US008679735B2

(12) United States Patent (10) Patent N0.: US 8,679,735 B2 Fahy et a]. (45) Date of Patent: Mar. 25, 2014

(54) METHODS AND COMPOSITIONS FOR THE Fahy, et al., Some emerging principles underlying the physical prop OF ORGANS erties, biological actions, and utility of vitri?cation solutions. , 1987. 24: 196-213. (75) Inventors: Gregory M. Fahy, Corona, CA (U S); Fahy, et a1., Vitri?cation as an approach to cryopreservation. , Corona, CA (U S) Cryobiology, 1984. 21: 407-426. (73) Assignee: 21st Century Medicine Inc., Rancho Fahy, et al., Improved vitri?cation solutions based on predictability of Cucamonga, CA (US) vitri?cation solution toxicity. Cryobiology, 2004. 48: 22-35. Jacobsen, et al., Introduction and removal of cryoprotective agents ( * ) Notice: Subject to any disclaimer, the term of this with rabbit kidneys: assessment by transplantation. Cryobiology, patent is extended or adjusted under 35 1988.25: 285-299. U.S.C. 154(b) by 1468 days. Karlsson, J .O. and M. Toner, Cryopreservation, in Principles of Tissue Engineering, Second Edition, R.P. Lanza, R. Langer, and J. (21) Appl.No.: 10/571,968 Vacanti, Editors. 2000, Academic Press: San Diego. p. 293-307. (22) PCT Filed: Sep. 16, 2004 Karow, A.M., Jr, The organ bank concept, in Organ Preservation for Transplantation, A.M. Karow, Jr, G.J.M. Abouna, and AL. (86) PCT No.: PCT/US2004/030544 Humphries, Jr, Editors. 1974, Little, Brown and Company: Boston. p. 3-8. § 371 (00)’ Kheirabadi, B. and GM. Fahy, Permanent life support by kidneys (2), (4) Date: Nov. 8, 2006 perfused with a vitri?able(7.5 molar) cryoprotectantsolution. Trans plantation, 2000. 70(1): 51-57. (87) PCT Pub. No.: WO2005/027633 Khirabadi, BS. and GM. Fahy, Cryopreservation of the mammalian kidney. 1. Transplantation of rabbit kidneys perfused with EC and PCT Pub. Date: Mar. 31, 2005 RPS-2 at 2-4°C. Cryobiology, 1994. 31: 10-25. (65) Prior Publication Data Kheirabadi, B.S., G.M. Fahy and LS. Ewing, Survival of rabbit kidneys perfused with 8.4 M . Cryobiology, 1995. US 2007/0190517 A1 Aug. 16,2007 32:543-544. Related US. Application Data Kheirabadi, B.S., F. Arnaud and E. Kapnik, The ejfect of vitri?cation (60) Provisional application No. 60/503,551, ?led on Sep. on viability ofrabbit renal tissue. Cryobiology, 1998. 37: 447. Kheirabadi, B.S ., G.M. Fahy, J. Saur and HT Meryman, Perfusion of 16, 2003. rabbit kidneys with 8 molar cryoprotectant (V52). Cryobiology, (51) Int. Cl. 1993.30: 611-612. A01N1/00 (2006.01) Kheirabadi, B.S., G.M. Fahy, P. Nannini, J. Saur and HT Meryrnan, (52) US. Cl. Life support function of rabbit kidneys perfused with 8 molar cryoprotectant. Cryobiology, 1993. 30: 612. USPC ...... 435/12; 435/13; 435/l.l Kheirabadi, B.S., G.M. Fahy, J. Saur, L. Ewing and HT Meryman, (58) Field of Classi?cation Search Failure of rabbit kidneys to survive chilling to -300 C. after perfusion None with 8 Mcryoprotectant at -3° C. Cryobiology, 1994. 31: 596-597. See application ?le for complete search history. (Continued) (56) References Cited U.S. PATENT DOCUMENTS Primary Examiner * Allison Ford (74) Attorney, Agent, or Firm * Finnegan, Henderson, 5,723,282 A 3/1998 Fahy et a1. Farabow, Garrett and Dunner, LLP 5,821,045 A 10/1998 Fahy et a1. 5,962,214 A 10/1999 Fahy et a1. 6,187,529 B1 2/2001 Fahy et a1. (57) ABSTRACT 6,274,303 B1 * 8/2001 Wowk et al...... 435/13 6,395,467 B1 5/2002 Fahy et a1. Methods and compositions are provided for the cryopreser 2002/0042042 A1 * 4/2002 Fahy ...... 435/13 vation of human organs and tissues. In certain embodiments, FOREIGN PATENT DOCUMENTS Step 1 comprises perfusion with a vitri?able cryoprotectant solution at a temperature above —100 C. for a time insuf?cient W0 WO 96/05727 2/1996 for the approximate osmotic equilibration of the organ with W0 WO 01/42388 6/2001 the solution, followed by cooling the organ to below —100 C. W0 WO 02/09516 6/2001 by perfusion with said solution at a reduced temperature. In W0 W0 03/009743 2/2003 W0 W0 03/065801 8/2003 certain embodiments, Step 2 comprises increasing the con centration of cryoprotectant further at a temperature from — 10 OTHER PUBLICATIONS to —400 C. In certain embodiments, Step 3 comprises cooling and vitrifying the organ, rewarming it, and perfusing the Arnaud, et al., Physiological evaluation of a rabbit kidney perfused organ with a vitri?able concentration of cryoprotectant with VS41A. Cryobiology, 2003. 46: 289-294. whose temperature is either raised gradually or is held at Fahy, G.M.,Analysis of “solution ejfects ” injury: cooling rate depen dence of the functional and morphological sequellae offreezing in 2—150 C. Compositions are provided that allow safe organ rabbit renal cortex protected with dimethyl sulfoxide. Cryobiology, perfusion with vitri?able media at >—10o C. and almost com 1981.18:550-570. plete avoidance of chilling injury at —20 to —25° C. and that Fahy, G.M., Prospectsfor vitri?cation of Whole organs. Cryobiology, allow slow warming after vitri?cation without freezing. 1981. 18: 617. Fahy, G.M., Cryoprotectant toxicity: biochemical or osmotic? Cryo Letters, 1984. 5: 79-90. 42 Claims, 11 Drawing Sheets US 8,679,735 B2 Page 2

(56) References Cited AppliedAspects, D.E. Pegg, I.A. Jacobsen, andN. A. HalasZ, Editors. 1982, MTP Press, Ltd: Lancaster. p. 389-393. OTHER PUBLICATIONS Rall, W.F. And G.M. Fahy, Ice-free cryopreservation of mouse embryos at -1960 C. by vitri?cation. Nature, 1985. 313:573-575. Kheirabadi, B.S., G.M. Fahy, L. Ewing, J. Saur and H.T. Meryman, Staer, T.E., A look ahead at transplantation. Journal of surgical 100% survival ofrabbit kidneys chilled to -32° C. after perfusion with research, 1970. 10:291-297. 8 M cryoprotectant at -22° C. Cryobiology, 1994. 31:597. Pegg, D.E ., Banking ofcells, tissues, and organs at low temperatures, Wang, X., H. Chen, H. Yin, S. Kim, S. Lin Tan, and R. Gosden, in Current Trends in Cryobiology, A.U. Smith, Editor. 1970, Plenum Fertility after intact ovary transplantation. Nature, 2002. 415: 385. Press: NeWYork. p. 153-180. International Search Report for PCT Patent Application No. PCT/ Pegg, D.E., Theory and experiments towards subzero organ preser US04/30544. vation, in Organ Preservation, D.E. Pegg, Editor. 1973, Churchill Supplementary European Search Report for EPO Patent Application Livingstone: London. p. 108-122. No. EP 04784413. Pegg, DE. and M.P. Diaper, The mechanism of cryoinjury in glyc erol-treated rabbit kidneys, in Organ Preservation, Basic and * cited by examiner US. Patent Mar. 25, 2014 Sheet 1 0f 11 US 8,679,735 B2

Figure 1

