WO2002089571A1 - Compartiment pour organe destine a un systeme de support metabolique exsanguin - Google Patents

Compartiment pour organe destine a un systeme de support metabolique exsanguin Download PDF

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Publication number
WO2002089571A1
WO2002089571A1 PCT/US2001/040679 US0140679W WO02089571A1 WO 2002089571 A1 WO2002089571 A1 WO 2002089571A1 US 0140679 W US0140679 W US 0140679W WO 02089571 A1 WO02089571 A1 WO 02089571A1
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WIPO (PCT)
Prior art keywords
organ
chamber
perfusion
support member
ems
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PCT/US2001/040679
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English (en)
Inventor
Lauren Brasile
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Breonics, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Breonics, Inc. filed Critical Breonics, Inc.
Priority to PCT/US2001/040679 priority Critical patent/WO2002089571A1/fr
Priority to JP2002586724A priority patent/JP2004529938A/ja
Priority to EP01933402A priority patent/EP1392113A1/fr
Priority to CA002446228A priority patent/CA2446228A1/fr
Publication of WO2002089571A1 publication Critical patent/WO2002089571A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0236—Mechanical aspects
    • A01N1/0242—Apparatuses, i.e. devices used in the process of preservation of living parts, such as pumps, refrigeration devices or any other devices featuring moving parts and/or temperature controlling components
    • A01N1/0247—Apparatuses, i.e. devices used in the process of preservation of living parts, such as pumps, refrigeration devices or any other devices featuring moving parts and/or temperature controlling components for perfusion, i.e. for circulating fluid through organs, blood vessels or other living parts

Definitions

  • the invention relates to a metabolic support system including a solution, method and apparatus for sustaining organs for transplantation under near-physiologic conditions. More particularly, the invention relates to the organ chamber of the system and its use in supporting synthetic functions required for active repair and/or long-term maintenance of organs for transplantation, prognostication of posttransplantation organ function, preparation of an organ for temporary cold storage and transport of an organ intended for transplantation.
  • organs for transplantation There continues to be an extreme shortage of organs for transplantation.
  • kidney transplantation is largely dependent upon the availability of organs retrieved from heart-beating cadaver donors.
  • organs for transplantation There exists, however, a large and as yet untapped source of organs for transplantation, namely, non- heart-beating cadavers.
  • Non-heart-beating cadavers are accident victims who succumb at the site of an injury and those having short post-trauma survival times.
  • non-heart-beating cadavers result when families are emotionally unable to make the decision to donate the organs of a loved one contemporaneously with making the decision to terminate life support. In these situations, the organs are not used because the lack of circulating blood supply (warm ischemia) once the heart stops beating, results in an injury cascade.
  • Hypothermic storage can also produce vasospasm and subsequent edema in an organ.
  • Hypothermically preserved organs can experience glomerular endothelial cell swelling and loss of vascular integrity along with tubular necrosis; phenomenon attributable to the hypothermic conditions employed.
  • Hypothermia can also inhibit the Na/K dependent ATPase and result in the loss of the cell volume regulating capacity.
  • the loss of volume regulation is what causes the cellular swelling and damage.
  • An ample supply of oxygen does not actively diminish the amount of this swelling because the cell membrane is essentially frozen, preventing the effective utilization of oxygen. Without adequate oxygen delivery, the anoxia leads to disintegration of the smaller vessels after several hours of perfusion.
  • ischemia is an injury cascade of events that can be characterized as a prelethal phase, and a lethal phase.
  • the prelethal phase produces harmful effects in three ways: hypoxia; malnutrition; and failure to remove toxic metabolic wastes.
  • hypoxia With the lack of circulating blood comes a lack of molecular oxygen.
  • the resulting hypoxia induces depletion of energy stores such as the depletion of ATP stores in mitochondria.
  • Depletion of ATP leads to cellular changes including edema, loss of normal cellular integrity, and loss of membrane polarity.
  • the cellular changes induces the lethal phase of ischemia resulting in accumulation of metabolic wastes, activation of proteases, and cell death.
  • the perfusate solution that represents the current state-of-the-art in hypothermic organ preservation, and provides for optimized organ preservation under hypothermic conditions, contains components which prevent hypothermic induced tissue edema; metabolites which facilitate organ function upon transplantation; antioxidants; membrane stabilizers; colloids; ions; and salts (Southard et al., 1990, Transpl. 49:251; and Southhard, 1989, Transpl. Proc. 21: 1195.
  • the formulation of this perfusate is designed to preserve the organs by hypothermic induced depression of metabolism. While it minimizes the edema and vasospasm normally encountered during hypothermic storage, it does not provide for the utilization of a substantially expanded donor pool.
  • a protective solution disclosed in U.S. Pat. No. 4,415,556, is used during surgical techniques or for organs to be transplanted for preventing ischemic damage to the organ.
  • the protective solution is used as a perfusate to improve aerobic metabolism during the perfusion of the organ.
  • U.S. Patent No. 5,395,314 describes a method of resuscitating a brain by circulating, after interruption of the blood supply, through the brain a hypothermic preservation solution (approximately 8°-10°C) designed to lower organ metabolism, deliver oxygen, and inhibit free radical damage.
  • a preservation solution useful for initial organ flushing and as a perfusate for in situ or ex vivo preservation of organs for transplantation which employs a warm preservation technology which minimizes, and, in fact, repairs damage due to warm ischemia, and which supports the organ at near normal metabolic rate.
  • Portability and automation of the system is important, particularly in situations where the system is used to initiate organ preservation in situ either prior to or immediately following termination of life support or at external sites following an accident where cardiac arrest has occurred.
  • the invention relates to an exsanguinous metabolic support system for maintaining an organ, tissue or section of anatomy in a near normal metabolic state outside of, or at least isolated from the circulatory system of the body.
  • the system comprises an organ chamber for holding an organ, having means to collect organ product generated during perfusion; a perfusion delivery subsystem comprising one or more perfusion fluid paths for circulating and regenerating a warm perfusion solution capable of supporting the organ in a near normal metabolic state; a controlled gassing subsystem for regulation of respiratory gases and pH of the perfusate; a temperature controller for controlling temperature of the perfusate; and a monitoring subsystem for monitoring various parameters of the perfusate.
  • the invention relates to a monitoring subsystem in which the monitoring of various parameters of the perfusion solution is microprocessor controlled.
  • a monitoring subsystem in which the monitoring of various parameters of the perfusion solution is microprocessor controlled.
  • Such a system would include a microprocessor, and sensors disposed in the perfusate flow path, and coupled to the microprocessor for sensing at least one of the temperature, pH, pressure, flow rate, Pa0 2 PaC0 2 and osmolarity of the perfusion solution and providing the sensed information to the microprocessor.
  • the invention relates to an organ chamber for use in an exsanguinous metabolic support system for preserving an organ, comprising a container, at least one support member positionable within the container for supporting the organ within the container, where the support member is adapted to inhibit movement of the organ in the organ chamber.
  • the support member of the present invention is a resilient support member conformable to an outer surface portion of the organ, such as a gas-, fluid- or gel-filled sac.
  • it is a rigid support member comprising a cavity generally contoured to accommodate an outer surface portion of the organ.
  • the organ may be a heart, liver, kidney, pancreas, lung or other tissue from an adult or pediatric donor.
  • the organ chamber further comprises a conduit for delivering venous outflow of a perfusion solution being circulated through the organ from the organ directly to a reservoir, a conduit for collecting organ product separately from perfusate; at least one sensor for monitoring at least one parameter of the perfusion solution selected from flow rate, pH, Pa0 2 , PaC0 2 , temperature, vascular pressure, and a metabolic indicator such as oxygen consumption, glucose consumption, consumption of at least one citric acid cycle component, C0 2 production and the like.
  • the invention relates to an organ chamber further comprising at least one warm preservation system component, for example, a reservoir, a heat exchanger, an oxygenator, and/or a pump.
  • the organ chamber of the present invention comprises connectors for releasably connecting the organ chamber to an external warm preservation system.
  • the invention relates to a method for preserving an organ comprising placing the organ within a container on a resilient support member adapted to inhibit movement of the organ within the container. The organ is then connected to a warm preservation system such as the metabolic support system of the present invention and perfused with a warm preservation solution capable of maintaining the organ at a near normal rate of metabolism.
  • a warm preservation system such as the metabolic support system of the present invention and perfused with a warm preservation solution capable of maintaining the organ at a near normal rate of metabolism.
  • the invention relates to a method for the maintenance of an organ or tissue for transplantation, comprising the steps of establishing and maintaining the organ in a warm preservation system comprising the organ chamber, such as the exsanguinous metabolic support system of the present invention, and monitoring the functional integrity of the organ. 0018.
  • the invention relates to the use of the organ chamber of the present invention in conjunction with a warm perufsion system to support continued de novo syntheses sufficiently for an active repair process to ensue.
  • the invention relates to a method for transporting an organ comprising establishing the organ in an organ chamber such as the one described herein, perfusing the organ in a first warm preservation system comprising the organ chamber, such as the exsanguinous metabolic support system described herein, capable of maintaining the organ at a near normal rate of metabolism for a period of time sufficient to inhibit damage to the organ, and removing the organ chamber from the warm preservation system and refrigerating or cold-packing it for shipment and transporting the organ chamber containing the organ.
