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New approaches for characterizing and monitoring mammalian cell cycle and specific growth rate in production cell lines
Citation Link: https://doi.org/10.15480/882.1556
Publikationstyp
Doctoral Thesis
Publikationsdatum
2018
Sprache
English
Author
Advisor
Referee
Title Granting Institution
Technische Universität Hamburg-Harburg
Place of Title Granting Institution
Hamburg
Examination Date
2017-12-11
This study focuses on the different putative interdependencies between the cell cycle and the behaviour of mammalian production cell lines, based on a controlled physiological synchronization and cultivation method, as well as the corresponding implications for reliable process analysis and control.
In the first part, an established experimental setup for the generation of synchronized suspension cultures was adapted for the Human Embryonic Kidney cell line 293. This cell line could then be synchronized under near-physiological conditions. Extensive optimisation efforts were required in order to achieve and maintain high viabilities as well as aggregation free growth.
Subsequently, this method was used to examine the putative dependency of the transfection efficiency from the cell cycle state at the time of transfection. These studies are based on synchronization methods under near-physiological conditions. The following synchronized growth was confirmed by DNA based cell cycle analysis and specific growth rate determination. The results indicated that the so-called cell cycle dependencies reported by other groups, cannot be confirmed when near-physiological synchronised cultures are used. Hence it is likely that the putative cell cycle dependencies, which were reported before, are artefacts of the non-physiological synchronisation methods used in those studies.
Note that this study doesn't draw conclusions concerning potential cell cycle dependencies of production rates; they require additional studies possibly utilizing the established methods described here.
In order to facilitate the follow-up cell cycle specific research and future on-line measurements, a novel approach was chosen. Two new derivatives of the widely used Chinese Hamster Ovary (CHO-K1) production cell line were established. They indicate their cell cycle as well as growth state in an on-line compatible manner by fluorescence. For this purpose, three different genetic FUCCI (Fluorescence Ubiquitination Cell Cycle Indicator) constructs were used in two different combinations. In coherence with the original nomenclature by Miyawaki et al. [C: mKO2-hCdt1(30/120), M: mVenus-hGeminin(1/110) and N: mVenus-hGeminin(1/60)] they were denoted CHO-K1 FUCCI CM und CN.
In order to enable quantitative and reproducible conclusions based on the fluorescence properties, two quantitative parameters were introduced. The parameter <i>iredn</i> indicates the percentage of cells with red fluorescence in relation to the total number of fluorescent cells, composed of red and green fluorescent cells. This relative parameter is generally independent from possibly altering total fluorescence intensities per single cell. Quantitative analyses revealed that <i>iredn</i> correlates well with the cell cycle state (expressed as percentage of cells in the G1 cell cycle phase) and is furthermore directly connected to the specific growth rate (μ). Hence the parameter <i>iredn</i> has a high validity but requires measuring the fluorescence of single cells in order to calculate their numerical ratio. Therefore, flow cytometry is the method of choice.
Correspondingly the parameter <i>iredtotal</i> is based on the total fluorescence intensity of the culture and is determined by the total red fluorescence in relation to the sum of total red and green fluorescence. This relative determination is robust with regard to variations in cell density and correspondingly changing total fluorescence intensities. Hence <i>iredtotal</i> is determined by the number of cells in the different cell cycle phases as well as the fluorescence intensity per cell. Consequently <i>iredtotal</i> is less exact compared to <i>iredn</i> but can be determined using simpler methods (fluorescence plate reader, online measurement).
It is pivotal for both approaches to detect and quantify the fluorescence intensities (red vs. green) with sufficient specificity (low cross talk) and accuracy. It was demonstrated that <i>iredtotal</i> can be used to indicate changes in the growth behaviour as well as limiting growth conditions, here in the form of L-glutamine limitation, early on. This was confirmed in shaking flask and bioreactor cultures alike. Significant signals, indicating emerging limitation were detected several hours earlier compared to traditional indicators (cell counts, oxygen uptake rate). Ultimately, on-line or at-line fluorescent probes are highly recommended for further analyses and process control of mammalian cell cultures.
In the first part, an established experimental setup for the generation of synchronized suspension cultures was adapted for the Human Embryonic Kidney cell line 293. This cell line could then be synchronized under near-physiological conditions. Extensive optimisation efforts were required in order to achieve and maintain high viabilities as well as aggregation free growth.
Subsequently, this method was used to examine the putative dependency of the transfection efficiency from the cell cycle state at the time of transfection. These studies are based on synchronization methods under near-physiological conditions. The following synchronized growth was confirmed by DNA based cell cycle analysis and specific growth rate determination. The results indicated that the so-called cell cycle dependencies reported by other groups, cannot be confirmed when near-physiological synchronised cultures are used. Hence it is likely that the putative cell cycle dependencies, which were reported before, are artefacts of the non-physiological synchronisation methods used in those studies.
Note that this study doesn't draw conclusions concerning potential cell cycle dependencies of production rates; they require additional studies possibly utilizing the established methods described here.
In order to facilitate the follow-up cell cycle specific research and future on-line measurements, a novel approach was chosen. Two new derivatives of the widely used Chinese Hamster Ovary (CHO-K1) production cell line were established. They indicate their cell cycle as well as growth state in an on-line compatible manner by fluorescence. For this purpose, three different genetic FUCCI (Fluorescence Ubiquitination Cell Cycle Indicator) constructs were used in two different combinations. In coherence with the original nomenclature by Miyawaki et al. [C: mKO2-hCdt1(30/120), M: mVenus-hGeminin(1/110) and N: mVenus-hGeminin(1/60)] they were denoted CHO-K1 FUCCI CM und CN.
In order to enable quantitative and reproducible conclusions based on the fluorescence properties, two quantitative parameters were introduced. The parameter <i>iredn</i> indicates the percentage of cells with red fluorescence in relation to the total number of fluorescent cells, composed of red and green fluorescent cells. This relative parameter is generally independent from possibly altering total fluorescence intensities per single cell. Quantitative analyses revealed that <i>iredn</i> correlates well with the cell cycle state (expressed as percentage of cells in the G1 cell cycle phase) and is furthermore directly connected to the specific growth rate (μ). Hence the parameter <i>iredn</i> has a high validity but requires measuring the fluorescence of single cells in order to calculate their numerical ratio. Therefore, flow cytometry is the method of choice.
Correspondingly the parameter <i>iredtotal</i> is based on the total fluorescence intensity of the culture and is determined by the total red fluorescence in relation to the sum of total red and green fluorescence. This relative determination is robust with regard to variations in cell density and correspondingly changing total fluorescence intensities. Hence <i>iredtotal</i> is determined by the number of cells in the different cell cycle phases as well as the fluorescence intensity per cell. Consequently <i>iredtotal</i> is less exact compared to <i>iredn</i> but can be determined using simpler methods (fluorescence plate reader, online measurement).
It is pivotal for both approaches to detect and quantify the fluorescence intensities (red vs. green) with sufficient specificity (low cross talk) and accuracy. It was demonstrated that <i>iredtotal</i> can be used to indicate changes in the growth behaviour as well as limiting growth conditions, here in the form of L-glutamine limitation, early on. This was confirmed in shaking flask and bioreactor cultures alike. Significant signals, indicating emerging limitation were detected several hours earlier compared to traditional indicators (cell counts, oxygen uptake rate). Ultimately, on-line or at-line fluorescent probes are highly recommended for further analyses and process control of mammalian cell cultures.
Schlagworte
process control
biotechnology
cell culture
fluorescence
FUCCI
DDC Class
570: Biowissenschaften, Biologie
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