NO883900L - PROCEDURE FOR CRYOPRESERVATION OF CARDIOVES. - Google Patents

Description

Technical area

The present invention relates to a method for cryopreservation of heart valves and more specifically to a method for freezing heart valves to ultra-cold temperatures, under which the valves can be stored for long periods of time. Valves cryopreserved according to the present invention are suitable for use as replacement valves in hearts with diseased or poorly functioning valves.

Background for the invention

Biological aortic valve replacement began around 1962.

This early work was initiated because artificial heart valve replacements were plagued by mechanical failure and the need to anticoagulate the patient.

Biological valves, normally from pigs or cattle, had

the overall advantage of identical shape and structure to the flap that was replaced. Today is approx. 18,000 of 36,000 total valves replaced in the U.S. every year tissue flaps. Virtually all of the 18,000 biological valves are of bovine or porcine origin, glutaraldehyde-fixed and mounted on a mechanical stent.

However, these types of biological valves are not living. Consequently, time and biological reaction act on the non-living material so that the tissue breaks down until it eventually malfunctions.

Because of the accompanying problems of using by to

Instead of using non-living heart valves of animal origin, researchers have begun to use human alloimplantation heart valves to replace defective heart valves. Human alloimplantation valves are useful either by themselves in conjunction with their conduit, i.e. the aorta with the aortic valve or the pulmonary valve with the pulmonary artery for use as a crucial component in congenital heart repair. Respiratory outflow tract reconstruction using aortic and respiratory valve leads is routinely used for complex tetralogy of fallot, respiratory atresia, truncus arteriosus, and complex transposition of the great arteries. In the past, congenital defects such as

occurs in eight out of every thousand births, artificial valves i

connection with artificial vascular lines. These reconstruction methods are subject to the same clinically harmful effects of calcific degeneration and thrombosis cases as with a non-implantation flap used alone.

Researchers have generally agreed that healthy tissue works better than old or dead tissue, and viable human tissue works better than bovine/pig xenoimplantation flaps. However, viable tissue remains alive only for short periods of time. The fibroblast cells, which are the main component of the valve leaf and are the cells most important for proper function and duration, will be viable for 2 or 3 days in a life-sustaining medium.

Storage for 2 or 3 days is impractical except for a few of the flap recipients because the flap size of the donor tissue is probably not the right size for the waiting recipient. Consequently, much of the tissue cannot be used for flap replacement due to a significant loss of cell viability over time. In addition, the preservation time is becoming increasingly important in light of the fact that increasing signs are emerging of improved tissue effect if the donor and recipient are of the same ABO blood group.

A number of researchers have described different methods for extending the storage life of heart valves. Previously known techniques such as freeze-drying, glutaraldehyde fixation and mechanical freezing combined with sterilization techniques with irradiation and antibiotics generally lead to early tissue failure.

The storage of cells and tissues became possible after the discovery in 1949 by Polge, Fmith and Parks that glycerol could be used to protect the cells from damage during freezing. In the development of low-temperature biology, professionals in the medical and biological fields have searched for better ways to maintain the viability of frozen donor cells or tissues.

Several methods for freezing cells and cell aggregates have been reported. U.S. patent no. 3,303,662 describes a method for cell preservation that uses a cryoprotectant during the freezing process. U.S. patent no. 4,423,600 describes a method for preserving living organic tissue by freezing which entails a lowering of the atmospheric pressure surrounding the tissue before freezing.

Several attempts have been made to freeze heart valves

to ultracold temperatures (e.g. Van der Kemp, et al.,

Journal of Surgical Research, Vol. 30., 47-56, 1981). However, even with the understanding that enzymatic degradation of the tissue during long-term storage is minimal at the ultra-cold temperatures of liquid nitrogen, a number of problems have been found in the implementation of this technology. What is required is a method of freezing heart valves to ultra-cold temperatures that will maintain maximum viability of individual cells within the tissue with minimal tissue degradation. The procedure should allow long-term storage of the biological tissue.

Summary of the Invention

The present invention is a method for freezing, storing and thawing collagen-rich tissue, such as heart valves with a cell viability of at least 70% after thawing. The collagen-rich tissues that are frozen according to the present invention can be stored over long periods of time at super-cold temperatures with minimal loss of cell viability. The present invention provides a unique freezing profile that allows a collagen-rich tissue, such as heart valves, to be frozen down to liquid nitrogen temperatures, approx. -196°C, with minimal tissue damage due to ice crystal formation. The present invention also contains a thawing scheme whereby the frozen tissue can be thawed quickly with minimal tissue damage. Heart valves that are cryopreserved according to the present invention are alive after thawing, and are ideally suited to replace diseased or poorly functioning heart valves in heart patients.

