Effect of cryopreservation on fusion efficiency and in vitro development into blastocysts of bovine cell lines used in somatic cell cloning

The outcome of the process of cloning by nuclear transfer depends on multiple factors that affect its efficiency. Donor cells should be carefully selected for their use in somatic nuclear transfer, and the protocols used for keeping frozen cell banks are of cardinal importance. Here we studied the e...

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Bibliographic Details
Published in:Zygote (Cambridge) 2005-11, Vol.13 (4), p.277-282
Main Authors: Hayes, O., Rodríguez, LL, González, A., Falcón, V., Aguilar, A., Castro, F.O.
Format: Article
Language:English
Subjects:
Animals
Blastocyst - physiology
Blastocyst - ultrastructure
Bovine
Cattle
Cell Fusion
Cell Line
Cloning, Organism
Cryopreservation
Female
Fibroblasts
Freezingy
Hybrid Cells - physiology
In Vitro Techniques
Micromanipulation
Nuclear transfer
Nuclear Transfer Techniques
Oocytes
Pregnancy
Skin - cytology
Tissue Preservation - methods
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Summary:The outcome of the process of cloning by nuclear transfer depends on multiple factors that affect its efficiency. Donor cells should be carefully selected for their use in somatic nuclear transfer, and the protocols used for keeping frozen cell banks are of cardinal importance. Here we studied the effect of two protocols for freezing donor cells on fusion rate and development into blastocysts. Our hypothesis is that freezing affects cell membranes in a way that interferes with the fusion process upon cloning but without hampering normal cell development in vitro. We found that freezing cell lines without controlling the cooling rate gives lower yields in the fusion step and in the final development into blastocysts, compared with cells frozen with a controlled cooling rate of approximately 1°C/min. Transmission electron microscopy of the cells subjected to different freezing procedures showed major damage to the cells frozen with a non-controlled protocol. We conclude that freezing of donor cells for cloning is a critical step in the procedure and should be monitored carefully using a method that allows for a step-wise, controlled cooling rate.
ISSN:0967-1994
1469-8730
DOI:10.1017/S0967199405003278