Method of preserving early embryos of experimental animals by vitrification

a technology of experimental animals and early embryos, which is applied in the field of vitrification of mammalian early embryos or es cells, can solve the problems of poor retrieval rate, difficult to solve problems, and cell culture usually involves the risk of a mutation of traits, and achieves high cell survival rates

Inactive Publication Date: 2007-04-19
CENT INST FOR EXPERIMENTAL ANIMALS
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  • Abstract
  • Description
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AI Technical Summary

Benefits of technology

[0025] The solution for preservation by vitrification according to the present invention is suitably used for preserving animal early embryos or ES cells, such as rat 2cell-stage embryos or primate ES cells. When animal early embryos or ES cells are cryopreserved via the method of preservation by vitrification according to the present invention that employs the solution for preservation by vitrification according to the present invention, high rates of cell survival and of cell retrieval can be achieved after thaw.

Problems solved by technology

When early embryos, such as rat 2cell-stage embryos, are preserved, however, the rate of retrieval is poor.
Thus, there are many issues to be resolved regarding techniques for cryopreserving experimental animal embryos.
Further, cell culture usually involves the risk of a mutation of traits, which necessitates preservation of some established cells.
However, there has been no preservation technique for rat early embryos or mammalian ES cells that can yield a high rate of cell retrieval.

Method used

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  • Method of preserving early embryos of experimental animals by vitrification
  • Method of preserving early embryos of experimental animals by vitrification
  • Method of preserving early embryos of experimental animals by vitrification

Examples

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example 1

Preservation of Rat 2Cell-Stage Embryos by Vitrification

[0045] Rat 2cell-stage embryos were used. Brl Han:Wist@Jcl (GALAS) rats were employed. 8-12-week-old female rats and 12-24-week-old male rats were employed. As test embryos, 2cell-stage embryos obtained from female rats that had been subjected to superovulation followed by natural mating were employed. Specifically, 150 IU / kg of PMGS was injected intraperitoneally into female rats and 75 IU / kg of hCG was injected intraperitoneally 48 hours thereafter. Rats were subjected to mating immediately after the injection of hCG, smears were sampled the following morning, and the estrous cycle and sperm impregnation were confirmed. The 2cell-stage embryos were sampled from female rats having positive smears via perfusion of the oviducts 45 hours after the injection of hCG. The sampled embryos were cultured at 37° C. in 5% CO2 and 95% air for approximately 1 hour and then subjected to the test.

[0046] The P10 solution, which comprises th...

example 2

Preservation of Marmoset ES Cells by Vitrification

[0056] As marmoset ES cells, two cell lines, i.e., No. 40 and No. 20, which were established by the Central Institute for Experimental Animals (Japan), were employed (Sasaki E. et al., 2005, Establishment of Novel Embryonic Stem Cell Lines Derived from the Common Marmoset (Callithrix jacchus), Stem Cells. (in press)). The cell clumps seperated via trypsin treatment were recovered with an egg-sampling pipette and used in the preservation experiment.

[0057] In the method of preservation by vitrification according to the present invention, the P10 pretreatment solution comprising PB13 and 10% propylene glycol and the PEPeS solution comprising PB1, 10% propylene glycol, 30% ethylene glycol, 20% Percoll®, and 0.3 M sucrose were used. In thaw, the SPB1 solution comprising PB1 and 0.3 M sucrose was used.

[0058] In preservation of the control embryos by vitrification (Fujioka T. et al., Int. J. Dev. Biol. 48: 1149-1154), a DAP213 solution c...

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Abstract

This invention provides a solution for preserving mammalian early embryos or ES cells by vitrification, which comprises, as a base material, a phosphate buffer that exclusively contains 10% to 15% (v/v) propylene glycol as polyhydric alcohol or a phosphate buffer that exclusively contains 10% to 15% (v/v) propylene glycol and 25% to 35% (v/v) ethylene glycol as polyhydric alcohols and further contains 15% to 25% (v/v) Percoll® and 0.2 M to 0.5 M sucrose. This invention also provides a method for preserving mammalian early embryos or ES cells by vitrification using such solution.

Description

TECHNICAL FIELD [0001] The present invention relates to a method of preserving mammalian early embryos or ES cells by vitrification. BACKGROUND ART [0002] In experimental animal sciences and related industries, techniques for cryopreserving embryos are critical and fundamental for the production of animals or the preservation of cell lines via reproduction technology or the like. In the case of mice, the preservation of 2cell-stage embryos has been already implemented because of the ease of sampling (see Nakao K. et al., Exp. Anim. 46(3), 231-234, 1997). [0003] When early embryos, such as rat 2cell-stage embryos, are preserved, however, the rate of retrieval is poor. Such preservation does not yield a rate of fetal development similar to that obtained from untreated embryos (see D. G. Whittingham, J. Reprod. Fert, 1975, 43, 575-578; and M. S. Han et al., Theriogenology 59, 2003, 1851-1863). Thus, there are many issues to be resolved regarding techniques for cryopreserving experiment...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N5/08C12N5/06A01N1/02C12N5/07C12N5/073C12N5/0735
CPCA01N1/02A01N1/0221A01N1/00
Inventor ETO, TOMOOSASAKI, ERIKA
Owner CENT INST FOR EXPERIMENTAL ANIMALS
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