Patent Application
20240373840
In one aspect, the invention provides an isolated perfusate mixture comprising: an inorganic salt solution; an artificial oxygen carrier; and autologous blood.
In another aspect, the invention provides a system for the hypothermic preservation of organs in a mammal, the system comprising:
a perfusion device for the perfusion of an isolated perfusate mixture into the mammal, the perfusion device comprising: a perfusion loop; and a controller programmed to regulate at least a perfusate temperature within the perfusion loop to maintain hypothermic conditions; and the isolated perfusate mixture as described elsewhere herein.
In yet another aspect, the invention provides a mammal perfused with the isolated perfusate composition as described elsewhere herein, wherein mammalian organs are perfused under hypothermic conditions.
In yet another aspect, the invention provides perfused organs in a diseased mammal, wherein the perfused organs maintain one or more properties selected from the group consisting of an in vivo level of cell function and viability, and an in vivo level of morphology
In certain embodiments, the one or more artificial oxygen carriers is selected from the group consisting of hemoglobin glutamer-250, isolated cell-free hemoglobin, cross-linked hemoglobin, polymerized hemoglobin, encapsulated hemoglobin, and perfluorocarbon oxygen carriers. In certain embodiments, the artificial oxygen carrier is hemoglobin glutamer-250.
In certain embodiments, the one or more inorganic salts are selected from the group consisting of sodium chloride, sodium bicarbonate, magnesium chloride, and calcium chloride.
In certain embodiments, the perfusate mixture comprises a priming solution containing one or more sugars. In certain embodiments, the one or more sugars are glucose or dextrane.
In certain embodiments, the isolated perfusate mixture further comprises one or more amino acids. In certain embodiments, the one or more amino acids are selected from the group consisting of glycine, L-alanyl-glutamine, L-arginine, L-cysteine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine and salts and solvates thereof.
In certain embodiments, the perfusate mixture further comprises one or more vitamins.
In certain embodiments, the one or more vitamins are selected from the group consisting of choline, D-calcium pantothenate, folic acid, niacinamide, pyridoxine, riboflavin, thiamine, i-inositol and salts and solvates thereof.
In certain embodiments, the perfusate mixture further comprises, ferric nitrate, magnesium sulfate, potassium chloride, sodium phosphate, and derivatives thereof.
In certain embodiments, the perfusate mixture further comprises an anti-clotting agent. In certain embodiments, the anti-clotting agent is heparin.
In certain embodiments, the percentage of autologous blood in the mixture is between 10% and 50%. In certain embodiments, the percentage of autologous blood in the mixture is approximately 28%.
In certain embodiments, the mixture is dialyzed against a solution comprising inorganic salts. In certain embodiments, the mixture is dialyzed against plasma.
In certain embodiments, the mixture comprises electrolytes and oncotic agents at levels comparable to those in autologous blood. In certain embodiments, the perfusate further comprises cytoprotective agents.
In certain embodiments, the cytoprotective agents are selected from the group consisting of 2-Iminobiotin, Necrostatin-1, sodium 3-hydroxybutryate, glutathione, minocycline, lamotrigine, QVE-Oph, methylene blue, and/or any salts, solvates, tautomers, and prodrugs thereof.
In certain embodiments, the mixture further comprises antibiotics. In certain embodiments, the antibiotic is ceftriazone. In certain embodiments, the mixture comprises one or more anti-inflammatory agents.
In certain embodiments, the one or more the anti-inflammatory agents is dexamathazone or cetirizine.
In certain embodiments, the temperature of the mixture is approximately 28° C.
In certain embodiments, the perfusion loop further comprises at least one pulse generator programmed to generate a pressure pulse within the perfusate within the perfusion loop.
In certain embodiments, the perfusion loop comprises a venous loop, a filtration loop and an arterial loop, wherein:
the venous loop comprises at least one perfusion pump;
the filtration loop comprises at least one perfusion pump, and at least one hemodiafiltration unit adapted and configured to equilibrate the perfusate;
the arterial loop comprises at least one gas exchange source and at least one gas mixer adapted and configured to supply oxygen and carbon dioxide to the perfusate;
wherein the mammal, the venous loop, the filtration loop and the arterial loop are in fluidic communication such that the perfusate can be carried from the mammal, through the venous loop, through the filtration loop, through the arterial loop and back to the mammal.
In certain embodiments, the one or more components selected from the group consisting of the venous loop, the filtration loop and the arterial loop further comprise a reservoir containing excess perfusate.
In certain embodiments, the one or more components selected from the group consisting of the mammal, the venous loop, the filtration loop and the arterial loop further comprise one or more elements selected from the group consisting of:
one or more valves adapted and configured to regulate the flow of the perfusate;
one or more filters adapted and configured to filter the perfusate; and
one or more sensors for measuring one or more properties of the perfusate selected from the group consisting of pH, dissolved oxygen concentration, dissolved carbon dioxide concentration, dissolved metabolite concentration, temperature, pressure, and flow rate.
In certain embodiments, the one or more sensors measure the concentration of at least one dissolved metabolite selected from the group consisting of nitric oxide, lactate, bicarbonate, oxygen, carbon dioxide, total hemoglobin, methemoglobin, oxyhemoglobin, carboxyhemoglobin, sodium, potassium, chloride, calcium, glucose, urea, ammonia, and creatinine.
In certain embodiments, the mammal perfusion apparatus comprises one or more sensors for measuring one or more properties of the perfusate selected from the group consisting of a pressure and a flow rate.
In certain embodiments, the one or more components selected from the group consisting of the mammal, the venous loop, the filtration loop and the arterial loop comprise one or more heat exchange units comprising:
one or more heat exchangers;
one or more temperature regulation units;
one or more temperature regulating pumps;
a thermoregulation fluid; and
one or more pipes configured and adapted to transport the thermoregulation fluid, wherein the one or more pipes are in fluidic communication with the one or more heat exchangers, the one or more temperature regulation units and the one or more temperature regulating pumps.
In certain embodiments, the one or more components selected from the group consisting of the brain enclosure unit, the venous loop, the filtration loop and the arterial loop comprise one or more sensors adapted and configured to measure the temperature within the perfusion device.
In certain embodiments, the one or more sensors are adapted and configured to measure the temperature within the perfusion device, the one or more temperature regulation units and the one or more temperature regulating pumps are in electronic communication with a computer programmed to regulate the temperature of the thermoregulation fluid and the specified flow rate of the one or more temperature regulating pumps to maintain a specified temperature within the perfusion device.
In certain embodiments, the hemodiafiltration unit is adapted and configured to supply one or more nutrients to the perfusate, selected from the group consisting of Glycine, L-Alanyl-Glutamine, L-Arginine hydrochloride, L-Cystine, L-Histidine hydrochloride-H2O, L-Isoleucine, L-Leucine, L-Lysine hydrochloride, L-Methionine, L-Phenylalanine, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine, L-Valine, Choline chloride, D-Calcium pantothenate, Folic Acid, Niacinamide, Pyridoxine hydrochloride, Riboflavin, Thiamine hydrochloride, i-Inositol, Calcium Chloride (CaCl2)-2H2O), Ferric Nitrate (Fe(NO3)3 9H2O), Magnesium Sulfate (MgSO4-7H2O), Potassium Chloride (KCl), Sodium Bicarbonate (NaHCO3), Sodium Chloride (NaCl), Sodium Phosphate monobasic (NaH2PO4-2H2O), D-Glucose (Dextrose), Phenol Red, Sodium Pyruvate, free fatty acids, cholesterol and nucleic acid constitutes.
In certain embodiments, the system is configured to perfuse the mammal with the perfusate at a cardiac pulsatile pressure of about 20 mmHg to about 140 mmHg.
In certain embodiments, the system is configured to perfuse the organs in the mammal with the perfusate through the pulse generator at a rate of about 40 to about 180 beats per minute.
In certain embodiments, the system further comprises a controller in electronic communication with one or more elements of the system.
In certain embodiments, the mammal is a deceased mammal. In certain embodiments, the mammal is a human. In certain embodiments, the deceased mammal is deceased for longer than 1 hour. In certain embodiments, the deceased mammal has been deceased for longer than 4 hours. In certain embodiments, the mammal died of cardiac arrest.
In certain embodiments, the organs in the deceased mammal are ischemic prior to perfusion with the isolated perfusate mixture.
In certain embodiments, rigor mortis is prevented. In certain embodiments, rigor mortis is reversed.
In certain embodiments, the perfusate mixture flows into the ophthalmic artery.
In certain embodiments, the perfusate mixture flows into the renal intralobular arteries.
Cells lack meaningful oxygen-storage capacity, leading to obligate oxygen-dependence. At the cellular level, in just minutes following ischemia, intracellular acidosis and edema develop, and cause secondary damage to cellular membranes, organelles, and ultimately cell death. At the whole-body scale, there is a systemic release of hormones and cytokines, and activation of autonomic nervous, immune, and coagulation systems, leading to end-organ injury and feedback onto cellular injury cascades that culminate in systemic metabolic acidosis and hyperkalemia.
Reperfusion of the whole-body with autologous blood has several deleterious issues, including coagulation, microvascular plugging, inflammation, and blood-intrinsic cellular dysfunction. These obstacles have limited whole-body reperfusion and recovery of large mammals to 20 minutes of warm ischemia. Appropriate interventions are needed for molecular and cellular recovery across all vital organs in the large mammalian body following prolonged warm ischemia. The present invention addresses this need.
The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.
The invention provides a novel system for restoration and preservation of an intact mammalian organs. In certain aspects, the system is capable of preserving organs in the mammalian body and restoring and maintaining cellular integrity and cellular function for hours post mortem or after global ischemia. The invention also provides novel synthetic organ perfusate formulations, and methods of mixing the perfusate with blood, for example, autologous blood, derived from the mammal.
The invention includes surgical methods and procedures to connect the mammal to the OrganEx system. In combination, the system, perfusate, and surgical method attenuate organ cell death, preserve anatomical and cellular integrity and restore cellular function as indicated by active metabolism. The invention also provides means to reduce reperfusion injury, stimulate recovery from hypoxia, and metabolically support the energy needs of organ function. The invention further provides methods of using the system and blood perfusate mixture, to prevent the collapse of organ vasculature and to allow for better perfusion of the organs. In addition, the invention provides methods to prevent the onset of rigor mortis.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described. As used herein, each of the following terms has the meaning associated with it in this section.
The instant invention is most clearly understood with reference to the following definitions.
As used herein, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.
Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. “About” can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
As used herein, the term “hypoxic” refers to a concentration of dissolved oxygen less than about 13%, corresponding to a partial pressure of about 100 mmHg, the physiologic partial pressure of oxygen in the alveoli of the lung.
As used herein, the term “cellular hypoxia” refers to a cellular response to exposure to a hypoxic environment, often resulting in apoptosis, or cellular death.
As used herein, the term “anaerobic metabolism” refers to the cellular consumption of glucose to produce two molecules of lactate, with the lactate remaining in dissolved in solution. The ratio of lactate produced to glucose consumed will be 2:1.