  • the organ chamber containing the organ can be established in a second warm preservation system so that the organ can be warm-perfused and the organ's functional integrity monitored prior to being transplanted into the recipient.
  • the invention relates to a method for preserving an organ comprising perfusing the organ with a warm preservation solution and directing venous outflow of the preservation solution away from the organ so as to inhibit contact of the preservation solution with the outer surface of the organ.
  • the invention relates to a warm preservation solution to be employed in an exsanguinous metabolic support system comprising a polyethylene- glycol modified hemoglobin.
  • the hemoglobin may be human, animal or recombinant in origin.
  • FIGURE 1 shows an embodiment of the exsanguinous metabolic support system having a closed loop perfusion subsystem for circulating the perfusion fluid and a dialysis subsystem for reprocessing it.
  • FIGURE 2 shows an embodiment, having a perfusion exchange subsystem and two perfusion paths, which is suitable for preservation of a liver.
  • FIGURE 3 shows a perspective view of one embodiment of the organ chamber of the invention.
  • FIGURE 4 shows a perspective view of one embodiment of the organ chamber of the invention wherein the organ chamber further comprises a reservoir.
  • FIGURE 5 shows a cross sectional view of one embodiment of the organ chamber of the invention.
  • FIGURE 6 shows a cross sectional view of a vein support for supporting the vein of the organ and holding it securely in contact with a conduit for directing the perfusate from the organ directly to the reservoir.
  • FIGURE 7 shows a perspective view of a vein support.
  • EMS metabolic support system
  • the EMS perfusion system of the present invention delivers a warm perfusion solution containing all the constituents necessary to reestablish, where necessary, and support oxidative metabolism by the organ.
  • the perfusion system may also reprocess the perfusion solution to ensure a continuous supply of nutrients and chemical energy substrates and remove metabolic by-products. Additionally, the EMS monitors and controls various parameters of the perfusion including temperature, vascular pressures, perfusion flow rate, OsM, pH, Pa0 2 , PaC0 2 , nutrient delivery and the removal of waste products.
  • organ, tissue or section of anatomy refers to an excised viable and whole section of the body to be maintained as such in the EMS of this invention, and refers to an intact organ including, but not limited to, a kidney, heart, liver, lung, small bowel, pancreas, brain, eye, skin, limb or anatomic quadrant.
  • organ product refers to any substance generated as the result of the secretory function of an organ, frequently a fluid, for example, bile from liver, urine from kidneys, but also includes mechanical functions such as kidney filtration or heart pumping.
  • preservation solution refers to a non-blood buffered physiologic solution that provides means for reestablishing cellular integrity and function in organs which may have experienced ischemic damage prior to or during isolation and further, enables an organ or tissue to be maintained at a near normal rate of metabolism.
  • non- blood is intended to exclude perfusates comprising substantially whole blood or its individual components.
  • the perfusion solution of the present invention may, however, contain a minimal amount of whole blood or a blood component, for example, red blood cells, serum or plasma.
  • tissue normal rate of metabolism and “near normal metabolic rate” are defined as about 70-100% of the normal rate of metabolism for a particular organ as determined by measuring and evaluating whether functional characteristics of an organ, such as those described in U.S. Patent No. 5,699,793, are within the range associated with normal function for that particular organ.
  • functional characteristics include, but are not limited to, electrical activity in a heart as measured by electrocardiogram; physical and chemical parameters of organ product, for example, oxygen consumption and glucose utilization which can be ascertained from perfusate concentrations; pancreatic enzymes; heart enzymes; creatinine clearance and filtration functions, and specific gravity of urine and so on.
  • therapeutic agent refers to any molecule used to effect a functional, synthetic, metabolic, immunogenic or genomic change in an organ or tissue. These include but are not limited to viral vectors, liposomes, episomes, naked DNA, either sense or anti-sense molecules, RNA, chemotherapeutic agents, biologies, such as cyokines and chemokines and the like.
  • the process according to the present invention involves isolating an organ, tissue or specific area of anatomy from the rest of the physiologic system by removing or interrupting the arterial source of blood feeding the desired tissue(s). Likewise, the venous outflow from the organ or section of anatomy is interrupted and the venous effluent is collected. If the tissue is completely excised from the body, then the enervation and lymphatics of the tissue(s) are also isolated. Next, the organ or tissue is flushed through the arterial system with the solution of the present invention at a temperature of about 25° - 37°C to remove blood and blood products.
  • Organ function is also monitored, for example, by collecting an organ product, such as urine or bile, and evaluating whether physical and chemical parameters of the organ product are within the range associated with normal function for that particular organ.
  • the invention may optionally include, therefore, a subsystem for evaluating the functional status of the organ. In this way, organ function can be monitored using in-line detection means and in-line testing methodology.
  • Perfusion of the isolated organ or section of anatomy with a solution at near physiologic temperature of about 25°C to 37°C performs a number of functions. It maintains the cellular environment at physiologic pH and maintains near normal oxygenation, temperature, and osmolarity. It maintains the normal barrier function of the tissue to macromolecules, thereby resulting in stable perfusion pressures and stable vasculature flow rates. It adequately dilates and fills the vasculature, delivers adequate trophic factors to maintain a near normal level of metabolism in the isolated organ or section of anatomy and supports the artificially interrupted aerobic metabolism by providing high energy compounds.
  • oxidative metabolism with supplemental substrates that may include, but are not limited to, glucose, pyruvate, and uridine 5-triphosphate (UTP).
  • the ongoing oxidative metabolism is further supported by maintaining the adenine compound pool.
  • the citric acid cycle and the electron transport chain are supported by providing adequate substrate delivery to continue metabolic support and function in the isolated organ and tissues.
  • the ongoing metabolism supported by the method and solution of the invention provides adequate metabolites and nutrients to maintain the tissue integrity with tight cellular functions and normal membrane polarity.
  • the method and system of the invention allows for the removal of blood within the organ or section of anatomy and refills the vascular and pericellular spaces with the solution of this invention. Further, the system maintains pH, Pa0 2 , temperature, osmolarity, and hydrostatic pressures and delivers adequate substrate to support the metabolism necessary for cellular integrity.
  • the ongoing metabolism provided by the method, solution and system, in combination, is of sufficient level to support the ongoing function of the specific organ or section of anatomy during the time the tissue(s) are isolated from the body or the circulatory system.
  • the solution is perfused at a systolic pressure appropriate for the tissue(s) being maintained with the EMS organ culture technology of this invention until a flow rate is achieved which is near normal for that particular organ or tissue.
  • a human kidney may be perfused with the solution at a systolic pressure of ⁇ 80 mmHg with a flow rate >80 cc/min.
  • the pH is maintained in a physiologic range by the injection of C0 2 or 0 2 via an oxygenator. Adequate oxygen is provided to the organ by including an oxygen transporting compound as a component of the solution. 0038.
  • the method, solution and system according to the present invention provides the necessary oxygen delivery, nutrients for metabolism, oncotic pressure, pH, perfusion pressures, temperature, and flow rates to support adequate organ metabolism near the respective physiologic range.
  • a near normal rate of metabolism as defined above is about 70-100% of normal rates of metabolism.
  • the method, solution and system according to the present invention supports a level of metabolism during the period of EMS organ culture which supports sufficient oxidative metabolism to result in the normal functional product of the organ or section of anatomy.
  • EMS exsanguinous metabolic support
  • Organ preservation and perfusate solutions are known in the art as comprising a base solution that consists of a buffered physiological solution, such as a salt solution or a cell culture-like basal medium, to which is added a variety of defined supplements.
  • the perfusion solution of the present invention employs such a base solution containing amino acids in quantities sufficient to support protein synthesis by the metabolizing organ, ions, physiologic salts, serum proteins, carbohydrates, and a buffering system for maintaining pH at physiologic levels.
  • the perfusion solution of the present invention has been designed to support the nutritional and metabolic needs of the vascular endothelium within a graft, thereby maintaining the integrity of the vasculature and, subsequently, the normal permeability of the organ.
  • the buffered basal medium may be any commercially available salt solution or cell culture medium, (e.g., Hank's BSS, Earle's BSS, Ham's F12, DMEM, Iscove's, MEM, Ml 99, RPMI 1640, RSM-210.)
  • a bicarbonate buffer system is employed.
  • the bicarbonate buffer works in concert with the respiratory gas controller subsystem of the EMS system to automatically maintain the pH of the perfusion solution in a narrow range, 7.0 to 7.6, and more preferably, 7.30 - 7.45, which approximates respiratory control of blood pH by the lungs.
  • a number of supplements including, but not limited to, essential and non-essential amino acids, growth factors, vasodilators, vitamins, and chemical energy substrates, in a physiologially effective amount to support oxidative metabolism by the organ or tissue.
  • Amino acids to be included in the perfusion solution of the present invention include the basic set of 20 amino acids and may be D- or L-amino acids, or a combination thereof, or may be modified amino acids, such as citrulline, ornithine, homocysteine, homoserine, ⁇ -alanine, amino-caproic acid and the like, or a combination thereof.
  • Chemical energy substrates added to the perfusion solution may include pyruvate, glucose, ATP, AMP, coenzyme A, flavin adenine dinucleotide (FAD), thiamine pyrophosphate chloride (cocarboxylase), ⁇ -nicotinamide adenine dinucleotide (DPN), ⁇ -nicotinamide adenine dinucleotide phosphate (TPN), uridine 5' triphosphate (UTP) chloride.
