DE102012112709B4 - System for fluid perfusion or persufflation of biological material comprising tissue - Google Patents

Abstract

A system (1000) for fluid perfusion or persufflation of biological material comprising tissue, the system (1000) comprising: (a) a persufflation system (1100) for use in persufflation of biological material, the persufflation system (1100) having a gas generator for generating a preservation gas;(b) a liquid perfusion system (1600) for use in perfusing biological material, the liquid perfusion system (1600) comprising a reservoir for liquid perfusate;(c) a delivery cannula, the delivery cannula having a first end and a second end, the first end being insertable into an inlet of the biological material; andd) a switching system (1700) which is coupled to the second end of the delivery cannula and to the persufflation system (1100) and to the liquid perfusion system (1600), the switching system (1700) being configured in such a way that the biological material comprises tissue , undergo fluid perfusion or persufflation.

Description

BACKGROUND OF THE INVENTION

The present invention relates generally to the preservation of biological material, including tissue, for transplantation or other purposes, and more particularly relates to the preservation of biological material, including tissue, using fluid perfusion.

Islet of Langerhans transplantation has been studied for over 30 years for its potential as a treatment for type 1 diabetes mellitus because rat islet allotransplantation has been shown to reverse chemically induced diabetes (see Ballinger et al., " Transplantation of intact pancreatic islets in rats," Surgery, 72(2):175-86 (1972), incorporated herein by reference). Several years later, it was shown that islet autotransplantation back to a donor was a possible treatment to prevent the development of diabetes in people with pancreatitis (see Najarian et al., "Total or near total pancreatectomy and islet autotransplantation for treatment of chronic pancreatitis," Ann Surg., 192(4): 526-42 (1980), incorporated herein by reference). However, successful islet allotransplantation remained difficult to achieve until 2000, when development of the Edmonton protocol was successful in seven consecutive patents transplanting islets from multiple pancreas (see Shapiro et al., "Islet transplantation in seven patients with type I diabetes mellitus using a glucocorticoid-free immunosuppressive regimen', New England Journal of Medicine, 343(4):230-8 (2000), incorporated herein by reference). The above success has been largely attributed to the use of glucocorticoid-free immunosuppression. This success has then been replicated at other institutions, including the University of Minnesota, with over 500 transplants worldwide from 2000-2006 using variations on the Edmonton protocol (see Shapiro et al., "Edmonton's islet success has indeed been replicated elsewhere, " The Lancet, 362(9391), 1242 (2003); Emamaullee et al., "Factors influencing the loss of beta-cell mass in islet transplantation," Cell Transplantation, 16(1): 1-8 (2007), the both are incorporated herein by reference). Islet transplantation offers several advantages over other currently used treatments for diabetes. Because of the islets' inherent ability to monitor glucose levels and regulate insulin production, transplantation allows for constant, tight control of blood glucose levels that cannot be achieved through patient self-monitoring. Although tight glucose control is not fully achieved, islet transplantation is particularly important in patients who present with hypoglycemia unawareness, the lack of physical symptoms that indicate low blood sugar. In addition, compared to other graft treatments, transplantation minimizes the possibility of infection because infusion of the islets into the portal vein does not require open surgery.

Although the use of islet transplantation for sustained diabetes reversal has been demonstrated, several obstacles remain to the widespread implementation of this therapy in the clinical setting. Although there has been some success with single-donor transplants, most centers continue to need multiple donor organs for a single patient for a variety of reasons, including poor islet yield and/or quality per donor (see Hering et al., "Single-donor, marginal-dose islet transplantation in patients with type I diabetes,” JAMA, 293(7):830-5 (2005), incorporated herein by reference). There is currently a shortage of quality donor organs, which limits the number of possible transplants. With increasing interest in islet transplantation and with new centers competing for U.S. Food and Drug Administration (FDA) for Investigational New Drug (IND) applications, it is more important than ever to maximize both the number and quality of organs available. In order to do this, it is necessary to protect the islets from damage from the time of organ procurement and islet preparation through later implantation in the transplant recipient.

One of the major challenges of pancreatic islet transplantation is to obtain sufficient viable, functional graft tissue for successful treatment (see Iwanaga et al., Pancreas preservation for pancreas and islet transplantation, invited review, Current Opinion in Organ Transplantation, 13(2):135-141 (2008), incorporated herein by reference). As such, it is important to: (1) maximize donor organs that are acceptable for clinical use; (2) improve the preservation, storage and transportation of these organs so that they remain acceptable; and (3) improve the yield and quality of islets harvested from the organ by improving isolation, culture and storage. There appears to be a critical mass of living islets for the success of single donor transplants, and current protocols yield graft tissue that is generally on the right border of this critical mass.

The current protocol for pancreatic harvesting includes brain dead, heart beating donors with virtually no warm ischemia time (WI). Pancreas preservation (including transportation and storage time) must be eight hours or less long (4-8°C) cold storage. Cold storage protocols vary, with some using established cold conservation solutions (CPS) such as UW (University of Wisconsin) solution, and others using experimental CPS or combinations of CPS. Since 2002, the two-layer method (TLM) has received a lot of attention. TLM was developed as a method for pancreas preservation in the late 1980s and early 1990s (see Kuroda et al., "A new simple method for cold-storage of the pancreas using perfluorochemical," Transplantation, 46(3):457-60 (1988) Fujino et al., "Preservation of canine pancreas for 96 hours by a modified two-layer (UW solution/perfluorochemical) cold storage method," Transplantation, 51(5):1133-5 (1991);Kuroda et al., "Oxygenation of the human pancreas during preservation by a two-layer (University of Wisconsin solution/perfluorochemical) cold-storage method," Transplantation, 54(3):561-2 (1992), all incorporated herein by reference will).

TLM involves suspending a pancreas between layers of CPS and oxygenated perfluorocarbon (PFC). The basic concept is to increase tissue oxygenation during storage by delivering greater amounts of oxygen to the organ surface due to the increased oxygen transport capacity of PFC compared to CPS. TLM as the state of the art for pancreas preservation received a major boost in the early 2000's after the development of the Edmonton protocol with many islet processing centers publicizing the advantages of this approach to organ preservation compared to classical methods ( see Hering et al., "Impact of two-layer pancreas preservation on islet isolation and transplantation", Transplantation, 74(12):1813-6 (2002), Fraker et al., "Use of oxygenated perfluorocarbon toward making every pancreas count ," Transplantation, 74(12): 1811-2 (2002); Tsujimura et al., "Human islet transplantation from pancreases with prolonged cold ischemia using additional preservation by the two-layer (UW solution/perfluorochemical) cold-storage method, Transplantation, 74(12):1687-91 (2002); Lakey et al., "Preservation of the human pancreas before islet isolation using a two-layer (UW solutionperfluorochemical) cold storage method," Transplantation, 74(12): 1809-11 (2002); Ricordi et al., "Improved human islet isolation outcome from marginal donors following addition of oxygenated perfluorocarbon to the cold-storage solution," Transplantation, 75(9):1524-7 (2003); Matsumoto et al., "The effect of two-layer (University of Wisconsin solution/perfluorochemical) preservation method on clinical grade pancreata prior to islet isolation and transplantation," Transplantation Proceedings, 36(4):1037-9 2004; Witkowski et al., "Two-layer method in short-term pancreas preservation for successful islet isolation," Transplantation Proceedings. 37(8), 3398-401 (2005), all of which are incorporated herein by reference).

Much of the interest in TLM has been due to the potential to use scarce donor organs for successful transplants. There is currently a shortage of suitable donor organs and in some countries the use of heartbeat donors is prohibited due to cultural taboos. However, it has recently been shown that oxygenation, which depends on surface diffusion, as is the case in TLM, may be insufficient to oxygenate the main part of the human pancreas. Modeling of pancreatic diffusion by a group at the University of Minnesota has shown that oxygen can penetrate only the outer 1 mm of the pancreas (see Papas et al., "Pancreas oxygenation is limited during preservation with the two-layer method," Transplantation Proceedings, 37(8), 3501-4 (2005), incorporated herein by reference). In addition, several islet transplantation centers have very recently released retrospective data showing that there is no significant improvement in the outcome of islet isolation or transplantation when the pancreas is preserved by TLM compared to classical storage in CPS (UW) alone (see Kin et al., "Islet isolation and transplantation outcomes of pancreas preserved with University of Wisconsin solution versus two-layer method using preoxygenated perfluorocarbon," Transplantation, 82(10):1286-90 (2006); Caballero-Corbalan et al., "No beneficial effect of two-layer storage compared with UW-storage on human islet isolation and transplantation," Transplantation, 84(7):864-9 (2007), both of which are incorporated herein by reference).

In view of the above, there clearly remains a compelling need for improved methods of pancreas preservation.

For other human organs, passive cold storage in CPS is common and reported in the peer-reviewed literature. There are also some published protocols and commercial equipment for liquid perfusion preservation of organs. In both of these procedures, the supply of oxygen is minimal and may be unsuitable for optimal organ preservation.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a system for fluid perfusion of biological material comprising tissue.

