AU627436B2 - Device and method for cryopreserving blood vessels - Google Patents

Description

-7 AU-AI-24210/81

PCT

3 WORLD INTELLECTUAL PROPERTY O NZ XO International Bureau INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 4 International Publication Number: WO 89/ 01286 A01N 1/02 Al (43) International Publication Date: 23 February 1989 (23.02.89) I (21) International Application Number: PCT/US88/02832 (22) International Filing Date: (31) Priority Application Number: (32) Priority Date (33) Priority Country: 18 August 1988 (18.08.88) 088,092 21 August 1987 (21.08.87) (71) Applicants: CRYOLIFE, INC. [US/US]; 2111 New Market Parkway, Suite 142, Marietta, GA 30067 (US).

MEDICAL UNIVERSITY OF SOUTH CAROLINA [US/US]; 171 Ashley Avenue, Charleston, SC 29401

(US).

(72) Inventors: McNALLY, Robert, T. 4693 Karlsgate Drive, Marietta, GA 30068 HEACOX, Albert 4349 Arbor Ridge Drive, Marietta, GA 30066 (US).

BROCKBANK, Kelvin, M. 3544 W. Hampton Drive, Marietta, GA 30064 McCAA, Cameron 1550 Terrell Mil Road #165, Marietta, GA 30067 BANK, H;rNey, L. 1315 Winchester Drive, Charleston, SC 294C7 (US).

(74) Agent: FROST, Roger, Jones, Askew Lunsford, 230 Peachtree Street, Suite 2000, Atlanta, GA 30303

(US).

(81) Designated States: AT, AT (European patent), AU, BE (European patent), CH (European patent), DE (European patent), DK, FI, FR (European patent), GB (Furopean patent), IT (European patent), JP, LU (buropean patent), NL (European patent), NO, SE (European patent).

Published With international search report.

Before the expiration of the time limit for at.'ending the claims and to he republished in the event of the receipt of amendments.

A.O.J.P. 2 7 APR 1989

AUS

T

RALIAN

9 MAl 1989 PATENT Ol-.FICE (54)Title: DEVICE AND METHOD FOR CRYOPRESERVING BLOOD VESSELS (57)Abstract A device for use in cryopteservation of blood vcsels comprising a pair of stylets insertabie into the ends of a dissected blod vessel segment. The stylets are mountable on a support track whereby the blood vessel can be distended and supported during cryogreservation procedures. Also disclosed is a freezing and thawing profile capable of maximizing endothelial cell survival. The use of chondroiting sulfate or similar compound is discussed as a novel crvoprotectant, i- Ii PCT/US88/02832 WO 89/01286 "Device and Method for Cryopreserving Blood Vessels" Technicaleld The present invention relates to a device for stabilizing blood vessels and more particularly to a preservation procedure for use during freezing blood vessels to ultra-cold temperatures whereby the blood vessels can be preserved for extended periods of time. Also disclosed is a method utilizing the device for freezing and thawing of blood vessels. Cryopreserved bi;od vessels are useful for providing grafts to patients who cannot provide their own blood vessel grafts or where fresh blood vessels are unavailable.

i Backgrau ,f the Invention "Cryopreservation" is a technique for freezing and storing cellular and tissue matter such as blood vessels, which include veins and arteries, at extremely low temperatures while preserving the viability and function of the tissue. Each year, 360,000 sri.aU vessel coronary bypass "jumps" are performed in the U.S. alone. Another 100,00 peripheral vascular procedures, below the umbilicus, are also performed. Of Vhe small vessel procedures, 15% are performed on patients whe have already had a previous operation resulting in a lack of suitable available tissue or on patients Who are diabetic or have a disease which renders the tissue less than adequate. Clinically, the only alternative is to use less than optimal tissue or use artificial vessels which are prone to c.,clusion and thus are less than ideal. Because of the successes resulting from the cryopreservation of heart valve tisue PCTIUS88/0 2 8 3 2 WO 89/01286 2 (see copending application Serial Number 000,095 filed January 2, 1987, which is incorporated by reference in its entirety herein), and to date, more than 3,000 cryopreserved valves and approximately 2,200 implants, it is the intention to expand this technology to vein and artery tissue as well. Thus, in the clinical setting, cryopreserved tissue would fill a need for the aforementioned patents and would in addition lead to less trauma for the patient and reduce surgical time and expense.

Previous attempts at the use of Ilograft vessels have met with a variety of problems. The primary concern was inconsisr'ncy in the method of harvest coupled with an inability to freeze and store the tissue properly untii its intended use. In addition, previous investigators failed to perform the freezing process using state of the art techniques, and consequently, the viability of the tissue was low and inconsistent and resulted in early loss of patency.

Although there have been a few published reports on the cryogenic preservation of veins and arteries, there has been no published systematic examination for the cryobiological variables involved in the preservation procedure. Most investigators have simply infiltrated the vessel with dimethyl sulfoxide (DMSO) and rapidly frozen the tissue in liquid nitrogen. Several other investigators have used uncontroUed and unmeasured freezing rates. When dissecrted from the body, blood vessel tissue has a natural tendency to constrict.

Investigations to date show that under such conditions the endothelial lining of the vessel may be denuded; therefore, if such a vessel is transplanted, it may be prone to thrombosis.

Preservation of the endothelial lining of these vessels is of particular importance, because the internal endothelial lining of the blood vessels actively inhibit thrombosis. Previous studies of saphenous vein cryopreservation indicate that the major abnormality in the frozen and thawed tissue was destruction and loss of this tissue layer. A primary goal of cryopreservation of the tissue is the prevention of ice crystals which damage or destroy cellular structme.

Different freezing methods are applicable to particular ti.Nsues; not all tissues are alike in their ability to withstand cryopreservation and thawing yet maintain effective viability. No investigator is known to

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WO 6 PCT/US88/02 8 3 2 WO 89/01286 3 have successfully applied this technology to the internal mammary artery or other arterial tissue.

i Summary of the Invention The device of the present invention is a structure for supporting and distending a blood vessel while permitting fluids to Sinfiltrate the vessel to facilitate cryopreservation. More particularly Sdisclosed is a blood vessel stent for use in cryopreserving blood vessels, the sent comprising: first and second elongated stylets each having an end capable of insertion within a portion of a blood vessel I from a donor, nmeans on said stylets operative to engage the interior of the blood vessel and thereby facilitate fluid tight ligation of the blood i vessel on the stylets; and support means receiving the stylets in Sselectively adjustable mutually confronting relation whereby the blood vessel is distended between the stylets to prevent contraction of the blood vessel, so that the stent supports the blood vessel through the Sstages of procurement and cryopreservation. The method of the present invention using the device involves the technique for preparation of the jj vessel prior to stenting, removal, shipping to the processing laboratory, processing (including freezing), thawing and dilution. A particular 1 emphasis is made for the preservation of the endothelimu (inner lining) of the vessels in addiion to keeping the basic integrity of the vessel walls intact. This involves a "no touch" surgical technique coupled with vasodilation, the use of a stent, and the use of a unique freezing profile that allows veins or arteries to be frozen down to the temperature of liquid nitrogen, approximately -196C, with minimal tissue damage due to ice crystal formation or osmotic shock. The present invention also includes a thawing schedule whereby the frozen tisue can be rapidly thawed with minimal tissue damage. Vessels that are cryopreserved according to the present invention are alive when thawed and are ideally suited for replacing diseased or damaged vessels in patients who for whatever reason do not have suitable vessels for cardiac or peripheral vascular reconstruction.

