Figure 1.

How well does absorbance correlate with cell density and viability measurements in batch and fed-batch cell culture? As expected in science and nature, the answer depends on a set of factors.

Many people working with batch or fed-batch cell culture will reference discrete offline measures of cell activity. Total cell density (TCD), viable cell density (VCD), and percent viability can be evaluated offline using a hemocytometer and accepted trypan blue exclusion method. Cedex® and Vi-CELL™ are two examples of automated, offline cell counters.

With introduction of inline NIR probes used to monitor cell culture, characterization is provided in real time. A common initiative is to correlate inline measurement with offline results to eliminate duplication and improve controlled response time. If results are viewed as a collection over the entire run, data correlations may appear baffling. Relationships over a complete cell culture run need to be segmented depending on certain conditions.

Condition 1: No Cell Aggregates, Viability Stable

Correlation of near-infrared (NIR) absorbance to TCD can be easily proven by performing a dilution series of cells. In process, as long as cells do not aggregate or yield to apoptosis, good correlation with offline TCD and VCD should be expected (Figure 1).

Figure 1. ()

Conditions 2 and 3: Cells Aggregate (2) and Viability Drops (3)

Offline methods selectively ignore cell aggregates and discount cells identified as nonviable; inline NIR probes report an overall global measurement. Therein lie differences that must be considered in interpretation of the data. If cells start to clump or aggregate, inline/offline TCD and VCD correlations valid to that point need to be reconsidered as the run continues. Established correlation to VCD is affected as viability drops (Figure 2).

Figure 2. ()

When viability declines, absorbance can be used to trend percent viability and viable cell density. Correlation of absorbance to VCD may be dependent on programmed logic in the offline device to count or ignore clumps of cells. Lab methods used to break up aggregated cells may be required to compare VCD with absorbance, however percent viability can be observed by points of inflection in growth rate reported as absorbance. (See Figures 3 and 4)

Figure 3. ()

Figure 4. ()

Real-time analysis of growth rate is made by monitoring slope of the inline absorbance measurement measured over time. Using optek inline absorbance probes, not only to correlate with offline measurements of cell culture performance, but also to characterize what occurs in the bioreactor is a powerful tool for control.