WO2000060935A1 - Method of cryopreservation of blood vessels by vitrification - Google Patents
Method of cryopreservation of blood vessels by vitrification Download PDFInfo
- Publication number
- WO2000060935A1 WO2000060935A1 PCT/US2000/009652 US0009652W WO0060935A1 WO 2000060935 A1 WO2000060935 A1 WO 2000060935A1 US 0009652 W US0009652 W US 0009652W WO 0060935 A1 WO0060935 A1 WO 0060935A1
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- WIPO (PCT)
- Prior art keywords
- blood vessel
- cryoprotectant
- solution
- temperature
- solutions
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- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 208000037974 severe injury Diseases 0.000 description 1
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- 239000011734 sodium Substances 0.000 description 1
- 229940083542 sodium Drugs 0.000 description 1
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- 239000000176 sodium gluconate Substances 0.000 description 1
- 235000012207 sodium gluconate Nutrition 0.000 description 1
- 229940005574 sodium gluconate Drugs 0.000 description 1
- 229960002901 sodium glycerophosphate Drugs 0.000 description 1
- AQKFVNLHZYOMKQ-WWNCWODVSA-M sodium;(2r,3r,4r,5r)-2,3,5,6-tetrahydroxy-4-[(2s,3r,4s,5r,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyhexanoate Chemical compound [Na+].[O-]C(=O)[C@H](O)[C@@H](O)[C@@H]([C@H](O)CO)O[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O AQKFVNLHZYOMKQ-WWNCWODVSA-M 0.000 description 1
- REULQIKBNNDNDX-UHFFFAOYSA-M sodium;2,3-dihydroxypropyl hydrogen phosphate Chemical compound [Na+].OCC(O)COP(O)([O-])=O REULQIKBNNDNDX-UHFFFAOYSA-M 0.000 description 1
- HBYYJIPCYANMBX-BAOOBMCLSA-M sodium;[(3s,4r,5r)-3,4,5-trihydroxy-2-oxo-6-phosphonooxyhexyl] hydrogen phosphate Chemical compound [Na+].OP(=O)(O)OC[C@@H](O)[C@@H](O)[C@H](O)C(=O)COP(O)([O-])=O HBYYJIPCYANMBX-BAOOBMCLSA-M 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- UQZIYBXSHAGNOE-XNSRJBNMSA-N stachyose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO[C@@H]3[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O3)O)O2)O)O1 UQZIYBXSHAGNOE-XNSRJBNMSA-N 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
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- 208000019553 vascular disease Diseases 0.000 description 1
- 208000020854 vein disease Diseases 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- BPICBUSOMSTKRF-UHFFFAOYSA-N xylazine Chemical compound CC1=CC=CC(C)=C1NC1=NCCCS1 BPICBUSOMSTKRF-UHFFFAOYSA-N 0.000 description 1
- 229960001600 xylazine Drugs 0.000 description 1
- FYGDTMLNYKFZSV-BYLHFPJWSA-N β-1,4-galactotrioside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@H](CO)O[C@@H](O[C@@H]2[C@@H](O[C@@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-BYLHFPJWSA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/44—Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
Definitions
- Vitrified arterial grafts may also have a market as a scaffold for the seeding and adhesion of autologous endothelial cells or genetically modified endothelial cells.
- Prosthetic grafts are currently employed for large diameter (greater than 6mm internal diameter) non-coronary applications. Between 1985 and 1990, approximately 1,200 allogeneic vein segments vvere employed for arterial bypass. Brockbank KGM, McNally
- Blood vessels are also a ubiquitous component of vascularized tissues and organs, both human and animal, which may one day be successfully stored by vitrification for transplantation. Providing that significant immunological issues can be overcome, animal-derived grafts may, one day, provide an unlimited supply of blood vessels and vascularized tissues and organs that could be stored in a vitrified state prior to transplantation.
- cryopreservation Low temperature preservation of biological tissues and organs, i.e., cryopreservation, has been the subject of much research effort. Cryopreservation can be approached by freezing or by vitrification. If the organ or tissue is frozen, ice crystals may form within the organ or tissue that may mechanically disrupt its structure and thus damage its ability to function correctly when it is transplanted into a recipient. Organized tissues and organs are particularly susceptible to mechanical damage from ice crystals formed during freezing. Vitrification, by contrast, means solidification, as in a glass, without ice crystal formation. The principles of vitrification are well-known.