  • the chemical energy substrates comprise from about 0.01% to about 90% by volume of the combination of supplements added to the base solution in preparing the perfusion solution of the present invention.
  • nucleic acids for DNA repair and synthesis including 2' deoxyadenosine, 2'deoxyguanosine, 2'deoxycytidine, adenosine, thymidine, guanosine, cytidine and uridine.
  • the solution of the present invention may further comprise hormones, such as insulin, and thyroid stimulating hormone (TSH) and growth factors (GF), such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF-1, FGF-2), insulin-like GF I and II, epithelial GF, epidermal GF, brain- derived FGF, somatomedins Al, A2, B and C, nerve growth factor (NGF), vascular endothelial growth factor (VEGF), heparin-binding growth factor (HBGF), endothelial cell growth factor (ECGF), transforming growth factor (TGF), glucocorticoids and urogastone.
  • hormones such as insulin, and thyroid stimulating hormone (TSH) and growth factors (GF), such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF-1, FGF-2), insulin-like GF I and II, epithelial GF, epidermal GF, brain- derived FGF, somatomedins Al, A2, B and C
  • the perfusion solution of the present invention also comprises serum albumin and/or mucopolysaccharides such as chondroitin sulfate B, heparin, petastarch, hetastarch, and plasma expanders as a source of colloid, and lipids, such as linoleic acid, arachidonic acid, linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, oils. Additionally, attachment factors, antioxidants, vasodilators and impermeants may be included in the perfusion solution of the present invention.
  • serum albumin and/or mucopolysaccharides such as chondroitin sulfate B, heparin, petastarch, hetastarch, and plasma expanders as a source of colloid
  • lipids such as linoleic acid, arachidonic acid, linolenic acid, eicosapentaenoic acid, do
  • the high osmolar solution of the present invention is used for the initial organ flushing, and as a perfusate for long term maintenance of an organ in the EMS system using warm preservation technology (18°- 35°C) without extreme hypothermia.
  • the solution has been designed to support the nutritional, synthetic and metabolic needs of the vascular endothelium within a graft, thereby maintaining the integrity of the vasculature and subsequently the normal permeability of the organ.
  • the solution of the present invention was specifically designed to potentiate the simultaneous growth of microvessel and large vessel endothelial cells, to support the integrity of vascular endothelium within a graft; and to support normal permeability and metabolism without extreme hypothermia.
  • the enhanced ability of the solution to serve as a preservation solution for organs for transplantation using a warm preservation technology may be attributed to supplementation with serum albumin as a source of protein and colloid, vasodilators to ensure adequate dilation of the vasculature, trace elements to potentiate viability and cellular function, pyruvate and adenosine for oxidative phosphorylation support; transferrin as an attachment factor; insulin and sugars for metabolic support; and glutathione to scavenge toxic free radicals as well as a source of impermeant; cyclodextrin as a source of impermeant, scavenger and potentiator of cell attachment and growth factors; a high Mg concentration for microvessel metabolism support; mucopolysaccharides, comprising primarily chondroitin sulfates and heparin sulfates, for growth factor potentiation and hemostasis; and ENDO GROTM as a source of colloid, imper
  • the preservation solution of the present invention has been found to preserve organs without extreme hypothermia, and does not present the common problems encountered with cold perfusates, namely, edema, vasospasm, depletion of ATP stores, shutdown of ion pumps, glycolysis, and the generation of cold- induced toxic free radical intermediates.
  • the preservation solution of the present invention provides for more efficacious preservation thereby presenting the potential to utilize an expanded donor pool, namely, the non-heart beating cadaver donors.
  • Table 1 lists components of one embodiment of the perfusion solution of the present invention.
  • the perfusion solution contains one or more oxygen transporting compounds ("oxygen carrying agents") that function to provide molecular oxygen for oxidative metabolism to the organ.
  • oxygen carrying agents are well known to those skilled in the art and include, but are not limited to, hemoglobin, stabilized hemoglobin derivatives including crosslinked polyhemoglobin such as diaspirin cross-linked hemoglobin (DCLHb), o-raffinose cross-linked hemoglobin and gluteraldehyde cross- linked hemoglobin; conjugated hemoglobins such as polyoxyethylene conjugates (PHP), PEG-conjugated hemoglobin; recombinant hemoglobin products, perfluorochemical (PFC) emulsions and/or perfluorochemical microbubbles (collectively referred to as "perfluorochemical”).
  • PFC perfluorochemical
  • perfluorochemical microbubbles collectively referred to as "perfluorochemical”
  • perfluorochemical such as perflubron emulsion (perfluoroocytl bromide, PFOB).
  • perfluorochemical emulsions said to be useful as oxygen carrying agents are described, for example, in U.S. Pat. Nos. 5,403,575; 4,868,318; 4,866,096; 4,865,836; 4,686,024; 4,534,978; 4,443,480; 4,423,077; 4,252,827; 4,187,252; 4,186,253; 4,1 10,474; and 3,962,439.
  • Such liquid PFC emulsions include, but are not limited to perfluorooctyl bromide, perfluorooctyl dibromide, bromofluorocarbons, perfluoroethers, Fluosol DATM, F-44E, 1,2-bisperfluorobutyl-ethylene, F-4-methyl octahydroquinol-idizine, 9 to 12 carbon perfluoro amines, perfluorodecalin, perfluoroindane, perfluorotrimethyl bicycle [3,3,1] inane, perfluoromethyl adamante, perfluorodimethyl adamantine.
  • oxygen carrying agents comprise from about 0% to about 59% by volume of the supplements which are added to, and dissolved in, the base solution in preparing the perfusion solution of the present invention; or about 0% to about 20% of the total perfusion solution (v/v).
  • red blood cells may be used as an oxygen carrier in an effective amount to support metabolism by the organ being perfused.
  • RBC red blood cells
  • about 5cc of RBC per 500ml of perfusion solution that is, about 1%) is an effective amount.
  • RBC provides oxygen concentrations equivalent to the oxygen consumption by the metabolizing organ. This generally occurs at the rate of 0.1 - 0.3cc/min/gm. Additionally, after 24 to 48 hours of perfusion, there was no evidence that the RBC were cretinated. Therefore, an amount of RBC in this range does not present the problem of mechanical damage to the organ associated with blood-based perfusates.
  • chemically modified hemoglobin for example, polyethylene glycol-modified hemoglobin may be used to maintain oxygen tension of the perfusate at a level sufficient to support metabolism by the organ being perfused. Generally, about O.Olg to less than 0.5g per 500ml of perfusion solution is an effective amount.
  • Chemically modified hemoglobin refers to hemoglobin that is either recombinant or has been isolated from the blood of a human or other mammal, deoxygenated, and chemically altered, for example, by cross-linking or polymerization or through the addition of adducts such as polyethylene glycol or polyoxyethylene.
  • Polyethylene glycol-modified hemoglobin refers to hemoglobin that has been modified such that it is associated with polytheylene glycol ( ⁇ -hydro- ⁇ -hydroxypoly-(oxy- 1 ,2- thanediyl); methods for preparation of chemically modified hemoglobin are known to those of skill in the art, for example, U.S. Patent Nos. 5,234,903; 5,386,014; 6,054,427; 5,985,825; and 5,814,601 , the contents of which are hereby incorporated by reference herein in their entirety.
  • the perfusion solution of the present invention may additionally comprise vasodilators, in a physiologically effective amount, which provide a means to adequately dilate large vessels via smooth muscle cell relaxation, as well as to adequately dilate microvessels. To insure that normal permeability of the vasculature is maintained, the vasodilation is controlled in an endothelial cell-dependent manner.
  • Vasodilatory+ components for use in the perfusion solution of the present invention include (i) substrates for endothelial cell mediated vasodilation, such as acetylcholine, dopamine, bradykinin, and arginine; (ii) substrates for microvessel vasodilation, such as prostacyclin (and analogs, e.g. carbacyclin) and Mg + ; and (iii) adenosine (and analogs, e.g. cyclohexyladenosine), and verapamil for their combined effects on vascular dilation mediated by calcium channel blocking.
  • substrates for endothelial cell mediated vasodilation such as acetylcholine, dopamine, bradykinin, and arginine
  • substrates for microvessel vasodilation such as prostacyclin (and analogs, e.g. carbacyclin) and Mg +
  • vasodilators Use of the aforementioned vasodilators ensures that the vasculature is well dilated while simultaneously retaining its integrity and normal barrier function.
  • the vasodilators comprise from about 1% to about 50% by volume (w/v) of the combination of supplements which are added to the base solution in preparing the perfusion solution of the present invention.
  • FIGURE 1 is a diagram of one embodiment of the organ perfusion circulation path of the present invention. In most instances, only one perfusion path is necessary. When the organ to be metabolically maintained is a liver, however, two perfusion circuits are required. These are illustrated in FIGURE 2 and described in more detail below. All perfusate available for circulation through the system is propelled through the circulation path 10 by a pump 12. The direction of flow of the perfusion solution is indicated by arrows within the flow path. While any pump may be used to circulate the perfusion fluid of the instant invention, a pulsatile pump, for example, Model No. MOX-100TM, (Waters Instruments, Inc., Rochester, Minn.) is preferred.
