Vitrification of Heart Valve Tissues

  • Kelvin G. M. BrockbankEmail author
  • Zhenzhen Chen
  • Elizabeth D. Greene
  • Lia H. Campbell
Part of the Methods in Molecular Biology book series (MIMB, volume 1257)


Application of the original vitrification protocol used for pieces of heart valves to intact heart valves has evolved over time. Ice-free cryopreservation by Protocol 1 using VS55 is limited to small samples where relatively rapid cooling and warming rates are possible. VS55 cryopreservation typically provides extracellular matrix preservation with approximately 80 % cell viability and tissue function compared with fresh untreated tissues. In contrast, ice-free cryopreservation using VS83, Protocols 2 and 3, has several advantages over conventional cryopreservation methods and VS55 preservation, including long-term preservation capability at −80 °C; better matrix preservation than freezing with retention of material properties; very low cell viability, reducing the risks of an immune reaction in vivo; reduced risks of microbial contamination associated with use of liquid nitrogen; improved in vivo functions; no significant recipient allogeneic immune response; simplified manufacturing process; increased operator safety because liquid nitrogen is not used; and reduced manufacturing costs.

Key words

Heart valves Vitrification Tissue banking 



This work was supported in part by a US Public Health Grant from the National Institute of Biomedical Imaging and Bioengineering, Grant # R43 EB014614, to KGMB. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Biomedical Imaging and Bioengineering or the National Institutes of Health. Commercial use of protocols disclosed in this work is subject to several issued US Patents (6,194,137; 6,596,531; 6,740,484; 7,157,222; 8,440,390) and International Patents (available upon request).


