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Journal Article

# Continuous Cultivation of Trypanosoma brucei Blood Stream Forms in a Medium Containing a Low Concentration of Serum Protein without Feeder Cell Layers

Hiroyuki Hirumi and Kazuko Hirumi
The Journal of Parasitology
Vol. 75, No. 6 (Dec., 1989), pp. 985-989
DOI: 10.2307/3282883
Stable URL: https://www.jstor.org/stable/3282883
Page Count: 5

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Blood stream forms (BSF) of Trypanosoma brucei brucei GUTat3.1 were propagated in vitro in the absence of feeder layer cells at 37 C, using a modified Iscove's medium (HMI-18). The medium was supplemented with 0.05 mM bathocuproine sulfonate, 1.5 mM L-cysteine, 1 mM hypoxanthine, 0.2 mM 2-mercaptoethanol, 1 mM sodium pyruvate, 0.16 mM thymidine, and 20% (v/v) Serum [Circled Trade Mark Sign] (SP) (Hazleton Biologies, Lenexa, Kansas). The latter contained a low level of serum proteins (13 μ/ml). Each primary culture was initiated by placing $3.5\minus4 \times 10^6$ BSFs isolated from infected mice in a flask containing 5 ml of the medium (HMI-9) supplemented with 10% fetal bovine serum (FBS) and 10% SP. The cultures were maintained by replacing the medium every 24 hr for 5-7 days. During this period, many BSFs died. However, from day 4 onward, long slender BSFs increased in number. On days 5-7, trypanosome suspensions were pooled and cell debris was removed by means of diethylaminoethyl cellulose (DE52) column chromatography. Blood stream forms then were collected by centrifugation, resuspended in fresh medium at $7\minus9 \times 10^5$/ml, and transferred to new flasks. Subcultures were maintained by readjusting the BSF density to $7\minus9 \times 10^5$/ml every 24 hr. Concentrations of FBS were reduced gradually at 5-7-day intervals by alternating the amounts of FBS and SP in HMI-9 with 5% FBS and 15% SP, with 2% FBS and 18% SP, and finally with 20% SP (HMI-18). By this method, $2\minus3 \times 10^6$ BSFs/ml were obtained consistently every 24 hr, for more than 80 days. This system also has been applied successfully to the cultivation of BSFs of Trypanosoma brucei gambiense IL2343.