  • a pulsatile pump for example, Model No. MOX-100TM, (Waters Instruments, Inc., Rochester, Minn.) is preferred.
  • the perfusate Before the perfusate enters the organ 40, it passes through a heat exchanger 14, to bring the temperature within the range of 25° - 37°C, the preferred temperature for optimal metabolism by the organ being in that range.
  • the heat exchanger 14 is controlled by a temperature controller 16 which receives input from a temperature sensor 18 situated in the perfusate path 10.
  • the temperature controller is a single unit comprising a thermocouple which senses the temperature of the perfusate and a heat exchanger which is activated by the thermocouple, when required, to maintain the temperature in the desired range.
  • the temperature may be controlled by means of a water heater for circulating warmed water around the perfusate reservoir or oxygenator.
  • a temperature sensor situated within the organ chamber is used to monitor and control the temperature of the perfusate.
  • the perfusate which contains an oxygen carrier, is also oxygenated prior to contact with the organ via a hollow fiber or membrane oxygenator 20, for example, a pediatric size oxygenator, such as those available from Sarnes, to provide a partial pressure of oxygen, in most cases, in the range of 100 - 240 mmHg.
  • a pediatric size oxygenator such as those available from Sarnes
  • the partial pressure is maintained in the range of 80 - 140 mmHg.
  • the oxygenator 20 is situated in the perfusion solution flow path 10 between the heat exchanger 14 and pH sensor 22.
  • the oxygenator may be an independent unit or may be combined with another component of the system to form a single unit, for example, combined with the heat exchanger, or as part of the organ chamber.
  • the pH and PaC0 2 of the perfusion solution are controlled by automatic intermittent gassing of the perfusate with C0 2 to provide a circulating perfusate having a pH in the range of 7.30 - 7.45 and a partial pressure of C0 2 of 30-60 mmHg.
  • the perfusate is debubbled in a conventional bubble trap 30 just prior to entering the organ chamber 32.
  • the perfusate leaves the organ as venous effluent and, in most perfusion systems, drains by gravity directly into an effluent reservoir 38 situated beneath the compartment of the organ chamber 32 which holds the organ.
  • the effluent is directed to the effluent reservoir 38 by means of conduit 43 which is connected to the vein either by a conventional non-traumatic cannula which has been inserted into the vein of the organ 40 or by means of a device 49 for supporting the vein so as to keep the vein in communication with the conduit 43 as described in more detail below.
  • the specifications of the organ chamber 32 used depend on the organ to be maintained. For example, when the organ is a liver, the organ chamber must have more than one perfusate inlet to accommodate two simultaneously operating perfusion flow paths (FIGURE 2).
  • FOGURE 2 perfusion flow paths
  • an open vertical tube extending upward from the perfusate flow path above the point of entry of perfusate into the heart, provides a column of perfusate, such that sufficient pressure is maintained to counter the expulsion of the perfusate by the beating heart.
  • the organ chamber 32 (as shown in FIGURES 1-4) comprises a container 33, comprising a lower portion, and an upper portion adequate in size to accommodate the organ 40 to be maintained, and having a cover or lid 34.
  • the entire chamber is made of a rigid material which is of a medical grade, non-toxic, non- leeching and sterilizable.
  • the organ chamber is made of stainless steel and has means, such as one or more transparent or translucent panels, for viewing the interior of the chamber. The ability to observe the organ during perfusion is highly desirable; thus, in a preferred embodiment, the entire organ chamber is made of a rigid material that is transparent.
  • a variety of thermoplastics are commercially available, for example, lucite, or polycarbonate materials, such as LEXAN®.
  • the organ chamber of the present invention may also comprise a combination of suitable materials.
  • the organ chamber of the present invention provides comprises at least one support member 36, positionable within the upper portion of the organ chamber container 33 for supporting the organ 40 within the container 33 which provides mechanical restraint of the organ during perfusion, thereby minimizing injury or damage to the organ or its blood vessels due to accidental movement of the organ, for example during transport.
  • the organ chamber 32 further comprises one or more perfusate inlets 42, conduit 43 for delivering the venous outflow of perfusate from the organ directly to a reservoir of the warm preservation system to minimize the risk of contamination by avoiding contact of the perfusate with the external surfaces of the organ and the internal surfaces of the organ chamber.
  • the organ chamber comprises a conduit 48 for collecting organ product from the organ 40.
  • the lower portion of the container 33 comprises a perfusate reservoir 38 and/or other components of the perfusion system.
  • Organ support member 36 is adapted to inhibit movement of the organ within the container.
  • Organ support member 36 may be made of a rigid or semi-rigid material, or of a mesh-like fabric which suspends the organ in a sling-like fashion.
  • the support member is a rigid or semi-rigid material having a cavity therein contoured to the shape of a portion of the organ so that the organ sits within the cavity.
  • the support member is a resilient material, such as a gas-(an inert gas), fluid- (for example, saline or other suitable non-toxic liquid) or gel- filled (for example, silicone) pouch or sac, capable of conforming to the shape of an organ which is placed on it.
  • Both the exterior surface of the support member and the interior material must be made of an inert, biocompatible and sterilizable material.
  • the support member contacts the organ on the bottom and partially on the sides, leaving a portion of the organ visible through the sides or cover of the organ chamber container so that it can be observed during perfusion. Visual observation of the organ is necessary to detect early signs of edema, to detect areas of the organ not adequately perfused or other indications of organ failure.
  • the support member supports the organ within the organ chamber and importantly, inhibits movement of the organ within the chamber, thereby preventing damage to the organ.
  • this type of organ support more closely approximates the normal physiological environment in which the organ is supported by and in contact with neighboring tissues in the body.
  • a formable support member allows the organ to be oriented in a variety of positions (including physiological), not just lain on its side.
  • Figure 3 depicts an embodiment in which the organ chamber 32 has a perfusate outlet 43 having means to directly conduct the perfusate from the vein 41 of the organ 40 to a perfusate reservoir 38 which is either a part of the organ chamber (as shown in Figure 3) or separate from the organ chamber ( Figures 1 and 2).
  • the organ chamber of the present invention substantially reduces the potential for contamination of the perfusate by preventing contact of the perfusate with the exterior surfaces of the organ.
  • the organ chamber further comprises a vein support 48 as depicted in Figures 3 and 5.
  • a further advantage of this embodiment is the ability to avoid the necessity of cannulating the vein and the cannulation-associated injury or damage, by a venous support means, such as a small sterilizable pronged member (shown in Figures 3 and 5) disposed adjacent to the organ.
  • the vein support 48 supports the vein at a position proximal the intersection of the vein with the exterior wall of the organ and is capable of holding the open end of the vein securely against a piece of tubing which conducts the venous effluent away from the organ and into a reservoir 38.
  • the organ chamber of the present invention may be a disposable single-use unit or of a suitable material, such as stainless steel or polycarbonate, which can be sterilized and reused.
  • the organ chamber of the present invention comprises connectors for releasably connecting the organ chamber to an external warm preservation system. Suitable connectors include, without limitation, lengths of inert biocompatible tubing or more complex mechanic devices for ensuring the integrity of the fluid path once the organ chamber has been disposed therein.
  • the organ chamber may incorporate various components of the warm perfusion system, including but not limited to, an oxygenator, a reservoir, a bubble trap, and flow meter. Additionally, means for measuring pressure, temperature, pH, Pa0 2 , PaC0 2 , indicators of metabolism, such as glucose, and markers of synthetic functions, such as NO (stable end products) PCNA or ZO-1, may be integrated into the organ chamber. Additionally, the organ chamber of the present invention includes one or more ports for sampling perfusate and one or more ports for perfusion regeneration.
  • Figure 3 illustrates an embodiment of an organ chamber of the present invention having a perfusate reservoir in the lower portion of the organ chamber container.
  • Sensors for monitoring parameters of the perfusate which are indicative of organ metabolism for example, glucose concentration, Pa0 2 , PaC0 2 , and the like are disposed to measure the characteristics of the perfusate just prior to entry of the perfusate into the organ and as it exits the organ. The change in the values of the pre- and post-organ perfusate is indicative of organ metabolism.
  • Sensors for other parameters of the perfusate, including pH and temperature may also be situated within the organ chamber and allow for optimal control of perfusate parameters during perfusion.
  • the venous effluent from the organ is collected by gravity flow directly into the reservoir 38 of the organ chamber 32 either through openings 37 in the support means 36.
  • the venous outflow is collected by gravity flow, but the venous outflow is conducted away from the organ, by connecting the vein with, for example, a length of tubing 43. In one embodiment this is done by cannulating the vein using a non-traumatic cannula. The length of vein available for resection upon reimplantation, however, is reduced by any damage that occurs during cannulation.
  • a venous support member 46 for supporting the vein 41 within a conduit is disposed beneath the vein, adjacent to the organ at a point where the vein 41 intersects the external wall of the organ 40.
  • the upper portion of the support is wide enough to support the vein and the conduit at the juncture of the two. This enables the ligated end of the vein 41 to be connected with a conduit, for example, a length of tubing 43 without the necessity of cannulation, for example, by simply slipping the end of the tubing 43 over the vein 41.
  • the vein may rest in a partially open conduit. Further injury to the vein is, thereby, avoided.
  • the organ chamber of the present invention allows for either collection of organ product separate from perfusate or recirculation of organ product as part of the perfusate.
