1 Interuniversitäres Forschungsinstitut für Agrarbiotechnolgie, Tulln and 2 Ludwig Boltzmann Institute für immuno-, zyto- und molekulargenetische Forschung, Vienna, Austria
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: cryopreservation/embryo/mice/rapid freezing/vitrification
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Exposure time to the high concentration of cryoprotectant(s) during the rapid freezing procedure and vitrification has been proved to be one of the main factors influencing the survival of embryos and oocytes (Scheffen et al., 1986; Vanderzwalmen et al., 1988
; Ishimori et al., 1992
; Shaw et al., 1992
; Nowshari et al., 1994
). However, there are practical limitations in reducing the exposure time beyond a certain minimum, because once embryos/oocytes are exposed to high concentrations of cryoprotectant, time is required to load them in to straws and to seal the straws before plunging them in liquid nitrogen. Another factor which has been less studied and which may influence the survival of embryos/oocytes during rapid freezing or vitrification is the rate of freezing. A freezing rate of about 1200°C/min can be achieved by directly plunging straws into liquid nitrogen. Recently, different approaches have been suggested and applied to increase the freezing rate of embryos/oocytes, however, nearly all the cooling techniques are based on direct contact between the cryoprotectant solution and the liquid nitrogen. The simplest way to establish this contact is to immerse embryos directly into liquid nitrogen (Landa and Tepla, 1990
). This procedure developed for mouse embryos was then successfully used for bovine embryos, zygotes and oocytes (Riha et al., 1991
; Yang and Leibo, 1999
; Papis et al., 1999
). A similar effect was achieved by using a cryoloop for freezing of mouse and human blastocysts (Lane et al., 1999
). The surface of an electron microscope grid (Martino et al., 1996
) or a metal slab (Dinnyés et al., 2000
) have both been used for placing the bovine oocytes/embryos before submerging them into liquid nitrogen. Using an electron microscope grid, the estimated cooling rate was 11 00014 000 and 24 000°C/min in liquid nitrogen or in the slush of liquid nitrogen respectively (Arav et al., 2000
). Open pulled straw (OPS) technology (Vajta et al., 1998a
) involves the use of a narrow plastic tube and an approximate volume of 12 µl cryoprotectant solution, loaded into the open end of the straw and directly plunged into liquid nitrogen. A device (Vit-Master®; Mini Tübe, Landeshut, Germany) with which the temperature of liquid nitrogen can be decreased down to 212°C by creating a low atmospheric pressure (Arav et al., 2000
) has been introduced. Using this device the freezing rate of straws from + 20°C to 10°C was increased from 12004000°C and, when using 2 µl of cryoprotectant and an OPS, the freezing rate increased from 5300 to 10 300°C/min.
In this study we examined the effect of decreasing the exposure time (from 4520 s) of pronucleate embryos to a high concentration of cryoprotectant and increasing the freezing rate from 1200°C to about 10 300°C by using two different freezing vessels, i.e. straws and OPS, and plunging them either directly in to liquid nitrogen or after immersion in Vit-Master® (212°C) before final plunging in liquid nitrogen. The mouse was used as a model for development and testing of freezing methods to assess the effect of factors which may influence the viability of human embryos in a cryopreservation procedure, though the freezing sensitivities of different species cannot be expected to be identical. The rapid procedure used here has previously been shown by us to be suitable for cryopreservation of pronuclear stage mouse embryos (Nowshari et al., 1995). Intact and biopsied mouse embryos at different stages frozen with this freezing procedure have resulted in the birth of live animals (Nowshari et al., 1995
; Nowsahri and Brem, 1998 Nowsahri and Brem, 2000a,b).
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Freezing
Freezing solutions of 1.5 mol/l ethylene glycol (Sigma) with 0.25 mol/l sucrose (Sigma) and 7.0 mol/l ethylene glycol with 0.5 mol/l sucrose and thawing solution of 0.5 mol/l sucrose were made with PBS supplemented with 10% heat-inactivated FCS. All manipulations were performed in 35 mm plastic Petri dishes (Nunc, Denmark).
Embryos were frozen using a rapid freezing procedure described earlier (Nowshari and Brem, 1998). However, in this experiment either normal 0.25 ml straws (IMV, L'Aigle, France) or OPS (Vajta et al., 1998a
), a narrow plastic tube (i.d. 0.8 mm) manually pulled out from a heat softened straw, were used.
Freezing and thawing protocol
For freezing embryos, using a 0.25ml straw or OPS, embryos were pre-equilibrated at room temperature (2223°C) in 1.5 mol/l ethylene glycol with 0.25 mol/l sucrose in medium PBS for 5 min.
