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SOP-008: Passaging Suspension Mammalian Cells

Purpose​

To maintain healthy suspension mammalian cell cultures by routinely diluting or subculturing cells while preserving viability, optimal cell density, and sterility.

Scope​

This SOP applies to mammalian cell lines that grow in suspension, including hematopoietic, lymphoid, hybridoma, and other non-adherent cell cultures.

Responsibilities​

The operator is responsible for:

  • Monitoring cell density and viability.
  • Maintaining aseptic technique throughout the procedure.
  • Preparing the appropriate complete culture medium.
  • Recording passage information and observations.

Required Materials​

Reagents​

  • Suspension cell culture
  • Fresh complete culture medium
  • Sterile PBS (if required)

Equipment​

  • Class II Biosafety Cabinet
  • Sterile centrifuge tubes
  • Serological pipettes
  • Pipette aid
  • Micropipettes and sterile tips
  • Centrifuge (if required)
  • Inverted microscope

Procedure​

1. Inspect the Culture​

Examine the culture for:

  • Cell density
  • Cell morphology
  • Cell aggregation
  • Evidence of contamination

Proceed only if the culture appears healthy.

2. Determine Cell Density​

Determine cell concentration and viability using the laboratory's approved counting method.

Refer to SOP-004: Cell Counting and Viability Assessment if required.

3. Prepare the Culture​

Gently mix the culture to obtain a homogeneous cell suspension.

Avoid vigorous agitation that may damage cells.

4. Subculture the Cells​

Transfer the required volume of the cell suspension into a new sterile culture vessel.

Add fresh complete medium to obtain the desired cell density.

If required by the protocol, pellet the cells by centrifugation before resuspending them in fresh medium.

5. Mix the Culture​

Gently invert or swirl the vessel to distribute cells evenly.

Avoid excessive shaking.

6. Return to Incubator​

Label the culture vessel appropriately and return it promptly to the incubator.

Acceptance Criteria​

ParameterAcceptance Criterion
Cell suspensionHomogeneous with minimal aggregation
Cell viabilityMeets laboratory requirements for continued culture
Cell densityAppropriate for the cell line
Culture appearanceNo evidence of contamination

Critical Control Points​

  • Mix the suspension thoroughly before sampling or transferring cells.
  • Maintain aseptic technique throughout the procedure.
  • Avoid excessive centrifugation or pipetting that may reduce viability.
  • Use the recommended seeding density for the specific cell line.
  • Minimize the time cells remain outside the incubator.

Documentation​

Record the following:

  • Date
  • Cell line
  • Passage number
  • Cell concentration
  • Viability
  • Split ratio or dilution
  • Medium used
  • Operator initials

Quality Control​

Verify:

  • Cell viability is acceptable.
  • Cell density is appropriate.
  • No microbial contamination is present.
  • Culture vessels are correctly labeled.
  • Fresh medium has been prepared correctly.

Troubleshooting​

ObservationPossible CauseCorrective Action
Excessive cell aggregationInadequate mixingGently resuspend cells before passaging
Poor cell growthCell density outside the recommended rangeAdjust the seeding density during subculture
Low viabilityDelayed passaging or harsh handlingPassage cultures at appropriate intervals and minimize mechanical stress
Cloudy mediumPossible contaminationDiscontinue the culture and investigate the source of contamination

Safety Considerations​

  • Handle all cultures as potentially biohazardous material.
  • Wear appropriate personal protective equipment (PPE).
  • Perform all procedures in a certified Class II Biosafety Cabinet.
  • Dispose of biological waste according to institutional biosafety guidelines.
  • Disinfect work surfaces before and after use.