  • Means 48 for collecting organ product includes a valve and/or port by which all or some portion of the organ product generated by the organ during the perfusion period can be sampled and assayed for parameters indicative of normal organ function for that organ.
  • the organ chamber of the present invention additionally comprises means for optionally collecting organ product separately from perfusate. For example, urine being produced by a perfused kidney exits the ureter into an organ product fluid pathway. Disposed within the organ product pathway is a valve which when open allows the organ product pathway to be in fluid communication with the venous perfusate pathway.
  • organ product When the valve is in the closed position, organ product is diverted away from the perfusate pathway and may be removed from the system by means of a sampling or collection port. Functional characteristics of the organ can be assessed by evaluating chemical parameters of the collected organ product, its rate of production or concentration of an added tracer molecule to determine clearance rates. Alternatively, organ product is recombined with perfusate so as to preserve perfusate volume, osmolarity and chemical integrity. In this way, organ metabolism, perfusion and function can be measured and the condition of the preserved organ can be evaluated prospectively.
  • the effluent is filtered through a filter 44 having a pore size of 0.22 microns, such as those available from Millipore Corp., to remove debris and any bacterial contaminants. Collection of effluent, in this manner, provides the additional benefit of minimizing perfusion contamination due to contact with air.
  • the organ chamber of the present invention further comprises a suitable microprocessor and sensors for monitoring and control of parameters of the perfusion solution.
  • the perfusate would eventually be exhausted without some replenishment or regeneration.
  • the ingredients necessary to support metabolism can be replenished by perfusate exchange, that is, removing a portion of the spent perfusate via means 46 in the reservoir and replacing it with fresh perfusate.
  • the perfusate is exchanged by means of two volume- regulatable pumps 47 and 49; pump 47 is connected to means 46 in the reservoir to extract depleted perfusate, and pump 49 is situated in the perfusate path 10 just prior to the oxygenator 20. Removal of depleted perfusion solution by pump 47 is immediately followed by introduction of an equal volume of fresh perfusate into the system by pump 49. Exchange in this manner can be continuous or intermittent. The rate of exchange is dependent upon temperature and/or metabolic rate of the organ being perfused.
  • the perfusate is continuously regenerated by dialysis, alone or in combination with perfusate exchange.
  • the perfusion dialysis subsystem of the EMS comprises an effluent reservoir 50 in fluid communication with the perfusion flow path 10 and one or more nutrient reservoirs Dl, D2, D3, Dn.
  • Nutrient reservoirs, Dl, D2, D3, Dn are in communication with the effluent reservoir 50 via semi-permeable membranes Ml, M2, M3, Mn of varying molecular weight cut-offs in the range of 1,000 - 80,000 daltons. These nutrient reservoirs contain concentrated amounts of components necessary to regenerate the perfusion solution, which are allowed to diffuse into perfusate in the effluent reservoir 50.
  • the nutrient reservoirs are arranged in order of decreasing molecular size, so that the perfusate flowing by is contacted first, by the nutrient with the largest molecular size, followed by nutrients with increasingly smaller molecular weight. This ensures that any components which might be lost at the first dialyzer unit are replenished by exposure to subsequent units before leaving the perfusion dialysis system.
  • the system further comprises means for regulating the volume of the perfusion solution, so that an equilibrium is maintained between the effluent reservoir and the nutrient reservoirs.
  • the perfusion solution monitor subsystem performs a plurality of functions, using standard components and hardware to monitor various parameters of the perfusion solution.
  • temperature is sensed and controlled by a thermocouple or thermosensor 18 linked to the heat exchanger.
  • One or more manometers 31 situated in the perfusion solution flow path are used to monitor the vascular resistance to the perfusate and the partial pressures of the respiratory gases.
  • a flowmeter 52 for example, an ultrasonic device, such as those available from Transonic, for detecting flow rate having a sensor 54, which clips onto the tubing carrying the perfusate, is used to monitor the flow rate of the perfusate.
  • the flowmeter is situated in the flow path between the bubble trap and arterial inlet of the organ chamber. Osmolarity of the perfusate is assessed using the conventional freezing point depression method. Alternatively, osmolarity may be monitored using in-line detection means to assess the concentration of major constituents of the perfusion solution. A sample of perfusate may be removed via the bubbletrap 30 or some other means situated in the perfusion flow path for this determination.
  • monitoring and control of parameters of the perfusion solution are accomplished by the use of sensors and a microprocessor integrated into the organ chamber; all within the skill of the ordinary artisan.
  • Components of the control subsystem may be combined or provided as separate parts which are implemented in the system as a matter of design choice.
  • the perfusion solution is continuously oxygenated by introducing 100% oxygen 28 via a membrane or hollow fiber oxygenator 20 to maintain a partial pressure of 0 2 (Pa 0 2 ) of 100-250mmHg.
  • the system of the instant invention includes a novel mechanism for maintaining a perfusate pH between 7.32 and 7.38 and the level of C0 2 in the perfusate at a partial pressure of 30-60mmHg. Regulation of pH and C0 2 levels of the perfusate is achieved by the controlled intermittent gassing of the perfusion solution with C0 2 .
  • the system continuously monitors the pH, for example, by a pH electrode or sensor 22 in the perfusion path 10.
  • the pH sensor 22 is operatively connected to a controller 24 and solenoid 25 for regulation of the C0 2 26 gassing required to maintain tight control of the pH.
  • the pH rises above 7.35, thereby exceeding the set point of the controller 24 the gassing system is activated by way of valve interface 23 and solenoid 25, and C0 2 26 is injected into the perfusion solution until a pH of 7.35 again is reached, at which point the system is deactivated and the gas is turned off.
  • the intermittent gassing of the perfusion solution with C0 2 in this manner, when used in conjunction with a perfusion solution having a bicarbonate buffer system is especially effective in mimicking the tight physiologic control of blood pH by the respiratory system.
  • the pH and respiratory gases may be monitored and controlled using sensors and microprocessors known to one of skill in the art for this purpose.
  • a sufficient amount of the perfusion solution is slowly introduced by infusion via a cannula into the major arterial blood supply for the particular organ to be perfused until the effluent is free of blood.
  • the amount of the perfusion solution sufficient for use in flushing the organ may depend on the particular organ type and size, as well as the length of time the organ was deprived of blood flow. For example, 200 to 600 mis of the perfusion solution may be sufficient to flush a human kidney which has been deprived of blood flow for a period of 1-3 hours. In this way, any ischemic blood and acidotic products which have accumulated in the vascular space are removed. Further, pH is restored and fresh substrate is delivered to support anaerobic metabolism and other cellular pathways necessary for cellular integrity and function.
  • the organ is immobilized within the interior space of the organ chamber, for example, by placing the organ on a support member positionable within the organ chamber and adapted to inhibit movement of the organ.
  • the support member may be a resilient support member conformable to a portion of the outer surface of the organ such as a fluid-, gas- or gel-filed sac; alternatively it may be a rigid or semi-rigid support member comprising a cavity generally contoured to an outer surface portion of the organ, so as to prevent the organ from moving around in the chamber, particularly during transport.
  • the organ is then connected to the perfusion solution path by a short length of tubing connecting the cannulated artery to an inlet port of the organ chamber. It is desirable that all the tubing used in the instant invention be made of an inert, sterilizable material, for example, silicon or Tygon ® tubing.
  • the organ is perfused, while the perfusion solution is maintained at a temperature in the range of 25°C to 37°C.
  • the system regulates the Pa0 2 PaC0 2 and pH of the perfusate with the gassing subsystem, and monitors the flow rate vascular pressure and osmolarity.
  • a perfusate of a known formulation such as that illustrated in Table 1, for example, can facilitate measurement of certain aspects of metabolic function.
  • the rate at which the glucose concentration is depleted, in relation to time, in the perfusate as it circulates through the organ is one measure of organ function. Relative to a selected time point, a glucose concentration that is below the range of a normal level of glucose utilization may be an indicator of organ damage or inhibited metabolism.
  • Another example of a measure of organ function is the amount of oxygen consumption as the perfusate circulates through the organ. Inadequate oxygen consumption, i.e.
  • a low oxygen consumption compared to the normal range may be indicative of tissue hypoxia.
  • a measure of organ function is the vascular flow rate of perfusate circulating through the organ using a standard perfusion pressure.
  • a low flow rate, compared to the normal range may be indicative of vasoconstriction, edema, vascular endothelial cell swelling and loss of vascular integrity in a damaged organ.
  • the system of the present invention also provides a second monitoring subsystem for evaluation of other indicators of an organ's functional integrity.
  • a conduit of the organ chamber for extracting organ product from the organ during the perfusion period makes it possible to monitor organ function by tracking organ output and evaluating physical and chemical parameters of the product of the perfused organ relative to ranges indicative of normal organ function.
  • One of skill in the art can readily determine, based on the organ to be preserved, what characteristics to monitor. For example, where the organ is a kidney, overall renal function, as well as individual aspects of kidney physiology, can be determined by measuring parameters including the rate of diuresis, urine osmolality, specific gravity, creatinine clearance and so on.
  • the nature of the monitoring subsystem will be dependent on the organ being perfused and may include means for measuring bile enzymes, heart enzymes, electrocardiographic activity, and so on.
