The effect of cooling rate, warming rate, cryoprotective agent and stage of development of survival of mouse embryos during freezing and thawing

Life Sciences ◽  
1972 ◽  
Vol 11 (22) ◽  
pp. 1071-1079 ◽  
Author(s):  
I. Wilmut
Keyword(s):  
Cooling Rate ◽  
Mouse Embryos ◽  
Freezing And Thawing ◽  
Cryoprotective Agent ◽  
Stage Of Development ◽  
Warming Rate

Studies on the cryopreservation of eggs of Angiostrongylus cantonensis

1983 ◽  
Vol 57 (4) ◽  
pp. 297-303 ◽  
Author(s):  
Shoji Uga ◽  
Kazuyoshi Araki ◽  
Takeo Matsumura ◽  
Noboru Iwamura
Keyword(s):  
Cooling Rate ◽  
Final Concentration ◽  
Angiostrongylus Cantonensis ◽  
Growth Stages ◽  
Optimum Conditions ◽  
Rat Serum ◽  
Cryoprotective Agent ◽  
Warming Rate ◽  
The Relationship

AbstractThe possibility of cryopreserving the eggs of Angiostrongylus cantonensis collected from the uterus of female worms was investigated. Eggs were cultured in NCTC 109 medium containing 50% rat serum, and various growth stages, from one-cell eggs to embryonated eggs, were used in this study.As a cryoprotective agent, dimethylsulphoxide (Me2SO) was added to the medium at a final concentration of 1 M. Eggs suspended in 0·2 ml of the medium at 37°C were cooled to 0°C at a rate of l°C min−1, then an equal volume of 2M-Me2SO solution was added. After equilibration for 15min, the freezing procedures were started. In the freezing procedures, the effectiveness of (i) a seeding process, (ii) different cooling and warming rates and (iii) the relationship between the growth stages of the eggs and their tolerance to freezing at −20°C were investigated.It was found the highest level of survival could be obtained with 32-cell eggs cooled at a rate of 0·3° C min−1 or more slowly with seeding at −4°C and warming at a rate of 5°C min−1. Survival was influenced more by cooling rate than by warming rate.Using these optimum conditions, the survival of eggs was then investigated following cooling to various temperatures. While more than 50% of eggs were found to survive cooling to −30–C, extremely low survival was noted from lower temperatures.

Small-volume vitrification and rapid warming yield high survivals of one-cell rat embryos in cryotubes

2021 ◽  
Author(s):  
Yasuyoshi Fukuda ◽  
Misako Higashiya ◽  
Takahiro Obata ◽  
Keita Basaki ◽  
Megumi Yano ◽  
...  
Keyword(s):  
Cooling Rate ◽  
Small Volume ◽  
Sucrose Solution ◽  
High Concentration ◽  
Rat Embryos ◽  
Low Concentrations ◽  
Intracellular Ice Formation ◽  
Intracellular Ice ◽  
Warming Rate ◽  
Very High

Abstract To cryopreserve cells, it is essential to avoid intracellular ice formation during cooling and warming. One way to achieve this is to convert the water inside the cells into a non-crystalline glass. It is currently believed that to accomplish this vitrification, the cells must be suspended in a very high concentration (20–40%) of a glass-inducing solute, and subsequently cooled very rapidly. Herein, we report that this belief is erroneous with respect to the vitrification of one-cell rat embryos. In the present study, one-cell rat embryos were vitrified with 5 μL of EFS10 (a mixture of 10% ethylene glycol, 27% Ficoll, and 0.45 M sucrose) in cryotubes at a moderate cooling rate, and warmed at various rates. Survival was assessed according to the ability of the cells to develop into blastocysts and to develop to term. When embryos were vitrified at a 2,613 °C/min cooling rate and thawed by adding 1 mL of sucrose solution (0.3 M, 50 °C) at a warming rate of 18,467 °C/min, 58.1 ± 3.5% of the EFS10-vitrified embryos developed into blastocysts, and 50.0 ± 4.7% developed to term. These rates were similar to those of non-treated intact embryos. Using a conventional cryotube, we achieved developmental capabilities in one-cell rat embryos by rapid warming that were comparable to those of intact embryos, even using low concentrations (10%) of cell-permeating cryoprotectant and at low cooling rates.

