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Mansonella perstans infections are widespread in Sub-Saharan Africa and Central and South America and thus can be considered as the most prevalent parasite of man in tropical Africa. In contrast to the high prevalence, knowledge about the biology of this filarial nematode is restricted and no effective treatment regimens of this ivermectin-resistant parasite is lacking. An obstacle for the research is that M. perstans resides in body cavities and thus have been only rarely recovered during surgery or autopsy. Therefore, alternative methods like in vitro culture systems need to be implemented to decipher the nature of mansonellosis and effective drugs. Previously, we have established a monkey kidney epithelial cell-based in vitro culture for the maintenance of M. perstans infective larvae (L3) up to 77 days. However, no alternative for this culture system have been postulated to allow longer survival rates and development of adult worms in vitro. Thus, we aim to establish an alternative in vitro culture system for M. perstans L3. M. perstans L3 were isolated from engorged and laboratory reared Culicoides midges. The larvae were then cultured in Dulbecco's Modified Eagle Medium supplemented with either 10% foetal bovine serum (FBS), 10% newborn calf serum (NCS) or 1% bovine serum albumin (BSA) together with human colon carcinoma cells (HCT-8) as feeder cells. Survival and growth were recorded. We obtained that the 10% NCS culture condition was superior allowing long-term maintenance of M. perstans L3 for up to 100 days and boosted growth of the parasites for up to 5-folds compared to the initial size at culture inception. Although no moulting of the L3 into L4 or adult worms could be overserved, the human colon carcinoma cell-based in vitro culture provides an alternative platform to analyse M. perstans biology and screen for novel drugs against M. perstans., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)