  • bovine kidneys were also tested. Bovine kidneys were placed in EMS organ culture for three days using the same techniques described in Example 1. Similar to the results using canine kidneys, the bovine kidneys could be maintained in EMS organ culture, intact for three days without loss of ongoing metabolism or resulting function. Similar to the results obtained with canine kidneys, the bovine kidneys exhibited stable perfusion pressures, flow rates and lack of edema during the period of organ culture. Upon histologic evaluation, the bovine kidneys appeared normal, with excellent preservation of all components of the kidney architecture.
  • Viable cells could be isolated from the whole organ following 22 hours in EMS organ culture. Tissue from both the umbilical cord vein and the intact placenta were isolated by collagenase digestion. The cells isolated from the intact organ retained their ability to attach to the culture flask surface and the ability to replicate in standard tissue culture. The isolated cells continued to replicate in standard tissue culture and were eventually passaged three times. Therefore, the intact organ was successfully maintained in the EMS organ culture and was therefore metabolically active. The subsequent viability and function in cells isolated from the whole organ following a period of organ culture supports this interpretation.
  • the EMS organ culture technology of the present invention was used to maintain human umbilical cords measuring approximately 2 - 2.5 feet in length.
  • the human umbilical cords were maintained intact in EMS organ culture for at least seven days.
  • the umbilical cord vein was cannulated on one end. On the other end, the vein and the two arteries were connected with silicon tubing connected to a y-connector to form a closed circuit.
  • the luminal surface of the umbilical cord blood vessels were digested with collagenase.
  • the isolated endothelial cells were viable and attached to the flask surface in standard tissue culture. The isolated cells demonstrated normal characteristics of endothelial cells in tissue culture; i.e. they attached, replicated and expressed factor VIII antigen.
  • the EMS whole organ culture technology of the present invention can be used for in vivo applications.
  • a specific organ can be isolated via isolation of the major arterial source feeding the organ and simultaneous isolation and collection of the venous outflow.
  • An in vivo application of the organ culture technology entails physiologic maintenance of the enervation and lymphatic systems.
  • the artery feeding the targeted organ or section of anatomy is cannulated, the vein receiving the organ effluent is likewise cannulated, and blood is flushed from within the tissues.
  • the vascular compartment is refilled with the solution of the present invention.
  • the organ or section of anatomy is maintained in vivo but is "off-line" from the physiologic system and is maintained by the EMS organ culture technology.
  • Canine kidneys were isolated by cannulating the renal arteries and veins, flushing the kidneys of blood and refilling with the solution of the present invention.
  • the kidneys were maintained in situ at 37 °C with the EMS organ culture technology for 8 hours.
  • the kidneys continued to metabolize as determined by oxygen and glucose consumption calculations. Likewise, the metabolism was of sufficient levels to result in continuous diuresis.
  • the kidneys while isolated from the rest of the vascular system and maintained with EMS organ culture technology, continued to produce urine and fill the connected bladder.
  • the cannulas were removed and the kidneys reperfused with blood. The kidneys continued to function normally. All 10 canines demonstrated normal serum chemistries following EMS organ culture of the kidneys.
  • Isolated organ or regionalized tissue perfusion is well suited for high-dose chemotherapy delivery while attempting to reduce whole body toxicity.
  • Recent attempts at several cancer institutes to perform isolated organ perfusion to deliver chemotherapeutic agents have carried a high risk of causing damage to non-target tissues.
  • Target organs can be maintained with the organ culture technology of the present invention, without causing any damage.
  • the advantage of using the organ culture technology to deliver chemotherapy is the opportunity to deliver much higher doses while simultaneously reducing or even eliminating the usual systemic side- effects.
  • the efficacy of the EMS organ culture as a drug delivery technology was established with nontransplantable human kidneys.
  • the kidneys were nontransplantable because of renal tumors identified as renal cell carcinoma.
  • the human kidneys demonstrated stable perfusion pressures, vascular flow rates and oxidative metabolism during the EMS perfusion.
  • the human kidneys also consumed oxygen and glucose, produced urine and cleared creatinine.
  • Biopsies of the kidneys were taken both pre- and post-EMS whole organ culture perfusion for histologic evaluations. There were no observed histologic changes following 18 hours of EMS organ culture. Therefore, the EMS organ culture maintains kidneys with renal cell carcinoma without causing damage, thereby providing a delivery system for targeted drug therapies.
  • Ischemic insult secondary to occlusive disease such as heart attack and stroke represents another in vivo application of the EMS organ culture technology.
  • Recently developed thrombolytic agents hold great promise for patients suffering a stroke or heart attack.
  • the efficacy of these therapies is dependent upon being able to administer the drugs within a short period of time following the occlusive phenomenon.
  • the tissues downstream from the obstruction are deprived of blood resulting in ischemic damage.
  • the EMS organ culture technology can be used to reperfuse and maintain the tissue downstream from an obstruction, thereby providing an expanded window of opportunity to implement thrombolytic therapies.
  • EMS organ culture technology of the present invention can be implemented to sustain tissue integrity and function in vivo in the resuscitation from shock. Normovolemia can be restored with the acellular solution of the present invention at physiologic temperatures, thereby minimizing the development of a secondary reperfusion injury in patients experiencing a life-threatening hemorrhage.
  • the EMS organ culture technology maintains isolated organs or may be used to maintain the whole patient.
  • kidneys experiencing 60-120 min of warm ischemia were maintained in the EMS whole organ culture technology of the present invention.
  • mitotic figures were observed in the distal tubules of the thick ascending limb of the loop of Henle. There was no evidence of reparative procedure in the areas in the kidney without apparent damage, i.e., the proximal tubules and glomeruli.
  • PNF primary nonfunction
  • EMS technology can resuscitate and repair organs from an ischemic insult that today in clinical transplantation is considered to be irreparable damage.
  • the period of EMS perfusion directly correlates with the degree of repair, in that the longer a kidney is perfused the more repaired it becomes.
  • Eight hours of EMS perfusion repaired PNF kidneys sufficiently to become transplantable with peak serum creatinine values on the fourth posttransplant day and normalization on day 12 & 13.
  • Eighteen hours of EMS perfusion provided additional repair that resulted in a slight elevation in serum creatinine value, peaking on day 2 and was normal on the third posttransplant day; more than a week sooner than the kidneys perfused for 8 hours prior to reimplantation.
  • the liver perfusion system described in FIGURE 2 differs from the kidney and heart system because of the need to support both the arterial and venous vascular systems.
  • the system described in this invention is unique in delivering a high PaO 2 ,(100 - 240 mmHg) via the hepatic artery with a lower vascular flow rate ( ⁇ 250cc/min) and simultaneously providing a low Pa0 2 , ((140mmHg) and correspondingly high vascular flow rate (>240cc/min) via the portal system.
  • the net effect is to provide a more physiologic perfusion, such that the biliary tree is protected and bile production during perfusion is maximized.
  • the effluent reservoir 38 has a second effluent outlet 62 which allows perfusate to be drawn by means of a pump 64, into a second perfusion path 60 carrying perfusate to the portal vein 66 of the liver.
  • the EMS system controls the temperature, perfusion pressure, vascular flow rate, osmolarity, pH, Pa0 2 , PaC0 2 , nutrient delivery and the removal of waste by-products of the perfusion. This may be done by monitoring and controlling perfusate parameters in the two flow paths independently of each other or by regulation of the perfusion path supplying the hepatic artery only.
  • the flow path for the portal system may further comprises its own temperature controller, oxygenator and respiratory gas controller, pH controller, bubbletraps, and means for sensing flow rate, vascular resistance, OsM, Pa0 2 , and PaC0 2
  • the perfusion temperature of the portal vein flow path is maintained in the range of 25 -37°C.
  • One or more in-line oxygenators and oxygen carrier provides the oxygen tension described above for the hepatic artery and portal supplies.
  • One or more pH electrodes, controllers and solenoid systems control the entry of C0 2 to maintain pH of perfusate in both flow paths in the range of 7.30 - 7.45.
  • liver preservation is achieved by replenishing the depleted nutrients in the perfusate by perfusate exchange, dialysis, or a combination thereof, and by removing the accumulation of metabolic waste products by dialysis.
  • the synthetic properties unique to the liver provide regenerative opportunities by synthesizing protein and generating new cell growth.
  • livers were excised.
  • the suprahepatic vena cava was isolated at the diaphragm. Starting from the top left side, the livers were dissected cutting the lymphatic tissue, gastric artery and common bile duct. The portal vein and hepatic artery were isolated and cannulated with a 12 and 5 mm cannula, respectively.
  • the livers were perfused for approximately 4 hours at 34°C. During the period of perfusion, samples of the perfusate were collected at 2 and 4 hours of perfusion for measurements of bilirubin, ALT, AST, ALP and blood gases. The contents of the gallbladder were likewise collected at 2 and 4 hours of perfusion. There was no edema or discoloration demonstrating the ability of the EMS organ culture technology to repair livers.
  • Livers subjected to 30 minutes of postmortem WI all demonstrated high rates of oxygen consumption following resuscitation with EMS. When the WI insult was extended to 60 minutes, no significant difference in the rates of oxygen consumption were detected.
  • Livers subjected to 30 minutes of postmortem WI demonstrated mean arterial pressures (MAP) of 31 mmHg (combined hepatic artery and portal vein) with corresponding combined flow rates of 320 cc/min following re-establishment of metabolism. The vascular resistance was 0. 1.
