Cell and Tissue Banking

, Volume 14, Issue 1, pp 1–10 | Cite as

Tissue cryobanking for conservation programs: effect of tissue type and storage time after death

  • Arzu Tas Caputcu
  • Tolga Akkoc
  • Gaye Cetinkaya
  • Sezen Arat


In this study, we investigated the temporal post-mortem limits, within which there will be guarantees of obtaining living cells from several tissues of sheep and cattle and the effect of vitrification on the ability of cells from tissue stored at different times. Muscle tissue and auricular cartilage were stored at 4°C for 5, 48, 72, 96 and 216 h post-mortem (hpm). Tissue samples were sorted into two groups: one group was in vitro cultured immediately after storage and the other was vitrified after storage and then in vitro cultured. In cattle and sheep, no differences in subconfluence rates were observed between the two experimental groups. At the same time, no significant differences were observed in the number of days required in culture to reach confluence between non-vitrified and vitrified groups when tissues were stored at 4°C for different times. In sheep, while the population doubling times (PDT) were similar in cartilage cells from vitrified and non-vitrified tissues and stored at 4°C for 5 and 216 hpm, PDT of muscle cells were longer in 216 hpm stored groups than in 5 hpm stored groups. In bovine, although the PDT of muscle cells were similar for 5 and 216 hpm and both vitrified and non-vitrified tissues and the PDT were longer in cartilage cells from vitrified than from non-vitrified tissues. In conclusion, although storage times and vitrification have different effects on tissues from cattle and sheep, this study showed that living cells could be obtained from all groups. Therefore, cartilage and muscle tissues can be stored at 4°C for 216 hpm and used for cyrobanking.


Cryopreservation Vitrification Cartilage tissue Muscle tissue Cryobanking 



We would like to thank Dr. Digdem Aktoprakligil Aksu for manuscript review; Fatih Karakaya, Erman Ates and Ozlem Celasin for technical assistance. The manuscript has been edited by native speaker Dr. Anita L. Akkas, who has PhD degree in English Literature and MA degree in Linguistics Engineering and Science. This research was funded by the Scientific and Technological Research Council of Turkey (TUBITAK) (Project no. KAMAG-106G005).


