Prior to thawing the cells, prepare cell growth medium (GM) for use.
NOTE: Schneider’s Insect growth medium is light-sensitive. Please handle the media and the cell line culture in the dark. Failure to do so may result in the death of the cells.
Cu. sonorensis cell line CuVaW8a is grown in a customized media as follows:
1. Measure 400 mL of Schneider’s Insect Medium (Sigma-Aldrich S0146-iL) into a large beaker and add a stirring rod.
2. Add 3 mg sodium bicarbonate
3. Add 3 mg reduced glutathione (Sigma-Aldrich G6013)
4. Add 15 mg L-asparagine (Sigma-Aldrich A4159)
5. Add 9 µl of 10 mg/mL bovine insulin, pH 8.2 (Sigma-Aldrich I0516)
6. Adjust pH to approximately 6.7 first by adding NaOH dropwise, until the pH is 9.2, then adding HCl dropwise until pH is 6.7
7. Filter through 0.2 µm membrane
8. Add 88 mL heat-inactivated insect-tested fetal bovine serum (Sigma-Aldrich F4135) (MUST USE INSECT TESTED FBS) to complete the media
This GM is formulated for use with 5% CO2 in air atmosphere.
Rapidly thaw the vial of cells in a 37°C water bath with gentle agitation. To reduce the risk of contamination, keep the cap and O-ring of the vial out of the water and repeatedly check the cap for tightness during thawing. Remove from the water bath immediately when thawed. Dry the vial with a sterile wiper, decontaminate using a wiper soaked with 70% isopropyl alcohol, and let the vial air dry. Aseptically open the vial, remove the vial contents, and add to 4 mL of GM in a centrifuge tube. Centrifuge the cell suspension at 125 to 200 × g for 8 to 10 minutes at 18 to 25°C. Discard the supernatant and resuspend the cell pellet in 10 mL of pre-warmed GM. Transfer the cell suspension into a 75 cm2 tissue culture flask. Incubate the new culture at 27°C and 5% CO2. Incubate until the cell sheet is approximately 80% confluent.
Sub-culture procedure:
Aseptically remove the GM and discard. Briefly rinse the cell layer with 4 to 15 mL of Ca2+- and Mg2+-free Dulbecco’s phosphate-buffered saline (PBS) to remove all traces of serum. Discard the PBS. Add 2 to 8 mL of 0.05% trypsin-EDTA to the culture flask and incubate the flask at 37°C until cell layer is dispersed (usually within 3 minutes but no longer than 15 minutes). Note: To avoid clumping, do not agitate the cells by hitting or shaking the flask. Add an equal volume of GM and aspirate cells by gently pipetting. Once a week, passage at 1:10 split ratio. Adjust the volume of GM to 15 to 20 mL for a 75 cm2 flask. Incubate cultures at 27°C and 5% CO2 until the cell sheet is approximately 80% confluent.