Accordingly, it is an aim of the present invention to provide a method for cryopreservation of a human heart valve, so that the valve is viable and functional after thawing.

It is another aim of the present invention to provide a method for preserving viable human heart valves over long periods of time.

It is yet another object of the present invention to provide a unique freezing scheme for freezing a heart valve so that the heart valve cells retain a high level of viability after it is thawed.

It is yet another object of the present invention to provide a method for cryopreservation of a human heart valve that allows rapid thawing of the heart valve while maintaining maximum cell viability.

It is yet another object of the present invention to provide a viable heart valve suitable for transplantation after long-term storage at ultra-cold temperatures.

These and other aims, features and advantages of the present invention will become clear after reviewing the following detailed description of the presented embodiment and the accompanying claims.

Brief description of the figure

Fig. 1 is a schematic representation of the freezing profile for freezing a heart valve according to a preferred embodiment of the present invention.

Detailed description of the preferred embodiment

The preferred embodiment is a method for freezing, storing and thawing collagen-rich tissue, such as heart valves, with a cell viability of at least 70% after thawing. The collagen-rich tissue frozen according to the present invention can be stored over long periods of time at super-cold temperatures with minimal loss of cell viability. The present invention contains a unique freezing profile that allows a collagen-rich tissue, such as heart valves, to

frozen to the temperature of liquid nitrogen, approx. -196°C, with minimal tissue damage due to ice crystal formation. The present invention also contains a thawing plan, whereby the frozen tissue can be thawed quickly with minimal tissue damage. Heart valves cryopreserved according to the present invention are biologically viable after thawing and are ideally suited for

replacement of diseased or poorly functioning heart valves in heart patients.

The tissue to be preserved is only as good as it was received in the laboratory. Consideration must be given to the donor's age, health and history of cardiovascular disease. Another important consideration is the time between death and removal of the valves (warm ischaemia)

and the time from withdrawal from the valves to dissection (cold ischemia). Care must be taken in the handling method of the tissue during preparation and the medium used to ship the tissue.

A donor heart that can be used as a source of human heart valves to be frozen according to the present invention should be from donors between the ages of newborn and 55 years, and the donor should not have suffered from any significant high blood pressure, diabetes, infectious disease or cardiac tissue abnormality.

Dissection of the heart must be performed within the 24 hour time limit to maximize cell viability. In addition, the dissection must be performed in such a way that the tissue is torn up as little as possible and only the unnecessary adventitia is removed. The procedure should also involve minimal handling and the tissue must be kept moist.

In the practice of the present invention, there are only two aspects of sterilizing the tissue that require attention. First, the combination of sterilization medium must not be

toxic to the cells; and secondly, it must sterilize the tissue. The effects of antibiotics will vary with the type of cell or cell suspension being preserved and the type of antibiotic used. The effects of a particular antibiotic on a particular cell must be well documented. The time and temperature at which the antibiotic will be used is also of critical importance. It must be clear that if the tissues remain sterile during the sampling and dissection process, no antibiotics are necessary.

The medium in which the tissue is frozen is of great importance

to maintain a balanced cellular environment. Time and temperature also contribute to whether a single medium will be successful.

In general, a protein suspension such as blood serum or

artificial serum also be present for maximum cell viability.

A variety of freezing media can be successfully used in the practice of the present invention. Media such as balanced tissue culture media or simple phosphate-buffered saline can be used for most tissue types. RPMI 1640 (KC Biologicals, Kansas City, Missouri) with 10% fetal calf serum is the preferred combination for heart valves. Other media with a somewhat less acceptable nature are: Eagle's minimum essential medium, TC199, and others.

A cryoprotectant is added to the freezing medium to protect the cell during the freezing and thawing phases of the present invention. Care must be taken to prevent osmotic shock and cytotoxicity when using these cryoprotectants. Cryoprotectants serve to protect the cells, especially during the ice crystallization phase, and during the cell shrinkage phase immediately before crystallization. Factors such as tissue toxicity in conjunction with time, temperature, pressure and mixing speed to reach osmotic equilibrium must be taken into account in a successful freezing method. Commonly used cryoprotectants include, but are not limited to, glycerol, ethylene glycol, dimethyl sulfoxide with acetamide or propylene glycol, or propylene glycol alone, trimethylamine acetate, and a variety of aldoses and ketones, such as xylose, erythrose, arabinose, ribose, glucose, fructose, and galactose or combinations thereof.

