A Simple Apparatus for Large-Scale Production of Plasmodium falciparum In vitro (1981)

Current techniques for culturing Plasmodium falciparum in vitro are based on the original methods of Trager and Jensen (1976, Science 193: 673-675) and can be divided conveniently into those techniques by which (1) the settled layer of infected erythrocytes are supplied continuously with fresh culture medium such as in Trager's flow vessel (Trager, 1979, J. Protozool. 26: 125-129) or Chin's adaptation using plastic tissue culture flasks (Chin, 1979, Trans. R. Soc. Trop. Med. Hyg. 73: 334-335); and (2) those by which the medium is changed intermittently. The latter include the semiautomated tipper apparatus (Jensen et al., 1979, Exp. Parasitol. 48: 3641) manually changed Erlenmeyer flask cultures (Siddiqui, 1979. In Practical tissue culture applications, Maramorosch and Hirumi [eds.], Academic Press, New York, pp. 267277), or the petri dish-candle jar technique (Jensen and Trager, 1977, J. Parasitol. 63: 883-886). Of these techniques the simplest and most widely used is the petri dish-candle jar method, or a variation of it using multiwell culture plates in place of the petri dishes. This method is excellent for experimental work for which numerous replicas are needed and small volumes are not a problem, such as parasite inhibition assays using antimalarial compounds or immunological factors, or in studies on parasite biology, ultrastructure, physiology, etc. Although it can be expanded to a limited degree, the manual changing of the medium quickly becomes a constraint when large volumes of parasite material are needed for antigen preparations or biochemical studies. The semiautomated methods, such as the continuous flow method or the tipper apparatus, are expensive to assemble or manufacture and, unfortunately, in their present state are not readily expandable and simply do not produce enough parasites. The present report describes a laboratory method for cultivation of P. falciparum that, although requiring custom built (but readily available) glassware and manual changing of culture medium, is an expandable and inexpensive method for large-scale production of parasites. It requires no peristaltic pumps and the medium can be changed without exposing the parasites to possible bacterial or fungal contamination. The culture vessels (Fig. 1), made from rectangular glass tubing (Vitro Dynamic, Inc., Rockaway, New Jersey 07866) 22 x 75 x 400 mm, with a silicone-rubber-stoppered, cylindrical chimney (21 mm I.D.) (d) and vaccinetype, rubber-stoppered side port (40 x 14 mm I.D.) (e) at each end are connected by glass and silicone-rubber tubing (Ace, Linden, New Jersey) (c) to a four-place glass manifold whose arms (150 x 28 mm I.D.) (a) are to premeasure and prewarm the culture medium before it enters the vessels. Each vessel will hold 5 ml of packed human erythrocytes and each arm of the manifold 75 ml of medium so that the final cell suspension in the vessels is 6.7%. The culture medium is stored in a reservoir (n) in a small refrigerator (m) above a forced-air, low-temperature incubator (Freas 815) containing the culture vessels. Approximately 1 to 2 hr before the medium is to be changed in the vessels, the manifold (a) is filled via gravity by opening a clamp on the silicone-rubber tube that acts as a joint between the teflon-lined, plastic, delivery tubing (3/16 in. Teflon-TFE, Ace, Linden, New Jersey) (1) and the manifold. This tubing which delivers media is impermeable to CO2 and 02, an important characteristic to maintain proper pH and gas concentrations. To change the culture medium, it is allowed to