindrical projection having a central longitudinal axis aligned with a central longitudinal axis of the diffusion stone 14. As shown in Figure 19, the nipple 16 on the diffuser 12 has a barbed profile for connection of a gas conduit. Preferably, the gas delivered to the diffusion stone 14 is oxygen or oxygen-enriched gas, which is delivered from an oxygen source 31, such as an oxygen concentrator, by oxygen line 32 (see Figure 5).

[0109] The oxygen line 32 delivers the gas under pressure to the nanoscale pore diffuser 12 where the gas diffuses through the diffusion stone 14. When the diffusion stone 14 is submerged in a fluid, such as the organ preservation solution 56 discussed further below, the diffusion stone 14 will produce gas bubbles which become entrained in the fluid. The preferred gas pressure is in the range of approximately 1-5 psi. This pressure is suitable for the production of nanobubbles. Otherwise, outside of this range, microbubbles and larger sized bubbles are produced which are undesirable for organ preservation.

[0110] The nanoscale pore diffuser 12 is housed within a flow channel in the form of a tube 18, shown in Figure 2 having aligned transverse circular apertures 20 to receive the diffusion stone 14. The inside diameter of the transverse circular apertures 20 is 13 mm. Accordingly, with the diffusion stone 14 placed with its longitudinal axis aligned with the longitudinal axis of the transverse aperture 20, there is a 0.2mm radial gap (not shown) between the diffusion stone 14 and the side wall of the tube 18.

[0111] The tube 18 is closed at one end. For instance, the tube 18 may have a cap which is approximately 3-4 mm in thickness. The cap may be a threaded closure which engages with threads provided on the tube 18. The flow channel in the form of the tube 18 has an inside diameter of 20 mm.

[0112] The oxygenation apparatus 10 is installed within the organ preservation apparatus 50 illustrated in Figure 5. The nanobubble generator 11 is configured to oxygenate at least a part of the organ preservation solution 56 with oxygen nanobubbles. The nanobubble generator 11 is housed and centrally disposed within an inner container 24 and, in turn, the inner container 24 is housed centrally within an outer container 26. The height of walls of the inner container 24 is less than the height of the walls of the outer container 26, such that fluid, such as the organ preservation solution 100581492756, is able to spill over into the outer container 26. The inner container 24 is thus full of the organ preservation solution 56. The benefit of this spillover arrangement is that it permits microbubbles or larger which may be produced by the nanobubble generator 11 to be allowed to escape. The outer container 26 thus only contains organ preseravtion solution comprising nanobubbles including oxygen gas 56’. The inner container 24 thus acts as a bubble trap for microbubbles and larger sized bubbles which leave the fluid at the surface and are dispersed to atmosphere.

[0113] The 0.5-micron nanoporous diffusion stone 14 measuring 12.6mm (outside diameter) is positioned in the 20 mm (inside diameter) rigid tube 18, with the central longitudinal axis of the diffusion stone 14 aligned with the central longitudinal axis of the aligned circular apertures 20 in the tube 18. The organ preservation solution 56 is pumped through the tube 18.

[0114] Meanwhile, gas including oxygen is pumped from the oxygen concentrator 31 via delivery line 32 through the diffusion stone 14.

[0115] As will be appreciated from the dimensions, there is a drop in diameter of the tube 18 due to the presence of the diffusion stone by 7.4 mm. This is a first constricted portion of the flow path. Additionally, due to the diffusion stone being received within the circular apertures 20, there will be a 0.4mm diameter outflow from the inside of the tube 18 through circular apertures 20. This is a second constricted portion of the flow path. The exit from the tube 18 opens into a 50mm width / diameter inner container 24. This is an expanded portion of the flow path. Thus, the organ preservation solution 56 passes through 2 successive constricted portions in the tube 18, each of increased constriction compared to the preceding segment, before it passes into the inner container 24, which is unconstricted.

[0116] Due to the venturi effect, this causes a drop in pressure in the constricted portions of the tube 18 proportional to the ratio of the squares of the cross-sectional areas, followed by a sudden rise in pressure at the final exit to the inner container 24. The continuous flow of nanobubbles from the nanoporous diffuser 12 in combination with hydrodynamic cavitation as a result of the Venturi generates a large volume of nanobubbles in the organ preservation solution 56 (perfusate). The pressure drop through the two stages was 11.7 times and 34 times, respectively, followed by an increase of pressure by 6943 times. 1005814927

[0117] The presence of nanobubbles in fluid 56 treated in the apparatus 10 was confirmed using a green 512nm green light laser to observe for a diffraction pattern (Figures 4 and 20).

[0118] The spillover illustrated in Figure 5 is one proposal to remove microbubbles from the organ preservation solution. Another proposal is provided by Figure 18 discussed below.

[0119] The oxygenation apparatus10 disclosed herein may be a module of an organ preservation apparatus 50 shown in Figure 5. The organ preservation apparatus 50 further comprises a container 52 for containing the organ 54 (or organ block) in communication with the organ preservation solution 56.

[0120] The organ preservation solution comprising nanobubbles of gas including oxygen 56 is delivered from the oxygenation apparatus 10. A delivery apparatus 58 is configured to deliver the organ preservation solution 56 to at least one major vessel of the organ 54 in the container 52. A fluid removal apparatus 60 in the form of a pump is configured to carry fluid away from the organ 54 / container 52. The fluid removal apparatus 60 may be connected to circulate organ preservation solution 56 back to the oxygenation apparatus 10, where the organ preservation solution 56 is pumped into the tube 18. Alternatively, the fluid removal apparatus 60 may carry the fluid to waste (not shown).

