JP5650191B2 - Fluid perfusion system for biological material containing tissue - Google Patents

Description

(Cross-reference of related applications)
This application is a continuation-in-part of US patent application Ser. No. 12 / 803,083 filed on June 18, 2010 by inventor Tempelman et al., Under 35 USC § 119 (e). Claims the benefit of US Provisional Patent Application No. 61 / 268,973, filed Jun. 18, 2009, the disclosures of both applications being incorporated herein by reference.

(Description of research or development sponsored by the US federal government)
The U.S. Government is as prescribed by the paid licenses in the present invention and the terms of the US Small Business Innovation Research Program First Term Contract Numbers R43DK070400, R43DK070400-02S1, and R44DK070400 awarded by the National Institutes of Health. With rights in limited circumstances that require the patentee to grant a license to others on reasonable terms.

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

  Since it has been shown that rat allograft islet transplantation reverses chemically-induced diabetes, Langerhans islet transplantation has been studied for its potential as a treatment for type 1 diabetes mellitus for over 30 years. (See Non-Patent Document 1, which is incorporated herein by reference).

  Several years later, islet autotransplantation to donors has been shown to be a treatment that can prevent the onset of diabetes in people with pancreatitis (see Non-Patent Document 2, which is incorporated herein by reference). ).

  However, islet allogeneic transplantation remained difficult to achieve until 2000, when it was successful in seven secondary patients transplanted with multiple pancreatic islets by the development of the Edmonton protocol ( See Non-Patent Document 3. This is incorporated herein by reference.)

  The aforementioned success has been attributed largely to the use of immunosuppression without glucocorticoids. This success was subsequently repeated at other institutions, including the University of Minnesota, and a variant of the Edmonton protocol was used for more than 500 transplants worldwide in 2000-2006 (Non-Patent Document 4, Non-Patent Document 4). See 5, all of which are incorporated herein by reference.)

  Islet transplantation offers several advantages over other currently available treatments for diabetes. Due to the innate ability of islets to monitor and regulate glucose levels through insulin production, transplantation allows steady and tight control of blood glucose levels, which cannot be done by patient self-monitoring. Islet transplantation is particularly important in patients with subconscious hypoglycemia, which lacks physical symptoms indicative of hypoglycemia, even when strict glucose control cannot be fully achieved. In addition, as compared to other transplant procedures, injection into the islet portal vein does not require open surgery, so this transplant minimizes the likelihood of infection.

  Although the use of islet transplantation for the consistent reversal of diabetes has been demonstrated, some obstacles remain for the widespread implementation of this therapy in the clinical setting. Although some have been successful with transplants from a single donor, many institutions have a variety of reasons, including low islet yield and / or low quality per donor. It continues to require multiple donor organs for one patient (see Non-Patent Document 6, which is hereby incorporated by reference).

  Currently there is a shortage of high quality donor organs, which limits the number of transplants possible. As the interest in islet transplantation grows, and as new institutions apply to the US Food and Drug Administration (FDA) to qualify for investigational drugs (IND), both the number and quality of available organs Maximizing it is more important than ever. In order to maximize both the number and quality of the organs, it is necessary to protect the islets from damage starting at the time from organ procurement and islet processing to final engraftment in the transplant recipient.

  One of the main challenges of islet transplantation is to obtain viable functional transplant tissue in an amount sufficient for successful treatment (see Non-Patent Document 7, which is incorporated herein by reference). ). Thus, (1) maximizing the acceptable donor organ for clinical use, (2) improving the preservation, storage, and transport of the organ to keep the donor organ in an acceptable state, and ( 3) It is important to increase the yield and quality of islets collected from organs by improving islet isolation, culture, and storage. There appears to be a critical amount of viable islets for successful transplantation from a single donor, and current protocols produce this critical amount of generally just marginal transplant tissue .

  Current protocols for pancreatic procurement include donors who actually have brain death without warm ischemia (WI) time and the heart is beating. Pancreas preservation (including transportation and storage time) must be refrigerated (4-8 ° C.) within 8 hours. Refrigeration protocols differ in that some use established cryopreservation solutions (CPS) such as UW (University of Wisconsin) solutions and others use a combination of experimental CPS or CPS. Since 2002, the two-layer method (TLM) has received more attention. TLM was developed as a method of pancreas preservation from the late 1980s to the early 1990s (see Non-Patent Document 8, Non-Patent Document 9, and Non-Patent Document 10. All of which are incorporated herein by reference. ).

  TLM involves floating the pancreas between a layer of CPS and a layer of oxygen-containing perfluorocarbon (PFC). The basic concept is to increase the oxygenation of the tissue during storage by supplying a large amount of oxygen to the organ surface compared to CPS due to the high oxygen carrying capacity of PFC. TLM has made great strides as a state-of-the-art technology for pancreatic preservation in the early 2000s following the development of the Edmonton Protocol, and the superiority of this approach to organ preservation when compared to classical methods by many islet processing facilities (See Non-Patent Document 11, Non-Patent Document 12, Non-Patent Document 13, Non-Patent Document 14, Non-Patent Document 15, Non-Patent Document 16, and Non-Patent Document 17. All of which are incorporated herein by reference. ).

  Much of the interest in TLM was due to the possibility of using minimal donor organs for successful transplantation. Currently, there is a lack of suitable donor organs and in some countries the use of beating heart donors is prohibited by cultural taboos. Recently, however, as with TLM, it has become clear that oxygenation dependent on surface diffusion may be insufficient to oxygenate the majority of human pancreas. Pancreatic diffusion modeling by a group at the University of Minnesota has demonstrated that oxygen can only penetrate up to 1 mm of the external surface of the pancreas (see Non-Patent Document 18, which is incorporated herein by reference). In addition, multiple islet transplantation facilities have only recently been used for islet isolation and transplantation, whether the pancreas is preserved by TLM or classical storage in CPS (UW) only. Retrospective data demonstrating that there is no significant improvement in the results are also disclosed (see Non-Patent Document 19 and Non-Patent Document 20, both of which are incorporated herein by reference).

  In view of the above, it is clear that there is still an urgent need for improved pancreas preservation methods.

  For other human organs, passive refrigeration in CPS is common and has been reported in peer-reviewed literature. There are also several protocols and commercially available equipment for fluid perfusion preservation of organs. In any of these methods, the supply of oxygen is negligible and may be inappropriate for optimal organ preservation.

Surgery, 72 (2): 175-86 (1972), “Transplantation of intact pancreatic islets in rats,” Ballinger et al. Ann Surg, 192 (4): 526-42 (1980), “Total or near total pancreatic tom and autotranslation for treatment of chronic pancreatis, et al.” New England Journal of Medicine, 343 (4): 230-8 (2000), “Islet transplantation in seven patients with type of Ibetre The Lancet, 362 (9391), 1242 (2003), "Edmonton's issuture success has been replicated elsewere," Shapiro et al. Cell Transplantation, 16 (1): 1-8 (2007), "Factors influencing the loss of beta-cell mass in islet translation," Emmaulele et al. JAMA, 293 (7): 830- (5) (2005), “Single-donor, marginal-dose islet translation in partnership with type I diabets,” Hering et al. Current Opinion in Organ Transplantation, 13 (2): 135-141 (2008), “Pancreas preservation for pancreas and issuance translation, invited review,” Iwanaga et al. Transplantation, 46 (3): 457-60 (1988), “A new simple method for cold-storage of the pancreasusing perfluorochemical,” Kuroda et al. Transplantation, 51 (5): 1133-5 (1991), “Preservation of canine pancreas for 96 hours by a modified two-layered, UW solution / perfluorochemical, et al. Transplantation, 54 (3): 561-2 (1992), “Oxygenation of the human pancreating preservation by a two-threshold, a universality of Wisconsin solution / perfusion. Transplantation, 74 (12): 1813-6 (2002), “Impact of two-layer pancreas preservation on islet isolation and translation,” Hering et al. Transplantation, 74 (12): 1811-2 (2002), “Use of oxygenated perfluorocarbon manufacturing making pancreas count,” Fraker et al. Transplantation, 74 (12): 1687-91 (2002), “Human islet transplantation, and the first generation of the human population, and the prevalence of the prevalence of the human population. Transplantation, 74 (12): 1809-11 (2002), “Preservation of the human pantheas before isolation using a two-layered (UW solution-perfluorochemical),” Transplantation, 75 (9): 1524-7 (2003), “Improved human isolation outcome from marginal donors, overflowing, oxidative, ol- forded, and the like. Transplantation Proceedings, 36 (4): 1037-9 2004, "The effect of two-layer (University of Wisconsin solution / perfluorochemical) preservation method on clinical grade pancreata prior to islet isolation and transplantation," Matsumoto et al. Transplantation Proceedings, 37 (8), 3398-401 (2005), “Two-layer method in short- pancreas preservation for successful isolation,” Witwski et al. Transplantation Proceedings, 37 (8), 3501-4 (2005), “Pancreas oxidation generation limited with the two-layer method,” Papas et al. Transplantation, 82 (10): 1286-90 (2006), “Islet isolation and translation outcome in the public and preserved with the usage of the public population. Transplantation, 84 (7): 864-9 (2007), “No beneficial effect of two-layer storage compared with UW-storage on human isolation and translation,” Calibration, Trans. Liver Transpl, 14 (3): 358-64 (March 2008), “Retrograde oxygen persufflation preservation of human libers: a pilot study,” Trekmann et al. Nature Medicine, 13 (6): 673-4 (2007), “The hydrogen highway to reperfusion therapy,” Wood et al. Nature Medicine, 13 (6): 688-94 (2007), “Hydrogen acts as a therapeutic antioxidant by selective reducing oxytoxic radicals,” Ohashi, et al. Diabetes, 54 (5): 1400-6 (2005), “Donor Treatment with carbon monoxide canal salt allograft survival and tolerance,” Wang et al. Transplantation, 71 (4): 498-502 (2001), “Extension of ischemic tolerance of porcible preserving indication post-constituting post-growth post-situation, with-growth-post-conduction-with-gassing-as-usage American Journal of Transplantation, 7 (3): 707-13 (2007), “Human Isle Oxygen Consumption Rate and DNA measurements preparative dialects reversal in innumerables”. Biotechnology and Bioengineering, 98: 1071-82 (2007), “A stirred microchamber for oxygen consumption rate measurements with islets,” Papas et al. Xenotransplantation, 10 (5): 484 (2003), “Islet oxygen consumption rate as a predictor of in vivo post-transplantation,” Koulmana et al. Cell Transplantation, 12: 176-176 (2003), “Issue quality assessment based on oxygen consumption rate and DNA measurement prescriptions graph function in mice,” et al. Cell Transplantation, 10 (6): 519 (2001), “Rapid Isle Quality Assessment Priority to Transplantation,” Papas et al. Transplant Proc, 33: 1709 (2001), “Challenge tower standardization of islet technology,” Ricordi et al. Am. J. et al. Transplant, 5 (7): 1635-45 (2005), “A novel method for the assessment of cellular composition and beta-cell viability in human islet preparations,” et al. Am. J. et al. Ther, 12: 600-4 (2005), “Update on regulatory issues in pancreatic islet translation,” Wonnacott et al. American Journal of Transplantation, 7 (3): 707-13 (2007), “Human Isle Oxygen Consumption Rate and DNA measurements preparatory diabetics reversal in innumerables”.