  • the MAP was lowered to 20 mmHg and the flow rates increased to a mean of 420 cc/min.
  • the resulting vascular resistance was 0.06.
  • liver function screen was performed testing for ALT, AST, Alkaline phosphatase (ALP) and total/direct bilirubin. Following resuscitation with the EMS organ culture technology, livers injured by 30 minutes of postmortem WI, except in one liver at 2 hours, demonstrated normal values of ALT, ALP and bilirubin at both time points tested (Table IV). Likewise, when the WI insult was increased to 60 minutes postmortem, after 2 and 4 hours of EMS perfusion, the ALT, ALP and bilirubin values were normal.
  • the mean values were close to the normal bile concentration of bilirubin.
  • the concentration of ALP in the bile of the test livers were similar to that of normal bile. Similar to the results observed for the concentration of liver enzymes in the EMS perfusate described above, the bile AST levels decreased by half between 2 & 4 hours of perfusion in livers with 30 min WI, while the bile AST levels remained constant in the livers damaged by 60 minutes of WI. ALT was found in the bile from two of the livers following 30 minutes of WI insult at 2 hrs. of perfusion. The bile produced by livers subjected to 60 minutes of postmortem WI also contained ALT at both 2 & 4 hours of EMS perfusion. Likewise, the restored liver function was sufficient to support continuous bile production.
  • EMS perfusion prior to exposure to cold ischemia provides tissue protection and allows for extended periods of cold storage of an allograft.
  • an allograft is routinely stored clinically for only 24 hours by cold storage. Extending the period of cold storage leads to a high rate in delayed function, in many cases necessitating support of the patient by dialysis until the kidney can recover function.
  • cellular protection from the cold damage is provided. 0121.
  • the present invention therefore, also provides a method for storing a tissue, explant or organ intended for transplantation comprising the steps of flushing the tissue, explant or organ with a non-blood buffered physiological solution to remove blood and blood products; perfusing the tissue, explant or organ in a warm preservation system, such as the one described herein, capable of maintaining the tissue, explant or organ at a near normal rate of metabolism for a period of time sufficient to inhibit damage to the tissue, explant or organ; and storing the organ at 4-8°C.
  • a period of time sufficient to inhibit damage is one in which the isolated organ has had the opportunity to normalize perfusion parameters, for example, flow rate and vascular resistance. Generally, a perfusion period in the range of 30 mins to 24 hours is sufficient.
  • the organ chamber of the present invention also is particularly well suited for transporting the organ.
  • the organ can be prepared as described above for storage. Subsequently, the organ is transported by simply removing the organ chamber from the warm preservation system and refrigerating or cold-packing it for shipment. If desired, upon arrival at the transplantation site, the organ chamber containing the organ can be reestablished in a compatible warm preservation system so that the organ can be warm-perfused prior to being transplanted into the recipient. Additionally, the period of warm perfusion can be used to monitor the functional characteristics of the organ prior to transplantation.
  • perfusion of the organ can be continued during transport using a suitable power source.
  • the test group kidneys were placed on EMS perfusion upon excision, prior to cold storage.
  • the excised kidneys were flushed of blood with EMS perfusion solution and placed on EMS perfusion at 32°C - 34°C for 6 hours.
  • the kidneys were flushed again with approximately 200cc of ViaSpanTM at 4°C double bagged and stored statically packed in ice for 48 hours. 0125.
  • the contralateral kidneys were nephrectomized.
  • test and control kidneys were then autotransplanted using an end-to-side anastomosis made between the renal artery and the aorta, and the renal vein to the vena cava. Following transplantation the canine was closed and allowed to recover. Each morning the canine had blood drawn from the forelimb and chemistries were performed to determine the clinical course.
  • EMS perfusion prior to cold storage provided for better outcomes in that the time to recovery of normal function was halved following 48 hours of static cold storage.
  • the kidneys of the control group reperfused slowly and no urine was produced on the table. Very little urine was produced during the first evening posttransplantation.
  • Control group dogs experienced a period of reversible acute tubular necrosis (ATN).
  • ATN reversible acute tubular necrosis
  • the 24 hour posttransplant serum creatinine values were all elevated above normal values, 5.3, 2.6, & 2.8 mg/dL, respectively.
  • the serum creatinine values continued to rise, peaked and then slowly declined, normalizing on day 7, 9 &12 respectively.
  • kidneys produced urine on the table within minutes of reperfusion. The kidneys continued to produce urine throughout the posttransplant period.
  • the 24-hour posttransplant serum chemistries were elevated, with serum creatinine values of 2.2 & 2.5 mg/dL.
  • serum creatinine values normalized on day 5 and 6, respectively.
  • the kidneys appeared normal macroscopically. Histologic studies also revealed normal renal pathology.
  • EMS perfusion prior to reimplantation therefore, provides protection against cold ischemia induced damage, in that the warm perfused test kidneys experience ATN that is less severe and of shorter duration.
  • EMS perfused test group kidneys also had lower 24-hour posttransplant serum creatinine values when compared to the control group kidneys without EMS perfusion.
  • EMS perfused kidneys also had normalized serum creatinine values sooner (5.5 vs. 9.3 days) than the control group kidneys that were immediately placed into cold storage.
  • EMS perfusion can be used to deliver a drug to an organ, section of anatomy or tissue in a targeted fashion.
  • the EMS supported ongoing metabolism and function can be monitored providing a mechanism to quantify the impact of the drug and any corresponding cellular response to it.
  • a compound known to cause the up-regulation of the heat-shock protein, hemeoxygenase- 1 was administered to an isolated kidney being perfused in accordance with the technology of the present invention.
  • the EMS technology provided adequate support of renal metabolism and function during ex vivo perfusion to increase expression of the hemeoxygenase-1 (HO-1) enzyme within six hours.
  • Metabolic and functional evaluations consisted of quantification of the ex vivo kidney function during the period of HO-1 induction. Using the EMS technology, kidneys were maintained in a metabolically active state by perfusion at near physiologic temperature. Kidneys weighing approximately 70 - 90 gm were procured following brain death with concomitant warm ischemia of no more than 15 minutes.
  • the kidneys were then transitioned to EMS perfusion at 32°C to 34°C.
  • the kidneys were evaluated for oxidative metabolism, vascular dynamics and organ function.
  • the organ parameters were allowed to stabilize for one hour prior to the evaluation of function.
  • Samples from the arterial and venous lines were collected, along with the urine produced during the perfusions.
  • PO2 measurements for the arterial and venous samples were made with a Radiometer ABL5.
  • Creatinine was added to the perfusate as a tracer.
  • the urine made during the EMS perfusions at 32°C to 34°C was collected and tested.
  • the concentration of creatinine was measured on an IDEXX Vet Lab chemistry analyzer and the glomerular filtration rate (GFR) was calculated as:
  • GFR (urinary creatinine) X (urine flow) . (perfusate creatinine) X (time )
  • EMS perfusion effectively supported ongoing metabolism and function sufficiently for a cellular response to the CoPP leading to an increased expression of hemeoxygenase-1. Furthermore, during EMS perfusion the administration of CoPP did not adversely affect perfusion characteristics or cellular metabolism. However, organ function was found to be affected, in a dose dependant fashion as the concentration of the CoPP was increased, in terms of compromised urine flow, glomerular filtration rate and development of proteinuria. Therefore, any drug- induced toxicity can be detected during administration via EMS perfusion. At therapeutic doses, EMS perfusion was effective in supporting CoPP induction of HO-1 expression.
  • a related use of EMS technology is as a targeted gene delivery system.
  • the long-term benefit of gene therapies is hampered by the problem of delivering the desired gene to a specific location.
  • Organ culture technology provides a mechanism for delivering genes to the desired location.
  • Gene therapy is particularly appealing in the context of transplanted organs because an exogenous gene can be transferred prior to transplantation.
  • the pharmaceutical agent is a gene therapy vector
  • the construct may functionally encode for endogenous or exogenous proteins, which can then be expressed in the target after transplantation. Such gene transfer will allow for the expression of various proteins by the target tissues.
  • a perfusion system is particularly applicable for gene transfer and pharmaceutical administration into a number or organs, as long as the target organ has a suitable blood circulation system, for example, kidney, liver, heart and so on.
  • the most obvious benefit of gene delivery via a perfusion system is the enhanced efficiency, target specificity for gene transfer, and the possibility of using only a small amount of vector material.
  • extracorporeal perfusion systems diminish the risk of administering a large amount of foreign genetic material into the general circulation of the subject, especially important for immunocompetent individuals.
  • the EMS technology of the present invention can be used to achieve targeted gene transfer, either ex vivo or in situ.
  • the EMS maintains the organ or tissue to be transfected in a metabolically active state before, during and following the transfection period.
  • the ability to interface directly with targeted tissue over extended periods of time under near physiologic conditions without exposing non-target tissues presents the opportunity to use alternative transfection procedures; including eliminating the need for viral vectors.
  • liposome complexes or viral vectors and vascular endothelial cell-specific promoters can be introduced in the perfusion system to transfer the therapeutic DNA at a near physiologic temperature to facilitate higher incorporation rates.
