title: Optimization of the cryopreservation of biological resources, Toxoplasma gondii tachyzoites, using flow cytometry.
abstract: 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.
authors.affiliation: EA 3800, SFR CAP-Santé, University of Reims Champagne-Ardenne, Reims, France; Laboratory of Parasitology-Mycology, National Reference Centre for Toxoplasmosis, Biological Resource Centre Toxoplasma, Maison Blanche Hospital, Reims, France. Electronic address: amzabi@chu-reims.fr.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Mzabi
authors.affiliation: EA 3800, SFR CAP-Santé, University of Reims Champagne-Ardenne, Reims, France; Laboratory of Parasitology-Mycology, National Reference Centre for Toxoplasmosis, Biological Resource Centre Toxoplasma, Maison Blanche Hospital, Reims, France.
authors.forename: Sandie
authors.initials: S
authors.lastname: Escotte-Binet
authors.affiliation: EA 4683, SFR CAP-Santé, University of Reims Champagne-Ardenne, Reims, France; Flow Cytometry Platform URCACyt, University of Reims Champagne-Ardenne, Reims, France.
authors.forename: Richard
authors.initials: R
authors.lastname: Le Naour
authors.affiliation: Laboratory of Parasitology-Mycology, National Reference Centre for Toxoplasmosis, Biological Resource Centre Toxoplasma, Maison Blanche Hospital, Reims, France.
authors.forename: Naïma
authors.initials: N
authors.lastname: Ortis
authors.affiliation: Flow Cytometry Platform URCACyt, University of Reims Champagne-Ardenne, Reims, France.
authors.forename: Sandra
authors.initials: S
authors.lastname: Audonnet
authors.affiliation: INSERM, University of Limoges, CHU Limoges, Parasitology Department, UMR-S 1094, Tropical Neuroepidemiology, Institute of Neuroepidemiology and Tropical Neurology, Limoges, France; National Reference Centre for Toxoplasmosis, Biological Resource Centre Toxoplasma, Limoges University Hospital, University of Limoges, Limoges, France.
authors.forename: Marie-Laure
authors.initials: ML
authors.lastname: Dardé
authors.affiliation: EA 3800, SFR CAP-Santé, University of Reims Champagne-Ardenne, Reims, France; Laboratory of Parasitology-Mycology, National Reference Centre for Toxoplasmosis, Biological Resource Centre Toxoplasma, Maison Blanche Hospital, Reims, France.
authors.forename: Dominique
authors.initials: D
authors.lastname: Aubert
authors.affiliation: EA 3800, SFR CAP-Santé, University of Reims Champagne-Ardenne, Reims, France; Laboratory of Parasitology-Mycology, National Reference Centre for Toxoplasmosis, Biological Resource Centre Toxoplasma, Maison Blanche Hospital, Reims, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Villena
mesh_terms: D000818:Animals; D002417:Cattle; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D014122:Toxoplasma
chemical_list: D003451:Cryoprotective Agents
keywords: Biological resource centre; Cryopreservation; Cryoprotector; Flow cytometry; Foetal calf serum; Me(2)SO; Tachyzoites; Toxoplasma gondii