A cell sorting protocol for selecting high-producing sub-populations of Sf9 and High Five™ cells

Vidigal, João; Dias, Mafalda M.; Fernandes, Fabiana; Patrone, Marco; Bispo, Cláudia; Andrade, Claúdia; Gardner, Rui; Carrondo, Manuel J.T.; Alves, Paula M.; Teixeira, Augusto

doi:10.1016/j.jbiotec.2013.10.020

Cited by 13 publications

(13 citation statements)

References 26 publications

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“…Unfortunately, the DNA quality of isolated cells turned out to be quite low or had too few reads to draw conclusions. A possible explanation is that the CTCs were unable to withstand the shearing stress of the sorter, resulting in their destruction [22]. However, it has been shown before that FISH can be used on ISET filters to identify rearrangements, proving the malignant origin of cells identified in this manner [23].…”

Section: Discussionmentioning

confidence: 99%

Detection of Circulating Tumor Cells in the Diagnostic Leukapheresis Product of Non-Small-Cell Lung Cancer Patients Comparing CellSearch® and ISET

Tamminga

Andree

Hiltermann

et al. 2020

Cancers

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Circulating tumor cells (CTCs) detected by CellSearch are prognostic in non-small-cell lung cancer (NSCLC), but rarely found. CTCs can be extracted from the blood together with mononuclear cell populations by diagnostic leukapheresis (DLA), therefore concentrating them. However, CellSearch can only process limited DLA volumes (≈2 mL). Therefore, we established a protocol to enumerate CTCs in DLA products with Isolation by SizE of Tumor cells (ISET), and compared CTC counts between CellSearch® and ISET. DLA was performed in NSCLC patients who started a new therapy. With an adapted protocol, ISET could process 10 mL of DLA. CellSearch detected CTCs in a volume equaling 2 × 108 leukocytes (mean 2 mL). CTC counts per mL were compared. Furthermore, the live cell protocol of ISET was tested in eight patients. ISET successfully processed all DLA products—16 with the fixed cell protocol and 8 with the live cell protocol. In total, 10–20 mL of DLA was processed. ISET detected CTCs in 88% (14/16), compared to 69% (11/16, p < 0.05) with CellSearch. ISET also detected higher number of CTCs (ISET median CTC/mL = 4, interquartile range [IQR] = 2–6, CellSearch median CTC/mL = 0.9, IQR = 0–1.8, p < 0.01). Cells positive for the epithelial cell adhesion molecule (EpCAM+) per mL were detected in similar counts by both methods. Eight patients were processed with the live cell protocol. All had EpCAM+, CD45−, CD235- cells isolated by fluorescence-activated cell sorting (FACS). Overall, ISET processed larger volumes and detected higher CTC counts compared to CellSearch. EpCAM+ CTCs were detected in comparable rates.

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Section: Discussionmentioning

confidence: 99%

Detection of Circulating Tumor Cells in the Diagnostic Leukapheresis Product of Non-Small-Cell Lung Cancer Patients Comparing CellSearch® and ISET

Tamminga

Andree

Hiltermann

et al. 2020

Cancers

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“…FACS was used to select cells exhibiting a phenotype of high mCherry/Gag-eGFP expression during the zeocin selection process and remove non-producing cells ( Figure 1B). Cell enrichment by FACS has been successfully applied for the generation of stable CHO cells [35] and insect cell lines [28] to produce a variety of recombinant products, (Table 1).…”

Section: Stable Cell Pool Generationmentioning

confidence: 99%

Accelerating HIV‐1 VLP production using stable High Five insect cell pools

Puente‐Massaguer

Grau-Garcia

Strobl

et al. 2020

Biotechnology Journal

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Stable cell pools are receiving a renewed interest as a potential alternative system to clonal cell lines. The shorter development timelines and the capacity to achieve high product yields make them an interesting approach for recombinant protein production. In this study, stable High Five cell pools are assessed for the production of a simple protein, mCherry, and the more complex HIV-1 Gag-eGFP virus-like particles (VLPs). Random integration coupled to fluorescence-activated cell sorting (FACS) in suspension conditions is applied to accelerate the stable cell pool generation process and enrich it with high producer cells. This methodology is successfully transferred to a bioreactor for VLP production, resulting in a 2-fold increase in VLP yields with respect to shake flask cultures. In these conditions, maximum viable cell concentration improves by 1.5fold, and by-product formation is significantly reduced. Remarkably, a global increase in the uptake of amino acids in the Gag-eGFP stable cell pool is observed when compared with parental High Five cells, reflecting the additional metabolic burden associated with VLP production. These results suggest that stable High Five cell pools are a robust and powerful approach to produce VLPs and other recombinant proteins, and put the basis for future studies aiming to scale up this system.

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“…As a general strategy to monitor site-specific donor integration in a preselected chromosomal target, the landing pad, we chose to follow the expression of two spectral distinct fluorescent proteins. The favourable properties of stable cell lines isolated via cell sorting according to fluorescent protein gene expression are well established 17 , 18 . Here, the switch was from the landing pad vector (LPV)-encoded green fluorescent protein (GFP) to a red fluorescent protein (RFP).…”

Section: Resultsmentioning

confidence: 99%

Site-specific chromosomal gene insertion: Flp recombinase versus Cas9 nuclease

Phan

Contzen

Seemann

et al. 2017

Sci Rep

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Site-specific recombination systems like those based on the Flp recombinase proved themselves as efficient tools for cell line engineering. The recent emergence of designer nucleases, especially RNA guided endonucleases like Cas9, has considerably broadened the available toolbox for applications like targeted transgene insertions. Here we established a recombinase-mediated cassette exchange (RMCE) protocol for the fast and effective, drug-free isolation of recombinant cells. Distinct fluorescent protein patterns identified the recombination status of individual cells. In derivatives of a CHO master cell line the expression of the introduced transgene of interest could be dramatically increased almost 20-fold by subsequent deletion of the fluorescent protein gene that provided the initial isolation principle. The same master cell line was employed in a comparative analysis using CRISPR/Cas9 for transgene integration in identical loci. Even though the overall targeting efficacy was comparable, multi-loci targeting was considerably more effective for Cas9-mediated transgene insertion when compared to RMCE. While Cas9 is inherently more flexible, our results also alert to the risk of aberrant recombination events around the cut site. Together, this study points at the individual strengths in performance of both systems and provides guidance for their appropriate use.

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A cell sorting protocol for selecting high-producing sub-populations of Sf9 and High Five™ cells

Cited by 13 publications

References 26 publications

Detection of Circulating Tumor Cells in the Diagnostic Leukapheresis Product of Non-Small-Cell Lung Cancer Patients Comparing CellSearch® and ISET

Detection of Circulating Tumor Cells in the Diagnostic Leukapheresis Product of Non-Small-Cell Lung Cancer Patients Comparing CellSearch® and ISET

Accelerating HIV‐1 VLP production using stable High Five insect cell pools

Site-specific chromosomal gene insertion: Flp recombinase versus Cas9 nuclease

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