- the lowest temperature a solution can possibly supercool to without freezing is the homogeneous nucleation temperature T h , at which temperature ice crystals nucleate and grow, and a crystalline solid is formed from the solution.
- Vitrification solutions have a glass transition temperature T g , at which temperature the solute vitrifies, or becomes a non- crystalline solid. Owing to the kinetics of nucleation and crystal growth, it is effectively impossible for water molecules to align for crystal formation at temperatures much below T g .
- cryoprotectants One type of damage caused by cryoprotectants is osmotic damage. Cryobiologists learned of the osmotic effects of cryoprotectants in the 1950's and of the necessity of controlling these effects so as to prevent damage during the addition and removal of cryoprotectants to isolated cells and tissues. Similar lessons were learned when cryobiologists moved on to studies of whole organ perfusion with cryoprotectants
- cryoprotectants Despite efforts to control the delete ⁇ ous osmotic effects of cryoprotectants, limits of tolerance to cryoprotectants are still observed. There appear to be genuine, inherent toxic effects of cryoprotectants that are independent of the transient osmotic effects of these chemical agents.
- the present invention is directed to a method for vit ⁇ fication of a blood vessel
- the method comp ⁇ ses immersing the blood vessel in increasing concentrations of cryoprotectant solution at a temperature greater than -5°C to a cryoprotectant concentration sufficient for vit ⁇ fication, rapidly cooling the blood vessel to a temperature between -80°C and the glass transition temperature (T g ), and further cooling the blood vessel from a temperature above the glass transition temperature to a temperature below the glass transition temperature to vit ⁇ fy the blood vessel.
- the present invention is also directed to a method for removing a blood vessel from vit ⁇ fication in a cryoprotectant solution.
- the method comp ⁇ ses slowly warming a vit ⁇ fied blood vessel in the cryoprotectant solution to a temperature between -80°C and the glass transition temperature; rapidly warming the blood vessel in the cryoprotectant solution to a temperature above -75°C, and reducing the concentration of the cryoprotectant by immersing the blood vessel in decreasing concentrations of cryoprotectant.
- the present invention is also directed to a method for treating blood vessels such that smooth muscle functions and graft patency rate are maintained and intimal hyperplasia is reduced relative to fresh untreated controls.
- the present invention is directed to a method in which at least 70%, preferably at least 80%, of smooth muscle function and graft patency rate are maintained relative to fresh untreated controls.
- Fig. 1 shows an example of a perfusion system that can be used in the present invention.
- Fig. 2 shows a device that can be used for rapidly cooling the blood vessels.
- Fig. 3 shows the cooling profile generated by placing the glass scintillation vial
- Fig. 4 shows the warming profile generated by placing the glass scintillation vial in cold air and then in a mixture of 30% DMSO H,0 at room temperature.
- Fig. 5 shows the structural integrity of vein segments following preservation by vitrification.
- Figs. 6A-F show the fresh and vitrified grafts at implantation, after perfusion, fixation and removal, and after graft dissection.
- Figs. 7A-F show the morphology of non-operated veins, fresh vein grafts and vitrified vein grafts in Mikat or H&E staining.
- the present invention is directed to a method for vitrification of a blood vessel in a cryoprotectant solution and for subsequently removing the blood vessel from vitrification.
- Blood vessel is used herein to refer to any biological tube conveying blood. Thus, the phrase refers to an artery, capillary, vein, sinus or engineered construct.
- vitrification refers to solidification without ice crystal formation.
- a tissue is vitrified when it reaches the glass transition temperature (Tg).
- the "glass transition temperature” refers to the glass transition temperature of a solution under the conditions at which the process is being conducted. In general, the process of the present invention is conducted at physiological pressures.
- physiological pressures refer to pressures that blood vessels undergo during normal function.
- the term “physiological pressures” thus includes normal atmospheric conditions, as well as the higher pressures blood vessels undergo under diastolic and systolic conditions.
- perfusion means the flowing of a fluid through the blood vessel. Techniques for perfusing organs and tissues are discussed in, for example,
- cryoprotectant means a chemical that inhibits ice crystal formation in a tissue or organ when the tissue or organ is cooled to subzero temperatures and results in an increase in viability after warming, in comparison to the effect of cooling without cryoprotectant.
