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Production of bovine cloned embryos with donor cells frozen at a slow cooling rate in a conventional freezer (−20 °C)

  • Liliana Chacón (a1) (a2) (a3), Martha C. Gómez (a4) (a2) (a5), Jill A. Jenkins (a6), Stanley P. Leibo (a2) (a3), Gemechu Wirtu (a2), Betsy L. Dresser (a2) (a3) and C. Earle Pope (a2)...
Summary

Usually, fibroblasts are frozen in dimethyl sulphoxide (DMSO, 10% v/v) at a cooling rate of 1 °C/min in a low-temperature (−80 °C) freezer (LTF) before storage in liquid nitrogen (LN2); however, a LTF is not always available. The purpose of the present study was to evaluate apoptosis and viability of bovine fibroblasts frozen in a LTF or conventional freezer (CF; −20 °C) and their subsequent ability for development to blastocyst stage after fusion with enucleated bovine oocytes. Percentages of live cells frozen in LTF (49.5%) and CF (50.6%) were similar, but significantly less than non-frozen control (88%). In both CF and LTF, percentages of live apoptotic cells exposed to LN2 after freezing were lower (4% and 5%, respectively) as compared with unexposed cells (10% and 18%, respectively). Cells frozen in a CF had fewer cell doublings/24 h (0.45) and required more days (9.1) to reach 100% confluence at the first passage (P) after thawing and plating as compared with cells frozen in a LTF (0.96 and 4.0 days, respectively). Hypoploidy at P12 was higher than at P4 in cells frozen in either a CF (37.5% vs. 19.2%) or in a LTF (30.0% vs. 15.4%). A second-generation cryo-solution reduced the incidence of necrosis (29.4%) at 0 h after thawing as compared with that of a first generation cryo-solution (DMEM + DMSO, 60.2%). The percentage of apoptosis in live cells was affected by cooling rate (CF = 1.9% vs. LFT = 0.7%). Development of bovine cloned embryos to the blastocyst stage was not affected by cooling rate or freezer type.

Copyright
Corresponding author
All correspondence to: Martha C. Gómez. Audubon Center for Research of Endangered Species, 14001 River Road, New Orleans, Louisiana 70131, USA. Tel: +1 504 398 3159. Fax: +1 504 391 7707. e-mail: mgomez@auduboninstitute.org
References
Hide All
Adams, R.M., Wang, M., Crane, A.M., Brown, B., Darlington, G.J. & Ledley, F.D. (1995). Effective cryopreservation and long-term storage of primary human hepatocytes with recovery of viability, differentiation and replicative potential. Cell Transplant 4, 579–86.
Baust, J.M., Baust, J.G. & Van Buskirk, R. (1998). Cryopreservation outcome is enhanced by intracellular type medium and inhibition of apoptosis. Cryobiology 37, 410–1.
Baust, J.M., Van Buskirk, R. & Baust, J.G. (2000a). Cryopreservation-induced apoptotic gene activation. Cryobiology 41, 338 [abstract].
Baust, J.M., Van Buskirk, R. & Baust, J.G. (2000b). Cell viability improves following inhibition of cryopreservation-induced apoptosis. In Vitro Cell. Dev. Biol. Anim. 36, 262–70.
Baust, J.M., Van Buskirk, R. & Baust, J.G. (2002). Modulation of the cryopreservation cap: elevated survival with reduced dimethyl sulphoxide concentration. Cryobiology 45, 97108.
Baust, J.M. (2002). Molecular mechanisms of cellular demise associated with cryopreservation failure. Cell.Preserv. Technol. 1, 1731.
Bhojwani, S., Vajta, G., Callesen, H., Roschlau, K., Kuwer, A., Becker, F., Alm, H., Torner, H., Kanitz, W. & Poehland, R. (2005). Developmental competence of HMC™ derived bovine cloned embryos obtained from somatic cell nuclear transfer of adult fibroblasts and granulosa cells. J. Reprod. Dev. 51, 465–75.
Coundouris, J.A., Grant, M.H., Engeset, J., Petrie, J.C. & Hawksworth, G.M. (1993). Cryopreservation of human adult hepatocytes for use in drug metabolism and toxicity studies. Xenobiotica 23, 1399–409.
De Loecker, W., Koptelov, V.A., Grischenko, V.I. & De Loecker, P. (1998). Effects of cell concentration on viability and metabolic activity during cryopreservation. Cryobiology 37, 103–9.
