Packed Cell Volume is an Overestimate in Common Cancer Cell Lines

Background

Accurate measurements of cell volume are essential for several biological research applications, particularly for normalizing material loading when calculating the absolute concentration of intracellular molecules. Systemic errors in cell volume measurements could therefore skew intracellular molarity calculations and substantially alter interpretations. For example, intracellular concentrations of central carbon metabolites are often near the Km of the enzymes that consume them (Park et al., 2016), meaning that changes in steady-state metabolite levels can proportionally change metabolic fluxes across many metabolic reactions. Thus, systemic errors in cell volume calculations skew metabolite concentration calculations and thereby alter interpretations of metabolic state based on calculated molarities. Accurate cell volume measurements are therefore critical for quantitative measurements of cell content and biological interpretation.

Packed cell volume (PCV) is a common and convenient technique used to measure the total cell volume of a cell suspension. These measurements typically use a microcentrifuge tube with a narrow, calibrated capillary at the bottom. Upon centrifugation, cells pellet in the capillary, and PCV is determined by the height of the cell pellet from the bottom of the tube. This technique has been well validated as an accurate method to measure the volume of red blood cells from blood, but whether it is accurate for cell volume measurements for other cell types has not been widely determined. While red blood cells are smooth and regularly shaped, other cell types have differences in expression of extracellular facing molecules and distinct biophysical properties that may alter packing efficiency.

To evaluate the accuracy of PCV-based measurements in common cell culture cell lines, we compare standard and modified PCV measurement protocols with Coulter counter-based impedance measurements. The Coulter counter is widely accepted as a gold standard method to measure particle volume but is less accessible than PCV since it requires specialized equipment. Direct comparison of these protocols here indicates that standard PCV protocols can lead to an overestimate of cell volume in all tested cell lines compared with Coulter Counter measurements, which can be partially or fully corrected by fast and prolonged centrifugation depending on cell line.