Cryopreservation and long-term maintenance of bovine embryo-derived cell lines

Pashaiasl, Maryam, Khodadadi, Khodadad, Richings, Nadine M., Holland, Michael K. and Verma, Paul J. (2013) Cryopreservation and long-term maintenance of bovine embryo-derived cell lines. Reproduction, Fertility and Development, 25 4: 707-718. doi:10.1071/RD12018


Author Pashaiasl, Maryam
Khodadadi, Khodadad
Richings, Nadine M.
Holland, Michael K.
Verma, Paul J.
Title Cryopreservation and long-term maintenance of bovine embryo-derived cell lines
Journal name Reproduction, Fertility and Development   Check publisher's open access policy
ISSN 1031-3613
1448-5990
Publication date 2013
Year available 2012
Sub-type Article (original research)
DOI 10.1071/RD12018
Volume 25
Issue 4
Start page 707
End page 718
Total pages 12
Place of publication Collingwood, Vic., Australia
Publisher CSIRO Publishing
Collection year 2013
Language eng
Formatted abstract
The aim of this study was to develop methods for cryopreservation and long-term maintenance of putative bovine embryonic stem cells (ESCs). Putative bovine ESC (bESC) lines (n = 3) isolated in conventional medium were used to compare slow-freezing and vitrification. After warming, vitrified cells (96.9%) demonstrated significantly (P < 0.05) better survival than frozen–thawed cells (81.5%) and formed significantly more colonies with good morphology (vitrification: 93/93, 100.0%; slow-freezing: 74/106, 69.81%; P < 0.05). The effect of inhibitors of differentiation (PD184352, SU5402, CHIR99021) on ESC maintenance was assessed on putative bESC lines established in N2B27–3i medium (n = 8) or conventional medium (n = 1) after culture over 30 passages (>240 days). All cell lines expressed ALP, SSEA1, SSEA4, OCT4, REX1 and SSEA1. OCT4 expression was confirmed by relative real-time PCR and was upregulated in early passages of putative bESCs cultured in N2B27–3i (2.9 ± 0.89-fold higher at Passage (P) 2–4), whereas the converse was observed later (P22–26; 2.2 ± 0.1-fold increase in conventional medium). Putative bESC lines isolated in N2B27–3i medium (n = 3) or conventional medium (n = 1) were vitrified at P18 and, after warming, were cultured for a further 12 passages. These cells survived vitrification and expressed OCT4, REX1, SSEA1, ALP, SSEA1 and SSEA4. These results demonstrate that putative bESC lines that express pluripotent markers can be cultured long term and retain expression of pluripotent markers after vitrification.
Keyword Cell signalling
ESCs
Three inhibitors (3i)
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ
Additional Notes Published online 23 July 2012

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2013 Collection
School of Veterinary Science Publications
 
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Created: Mon, 15 Apr 2013, 15:10:29 EST by Dr Michael Holland on behalf of School of Veterinary Science