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US 8,679,735 B2 1 2 METHODS AND COMPOSITIONS FOR THE exchange media would relax the de?nition of “vitri?able CRYOPRESERVATION OF ORGANS concentration” to slightly lower concentrations, but very high concentrations would still be required. Moreover, in the con RELATED APPLICATIONS text of organ vitri?cation, it will generally be true that a vitri?able concentration that does not permit the complete or This application claims priority under 35 U.S.C. 119 to near-complete suppression of devitri?cation on rewarrning at US. Provisional Application Ser. No. 60/503,551, ?led Sep. practicable warming rates will not be useful because devitri 1 6, 2003, the entire contents of which are hereby incorporated ?cation on warming may be unacceptably damaging. As used by reference herein. herein, a “vitri?able concentration” is de?ned as a concentra tion that is capable of allowing vitri?cation at a cooling rate of FIELD OF THE INVENTION 520° C./min as judged by visual absence of ice in a 10 ml sample after cooling to below the glass transition temperature This invention is related to compositions and methods for (T8) or by absence of detectable exotherrns when the solution the cryopreservation of organs. is cooled to below T8 in a differential scanning calorimeter (DSC). BACKGROUND OF THE INVENTION Since the introduction of the concept of organ vitri?cation, many advances have been made in the art. However, as of One of the greatest challenges in cryobiology is the cryo 2004, 23 years have passed since the concept of organ vitri preservation of entire organs. Although dif?cult, this goal is ?cation was ?rst suggested [3], and 19 years have passed important [14, 15, 24, 25, 28, 29], in part because present 20 since the ?rst proof-of-principle experiment was published limits on human organ storage times after procurement for showing that mammalian embryos can be vitri?ed and transplantation substantially reduce the effectiveness and rewarmed with high survival [27], yet the original goal of increase the cost of organ replacement [15]. These problems successfully vitrifying organs remains elusive. could be eliminated if organs could be banked [9, 24, 25] and Processes related to the cryopreservation of organs, includ stored for times that are shorter than current organ recipient 25 ing methods and compositions for the introduction and waiting times. Although organ cryopreservation has usually removal of vitri?able concentrations of cryoprotective been conceptualized as a way of facilitating the replacement agents, have been described in the prior art. For example, US. of vital organs by allografts or xenografts, there is also con Pat. Nos. 5,723,282 and 5,962,214 claim the following siderable current interest in using the technique to preserve method for preparing organs, tissues, or cells for vitri?cation: gonads during chemotherapy and then return them to the 30 a) cryoprotectant concentration is gradually elevated to a donor after the completion of treatment [29]. Inde?nite-term ?rst concentration while the temperature is mildly cryopreservation is probably also essential for solving the reduced; largest problem in transplantation medicine, which is the b) the ?rst concentration is maintained for a suf?cient time shortfall in organ availability in relation to the total number of to permit the approximate osmotic equilibration of the transplants that are needed. To address this need, a multi 35 organ or tissue (de?ned as <50-200 mM difference billion dollar investment in the ?eld of tissue engineering has between arterial and venous concentrations for organs) been made [23], but this approach will also require cryo to occur; preservation in order to achieve inventory control and ef? c) concentration is raised to a ?rst intermediate concentra cient supply chain management of the tissue-engineered tion that is not suf?cient to permit vitri?cation (is not products [13]. 40 vitri?able); The cryopreservation of organs was ?rst seriously investi d) the ?rst intermediate concentration is maintained for a gated in the 1950s as a result of the rediscovery of the cryo suf?cient time to permit the approximate osmotic equili protective properties of glycerol by Polge, Smith, and Parkes bration of the organ or tissue with the non-vitri?able in 1949. Until 1981 , it was assumed that freezing was the only intermediate concentration (<50-200 mM difference option for cryopreservation, but in 1981, Fahy introduced the 45 between arterial and venous concentrations for organs); radically different concept of vitri?cation, in which no ice is e) the temperature is further reduced; and allowed to form in the organ during either cooling or warm f) the concentration of cryoprotectant is increased to a level ing, thus eliminating mechanical injury from ice. In 1985, suf?cient for vitri?cation, or to a level still insuf?cient Rall and Fahy [27] coined the term “vitri?cation solution,” for vitri?cation followed by an additional cooling step which is a cryoprotectant solution concentrated enough to 50 and a ?nal step of increasing concentration to a ?nal, permit vitri?cation on cooling and, preferably, no devitri?ca vitri?able concentration. tion (freezing) on rewarrning after previous vitri?cation. US. Pat. Nos. 5,821,045 and 6,187,529 claim a method in Although it is thought that any aqueous sample that can be which a previously cryopreserved organ is: cooled at ultrarapid rates can be vitri?ed in principle, in the a) warmed without perfusion to a temperature high enough context of organ vitri?cation, or even in the context of the 55 to permit reperfusion of the organ wherein damage is vitri?cation of small biological systems like embryos that are minimized, and then to be cooled and warmed in containers, a vitri?cation solution b) perfused directly with a composition comprising a non must be concentrated enough to vitrify when cooled at, gen vitri?able concentration of cryoprotectant that is less erally, less than 3,000° C./min. than the concentration of cryoprotectant used for cryo Human kidneys, for example, can be cooled no more rap 60 preservation, and further comprising one or two osmotic idly than 2° C./min in their core, and for such a case a vitri buffering agents, where an osmotic buffering agent is ?able concentration of cryoprotectant would be de?ned as a de?ned as an extracellular solute that counteracts the concentration that allows vitri?cation in a kidney-sized object osmotic effects of greater intracellular and extracellular cooled at 2° C./min or less. Generally, “vitri?cation” in this concentrations of during the cryopro context means that no, or at most very few, visible ice crystals 65 tectant e?lux process. When a liver is being treated, would form in such a volume on cooling. Means of cooling osmotic buffering agents are omitted, but step b) still organs more rapidly by vascular perfusion with cold heat requires perfusing the liver with a non-vitri?able con US 8,679,735 B2 3 4 centration of cryoprotectant immediately after attaining higher subzero temperatures [12, 26]. This phenomenon is the target reperfusion temperature. According to the pro also readily detectable in renal cortical slices [11]. Although cess limits of the prior art, the concentration during step it was found that this injury could be avoided by cooling to b) is limited to 20-40% w/v or to about 3-6M, or 60% of about —24° C. in the presence of only 6.1M cryoprotectant the highest concentration perfused. [11, 22], this approach was later found to result in so much Clearly, the prior art of adding and removing cryopro more chilling injury when additional cryoprotectant was tectants and for cooling and warming has proven inadequate added and the temperature was further reduced as to be worse for organs as evidenced by the lack of any actual demon than simple cooling from 0° C. [18]. A completely different