- approximately osmotic equilibration means that there is no more than a 10% difference between the intracellular and extracellular solute concentrations.
- a difference of no more than 10% means, for example, that, if the extracellular concentration is 4M, the intracellular solute concentration is between 3.6 and 4.4M.
- the blood vessel is immersed in increasing concentrations of cryoprotectant solution at a temperature greater than -5°C.
- the temperature is preferably between 0°C and 15°C, more preferably between 0°C and 10°C.
- the blood vessel is also perfused with the increasing concentrations of cryoprotectant.
- the increase in concentration is preferably conducted in a step-wise manner. That is, cryoprotectant is added to the extracellular solution to achieve a particular concentration of cryoprotectant. After this concentration level is obtained, the concentration of the solution is then substantially maintained for a period of time. In particular, the concentration level is generally maintained for a sufficient time to permit approximate osmotic equilibration of the blood vessel in the solution. To obtain approximate osmotic equilibration, the concentration level is generally maintained for at least 10 minutes. In a preferred embodiment, the concentration level is maintained for at least 15 minutes. This process is repeated as many times as necessary, with the final concentration being sufficient for vitrification at physiological pressures, particularly under normal atmospheric conditions. In addition, the blood vessel is generally maintained at each concentration level for no more than an hour, preferably for no more than 30 minutes.
- the blood vessel is first immersed in a cryoprotectant- free solution.
- the blood vessel may also be perfused with the cryoprotectant- free solution.
- This solution can be any type of solution that maintains cellular integ ⁇ ty under in vitro conditions as typified by synthetic physical buffers at normal temperatures, and organ preservation solutions at hypothermic temperatures.
- the initial cryoprotectant- free solution will be the same as the vehicle solution used to add and remove cryoprotectants in the blood vessel.
- the cryoprotectant- free solution can be a Euro-Collins solution, which is an aqueous solution desc ⁇ bed in Table 1 below.
- RPS-2TM solution is modified RPS-2 without CaCl 2 and also without MgCL
- a cryoprotectant concentration sufficient for vit ⁇ fication generally contains from 6.0 to 9.5M cryoprotectant, preferably from 7 to 9M cryoprotectant, more preferably from 8 to 9M cryoprotectant.
- the cryoprotectant solution may contain any combination of cryoprotectants sufficient for vit ⁇ fication.
- Cryoprotectants include, but are not limited to, dimethyl sulfoxide, formamide, 1,2-propanediol, 2,3-butanediol, glycerol, ethylene glycol, n-dimethyl formamide and 1,3-propanediol.
- Impermeable cryoprotectant agents such as polyvinylpyrrolidone or hydroxyethyl starch may be more effective at protecting biological systems cooled at rapid rates. Such agents are often large macromolecules, which effect the properties of the solution to a greater extent than would be expected from their osmotic pressure. Some of these non- permeating cryoprotectant agents have direct protective effects on the cell membrane. However, the primary mechanism of action appears to be the induction of extracellular glass formation. When such cryoprotectants are used in extremely high concentrations, ice formation may be eliminated entirely during cooling to and warming from cryogenic temperatures.
- Impermeable chemicals with demonstrated cryoprotective activity include agarose, dextrans, glucose, hydroxyethylstarch, inositol, lactose, methyl glucose, polyvinylpyrrolidone, sorbitol, sucrose and urea.
- the cryoprotectant solution contains dimethyl sulfoxide, formamide, and 1,2-propanediol.
- the cryoprotectant solution may be a solution called VS55.
- VS55 is a solution containing 24.2% w/v (3.1M) dimethyl sulfoxide, 16.8% w/v (2.2M) 1,2-propanediol, and 14.0% w/v (3.1M) formamide.
- the solution contains about 55% w/v cryoprotectant or 8.4M cryoprotectant.
- the amount of dimethyl sulfoxide may be varied from 20 to 30% w/v.
- the amount of 1 ,2-propanediol and formamide may each be varied from about 10 to 20%) w/v.
- the total amount of cryoprotectant in the full strength solution should be between 45% w/v to 60%> w/v, preferably from about 50%> w/v to 55% w/v.
- 1,2-propanediol may be replaced by similar concentrations of 2,3-butanediol.