Modification of cell division by phorbol ester in preimplantation mouse embryos

Development ◽  
1987 ◽  
Vol 101 (2) ◽  
pp. 403-408
Author(s):  
E.T. Mystkowska ◽  
W. Sawicki
Keyword(s):  
Cell Division ◽  
Video Recording ◽  
Mouse Embryos ◽  
Time Intervals ◽  
Cell Divisions ◽  
Stage Of Development ◽  
Preimplantation Mouse Embryos ◽  
Preimplantation Mouse ◽  
Single Blastomeres

2-cell mouse embryos were treated in vitro with a 2 h pulse of phorbol myristate acetate (PMA) at 32nd, 38th and 50th h after hCG, then chased in culture for up to 46 h. Embryos were fixed at various time intervals of chasing, then stained and inspected. Some embryos were carefully inspected with a video recording system, every 1.44s and the cell divisions (cytokinesis) as well as formation of large, single blastomeres, each from two smaller ones, were recorded. PMA pulse let to the suppression of cell divisions. The rate of the suppression was time dependent: with a delay of 0–1, 12 and 18 h between the PMA pulse and time of scheduled cell division about 99, 87 and 44% of 2-cell embryos remained at this stage of development, for at least 10 h, respectively, and 90, 58 and 12% of their blastomeres revealed binuclearity. Since we found that PMA-mediated formation of binuclearity was not the effect of cell fusions, it was assumed that the inhibition of cytokinesis preceded by karyokinesis was responsible for binuclearity. PMA effect on cell divisions was reversible. PMA-treated embryos revealed formation of large, single blastomeres, each from two smaller ones. If cell division appeared after PMA pulse, in about 52% of 3- to 6-cell embryos, the large blastomere formation was recorded in the course of the subsequent 38 h. Large blastomere formation was concluded to be the result of either cell fusion or reversion of incompleted cytokinesis brought about by PMA.

Effect of stage of development on survival of mouse embryos frozen-thawed rapidly

Cryobiology ◽  
1984 ◽  
Vol 21 (5) ◽  
pp. 574-577 ◽  
Author(s):  
A. Massip ◽  
P. Van Der Zwalmen ◽  
F. Leroy
Keyword(s):  
Mouse Embryos ◽  
Stage Of Development

Hardening of the Porcine Thoracic Aorta Subjected to Freezing and Thawing: Experimental Evaluation and Mathematical Modeling

2008 ◽  
Author(s):  
Hiroshi Yamada ◽  
Kousuke Yasuno ◽  
Kensuke Fujisaki ◽  
Hiroshi Ishiguro
Keyword(s):  
Cooling Rate ◽  
Physiological Stress ◽  
Strain Curve ◽  
Fluid Phase ◽  
Collagen Fibers ◽  
Uniaxial Tensile ◽  
Freezing And Thawing ◽  
Loading Test ◽  
Stress Strain ◽  
Collagen Molecules

Identifying changes in the mechanical behavior of blood vessels subjected to freezing and thawing, such as occur with cryopreservation, are of key importance. Excising pairs of fresh ring specimens from identical porcine thoracic aortas (n = 8 for each cooling rate), we carried out uniaxial tensile loading and unloading tests over the physiological stress range (first and second tests) and performed a loading test until the breaking point within the range of a load cell (third test). After the first test, one specimen of the pair was frozen at −80°C at a cooling rate of −1°C or −50°C/min and thawed, while the other was held at 5°C as a control. At both cooling rates, for the specimens subjected to freezing, the ratios of the tangential modulus in the stress-strain curve (between 130 and 150 kPa) in the second test to that in the first test differed significantly (p < 0.01) from the respective ratios of the control specimens. We formulated a mathematical model of the stress–strain relationship considering elastic and collagen fibers and an incompressible fluid phase. We evaluated the working hypothesis that collagen fibers reduce their extensibility either by hardening as a mechanical change or by shortening as a geometric change. We attributed this response to the formation of dehydration-induced cross-linking in collagen molecules at the microscopic level.

