Best Practices in Cell Counting for Regenerative Medicine

Regenerative medicine has emerged as an innovative means to heal tissues and organs when few other methods work. It relies on our body's ability to heal itself. Stem cell research and cell therapy are major areas of focus for scientists in this field.

However, any scientist working in regenerative medicine understands how relevant and critical cell measurements and characterization are. Cell counting and cell analysis are required for various experiments, quality control, and therapeutic applications.

Assurance in the accuracy of measurements gives greater confidence in downstream processes and better therapy outcomes. Hence, we have compiled a set of best practices for cell counting in regenerative medicine to help scientists achieve better success rates in their workflows.

While there are many techniques for cell counting, such as hemocytometers, flow cytometry, Coulter Principle-based counting, and image analysis, choose a method that best suits your specific needs. Each method has its own advantages and limitations. Thus, factors such as cell type, sample size, and desired accuracy should guide your choice. One could refer to the latest ISO Cell Counting Standard Part 1 for selection of counting methods.

Optimize sample preparation

Needless to say, proper sample preparation is crucial. Pay attention to the timing of sample collection as well. Ensure that your cell suspension is well-mixed and free of clumps. Trypan blue staining or similar viability dyes can help distinguish between live and dead cells. Consistent and accurate pipetting techniques are important to avoid variations in cell concentration.

Calibrate equipment regularly

If you're using automated cell counting equipment, calibrate it regularly according to the manufacturer's instructions. Calibration ensures accurate measurements and prevents errors. Update to the latest available software or parts, and follow maintenance procedures to ensure accuracy.

Pause, verify, and refine

Periodically validate your cell counting method by comparing results with alternate methods or manual counts. This helps identify any discrepancies and maintains the accuracy of your counting technique. Refine your procedures for sample preparation and testing based on results and feedback.

Use proper cell counting techniques

When using a hemocytometer, follow proper counting techniques, such as counting cells in multiple squares and averaging the results. For flow cytometry, set appropriate gating parameters to accurately identify the cell population of interest.

Account for cell aggregation

Some cell types tend to form clusters or aggregates, which can lead to inaccurate counting. Try using enzymatic treatments or gentle pipetting to break up aggregates and obtain a more accurate count. Cell debris may also get counted, leading to inaccurate results. Avoid that by using instruments that enable differentiation of cells from debris or aggregates.

Consider cell viability

In regenerative medicine, cell viability is often critical. Differentiate between live and dead cells using dyes or other methods, especially if you're working with transplanted cells. Proper storage of samples and speedy analysis are also effective measures to ensure viability at the time of analysis.

Standardize protocols

Maintain standardized protocols for sample collection, processing, and counting. Consistency in procedures minimizes variability in results. Strictly follow the manufacturer's instructions for the use of reagents, dyes, duration of tests, etc.

Document and track data

Keep a detailed record of all cell counting procedures, including the method used, equipment settings, and results. This documentation is essential for quality control, troubleshooting, and future reference. It will also help you to ensure reproducibility in future tests or to refine the procedure for more accurate results.

Quality control for cell cultures

Regularly check cell cultures for contamination, growth rate, and overall health. Healthy cultures lead to accurate cell counts and reliable experimental outcomes. Keeping cells free of contamination requires correct procedures in place for storage and handling. Poor quality reagents can also affect the quality of the culture.

Train personnel

Ensure that personnel involved in cell counting are properly trained in the chosen method, instrument, and protocols. Mistakes in counting can lead to inaccurate data, skewed results, and potentially poor outcomes.

Perform cell counting in replicates

Whenever possible, perform cell counting in replicates. This helps identify any variations or inconsistencies in the counting process. Some of this can be reduced with the use of a high-accuracy cell counter, such as one based on the Coulter Principle, that counts 10,000s of cells per sample.

Be aware of limitations

Each cell counting method has its limitations. Understand the limitations of your chosen method and adjust your experimental design accordingly.

Stay updated

Keep abreast of new advances in cell counting technologies and new standards. Newer methods might offer improved accuracy and efficiency for your specific needs.