- the vehicle for the cryoprotectant solution may be any type of solution that maintains cellular integ ⁇ ty under in vitro conditions.
- the vehicle generally comprises slowly penetrating solutes.
- the vehicle solution is a Euro-Collins solution containing lOmM HEPES.
- HEPES is included as a buffer and may be included in any effective amount.
- other buffers, as well as no buffer, may be used.
- Alternative vehicles include, but are not limited to, the solutions discussed in Tables 2 and 3 above.
- the final concentration of the perfusion solution is sufficient to vit ⁇ fy the blood vessel.
- the concentration of the solution is gradually increased to achieve this level, preferably in a step-wise manner.
- cryoprotectant is added to achieve a particular plateau, which is maintained for a sufficient time to achieve approximate osmotic equilibration, in particular for at least 10 minutes and preferably for about 15 minutes.
- further cryoprotectant is added to increase the cryoprotectant concentration, which may or may not be a level sufficient for vit ⁇ fication. If not, after maintaining the concentration for sufficient time to achieve approximate osmotic equilibration, further cryoprotectant is added in one or more steps to achieve a concentration sufficient for vitrification.
- cryoprotectant concentration plateaus before reaching the concentration sufficient for vit ⁇ fication there are four cryoprotectant concentration plateaus before reaching the concentration sufficient for vit ⁇ fication.
- step 1 no cryoprotectant is used; in step 2, 5 to 20%, preferably 10 to 15%, of the final cryoprotectant concentration is used; m step 3, 15 to 35%), preferably 20 to 30%, of the final cryoprotectant concentration is used; in step 4, 40 to 60%), preferably 45 to 55%, of the final cryoprotectant concentration is used; in step 5, 65 to 85%o, preferably 70 to 80%>, of the final cryoprotectant concentration is used; and in step 6, the final cryoprotectant concentration, which is sufficient for vit ⁇ fication, is used.
- Each cryoprotectant concentration step is maintained for a sufficient time to achieve approximate osmotic equilibration.
- the blood vessel is perfused with the solution at each step.
- the blood vessel After the blood vessel has been immersed in a solution containing a concentration of cryoprotectant sufficient for vit ⁇ fication, the blood vessel, which is maintained in a solution containing a concentration of cryoprotectant sufficient for v it ⁇ fication, is rapidlv cooled to a temperature between -80°C and the glass transition temperature
- the rapid cooling rate is generally at least 25°C per minute
- the average cooling rate du ⁇ ng this step is preferably from 30-60°C per minute, more preferably from 35-50°C per minute, and ev en more preferably from 40-45°C per minute
- the temperature to which the blood vessel is cooled du ⁇ ng this rapid cooling process is preferably between -90 and - 1 10°C, more preferably between -95 and -105°C
- the blood vessel After the blood vessel undergoes this rapid cooling process, the blood vessel then undergoes a slow cooling process in which the blood vessel is cooled at an average rate less than 10°C per minute from a temperature above the glass transition temperature to a temperature below the glass transition temperature to vit ⁇ fy the blood vessel
- the cooling process is preferably conducted at an average rate less than 5°C per minute
- the rate of cooling du ⁇ ng this entire step does not increase above 10°C per minute, more preferably the rate of cooling does not increase above 5°C per minute.
- the glass transition temperature is generally about -120°C to -135°C under normal atmosphe ⁇ c conditions.
- the blood vessel can then be stored for long pe ⁇ od of time at a temperature below the glass transition temperature.
- the slow cooling rate is achieved by changing the environment in which the container containing the solution is placed
- the rapid cooling rate is achieved by placing the container in a liquid, such as 2-methylbutane, that has been pre-cooled to a temperature below -100°C, preferably near or below the glass transition temperature of the solution to be cooled. Then, to achieve the slow cooling rate, the container is removed from the liquid and cooled in a gaseous environment at a temperature below the glass transition temperature.
- the present invention is also directed to a method for removing a blood vessel from vit ⁇ fication in a cryoprotectant solution.
- the method comp ⁇ ses slowly warming a vit ⁇ fied blood vessel in the cryoprotectant solution to a temperature between -80°C and the glass transition temperature.
- the slow warming rate is generally below 50°C per minute.
- the average warming rate du ⁇ ng this stage is generally from 20-40°C per minute, preferably from 25-35°C per minute.
