Monitor Production Runs for Better Process Control
DebrisIndex™ is a tool for monitoring production runs, providing a read-out of the amount of debris in your culture, relative to your cell concentration. The tool is a feature of the NucleoCounter® NC-202™ and its accompanying NC-View™ software.
Include DebrisIndex™ as an additional process parameter for making key decisions: When do you take your cell culture to the next step of the production process? Along with measurements of cell concentration, live and dead cells, DebrisIndex™ indicates the fitness of your bioreactor cell culture.
How DebrisIndex™ Works
Conventional automated cell counters cannot easily distinguish between living cells and non-viable cell material such as apoptotic bodies or denucleated cells. This cell material is counted as total cells, as they may morphologically resemble intact cells, resulting in an overestimation of the total number of cells.
DebrisIndex™ makes use of the powerful cellular stains, acridine orange (AO) and 4′,6-Diamidino-2-Phenylindole (DAPI), which stain cellular material and dead cell nuclei, respectively. The NucleoCounter® NC-202™ automated cell counter with its NC-View™ software, records and analyzes staining intensities for a triple readout: Viable cells, dead cells, and debris. In addition, it allows for multiple useful parameters: Cell culture viability, cell concentration, cell size (diameter), aggregation percentage, and DebrisIndex™.
Assume that in a cell culture, there are nine cells in total, as shown as an example in the diagram. Depending on the cell counting method, these cells may be analyzed as four viable cells (shown as yellow nuclei) and two dead cells. Certain software algorithms would analyze the same sample and show results as five dead cells (recording the grey nucleus and all irregularly shaped cells). Mistaking debris for dead cells will result in an increase in cell number even if your cell culture has a low viability.
The NucleoCounter® NC-202™ provides more detailed results when analyzing non-viable cells, as it records one dead cell (shown as the round shaped cell, with a grey nucleus) and four debris events (the irregularly shaped cells), for a DebrisIndex™ of 80 (= debris events/total cell number × 100).
In conclusion, viable and dead cells are more precisely defined by the NucleoCounter® NC-202™ as a) intact viable cell nuclei; b) dead cell nuclei and c) debris, i.e. anything resembling a cell but without an intact nucleus.
Using DebrisIndex™ in Production Processes
You will get more reliable results when separating the dead cells from apoptotic bodies and other cell-like cellular remains. The DebrisIndex™ provides a read-out of your cell culture’s overall fitness and performance. You can therefore benefit from the DebrisIndex™ as an additional process parameter to help you take your cell culture to the next step in the production process.
Starvation Culture of HEK293 Cells
The usefulness of the DebrisIndex™ is best demonstrated in a HEK293 cell culture; an increase in dead cell count and DebrisIndex™ data correlate with a decrease in the viable cell count. This indicates that the cell culture growth has reached its growth maximum and nutrient competition and cell debris is inhibiting further cell growth.
The diagram shows the development of viable and dead cell counts and DebrisIndex™ data in a HEK293F cell line during 18 days of continuous culture under conditions of starvation.
Bioreactor Culture of CHO Cells
The DebrisIndex™ can also be applied in high viability cell cultures. The CHO cell culture density grows until a steady state is reached after the first eight days in culture. The dead cell count increases slowly throughout the culture period but remains low due to good culture conditions. DebrisIndex™ data are not detectable until day 12, at which point the dead cell count slowly rises, indicating that the cell density has reached its maximum capacity and cells are starting to break down, evident through the emergence of debris.
The diagram shows the development of viable and dead cell counts and DebrisIndex™ data in a CHO cell line during 14 days of culture in a bioreactor (fed-batch culture).
Detecting Yeast Infection in Cell Cultures
If the growth pattern of a cell culture is well-defined, DebrisIndex™ can be used to detect yeast infections. Unusual timing or large fluctuations in the DebrisIndex™ in standard production runs will indicate an irregular build-up of debris, which may stem from cell culture contamination and further investigated is needed. On the contrary, when observing a minimal and recognizable pattern in your DebrisIndex™, you can have confidence that your production process is a success.