  • An additional benefit of preserving metabolism during EMS is the feasibility of establishing the functional status of the organ or tissue prior to transfection, in contrast to using hypothermia, which inhibits metabolism and alters cell membrane potential. This is of particular importance where the organ was obtained from a donor other than a beating heart donor and may have experienced some ischemic injury. Furthermore, it is feasible to monitor the organ's metabolism during transfection, and in the case of an intact organ, to quantify the resulting organ function, i.e. diuresis, etc. Because the cell membranes are in their normal fluid state, normal barrier function is maintained providing the ability to target a gene(s) therapeutic directly to the vascular cells.
  • the ability of the EMS perfusion to preserve the integrity and normal barrier functions of the vessel wall within kidneys provides a mechanism for effecting transfection restricted to the vascular cells.
  • the ability to target a gene(s) therapeutic to the vascular cells within an organ provides several important benefits.
  • the reporter gene product is in direct contact with the bloodstream, thereby providing an efficient gene therapy delivery system. Since the vascular endothelium represents the immunologic interface in organs, gene therapies present opportunities for organ specific immunomodulating therapies, in both transplantation and autoimmune diseases.
  • the EMS is acellular and provides the milieu for optimal transfection of a gene(s) therapeutic to the nucleus without eliciting an early immunological response. Therefore, the EMS represents a high efficiency delivery vehicle for targeted gene delivery for in situ or ex vivo application and allows for the simultaneous monitoring of metabolism and function.
  • a gene of interest may be delivered to the targeted tissue via a host of currently known vehicles.
  • Gene therapy by the homogenous recombination between exogenous DNA and the genome of the target tissue results in the modification of a particular locus within the genome.
  • Some methods of transfection include delivery by viral vector containing the gene, liposomes, plasmid DNA and the like.
  • delivery of episomal DNA by the method of the invention is feasible and circumvents the need for genomic integration of the delivered gene due to episomal replication of the targeted gene vector independent of the genome. 0143.
  • an organ containing the target tissue is isolated from a living body, is flushed of blood with warm EMS solution and then placed on EMS perfusion. The perfusion is conducted for a period of time sufficient to establish baselines of function.
  • An exogenous molecule for example, a viral vector, is infused into the system and perfusion continued for a period of time sufficient to deliver a pharmaceutically effective amount of the exogenous molecule.
  • Organ function is monitored for a period of time after delivery, prior to returning the organ to a recipient.
  • a replication defective adenovirus (Adeno CMV5-GFP) encoding a green fluorescence protein (GFP) gene was used to evaluate the effectiveness of the EMS perfusion system to support gene transfer to intact bovine kidneys during ex vivo perfusion at 34°C. Bovine kidneys were flushed of blood with warm EMS solution and then placed on EMS perfusion. The perfusion was conducted for approximately 60 minutes to establish baselines of function. The viral vector (1 x 10 9 PFU) was infused into the system and perfusion was continued for 2 hours. Administration of the viral vector and the resulting infectivity during EMS perfusion did not adversely affect organ metabolism or function.
  • Control kidneys consisted of kidneys flushed and perfused at 4°C using a ViaSpan-based cold perfusate. Since metabolism and function are inhibited at 4°C, any effect of the viral vector on the metabolism or function of the control kidneys could not be determined.
  • the kidneys were copiously flushed via the renal artery to remove any residual circulating viral particles. The lumen of the vasculature was then filled with a collagenase solution to isolate the vascular cells within each kidney and the identity of these cells was confirmed morphologically. Once the digested and isolated vascular endothelial cells were flushed from the kidneys, they were washed and placed into primary cultures at a concentration of 1 xlO 6 per ml. The expression of the transfected gene encoding the green fluorescence protein is described in TABLE 7.
  • the EMS perfusion provided for enhanced infection rates, with 40% of the vascular cells in culture expressing the GFP by 18 hours versus 17% in the cold perfused control kidneys.
  • the endothelial cells isolated from the EMS perfused kidneys demonstrated strong expression of the GFP in 60% of the cells. In contrast, no increased level of expression was detected in the control kidneys were the transfection was conducted in the cold.
  • Parenchymal cells were isolated from the EMS perfused kidneys, following the isolation of the vascular endothelial cells by collagenase digestion. The isolated parenchymal cells were found to be negative for the expression of GFP at 24 and 48 hours in tissue culture. These results suggest that the infectivity and transfer of the GFP encoding gene during EMS perfusion were limited to the vascular cells within the blood vessels of the kidneys. Therefore, better transfection rates and higher reporter gene expression was observed at both time points in the test kidneys perfused with the EMS and evidence of transfection was only found in the vascular cells.
  • the EMS warm preservation technology of the present invention preserves organs without inflicting damage and supports metabolism at a level sufficient to result in immediate normal function upon organ reimplantation (following ex vivo organ perfusion), or reperfusion (following in situ perfusion). For these reasons, EMS perfusion is particularly well suited for delivery of a therapeutic agent including, for example, in gene therapy, where delivery of a nucleotide is desired. Furthermore, the EMS warm preservation technology of the present invention provides a gene delivery/transfection system that is superior to current methodologies.
  • EMS perfusion The improvement of EMS perfusion over that of previously reported work is that the organ is maintained at near physiologic temperature with supported organ metabolism that imparts no damage and will result in immediate normal function when reimplanted following ex vivo perfusion or reperfused following in situ perfusion. Furthermore, EMS perfusion can be performed for several days, allowing for not only efficient gene transfection while imparting no damage, but EMS can also support the organ long enough for the full expression of the gene product. The ability to support organs at near physiologic metabolism for days represents a unique ability to synthesis the protein product encoded by the transfected gene prior to reimplantation or reperfusion, something not possible with existing technology.
  • the following examples demonstrates the superior ability of the EMS technology to preserve organ integrity during gene delivery and transfer.
  • Paired kidneys were excised from calves. The excised kidneys were flushed of blood, and one of the pair was placed on EMS perfusion and the other was placed on perfusion using the system and solution described by Tryggvason. During the perfusion, the oxidative metabolism was evaluated by calculating the oxygen consumption in terms of 0 2 consumed per minute per gram, diuresis and corresponding organ function. Organ function was determined as the ability of the glomeruli to retain its normal barrier function and retain large molecules in the vascular compartment.
  • organ function was the ability to produce urine without protein leak of the colloid, serum albumin, and since the Tryggvason patent does not include a colloid, the ability to retain the red blood cells in the vascular compartment, i.e. urine negative for red blood cells.
  • EMS technology can be used to preserve organs ex vivo for extended periods in a metabolically active state and at near physiologic temperature.
  • 10 canine kidneys were preserved ex vivo for 24, 48 & 72 hours.
  • perfusion pressures, vascular flow rate, vascular resistance, oxygen consumption, glucose consumption and diuresis remained stable.
  • the kidneys appeared to be normal upon histologic evaluations.
  • the present invention provides a useful method for storing organs and preparing organs for transport to the transplantation site.
  • a method for storing a tissue, explant or organ intended for transplantation is described above.
  • the organ chamber and warm preservation system of the present invention are also advantageous for preparing organs for transport to the transplantation site. Basically, the organ is established in the organ chamber and perfused in the warm preservation system to repair damage to the organ and to impart protection from further damage which may result from cold storage or shipping.
  • the method comprises the steps of (a) flushing the tissue, explant or organ with a non-blood buffered physiological solution to remove blood and blood products; (b) perfusing the tissue, explant or organ in an organ chamber having a support member capable of supporting the organ, tissue or explant and inhibiting the movement of the organ, tissue or explant within the organ chamber during transport wherein the organ chamber is part of a warm preservation system capable of maintaining the tissue, explant or organ at a near normal rate of metabolism for a period of time sufficient to impart protection to the tissue, explant or organ; (c) perfusing the tissue, explant or organ with a cold perfusion solution, for example, having a temperature in the range of 4-8°C; (d) removing the entire organ chamber containing the organ from the warm preservation system; cold-packing the organ chamber containing the organ and (e) transporting the organ chamber containing the organ to the transplantation site.

Abstract

L'invention concerne un système de support métabolique exsanguin permettant de maintenir un organe (40) ou un tissu à un rythme métabolique sensiblement normal. Ce système comprend un compartiment pour organe (32) renfermant un récipient (33) et un élément de support (36) conçu pour empêcher tout mouvement de l'organe (40) dans le récipient (33) lors d'une perfusion et/ou d'un transport. Le compartiment pour organe (32) comprend également un conduit (43) destiné à recevoir le débit veineux sortant d'une solution de perfusion et à empêcher tout contact de cette dernière avec les surfaces externes de l'organe (40). Un conduit (47) destiné à recevoir le produit organique permet de récupérer le produit organique d'un organe fonctionnel (40) lors d'une perfusion. L'invention concerne également l'utilisation de ce compartiment pour organe (32) pour une synthèse de novo ou continue des constituants nécessaires à un entretien à long terme des organes, pour la transplantation, la réanimation et la réparation active d'organes ayant subi des lésions ischémiques chaudes durables, et pour le transport d'organes isolés.
PCT/US2001/040679 2001-05-04 2001-05-04 Compartiment pour organe destine a un systeme de support metabolique exsanguin WO2002089571A1 (fr)

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JP2002586724A JP2004529938A (ja) 2001-05-04 2001-05-04 血液を含まない代謝支持システム用の器官室
EP01933402A EP1392113A1 (fr) 2001-05-04 2001-05-04 Compartiment pour organe destine a un systeme de support metabolique exsanguin
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