Accordingly, it is an object of the present invention to provide a device and method for retrieval handling of human or

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pCl'US88/02832 WO 89/01286 WO 89/01286 PCT/US88/02832 4 animi vein and artery tissue.

It is another object the present invention to provide a device; and method for preserving a living vessel for long periods of time.

It is yet another object of the present invention to providi: a unique set of chemical constituents prior to the freezing process.

It is yet another method of the present invention to provide a device for supporting and distending a dissected blood vessel.

It is yet another object of the present invention to provide a unique cooling schedule for freezing a vessel so that the vessel cells, including the endothelium, maintain high viability after it is thawed.

It is yet another object of the present invention to provide a method for cryopreserving a human vessel which allows rapid thawing of the vessel while rnairnainbig maximmn cell viability.

It is yet another object of thco present invention to provide a living vessel such that it is srniteble for transplantation after long term storage at super-cold temperaturs.

It is yet another object of the present inivention to provide a living vessel with a specific and unique mnethod for thatwing.

These and other objects, feature,,) and advantages of the ti 25present invention will become apparent after review of the following t' 25detailed description of the disclosed embodiment and the appended claims.

Brief Description of the Drawi=~ Figure 1 is a schematic representation of a stent apparatus according to a preferred embodiment of the present invention.

Figure 2 is an exploded isolated prespective of a stent apparatus according to a preferred embodiment of the present invention.

Figure 3 is a schematic representation of a freezing profile for freezing a vein.

Figure 4 is a representation of a thawing curve for a WO 89/01286 stented vein in approximately 80-90 r preferred embodiment of the present inve PCT/US88/0283 2 nl of solution according to a ntion.

Detailed Descrintion of the Freferred Embodiment Referring now in more detail to the drawings, in which like nun "als indicate like parts throughout the several views, Fig. 1 shows a bil od vessel stent 10 which is composed of three main parts: a support track 14, sliding proximal stopcock assembly 18, and fixed distal stopcock assembly 22. All three parts can be manufactured from any appropriate material which is freezable to ultra low temperatures without adverse deforming or cracking, maintains flexibility at ultra low temperatures, is chemically inert and will not contaminate the vessel by leaching chemicals such as plasticizers into the vessel, and (d) can withstand and not react with dimethylsulfoxide ("DMSO"), ethylene or propylene glycol, glycerol or any other chemicals or solvents used in the cryopreservation process. Track 14 is a substantially straight elongated member used to mount stopcock assemblies 18 and 22. The track can be any convenient shape, but is preferably a parallelepiped and has on one side a groove 26 running at least part of its length for the purpose of stabilizing the stopcock assemblies 18 and 22 when mounted on track 14. On the surface at the proximal end of track 14 is located a ramp 30 with a flange having its larger portion facing toward the distal end. Ramp 30 inhibits the mount from sliding off the end of track 14 once the mount is slid on the track.

Proxirmal slidable mount 34 is a cylindrical member having a recessed cylindrical opening axial at one end capable of matably receiving a mounting pin. At the ether end of the mount 34 is an opening radial to the cylinder, capable of operatively receiving the track 14. Mount 34 is able to slide along track 14 so as to be adjustable for a given length of blood vessel segment.

Proximal stopcock assembly 18 includes a cylindrical mounting pin 38 which removably mates coaxially within the axial hole of the proximal mount 34 and is preferably able to rotate about the cylindrical axiL, Pin 38 of the proximal mount is orthogonally

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O8928PCT/US88/02 8 3 2 ii WO 89/01286 6 integrated with the body 42, which is a hollow cylinder having a hole 46 extending through both sides of the cylinder, positioned orthogonal to the pin. Along one end of body 42, positioned radially opposite each other are a pair of protrusions 50 which act as stops. Stopcock barrel 54, which fits within the cylindrical interior of the body 42, is a cylinder rotatably positioned in a longitudinal orientation within body 42. A barrel hole 58 is positioned perpendicular to the barrel 54 axis through the barrel and selectively aligns with hole 46 in body 42 so as to allow a passageway through the stopcock. Lever 62 is a substantially rectangular member perpendicularly integrated with stopccck barrel 54 at one end. When rotated so that the lever is parallel with track 14, barrel hole 58 and body hole 46 are aligned to form a passageway through the stopcock while, when the barrel 54 is rotated clockwise to a position perpendicular to track 14, the holes are not in alignment, thereby blocking the passage of fluid or air dh'ough the stopcock.

Stylet 66 is a hollow substantially straight elongated tube aligned parallel to the track 14, with a port 70 at the free end ofthe styler and with the other end of the stylet abutting the body 42. Stylet 66 is integrated with body 42 and is orthogonal to the pin 38. Stylet 66 is aligned with and opposite hole 46. The hollow interior of the stylet 66 communicates with the hole 46 in one side of the stopcock body 42.

On the surface of styler 66 is at least one and preferably two pr more forwardly tapering ramp surfaces 70 merging with stylet 66. Stylet 66 is designed to accommodate coupling with a vessel which may be press fit upon the stylet and can abut body 42. Stylet port 68 permits fluid or air to enter the blood vessel when the stylet 66 is coupled with the blood vessel. The stylet tube is sized so as to be essentially parallel with theinternal hollow bore of the tube. Rap surface 70 terminates in a shoulder 74 which permits pressure-tight ligation as described mor 5ally hereinbelow.

Fixed distal stopcock assembly 22 is essentially the same as sliding proximal stopcock assembly 18 except with the following notable modifications. The bore of the axial mount hole is different than that of the proximal mount hole and the diameter of the i PCT/US88/ 02 8 3 2 WO 89/01286 7 mounting pin 78 is correspondingly different from the diameter of pin i 38 so that the two stopcock assemblies cannot be interchangeably mounted. This avoids confusion of which stopcock assembly is distal and which is proximal. Distal mount 82 is preferably permanently mounted on the distal end of track 14.

Distal body 86 also has integrated on its surface in 1 alignment with the hole (as in hole 46) a Luer fitting 86 for detachably receiving a syringe. Alternatively, a hub 90 can be used without the SLuer lock fitting for detectably receiving a fluid delivery device such as a syringe. Standard Luer fittings are commonly known to those skilled in the art. In an alternative embodiment a hub 90 can be integrated in a similar with the proximal stopcock assembly 18 to permit bask flushing of the blood vessel before or after being mounted on track 14.