  1. 1.
    Angell WW, DeLanerolle P, Shumway NE (1973) Valve replacement: present status of homograft valves. Prog Cardiovasc Dis 15:589–622CrossRefGoogle Scholar
  2. 2.
    Stelzer P, Jones DJ, Elkins RC (1989) Aortic root replacement with pulmonary autograft. Circulation 80:209–213Google Scholar
  3. 3.
    Angell WW, Oury JH, Lamberti JJ, Koziol J (1989) Durability of the viable aortic allograft. J Thorac Cardiovasc Surg 98:48–56Google Scholar
  4. 4.
    O’Brien MF, McGiffin DC, Stafford EG, Gardner MA, Pohlner PF, McLachlan GJ, Gall K, Smith S, Murphy E (1991) Allograft aortic valve replacement: long-term comparative clinical analysis of the viable cryopreserved and antibiotic 4C stored valves. J Card Surg 6:534–543Google Scholar
  5. 5.
    O'Brien MF, Stafford EG, Gardner MAH, Pohlner PF, Tesar PJ, Cochrane AD, Mau TK, Gall KL, Smith SE (1995) Allograft aortic valve replacement: long-term follow-up. Ann Thorac Surg 60:565–570Google Scholar
  6. 6.
    Clarke DR, Campbell DN, Hayward AR, Bishop DA (1993) Degeneration of aortic valve allografts in young recipients. J Thorac Cardiovasc Surg 105:934–942Google Scholar
  7. 7.
    Yankah AC, Alexi-Meskhishvili V, Weng Y, Schorn K, Lange RE, Hetzer R (1995) Accelerated degeneration of allografts in the first two years of life. Ann Thorac Surg 60:71–77CrossRefGoogle Scholar
  8. 8.
    Wolfinbarger L Jr, Hopkins RA (1989) Biology of heart valve cryopreservation. In: Hopkins PA (ed) Cardiac reconstructions with allograft valves. Springer, New York, pp 21–36CrossRefGoogle Scholar
  9. 9.
    Mitchell RN, Jonas RA, Schoen FJ (1995) Structure-function correlations in cryopreserved allograft cardiac valves. Ann Thorac Surg 60:8108–8113Google Scholar
  10. 10.
    Mitchell RN, Jonas RA, Schoen FJ (1998) Pathology of explanted cryopreserved allograft heart valves: comparison with aortic valves from orthotopic heart transplants. J Thorac Cardiovasc Surg 115:118–127CrossRefGoogle Scholar
  11. 11.
    Brockbank KGM, Lightfoot FG, Song YC, Taylor MJ (2000) Interstitial ice formation in cryopreserved homografts: a possible cause of tissue deterioration and calcification in vivo. J Heart Valve Dis 9:200–206Google Scholar
  12. 12.
    Taylor MJ, Song YC, Brockbank KGM (2004) Vitrification in tissue preservation: new developments. In: Benson E, Fuller B, Lane N (eds) Life in the frozen state. Taylor and Francis Books, London, pp 603–641CrossRefGoogle Scholar
  13. 13.
    Fahy GM, MacFarlane DR, Angell CA, Meryman HT (1984) Vitrification as an approach to cryopreservation. Cryobiology 21:407–426CrossRefGoogle Scholar
  14. 14.
    Rall WF, Fahy GM (1985) Ice-free cryopreservation of mouse embryos at –196 °C by vitrification. Nature 313:573–575CrossRefGoogle Scholar
  15. 15.
    Rall WF (1987) Factors affecting the survival of mouse embryos cryopreserved by vitrification. Cryobiology 24:387–402CrossRefGoogle Scholar
  16. 16.
    Fahy GM (1988) Vitrification. In: McGrath JJ, Diller KR (eds) Low temperature biotechnology: emerging applications and engineering contributions. The American Society of Mechanical Engineers, New York, pp 113–146Google Scholar
  17. 17.
    Fahy GM (1989) Vitrification as an approach to organ cryopreservation: past, present, and future. In: Smit Sibinga CT, Das PC, Meryman HT (eds) Cryopreservation and low temperature biology in blood transfusion. Kluwer Academic Publishers, Dordrecht, pp 255–268Google Scholar
  18. 18.
    Armitage WJ, Rich SJ (1990) Vitrification of organized tissues. Cryobiology 27:483–491CrossRefGoogle Scholar
  19. 19.
    Pegg DE, Diaper MP (1990) Freezing versus vitrification: basic principles. In: Smit Sibinga CT, Das PC, Meryman HT (eds) Cryopreservation and low temperature biology in blood transfusion, vol 24. Kluwer Academic Publishers, Dordrecht, pp 55–69CrossRefGoogle Scholar
  20. 20.
    MacFarlane DR, Forsyth M, Barton CA (1992) Vitrification and devitrification in cryopreservation. In: Steponkus PL (ed) Advances in low temperature biology, vol 1. JAI Press, Greenwich, CT, pp 221–278Google Scholar
  21. 21.
    Song YC, Khirabadi BS, Lightfoot F, Brockbank KGM, Taylor MJ (2000) Vitreous cryopreservation maintains the function of vascular grafts. Nat Biotechnol 18:296–299CrossRefGoogle Scholar
  22. 22.
    Rabin Y, Taylor MJ, Walsh JR, Baicu S, Steif PS (2005) Cryomacroscopy of vitrification, Part I: a prototype and experimental observations on the cocktails VS55 and DP6. Cell Preserv Technol 3:169–183CrossRefGoogle Scholar
