2021
DOI: 10.3390/mps4010016 View full text |Buy / Rent full text
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Abstract: Stable cell lines are widely used in laboratory research and pharmaceutical industry. They are mainly applied in recombinant protein and antibody productions, gene function studies, drug screens, toxicity assessments, and for cancer therapy investigation. There are two types of cell lines, polyclonal and monoclonal origin, that differ regarding their homogeneity and heterogeneity. Generating a high-quality stable cell line, which can grow continuously and carry a stable genetic modification without alteration … Show more

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“…We set out to test for variations from a single cell using a FT 13,16,17 . Cultures were diluted to about 0.5 cell/well (Limiting Dilution assay 28,29 ) in a 96-well microplate; on average, 48 wells had growth, and 48 wells did not (Fig. 1e).…”
Section: Resultmentioning
“…This sequence led to a double purification of cells, which are colony forming and express STRO-1. Two-step purification protocols for SCs or stable cell lines have also been established in other contexts [ 108 , 109 ]. The sequential purification protocol should clarify the question, if a double selection and thus a DPSC exhibiting both biological properties is favorable in terms of basic SC properties, because the repeated singulation of the cells may prevents them from influencing each other in a paracrine manner [ 110 , 111 ].…”
Section: Discussionmentioning
“…The clonal purity of cell lines established by a single round of LDC has been questioned due to the statistical nature of the method and cellular tendency to form aggregates. Therefore, it is recommended that two consecutive rounds of LDC be performed to ensure clonality of the resulting cell line(s) (5, 6). However, even after two consecutive rounds of cloning at 0.3 cell/well density, only 75% of the clones can be considered monoclonal at 95% confidence, as estimated by Poisson statistical analysis (4).…”
Section: Introductionmentioning
“…While FACS requires the addition of a viability marker or stable co-expression of a fluorescent reporter and was further reported to negatively affect cell viability [ 10 ], limiting dilution cloning is suggested to induce less cellular stress and does not require sophisticated instruments or reagents, other than standard or automated pipetting tools [ 11 , 12 ]. During limiting dilution, the initial cell suspension is highly diluted and plated on 96-well plates with the aim to obtain single cell-derived clones that are isolated and expanded subsequently [ 13 , 14 ]. Due to the statistical nature of obtaining a single cell after dilution, this clonal isolation method is comparatively inefficient and laborious [ 9 ].…”
Section: Introductionmentioning
“…In this work, we outline a novel approach for the plaque assay that involves the multi-well plate imaging system IncuCyte ® S3 and does not require the traditional cell fixation and staining steps. The IncuCyte ® S3 is a machine that can provide instant and real-time cell images (including whole well cell images) and thereby help to derive more profound and physiologically relevant information about the cells, such as confluence and fluorescence intensity [30].…”
Section: Introductionmentioning