strated success after cooling organs to cryogenic tempera and far superior method of avoiding chilling injury was later tures and rewarming them. Thus, while US. Pat. No. 6,395, put forward (US. patent application Ser. No. 09/916,032), but 467 B1 and US. patent application Ser. No. 09/916,396 this method does not provide explicit means for applying the provide extraordinary vitri?cation solutions and an excellent technique to whole organs. Furthermore, the method for carrier solution for enhancing their effectiveness, there is still avoiding chilling injury does not include critically important a need in the art for further improvements in the methods and compositions employed for adding and removing cryopro methods for rewarming organs after prior cooling and for tectants and for cooling and warming organs and tissues. reperfusing them in such a way as to avoid damage following exposure to vitri?able concentrations of cryoprotectant such SUMMARY OF THE INVENTION as M22. In accordance with the present invention, new processes In accordance with the present invention, there are pro 20 have been discovered that overcome all of these problems and vided superior new methods for cooling, cryoprotecting, are highly advantageous for the introduction and washout of rewarming, and reperfusing organs and tissues. The present cryoprotectants and for cooling and warming organs during invention extends the teachings of the prior art by providing continuous perfusion. even more extraordinary and unprecedentedly effective vitri Invention methods for cooling, stabilizing, rewarming, and ?cation solutions. It is, therefore, a purpose of the present 25 diluting cryoprotectants in perfused organs have been suc disclosure to describe new methods and compositions cessfully testedusing M22 as a model solution as described in capable of allowing organs to survive and provide life support the Examples given below, but will be equally applicable to after cryopreservation by vitri?cation and transplantation. any solution intended for the vitri?cation of whole organs. Organ vitri?cation requires the use of a perfusion solution They are not restricted to the rewarming and dilution of that is suf?ciently stable against ice formation (suf?ciently 30 organs following perfusion above —15° C. and will in fact be able to resist both ice nucleation and ice crystal growth) to particularly helpful following the perfusion of organs at tem allow ice formation to be prevented or adequately limited during the cooling and warming of an organ after perfusion peratures of — 1 5° C. and below, and still more helpful follow with the solution. Solutions having the required stability, ing the perfusion of organs at temperatures between —20° C. and —30° C. They are not restricted to the safe cooling of however, tend to be toxic when perfused at 0° C., thus, to 35 reduce the toxicity, perfusion below 0° C. is often desirable. organs to —15° C. or above, but are particularly helpful for the In accordance with one aspect of the present invention, an safe cooling of organs to —15° C. or below, and particularly especially advantageous solution (referred to herein as M22) for the safe cooling of organs to —20° C. to —30° C. They have has been developed. M22 is characterized by the simulta been shown to be compatible with the survival and life sup neous presence of dimethyl sulfoxide, ethylene glycol, for 40 port function of a mammalian organ after vitri?cation and mamide, N-methylformamide, 3-methoxy-1,2,propanediol, rewarming, an accomplishment not previously achieved. polyglycerol, polyvinyl alcohol or a polyvinyl alcohol-poly Although the invention is speci?cally directed toward organs vinyl acetate copolymer, and low molecular mass polyvi perfused primarily through the vascular system (perfusion nylpyrrolidone, wherein the solution is suf?ciently concen through internal cavities other than blood vessels, such as the trated to remain ice-free based on differential scanning 45 chambers of the heart or the ventricles of the brain is also calorimetry when vitri?ed and then rewarmed at less than 1° included within the scope of the invention, usually as a C./min. Although the composition can vary, as discussed in supplement to vascular perfusion), the same concentration greater detail herein, in accordance with a presently preferred time-temperature protocols can also be effectively applied to embodiment of the invention, M22 has a total concentration unperfused tissues treated with cryoprotectants by immersion of about 9.3 molar, or about 64.8% w/v. M22 is so named 50 or superfusion. because it is intended to be exposed to living systems pre dominantly near —22° C. to minimize the potential for toxicity BRIEF DESCRIPTION OF THE FIGURES that may be produced at higher temperatures. However, there are no methods known in the prior art by FIG. 1 presents a “Viability-stability plot” for rabbit renal which an organ can be continuously perfused starting with no 55 cortical slices exposed to the vitri?cation solutions described cryoprotectant at above 0° C. and ending with a solution like in Tables 1 and 2. M22 at —22° C. with subsequent retention of life support FIG. 2 collectively illustrates hypertonic modi?cation of capability after transplantation, and there is no guidance in chilling injury caused by abrupt transfer of slices from solu the art about how such a continuous perfusion protocol can be tions at 0° C. to solutions at —20° C. FIG. 2A illustrates the successfully reversed, returning the organ to zero percent 60 occurrence of injury on slices transferred to —20° C. at the cryoprotectant at above 0° C. after prior perfusion at —22° C. stated tonicity. FIG. 2B presents a comparison of constant Cooling to —22° C. is complicated by the need to avoid so tonicity protocols to non-equilibrium tonicity protocols. called chilling injury, which can be de?ned as injury caused Slices transferred under constant isotonic conditions by cooling per se. Directly cooling organs perfused with 7.5M (1><—>1><) experienced considerable damage, slices trans to 8M cryoprotectant from —3° C. to —30° C. produced 65 ferred under constant hypertonic conditions (2><) sus marked injury [21], consistent with earlier observations on tained practically no injury, and slices equilibrated under kidneys perfused with other cryoprotectants and cooled to isotonic conditions (1><) at 0° C. and transferred to a twice US 8,679,735 B2 5 6 isotonic (2><) precooled solution at —200 C. (l><—>2><) showed left of each schematic temperature. Bold text in the protocol injury intermediate between the le1x and the 2><—>2>< schematics indicates key differences between the tested pro treatments. tocols. In each case, the preserved kidney served as the sole FIG. 3A summarizes a baseline method onto which cooling renal support immediately after transplantation. For each and warming methods according to the present invention can 5 group, n:3. Meanszl standard error of the mean (SEM). be superimposed. FIG. 3A also establishes the lack of toxicity FIG. 5 describes a speci?c experiment showing an exem of VMP at —3° C., and provides a useful data format for later plary continuous perfusion protocol according to the present ?gures.All complete data sets are plotted horizontally against invention for cooling after insuf?cient time for approximate a common time base in three separate panels (upper, middle, osmotic equilibrium between the organ and the perfusate and lower). More focused attention is given to narrow time prior to cooling, with warming by reperfusion with a concen range data in the insets. All insets show response vs. time tration not less concentrated than the concentration present plots. below —15° C. The left upper inset shows that at the time of FIG. 3A, Upper panel: arterial molarity (M; heavy line) and onset of cooling (marked by the vertical line), nominally 5 the arteriovenous concentration difference across the kidney min after the onset of VMP perfusion, the A-V concentration (A-V) in molar (M) units. (Note: upon switching to 0 mM difference was extreme at about 2.25M, which is very far cryoprotectant, the display mode changes to plot the concen from approximate osmotic equilibrium. The middle upper tration of mannitol being perfused, causing an apparent inset shows that at this time, not only the venous concentra increase in concentration on the record.) tion but also the arterial concentration (plotted on a molar FIG. 3A, Middle panel: arterial (heavy line) and venous concentration scale) has failed to reach the target 8.4M con temperatures (T) in o C. as measured using an arterial in-line 20 centration ofV MP, arterial concentration having reached only needle thermocouple and a second ?ne thermocouple inserted 8.15M. This emphasizes the nominal nature of organ perfu directly in the venous e?iuent underneath the kidney. sion protocols, in which programmed step changes in time FIG. 3A, Lowest panel: arterial perfusion pressure (P) in and temperature do not necessarily occur instantaneously. For mmHg and perfusate ?ow rate (heavy line) in ml/min per this reason, both literal and non-literal interpretation of pro gram of post-?ush, pre-perfusion kidney weight. Perfusion 25 cess descriptions such as “perfuseVMP for 5 min” are appro pressure was divided by 40 to permit it to be plotted on the priate, not just literal interpretations. The right upper inset same scale as the ?ow rate. Prior to introducing cryopro illustrates the method of constant-concentration continuous tectant, the perfusate was TransSend-B ([10], and formula 7 perfusion-rewarming (perfused arterial molarity indicated by of [7]) plus 2% hydroxyethyl starch (HES, of relative molecu heavy line and left vertical axis, arterial perfusate temperature lar mass Mr~450 kilodaltons, obtained from B. Braun, Irvine, 30 indicated by the light line and right vertical axis). Before Calif.). As VMP was introduced (V MP molarity plotted in warming, the organ is already at a temperature permitting upper panel, heavy line; VMP prepared in LM5 carrier), the reperfusion of the organ wherein damage is minimized HES and TransSend-B were diluted gradually to zero. This (~—23o C.), but it is not perfused with non-vitri?able solution procedure was followed in all perfusions. VMP was washed containing osmotic buffers at —200 C. Instead, it is perfused out initially with 3% HES plus 300 mM mannitol using half 35 with rapidly warming, undiluted, vitri?able, and osmotic strength VMP in LM5. As washout proceeded, the carrier buffer-free solution until the arterial temperature exceeds solution was gradually transitioned back from LM5 to Trans —10° C. and approaches —50 C., and only then is perfused with Send-B. Note that at the end of the VMP plateau, suf?cient more dilute, non-vitri?able solution. The left and right insets time has been allowed for approximate osmotic equilibration in the middle panel represent postoperative creatinine levels of the organ with VMP as indicated by an A-V difference of 40 and the concentrations of cryoprotectant being perfused just only 50 mM (middle inset in middle panel). Upper right inset: before switching to an entirely cryoprotectant-free perfusate, postoperative serum creatinine levels (Cr); “Day” refers to respectively. postoperative day; time zero Cr values represent serum crea FIG. 6 summarizes results of another exemplary method tinines at nephrectomy and at transplant. Upper left inset: according to the present invention for continuous perfusion “URI” refers to urine refractive index and is plotted against 45 rewarming from below —10° C. with simultaneous dilution of the duration of VMP perfusion. highly vitri?able concentrations of cryoprotectant and its FIG. 3B monitors serum creatinine in seven consecutive effect on postoperative creatinine levels. The inset shows transplants following use of the protocol described in FIG. three arterial temperature vs. time histories and the times and 3A. All transplants resulted in no detectable injury to the temperatures at which the measured arterial concentration kidney, and no use of iloprost, aspirin, or heparin was needed 50 ?rst began to drop (indicated by the open triangles labeled or used. 9.3M cryoprotectant) and ?nally began to approach the con FIG. 4 is a schematic diagram of the programmed continu centration of VMP (indicated by the ?lled triangles designat ous perfusion, osmotic non-equilibrium method for minimiz ing the attainment of 8.5M cryoprotectant). Time zero in the ing chilling injury and cold dilution damage in whole organs. inset represents the time the perfusion machine was set to Rabbit kidneys were perfused with VMP for a nominal 5 or 10 55 switch from M22 to VMP; about 2.5 min was required for the min period at about —3° C. according to FIG. 3 and then for programmed switch to result in an actual fall in concentration another 10 min while the temperature of the arterial perfusate in these experiments. The line types in the protocol inset was set to about —220 C. as rapidly as possible. Each protocol match the line types for the same groups in the main panel schematic represents the time (horizontal direction) and tem showing postoperative results. The M22 perfusion time prior perature (vertical direction) of the initial VMP perfusion step 60 to washout at —220 C. was 15-25 min; there was no apparent at —3° C., the cooling step involving the perfusion of VMP at in?uence of M22 perfusion time on postoperative creatinine temperatures down to —22.5:2.5o C., and the subsequent levels (data not shown). Meanszl SEM. The n:ll notation warming and dilution steps. “1/2 VMP+” refers to half for the rapid-warming, “warm dilution” group pertains to the strength VMP plus 300 mM mannitol as an osmotic buffering thermal data in the inset only, with n:8 for the creatinine data agent; 3% HES was also present. The concentration perfused 65 for the same group (some kidneys were not transplanted for at each step is indicated above the schematic line and the reasons unrelated to the ef?cacy of the method described for nominal temperature perfused at each step is indicated to the this group). US 8,679,735 B2 7 8 FIG. 7 summarizes results with another exemplary proto cooled to —450 C. as in Method 1, and reperfused at —3° C. col according to the present invention for the successful with M22MX after prior external warming to a surface tem recovery of organs after perfusion with vitri?able concentra