Cryopreservation method for Entamoeba histolytica trophozoites

Entamoeba histolytica, a protozoan parasite, causes amebiasis, which is a global public health problem. Clinical manifestation and pathogenesis of amebiasis are closely associated with the proliferation and tissue invasion abilities of E. histolytica trophozoites. At the same time, E. histolytica trophozoites are studied in a broad range of biology research fields. As such, the development of in vitro culture of E. histolytica trophozoites greatly facilitated amebic research. Now, a standard method for cryopreservation of E. histolytica trophozoites is required because available methods either do not give a high enough revival rate, which can significantly delay projects, or they are not widely adopted. Here, we attempted to optimize the conditions for E. histolytica trophozoite cryopreservation, including cell density, cooling rate, cell freezing reagent, and freezing profile. We found an optimized condition that reproducibly yielded >30% revival, regardless of storage period in liquid nitrogen (up to 365 days). This optimized condition is that E. histolytica trophozoites are suspended in 0.5 mL CELLBANKER 2 in a 1 mL cryotube at 2 × 10⁶ cells/mL and frozen from 4 to -40°C at a rate of -0.2°C/min using VIA Freeze Uno. This cryopreservation method can minimize the risk of losing E. histolytica trophozoite lines required to continue projects, facilitating amebic research.

Amebiasis, which is caused by Entamoeba histolytica infection, is the third deadliest parasitic disease globally. Proliferation of E. histolytica trophozoites and their invasion into the host tissues cause amebiasis symptoms and pathogenesis. E. histolytica trophozoites are also important in multiple biology research topics. Therefore, E. histolytica trophozoites are a common subject in academic as well as clinical fields. A standard method for in vitro culture of E. histolytica trophozoites is well established. By contrast, a widely adopted practical method for cryopreservation of E. histolytica trophozoites is not yet available. This hampers the advancement of amebic research, as the required E. histolytica trophozoite lines sometimes cannot be revived from cryopreservation. In this study, we varied parameters critical to the revival rate, namely, cell density, cooling rate, freezing reagent, and freezing profile, and present an optimized cryopreservation method for E. histolytica trophozoites, which gives reproducibly high revival rates.