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(12) United States Patent (10) Patent No.: US 8,679,735 B2 Fahy Et Al

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(12) United States Patent (10) Patent No.: US 8,679,735 B2 Fahy Et Al US0086,79735B2 (12) United States Patent (10) Patent No.: US 8,679,735 B2 Fahy et al. (45) Date of Patent: Mar. 25, 2014 (54) METHODS AND COMPOSITIONS FOR THE Fahy, et al.. Some emerging principles underlying the physical prop CRYOPRESERVATION OF ORGANS erties, biological actions, and utility of vitrification solutions. Cryobiology, 1987. 24: 196-213. (75) Inventors: Gregory M. Fahy, Corona, CA (US); Fahy, et al., Vitrification as an approach to cryopreservation. Brian Wowk, Corona, CA (US) Cryobiology, 1984. 21: 407-426. (73) Assignee: 21st Century Medicine Inc., Rancho Fahy, et al., Improved vitrification solutions based on predictability of Cucamonga, CA (US) vitrification 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 A.L. (86). PCT No.: PCT/US2004/030544 Humphries, Jr, Editors. 1974, Little, Brown and Company: Boston. p. 3-8. S371 (c)(1), Kheirabadi, B. and G.M. Fahy, Permanent life support by kidney's (2), (4) Date: Nov. 8, 2006 perfused with a vitrifiable (7.5 molar) cryoprotectant solution. Trans plantation, 2000. 70(1): 51-57. (87) PCT Pub. No.: WO2005/027633 Khirabadi, B.S. and G.M. Fahy, Cryopreservation of the mammalian kidney. I. Transplantation of rabbit kidney's 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 L.S. Ewing, Survival of rabbit kidney's perfused with 8.4 M cryoprotectant. Cryobiology, 1995. US 2007/O190517 A1 Aug. 16, 2007 32:543-544. Related U.S. Application Data Kheirabadi, B.S., F. Arnaud and E. Kapnik, The effect of vitrification on viability of rabbit renal tissue. Cryobiology, 1998. 37:447. (60) Provisional application No. 60/503,551, filed on Sep. Kheirabadi, B.S., G.M. Fahy, J. Saurand H.T. Meryman, Perfusion of 16, 2003. rabbit kidneys with 8 molar cryoprotectant (V52). Cryobiology, (51) Int. Cl. 1993. 30: 611-612. AOIN I/00 (2006.01) Kheirabadi, B.S., G.M. Fahy, P. Nannini, J. Saur and H.T. Meryman, Life support function of rabbit kidney's perfused with 8 molar (52) U.S. Cl. cryoprotectant. Cryobiology, 1993. 30: 612. USPC ............................... 435/1.2: 435/13:435/1.1 Kheirabadi, B.S., G.M. Fahy, J. Saur, L. Ewing and H.T. Meryman, (58) Field of Classification Search Failure of rabbit kidneys to survive chilling to -30°C. afier perfusion None with 8M cryoprotectant at -3°C. Cryobiology, 1994. 31:596-597. See application file 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 al. Farabow, Garrett and Dunner, LLP 5,821,045 A 10/1998 Fahy et al. 5,962,214 A 10/1999 Fahy et al. 6,187,529 B1 2/2001 Fahy et al. (57) ABSTRACT 6,274,303 B1* 8/2001 Wowk et al. ................... 435/13 6,395,467 B1 5/2002 Fahy et al. 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 vitrifiable cryoprotectant solutionata temperature above -10°C. for a time insufficient WO WO 96/O5727 2, 1996 for the approximate osmotic equilibration of the organ with WO WOO1/42388 6, 2001 the solution, followed by cooling the organ to below -10°C. WO WOO2,09516 6, 2001 WO WOO3,OO9743 2, 2003 by perfusion with said solution at a reduced temperature. In WO WOO3,0658O1 8, 2003 certain embodiments, Step 2 comprises increasing the con centration of cryoprotectant further at a temperature from -10 OTHER PUBLICATIONS to -40°C. In certain embodiments, Step 3 comprises cooling Arnaud, et al., Physiological evaluation of a rabbit kidney perfised and vitrifying the organ, rewarming it, and perfusing the with VS4 IA. Cryobiology, 2003. 