Methods and compositions for preserving tissues and organs
The present invention generally relates to methods and compositions to determine viability of an organ for transplantation and other medical purposes. One aspect of the invention relates to a method for assessing the viability of an organ by measuring the energy parameters to determine the energy level of the organ by determining the stored cellular energy (e.g., ATP levels), and/or energy consumption over a particular time period of viability. The energy parameters can be compared to reference energy parameters as a highly accurate and reliable prediction of viable cell yield, and organ viability. Another aspect of the invention relates methods to preserve or extend the time period of viability of an organ any combination of (i) preservation perfusion of the organ to prevent ischemic damage, (ii) chemical metabolic suppression of the organ e.g., using metabolic suppressants, (iii) metabolic suppression by physical or environmental conditions, e.g., sub-zero non-freezing storage.
1. A method of preserving a liver comprising:
a. contacting a liver with a media comprising 3-O-methyl glucose;
b. cooling said liver to a predetermined sub-zero temperature; and
c. storing said liver at the predetermined sub-zero temperature, wherein the liver is cooled and stored at the predetermined sub-zero temperature without freezing,
and wherein cooling said liver to the predetermined sub-zero temperature in step (b) comprises:
i. cooling said liver to a hypothermic temperature;
ii. perfusing said liver with a preservation solution; and
iii. cooling said liver to the predetermined sub-zero temperature
wherein said contacting with the media comprising 3-O-methyl-glucose is subnormothermic perfusion at about 20° C., cooling to the hypothermic temperature is at a rate of about 1° C. per minute, the hypothermic temperature is between about 3° C. and about 5° C., cooling said liver to a predetermined sub-zero temperature is at a rate of about 0.1° C. per minute and said predetermined sub-zero temperature is below about −5° C.
2. The method of claim 1 , further comprising re-warming the liver.
3. The method of claim 1 , wherein said storing at a predetermined sub-zero temperature is for at least about 1 day.
4. The method of claim 1 , further comprising measuring at least one energy parameter, and determining a measure of viability as a function of the at least one energy parameter.
5. The method of claim 4 , wherein the energy parameter is selected from at least one or a combination of: oxygen consumption by the liver, the level of gluconeogenesis of the liver or the level of nitrogen metabolism by the liver.
6. The method of claim 4 , wherein determining a measure of viability comprises comparing the measured energy parameter to a transplantability threshold of a liver, and/or a cell harvesting threshold of a liver, and/or measuring the metabolic activity of the liver, or a combination thereof.
7. The method of claim 6 , wherein measuring the metabolic activity of the liver is measured by measuring the change in the levels of a plurality of metabolites over a predefined period of time, wherein the change in the plurality of metabolites is selected from any or a combination from the group consisting of: Oxygen uptake, carbon dioxide output, glucose output, lactate uptake, acetoacetate output, β-Hydroxybutyrate output, urea output, ammonia uptake, alanine uptake, arginine uptake, ornithine uptake, asparagine uptake, aspartate uptake, cysteine output, glutamate output, glutamine uptake, glycine uptake, histidine uptake, proline uptake, serine uptake, and methionine uptake.
8. The method of claim 4 , wherein measuring at least one energy parameter comprises measuring for at least one or any combination of: the level of ATP, cellular energy status, cellular energy status during normothermic perfusion of liver or the level of a plurality of metabolites.
9. The method of claim 8 , wherein the plurality of metabolites is selected from any or a combination from the group consisting of: Glucose, Urea, Nitrogen, Total Carbon dioxide, AST, ALT, ALP, total protein, total bilirubin, Creatinine, Sodium, Potassium, Calcium, Chloride, Total Cholesterol, triglycerides (high density and/or low density), very low density lipoproteins, Amino acids, Lactate, Free fatty acids, Glycerol, Insulin, Glucagon, β-hydroxybutyrate, Acetatoacetate, Nitric Oxide, Gluthatione, Glutathione disulfide, bile, principle bile acids, steroids, O 2 and CO 2 , Hematocrit, Hemoglobin (Free, oxygenated), electrolytes, and TNF-α.
10. The method of claim 9 , wherein the plurality of metabolites is selected from amino acids.