perature of about —22°. Meansil SEM. tions ofcryoprotectant below —100 C. using the simultaneous FIG. 10 is a graph of the effect of arterial perfusion pressure rewarming and dilution method of FIG. 6. The same format during M22 perfusion on tissue equilibration with cryopro and abbreviations are employed herein as in FIGS. 3 and 5. tectant as measured by arteriovenous (A-V) concentration Insets show lack of approximate osmotic equilibrium at onset difference. In all cases, A-V differences were measured at the of cooling (upper left inset), lack of approximate osmotic end of 25 minutes of perfusion with M22, the arterial perfu equilibrium at onset of perfusion with the ?nal vitri?cation sion pressure was 40 mmHg prior to perfusion with M22, and solution (upper middle inset), lack of approximate osmotic pressure was set to the value plotted at the time of onset of equilibrium at the time of onset of warming, which is the M22 perfusion. The A-V difference is the arterial perfusate nominal time to cool and vitrify the organ (middle left inset), concentration minus the organ ef?uent concentration (venous the relationship between arterial molarity and temperature concentration). during perfusion-rewarming (middle right inset), and postop FIG. 11 summarizes the perfusion record and postopera erative serum creatinine levels (upper right inset). The tem tive recovery data for a rabbit kidney that was perfused with perature control instabilities shown immediately after rapid M22, vitri?ed, rewarrned, transplanted, and supported life as warming from —220 C. were not typical of this protocol, but the sole kidney until the recipient was euthanized 40 days illustrate the tolerance of the organ to mild temperature ?uc after transplantation. tuations within this range. FIG. 8 presents the environmental and intrarenal thermal 20 DETAILED DESCRIPTION OF THE INVENTION history of a rabbit kidney exposed to —500 C. by forced convection for 6 min and then rewarmed. C:cortical tempera In accordance with one embodiment of the present inven ture (2 mm below the renal surface); MImedullary tempera tion, there are provided methods for the cooling of organs ture (7 mm below the renal surface); Prpapillary/pelvic tem from a temperature above —10° C. to a desired low tempera perature (12 mm below the renal surface); BFl?emperature 25 ture below —100 C. by continuous vascular perfusion with of rapidly-moving air in contact with the renal surface in a minimal injury, the methods comprising: Linde BF-l Biological Freezer; AIarterial temperature dur perfusing the organ with a cryoprotectant solution (e.g., a ing reperfusion, qunous temperature during reperfusion. ?rst mixture of permeating and non-permeating cryo Intra-renal temperatures were monitored using a PhysiTemp protectants having a freezing point lower than the said (Huron, Pa.) triple bead needle probe. Horizontal lines illus 30 desired low temperature) for a time insuf?cient to permit trate that all parts of the kidney were between —400 C. and the approximate osmotic equilibrium of the organ with —500 C. at the time of onset of warming. Reperfusion for said mixture of cryoprotectants to take place; rewarming was accomplished using Method 1 described in lowering the temperature of the arterial perfusate to or conjunction with FIG. 9. below the said desired low temperature; and FIG. 9 illustrates the effects of two exemplary methods 35 continuing to perfuse said mixture until the temperature of according to the present invention for the prevention of dam at least a portion of the organ is considered to reach the age after cooling an organ to —450 C. Rewarrn Method 1 target temperature. (diamonds) employed the M22 washout method described An exemplary ?rst mixture of permeating and non-perme with reference to FIGS. 6 and 7. In this method, M22 in the ating cryoprotectants, VMP, which can be effectively used in arterial perfusion line was held at —220 C. until the cooled 40 the above-described method is as follows. VMP is usually organ was reattached to the perfusion machine and reper prepared in a carrier solution whose tonicity in the absence of fused, after which rapid warming of the arterial perfusate the cryoprotectants of VMP is de?ned to be isotonic, or 1>< proceeded as in FIGS. 6 and 7. In Rewarrn Method 2 (inverted isotonic. VMP in the carrier solution LM5 (the composition triangles), the perfusate temperature was increased to —3° C. of which is provided in [5, 10] and in the footnotes of Table 1) during the time the organ was not perfused. When the organ 45 has an effective tonicity of 1.2 times isotonic, where tonicity was reattached to the perfusion machine, it was reperfused is de?ned as the ratio of the osmolality of the components of directly with vitri?able concentrations of cryoprotectant at VMP minus the permeating cryoprotectants (permeating —3° C. for a total of 2.4107 min before beginning to experi cryoprotectants de?ned as those cryoprotectants whose ence dilution of the arterial perfusate. The inset shows a molecular mass is less than 150 daltons) to the osmolality of vertical line marking the discontinuity in perfusion required 50 the cryoprotectant-free carrier solution (preferably, the solu for cooling to and rewarming from about —450 C.; except for tion known as LM5). The increase in tonicity over isotonic is calibration drift in the arterial refractometer, there is no due to the presence in VMP of two antinucleating agents or change in arterial concentration but there is a step change in “ice blockers,” polyglycerol (sometimes referred to as PGL or temperature when the organ is reperfused after having previ “Z-1000”) and polyvinyl alcohol or a copolymer of polyvinyl ously been cooled to —450 C. At the time of onset of reduction 55 alcohol and polyvinyl acetate (sometimes referred to as PVA in arterial concentration, venous temperature is around —170 or “X-1000”) of mass >150 daltons. Including the ice block C. despite the more rapid warming in Method 2, but rises to ers in VMP provides protection from chilling injury during above —10° C. in less than 10 min, suggesting that 10 min of cooling of the organ to —1 5 to —400 C. and also provides more —3° C. perfusion will be suf?cient to return all parts of an time for these polymers to permeate through the extracellular organ to a temperature at which dilution to lower concentra 60 spaces, including cavities such as urinary spaces or ventricles, tions is safe. The Method 1 —450 C. group was perfused with of the organ, thus further ensuring their adequate distribution M22 at ~—22° C. (arterial temperature, —22.5:2.5o C.) for 20 prior to vitri?cation. (n:2) or 25 (n:6) min before further cooling and rewarming The non-ice-blocker components of VMP can each vary by as in FIG. 7. The Method 2 —450 C. group was perfused with 125% without loss of the effectiveness of the VMP variant “M22MX”, which consists of M22 lacking “X1000” (a 65 solution. VMP can also be modi?ed to include otherpolymers copolymer of polyvinyl alcohol and polyvinyl acetate intended to contribute to ice blocking or ice growth inhibiting described in detail elsewhere), for 25 min as in Method 1, properties of the subsequently-perfused primary or ?nal