It is assumed that the function of the cryoprotectants is to partially replace water inside the cell. As water crystals form in the extracellular medium, the hypertonic shrunken state of the cell without the cryoprotectant could lead to disruption of cellular components leading to cell death.

Therefore, the speed at which the cryoprotectant is added, the concentration of the cryoprotectant and the temperature at which the cryoprotectant is added must be taken into account.

The freezing profile is of critical importance for successful cryopreservation of a tissue. A number of variables exist that maximize tissue survival. For example, the fluid volume, the size of the tissue and combinations of properties that include cryoprotectant, tissue and freezing media, which all contribute to an optimal freezing profile. It will be clear that previously known freezing profiles available for cell suspensions are not suitable for freezing tissues such as heart valves. It has been found that each tissue has its own unique and unpredictable freezing profile. The freezing profile required to successfully cryopreserve one tissue is unexpectedly different from the freezing profile required to successfully cryopreserve another tissue.

A number of factors must be taken into account when freezing a tissue. Among these factors are: The temperature around the equilibrium point, (generally +4°C, to the temperature at the freezing point); release and control of the exothermic heat given off at the freezing point; optimal cooling rate determined by the permeability of the cell membrane to water; the surface-to-volume ratio of the cell; the type and concentration of cryoprotectants in the medium; and finally removal of the cryopreserved tissue from the controlled-rate freeze to an immersion in a liquid nitrogen refrigerator.

The thawing and dilution steps with an allo-implant must be clearly defined, as crystal growth and osmotic shock can still damage the tissue. It has been found that a thawing rate of 50°C per minute is appropriate for human heart valves. Immediately after thawing, the cryoprotectant used must be removed, normally in a stepwise manner, to reduce the effects of osmotic shock on the cells and thus enable a regulated equilibration of the cell with the surrounding medium. Time and temperature are main factors.

Thus, the method for cryopreservation of heart valves according to the present invention comprises dissecting the heart valve from the donor hearts, sterilizing the tissue with a mixture of antibiotics, placing the valves in a medium with the correct cryopreservation agents, freezing the valves according to a precise freezing plan. The freezing plan used to cryopreserve the heart valves in the present invention comprises placing the sample in a sample freezing chamber in a cryopreservation apparatus. The initial temperature in the test chamber is above freezing and is normally approx. +22°C. The temperature in the sample chamber is reduced at a rate of 0.01°C per min until the sample chamber reaches a temperature of -2°C. The temperature in the sample chamber is then reduced at a rate of 1°C per min., until the sample chamber reaches a temperature of -80°C. The heart valve is then transferred to a liquid nitrogen storage device where the valve is preserved at a temperature of approx. -196°C until the valve is to be transplanted into a living heart.

The frozen heart valve is thawed by the following general procedure. A foil bag containing the frozen heart valve is placed in two liters of sterile saline or water at 37°C to 42°C. The bag is left in the water bath until the ice crystals have mostly dissolved. Due to the toxic effects of the dimethyl sulfoxy cryo-preservative at temperatures above 4°C, under no circumstances should the bag remain in the heat bath for more than 14 minutes. After the tissue has been thawed to 4°C, the tissue is placed in an appropriate container where a series of precise dilution steps are performed.

The following specific examples will illustrate the invention with respect to harvesting, freezing to ultra-cold temperatures and thawing a human heart valve. It will be obvious that other examples will be obvious to a person skilled in the art, and that the invention is not limited to these specific illustrative examples.

EXAMPLE 1

The heart was anesthetized in toto using strictly sterile conditions. The initial preparation and draping of the donor extended over the pit of the neck and laterally to the nipples. A midline sternotomy incision is used to expose the heart. Lack of significant aortic and mitral valve regurgitation should be confirmed by palpitation on tension. The aorta was dissected around just distal to the beginning of the innominant artery. Cavae were ligated with umbilligal tape. Then, forced inflation of the lungs helped empty the heart. The heart was turned outward from the pericardium and the pulmonary veins transected. After the heart was returned to the pericardium, the pulmonary veins were transected as were the cavae proximal to the ligaments. The aorta was ligated distal to the innominant artery and transected. The heart was placed in a basin containing 200 to 300 ml of Ringer's solution at 4°C, and as much blood as possible flushed from the heart by gentle massage of the ventricles.