Dong, Y.J., Bai, X.J., Barisanga, M.D., Matango, N.R. & Suzuki, T. (2003). Production of cloned calves following nuclear transfer with cultured frozen–thawed somatic cells using simple portable CO2 incubator. Theriogenology 59, 246 [abstract].
Du, F., Sung, L.Y., Tian, X.C. & Yang, X. (2002). Differential cytoplast requirement for embryonic and somatic cell nuclear transfer in cattle. Mol. Reprod. Dev. 63, 183–91.
Estrada, J., Lee, E. & Piedrahita, J.A. (2006). Cryopreservation of donor cells for nuclear transfer: effect of cell freezing method on the efficiency of somatic cell nuclear transfer. Reprod. Fertil. Dev. 18, 125 [abstract].
Freshney, R.I. & Freshney, M. (2002). Culture of Epithelial Cells. 2nd edn, p. 461New York: J. Wiley & Sons, Inc.
Frim, J., Snyder, R.A., McGann, L.E. & Kruuv, J. (1978). Growth kinetics of cells following freezing in liquid nitrogen. Cryobiology 15, 502–16.
Giraldo, A.M., Lynn, J.W., Godke, R.A. & Bondioli, K.R. (2006). Proliferative characteristics and chromosomal stability of bovine donor cells for nuclear transfer. Mol. Reprod. Dev. 73, 1230–8.
Gómez, M.C., Pope, C.E., Lopez, M., Dumas, C., Giraldo, A. & Dresser, B.L. (2006). Chromosomal aneuploidy in African Wildcat somatic cells and cloned embryos. Cloning Stem Cells 8, 6978.
Green, A.L., Wells, D.N. & Oback, B. (2007). Cattle cloned from increasingly differentiated muscle cells. Biol. Reprod. 77, 395406.
Hayes, O., Rodriguez, L. L., Gonzalez, A., Falcon, V., Aguilar, A. & Castro, F.O. (2005). Effect of cryopreservation on fusion efficiency and in vitro development into blastocysts of bovine cell lines used in somatic cell cloning. Zygote 13, 277–82.
Hill, J.R., Winger, Q.A., Long, C.R., Looney, C.R., Thompson, J.A. & Westhusin, M.E. (2000). Development rates of male bovine nuclear transfer embryos derived from adult and fetal cells. Biol. Reprod. 62, 1135–40.
Holm, P., Booth, P.J., Schmidt, M.H., Greve, T. & Callesen, H. (1999). High bovine blastocyst development in a static in vitro production system using SOFaa medium supplemented with sodium citrate and myo-inositol with or without serum-proteins. Theriogenology 52, 683700.
Jacobs, P.A., Court, B. & Doll, R. (1961). Distribution of human chromosome counts in relation to age. Nature 191, 1178–80.
Karlsson, J.O., Cravalho, E.G., Borel Rinkes, I.H., Tompkins, R.G., Yarmush, M.L. & Toner, M. (1993). Nucleation and growth of ice crystals inside cultured hepatocytes during freezing in the presence of dimethyl sulfoxide. Biophys. J. 65, 2524–36.
Kato, Y., Tani, T. & Tsunoda, Y. (2000). Cloning of calves from various somatic cell types of male and female adult, newborn and fetal cows. J. Reprod. Fertil. 120, 231–7.
Kerr, J.F., Wyllie, A.H. & Currie, A.R. (1972). Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239–57.
Kragh, P.M., Du, Y., Corydon, T.J., Purup, S., Bolund, L. & Vajta, G. (2005). Efficient in vitro production of porcine blastocysts by handmade cloning with a combined electrical and chemical activation. Theriogenology 64, 1536–45.
Kragh, P.M., Vajta, G., Corydon, T.J., Purup, S., Bolund, L. & Callesen, H. (2004). Production of transgenic porcine blastocysts by hand-made cloning. Reprod. Fertil. Dev. 16, 315–8.
Li, J. and Mombaerts, P. (2008). Nuclear transfer-mediated rescue of the nuclear genome of nonviable mouse cells frozen without cryoprotectant. Biol. Reprod. 79, 588–93.
