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Protocols for growing Caco2 cells

Background

Caco2 cells are an ideal model for studying the small intestine which is ironic because they are derived from the colon. Caco2 cells grow relatively slowly and should be cultured for at least 21 days after confluence before doing an experiment to ensure they have differentiated to resemble the SI with a polarized cell layer for transport studies.

Materials

  • DMEM high glucose (11995-065 Life Tech or Sigma D6429-500mL)
  • IMDM (Thermo 12440-053)
  • Fetal Bovine Serum (heat-inactivated)
  • Pen/Strep (Thermo 15140-122)
  • MEM Non-essential amino acids (Thermo 11140050)
  • TrypLE (Thermo 12605010)
  • D-PBS (w/o calcium or magnesium salts, Thermo 14190144)
  • T75 Flask (USA Scientific CC7682-4875)
  • Trypan blue (Thermo 15250061)
  • Millicell ERS-2 or equivalent
  • Millicell Cell Culture Inserts 0.4µM PCF 12mm Diameter- PIHP01250 (400µL internal and 600µL volume external)
  • DMSO

Media Recipes

Caco2 routine growth media

Item Volume
DMEM 500 mL
FBS 50 mL
MEM NEAA 5 mL
Pen/Strep 5 mL

Caco2 Transwell Seeding Media

Item Volume
IMDM 80 mL
FBS (20%) 20 mL
Pen/Strep 5 mL

Caco2 Transwell Growth Media

Item Volume
IMDM 420 mL
FBS (10%) 50 mL
Pen/Strep 4.7 mL

Thawing Preserved Cells

  • Warm up media in 37˚C water bath.
  • Add 15 ml of media to new T75 flask.
  • Obtain cryovial from LN2 storage and thaw as quickly as possible in hand or water bath.
  • Add 1 ml of media to cryovial and aspirate all contents. *No need to centrifuge cells because DMSO will be diluted
  • Add 2 ml of cells plus media to flask.
  • Incubate at 37˚C, 5% CO2.
  • Change with 17 ml of fresh media every other day (depending on color—if yellow tinted, must change). Check confluence under microscope.

Passaging Cells

  • Wait for cells to be ~80% confluent
  • Warm up media, D-PBS, and TrypLE.
  • Add ~5 ml D-PBS to flask and aspirate to wash cells.
  • Add 5 ml trypLE and make sure it covers all cells
  • Incubate at 37˚C for 10-15 minutes, periodically checking under microscope and tapping.
  • Add 15 mL of Caco2 growth media
  • Pipet cells up and down at least five times to mix.
  • Use a splitting ratio of 1:5 (i.e. dilute 5x to seed a new flask)

Freezing:

  • Carry out steps 1-5 of passaging.
  • Transfer cells in media to 15-ml conical. (Should be 4mL from T75 flask per cryofile)
  • Centrifuge at ~500g for 5 minutes.
  • Aspirate supernatant.
  • Resuspend cells in enough freezing media (growth media + 5% DMSO) such that there will be ~1e6 cells (in 1 or 1.5 ml) per cryovial, and aliquot.
  • Store cryovials isopropanol device and after at least 24 hours, transfer to LN2 storage.

Seeding Transwells

  • When cells are 80% confluent
  • Warm up seeding media, D-PBS, and tryple.
  • Add 200 µL of seeding media to each well of the Transwell insert.
  • Remove Transwell insert from reservoir and add 600 ml of seeding medium.
  • Incubate at 37˚C, 5% CO2 for at least 1 hour.
  • Add ~5 ml D-PBS to flask and aspirate to wash cells.
  • Add 5 ml tryple and make sure it covers whole flask.
  • Incubate at 37˚C for 10-15 minutes, periodically checking under microscope and tapping.
  • Add enough media for the volume to be 10 ml total.
  • Pipet cells up and down at least five times to mix.
  • Transfer cells to a conical tube and pellet by centrifugation at 250xg for 5 minutes.
  • Aspirate media and resuspend in seeding media.
  • Remove 10 µL of cells into eppendorf tube and mix 1:1 with Trypan blue.
  • Count cells on hemocytometer slide.
  • Add enough seeding media to cells so that they are at a density of 112,000 cells/ml (3.9x104 cells/cm2), and resuspend well.
  • Add 200 µL of cell suspension to each well in previously prepared Transwell plate.
  • Incubate at 37˚C, 5% CO2.

Feeding Transwells

  • Aspirate media from lower chamber (receiver or reservoir plate).
  • Aspirate media from wells in upper chamber
  • Add at least 400 µL of growth media to upper chamber wells.
  • Add 600µL of growth media to lower chamber wells.

Checking TEER

  • Immerse electrode so shorter tip is inside the insert and longer tip is in the outer well with the longer part just touching the bottom of the plate
  • Also measure a well without any cells in it for a blank bacground.
  • Measure the resistance and adjust to unit area resistance by Resistance (ohms) x effective membrane area which for the 24-well standing insert is 0.6cm2
  • Unit Area Resistance = Resistance (ohms - control) x Effective Membrane Area (cm2)
  • Expecting the TEER to be >250 ohms*cm2