46: 289-294. organ with a vitrifiable concentration of cryoprotectant Fahy, G.M., Analysis of "solution effects' injury: cooling rate depen whose temperature is either raised gradually or is held at dence of the fianctional and morphological sequellae of freezing in >-15° C. Compositions are provided that allow safe organ rabbit renal cortex protected with dimethylsulfoxide. Cryobiology, perfusion with vitrifiable media at >-10°C. and almost com 1981. 18: 550-570. plete avoidance of chilling injury at -20 to -25° C. and that Fahy, G.M., Prospects for vitrification of whole Organs. Cryobiology, allow slow warming after vitrification 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 Applied Aspects, D.E. Pegg, I.A. Jacobsen, and N. 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 -196° C. by vitrification. Nature, 1985.313:573-575. Kheirabadi, B.S., G.M. Fahy, L. Ewing, J. Saur and H.T. Meryman, Starzl, T.E., A look ahead at transplantation. Journal of Surgical 100% survival of rabbit kidney's chilled to -32 C. after perfusion with 8 M cryoprotectant at -22°C. Cryobiology, 1994, 31:597. research, 1970. 10:291-297. Pegg, D.E., Banking of cells, 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: New York. p. 153-180. International Search Report for PCT Patent Application No. PCT/ Pegg, D.E., Theory and experiments towards subzero organ preser USO4, 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, D.E. and M.P. Diaper, The mechanism of cryoinjury in glyc erol-treated rabbit kidneys, in Organ Preservation, Basic and * cited by examiner U.S. Patent Mar. 25, 2014 Sheet 1 of 11 US 8,679,735 B2 Figure 1 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 O, 40 min exposure, O'C KX 30 min exposure, O'C O 30 min exposure, -22°C O 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Warming Rate Yielding 0.2% Whw Crystallization (C/min) U.S. Patent Mar. 25, 2014 Sheet 2 of 11 US 8,679,735 B2 Figure 2 cu as 2 9 S. s 5 S E O O O O as as - E 3 P 2 2S-XW 10 15 2.0 1X- 1X -- 2X -- Relative Tonicity (X) 1X 2X 2X Between 0°C and -22°C Relative Tonicity (X) U.S. Patent Mar. 25, 2014 Sheet 3 of 11 US 8,679,735 B2 F.G. 3A 8 . .480 ty. r 3 ci iwi (arteria)M 8 5 335/ser tra - M. - Riri: 8 is 282- s A-W (ii) s * ^ 2 & 3 is 8 - is ess seei:53 . " rig yy ^ - wax (iiirii; fg ... re S s R s t 8 s: 8: $3. 3: Perfusior its (viii. F.C. 3 as S. : & S. Sima 33 f is 8 : 8 & 8 °ost operative Day U.S. Patent Mar. 25, 2014 Sheet 4 of 11 US 8,679,735 B2 Figure 4 10 in 10 min WMP 1/2 VMP+ -3 10 min WMP 22 10 min 10 min 10 min WMP WMP 1/2 VMP+ 5 min 10 min 10 nin 10 min WMP WMP 1/2 WMP-- WMP -3 Ontin WMP Postoperative Day U.S. Patent Mar. 25, 2014 Sheet 5 of 11 US 8,679,735 B2 t: 123.0 123.8 M (arterial) A-V(M) EEN2. N 'o"10 is 7.5 A-V (M) T (arterial) T (venous) Perf Time 271.5 273.5 Pressure/40 (mm Hg) (m/min)fgFlow O 60 120 18O 240 300 Perfusion Time (Min) U.S. Patent Mar. 25, 2014 Sheet 6 of 11 US 8,679,735 B2 Figure 6 2 4 -O- wilo at-22OC (n=2) -0- slow rewarming (n=6) -0- fast rewarming (n=11) O 2 4 6 8 101214 Time (min) 0 2 4 6 8 10 12 14 16 18 Postoperative Day U.S. Patent Mar. 25, 2014 Sheet 7 of 11 US 8,679,735 B2 Figure 7 M2210; ya 1.90 EN M (arterial) (M) is A-V (M) T (arterial) M22 0.6 annum- A-W O.5 T (venous) (M) his Pressure?40 (mmHg) (ml/min/g) Perfusion Time (Min) U.S. Patent Mar. 25, 2014 Sheet 8 of 11 US 8,679,735 B2 Figure 8 0.0 5.0 10.0 15.0 20.0 25.0 Time after M22 Perf. U.S. Patent Mar.
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