  An object of the present invention is to provide a fluid perfusion system for biological material containing tissue.

  According to one aspect of the present invention, (a) a gas generator for producing a storage gas, and (b) a fluid tube that is in communication with the gas generator and can be inserted into the tissue to supply the storage gas to the tissue. A biological material fluid perfusion system comprising a tissue is provided.

According to the aforementioned system characteristics, the storage gas may comprise gaseous oxygen.
According to another feature of the aforementioned system, the gas generator may comprise an electrochemical means for generating gaseous oxygen.
According to a further feature of the aforementioned system, the electrochemical means for generating gaseous oxygen may comprise an electrochemical oxygen concentrator.
According to a further feature of the aforementioned system, the storage gas may further include water vapor.
According to a further feature of the aforementioned system, the gas generator may comprise means for adjusting the concentration of gaseous oxygen in the storage gas.
According to a further feature of the aforementioned system, the storage gas may consist of gaseous oxygen.
According to a further feature of the aforementioned system, the gas generator may comprise at least one outlet for outputting a gas stream containing the storage gas.
According to a further feature of the aforementioned system, the gas generator may comprise a plurality of outlets for outputting a corresponding plurality of gas streams containing the storage gas.
According to a further feature of the aforementioned system, the gas flow may have an independently adjustable flow rate.

According to a further feature of the aforementioned system, the fluid line may comprise a cannula.
According to further features of the aforementioned system, the system may further comprise a container sized to receive biological material including tissue.
According to a further feature of the aforementioned system, the container may be an insulated container.
According to a further feature of the aforementioned system, the system may further comprise temperature control means for maintaining the contents of the container at a desired temperature.
According to a further feature of the aforementioned system, the temperature control means includes a temperature sensor that senses a temperature in the container, a temperature control device that changes the temperature in the container, and a temperature control device in response to the temperature sensor. And a process controller that controls operation.

According to further features of the aforementioned system, the system may further comprise a liquid perfusion system.
According to a further feature of the aforementioned system, the liquid perfusion system includes a liquid perfusate bath, a fluid supply tube for supplying liquid perfusate from the bath to the biological material containing tissue, and a liquid perfusion perfused with the biological material containing tissue. And a fluid discharge pipe for discharging the liquid.
According to further features of the aforementioned system, the system may further comprise a larger volume of liquid, in which the storage gas is in the liquid to form a gas / liquid solution that is supplied to the tissue by the fluid tube. Is dissolved.
According to further features in the aforementioned system, the liquid may 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 further features of the aforementioned system, the liquid further comprises an antioxidant, an anti-apoptotic agent, a vasodilator / vasoconstrictor, an oxygen carrier, a chelator, a toxin binder, and an anticoagulant. An additive selected from the group may be included.

According to another aspect of the aforementioned system, a fluid perfusion system for biological material comprising tissue is provided, the system comprising: (a) a storage container for storing biological material comprising tissue; and (b) the contents of the storage container desired. Temperature control means for maintaining the temperature; (c) a gas generator for producing a storage gas; and (d) a fluid tube that communicates with the gas generator and can be inserted into the tissue to supply the storage gas to the tissue. .
According to one feature of the aforementioned system, the storage gas may include gaseous oxygen.
According to another feature of the aforementioned system, the gas generator may comprise means for generating gaseous oxygen.
According to a further feature of the aforementioned system, the gaseous oxygen generating means may comprise an electrochemical means for generating gaseous oxygen.
According to a further feature of the aforementioned system, the electrochemical means for generating gaseous oxygen may comprise an electrochemical oxygen concentrator.
According to further features of the aforementioned system, the system may further comprise means for diluting the preservation gas to form a gas / gas mixture that is transported to the tissue through the fluid conduit.

According to further features of the aforementioned system, the system may further comprise a liquid perfusion system.
According to a further feature of the aforementioned system, the liquid perfusion system includes a liquid perfusate bath, a fluid supply tube for supplying liquid perfusate from the bath to the biological material containing tissue, and a liquid perfusion perfused with the biological material containing tissue. And a fluid discharge pipe for discharging the liquid.
According to further features in the aforementioned system, the system further comprises at least one gas sensor that monitors the gas concentration in at least one of the fluid exiting the gas generator or tank or the fluid entering or leaving the container. It may be.

According to another aspect of the present invention, there is provided a system for use in preserving biological material including tissue, the system comprising: (a) a storage container for storing biological material including tissue; and (b) contents of the storage container. Temperature control means for maintaining the temperature at a desired temperature, (c) a gas generator for generating a storage gas, and (d) means for supplying the storage gas to the contents of the storage vessel in at least one gas stream. Prepare.
According to the features of the system described above, the at least one gas stream may include a plurality of gas streams, and the plurality of gas streams may have independently adjustable flow rates.
According to another feature of the aforementioned system, the system may further comprise means for controlling the operation of the gas generator.
According to further features of the aforementioned system, the system may further comprise a fluid perfusion system.

According to another aspect of the invention, there is provided a fluid perfusion system of biological material comprising tissue, the system comprising: (a) means for generating a preservative gas in situ; and (b) in situ in vivo. Means for perfusing the tissue with the generated storage gas.
According to the features of the system described above, the system may further comprise means for diluting the preserved gas generated in-vivo with a fluid, wherein said means for perfusing is a diluted in-vivo source. Including perfusing tissue with preservative gas generated at the location.

  For the purposes of this specification and the claims, the term “fluid” refers to a pure substance in a liquid state, a pure substance in a gaseous state, and a mixture of two or more liquids, two or It is intended to include, but is not limited to, mixtures of three or more gases, and mixtures of liquid and / or gaseous substances such as, but not limited to, mixtures of at least one liquid and at least one gas.

  Also, for purposes of this specification and claims, the term “biological material comprising tissue” is intended to encompass natural biological material comprising tissue, artificially generated biological material comprising tissue, and composites thereof. Intended. In addition, the term “biological material comprising tissue” is intended to encompass biological material comprising tissue isolated from a human or biological material comprising tissue isolated from an animal. In addition, the term “biological material including tissue” refers to a single biological tissue, organ, partial organ, compound organ, compound organ within an organ system, complete organ system, compound partial organ system (generally compound tissue Intended to encompass biological material consisting of or including one or more biological tissues, including but not limited to, but not limited to, multiple complete organ systems including the entire tissue. The

  In addition, for the purposes of this specification and claims, the term “fluid perfusion” refers to one or more natural and / or artificial fluid distribution networks within a biological material containing tissue. It is intended to refer to supplying one or more fluids. Examples of natural fluid distribution networks include at least a portion of the vasculature in a biological material containing tissue, at least a portion of a conduit system in a biological material containing tissue, and at least a portion of the lymphatic system in the biological material containing tissue. However, it is not limited to these. Examples of artificial fluid distribution networks include, but are not limited to, artificial or mechanical channels created or provided in biological material including tissue. “Fluid perfusion” may be alternatively referred to herein as “gas perfusion” when used to refer to the administration of pure gas or a mixture of gases to biological material, including tissue. .

