American National Red Cross

Organ Recovery Systems, Inc.

The United States of America as Represented by the American National Red

Bijan S Khirabadi

Gregory M Fahy

Yasumitsu Okouchi

Kelvin G M Brockbank

Thomas Maciag

Ying C Song

A method for vitrification of a tissue or organ includes immersing the tissue or organ in increasing concentrations of cryoprotectant solution at a temperature greater than &#x2212;15&#xb0; C. to a cryoprotectant concentration sufficient for vitrification; cooling the tissue or organ at an average rate of from 2.5&#x2013;100&#xb0; C. per minute to a temperature between &#x2212;80&#xb0; C. and the glass transition temperature; and further cooling the tissue or organ at an average rate less than 30&#xb0; C. per minute to a temperature below the glass transition temperature to vitrify the tissue or organ. After the vitrified tissue or organ has been stored, the tissue or organ may be removed from vitrification by warming the tissue or organ at an average rate of from 20&#x2013;40&#xb0; C. per minute to a temperature between &#x2212;80&#xb0; C. and the glass transition temperature; further warming the tissue or organ at a rate greater than 80&#xb0; C. per minute to a temperature above &#x2212;75&#xb0; C.; and reducing the concentration of the cryoprotectant. Tissues or organs treated in this manner exhibit near normal functions, for example, blood vessels exhibit near normal smooth muscle contractility and normal graft functions.

Method of cryopreservation of tissues by vitrification

A method for vitrification of a tissue or organ includes immersing the tissue or organ in increasing concentrations of cryoprotectant solution at a temperature greater than &minus;15&deg; C. to a cryoprotectant concentration sufficient for vitrification; cooling the tissue or organ at an average rate of from 2.5-100&deg; C. per minute to a temperature between &minus;80&deg; C. and the glass transition temperature; and further cooling the tissue or organ at an average rate less than 30&deg; C. per minute to a temperature below the glass transition temperature to vitrify the tissue or organ. After the vitrified tissue or organ has been stored, the tissue or organ may be removed from vitrification by warming the tissue or organ at an average rate of from 20-40&deg; C. per minute to a temperature between &minus;80&deg; C. and the glass transition temperature; further warming the tissue or organ at a rate of from 200-300&deg; C. per minute to a temperature above &minus;75&deg; C.; and reducing the concentration of the cryoprotectant. Tissues or organs treated in this manner exhibit near normal functions, for example, blood vessels exhibit near normal smooth muscle contractility and normal graft functions.

A method for vitrification of a blood vessel includes immersing the blood vessel in increasing concentrations of cryoprotectant solution at a temperature greater than &minus;15&deg; C. to a cryoprotectant concentration sufficient for vitrification; cooling the blood vessel at an average rate of from 30-60&deg; C. per minute to a temperature between &minus;80&deg; C. and the glass transition temperature; and further cooling the blood vessel at an average rate less than 10&deg; C. per minute to a temperature below the glass transition temperature to vitrify the blood vessel. After the vitrified blood vessel has been stored, the blood vessel may be removed from vitrification by warming the blood vessel at an average rate of from 20-40&deg; C. per minute to a temperature between &minus;80&deg; C. and the glass transition temperature; further warming the blood vessel at a rate of from 200-300&deg; C. per minute to a temperature above &minus;15&deg; C.; and reducing the concentration of the cryoprotectant. Blood vessels treated in this manner exhibit near normal smooth muscle function, normal graft patency rates, and reduced levels of intimal hyperplasia.

Method of cryopreservation of blood vessels by vitrification

The invention relates to the field of organ and tissue perfusion. More particularly, the present invention relates to a method for preparing organs, such as the kidney and liver, for cryopreservation through the introduction of vitrifiable concentrations of cryoprotectant into them. To prepare the organ for cryopreservation, the donor human or animal, is treated in the usual manner and may also be treated with iloprost, or other vasodilators, and/or transforming growth factor &bgr;1. Alternatively, or additionally, the organ which is to be cryopreserved can be administered iloprost, or other vasodilators, and/or transforming growth factor &bgr;1 directly into its artery. The invention also relates to preparing organs for transplantation by a method for the removal of the cryoprotectant therefrom using low (such as raffinose, sucrose, mannitol, etc.), medium (such as agents with intermediate molecular weights of around 600-2,000) and high (such as hydroxyethyl starch) molecular weight agents osmotic buffering agents. The invention is also directed to new post-transplantation treatments such as the use of transforming growth factor &bgr;1, N-acetylcysteine and aurothioglucose.