- the temperature to which the vit ⁇ fied blood vessel is slowly warmed is preferably between -90 and -1 10°C, more preferably between -95 and -105°C After the blood vessel has undergone this slow warming process, the blood essel is then rapidly warmed to a temperature above -75°C The temperature should be sufficiently high that the solution is suffifienctly fluid that the blood vessel can be removed therefrom.
- the rapid warming process is generally conducted at a rate above 80°C per minute, preferably above 100°C per minute
- the average warming rate du ⁇ ng this step is preferably from 200-300°C per minute, more preferably from 215-250°C per minute.
- the blood vessel is preferably warmed in this process to a temperature between 80°C per minute and 100°C per minute.
- the blood vessel is warmed in this step to a temperature above -5°C, preferably between -5°C and +5°C
- the rapid warming rate is achieved by changing the environment in which the container containing the solution is placed.
- the slow warming rate is achieved by placing the container in a gaseous environment at a temperature above the temperature at which the blood vessel has been stored.
- the container is placed in a liquid, such as an aqueous solution of, for example, dimethyl sulfoxide (DMSO), at a temperature above -75 °C, preferably above 0°C, and more preferably at normal atmosphe ⁇ c temperatures.
- a liquid such as an aqueous solution of, for example, dimethyl sulfoxide (DMSO), at a temperature above -75 °C, preferably above 0°C, and more preferably at normal atmosphe ⁇ c temperatures.
- DMSO dimethyl sulfoxide
- the concentration of the cryoprotectant in the solution is gradually reduced.
- the cryoprotectant concentration is reduced in a step-wise manner
- decreasing cryoprotectant solutions are achieved by immersing the blood vessels in a se ⁇ es of decreasing cryoprotectant concentration solutions to facilitate elution of cryoprotectants from the blood vessel.
- the blood vessel may also be perfused with the solutions.
- the solutions are generally at a temperature above -15°C, preferably between -15 and +15°C, more preferably between 0°C and 10°C.
- the cryoprotectant concentration is preferably reduced to achieve a particular plateau, which is maintained for a sufficient time to achieve approximate osmotic equilibration, in particular for at least 10 minutes and preferably for about 15 minutes. Then, the cryoprotectant concentration is further reduced, which may or may not provide for a cryoprotectant- free solution. If not, after maintaining the concentration for sufficient time to achieve approximate osmotic equilibration, the cryoprotectant concentration is again further reduced in one or more steps to eventually provide a cryoprotectant-free solution.
- the blood vessel is generally immersed in each solution for no longer than an hour, preferably no longer than 30 minutes.
- the cryoprotectant solution may be mixed with a solution of a type similar to the cryoprotectant-free solution utilized in adding cryoprotectant to the blood vessel.
- the solution preferably comprises at least one osmotic buffering agent.
- osmotic buffering agent means a low or high molecular weight non-penetrating extracellular solute that counteracts the osmotic effects of the greater intracellular than extracellular concentrations of cryoprotectant during the cryoprotectant efflux process.
- non-penetrating means that the great majority of molecules of the chemical do not penetrate into the cells of the blood vessel but instead remain in the extracellular fluid of the tissue.
- low molecular weight osmotic buffering agents have a relative molecular mass of 1,000 daltons or less.
- Low molecular weight osmotic buffering agents include, but are not limited to, maltose, potassium and sodium fructose 1,6-diphosphate, potassium and sodium lactobionate, potassium and sodium glycerophosphate, maltopentose, stachyose, mannitol, sucrose, glucose, maltotriose, sodium and potassium gluconate, sodium and potassium glucose 6-phosphate, and raffmose.
- the low molecular weight osmotic buffering agent is at least one of mannitol, sucrose and raffmose.
- high molecular weight osmotic buffering agents generally have a relative molecular mass of from greater than 1,000 to 500,000 daltons.
- High molecular weight osmotic buffering agents include, but are not limited to, hydroxyethyl starch (HES), polyvinylpyrrolidone (PVP), potassium raffmose undecaacetate (> 1 ,000 daltons) and Ficoll (greater than 1,000 to 100,000 daltons).
- HES hydroxyethyl starch
- PVP polyvinylpyrrolidone
- PVP potassium raffmose undecaacetate
- Ficoll greater than 1,000 to 100,000 daltons.
