title: Reticulocytes from cryopreserved erythroblasts support Plasmodium vivax infection in vitro.
abstract: Plasmodium vivax is the most widely distributed human malaria parasite. Despite its importance, both clinical research and basic research have been hampered by lack of a convenient in vitro culture system, in part due to the parasite's infection preference of reticulocytes rather than mature erythrocytes. The use of reticulocyte-producing hematopoietic stem cell culture has been proposed for the maintenance of the parasite, but good numbers of reticulocytes and P. vivax parasites sufficient for practical use in research have been difficult to produce from this system. Here, we report an improved method of hematopoietic stem cell culture for P. vivax infection, which requires less time and produces higher or equivalent percentage of reticulocytes than previously reported systems. Reticulocytes were cultured from cryopreserved erythroblasts that were frozen after 8day-cultivation of purified CD34+ cells from human umbilical cord blood. This method of production allowed the recovery of reticulocytes in a shorter time than with continuous stem cell culture. We obtained a relatively high percentage of peak reticulocyte production by using co-cultivation with a mouse stromal cell line. Using P. vivax mature stage parasites obtained from infected Aotus monkeys, we observed substantial numbers (up to 0.8% of the total number of the cells) of newly invaded reticulocytes 24h after initial cultivation. The addition of fresh reticulocytes after 48h culture, however, did not result in significant increase of second cycle reticulocyte invasion. Assays of invasion inhibition with specific antibodies were successful with this system, demonstrating potential for study of biological processes as well as the conditions necessary for long-term maintenance of P. vivax in vitro.
authors.affiliation: BioService, Malaria Research and Reference Reagent Resource Center (MR4), American Type Culture Collection (ATCC), Manassas, VA 20110-2209, USA. Electronic address: furuyat@cc.tuat.ac.jp.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Furuya
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20852, USA.
authors.forename: Juliana M
authors.initials: JM
authors.lastname: Sá
authors.affiliation: Malaria Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi, India.
authors.forename: Chetan E
authors.initials: CE
authors.lastname: Chitnis
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20852, USA.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Wellems
authors.affiliation: BioService, Malaria Research and Reference Reagent Resource Center (MR4), American Type Culture Collection (ATCC), Manassas, VA 20110-2209, USA.
authors.forename: Timothy T
authors.initials: TT
authors.lastname: Stedman
mesh_terms: D000818:Animals; D016645:Aotidae; D018929:Cell Culture Techniques; D002460:Cell Line; D015925:Cryopreservation; D004900:Erythroblasts / Q000166:cytology*; D006801:Humans; D016780:Malaria, Vivax / Q000469:parasitology; D051379:Mice; D010966:Plasmodium vivax / Q000166:cytology* / Q000502:physiology*; D012156:Reticulocytes / Q000469:parasitology*
keywords: Culture system; Hematopoietic stem cells; Plasmodium vivax