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“Cryopreservation Of Human Body Organs

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Introduction

Cryopreservation is a process where cell or organ preserved by cooling to sub zero temperatures.

Cryoprotectant solutions are used in cryopreservation.

e.g. Phosphate buffered sucrose solution.

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Defination

Cryonics: It is the technique used to store human bodies at extremely low temperatures.

Cryogenics:

It is defined as, "The branches of physics and engineering that involve the study of very low temperatures, how to produce them, and how materials behave at those temperatures."

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Principles of organ preservation

Remove the organ

Organ is cooled rapidly and warm ischemia time is minimized

Transferred in cryoprotectant solution

The organ is maintained in hypothermic state and stored until it is ready to be transplant

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Techniques of organ preservation

Hypothermic preservation.

Freezing and thawing.

Vitrification.

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Hypothermic preservation

Two techniques of hypothermic preservation:

simple cold storage:

continuous hypothermic perfusion:

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Freezing and thawing When ice is used in this tech. it can damge to structual integrity of the organ.

Thermal strees occurin at low temp.

Osmotic movement can also causes mechanical stresses.

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Vitrification

This is the process of taking an aqueous solution and making it into an amorphous solid.

Vitrification can also be achieved by adding solutes that develop a structure in water that must be broken down for crystalline growth.

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Preservation solutions

Euro-Collins solutions

Ross-Marshall citrate solutions

Phosphate-buffered sucrose solution

University of Wisconsin solution

Celsior solution

Kyoto ET solution

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Euro-Collins solutions

Containing high concentrations of potassium, magnesium, phosphate, sulphate, and glucose.

The solution is adequate for use in preserving the heart, liver, and lung.

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Ross-Marshall citrate solutions Ross-Marshall citrate solutions were developed as alternatives to the Collins solutions. Their electrolytic compositions are similar except that citrate replaces phosphate, and mannitol replaces glucose. The citrate acts as a buffer and chelates with

magnesium to form an impermeable molecule that helps in stabilizing the extracellular environment.

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Phosphate-buffered sucrose solution

This solution contains sucrose 140 mmol/L and sodium hydrogen and dihydrogen phosphate as buffers.

In experimental studies, it preserve human kidneys for 3 days.

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University of Wisconsin solution

developed for liver, kidney, and pancreas

preservation.

The solution has an osmolality of 320 mmol/kg

and pH 7.4 at room temperature and is composed of

the following:

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Composition of UW solutionPotassium ……………………..135 mmol/L

Sodium………………………….. 35 mmol/L

Magnesium……………………. 5 mmol/L

Lactobionate ………………...100 mmol/L

Phosphate ……………………..25 mmol/L

Sulphate ………………………..5 mmol/L

Raffinose ……………………….30 mmol/L

Adenosine ……………………..5 mmol/L

Allopurinol ……………………1 mmol/L

Glutathione …………………..3 mmol/L

Insulin …………………………..100 U/L

Dexamethasone …………....8 mg/L

Hydroxyethyl starch (HES)50 g/L

Bactrim …………………………0.5 ml/L

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Celsior solution

Celsior is a recently developed extracellular-type, low-viscosity.

Composition:

Sodium…………………………………….. 100 mmol/L

Potassium…………………………………. 15 mmol/L

Magnesium………………………………. 13 mmol/L

Calcium …………………………………….0.25 mmol/L

Lactobionate ……………………………..80 mmol/L

Glutathione ……………………………….3 mmol/L

Glutamate…………………………………. 20 mmol/L

Mannitol ……………………………………60 mmol/L

Histidine…………………………………… 30 mmol/L

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Kyoto ET solution

Researchers at Kyoto University developed a new solution that contains a high sodium concentration, a low potassium concentration, trehalose, and gluconate. Composition:Sodium ………………..100 mmol/LPotassium……………. 44 mmol/LPhosphate……………. 25 mmol/LTrehalose ……………..41 mmol/LHES ………………………30 gm/LGluconate …………….100 mmol/L

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Cryopreservation of liver:. Pathological area of P3's liver,

showing numerous cavities reminiscent of ice crystal spaces. Scale bar = 40 microns. H&E (hematoxylin and eosin stain), Karnovsky's.

"Island" of well-preserved cellular structure in the liver. H&E, Karnovsky's primary fixative. Scale bar = 40 microns.

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Cryopreservation of kidney(Renal medula):

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Cryopreservation of heart:Cross-section of myofibrils, displaying normal shape and density.

Cardiac blood vessel with fundamentally preserved structure.

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Needs and benefits of organ Cryopreservation:

In organ transplantation to store the isolated organ until it s transplantation without damage.

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Major problems in Cryopreser- vation : Vascular damage may be caused due ice formation. Fracture of frozen tissue may be due thermal stress during warming. Formation of cytoplasm glass due to ultra -rapid cooling.

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References: Day J.G and McLellan M.R.(eds) (1995) Cryopreservation and freeze-drying protocols. Meth. Mol. Biol. 38: 254pp. Meryman, H.T., Ice Crystal Formation in Frozen Tissues. Lecture and Review Series, Naval Medical Research Institute, No. 53-3, 25-48,1953. Lillehei, R.C., et al., In vitro preservation of whole organs by hypothermia and hyperbaric oxygenation, Cryobiology, 1, 181-193, 1964. Toledo-Pereyra, L.H., and MacKenzie, G.H., Freezing of human kidneys, initial in vitro observations, The American Surgeon, 48, 232-236, 1982.

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