Freezing and thawing of straws
Pre-equilibrated embryos were directly loaded with a mouth controlled glass pipette into the middle of a 0.25 ml straw containing 40 µl of 7.0 mol/l ethylene glycol with 0.5 mol/l sucrose (Figure 1). The straws were capped (no heat sealing) and after 20 or 45 s either dipped slowly and vertically into liquid nitrogen (rapid freezing) or immersed first in a super-cooled (212°C) liquid nitrogen chamber (Vit-Master®) and then transferred to liquid nitrogen for further storage (super rapid freezing). Embryos were stored in liquid nitrogen for periods not less than 24 h.
|
Freezing and thawing of OPS
After pre-equilibration in 1.5 mol/l ethylene glycol with 0.25 mol/l sucrose for 5 min, 510 embryos in 2 µl of medium were transferred to a drop of 40 µl of 7.0 mol/l ethylene glycol with 0.5 mol/l sucrose and mixed together. Embryos were immediately taken out with a mouth controlled pipette with 2 µl of medium and pushed out to make a droplet on the surface of a Petri dish. Embryos were then loaded into an OPS by simply touching the droplet thus using the capillary effect of the straw. The straws were then either plunged directly into liquid nitrogen (OPS freezing) or were first transferred to the super cooled liquid nitrogen chamber (Vit-Master®) maintained at 212°C (super OPS) and then plunged into liquid nitrogen. The straws were later transferred to a liquid nitrogen container for further storage.
The OPS were thawed by leaving the straws in air for 5 s and then releasing the embryos into a drop of 0.5 mol/ l sucrose as described by Vajta et al. (1998a). Embryos were left in this medium for 5 min and then washed and cultured as described for those frozen and thawed in straws.
Statistical analysis
Differences in rates of survival and development between treatment groups were tested for significance with 2 test. The level of significance was set at 5%.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this experiment we have also shown that increasing the freezing rate of mouse embryos from 1200°C/min, achieved by direct plunging of straws in to liquid nitrogen, to higher freezing rates may not be of any advantage, at least while using the cryoprotectant and freezing protocol used in this study. Freezing by loading the embryos in a minimal amount of freezing medium in an OPS, thus increasing the freezing rate from about 12005300°C/min, or by plunging the straws or OPS first in super cooled liquid nitrogen (212°C), thus increasing the freezing rate to about 4000 and 10 300°C/min, may also not have a positive influence on the survival and development of embryos. A higher survival of embryos, exposed for 20 s and frozen at 1200°C/min compared with those frozen at higher rates (400010 500°C/min), indicates that just increasing the freezing rate may be at times rather disadvantageous. To obtain higher survival and development of embryos and oocytes after freezing and thawing, in addition to the rate of freezing, other factors like exposure time, type and concentration of cryoprotectant and freezing sensitivity of oocytes/embryos ought to be taken into consideration. The use of OPS for vitrification of embryos and oocytes in different species has been reported to improve their survival and development (Vajta, 2000, a review). However, there are a few drawbacks in using this freezing procedure. One of the main disadvantages is the possibility of contamination of the biological material from contaminated liquid nitrogen and vice versa, thus compromising the sanitary status of the embryos. Recently, embryos loaded in OPS exposed to contaminated liquid nitrogen with bovine viral diarrhoea virus (BVDV) and bovine herpesvirus-1 (BHV) were tested positive for viral association (Bielanski et al., 2000
). It has also been acknowledged that many viral and bacterial pathogenic agents may adhere to the intact zona pellucida and that most infectious viral and bacterial agents easily survive in cryoprotectants and liquid nitrogen (Melnick, 1965
; Wallis and Melnick, 1968
; Fountain et al., 1997
). As alternative preventive steps against contamination, measures such as liquid nitrogen filtration and the application of accessory protective storage containers have been proposed (Vajta et al., 1998b
). However, the practicability of such measures are questionable and the method can then no longer be considered a simple method.
The rapid freezing procedure used in the present experiment has been shown by us previously to be very efficient for freezing different stages of mouse embryos and oocytes (Nowshari et al., 1994, 1995
; Nowshari and Brem, 1998
, 2000a
). In the present experiment only in-vitro development of embryos was investigated; however, we have previously reported the in-vivo development of pronuclear stage embryos collected at a similar stage (Nowshari et al., 1995
), embryos frozen once or twice (Nowshari and Brem, 1998
) and those frozen in solutions supplemented with chemically defined macromolecules (e.g. PVA) using this freezing procedure (Nowshari and Brem, 2000c
).