As used herein, the term “aerobic metabolism” refers to the cellular consumption of glucose to produce two molecules of lactate, both of which will be consumed through the Krebs cycle in the presence of sufficient levels of oxygen. The ratio of lactate produced to glucose consumed will be 0:1.
As used herein, the term “hypothermic” refers to a body temperature substantially below normal bounds. Hypothermic temperatures include, but are not limited to, temperatures between 10° and 32° C., between 20° C. and 30° C., and about 28° C.
As used herein, the term “salt” embraces addition salts of free acids or free bases that are compounds useful within the invention. Suitable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, phosphoric acids, perchloric and tetrafluoroboronic acids. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, b-hydroxybutyric, salicylic, galactaric and galacturonic acid. Suitable base addition salts of compounds useful within the invention include, for example, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, lithium, calcium, magnesium, potassium, sodium and zinc salts. Acceptable base addition salts also include organic salts made from basic amines such as, for example, N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methyl-glucamine) and procaine. All of these salts may be prepared by conventional means from the corresponding free base compound by reacting, for example, the appropriate acid or base with the corresponding free base.
As used in the specification and claims, the terms “comprises,” “comprising,” “containing,” “having,” and the like can have the meaning ascribed to them in U.S. patent law and can mean “includes,” “including,” and the like.
Unless specifically stated or obvious from context, the term “or,” as used herein, is understood to be inclusive.
Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (as well as fractions thereof unless the context clearly dictates otherwise).
As described herein, rigor mortis refers to process that sets in upon the death of a mammal. Rigor mortis is characterized by stiffening of the muscles. Stiffening occurs as a result of ATP depletion. ATP is depleted in ischemic tissues where there is insufficient oxygen available for mitochondria to drive ATP formation. As a result, the bridges between actin and myosin fibers are no longer broken, causing muscle stiffness.
As used herein, OrganEx refers to a system that consists of a perfusion system and synthetic perfusate. The perfusion system consists of a computer driven custom-made pulse generator connected to a centrifugal pump, which enables reproduction of physiological pressure and flow waveforms, together with automated hemodiafiltration, gas mixer, and drug delivery systems which allow control of blood coagulation and supplementation of the perfusate. To ensure homeostasis and maintain the targeted perfusion parameters, the perfusion system is also equipped with sensors for electrolytes, blood gases, metabolic parameters, hemoglobin, vessels and cannulas pressures, and total circulatory flow rate. In the OrganEx system, prior to the initiation of the perfusion protocol, autologous blood of the mammal is drained into the OrganEx system and mixed with the perfusate, which is then used to perfuse the animal.
As used herein, ECMO refers to a clinical standard of a heart-and-lung substitution perfusion device—extracorporeal membrane oxygenation system (ECMO). In the ECMO system, the animals are perfused with autologous blood.
The following abbreviations are used herein:
The invention provides a novel OrganEx technology and its experimental application that has the potential for recovery of key molecular and cellular processes in multiple porcine organs after prolonged warm ischemia.
The data presented herein demonstrate that mammalian cells are more resilient to ischemic injury than previously understood. Further, the data establish that cellular deterioration is a more protracted process that is not scripted within narrowly-defined sequences or timeframes.
In one embodiment, the application of the OrganEx technology can halt the process of cell demise. In another embodiment, the application of the OrganEx technology can shift cellular states towards recovery at molecular and cellular levels. In another embodiment, the application of the present invention can shift cellular states towards recovery, even following prolonged warm ischemia. In another embodiment, the invention provides a comprehensive single-cell transcriptomic analysis of the brain and vital peripheral organs over varying warm ischemic intervals. In another embodiment, the comprehensive single-cell transcriptomic analysis of the brain and vital peripheral organs over varying warm ischemic intervals is obtained utilizing perfusion with either autologous blood, the OrganEx perfusate, or a mixture of autologous blood and the OrganEx perfusate.
In some embodiments, the invention provides a transcriptome dataset and a unique resource for future basic and translational studies on cell-types, organs and ischemia.
In one embodiment, the OrganEx platform and acellular perfusate, connected to an intact dead mammal, provides a solution to the problem of ischemic stress in tissue culture and isolated organs.
The invention provides a means to reinstate circulation and systemic metabolic parameters across multiple organs in an intact animal. In one embodiment, the invention provides for the removal of deleterious processes and lack of oxygen, crucial for the control of multiple non-specific injury mechanisms affecting end-organ recovery and overall prognosis after global ischemia.
In one embodiment, the invention facilitates or enables repair responses at the molecular and cellular level in several or all organs. In another embodiment, the repair at the molecular and cellular level translate to processes supporting recovery of organs. In another embodiment, recovery of organs last for an extended period of time. In another embodiment, rigor mortis can be prevented in an animal following warm ischemia. In another embodiment, rigor mortis can be reversed. In another embodiment, dead spots in the heart can be prevented. In another embodiment, electrical activity can be measured in the heart. In another embodiment, contractile activity can be measured in the heart. In another embodiment, electrical activity can be measured in the brain. In another embodiment, the movements can be initiated in the animal. In another embodiment, the animal may regain consciousness. In another embodiment, the animal may regain the ability to move.
In one embodiment, enduring effects on cellular recovery post perfusion occurs in slices from the tissue most susceptible to ischemia, the hippocampus. In another embodiment, long term in vivo recovery of the hippocampus, and other organs is observed at the organ level.
In one embodiment, long OrganEx perfusions of the whole-body can prevent organs from undergoing ischemic injury. In another embodiment, long OrganEx perfusion can lead to recovery of vital organs, including the brain, the hearth, the kidney, the pancreas, or the liver.
In another embodiment, the OrganEx technology can be used in combination with mammals that are still alive. In one embodiment, the live mammal is a human. In one embodiment, the live mammal being treated has experienced a stroke or a heart attack prior to perfusion with the blood perfusate mixture. In one embodiment, the human recovers and/or recovers more quickly from the stroke or the heart attack. In one embodiment, the invention preserves and recovers brain function. In one embodiment, the recovery from ischemic injury in the brain is measurable by magnetic resonance imaging (MRI). In one embodiment, long OrganEx perfusion can be applied to the mammal, e.g., a human, following a cardiac event, e.g., a heart attack. In one embodiment, the invention preserves and recovers heart function. In one embodiment, the mammal recovers and/or recovers more quickly from the heart attack. In one embodiment, the recovery from ischemic injury in the heart is measurable with an electrocardiogram (EKG). In another embodiment, death of a mammal may be reversed.
In one embodiment, the technology provides for new avenues for whole-body global ischemia research. In another embodiment, the invention provides a means to conduct clinical resuscitation science or transplantation medicine. In one embodiment, the invention provides for larger donor organ pools by recovering previously marginalized organs.
The invention includes a novel perfusion composition for the preservation of organs in the mammalian body. In certain embodiments, the perfusion composition can be used to preserve organs in a mammal after warm ischemia. In certain embodiments, the blood perfusate mixture can preserve the brain, the liver, lung, heart, pancreas, kidney, and the like.
In certain embodiments, the perfusion composition is a perfusate comprising a solution comprising one or more artificial oxygen carrier compounds and one or more compounds selected from the group consisting of anti-cytotoxic compounds, antioxidants, anti-inflammatory compounds, antiepileptic compounds, anti-apoptotic compounds, antibiotics, cell death inhibitors, neuroprotectants and oxidative/nitrosative stress inhibitors.
In certain embodiments, the perfusate comprises priming solution. In certain embodiments, the priming solution comprises sodium chloride, sodium bicarbonate, magnesium chloride, calcium chloride, glucose and dextrane. In certain embodiments, the concentrations of the components in the priming solutions is as shown in Table 1.
In certain embodiments, the perfusate comprises hemodiafiltration exchange solution. In certain embodiments, the hemodiafiltration exchange solution comprises one or more amino acids selected from the group consisting of glycine, L-alanyl-glutamine, L-arginine, L-cysteine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine and salts and solvates thereof; one or more vitamins selected from the group consisting of choline, D-calcium pantothenate, folic acid, niacinamide, pyridoxine, riboflavin, thiamine, i-inositol and salts and solvates thereof; and one or more inorganic salts selected from the group consisting of calcium chloride, ferric nitrate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium phosphate and salts and solvates thereof. In certain embodiments, the concentration of the components in the hemodiafiltration solution is as shown in Table 2.
In certain embodiments, the perfusate contains hemoglobin glutamer-250 (Hemopure @(HBOC-250)) or an alternative oxygen carrier, one or more cytoprotective agents selected from Hexahydro-2-imino-1H-thieno[3,4-d]imidazole-4-pentanoic acid (2-Iminobiotin), 5-(1H-Indol-3-ylmethyl)-3-methyl-2-thioxo-4-Imidazolidinone (Necrostatin-1), Sodium 3-Hydroxybutyric Acid, Glutathione Monoethyl Ester, Minocycline, Lamotrigine, 5-(2,6-Difluorophenoxy)-3-[[3-methyl-I-oxo-2-[(2-quinolinylcarbonyl)amino]butyl]amino]-4-oxo-pentanoic acid hydrate (QVD-Oph), Methylene Blue, and one or more antibiotics and anti-inflammatory agents selected from Ceftriaxone, Dexamethasone, and Cetirizine. In certain embodiments, the concentrations of the components recited above is listed in Table 3.
In certain embodiments, the perfusate comprises one or more artificial oxygen carrier compounds. In certain embodiments, the one or more artificial oxygen carrier compounds are hemoglobin derivatives. The hemoglobin derivatives can be one or more compounds selected from the group consisting of isolated, cell-free hemoglobin, cross-linked hemoglobin, polymerized hemoglobin, encapsulated hemoglobin and functionalized hemoglobin. In certain embodiments, the artificial oxygen carrier compound can be hemoglobin glutamer-250 (HEMOPURE®), a cross-linked hemoglobin tetramer comprising two alpha hemoglobin and two beta hemoglobin subunits cross-linked by a carbon linker. In other embodiments, the one or more artificial oxygen carrier compounds can be artificial red blood cell substitutes, such as ERYTHROMER™, PolyHeme, Oxyglobin, PolyHb-SOD-CAT-CA, PolyHb-Fibrinogen, Hemspan or MP4. In other embodiments, the artificial oxygen carrier compound can be any blood substitute compound known in the art.
In certain embodiments, the perfusion composition comprises one or more compounds selected from the group consisting of anti-cytotoxic compounds, antioxidants, anti-inflammatory compounds, antiepileptic compounds, anti-apoptotic compounds, antibiotics, cell death inhibitors, neuroprotectants and nitrite stress inhibitors. In certain embodiments, the perfusion composition comprises at least one imaging contrast agent. In other embodiments, the perfusion composition further comprises one or more ultrasound contrast agents, that also can be used under certain ultrasound settings as clot disintegrator and blood-brain barrier opener. In some embodiments, the one or more ultrasound contrast agents can be micrometer-sized air-filled polymeric particles. In yet other embodiments, the perfusion composition comprises at least one MRI contrast agent or CT contrast agent. In other embodiments, the perfusion composition comprises one or more compounds selected from the group consisting of the compounds of Tables. 1-3, or salts, solvates, tautomers, and prodrugs thereof.