Method for preparing organs for transplantation after cryopreservation

The invention relates to the field of organ and tissue perfusion. More particularly, the present invention relates to a method for preparing organs, such as the kidney and liver, for cryopreservation through the introduction of vitrifiable concentrations of cryoprotectant into them. To prepare the organ for cryopreservation, the donor human or animal, is treated in the usual manner and may also be treated with iloprost, or other vasodilators, and/or transforming growth factor .beta.1. Alternatively, or additionally, the organ which is to be cryopreserved can be administered iloprost, or other vasodilators, and/or transforming growth factor .beta.1 directly into its artery. The invention also relates to preparing organs for transplantation by a method for the removal of the cryoprotectant therefrom using low (such as raffi nose, sucrose, mannitol, etc.), medium (such as agents with intermediate molecular weights of around 600-2,000) and high (such as hydroxyethyl starch) molecular weight agents osmotic buffering agents. The invention is also directed to new post-transplantation treatments such as the use of transforming growth factor .beta.1, N-acetylcysteine and aurothioglucose.

Method of preparing tissues and cells for vitrification

The invention relates to the field of organ and tissue perfusion. More particularly, the present invention relates to a method for preparing organs, such as the kidney and liver, for cryopreservation through the introduction of vitrifiable concentrations of cryoprotectant into them. To prepare the organ for cryopreservation, the donor human or animal, is treated in the usual manner and may also be treated with iloprost, or other vasodilators, and/or transforming growth factor .beta.1. Alternatively, or additionally, the organ which is to be cryopreserved can be administered iloprost, or other vasodilators, and/or transforming growth factor .beta.1 directly into its artery. The invention also relates to preparing organs for transplantation by a method for the removal of the cryoprotectant therefrom using low (such as raffinose, sucrose, mannitol, etc.), medium (such as agents with intermediate molecular weights of around 600-2,000) and high (such as hydroxyethyl starch) molecular weight agents osmotic buffering agents. The invention is also directed to new post-transplantation treatments such as the use of transforming growth factor .beta.1, N-acetylcysteine and aurothioglucose.

Methods for removal of cryoprotectant from organs prior to

The invention relates to the field of organ and tissue perfusion. More particularly, the present invention relates to a method for preparing organs, such as the kidney and liver, for cryopreservation through the introduction of vitrifiable concentrations of cryoprotectant into them. To prepare the organ for cryopreservation, the donor human or animal, is treated in the usual manner and may also be treated with iloprost, or other vasodilators, and/or transforming growth factor .beta.1. Alternatively, or additionally, the organ which is to be cryopreserved can be administered iloprost, or other vasodilators, and/or transforming growth factor .beta.1 directly into its artery. The invention also relates to preparing organs for transplantation by a method for the removal of the cryoprotectant therefrom using low (such as raffinose, sucrose, mannitol, etc.), medium (such as agents with intermediate molecular weights of around 600-2,000) and high (such as hydroxyethyl starch) molecular weight agents osmotic buffering agents. The invention is also directed to new post-transplantation treatments such as the use of transforming growth factor .beta.1, N-acetylcysteine and aurothioglucose. Further, the invention relates to a method of vitrification of organs comprising a multi-step method of introducing increasing concentrations of cryoprotectant with osmotic equilibration of the organ and decreases in temperature prior to perfusion with vitrifiable concentrations of cryoprotectant at about -5.degree. C. to -35.degree. C.

Method of preparing organs for vitrification

A computer-controlled apparatus and method for perfusing a biological organ, such as a heart, kidney, liver, etc. The apparatus comprises a plurality of fluid reservoirs and an organ container for holding the biological organ. A first fluid flow path is defined as a loop from the plurality of reservoirs to necessary sensors and temperature conditioning means and back to the plurality of reservoirs. The reservoirs are selectively connectable to the first fluid flow path. Pump means are interposed in the first fluid flow path for pumping fluid from the first fluid flow path to a second fluid flow path. The organ container is located in this second fluid flow path. Pump means may also be included in the second fluid flow path for pumping fluid from the organ container to one or more of the reservoirs or to waste. One or more sensors are interposed in the fluid flow paths for sensing at least one of the concentration, temperature, pH, and pressure of the fluid flowing in the first and second fluid flow paths. Measuring means are interposed in the first and second fluid flow paths for measuring concentration and temperature differences between the upstream and downstream sides, in the fluid flow direction, of the organ container. The sensor(s) and the measuring means are connected to a programmable computer for providing a continuous information stream from the sensor(s) to the computer. The computer is coupled to the selection means and the pump means to continuously selectively control (a) the flow of fluid from each of the reservoirs individually to the fluid flow paths, and (b) at least one of the concentration, temperature, pressure and pH of the fluid flowing in the second fluid flow path, in accordance with a predetermined computer program without operator intervention.

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Rockville, MD, United States of America

Bijan S Khirabadi