[0121] The delivery apparatus 58 may include a fluid pump (not shown) and a flow controller 62 which is configured to control the flow of at least a part of the organ preservation solution 56. If desired, an additive supply apparatus 64 can be used to supply chemical substances, e.g. nutrients to the organ preservation solution 56. The delivery apparatus 58 may further include a cannula 61 which delivers the organ preservation solution 56 into at least one major vessel of the organ 54. The organ preservation apparatus 50 is configured to simulate the donor physiologic system for maintaining the organ in a functioning and viable state at normothermia.

[0122] The container 52 for containing the organ may be configured to adjustably cradle the organ 54. It may comprise a removable top / lid 68 for sealing the container. The cannula 61 for attachment to the organ 54 may extend through the removable top 100581492768, the walls (as shown) or the base of the container 52. The cannula may contain at least one flange (not shown) at the inferior end for attachment of the organ 54. Organ preservation apparatus

[0123] The organ preservation apparatus 50 may further comprise an organ preservation solution source (not shown) containing the organ preservation solution 56. The organ preservation apparatus 50 may further comprise a pressure regulator (not shown) to control the flow of the organ preservation solution 56.

[0124] The organ preservation apparatus 50 may allow organ preservation at a user defined temperature or environmental temperature. For a user defined temperature, the organ preservation apparatus 50 may comprise a temperature regulator (not shown) for regulating the temperature of the organ preservation solution 56. For example, a temperature regulator may be used for normothermic, sub normothermic, hypothermic, or isothermic organ preservation.

[0125] The organ preservation apparatus 50 is configured such that, in operation, the organ preservation solution 56 is routed from the oxygenation apparatus 10 to the cannula 61, where the organ preservation solution 56 is introduced and flows through the organ 54, and residual / waste organ preservation solution is siphoned / drained to the waste receptacle or circulated back to the nanobubble generator 11. Before circulation back to the nanobubble generator 11, the waste organ preservation solution may be dialyzed using standard dialysis methods known to those in the art.

[0126] Figure 18 shows a schematic diagram of an alternative exemplary continuous flow oxygenation apparatus 10’ suitable for the organ preservation apparatus 50 of the invention. The oxygenation apparatus 10’ comprises a flow path for passage of a precursor organ preservation solution 56. A nanobubble generator 11’ is in fluid contact with the precursor organ preservation solution 56 in the flow path. The nanobubble generator 11’ is configured such that, in operation, oxygen nanobubbles are introduced into the precursor organ preservation solution 56 to produce an organ preservation solution comprising nanobubbles including oxygen gas 56’ suitable for preservation of an organ.

[0127] The nanobubble generator 11’ combines a nanoscale pore diffuser 12’ with diffusion stone 14’ with induced hydrodynamic cavitation in the flow path to reliably and 1005814927stably produce nanobubbles, preferably bulk nanobubbles (ie nanobubbles in bulk phase in the solution or a homogeneous dispersion of nanobubbles consistently throughout the solution). The diffusion stone 14’ has a smaller outside diameter A compared to the inside diameter B of the tube 18’ it is disposed within (for example, 13 mm outside diameter diffusion stone 14’ positioned in a 15 mm inside diameter rigid tube 18’). The precursor organ preservation solution 56 flowing into the tube 18’ experiences the drop in diameter (constricted portion) because of the presence of the 13 mm diffusion stone 14’ which creates a constricted path of 1 mm wide radial gap, surrounding the diffusion stone 14’. The constricted path subsequently opens into a 50mm diameter (C) inner container 24’. Due to the Venturi effect, this causes a drop in pressure in the constricted segment of the tube 18’ proportional to the ratio of the squares of the cross-sectional areas, followed by a sudden rise in pressure at the final exit, resulting in hydrodynamic cavitation. Thus, a continuous flow of nanobubbles through the nanoporous diffuser 12’ into the organ preservation solution 56 which substantially simultaneously undergoes successive pressure variation leading to hydrodynamic cavitation generates a large volume of nanobubbles in the organ preservation solution 56’. The inclusion of a p trap 70 allows the escape of gases and removes any microbubbles in the perfusate. Other kinds of traps may also be used. Such traps may include an arcuate path such as the p trap 70 or and S trap.

[0128] Figure 15A is a flow diagram of a single flow perfusion system 80, in which there is single flow of perfusate 56 into the organ 24. Such organs requiring single flow include kidney, pancreas and intestines. As shown, the control system includes microprocessor 82 which sends control signals to the stepper motor controller 84 which in turn drives the stepper motor peristaltic pump 86. The pump 86 pumps perfusate 56 from the nanofuser (oxygenation apparatus) 10 to the organ 24. The microprocessor unit 82 controls the stepper motor controller 84 to operate according to a signals which the MPU 82 receives from the pressure transducer 88. Given that the pressure transducer 88 generates an electrical signal proportional to the amount of pressure, increased accuracy of the perfusate pressure to the organ can be obtained by calibrating the pressure transducer with the pressure calibrator 90.

[0129] In the dual flow perfusion system of Figure 15B, there are two flows of perfusate 56 to the organ 24. Organs that are better preserved with two inflows and one outflow include the liver and heart. 1005814927

[0130] Figure 16 shows an exemplary configuration for conversion of a standard hemodialysis circuit to the organ preservation apparatus of the invention, comprising an oxygenation apparatus 10 (nanofuser) for the generation of nanobubbles. The standard hemodialysis machine 100 includes a dialyser pathway and an extracorporeal circuit, as will be known to those skilled in the art. The standard hemodialysis machine 100 incorporates into the dialyser pathway, the oxygenation apparatus (nanofuser) 10, which may be according to any of the embodiments described herein. The oxygenation apparatus 10 diffuses oxygen nanobubbles into the dialysate 102 (which could be known dialysis fluid but is not limited thereto, any organ preservation solution as describes elsewhere in this disclosure could be used but acellular solutions are preferred). Accordingly, dialysate 102’, comprising nanobubbles of gas including oxygen, is introduced to the dialysis cartridge 104 where it interacts with the extracorporeal fluid in the known manner for dialysis, and passes to waste 105 in the known manner.