Distal lever 94 rotates in a manner similar but opposite to that of proximal lever 62; when parallel to track 14 the distal lever 94 is in the "open" position and when rotated perpendicular to the i track is in the "closed" position. The stopcock levers are positioned so that when both are parallel to track 14 they point in toward each other and will not stretch the vessel during closure. In this way they do not i 20 add to the overall length of the structure, nor does the distal lever 94 Iinterfere with distal hub or Luer fitting 86 or The stent is used to support and distend a dissected vessel. The dissection procedure is described in detail in the examples set forth hereinbelow. A specially designed perfusion solution containing a suitable vascular smooth muscle relaxant drug is applied along the length of the vessel. After a period of time, usually about minutes, the vessel is carefully dissected using what is commonly termed as "no touch" technique, whereupon an appropriate incision is made on the distal and proximal end of the vessel. If the donor's heart is beating, the portion of the vessel which was transected will be tied with ligature. The removable stopcock assemblies 18 and 22 of stent apparatus 10 are placed one on each end of the vessel and a portion of a flushing/distention solution is perfused through the vessel. The dissection ca_ then be completed. The perfusion medium ,an be any appropriate medium, preferably Delbecco's Minimal Essential Media

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PCT/US88/02832 WO 89/01286 8 Other media include, but are not limited to, Medium 199, Eagle media, Hank's media, Delbecco's modified Eagle media, Iscoves modified Delbecco's media, Defined media A2, CMRL-1066, RPMI- 1640 (also 1603, 1630, or 1634), F10, F12, alpha media, or the like.

To this media is added serum such as but not limited to human serum, fetal calf serum serum substrate, or the like, and a vasodilator such as but not limited to nitroprusside, dantrolene, nifedipine, verapamil, phentolamine, tolazoline, procardia, or the like. It is preferable to use papaverine having a concentration of from about lxlOE-4 to about 30xlOE-4M, more preferably about 3xlOE-4M. This media solution also has certain additives: bicarbonate, -PES or similar buffer, glutamine, D-glucose and sodium pyruvate.

Before ute vessel is completely removed, the stopcock ends are attached to the support track 14 of the stent appararts 10 in order that the vessel does not have the opportunity to contract. It is this natural contraction of the vessel that is deleterious to the inner endothelial lining of the vessel and for which the stent is designed to protect. Once the remaining portions of the vessel are dissected, the stent 10 with the attached vessel is ready for transport in an outer container.

The stent 10 continues to provide support and protection for the vessel while progressing through other stages in the cryopreservation process. Upon arrival of the stented vessel for cryopreservation, the stent will provide the support for continued flushing and inspection for tying of collateral vessels. During the freezing process the stent will be the support necessary to prevent the vessel side walls from collapsing and at the final phase during thawing and dilution the stent again keeps the vessel from collapse and facilitates the addition and removal of cryoprotectant agents.

The present invention provides a method of freezing, storing and thawing endothelial lined tissue, such as a vein and artey.

The tissue that is frozen according to the present invention can be stored for long periods of time at ultra cold temperatures with minimal loss of cell viability. The present invention includes a unique freezing profile that allows a tissue such as vein and artery to be froze down to the O 2 PCT/US88/ 0 283 2 WO 89101286 9 temperature of liquid nitrogen, approximately -196°C, with minimal tissue damage due to ice crystal formation or solution effects due to slow cooling. The present invention also includes a thawing schedule whereby the frozen tissue can be rapidly thawed with minimal tissue damage. Veins and arteries that are cryopreserved according to the present invention are biologically viable when thawed and are ideally suited for replacing diseased or mechanically damaged vessels.

The tissue to be preserved is only as good as that which is received into the laboratory. Consideration must be given to donor age, health and history of vascular disease. Another important consideration is the time between death and the harvest of the vessels (warm ischemia) and the time from the harvest of the vessels to laboratory processing (cold ischemia). Attention must be. paid to the method of handling the tiisue during procurement and the medium used to ship the tissue.

A donor dhat can be used as a source of human vessels which are frozen according to the present invention should be in the age range of up to about 55 years of age and the donor should not have suffered from significant atherosclerosis, diabetes, circulatory disorders, severe hypertension, veicose veins, or communicable disease.

All procurement is to be performed under sterile conditions. Time delay between death and harvest will have a deliterious effect on the endothelial cell layer and therefore should be completed immediately after expiration of the donor but in any case not longer than about 10 hours post mortem. For example, the ideal length for coronary bypass procedure would be to procure a vessel of approximately 17cm with at least a diameter of 4mm. It is to be understood that other diameters and lengths are usable and are within the scope of this invention.

Stelization It was discovered that many antibiotics were extremely toxic to the endothelial layer of the vessels. This toxicity is the result of a number of factors including time, temperattre and mode of action. In WO89/0128 PCr/US88/02832 addition to the antibiotics, the antimycotic ("antifungal) agents may be deleterious to the tissue endothelium. It is important to continually test new antibiotics and fungicides for cell toxicity and sterilization efficacy, in order to improve cell viability and kill microbes resistant to previous agents.

H A mixture o~f an antibiotic and an antimycotic were found to provide suitable steriization results. A mixture of Ixnipenem and Ancoban were found to be particularly suitable. Table 1 shows the effect of antibio tic incubation on endothelial viability in virlo.

TABLE I Effect of Anticogiotic Incubation on Endothelial Viability In Vitro Antibiotic Time Significance VAPCVL 4 hrs. NS* Imipenem gentamycin 4 hrs. NS APCVL 12 hrs P< PSA 12 hrs. NS Imnipenemn gentamycmn 12 brs. P< limipenem without 12 hrv, NS gentamnycin Irnipernm Ancoban 18 hrs. NS V *NS =Not Significant APCVL:: Amphotericin B, 25 microgramns/mil, Polymnixin B Sulfate, 100 maicrogramns/mI Cefoxitin, 240 micrograms/mi Vancomycin, 50 mnicrograms/mi Lincomycin, 120 microgramns/mi PSA =Penicillin (501(J/mI) Streptomycin Amphotericin B WO 89101286 PCT/US88/02 8 3 2

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i i Freezing Media The medium in he_ tissue is frozen is of great importance for maintaining a balanced cell environment. Time and temperature also contribute to whether a particular medium will be successful. Generally, a protein suspension, such as blood serum or artificial serum, must also be present for maximum cell viability.

A number of freezing media can be successfully used in practicing the present invention. Media, such as balanced tissue culture medium or simple phesphate buffered saline, can be used for most tissue types. For this particular tissue type DMEM is the preferred medium with the associated additive components discussed previously.

The freezing media is composed of the enriched DMEM plus FCS from about 1% to 30%, more preferably 10% fetal calf serum; plus the range of papaverine discussed above, preferably about 0.012% papaverine; and, chondroitin sulphate having a concentration of from about 1% to 10%, preferably 2.5% to more preferably Dimethylsulphoxide ("DMSO") is also added either in at least one step of 1M or preferably in three steps of .25M, .5M and 1M titrations at 4'C. Concentrations of DMSO can range from abou: 0.5 to 3 molar. The increase in molarity of DMSO should preferably be gradual so as not to traumatize the blood vessel. DMSO can be added at higher temperatures but timing becomes far more critical and toxicity may result in some tissues.