The invention provides a novel system for in situ hypothermic preservation of organs in a mammalian body.
In certain embodiments, the invention provides a system for the hypothermic, in situ preservation system, the system comprising: a perfusion device for the perfusion of a mammalian body, comprising a means for regulating the temperature, flow, pressure, dissolved gases, and concentration of metabolites in the system; and the perfusate composition of the invention. In certain embodiments, the means for regulating the temperature of the system comprises a controller programmed to regulate at least a perfusate temperature within the system to maintain hypothermic conditions. In certain embodiments, the means for regulating the flow, pressure, dissolved gases, and metabolite concentrations in the system comprises a controller programmed to regulate these parameters within the system to maintain constant or alterable levels/concentrations.
In certain embodiments, the system is configured to introduce oxygen to a perfusate of the invention and circulate the perfusate through mammal. In certain embodiments, the perfusion unit is adapted and configured to introduce oxygen and carbon dioxide to the perfusate. In other embodiments, the perfusion unit is adapted and configured to dialyze the perfusate.
In certain embodiments, the system comprises an intrarenal arterial cannula, an animal input line, a pressure sensor, a flow sensor, a pulse generator, an arterial oxygenator, an exchange solution, one or more roller pumps, a hemodiafiltration membrane, one or more reservoirs with perfusate drugs, one or more perfusate reservoirs, a pressure senor, a centrifugal pump, a hemoglobin sensor a return line from the mammal.
In certain embodiments, the electronic components of the perfusion system are built in a modular manner. Each sensor, motor or electronic component that requires external control has a separate logical controller built on Arduino Uno platform, local circuits and software. Each logical controller has a serial output and input and is connected via com port to the computer. In certain embodiments, the computer has modulus, scripts and functions written in Python that either control or collect data from these logical controllers.
In certain embodiments, the pulse generator consists of (1) 3D printed flow chamber (fluid space for blood/perfusate passage, and air space that is separated by a membrane from the fluid space and can change volume against fluid space) connected to the main circuit, downstream from the centrifugal pump, (2) high resolution electronic pressure regulator (0-10 PSI) which is connected to the air source on one end and to the flow chamber on the opposite end, (3) logical controller regulating electronic pressure regulator, (4) and a computer with control software. The Logical controller is made out of Arduino Uno microprocessor board and has a DAC converter (MCP 4725) with an operational amplifier (LT1215 CN8); it produces continuous analog output (0-10V) for electronic pressure regulator. Logical controller has a script which runs the electronic pressure regulator, based on sinusoidal function, and it can receive input from a computer via serial port to regulate variables within sinusoidal function (e.g. amplitude, iteration/looping speed, baseline value). In certain embodiments, continuous output is produced and sent to the electronic pressure regulator which then controls flow chamber volume, by moving a membrane which separates fluid space from the air space, thus producing pulsatility and oscillations in venous and arterial pressures throughout the perfusion system. Computer is connected to the logical controller, and custom-made scripts in Python allow for manual or automatic control. In certain embodiments, the software also monitors flow and pressure in the arterial and venous cannula and stores the data.
In certain embodiments, the automated hemodiafiltration system consists of (1) two peristaltic pumps which have separate logical controllers and are connected via serial ports to the computer, (2) liquid level solid-state sensor with a resistive output (0-5V), in a clear, elliptical polycarbonate tube integrated with logical controller, for monitoring fluid level in exchange solution canister, connected to the computer. Logical controllers for peristaltic pumps and level sensor have custom made scripts which allow for higher-order language control. In certain embodiments, the computer governs two peristaltic pumps and collects data from the sensor via Python script. The script performs proportional control over peristaltic pumps and allows for continuous dialysis while keeping the animal euvolemic.
The invention provides methods of preserving organs in the mammalian body. In some embodiments, the organs are perfused with a solution comprising a priming solution, a hemodialysis solution and a solution comprising pharmacological components. The constituents and concentrations of the components in the priming solution, hemodialysis solution and solution comprising the pharmacological components are as shown in Tables 1-3. In certain embodiments, the mammalian body is perfused with a mixture of the perfusate and autologous blood. In some embodiments, the autologous blood is mixed with any of the components of the perfusate solution before perfusion of the mammalian body. In some embodiments, one or more artificial oxygen carriers are present in the mixture.
In some embodiments, the mammalian organs maintain morphofunctional integrity under hypothermic conditions after perfusion with the blood perfusate mixture.
In certain embodiments, the organs in the mammal are ischemic prior to perfusion with the blood perfusate mixture.
In certain embodiments, the organs are perfused while the mammal is still alive. In other embodiments, the organs are perfused immediately upon death of the mammal. In other embodiments, there is a 20 minute delay between death and perfusion of the organs. In other embodiments, the time between death and perfusion is at least one hour. In other embodiments, the time between death and perfusion is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours.
In certain embodiments, the perfused organs are organs that can be transplanted from one mammal to another, including, but not limited to the kidney, the pancreas, the heart, the lung, the intestine, the corneas, the middle ear, bone, bone marrow, heart valves, connective tissue, skin, uterus, muscles, blood vessels, nerves and connective tissue. In some embodiments, the organs are removed from the mammal following perfusion with the blood perfusate mixture.
In certain embodiments, perfusion with the blood perfusate mixture helps maintain an in vivo rate of cellular metabolism and preserves functional responses of cells. The blood perfusate perfused organs can maintain longer viability than organs perfused with the ECMO system.
In certain embodiments, the perfused organs belong to any mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. Mammals can also include primates, including humans. In certain embodiments, the perfused mammal is a human.
In some embodiments, rigor mortis is prevented by perfusion of the deceased mammal. In one embodiment, perfusion of the deceased mammal with the technologies described herein prevents stiffening of the muscles. In another embodiment, the tissues in the perfused mammal continue or regain the consumption of. In another embodiment, the tissues in the perfused mammalian continue or regain the ability to produce ATP.
The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.
The materials and methods employed in practicing the following examples are here described:
The perfusion system consists of the main closed-loop circuit directly connected to an animal, and it includes a centrifugal pump (Medtronic Bio-Console 560, Medtronic, Minneapolis, MN) that drives the mixture of autologous blood and OrganEx perfusate through the oxygenator (Affinity Fusion, Medtronic), and custom-made pulsatility generator into animal arterial system. The oxygenator is connected to a refrigerated bath (Polystat, Cole-Parmer, Niles, IL) for temperature control and the gas blender (Sechrist Industries, Anaheim, CA), for control of dissolved gases and anesthesia infusion. The perfusion system has a fluid reservoir, which is used to prime the system and hold the supplement fluid. In parallel, an automated hemodiafiltration system and a reservoir are connected to the main circuit (
The OrganEx perfusate is a final mixture of a custom-made priming solution (Table 1), Hemopure (HbO2 Therapeutics, Waltham, MA), custom-made dialysis exchange solution (Table 2) and the solution of pharmacological compounds (Table 3).
In detail, prior to connecting an animal, the OrganEx perfusion system is flooded and primed with 2200 mL of custom-made priming solution (5000 mL), followed by infusion of 1000 mL of Hemopure into the system. These solutions are left to mix and equilibrate throughout the perfusion system, after which 600 mL is extracted from the perfusion system to achieve desired concentrations of electrolytes and oncotic agents in the perfusion system and prepare it for addition of the autologous blood.
Prior to initiation of the perfusion protocol, animal femoral vessels are cannulated and connected to the main circuit. At 30 minutes of WIT, 5 000 USP units of heparin (Sigma-Aldrich, St Louis, MO) is administered into the system, followed by approximately 1000 mL of venous blood from the dead animal, which is drained into the perfusion system. At this point, circulatory volume in the OrganEx perfusion system is approximately 3600 mL, out of which 2600 mL is the priming solution and 1000 mL of autologous blood. Next, the mixture of the perfusate and autologous blood is left to equilibrate, and counter dialyzed against the residual priming solution over 30 minutes to allow for correction of metabolic derangements in the drained venous blood. In parallel, approximately 1600 mL of fluid is filtered out of the perfusion system over 30 minutes while the residual fluid is cooled to 28° C., yielding a final volume of 2000 mL in the OrganEx perfusion system. Next, at 1h of WIT, 1000 mL of the perfusate and autologous blood mixture is infused back into the animal, ensuring circulatory system filling following venous drainage, and the perfusion protocol is initiated. The remaining 1000 mL of the mixture is stored in the reservoir and used for fluid supplementation, if required. Following infusion of the perfusate and autologous blood mixture, pharmacological compounds and dialysis exchange solution, containing amino acids, vitamins and inorganic salts, are continuously infused into the main perfusion circuit by infusion pump and hemodiafiltration system, respectively. The OrganEx perfusion system utilizes automated hemodiafiltration circuit which corrects and maintains certain metabolic and electrolyte parameters by performing 1:1 (vol:vol) exchange of solutes and particles smaller than 40 kDa against a custom dialysis exchange solution (20,000 mL), while maintaining euvolemia. Hemodiafiltration flux was kept at 30-35 mL/kg/hr throughout 6-hour perfusion.
The ECMO perfusion system was assembled according to clinical standard. ECMO perfusion system has the main closed-loop circuit directly connected to an animal and consists of a centrifugal pump (Bio-Console 560, Medtronic) that drives autologous blood through the oxygenator (Affinity Fusion, Medtronic) into animal arterial system. The oxygenator is connected to a refrigerated bath (Polystat, Cole-Parmer) for temperature control and the gas blender (Sechrist Industries), for control of dissolved gases and anesthesia infusion. The perfusion system has a fluid reservoir, which is used to prime the system and hold the supplement fluid. Furthermore, ECMO perfusion system contained the CDI blood parameter module, and the hematocrit/oxygen saturation probe (Terumo Cardiovascular Systems Corp.) are connected on the arterial and venous side, respectively, along with pressure (PendoTECH) and flow sensors (Bio-Probe TX50, Medtronic). All probes and sensors from the ECMO perfusion system are connected to a computer to allow data gathering. Detailed schematics available upon request.
The ECMO perfusion system is primed with 1000 mL 0.9% Sodium Chloride (Baxter Healthcare Corporation, Deerfield, IL) and 5000 USP units of heparin (Sigma-Aldrich). Upon initiation of the perfusion protocol, the reservoir is taken out of the main circuit and the residual priming solution is stored for later supplementation.
This research project was approved and overseen by Yale's Institutional Animal Care and Use Committee (IACUC) and guided by an external advisory and ethics committee. Experimental animals were procured from the local farm breeder, female domestic pigs (Sus scrofa domesticus; ˜30-35 kg). All animals were housed at Yale School of Medicine Division of Animal Care's facilities at a minimum of 3 days before the experiment.