[0131] In the extracorporeal circuit, the extracorporeal fluid passes in a circuit. The extracorporeal fluid may be an organ preservation solution as described elsewhere in the specification. The input from Reservoir 1 (106) to the dialysis cartridge 104 is the fluid exit from the organ 54. Similarly, the output from the dialysis cartridge 104 flows to Reservoir 2 (108) and is directed towards the organ 54. In the dialysis cartridge, it is understood that the extracorporeal fluid obtains nanobubbles of gas including oxygen via the membrane in the dialysis cartridge 104.

[0132] From Reservoir 2, the extracorporeal fluid comprising oxygen nanobubbles solution 56 flows to the stepper motor peristaltic pump 86’ which is controlled by a stepper motor controller 84’ which is in turn controlled by the microprocessor 82’ receiving signals indicative of pressure from the pressure transducer 88’. As described above, the accuracy of the pressure may be improved by use of the pressure calibrator 90’. From the pump 86’, the extracorporeal fluid 56 flows to the organ 54. In the specification, like numerals represent like parts, although parts may be adapted for different embodiments and this is indicated by the inclusion of a prime (‘) symbol. Appropriate nutrition and other supplements may be added to the extracorporeal circuit.

[0133] Another embodiment of an organ preservation apparatus 50’ is illustrated in Figures 22 to 24D. The organ preservation apparatus 50’ is similar to that illustrated in 1005814927Figure 5 and like numerals represent like parts with the inclusion of the prime symbol (‘) to indicate the modified parts.

[0134] The nanobubble generator 11’ is configured to oxygenate at least a part of the organ preservation solution with oxygen nanobubbles. The nanobubble generator 11’ is incorporated into T-fitting 120 as shown in more detail in Figure 24. As shown in particular in Figure 24B, the T-fitting comprises a plastic fitting with three internally threaded apertures. The T-fitting has a main tubular portion in which two of the threaded apertures are axially aligned and a branch tubular portion for the third threaded aperture. The branch tubular portion intersects the main tubular portion.

[0135] The diffuser 12’ is provided with a nano-porous diffusion stone 14’, exterior thread 126, hex nut portion 124 and nipple 16’. Accordingly, the diffuser 12’ is threaded into the T-fitting as best shown in Figure 24C and Figure 24D, with the longitudinal axis of the diffuser 12’ aligned with the longitudinal axis of the main tubular portion. As can be best seen in Figure 24D, there is a peripheral gap around the cylindrical diffusion stone with 14’, between the diffusion stone 14’ and the interior periphery of the T-fitting 120.

[0136] As can be seen in Figure 23, the branch tubular portion of the T-fitting 120 is provided with the fluid inlet (for the organ preservation solution) into the nanobubble generator 11’. Accordingly, the flow path through the nanobubble generator 11’ will encounter a constricted portion, being the gap around the diffuser 12’, followed by an expanded portion as the fluid passes into the opposite end of the main tubular portion to that which is threaded with the diffuser 12’, and finally opening out into the container 24’. This contorted flow path leads to hydrodynamic cavitation enhancing the production of nanobubbles in the organ preservation solution.

[0137] The diffuser 12’ of the nanobubble generator 11’ is attached to a source (not shown) of oxygen or oxygen-enriched gas at nipple 16’, also resulting in the production of nanobubbles in the organ preservation solution.

[0138] As the organ preservation solution exits the nanobubble generator 11’, it flows into a container 24’. The outlet 130 from the nanobubble generator 11’ (which is the inlet into the container 24’) is disposed above the outlet 132 from the container 24’. The fluid (being the organ preservation solution) in the container 24’ forms a fluid seal between 1005814927the fluid inlet 130 and the fluid outlet 132, serving as a form of bubble trap which aims to disburse bubbles larger than nanobubble size to atmosphere via gas escape 110. Additionally or alternatively, the fluid level in the container 24’ should be controlled to be above the fluid outlet 132 to create a bubble trap. A controller (not shown) may ensure the maintenance of the required fluid level to create a bubble trap within the container 24’.

[0139] The solution / fluid comprising oxygen nanobubbles is drawn from the container 24’ by the peristaltic pump 60’ and onto the perfusion cannula 61’ in a manner similar to that described in connection with Figure 5. The organ (not shown) is received in the reservoir and organ chamber 52’. The organ is held in the chamber 52’ by being placed upon the perforated metal draining rack 55 as shown in Figure 22. Accordingly, the organ is treated ex vivo.

[0140] The organ preservation solution / fluid which drains to the bottom of the chamber 52’ is collected at reservoir outlet 112. The fluid passes under gravity to the branch portion of the T-fitting 120 where it passes through the nanobubble generator 11’ as explained above. The passage of the fluid along the flow path through the T-fitting and beyond may be enhanced by the pressurised gas flow through the diffuser 12’ and T-fitting 120.

[0141] Organ Preservation Solution “perfusate”

[0142] The terms ‘organ preservation solution’, and ‘perfusate’ are used interchangeably herein to define the solution that is perfused into the organ or organ block during the organ preservation method. The solution is supplied to the organ or organ block under such conditions that the solution permeates the organ or organ block. The solution may circulate and distribute through the organ or organ block via blood vessels or other natural channels in the tissue. Preferably, the organ preservation solution is provided to an organ via an artery.

