Viable Cell Density monitoring in bioreactor

Introduction

Monitoring cell density and viability of mammalian cell culture bioreactors is a necessary task that presents today a number of remaining challenges. Traditional measure for cell count and cell viability still relies on sampling and staining protocol where the trypan blue exclusion method is performed once a day. While automatic cell counters have reduced the statistical error of the original manual method, daily sampling is still a challenge for small scale bioreactor because the sampled volume becomes significant.

NORMA analyzers integrate a new breakthrough method for accurately determination of cell concentration and viability without staining nor sample preparation or dilution. At first, we demonstrate, how the measurements correlate with a reference counter from different bioreactor samples. We assess the very high reproducibility of such technique with a low sample volume of 3 µL.

Material and methods

NORMA technique directly acquires the light diffraction properties of each individual cells through their hologram images without any objective, lens or focus settings. Living and dead cells have significant holographic patterns that can be distinguished and accurately counted. We compare cell counts and viability between the reference method and our lensless imaging device called the NORMA XS. Measures are performed once a day on samples from 12 bioreactors, from the inoculation to the end of the culture. We also assessed the repeatability of our method.

Results

With a concentration range up to 40 x 10^6 cells /mL and viability range at 75-100%, we obtained a correlation factor of 0.98 between the two compared methods. The large field of view allows the analysis of several thousand cells within a single image, keeping the statistical variability of the measure as low as 3%.

Conclusion

NORMA technology is capable of accurately monitoring VCD and viability with a combination of significant advantages like low sample volume, label free detection, quick measure, simple device which let us think that it is a good candidate for very small-scale bioreactor and high-throughput measures. Its high repeatability is also a key parameter in the effort to narrow batch to batch deviations. We envision NORMA to become the future method of choice for on-line monitoring of parallel suspension cell culture. It will be a perfect tool for process control in fed-batch or perfusion mode in single-use bioreactors or traditional steam sterilized vessels. It can certainly become the first VCD measurement technique from cell line engineering to process development, to pilot scale and to manufacturing scale.

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