vit US 8,679,735 B2 1 0 ri?cation solution, and can optionally include slightly (0-50% both low (<100-200 daltons) and high (2150 daltons) mass. more) concentrated carrier solution provided the desirable The phrase “vitri?able concentration of cryoprotectant” as tonicity of the modi?ed VMP in carrier solution, as elevated employed herein refers to a concentration that will allow by the sum of the presence of the ice blockers, the other regions of the organ that are saturated with the cryoprotectant polymer(s) intended to inhibit ice growth, and more than an to vitrify when the organ is cooled at a rate of 200 C./min or isotonic amount of carrier solution, has a tonicity usually below. A particularly preferred solution for use in this method within the generally more preferred range of 1.1 to 1.6 times contains DMSO, formamide, ethylene glycol, more than 1% isotonic. In other words, the overall mo st preferred mixture of w/v polyglycerol, polyvinylalcohol, polyvinylpyrrolidone, permeating and non-permeating cryoprotectants (in carrier N-methylformamide, and 3-O-methyl-rac-glycerol. A spe solution) for use in this method has a tonicity after full equili ci?c embodiment of this solution is the formula known as bration with living cells, tissues or organs that ranges from M22 whose composition is disclosed herein (see Table 2). approximately 1.1 to about 1.6 times isotonic. The total The components of M22 can be varied by 125% without loss amount of ice blocker inVMP can be varied from 0% to about of effectiveness of the resulting M22 variants in this method. 7% w/v, consistent with the above tonicity limitations, but the Also, M22 can be modi?ed to contain polyethylene glycol, mo st preferred amount is 1 -4% w/v. Typically, the desired low preferably at a concentration of about 0.5-4% w/v. temperature below —10° C. will range from about —150 C. to Also in this method, the perfusion can be conducted at a about —400 C., and is most preferably from about —200 C. to ?rst pressure, which can be about 40 mmHg, before perfusion —30° C. with said vitri?able concentration of cryoprotectant and can The perfusion pressure during the above-described method be raised to a second pressure, which is preferably 41-110 may be constant or variable but should be within about 20 mmHg, and most preferably to 55-85 mmHg, when perfusion 40-1 10 mmHg, or more preferably within about 50-90 mmHg with said solution begins. Constant pressure perfusion within or still more preferably within about 55-85 mmHg. In one the range of 32-110 mmHg can also be effective in accor variant of the method, the perfusion pressure is at 4018 dance with the present invention. mmHg before cooling and is elevated to 50-80 mmHg during In accordance with still another embodiment of the present cooling. The desirable perfusion pressure range for larger 25 invention, there are provided methods for initially diluting organs will be higher than for smaller organs, and may range cryoprotectants in organs previously perfused with cryopro from 50-110 mmHg, or more preferably from 60-100 mmHg. tectant at a temperature of —10° C. or below, the methods As used in this speci?cation, “arterial” means “?owing into comprising: the organ,” and is intended to include perfusion of the liver via raising the arterial perfusate temperature to or above a the portal vein and retrograde perfusion of organs through 30 desired high temperature above —10° C. during perfu their veins. sion of the organ without changing the concentration of In accordance with another embodiment of the present cryoprotectant; invention, there are provided methods for warming organs continuing to perfuse the organ without changing cryopro from below —10° C. to a desired high temperature of —10° C. tectant concentration for a time that is suf?ciently long or above by continuous vascular perfusion with minimal 35 to protect the organ from injury resulting from subse injury, the methods comprising: quent perfusate dilution but not so long as to cause raising organ temperature by raising the temperature of the undesired injury from continued exposure to the said arterial perfusate to or above the desired high tempera undiluted cryoprotectant at the desired high tempera ture without changing arterial concentration and while ture; and continuing to perfuse the organ; and 40 diluting the cryoprotectant. continuing to perfuse the organ without changing arterial Typically, the time that is suf?ciently long to protect the concentration until the organ temperature reaches the organ from osmotic dilution injury but not so long as to cause desired high temperature. undesired injury from exposure to the solution at the desired Typical desired high temperatures will range from about high temperature is 1 to 10 minutes. In a variation of this —9.9° C. to about +5o C., the most preferred range being —8° 45 method, perfusion below —10° C. can be interrupted for C. to —20 C. The organ is optimally considered to have example by disconnecting the organ from the perfusion reached the desired high temperature when the coldest parts machine, and the arterial perfusate can be warmed to the of the organ are suf?ciently close to the desired high tempera desired high temperature above —10° C. while the organ ture to minimize injury upon subsequent dilution of the cryo remains disconnected and unperfused, after which the organ protectant while also minimiZing toxicity caused by delaying 50 can be reattached to the perfusion machine and perfused with dilution. This method can be used for warming organs con arterial perfusate at the desired high temperature. taining non-vitri?able concentrations of cryoprotectant and In either variant method, perfusion pressure at the onset of, for organs containing vitri?able concentrations of cryopro during, or after cryoprotectant dilution can be lowered to a tectant that can vitrify at cooling rates of 200 C./min or below. pressure below that used during perfusion below —10° C. and In the latter case, the organ is perfused for a time with vitri 55 before the onset of perfusion temperature elevation and/ or the ?able media at the desired high temperature or above before onset of dilution. In a presently preferred embodiment, the any subsequent dilution. In this method, no inclusion of lowered perfusion pressure is preferably in the range of 40-85 osmotic buffering agents in a diluent is involved due to the mmHg. lack of cryoprotectant dilution in the warming method. In either variant method, a preferred means of dilution is to In accordance with yet another embodiment of the present 60 use a diluent that contains no added osmotic buffers. The invention, there are provided methods for perfusing organs solution known as VMP, which contains only permeating and with a vitri?able concentration of cryoprotectant, wherein the non-permeating cryoprotectants and no osmotic buffers, is a organ is ?rst cooled by the cooling method described above, useful prototypical diluent for use in the described dilution to the desired low temperature below —10° C. and the organ is method. The method is particularly useful when the concen then perfused with the vitri?able concentration of cryopro 65 tration perfused below —10° C. and