Cell Isolation tutorial


Cell Isolation Procedure


The isolation technique aims to isolate cells without unduly damaging cell membranes. Since enzyme activity is enhanced and less specific in the presence of divalent cations, all calcium and magnesium are removed from the culture flask before enzyme addition. Once cells are released from the flask bottom, the enzyme must be quenched by simultaneously diluting it with culture medium and occupying it with serum proteins to prevent excessive digestion of cell membranes. Centrifugation is then performed to remove the enzyme. All steps of this procedure should be carefully timed and carried out continuously in order to obtain optimal results.


The voltage-clamp technique requires formation of gigaohm seals to achieve high quality, high fidelity recordings without voltage escape. To achieve this, the cells must have clean membranes able to approach the rim of the voltage clamp aperture. In addition, the resulting cell suspension must be free from cell fragments and other debris that often results from cell harvesting. Harvesting cells for voltage clamp experiments is therefore a more cautious one than that used simply for fractional passaging of cells into a new flask for cell growth. This harvesting technique has proven less universal. We have experienced best results with two different procedures, each specific for either Chinese Hamster Ovary cells (CHO-K1) or Human Embryonic Kidney cells (HEK-293). The details follow.


Note that when storing cells in the incubator, the cell culture flasks have caps that are permeable to the passage of air (O2/CO2) through a molded-in filter, however the 50 mL tubes typically do not contain the filter-fitted gas permeable caps. In this case, the cells that are stored in the incubator within the 50mL tubes will die unless the cap of the tube is left very loose to allow air exchange.





Best Method for CHO Cell Lines


Note: For best performance, selective antibiotics should be removed from culture media 24 hours prior to use in experiments. Isolation medium contains: DMEM/F12 (Invitrogen #11330), 10% FBS (Invitrogen #10082) & 1% penicillin/streptomycin (Invitrogen #15140). The following amounts are for isolating cells from culture flasks with 75cm2 growth area.


  1. Warm medium to 37°C prior to isolating cells.
  2. View cells under microscope to ensure cell health and 70-85% confluence.
  3. Aspirate medium from culture being careful not to disturb cells.
  4. Add 10 mL of DPBS without Ca2+ and Mg2+ (VWR #82020-066).
  5. Tilt 1 to 3 times gently to wash the cell monolayer.
  6. Aspirate the DPBS.
  7. Repeat steps 4 through 6.
  8. Add 9 ml of DPBS and 3 ml of Accumax (Innovative Cell Technologies #AM105) to the flask. Tilt gently 1-3 times to mix and cover the cell monolayer.
  9. Leave flask undisturbed for 1 minute at room temperature.
  10. Aspirate the supernatant, repeat step 8 and incubate flask at 37°C for 2 minutes.
  11. View under a microscope to ensure that >95% of cells have dislodged from the flask bottom.
    1. Cells will have a round appearance and can be seen floating in solution when they have dislodged from the flask bottom.
    2. If needed, leave cells to incubate at room temperature until they fully dislodge, checking every 15-30sec.
  12. Add 9 mL of medium to stop enzyme digestion.
  13. Triturate cells to mix and dissociate clumps. Transfer to a 50 mL centrifuge tube.
  14. Centrifuge at 1000 rpm for 2 minutes.
  15. Carefully aspirate supernatant without disturbing the cell pellet.
  16. Re-suspend cells in medium to a final concentration of 0.5 x 106 cells/mL, or approximately 5 mL.
  17. Incubate cells at 37°C and 5% CO2 until ready to use in experiments.
    1. Allow 30-minute recovery time before use in electrophysiology experiment.
    2. To prevent excessive rundown, cells should not be used longer than four (4) hours after time of isolation.




Best Method for HEK Cell Lines


Note: For best performance, selective antibiotics should be removed from culture media 24 hours prior to use in experiments. Isolation medium contains: DMEM/high glucose (Invitrogen #10569), 10% FBS (Invitrogen #10082) & 1% penicillin/streptomycin (Invitrogen #15140). The following amounts are for isolating cells from culture flasks with 75cm2 growth area.


  1. Warm medium to 37°C prior to isolating cells.
  2. View cells under microscope to ensure cell health and 70-85% confluence.
  3. Aspirate medium from culture being careful not to disturb cells.
  4. Add 10 mL of DPBS without Ca2+ and Mg2+ (VWR #82020-066).
  5. Tilt 1 to 3 times gently to wash the cell monolayer.
  6. Aspirate the DPBS.
  7. Repeat steps 4 through 6.
  8. Add 9 ml of DPBS and 1 ml of trypsin (Invitrogen #25300) to the flask. Tilt gently 1-3 times to mix and cover the cell monolayer.
  9. Leave flask undisturbed for 1 minute at room temperature.
  10. Aspirate the supernatant, repeat step 8 and incubate flask at 37°C for 2 minutes.
  11. View under a microscope to ensure that >95% of cells have dislodged from the flask bottom.
    1. Cells will have a round appearance and can be seen floating in solution when they have dislodged from the flask bottom.
    2. If needed, leave cells to incubate at room temperature until they fully dislodge, checking every 15-30sec.
  12. Add 9 mL of medium to stop enzyme digestion.
  13. Triturate cells to mix and dissociate clumps. Transfer to a 50 mL centrifuge tube.
  14. Centrifuge at 1000 rpm for 2 minutes.
  15. Carefully aspirate supernatant without disturbing the cell pellet.
  16. Re-suspend cells in medium to a final concentration of 0.5 x 106 cells/mL, or approximately 5 mL.
  17. Incubate cells at 37°C and 5% CO2 until ready to use in experiments.
    1. Allow 15-minute recovery time before use in electrophysiology experiment.
    2. To prevent excessive rundown, cells should not be used longer than four (4) hours after time of isolation.




Feedback Adjustments:


ProblemSymptoms
Over digested cells (Too long in presence of enzyme.) Seen on PatchXpress as early prematures, lost seals, access resistance < 60 torr and/or poor duration.
Under digested cells (Enzyme isolation activity stopped too soon.) Seen on PatchXpress as slow or no seals and/or late prematures.
Pelleted too long Blebs visible on cell membrane when viewed under microscope; PatchXpress will show lost Rm, no seals, lost seals or seals <100mΩ.
Cell attraction pressure Decrease pressure when prematures are present; increase when prematures are infrequent. Setting can be adjusted in Patch Settings, Step 3.3.
Ra correction Turn on Ra pneumatic optimization in Step 6.2 of Patch Settings when Ra creeps upward. Turn off if loosing cells.