[0143] The solution, once perfused, may subsequently exit or defuse out of the organ or organ block. In some embodiments, the perfusate may flow into the organ or organ block, through the organ or organ block, exit the organ or organ block, and be recycled to perfuse again. This cyclical process of flowing perfusate into and out of the one or 1005814927more organs and recycling the perfusate each cycle may happen for multiple cycles. Recycling the perfusate may comprise dialysing the exit perfusate before it is supplied to flow into the organ or organ block again. Alternatively, the perfusate may flow into the organ or organ block (inflow), and any flow out of the organ or organ block (outflow) is discarded as waste organ preservation solution.

[0144] CSF is the oxygen carrier for the brain and spinal cord. CSF is separated from systemic circulation by the blood-CSF barrier, a component of the blood-brain barrier. It is formed by ultrafiltration of blood through the ependymal cells of the choroid plexus. There is no appreciable barrier between CSF and the extracellular space of the brain, and as such, CSF directly provides oxygenation and nutrition to neural tissue. CSF has higher sodium, chloride, and magnesium concentrations than plasma, and lower potassium and calcium concentrations (SakkaColl and Chazal, 2011). The average oxygen content of CSF in normal healthy humans is between 25 and 53 mmHg (Zaharchuk et al., 2005).

[0145] In certain embodiments, the organ preservation solution comprises an electrolyte solution. In some embodiments, the preservation solution comprises an acellular perfusate comprising an electrolyte composition. Preferably, the perfusate is acellular.

[0146] In certain embodiments, the organ preservation solution comprises a nutrition component.

[0147] In certain embodiments, the organ preservation solution is CSF-like. In other embodiments, the organ preservation solution is blood comprising oxygen nanobubbles. In certain embodiments, the organ preservation solution comprises one or more components selected from the group consisting of albumin, nutrient(s), and creatinine.

[0148] In the present disclosure, oxygen-enriched, balanced buffered electrolyte solution with albumin at a concentration of 4g / dl and total parenteral nutrition (TPN) (Olimel, Baxter™) @25ml / kg was used to perfuse composite organ blocks.10mg / dL of creatinine was added. The mean PaO2achieved was 318mmHg (SD = 119). Theelectrolytesolution composition is shown in Table 3. Table 3 - Electrolyte solution composition that is combined with albumin and creatinine before oxygen nanobubbles are generated and the final solution comprising 1005814927nanobubbles including oxygen is used to perfuse an organ Electrolyte Concentration Electrolyte Concentration Na+ 138.00 mmol / lCa++ 1.25 mmol / lHCO332.00 mmol / lK+ 3.00 mmol / l Mg++ 0.50 mmol / l CH3COO- 3.00 mmol / l Cl- 109.50 mmol / l C6H12O65.6 g / dlOsmolarity 293mosml / l Organ preservation method

[0149] ‘Organ preservation’ refers to maintaining, extending or otherwise prolonging the viability of an organ or organs ex vivo, such as during the time period after an organ or organ block has been removed from a body or cadaver and whilst it is being stored before transplantation surgery. In some embodiments, the organ or organs may be preserved for at least about 30 minutes, at least about an hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 12 hours, at least about 14 hours, at least about 1 day, at least about 2 days, at least about 3 days, or at least about 4 days.

[0150] Advantageously, the flow of an organ preservation solution comprising oxygen nanobubbles into an organ used the methods described herein can achieve higher PaO2 compared to room temperature oxygenation, preferably at least 3 times that of room temperature tissue oxygenation achieved by standard methods. In a preferred embodiment, the mean PaO2achieved is at least about 265mmHG.

[0151] ‘Flowed through the organ’ as used herein refers to perfusion of the preservation solution into the organ or organ block to a sufficient degree for the organ or organ block to be preserved. In some embodiments, the at least one organ is perfused used a pump, preferably a peristaltic pump. The perfusion pressure may be modulated depending upon the size of the organ or organ block, and may for example, be between at least about 30 to 60mmHG.

[0152] In some embodiments, the method of the invention comprises using an oxygenation apparatus or organ preservation apparatus as described herein. 1005814927

[0153] The oxygenation apparatus 10, the organ preservation apparatus 50 and / or the organ preservation solution 56 disclosed herein can be used in a method for normothermic, subnormothermic, hypothermic or isothermic organ preservation. The method comprises flowing the organ preservation solution 56 comprising oxygen nanobubbles through the organ or organs.

[0154] In some embodiments, one or more organs are maintained at between 2 and 40 °C. Preferred temperature ranges are explained and their context provided throughout this specification. The skilled person will be aware that, if oxygenation of an organ is to rely on the activity of red blood cells, then normothermic or body temperature conditions will be needed. Blood comprising oxygen nanobubbles, where oxygenation does not rely on red blood cell activity, could be effective at other temperatures.

[0155] In certain embodiments, the method is used for isothermic organ preservation. Isothermic organ preservation means the organ is maintained at ambient temperature. Advantageously, the methods of the invention described herein permits normothermic, subnormothermic, isothermic or hypothermic oxygenated organ preservation that is similarly effective or more effective than organ preservation requiring static cold storage (SCS) hypothermic conditions. A similar advantage can be achieved in subnormothermic conditions and at the preferred temperatures of 15°C to 25°C or 15°C to 20°C.

