Preliminary evaluations of 3-dimensional human skin models for their ability to facilitate in vitro the long-term development of the debilitating obligatory human parasite Onchocerca volvulus

Abstract

Onchocerciasis also known as river blindness is a neglected tropical disease and the world's second-leading infectious cause of blindness in humans; it is caused by Onchocerca volvulus. Current treatment with ivermectin targets microfilariae and transmission and does not kill the adult parasites, which reside within subcutaneous nodules. To support the development of macrofilaricidal drugs that target the adult worm to further support the elimination of onchocerciasis, an in-depth understanding of O. volvulus biology especially the factors that support the longevity of these worms in the human host (>10 years) is required. However, research is hampered by a lack of access to adult worms. O. volvulus is an obligatory human parasite and no small animal models that can propagate this parasite were successfully developed. The current optimized 2-dimensional (2-D) in vitro culturing method starting with O. volvulus infective larvae does not yet support the development of mature adult worms. To overcome these limitations, we have developed and applied 3-dimensional (3-D) culture systems with O. volvulus larvae that simulate the human in vivo niche using in vitro engineered skin and adipose tissue. Our proof of concept studies have shown that an optimized indirect co-culture of in vitro skin tissue supported a significant increase in growth of the fourth-stage larvae to the pre-adult stage with a median length of 816–831 μm as compared to 767 μm of 2-D cultured developed larvae. Notably, when larvae were co-cultured directly with adipose tissue models, a significant improvement for larval motility and thus fitness was observed; 95% compared to 26% in the 2-D system. These promising co-culture concepts are a first step to further optimize the culturing conditions and improve the long-term development of adult worms in vitro. Ultimately, it could provide the filarial research community with a valuable source of O. volvulus worms at various developmental stages, which may accelerate innovative unsolved biomedical inquiries into the parasite’s biology.

Establishment of an in vitro culture system to study the developmental biology of Onchocerca volvulus with implications for anti-Onchocerca drug discovery and screening

“…volvulus worm attachment and triggered apparent early nodulogenesis. Malkmus et al , [ 40 ] evaluated the ability of engineered human skin and adipose tissue to facilitate the in vitro long-term development of O . volvulus and reported culturing L4 larval stage until they were 92 days old with a clear benefit of tissue-engineered skin models although these co-culture experiments do not yet represent methodologically sound culture systems for O .…”

“…The serum-based culture systems have been reported to support parasite longevity in vitro and cuticle casting [ 11 , 17 – 26 ]. Due to the inconsistency of serum composition, serum-free culture systems and co-culture systems using eukaryotic cells as feeder layers have been found successful [ 27 – 37 , 38 – 40 ]. Moreover, feeder cells were already shown to be crucial for in vitro cultivation and growth of O .…”

“…Moreover, feeder cells were already shown to be crucial for in vitro cultivation and growth of O . volvulus [ 40 , 41 ]. From our previous observations on the in vitro growth and development of the filarial nematode Loa loa [ 36 ], among the three most used supplements (feeder layer, serum, and basic culture medium) for filarial parasite in vitro culture, feeder cells were classified as the most important requirement followed by the serum type and finally the nature of the basic culture medium.…”

Adoptive Transfer of Immune Cells Into RAG2IL-2Rγ-Deficient Mice During Litomosoides sigmodontis Infection: A Novel Approach to Investigate Filarial-Specific Immune Responses

“…Nevertheless, it is impossible to investigate the complexity of the filarial-driven modulation and survival tactics solely in humans due to limited access to parasitic life stages, unknown infection time point, possible re-and co-infections and most importantly ethical concerns. Although in vitro culture models of human filariae are promising (25)(26)(27)(28)(29)(30) and experimental infections in monkeys have been established (64,65), these approaches do not reflect the complexity of the human host and thus cannot be used to study filarial-driven modulation of host immunity and are difficult to perform accompanied with ethical concerns, respectively. Therefore, the rodent model of filariasis, namely L. sigmodontis, is used for filarial research and expanded the knowledge about filarial-driven immune regulation and is also a suitable platform for anti-filarial drug testing (36,37).…”

“…However, human studies are hindered especially due to the inability to obtain adult worms and the insufficient possibility to analyse infection kinetics and immune signalling. Although, in vitro culture models for human filariae have been developed (24)(25)(26)(27)(28)(29)(30), so far, it is impossible to obtain all life stages and mimic filarial life cycle, especially parasitic reproduction in vitro. Thus, several studies aimed to establish human filarial infections in rodent models (31)(32)(33)(34)(35), but so far, no complete life cycle could be established in vivo.…”