Prior to experimental protocol, all animals received a Fentanyl patch, 50 ug/hr (Duragesic, Henry Schein, Melville, New York, NY, USA) for sedation. To induce anesthesia, 6 mg/kg of Telazol (Henry Schein) and 2.2 mg/kg of Xylazine (Henry Schein) were administered. Next, animals were intubated and connected to the ventilator utilizing FiO2 of 40% and FiN2 of 60% with standard parameters of tidal volume 10-15 ml/kg and frequency of 14-16 breaths per minute, along with 1-2% isoflurane (Henry Schein). Following ventricular fibrillation and induction of cardiac arrest, ventilation was stopped for 1h. Upon initiation of the perfusion protocol, in ECMO and OrganEx groups, ventilation was continued utilizing a tidal volume of 3-4 ml/kg, frequency 5 breaths per minute, positive end-expiratory pressure (PEEP) of 10 cm H2O, low inflation pressure, FiO2 50%, FiN2 50%. During the 6-hour perfusion protocol, 0.5% isoflurane was administered through the vaporizer connected to the gas blender.
Cardiac arrest and subsequent circulatory collapse were induced by ventricular fibrillation through the substernal window by applying a 9V battery to the myocardial wall. Prior to the ventricular fibrillation, animals received 7 000 USP units of heparin (Sigma-Aldrich). In order to connect the circulatory system of the animal to ECMO or OrganEx perfusion system, an incision was made in the right inguinal region exposing femoral artery and vein (
At the start of the perfusion protocol in the OrganEx group, a mixture of the perfusate and autologous blood was slowly infused over 5 minutes resulting in an average flow rate of 600 mL/min at the end of infusion. Following this step, the flow rate was gradually increased over the next 20 minutes to a targeted flow rate of 80-100 mL/kg/min or highest possible flow rate without introducing overspinning of the centrifugal pump. Throughout the flow rate ramping-up process, pulsatility was set to oscillate around the mean flow rate at approximately ±10% of the given total flow rate. Residual mixture of the synthetic perfusate and autologous blood which was stored in the reservoir was used for fluid supplementation at 1-2 mL/kg/hr. Targeted arterial pressure was set to 50-80 mmHg, and it was controlled with phenylephrine, not more than 2 mg/hr. Similarly, in the ECMO group, flow rate was gradually increased over 25 minutes, targeting flow rates and arterial pressures as in the OrganEx group. Ringer's lactate (Baxter Healthcare Corporation) was used as a supplementation fluid at 3-4 ml/kg/hr. In both, ECMO and OrganEx group hypothermic perfusion protocol at 28° C. was utilized throughout the entire 6-hours of the perfusion protocol.
The animals in both, ECMO and OrganEx groups received 50 mL of 8.4% sodium bicarbonate (Henry Schein) during the first hour of perfusion. Glucose was supplemented according to the blood levels with the goal of maintaining euglycemia. Protamine, 25 mg, was administered immediately following initiation of the perfusion protocol to control activated clotting time, which was maintained between 180 and 220 seconds with titrated heparin administration. In both perfusion groups, partial pressure of arterial CO2 and O2 were targeted to 35 and 250 mmHg via gas blender, respectively.
Electrocardiogram (EKG) assessment was done with 4 leads placed at each corner of the trunk. Real time arterial and central venous pressure monitoring was done through cutdown of the brachial artery and jugular vein, respectively. Urine output was measured via Foley catheter. Animal core temperature was continuously monitored with a rectal probe. Monitoring of EKG, pressure and temperature was done utilizing Philips IntelliVue MP50 (Philips, Eindhoven, NL). During the preoperative procedure temperature was kept at 37° C. using a heating pad, which was turned off following ventricular fibrillation. Electroencephalogram (EEG) was monitored with Natus long-term monitoring (LTM) system and EMU40 breakout box (Natus Medical Inc., San Carlos, CA). Six electrodes were placed subcutaneously along the scalp (
Imaging of the abdominal and head blood vessels was performed using Philips Allura Xper FD20 system. In selected animals that underwent fluoroscopy, baseline physiological imaging was performed prior to the induction of ventricular fibrillation in both ECMO and OrganEx experimental protocol. The contrast-injecting catheter was introduced through the femoral artery cutdown and positioned in suprarenal aorta for renal and in the common carotid artery for brain imaging. Omnipaque Contrast 350 mg/mL (General Electric Inc., Boston, MA), 24 mL and 45 mL were introduced utilizing Medrad power injector (Bayer Vital GmbH, Leverkusen, Germany) for brain and kidney imaging acquisition. Following baseline imaging, all animals underwent additional fluoroscopy at hour 3 of perfusion. In both, ECMO and OrganEx group, imaging of abdominal blood vessels was modified by placing the contrast-injecting catheter in the infrarenal aorta due to the reversal of arterial flow direction, a consequence of femoral artery/vein perfusion approach in both, ECMO and OrganEx groups. The reconstructed images were saved in DICOM format and further post-processed using RadiAnt DICOM Viewer software (Medixant; Poznan, Poland).
Perfusion dynamics were monitored via Triplex Ultrasonography (Spectral Doppler, Colour Doppler, and B-mode) using the LOGIQe portable ultrasound system (General Electric) and an 8L-RS linear array probe (General Electric). In all assessed animals, left ophthalmic artery, common carotid artery and intrarenal arteries were used to profile perfusion dynamics. Power waveform analysis was done using Frq 4.4 MHz, Gn 17, SV 2 and DR 40.
Following the appropriate experimental workflow, regions of interest were extracted 30 from each organ and frozen at −80° C. To ensure consistency between the specimens, all dissections were performed by the same person. Cell nuclei isolation from each organ (brain, heart, liver, kidney) were treated the same according to our already established protocol with some modifications in order to acknowledge each organ's specific structural qualities and to have identical buffers to enable inter-organ comparison within the same experimental animal. To avoid experimental bias nuclei isolation was done by the same person blinded for the replicates of experimental conditions. Furthermore, to randomly and fully represent the full tissue section, each tissue was pulverized to fine powder in liquid nitrogen with mortar and pestle (Coorstek, Golden, CO). All reagents were molecular biology grade and sourced from Sigma unless stated otherwise. Small amounts of pulverized tissue (5-10 mg) were then added into 1 ml of ice-cold lysis buffer (“Buffer A” is 250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM NaCl, 10 mM Tris-HCl (pH 7.4), protease inhibitors w/o EDTA (Roche), RNAse inhibitor (80 U/ml) (Roche), 1 mM DTT, 1% BSA (m/v) (Gemini Bio-Products, Woodland, CA), 0.1% NP-40 (v/v), 0.1% Tween-20 (v/v) (Bio-Rad), 0.01% Digitonin (m/v) (Thermo-Fisher, Cleveland, OH). For lysis of heart, 0.1% TX-100 (v/v) was additionally added. DTT, RNAse Protector, protease inhibitors, and all detergents were added immediately before use. The suspension was transferred to 2 ml Dounce tissue homogenizer and lysed with constant pressure and without introduction of air with pestle A (30×) and pestle B (30×). The homogenate was strained through pre-wetted 40 μm tube top cell strainer (Thermo-Fisher). All subsequent centrifugation was performed in a refrigerated, bench-top centrifuge with swing-out rotor (Eppendorf, Hamburg, Germany). Heart lysate was centrifuged at 100 g for 5 min at 4° C., pellet of myofibrils and non-dissociated connective tissue was discarded, and supernatant saved. All lysates (brain, liver, kidney) and heart supernatant (post 100 g) were centrifuged at 1000 g, 10 min, 4° C., pellets were saved, and resuspended in 0.4 ml resuspension buffer (“Buffer B” is “Buffer A” w/o detergents). Final 0.4 ml of solution was mixed with 0.4 ml (1:1) of Optiprep solution (Buffer “C” is iodixanol 50% (v/v), 25 mM KCl, 5 mM MgCl2, 10 mM NaCl, 10 mM Tris-HCl (pH 7.4), protease inhibitors w/o EDTA, RNAse inhibitor (80U/ml), 1 mM DTT, 1% BSA (m/v)). The suspension (25% iodixanol final) was mixed 10× head over head and overlayed on 0.6 ml of 29% iodixanol cushion (appropriate mix of Buffer “B” and “Buffer “C”). The tubes were then centrifuged at 3000 g, for 30 min at 4° C. Following centrifugation, the supernatant was removed and total of 1 ml of wash buffer (“Buffer D” is 25 mM KCl, 5 mM MgCl2, 10 mM NaCl, 10 mM Tris-HCl (pH 7.4), RNAse inhibitor (80 U/ml), 1 mM DTT, 1% BSA (m/v), 0.1% Tween 20 (v/v), in DPBS (w/o Ca2+ and Mg2+) (Gibco)) was added in tubes and centrifuged at 1000 g, for 10 min at 4° C. Supernatants were then completely removed, pellets were gently dissolved by adding 100 ul of resuspension buffer (“Buffer E” is “Buffer D” w/o detergent) and pipetting 30× with lml pipette tip, pooled and filtered through 20 micrometer cell strainer. Finally, nuclei were counted on hemocytometer and diluted to 1 million/ml.
Single Nuclei Microfluidic Capture and cDNA Synthesis
Extracted nuclei samples were placed on ice and processed in the laboratory within 15 minutes for single nucleus RNA sequencing with targeted nuclei recovery of 10000 nuclei, respectively, on microfluidic Chromium System (10× Genomics, Pleasanton, CA) by following the manufacturer's protocol (CG000315_ChromiumNextGEMSingleCell3′_GeneExpression_v3.1 (DualIndex)_RevA), with Chromium Single Cell 3′ GEM, Library & Gel Bead Kit v3.1, (PN-1000268, 10×Genomics) and Chromium Single Cell G Chip Kit (PN-1000120, 10× Genomics), Dual Index Kit TT Set A (PN-1000215, 10×Genomics) on Chromium Controller (10×Genomics). Since the input material were nuclei, cDNA was amplified for 14 cycles.
Post cDNA amplification cleanup and construction of sample-indexed libraries and their amplification followed manufacturer's directions (CG000315_ChromiumNextGEMSingleCell3′_GeneExpression_v3.1 (DualIndex)_RevA), with the amplification step directly dependent on the quantity of input cDNA.
In order to reach sequencing depth of 20000 raw reads per nucleus, single nucleus libraries were run using paired end sequencing with single indexing on the NovaSeq 6000 S4 (Illumina) by following manufacturer's instructions (Illumina, San Diego, CA; 10× Genomics). To avoid lane bias, multiple uniquely indexed samples were mixed and distributed over several lanes.
