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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
Stable URL: https://www.jstor.org/stable/3282883
Page Count: 5
You can always find the topics here!Topics: Trypanosome, Flasks, Subcultures, Microelectromechanical systems, Blood, Feeder cells, Parasites, Sodium, Parasitology, Blood proteins
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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.
The Journal of Parasitology © 1989 The American Society of Parasitologists