  Additional objects of the present invention, as well as aspects, features, and advantages of the present invention, are set forth in part in the description that follows, and in part will be apparent from the description, or in part by the practice of the invention. You may understand. In the description, reference is made to the accompanying drawings that form a part hereof, and in which are shown by way of illustration various embodiments for carrying out the invention. The embodiments are described in as much detail as possible to enable those skilled in the art to practice the invention. The embodiments have been described in sufficient detail to enable those skilled in the art to practice the invention, and other embodiments may be utilized and structural changes may be made without departing from the scope of the invention. It should be understood that it is good. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present invention is best defined by the appended claims.

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate various embodiments of the invention and, together with the description, serve to explain the principles of the invention. In the drawings, like reference numerals designate like parts.
1 is a block diagram of a first embodiment of a biological material fluid perfusion system comprising tissue, the system being configured in accordance with the teachings of the present invention. FIG. FIG. 2 is a circuit diagram of one embodiment of the gas perfusion system shown in FIG. 1, wherein the gas perfusion system is shown with a power source. FIG. 3 is a partially exploded perspective view of one embodiment of the electrochemical oxygen concentrator shown in FIG. 2. FIG. 6 is a perspective view of a second embodiment of a fluid perfusion system for biological material containing tissue, the system being configured in accordance with the teachings of the present invention. FIG. 7 is a block diagram of a third embodiment of a fluid perfusion system for biological material containing tissue, the system being configured in accordance with the teachings of the present invention. FIG. 7 is a simplified circuit diagram of a fourth embodiment of a biological material fluid perfusion system including tissue, the system being configured in accordance with the teachings of the present invention; FIG. 5 is an image of islets isolated from dilated pancreas and exposed to dithizone without storage, as described in Example 1. FIG. 5 is an image of islets isolated and dithizone stained from pancreas exposed to 6 hours of gas perfusion storage, as described in Example 1. FIG. 5 is an image of islets isolated from dipsone stained and dithizone stained from 6 hours of TLM storage as described in Example 1. FIG. 8 is a table comparing storage methods based on islet yield and quality. It is a microscope picture of the section | slice of the porcine pancreas after 24 hours preservation | save by TLM. It is a microscope picture of the section | slice of the pig pancreas 24 hours after by gas perfusion. It is a microscope picture of a section of porcine pancreas at time = 0 (immediately after procurement). It is a table | surface which compares a preservation | save method based on a nude mouse bioassay. Table comparing pancreatic islet isolation results between 24-hour storage with gas perfusion, 24-hour storage with TLM, and no storage (2 days after is defined as 48-72 hours after isolation. The first three rows are The average value of the lower three sets of data.) FIG. 6 is a graph showing TLM results as a percentage of gas perfusion results for the preparation described in Example 2 (error bars indicate standard error of the mean).

  The present invention relates generally to the discovery that biological material containing tissue can be preserved by perfusion of the tissue with a preservation 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 present invention, there is provided a fluid perfusion system of biological material comprising tissue, the system comprising: (a) means for generating a preservative gas in situ; Means for perfusing the tissue using a preservative gas generated in situ in the body. The preserving gas generating means may comprise electrochemical means and / or other means. For example, the storage gas generating means may comprise an electrochemical oxygen concentrator and / or a water electrolysis layer. Alternatively, the storage gas generating means additionally or alternatively generates such gas by pressure swing adsorption and / or by chemical oxygen release (eg by burning a perchlorate or oxygen candle) You may provide the means to make. The means for perfusing the tissue with the stored gas generated in situ in the living body may comprise, for example, a cannula or other fluid guiding means that can be inserted into the tissue in communication with the gas generating means.

  The preservation gas used to perfuse the tissue may be administered to the tissue as a gas / liquid solution after being dissolved in the liquid, or dissolved in the atmosphere or in one or more other gases Later, it may be administered to the tissue as a gas / gas mixture. Perfusion with a gas or with a gas / gas mixture may alternatively be referred to as “gas perfusion”. Gas perfusion can be applied to tissue having a distribution network capable of distributing gas. The most common distribution network that distributes gas during gas perfusion is the vascular system consisting of arteries, veins, and capillaries. Another distribution network is a conduit system including, but not limited to, a wide range of pancreatic and mammary gland conduit systems. The lymphatic system has an induction system consisting of tubular containers in the body, which can also be a gas perfusion distribution network. In addition, the distribution network can be made by piercing the tissue, or by “piercing” the tissue (see Non-Patent Document 21, which is hereby incorporated by reference). In the case of a prosthetic tissue composition, it can be a natural or artificial distribution network, such as a gas permeable tube that functions as an artificial or surrogate vasculature or conduit system. The gas distribution network may comprise any combination of the aforementioned distribution network types. Distribution by these distribution networks can be in a physiologically occurring direction (ie, antegrade to the vasculature) or utilized in other manners (eg, retrograde to the vasculature) Can be done.

  Biological materials including tissues with distribution networks suitable for gas perfusion are tissues, organs, partial organs, complex organs, complex organs within organ systems, complete organ systems, complex partial organ systems (commonly referred to as complex tissues). ), And less complex complete organ systems including the whole organism. Examples of organs include, but are not limited to, the pancreas, liver, kidney, heart, lung, large and small intestine, eye, gallbladder, stomach, skin, and male and female genital organs. An example of a partial organ includes, but is not limited to, one lobe of the pancreas. Examples of dual organs include, but are not limited to, two or more of the aforementioned organs, such as two kidneys, one kidney and one pancreas, and two lungs. Examples of dual organs within an organ system may include, but are not limited to, the kidney and urinary bladder, which are part of the excretory system consisting of the kidney, ureter, bladder, and urethra as a whole. Examples of compound partial organ systems include, but are not limited to, fingers, hands, feet, limbs, ears, nose, face, skin graft, genital organs, abdominal wall, and other complex tissues.

  Examples of preservative gases include gaseous oxygen (which is a nutrient required by cells), gaseous hydrogen (which may act to protect cells by its antioxidant and anti-apoptotic properties (Non-Patent Document 22, Non-Patent Document 22). Patent Literature 23), gaseous carbon dioxide (which may regulate metabolism), gaseous carbon monoxide (which may have anti-inflammatory and anti-apoptotic effects (see Non-Patent Literature 24)). ), And water vapor may be included, but the storage gas may be generated in-situ by electrochemical or other means, for example, when the storage gas is oxygen, storage The gas may be generated by using an electrochemical oxygen concentrator in situ in the living body, or when the storage gas is oxygen and / or hydrogen, the storage gas uses an electrolytic cell. Or if the storage gas is oxygen, the storage gas may be burned by pressure swing adsorption or by chemical oxygen release (eg, perchlorate or oxygen candles). It may occur in situ).

  In addition, the use of a replacement purge or bath with cell culture medium or organ preservation solution for the surface of the tissue or organ or for the conduit system or vascular system at the surface to enhance the quality of the biological material during preservation by gas perfusion. (See Non-Patent Document 25, which is incorporated herein by reference). Also, the addition of a purge purge of antioxidants to the gas perfusion protocol can be used to improve the quality of biological materials during storage. In addition, nutrients, antioxidants, or other preservatives can be added in aerosol form during gas perfusion.

  A preservative solution and additives to the preservative solution can be used to immerse the exterior of the stored biological material during liquid or gas perfusion. Also, preservatives and additives to preservatives can be utilized in liquids that perfuse stored biological material for a period of time before or after gas perfusion, or instead of gas perfusion.

  The preservation solution can be a cell culture solution (eg, cryopreserved hepatocyte collection medium (CPRM)), organ preservation solution (eg, University of Wisconsin (UW) solution or histidine-tryptophan-ketoglutarate (HTK) solution), or medical procedure. Other solutions utilized in (eg, saline or 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid (HEPES) buffer). To enhance preservation, antioxidants (e.g., vitamins C and E and their derivatives, catalytic antioxidants CA: Redox Modulation Protection Isles From Transplant-Related Injury, Martha M. Sklavos, Subanne Berbert, Huber. Rita Bottino, Jing He, Joshua N. Beilke, Marilyn G. Coulombe, Ronald G. Gill, James D. Crapo, Massimo Trucco, and Jon D. Piganelli. , Anti-apoptotic agents (eg caspase selection 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, 6 May 2008 p.1556-1566 , “Caspase Inhibitor Therapeutic Synthesis With Costation Blockade To Promote Independent Isle Allograf Survival, Juliet A. Emmaullee, Joy Davis, Rena Pawlick, Christian Toso, Shaheed Merani, Sui-Xiong Cai, Ben Tseng, and A. M. James Shapiro, DIABET 59, DIABET59. / Vasoconstrictors (eg alpha adrenergic receptor antagonists (alpha blockers, arginine vasopressin)), oxygen carriers (eg perfluorocarbons, artificial blood components), chelators / other toxin binding agents (eg ethylenediaminetetraacetic acid (EDTA)), anticoagulants (eg, heparin), and other nutrients (eg, sugars, amino acids, vitamins)) may be included in the liquid.