  1. Akkoc T, Taskin C, Caputcu TA, Arat S, Bagis H (2011) The effect of solid surface vitrificaiton (SSV) versus classic vitrification technique on survive rate of in vitro produced bovine blastocysts. J Anim Vet Adv 10(22):2885–2891Google Scholar
  2. Andrabi SMH, Maxwell WMC (2007) A review of reproductive biotechnologies for conservation of endangered mammalian species. Anim Reprod Sci 99:223–243PubMedCrossRefGoogle Scholar
  3. Arat S, Gibbons J, Rzucidlo SJ, Respess DS, Tumlin M, Stice SL (2002) In vitro development of bovine nuclear transfer embryos from clonal lines of transgenic adult and fetal fibroblast cells of the same genotype. Biol Reprod 66:1768–1774PubMedCrossRefGoogle Scholar
  4. Arat S, Bagis H, Ergin F, Sagirkaya H, Mercan Odaman H, Dinnyes A (2004) Cold storage of tissue as source for donor cells does not reduce the in vitro development of bovine embryos following nuclear transfer. Reprod Fertil Dev 16(1,2):135CrossRefGoogle Scholar
  5. Arat S, Bagis H, Mercan Odaman H, Dinnyes A (2005) Cloned embryos can be produced using donor cells obtained from 72-hour cooled carcass. Reprod Fertil Dev 17:164CrossRefGoogle Scholar
  6. Arat S, Tas A, Akkoc T, Cetinkaya G, Bagis H, Sekmen S, Ates E, Soysal D (2009) Effect of growth factors on development of nuclear transfer embryos from cartilage cell of an Anatolian native cow. Reprod Domes Anim 44:93Google Scholar
  7. Arat S, Caputcu AT, Akkoc T, Pabuccuoglu S, Sagirkaya H, Cirit U, Nak Y, Koban E, Bagis H, Demir K, Nak D, Senunver A, Kilicaslan R, Tuna B, Cetinkaya G, Denizci M, Aslan O (2011) Using cell banks as a tool in conservation programmes of native domestic breeds: the production of the first cloned Anatolian grey cattle. Reprod Fertil Dev 23(8):1012–1023PubMedCrossRefGoogle Scholar
  8. Asawa Y, Ogasawara T, Takahashi T, Yamaoka H, Nishizawa S, Matsudaira K, Mori Y, Takato T, Hoshi K (2009) Aptitude of auricular and nasoseptal chondrocytes cultured under a monolayer or three-dimensional condition for cartilage tissue engineering. Tissue Eng Part A 15:1109–1118PubMedCrossRefGoogle Scholar
  9. Bagis H, Akkoc T, Tas A, Aktoprakligil D (2008) Cryogenic effect of antifreeze protein on transgenic mouse ovaries and the production of live offspring by orthotopic transplantation of cryopreserved mouse ovaries. Mol Reprod Dev 75:608–613PubMedCrossRefGoogle Scholar
  10. Bagis H, Akkoc T, Taskin C, Arat S (2010) Comparison of different cryopreservation techniques: higher survival and implantation rate of frozen-thawed mouse pronuclear embryos in the presence of beta-mercaptoethanol in post-thaw culture. Reprod Domest Anim 45:332–337CrossRefGoogle Scholar
  11. Brem G, Kuhholzer B (2002) The recent history of somatic cloning in mammals. Cloning Stem Cells 4(1):57–63PubMedCrossRefGoogle Scholar
  12. Cetinkaya G, Arat S (2011) Cryopreservation of cartilage cell and tissue for biobanking. Cyrobiology 63:292–297CrossRefGoogle Scholar
  13. Day JG, Stacey GN (2007a) Cryopreservation and freeze-drying protocols. In: Day JG, Stacey GN (eds) Long-term ex situ conservation of biological resources and the role of biological resource centers, 2nd edn. Humana Press, New Jersey, pp 1–14Google Scholar
  14. Day JG, Stacey GN (2007b) Cryopreservation and freeze-drying protocols. In: Sputtek A (ed) Cryopreservation of Red Blood Cells and Platelets, 2nd edn. Humana Press, New Jersey, pp 283–301Google Scholar
  15. Fahy GM, Wowk B, Wu J, Phan J, Rasch C, Chang A, Zendejas E (2004) Cryopreservation of organs by vitrification: perspectives and recent advances. Cryobiology 48:157–178PubMedCrossRefGoogle Scholar
  16. Fröhlich M, Malicev E, Gorensek M, Knezevic M, Velikonja NK (2007) Evaluation of rabbit auricular chondrocyte isolation and growth parameters in cell culture. Cell Biol Int 31:620–625PubMedCrossRefGoogle Scholar
  17. Gajda B, Katska-Ksiazkiewicz L, Rynska B, Bochenek M, Smorag Z (2007) Survival of bovine fibroblasts and cumulus cells after vitrification. Cryo Lett 28(4):271–279Google Scholar
  18. Gañán N, Sestelo A, Garde JJ, Martínez F, Vargas A, Sánchez I, Pérez-Aspa MJ, López-Bao JV, Palomares F, Gomendio M, Roldan ER (2010) Reproductive traits in captive and free-ranging males of the critically endangered Iberian lynx (Lynx pardinus). Reproduction 139:275–285PubMedCrossRefGoogle Scholar
  19. Gibbons J, Arat S, Rzucidlo SJ, Waltenburg R, Respess DS, Venable AM, Stice SL (2002) Enhanced survivability of cloned calves derived from roscovitine-treated adult somatic cells. Biol Reprod 66:895–900PubMedCrossRefGoogle Scholar
  20. Hay RJ (1992) Cell line preservation and characterization. In: Freshney RI (ed) Animal Cell Culture. Oxford University Press, New York, A Practical Approach, pp 95–148Google Scholar
  21. Ideta A, Hayama K, Urakawa M, Tsuchiya K, Aoyagi Y, Saeki K (2010) Comparison of early development in utero of cloned fetuses derived from bovine fetal fibroblasts at the G1 and G0/G1 phases. Anim Reprod Sci 119:191–197PubMedCrossRefGoogle Scholar