An important innovation in endothelial pxot'to4 i used to further refine and preserve tissue integrity is to use chondroitin sulfate. This glycosaminoglycan (G AGS) is a major component of the extracellular matrix. The molecular weight of chondroitin sulfate can vary from 5,000 to 50,000 and it is a sulphated dissacharide consisting of repeat units of D-glucuronic acid and N-acetyl-D-galactosamine.

Currently, this material is an additive in K-sol, a solution used for the short term (40C) storage of corneas.

Examples of other suitable glycosaminoglycans include but are not limited to hyaluronic acid, dermatan sulfate, heparin sul.ate, heparin, arid the like. Other cryoprotectants include but are not limited IO PCT/US88/0283 2 WO 89/01286 12 to glycerol, polyvinylpyrolidone, hydroxyethyl starch, and polyethylene glycol, dimethylformamide, ethyl glycol, and the like.

i Table II shows a series of experiments using this additive to the freezing solution. Groups 1-6 used a protocol which differed from Groups 7 and 8 in that a two hour 37'C incubation was performed. The results indicate that the addition of chondroitin sulfate to the freeze mixture significantly improved endothelial viability.

SThese materials can be used in cryopreservation procedures with or without the stent apparatus 10. As a cryoprotectant chondroitin sulfate or its alternatives(GAGS) can be employed in procedures for cryoprotection of cells, tissues and organs in addition to blood vessels.

PCT/US88/028 32 WO 89/01286 TABLE II Influence of Chonjo.itin Sulphate unon Endoth Ce Viabilit Cell Viability (Regnrssionl T-telt Treated Chnroitin sfate T Signif r f l I ud Refr /Contl ession (CS) addion oLMM La..

1) Without vs with 6 chondroitin sulfate a) Saphenous veins only 3 b) Femoral veins only 3 2) Without vs with CS 4 in mannitol during dilution (no CS during freeze) 3) Without vs with CS 5 during 2 hr incubation (with CS during freeze and dilution) a) Saphenous veins only 3 b) Femoral veins only 2 4) Unfrozen vs frozen 4 without CS, with CS during dilution and 2 hr incubation Without vs with CS 6 during all steps except no CS during freeze 6) Unfrozen vs with CS 2 during dilution, no CS during freeze and no incubation 3.309 3.426 '4.091 42) 51 (124) 174 (147) 95 (391) 429 (25) 29 (61) 71 2.95 2.66 2.44 86 .457 ns (217) 203 (187) 171 ns (109) 63 (60) 47 .606 ns .603 ns 12.171 (94) 83) (89) (300)327 (57) 56 2.069 ns 69) 48 (32) 28 5.752 (390)457 (177) 163 A ternaive CS Concentations 7) Without vs with 10g% 4 chondroitin sulfate (CS) 2.415 (118) 172 (319) 334 8) Without vs w/lg% chondroitin sulfate 4 1.704 ns (207) 131 (69) 82 I ns not significant; (P P .05; P .01 tt Groups 1-6 were performed using Protocol II. Protocol I1 differed from PrWtool I by Lhe addition of post-thaw 2 hr. 37C incoation in culture medium 4i

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O PCT/US88/02832 wo 89/01286 14 Freeze Profile The freezing profile is of critical importance to successful cryopreservation of a tisue. A multitude of variables exist to maximize tissue survival. For instance, the volume of fluid, the size of the tissue, geometry of the package and the combination of characteristics incorporating cryoprotectant, tissue, and freezing mtl: all contribute to an optimal freezing profile. It is to be understood that the prior art freezing profiles available for cell suspensions may not be suitable for freezing blood vessels, and that prior art freezing profiles for heart valve tissue also may not be suitable. It has been determined that each tissue has its own unique and optimal freezing profile. The I freezing profile required to successfully cryopreserve one tissue may be different from the freezing profile required to successfully cryopreserve another tissue.

I 15 A number of factors need to be considered when |i freezing a tissue. Among these factors are: the temperature anrund the equilibrium point, (generally +4 0 C, to the temperature at the freezing point); release and control of the exothermic heat given off at the freezing point; optimum cooling rate as which is determined by the 20 permeability of the cell membrane to water; the surface to volume ratio of the cells; the type and concentration of cryoprotective agents in the ztedia; temperature and time of exposure to Lhose agents, cooling rate removal of the cryopreserved tissue from the controlled rate freezer and immersing the tissue into a liquid nitrogen refrigerator, and, warming rate and the thickness of the tissue.

Detail of Freezing Profile Thus, the method of harvesting veins from a donor, placing the vessel into a medium with the proper tissue preserving characteristics for transportation, and the use of proper cryopreservation agents prior to the freezing of the vessel according to a freezing schedule is desirable for proper cryopreservation. To accomplish this the chamber temperature and cooling rate is controlled so as to produce the desired effect on the sample. Since the blood vessel tissues cool at slightly different rates, and phase changes occur at

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PCT/US88/0283 2 WO 89/01286 particular temperatures, careful coinrol over the rates of freezing should be maintained.

The range of the freezing rate is also a function of the fluid volume in the package containing the blood vessel as well as the geometry of the package. While the freezing profiles described herein are related to the volume and geometry of the package, it is to be understood that the present invention encompasses those modifications of package design which rsult in a change in the volume and geometry, which in turn, resul: in a variance in the freezing rate.

Freezing rates can vary to a certain amount at a given temperature. A suitable range is from about 0.01 -10OC/rain, about 0.1-30'C/min, preferably 'bout 0.3-1 C/min and more preferably 0.5-1'C/min. In a preferred embodiment of the present invention, the overall rate of freezing of the blood vessel is kept at approximately 0.5'C per mintue.

A preferred freezing schedule used to cryopreserve the veins and airteries in the present invention is comprised of placing the packaged tissue, having a total volume of about 2.5 cm x 25 cm, into a freezing apparatus. A typical profile for a specific fluid volume of 75-85mi would have the initial temperature of The chamber set to -10'C. The chamber is set to cool at a rate of 0.01'C/min. until the sample(s) reaches At this point the tissue cools at a. rate of 0.5 02'C/min. until the sample reaches -2 0.5'C, at which time a phase change is initiated. At this point, in order to prepare for the exothermic heat of fusion, the cooling rate is increased to -30C until the chamber reaches -70'C. Immediately after the chamber reaches -70*C, the chamber is warmed at a rate of 20C/min. until the chamber reaches whereupon this temperatur is held for a period of 17 minutes.

