Using best practices at every step of cell isolation, starting from PBMC separation and characterisation to cryopreservation and thawing, will maximise the viability and functionality of isolated cells, contributing to more consistent and reliable results.
Optimal PBMC isolation
Density gradient centrifugation is the most widely used method to isolate PBMCs, but it needs to be optimised to provide ideal separation for the blood source. Centrifugal acceleration, duration and density gradient all contribute to a successful PBMC isolation and require proper optimisation to reduce variability and increase yield.
In addition, the removal of contaminating components, such as red blood cells and platelets, is essential to maximise the purity of a PBMC isolation. Removing these components eliminates interference or artefacts that can hamper downstream applications.
Essential automated characterisation
The use of dependable, automated characterisation eliminates inconsistencies that can arise from subjective, user-dependent methods. Using manual methods such as a haemocytometer and trypan blue exclusion to count viable cells is an accepted method … but can be both laborious and prone to errors (owing to dilutions or an untrained eye). Employing automated cell counters or flow cytometers can remove operator bias.
After isolating cell subsets such as T cells or B cells, it is best practice to measure the purity of the isolated population to ensure that the correct subset has been isolated at an acceptable purity level. Evaluating cells with differentiation markers in combination with a flow cytometer, or another advanced quantitative method, is essential for the proper characterisation of cell subsets.
Proper cryopreservation and thawing
Using cryopreserved cells is often mandatory in applications when consistency is needed and large batch sizes are required across multiple assessments or time points. PBMCs should ideally be cryopreserved with an appropriate cryoprotective medium at a controlled freezing rate, typically about 1°C per minute. Proper controlled freezing prevents the formation of lethal ice crystals that can mechanically damage cells and reduce their viability.
Appropriate thawing procedures are equally important and essential to preserving cell viability. Standardising thawing procedures will eliminate variability and reduce the potential for compromised sterility. Overall, starting bioprocessing applications with meticulously prepared and characterised PBMCs and cell subsets eliminates variability and simplifies root cause analysis for downstream applications.
