Volumetric mass density measurements of mesenchymal stem cells in suspension using a density meter

Abstract

To use regeneratively active cells for cell therapeutic applications, the cells must be isolated from their resident tissues. Different isolation procedures subject these cells to varying degrees of mechanical strain, which can affect the yield of cell number and viability. Knowledge of cell volumetric mass density is important for experimental and numerical optimization of these procedures. Although methods for measuring cell volumetric mass density already exist, they either consume much time and cell material or require a special setup. Therefore, we developed a user-friendly method that is based on the use of readily available instrumentation. The newly developed method is predicated on the linear relationship between the volumetric mass density of the cell suspension and the volumetric mass density, number, and diameter of the cells in the suspension. We used this method to determine the volumetric mass density of mesenchymal stem cells (MSCs) and compared it to results from the established density centrifugation.

Keywords: Biology experimental methods; Biophysics; Stem cells research.

Graphical abstract

Figure 1

Cell suspension density measurement Volumetric mass density of cell suspension (n = 6) and DPBS plotted (A) against the cell number and (B) against the total cellular volume for donors 1–6. R² of the linear regression (dashed line) increased by plotting against the total cellular volume. The total cellular volume accounts not only for the cell number but also for the diameter of the cells. The better regression underlined the importance of the proper calculation of the average diameter (using a volume average) from the diameter distribution. (C) Volumetric mass density of the cells (n = 6). The fluctuations in the individual cell samples 1–3 for each donor are by factor 3 smaller than the fluctuation over all cell samples of all six donors. It is also noteworthy that the determined cell volumetric mass densities of donor 4 are 0.004 g/cm³ lower than the lowest determined volumetric mass density of the other five donors.

Figure 2

Density centrifugation (A) Total number of cells after centrifugation normalized to total number of cells before centrifugation (n = 6). Shown are the technical replicates of all individual cell samples (black dots) and their median (dashed black line) for the cell pellet and the supernatant (red dots and dashed red line) as well as the average viability of the cells in the pellet (dotted black line) and in the supernatant (dotted red line). (B) Change in the normalized cell number for pellet (black) and supernatant (red) for n = 6. The peak of change occurs both for pellet and supernatant at a volumetric mass density of 1.045 g/cm³.

Figure 3

Measurement principle of DSA 5000 M density meter (Anton Paar GmbH, Austria) The resonance frequency of the oscillation of the glass tube is dependent on the density of the fluid in the glass tube. For a cell suspension, it is a mixture of a continuous fluid (the cell suspension medium) and a dispersed phase (the cells) that displaces the medium.

Figure 4

Volumetric mass density, dynamic viscosity, osmolality, and pH of different DPBS-LSM mixtures The exact fluid properties are listed in Table S2.

Figure 5

Concept of density centrifugation With the volumetric mass density of the cells being higher than the volumetric mass density of the DPBS (1.0052 g/cm³) that carries the cells, they always sink to the interface of DPBS and DPBS-LSM during centrifugation. (A) If the cell’s volumetric mass density is lower than the volumetric mass density of the respective DPBS-LSM mixture, the cells will remain at this interface. (B) If the volumetric mass density of the cells matches the volumetric mass density of the DPBS-LSM mixture, the cells will disperse throughout the DPBS-LSM mixture nearly homogeneously. (C) If the cells have even a higher volumetric mass density than the DPBS-LSM mixture, they will transfer through the DPBS-LSM and sink to form a pellet at the bottom of the centrifugation tube.