title: Endothelial cells release soluble factors that support the long-term survival of filarial worms in vitro.
abstract: The inability to maintain filarial nematodes in long-term in vitro culture greatly limits research into the basic biology of these parasites and hinders in vitro screening of novel anti-filarial agents. In this study, we sought to characterize nutrients that promote the long-term survival of filarial worms in vitro. Using microfilariae (MF) obtained from gerbils infected with Litomosoides sigmodontis, a filarial parasite of rodents, we found that Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) resulted in MF survival of only 5 days. However, co-culturing MF with a mouse endothelial cell line (EOMA) enabled survival for 40 days. Culturing EOMA cells in transwell plates extended MF survival to the same degree as direct co-culture, suggesting that the factors microfilariae require are soluble in nature. Heat inactivation of EOMA conditioned media at 56 °C reduced MF survival by approximately 50%, and heat inactivation at 100 °C reduced survival to 3 days, demonstrating that both heat labile and heat stable factors are involved. EOMA cells require FBS to produce these factors, as conditioned media collected from EOMA cells grown in the absence of FBS failed to prolong survival. The removal of lipids also abrogated survival, indicating MF are likely utilizing lipid factors released by EOMA cells. Dialysis experiments demonstrate that at least some of the required factors are between 0.1 and 1 kDa in size. Importantly, L. sigmodontis adult worms also show significantly extended survival when cultured in EOMA conditioned media. Together, these results suggest that EOMA-produced factors include lipid-containing molecules, heat labile molecules (likely a protein), and micronutrients between 0.1 and 1 kDa in size. These studies have established a cell-free approach to maintaining MF and adult stage filarial worms in long-term in vitro culture and have taken important steps towards biochemically characterizing host-derived nutrients required for parasite survival.
authors: Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814, United States.
authors: Holly
authors: H
authors: Evans
authors: Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814, United States.
authors: Alexander Francis
authors: AF
authors: Flynn
authors: Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814, United States. Electronic address: edward.mitre@usuhs.edu.
authors: Edward
authors: E
authors: Mitre
mesh_terms: D000818:Animals; D002460:Cell Line; D016000:Cluster Analysis; D018920:Coculture Techniques; D009033:Culicidae; D017077:Culture Media, Conditioned; D042783:Endothelial Cells / Q000378:metabolism* / Q000469:parasitology; D005260:Female; D005368:Filariasis / Q000469:parasitology*; D005370:Filarioidea / Q000302:isolation & purification / Q000502:physiology*; D005849:Gerbillinae; D006358:Hot Temperature; D008055:Lipids / Q000737:chemistry; D013058:Mass Spectrometry; D051379:Mice; D008842:Microfilariae / Q000502:physiology; D009705:Nucleosides / Q000378:metabolism; D035422:Pleural Cavity / Q000469:parasitology; D051381:Rats; D013997:Time Factors; D015854:Up-Regulation
chemical_list: D017077:Culture Media, Conditioned; D008055:Lipids; D009705:Nucleosides
keywords: Endothelial cells; Filariae; In vitro cell culture; Life cycle