Dudng this time, the actual rate of vessel cooling is approximately 0.2 C/min. At the end of this 17 minute period, the chamber is again warmed at a rate of 10'C /min. until the chamber reaches a level of 0 C. The level of -30 0 C is held for one minute and then the cooling of the chamber commences at a rate of 0.01*C/min. until the sample reaches -20*C. During this time, the actual rate of cooling of the sample is approximately 0.5 0.2*C/min. The final rate adjustment step is to continue cooling at 0.5 0.2*C/min. until the sample reaches -65*C or (i

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WO 89/01286 PTU812 3 16 below. The result of this freezing profile is a rate of freezing from the start of the procedure until the end of about 0.5 0.2 0 CQmin. This rate of cooling has been optimized for vein tissue. T'he package containing the vein is removed from the chamber and placed in the storage liquid nitrogen refrigerator at -1 96 0 C. Figure 3 illustrates a typical freeze profile of the present invention.

At such time that the vessel is requested by an implanting institution, the tissue will remain in the liquid nitrogen A refrigerator.

Shipping At the request of the implanting hospital, the tissue may be returned in a suitable insulated shipping container such as the container disclosed in U.S. Patent No. 4,597,266 which is incorporated by reference in its entirely herein, which includes a cardboard container with four inches of foamn insulation. A preferred embodiment is the use of dry ice whicn has been stored in liquid nitrogen whereupon liquid nitrogen infused dry ice is placed around the package containing the vessel. The package is then placed into the box.

Appropriate protocols and other papers necessary to document clinical implants are included in the shipment Upon arrival at the hospital, the vessel and its associated package are placed into a liquid nitrogen freezer. The tissue cannot tolerate storage at temperatures above 130TC since repeated cycling of temperatuires has a tendency to lessen the viability of the cells. Storage at temperatues equivalent to dry ice is not considered sufficiently cold to prevent enzyme molecular degradation of the tissue and thus the storage time is significantly reduced.

nawig The thawing and diluting steps with an allograft must be clea~rly defned, since ice crystal growth and osmotic shock can still harm the tissue. Venous blood vessels should be thawed by being placed in a warm water bath. It has been deternined that a thawing rate of I I000 0 /min., preferably, I 0*-50*Q/min. is appropriate for these PCI/US88/02832 WO 89/01286 17 vessels, depending upon the volume of the sample. Once thawed, the cryoprotectant of choice must be removed, usually in a step-wise fashion, to lessen the effects of osmotic shock to the cells and thus allow for an orderly equilibration of the cell with the surrounding medium. Time and temperature are major considerations.

Immediately prior to the time that the vessel is to be used, it is to be thawed and the cryopreservation additives are to be removed using a gradual dilution procedure to minimize osmotic damage. This thawing and dilution procedure is considered as important as the actual freezing procedure since ice crystal formation can occur during this phase of the procedure as well. In addition, inattention to proper temperature and timing can and will either reduce the number of viable cells due to the toxicity of the cryoprotectant ingredients and can on occasion actually lead to the cracking of the vessels into mlnusable pieces.

The following specifc examples will illustrate the method of the present invention as it applies to harvesting, freezing to ultra-cold temperatures, and thawing of a blood vessel. It will be appreciated that the example will be apparent to those of ordinary skill in the art and that the invention is not limited to this specific illustrative example.

Examnle 1 The dissection is performed using sterile "no-touch" technique. The vessel and adventitia are bathed in perfusion medium (DMEM, 10% fetal calf serum and 0.12 mg of papaverine/ml) throughout the procedure. This medium also has as an additive: Hepes buffer, glutamine, 1000mg D glucose/L and sodium pyruvate at at pH 7.3 0.5(GIBCO Lab Cat #380-2320). Following removal of the adventitia, a caudal venotomy is made and the stopcock assembly is inserted and ligated into place. The vessel is gently perfused with the perfusion medium. The collateral vessels are identified and ligated approximately 1-2mm from the main vesseL Before complete excision of the vessel, the support track 14 of the stent apparatus 10 is affixed to the distal and proximal 1 PCT/US88/0283 2 WO 89/01286 18 stopcock assemblies 18 and 22. The dissection is completed, the proximal stopcock is closed and the vessel is infused with the perfusion medium up to approximately 100mmHg, whereupon tie distal stopcock is closed in order to keep the vessel distended The vessel with the remaining perfusion solution is placed into a shipping container such as a plastic tube and double sterile wrapped. The transportation box is commonly made of styrofoam and the tube containing the vessel is placed in water and ice at approximately 4C. Hereafter, a courier service is commonly used to speed the delivery to the laboratory, since the vessel, in order to remain living, should arrive within about 24 hours after the cessation of the donor heartbeat.

Upon arrival at the processing laboratory, die- vessel is checked for proper packaging to verify that ice is stiU present. In a clean room, a sterile field is established to inspect the vessel and complete the processing steps, the first of which is to inspect and trim extraneous tissue. After all collateral branches have been checked and there are no leaks, the stented vessel is ready to start antibiotic sterilization.

Stelization In order to prophylactically sterilize the vessels, the following critical procedure must be observed.

1. Imipenem (12gg/ml) is placed into a solution of DMEM, which is a tissue culture media; 2. Ancoban (antimycotic) (50p4g/rnl) is also added to the solution; 3. the vessel is perfused and bathed in this solution is placed into a 37 0 C incubator for four hours.

Following the titration of the cryoprotectant, the vessels are packaged in pouches which are capable of withstanding the rigors of ultra cold cryopreservation. Normally, several successive layers of packaging are used in order to preserve sterility of the inner package containing the vessel. Finally, the vessel is ready for cryopreservation.

The freezing medium is composed of the enriched PCT/US88/028 32 WO 89/01286 19 DMEM 10% fetal calf serum 0.12mg/mi papaverine chondroitin sulphate IM DMSO or other suitable protectant.

Detail of Freezing Profile The freezing schedule used to cryopreserve the veins and arteries in the present invention is comprised of placing the packaged tissue with 75-85rrm of fluid volume and cylindrical shape of x 25cm into a suitable freezing apparatus (such as Cryomed Model #1010 (990C)). Temperatures provided are approximate and a certain amount of latitude must be provided to account for machine and instrument variance. The initial temperature of the chamber is set to 0 C. The chamber is set to cool at a rate of 0.01oC/minute until the 1 sample reaches +4 0 C. At this point the tissue cools at a rate of 0.3 S0.2 0 C/min. until the sample reaches which initiates a phase change. At this point, in order to prepare for the exothermic heat of fusion, the cooling rate is increased to -30°C until the chamber reaches 0 C. Immediately after the chamber reaches -70 0 C, the chamber is warmed at a rate of 20°C/min. until the chamber reaches whereupon this temperature is held for a period of 17 minutes. During this time, the actual rate of vessel cooling is approximately 0.5 .2 0 C/min. At the end of this 17 minute period, the chamber is again warmed at a rate of 10 0 C/min. until the chamber reaches a level of The level of -30 0 C is held for one minute and then the cooling of the chamber commences at a rate of 0.01oC/min. until the sample reaches -20*C. During this time, the actual rate of cooling of the sample is approximitely 0.5 0.2 0 C/min. The final rate adjustment step is to continue cooling at 0.5 0.2 0 C/min. until the sample reaches -65 0 C or below. The result of this freezing prwfiie is a rate of freezing from the start of the procedure until the end of 0.5 0.2 0 C/min. This rate of cooling has been optimized for vein tissue. The package containing the vein is removed from the chamber and placed in the storage liquid nitrogen refrigerator at 196°C.

