Methods in Anopheles Research - MR4

Chapter 3 : Specific Anopheles Techniques3.1 Embryonic Techniques3.1.3 Establishing Cell Lines from Anopheles spp. Embryonic TissuesPage 2 of 2sprouting from embryonic fragments. Once cells in a primary culture have replicated sufficiently, a portion(~1/2 to 1/3) of the tissues can be removed by pipetting, and transferred to a new tube to set up asubculture. It is advisable to keep the other portion of cells in the parent flask or tube, as it is common thatthey will remain vigorous even if the subculture should fail.This process is repeated many times until cultures can be subcultured or split on a regular basis, and theculture is considered established. During this process increasingly larger culture vessels will be used,e.g., 12.5-cm 2 flasks, then 25-cm 2 flasks, etc. Although the first several subcultures are usually made bytransferring 30 – 50% of the cells to a new culture vessel, with time it is advisable to “push” a cell line byusing higher dilutions of 1:10 or more. Some mosquito cell lines can be diluted up to 100-fold. Seedingsubcultures at relatively high densities (dilutions of 1:2 or 1:3) will depress cell replication and slowgrowth, often resulting in cultures of poor condition.It is not uncommon that a single primary culture will give rise to sublines displaying differingmorphologies. Some sublines may continue growth as adherent cells, and others may becomeestablished as suspension cultures. In particular, the “hollow ball” or “vesicle” phenotype frequentlydevelops, and may become fixed. Sublines with particular, desired characteristics can also be selected byculture manipulation (e.g., adherent lines can be developed by continuously discarding non-adherent cellsduring medium changes). Once cells are growing reliably, it is a good idea to try to reduce the amount ofFBS, and TPB. Established mosquito cell lines commonly grow quite well with only 5% of FBS. Adding alipoprotein supplement (such as the one from Rocky Mountain Biologicals, Missoula, MT, or the CellPro-LPS from Fisher Scientific), if available, can further reduce the requirement for FBS, and reduce cost.These methods can equally be applied to other mosquito species, keeping in mind the length of timerequired for embryonic development. Eggs should be at least at their half point before hatching, all theway up to just before hatching. Although open culture vessels can be used, such as small Petri dishes ormulti-well plates, they require a humidified atmosphere as well as a CO 2 incubator when media containingbicarbonate are employed. Also, open culture vessels are far more susceptible to contamination thanclosed ones.Additional references: (Munderloh et al. 1982) (Mazzacano et al. 1991)ReferencesMazzacano CA, Munderloh UG, Kurtti TJ (1991) Characterization of a New Continuous Cell Line from theFlood Water Mosquito, Aedes vexans. Cytotechnology 5:147-154Munderloh UG et al. (1999) Invasion and intracellular development of the human granulocytic ehrlichiosisagent in tick cell culture. J Clin Microbiol 37:2518-2524Munderloh UG, Kurtti TJ (1989) Formulation of medium for tick cell culture. Exp Appl Acarol 7:219-229Munderloh UG, Kurtti TJ, Maramorosch K (1982) Anopheles stephensi and Toxorhynchites amboinensis:aseptic rearing of mosquito larvae on cultured cells. Journal of Parasitology 68:1085-1091