Sequencing reads were aligned to the reference pig genome (susScr11) with the combined exon-intron gene annotations from NCBI RefSeq using CellRanger 5.0.1. pipeline, which also performed UMI counting, barcode counting and distinguishing true cells from background. The filtered count matrices were then moralized by library size using the “NormalizeData” function in Seurat. To compare cellular and transcriptomic changes across conditions, feature selection was first performed for each batch and the features from the same conditions were summarized using “SelectIntegrationFeatures”. The union of the highly variable genes across conditions were then passed to the data integration pipeline in Seurat to generate a batch-corrected expression matrix. To reveal the cellular diversity among the cells, we scaled the integrated data followed by dimension reduction using principal component analysis (PCA) and selecting principal components via elbow plot. The cell clusters in the k-nearest neighbor graph were then identified and visualized clustering results by Uniform Manifold Approximation and Projection (UMAP). The initial clustering analysis revealed some low-quality clusters with low number of unique molecular identifiers (UMIs), and normally high mitochondria percentage and no meaningful cluster markers, which were all removed for downstream analysis. To annotate the identity of the cell clusters, the pig data was then integrated with published human data from the same organ using the same data integration pipeline described above. The cluster markers were calculated using “FindMarkers” function and manually removed the doublet clusters that showing high expression of markers of two different type of cells. To gain more accurate cell annotations and clearer UMAP visualizations, the same pipelines of data integration, dimension reduction and cell clustering were reperformed on the filtered data.
Global Transcriptomic Comparisons with Public Datasets.
To validate the cluster annotation of the data, global transcriptomic comparison with public reference datasets for the four organs was performed. For each organ, the log-transformed average expression for each cell clusters was calculated followed by performing pairwise Pearson correlation between the clusters in the present dataset and the corresponding dataset using the highly variable genes. The resulted correlation coefficients were visualized on gradient heatmaps (
The cell type classification for hippocampus data (
The heart data (
The kidney data (
The liver data (
FindMarkers function from Seurat was used to determine marker genes for high resolution clusters. P-value adjustment is performed using Bonferroni correction with the cutoff set at 0.05. Top 10 genes for each cluster were ranked by fold changes and were visualized on a heatmap by using DoHeatmap function (Seurat). The dataset was randomly sampled to have 1000 cells per condition for each t-type prior to differential expression analysis.
In order to find the cell populations that exhibit high degree of transcriptomic changes, Augur was applied to prioritize the cell types between each pair of conditions. Since there are three samples per condition, the Augur analysis was performed on all of the nine sample pairs in each condition pair using the high-resolution cell clusters identified via Seurat. The median of the calculated area under curve (AUC) scores of each cluster were then visualized on the UMAP layout. Comparison of the AUC scores for each specific cell type and a given condition pair were done by comparing the specific cell type of interest AUC score and AUC scores of all the other cell types in that given condition pair by using Wilcoxon Rank Sum test (one tailed).
In order to find differentially expressed genes (DEGs) between OrganEx and other conditions (0h PMI, 1h PMI, 7h PMI, ECMO) in major cell-types for each organ Seurat “FindMarkers” function was used. DEGs were defined at cut-off criteria of adjusted P-value (Bonferroni)<0.05, expression ratio greater than 0.1 in one condition and average log 2 fold change (log 2FC) greater than 0.2 in the same condition. Top 15 DEGs ranked by absolute values of log 2FC, were visualized using Bioconductor EnhancedVolcano package. Top 100 DEGs were used for HumanBase Functional Module Detection. Gene symbols starting with ma and LOC were excluded since HumanBase Functional Module Detection does not recognize those gene symbols. Identifying enriched biological pathways between OrganEx and all other groups in major cell-types for each organ, was performed using “enrichGO” function from clusterProfiler. Multiple testing was adjusted by false discovery rate (FDR) with the cutoff set at 0.2. Top 15 biological processes ranked by P-value were visualized by in-house made ggplot2 script.
In order to assess whether gene sets of interest are upregulated in a specific condition (e.g., 0h WIT, 1h WIT, 7h WIT, ECMO and OrganEx), gene set enrichment analysis was performed in hippocampal neurons, astrocytes and microglial cells, cardiomyocytes in the heart, hepatocytes in the liver and proximal convoluted tubule cells in the kidney. This method is commonly used in Gene Ontology enrichment analysis and has been widely applied in multiple published studies. Specifically, all the expressed genes (expressed in at least one cell) were set as the gene universe and considered each set of condition-enriched genes as a sampling from the gene universe. The gene set enrichment, performed by Hypergeometric test (also named one-tailed Fisher's Exact test), is an assessment of whether genes from a given gene sets are overrepresented in condition-enriched genes than drawing from the gene universe by chance. To identify condition-enriched genes in the above-mentioned t-types, differential expression (DE) analysis was performed using Seurat FindMarkers function. In brief, one condition group was taken, its expression profiles were compared with the rest of the conditions using Wilcoxon Rank Sum test. For any given comparison, genes with false discovery rate (FDR) smaller than 0.01 were considered statistically significant and were kept. Because Wilcoxon Rank Sum test can be biased by the differences of cell numbers, that is, more cells lead to more differentially expressed genes, the datasets were randomly sampled to have 1000 cells per condition in each t-type prior to differential expression. With the condition-enriched genes and certain selected gene sets downloaded from GeneOntology (http://geneontology.org/), it was possible to assess the significance of gene set enrichment in a given condition. P-value of less than 0.05 was considered significant. Significance of the enrichment was visualized in a dot plot where size and color of the dot shows significance as −log 10(p-value). As shown in the
The expression dynamics of selected cell death-related gene set across conditions was also evaluated. For each gene of the given gene set, its expression enrichment was tested in each condition by comparing its expression in the given condition to that of other conditions. Similarly, Wilcoxon Rank Sum test was used to measure the significance. The resulted log-transformed P-values (−log 10[P-value]) across conditions were visualized in a bar plot.
To acquire the dynamic changes of transcriptome among different conditions and time points for each tissue, the top differential expressed genes for all the paired conditions were identified: 0h vs 1h, 0h vs 7h, 0h vs ECMO, 0h vs OrganEx, 1h vs 7h, 1h vs ECMO, 1h vs OrganEx, 7h vs ECMO, 7h vs OrganEx, and ECMO vs OrganEx using FindMarkers( ) function in Seurat. For hippocampus, significant DEG was selected with average log 2FC greater than 0.5 or less than −0.5. The top 50 upregulated genes were merged with 50 downregulated differentially expressed genes (DEGs, in total 100 genes) from each paired condition for the following analysis. To identify gene expression patterns, the average expression of the merged DEGs across t-types and experimental groups was calculated and the correlation coefficients subtracted from 1 as gene-gene distances was defined, which was passed to hierarchical clustering using the hclust function in R with ward.D2 algorithm. To define gene modules (clusters), the genes were parcellated using the cutreeDynamic function from R WGCNA package with a setting minClusterSize=45, sensitivity=2. The scaled eigengenes were the plotted, the first principal component of the expression matrix of each module to show the expression trend of the genes in each module. Using TopGO, the gene ontology analysis was performed for the genes in each module, and used fisher's exact test to calculate the P values. For heart, kidney and liver, the same methods with hippocampal data to select the genes were used. To keep the numbers of selected gene are comparable with hippocampus samples, significantly DEG with average log 2FC greater than 0.75 or less than −0.75 in heart data, significantly DEG with average log 2FC greater than 1.00 or less than −1.00 in kidney data and selected significantly DEG with average log 2FC greater than 1.75 or less than −1.75 in liver data. Then the same setting was used to build the gene expression network using hierarchical clustering. The scaled eigengenes of each module was plotted and used the same method (TopGO) for gene ontology analysis.
To evaluate the effects of the perfusate components on OrganEx, the expression enrichment of the related pathways (cell death, inflammation, and oxidative response) between OrganEx and ECMO conditions (Tables 4, 5, and 6) were compared. Specifically, the AUC (area under curve) scores were calculated using the AUCell package and a one-sided Wilcoxon Rank-Sum test was performed to evaluate the significance of the pathway enrichment.
The monocle2 was used and in-house R scripts to conduct pseudotime analysis for hippocampus, heart, liver, and kidney. The recommended analysis protocol was followed, except using FindMarkers function from Seurat package to perform pairwise comparison across different conditions to find the statistically significant up- and down-regulated genes. To reduce false positive results, some parameters were customized based on computational permutation. For examples, it was required that minimum percentage of expressed cells for each gene in either condition is larger than 0.1, and fold change larger than 1.25. Maximum number of cells in either condition was down sampled to 1000 cells to balance the comparison. Consequently, the identified differentially expressed genes by Seurat were used as the informative genes to order cells using setOrderingFilter function from monocle2, and the advanced nonlinear reconstruction algorithm called DDRTree was chosen to execute data dimensional reduction.
Cell-cell interactions based on the expression of known ligand-receptor pairs in different t-types were inferred using CellChat (v.1.1.3). The official CellChat workflow was followed for analyzing multiple datasets (0h WIT, 1h WIT, 7h WIT, ECMO and OrganEx). each dataset was first randomly down sampled to 1000 cells per t-type to to balance the comparison. Next, normalized counts were loaded into CellChat. After that, CellChatDB human database was selected for cell-cell communication analysis. The preprocessing was then applied as functions identifyOverExpressedGenes and identifyOverExpressedInteractions with standard parameters set. Next, communication probability was computed between interacting cell groups with truncated mean set at 0.1. After that filterCommunication, computeCommunProbPathway, and aggregateNet were applied using standard parameters. To identify conserved and context-specific signaling pathways rankNet function was applied on the netP data slot which showed in a stacked bar plot overall information flow of each signaling pathway. To determine strength of the reactions and t-types involved in each signaling pathway, netAnalysis_signalingRole_heatmap was performed and visualized overall signaling by aggregating outgoing and incoming signaling together.
The monocle2 and in-house R scripts were used to conduct pseudotime analysis for all major organs, including hippocampus, heart, liver, and kidney. The recommended analysis protocol was followed, except using FindMarkers function from Seurat package to perform pairwise comparison across different conditions to find the statistically significant up- and down-regulated genes. To reduce false positive results, some parameters were customized based on computational permutation. For examples, it was required that minimum percentage of expressed cells for each gene in either condition is larger than 0.1, and fold change larger than 1.25. Maximum number of cells in either condition was down sampled to 1000 cells to balance the comparison. Consequently, the identified differentially expressed genes by Seurat were used as the informative genes to order cells using setOrderingFilter function from monocle2, and the advanced nonlinear reconstruction algorithm called DDRTree was chosen to execute data dimensional reduction.
Following the completion of each experimental protocol brain, heart, lungs, kidney, liver, and pancreas were extracted and immersion-fixed in a solution containing 10% (w/v) neutral buffered formalin with gentle shaking. After fixation each tissue piece was processed and embedded into a paraffin block using the Excelsior tissue processor (Thermo Scientific, Waltham, MA).
Tissue Staining with Hematoxylin and Eosin (H&E) and TUNEL Assay
Heart, lungs, kidney, liver and pancreas paraffin blocks were trimmed on the Shandon Finesse 325 microtome (Thermo Scientific) to 5 μm sections. Sections were mounted on TruBond 380 adhesive slides and allowed to dry overnight at room temperature. All slide were then stained simultaneously for H&E using the automatic Shandon Linistain slide stainer (Thermo Scientific).
For Nissl staining, brain sections (hippocampus and prefrontal cortex) were stained with 0.1% cresyl violet solution (Abcam, ab246816) for 5 min and were rinsed quickly in 1 change of distilled water. Then these sections were dehydrated quickly in absolute alcohol and later cleared in Histo-Clear II and finally cover-slipped with Prolong Gold Antifade Mountant (ThermoFisher, P36934).
