Home / Wet Lab

Splitting CaCo2 Cells

Use this page when cells have reached confluence and are ready to be split.

Purpose

When cells have reached confluence, it is important to split cells to maintain their growth. It is also important to keep a constant stash of live cells in case of contamination.

CaCo2 cells look confluent when there is clear tight junction formation, cell morphology changes into non-circular shapes, and the entire monolayer is seeded. Extra cell debris and dead cells, dark spots from doming, and movement of particulates on the surface of the monolayer are all additional indicators.

Safety

• Complete work in the BSC.
• Spray down all materials brought into the BSC with 70% Ethanol.
• Try not to touch the sides of the flask opening with pipette tips.
• Do not touch the rims of the flasks or tubes to work with.

Materials

• Trypsin
• DMEM Complete Cell Culture Medium
• Aspirator pipette
• Motorized pipette/large 15mL pipette.
• Centrifuge eppendorf tube (+ balance eppendorf)
• Centrifuge
• 37°C Water Bath
• Fresh cell culture flasks

Procedure

1. Warm up an alliquot of DMEM complete cell medium and trypsin for 15 minutes before beginning.
2. Use vacuuation pipette (tips in metal container) to aspirate the media from the culture flask containing the confluent cells.
3. Spray down motorized pipette with 70% Ethanol and pipette 2mL of trypsin into the flask, gently tilting and spreading the solution over the cells.
4. Place in the incubator for 2-10 minutes, until all of the cells are detatched (as indicated by the waterfalling of cell clumps on the base of the flask).
5. Once all cells are fully detached, add 3mL of DMEM media to the culture flask. Suspend the cells in the solution. Aspirate and release multiple times to wash the base of the flask evenly until fully clear.
6. Collect the complete solution from the bottom of the flask and place in a centrifuge eppendorf tube. Centrifuge with a balance for 4 minutes at 0.4 rcf.
7. Once complete, verify that there is a pellet at the base of the tube containing the cells. Aspirate the supernatant from the tube using the vacuum pipette, leaving the pellet.
8. Resuspend the cells in roughly 2-3mL of complete DMEM media per new flask. (ie. if splitting into 3 flasks, use ~6mL to resuspend). Make sure no clumps remain and the solution is homogenous.
9. Pipette 10-11mL of complete DMEM media into each new flask. The total volume per flask including cells/media should be 12-15mL.
10. Spread the media containing the resuspended cells throughout the flask by sliding the flask in a figure 8 motion on the surface of the table.
11. Place in the incubator.

Quality Control

• Check the clarity of the supernatant and the visibility of the pellet.
• Make sure to complete the methods quickly and accurately.
• Confirm all solutions to be used with the cells is adequately warmed-up.
• Follow standard sterile protocols.

Revision History