- the high molecular weight osmotic buffering agent is HES, more preferably having a molecular weight of about 450,000.
- the cryoprotectant-free solution preferably contains less than about 500mM of an osmotic buffering agent, more preferably from about 200 to 400mM osmotic buffering agent.
- an osmotic buffering agent preferably a low molecular weight osmotic buffering agent is used. Most preferably, the low molecular weight osmotic buffering agent is mannitol.
- the cr oprotectant is removed in seven steps In the preferred embodiment, in step I , the cryoprotectant concentration is 40 to
- the cryoprotectant concentration is 30 to 45%, preferably 35 to 40%, of the cryoprotectant concentration used for vit ⁇ fication, in step 3, the cryoprotectant concentration is 15 to 35%, preferably 20 to 30%, of the cryoprotectant concentration used for vit ⁇ fication; in step 4, the cryoprotectant concentration is 5 to 20%, preferably
- the cryoprotectant concentration is 2 5 to 10%, preferably 5 to 7 5%, of the cryoprotectant concentration used for vit ⁇ fication
- the remainder of the solution is a cryoprotectant-free solution containing osmotic buffe ⁇ ng agent.
- step 6 essentially all of the cryoprotectant is removed. However, the osmotic buffe ⁇ ng agent is retained
- the osmotic buffe ⁇ ng agent is removed.
- steps 6 and 7 can be combined in a single step That is, the osmotic buffe ⁇ ng agent can be removed at the same time as the remainder of the cryoprotectant.
- step 7 can be eliminated.
- Each of these concentration steps is maintained for a sufficient time to achieve approximate osmotic equilibration.
- the temperature of the se ⁇ es of solutions is generally above -15°C , preferably between -15 and +15°C, and more preferably between 0°C and + 10°C.
- the blood vessel is at a temperature above -75°C, preferably above -65°C.
- the external jugular vein was obtained from New Zealand white rabbits.
- the distal site of each vein segment above the bifurcation was cannulated in situ with a si cone tube.
- the proximal site was left open for fluid outflow.
- the vein segments, which were about 40-60mm long, were perfused at 0°C to 4°C
- the perfusion system of Fig. 1 was used.
- the perfusion system comp ⁇ ses a reservoir 1 (a 60CC sy ⁇ nge) containing perfusion solution 5 connected to the cannula 2 with a 3-way stopcock.
- the reservoir 1 was adjusted to physiologic pressure.
- the vein 3 was placed in a pet ⁇ dish 4 (Dia. x H, 50x15 mm) containing perfusion solution 5.
- the perfusion solution 5 in both reservoir I and petri dish 4 was the same and was pre-cooled (0°C-4°C) and the petri dish 4 was placed in ice
- the vitrification solution used was VS55.
- the full strength VS55 solution was introduced in six serial steps. In the first step, the blood vessels were perfused with
- Euro-Collins solution which is the carrier of VS55. In steps two to five, respectively, the amount of full strength VS55 in the solution was as follows: 1/8 VS55, 2/8 VS55;
- the vein segments were rapidly cooled using the device demonstrated in Fig. 2.
- the vein segments 3, together with the silicone tube, were placed in a glass scintillation vial 6 (Dia. x H, 25x60 mm) containing 1 ml of pre-cooled full strength VS55 solution 7 to form the sample 8.
- the top of the vitrification solution 7 was covered with 0.7 ml of 2-methylbutane 9 (isopentane, freezing point: -160°C, density: 0.62) at 0°C to 4°C to prevent direct contact with air.
- a thermocouple 10 was inserted into a dummy sample 1 1 of the vitrification solution 7, and its output was monitored on a digital thermometer 12. Temperature was recorded throughout the cooling process.
- the cooling apparatus was set up inside a -135°C freezer.
- the cooling rates were adjusted by placing the sample in a container 13 containing precooled 2-methylbutane 14.
- the cooling rates could be varied depending on the depth the vial was placed in 2-methylbutane (30 mm generate a cooling rate of 43°C/min; 60 mm generate cooling rate 71°C/min).
- Fig. 3 shows the cooling profile using the technique of placing the glass scintillation vial 30 mm deep in precooled (-135°C) 2-methylbutane.
- the sample was then stored in the -135°C freezer for at least 24 hours.