All slides for the TUNEL Assay (Millipore, S7101, 3542625) were processed simultaneously. They were fist deparaffinized, rehydrated and incubated with proteinase K (20 g/mL in PBS) for 30 min at 37° C. Slides were rinsed with PBS and incubated with 3% H2O2 in PBS for 10 min at room temperature to block endogenous peroxidase activity, followed by PBS washing and incubation in 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice (4° C.). Sections were incubated with a mixture of TdT solution and fluorescein isothiocyanate dUTP solution in a humidified chamber at 37° C. for 60 min. This was followed by washings with PBS and incubation with antifluorescein antibody Fab fragments conjugated with horseradish peroxidase in a humidified chamber at 37° C. for 30 min. After washing with PBS, methyl green counterstain was applied to stain for nuclei.
All slides used for a specific immunohistochemistry staining were processed simultaneously. Slides containing formalin-fixed paraffin-embedded histological sections were first deparaffinized in 2 changes of Histo-Clear II (64111-04, Electron Microscopy Sciences, Hatfield, PA) for 10 minutes each. Slides were then transferred to 100% alcohol, for two changes, 10 minutes each, and then transferred once through 95%, 70%, and 50% alcohol respectively for 5 minutes each. Slides were then rinsed in water and washed in wash buffer (0.05% Tween 20 in 1×PBS) for 10 minutes. Slides were then placed into a chamber filled with antigen retrieval buffer (lOX R-Buffer A diluted to 1× by water, pH 6, 62706-10, Electron Microscopy Sciences). Slides then underwent heat-mediated antigen retrieval in the Unique Retriever system (Electron Microscopy Sciences). After antigen retrieval, slides were washed in wash buffer and blocked for 1 hour in 10% normal goat serum diluted in wash buffer at room temperature. Slides were then incubated in primary antibodies at 4 degrees C. overnight at the following dilutions: rabbit anti-NeuN (1/1000, Abcam, ab177487, GR3275112-10), mouse anti-GFAP (1/1000, Sigma-Aldrich, G3893-100UL, 0000082460), rabbit anti-Iba1 (1/500, Wako, 019-19741, PTR2404), mouse anti-albumin (1/500, Abcam, 4A1C11, GR3215248-15), mouse anti-beta actin (1/500, Invitrogen, AC-15, 01003256), rabbit cleaved caspase-3 (1/50, R&D Systems, MAB835, KHK0821021). The next day, slides were washed and then incubated with fluorescently tagged secondary anti-rabbit (Cell Signaling Technology, 8889S, 12) and anti-mouse (Abcam, ab150113, GR3370569-1) antibodies at a dilution of 1/500 for 1 hour at room temperature. After staining with secondary antibodies, slides were washed and incubated in DAPI (1/1000 for 5 minutes) at room temperature. Finally, slides were washed and mounted with coverslips using Prolong Gold Antifade Mountant (P36934, ThermoFisher).
Tissue sections were imaged using an LSM880 confocal microscope (Zeiss; Jena, Germany) equipped with a motorized stage using 10× (0.3 NA) or 20× (0.8 NA) objective lenses with identical settings across all experimental conditions. Lasers used: argon 458, 488, and 514; diode 405; and DPSS 561-10. The DPSS 561-10 laser intensity was increased during imaging of the control perfusate samples for the intravascular hemoglobin fluorescence study in order to obtain a background signal comparable to other groups. Images were acquired at either 1,024×1,024 or 2,048×2,048-pixel resolution. Images are either representative confocal tile scans, high-magnification maximum intensity Z-stack projections (approximately 7-9-μm stacks; −1 μm per Z-step), or high magnification confocal images. Alternatively, histological images were acquired using an Aperio CS2 Pathology Slide Scanner (Leica; Wetzlar, Germany) as described above. Image adjustments were uniformly applied to all experimental conditions in Zeiss Zen. Digitized images were assembled in Zeiss Zen, ImageScope, and Adobe Illustrator.
All H&E slides were scanned. Four images were randomly selected from each slide and from the corresponding areas. Blinded observers scored each image accordingly. Criteria for heart were nuclear damage, myocyte vacuolization, widened spaces between myofibers and edema. Criteria for lungs were nuclear damage, pneumocyte vacuolization and hemorrhage. Criteria for kidney were nuclear damage, tubule vacuolization, hemorrhage, and tubule damage. Criteria for liver were nuclear damage, tissue vacuolization, hepatocyte vacuolization, and congestion. Criteria for pancreas were nuclear damage, cell vacuolization, hemorrhage, edema.
Two images were taken per region of interest (hippocampal CA1 region or PFC) and were evaluated by blinded observer using the cell counter function in ImageJ according to already established criteria.
All kidney PAS-stained slides were examined by blinded kidney pathologist. Score (0-3) was assigned depending on the severity of the damage which included: Bowman space dilation, tubular dilation, tubular vacuolization, brush border disruption and presence of the casts.
All images were normalized to the regions of interests (hippocampal CA1, CA3 and DG) using ImageJ and randomized. The number of labeled astrocytes (GFAP+ cells), microglia (IBA1+ cells) cells were quantified manually by a blinded observer using the cell counter plugin and averaged based on the acquired area/cells. The particle analysis of astrocytes was performed using a custom pipeline in the open-source software, CellProfiler56, where a uniform threshold was set on GFAP to identify GFAP+ skeleton objects. The number of GFAP+ fragments in each area were then auto-counted and averaged according to the number of the astrocytes. The mean intensity of NeuN per neuron were quantified by a custom pipeline where neurons were identified based on the DAPI segmented objects expressing NeuN. Data were analyzed and plotted in GraphPad Prism9 software. All data are shown as mean±SEM. “n” refers to the number of biological replicates.
In the kidney, three images were taken randomly of the kidney cortex. All images were randomized and analyzed by a blinded observer. Glomerulus and the proximal convoluted tubule were manually selected as the regions of interest in the four same-size circles respectively in corresponding regions across images. The mean intensity of ACTB and TIMI was calculated using ImageJ software. The number of Ki-67 positive cells were manually counted.
In the liver, immunolabelling quantification was done the same for albumin and factor V. Three images were randomly taken from the same region of the liver. The mean intensity of albumin and factor V was automatically quantified using ImageJ across the whole slide.
In the heart, three images were taken randomly from the left ventricle just slightly below the epicardium. All images were randomized, after which a blinded observer selected cardiac muscle tissue as a region of interest. The amount of cytoplasmic troponin I was then quantified using mean intensity function in ImageJ.
Cell Death Pathway Quantification and Analysis (actCASP3, IL1B, RIPK3, and GPX4).
For kidney, liver, heart, and pancreas, three images were taken randomly from each slide and from the corresponding areas of each organ. actCASP3+ cell intensity was quantified manually by a blinded observer using ImageJ. Mean immunolabelling intensity of IL1B, RIPK3 and GPX4 in kidney, liver and heart was quantified using ImageJ with background subtraction and measure functions. In the brain, the number of actCASP3+ and RIPK3+ cells were counted manually in the hippocampal CA1 region and prefrontal cortex. Expression of ILIB and GPX4 in hippocampal CA1 was measured in cells that were manually selected in the granule cell layer using ImageJ measure function.
All images were taken with a bright field microscope followed by a random same-size snapshots of the slides. A blinded observer analyzed randomized images using Fiji to separate channels of nuclei staining (hematoxylin) and DNA fragments (DAB). Total DNA fragments were quantified based on the total DAB intensity using CellProfiler.
Small piece of the apical portion of the hearts' left ventricle was excised and placed in the carbonated Tyrode's solution (140 mM NaCl, 6 mM KCl, 10 mM glucose, 10 mM HEPES, 1 mM MgCl2, 1.8 mM CaCl2), pH=7.4). The tissue was then dissected into 1-2 cm3 cubes and placed on a holder with the epicardium facing down so the cutting align with the cardiac myofibril orientation. Using a vibratome, the heart was sliced at 150 μm thickness at 4° C. After slicing, spontaneous rate and rhythm of heart contraction were characterized and recorded using a slice microscope with temperature control set to 37° C. (Scientifica SliceScope, Uckfield, UK). Beating frequency was quantified within the time course of 30 seconds.
At the end of each perfusion, two identical tissue samples with 5 mm length were acquired from each organ (kidney, heart, and cerebral cortex) using a 3 mm biopsy punch (Miltex, Integra, York, PA) and were placed in cold PBS (fresh) and PFA (fix overnight) solutions respectively. The fresh tissues and the fixed controls were incubated in 2-NBDG working solution (Cayman 11046, 100 μM, Ann Arbor, MI) for 30 min at 37° C. followed by 15 min of wash in PBS. The images were acquired immediately using widefield microscope (Zeiss) under 2.5× objective (Ex: 465-495 nm, Em: 5210-560 nm) with exposure time of 295 ms (kidney and heart) and 50 ms (cerebral cortex). Images were analyzed using ImageJ to quantify the mean intensity of the ROI with normalization based on the fixed controls.
Hippocampus was isolated at the end of appropriate experimental protocol and sectioned with a vibratome at 250 μm thickness in carbonated NMDG-HEPES aCSF solution (92 mM NMDG, 2.5 mM KCl, 1.25 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 2 mM thiourea, 5 mM Sodium ascorbate, 3 mM Sodium pyruvate, 0.5 mM CaCl2)·2H2O, and 10 mM MgSO4·7H2O, pH=7.3). Hippocampal slices were then collected and transferred onto culture membranes (Falcon culture insert, 0.4 m) and cultured in a six-well culture dish with 1.5 ml medium (48% DMEM/F-12 (Gibco), 48% Neurobasal (Gibco), 1× N−2 (Gibco), 1×B−27 (Gibco), 1× Glutamax (Gibco), 1×NEAA (Gibco), 1× Pen Strep (Gibco). The plates were maintained in an incubator at 37° C. with 5% CO2 for up to 2 weeks. The amino acid methionine analog, azidohomoalanine (AHA, ThermoFisher), was added to the hippocampal slice at a final concentration of 50 μM in HEPES buffered solution (HBS) followed by the incubation of 6 hours at 37° C. Slices were washed with PBS and immediately fixed in 4% paraformaldehyde at 4° C. overnight. Detection of nascent protein synthesis was performed with the modified Click-iT 1-azidohomoalanine (AHA) Alexa Fluor 488 Protein Synthesis HCS Assay kit (ThermoFisher). To visualize neurons simultaneously, the slices were then blocked with 5% NDS for 1 hour at room temperature, and incubated with Rabbit anti-NeuN antibody (abcam, 1:1000) overnight at 4° C. followed by the incubation of anti-rabbit AlexaFluor647 (1:500) for 1 hour at room temperature. The slices were then co-stained with DAPI to visualize nuclei. Images were acquired using Zeiss LSM800 confocal microscope with 20× objective. The combined Z-stack images of DAPI, AlexaFluor 488 and AlexaFluor 647 channels were acquired with optical slice thickness of around 20 μm and images of maximum projection are shown. Proteins detected around the nucleus (perinuclear) are the most abundant newly synthesized proteins in cells28, and were quantified using ImageJ intensity measurement function.