  For example, if the stored biological material is human or porcine pancreas, the combination of artery and conduit through one or more arteries, one or more veins, one or more conduits The pancreas may be gas perfused through or through a combination of veins and conduits. For example, in the case of the human pancreas, gas perfusion is intubated with one or more of the following blood vessels: (i) bulk pancreaticoduodenectomy with aortic detachment to include the celiac and superior mesenteric arteries And / or splenectomy, (ii) bulk pancreatoduodenectomy and / or splenectomy with direct intubation of the celiac and superior mesenteric arteries, and (iii) between the proximal splenic artery and superior intestine It may be antegrade via bulk pancreatoduodenectomy and / or splenectomy with direct intubation of the membranous artery. Alternatively, gas perfusion involves the following vascular intubations: (i) bulk pancreatoduodenectomy and / or splenectomy with intubation of the inferior mesenteric vein or splenic vein, (ii) superior mesenteric vein May be retrograde via bulk pancreatoduodenectomy and / or splenectomy with intubation of the liver, and (iii) bulk pancreatoduodenectomy and / or splenectomy with portal intubation .

  Alternatively, in the case of porcine pancreas, gas perfusion is intubated with one or more of the following blood vessels: (i) bulk pancreaticoduodenectomy with aortic detachment to include the celiac and superior mesenteric arteries And / or splenectomy, (ii) bulk pancreatoduodenectomy and / or splenectomy with direct intubation of the celiac and superior mesenteric arteries, and (iii) between the proximal splenic artery and superior intestine It may be antegrade via bulk pancreaticoduodenectomy and / or splenectomy with direct intubation of the membranous artery. Alternatively, gas perfusion involves the following vascular intubations: (i) bulk pancreatoduodenectomy and / or splenectomy with splenic vein intubation, (ii) bulk tubule intubation with superior mesenteric vein intubation Pancreatoduodenectomy and / or splenectomy; and (iii) bulk pancreatoduodenectomy and / or splenectomy with intubation of the inferior mesenteric vein, and (iv) bulk pancreas with portal vein intubation It may be retrograde via duodenal resection and / or splenectomy.

  In the case of the liver, gas perfusion was antegrade via intubation of one or more of the following blood vessels: (i) common hepatic artery, (ii) intrinsic hepatic artery, (iii) portal vein. May be. It is. Alternatively, gas perfusion may be retrograde via intubation of one or more of the following blood vessels: (i) hepatic vein, and (ii) inferior inferior vena cava or inferior hepatic vena cava. Also good.

  For example, if the preservative gas generated in situ consists of pure gaseous oxygen, dilute the gaseous oxygen with a liquid solvent such as water or an aqueous solvent, and use a gas / liquid solution to It may be desirable to perfuse, or it may be desirable to dilute gaseous oxygen with another gas or other gas, such as the atmosphere, and to gas perfuse biological material with a gas / gas mixture. In the case of perfusion using a gas / liquid solution, the concentration of gaseous oxygen in the solution may be approximately at the liquid saturation point and may be a partial pressure of approximately 0.3 to 1 atmosphere. In addition, the flow rate of the solution relative to the biological material may be, for example, about 200 cc / min or more. For gas perfusion, the concentration of gaseous oxygen in the mixture may be about 20-100%, more preferably about 30-80%, even more preferably about 40%. In addition, the flow rate of the mixture relative to the biological material may be, for example, approximately 10-60 cc / min, preferably 15-22 cc / min, and the gas pressure of the administered solution is, for example, about 22 mmHg or less. Also good. In the inevitable case, storage may increase if gas perfusion is performed at a low temperature, such as approximately 4-8 ° C.

  When the biological material containing the preserved tissue is the pancreas, systemic heparinization of the pancreas using 100,000 units at least 5 minutes before death to prevent the formation of small clots throughout the pancreas Is preferably implemented. In addition, the organ is exposed to ischemia in the central region of the pancreas to prepare the organ for storage, to remove any residual blood from the vasculature, and at high temperatures (> 8 ° C.). In order to accelerate the cooling of the whole organ that lowers, it should be flushed with 5 liters of heparinized cold storage solution during procurement. After procurement, the organ should be extensively tested for leaks by rinsing the vasculature with a cold preservation solution and any leaks discovered should be plugged. The pancreas and accompanying vasculature should then be left free in cold storage solution to prevent any bending of the vasculature, especially at the site of intubation. Any remaining small leaks should be located by the presence of bubbles emerging from the vasculature to ensure that all gas is flowing through the pancreas, and these leaks should be plugged. Access points for intubation are described above. Other organs may also benefit from heparinization.

  Using this procurement method, the majority of the pancreas is washed away with a cold preservation solution, thereby clearing the path for gas perfusion throughout the organ. Tissue sections of organs procured using this technique showed that red blood cells were not present in the preserved tissue by more thorough washing, either by gas perfusion or by TLM. No significant histological differences were observed between organ samples collected immediately after procurement (t = 0) and organ samples collected 6 or 24 hours after gas perfusion. However, tissues stored using TLM exhibited high necrosis / autolysis and a high incidence of nucleus enriched nuclei, especially compared to gas perfused samples after 24 hours storage.

  The foregoing methods may allow a means to use human pancreas and a means to use porcine pancreas for xenotransplantation. Methods that enhance pancreatic preservation and result in improved islet yield and quality, and ultimately improved clinical outcome, are urgently needed to advance the field of islet transplantation into established medical practice. ing. It is shown herein that pancreatic arterial gas perfusion during storage to supply oxygen throughout the organ at the capillary level is a feasible technique. In particular, the feasibility of supplying electrochemically generated oxygen at a flow rate and pressure suitable for gas perfusion of porcine pancreas and human pancreas has been demonstrated. The results show the promise of this technology compared to no storage and as an improvement over TLM storage.

  Two applications of this method in pancreatic preservation are as follows. (1) In order to increase donor pool and facilitate logistics of organ and islet procurement, the goal is to extend cryopreservation time beyond the current 8-hour standard (isolation of donor organs from islet removal) Oxygen gas perfusion through cryopreservation (up to) and (2) oxygen gas after organ transport with standard refrigeration (about 12 hours TLM) as a recovery method to increase islet yield and quality in the islet processing facility Perfusion (about 3 hours). These two applications involve improvements in islet yield and quality and / or storage time for pancreas from standard donors. In one application, the pancreas is gas perfused for the entire storage time, and in the second application, gas perfusion is used as a recovery procedure after standard refrigeration (for pancreas preservation, between storage time and islet yield and quality. Note that there is almost always a tradeoff. Extended storage time (in similar islet yield and quality) can lead to procedural flexibility and use of more organs. The high yield and quality of islets (in current storage time) may have a greater impact on individual transplant results. A tributary of completely independent applications that would also be very useful if successful is to increase the quality of the organs and thus improve the organ supply in exchange for donors after entering the category of cardiac death. It would be to use perfusion.

  Referring now to FIG. 1, there is shown a block diagram of a first embodiment of a biological material fluid perfusion system comprising tissue, the system being constructed in accordance with the teachings of the present invention, generally designated by reference numeral 11. It is represented by

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

  Referring now to FIG. 2, the gas perfusion system 100 is shown in more detail and the gas perfusion system 100 is shown with a power source 500. The gas perfusion system 100 may include an electrochemical oxygen concentrator (EOC) 101. The EOC 101, the details of which are described in more detail below, may comprise a single oxygen concentrator cell or a plurality of series or parallel connected oxygen concentrator cells. Each of the one or more oxygen concentrator cells may include an anode 103 and a cathode 105, where the anode 103 and the cathode 105 are in direct contact with and separated by the ion conductive separator 107. ing. Separator 107 may be, for example, a solid proton exchange membrane (PEM) such as a NAFION® NRE-1135 membrane (EI du Pont de Nemours & Company, Wilmington, DE). Anode 103 and cathode 105 may be electrically coupled to power supply 500 using leads 111-1 and 111-2, respectively, and may be process controller 300 (shown in FIG. 2) by means not shown. (Additional components of the gas perfusion system 100 may be coupled to the power source 500 and / or the process controller 300, but these connections are described herein for clarity.) Is not shown.)

