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

Procedure

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°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).
   • 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°C to facilitate dispersal.
3. Add 6.0 to 8.0 mL of media and aspirate cells by gently pipetting.
4. Transfer cell suspension to centrifuge tube. Spin at 3000 rpm for 5 minutes.
5. Discard supernatant and resuspend cells in fresh media. Aliquot suspension to new culture vessels.
6. Place T25 flasks in incubator at 37°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).
   • 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°C to facilitate dispersal.
3. Add 6.0 to 8.0 mL of media and aspirate cells by gently pipetting.
4. Transfer cell suspension to centrifuge tube. Remove 20uL from tube and set aside.
5. Spin at 3000 rpm for 5 minutes.
6. Count cells from 20 uL sample by adding Trypan Blue and viewing on hematocytometer.
   • Multiply average count by 200,000 to get numbers of cells in pellet.
7. Prepare enough freezing media to dilute cells to 1E6 cells/mL.
   • 75% DMEM with L-glutamine, dextrose, and sodium bicarbonate
   • 20% Fetal Bovine Serum
   • 5% DMSO
8. Discard supernatant and resuspend cells in freezing media to 1E6 cells/mL. Vortex to break up pellet.
9. Aliquot 1 mL of cell suspension into each cryo-vial.
10. Place vials in -80°C freezer in cryo-box lined with a paper towel for 3 hours.
11. Move vials to LN2 storage. Mark location.

Contact

Jessica Kaufman