According to one aspect of the invention there is provided a system for fluid perfusion or persufflation of biological material comprising tissue, the system comprising (a) a persufflation system for use in persufflation of biological material, the persufflation system comprising a gas generator for generating a preservative gas; (b) a liquid perfusion system for use in perfusing biological material, the liquid perfusion system comprising a reservoir for liquid perfusate; (c) a delivery cannula, the delivery cannula having a first end and a second end, the first end being insertable into an inlet of the biological material comprising tissue; and (d) a switching system coupled to the second end of the delivery cannula and to the persufflation system and to the liquid perfusion system, the switching system being configured to fluidly perfuse or persufflate the biological material, which includes tissue.

According to a feature of the above system, the preservation gas may comprise gaseous oxygen.

According to another characteristic of the above system, the gas generator may include electrochemical means for generating gaseous oxygen.

According to a further feature of the above system, the electrochemical means for generating gaseous oxygen may comprise an electrochemical oxygen concentrator.

According to another feature of the above system, the preservative gas may further comprise water vapour.

According to another feature of the above system, the gas generator may include means for adjusting the concentration of gaseous oxygen in the conservation gas.

According to another feature of the above system, the preservative gas may consist of gaseous oxygen.

According to a further feature of the above system, the gas generator may comprise at least one outlet for delivering a gas flow comprising the preservative gas.

According to a further feature of the above system, the gas generator may include a plurality of outlets for discharging a corresponding plurality of gas streams comprising the preservative gas.

According to a further feature of the above system, the gas streams can have independently adjustable flow rates.

According to a further feature of the above system, the system may further comprise a container, the container being suitably sized to receive biological material comprising tissue.

According to another feature of the above system, the container can be a thermally insulated container.

According to a further feature of the above system, the system may further comprise temperature control means for maintaining the contents of said container at a desired temperature.

According to a further feature of the above system, the temperature control means may include a temperature sensor sensing the temperature within said container, a temperature control device for changing the temperature within said container, and a process controller ler responsive to said temperature sensor for controlling the operation of said temperature control device.

According to a further feature of the above system, the liquid perfusion system may comprise a fluid drain line for draining the liquid perfusate perfused through the biological material comprising tissue.

According to a further feature of the above system, the liquid can be selected from the group consisting of a liquid cell culture medium, an organ preservation solution, a saline solution, a HEPES buffer and combinations thereof.

According to a further feature of the above system, the liquid may further comprise an additive selected from the group consisting of antioxidants, anti-apoptotic agents, vasodilators/vasoconstrictors, oxygen carriers, chelators, toxin binders and anticoagulants.

According to a further feature of the above system, the system can further include at least one gas sensor for monitoring a gas concentration in at least one fluid exiting the gas generator or the reservoir, or one fluid entering or exiting the container.

For the purposes of the present description and claims, the term "fluid" shall mean a pure substance in liquid state, a pure substance in gaseous state and a mixture of liquid substances and/or gaseous substances such as, but not limited to, a mixture of two or include, but not be limited to, more liquids, a mixture of two or more gases, and a mixture of at least one liquid and at least one gas.

Also for the purposes of the present specification and claims, the term "biological material comprising tissue" is intended to include naturally occurring biological material comprising tissue, engineered biological material comprising tissue, and mixtures thereof. Additionally, the term "biological material comprising tissue" is intended to include biological material comprising animal tissue, including biological material comprising human tissue. Furthermore, the term "biological material comprising tissue" is intended to include biological material consisting of or comprising one or more biological tissues such as, but not limited to, a single biological tissue, an organ, a sub-organ, multiple organs, multiple organs within an organ system, a complete organ system, multiple sub-organ systems (commonly referred to as complex tissue), and multiple complete organ systems up to and including a complete organism.

Additionally, for the purposes of the present specification and claims, the term "fluid perfusion" is intended to refer to the delivery of one or more liquids to one or more natural and/or artificial fluid distribution networks within biological material, which includes tissue. Examples of natural fluid distribution networks include, but are not limited to, at least a portion of a vascular system within biological material that includes tissue, at least a portion of a ductal system within biological material that includes tissue, and at least a portion of a lymphatic system within of biological material, which includes tissue. Examples of artificial fluid distribution networks include, but are not limited to, man-made or machine-made fluid channels created or provided in biological material, including tissue. When "fluid perfusion" is used to refer to the administration of a pure gas or the administration of a mixture of gases to biological material comprising tissue, it may alternatively be referred to herein as "persufflation".

character list

• ist 1 ein Blockdiagramm eines Ausführungsbeispiels zur Fluidperfusion von biologischem Material, das Gewebe umfasst, wobei das System entsprechend der Lehren der vorliegenden Erfindung konstruiert wurde;
• ist 2 ein schematisches Diagramm eines Ausführungsbeispiels des in 1 gezeigten Persufflationssystems, wobei das Persufflationssystem zusammen mit der Energieversorgung gezeigt ist;
• ist 3 eine perspektivische Explosionsteilansicht eines Ausführungsbeispiels des elektrochemischen Sauerstoffkonzentrators, der in 2 gezeigt ist;
• ist 4 eine perspektivische Ansicht eines Ausführungsbeispiels eines Systems zur Fluidperfusion von biologischem Material, das Gewebe umfasst;
• ist 5 ein Blockdiagramm eines Ausführungsbeispiels eines Systems zur Fluidperfusion von biologischem Material, das Gewebe umfasst, wobei das System entsprechend der Lehren der vorliegenden Erfindung konstruiert wurde;
• ist 6 ein vereinfachtes schematisches Diagramm eines weiteren Ausführungsbeispiels zur Fluidperfusion von biologischem Material, das Gewebe umfasst, wobei das System entsprechend der Lehren der vorliegenden Erfindung konstruiert wurde;
• sind die 7(a) bis 7(c) Bilder von Dithizon-gefärbten Inseln, isoliert aus Bauchspeicheldrüsen, wie in Beispiel 1 diskutiert, welche (a) keiner Konservierung, (b) 6 Stunden lang Persufflationskonservierung und (c) 6 Stunden lang TLM-Konservierung ausgesetzt wurden;
• ist 8 ist eine Tabelle, in welcher Konservierungsverfahren, basierend auf Insel-Ausbeute und -Qualität verglichen werden;
• sind die 9(a) bis 9(c) Mikroaufnahmen von Sektionen von Schweine-Bauchspeicheldrüsen (a) nach 24 Stunden langer Konservierung mit dem TLM, (b) nach 24 Stunden mit Persufflation; beziehungsweise (c) zur Zeit = 0 (sofort nach Entnahme);
• ist 10 eine Tabelle, in welcher Konservierungsverfahren basierend auf dem Nacktmaus-Bioassay verglichen werden;
• ist 11 eine Tabelle, in welcher Resultate von Insel-Isolierung zwischen 24 Stunden langer Konservierung mittels Persufflation, 24 Stunden langer Konservierung mittels TLM und ohne Konservierung (Tag 2 ist definiert als 48-72 Stunden nach der Isolierung) verglichen werden. Die ersten drei Zeilen sind Mittelwerte der folgenden drei Datensätze); und
• ist 12 eine graphische Darstellung, welche TLM-Resultate als Prozentsätze von Persufflations-Resultaten für die Präparationen zeigt, die in Beispiel 2 diskutiert werden (die Fehlerbalken zeigen den Standardfehler des Mittelwertes).

The accompanying drawings, which are hereby incorporated into and constitute a part of this specification, illustrate various embodiments of the invention and, together with this description, serve to explain the principles of the invention. In the drawings, where like reference numbers represent like parts:

• is 1 Figure 12 is a block diagram of an embodiment for fluid perfusion of biological material, including tissue, constructed in accordance with the teachings of the present invention;
• is 2 a schematic diagram of an embodiment of the in 1 persufflation system shown, the persufflation system being shown together with the power supply;
• is 3 12 is a partial exploded perspective view of an embodiment of the electrochemical oxygen concentrator disclosed in FIG 2 is shown;
• is 4 Figure 12 is a perspective view of one embodiment of a system for fluid perfusion of biological material that includes tissue;
• is 5 Figure 12 is a block diagram of one embodiment of a system for fluid perfusion of biological material that includes tissue, the system being constructed in accordance with the teachings of the present invention;
• is 6 Figure 12 is a simplified schematic diagram of another embodiment for fluid perfusion of biological material, including tissue, constructed in accordance with the teachings of the present invention;
• are the 7(a) until 7(c) Images of dithizone-stained islets isolated from pancreases as discussed in Example 1 that were exposed to (a) no preservation, (b) persufflation preservation for 6 hours, and (c) TLM preservation for 6 hours;
• is 8th Figure 12 is a table comparing preservation methods based on islet yield and quality;
• are the 9(a) until 9(c) Photomicrographs of sections of porcine pancreas (a) after 24 hours of preservation with the TLM, (b) after 24 hours of persufflation; respectively (c) at time = 0 (immediately after extraction);
• is 10 a table comparing preservation methods based on the nude mouse bioassay;
• is 11 a table comparing results of islet isolation between 24 hour preservation by persufflation, 24 hour preservation by TLM and no preservation (day 2 is defined as 48-72 hours after isolation). The first three rows are mean values of the following three data sets); and
• is 12 Figure 12 is a graph showing TLM results as percentages of persufflation results for the preparations discussed in Example 2 (the error bars indicate the standard error of the mean).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates generally to the discovery that biological material, which includes tissue, can be preserved by perfusing the tissue with a preservative fluid. In particular, the present invention is directed to a system designed for fluid perfusion of biological material, including tissue.