When preparing for transport, the heart was placed in a sterile inner bag with approx. 350 ml Ringer's 1actate at 4°C. The bag was secured with a plastic band or umbilical tape and was placed in a second gut bag which was also secured. Now the heart in a double bag was placed in a nalgin or a plastic container and the lid fixed. The container was then placed in a third sterile gut bag and placed in a styrofoam shipping container with wet ice.

EXAMPLE 2

The pericardial reflection was removed by creating a plane in the adventitia on the most distal surface of the aorta, and remaining internally, dissected toward the right ventricle until the right coronary artery was identified and dissected free. The right coronary artery and coronal branch are carefully dissected away from the right ventricle 1 to 2 cm and transected. By using "crile forceps", the pericardial reflection is anchored to the incision, and with the clockwise direction the adventitia is dissected circularly circumscribing away from the entire aorta to the height of the ventricular ring. This dissection is continued to the right using the hemostats for traction until the left coronary artery is identified or approx. 100° of the circumference of the aortic pathway was identified.

The hemostats were removed and the distal pulmonary artery was separated from the pericardial reflection. The pulmonary artery was inverted and secured with a Kelly clamp to the left ventricle. The pulmonary artery was cut clockwise from the beginning of the right coronary artery between the base of the pulmonary artery and the aortic annu.lr.s. Dissection continued counterclockwise until the left coronary artery was identified, dissected free 1 to 2 cm, and transected. The pulmonary outflow system was removed by leading the right ventricle at least 1.5 cm within the base of the pulmonary artery. On entering the right ventricular chamber, the scissors were directed toward the origin of the right coronary artery, and a full-thickness incision was made through the inner wall of the right ventricle. The dissection was completed 3 mm inside the aortic arch. A shallow funnel was then made 1 cm distal to the beginning of the lung apices and 2 mm deep across the inner surface of the posterior wall of the right ventricle.

The lower jaw point and blade of the Metzenbaum dissecting scissors

was used to form the funnel. The respiratory valve and artery were dissected free of the right ventricle by following the funnel over a depth of 2 to 3 mm. After removal, the entire respiratory outlet system was placed in the bath of cold Ringer's solution.

The right ventricle was dissected away from the aorta in a clockwise direction until the membranous septum was encountered. Chordae attached to the medial and medial tricuspid tips were transected, and keeping 2 to 3 mm away from the aortic pathway

one into the left atrium. The left atrium was dissected away, leaving 1 to 2 mm of attachment to the left and right fibrous trigone.

The left ventricle was entered by making a full-thickness cut between the mitral tip at the commissural tips.

This exposed the left ventricle. The dissection continued

to apex. Chordae tendinae which are attached to the internal mitral leaflet were transected and draped over the posterior aortic pathway. The aortic tips were inspected for damage or defect.

Using the lower jaw of the Metzenbaum dissecting scissors,

a horizontal funnel was made 1 cm inside the posterior and left aortic apex. Stretchers were placed on the aortic pathway and the aortic homoimplant dissected free from the left ventricle yielding 2 to 3 mm thickness. Ventricular excess was carefully dissected from the aortic tract, and the internal diameter of the alloimplant was sized using Hegar dilators. The coronary arteries were ligated 2 mm from the beginning with 20 silk. The tube of the valve was placed in nutrient medium RPMI 1640, and was

ready for antibiotic sterilization.

For the pulmonary tube, the adventitia was removed from the surface of the artery in a manner similar to that used for the aorta with dissection beginning distally and working toward the ventricular muscle band. Finally, excess fat was trimmed from the muscle band, the lung alloimplant was measured and then placed in antibiotic solution with the aortic alloimplant.

EXAMPLE 3

The following commonly used antibiotics were added to RPMI medium and applied to the dissected tissue:

1. Amphotericin B, 25 micrograms per ml; 2. Polymixin B Sulphate, 100 micrograms per ml; 3. Cefoxitin, 240 micrograms per ml;

4. Vancomycin, 50 micrograms per ml; and

5. Lincomycin, 120 micrograms per ml.

Investigations into the use of these antibiotics show that for every 24-hour period at 4°C, approx. 10% or more of the cells. Furthermore, it is known that time and temperature can also strongly affect the survival of the cells.