at the desired high tem tectant. As used herein, the word “cryoprotectant” is generic perature above —10° C. before dilution is vitri?able at a cool and can refer to a mixture of individual cryoprotectants of ing rate of 20° C./min or less, and preferably at a cooling rate US 8,679,735 B2 11 12 of 2° C./min or less. Further, the dilution step can be con reduced concentration of cryoprotectant that does not ducted using a concentration of cryoprotectant that is lower contain a non-penetrating osmotic buffering agent. than the highest concentration added at below — 10° C. but still In this method, the reduced concentration of cryopro high enough to be vitri?able at a cooling rate of 520° C./min. tectant can be either non-vitri?able (for organs other than the In accordance with still another embodiment of the present liver) or vitri?able (for all organs). invention, there are provided methods for diluting cryopro Invention methods and compositions will now be tectant in and warming organs from below —10° C. to a described in greater detail with reference to the following desired high temperature of —10° C. or above by continuous non-limiting Examples that describe each inventive compo vascular perfusion with minimal injury, the methods compris nent of the invention, as follows. ing: raising the temperature of the arterial perfusate to or above Example 1 the said desired high temperature while continuously perfusing the organ; and M22 and Other Preferred Vitri?cation Solutions simultaneously reducing arterial concentration while the organ temperature is rising toward said desired high Several preferred solutions of utility in the present inven temperature during continuous arterial perfusion of the tion are described in Table 1, and their effects on ice formation organ. and kidney slice viability are described in FIG. 1. In this type Typically, the desired high temperature contemplated for of plot, the biological recovery of the system after exposure to use in this method will range from about —9.9° C. to about 0° a vitri?cation solution is plotted against the critical warming C., the most preferred range being —8° C. to —2° C. Typically 20 rate of the tested vitri?cation solution. In this ?gure, the in this method the concentration of the arterial perfusate is critical warming rate was de?ned as the rate that was suf? lowered by 05-15 molar while temperature is raised from a cient to suppress crystallization of all but 0.2% of the test temperature below —10° C. to a temperature above —10° C. solution mass, as measured by the mean enthalpy of melting Also typically in this method, the warming rate from below of triplicate samples cooled and warmed in a differential —10° C. to above —10° C. is between 05° C./min and 20° 25 scanning calorimeter (DSC). The numbers inside the plotted C./min and is preferably greater than 1° C./min, with two symbols refer to the numbers of the corresponding solutions satisfactory rates being about 2° C./min and about 10° C./min. listed in Table 1, except that point 13 refers to data obtained Typically, the rate of arterial concentration dilution during for a new solution, M22, whose formula is provided in Table warming from below —10° C. to above —10° C. is between 50 2. Viability is assessed using the steady-state K+/Na+ ratio mM/min and 1000 mM/min. This method of simultaneous achieved by the slices after cryoprotectant washout and 90 perfusion warming and perfusion dilution can be effectively 30 min of incubation at 25° C. followed by washout of most and desirably accomplished without the inclusion of osmotic extracellular cations with isotonic mannitol [2, 4, 8]. The buffering agents during dilution of the cryoprotectant. The methods used for addition and washout of cryoprotectant are reduction of cryoprotectant concentration in this method need believed to avoid osmotic injury and allow only the intrinsic not result in a ?nal concentration that is non-vitri?able. Desir effects of the tested solutions to be measured, and follow the ably in the method, cryoprotectant concentration is reduced to 35 basic methodology of FIG. 5 or 7 below but without perfu a level that is still vitri?able. sion. In accordance with a still further embodiment of the The preferred methodology of the present invention is present invention, there are provided methods wherein an illustrated below using examples involving the particularly organ is perfused with a solution capable of allowing the preferred M22 vitri?cation solution and the VMP transitional organ to vitrify when the organ is cooled at a rate of 20° C./min or less at the desired low temperature at or below —10° 40 solution. FIG. 1 shows the position of M22 as a particularly C. according to the cooling method described above but fur advantageous new vitri?cation solution. One of the advan ther comprising: tages of M22, as noted above, it that it has a very low critical interrupting continuous perfusion at the desired low tem warming rate. The critical warming rate, referred to here as perature, and; vWCR, is de?ned here as the warming rate needed to cooling the organ to a temperature below the said desired 45 adequately or fully suppress detectable crystallization. FIG. 1 low temperature at or below —10° C. plots the recovery of functionality of rabbit renal cortical Interruption of perfusion as contemplated herein may con slices against the vWCR of the solution to which they were tinue until the organ is rewarmed to a temperature suf?ciently exposed (Table 1). Data for exposure to the prior art solution high to allow the organ to be perfused at an arterial perfusate known as VS41A or V855, which consists of 3.1M dimethyl temperature equal to or greater than the said desired low 50 sulfoxide plus 3.1M formamide plus 2.6M 1,2-propanediol, temperature. The interruption of perfusion may also continue are included as a point of reference (circles). As can be seen, while the organ is stored at a temperature below the desired by judicious selection of compositional factors and exposure low temperature. In addition, the interruption of perfusion conditions, it is possible to travel closer and closer to, and prior to cooling can occur after a previous perfusion time perhaps even to literally reach, a solution that does not devit insuf?cient to permit the approximate osmotic equilibration 55 rify (critical warming rate, ~0° C./min) while retaining high of the organ. In addition, the organ can be vitri?ed by cooling functional viability. For example, adding 0.5% w/v of each of following perfusion with said solution capable of allowing the organ to vitrify when the organ is cooled at a rate of 20° the antinucleators (“ice blockers”) polyvinyl alcohol (PVA) C./min or less at the said desired low temperature at or below and polyglycerol (PGL) and changing the carrier solution —10° C. for a time insuf?cient to permit the approximate from a high glucose to a lower glucose carrier known as LM5 osmotic equilibration of the organ with said solution. 