Preparing a Cell Pellet

Overview

Preparing a cell pellet is a necessary step for a variety of experimental methods, including encapsulating cells in a bioink, cell counting and cell passaging. This protocol will walk you through the process of preparing a cell pellet. 

Materials

• Cell Flask
• Cell Culture Medium
• ReagentPack™ Subculture Reagents
  • Trypsin/EDTA
  • Trypsin Neutralizing Solution (TNS)
  • HEPES Buffered Saline Solution (HBSS)
• Centrifuge Tubes 
• Centrifuge
• Micropipettes
• Micropipette Tips
• Microscope
• Incubator (37˚C, 5% CO₂, 90% humidity)
• Timer
• 70% Ethanol
• Kimwipes
• Vacuum Bottle

Methods

1. Warm cell culture medium and ReagentPack™ reagents to 37˚C in the water/bead bath;
2. Spray them down with ethanol and place them inside a biosafety cabinet (BSC);
3. Spray down incubator doors with ethanol and minimize breathing to diminish contamination risk;
4. Remove cell culture flasks from incubator and place them under microscope to ensure there is no contamination and to check for confluency;
   • Note: It is good practice to use cells that are ~80% confluent to ensure a high yield.
5. Spray down flasks with ethanol, wipe them with Kimwipes and place them in BSC;
6. Aspirate cell media from culture flasks, ensuring to avoid touching the bottom of the flask;
7. Wash flask with HBSS, using the amount needed to cover your cell culture flask surface;
8. Aspirate HBSS;
9. Add trypsin/EDTA to the flask, using the amount needed to cover your cell culture flask surface (write down this volume);
   • Note: the recommended volume/flask surface area is 0.5 mL/10 cm².
10.  Gently rock your flask and place it back into the incubator for 5 minutes;
11.  Check cells under the microscope to see if they have released from the bottom of the flask (are floating). If so, go to the next step. If not, spray the flask down with ethanol, wipe it with Kimwipes and place it into the incubator for another 5 minutes. Repeat this step until cells are released, ensuring to not exceed 15 minutes of trypsin/EDTA exposure, as it may be toxic to cells;
12.  Add the same volume of TNS to your flask as trypsin/EDTA in order to neutralize trypsin/EDTA activity;
13.  Pipette cell suspension into a centrifuge tube; 
14.  Centrifuge for the time and speed indicated for you cell type;
    • Note: usually, centrifugation time is 5-10 minutes, and speed is 100-200 X g. 
    • Note: to convert from g to rpm, use the following formula: g = 1.12 x rotor radius (in mm) x (rpm/1000)²
15.  Spray down your centrifuge tube with ethanol and wipe it with Kimwipes before going back into the BSC;
16.  After centrifuging, you should be able to see a cell pellet at the bottom of the flask. Gently aspirate supernatant, ensuring to not disturb the pellet;
17.  Your cells are now ready for the cell-counting protocol, cell encapsulation protocol, or cell-bioink mixing protocol.