As shown below, EOC 101 may be used to concentrate oxygen from the atmosphere.
Anode: 2H ₂ O ← → 4H ⁺ + 4e ⁻ + O ₂ (pure) [1]
Cathode: O ₂ (air) + 4H ⁺ + 4e ⁻ ← → 2H ₂ O [2]
Net: O ₂ (air) at the cathode ← → O ₂ (pure) at the anode [3]

  The EOC 101 may additionally include a water tank 113 for storing a large amount of water to be supplied to the anode 103. As described further below, a pervaporation membrane 115 (which may be, for example, a NAFION® NRE-1135 membrane) is installed between the bottom outlet of the water tank 113 and the anode 103. Alternatively, the pervaporation membrane 115 functions to steadily supply vapor phase water to the anode 103 and at the same time prevents oxygen produced in the anode 105 from mixing with water. In other words, this arrangement allows water to be passively supplied to the anode 103 while at the same time the product oxygen can flow out of the anode along the edges through the gap in the flow field. The steam supply system described above is advantageous over the corresponding liquid supply system in that it does not supply the anode 103 with many impurities that may be present in the feed water. Conceivable. For example, when tap water is used as feed water, halogens, 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 can result in degradation of EOC performance and efficiency due to, for example, membrane contamination or catalyst poisoning when carried to an active device of EOC 101. These materials may also be harmful to stored biological material. The pervaporation membrane 115 effectively restricts the entry and exit of these species into the active element of the EOC 101 and the stored biological material downstream of it to effectively distill the stored water in situ. to enable.

  The EOC 101 may additionally include a recovery pipe 117 having one end connected to the cathode 105 and the other end connected to the water storage tank 113, and the recovery pipe 117 supplies water produced by the cathode 105 to the water storage tank 113. Used to guide. Thus, the frequency with which water needs to be added to the system is reduced.

  The gas perfusion system 100 may further comprise means for cooling and drying the gaseous oxygen produced at the anode 103. In this embodiment, the cooling and drying means may include an oxygen cooler 121, an oxygen / water separator 123, and an oxygen dryer or desiccant 125. The fluid line 127 may have one end connected to the outlet of the anode 103 and the other end connected to the inlet of the oxygen cooler 121. In addition, the fluid line 129 may have one end connected to the outlet of the oxygen cooler 121 and the other end connected to the inlet of the oxygen / water separator 123. In addition, the fluid line 131 may have one end connected to the outlet of the oxygen / water separator 123 and the other end connected to the inlet of the oxygen dryer 125. Moreover, one end of the fluid line 133 may be connected to the outlet of the oxygen dryer 125. The fluid line 135 for draining the oxygen / water separator 123 may be connected to the bottom of the oxygen / water separator 123, and the drain plug 137 may be connected to the outlet end of the fluid line 135.

  The gas perfusion system 100 may further include air supply means. In the present embodiment, the air supply means may include a fluid line 141, and the fluid line 141 has an inlet 143 that takes air into the fluid line 141. An air filter 145 may be installed along the line 141 to remove certain impurities from the atmosphere. The air filter 145 may be of a type that physically captures particles (eg, a 10μ pore size filter), or a type that chemically captures or modifies particles (eg, carbon filters or permanganic acid). Salt filter). An air pump 147 that sends air through line 141 (to supply air to cathode 105 and dilute the oxygen produced by EOC 101) may be installed along line 141 downstream of air filter 145. Downstream of the air pump 147, the line 141 may branch into a fluid line 149 and a fluid line 151. Valve 153 may also include a flow indicator and may be installed along line 149, and the outlet of line 149 is appropriately installed to supply air to cathode 105. Variable along line 151 to adjust the flow of air in line 151 so that the desired oxygen concentration may be obtained, and thereby adjust the rate at which the atmosphere should be mixed with pure oxygen. A flow valve 155 may be installed. In this manner, for example, a continuous adjustment may be made to the concentration of oxygen present in the resulting oxygen / air solution. This may be desirable, for example, in situations where it is desired to gas perfuse biological tissue with a gas having a first oxygen concentration, followed by gas perfusion of biological tissue with a gas having a different oxygen concentration. A check valve 157 may be installed along a line 151 downstream of the valve 155.

  The gas perfusion system 100 may further comprise a fluid line 161 connected to the outlets of lines 133 and 151 for mixing pure oxygen from line 133 with the atmosphere of line 151. For example, a medical grade filter 163 that may have a pore size of 0.2μ may be placed along line 161. An oxygen concentration sensor 165 may be installed along the line 161 downstream of the filter 163. A pressure transducer / gauge 167 and pressure relief valve 169 may be installed along line 161 downstream of sensor 165. A pressure regulating valve 171 may be installed along line 161 downstream of the transducer / gauge 167 and valve 169. A pressure transducer gauge 173 may be installed along line 161 downstream of gauge 173. Downstream of the gauge 173, the line 161 may branch into a line 175-1, 175-2, and 175-3. A valve 177-1 with a flow indicator may be installed at the outlet end of line 175-1, and a valve 177-2 with a flow indicator may be installed at the outlet end of line 175-2, the flow indicator A valve 177-3 having the following may be installed at the outlet end of the line 175-3.

  Thus, as can be seen, the gas perfusion system 100 can administer up to three downstreams of the storage gas to the biological material, each of the three downstreams having an independently adjustable flow rate. . In addition, it should also be noted that the three downstream oxygen concentrations and the maximum supply pressure can be adjusted despite being related independently of each other. Additional pressure regulating valves or mass flow controllers may be newly employed downstream of these to provide further independent pressure and flow regulation for each downstream. Moreover, if desired, the gas perfusion system system 100 has the ability to produce humidified gas, for example, by omitting the oxygen dryer 125.

  Referring now to FIG. 3, the EOC 101 is shown in more detail. As can be seen, the EOC 101 may include a membrane electrode assembly 181. The assembly 181 may further comprise an anode 103, a separator 107, and a cathode 105 (which is not shown in FIG. 3 but is located on the opposite side of the anode 103 below the separator 107). . The EOC 101 may further include an anode collector 182 and a cathode collector 183. The anode collector 182 may have a porous central region 182-1 that defines a fluid diffusion chamber and may be located adjacent to the top surface of the anode 103, and the cathode collector 183 defines a fluid diffusion chamber. May have a porous central region 183-1 that may be placed adjacent to the bottom surface of the cathode. The EOC 101 may further include a pervaporation film 115, which may be disposed adjacent to the upper surface of the anode collector 182. As explained above, the pervaporation membrane 115 may be used to supply water in the gas phase to the anode 103.

  The EOC 101 may further include an annular anode gasket 185 and a cathode insulator 186. The gasket 185 may be installed adjacent to the upper surface of the pervaporation membrane 115, and the cathode insulator 186 may be installed adjacent to the bottom surface of the cathode collector 183. The EOC 101 may further include an anode end plate 187 and a cathode end plate 188. The anode end plate 187 may include a porous central region 187-1 and may be disposed adjacent to the anode gasket 185, and the cathode end plate 188 may be disposed adjacent to the cathode insulator 186. Also good.

  The EOC 101 may further include a cathode inlet port 189 and a cathode outlet port 190, the inlet port 189 and outlet port 190 are mechanically coupled to the cathode end plate 188 and are in communication with the cathode.

  The EOC 101 may further include a water storage tank 113, and the water storage tank 113 may be installed on the anode end plate 187. A reservoir O-ring 191 may be fitted to the anode end plate 187 and may be used to form a fluid seal against water flowing from the reservoir 113 to the porous central region 187-1 of the anode end plate. An anode fastening ring 192 may be attached to the peripheral flange 113-1 of the water storage tank 113.

  The EOC 101 may further include an oxygen outlet pipe 193 that guides gaseous oxygen exiting the anode 103, the oxygen outlet pipe 193 having one end attached to the anode end plate 187 and the flange 113-1 of the water storage tank 113. And the anode clamp ring 192.

  The EOC 101 further includes a water injection port 194 that may be connected to the tank 113 via the opening 113-2, a tank vent port 195-1 that may be connected to the tank 113 via the opening 113-3, and an opening 113-4. A water recovery port 117 that may be connected to the tank 113 via the

  The EOC 101 may further comprise hardware that mechanically couples many of the aforementioned components together. Such hardware may include a plurality of unit cell assembly bolts 196, a plurality of corresponding washers 197, a Belleville washer 198, and a nut 199.

  Referring back to FIG. 1, the container 200 may be suitably sized to store the biological material, and a natural or artificial fluid distribution within the biological material stored in the container 200. One or more fluid lines 550 connected to the outlets of valves 177-1, 177-2, and 177-3 are passed through to supply the gaseous oxygen solution produced by gas perfusion system 100 to the net. You may provide the opening (not shown) which may be. The fluid pipe 550 includes, for example, a cannula whose one end can be inserted into a biological material, and one pipe connected at one end to one of the valves 177-1 to 177-3. And may be provided. Further, one end of the fluid pipe 550 is connected to one or more of the valves 177-1 to 177-3, and the other end thereof is connected to one or two of the valves 177-1 to 177-3 on the surface of the biological material. From the above, one pipe or a similar structure adapted to allow the gas output to flow may be provided. An oxygen sensor 203 or other gas sensor and a temperature sensor 205 may be provided inside the container 200, for sensors 203 and 205 for feedback control of oxygen, gas, and / or temperature readings, respectively. To be provided to the process controller 300.