More specifically, according to one aspect of the invention, a system for fluid perfusion of biological material comprising tissue is provided, the system comprising (a) means for generating a preservative gas in situ and (b) means for perfusing the tissue with the in includes preservative gas generated in situ. The means for generating the preservation gas may include electrochemical and/or other means. For example, the means for generating the preservation gas may comprise an electrochemical oxygen concentrator and/or a water electrolyzer. Alternatively, the means for generating the preservation gas can additionally or alternatively comprise means for generating such a gas by means of pressure swing adsorption and/or by means of chemical oxygen release (e.g. by burning perchlorate or by means of an oxygen candle). The means for perfusing the tissue with the preservative gas generated in situ may comprise, for example, a cannula or other fluid-conducting means fluidly coupled to the gas-generating means and insertable into the tissue.

The preservative gas used to perfuse the tissue can be dissolved in a liquid and then administered to the tissue as a gas/liquid solution, or it can be dissolved in ambient air or in one or more other gases and then the tissue be administered as a gas/gas mixture. Perfusion with a gas or with a gas/gas mixture may alternatively be referred to as "persufflation". Persufflation can be applied to tissues that have networks through which gas can be distributed. The most common network for distributing gas during persufflation is the vascular system of arteries, veins, and capillaries. Another network is the ductal systems, including but not limited to the extensive ductal system of the pancreas and mammary glands. The lymphatic system has a conducting system of tubular vessels throughout the body, which can also be a network for persufflation. In addition, networks can be created by perforating or "needling" the tissue (see Treckmann et al., "Retrograde oxygen persufflation preservation of human livers: a pilot study," Liver Transpl., 14(3):358-64 (March 2008 ), incorporated herein by reference). In the case of artificial tissue constructs, there could be natural or artificial networks, such as a gas permeable tube, serving as an artificial vascular or ductal system or a replacement thereof. The gas distribution network could include any combination of the above network types. Distribution through these networks can be in the direction that occurs physiologically (eg, anterograde in the vasculature) or can be accomplished in other ways (eg, retrograde in the vasculature).

Biological material, which includes tissue that has networks suitable for persufflation, includes a tissue, an organ, a sub-organ, multiple organs, multiple organs within an organ system, a complete organ system, multiple sub-organ systems (commonly referred to as complex tissue), and multiple complete organ systems up to and including a complete organism. Examples of organs include, but are not limited to, pancreas, liver, kidney, heart, lung, colon and small intestine, eye, gallbladder, stomach, skin, and male and female reproductive organs. An example of a sub-organ could include, but is not limited to, a lobe of the pancreas. Examples of multiple organs include, but are not limited to, two or more of the above organs, such as two kidneys, one kidney and one pancreas, and two lungs. Examples of multiple organs within an organ system might include, but are not limited to, the kidney and bladder, which are part of the excretory organ system, which in its entirety consists of the kidneys, ureters, bladder, and urethra. Examples of multiple sub-organ systems include, but are not limited to, finger, hand, foot, extremity, ear, nose, face, skin graft, genitalia, abdominal wall, and other complex tissues.

Examples of the preservative gas may include, but are not limited to, gaseous oxygen (which is a nutrient that cells need), gaseous hydrogen (which may act to protect cells via its antioxidant and antiapoptotic properties (see Wood et al. , "The hydrogen highway to reperfusion therapy," Nature Medicine, 13(6):673-4 (2007);Ohsawa et al., "Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals," Nature Medicine, 13( 6):688-94 (2007)), gaseous carbon dioxide (which may regulate metabolism), gaseous carbon monoxide (which may have anti-inflammatory and anti-apoptotic effects (see Wang et al., "Donor Treatment with carbon monoxide can yield islet allograft survival and tolerance," Diabetes, 54(5):1400-6 (2005)), and water vapor. The preservative gas may be generated in situ by electrochemical or other means. If, for example, there s preservation gas is oxygen, the preservation gas can be generated in situ using an electrochemical oxygen concentrator. Alternatively, when the preservation gas is oxygen and/or hydrogen, the preservation gas can be generated in situ using an electrolyzer. Alternatively, when the preservation gas is oxygen, the preservation gas can be generated in situ by pressure swing adsorption or by oxygen release (e.g., by burning perchlorate or by an oxygen candle).

The additional application of cell culture media or organ preservation solution rinses or baths to the tissue or organ surface or to ductal or vascular systems therein may also be included to enhance the quality of the biological material during preservation by persufflation (see Saad et al., "Extension of ischemic tolerance of porcine livers by cold preservation including postconditioning with gaseous oxygen," Transplantation, 71(4):498-502 (2001), incorporated herein by reference). Adding antioxidant rinses to the persufflation protocol can also be used to improve the quality of biological material during preservation. In addition, nutrients, antioxidants or other preservatives can be added in aerosol form during persufflation.

Preservative liquids and additives to the preservative liquids can be used to wet the exterior of the preserved biological material during liquid perfusion or persufflation. Preserving liquids and additives to the preserving liquids can also be used in liquids that preserve the preserved biological material for a period of time perfuse before, after or instead of persufflation. The preserving fluid can be a cell culture solution (e.g., cryopreserved hepatocyte recovery medium (CPRM)), an organ preservation solution (e.g., University of Wisconsin (UW) solution, or histidine-tryptophan ketoglutarate (HTK) solution), or other solution used in medical procedures (e.g., saline or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer). Additives such as antioxidants (e.g. vitamins C and E and derivatives, catalytic antioxidants, CA: "Redox Modulation Protects Isles From Transplant-Related Injury", Martha M. Sklavos, Suzanne Bertera, Hubert M. Tse, Rita Bottino, Jing He , Joshua N. Beilke, Marilyne G. Coulombe, Ronald G. Gill, James D. Crapo, Massimo Trucco and Jon D. Piganelli, DIABETES, VOL 59, July 2010 1731-1738), anti-apoptotic agents (e.g Caspase-Selective Inhibitors, EP1013: "The Caspase Selective Inhibitor EP1013 Augments Human Islet Graft Function and Longevity in Marginal Mass Islet Transplantation in Mice", Juliet A. Emamaullee, Joy Davis, Rena Pawlick, DIABETES, VOL 57, June 2008 p 1556-1566, "Caspase Inhibitor Therapy Synergizes With Costimulation Blockade to Promote Indefinite Islet Allograft Survival," Juliet A. Emamaullee, Joy Davis, Rena Pawlick, Christian Toso, Shaheed Merani, Sui-Xiong Cai, Ben Tseng, and AM James Shapiro, DIABETES, VOL 59, June 2010 1469-1477), vasodilators/vasoconstrictors (e.g. alpha-adrenoceptor antagonists (alpha-blockers; arginine vasopressin), oxygen carriers (eg, perfluorocarbons, artificial blood components), chelators/other toxin binders (eg, ethylenediaminetetraacetic acid (EDTA), anticoagulants (eg, heparin), and other nutrients (eg, sugars, Amino acids, vitamins) can be included in the liquid to improve preservation.

For example, where the material being preserved is a human or porcine pancreas, one may persufflate the pancreas through one or more arteries, through one or more veins, through one or more ducts, or through a combination of veins and ducts. For example, in the case of the human pancreas, persufflation may be anterograde via cannulation of one or more of the following vessels: (i) en bloc duodenopancreatectomy and/or splenectomy with division of the aorta to include the celiac trunk and superior mesenteric artery; (ii) en bloc duodenopancreatectomy and/or splenectomy with direct cannulation of the celiac trunk and superior mesenteric artery; and (iii) en bloc duodenopancreatectomy and/or splenectomy with direct cannulation of the proximal splenic artery and superior mesenteric artery. Alternatively, persufflation can be done retrograde via cannulation of the following vessels: (i) en bloc duodenopancreatectomy and/or splenectomy with cannulation of the inferior mesenteric vein or splenic vein; (ii) en bloc duodenopancreatectomy and/or splenectomy with superior mesenteric vein cannulation; and (iii) en bloc duodenopancreatectomy and/or splenectomy with portal vein cannulation.

Alternatively, in the case of the porcine pancreas, persufflation may be anterograde via cannulation of one or more of the following vessels: en bloc duodenopancreatectomy and/or splenectomy with division of the aorta to include the celiac trunk and superior mesenteric artery; (ii) en bloc duodenopancreatectomy and/or splenectomy with direct cannulation of the celiac trunk and superior mesenteric artery; and (iii) en bloc duodenopancreatectomy and/or splenectomy with direct cannulation of the proximal splenic artery and superior mesenteric artery. Alternatively, persufflation may be retrograde via cannulation of the following vessels: (i) en bloc duodenopancreatectomy and/or splenectomy with splenic vein cannulation; (ii) en bloc duodenopancreatectomy and/or splenectomy with superior mesenteric vein cannulation; and (iii) en bloc duodenopancreatectomy and/or splenectomy with inferior mesenteric vein cannulation; and (iv) en bloc duodenopancreatectomy and/or splenectomy with portal vein cannulation.