  23. 23.
    Baicu S, Taylor MJ, Chen Z, Rabin Y (2006) Vitrification of carotid artery segments: an integrated study of thermophysical events and functional recovery towards scale-up for clinical applications. Cell Preserv Technol 4:236–244CrossRefGoogle Scholar
  24. 24.
    Brockbank KGM, Taylor MJ (2007) Tissue preservation. In: Baust JG (ed) Advances in biopreservation. CRC Press, Boca Raton, pp 157–196Google Scholar
  25. 25.
    Song YC, An YH, Kang QK, Li C, Boggs JM, Chen Z, Taylor MJ, Brockbank KGM (2004) Vitreous preservation of articular cartilage grafts. J Invest Surg 17:65–70CrossRefGoogle Scholar
  26. 26.
    Song YC, Lightfoot FG, Chen Z, Taylor MJ, Brockbank KGM (2004) Vitreous preservation of rabbit articular cartilage. Cell Preserv Technol 2:67–74CrossRefGoogle Scholar
  27. 27.
    Brockbank KGM, MacLellan WR, Xie J, Hamm-Alvarez SF, Chen ZZ, Schenke-Layland K (2008) Quantitative second harmonic generation imaging of cartilage damage. Cell Tissue Bank 9:299–308CrossRefGoogle Scholar
  28. 28.
    Brockbank KGM, Chen ZZ, Song YC (2010) Vitrification of porcine articular cartilage. Cryobiology 60:217–221CrossRefGoogle Scholar
  29. 29.
    Song YC, Chen ZZ, Mukherjee N, Lightfoot FG, Taylor MJ, Brockbank KGM, Sambanis A (2005) Vitrification of tissue engineered pancreatic substitute. Transplant Proc 37:253–255CrossRefGoogle Scholar
  30. 30.
    Elder E, Chen Z, Ensley A, Nerem R, Brockbank K, Song Y (2005) Enhanced tissue strength in cryopreserved, collagen-based blood vessel constructs. Transplant Proc 37:4625–4629CrossRefGoogle Scholar
  31. 31.
    Dahl S, Chen Z, Solan A, Lightfoot F, Li C, Brockbank KGM, Niklason L, Song YC (2006) Tissue engineered blood vessels. Tissue Eng 12:291–300CrossRefGoogle Scholar
  32. 32.
    Farooque TM, Chen ZZ, Schwartz Z, Wick TM, Boyan BD, Brockbank KGM (2009) Protocol development for vitrification of tissue-engineered cartilage. Bioprocessing (Williamsbg Va) 8:29–36Google Scholar
  33. 33.
    Khirabadi BS, Song YC, Brockbank KGM (2004) Method of cryopreservation of tissues by vitrification. United States Patent #6,740,484, 2004Google Scholar
  34. 34.
    Khirabadi BS, Song YC, Brockbank KGM (2007) Method of cryopreservation of tissues by vitrification. United States Patent #7,157,222Google Scholar
  35. 35.
    Brockbank KGM, Song YC (2004) Morphological analyses of ice-free and frozen cryopreserved heart valve explants. J Heart Valve Dis 13:297–301Google Scholar
  36. 36.
    Schenke-Layland K, Madershahian N, Riemann I, Starcher B, Halbhuber KJ, König K, Stock UA (2006) Impact of cryopreservation on extracellular matrix structures of heart valve leaflets. Ann Thorac Surg 81:918–926CrossRefGoogle Scholar
  37. 37.
    Schenke-Layland K, Riemann I, Damour O, Stock UA, König K (2006) Two-photon microscopes and in vivo multiphoton tomographs – powerful diagnostic tools for tissue engineering and drug delivery. Adv Drug Deliv Rev 58:878–896CrossRefGoogle Scholar
  38. 38.
    Schenke-Layland K, Xie J, Haydarkhan-Hagvall S, Hamm-Alvarez SF, Stock UA, Brockbank KGM, MacLellan WR (2007) Optimized preservation of extracellular matrix in cardiac tissues: implications for long-term graft durability. Ann Thorac Surg 83:1641–1650CrossRefGoogle Scholar
  39. 39.
    Brockbank KGM, Wright GJ, Yao H, Greene ED, Chen ZZ, Schenke-Layland K (2011) Allogeneic heart valve preservation – allogeneic heart valve storage above the glass transition at −80 °C. Ann Thorac Surg 91:1829–1835CrossRefGoogle Scholar
  40. 40.
    Hazekamp MG, Koolbergen DR, Braun J, Sugihara H, Cornelisse CJ, Goffin YA, Huysmans HA (1995) In situ hybridization: a new technique to determine the origin of fibroblasts in cryopreserved aortic homograft valve explants. J Thorac Cardiovasc Surg 110:248–257CrossRefGoogle Scholar
  41. 41.
    Koolbergen DR, Hazekamp MG, de Heer E, Bruggemans EF, Huysmans HA, Dion RA, Bruijn JA (2002) The pathology of fresh and cryopreserved homograft heart valves: an analysis of forty explanted homograft valves. J Thorac Cardiovasc Surg 124:689–697CrossRefGoogle Scholar
  42. 42.
    Armiger LC (1998) Postimplantation leaflet cellularity of valve allografts: are donor cells beneficial or detrimental? Ann Thorac Surg 66(suppl):S233–S235CrossRefGoogle Scholar
  43. 43.