60 (see Table 1) Cut vWCR from about 26° C./min to about 12° In accordance with yet another embodiment of the present C./min (point 9 vs. point 4) with no penalty in toxicity. The invention, there are provided methods of diluting cryopro same maneuver plus the replacement of PVP K30 with PVP tectant in an organ after the organ has been perfused at a K12 loweredvWCR from 63° C./min to 14° C./min (point 8 vs. temperature below —10° C., the methods comprising: point 1). Elevating the ice blockers by another 0.5% w/v each warming the organ externally; and 65 and increasing permeating cryoprotectant level by 1% w/v perfusing the organ at an arterial temperature equal to or (point 1 1 vs. point 8) yielded an additional 3 .7-fold gain with, above a desired high temperature of 210° C. with a again, no reduction in functional recovery. Overall, compared US 8,679,735 B2 13 14 to VS41A, the use of ice blockers, PVP K12, and LM5 in TABLE 2-continued combination with permeating cryoprotectant mixtures based on the combination of dimethyl sulfoxide, formamide, and Properties of M22 ethylene glycol [10] permits approximately a 20-fold Componentl Concentration or Property improvement in vWCR with a simultaneous improvement in K+/Na+ ratio from about 55% of control to about 85% of 'TPVA (also called “Supercool X-1000”) and PGL (also called “Supercool Z-1000”) are commercially available ice blockers obtainable from 21st Century Medicine, Inc. and control in slices exposed to the ?nal vitri?cation solution for consist of a polyvinylalcohol-polyvinylacetate copolymer (in which approximately 80% of the alcohol or acetate moieties are hydroxyl groups and 20% are acetyl groups) and polyg 40 min at 0° C. (FIG. 1, squares vs. circles). Reducing expo lycerol, respectively. sure by just 10 min, to 30 min, resulted in still less toxicity bFinal polymer concentrations. (diamond-shaped points). TABLE 1

Some Biologically Acceptable Highly Stable Vitri?cation Solutions”

Point Name vWCR D F E A PK30 PK12 PVA PGL Carrier % w/v

l VEG — 4% D(1)F + 7PK30 63 21.671 12.492 16.837 0 7 0 0 0 RPS-2 58 2 VEG + 1% PVA 60.3 24.208 13.955 16.837 0 0 0 l 0 56 3 VEG + 2% D 53.3 24.208 13.955 16.837 0 0 0 0 0 RPS-T 57 4 E[D(.7)F]38_16+ 6PK12 26.3 20.926 17.234 16.840 0 0 6 0 0 GHP-2 61 5 52% VEGS + 6PK12 + 0.5 + 0.5 22.2 22.887 13.194 15.919 0 0 6 0.5 0.5 LM5 59 6 52% VEGS + 6PK12 + 1% PVA 16.7 22.887 13.194 15.919 0 0 6 l 0 RPS-2 59 7 50% VEGS + 8PK12 + 1% PVA 15.6 22.007 12.686 15.307 0 0 8 l 0 RPS-2 59 8 VEG—4% D(1)F+7PK12+ .5 + .5 14.1 21.671 12.492 16.837 0 0 7 0.5 0.5 LM5 59 9 E[D(.7)F]38_16 + 6PK12 + .5 + .5 11.7 20.926 17.234 16.840 0 0 6 0.5 0.5 LM5 62 10 VEG— 3% D(1)F+7A+ lPVA+4 5.1 22.305 12.858 16.837 7 0 0 l 4 LM5 64 11 VEG—3% D(1)F+7PK12+ 1 +1 3.8 22.305 12.858 16.837 0 0 7 l l LM5 61 12 VEG— 3% D(1)F+7E+ lPVA+4 2.9 22.305 12.858 23.837 0 0 0 l 4 LM5 64

“Solution numbers refer to points in FIG. 1. vWCR is the warming rate required to suppress ice formation to 0.667joules/100 g, which is equivalent to the amount ofheat that would be produced bythe crystallization of0.2 grams ofwater per 100 grams ofsolution at 0° C. vWCR was measured alter cooling to —150° C. at 100° C./min. Cryoprotectant concentrations are given in % w/v (g/dl) units. Abbreviations: D = dimethyl sulfoxide; F = formamide; E = ethylene glycol; A = acetol; PK30 = PVP K30, ofl\¢r 40,000 daltons; PK12 = PVP K12, ofM, 5000 daltons; PVA = polyvinyl alcohol or a copolymer ofpolyvinyl alcohol and polyvinyl acetate, particularly the Supercool X- 1000 ice blocker commercially available from 21” Century Medicine (21 CM) in Rancho Cucamonga, California; PGL = polyglycerol, speci?cally decaglycerol, particularly the commercially-available Supercool Z-1000 ice blocker from 21 CM; (1) and (.7) refer to the molar ratio ofdimethyl sulfoxide to formamide in the solution; VEG = a cryoprotectant solution described elsewhere [9, 10] and recapitulated for point 2; VEgS = “VEG Solutes,” which refers to the total amount ofthe sum ofthe D, F, and E components ofVEG (in the proportions found in V53); the carrier solutions RPS-2, RPS-T, and GHP-2 are described elsewhere [5, 9]. The LM5 carrier solution [5] consists of90 mM glucose, 45 mM mannitol, 45 mM lactose, 28.2 mM KCl, 7.2 mM KZHIPO4, 5 mM reduced glutathione, 1 mM adenine HCl, 10 mM NaHCO;, and, when cryoprotectant is absent, 1 mM CaClZ and 2 mM MgC12. Solution 11 is also known as VM3. Solution 10 is also known as “1.5X”. The absolute amounts and relative proportions of all components of solutions 1-12 can be varied by :20% without losing effectiveness in the invention.

45 TABLE 2 TABLE 2-continued

Properties of M22 Properties of M22

Componentl Concentration or Property Compon?ntl commutation or Prop?lty 50 ' ' o ClX LM5 (see Table 1 for formula) contains 1 mM CaClZ and 2 mM MgC12, but these are Dlm?thyll SHlfOXMk 2' 855 M (22305 A) W/v) omitted from the 5X LM5 to avoid the formation ofprecipitates. “5X LM5” refers to a 5-fold FOITH?-l'nld? 2-855 M (12-858% W/V) increase in the molar concentrations ofthe components ofLM5. The use of5X LM5 is for Ethyl?n? glycol 2_713 M (16_837% W/v) convenience in preparing the solution but the components of IX LM5 can be weighed out . 0 directly and used instead ifdesired. Although LM5 is the preferred carrier solution for M22, N'mEthylforl'na-rn1d6 0508 M (3 A) W/V) it is not part ofthe de?nition ofM22, as M22 can also be made up effectively in other carrier 3-me?10Xy,1,2-propanediol* (1377 M (4% W/v) solutions. Also, the amount of5X LM5, or ofdry 1X LM5 components ifan LM5 concen PVP K12 2 80/ / (N0 005 6 M) 55 trate is not used to prepare the solution, can be varied. Tonicity can be kept constant at 1.5X ' o va ' by increasing the amount ofLM5 and decreasing the amount ofpolymer(s) present (PVP PVA“ 1% W/V (~0.005 M) K12, PVA, and PGL), or by reducing the amount of LM5 and increasing the amount of a 0 b N polymer(s) present (see Table 3 for tonicity equivalents of LM5 and polymers). Total PGL C 2 A) “IN ( 00267 M) solution tonicity can also be varied from 1 to 3 times isotonic. However, 1X LM5 and 1.5 5X LM5 20 ml/dl times isotonic are preferred in the best mode invention. Total cryoprotgctant concgntration 9345 M (648% W/v) “This solution could not be frozen and therefore a theoretical melting point could only be H 8 0 obtained by extrapolation of data for 94% v/v and 97% v/v of full-strength M22. Note: p ' polyethylene glycol (PEG) can also be used effectively in this solution at about 0.5-4% w/v Nominal tonicity 1.5 times isotonic 60 in place ofand/or in addition to other polymer (PGL and PVP). PEG of 600 or more daltons Melting pointd ~_54_9o C_ (estimat?d) is preferred, and especially of 600-4000 daltons. Cnncal- - Wmmg- rm <1 o cm“- Finally,. exposure at —220 C. for 30 min. allowed good recov 1The ?rst 5 components listed can be varied by :25% without losing effectiveness in the (hexagon-Shaped pelnts) after eXposuge to Sqlunons hav rhvehtroh. The next 3 Components (polymers) Can be varied up to three fold without losing ing critical warming rates of about 0 to 2.9 C./min. The point effectiveness in the invention. However, the amounts ofcomponents as listed are the best 65 plotted at 00 represents solution mode amounts. PVP K12 ~1300-5000 daltons. *This molecule is also called 3-O-methyl-rac-glycerol. from below its T8 at 10 C./min (the lower warming rate limit of our differential scanning calorimeter) failed to reveal any