- the VS55 vit ⁇ fication solution was then removed in seven steps, using the perfusion system of Fig 1
- the perfusion solution 5 in both reservior 1 and pet ⁇ dish 4 was the same and was pre-cooled (0°C-4°C) and the pet ⁇ dish 4 was placed in ice (0°C to 4°C) du ⁇ ng the perfusion process
- du ⁇ ng the first step the blood vessel is further warmed to a temperature between 0°C and 4°C
- the solution contained, in addition to the cryoprotectant solution, 400 to 200mM mannitol
- the amount of full strength VS55 in the solution was as follows 4/8 VS55, 3/8 VS55, 2/8 VS55, 1/8 VS55, and 0 5/8 VS55, with the remainder of the solution being a mannitol-containing Euro- Collins solution (The 4/8 strength VS55 solution contained 400mM mannitol and the cryoprotectant-free Euro-Collins solution that was mixed therewith to form the lower cryoprotectant concentration solutions contained 200mM mannitol Thus, as the amount of VS55 was decreased, the amount of mannitol was decreased between 400 and 200 mM )
- step six Euro-Collins solution contaimng 200mM mannitol was used
- step 7 a Euro-Collins solution that did not contain mannitol was used.
- FIG. 5 demonstrates a histological section of a vit ⁇ fied rabbit jugular vein showing intact morphological features, including endothelial cells, smooth muscle and the connective tissue of the adventitia.
- Vein graft implantation expe ⁇ ments demonstrate the viability of the vein segments after vit ⁇ fication as compared to fresh autologous veins into the carotid position as a control procedure.
- New Zealand white rabbits (average weight 2.0 to 2 5 kg) underwent a ⁇ ght common carotid interposition bypass graft.
- the fresh, or vit ⁇ fied, reversed ipsilateral external jugular veins were used as syngeneic grafts. Animals were sacnficed at either two or four weeks after implantation Vein grafts were harvested for histology studies.
- Anesthesia was induced in New Zealand white rabbits with an injection of a mixture of ketamine hydrochlo ⁇ de (60mg kg) and xylazine (6mg/kg) and maintained in intubated animals using isoflurane delivered in oxygen
- a single-dose antibiotic prophylaxis in the form of enrofloxacin (5 mg'kg) was gi en intramuscularly at the time of induction.
- the operation was performed with an operating microscope under ste ⁇ le conditions. After exposure through a ⁇ ght longitudinal neck incision, the ⁇ ght external jugular vein was identified, its branches vvere caute ⁇ zed and then removed. Fresh veins vvere implanted immediately Vit ⁇ fied veins were rewarmed in the laboratory and transported to the operation room in DMEM medium on ice.
- the ⁇ ght common carotid artery was identified and dissected. Hepa ⁇ n (200 lU kg) was administered intravenously. A proximal longitudinal arte ⁇ otomy was made, and one end of the reversed jugular vein was anastomosed to the artery end-to-side with a continuous 8-0 microvascular prolene suture The distal anastomosis was performed similarly (Fig. 6, A-B). Throughout the procedure, care was taken to avoid unnecessary instrumentation of the vein graft. The ⁇ ght common carotid was ligated and divided between the two anastomoses with 4-0 silk ligatures. Hemostasis was achieved, and the wound was subsequently closed in layers.
- analgesic (buprenorphine 0.05 mg/kg, S.C.) was provided as necessary. Animals were observed daily for signs of infection, illness, injury, or abnormal behavior. Sick or injured animals were referred immediately for vete ⁇ nary care or euthanized. At the time of graft harvest, under the same anesthetic regimen desc ⁇ bed above, the o ⁇ ginal incision was reopened and both the vein grafts and the non- operated contralateral veins were isolated. Following hepa ⁇ nisation, the vein grafts and contralateral controls were perfusion fixed in situ at 80 mm Hg (Fig. 6, C-D).
- Grafts were perfused with a standardized initial infusion of lactated Ringer's solution followed by 2% glutaraldehyde made up in 0.1M cacodylate buffer supplemented with 0.1M sucrose to give an osmolahty of approximately 300mOsm/kg. After immersion in fixative for 24-48 hours, the graft was divided into a proximal, middle and distal parts (Fig. 6, E-F). Cross-sections from the central region and longitudinal-sections from proximal and distal anastomosis regions were taken for histology studies. Graft patency: Using these techniques, 10 fresh grafts have been harvested, and 9 grafts were patent two or four weeks post-operatively.