Until such time that the vessel is requested by an implanting institution, the tissue will remain in the liquid nitrogen refrigerator.

4 PCT/U8/83 WO089101286 _Sh ingfl The frozen vessel is shipped in an appropriate container in the manner as described above.

Thawin a The thawing and dilution procedure can be performed as follows: 1, In a sterile field and with all sterile components, at least two liters of sterile water warmed to about 371- 42'C are placed into an instrument tray or other vessel to accommodate the length of the stented tissue.

2. The packaged vessel is removed, from the protective cardboard box and placed into the water bath. The package is to remain in this bath, where it is manually or automatically agitated for approximately eight.minutes, or until such tirne that gentle palpation of the package reveals that no further ice crysta1z are present.

3. Once it has been determined that no ice crystals are present, the package is removed from the bath and the outer foil package is carefully wiped dry in the area between the two notches and with a pair of scissors, the foil package is cut between the two notches.

Sterile forceps are used to retrieve the inner clear pouch from the foil pouch and the process is repeated using sterile scissor to cut the inner pouch.

4. The stented vessel is carefully removed and 1? placed into a clean sterile instrument tray or other suitable container whereupon the first of the dilution steps takes place. A syringe is used to add to the vessel 50cc of a solution ("~Bottle containingmannitol; 10% fetal calf serum; and

PMEM

71,e vessel is gently perfused with this solution and the washout material is allowed to rest in the tray whereupon the vessel is allowed to soak for five minutes. Instead of mannizol, any non-permeable biocompatible sugar can be substituted, such as but not limited to,

I

I

PCT

WO 89/01286 21 'US88/02832 1 Jl sucrose, sorbitol, trehalose, glucose or the like. The dilution of DMSO concentration should be in decreasing steps of no more than 1/2 the molarity of the previous step. Thus, if the original DMSO concentration is 1M, the first dilution step should be 1/2M sugar, followed by 1/4 M sugar and finally zero molar sugar.

At the completion of step 4, the solution in the tray is discarded into another suitable container and the following is added to the tray and mixed: remaining in the bottle A from step 4; plus 50cc from a solution ("Bonte containing: fetal calf serum; and

DMEM

The mixture is now 0.25M mannitol. A syringe is used to gently perfuse the vessel with this mixture, and it is bathed in this solution for approximately 5 minutes.

6. At the completion of step 5, the contents of the tray are again discarded and the remainder of bottle B is added to the tray. The vessel is gently perfused and agitated in this solution for five minutes.

7. The vessel is now ready for transplant, but should not be removed from the stent until just prior to its intended use.

Fig. 4 illustrates a typical thawing curve based upon an ml (1M DMSO DMEM) sample containing a 20 cm. segment of vein being placed in 5 liters of 42"C water and package was agitated by hand twice per minute for 15 seconcds for each agitation until the vessel is thawed. The water bath was allowed to cool as the thaw progressed.

The thawing rate was approximately 25'C/min. A typical thawing time is about eight minutes.

Transolant The vessels preserved by the method just described are intended for use as arterial substitutes for the coronary arteries or for peripheral vascular reconstruction. Accordingly, since the graft tissue is antigenic tissue several precautions and recommendations are suggested:

J

WO 89/012 PCT/US88/02832 WO 89/01286 22 1. The donor/recipient blood groups should be compatible; 2, A postoperative course of antipialelet therapy may include but not be limited to Dipyridamole or aspirin; and 3. Low dose short duration immunosuppression which may include but not be limited to cyclosporin, prednisolone, and azathioprine to minimize the possibility of graft rejection.

By use of the above technique, platelet deposition is depressed, thus lessening the possibility for thrombus formation and eventual loss of patency. Other forms of vessel pretreatment to lessen immunologic effects such as incubation in an immunosuppressive agent could be I considered.

Studies to Support the Claim that Vessel Endothelial Cells are Preserved S|An endothelial cell line was comprised of clonal bovine endothelium (BFA-lc). These cells were cryopreserved in situ while attached to plastic tissue culture substrate. Viability was determined by using a combination of acridine orange and propi'dium iodide, a dye Sinclusion/exclusion assay. Dimethylsulfoxide was found to be more Seffective than other cryoprotective agents tested including glycerol, hydroxyethyl starch and polyvinylprolidone. It was determined that 1M DMSO was optimal when used in combination with slow cooling rates. See Table II.

PCT/US88/02832 WO 89101286 23 TABLE III Studies on Freezing P~vcedures for Veins (using limiting dilution assay on endothelial cells) ICeU Viability r~ I 5ignd Control Treate Trete/ntrol Frezinrae 1) Unfrozen vs O,S 0 /min, 6 3 step DMSO addition 2) Unfrozen vs 3 0 /min, 3 3 step DMSO addition 3) OX 0 vs 3 0 /min, 3 step DMSO addition 3 4) Without vs with DMSO (3 ,ep) 6.696 3,554 1.679 4.5 12 (258) 338 (153) 138 (248) 228 96) 56 ns (119) 95 96) 56 (20)18S (109) 84 1. ns= not significant, p 0.0 :501 8/2PCT/US88/028 3 2 WO 89/01286 24 At 0.5°C/min., more than 70% of the endothelial cells survived, as compared with a cooling rate of 1 0 C/min. or more where less than of the cells survived. See Table IV.

Iu e TABLE IV Influence of Cooling Rate Upon Survival of a Bovine Endothelial Cel Line Coolin_ Rate °C/min Experiment 1/2 3 10 Viable Cells 1 68.8 58.7 17.5 15.3 (%Total) 2 78.9 63.1 3.1 16.1 3 76.7 56.4 17.5 4 80.3 28.4 24.5 5.3 Mean plus one 75.68 51'f1.6 5 11.68 standard deviation 54 3. .0 The previous experiment was repeated using actual saphfious vein vessels and utilizing the protocol for preparation and titration of the DMSO discussed earlier. In essence, DMSO is added in three steps of ten minutes each at 4C, such that the concentration increase from 1/4, 1/2 to IM. The DMSO is mixed with DMEM containing HEPES buffer and 10% fetal calf serum. The freezing rate was varied h from either 0.50 or 3C/min, The experiments show that the freezing rate of 0.5*C resulted in a higher percentage of endothelial integrity than the faster rate of 3C. Sec Table V.

WO089101286 PCT/US88/02832 TABLE V Endothelial Integrity After Cryopreservation at 0.5' or 31C/miri.