    Navarro FB, Costa FD, Mulinari LA, Pimentel GK, Roderjan JG, Vieira ED, Noronha L, Miyague NI (2010) Evaluation of the biological behavior of decellularized pulmonary homografts: an experimental sheep model. Rev Bras Cir Cardiovasc 25:377–387CrossRefGoogle Scholar
  44. 44.
    da Costa FD, Costa AC, Prestes R, Domanski AC, Balbi EM, Ferreira AD, Lopes SV (2010) The early and midterm function of decellularized aortic valve allografts. Ann Thorac Surg 90:1854–1860CrossRefGoogle Scholar
  45. 45.
    Campbell LH, Brockbank KGM (2010) Cryopreservation of porcine aortic heart valve leaflet-derived myofibroblasts. Biopreserv Biobank 8:211–217CrossRefGoogle Scholar
  46. 46.
    Ketchedjian A, Jones AL, Krueger P, Robinson E, Crouch K, Wolfinbarger L Jr, Hopkins R (2005) Recellularization of decellularized allograft scaffolds in ovine great vessel reconstructions. Ann Thorac Surg 79:888–896CrossRefGoogle Scholar
  47. 47.
    Ketchedjian A, Krueger P, Lukoff H, Robinson E, Jones A, Crouch K, Wolfinbarger L, Hopkins RA (2005) Ovine panel reactive antibody assay of HLA responsivity to allograft bioengineered vascular scaffolds. J Thorac Surg 129:155–166CrossRefGoogle Scholar
  48. 48.
    Linthurst Jones A, Moore M (2009) MATRACELLTM Decellularized allograft bio-implants – critical applications for cardiovascular surgery a bio-implants brief. LifeNet Health Nosfolk, VA, USAGoogle Scholar
  49. 49.
    Lisy M, Pennecke J, Brockbank KGM, Fritze O, Schleicher M, Schenke-Layland K, Kaulitz R, Riemann I, Weber CN, Braun J, Mueller KE, Fend F, Scheunert T, Gruber AD, Albes JM, Ziemer G, Stock UA (2010) The performance of ice-free cryopreserved heart valve allografts in an orthotopic pulmonary sheep model. Biomaterials 31:5306–5311CrossRefGoogle Scholar
  50. 50.
    Brockbank KGM, Schenke-Layland K, Greene ED, Chen Z, Fritze O, Schleicher M, Kaulitz R, Riemann I, Fend F, Albes JM, Stock UA, Lisy M (2012) Ice-free cryopreservation of heart valve allografts: better extracellular matrix preservation in vivo and preclinical results. Cell Tissue Bank 13:663–671CrossRefGoogle Scholar
  51. 51.
    Brockbank KGM (2013) Methods for ice-free preservation of tissues. US Patent #8,440,390Google Scholar
  52. 52.
    Thakrar RR, Patel VP, Hamilton G, Fuller BJ, Seifalian AM (2006) Vitreous cryopreservation maintains the viscoelastic property of human vascular grafts. FASEB J 20:874–881CrossRefGoogle Scholar
  53. 53.
    Huber AJ, Brockbank KGM, Aberle T, Schleicher M, Chen Z, Greene ED, Lisy M, Stock UA (2012) Development of a simplified ice-free cryopreservation method for heart valves. Biopreserv Biobank 10:479–484CrossRefGoogle Scholar
  54. 54.
    Huber AJT, Brockbank KGM, Riemann I, Schleicher M, Fritze O, Wendel H, Stock UA (2012) Preclinical evaluation of ice-free cryopreserved arteries: structural integrity and hemocompatibility. Cells Tissues Organs 196:262–270CrossRefGoogle Scholar
  55. 55.
    Brockbank KGM, Song YC, Greene ED, Taylor MJ (2007) Quantitative analyses of vitrified autologous venous arterial bypass graft explants. Cell Preserv Technol 5:68–76CrossRefGoogle Scholar
  56. 56.
    Rajani B, Mee RB, Ratliff NB (1998) Evidence for rejection of homograft cardiac valves in infants. J Thorac Cardiovasc Surg 115:111–117CrossRefGoogle Scholar
  57. 57.
    Bechtel JF, Stierle U, Sievers HH (2008) Fifty-two months mean follow up of decellularized SynerGraft-treated pulmonary valve allografts. J Heart Valve Dis 17:98–104Google Scholar
  58. 58.
    Brockbank KGM, Campbell LH, Chen Z, Greene ED, Stock UA, Seifert M (2013) Minimization of allogeneic tissue immunogenicity by cryopreservation. Presented at the 22nd annual congress of the European Association of Tissue Banks, November (abstract)Google Scholar
  59. 59.
    Valente M, Faggian G, Billingham ME, Talentie E, Calabrese F, Casula R, Shumway NE, Thiene G (1995) The aortic valve after heart transplantation. Ann Thorac Surg 60(suppl):S135–S140CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Kelvin G. M. Brockbank
    • 1
    • 2
    • 3
    Email author
  • Zhenzhen Chen
    • 1
  • Elizabeth D. Greene
    • 1
  • Lia H. Campbell
    • 1
  1. 1.Cell & Tissue Systems, Inc.North CharlestonUSA
  2. 2.Department of BioengineeringClemson UniversityClemsonUSA
  3. 3.Department of Regenerative Medicine and Cell BiologyMedical University of South CarolinaCharlestonUSA

Personalised recommendations