In the case of a liver, persufflation may be anterograde via cannulation of one or more of the following vessels: (i) common hepatic artery; (ii) hepatic artery and (iii) portal vein. Alternatively, persufflation may be retrograde via cannulation of one or more of the following vessels: (i) hepatic vein(s) and (ii) infra- or suprahepatic inferior vena cava.

For example, where the in situ generated preservation gas consists of pure gaseous oxygen, it may be desirable to dilute the gaseous oxygen in a liquid solvent such as water or a water-based solvent and the biological material, including tissue, with the gas/liquid solution or it may be desirable to dilute the gaseous oxygen with another gas or gases such as ambient air and perfuse the biological material with the gas/gas mixture. In the case of perfusion with a gas/liquid solution, the concentration of gaseous oxygen in the solution may be around the saturation point of the liquid, which may be in the range of 0.3-1 atm partial pressure. In addition, the Flow rate of the solution to the biological material can be, for example, about 200 cm ³ /min or greater. In the case of persufflation, the concentration of gaseous oxygen in the mixture can be about 20-100%, more preferably about 30-80%, even more preferably about 40%. In addition, the flow rate of the mixture to the biological material can be, for example, about 10-60 cm ³ /min, preferably 15-22 cm ³ /min, and the gas pressure of the applied solution can be, for example, no more than about 22 mm Hg. In certain cases, preservation can be improved if persufflation is carried out at reduced temperatures, such as around 4-8 °C.

Where the tissue-comprising biological material being preserved is a pancreas, systemic heparinization of the pancreas with 100,000 units is preferably performed at least 5 minutes prior to death to prevent small clots from forming throughout the pancreas. Additionally, during harvesting, the organ should be irrigated with 5 liters of a heparinized cold preservation solution to prepare it for preservation, in order to flush any remaining blood from the blood vessels and accelerate the cooling of the organ mass, reducing exposure of the core regions of the pancreas to ischemia reduced at elevated temperatures (>8 °C). Following harvest, the organ should be examined extensively for leakage by flushing cold preservation solution through the blood vessels, and any leakage found should be ligated. The pancreas and attached blood vessels should then be allowed to float freely in the cold preservative solution to prevent any kinking of the blood vessels, particularly at the sites of cannulation. Any small leaks that remain should be identified by the presence of bubbles emerging from the blood vessels to ensure that all the gas is flowing through the pancreas, and these leaks should be tied off.

Access points for cannulation are discussed above. Other organs can also benefit from heparinization.

With the present method of harvesting, the vast majority of the pancreas is irrigated with cold preservative solution, clearing a duct for persufflation of the entire organ. Histological sectioning of organs harvested using this technique showed that due to more extensive lavage, no red blood cells are present in the tissue preserved by either persufflation or TLM. No discernible differences were observed by histology between organ samples collected immediately following collection (t=0) and those collected following 6 or 24 hours of persufflation. However, tissue preserved with TLM showed increased necrosis/autolysis and a high incidence of pyknotic nuclei compared to persufflated samples, particularly after 24 hours of preservation.

The method described above can make it possible to carry out xenotransplantation with human pancreas and with porcine pancreas. Methods to improve the preservation of pancreas with consequent improvements in islet yield and quality, and ultimately clinical outcome, are urgently needed to advance the field of islet transplantation to an established medical application. Herein it is shown that arterial persufflation of the pancreas to provide oxygen throughout the organ at the vascular level during storage is a viable technique. In particular, the feasibility of delivering electrochemically generated oxygen at adequate flow rates and pressures for persufflation of the porcine pancreas and the human pancreas has been demonstrated. The results show the promise of this technique compared to no preservation and as an improvement over TLM storage.

Two applications of the present method of pancreas preservation are as follows: (1) Oxygen persufflation throughout cryopreservation (from donor organ harvest to islet isolation) with the goal of extending the period of cryopreservation beyond the current standard of 8 Extend hours to increase the donor pool and ease the logistics of organ and islet harvesting; and (2) oxygen persufflation (~3 hours) after organ transport with standard cold preservation (-12 hours for TLM) as a recovery procedure to improve islet yield and quality at the islet processing center. These two uses include improving islet yield and quality and/or shelf life for pancreases from standard donors. In one application, the pancreas is persufflated throughout the storage period; in the second application, persufflation is applied as a recovery treatment after standard cold storage (Note that in pancreas preservation, almost always there is a trade-off between preservation time and island yield and quality). Increased storage times (with the same islet yield and quality) can lead to flexibility in operation and the use of more organs. Improved islet yield and quality (with common storage times) may have a greater impact on individual transplant outcomes. An entirely separate application branch, which would also be extremely valuable if successful, would be to use persufflation to improve organ quality and thus improve the donor's organ supply after the cardiac death category.

Now referring to 1 , there is shown a block diagram of one embodiment of a system for fluid perfusion of biological material, including tissue, constructed in accordance with the teachings of the present invention and represented generally by reference numeral 11 .

System 11 may include a gas generator or persufflation system 100, a biological material container 200, a process controller 300, a temperature control device 400, and a power supply 500.

Now referring to 2 , the persufflation system 100 is shown in more detail, with the persufflation system 100 being shown along with the power supply 500. FIG. Persufflation system 100 may include an electrochemical oxygen concentrator (EOC) 101 . EOC 101, the details of which are discussed in more detail below, may comprise a single oxygen concentrator cell or a plurality of oxygen concentrator cells connected in series or in parallel. Each of said one or more oxygen concentrator cells may comprise an anode 103 and a cathode 105, with the anode 103 and cathode 105 being in intimate contact with and separated from each other by an ionically conductive separator. Separator 107 can be, for example, a solid proton exchange membrane (PEM) such as a " ^NAFION® NRE-1135" membrane (EI duPont de Nemours & Company, Wilmington, DE). Anode 103 and cathode 105 may be electrically coupled to power supply 500 using electrical connectors 111-1 and 111-2, respectively, and may also be coupled to process controller 300 by means not shown (not shown in 2 shown). (Additional components of persufflation system 100 may be coupled to power supply 500 and/or process controller 300, but these connections are not shown herein for clarity.)

• Anode: 2H₂O ↔ 4H⁺ + 4e^- + O₂ (rein) [1]
• Kathode: O₂ (Luft) + 4H⁺ + 4e^- ↔ 2H₂O [2]
• Netto: O₂ (Luft) @ Kathode ↔ O₂ (rein) @ Anode [3]

As shown below, EOC 101 can be used to concentrate oxygen from ambient air.

• Anode: 2H ₂ O ↔ 4H ⁺ + 4e ^- + O ₂ (neat) [1]
• Cathode: O ₂ (air) + 4H ⁺ + 4e ^- ↔ 2H ₂ O [2]
• Net: O ₂ (air) @ cathode ↔ O ₂ (pure) @ anode [3]

EOC 101 may additionally include a water reservoir 113 to hold a quantity of water that is supplied to the anode 103 . As further described below, a pervaporation membrane 115, which may be, for example, a " ^NAFION® NRE-1135" membrane, may be positioned between a bottom outlet of water reservoir 113 and anode 103, with pervaporation membrane 115 serving to steadily supply gas-phase water to anode 103 while preventing oxygen generated at anode 105 from mixing with the water. In other words, this arrangement allows water to be passively supplied to anode 103 while allowing product oxygen to flow laterally out of the anode through a flow field gap. It is believed that the vapor feed system described above is advantageous compared to a corresponding liquid feed system in that the present vapor feed system prevents many contaminants that may be present in the water supply from being fed to anode 103 . For example, where tap water is used as the water supply, halogen, alkali metals, alkaline earth metals, transition metal elements, organic compounds, and/or microorganisms may be present in the water. These elements, compounds and/or microorganisms, when they belong to the active elements of the EOC 101 lead to a reduction in performance and efficiency of the EOC, for example due to membrane contamination or catalyst poisoning. These materials could also be harmful to the biological material being preserved. Pervaporation membrane 115 effectively limits the access of these species to the active elements of EOC 101 and the biological material conserved downstream, and effectively allows for in situ distillation of the reservoir water.

EOC 101 may additionally include a return tube 117 coupled to cathode 105 at one end and to reservoir 113 at the opposite end, where return tube 117 is used to direct water produced at cathode 105 to reservoir 113 . In this way, the frequency with which water must be added to the system is reduced.

Persufflation system 100 may further include means for cooling and drying the gaseous oxygen produced at anode 103 . In the present embodiment, said cooling and desiccant means may include an oxygen cooler 121, an oxygen/water separator 123, and an oxygen dryer or desiccant means 125. A fluid line 127 may be connected to an outlet of anode 103 at one end and to an inlet of oxygen cooler 121 at the other end. Additionally, a fluid line 129 may be connected to an outlet of oxygen cooler 121 at one end and to an inlet of oxygen/water separator 123 at the other end. Additionally, a fluid line 131 may be connected to an outlet of oxygen/water separator 123 at one end and to an inlet of oxygen dryer 125 at the opposite end. Furthermore, a fluid line 133 may be connected at one end to the outlet of oxygen dryer 125 . A fluid line 135 for draining oxygen/water separator 123 may be connected to the bottom of oxygen/water separator 123 and a drain valve 137 may be connected to the outlet end of fluid line 135 .

