Babesia bigemina: Advances in continuous in vitro culture using serum-free medium supplemented with insulin, transferrin, selenite, and putrescine

• Published in: Parasitology international 2018-06, Vol.67 (3), p.294-301
• Main Authors: Rojas-Martínez, Carmen, Rodríguez-Vivas, Roger I., Figueroa Millán, Julio V., Acosta Viana, Karla Y., Gutiérrez Ruíz, Edwin J., Bautista-Garfias, Carlos R., Lira-Amaya, José J., Polanco-Martínez, Diego J., Álvarez Martínez, Jesús A.
• Format: Article
• Language: English
• Subjects: B. bigemina; in vitro culture; Insulin; Perfusion bioreactor; Putrescine; Selenite; Serum-free; Transferrin
• ISSN: 1383-5769; 1873-0329
• DOI: 10.1016/j.parint.2017.11.003

This study reported that Babesia bigemina (Bbig-SF) was continuously cultured in vitro in a serum-free medium supplemented with a mixture of insulin-transferrin-selenite (M-ITS) and putrescine (Pu). Firstly, the effect of five different types of basal culture media supplemented with 40% bovine serum was evaluated regarding the proliferation of the protozoan parasite. Cultures with the advanced DMEM/F12 medium (A-DMEM/F12) showed the highest percentage of parasitized erythrocytes (PPE) at 8.37%. Using A-DMEM/F12, a strain of B. bigemina (Bbig-SF) was adapted for growth in bovine serum-free medium by a sequential reduction of serum and demonstrated a maximum PPE of 7.18% in the absence of serum. The next study was the evaluation of the effect of adding four different concentrations of M-ITS to the serum-free A-DMEM/F12 medium on Bbig-SF; the optimal concentrations of M-ITS were 2000, 1100, and 1.34mg/L, which yielded a PPE of 7.23%. Next, eight levels of Pu were evaluated on Bbig-SF cultured in serum-free A-DMEM/F12. After the addition of 0.1012mg/L of Pu, the maximum PPE was 7.61%. When the combination of serum-free A-DMEM/F12+M-ITS (2000, 1100, and 1.34mg/L)+Pu (0.1012mg/L) was evaluated, it yielded a maximum PPE of 14.80%. Finally, the combination of M-ITS+Pu in A-DMEM/F12 without serum and incorporation of a perfusion bioreactor yielded a maximum PPE of 33.45%. We concluded these culturing innovations for B. bigemina in vitro allow the optimization of small- and large-scale proliferation as a source of this protozoan parasite for future studies. [Display omitted] •Serum-free A-DMEM/F12 with insulin, transferrin, selenite and putrescine increased proliferation of B. bigemina in vitro.•The putrescine is essential for proliferation of B. bigemina in vitro.•Higher density of erythrocytes and a greater harvest of B. bigemina infected erythrocytes were yielded in a bioreactor.