[0156] In certain other embodiments, the method is used for normothermic organ preservation. Normothermic organ preservation means the organ is maintained at or about normal body temperature, such as between about 34 to about 38 degrees Celsius, preferably 35.5°C to about 37.5°C.

[0157] In still certain other embodiments, the method is used for hypothermic organ preservation. Hypothermic organ preservation means the organ is maintained at a temperature that is less than normal body temperature or less than about 35 °C, such as between about 2°C to about 8°C. Organ viability

[0158] In some embodiments of other methods defined herein, there is provided a method for determining the suitability of one or more organs for transplantation, comprising determining the viability of one or more organs. The skilled person will be 1005814927aware of suitable methods known in the art to assess the viability of an organ being prepared for transplantation. The known methods are largely comparisons with circumstances that have previously resulted in successful transplant of the relevant organ or organ block. Predominantly, known methods rely upon the time that has elapsed since the organ for transplant was removed from the donor body.

[0159] Assessing organ viability may comprise assessing organ function. In turn, organ viability and function can be taken as measures to assess ex vivo preservation quality and suitability for transplant. Both assessing organ function and assessing preservation quality may be embodiments of other methods defined herein. Organ viability is preferably tested ex vivo and during the organ preservation method of the invention and / or with an apparatus of the invention. Most analysis is determined by testing the fluid leaving the organ and comparing to the composition of the organ preservation solution.

[0160] Exemplary methods for assessing viability include, but are not limited to: - measuring aerobic respiration via consumption of oxygen and levels of carbon dioxide (levels can be determined by a blood gas analyser) - measuring tissue ATP generation as evidence of aerobic respiration (such as luciferase assay to detect ATP) - measuring metabolic activity eg glucose consumption. Glucose utilisation may be assessed using commercially-available glucose strips; or a combination thereof.

[0161] Determining organ viability may further comprise: - measuring platelet-activating factor (PAF) levels in plasma, to assess reperfusion injury and hyperoxia. PAF levels may be measured up to 5- hours post-preservation. - assessing insulin and GLP-1 response to enteral glucose stimulation (for pancreas, as an assessment of function and usually completed using Eliza assays) - measuring ICG clearance (for liver, as an assessment of function often using an RD15 test) 1005814927- measuring creatinine clearance (for kidneys, as an assessment of function using a creatinine in vitro test) or a combination thereof.

[0162] Suitable methods are exemplified in the Examples provided herein. Said methods may also be used to determine the suitability of an organ preservation, produced as described herein, and for the methods of organ preservation described herein.

[0163] Assessing the viability of an organ or organ block preserved using a method defined herein may further require a tissue biopsy or biopsies to obtain tissue for analysis. The skilled person will be aware of suitable methods to sample organ tissue to determine organ viability, without compromising organ viability. Organ viability may be assessed by performing histology on tissues to assess for reperfusion injury. For example, histology could be performed on tissue sections stained with hematoxylin and eosin (H&E staining), then the tissue could be scored for injuries and specific cell types. In the Examples provided herein, for H&E staining, a score of 0-2 was considered mild damage, 3-5 moderate, and >6 severe. An angiography may be performed (using a contrast agent such as fluorescein) to visualise and qualitatively assess vascular integrity eg of mesenteric arcades in the small intestine.

[0164] An organ or organ block preserved using a method defined herein may be compared to a similar organ or organ block stored in the same manner but reperfused differently.

[0165] In any embodiment of a method described herein, the method may further comprise one or more steps for determining the one or more organs is free from a pathogen or pathogens, such as blood-borne pathogens. For example, one or more preserved organs may be tested for the presence of HIV, Sar-Cov2 coronavirus, influenza, Hepatitis B, Hepatitis C, Epstein-Barr virus (EBC), Cytomegalovirus (CMV), syphilis, toxoplasma. The skilled person will be aware of suitable pathogens to detect that may be transmitted by organ transplantation to the donor, and suitable methods to detect said pathogens without compromising organ viability. 1005814927

[0166] The present disclosure further provides for a preserved organ or organs for transplantation into a human subject, wherein the preserved organ or organs have been preserved by a method described herein.

[0167] The present disclosure further provides for a method of treating a subject in need of organ transplantation, by administering an organ preserved by a method described herein. Examples Example 1 – Pilot study to determine if a dialysis system could be successfully modified to synchronously oxygenate and dialyze blood to create a normothermic perfusion machine

[0168] A Baxter PrismaflexTMDialysis System was modified to use oxygenated dialysate (Figure 16). Whole blood was used to perfuse 12 composite porcine abdominal blocks before oxygenation of the organs using the modified dialysis system. Serial arterial and venous blood gas samples were taken to assess oxygenation across the dialysis membrane, oxygen consumption, carbon dioxide production and pH maintenance.

[0169] Results of the study are shown in Figure 14. Oxygen transfer from the oxygen nanobubbles across the dialysis cartridge was effective, with significantly higher PaO2 (M = 318 mmHg, SD = 119) as compared to room air oxygenation (p<0.05). Oxygen consumption alongside carbon dioxide production was observed, with a baseline approximately three times that of normal resting tissue oxygen consumption. The dialysis function was able to maintain pH with a range of normality and the blocks were successfully perfused and preserved for 6 hours.

