Evaluation of in vitro culture systems for the maintenance of microfilariae and infective larvae of Loa loa

Abstract

BackgroundSuitable and scalable in vitro culture conditions for parasite maintenance are needed to foster drug research for loiasis, one of the neglected tropical diseases which has attracted only limited attention over recent years, despite having important public health impacts. The present work aims to develop adequate in vitro culture systems for drug screening against both microfilariae (mf) and infective third-stage larvae (L3) of Loa loa.MethodsIn vitro culture conditions were evaluated by varying three basic culture media: Roswell Park Memorial Institute (RPMI-1640), Dulbecco’s modified Eagle’s medium (DMEM) and Iscove’s modified Dulbecco’s medium (IMDM); four sera/proteins: newborn calf serum (NCS), foetal bovine serum (FBS), bovine serum albumin (BSA) and the lipid-enriched BSA (AlbuMax® II, ALB); and co-culture with the Monkey Kidney Epithelial Cell line (LLC-MK2) as a feeder layer. The various culture systems were tested on both mf and L3, using survival (% motile), motility (T90 = mean duration (days) at which at least 90% of parasites were fully active) and moulting rates of L3 as the major criteria. The general linear model regression analysis was performed to assess the contribution of each variable on the viability of Loa loa L3 and microfilarie. All statistical tests were performed at 95% confidence interval.ResultsOf the three different media tested, DMEM and IMDM were the most suitable sustaining the maintenance of both L. loa L3 and mf. IMDM alone could sustain L3 for more than 5 days (T90 = 6.5 ± 1.1 day). Serum supplements and LLC-MK2 co-cultures significantly improved the survival of parasites in DMEM and IMDM. In co-cultures with LLC-MK2 cells, L. loa mf were maintained in each of the three basic media (T90 of 16.4–19.5 days) without any serum supplement. The most effective culture systems promoting significant moulting rate of L3 into L4 (at least 25%) with substantial maintenance time were: DMEM + BSA, DMEM + NCS, DMEM-AlbuMax®II, DMEM + FBS all in co-culture with LLC-MK2, and IMDM + BSA (1.5%), DMEM + FBS (10%) and DMEM + NCS (5%) without feeder cells. DMEM + 1% BSA in co-culture scored the highest moulting rate of 57 of 81 (70.37%). The factors that promoted L. loa mf viability included feeder cells (β = 0.490), both IMDM (β = 0.256) and DMEM (β = 0.198) media and the protein supplements NCS (β = 0.052) and FBS (β = 0.022); while for L. loa L3, in addition to feeder cells (β = 0.259) and both IMDM (β = 0.401) and DMEM (β = 0.385) media, the protein supplements BSA (β = 0.029) were found important in maintaining the worm motility.ConclusionsThe findings from this work display a range of culture requirements for the maintenance of Loa loa stages, which are suitable for developing an effective platform for drug screening.Electronic supplementary materialThe online version of this article (10.1186/s13071-018-2852-2) contains supplementary material, which is available to authorized users.

Preclinical development of an oral anti- Wolbachia macrolide drug for the treatment of lymphatic filariasis and onchocerciasis

“…Testing L. loa in vitro L. loa microfilariae were extracted from baboon peripheral blood and placed in culture in vitro, as previously described (30). Between 20 and 30 microfilariae per well were seeded onto a 96-well plate containing test compounds prepared in DMSO to a final concentration of 1% in culture medium (10% FBS-supplemented Dulbecco's modified Eagle's medium).…”

Generation of Loa loa infective larvae by experimental infection of the vector, Chrysops silacea

“…The L3 were then cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco Life Technologies, Cergy-Pontoise, France) supplemented with 10% foetal bovine serum (FBS; Lonza Verviers, Belgium) on monkey kidney epithelial cells (LLC-MK2; ATCC, Virginia, USA) in a flat bottom 48 well plates (Corning, Kennebunk-ME, USA) containing lids for a maximum period of 30 days at 37˚C, 5% CO 2 in an incubator (CO 2 series Sheldon Mfg. Inch, Cornelius, OR, USA) while monitoring for parasite motility, survival and moulting [12]. Parasite viability during culture was assessed daily by visual inspection under an inverted microscope for 30 days.…”

“…Parasite viability during culture was assessed daily by visual inspection under an inverted microscope for 30 days. Their motility was scored on a 4 point scale: 0; no movement or immotile, 1; intermittent shaking of head and tail, 2; sluggish (shaking of the whole worm on a spot), 3; vigorous movement (shaking of the whole worm and migration from one spot to another) as previously described [12,22].…”

“…We have recently developed a long-term mammalian cell co-culture system whereby L. loa L3 isolated from wild-caught C. silacea remain �90% viable for 18 days, with growth and 20-40% moulting success rate to the L4 stage [12]. We exploited these defined in vitro culture conditions to evaluate the in vitro fitness of experimentally-derived L. loa L3.…”

“…Further, high yielding production of L3 following experimental infections of Chrysops would be a significant improvement in throughput for translational medicine applications compared to standard methods. In this study, we assessed if intrathoracic L. loa mf injected C. silacea can survive under laboratory conditions for a minimum of two weeks and whether L3 infective larvae recovered from lab maintained flies could maintain their viability and development both in vitro and in vivo taking advantage of recent advances in L. loa culture systems and mouse models developed by our laboratories [12,18].…”