Persufflation system 100 may further include ambient air delivery means. In the present embodiment, said ambient air supply means may comprise a fluid line 141, fluid line 141 having an inlet 143 for ambient air to enter therein. An air filter 145 may be positioned along line 141 to remove certain contaminants from the ambient air. Air filter 145 may be of the type that physically traps particulates (e.g., a 10 micron pore size filter), or may be of the type that chemically traps or modifies particulates (e.g., a carbon or permanganate filter). An air pump 147 to pump air through line 141 (both to supply air to cathode 105 and to dilute oxygen generated by EOC 101) may be positioned along line 141 downstream of air filter 145. Downstream of air pump 147 line 141 may branch into a fluid line 149 and a fluid line 151 . A valve 153, which may also include a flow rate indicator, may be positioned along line 149, with the outlet of line 149 suitably positioned to supply cathode 105 with air. An adjustable flow valve 155 may be positioned along line 151 to regulate the flow of air through line 151 and, in doing so, adjust the rate at which ambient air is to be mixed with pure oxygen so that a desired oxygen concentration can be obtained. In this way, for example, one can make sequential adjustments to the concentration of oxygen present in the resulting oxygen/ambient air solution. This may be desirable, for example, in situations where one wishes to persufflate biological tissue with gas having a first oxygen concentration and then later persufflate the biological tissue with gas having a different oxygen concentration. A check valve 157 may be positioned along line 151 downstream of valve 155 .

Persufflation system 100 may further include a fluid line 161 coupled to the outlets of lines 133 and 151 for combining the pure oxygen from line 133 with the ambient air from line 151 . A medical grade filter 163 , which may have a pore size of 0.2 microns, for example, may be positioned along line 161 . An oxygen concentration sensor 165 may be positioned along line 161 downstream of filter 163 . A pressure sensor/indicator 167 and pressure relief valve 169 may be positioned along line 161 downstream of sensor 165 . A pressure regulator 171 may be positioned along line 161 downstream of pressure sensor/gauge 167 and valve 169 . A pressure sensor/indicator 173 may be positioned along line 161 downstream of sensor 173 . Downstream of sensor 173, line 161 may branch into lines 175-1, 175-2 and 175-3. A flow rate indicator valve 177-1 may be positioned at the outlet end of line 175-1, a flow rate indicator valve 177-2 may be positioned at the outlet end of line 175-2, and valve with flow rate indicator 177-3 may be positioned at the outlet end of line 175-3.

As can be seen, therefore, the persufflation system 100 allows up to three different substreams of preservation gas to be administered to the biological material, each of the three different substreams having an independently adjustable flow rate. In addition, it should also be mentioned that the oxygen concentration and the maximum feed pressure of the three sub-streams can be adjusted, although not independently of each other. Additionally, pressure controllers or mass flow controllers can be used in these substreams to provide further independent pressure and flow control for each substream. In addition, the persufflation system 100 has the capability to produce gas that is humidified, for example by omitting the oxygen dryer 125, if desired.

Now referring to 3 , EOC 101 is shown in more detail. As can be seen, EOC 101 may include a membrane electrode assembly 181 . Assembly 181 may in turn include anode 103, separator 107 and cathode 105 (which are not included in 3 shown but positioned opposite anode 103 on the underside of separator 107). EOC 101 may further include an anode collector 182 and a cathode collector 183 . Anode collector 182, which may have a porous central region 182-1 defining a fluid diffusion chamber, may be positioned against the top surface of anode 103, and cathode collector 183, which may have a porous central region 183-1 defining a fluid diffusion chamber, may be positioned against the bottom surface of the cathode. EOC 101 may further include pervaporation membrane 115 which may be positioned against the top surface of anode collector 182 . As explained above, pervaporation element 115 can be used to supply water to anode 103 in a vapor phase.

EOC 101 may further include annular anode seal 185 and cathode insulation 186 . Gasket 185 may be positioned against the top surface of pervaporation membrane 115 and cathode insulation 186 may be positioned against the bottom surface of cathode collector 183 . EOC 101 may further include an anode end-plate 187 and a cathode end-plate 188 . Anode end plate 187, which may include a porous central region 187-1, may be positioned against anode gasket 185 and cathode end plate 188 may be positioned against cathode insulator 186.

EOC 101 may further include a cathode inlet port 189 and a cathode outlet port 190, where inlet port 189 and outlet port 190 are mechanically coupled to cathode endplate 188 and fluidly coupled to the cathode.

EOC 101 may further include water reservoir 113 which may be positioned over anode end plate 187 . A water reservoir O-ring 191 may be placed on the anode endplate 187 and may be used to form a fluid seal for the water passing from reservoir 113 to the porous central region 187-1 of the anode endplate. An anode clamp ring 192 may be mounted on a peripheral flange 113 - 1 of reservoir 113 .

EOC 101 may further include an oxygen outlet tube 193 to conduct gaseous oxygen away from anode 103, tube 193 being attached at one end to anode end plate 187 and extending through flange 113-1 of reservoir 113 and anode clamp ring 192.

EOC 101 may further include a water fill port 194, which may be coupled to reservoir 113 through an opening 113-2, a reservoir vent port 195-1, which may be coupled to reservoir 113 through an opening 113-3, and water recovery port 117, which may be coupled to reservoir 113 may be coupled through an opening 113-4.

EOC 101 may further include components to mechanically couple many of the components described above. Such components may include a plurality of cell assembly bolts 196 as well as a corresponding plurality of washers 197, disc springs 198 and nuts 199.

Again referring to 1 , container 200 may be suitably sized to store the biological material of interest and may include an opening (not shown) through which one or more fluid lines 550 coupled to the outlets of valves 177-1, 177-2 and 177-3 may be conducted to the solution of gaseous oxygen generated by persufflator system 100, a natural or artificial fluid distribution network within the biological material contained in container 200 stores to supply. Fluid line 550 may include, for example, a cannula insertable into the biological material at one end and a length of tubing coupled to the cannula at one end and to one of valves 177-1 through 177-3 at the opposite end. include. Fluid line 550 may also include a length of tubing or similar structure coupled at one end to one or more of valves 177-1 through 177-3 and adapted at the opposite end to allow one to exhaust gaseous from one or more valves 177-1 to 177-3 to flow over the surface of the biological material. The interior of vessel 200 may be equipped with an oxygen sensor 203 or other gas sensors and a temperature sensor 205, with sensors 203 and 205 being used to provide oxygen, gas and/or temperature readings to process controller 300 for feedback control.

Controller 300 may include an embedded controller with a keyboard/LCD user interface to operate the various system components and facilitate user control. Two control loops may be included to allow self-regulation of process conditions, one for EOC cell current and the other for post-dilution oxygen concentration. A current sensor and variable voltage DC-to-DC converter can be used in conjunction with a software-based proportional-integral control algorithm to regulate the EOC cell current (and therefore the rate of pure oxygen production) to the amount required due to the user's total current and oxygen concentration requirements. Similarly, controller 300 may also be used in conjunction with oxygen sensor 203 and valve 155 to control the amount of air/oxygen mixing to achieve the desired oxygen concentration setpoint. The embedded controller can also be used to provide the user with real-time readings of system line pressure and pressure manifold supply pressures

For example, temperature control device 400 may be a commercially available thermoelectric (or Peltier) based chest, such as the ^VECTOR® thermoelectric cooler. Vapor compression refrigeration or physical cooling via ice or other coolants can be used in place of thermoelectrics; however, in the event one desires to add heat to vessel 200, as opposed to lowering the temperature in vessel 200, this enables the potential of bi-directional heat pumping of the thermoelectrics in a way that vapor compression refrigeration or physical cooling cannot. In the case of passive or active cooling, the insulation of vessel 200 can be enhanced through the use of highly efficient vacuum panels.

For example, power supply 500 may include a battery pack module housing, two 24V nickel-metal hydride (NiMH) batteries, a charger for those batteries, a 24V DC-to-DC converter (for regulation), and a battery shutdown circuit to to prevent deep battery discharge.

To use system 11, the biological material comprising tissue that one desires to preserve can be placed in container 200 and one or more outlets 177 - 1 through 177 - 3 of persufflation system 100 can be fluidically coupled to the biological material via a fluid line 550 , which is fluidly coupled at one end to one of the outlets 177-1 to 177-3 and at the counterpart is introduced into an appropriate access of a fluid distribution network (e.g. vascular, ductal, lymphatic) of the tissue for anterograde or retrograde persufflation of the allow tissue. Persufflation system 100 may then be powered by power supply 500 and operated by process controller 300 to persufflate the tissue according to predetermined parameters. In addition, temperature controller 400, which is powered by power supply 500, can be used to maintain container 200, as well as the biological material contained therein, at a preselected temperature. Oxygen sensor 203 and temperature sensor 205 may be used in conjunction with process controller 300 to provide feedback control for persufflation system 100 and temperature control device 400 .

