Optimization of the cryopreservation of biological resources, Toxoplasma gondii tachyzoites, using flow cytometry.

Fiche publication

Date publication

décembre 2015

Journal

Cryobiology

Auteurs

Membres identifiés du Cancéropôle Est :
Pr LE NAOUR Richard , Dr AUDONNET Sandra , Pr VILLENA Isabelle

Tous les auteurs :
Mzabi A, Escotte-Binet S, Le Naour R, Ortis N, Audonnet S, Dardé ML, Aubert D, Villena I

Lien Pubmed

https://www.ncbi.nlm.nih.gov/pubmed/26408852

Résumé

The conservation of Toxoplasma gondii strains isolated from humans and animals is essential for conducting studies on Toxoplasma. Conservation is the main function of the French Biological Toxoplasma Resource Centre (BRC Toxoplasma, France, http://www.toxocrb.com/). In this study, we have determined the suitability of a standard cryopreservation methodology for different Toxoplasma strains using the viability of tachyzoites assayed by flow cytometry with dual fluorescent labelling (calcein acetoxymethyl ester and propidium iodide) of tachyzoites. This method provides a comparative quantitative assessment of viability after thawing. The results helped to define and refine quality criteria before tachyzoite cryopreservation and optimization of the cryopreservation parameters. The optimized cryopreservation method uses a volume of 1.0 mL containing 8 × 10(6) tachyzoites, in Iscove's Modified Dulbecco's Medium (IMDM) containing 10% foetal calf serum (FCS). The cryoprotectant additive is 10% v/v Me2SO without incubation. A cooling rate of ∼1 °C/min to -80 °C followed, after 48 h, by storage in liquid nitrogen. Thawing was performed using a 37 °C water bath that produced a warming rate of ∼100 °C/min, and samples were then diluted 1:5 in IMDM with 5% FCS, and centrifuged and resuspended for viability assessment.