A measurement to determine whether the cells that form the heart valve leaflet are viable is a<3>H-proline uptake measurement. A description of this procedure is as follows: The flap piece was cut into three equal parts. The pieces were used for histology and for incubation in<3>H-proline. Flap pieces were incubated for 6 hours in 5 ml F10 medium containing 10% fetal calf serum and 15jjC<3>H-proline per ml (specific activity 1.0 mCI/mmol). Then the valves were incubated with cold proline and fixed in formaldehyde. Paraffin sections (5 µm) were made for immersion autoradiography using Kodak emulsion. After two weeks, the film is fixed and sections are stained with hematoxylin and eosin. These autoradiographs show transcellular and intracellular labeling representing proline- or hydroxyproline-containing proteins that have been secreted by the cells. Collagen is a good candidate for such a measurement, because collagen contains a high concentration of proline and hydroxyproline and is the main component of valve fibroblasts. Cells not labeled with <3>H-proline are considered dead. The calculation to determine viability is as follows: (% labeled cells/% labeled control) x 100% = % viable cells)

The viability of a heart valve is not only dependent on the percentage of labeled cells, but even more so on the absolute number of labeled cells. A flap with a high cell count and low viability percentage may still contain a reasonable number of viable cells, and a flap with a low cell count and high viability percentage may contain an unacceptably low number of viable cells. According to the literature, a cell is alive if 200 or more cells are labeled and counted per field of vision.

Using the above-described <3>H-proline uptake assay for cell viability, the effect of antibiotics on human heart valves was determined after a 48 hour incubation and a 24 hour incubation in the aforementioned antibiotic mixture. The results of this test are summarized in Table A.

The data show that there is an approximately 25% improvement in cell viability when cells were incubated in the antibiotic solution for 24 hours over when cells were incubated for 48 hours in the antibiotic solution. The control tissue was subjected to the same dissection and freezing, except that antibiotics were not added during the incubation in nutrient medium RPMI 1640. The examination was carried out using tritiated proline introduced into the fibroblast cells of the valve leaflets.

EXAMPLE 4

For cryopreservation of heart valves according to the present invention, it has been measured that 10% dimethylsulfoxide was added

RPMI 1640 with 10% fetal calf cells is sufficient to cryoprotect heart valves during freezing if the dimethyl sulfoxide is added at temperatures of 4°C or lower. In the case of human heart valves, the mixture of RPMI 1640 with 10% fetal calf serum and 10% dimethyl sulfoxide was added without the need for titration, but it must be performed at 4°C or lower. A half-hour equilibration time is required before starting the freezing cycle.

Once the cryoprotectant was added to the freezing medium, the tissue was allowed to equilibrate at 4°C. The freeze cycle in a controlled speed freezer, Model 101A (Cryomed, Mt. Clemens, MI) was started. Other standard cryogenic freezers well known to one skilled in the art may be used. Selection of the freezing profile, i.e. the cooling rate and variations in the cooling rate, are very important for maintaining the viability of the heart valve cells during the freezing process.

Referring now to fig. 1, which graphically shows the optimal freezing profile for human heart valves, the vertical bars represent the chamber temperatures and the horizontal solid line represents the temperature of the tissue sample. It is the horizontal factor that requires precise manipulation through the use and regulation of liquid nitrogen entering the system. Freezers using a platform Dewar system would have similar regulation through raising or lowering the platform on which the freezing chamber is placed instead of opening or closing the liquid nitrogen valve. The freezing profile can be summarized as follows and is shown graphically in fig. 1. The sample was placed in the sample chamber at approx. 22°C. The temperature in the sample chamber was increased at a rate of 10°C per my.,

until the sample chamber had reached a temperature of -10°C. The chamber temperature was then reduced at a rate of 0.01°C per min., until the sample temperature reaches 4°C. The chamber temperature then cools by 1.5°C per my. until the sample is -3°C. When the sample reaches -3°C, the sample chamber was rapidly cooled to -140°C at a rate of 95°C per second. my. When the temperature in the sample chamber reaches -140°C, the temperature was kept constant for 1 min. After the one-minute period, the sample chamber was heated to -100°C at a rate of 20°C per minute. my. When the sample chamber

reaching a temperature of -100°C, the temperature was kept constant for 6 min. At the end of the six-minute constant temperature, the sample chamber is warmed to -32°C at a rate of 20°C per minute. my. The sample chamber was then kept at -32°C for a period of two minutes. The temperature in the test chamber is then lowered by 1°C per my. to a temperature of -80°C.