Now referring to 4 , there is shown a perspective view of one embodiment of a system for fluid perfusion of biological material, including tissue, and represented generally by reference numeral 600. FIG. (Certain components of system 600 are not shown for the sake of clarity and simplicity.) This embodiment is shown as a portable system.

System 600 is similar in many respects to system 11, with system 600 including persufflation system 100, tank 200, process controller 300, temperature control device 400, and power supply 500. System 600 may further include a thermally insulated enclosure 610 in which persufflation system 100, tank 200, process controller 300, temperature control device 400, and power supply supply 500 can be arranged. (A sidewall of housing 610 is not shown to reveal some components located inside housing 610, and an insulated lid removably attached to housing 610 is not shown to reveal the top of container 200 and an opening 620 through which container 200 can be removably inserted.) Without wishing to be bound by any specific dimensions, housing 610 can have a footprint similar to that of a picnic cooler (eg, 12"H x 10"D x 22"L). Casters 630 and handles 640 may be attached to housing 610 to facilitate transport of system 600. System 600 may also include fluid lines (not shown) similar to fluid lines 550 to transport fluid from persufflation system 100 to the biological material.

System 600 may further include logging/diagnostics functionality. This feature would allow a system user to view and record the system process readings (pressure, concentration, temperature, etc. which may not be available on the onboard controller interface) via downloading to an external computer at the conclusion of the storage period. Power conditioning circuitry, relays, and certain sensor transducers can all be integrated into a modular circuit board. A heat pump may require non-standard or variable DC electrical power to operate the Peltier devices, which would require identification of a suitable power supply. This design may include a 24 hour battery design with an optional AC converter and on-board battery charger. The batteries can be rechargeable NiMH batteries because they cost less, are safer and more convenient than lithium ion batteries, and have reasonable energy storage densities.

Now referring to again 5 1, a block diagram of one embodiment of a system for fluid perfusion of biological material comprising tissue according to the present invention, constructed in accordance with the teachings of the present invention and represented generally by reference numeral 1000, is shown. For the sake of simplicity and clarity, specific components of system 1000 are not shown or discussed herein.

System 1000 may include a persufflation system 1100, a liquid perfusion system 1600, a switching system 1700, a container 1200, a process controller 1300, a temperature control device 1400, and a power supply 1500.

Persufflation system 1100, tank 1200, process controller 1300, temperature control device 1400, and power supply 1500 may be structurally and functionally similar to persufflation system 100, tank 200, process controller 300, temperature control device 400, and power supply 500 of system 11, respectively.

The liquid perfusion system 1600 can comprise a reservoir for storing a volume of preservation liquid (“perfusate”), means for supplying the perfusate to the organ container 1200 and means for draining the perfusate from the organ container 1200 . The reservoir may comprise a fluid-containing bag, such as those used to hold fluids for intravenous administration. The means for delivering the perfusate to organ container 1200 may comprise a tube connected from the reservoir to organ container 1200, whereby the perfusate flows through the tube due to gravity. Where the perfusate flows from the reservoir to the organ container 1200 via a connecting tube, the liquid perfusion system 1600 may additionally include means to support the reservoir at a height above that of the container 1200. Alternatively, an electrically or manually operated pump can be used to convey the perfusate from the reservoir to container 1200 via the connecting tube. An alternative means of delivering the perfusate may include a tube connected from persufflation system 1100 to the reservoir whereby gas pressure exerted by persufflation system 1100 forces liquid in the reservoir to flow from the reservoir to container 1200 through the connecting tube to flow. Means for draining perfusate from container 1200 may include a tube that allows the perfusate to flow out of container 1200 by gravity, an electrically operated or manual pump, or gas pressure from persufflation system 1100, whereby the liquid then collects in a receptacle or channel becomes.

Switching system 1700 may include tube connectors designed to connect to tubes leading from persufflation system 1100, liquid perfusion system 1600, and container 1200. Switching system 1700 may further include an electrically actuated valve activated by signals from process controller 1300, whereby process controller 1300 controls whether the flow directed to container 1200 is from persufflation system 1100 or liquid perfusion system 1600. Alternatively Switching system 1700 may include a manual valve that allows the user to manually control whether the flow going to container 1200 is from persufflation system 1100 or liquid perfusion system 1600. Alternatively, switching system 1700 may include one or more electrically actuated and manual valves, whereby the flow to tank 1200 may be controlled manually or by electrical triggering.

System 1000 may further include gas sensors, such as an oxygen sensor 1203 and a carbon dioxide gas sensor 1207, used to measure the concentrations of oxygen and carbon dioxide gas, respectively, in inlet and outlet streams. Gas sensors may be connected to the persufflation system 1100 and liquid perfusion system 1600 outlet streams. Gas sensors may also be connected to the inlet and outlet streams of container 1200. Process controller 1300 may be electrically connected to gas sensors 1203 and 1207 and may provide both oxygen sensor 1203 and carbon dioxide sensor 1207 with data acquisition capabilities. Additionally, process controller 1300 can use the signals collected from the gas sensors to make process decisions, such as triggering an alarm or shutoff valve, when the concentration of a particular gas falls outside of a predetermined range.

System 1000 may further include a temperature sensor 1205 for measuring the temperature within container 1200 . Process controller 1300 may be electrically connected to temperature sensor 1205 and may provide temperature sensor 1205 data collection capability. Additionally, process controller 1300 may use the signals collected by temperature sensor 1205 to make a process decision such as activating or deactivating temperature control device 1400 when the temperature within container 1200 falls within or outside of a predetermined range.

System 1000 may further include filters, such as filters used to separate liquids from gas streams, to prevent contaminants from entering critical system components, to prevent solids from entering the preserved organ or tissue, or to prevent that liquids interfere with gas concentration monitoring. Filters may be located in the tubing at the outlets of persufflation system 1100 and liquid perfusion system 1600, on the tubing connecting the gas sensors, and at the inlet and outlet of organ container 1200.

System 1000 may further include electro-optical sensors used to measure the presence of liquid or gas in pipes. Electro-optical sensors may be connected to the inlet and outlet tubes of container 1200 and to the outlet tubes of persufflation system 1100 and liquid perfusion system 1600. Process controller 1300 may be used to process the signals provided by the electro-optical sensors and actuate switching device 1700 to switch between persufflation system 1100 and liquid perfusion system 1600.

System 1000 can be used in a manner similar to that discussed above for System 11, the key difference between the two systems being that with System 1000 the biological material, which includes tissue, is in liquid perfusate from Liquid Perfusion System 1600 may be bathed before, during, or after being persufflated using persufflation system 1100, or perfused with liquid perfusate instead of being persufflated using persufflation system 1100.

Now referring to 6 , there is shown a simplified schematic diagram of a system for fluid perfusion of biological material comprising tissue in accordance with one embodiment of the present invention, the system being constructed in accordance with the teachings of the present invention and represented generally by reference numeral 2000. FIG. For the sake of simplicity and clarity, specific components of System 2000 are not shown or discussed herein.

System 2000 may include a persufflation system 2100, which may be similar in construction and function to persufflation system 100, except that system 2100 may include a single outlet 2101 instead of three outlets 177-1 through 177-3. A length of tube 2103 may be fluidly connected to outlet 2101 at one end and may be connected to a port of valve 2105 at the opposite end. A length of tube 2107 may be fluidly connected at one end to another port of valve 2105 . The opposite end of tube 2107 may be fluidly connected to a connector 2108 . Link 2108 may be fluidly connected to a linkage member 2109 to be connected. Connector 2109, in turn, may be fluidly connected to one end of a delivery cannula 2111, with the opposite end of delivery cannula 2111 being inserted into an appropriate perfusion/persufflation inlet of an organ O, organ O being disposed within a container 2113, container 200 or 1200 similar is.

System 2000 may further include a fluid reservoir 2121, which may be in the form of a fluid bag for intravenous administration. Reservoir 2121 can contain a volume of liquid perfusate 2123 . A length of tube 2125 may be fluidly connected to an outlet 2127 of reservoir 2121 at one end and may be connected to a port of a valve 2129 at the opposite end. A length of tube 2131 may be fluidly connected at one end to another port of valve 2129 . The opposite end of tube 2131 may be fluidly connected to a connector 2132 . Link 2132 may be fluidly connected to link 2109 .

System 2000 may further include an exit cannula 2141, exit cannula 2141 being inserted at one end into an appropriate organ O perfusion/persufflation outlet. The opposite end of exit cannula 2141 may be fluidly connected to a connector 2143 .

System 2000 may further include a length of pipe 2151. Tube 2151 may be fluidly connected at one end to connector 2153 , which may be fluidly connected to connector 2143 . The opposite end of tube 2151 may be connected to a port of valve 2155 . A length of tube 2157 may be fluidly connected to another port of valve 2155 .

System 2000 may further include a length of pipe 2171 . Tube 2171 may be fluidly connected at one end to connector 2173 , which may be fluidly connected to connector 2143 . The opposite end of tube 2171 may be connected to a port of a valve 2175. A length of tube 2177 may be fluidly connected to another port of valve 2175 .