Endtepl Inert Rate 0 C/rniin) Sample Vein Mean se of Control Control 114 38 3 10 4 62+/-7 72.1 20 7 70+/-5 81.4

Claims (28)

1. A method for cryopreserving a blood vessel comprising the steps of: dissecting a blood vessel from a patient and mounting said blood vessel in a suitable frame or stent; placing the dissected blood vessel into an appropriate medium with effective amounts of at least one antibiotic; contacting the dissected blood vessel with an effective amount of at least one cryopreservative; freezing the blood vessel, mounted in the stent, according to a freezing schedule which comprises: placing the blood vessel into a freezing chamber; (ii) cooling the blood vessel at a suitable ra.to until the sample reaches about -2 0 C. (iii) cooling the blood vessel after substantial completion of phase change at a suitable rate; (iv) removing the blood vessel from the chamber; placing the blood vessel in a liquid nitrogen refrigerator at a temperature of about -196 0 C; and storing the blood vessel at a temperature below -lCOC.

2. The method of claim 1 wherein the antibiotic is an effective amount of a mixture of Imepenem and Ancoan.

3. The method of claim 1 or claim 2 wherein the vessel is titrated with the cryopreservative and the addition of dimethylsulfoxide.

4. The method of any one of claims 1 to 3 wherein the cryopreservative is an effective amount of a medium, HEPES buffer, glutamine, D-giu-ose and sodium pyruvate, fetal calf serum, papaverine chrondroltin sulfate and DMSO. The method of claim 4 wherein said medium is selected from the group consisting of Medium 199, Eagle media, Hank's media, Delbecco's modified Eagle media, Iscoves modified Delbecco's media, Defined media A2, CMRL-1066, RPMI-1640 (also 1603, 1630, or 1634), FlO, F12, alpha media, or the like.

6. The method of claim 5 wherein the medium is Delbecco's Minimal Essential Media.

7. The method of any one of claims 1 to 5 wherein the cryopreservatfve is an effective amount of a mixture comprising: Delbecco's Minimum Essential Medium, i I I t i1 I j 1 A 4i I CERj"~c~ cA RA 4 C 4 N f STA/1624w 1. I S.-*P INTERNATIONAL SEARCH REPORT Internatlonal AocIication No PCT/US 88/02832 1 I I CLASSIFICATION OF SUIJECT MATTER (it several clrIasslcjton synools a oly. v1dicate AlItI I i N I 4i I 1 11-1- mi 27 hepes bufrer, glutamine, 1000mg D-glucose/liter, sodium pyruvate at pH 7.3, 10% fetal calf serum, 0.012% papaverine, chrondroitin sulfate, and IM DMSO.

8. The method of any one of claims 1 to 7 wheriein the vessel is thrice titrated with an effective amount of a mixture of: Delbecco's Minimum Essential Medium, 25mmol hepes buffer, glutamine, 1000mg D-gluccse/liter, sodium pyruvate at pH 7.3, 10% fetal calf serum, 0.012% papaverine, chrondroitin sulfate, and either 1M DMSO, or successive addition of 1/4M, 1/2M and 1M dimethylsulfoxide.

9. The method of any one of claims 1 to 8 wherein the cooling rate is in a range of from about 0.1-100°C/min. The method of claim 9 wherein the cooling rate is in a range of from about 0.1-30 0 C/min.

11. The method of claim 10 wherein the cooling rate is in a range of from about 0.3-1°C/min.

12. The method of claim ii wherein the cooling rate is in a range of from about 0.5-1*C/min.

13. The method of claim 12 wherein the cooling rate is about 0 C/min.

14. The method of any one of claims 1 to 13 wherein the freezing schedule comprises: placing the blood vessel into a freezing chamber; then cooling the chamber from an Initial temperature of about at a rate of about 0.01 0 C per min. until the sample reaches about +4°C; then cooling the chamber at a rate of about 0.3°C per min. until the sample reaches about -2 0 C; then 'I 1 IE I I ;i i-J 28 cooling the chamber at a rate of about 30°C per min. until the chamber reaches a temperature of about -70°C; then warming the chambar at a rate of about 200C per min. until the chamber reaches a temperature of about -60oC; then holding the chamber at a temperature of abo, -60C for 17 minutes; then warming the chamber at a rate of about 10 0 C per min. until the chamber reaches a temperature of about -30 0 C; then holding the chamber at a temperature of about -30"C for one min.; then cooling the chamber at a rate of about 0.01°C per min. until the sample reaches a temperature of about -20OC; then cooling the chamber at a rate of about 0.5°C per min. until the sample reaches a temperature of about -65°C; then removing the blood vessel from the chamber; and then placing the blood vessel in a liquid nitrogen refrigerator at a temperature of about -196 0 C. The method of any one of claims 1 to 14 further comprising the step of thawing the frozen blood vessel,

16. The method of claim 15 wherein the thawing comprises the steps or: placing the vessel into an amount of sterile water warmed to 37*C until such time that gentle palpation or visual inspection of the vessel reveals that no further ice crystals are present; removing the cryopreserved vessel from the sterile water; perfusing the vessel with 50cc of a solution comprising effective amounts of: 0.5M mannitol; (11) 10% fetal calf serum; and (111) Delbecco's Minimal Essential Media ("DMEM") plus Hepes buffer, glutamine, 1000mg D-glucose/liter and sodium pyruvate: allowing the vessel to soak for five minutes in the washout material from step above; adding a solution comprising effective amounts of: 0.25M mannitol; (ii) 107. fetal calf serum; and (iii) DMEM plus 25mM Hepes buffer, glutamine, 1000mg D-glucose/llter and sodium pyruvate; STA/1624w I a. a a.. cc 4 R~ It 1 r, I I :i i r I i i i i i i 1 ir iii 29 perfusing the vessel with the mixture of: 0.25M mannitol; (ii) 10% fetal calf serum; and (1ii) DMEM plus 25mM Hepes buffer, perfusing and bathing the vessel in an effective amount of solution composed of effective amounts of: 10% fetal calf serum; (ii) DMEM plus 25mmol Hepes buffer, glutamine, 1000mg D-glucose/liter and sodium pyruvate; for five minutes.

17. The method of any one of claims 1 to 16, wherein said stent comprises: first and second elongated stylets each having an end capable of insertion within a portion of a blood vessel from a donor; the stylets comprise a fluid passage in selective communication with the interior of a blood vessel within which the stylet is inserted, so as to perfuse a fluid through the blood vessel extended between the stylets; valves associated with at least one fluid passage to selectively permit fluid flow through the stylets to the blood vessel; means on said stylets operative to engage the interior of the blood vessel and thereby facilitate fluid tight ligation of the blood vessel on the stylets; and support means receiving the stylets in selectively adjustable mutually confronting relation whereby the blood vessel is distended between the stylets to prevent contraction of the blood vessel, so that the stent supports the blood vessel through the stages of procurement and cryopreservation.

18. The method of claim 17, wherein the support means comprises: an elongated track; first and second stylet support members disposed on said track; at least one of stylet support members movably attached to the track, so that the distance between the stylet support members is adjusted by moving the one stylet support member relative to said track; means detachably receiving the stylets on corresponding the stylet support members; the stylets including a fluid passage accessible to the interior of a blood vessel disposed on the stylets, and STA/1624w a a *aa. a a V: (I I F ii "I 30 *9 0* cc a valve associated with the stylets and selectively operative to block said fluid passage, thereby controlling access to the interior of a blood vessel disposed on the stent.