  22. Leon-Quinto T, Simon MA, Cadenas R, Jones J, Martinez-Hernandez FJ, Moreno JM, Vargas A, Martinez F, Soria B (2009) Developing biological resource banks as a supporting tool for wildlife reproduction and conservation the Iberian lynx bank as a model for other endangered species. Anim Reprod Sci 112:347–361PubMedCrossRefGoogle Scholar
  23. Loi P, Ptak G, Barboni B, Fulka J Jr, Cappai P, Clinton M (2001) Genetic rescue of an endangered mammal by cross-species nuclear transfer using post-mortem somatic cells. Nat Biotech 19:962–964CrossRefGoogle Scholar
  24. Martin H, Bournique B, Sarsat JP, Albaladejo V, Lerche-Langand C (2000) Cryopreserved rat liver slices: a critical evaluation of cell viability, histological integrity, and drug metabolizing enzymes. Cryobiology 41:135–144PubMedCrossRefGoogle Scholar
  25. McLaren A (2000) Cloning: pathways to a pluripotent future. Science 288:1775–1780PubMedCrossRefGoogle Scholar
  26. Morgan SJ, Darling DC (1993) Animal cell culture, In: JM Graham, D Billington (eds), BIOS scientific publisher limited, UK, 161Google Scholar
  27. Nel-Themaat L, Gómez MC, Damiani P, Wirtu G, Dresser BL, Bondioli KR, Lyons LA, Pope CE, Godke RA (2007) Isolation, culture and characterisation of somatic cells derived from semen and milk of endangered sheep and eland antelope. Reprod Fertil Dev 19:576–584PubMedCrossRefGoogle Scholar
  28. Orief Y, Schultze-Masgau A, Dafopoulus K, Al-hasani S (2005) Vitrification: will it replace the conventional gamete cryopreservation techniques. Middle East Fertil Soc J 10(3):171–184Google Scholar
  29. Pegg DE, Wusteman MC, Wand L (2006) Cryopreservation of articular cartilage. Part 1: conventional cryopreservation methods. Cryobiology 52:335–346PubMedCrossRefGoogle Scholar
  30. Prentice JR, Anzar M (2011) Cryopreservation of mammalian oocyte for conservation of animal genetics. Vet Med Int. doi: 10.4061/2011/146405 Google Scholar
  31. Ryder OA (2002) Cloning advances and challenges for conservation. Trends Biotechnol 20:231–232PubMedCrossRefGoogle Scholar
  32. Saeed AM, Escriba MJ, Silvestre MA, Garcia-Ximenez F (2000) Vitrification and rapid-freezing of cumulus cells from rabbits and pigs. Theriogenology 54:1359–1371PubMedCrossRefGoogle Scholar
  33. Shiga K, Fujita T, Hirose K, Sasae Y, Nagai T (1999) Production of calves by transfer of nuclei from cultured somatic cells obtained from Japanese black bulls. Theriogenology 52(3):527–535PubMedCrossRefGoogle Scholar
  34. Shroeder AC, Johnston D, Epping JJ (1991) Reversal of post-mortem degeneration of mouse oocytes during meiotic maturation in vitro. J Exp Zool 258:240–245CrossRefGoogle Scholar
  35. Silvestre MA, Saeed AM, Escriba MJ, Garcia-Ximenez F (2000) Vitrification and rapid-freezing of cumulus cells from rabbits and pigs. Theriogenology 54(9):1359–1371PubMedCrossRefGoogle Scholar
  36. Silvestre MA, Saeed AM, Escribá MJ, García-Ximénez F (2002) Vitrification and rapid freezing of rabbit fetal tissues and skin samples from rabbits and pigs. Theriogenology 58(1):69–76PubMedCrossRefGoogle Scholar
  37. Silvestre MA, Saeed AM, Cervera RP, Escriba MJ, Garcia-Ximenez F (2003) Rabbit and pig ear skin sample cryobanking: effects of storage time and temperature of the whole ear extirpated immediately after death. Theriogenology 59:1469–1477PubMedCrossRefGoogle Scholar
  38. Silvestre MA, Sanchez JP, Gomez EA (2004) Vitrification of goat, sheep, and cattle skin samples from whole ear extirpated after death and maintained at different storage times and temperatures. Cryobiology 49:221–229PubMedCrossRefGoogle Scholar
  39. Smith LC, Bordignon V, Babkine M, Fecteau G, Keefer C (2000) Benetif and problems with cloning animals. Can Vet J 4:919–924Google Scholar
  40. Stolzing A, Scutt A (2006) Age-related impairment of mesenchymal progenitor cell function. Aging Cell 5:213–224PubMedCrossRefGoogle Scholar
  41. Vajta G, Nagy ZP, Cobo A, Conceicao J, Yovich J (2009) Vitrification in assisted reproduction: myths, mistakes, disbeliefs and confusion. Biomed Online 19:1–7CrossRefGoogle Scholar
  42. Wells DNA, Misica PM, Tervit HR, Vivanco WH (1998) Adult somatic cell NT in used to preserve the last surviving cow of Enderby Island cattle breed. Reprod Fertil Dev 10:369–378PubMedCrossRefGoogle Scholar
  43. Wildt DE, Wemmer C (1999) Sex and wildlife: the role of reproductive science in conservation. Biodivers Conserv 8:965–976CrossRefGoogle Scholar
  44. Wusteman M, Robinson M, Pegg D (2004) Vitrification of large tissues with dielectric warming: biological problems and some approaches to their solution. Cryobiology 48:179PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Arzu Tas Caputcu
    • 1
  • Tolga Akkoc
    • 1
  • Gaye Cetinkaya
    • 1
  • Sezen Arat
    • 2
  1. 1.TUBITAK MRC Genetic Engineering and Biotechnology InstituteGebzeTurkey
  2. 2.Department of Agricultural BiotechnologyNamik Kemal University Agriculture FacultyTekirdagTurkey

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