Preservation solution

Details Preservation solution Preservation solution

2000-01-18

2004-09-21

Saucier, Sandra (Department: 1651)

Chemistry: molecular biology and microbiology

Differentiated tissue or organ other than blood, per se, or...

C435S001200, C435S001300

Reexamination Certificate

active

06794124

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to an improved preservation solution for organs and tissues, or parts thereof, from humans and animals.
BACKGROUND ART
In coronary artery surgery (about 800 operations per one million inhabitants a year) and in peripheral vascular surgery (about 100 operations per one million inhabitants a year), so-called physiological saline solution (0.9% NaCl) is in use today in most European clinics as a solution for washing away blood from blood vessel transplants, and for storing blood vessel transplants before inserting them in their new positions. In coronary artery surgery, use is generally made of the vena sapena magna, i.e. the superficial vein extending from the inside of the foot over the inner ankle and along the inside of the lower leg and the thighbone to the groin, where it joins the thigh vein (vena femoralis). In a coronary artery operation, first the vena sapena magna in one leg is removed, while the breastbone is opened and preparatory measures are taken for connection to a heart-lung machine. After removal of the vena sapena magna, this blood vessel is flushed with saline solution of the above-mentioned type, on the one hand to wash away all blood from the inside of the vessel and, on the other hand, to ensure that one has not neglected to ligate any branch of the vein, i.e. to tie the branches with a thread with a view to preventing leakage therethrough. Subsequently, the removed vein is placed in a dish containing saline solution of room temperature, i.e. 20-25° C. Then the heart-lung machine is connected and cardioplegia is given to the heart. About 15-20 cm-long segments are cut off from the vein in the dish and are sewn as a so-called aortacoronary vein bypass to the sick coronary arteries. Before all vessel transplants are sewn and the blood again circulates through these, a period of up to 2 hours may have passed. For patients who are to have one or two cardiac valves inserted as well, this period can be still longer.
Instead of storing the vessel transplants in saline solution before being sewn, some surgeons use the patient's own blood. Blood is then drawn off from the patient and is placed in a dish. The transplant is then allowed to lie in this blood before being sewn to the heart. First the temperature is 37° C. but rapidly falls to room temperature. It is thought that since blood is the medium to which the vessel is exposed throughout life, this would be the ideal storage medium for a vascular transplant.
In heart surgery, the coronary artery surgery constitutes about 70% of the operations on adults. It is well known from studying experiments on animals that when a vascular transplant is used where the endothelium is destructed, so-called intimal hyperplasia is released and the transplants are occluded after some time (the vascular lumen becomes smaller and smaller and at last the flow of blood is stopped completely). In clinical follow-up studies, it has been found that 5 years after a coronary artery operation about 30-50% of the venous transplants have been occluded, and when these are studied histologically, a pronounced intimal hyperplasia will be discovered. This thus applies to venous transplants which have been rinsed and stored in the above-mentioned physiological saline solution.
The applicant's research team has intensely studied both short-term and long-term preservation of blood vessels. Regarding short-term preservation of blood vessels, i.e. up to 2 hours preservation, it has been found that a physiological saline solution is toxic to the vascular endothelium. If a saline solution is flushed through, for example, the arteria iliaca of a rat, intimal hyperplasia can be found in the vessel after about one month. If, on the other hand, serum is used for rinsing correspondingly, no intimal hyperplasia will be discovered. Thus, the use of a physiological saline solution as preservation solution is not favourable to the blood vessels. All the same and in the absence of a better alternative, the clinical use of physiological saline solution, however, continues in most thoracic surgery centres throughout the world.
The applicant's research team has also demonstrated that blood is not satisfactory as a preservation solution. Blood of room temperature which is stored in a dish and is not oxygenated is extremely toxic to the endothelia of the blood vessels and inhibits the endothelial function to a considerable extent. This may seem to be a paradox, but since blood is an organ that has its normal function only when it is moving and is continuously oxygenated in the lungs, it cannot function in the normal manner. Deoxygenated, non-moving blood contains, like all other blood, white and red blood corpuscles and thrombocytes. Indeed, it is well known that white blood corpuscles are activated in case of hypoxaemia (low concentration of oxygen), and that they then produce toxic substances.
The applicant's research team has also confirmed that extracellular solutions can preserve blood vessels, but only for limited times, at room temperature. Extracellular solutions, in the literature sometimes misleadingly called preservation solutions, are solutions having ionic concentrations similar to plasma. The classic extracellular solution is Ringer's solution, which has a normal extracellular concentration of sodium, potassium, calcium and magnesium. To match the positive ions for obtaining ionic equivalence, chloride, lactate or acetate are used in different types of Ringer's solution. For functional in vitro studies the classical organ bath solution is Krebs solution which is electrolytically constructed like Ringer's solution. However, Krebs solution also contains glucose for metabolism, and it contains phosphate and bicarbonate buffers to achieve a pH of 7.40 when this solution is bubbled with a mixture of 95% oxygen and 5% CO
2
at 37° C. If a cold perfusion is preferred, enough oxygen is physically dissolved to match the lowered metabolism caused by the cooling. However, neither of these two methods have been a success for extended preservation periods in experimental transplantation. During the cooling of hypothermia, rigidity develops in the cell endothelial membranes. This occurs because the fluidity of the lipids is diminished as an effect of the temperature reduction. The rigidity of the endothelium contributes to the endothelial injury described following prolonged cold perfusion with the intention to preserve, for example, the kidney and the liver.
Extracellular solutions exhibit what has been called the “calcium paradox.” If an organ is perfused with an extracellular solution without calcium for a while, and then the perfusion continues with the same solution but now including calcium, the organ may be destroyed more quickly compared to perfusing it only with the calcium free solution, i.e. perfusion without calcium is dangerous, and perfusion with calcium is dangerous—that is the paradox. In clinical organ preservation, the organ is immediately cooled down by flushing it with a cold preservation solution created, for example, for cold anaerobic storage.
It should be emphasized that the composition of preservation solutions used for cold anaerobic storage needs to be constructed in quite another way than conventional extracellular solutions. This is due in part, at least, to the effects of hypothermia.
In the first successful liver transplantation performed, Welch found that 33 minutes of warm ischemia of the dog liver was the upper limit, if the recipient animal was going to survive the operation (Goodrich E O, Welch H N, Nelson J A et al: Homotransplantation of the canine liver. Surgery 39:244, 1956. This reference is incorporated by reference herein.). With this approach, success was noted in 21 of the 49 cases, which survived for at least 5 days. Moore et al., were the first to describe the use of hypothermia in preservation of the liver, namely by surface cooling of the organ, but they did not attempt to prolong the ischemic time to more than half an hour (Moore

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