  The controller 300 may include an embedded controller with a keypad / LCD user interface to manage various system components and facilitate user control. Two closed loop controls, one for EOC cell current and the other for diluted oxygen concentration, may be incorporated to allow for self-regulation of processing conditions. Current sensors and variable voltage DC-DC converters are software-based to control EOC cell current (and hence pure oxygen production) to the amount required by all user flow and oxygen concentration requirements. It may be employed with a proportional-integral control algorithm. Similarly, the controller 300 may also be used with the oxygen sensor 203 and valve 155 to control the degree of air / oxygen mixing to achieve the desired oxygen concentration set point. An embedded controller may also be used to provide the user with a real-time indication of system release and manifold supply pressure.

  The temperature control device 400 may comprise a commercially available thermoelectric (or Peltier) box, such as, for example, a VECTOR® thermoelectric cooler. Vapor compression cooling or physical cooling from ice or other coolant may be used instead of thermoelectric means, but if it is desired to supply heat to the container 200 as opposed to lowering the temperature of the container 200, The bidirectional heat pumping capability of the thermoelectric means makes this possible in a way that cannot be done with vapor compression cooling or physical cooling. In the case of passive cooling or active cooling, the insulation of the container 200 can be increased by using a highly efficient vacuum panel.

  The power source 500 includes, for example, two 24V nickel metal hydride (NiMH) batteries, a battery charger for these batteries, a 24V DC-DC converter (for adjustment), and a battery cutoff circuit for preventing heavy discharge of the battery. You may provide the battery pack module to accommodate.

  In order to use the system 11, biological material containing the tissue to be stored may be placed in the container 200, and one or more of the outlets 177-1 to 177-3 of the gas perfusion system 100 may be antegrade of the tissue. One end communicates with one of the outlets 177-1 to 177-3 and the opposite end communicates with a tissue fluid distribution network (eg, vasculature, conduit system, lymphatic system) to allow for sexual or retrograde gas perfusion ) May be in communication with the biological material by a fluid tube 550 inserted in a suitable portal. The gas perfusion system 100 may then be powered from the power source 500 and operated by the process controller 300 in accordance with predetermined parameters to gas perfuse tissue, and in addition, a temperature control 400 powered from the power source 500. May be used to maintain the container 200 and the biological material contained in the container 200 at a preselected temperature. Oxygen sensor 203 and temperature sensor 205 may be used with process controller 300 to provide feedback control of gas perfusion 100 and temperature control device 400.

  Referring now to FIG. 4, there is shown a perspective view of a second embodiment of a biological material fluid perfusion system comprising tissue, the system being constructed in accordance with the teachings of the present invention, generally designated by reference numeral 600. (Some components of system 600 are not shown for clarity or simplification). This embodiment is shown as a portable system.

  System 600 is similar in many respects to system 11, and system 600 includes a gas perfusion system 100, a container 200, a process controller 300, a temperature control device 400, and a power supply 500. The system 600 may further include an insulated housing 610 in which the gas perfusion system 100, the container 200, the process controller 300, the temperature control device 400, and the power source 500 are disposed. (The side wall of the housing 610 is not shown in order to show some of the components disposed inside the housing 610, and the top of the container 200 and the opening 620 into which the container 200 can be removably inserted. In order to show this, the heat insulating lid removably attached to the housing 610 is not shown. While not intending to be limited to any particular dimensions, the housing 610 has a footprint equivalent to that of a picnic cooler (eg, about 30.5 cm (12 inches) high by about 25.4 cm (10 inches) wide by about 55 wide). .9 cm (22 inches)). Caster 630 and handle 640 may be attached to housing 610 to facilitate transport of system 600. The system 600 may also include a fluid tube (not shown) similar to the fluid tube 550 that transports fluid from the gas perfusion system 100 to the biological material.

  The system 600 may further include a logging / diagnostic function. This feature allows system users to view / record system process readings (pressure, concentration, temperature, etc. that may not be available on the controller interface on the circuit board) by downloading to an external computer at the end of the storage time. Would be able to. The power conditioning circuitry, relays, and some sensor transducers may all be integrated on the circuit board utilized. The heat pump may require a non-standard or variable DC power source to operate the Peltier element, which will require confirmation that the power source is appropriate. This design may include a 24-hour battery design using an optional AC converter and a battery charger on the circuit board. The battery may be a NiMH rechargeable battery that is cheaper, safer and more convenient than a lithium ion battery and has a reasonable energy storage density.

  Referring now to FIG. 5, a block diagram of a third embodiment of a biological material fluid perfusion system comprising tissue is shown, the system being constructed in accordance with the teachings of the present invention, generally designated by the reference numeral 1000. Has been. For simplicity and clarity, some components of the system 1000 are not shown and are not described herein.

  The system 1000 may include a gas perfusion 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 source 1500.

  The gas perfusion system 1100, the container 1200, the process controller 1300, the temperature control device 1400, and the power source 1500 are the structure and function of the gas perfusion system 100, the container 200, the process controller 300, and the temperature control. The device 400 and the power source 500 may be similar to each other.

  The liquid perfusion system 1600 may comprise a tank for storing a large amount of preservation solution (“perfusate”), means for supplying the perfusate to the organ container 1200, and means for discharging the perfusate from the organ container 1200. Good. The reservoir may comprise a liquid-containing bag, such as a bag used to contain fluid for intravenous administration. The means for supplying the perfusate to the organ container 1200 may comprise a tube connected from the tank to the organ container 1200, whereby the perfusate flows through the tube by gravity. Where the perfusate flows from the reservoir to the organ container 1200 via the interconnecting tube, the liquid perfusion system 1600 may additionally include means for supporting the reservoir above the height of the container 1200. Alternatively, an electrically or manually operated pump may be used to supply the perfusate from the tank to the container 1200 via an interconnecting tube. An alternative means of supplying the perfusate may comprise a tube connected from the gas perfusion system 1100 to the tank so that the gas pressure applied by the gas perfusion system 1100 causes the liquid in the tank to flow from the tank to the container 1200. Force to flow through interconnecting tubes. The means for draining the perfusate from the container 1200 may comprise a tube that causes the perfusate to flow out of the container 1200 by gravity, an electrically operated pump or manual pump, or by gas pressure from the gas perfusion system 1100, Thereby, the liquid is then collected in a receptacle or drain.

  The switching system 1700 may comprise a plumbing connector designed to connect to a pipe coming from the gas perfusion system 1100, the liquid perfusion system 1600, and the container 1200. The switching system 1700 may further comprise an electrically actuated valve that is actuated by a signal from the process controller 1300 so that the process controller 1300 allows the flow being supplied to the container 1200 to be a gas perfusion system 1100 or a liquid perfusion system. Which one is derived from 1600 is controlled. Alternatively, the switching system 1700 may include a manual valve that allows a user to manually control whether the flow supplied to the container 1200 comes from a gas perfusion system 1100 or a liquid perfusion system 1600. Alternatively, the switching system 1700 may include two or more electrically operated valves and manual valves, whereby the flow supplied to the container 1200 can be controlled manually or electrically.

  The system 1000 may further comprise gas sensors such as an oxygen sensor 1203 and a carbon dioxide gas sensor 1207 that are used to measure the concentration of oxygen gas and carbon dioxide gas in the inlet and outlet streams, respectively. The gas sensor may be connected to the outlet flow of the gas perfusion system 1100 and the liquid perfusion system 1600. The gas sensor may also be connected to the inlet and outlet flow of the container 1200. The process controller 1300 may be electrically connected to the gas sensors 1203 and 1207 and may have a data logging function for each of the oxygen sensor 1203 and the carbon dioxide sensor 1207. In addition, the process controller 1300 may use the signal collected from the gas sensor to make a process decision, such as actuating an alarm or shut-off valve when the concentration of a particular gas falls outside a specified range.

  The system 1000 may further include a temperature sensor 1205 that measures the temperature in the container 1200. The process controller 1300 may be electrically connected to the temperature sensor 1205 and may have a data logging function for the temperature sensor 1205. In addition, the process controller 1300 is collected from the temperature sensor 1205 to make process decisions such as activating or deactivating the temperature control device 1400 when the temperature in the container 1200 falls within or outside the specified range. Other signals may be used.

  The system 1000 further separates the liquid from the gas flow, prevents entry of contaminants into critical system components, prevents particulate matter from entering the stored organ or tissue, or the liquid is gas A filter, such as a filter used to prevent interference with concentration monitoring, may be provided. Filters may be installed in the outlet piping assembly of the gas perfusion system 1100 and the liquid perfusion system 1600, the piping assembly of the piping assembly connecting the gas sensors, and the inlet and outlet piping assemblies of the organ container 1200.

  System 1000 may further comprise an electro-optic sensor used to measure the presence of liquid or gas in the tube. The electro-optic sensor may be connected to the inlet and outlet tubes of the container 1200 and may be connected to the outlet tubes of the gas perfusion system 1100 and the liquid perfusion system 1600. The process controller 1300 can be used to process the signal provided by the electro-optic sensor and activate the switching system 1700 to switch between the gas perfusion system 1100 and the liquid perfusion system 1600.