In use, links 2108 and 2132 are connected to link 2109, links 2153 and 2173 are connected to link 2143, and cannulas 2111 and 2141 are inserted into organ O. To perfuse organ O with liquid perfusate 2123, which may be desirable prior to persufflation, after persufflation, during persufflation, and/or in lieu of persufflation, valves 2129 and 2155 are opened, whereby perfusate 2123 is delivered to organ O through cannula 2111 , circulates through organ O and drains from organ O through cannula 2141. To persufflate organ O with a preservation gas or with a gas/gas mixture, valves 2105 and 2175 are opened and persufflation system 2100 is actuated, whereby the gas or gas/gas mixture from persufflation system 2100 is delivered to organ O through cannula 2111 , circulates through organ O and drains from organ O through cannula 2141. As will be appreciated, when one wishes to perfuse organ O with liquid perfusate without simultaneously persufflating with a preservative gas or with a gas/gas mixture, valves 2105 and 2175 are closed, whereas valves 2129 and 2155 are opened. Conversely, if one wishes to persufflate organ O with the preservation gas or a gas/gas mixture without also perfusing with liquid perfusate, valves 2129 and 2155 are closed, whereas valves 2105 and 2175 are opened. If one wishes to perfuse and persufflate simultaneously, thereby delivering the gas dissolved in the liquid perfusate to organ O, valves 2105, 2129, 2155 and 2175 are opened.

The examples below only illustrate and do not limit the present invention.

Example 1: Demonstration of improved islet persufflation oxygenation with electrochemical oxygen generation,

The effect of persufflation on islet isolation was tested in five (5) organs by dividing each organ into its three lobes, using the duodenal lobe for immediate isolation, the spleen lobe being preserved for 6 hours with persufflation, and the conjoining flap was preserved with TLM for 6 hours. In one experiment, the preservation was extended to 24 hours. The persufflation system used an EOC that has an active catalyzed area of 40 cm ² and uses a "NAFION ^® NRE-112" membrane as separator 107 and a pervaporation membrane 115 . Anode 103 and cathode 105 comprised inked stickers applied to separator 107 and having a 4 mg/cm ² loading of platinum-iridium black catalyst and platinum black catalyst, respectively. With deionized water provided to the EOC reservoir, and Air provided to the cathode at 300 cc, both at 21°C, the performance of this EOC was 0.84 volts at 8 amps (200 mA/cm ² ) and 0.93 volts at 12 amps (300 mA/cm ² ). The persufflation system was still configured as in 2 shown. Persufflation of individual pancreatic lobes was done with 40% oxygen at 15-22 cc and a pressure of -20 mm Hg, requiring an EOC current of 0.94-1.4 amps. The method of separating each organ into its lobes was chosen to allow pairwise comparison of preservation methods by eliminating donor variability. Given the small sample size of the five organs, it was decided to standardize (rather than randomize) the lobe used for each condition.

Organs were evaluated macroscopically following harvest and immediately prior to islet isolation. All flaps showed complete perfusion and good consistency following collection. However, it was observed that the persufflated organs subjectively 'looked' and 'felt' better (plumper, fresher) to the surgeons who performed the enzymatic distension during isolation compared to flaps preserved with TLM. This was particularly evident for the organ that was preserved for 24 hours prior to isolation.

The first important observation and data showing improvement from persufflation storage concerned islet morphology. How to refer to the 7(a) until 7(c) As can be seen, the persufflation-preserved porcine pancreas yielded the most morphologically well-preserved islets; fragmentation was extensive in the islets isolated from the fragmentations stored in TLM. (Dithizone is an insulin stain that stains β-cells within islets.)

Table 1, below, shows that islets from persufflated organs had a better morphology mean than those from TLM-stored or even fresh (t=0) organs; by pairwise comparison t-test, the improvement in persufflation-conserved (PSF) versus TLM was statistically significant (p=.008). Table 1. Morphology scores for islets at day 0 after isolation. Pork pancreas ID preservation period t=0 PSF TLM P647 6 hours 3.0 2.0 P648 6 hours 7.0 7.5 7.0 P649 6 hours 5.0 7.5 6.0 P650 24 hours 6.0 6.0 4.0 P656 6 hours 6.0 9.0 7.0 Average 6.0 6.6 5.2 standard dev . 0.82 2.3 2.2 t-test for paired samples 0.134 0.0086

• Tag 0, IE-Ausbeute: Insel-Äquivalent mittels Anzahl pro Gramm verdautes Gewebe. Ein Insel-Äquivalent (IE) ist eine Volumeneinheit, gleich der einer Kugel mit einem Durchmesser von 150 µm.
• Tag 2, IE-Erholung. , %: % IE durch Anzahl an Tag 2, verglichen mit Tag 0 (Die Anzahl an Tag 2 wäre die IE-Ausbeute an Tag 0, multipliziert mit der IE-Erholung in %.).
• Tag 2, OCR/DNA: Die Insel-Sauerstoffverbrauchsrate (OCR) in (nmol/min) ist eine Messung der Quantität lebender Inseln; DNA (mg) ist eine Messung der gesamten Insel-Quantität; OCR/DNA ist folglich das Verhältnis der Insel-Quantität lebender Inseln zur gesamten Insel-Quantität und ist ein Maß für die Insel-Vialibilität.
• Tag 2, OCR-Ausbeute: Menge an lebendem Insel-Gewebe [Insel-OCR (nmol/min)] pro Gewicht verdautes Gewebe (g).
• Tag 2, Fragmentierungsverhältnis: Bruchteil der Insel-Äquivalente, berechnet aus der DNA-Messung (1 DNA IE = 10,4 ng DNA) und der IE durch Zählung. Eine Messung der Präparationsqualität; es gibt Anzeichen dafür, dass fragmentierte Inseln mit geringerer Wahrscheinlichkeit die Transplantation überleben und Insulin bilden.
• Tag 2, IE-basierte OCR-Ausbeute: Eine OCR-Ausbeute (OCR/g verdautes Gewebe) von unfragmentierten Inseln.

Overall, islets from porcine lobes stored with persufflation preservation showed better morphology compared to those isolated shortly after collection or those from lobes stored with TLM. 8th summarizes various measurements of islet quantity and quality for porcine pancreas preserved by various methods. Oxygen consumption rate (OCR) measurement has been shown to be a rapid, simple, quantitative, prospective assay for islet quality assessment (see Papas et al., "Human islet oxygen consumption rate and DNA measurements predict diabetes reversal in nude mice," American Journal of Transplantation," 7(3):707-13 (2007); Papas et al., "A stirred microchamber for oxygen consumption rate measurements with pancreatic islets," Biotechnology and Bioengineering, 98:1071-82 (2007), Koulmanda et al., Islet oxygen consumption rate as a predictor of in vivo efficacy post-transplantation, Xenotransplantation, 10(5):484 (2003), Papas et al. "Islet quality assay based on oxygen consumption rate and DNA measurements predicts graft function in mice," Cell Transplantation, 12:176-176 (2003); Papas et al., "Rapid Islet Quality Assessment Prior to Transplantation," Cell Transplantation, 10 (6): 519 (2001)) and was used as one of the measurements in the This work, performed together with standard islet equivalent count, DNA measurement and fragmentation assessment. In the 8th presented measurements are briefly defined below:

• Day 0, IE yield: islet equivalent by number per gram of tissue digested. An island equivalent (IU) is a unit volume equal to that of a 150 µm diameter sphere.
• Day 2, IE recovery. , %: % IE by count on day 2 compared to day 0 (The count on day 2 would be the IE yield on day 0 multiplied by the % IE recovery.).
• Day 2, OCR/DNA: The islet oxygen consumption rate (OCR) in (nmol/min) is a measure of the quantity of living islets; DNA (mg) is a measurement of total islet quantity; OCR/DNA is thus the ratio of islet quantity of living islets to total islet quantity and is a measure of islet viability.
• Day 2, OCR yield: amount of living islet tissue [islet OCR (nmol/min)] per weight digested tissue (g).
• Day 2, fragmentation ratio: fraction of islet equivalents calculated from DNA measurement (1 DNA IE = 10.4 ng DNA) and IE by counting. A measurement of the preparation quality; there is evidence that fragmented islets are less likely to survive engraftment and produce insulin.
• Day 2, IE-based OCR yield: An OCR yield (OCR/g digested tissue) of unfragmented islets.

As can be seen, the main results of persufflation storage were superior to those obtained with immediate isolation and TLM storage. However, due to the small number of pancreases and the intrinsic large variability from donor to donor (and/or isolation to isolation), most of the differences were not statistically significant. OCR/DNA, a measure of viability, was consistently higher with persufflation after 2 days in culture, reflecting a trend toward statistical significance versus TLM at 6 h preservation (p=0.090, n=4) and statistical significance combined 6 and 24 hour preservation (p=0.036, n=5). The last column in 8th Figure 1 shows the superiority of persufflation in OCR yield associated with islet counts (calculated using IE counts), which is a measure of total living, intact islet tissue after culture. This superiority is in part due to the lower islet fragmentation observed with persufflation as expressed by the ratio of DNA IE (1 DNA IE = 10.4 ng DNA) to IE counts. This fragmentation is reflected in photomicrographs taken of island census samples, as in Figs 9(a) until 9(c) shown. (Note that the numerous pyknotic nuclei (arrows) in the TLM-bearing pancreas ( 9(a) ) indicate apoptotic or necrotic cell death. Pyknotic nuclei were present in pancreatic islets as well as in exocrine tissue. This feature is less evident in the persufflated ( 9(b) ) and control (t = 0) pancreas ( 9(c) ).) Importantly, the benefits of persufflation over TLM were consistent and appeared to be even more pronounced after 24 hours of preservation (single experiment).

