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Published on: September 14, 2019
Long-Term Storage of Cryptosporidium parvum for In Vitro Culture
A Paziewska-Harris1, G Schoone1, H D F H Schallig1
1School of Biosciences, Cardiff University, The Sir Martin Evans Building, Museum Avenue, Cardiff CF10 3AX, U.K.
Abstract:
The long-term storage of Cryptosporidium life-cycle stages is a prerequisite for in vitro culture of the parasite. Cryptosporidium parvum oocysts, sporozoites, and intracellular forms inside infected host cells were stored for 6-12 mo in liquid nitrogen utilizing different cryoprotectants (dimethyl sulfoxide [DMSO], glycerol and fetal calf serum [FCS]), then cultured in vitro. Performance in vitro was quantified by estimating the total Cryptosporidium copy number with quantitative polymerase chain reaction (qPCR) in 3- and 7-day-old cultures. Although few parasites were recovered either from stored oocysts or from infected host cells, sporozoites stored in liquid nitrogen recovered from freezing successfully. More copies of parasite DNA were obtained from culturing those sporozoites than sporozoites excysted from oocysts kept at 4 C for the same period. The best performance was observed for sporozoites stored in Roswell Park Memorial Institute (RPMI) medium with 10% FCS and 5% DMSO, which generated 240% and 330% greater number of parasite DNA copies (on days 3 and 7 post-infection, respectively) compared to controls. Storage of sporozoites in liquid nitrogen is more effective than oocyst storage at 4 C and represents a more consistent approach for storage of viable infective Cryptosporidium aliquots for in vitro culture.
Insights
Long-term storage of Cryptosporidium sporozoites in liquid nitrogen offers a superior method for in vitro culture compared to oocyst storage. This approach ensures a more consistent supply of viable parasites for research.
Area of Science:
- Parasitology
- Microbiology
- Infectious Diseases
Background:
- Long-term storage of Cryptosporidium life-cycle stages is crucial for successful in vitro culture.
- Previous methods for storing Cryptosporidium parasites have limitations for maintaining viability for extended periods.
Purpose of the Study:
- To evaluate the effectiveness of liquid nitrogen cryopreservation for long-term storage of Cryptosporidium parvum oocysts, sporozoites, and intracellular forms.
- To compare the in vitro culture performance of parasites stored using different cryoprotectants and methods.
Main Methods:
- Cryptosporidium parvum oocysts, sporozoites, and infected host cells were stored in liquid nitrogen for 6-12 months with cryoprotectants (DMSO, glycerol, FCS).
- Stored parasites were cultured in vitro, and parasite DNA copy number was quantified using quantitative polymerase chain reaction (qPCR) at 3 and 7 days post-infection.
- Performance was compared to sporozoites excysted from oocysts stored at 4°C.
Main Results:
- Sporozoites stored in liquid nitrogen showed successful recovery and better in vitro culture performance compared to stored oocysts or intracellular forms.
- Sporozoites stored in RPMI medium with 10% FCS and 5% DMSO yielded significantly higher parasite DNA copy numbers (240% at day 3, 330% at day 7) compared to controls.
- Liquid nitrogen storage of sporozoites was more effective than 4°C oocyst storage for maintaining parasite viability for in vitro culture.
Conclusions:
- Liquid nitrogen cryopreservation of Cryptosporidium sporozoites is a highly effective method for long-term storage.
- This method provides a more consistent and reliable approach for obtaining viable Cryptosporidium aliquots for in vitro culture.
- Optimized cryoprotectant mixtures enhance parasite recovery and performance post-thawing.