Factors affecting survival of mouse embryos during freezing and thawing

1974 ◽  
Vol 89 (1) ◽  
pp. 79-88 ◽  
Author(s):  
S.P. Leibo ◽  
P. Mazur ◽  
S.C. Jackowski
Keyword(s):  
Mouse Embryos ◽  
Freezing And Thawing ◽  
Factors Affecting

57 GENOME ACTIVATION IN MOUSE EMBRYOS OF DIFFERENT ORIGIN

2008 ◽  
Vol 20 (1) ◽  
pp. 109
Author(s):  
O. Svarcova ◽  
A. Dinnyes ◽  
Z. Polgar ◽  
S. Bodo ◽  
M. Adorjan ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Embryonic Stem ◽  
Mouse Embryos ◽  
Ultrastructural Changes ◽  
Rrna Synthesis ◽  
Cell Stage ◽  
Stage Of Development ◽  
Nucleolar Proteins ◽  
Genome Activation ◽  
Different Origin

Major genome activation is a key event in early embryonic development occurring at the late 2-cell stage in the mouse. Concomitantly occurring molecular and ultrastructural changes in the nucleolus, where the ribosomal RNA genes are transcribed and their transcripts processed, enable the use of this organelle as a sensitive marker of genome activation in embryos produced by different techniques. The aim of this study was to evaluate and compare the genome activation in mouse embryos of different origin using the nucleolus as a marker. Early and late 2-cell- and late 4-cell-stage embryos, prepared by in vitro fertilization (IVF), parthenogenetic activation (PG), and somatic cell nuclear transfer of mouse embryonic fibroblast (MEF), and mouse HM1 embryonic stem cells (HM1) were processed for autoradiography following 3H-uridine incubation and transmission electron microscopy (5 embryos per group) and for immunofluorescence for detection of nucleolar proteins involved in rRNA synthesis (upstream binding factor; UBF) and processing (nucleophosmin; B23) (10–21 embryos per group). Early 2-cell embryos in all groups showed transcriptional activity in the nucleoplasm, but not over nucleolar precursor bodies (NPBs). UBF was localized diffusely in the cytoplasm. B23 was, likewise, localized in the cytoplasm and, in 30% of embryos, in the nucleoplasm. Late 2-cell IVF and PG embryos displayed transcriptional labelling over nucleoplasm and NPBs, which, ultrastructurally, were in the process of transformation into fibrillo-granular nucleoli presenting fibrillar centers, a dense fibrillar component, and a granular component. MEF and HM1 embryos displayed transcriptional labelling over nucleoplasm, but not over NPBs, and the transformation into functional nucleoli was never observed at this stage of development. UBF and B23 were in all groups localized in the nucleoplasm and, in 40–50% of cases, distinctly in the developing nucleoli. At the late 4-cell stage, all embryos presented transcriptional labelling over nucleoplasm and NPBs, which were at different levels of transformation into fibrillo-granular nucleoli. UBF and B23 were distinctly localized in these developing nucleoli. However, whereas fully transformed reticulated fibrillo-granular nucleoli without remnants of NPBs were found in IVF and PG embryos, despite the distinct localization of nucleolar proteins, the nucleoli in MEF and HM1 embryos were not reticulated and still displayed remnants of NPBs. Conclusively, embryos reconstructed by nuclear transfer, independent of cell origin, displayed well-timed extranucleolar genomic activation, but delayed transformation of NPBs into reticulated fibrillo-granular nucleoli. Moreover, the proper nucleolar activation noted in PG embryos activated in the same manner as MEF and HM1 embryos demonstrate that somatic and embryonic stem cell factors exert an influence on nucleolar activation and may cause reduced embryo viability. This work was supported by the Specific Targeted Project (MED-RAT; contract LSHG-CT-2006-518240) and Marie Curie ResearchTraining Networks (CLONET; contract 035468-2).

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