Characterization and improvement of cell line performance <i>via</i> flow cytometry and cell sorting

Moretti, Pierre; Behr, Larissa; Walter, Johanna‐Gabriela; Kasper, Cornelia; Stahl, Frank; Scheper, Thomas

doi:10.1002/elsc.200900076

Cited by 8 publications

(5 citation statements)

References 40 publications

(78 reference statements)

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“…Many newly-developed drugs have been withdrawn during animal trials, because the cell-based assays were not able to detect the hazards [ 2 ]. Flow cytometry (FCM) is a well-established and highly efficient technology for high throughput cell analysis [ 3 , 4 , 5 ]. Sample preparation for FCM is fast and simple, especially in comparison to other established techniques such as transmission electron microscopy (TEM) [ 6 ].…”

Section: Introductionmentioning

confidence: 99%

Revelation of Different Nanoparticle-Uptake Behavior in Two Standard Cell Lines NIH/3T3 and A549 by Flow Cytometry and Time-Lapse Imaging

Jochums

Friehs

Sambale

et al. 2017

Toxics

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The uptake of nanomaterials into different cell types is a central pharmacological issue for the determination of nanotoxicity as well as for the development of drug delivery strategies. Most responses of the cells depend on their intracellular interactions with nanoparticles (NPs). Uptake behavior can be precisely investigated in vitro, with sensitive high throughput methods such as flow cytometry. In this study, we investigated two different standard cell lines, human lung carcinoma (A549) and mouse fibroblast (NIH/3T3) cells, regarding their uptake behavior of titanium dioxide NPs. Cells were incubated with different concentrations of TiO2 NPs and samples were taken at certain time points to compare the uptake kinetics of both cell lines. Samples were analyzed with the help of flow cytometry by studying changes in the side and forward scattering signal. To additionally enable a detection via fluorescence, NPs were labeled with the fluorescent dye fluorescein isothiocyanate (FITC) and propidium iodide (PI). We found that NIH/3T3 cells take up the studied NPs more efficiently than A549 cells. These findings were supported by time-lapse microscopic imaging of the cells incubated with TiO2 NPs. Our results confirm that the uptake behavior of individual cell types has to be considered before interpreting any results of nanomaterial studies.

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Section: Introductionmentioning

confidence: 99%

Revelation of Different Nanoparticle-Uptake Behavior in Two Standard Cell Lines NIH/3T3 and A549 by Flow Cytometry and Time-Lapse Imaging

Jochums

Friehs

Sambale

et al. 2017

Toxics

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“…Their approach does not require any expensive equipment, but it is laborious, time-consuming and does not include the preparation of single cells, a prerequisite for the generation of a truly monoclonal BY-2 cell line. We addressed the challenge of culture heterogeneity by using FACS to generate homogeneous and highly productive monoclonal tobacco cell lines, a principle that has already been shown to work with mammalian cells (Carroll and Al-Rubeai, 2004;Moretti et al, 2010). We used BY-2 suspension culture MTED#18 as a model cell line because it produces two recombinant proteins, the human antibody M12 and the fluorescent marker protein DsRed.…”

Section: Discussionmentioning

confidence: 99%

Monoclonal tobacco cell lines with enhanced recombinant protein yields can be generated from heterogeneous cell suspension cultures by flow sorting

Kirchhoff

Raven

Boes

et al. 2012

Plant Biotechnology Journal

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SummaryPlant cell suspension cultures can be used for the production of recombinant pharmaceutical proteins, but their potential is limited by modest production levels that may be unstable over long culture periods, reflecting initial culture heterogeneity and subsequent genetic and epigenetic changes. We used flow sorting to generate highly productive monoclonal cell lines from a heterogeneous population of tobacco BY-2 cells expressing the human antibody M12 by selecting the co-expressed fluorescent marker protein DsRed located on the same T-DNA. Separation yielded 35% wells containing single protoplasts and 15% wells with monoclonal microcolonies that formed within 2 weeks. Thus, enriching the population of fluorescent cells from initially 24% to 90-96% in the six monoclonal lines resulted in an up to 13-fold increase in M12 production that remained stable for 10-12 months. This is the first straightforward procedure allowing the generation of monoclonal plant cell suspension cultures by flow sorting, greatly increasing the potential of plant cells as an economical platform for the manufacture of recombinant pharmaceutical proteins.

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“…As a result, carefully and accurately monitoring these two parameters is of importance in developing a high-yielding process. The most common method for achieving this is to determine product titer via either a high performance liquid chromatography (HPLC) analysis [7][8][9] or an enzyme-linked immunosorbent assay (ELISA) [2,[10][11][12]. However, both of these methods are regrettably time-consuming and laborious.…”

Section: Introductionmentioning

confidence: 99%

Monitoring cell productivity for the production of recombinant proteins by flow cytometry: An effective application using the cold capture assay

Meyer

Siller

Schellenberg

et al. 2021

Engineering in Life Sciences

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Due to the increasing economic and social relevance of biotherapeutics, their production processes are continually being reconsidered and reoptimized in an effort to secure higher product concentrations and qualities. Monitoring the productivity of cultured cells is therefore a critically important part of the cultivation process. Traditionally, this is achieved by determining the overall product titer by high performance liquid chromatography (HPLC), and then calculating the specific cell productivity based on this titer and an associated viable cell density. Unfortunately, this process is typically time-consuming and laborious. In this study, the productivity of Chinese Hamster Ovary (CHO) cells expressing a monoclonal antibody was analyzed over the course of the cultivation process. In addition to calculating the specific cell productivity based on the traditional product titer determined by HPLC analysis, culture productivity of single cells was also analyzed via flow cytometry using a cold capture assay. The cold capture assay is a cell surface labelling technique described by Brezinsky et al., which allows for the visualization of a product on the surface of the producing cell. The cell productivity results obtained via HPLC and the results of cold capture assay remained in great accordance over the whole cultivation process. Accordingly, our study demonstrates that the cold capture assay offers an interesting, comparatively time-effective, and potentially cheaper alternative for monitoring the productivity of a cell culture.

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Characterization and improvement of cell line performance via flow cytometry and cell sorting

Cited by 8 publications

References 40 publications

Revelation of Different Nanoparticle-Uptake Behavior in Two Standard Cell Lines NIH/3T3 and A549 by Flow Cytometry and Time-Lapse Imaging

Revelation of Different Nanoparticle-Uptake Behavior in Two Standard Cell Lines NIH/3T3 and A549 by Flow Cytometry and Time-Lapse Imaging

Monoclonal tobacco cell lines with enhanced recombinant protein yields can be generated from heterogeneous cell suspension cultures by flow sorting

Monitoring cell productivity for the production of recombinant proteins by flow cytometry: An effective application using the cold capture assay

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