Kafatos:Instructions for Anopheles gambiae cell lines

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Instructions for the culture of my Anopheles gambiae cell lines

by Hans-Michael Mueller

The cell lines were established with minced neonate A.gambiae larvae (within 1 hour of hatching) using modified protocols according to Pudney et al. (see also: Colin Leake, 1997, in The Molecular Biology of Insect Disease Vectors. Eds.: Crampton, J.M., Beard, C.B. and Louis, C. Chapman & Hall, London.). Three A.gambiae strains were used: Suakoko 2La, 4a r/r, and L35. One of the major modifications was that I made large primary cultures in 50ml polystyrene flasks using several thousand larvae (see also: Methods and results sections in Mueller et al., 1999). Further variation was achieved by differing splitting modes, media used etc. In one case, I used younger embryos as starting material (line SuaE). Initially I used the recommended MK/VP medium. But then I discovered that the lactalbumin hydrolysate used in this medium led to a constitutive "immune response" in the cells (e.g. defensin RT-PCR) - but Schneider medium did not (at least, inducibility upon addition of bacteria can be detected in this medium; naturally, a weak immune-stimulative effect of Schneider medium cannot be ruled out).

I grow the cells in Schneider medium (used: GibcoBRL-liquid, or Sigma-powder) supplemented with 10% FCS and 100U/ml Pen.+100µg/ml Strep. Note that the cell lines can easily be grown without antibiotics. The brand of plasticware seems not to matter (used: NUNC, Falcon, Greiner). CO2 is not necessary, as all my cell lines grow in Schneider medium whose buffer capacity is based on phosphate. However, close the bottles tight! Cells are in general grown at 27°C. However, you can keep them also at 25°C like Drosophila cells. You can also simply grow them on your bench, protected from direct sun light. On the other hand, the lines grow still at temperatures above 30°C without obvious suffering. If you are absent for a longer time and you do not want to thaw cells again upon arrival: Keep them at 18°C, with elevated medium (10ml instead 7ml per 50ml flask, 35ml instead 25ml per 250ml flask) and decreased FCS concentration (5%).

FCS: Just take what you have. When I tested the last time different batches, I observed only minor differences. My cells are meanwhile growing in about 20 labs, in Europe and the U.S., so it seems not to be a major point. I routinely grow the cells with 10% FCS. Important !!!! Heat-inactivate the FCS for one hour at 56°C! I am actually not sure if its necessary for all the lines. But the short 65°C heat-inactivation protocol sometimes used for Drosophila cell culture seems to be deleterious for the Anopheles cells.

Splitting is different from cell line to cell line! I split the cells not before they are piling up. Even if they are piled up, you can leave them a week longer, they usually do not suffer. Unless the cell layers detach from the plastic surface, the cells are fine. I usually do not use a scraper (and if, than the soft ones of NUNC), unless I need all the cells. What I usually do is to shake the bottle vigorously. This releases most of the cells. Especially in the case of Sua1B, there may be many attached cells left, those I release by scratching with the pipette.
I split usually diluting the cells 1:3-1:10 (cultures of piled up cells are up to 5-fold confluent). It depends also how fast I need the cells. If you need lots of cells, then split them at low ratio but in short intervals (few days). That is a much more efficient strategy than to prepare many, but highly diluted cultures from the beginning, where depending on cell line (especially Sua1B!) the following may happen: You may observe insulated, big cell clumps - in this case just resuspend them in the old medium vigorously with the pipette and let them grow further.
Strongly adherent cells like Sua1B can also be trypsinised using standard cell culture protocols. Just wash the cells often with PBS before, otherwise it does not work. Sometimes you may observe some debris, especially when splitting too old cultures, the cells will phagocytise it! The more FCS you add (up to 30%) the easier the cells are to resuspend.

Once the cultures do bad: scrape all the cells, put them in a smaller flask/dish so that they are confluent, and increase FCS to 25%. If the cell line grows too fast, decrease FCS to 5%.

Freezing/thawing: I use a densely grown big flask (250ml) for 2-3 aliquots. In this way, upon thawing up cells, there will be nearly no lag waiting for cells (you may also make 4-5 aliquots and thaw up in a 50ml flask).

  • remove ALL the medium (most cell lines are adherent enough, bit of loss doesn't matter)
  • add 2-3ml Schneider medium with 10% FCS and 10% DMSO and scrape the cells
  • aliquot 1ml in cryo tubes, place on ice for 30-45 minutes
  • put tubes in a cooled polystyrene box (in order not to cool down too fast in the freezer) and place overnight at -80°C.
  • next morning (or a day later) put tubes in liquid nitrogen
  • to thaw, take the tube in your warm hand, flame the top, flame again after opening, (I flame extensively, as all the cells any how settled on the ground of the tube during ice incubation)
  • take 1ml of medium and resuspend part of the tube contents, add to the medium in a 15ml tube
  • repeat until cryo tube is empty
  • centrifuge 5min at 1500 rpm, take away medium, resuspend cells in new medium and transfer in flask.

You may also thaw by simply adding the cryo tube contents in the flask with medium. After the majority of cells settled (you may wait few hours), take off the DMSO containing medium and replace it with fresh medium. You may increase FCS to 20% at the beginning to ensure a good start.

Good luck!