Cryoconservation of Chicken Blastodermal Cells: Effects of Slow Freezing, Vitrification, Cryoprotectant Type and Thawing Method During <I>In Vitro</I> Processing

Abstract

SAWICKA D., CHOJNACKA-PUCHTA L., BRZEZINSKA J., LAKOTA P., BEDNARCZYK M. 2015. Cryoconservation of chicken blastodermal cells: Effects of slow freezing, vitrification, cryoprotectant type and thawing method during in vitro processing. Folia Biologica (Kraków) 63: 129-134. Cryoconservation of blastodermal cells (BCs) can preserve genetic material for the future reconstruction of poultry breeds. The aim of our study was to compare the effects of three slow freezing programs and vitrification, different cryoprotectants (5% DMSO, 10% DMSO, or multi-component cryoprotectant (MC) and two thawing methods on the viability of chicken BCs. Significant differences in the survival of slowly frozen BCs using program 3 (2°C/min. to 0.4°C/min.) compared with programs 1 (1°C/min. to 0.3°C/min.) and 2 (4°C/min. to 0.3°C/min.) were observed. The percentage of live BCs was significantly higher after slow freezing in the presence of the MC compared with DMSO. The thawing method did not have a significant effect on the percentage of live BCs. We also observed significant differences in the survival rate of BCs after vitrification (81%) and slow freezing in the presence of 10% DMSO using program 3 (60%). The highest percentage of viable BCs was achieved by slow freezing with the MC using program 2 and thawing with method 1 (94%). The most unfavorable combination for BCs survival was slow freezing in 5% DMSO using program 3 and thawing with method 2 (58.3%). This is the first study to apply MC to the slow freezing of BCs. We also showed successful BCs vitrification.

Cryopreservation of chicken blastodermal cells and their quality assessment by flow cytometry and transmission electron microscopy

“…Cryopreservation is widely accepted technique for achieving long‐term storage and has been applied to an increasingly diverse range of biological materials. Slow and rapid freezing are the two conventional approaches utilized in practice for cell cryopreservation . In slow freezing method, cells are cooled down at a rate of −1°C/min and eventually stored at −80°C .…”

Efficiency of Chicken Blastoderm Cell Transfection Combining two Nonviral Methods: Electroporation and Synthetic Carriers

“…The mains steps of this strategy involve: plasmid construction, isolation of donor embryonic cells, their transfection in vitro, injection into recipient embryos, identification of somatic chimeras, raising chimeras and identification of germ line chimeras (ROYCHOUDHURY et al 2010). In our investigations, the production of chicken chimeras was conducted with a modified method of injection of BCs (PETITTE et al 1990;PERRY et al 1991;BEDNARCZYK et al 2000BEDNARCZYK et al , 2002SIWEK et al 2010;SAWICKA et al 2015). Manipulation of BCs is very useful for producing transgenic chickens, as stage X BCs are transferred into recipient embryos and form somatic and germline chimeras (PETITTE et al 1990;PERRY et al 1991;SIWEK et al 2010).…”

“…Manipulation of BCs is very useful for producing transgenic chickens, as stage X BCs are transferred into recipient embryos and form somatic and germline chimeras (PETITTE et al 1990;PERRY et al 1991;SIWEK et al 2010). BCs can be maintained in a culture for several days cryoproserved (SAWICKA et al 2011;SAWICKA et al 2015) and genetically modified in vitro (BEDNARCZYK et al 2003;WAWRZYÑ SKA & BEDNARCZYK 2007;WAWRZYÑSKA et al 2008).…”