Can frozen cells be used for SeekOne DD 3' scRNA-seq, and what are the best practices?

Answer:
Yes, frozen cells can be used, provided that appropriate cryopreservation procedures are followed.

Direct freezing of whole blood or cell pellets without cryoprotectants is not recommended, as this can damage cell membranes and significantly reduce cell viability.

Recommended cryopreservation workflow:

1. Cell collection:
   After red blood cell lysis, centrifuge the cell suspension at 300 × g for 5 minutes at 4°C. 
2. Resuspension:
   Remove the supernatant and resuspend cells in cryopreservation medium (commercial solution or 90% FBS + 10% DMSO) at a concentration of 1 × 10⁶ cells/mL. 
3. Controlled-rate freezing:
   4°C for 30 minutes → −20°C for 30 minutes → −80°C for 24 hours → transfer to liquid nitrogen for long-term storage.
   (The protocol may be optimized depending on cell type.) 

Best practices for thawed cells:

• Post-thaw viability is often reduced. Removal of dead cells and debris is recommended using methods such as FACS or dead cell removal kits (e.g., Miltenyi, #130-090-101).
  However, for positively selected samples (e.g., CD45⁺ cells), certain dead cell removal protocols (e.g., Miltenyi, #130-090-101) may not be suitable.  
• Wash cells thoroughly to remove residual cryoprotectants, which may interfere with downstream reactions. 
• Minimize the time between thawing and chip loading to prevent premature cell lysis. 
• Resuspend cells in a buffer containing 0.04% BSA or another validated buffer. 
• Perform cell counting and quality control prior to chip loading to confirm viability and concentration, and to reduce clogging risk. 

Important considerations:

• A decrease in viability and recovery is expected after freezing and thawing. 
• Cell loss of approximately 50% or more is common, depending on initial sample quality. 

Therefore, a higher starting cell number is recommended to compensate for this loss.