- Fig. 7 shows the morphology of non-operated veins, fresh vein grafts and vitrified vein grafts in Mikat or H&E staining.
- Non-operated control veins showed unaltered endothelial cells on the intimal surface and their walls were composed of a couple of layers of smooth muscle cells (Fig. 7, A-B).
- Fig. 7, A-B smooth muscle cell proliferative lesion-intimal hyperplasia and a thickened media appeared in fresh vein grafts
- the vitrified veins developed an intimal hyperplasia layer, which is however, much thinner than fresh vein grafts (Fig. 7, E-F).
- This study demonstrated that intimal hyperplasia has been reduced in vein grafts pretreated by vitrification. This reduced intimal hyperplasia was a particularly unexpected discovery.
- Vein rings which are about 4mm long segments of rabbit external jugular veins, are vitrified in VS55 by the method described above for longer external jugular vein segments, except that perfusion is not used. After being rewarmed, the vein rings are mounted between two stainless steel wire hooks and suspended in a vascular smooth muscle bath containing 5 ml of Krebs Henseleit (KH) solution, which is gassed continuously with 95% O 2 and 5%> CO 2 at 37°C. The baseline tension of all vein rings is adjusted to 0.25 to 0.75 g. Changes in tension are recorded by force transducers. After 1 hour of equilibration, the vein rings are pretested with potassium chloride for contraction.
- KH Krebs Henseleit
- the vein rings After rinsing with KH solution, the vein rings are equilibrated for another 30 minutes prior to the start of experiment. During a relaxation experiment, contraction of the vein rings is produced by 10 '6 M norepinephrine and, after the contractile response plateaus, cumulative concentrations of acetylcholine (10 '10 M to lO ⁇ M) are added to the bathing medium to induce the endothelium dependent relaxation response.
- Table 4 below demonstrates that the vit ⁇ fied vein ⁇ ngs produce similar contractile function as compared to fresh vein ⁇ ngs.
- Artery rings are vitrified in VS55 by the method desc ⁇ bed above for vein ⁇ ngs. After being rewarmed, norepinephrine and phenylephrine are tested in artery ⁇ ng segments vitrified with VS55 and fresh artery ⁇ ngs.
- Table 5 below demonstrates that the vit ⁇ fied artery rings produce similar contractile function as compared to fresh artery rings.
Abstract
Description
Claims
Priority Applications (5)
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CA002368884A CA2368884A1 (en) | 1999-04-13 | 2000-04-12 | Method of cryopreservation of blood vessels by vitrification |
JP2000610286A JP2002541160A (en) | 1999-04-13 | 2000-04-12 | Cryopreservation of blood vessels by vitrification |
EP00922059A EP1182926B1 (en) | 1999-04-13 | 2000-04-12 | Method of cryopreservation of blood vessels by vitrification |
AU42302/00A AU4230200A (en) | 1999-04-13 | 2000-04-12 | Method of cryopreservation of blood vessels by vitrification |
DE60007304T DE60007304T2 (en) | 1999-04-13 | 2000-04-12 | METHOD FOR CRYOPRESERVATING BLOOD VESSELS THROUGH GLASS FORMATION |
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US09/289,977 US6194137B1 (en) | 1999-04-13 | 1999-04-13 | Method of cryopreservation of blood vessels by vitrification |
US09/289,977 | 1999-04-13 |
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US (3) | US6194137B1 (en) |
EP (1) | EP1182926B1 (en) |
JP (1) | JP2002541160A (en) |
AU (1) | AU4230200A (en) |
CA (1) | CA2368884A1 (en) |
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WO (1) | WO2000060935A1 (en) |
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Also Published As
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EP1182926A1 (en) | 2002-03-06 |
DE60007304D1 (en) | 2004-01-29 |
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US20050100876A1 (en) | 2005-05-12 |
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US6194137B1 (en) | 2001-02-27 |
AU4230200A (en) | 2000-11-14 |
JP2002541160A (en) | 2002-12-03 |
CA2368884A1 (en) | 2000-10-19 |
EP1182926B1 (en) | 2003-12-17 |
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