  The system 1000 may be used in a manner similar to that previously described with respect to the system 11, the main difference between the two systems being that in the case of the system 1000, the biological material containing the tissue is used with the gas perfusion system 1100. May be immersed in liquid perfusate from liquid perfusion system 1600 before, during, or after gas perfusion, or may be perfused with liquid perfusate instead of gas perfusion using gas perfusion system 1100 It is.

  Referring now to FIG. 6, a simplified diagram of a fourth embodiment of a biological material fluid perfusion system containing tissue is shown, the system being constructed in accordance with the teachings of the present invention and generally designated by the reference numeral 2000. Has been. For simplicity and clarity, some components of the system 2000 are not shown and are not described herein.

  The system 2000 may include a gas perfusion system 2100 that may be similar in configuration and function to the gas perfusion system 100, except that the system 2100 replaces the three outlets 177-1 to 177-3. One outlet 2101 may be provided. One pipe 2103 may have one end fluidly connected to the outlet 2101 and the other end connected to one port of the valve 2105. One pipe 2107 may be fluidly connected at one end to another port of the valve 2105. The opposite end of the pipe 2107 may be fluidly connected to the connector 2108. Connector 2108 may be fluidly connected to connector 2109. The connector 2109 may further be fluidly connected to one end of the supply cannula 2111 with the opposite end of the supply cannula 2111 inserted into the appropriate perfusion / gas perfusion inlet of the organ O, which is in the container 2113. The container 2113 may be similar to the container 200 or 1200.

  The system 2000 may further include a fluid tank 2121 that may be in the form of an intravenous infusion bag. The tank 2121 may contain a large amount of liquid perfusate 2123. One pipe 2125 may have one end fluidly connected to the outlet 2127 of the tank 2121 and the other end connected to one port of the valve 2129. One pipe 2131 may be fluidly connected at one end to another port of the valve 2129. The opposite end of the pipe 2131 may be fluidly connected to the connector 2132. Connector 2132 may be fluidly connected to connector 2109.

  System 2000 may further include an outlet cannula 2141 that is inserted at one end into a suitable perfusion / gas perfusion outlet of organ O. The opposite end of the outlet cannula 2141 may be fluidly connected to the connector 2143.

  The system 2000 may further include a single pipe 2151. One end of the pipe 2151 may be fluidly connected to the connector 2153, and the connector 2153 may be fluidly connected to the connector 2143. The opposite end of the pipe 2151 may be connected to one port of the valve 2155. One pipe 2157 may be fluidly connected to another port of the valve 2155.

  The system 2000 may further include a single pipe 2171. One end of the pipe 2171 may be connected to the connector 2173, and the connector 2173 may be fluidly connected to the connector 2143. The opposite end of the pipe 2171 may be connected to one port of the valve 2175. One pipe 2177 may be fluidly connected to another port of the valve 2175.

  In use, connectors 2108 and 2132 are connected to connector 2109, connectors 2153 and 2173 are connected to connector 2143, and cannulas 2111 and 2141 are inserted into organ O. To perfuse organ O with perfusate 2123 (this perfusion may be desirable to be performed before gas perfusion, after gas perfusion, during gas perfusion, and / or instead of gas perfusion. ), Valves 2129 and 2155 are opened, whereby perfusate 2123 is supplied to organ O through cannula 2111, circulates through organ O, and is discharged from organ O through cannula 2141. In order to gas perfuse the organ O with a preserving gas or gas / gas mixture, valves 2105 and 2175 are opened and the gas perfusion system 2100 is activated so that the gas or gas / gas mixture is cannulated from the gas perfusion system 2100 to the cannula 2111. To the organ O, circulates through the organ O, and is discharged from the organ O through the cannula 2141. As will be appreciated, if it is desired to perfuse organ O with liquid perfusate without simultaneously perfusing with preservation gas or gas / gas mixture, valves 2105 and 2175 are closed, while valves 2129 and 2155 are opened. Conversely, if it is desired to perfuse organ O with a preservation gas or gas / gas mixture without perfusing with liquid perfusate, valves 2129 and 2155 are closed, while valves 2105 and 2175 are opened. Valves 2105, 2129, 2155, and 2175 are opened when it is desired to gas perfuse at the same time as perfusion, thereby providing gas to organ O that has been released to liquid perfusate.

The following examples are merely illustrative and do not limit the invention.
(Example)
Example 1:
Illustrative example of improved islet gas perfusion oxygenation with electrochemical oxygen generation The effect of gas perfusion on islet isolation is to divide each of the five organs into three lobes and the pancreatic head for immediate isolation They were collected and tested by storing the pancreatic tail for 6 hours with gas perfusion and storing the pancreatic body for 6 hours with TLM. In one experiment, storage was extended to 24 hours. The gas perfusion system utilized an EOC having an actively catalyzed area of 40 cm ² and employing NAFION® NRE-112 membrane as separator 107 and water pervaporation membrane 115. The anode 103 and the cathode 105 each contained a thermal spray ink decal applied to a separator 107 having a platinum-iridium black catalyst and a platinum black catalyst loaded at 4 mg / cm ² . In both cases, when deionized water is supplied to the EOC tank and air is supplied to the cathode at 300 cm ³ at 21 ° C., the performance of this EOC is 0.84 V at 8 A (200 mA / cm ² ) and 12 A (300 mA / cm ² ). ² ) 0.93V. The gas perfusion system was further configured as shown in FIG. Single pancreatic lobe gas perfusion was performed using 15-22 cm ³ of 40% oxygen and a pressure of about 20 mm Hg, which required an EOC current of 0.94-1.4A. The method of separating each organ into its leaves was chosen to allow a comparable comparison of preservation methods by eliminating donor variability. Since the number of 5 organs was small, we decided to standardize (rather than randomize) the leaves used for each condition.

  Organs were evaluated with the naked eye after procurement and immediately before islet isolation. All leaves were completely washed away after procurement and had good texture. However, when compared to leaves preserved by TLM, gas-perfused organs “seen” and “feel” subjectively as “good” to the surgeon performing enzymatic swelling during isolation (more tight and fresher) Is observed). This was particularly evident for organs that were stored for 24 hours prior to isolation.

  The first important observation and data showing improvement with gas perfusion storage was related to islet morphology. As can be seen with reference to FIGS. 7 (a) to 7 (c), porcine pancreas preserved by gas perfusion yield morphologically best preserved islets and fragmentation is from TLM stored It was widespread in isolated islets (dithizone is an insulin stain that stains β cells in islets).

Table 1 below shows that islets from gas-perfused organs had a better average morphological score than islets from organs that were TLM stored and even fresh (t = 0) In a t-test comparison, the improvement was statistically significant (p = 0.008), with gas perfusion (PSF) storage exceeding TLM.
Table 1. Pancreatic islet morphology score 0 days after isolation
Morphological score after 0 days Porcine pancreas ID Storage time 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 deviation 0.82 2.3 2.2
Comparison by paired t-test 0.134 | 0.0086

  Overall, islets from porcine lobes stored by gas perfusion preservation showed better morphology than islets isolated immediately after procurement or from leaves stored in TLM. FIG. 8 summarizes various measurements of islet quantity and quality from porcine pancreas preserved by various methods. Oxygen consumption (OCR) measurement has been shown to be a rapid, simple and quantitative expected assay for islet quality assessment (Non-patent document 26, Non-patent document 27, Non-patent document 28, Non-patent document (See Patent Document 29 and Non-Patent Document 30), standard islet equivalence counting, DNA measurement, and fragmentation evaluation were carried out as one of the scales in this study. The scale presented in FIG. 8 is briefly defined below.

  IE yield after 0 days: islet equivalent by counting per gram of digested tissue. Islet equivalent (IE) is a volume unit equal to the volume unit of a 150 μm diameter sphere.

  % IE recovery after 2 days:% IE by counting after 2 days compared to after 0 days (the count after 2 days is the IE yield after 0 days multiplied by the% IE recovery).

  Two days later OCR / DNA: islet oxygen consumption (OCR) (nmol / min) is a measure of the amount of viable islets, DNA (mg) is a measure of the total number of islets, and OCR / DNA is therefore the number of viable islets Is a ratio of the total number of islets and is a measure of islet viability.

  2 days later OCR amount: amount of viable islet tissue per weight (g) of digested tissue [islet OCR (nmol / min)]

  Fragmentation ratio after 2 days: Ratio of islet equivalent calculated from DNA measurement (1 DNA IE = 10.4 ng DNA) and IE by counting. Measurement of the quality of the preparation. There are indications that fragmented islets are less likely to survive transplant and provide insulin.

  2 days later IE-based OCR amount: OCR amount for unfragmented islets (OCR / digested tissue g).