Matsushita, T., Yagi, T., Hardin, J.A., Cragun, J.D., Crow, F.W., Bergen, H.R., Gores, G.J. & Nyberg, S.L. (2003). Apoptotic cell death and function of cryopreserved porcine hepatocytes in a bioartificial liver. Cell Transplant 12, 109–21.
Mazur, P. (1963). Kinetics of water loss from cells at subzero temperatures and the likelihood of intracellular freezing. J. Gen. Physiol. 47, 347–69.
Mazur, P. (1984). Freezing of living cells: mechanisms and implications. Am. J. Physiol. Cell. Physiol. 247, C125C142.
Mazur, P. (2004). Principles of cryobiology. In Life in the Frozen State (eds Fuller, B.J., Laneg, N. & Benson, E.E.) pp. 365. Boca Raton: CRS Press.
McGann, L.E. (1979). Optimal temperature ranges for control of cooling rate. Cryobiology 16, 211–6.
Muldrew, K., Acker, J.P., Elliot, J.A.W. & McGann, E. (2004). The water to ice transition: implications for living cells. In Life in the Frozen State (eds , B.J.Fuller, , Lane, N. & Benson, E.E.) pp. 67108. Boca Raton: CRS Press.
Pegg, D.E. and Diaper, M.P. (1990). Freezing versus vitrification: basic principles. In Cryopreservation and Low Temperature Biology in Blood Transfusion (eds Smit-Sibinga, C.T., Das, P.C. & Meryman, H.T.) pp. 5569. Dordrecht: Kluwer Academic Publishers.
Polge, C., Smith, A.U. & Parkes, A.S. (1949). Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 164, 666.
Rapatz, G. & Luyet, B. (1968). Combined effects of freezing rates and of various protective agents on the preservation of human erythrocytes. Cryobiology 4, 215–22.
Saksela, E. and Moorhead, P.S. (1963). Aneuploidy in the degenerative phase of serial cultivation of human cell strains. Proc. Natl. Acad. Sci. USA 50, 390–5.
Stroh, C., Cassens, U., Samraj, A.K., Sibrowski, W., Schulze-Osthoff, K. & Los, M. (2002). The role of caspases in cryoinjury: caspase inhibition strongly improves the recovery of cryopreserved hematopoietic and other cells. FASEB J. 16, 1651–3.
Stylianou, J., Vowels, M. & Hadfield, K. (2006). Novel cryoprotectant significantly improves the post-thaw recovery and quality of HSC from CB. Cytotherapy 8, 5761.
Tani, T., Kato, Y. & Tsunoda, Y. (2000). Developmental potential of cumulus cell-derived culture frozen in a quiescent state after nucleus transfer. Theriogenology 53, 1623–9.
Thornberry, N.A. and Lazebnik, Y. (1998). Caspases: enemies within. Science 281, 1312–6.
Vajta, G., Peura, T.T., Holm, P., Paldi, A., Greve, T., Trounson, A.O. & Callesen, H. (2000). New method for culture of zona-included or zona-free embryos: the well of the well (WOW) system. Mol. Reprod. Dev. 55, 256–64.
Vajta, G., Lewis, I.M., Hyttel, P., Thouas, G.A. & Trounson, A.O. (2001). Somatic cell cloning without micromanipulators. Cloning 3, 8995.
Vajta, G., Maddox-Hyttel, P., Skou, C.T., Tecirlioglu, R.T., Peura, T.T., Lai, L., Murphy, C.N., Prather, R.S., Kragh, P.M. & Callesen, H. (2005). Highly efficient and reliable chemically assisted enucleation method for handmade cloning in cattle. Reprod. Fertil. Develop. 17, 791–7.
Wakayama, S., Ohta, H., Hikichi, T., Mizutani, E., Iwaki, T., Kanagawa, O. & Wakayama, T. (2008). Production of healthy cloned mice from bodies frozen at –20 °C for 16 years. Proc. Natl. Acad. Sci. USA. 105, 17318–22.
Wells, D.N., Misica, P.M. & Tervit, H.R. (1999). Production of cloned calves following nuclear transfer with cultured adult mural granulosa cells. Biol. Reprod. 60, 9961005.
Wilmut, I., Schnieke, A.E., McWhir, J., Kind, A.J. & Campbell, K.H. (1997). Viable offspring derived from fetal and adult mammalian cells. Nature 385, 810–3.
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Zygote
  • ISSN: 0967-1994
  • EISSN: 1469-8730
  • URL: /core/journals/zygote
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