Depiction of the pig in
All data are reported as mean±standard error of mean with data analysis being conducted using one-way ANOVA with Dunnett's post hoc multiple comparisons in reference to the OrganEx perfusion group, or unpaired t-test for comparisons between two groups. Fisher's exact test was used to compare occurrence of QRS complexes in OrganEx and ECMO groups during the perfusion protocol. For the number of replicates in each experimental group together with appropriate statistical analyses please see below. Significance was set at P<0.05. All statistical analysis and plotting were done in GraphPad 9 (GraphPad Software, San Diego, CA) or in Python. All figures were created using Adobe Illustrator (Adobe Systems, San Jose, CA).
Further information regarding statistical values and reproducibility of the results is given below.
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The OrganEx technology consists of a perfusion system and synthetic perfusate (
To evaluate OrganEx technology in large mammals, a porcine global warm ischemia model induced by cardiac arrest on anesthetized and heparinized animals (
Since systemic circulation assures supply of oxygen, metabolites and potential therapeutic agents during recovery from ischemia, it was first queried whether circulation could be restored with external perfusion following one hour of warm ischemia. Perfusion of the whole-body with ECMO system invariantly resulted in low or no flow states due to circulatory collapse. In particular, utilizing fluoroscopic angiography, ECMO perfusion exhibited limited arterial filling of major conduit arteries and organs, such as kidney, liver and brain (
In contrast, robust whole-body perfusion in the OrganEx treated animals was observed, as indicated by contrast enhancement of major conduit arteries and organs, demonstrating patent circulation (
Following successful restoration of circulation, the extent that OrganEx is capable of recovering metabolic parameters was assessed. By measuring mixed venous oxygen saturation of blood returning to the venous circulation from peripheral tissues and organs, it was confirmed that the OrganEx technology was able to deliver adequate levels of oxygen to the whole body throughout the perfusion (
Upon restoration of systemic circulation and certain key metabolic parameters following OrganEx perfusion, next investigated was its effects on organ structural integrity and cytoarchitecture. The brain's three major cell types, neurons, astrocytes, and microglia in two regions most vulnerable to ischemia, the hippocampal CA1 subregion and the prefrontal cortex (PFC) were analyzed. The intensity of immunostaining for RBFOX3 (NeuN), a pan-neuronal marker, which has previously been shown to decrease in hypoxia, was lower in the ECMO and 7h WIT groups in both regions, as compared to OrganEx (
After assessing highly oxygen-sensitive brain structures and cell types, next investigated were the effects of OrganEx on tissue and cellular integrity in essential peripheral organs, including heart, lungs, liver, kidney, and pancreas. Utilizing hematoxylin and eosin (H&E) staining, hemorrhage, tissue edema, nuclear pyknosis, cell vacuolization, and cellular integrity were evaluated and were combined into a cumulative damage score according to the standard pathologic criteria. Notably, the OrganEx group showed a decrease in the H&E damage score, as compared to 7h WIT and ECMO groups (
To further assess recovery of cytoarchitecture in the OrganEx intervention group compared with possible injury promotion in ECMO group, the cytoskeletal features of the kidney that are well-studied in this regard were investigated. Expression of renal cytoskeletal 3-actin is known to be upregulated in ischemic states. However, the reintroduction of autologous blood causes risk for cell injury and cytoskeletal disruption leading to protein loss. This analysis revealed reduced 3-actin immunoreactivity in the ECMO, as compared to OrganEx and other groups, which exhibited preserved immunoreactivity (
Because the OrganEx perfusate contains pharmacological suppressors of cell death and decreased cellular demise based on the histopathological analysis in the OrganEx group (
The analysis of the CA1 and PFC revealed that the intensity of actCASP3 immunolabeling in the OrganEx group was lower than in the 0h and 1h WIT groups (
Next, to investigate pyroptosis, the cell death pathway triggered by proinflammatory signals, interleukin 1 beta (IL1B) immunohistochemistry was utilized. Across all investigated peripheral organs such as heart, liver, and kidney, IL1B immunolabeling intensity was comparable in 0h WIT and increased in ECMO when compared to OrganEx. In the brain, immunolabeling intensity was decreased in ECMO when compared to OrganEx group (
Finally, necroptosis and ferroptosis, two distinct cell death pathways were investigated by utilizing immunohistochemistry of their important proteins in the pathways, the receptor-interacting ser/thr kinase 3 (RIPK3) and the glutathione peroxidase 4 (GPX4), respectively. The results were consistent between the two cell death pathways and between all the organs evaluated such as brain, heart, liver, and kidney. Compared to OrganEx, immunolabeling intensity was comparable in 0h WIT and significantly decreased in 7h WIT and ECMO groups (
After observing improvements in metabolic function, tissue cytoarchitecture and cell death outcomes using OrganEx, cellular energy balance was next investigated in detail. The glucose uptake was measured in highly metabolic organs (brain, heart, kidney) using the fluorescent glucose analog, 2-NBDG23. This showed comparable levels of glucose uptake in the OrganEx and 0h WIT groups in all assessed organs and reduced cellular glucose capture in the ECMO group that may indicate impaired glucose utilization of cellular leakage (
Indicators of cell- and tissue-level recovery in relevant organs were next tested. Cardiac assessment with electrocardiography (ECG) demonstrated spontaneous reemergence of QRS complexes during OrganEx perfusion, indicating ventricular depolarization (
Liver cellular recovery was assessed using immunostaining for albumin and factor V, which are non-structural liver-synthesized proteins with great abundance and short half-life, respectively. Compared to OrganEx group, immunolabeling intensities of both proteins were comparable in 0h WIT and significantly diminished in 7h WIT and ECMO groups (
In the kidney, although many cellular features were preserved similar to 0h WIT in OrganEx groups, including tissue integrity (
Next, continuous electroencephalography (EEG) of the brain in OrganEx and ECMO groups was conducted, and no signs of global network activity were detected (
Next, it was sought to investigate the longer-term actions of OrganEx perfusion on cellular viability. Yet because of regulatory constraints and inability to extend the perfusion protocol beyond 6h, organotypic hippocampal brain slice cultures (BSCs) were utilized, to monitor features of tissues previously exposed to different perfusion interventions. BSCs were prepared at the beginning of experiments (0h WIT), at the end of ECMO and OrganEx perfusions, and from time-matched 7h WIT controls, and were cultured for 14 days while assessing tissue integrity and protein synthesis using Click-iT assay screening. BSCs from the 7h WIT group failed these measures due to severe tissue degradation. Based on visual inspection and DAPI staining, BSCs from the ECMO group were more fragile than OrganEx samples, and most disintegrated by day 14 (
To investigate transcriptomic responses to distinct ischemic exposures and the effects of the OrganEx intervention, single-nucleus RNA-sequencing (snRNA-seq, see Methods) was performed. The computational analysis of snRNA-seq data revealed major transcriptomically-defined cell types (t-types) that were comparable to publicly available human and mouse single-cell datasets (
To compare cell type responsiveness to ischemia based on transcriptomic changes across experimental groups, Augur prioritization was performed and t-types with the greatest transcriptomic divergence were highlighted. This identified prominent changes in neurons, cardiomyocytes, hepatocytes, and proximal convoluted tubule (PCT) cells, consistent with t-types validated in earlier studies and prompting detailed sub-analysis (
Further investigation of OrganEx actions upon glial inflammatory responses underlying brain injury progression after ischemia showed that hippocampal microglial pro-inflammatory transcriptional enhancement was absent in the OrganEx group. Conversely, microglial inflammatory and astrocytic pan-reactive transcriptomic signatures were upregulated in ECMO and 1h WIT groups, respectively. (
The transcriptomic signatures of tissue and cellular functioning in heart, liver, and kidney specimens were then evaluated. Cardiomyocytes in the OrganEx group exhibited enrichment of genes orchestrating action potential formation and the well-described shift28 toward glycolytic metabolism following ischemia signifying cardiomyocyte viability (
To identify transcriptomic patterns across experimental groups more systematically, and to determine functional gene modules, we performed co-expression analysis on differentially expressed genes across groups. Eigengenes of each module designated key gene expression trends, with subsequent gene ontology (GO) analyses highlighting relevant biological pathways (
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Here, the OrganEx technology and its potential to support recovery of key molecular and cellular processes in multiple porcine organs after prolonged global warm ischemia are described. This also demonstrates the underappreciated capacity of the large mammalian body for restoration of hemodynamic and metabolic parameters following circulatory arrest or other severe ischemic stress. This application of the OrganEx technology demonstrates that cellular demise can be halted, and their state be shifted towards recovery at molecular and cellular levels, even following prolonged warm ischemia. Additionally, a comprehensive single-cell transcriptomic analysis of multiple porcine organs was generated to provide a unique resource for future studies on cell-types, ischemia, and reperfusion.
Although some cellular viability can be restored following prolonged ischemia in tissue cultures or isolated organs, clinical scenarios typically involve shorter-duration ischemia in the setting of cardiac arrest or regional perfusions in the setting of organ transplant. By employing a rational polytherapy approach built upon optimized perfusion dynamics and augmentations to an acellular synthetic perfusate, the OrganEx technology was able to bridge prior clinical-translational gaps by restoring circulation and metabolic homeostasis across the whole body. This quells deleterious processes caused by disturbed cellular environments and lack of oxygen, representing a distinct feature of this technology and an essential control for multiple nonspecific injury mechanisms affecting end-organ recovery and overall prognosis after global ischemia. Potential applications of this technology are manifold and could provide novel pathways in ischemia research and advance related clinical disciplines. OrganEx has the potential to extend limits of allowable warm ischemia times through regional abdominal/thoracic reperfusion, thereby increasing organ availability for transplantation. This approach would require obligatory antecedent clamping of the aorta/carotid arteries to prevent brain recirculation in the organ donor. Conversely, if any future refinements of the technology could be aimed at the recovery of the brain function after injury, then the brain circulation would remain patent. In this regard, OrganEx technology may improve outcomes in extracorporeal cardiopulmonary resuscitation with the needed circulatory support, or the OrganEx perfusate could aid in recovery wherein cardiac function is preserved but the brain is damaged, as seen in stroke. Thus, a clear distinction should be made before possible utilization of OrganEx technology, relative to inclusion of the brain circulation.
While these studies demonstrate important cellular protection and repair processes in vital organs across meaningful timepoints, questions remain concerning organ recovery over an extended timeframe. Since repeating all experiments over additional, extended durations to comprise a full, longitudinal study were not feasible under current regulatory constraints, long-term organotypic slice culture preparations of hippocampus were employed to study post-perfusion cell survivability in the most ischemia-sensitive tissue possible. This demonstrated that OrganEx intervention provides enduring effects on cellular recovery following transfer to the extended survival conditions.