  As can be seen, the basic gas perfusion storage results were superior to those obtained by immediate separation and TLM storage. However, most differences were not statistically significant due to the small number of pancreas and the inherently large variability between donors (and / or separations). OCR / DNA, a measure of viability, is consistently higher in gas perfusion after 2 days of culture compared to TLM and tends to be statistically significant for 6 hour storage (p = 0.090, n = 4), and statistical significance (p = 0.036, n = 5) was obtained for 6 hour and 24 hour storage. The last column of FIG. 8 shows the superiority of gas perfusion in the amount of OCR associated with the counted islets (calculated using IE counts), which is a measure of total viable intact islet tissue after culture. It is a scale. This advantage is due in part to the low islet fragmentation observed with gas perfusion and is expressed as the ratio of DNA IE (1 DNA IE = 10.4 ng DNA) to IE count. This fragmentation is reflected in the micrographs taken from the islet count samples, as can be seen from FIGS. 9 (a) -9 (c) (the many nuclear enrichments in the TLM preserved pancreas (FIG. 9 (a)). Note that nuclei (arrows) show apoptotic or necrotic cell death, nuclei enriched nuclei were present in islets and exocrine tissues, a characteristic of gas-perfused pancreas (FIG. 9 (b) ) And control (t = 0) pancreas (few obvious in FIG. 9 (c)). Importantly, the benefits of gas perfusion over TLM were found to be consistent and more pronounced after 24 hours of storage (single experiment).

  Islet transplantation into nude mice in the Nude Mouse Bioassay (NMB) is an “excellent standard” test for islet function (Non-Patent Document 31, Non-Patent Document 32, Non-Patent Document 33, and Non-Patent Document 34). reference). NMB was used for experimental and control conditions for 5 porcine islet isolation after gas perfusion, as budget and islet availability were observed. All data is summarized in FIG. The main data set was diabetes reversal rate and the number of mice that sustained normoglycemia (blood glucose <200 mg / dL) was compared to the number of treated mice. It is important to note that only intact islets are selected (according to the assay protocol) for transplantation purposes and are therefore lost in this assay due to fragmentation (typical of TLM conditions). It does not take into account the low quality of islets (and therefore not transplanted). Despite these limitations, gas perfusion as a pancreatic preservation technique, when data from different conditions (storage time and incubation time) is classified, diabetes reversal rate, time to diabetes reversal, and mean blood glucose (curve) Conveniently compared for both immediate separation and TLM from the perspective of Although the results were not statistically significant, mean blood glucose still showed a statistically significant trend for gas perfusion and immediate separation (2-sample t-test, p = 0.076).

Example 2:
Additional gas perfusion experiments using an electrochemical oxygen concentrator (EOC) on porcine pancreas and human pancreas In these experiments, islet separation was included and results from unstored leaves (control) were The results were compared with leaves stored for 24 hours using the two-layer method (TLM) or by gas perfusion. As a result, the number of porcine pancreas used for the separation was 7, of which 4 were used for evaluation for storage for 6 hours and the remaining 3 were used for evaluation for storage for 24 hours. In addition to experiments performed on porcine pancreas, we have access to research grade human pancreas, which was used to test the gas perfusion technique. Some important discoveries are: That is, (1) 24-hour storage with gas perfusion using electrochemically generated oxygen gives superior results compared to 24-hour storage with TLM (see FIGS. 11 and 12), (2) Human pancreas gas perfusion is feasible and less surgically complex than porcine pancreas gas perfusion. In addition, gas perfusion of the human pancreas can be achieved in a portion of the pancreas when the pancreas is divided in two. This demonstration is important because future studies will allow the use of the same organ for a direct comparison between gas perfusion storage and TLM or between gas perfusion storage and no storage.

  In one experiment, the effects of gas perfusion on islet isolation were as follows: each porcine organ was divided into three lobes, the pancreatic head was used for immediate isolation, the pancreatic tail was stored with gas perfusion for 24 hours, and the pancreatic body was treated with TLM. Tested by storing for 24 hours. The method of separating each organ into its leaves was chosen to allow a comparable comparison of preservation methods by eliminating donor variability. Since the number of samples was small, we decided to standardize (rather than randomize) the leaves used for each condition. Data from our porcine isolation database (a total of about 650 times) show that there is no difference between the three lobes in the expected islet yield per gram of pancreatic tissue.

  There was no significant difference in histology between organ samples collected immediately after procurement (t = 0) and organ samples collected after 24-hour gas perfusion. However, TLM-stored tissues exhibited multiple nuclear-enriched nuclei as seen in FIGS. 7 (a) -7 (c), especially compared to gas-perfused samples after 24 hours storage. .

  FIG. 11 summarizes various measures (mean and standard deviation) of the amount and quality of islets from porcine pancreas preserved by various methods. Based on the paired two-tailed t-test, gas perfusion was determined by TLM for IE recovery after 2 days (p = 0.011, n = 2) and OCR / DNA after 2 days (p = 0.011, n = 3). Was significantly better than. Average values of all parameters suggested better results for gas perfusion, but there was no statistically significant difference between immediate separation and gas perfusion on any scale. FIG. 12 summarizes the TLM results as a percentage of gas perfusion after 24 hours storage. In addition, islets isolated from gas-perfused leaves and control leaves (no storage) were transplanted into diabetic athymic nude mice after culture. The leaves stored by TLM did not provide enough islets that could be transplanted into nude mice after culture. According to initial results (5 days after transplantation), 3 out of 4 mice transplanted with cultured islets obtained from leaves that had been gas perfused for 24 hours had a blood glucose of less than 200 mg / dL ( Suggests reversal of diabetes.) But only one of the three mice transplanted with islets isolated from unconserved control leaves had blood glucose below 200 mg / dL.

  The embodiments of the present invention described above are intended to be examples only, and those skilled in the art can make many changes and modifications to the embodiments of the present invention without departing from the spirit of the present invention. . All such changes and modifications are intended to be within the scope of the present invention as defined by the appended claims.

DESCRIPTION OF SYMBOLS 100 Gas perfusion system 103 Anode 105 Cathode 143 Atmosphere 200 Container 203 Oxygen sensor 205 Temperature sensor 300 Process controller 400 Temperature control device 500 Power supply 550 Fluid pipe 1100 Gas perfusion system 1200 Container 1203 Oxygen sensor 1205 Temperature sensor 1207 Carbon dioxide sensor 1300 Process controller 1400 Temperature control device 1500 Power supply 1600 Liquid perfusion system 1700 Switching system

Claims (16)

A fluid perfusion system for biological material including tissue,
(A) a gas generator for producing a storage gas;
(B) a fluid perfusion system comprising a large amount of fluid perfusate;
(C) is insertable into the tissue, coupled to said beauty before Symbol fluid perfusion system Oyo to the gas generator, a fluid supply tube operated according to at least two alternative embodiments, said at least two selected A typical aspect is the first aspect in which the preservation gas from the gas generator is supplied to the tissue and the fluid perfusate from the fluid perfusion system is not supplied to the tissue, and the fluid from the fluid perfusion system. A fluid supply tube comprising: a second mode in which a perfusate is supplied to the tissue and the storage gas from the gas generator is not supplied to the tissue;
Comprising a fluid perfusion system.   The fluid perfusion system of claim 1, wherein the storage gas comprises gaseous oxygen.   The fluid perfusion system according to claim 2, wherein the gas generator comprises electrochemical means for generating gaseous oxygen.   4. A fluid perfusion system according to claim 3, wherein the electrochemical means for generating gaseous oxygen comprises an electrochemical oxygen concentrator.   The fluid perfusion system of claim 2, wherein the storage gas further comprises water vapor.   The fluid perfusion system of claim 2, wherein the gas generator comprises means for adjusting the concentration of gaseous oxygen in the storage gas.   The fluid perfusion system of claim 1, wherein the storage gas comprises gaseous oxygen.   The fluid perfusion system of claim 1, further comprising a container sized to receive the biological material including tissue.   The fluid perfusion system of claim 8, wherein the container is an insulated container.   The fluid perfusion system of claim 9, further comprising temperature control means for maintaining the contents of the container at a desired temperature.   The temperature control means includes a temperature sensor that senses the temperature in the container, a temperature control device that changes the temperature in the container, and a process controller that controls operation of the temperature control device in response to the temperature sensor. The fluid perfusion system of claim 10, comprising:   The fluid perfusion system of claim 1, further comprising a fluid drain tube that drains the liquid perfusate perfused with the biological material including any preservative gas and / or tissue.   The fluid perfusion system of claim 1, wherein the liquid perfusate 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.   The liquid perfusate is further selected from the group consisting of an antioxidant, an anti-apoptotic agent, a vasodilator / vasoconstrictor, an oxygen carrier, a chelating agent, a toxin binding agent, and an anticoagulant. The fluid perfusion system of claim 13, comprising: The said at least two optional aspects include a third aspect, wherein both the storage gas from the gas generator and fluid perfusate from the fluid perfusion system are supplied to the tissue. Fluid perfusion system. The fluid perfusion system of claim 1, wherein the fluid supply tube comprises one or more valves for switching between two or more aspects of the at least two alternative aspects.

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