Harvesting cells from multilayer CellSTACK culture chambers that do not allow direct pipette access to the cells can be challenging. The inability to view the cells microscopically when working with the larger size (10 and 40 layer) vessel adds to the challenge. This protocol covers some basic techniques and suggestions for harvesting cells from CellSTACK culture chambers. For optimal cell yields and viability, it is important to optimize the harvesting protocol for each cell line.
Note: A phosphate-buffered saline is used for rinsing since it maintains a physiological pH without requiring a closed system (required by buffers based on Hanks’ saline) or gassing with carbon dioxide (required by buffers based on Earle’s saline). Calcium and magnesium are omitted because these play an important role in cell attachment.
The recommended amounts of CMF-PBS and dissociating solution are starting volumes only. If cells are relatively easy to harvest from the chambers, then the amounts used for future harvests may be reduced by up to 50% or more.
Cell Harvesting Procedure
The goal is to remove the cells from the plastic substrate and break cell-to-cell bonds as gently as possible. There are several variations of this protocol dependent on the methods (pouring, pumping, etc.) you prefer for removing and adding solutions to the Corning CellSTACK chambers. This protocol is designed for harvesting cells from a 10-layer CellSTACK chamber; amounts used for other chamber sizes should be changed accordingly. We recommend growing additional cell cultures under identical conditions (cell density and medium amounts) in a companion vessel (a flask or CellSTACK 1-Stack chamber) as an aid for monitoring both cell growth and harvesting.
a. Add the recommended volume of CMF-PBS to the 10-layer CellSTACK chamber. Recap tightly and distribute the CMF-PBS equally to each chamber layer by laying the vessel on its longest side (liquid will come in contact with cap).
b. Once the liquid has equilibrated, stand up the vessel to separate the layers, then slowly set vessel down to incubation position vessel. Tilt the chamber back and forth in both directions to thoroughly rinse each layer and remove all traces of the old medium.
c. Remove and discard the wash solution. A second rinse with CMF-PBS is highly recommended for cells that are difficult to harvest.
a. Add the recommended volume of prewarmed dissociating solution to the chamber and distribute equally to each chamber by following rinsing steps. Prewarming of the dissociating solution will decrease the required exposure period.
b. Tapping on the chamber’s sides may help detach the cells from the surface.
a. For easy to remove cells, this rinse step can be done with CMF-PBS or medium which is then added to the cell suspension from the first dissociation. If a substantial number of viable cells are found in the rinsing solution, then a second rinse should be done or the dissociating solution or harvesting procedure may need to be adjusted. (See step b below.) If the rinsing solution contains few viable cells, then this step can be omitted in the future.
b. For very difficult to remove cells, rinsing should be done with additional prewarmed dissociating solution. It may be necessary to incubate the cells for a few minutes to give the solution time to act on the remaining attached cells. If a substantial number of viable cells are found in the second harvest, then the original dissociating solution or harvesting procedure may need to be adjusted. If the second harvest contains few viable cells, then this step can be omitted in the future.
a. check if dissociating solution was prewarmed;
b. increase the concentration, volume or incubation time of the dissociating agent;
c. add a chelator, such as EDTA, to the dissociating solution;
d. add an additional washing step with CMF-PBS either prior to adding, or after removing the dissociating solution.
If cell viability is low, you need to re-examine the harvesting process to determine if:
a. the dissociating solution is too harsh;
b. the process for removing the cell suspension is causing damage;
c. the centrifugation step is too hard or too long;
d. the dissociating solution is not being removed or inactivated.