Protocols for the in vitro culture of the zoonotic malaria parasite Plasmodium knowlesi

Plasmodium knowlesi is an emerging zoonotic malaria parasite causing severe human infections in Southeast Asia, characterized by high parasitemia, a rapid 24‑hour erythrocytic cycle, and frequent coinfection with P. falciparum and P. vivax. Continuous in vitro culture of non‑falciparum human malaria parasites remains technically challenging, limiting experimental reproducibility and downstream applications. Here, we describe protocols for adapting two P. knowlesi strains to long‑term in vitro culture using host‑specific erythrocytes under defined hematocrit and gas conditions. Parasites were cultured at 2-4% hematocrit in RPMI‑based media supplemented with horse serum and/or AlbuMAX under low‑oxygen conditions. One strain was successfully adapted to human erythrocytes, demonstrating sustained asexual replication, stable growth across serial passages, parasitemia exceeding 11% by day 20, and the successful generation of cryopreserved seed stocks. In contrast, the second strain exhibited host‑cell restriction. It could not be maintained in human erythrocytes but was successfully propagated in rhesus macaque erythrocytes under reduced oxygen conditions, reaching parasitemia >10%. These findings highlight strain‑dependent host specificity and define optimized culture conditions that support robust parasite growth, long‑term maintenance, and preservation. These optimized culture conditions expand available resources for P. knowlesi research and enable its application in downstream experimental systems. Given the comparatively high reported transfection efficiencies of P. knowlesi relative to P. falciparum, these culture systems provide a robust platform for advancing molecular studies of zoonotic malaria parasites.