19. The method of claim 18, wherein: the valve comprises a valve body having an interior chamber and a port communicating with the chamber; a stylet connected to the body with the fluid passage of the stylet communicating with the port to the interior chamber; and a valve element operatively associated with the interior chamber to selectively allow fluid flow access to the interior of a blood vessel disposed on the stent. The method of claim 18 or claim 19, wherein: the detachable receiving means comprises a plug member on one of the valve body and the stylet support means, and a mating receptacle member on the other of said valve body and the stylet support means, so that the valve bodies and connected stylets can be removed from the remainder of the stent while connecting the stylets to the blood vessel, an thereafter attached to the stylet support means with the blood vessel extended between the stylets.

21. The method of any one of claims 1 to 16 wherein the dissected blood vessel is supported and distended on a stent as defined in any one of claims 17 to 20 inserting the proximal stylet into the proximal opening of the vessel and inserting the distal stylet into the distal opening of the vessel and ligating each end of the vessel to the respecti.e stylet and then mounting each stopcock assembly on the respective mount on the support track to prevent the contraction of the blood vessel. 22, The method of any one of claims 1 to 16, wherein the stent comprises; an slongated substantially straight support track means; a slidable stopcock assembly removably mountable on the support track means, comprising: a stopcock body; (1i) a hollow stylet communicating with the stopcock body; (ill) a mounting means for removably mounting the stopcock body on the support track means; a fixed stopcock assembly mounted on the support track means, comprising: STA/1624w I I S- 31 i a stopcock body; (ii) a hollow stylet communicating with the stopcock body; and (iii) a hub means communicating with the stopcock body for facilitating the passage of fluid from a container to the stylet.

23. The method of any one of claims 1 to 16, wherein the stent comprises: an elongated subtantially straight support track means; a sliding stopcock assembly adjustably mounted on the support j track, and comprising: 'J an elongated cylindrical mount connecting the stopcock with the track and having a hollow space bored radially through the mount and a second hole bored axially through a portion of the mount capable of I receiving a mounting pin; (ii) an elongated hollow stylet having a blunt open end and of a diameter capable of being inserted into a portion of blood vessel; Sand at least one raised annular flange situated along the exterior of the stylet, the flange having a rearwardly tapering ramp surface to facilitate insertion of the flange and stylet into a blood vessel, the flange terminating in a flange shoulder thereby serving to engage the interior of the blood vessel to facilitate fluid tight ligation of the i .i •blood vessel to facilitate fluid tight ligation of the blood vessel on the stylet; (ill) a hollow cylindrical body within which is coaxially and operationally positioned a cylindrical stopcock and lever containing a radial hole which can be aligned with a hole in the body so as to S. :selectively permit or block material from entering the stylet being sized to be removably accepted within a blood vessel; (iv) a hollow tubular stylet extending radially outward from the body in alignment with the body and stopcock holes; a cylindrical mounting pin extending outward from the body and orthogonal to the stylet capable of being operatively positioned in a coaxial orientation within the longitudinal hole of the stopcock mount; and a fixed stopcock assembly as in further comprising a hollow cylindrical hub terminating in a substantially planar surface and axially extending from the body so as to be linear with the stylet thereby forming an open passageway through the hub to the body to the c i ul STA/1624w I- 32 stylet, and into the blood vessel, the fixed stopcock being rotatably mounted perpendicular to and at one end of the support track and planar to the sliding stopcock by a fixed mount integrated with the support track means in a manner such that the port of the sliding stopcock stylet and the port of the fixed stopcock stylet can point toward one another.

24. A method of cryopreserving a blood vessel substantially as hereinbefore described with reference to the Example. A blood vessel stent for use in cryopreserving blood vessels substantially as hereinbefore described with reference to the accompanying drawings. 26, A kit for the cryopreservation of a blood vessel, wherein the components of said kit are adapted to be used in the manner defined in any one of claims 1 to 24, comprising, in combination: a blood vessel stent as described in any one of claims 17 to 23; an effective amount of a cryopreservative solution; and packaging means for maintaining the sterility and integrity of the blood vessel. I 27. The kit of claim 26 wherein the cryopreservative solution i comprises a mixture of: Delbecco's minimum Essential Medium, 25mmol hepes buffer, gl utamine, 000mg D-glucose/llter, sodium pyruvate at pH 7.3, fetal calf serum, S...0.012% papaverine, 2.5% chrondroitin sulfate, and I 1M DMSO.

28. A kit for the cryopreservation of a blood vessel, wherein the components of said kit are adapted to be used substantially as hereinbefore described with reference to the Example.

29. A cryoprotectant composition when used in cryopreserving a blood vessel, in a manner defined in any one of claims 1 to 24, comprising an effective amount of chrondroitin sulfate. c C. J STA/16,24w 'T ^T *y t 33 A cryoprotectant composition used in cryopreserving blood vessels, comprising a medium for freezing tissue, a cryoprotectively effective concentration of a cell penetrating cryoprotectant and a cryoprotectively effective concentration of a glycosaminoglycan.

31. The cryoprotectant composition of claim 30 wherein the glycosaminoglycan is selected from hyaluronic acid, dermatan sulfate, hparin sulfate, heparin, and chondroitin sulfate.

32. The cryoprotectant composition of claim 30 or claim 31, wherein the freezing medium comprises Delbecco's minimum essential medium and fetal calf serum.

33. A composition comprising: 1) a tissue storage medium comprising a cryoprotectively effective concentration of a cell penetrating cryoprotectant and a cryoprotectively effective concentraL on of a glycosaminoglycan, and ii) a segment of a blood vessel, wherein said segment of said blood vessel is submerged in said medium.

34. A method of maintaining intact a layer of endothelial cells present in a blood vessel during cryopreservation comprising: 1) contacting said blood vessel with an effective amount of a cryoprotectant composition comprising a medium for freezing said blood vessel, a cryoprotectively effective concentration of a cell penetrating i. cryoprotectant and a cryoprotectively effective concentration of a glycosaminoglycan; and ii) maintaining said blood vessel in contact with said composition 'at a temperature below -100 0 C. The method according to claim 34 wherein said composition further comprises an antibiotic.

36. The method according to claim 34 wherein the glycosaminoglycan is chondroitin sulfate.

37. The method according to claim 34 wherein said cell penetrating cryoprotectant is dimethylsulfoxide.

38. The composition of claim 33 or the method of maintaining intact a layer of endothelial cells present in a blood vessel during cryopreservation of claim 34 wherein the cryoprotectant comprises an effective amount of a mixture of: Delbecco's Minimum Essential Medium, hepes buffer, ,r ^/STA/LMM/1624w -34 gi utami ne, 1000mg D-glucose/liter, sodium pyruvate at pH 7.3, 1010 fetal calf serum, 0.012% papaverine, an effective amoun't. of chrondroitin sulfate, and IM DMSO. DATED this FIFTEENTH day of JUNE 1992 Medical University of South Carolina Cryolife, Inc. Patent Attorneys for the Applicants SPRUSON FERGUSON