How does cell viability impact Cytos™ performance?

Question

How does cell viability impact Cytos performance on the AVITI24™ system?

Answer

Cell viability, or the proportion of live, healthy cells, is an important factor to consider in cell culture optimization for Cytos runs. Greater than 85% cell viability is ideal as a general baseline for Cytos runs. Low cell viability can lead to altered morphology or high background in Cytos runs.

Summary

This article covers current guidelines and frequently asked questions regarding cell viability for immortal cell lines, including:

• Methods to determine cell viability
• Acceptable levels of cell viability for a Cytos run
• Impacts of sub-optimal cell viability
• Guidance to improve cell viability

Considerations

Methods to Determine Cell Viability

Viability assessment combines two methods: a dye or marker that distinguishes live cells from dead cells, and a counting method that quantifies cells. For routine pre-run checks, combine Trypan Blue dye exclusion with an automated cell counter. Measure viability as close to seeding as possible because readings can shift between counting and plating.

Dyes or Markers

• Trypan Blue exclusion: Live cells exclude the Trypan Blue dye, while dead cells stain blue. This method is inexpensive, fast, and widely compatible. Read within 3–5 minutes of staining to avoid underestimating viability.
• Fluorescent viability dyes (AO/PI [Acridine Orange/Propidium Iodide], calcein AM/EthD-1): These methods are more accurate in samples with heavy debris or for cell types where Trypan Blue is unreliable and require a fluorescence-capable counter or imager.

Counting Methods

• Manual counting by hemocytometer: Using 10 µl of sample, count the number of cells within a 1 mm² subgrid on a gridded slide. This method can be used in conjunction with a dye like Trypan Blue to count live and dead cells.
• Automated cell counter: Recommended for routine Cytos sample preparation. Check that your sample falls within the counter's concentration and cell-size ranges.
• Flow cytometry: Provides high accuracy with fluorescent viability dyes. Counts thousands of cells per sample and can assess additional markers simultaneously. This method is useful when characterizing a new cell line or troubleshooting persistent viability issues, but is not generally needed for routine checks.

When to Proceed with an Optimization or Cytos Run

Optimal cell viability varies by cell type. General recommendations for a Cytos run include:

• > 85% viability is ideal as a general baseline
• > 90% viability for Control wells for experiments with treatment factors
• For drug discovery experiments, aim for LC₃₀-LC₅₀ to help determine treatment conditions

Impacts of Sub-Optimal Cell Viability

Sub-optimal cell viability can result in fewer cells seeded and adhered to a Cytos Slide Kit leading to lower confluency and counts than expected.

Lower confluency can cause higher background during cell membrane and nuclear staining due to residuals left from non-adhered cells, reducing visual quality of the cell paint features. The high background may not always impact segmentation. Additionally, dead cells do not produce usable data.

Guidance to Improve Cell Viability

Always employ best practices for cell culture while maintaining optimal conditions for your specific cell line. You can review the user guides and cell culture protocols for general recommendations. Potential causes for reduced viability and tips for avoiding are as follows:

Culture Conditions

• Monitor osmolality: Drift in osmolality can stress cells significantly.
• Monitor environment: Ensure your incubation environment provides an accurate recording of CO₂ and humidity to identify sources of cell stress. Perform CO₂ and humidity calibration checks on incubators, particularly if multiple users share equipment.
• Mycoplasma testing: Contamination can reduce viability.
• Passage number: High passage numbers have higher cell death, resulting in more cell debris.

Media and Reagents

• Monitor media age and storage: Glutamine degrades over time, and expired or improperly stored media can reduce viability.
• Review antibiotic use: Antibiotic use can mask contamination and stress cells.

Handling

• Incubation: Minimize the time cells spend outside the incubator during passaging.
• Trypsin exposure time: Use the shortest exposure time possible to detach cells. Over-trypsinization is a frequent cause of reduced viability.
• Check centrifugation speed and duration: Choosing a centrifugation speed that is too aggressive can damage cells mechanically. Use soft/slow deceleration and brake-off settings on your centrifuge.

Seeding

• Seeding density: Too sparse or too dense seeding density for your cell line can cause viability issues.
• Temperature: Check the temperature of media and surfaces at the time of seeding.
• Recovery: Provide sufficient recovery time and passages for the cell line, including post cryo-preservation to ensure live cells adhere well to the Cytos Slide Kit.

Rescuing Low Viability

• Remove dead cells: Extra washes can help to remove dead cells.
  • FACS (fluorescence-activated cell-sorting): Use fluorescent markers to separate live cells from dead cells.
  • MACS (magnetic-activated cell-sorting): Use magnetic beads to separate live cells. Remove beads before proceeding with a Cytos run.
• Centrifugation to remove debris: When passaging cells, include an extra centrifugation step after resuspending the cells in media or PBS to help remove additional cell debris.