Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

These protocols will help any researcher incorporate the use of Drosophila cell lines to drive or complement their research agenda. Most if not all Drosophila cell cultures revive, proliferate, and cryo-preserve well when handled according to these guidelines. After wiping down the work surface of a laminar flow hood with 70%ethanol, dispense five milliliters of the appropriate medium into a 25 centimeters squared T-flask.

Wipe the container of frozen cell line with 70%ethanol and carefully loosen and unseal the lid. Using a Pasteur pipette, transfer one milliliter of room temperature medium from the flask to the cryo-vial and gently mix to thaw the frozen cells. Taking care that the cell suspension does not overflow, transfer the entire volume of the thawed cell suspension to the flask and rinse the cryo-vial with fresh medium.

Allow the cells to settle to the bottom of the flask for at least two hours in a 25 degree celsius incubator. Do not return frozen cells that have been packaged for travel back into minus 80 degrees celsius freezer for a prolonged period or into liquid nitrogen. Instead thaw the cells ASAP.

After confirming attachment under a light microscope, gently replace the supernatant with five milliliters of fresh medium before returning the flask to the incubator. Alternatively, after thawing, transfer the cell suspension into a 15 milliliter conical tube for centrifugation and re-suspend the pellet in five milliliters of fresh medium before seeding into a T-25 flask as just demonstrated. To determine whether the cells are ready to be passaged, examine the morphology and confluence of the culture under a microscope.

Considering the cell density and doubling time, the last time the cells were sub-cultured, and any signs of microorganismal contamination in the culture. If the culture appears highly confluent, use ten milliliters of the culture supernatant to gently dislodge the cells from the culture plate bottom and determine the cell density by counting the number of viable cells in the resulting single cell suspension. Sub-culture the cells if the cell density is between five to ten times ten to the sixth cells per milliliter in the appropriate medium.

Add in at least one times ten to the sixth cells per milliliter concentration in a new culture plate to achieve the desired seeding cell density. Then cover and label the plates with the operator initials, date, split ratio, seeding cell density, cell line identifier, medium, passage number, and any medium additions such as antibiotics, and place the plates into plastic container for their continued incubation in the 25 degree celsius incubator. To dislodge adherent cells form a 100 millimeter culture dish bottom, first transfer all of the supernatant into a sterile flask and rinse the cells with a slow addition of one milliliter of 0.05%trypsin EDTA.

Gently swirl to ensure that the trypsin solution covers the entire growth surface before discarding the solution. Next, gently add one to two milliliters of fresh 0.05%trypsin EDTA to the plate and place the plate in the 25 degree celsius incubator for three to ten minutes. When the cell layer can be observed detaching and sliding off of the growing surface, stop the trypsin activity with the addition of nine milliliters of the saved supernatant and mix the cell suspension thoroughly to dissociate the cell clumps.

When all of the cells have been dislodged, the growing surface will be clear. To manually count the cells using a Neubauer cell counting slide, first wipe the surface of a hemocytometer slide and cover slip with 70%alcohol. After mixing, add 15 microliters of the cells into each grooved edge of the hemocytometer to fill both chambers.

The cell suspension will be drawn into the counting chamber by capillary action. Using a 10x microscope objective, count between 100 to 200 cells within the one millimeter squared area in the middle of the grid bound by the parallel lines. Then repeat the enumeration with the second chamber and use the average of the two counts to determine the cell density according to the formula.

For Drosophila cell line cryo-preservation, re-suspend the cell pellet in a volume of freezing medium that will result in a final cell density of at least four times ten to the seventh cells per milliliter. Add an appropriate volume of the cryo-protectant, dimethyl sulfoxide or DMSO, into the cell suspension drop wise such that the final DMSO concentration is 10%and gently mix the cell suspension. Next, carefully add 0.5 milliliter aliquots of the cell suspension into pre-labeled cryo-vials and place the cryo-vials into a freezing container filled with isopropanol.

Then transfer the freezing container into a minus 80 degrees celsius freezer over night to allow the temperature of the cryo-vials to drop slowly to the freezer temperature. Next, quickly transferring the cryo-vials to canes for insertion into a liquid nitrogen canister. Alternatively, transfer the frozen cryo-vials into a pre-cooled freezing box and store the frozen cryo-vials in the liquid phase of a nitrogen freezer.

The confluence of a cell line can be determined by light microscope. Fast growing cell lines that reach confluence early need to be passaged regularly, up to twice a week. In contrast, slow growing cells can be passaged at least once every two weeks or longer, although these cells need to be fed fresh medium every week.

Cell lines derived from varying tissue sources differ in their morphology, adherence properties, media requirements, and doubling times. For quantitative experiments, cell counting is essential. The cells can be counted using a hemocytometer or an automated particle counter.

Cell confluence is a visual guide for when to subculture for the experienced operator. However, cell counting leads to a predictable subculturing schedule and facilitates the detection of growth anomalies. Be careful and use the appropriate protective gear when working with liquid nitrogen.