NEW DebrisIndex™: Monitor production runs
DebrisIndex™ is a new 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 accompanying NC-View™ software.
Implement 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, and key molecule output, DebrisIndex™ indicates the fitness of your bioreactor cell culture.
How DebrisIndex™ works
Conventional automated cell counters cannot distinguish between living cells and cells that are non-viable, but not yet dead, such as apoptotic or denucleated cells. These cells are counted as living cells, since they stain negative for cell counting markers, such as trypan blue. The result is 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 all cell membranes and dead cell nuclei, respectively. The NucleoCounter® NC-202™ automated cell counter with NC-View™ software, records and analyzes staining intensities for a triple readout: Living cells, dead cells, and debris, allowing 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. Depending on the counter’s algorithm settings, specifically the cut-off of cell definition and sensitivity, a conventional automated cell counter will record and analyze these cells as four living cells (yellow nuclei; see figure above) and two dead cells. Certain software algorithms would analyze the same sample and show results as five dead cells (recording grey nucleus and all irregularly shaped cells). Used with its accompanying NC-View™ software, the NucleoCounter® NC-202™ provides more granular results when analyzing non-living cells, as it records one dead cell (round shaped cell, grey nucleus) and also four debris events (irregularly shaped cells), for a DebrisIndex™ of 80 (= debris events/total cell number).
In conclusion, living and dead cells as defined by the NucleoCounter® NC-202™ comes from precisely recording a) intact living cell nuclei; b) dead cell nuclei and c) debris, i.e. anything resembling a cell but without an intact nucleus as debris.
Using DebrisIndex™ in production processes
While over-estimation of the number of living cells may not be an immediate problem, you will get much more reliable results when knowing the actual number of apoptotic/denucleated cells, since the DebrisIndex™ provides a read-out of your cell culture’s overall fitness and performance.
You can truly benefit from the DebrisIndex™ by using it as an additional process parameter to make crucial decisions, taking your cell culture to the next step of your production process. It indicates the fitness of your bioreactor cell culture and supports decision-making at each step along with measurements of cell concentration, live and dead cells and key molecule output.
Bioreactor culture of CHO cells
Development of living and dead cell counts and DebrisIndex™ data in a CHO cell line during 14 days of culture in a bioreactor (fed-batch culture).
The CHO cell culture density grows until a steady state after 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 they slowly rise, indicating that the cell density has reached its maximum capacity and is starting to show distress, i.e. debris.
Starvation culture of HEK293 cells
Development of living and dead cell counts and DebrisIndex™ data in a HEK293F cell line during 18 days of continuous culture under conditions of starvation.
In the HEK293 cell line, an increase in dead cell count and DebrisIndex™ data correlate with a decrease in living 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.
Detecting yeast infection in cell cultures
If the growth patterns of a cell culture vessel are well-defined, DebrisIndex™ can be used to detect yeast infections in a cell culture. Yeast cells are detected as debris by the NC-View™ software. Unusual timing or large fluctuations of DebrisIndex™ for standard production runs will indicate a build-up of debris, possibly contamination, that needs to be further investigated.
On the contrary, observing a minimal and recognizable pattern in your DebrisIndex™, gives you confidence of a successful production process.