The following enumerated embodiments are provided, the numbering of which is not to be construed as designating levels of importance.
Embodiment 1 provides an isolated perfusate mixture comprising:
an inorganic salt solution;
an artificial oxygen carrier; and
autologous blood.
Embodiment 2 provides the isolated perfusate mixture of embodiment 1, wherein the one or more artificial oxygen carriers is selected from the group consisting of hemoglobin glutamer-250, isolated cell-free hemoglobin, cross-linked hemoglobin, polymerized hemoglobin, encapsulated hemoglobin, and perfluorocarbon oxygen carriers.
Embodiment 3 provides the isolated perfusate mixture of embodiments 1-2, wherein the artificial oxygen carrier is hemoglobin glutamer-250.
Embodiment 4 provides the isolated perfusate mixture of embodiments 1-3, wherein the one or more inorganic salts are selected from the group consisting of sodium chloride, sodium bicarbonate, magnesium chloride, and calcium chloride.
Embodiment 5 provides the isolated perfusate mixture of embodiments 1-4, wherein the perfusate comprises a priming solution containing one or more sugars.
Embodiment 6 provides the isolated perfusate mixture of embodiments 1-5 wherein the one or more sugars are glucose or dextrane.
Embodiment 7 provides the isolated perfusate mixture of embodiments 1-6, further comprising one or more amino acids.
Embodiment 8 provides the isolated perfusate mixture of embodiments 1-7, wherein the one or more amino acids are selected from the group consisting of glycine, L-alanyl-glutamine, L-arginine, L-cysteine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine and salts and solvates thereof.
Embodiment 9 provides the isolated perfusate mixture of embodiments 1-8, wherein the mixture further comprises one or more vitamins.
Embodiment 10 provides the isolated perfusate mixture of embodiments 1-9, wherein the one or more vitamins are selected from the group consisting of choline, D-calcium pantothenate, folic acid, niacinamide, pyridoxine, riboflavin, thiamine, i-inositol and salts and solvates thereof.
Embodiment 11 provides the isolated perfusate mixture of embodiments 1-10, wherein the mixture further comprises, ferric nitrate, magnesium sulfate, potassium chloride, sodium phosphate, and derivatives thereof.
Embodiment 12 provides the isolated perfusate mixture of embodiments 1-11, wherein the mixture further comprises an anti-clotting agent.
Embodiment 13 provides the isolated perfusate mixture of embodiments 1-12, wherein the anti-clotting agent is heparin.
Embodiment 14 provides the isolated perfusate mixture of embodiments 1-13, wherein the percentage of autologous blood in the mixture is between 10% and 50%.
Embodiment 15 provides the isolated perfusate mixture of embodiments 1-14, wherein the percentage of autologous blood in the mixture is approximately 28%.
Embodiment 16 provides the isolated perfusate mixture of embodiments 1-15, wherein the mixture is dialyzed against a solution comprising inorganic salts.
Embodiment 17 provides the isolated perfusate mixture of embodiments 1-16, wherein the mixture is dialyzed against plasma.
Embodiment 18 provides the isolated perfusate mixture of embodiments 1-17, wherein the mixture comprises electrolytes and oncotic agents at levels comparable to those in autologous blood.
Embodiment 19 provides the isolated perfusate mixture of embodiments 1-18, wherein the perfusate further comprises cytoprotective agents.
Embodiment 20 provides the isolated perfusate mixture of embodiments 1-19, wherein the cytoprotective agents are selected from the group consisting of 2-Iminobiotin, Necrostatin-1, sodium 3-hydroxybutryate, glutathione, minocycline, lamotrigine, QVE-Oph, methylene blue, and or any salts, solvates, tautomers, and prodrugs thereof.
Embodiment 21 provides the isolated perfusate mixture of embodiments 1-20, wherein the mixture further comprises antibiotics.
Embodiment 22 provides the isolated perfusate mixture of embodiments 1-21, wherein the antibiotic is ceftriazone.
Embodiment 23 provides the isolated perfusate mixture of embodiments 1-22, wherein the mixture comprises one or more anti-inflammatory agents.
Embodiment 24 provides the isolated perfusate mixture of embodiments 1-23, wherein the one or more the anti-inflammatory agents is dexamathazone or cetirizine.
Embodiment 25 provides the isolated perfusate mixture of embodiments 1-24, wherein the temperature of the mixture is approximately 28° C.
Embodiment 26 provides a system for the hypothermic preservation of organs in a mammal, the system comprising:
a perfusion device for the perfusion of an isolated perfusate mixture into the mammal, the perfusion device comprising:
a perfusion loop; and
a controller programmed to regulate at least a perfusate temperature within the perfusion loop to maintain hypothermic conditions; and the isolated perfusate mixture of any of embodiments 1-25.
Embodiment 27 provides the system of embodiment 26, wherein the perfusion loop further comprises at least one pulse generator programmed to generate a pressure pulse within the perfusate within the perfusion loop.
Embodiment 28 provides the system of embodiments 26-27, wherein the perfusion loop comprises a venous loop, a filtration loop and an arterial loop, wherein:
the venous loop comprises at least one perfusion pump;
the filtration loop comprises at least one perfusion pump, and at least one hemodiafiltration unit adapted and configured to equilibrate the perfusate;
the arterial loop comprises at least one gas exchange source and at least one gas mixer adapted and configured to supply oxygen and carbon dioxide to the perfusate;
wherein the mammal, the venous loop, the filtration loop and the arterial loop are in fluidic communication such that the perfusate can be carried from the mammal, through the venous loop, through the filtration loop, through the arterial loop and back to the mammal.
Embodiment 29 provides the system of embodiments 26-28, wherein one or more components selected from the group consisting of the venous loop, the filtration loop and the arterial loop further comprise a reservoir containing excess perfusate.
Embodiment 30 provides the system of embodiments 26-29, wherein one or more components selected from the group consisting of the mammal, the venous loop, the filtration loop and the arterial loop further comprise one or more elements selected from the group consisting of:
Embodiment 31 provides the system of embodiments 26-30, wherein the one or more sensors measure the concentration of at least one dissolved metabolite selected from the group consisting of nitric oxide, lactate, bicarbonate, oxygen, carbon dioxide, total hemoglobin, methemoglobin, oxyhemoglobin, carboxyhemoglobin, sodium, potassium, chloride, calcium, glucose, urea, ammonia, and creatinine.
Embodiment 32 provides the system of embodiments 26-31, wherein the mammal perfusion apparatus comprises one or more sensors for measuring one or more properties of the perfusate selected from the group consisting of pressure and flow rate.
Embodiment 33 provides the system of embodiments 26-32, wherein one or more components selected from the group consisting of the mammal, the venous loop, the filtration loop and the arterial loop comprise one or more heat exchange units comprising:
one or more heat exchangers;
one or more temperature regulation units;
one or more temperature regulating pumps;
a thermoregulation fluid; and
one or more pipes configured and adapted to transport the thermoregulation fluid, wherein the one or more pipes are in fluidic communication with the one or more heat exchangers, the one or more temperature regulation units and the one or more temperature regulating pumps.
Embodiment 34 provides the system of embodiments 26-33, wherein the one or more components selected from the group consisting of the brain enclosure unit, the venous loop, the filtration loop and the arterial loop comprise one or more sensors adapted and configured to measure the temperature within the perfusion device.
Embodiment 35 provides the system of embodiments 26-34, wherein the one or more sensor(s) is/are adapted and configured to measure the temperature within the perfusion device, the one or more temperature regulation units and the one or more temperature regulating pumps are in electronic communication with a computer programmed to regulate the temperature of the thermoregulation fluid and the specified flow rate of the one or more temperature regulating pumps to maintain a specified temperature within the perfusion device.
Embodiment 36 provides the system of embodiments 26-35, wherein the hemodiafiltration unit is adapted and configured to supply one or more nutrients to the perfusate, selected from the group consisting of Glycine, L-Alanyl-Glutamine, L-Arginine hydrochloride, L-Cystine, L-Histidine hydrochloride-H2O, L-Isoleucine, L-Leucine, L-Lysine hydrochloride, L-Methionine, L-Phenylalanine, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine, L-Valine, Choline chloride, D-Calcium pantothenate, Folic Acid, Niacinamide, Pyridoxine hydrochloride, Riboflavin, Thiamine hydrochloride, i-Inositol, Calcium Chloride (CaCl2)-2H2O), Ferric Nitrate (Fe(N03)3 9H2O), Magnesium Sulfate (MgSO4-7H2O), Potassium Chloride (KCl), Sodium Bicarbonate (NaHCO3), Sodium Chloride (NaCl), Sodium Phosphate monobasic (NaH2PO4-2H2O), D-Glucose (Dextrose), Phenol Red, Sodium Pyruvate, free fatty acids, cholesterol and nucleic acid constitutes.
Embodiment 37 provides the system of embodiments 26-36, wherein the system is configured to perfuse the mammal with the perfusate at a cardiac pulsatile pressure of about 20 mmHg to about 140 mmHg.
Embodiment 38 provides the system of embodiments 26-37, wherein the system is configured to perfuse the organs in the mammal with the perfusate through the pulse generator at a rate of about 40 to about 180 beats per minute.
Embodiment 39 provides the system of embodiments 26-38, further comprising a controller in electronic communication with one or more elements of the system.
Embodiment 40 provides a mammal perfused with the isolated perfusate composition of any of embodiments 1-25, wherein mammalian organs are perfused under hypothermic conditions.
Embodiment 41 provides the mammal of embodiment 40, wherein the mammal is a deceased mammal.
Embodiment 42 provides the mammal of embodiments 40-41, wherein the mammal is a human.
Embodiment 43 provides the mammal of embodiments 40-42, wherein the deceased mammal is deceased for longer than 1 hour.
Embodiment 44 provides the mammal of embodiments 40-45, wherein the deceased mammal has been deceased for longer than 4 hours.
Embodiment 45 provides the mammal of embodiments 40-45, wherein the mammal died of cardiac arrest.
Embodiment 46 provides the mammal of embodiments 40-45, wherein the organs in the deceased mammal are ischemic prior to perfusion with the isolated perfusate mixture of any of claims 1-25.
Embodiment 47 provides the mammal of embodiments 40-46, wherein rigor mortis is prevented.
Embodiment 48 provides the mammal of embodiments 40-47, wherein rigor mortis is reversed.
Embodiment 49 provides the mammal of embodiments 40-48, wherein the perfusate mixture flows into the ophthalmic artery.
Embodiment 50 provides the mammal of embodiments 40-49, wherein the perfusate mixture flows into the renal intralobular arteries.
Embodiment 51 provides perfused organs in a diseased mammal, wherein the perfused organs maintain one or more properties selected from the group consisting of an in vivo level of cell function and viability, and an in vivo level of morphology.
The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.
The present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63/245,632, filed Sep. 17, 2021, which application is incorporated herein by reference in its entirety.
This invention was made with government support under MH117064 and MH117064-01S1, awarded by the National Institute of Mental Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/043816 | 9/16/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63245632 | Sep 2021 | US |
