Klapperich Lab:Culture of HCN-1a cells

Overview
This protocol outlines the steps for culturing HCN-1a cells.

Materials

 * T25 culture flasks
 * Collagen-GAG solution
 * HCN-1a media
 * DMEM
 * 4mM L-glutamine
 * 1.5 g/L sodium bicarbonate
 * 4.5 g/L glucose
 * 10% Fetal Bovine Serum
 * TrypLE Express
 * DMSO

Basic Aseptic Technique

 * Sanitize all work surfaces periodically with 10% bleach followed by 70% ethanol.
 * Clean all work surfaces (especially in the biosafety cabinet) with 70% ethanol before use.
 * Change or spray gloves with 70% ethanol after they touch any unclean surface.
 * Spray gloves with ethanol prior to placing objects in/ removing objects from incubator. Be sure to fully dry sprayed gloves before putting hands in incubator.  Ethanol can kill the cells.
 * When working with RNA, change gloves at least every 15 minutes.
 * Wipe down all objects with 70% ethanol prior to placing in biosafety cabinet or RNA work area.

Starting New Cells

 * 1) Coat 4 T25 flasks with collagen-GAG. Pour small amount of fluid into each flask.  Leave in BSC overnight with UV light on.
 * 2) Make a batch of supplemented DMEM as described above. Adjust pH to 7.35.
 * 3) Thaw cells by briefly placing in 37&deg;C water bath.
 * 4) Pipette 250 uL of cells into each T25 tissue culture flask. Add 10 mL media to each flask.
 * 5) As soon as cells attach (overnight), remove original media. Replace with new media.

Subculturing

 * 1) Remove media. Rinse cells with 10mL PBS.
 * 2) Add 3.0 mL of TrypLE Express to flask and observe cells under inverted microscope until cell layer is dispersed (5-15 minutes).
 * 3) *Note: To avoid clumping do not agitate cells by hitting or shaking the flask while waiting for cells to detach. Place flask in incubator at 37&deg;C to facilitate dispersal.
 * 4) Add 6.0 to 8.0 mL of media and aspirate cells by gently pipetting.
 * 5) Transfer cell suspension to centrifuge tube. Spin at 3000 rpm for 5 minutes.
 * 6) Discard supernatant and resuspend cells in fresh media. Aliquot suspension to new culture vessels.
 * 7) Place T25 flasks in incubator at 37&deg;C, 5% CO2.

Freezing cells

 * 1) Remove media. Rinse cells with 10mL PBS.
 * 2) Add 3.0 mL of TrypLE Express to flask and observe cells under inverted microscope until cell layer is dispersed (5-15 minutes).
 * 3) *Note: To avoid clumping do not agitate cells by hitting or shaking the flask while waiting for cells to detach. Place flask in incubator at 37&deg;C to facilitate dispersal.
 * 4) Add 6.0 to 8.0 mL of media and aspirate cells by gently pipetting.
 * 5) Transfer cell suspension to centrifuge tube. Remove 20uL from tube and set aside.
 * 6) Spin at 3000 rpm for 5 minutes.
 * 7) Count cells from 20 uL sample by adding Trypan Blue and viewing on hematocytometer.
 * 8) *Multiply average count by 200,000 to get numbers of cells in pellet.
 * 9) Prepare enough freezing media to dilute cells to 1E6 cells/mL.
 * 10) *75% DMEM with L-glutamine, dextrose, and sodium bicarbonate
 * 11) *20% Fetal Bovine Serum
 * 12) *5% DMSO
 * 13) Discard supernatant and resuspend cells in freezing media to 1E6 cells/mL. Vortex to break up pellet.
 * 14) Aliquot 1 mL of cell suspension into each cryo-vial.
 * 15) Place vials in -80&deg;C freezer in cryo-box lined with a paper towel for 3 hours.
 * 16) Move vials to LN2 storage. Mark location.

Contact
Jessica Kaufman