[0170] These results evidenced that existing dialysis medical equipment could be repurposed for organ preservation under normothermic or subnormothermic conditions, using a dialysate comprising oxygen nanobubbles. Advantageously, the approach of using an organ preservation apparatus that undergoes dialysis to remove waste whilst also providing oxygen nanobubbles, allows compensation for metabolic acidosis (as indicated by reduced pH) and permits further preservation of the one or more organs. 1005814927Example 2 – Experimental testing of organ preservation and viability

[0171] A large animal model of 70 – 80 Kg landrace pigs was used to test the organ preservation apparatus. The pigs were randomised into 3 groups of 4 each. Group 1 underwent static cold storage, group 2 underwent isothermic preservation, and group 3 underwent normothermic oxygenated perfusion using a modified dialysis machine with dialysate oxygenation using the organ preservation apparatus as shown in Figure 16. The organ preservation solution used is as described in the paragraph 148 including Table 3.

[0172] Abdominal organs were retrieved as a block including, the liver, small bowel, pancreas, and kidneys and were stored either with static cold storage or machine perfusion for 5 hours. The organs were then cooled and then re-perfused with blood on the modified dialysis machine as a surrogate to transplant.

[0173] The following tests of organ viability were performed: • Evidence of respiration • Evidence of ATP generation • Evidence of Glucose Utilization

[0174] The following tests of organ function were performed: • Insulin and GLP-1 response to enteral glucose stimulation • ICG clearance • Creatinine clearance • H&E Histology Results Evidence of respiration (Figures 6 and 7)

[0175] A comparison of arterial and venous oxygen content showed oxygen consumption approximately 3 times that of resting tissue oxygen consumption. In 1005814927conjunction with carbon dioxide production, this indicated that aerobic respiration was taking place during the preservation period. Evidence of ATP Generation (Figures 8 and 9)

[0176] Quantitative ATP measurement in the kidneys showed regeneration of ATP levels over a period of 5 hours, indicating intact metabolic pathways. The control SCS arm showed degeneration of ATP over this period. ATP levels in the isothermic preserved kidneys were non-inferior to the current gold- standard. ATP levels in the liver followed a similar trend. It is important to note that ATP levels in the NMP arms were not 0, they were an order of magnitude smaller than the IMP or SCS arms, and as such are difficult to display on the same graph. Evidence of Glucose Utilization (Figure 10)

[0177] A decreasing gap between hepatic venous and arterial glucose content, with increasing ATP levels suggest aerobic glucose utilization with arrest of glycolysis. Insulin and GLP-1 response to enteral glucose stimulation (Figure 11)

[0178] Post-enteral glucose stimulation, GLP-1 and insulin responses were observed. Also observed was the fact that insulin peaks corresponded to GLP-1 peaks, indicating intact entero-pancreatic signalling and function. Creatinine clearance (Figure 12)

[0179] Creatinine clearance was observed in all perfused kidneys, indicating intact function. Clearance present in perfusion is an advantage over SCS, as function can be determined and correlated to performance post-transplant. Histology (Figure 13)

[0180] A senior blinded pathologist reviewed all H&E-stained biopsies of the perfused organs. A novel scoring system was developed to quantify the degree of injury at end of preservation and after reperfusion.

[0181] Statistical analysis showed that there was no significant difference in the degree of injury at end of preservation or following reperfusion for organs preserved for 10058149275 hours. All modes of preservation over this period produced organs with minimal damage on histology.

[0182] This study showed that: • Oxygen nanobubbles can be introduced in a perfusion circuit and maintain continuous flow; • Preservation of an organ with an organ preservation solution comprising oxygen nanobubbles is non–inferior to static cold storage; and • It is possible to demonstrate organ viability and physiological and metabolic integrity during perfusion. REFERENCES SAKKA, L., COLL, G. & CHAZAL, J.2011. Anatomy and physiology of cerebrospinal fluid. Eur Ann Otorhinolaryngol Head Neck Dis, 128, 309-16. ZAHARCHUK, G., MARTIN, A. J., ROSENTHAL, G., MANLEY, G. T. & DILLON, W. P.2005. Measurement of cerebrospinal fluid oxygen partial pressure in humans using MRI. Magn Reson Med, 54, 113-21. Michailidi et al. J Colloid Interface Sci.2020 Mar 22:564:371-380. doi: 10.1016 / j.jcis.2019.12.093. Epub 2019 Dec 23. Wang, Yanwei & Wang, Tianxiang. (2023). Preparation Method and Application of Nanobubbles: A Review. Coatings.13.1510.10.3390 / coatings13091510.

[0183] The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that such prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and / or combined with other pieces of prior art by a skilled person in the art.

[0184] It will be understood that the terms “comprise” and “include” and any of their derivatives (e.g. comprises, comprising, includes, including) as used in this specification, and the claims that follow, is to be taken to be inclusive of features to 1005814927which the term refers, and is not meant to exclude the presence of any additional features unless otherwise stated or implied.

[0185] In some cases, a single embodiment may, for succinctness and / or to assist in understanding the scope of the disclosure, combine multiple features. It is to be understood that in such a case, these multiple features may be provided separately (in separate embodiments), or in any other suitable combination. Alternatively, where separate features are described in separate embodiments, these separate features may be combined into a single embodiment unless otherwise stated or implied. This also applies to the claims which can be recombined in any combination. That is a claim may be amended to include a feature defined in any other claim. Further a phrase referring to “at least one of” a list of items refers to any combination of those items, including single members. As an example, “at least one of: a, b, or c” is intended to cover: a, b, c, a-b, a-c, b-c, and a-b-c.

[0186] It will be appreciated by those skilled in the art that the disclosure is not restricted in its use to the particular application or applications described. Neither is the present disclosure restricted in its preferred embodiment with regard to the particular elements and / or features described or depicted herein. It will be appreciated that the disclosure is not limited to the embodiment or embodiments disclosed, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope as set forth and defined by the following claims.