In summary, a high proportion of pronuclear stage embryos survive freezing and retain their development capacity after using the simple rapid freezing protocols with a high concentration of ethylene glycol with sucrose. We have shown that reducing the time of exposure from 45 to 20 s does not significantly affect the survival and development of embryos. Further, we have shown that increasing the freezing rate either by direct exposure of embryos to liquid nitrogen in an OPS or by immersion in super-cooled liquid nitrogen may not be of any advantage, when using this freezing protocol for cryopreservation of mouse pronuclear stage embryos. These findings need to be given consideration while developing faster freezing procedures for embryos and oocytes of human as well as animal origin.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arav, A., Zeron, Y. and Ocheretny, A. (2000) A new device and method for vitrification increases the cooling rate and allows successful cryopreservation of bovine oocytes. Theriogenology, 53, 248 (abstract).
Ashwood-Smith, M.J. and Simons, R. (1986) The freezing of early human embryos and blastocysts. J. In vitro Fertil. Embryo Transfer, 3, 67 (abstract).
Bernart, W., Kamel, M., Neulen, J. and Breckwoldt, M. (1994) Influence of the developmental stage and the equilibration time on the outcome of ultrarapid cryopreservation of mouse embryos. Hum. Reprod., 9, 100102.[Abstract]
Bielanski, A., Nadin-Davis, S., Sapp, T. and Lutze-Wallace, C. (2000) Viral contamination of embryos cryopreserved in liquid nitrogen. Cryobiology, 40, 110116.[ISI][Medline]
Damario, M.A.., Hammit, D.G., Galantis, B.A. et al. (1999) Pronuclear stage cryopreservation after intracytoplasmic sperm injection and conventional IVF: implications for timing of the freeze. Fertil. Steril., 72, 10491054.[ISI][Medline]
Dinnyés, A., Dai, Y., Jiang, S. and Yang, X. (2000) High development rates of vitrified bovine oocytes following parthenogenetic activation, in vitro fertilisation, and somatic cell nuclear transfer. Bio. Reprod., 63, 513518.
Feichtinger, W., Hochfellner, C. and Ferstl, U. (1991) Clinical experience with ultra-rapid freezing of embryos. Hum. Reprod., 6, 735736.[Abstract]
Fountain, D., Ralston, M., Higgins, N. et al. (1997) Liquid nitrogen freezers: a potential source of microbial contamination of hematopoietic stem cell components. Transfusion, 37, 585591.[ISI][Medline]
Gordts, S., Roziers, P., Campo, R. and Noto, V. (1990) Survival and pregnancy outcome after ultrarapid freezing of human embryos. Fertil. Steril., 53, 469472.[ISI][Medline]
Hogan, B., Costantini, F. and Lucy, E. (1986). Manipulating the mouse embryo. Cold Spring Harbor Laboratory, Plainview, NY.
Ishimori, H., Takahashi, Y. and Kanagawa, H. (1992) Factors affecting survival of mouse blastocysts vitrified by a mixture of ethylene glycol and dimethylsulfoxide. Theriogenology, 38, 11751185.[ISI]
Landa, V. and Tepla, O. (1990) Cryopreservation of mouse 8-cell embryos in microdrops. Folia. Biol. (Praha.), 36, 153158.[ISI][Medline]
Lane, M., Schoolcraft, W.B. and Gardner, D.K. (1999) Vitrification of mouse and human blastocysts using a novel cryoloop container-less technique. Fertil. Steril., 72, 10731078.[ISI][Medline]
Mandelbaum, J., Belaisch-Allart, J., Junka, A-M. et al. (1998) Cryopreservation in human assisted reproduction is now routine for embryos but remains a research procedure for oocytes. Hum. Reprod., 13 (Suppl. 3), 161174.[Abstract]
Martino, A., Songsasan, N. and Leibo, S.P. (1996) Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Bio. Reprod., 54, 10591069.[Abstract]
Melnick, J.L. (1965) Preservation of viruses by freezing. Fed. Proc., 24 (Suppl. 15), 280283.
Mohr, L.R., Trounson, A.O. and Freeman, L. (1985) Deep freezing and transfer of human embryos. J. In vitro Fertil. Embryo Transfer, 2, 110.[Medline]
Nowshari, M.A. and Brem, G. (1998) Effect of cryoprotectants and their concentration on post-thaw survival and development of expanded mouse blastocysts. Theriogenology, 50, 10011013.[ISI][Medline]
Nowshari, M.A. and Brem, G. (2000a) Effect of thawing temperature and in-straw dilution of cryoprotectant on survival and development of murine embryos frozen by rapid freezing procedure. Theriogenology, 53, 162 (abstract).
Nowshari, M.A. and Brem, G. (2000b) Refreezing of murine intact and biopsied embryos by rapid freezing procedure. Hum. Reprod., 15, 25772581.