At the heat fusion point, -4°C, one must be careful not to overcompensate for the lowering of the tissue temperature, but allow a constant -1°C per my. fall. This reduction rate and temperature for this tissue continues to a temperature of -80°C. The tissue was then immersed in a liquid nitrogen freezer for long-term storage.

EXAMPLE 5

The heart valve frozen in Example 4 was thawed as follows: The foil bag containing the alloimplant was placed in two liters of sterile saline or water at 37 to 42°C. The bag was left in the water bath until the ice crystals were mostly dissolved. This can be determined by careful palpitation of the foil bag. Due to the toxic effects of dimethyl sulfoxide at temperatures above 4°C, under no circumstances should the bag remain in the bath for more than 14 minutes.

The tissue was placed in an appropriate container where the following dilution sequences were performed to rapidly remove the cryoprotectant: 1. RPMI plus 10% fetal calf serum plus 7.5% dimethyl sulfoxide for 1 minute; 2. < RPMI plus 10% fetal calf serum plus 5% dimethyl sulfoxide for 1 minute; 3. RPMI plus 10% fetal calf serum plus 2.5% dimethyl sulfoxide for 1 minute; and 4. RPMI plus 10% fetal calf serum plus 0% dimethyl sulfoxide until surgery.

The dilution sequence above depends on accuracy in detail in short periods of time.

As soon as the tissue is thawed and diluted, there was stabilization phase for the tissue which is beneficial for optimal accountability.

It should of course be clear that the foregoing only relates to a preferred embodiment of the present invention, and that numerous modifications or alternatives can be made therein without departing from the scope and idea of the invention as set forth in the accompanying claims.

Claims (11)

1. Procedure for cryopreservation of heart valves comprising the steps: a. Dissecting a heart valve from a heart; b. Placing the dissected heart valve in an isotonic medium with antibiotics and a cryopreservative; c. Freezing the heart valve according to a freezing schedule that will maintain an acceptable degree of cell viability; and d. Storage of the heart valve at a temperature below -132°C. 2. Method according to claim 1, wherein said antibiotic comprises an effective amount of a mixture of Amphotericin B, Polymixin B Sulfate, Cefoxitin, Vancomycin and Lincomycin. 3. Method according to claim 1, wherein the medium is a nutrient medium. 4. Method according to claim 1, wherein the cryopreservative is dimethyl sulfoxide. 5. Method according to claim 1, in which the freezing plan includes: a. Placement of the dissected heart valve in a freezing chamber; b. Lowering the temperature of the freezer chamber at a rate of approx. 0.01°C/min. to a test temperature of approx. +4°C; c. Lowering the temperature of the freezer chamber at a rate of approx. 1.5°C/min. to a test temperature of approx. -3°C. d. Lowering the temperature of the freezer chamber at a rate of approx. 95°C/min to a chamber temperature of approx. -140°C; e. Maintaining the temperature in the freezer chamber at approx. -140°C for approx. 1 minute; f. Raising the temperature of the freezer to approx. -100°C with a speed of approx. 20°C/min; g. Maintaining the temperature in the freezer at approx. -100°C for approx. 6 minutes; h. Raising the temperature in the freezer to approx. -70°C with a speed of approx. 10°C/min; i. Raising the temperature in the freezer to approx. -26°C at a speed of approx. 20°C/min. j. Maintaining the temperature in the freezer compartment at approx. -26°C for approx. 2 min.; and k. Lowering the temperature in the freezer chamber at a rate of l°C/min until the temperature in the freezer chamber is approx. -80°C. 6. Method according to claim 1, further comprising the step of thawing the frozen tissue. 7. Method according to claim 4, wherein the step of thawing the frozen tissue comprises the steps of: a. Placing the frozen tissue in a sterile saline bath at a temperature between approx. 32°C and 40°C for approx. 10 to 14 minutes; b. Transferring the frozen tissue to a medium with approx. 7.5 vol% cryopreservative for approx. 1 min.; c. Transferring the frozen tissue to a medium with approx. 5 vol% cryopreservative for approx. 1 min.; and d. Transferring the frozen tissue to a medium with approx. 0 vol% cryopreservative. 8. Method according to claim 6, wherein the heart valve has a cell viability of at least 70% after thawing. 9. Frozen viable heart valve with a cell viability of at least 70% prepared according to claim 1. 10. Heart valve with a cell viability of at least 70% produced according to claim 6. 11. Heart valve that has been frozen to at least -132°C with a cell viability of at least 70%.