Protocol: Cell culture

Materials

• VersaLive Starter Kit
• VersaLive chips
• Cell suspension (10 μL/chip or more, 1—5•10⁶ cells/mL)
• Cell medium
• 1X PBS
• Mineral oil for cell culture
• Stereomicroscope

Channel wetting

1. Add 10 μL of PBS in port B of the chip and observe at the stereomicroscope the wetting of all channels up to the remaining ports.
2. If capillary action is not enough to achieve the wetting of all channels:
   1. Fill all ports with 10 μL of PBS
   2. Place the chip in a desiccator
   3. Degas the chip applying vacuum for 15 minutes
   4. Check the result at the stereomicroscope and repeat if necessary
3. VersaLive chips can be sealed with adhesive tape to prevent evaporation and stored at 4˚C for at least one week. Do not use mineral oil before cell loading

This is a good pause point

Cell loading and static cell culture

1. Empty all ports from the PBS used for the channel wetting 
2. Add 10 μL of cell suspension in port B. Cells will start flowing through the main channel and entering the chambers
3. When a given chamber is filled with enough cells, add 10 μL of cell medium to the respective port to stop its filling
4. When all chambers are filled, fill port A with 20 μL of cell medium
5. Remove the cell suspension from port B and rinse it with cell medium or PBS
6. Add 20 μL of cell medium to port B
7. Fill up to 20 μL all chamber ports #1 to #5
8. Add 2.5 μL of mineral oil to each reservoir to prevent the evaporation of the cell medium
9. Place the chip in the incubator at 37˚C and 5% of CO₂ until the cells are adherent to the bottom of the chip (a few hours or days, depending on the cell line)

Perfusion cell culture

Perfusion in single-input mode

1. Remove content from ports #1 to #5 and B
2. Replace content from ports A with suitable inputs (according to experimental plan)
3. Add 2.5 μL of mineral oil in input port A

Perfusion in multi-input mode

1. Remove content from ports A and B
2. Replace content from ports #1 to #5 with suitable inputs (according to experimental plan)
3. Add 2.5 μL of mineral oil in input ports #1 to #5