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

Alexandre Mzabi; Sandie Escotte-Binet; Richard Le Naour; Naïma Ortis; Sandra Audonnet; Marie-Laure Dardé; Dominique Aubert; Isabelle Villena

doi:10.1016/j.cryobiol.2015.09.002

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

Cryobiology. 2015 Dec;71(3):459-63. doi: 10.1016/j.cryobiol.2015.09.002. Epub 2015 Sep 25.

Authors

Alexandre Mzabi¹, Sandie Escotte-Binet², Richard Le Naour³, Naïma Ortis⁴, Sandra Audonnet⁵, Marie-Laure Dardé⁶, Dominique Aubert², Isabelle Villena²

Affiliations

¹ 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.
² 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.
³ EA 4683, SFR CAP-Santé, University of Reims Champagne-Ardenne, Reims, France; Flow Cytometry Platform URCACyt, University of Reims Champagne-Ardenne, Reims, France.
⁴ Laboratory of Parasitology-Mycology, National Reference Centre for Toxoplasmosis, Biological Resource Centre Toxoplasma, Maison Blanche Hospital, Reims, France.
⁵ Flow Cytometry Platform URCACyt, University of Reims Champagne-Ardenne, Reims, France.
⁶ 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.

PMID: 26408852
DOI: 10.1016/j.cryobiol.2015.09.002

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.

Keywords: Biological resource centre; Cryopreservation; Cryoprotector; Flow cytometry; Foetal calf serum; Me(2)SO; Tachyzoites; Toxoplasma gondii.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cattle
Cryopreservation / methods*
Cryoprotective Agents / pharmacology
Flow Cytometry / methods*
Humans
Toxoplasma*

Substances

Cryoprotective Agents