Transient gene expression in CHO cells monitored with automated flow cytometry

Sitton, Greg; Hansgate, Ann; Srienc, Friedrich

doi:10.1007/s10616-006-9020-9

Cited by 16 publications

(11 citation statements)

References 13 publications

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“…These data suggest that the proliferation behavior of encapsulated cells is environment‐dependent. Given the limited space within microcapsules, it is expected that the proliferation behavior may also have a significant impact on cell viability and consequently their secretion rate 36. The findings from this study support our previous findings7, 10 and highlight the advantageous characteristic of myoblasts of differentiating into non‐proliferative myotubes upon nutrient or cell confluency stress.…”

Section: Discussionsupporting

confidence: 87%

Characterization of viability and proliferation of alginate‐poly‐L‐lysine–alginate encapsulated myoblasts using flow cytometry

et al. 2010

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Genetically modified cells encapsulated in alginate-poly-L-lysine-alginate (APA) are being developed to deliver therapeutic products to treat a variety of diseases. The characterization of the encapsulated cells thus becomes paramount. This study reports a novel method to assess the viability, granularity and proliferation of encapsulated cells based on flow cytometry. The in vitro viability of encapsulated G8 murine myoblasts secreting canine FVIII (cFVIII) measured by flow cytometry was comparable to the traditional trypan blue exclusion method and both correlated with cFVIII secretion levels. In contrast, after implantation into mice, only viability measured by flow cytometry correlated with cFVIII secretion. Further, flow cytometry analysis of encapsulated cells maintained in vitro and in vivo revealed a greater fraction of granular cells compared to free cells, suggesting that encapsulation influences the morphology (cytoplasmic composition) of cells within APA microcapsules. Interestingly, the proliferation study showed that encapsulated cells proliferate faster, on average, and were more heterogeneous in vivo compared to in vitro culture conditions, suggesting that encapsulated cell proliferation is complex and environment-dependent. In conclusion, we show that flow cytometry analysis allows for a more consistent and comprehensive examination of encapsulated cells to aid in the development of cell therapy protocols.

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Section: Discussionsupporting

confidence: 87%

Characterization of viability and proliferation of alginate‐poly‐L‐lysine–alginate encapsulated myoblasts using flow cytometry

et al. 2010

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“…Sitton et al (2006), did show the possibility of using forward scatter (FSC) and side scatter (SS) to discriminate between apoptotic and non-apoptotic cells using an online FC setup but this does not give any definitive information about early apoptotic cells within the population. In biologics manufacturing it is important to closely monitor culture conditions and it is beneficial to carefully track the progress of apoptosis; it can have an effect on final product quality and quantity, as the proteases activated by apoptosis may degrade the desired protein products.…”

Section: Stirred Suspension Cell Culturementioning

confidence: 99%

Online flow cytometry for monitoring apoptosis in mammalian cell cultures as an application for process analytical technology

2014

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Apoptosis is the main driver of cell death in bioreactor suspension cell cultures during the production of biopharmaceuticals from animal cell lines. It is known that apoptosis also has an effect on the quality and quantity of the expressed recombinant protein. This has raised the importance of studying apoptosis for implementing culture optimization strategies. The work here describes a novel approach to obtain near real time data on proportion of viable, early apoptotic, late apoptotic and necrotic cell populations in a suspension CHO culture using automated sample preparation in conjunction with flow cytometry. The resultant online flow cytometry data can track the progression of apoptotic events in culture, aligning with analogous manual methodologies and giving similar results. The obtained near-real time apoptosis data are a significant improvement in monitoring capabilities and can lead to improved control strategies and research data on complex biological systems in bioreactor cultures in both academic and industrial settings focused on process analytical technology applications.

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“…From a PAT perspective, culture feeding should be triggered in response to extant conditions. In mammalian cell culture, stationary phase is presaged by the onset of cell death during exponential phase but off-line measures of cell titer and viability occur too infrequently for precise timing of the feeding and would require manual operator control (Sitton et al, 2006). In-line probes for turbidity and capacitance provide unstructured data incapable of quantifying an absolute concentration of dead cells.…”

Section: Harvest Phasementioning

confidence: 99%

Process analytical technology (PAT) for biopharmaceutical products: Part I. concepts and applications

Read¹,

Park²,

Shah³

et al. 2009

Biotech & Bioengineering

197

118

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Process analytical technology (PAT) has been gaining momentum in the biotech community due to the potential for continuous real-time quality assurance resulting in improved operational control and compliance. In this two part series, we address PAT as it applies to processes that produce biotech therapeutic products. In the first part, we address evolution of the underlying concepts and applications in biopharmaceutical manufacturing. We also present a literature review of applications in the areas of upstream and downstream processing to illustrate how implementation of PAT can help realize advanced approaches to ensuring product quality in real time. In the second part, we will explore similar applications in the areas of drug product manufacturing, rapid microbiology, and chemometrics as well as evolution of PAT in biotech processing.

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Transient gene expression in CHO cells monitored with automated flow cytometry

Cited by 16 publications

References 13 publications

Characterization of viability and proliferation of alginate‐poly‐L‐lysine–alginate encapsulated myoblasts using flow cytometry

Characterization of viability and proliferation of alginate‐poly‐L‐lysine–alginate encapsulated myoblasts using flow cytometry

Online flow cytometry for monitoring apoptosis in mammalian cell cultures as an application for process analytical technology

Process analytical technology (PAT) for biopharmaceutical products: Part I. concepts and applications

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