Islet transplantation in nude mice by the nude mouse bioassay (NMB) is a "gold standard" test for pancreatic islet function (see Ricordi et al., "Challenges toward standardization of islet isolation technology," Transplant Proc., 33:1709 ( 2001); Ichii, et al., "A novel method for the assessment of cellular composition and beta-cell viability in human islet preparations," Am J Transplant, 5(7):1635-45 (2005); Wonnacott, "Update on regulatory issues in pancreatic islet transplantation," Am J Ther 12:600-4 (2005), Papas et al., "Human islet oxygen consumption rate and DNA measurements predict diabetes reversal in nude mice," American Journal of Transplantation," 7(3):707-13 (2007)). The five (5) porcine islet isolates used the NMB after persufflation for experimental and control conditions as budget and islet availability permitted. All values be in 10 summarized. The main data set is the diabetes reversal rate: number of mice with sustained normoglycemia (blood glucose <200 mg/dl) compared to the number of treated mice. It is important to note that only intact islets are selected (according to the assay protocol) for the purposes of transplantation; therefore this assay does not reflect the inferior quality of lost (and therefore not transplanted) islets due to fragmentation (typical of the TLM condition). Despite these limitations, persufflation as a pancreas preservation technique compared favorably to both immediate isolation and TLM in terms of diabetes reversal rates, time to diabetes reversal, and mean blood glucose (equivalent to area under the curve) when values from different conditions (storage duration and Culture duration) are grouped. While these results were not statistically significant, mean blood glucose showed a trend toward statistical significance for persufflation versus immediate isolation (2-sample t-test, p=0.076).

Example 2: Additional persufflation experiments using an electrochemical oxygen concentrator (EOC) with porcine pancreas and human pancreas.

These experiments included islet isolations and compared the results of unpreserved flaps (control) with those of flaps preserved for 24 hours by the two-layer method (TLM) or by persufflation. As a result, the number of porcine pancreases used for the isolation was seven, four of which were used for evaluation of preservation at 6 hours, and three of which were used for evaluation of preservation at 24 hours. In addition to the experiments performed with porcine pancreas, we were able to obtain research-grade human pancreas, which was used to test the persufflation technique. Some main results were as follows: (1) 24-hour preservation with persufflation using electrochemically generated oxygen resulted in a superior result when compared to 24-hour storage on TLM (see 11 and 12 ); (2) Human pancreas persufflation is feasible and surgically less complex than porcine pancreas persufflation. In addition, the persufflation of the human pancreas with a part of the pancreas can be performed when the pancreas is divided into 2 parts. This evidence is important because it will allow direct and pairwise comparisons between persufflation preservation and TLM or persufflation preservation and no preservation with the same organ in future work.

In one experiment, the effect of persufflation on islet isolation was studied by dividing each pig organ into its three lobes, using the duodenal lobe for immediate isolation, the spleen lobe being preserved for 24 hours with persufflation, and the conjunctive flap was preserved with TLM for 24 hours. The method of separating each organ into its lobes was chosen to allow pairwise comparison of preservation methods by eliminating donor variability. Because of the small sample size, it was decided to standardize (rather than randomize) the flap used for each condition. Data from our pig isolation database (-650 isolations total) shows that there is no difference in the expected islet yield per gram of pancreatic tissue between the 3 lobes.

No significant differences were observed by histology between organ samples collected immediately after collection (t=0) and those collected following 24 hours of persufflation. However, tissue preserved with TLM showed a high abundance of pyknotic nuclei compared to persufflated specimens, particularly after 24 hours of preservation, as shown in FIGS 7(a) until 7(c) sees.

11 summarizes various measures of islet quantity and quality (means and standard deviations) for the porcine pancreas preserved by various methods. Based on pairwise two-tailed t-test, persufflation was significantly better compared to TLM at day 2 IE recovery (p=0.011, n=2) and day 2 OCR/DNA (p=0.011, n=3). There was no statistically significant difference between immediate isolation and persufflation on any measure, although mean values for all parameters suggest better outcomes with persufflation. 12 summarizes the TLM results as percentage of persufflation after 24 hours of preservation. In addition, islets isolated from persufflated and control (no storage) lobes were implanted into diabetic, athymic nude mice after culture. The TLM-stored lobes did not yield enough islets to allow nude mouse transplantation after culture. Early results (5 days post-transplant) show that 3 of the 4 mice transplanted with cultured islets obtained from lobes persufflated for 24 hours had blood glucose below 200 mg/dL (assuming diabetes reversal), whereas only 1 of 3 mice transplanted with islets isolated from the unpreserved control lobe had blood glucose below 200 mg/dL.

Claims (19)

System (1000) for fluid perfusion or persufflation of biological material comprising tissue, the system (1000) comprising: (a) a persufflation system (1100) for use in persufflation of biological material, the persufflation system (1100) having a gas generator for generating a preservative gas; (b) a liquid perfusion system (1600) for use in perfusing biological material, the liquid perfusion system (1600) comprising a reservoir for liquid perfusate; (c) a delivery cannula, the delivery cannula having a first end and a second end, the first end being insertable into an inlet of the biological material; and d) a switching system (1700) which is coupled to the second end of the delivery cannula and to the persufflation system (1100) and to the liquid perfusion system (1600), the switching system (1700) being designed in such a way that the biological material, the tissue comprises subjecting to fluid perfusion or persufflation. System(1000), as in claim 1 claimed wherein the preservative gas comprises gaseous oxygen. System(1000), as in claim 1 Claimed wherein said gas generator comprises electrochemical means for generating gaseous oxygen. System(1000), as in claim 3 Claimed wherein said electrochemical means for generating gaseous oxygen is an electrochemical oxygen concentrator. System(1000), as in claim 2 claimed wherein the preservative gas further comprises water vapor. System(1000), as in claim 2 Claimed wherein the gas generator comprises means for adjusting the concentration of gaseous oxygen in the preservative gas. System(1000), as in claim 1 claimed, wherein the preservative gas consists of gaseous oxygen. System(1000), as in claim 1 Claimed wherein said gas generator comprises at least one outlet for discharging a gas stream comprising the preservative gas. System(1000), as in claim 8 as claimed, wherein said gas generator comprises a plurality of outlets for discharging a corresponding plurality of gas streams comprising the preservative gas. System(1000), as in claim 9 claimed, wherein said gas streams have independently adjustable flow rates. System(1000), as in claim 1 Claimed further comprising a container (1200), the container (1200) being sized to receive the biological material comprising tissue. System(1000), as in claim 11 claimed wherein said container (1200) is a thermally insulated container. System(1000), as in claim 12 as claimed, further comprising temperature control means for maintaining the contents of said container at a desired temperature. System(1000), as in Claim 13 Claimed wherein said temperature control means comprises a sensor (1205) for sensing the temperature within said container, a temperature control device (1400) for changing the temperature within said container, and a process controller responsive to said temperature sensor (1205), for controlling the operation of said temperature control device (1400). System(1000), as in claim 1 Claimed wherein said perfusion system (1600) comprises a fluid drain line for draining the liquid perfusate perfused through the biological material comprising tissue. System(1000), as in claim 1 claimed wherein the liquid is selected from the group consisting of a liquid cell culture medium, an organ preservation solution, a saline solution, a HEPES buffer, and combinations thereof. System(1000), as in Claim 16 claimed wherein the liquid further comprises an additive selected from the group consisting of antioxidants, anti-apoptotic agents, vasodilators/vasoconstrictors, oxygen carriers, chelators, toxin scavengers and anticoagulants. System (1000) as claimed in at least one of the preceding claims, further comprising at least one gas sensor (1203, 1207) for monitoring a gas concentration in at least one fluid exiting the gas generator or the reservoir, or a fluid entering the container or leave it. System (2000) for fluid perfusion and/or persufflation of biological material comprising tissue, the system (2000) comprising: (a) a persufflation system (2100) for use in persufflation of biological material, the persufflation system (2100) including a gas generator for generating a preservative gas and being fluidly connected to a valve (2105); (b) a reservoir (2121) for liquid perfusate (2123) for use in perfusion of biological material, the reservoir (2121) being fluidly connected to a valve (2129); (c) a delivery cannula (2111), the delivery cannula (2111) having a first end and a second end, the first end being insertable into an inlet of the biological material (O); and the second end is connected to the valves (2105, 2129) by a link (2109); so that by appropriately opening and closing the valves (2105, 2129), the biological material (O) can be perfused with liquid perfusate (2123) or persufflated with a preservation gas or with a gas/gas mixture, or perfused and persufflated simultaneously.

Images