Nowshari, M.A. and Brem, G. (2000c) The protective action of polyvinyl alcohol during rapid freezing of mouse embryos. Theriogenology, 53, 11571166.[ISI][Medline]
Nowshari, M.A., Nayudu, P.L. and Hodges, J.K. (1994) Effect of cryoprotectant concentration, equilibration time and thawing procedure on survival and development of rapid-frozenthawed mature mouse oocytes. Theriogenology, 42, 11931204.[ISI]
Nowshari, M.A., Nayudu, P.L. and Hodges, J.K. (1995) Effect of cryoprotectants and their concentration on post-thaw survival and development of rapid frozenthawed pronuclear stage mouse embryos. Hum. Reprod., 10, 32373242.[Abstract]
Papis, K., Schmizu, M. and Izaike, Y. (1999) The effect of gentle pre-equilibration on survival and development rates of bovine in vitro matured oocytes vitrified in droplets. Theriogenology, 51, 173 (abstract).[ISI]
Riha, J., Landa, V., Kneissl, J. et al. (1991) Vitrification of cattle embryos by direct dropping into liquid nitrogen and embryo survival after non-surgical transfer. Zivoc Vir., 36, 113120.
Scheffen, B., Van der Zwalmen, P. and Massip, A. (1986) A simple and efficient procedure for preservation of mouse embryos by vitrification. Cryo-Letters, 7, 260269.[ISI]
Shaw, J.M., Diotallevi, L. and Trounson, A.O. (1991) A simple rapid 4.5 M dimethylsulfoxide freezing technique for the cryopreservation of one-cell to blastocyst stage preimplantation mouse embryos. Reprod. Fertil. Dev., 3, 621626.[ISI][Medline]
Shaw, P.W., Bernard, A.G., Fuller, B.J. et al. (1992) Vitrification of mouse oocytes using short cryoprotectant exposure times on survival. Mol. Reprod. Dev., 33, 210214.[ISI][Medline]
Siebzehnrübl, E., Trotnow, S., Weigel, M. (1986) Pregnancy after in vitro fertilisation, cryopreservation and embryo transfer. J. In vitro Fertil. Embryo Transfer, 3, 261.[Medline]
Testart, J., Lassalle, B., Forman, R. et al. (1987) Factors influencing the success of human embryo freezing in an in vitro fertilisation program. Fertil. Steril., 48, 107.[ISI][Medline]
Trounson, A.O., Peura, A. and Kirby, C. (1987) Ultrarapid freezing: a new low cost and effective method of embryo cryopreservation. Fertil. Steril., 48, 843850.[ISI][Medline]
Vajta, G. (2000) Vitrification of the oocytes and embryos of domestic animals. Anim. Reprod. Sci., 6061, 357364.[ISI]
Vajta, G., Holm, P., Kuwayama, M. et al. (1998a) Open pulled straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryo. Mol. Reprod. Dev., 51, 5358.[ISI][Medline]
Vajta, G., Lewis, I.M., Kuwayama, M. et al. (1998b) Sterile application of the open pulled straw (OPS) vitrification method. Cryo-Letters, 19, 389392.[ISI]
Van den Abbeel, E., Camus, M., Van Waesberghe, L. et al. (1997) A randomized comparison of the cryopreservation of one cell human embryos with a slow controlled-rate cooling procedure or a rapid cooling procedure by direct plunging into liquid nitrogen. Hum. Reprod., 12, 15541560.[Abstract]
Van der Auwera, I., Cornillie, F., Ongkowidjojo, R. et al. (1990) Cryopreservation of pronucleate mouse ova: slow versus ultrarapid freezing. Hum. Reprod., 4, 619621.
Van der Elst, J., Van den Abbeel, E. and Van Steirteghem, A.C. (1995) The effect of equilibration temperature and time on the outcome of ultrarapid freezing of 1-cell mouse embryos. Hum. Reprod., 10, 379383.[Abstract]
Vanderzwalmen, P., Gaurois, B., Ectors, F.J. et al. (1988) Some factors affecting successful vitrification of mouse blastocysts. Theriogenology, 30, 11771183.[ISI]
Veeck, L.L., Amundson, C.H., Brothman, L.J. et al. (1993) Significantly enhanced pregnancy rates per cycle through cryopreservation and thaw of pronuclear stage oocytes. Fertil. Steril., 59, 12021207.[ISI][Medline]
Yang, B.S. and Leibo, S.P. (1999) Viability of in vitro-derived bovine zygotes cryopreserved in microdrops. Theriogenology, 51, 178.[ISI]
Wallis, C. and Melnick, J.L. (1968) Stabilisation of enveloped viruses by dimethylsulfoxide. J. Virol., 2, 953954.[ISI][Medline]
Whittingham, D.G. (1971) Culture of mouse ova. J. Reprod. Fertil., 14 (Suppl.), 721.
Submitted on March 16, 2001; accepted on August 2, 2001.