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Perspectives on the in vitro culture of filariae
1Department of Entomology, University of Arizona, Tucson 85721.
Abstract:
A primary constraint in the culture of human filariae is in obtaining starting material--either microfilariae (mfs), which infect invertebrates, or third stage larvae (L3s), which are infective to humans. Cryopreservation methods which partially overcome this difficulty have been developed for both mfs and L3s. Complete development of mfs to L3s outside an intact host was obtained recently when mosquito thoraces infected by Brugia malayi (24 h after the bloodmeal) were maintained in vitro. In another recent study in which no host tissues were present, a semidefined culture medium was used to investigate the properties of reduced glutathione (GSH) that stimulate early development of Onchocerca lienalis mfs. An extended cysteinyl backbone and a free sulfhydryl were identified as the key structural elements provided by GSH. Stimulation also required the presence of low and high molecular weight components of serum as well as oxygen. Molting of Onchocerca spp. L3s to the fourth stage at the rate of 50 to 70% has been reported by several researchers. Key factors identified in those successes have been temperature and serum lot. Improved long-term viability occurred with cellular co-culture. Beneficial effects of co-culture were shown to be due both to cellular conditioning of the medium as well as to lowered dissolved oxygen levels as a result of cellular metabolism. With the use of cell-conditioned medium and decreased incubator oxygen levels, long-term viability of Onchocerca larvae in culture exceeded that previously reported. Recently, Brugia malayi adults of both sexes were cultured from L3s using a semidefined medium supplemented with human serum. Many of these sexually matured adults mated and produced viable microfilariae.
Insights
Researchers developed new methods for culturing filarial worms like Brugia malayi and Onchocerca, overcoming challenges in obtaining starting materials. These advances enable complete larval development and adult reproduction in vitro, aiding parasitic disease research.
Area of Science:
- Parasitology and Tropical Medicine
- Invertebrate Cell Culture
- Molecular Biology
Background:
- Culturing human filariae (parasitic worms) is challenging due to difficulties in obtaining infectious microfilariae (mfs) or third-stage larvae (L3s).
- Existing cryopreservation methods offer partial solutions for mf and L3 preservation.
- Recent advancements have enabled in vitro development from mfs to L3s and successful molting of L3s.
Purpose of the Study:
- To investigate methods for overcoming constraints in culturing human filariae.
- To achieve complete development of microfilariae to infective larvae outside a host.
- To improve long-term viability and reproductive capacity of cultured filarial worms.
Main Methods:
- Maintenance of infected mosquito thoraces in vitro for Brugia malayi mf to L3 development.
- Utilizing a semi-defined culture medium with reduced glutathione (GSH) to study Onchocerca lienalis mf development.
- Employing cellular co-culture and cell-conditioned medium with controlled oxygen levels for Onchocerca spp. L3 molting and long-term viability.
Main Results:
- Complete development of Brugia malayi mfs to L3s was achieved in vitro using infected mosquito thoraces.
- Reduced glutathione (GSH) was identified to stimulate Onchocerca lienalis mf development, requiring specific structural elements, serum components, and oxygen.
- Successful molting of Onchocerca spp. L3s to the fourth stage (50-70%) was reported, with improved long-term viability using cell-conditioned medium and reduced oxygen.
- Brugia malayi adults were cultured from L3s in a semi-defined medium with human serum, leading to mating and production of viable microfilariae.
Conclusions:
- In vitro culture systems are advancing, enabling complete filarial worm development from early stages to reproductive adults.
- Key factors like reduced glutathione, serum components, temperature, serum lot, cellular co-culture, and controlled oxygen are crucial for successful cultivation.
- These improved culture techniques are vital for advancing research on filarial diseases and developing control strategies.