[0187] Please note that the following claims are provisional claims only, and are provided as examples of possible claims and are not intended to limit the scope of what may be claimed in any future patent applications based on the present application. Integers may be added to or omitted from the example claims at a later date so as to further define or re-define the scope. It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention. 1005814927

Claims

CLAIMS 1. An oxygenation apparatus for producing an organ preservation solution comprising nanobubbles of gas including oxygen, the apparatus including: a flow path for passage of a precursor organ preservation solution, wherein the flow path comprises at least one constricted portion followed by one or more expanded portion; a nanoporous gas diffuser adapted for connection to a source of gas including oxygen, the nanoporous gas diffuser disposed for fluid contact with the precursor organ preservation solution passing through the flow path.

2. The oxygenation apparatus as claimed in claim 1 wherein the one or more constricted portions including a first constricted portion and a second constricted portion, the second constricted portion being downstream of the first constricted portion.

3. The oxygenation apparatus as claimed in claim in claim 1 or 2, wherein the nanoporous gas diffuser is disposed at the one or more constricted portions.

4. The oxygenation apparatus as claimed in claim 2, wherein the nanoporous gas diffuser is positioned to create either or both of the first constricted portion and / or the second constricted portion.

5. The oxygenation apparatus as claimed in claim 4, wherein the nanoporous gas diffuser is positioned within the flow path.

6. The oxygenation apparatus as claimed in claim 5, wherein the flow path is in part defined by a tube in which the nanoporous gas diffuser is received.

7. The oxygenation apparatus as claimed in claim 6, wherein the tube is formed as a T- fitting having a main tubular portion and a branch tubular portion, the main tubular portion receiving the nanoporous gas diffuser, thereby creating a first constricted portion as a result of the nanoporous gas diffuser occluding part of the tube, wherein the branch tubular portion is adapted for fluid connection to a source of the organ preservation solution .

8. The oxygenation apparatus as claimed in claim 6 or claim 7, wherein the central 1005814927longitudinal axis of the nanoporous gas diffuser is aligned with the central longitudinal axis of the tube.

9. The oxygenation apparatus as claimed in any one of claims 1 to 8, further including a bubble trap for escape of undesirable bubbles.

10. The oxygenation apparatus as claimed in claim 9, wherein the bubble trap is in the form of a spillway or overflow.

11. The oxygenation apparatus as claimed in claim 9, wherein the bubble trap is in the form of a p-trap or s-trap or other arcuate conduit.

12. An oxygenation apparatus for producing an organ preservation solution comprising nanobubbles of gas including oxygen, the apparatus including: a flow path for passage of a precursor organ preservation solution; a nanobubble generator for producing nanobubbles of gas including oxygen in the organ preservation solution, the nanobubble generator comprising at least one of: a nanoporous gas diffuser adapted for connection to a source of gas including oxygen, and disposed for fluid contact with the precursor organ preservation solution; and the flow path comprising at least one constricted portion followed by at least one or more expanded portion; and the apparatus further including a bubble trap downstream of the nanobubble generator.

13. The oxygenation apparatus as claimed in claim 12, wherein the bubble trap comprises any one or more of: a spillway or overflow; a p-trap or s-trap or other arcuate conduit; and a liquid seal achieved from the organ preservation solution being maintained within a vessel at a level above an outlet to the container / vessel.

14. An organ preservation apparatus comprising: a receptacle for receiving one or more organs ex vivo; a delivery apparatus configured to deliver an organ preservation solution to one or more organs; an oxygenation apparatus for oxygenating at least a part of the organ preservation 1005814927solution comprising nanobubbles of gas including oxygen.

15. The organ preservation apparatus as claimed in claim 14 wherein the oxygenation apparatus is according to any of the preceding claims.

16. A method of producing an organ preservation solution comprising nanobubbles of gas including oxygen, the method including: directing a precursor organ preservation solution along a flow path which has one or more constricted portions followed by at least one expanded portion; and directing gas including oxygen through a nanoporous gas diffuser, the nanoporous gas diffuser being in fluid contact with the precursor organ preservation solution in the flow path to introduce nanobubbles of gas including oxygen into the precursor organ preservation solution; wherein the flow rate through, and the configuration of the flow path induces hydrodynamic cavitation.

17. A method of converting a hemodialysis circuit or a continuous veno-venous hemofiltration circuits for preservation of one or more organs ex vivo, wherein the circuit comprises a dialyser pathway for dialysate and an extracorporeal circuit for extracorporeal fluid, the method comprising: introducing an oxygenation apparatus into the dialyser pathway for oxygenating the dialysate with nanobubbles of gas including oxygen; and adapting the extracorporeal circuit for receipt of one or more organs for preservation.

18. The method according to claim 17, wherein the oxygenation apparatus is according to any of claims 1 to 13.

19. An ex vivo method for organ preservation using oxygenated perfusion, the method comprising: - flowing an organ preservation solution comprising nanobubbles of gas including oxygen through one or more organs, preferably the nanobubbles are bulk nanobubbles.

20. A method for normothermic, hypothermic or isothermic organ preservation ex vivo 1005814927comprising flowing an organ preservation solution comprising nanobubbles of gas including oxygen through one or more organs.

21. The method of claim 19, wherein the one or more organs is preserved in hypothermic, subnormothermic, normothermic, or isothermic conditions, or a combination thereof, preferably subnormothermic or isothermic conditions.

22. The method of any one of claims 19 to 21, wherein the organ is preserved at a temperature from 2 to 40 °C, preferably from 15 to 35 °C, 15 to 25 °C or 15 to 20°C.

23. The method of claim 22, wherein the organ is preserved ex vivo for a preservation period of about 30 minutes to 4 days, preferably from 1 hour to 12 hours.

