Abstract
The bioreactor conditions and cell diversity in mammalian cell cultures are often regarded as homogeneous. Recently, the influence of various kinds of heterogeneities on production rates receives increasing attention. Besides spatial gradients within the cultivation system, the variation between cell populations and the progress of the cells through the cell cycle can affect the dynamics of the cultivation process. Strong metabolic up- and down-regulations leading to variable productivities, even in exponentially growing cell cultures, have been identified in CHO cell cultivations. Consequently, scientific studies of cell cycle-related effects and metabolic regulations require experiments utilizing cell cycle-enriched subpopulations. Importantly, the enrichment procedure itself must not strongly interfere with the cell culture under investigation. Such subpopulations can be generated by near-physiological countercurrent centrifugal elutriation, which is described in the following chapter. At first, a brief overview regarding the cell cycle, currently identified effects and commonly used methods, and their applicability is outlined. Then, the experimental setup and the synchronization itself are explained.
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Abbreviations
- CHO:
-
Chinese hamster ovary
- DNA:
-
Deoxyribonucleic acid
- DAPI:
-
4′,6-Diamidino-2-phenylindole
- FSC:
-
Forward scatter
- G1 phase:
-
Gap phase 1
- G2 phase:
-
Gap phase 2
- HEK:
-
Human embryonic kidney
- M:
-
Mitosis
- PBS:
-
Phosphate-buffered saline
- S phase:
-
Synthesis phase
- SSC:
-
Side scatter
- %sync:
-
Percentage of cells in a cell cycle phase after synchronization
- %non-sync:
-
Percentage of cells in a cell cycle phase in the preculture
- YΩ :
-
Enrichment factor
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Möller, J., Korte, K., Pörtner, R., Zeng, AP., Jandt, U. (2020). Near-Physiological Cell Cycle Synchronization with Countercurrent Centrifugal Elutriation. In: Pörtner, R. (eds) Animal Cell Biotechnology. Methods in Molecular Biology, vol 2095. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0191-4_1
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DOI: https://doi.org/10.1007/978-1-0716-0191-4_1
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