24. The method of any one of claims 19 to 23, wherein the method is conducted under subnormothermic conditions such as from 15 to 25 °C, preferably at subnormothermic and isothermic conditions.

25. The method according to any one of claims 16 to 24, wherein the organ preservation solution is oxygen-enriched (ie nanobubble loaded) blood, CSF, fractionated blood product, plasma (ie blood-like, CSF-like, fractionated blood product-like or plasma- like) or acellular perfusate, alternatively the organ preservation solution is blood or a fractionated blood product.

26. The method according to claim 25, wherein the acellular perfusate is a nutritional solution.

27. The method according to claim 26, wherein the nutritional solution includes glucose, preferably the nutritional solution is a total parenteral nutrition solution.

28. The method of any one of claims 16 to 27, wherein the organ preservation solution comprises electrolytes.

29. The method of any one of claims 17 to 28, wherein the organ preservation solution is provided to the one or more organs via oxygenated machine perfusion, preferably subnormothermic machine perfusion.

30. The method of any one of claims 17 to 29, wherein the one or more organs conducts aerobic respiration during a preservation period. 100581492731. The method of any one of claims 16 to 30, wherein the one or more organs is selected from: a kidney, a small bowel, a pancreas, a liver, a heart, a cornea, a lung or combinations thereof such as an organ block.

32. The method of any one of claims 16 to 31, wherein the organ preservation solution comprises one or more components selected from the group consisting of albumin, nutrient(s), and creatinine.

33. The method of claim 32, wherein the albumin is present at from 3g / dl to 5 g / dl, preferably 4 g / dl.

34. The method of claim 32, wherein the organ preservation solution includes a total parenteral nutrition formula (such as that sold by Baxter), optionally at 20-30 ml / kg, preferably 25 mg / kg.

35. The method of claim 32, wherein the creatinine is present at from 5 to 15 mg / dL, preferably 10 mg / dL.

36. The method of any one of claims 16 to 35, wherein the organ preservation solution has a partial oxygen pressure of 275 to 375 mmHg, 300 to 350 mmHg, or 315 to 335 mmHg.

38. The method of any one of claims 16 to 37, wherein the osmolarity of the organ preservation solution is 200 to 400 mosml / l or 290-310 mosml / l.

39. The method of any one of claims 16 to 38, wherein the organ preservation solution comprises glucose, optionally 0.5-10 g / dl, preferably 5-10 mmol / l.

40. The method of any one of claims 16 to 39, wherein the organ preservation solution is buffered to pH 7 to 8, preferably about 7.

5.

41. The method of any one of claims 16 to 40, wherein the organ preservation solution flows at a flow rate from 15 to 450 ml / min, preferably 15 to 30 ml / min or about 25 ml / min for an acellular organ preservation solution or 350 to 450 ml / min or 400 ml / min for a blood organ preservation solution.

42. The method of any one of claims 16 to 41, wherein the organ preservation solution is at a pressure of 20 to 70 mmHg, preferably 30 to 40 mmHg for an acellular organ 1005814927preservation solution or 55 to 65 mmHg for a blood organ preservation solution.

43. The method of any one of claims 16 to 42, wherein the partial oxygen pressure of the organ preservation solution is 250 to 280 mmHg.

44. The method of any one of claims 17 to 43, wherein the organ preservation solution is supplied to one or more vessels of the one or more organs.

45. The method of claim 44, the one or more vessels are one or more arteries.

46. The method of any one of claims 17 to 45, wherein the organ preservation solution is directly supplied to the one or more organs via one or more catheters or cannulas.

47. The method of any one of claims 17 to 46 wherein after the organ preservation solution flows through one or more organs, the organ preservation solution is dialyzed before returning to the one or more organs.

48. The method according to any one of claims 17 to 47, wherein the organ preservation solution produced in accordance with the method of claim 16.

49. The method according to any one of claims 17 to 48, wherein the method further comprises one or more steps of: - determining the viability of the one or more organs; and / or - determining the one or more organs is free from a pathogen or pathogens; - obtaining the one or more organs to be preserved by harvesting the one or more organs from a human or a non-human animal.

50. The method according to any one of claims 19 to 49, wherein the method results in resuscitation or partial resuscitation of the organ.

51. The method according to any one of claims 17 to 50, wherein the method is performed using the oxygenation apparatus of any one of claims 1 to 14 or the organ preservation apparatus of claim 15.

52. A biological product comprising one or more organs preserved by the method according to any one of claims 17 to 51. 100581492753. A method for determining the suitability of one or more organs for transplantation ex vivo, comprising determining the viability of one or more organs by measuring one or more of: - aerobic respiration; - glucose utilisation; - ATP generation; - ICG clearance (where the one or more organs includes a liver); - creatinine clearance (where the one or more organs includes a kidney) - insulin and / or GLP-1 response to enteral glucose stimulation (where the one or more organs includes a pancreas), preferably measuring at least one or more of aerobic respiration, glucose utilisation, and ATP generation.

54. The method according to claim 53, further comprising transplanting into a subject in need thereof one or more organs determined as suitable for transplantation.

55. A method for treating a subject in need of organ transplantation, by transplanting into the subject one or more organs preserved by the method according to any one of claims 17 to 51, or the biological product of claim 52. 56.Use of the oxygen apparatus of any one of claims 1 to 14, or the organ preservation apparatus of claim 15, for the preservation of one or more organs ex vivo for organ transplantation.

57. The oxygen apparatus of any one of claims 1 to 14, or the organ preservation apparatus of claim 15, for use in the ex vivo preservation of one or more organs for organ transplantation. 1005814927

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