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Characterization of phenotypic and genotypic diversity in subclones derived from a clonal cell line.

ABSTRACT: Regulatory guidelines require the sponsors to provide assurance of clonality of the production cell line, and when such evidence is not available, additional studies are typically required to further ensure consistent long-term manufacturing of the product. One potential approach to provide such assurance of clonal derivation of a production cell line is to characterize subclones generated from the original cell line and assess their phenotypic and genotypic similarity with the hypothesis that cell lines derived from a clonal bank will share performance, productivity and product quality characteristics. In this study, a production cell line that was cloned by a validated FACS approach coupled with day 0 imaging for verification of single-cell deposition was subcloned using validated FACS and imaging methods. A total of 46 subclones were analyzed for growth, productivity, product quality, copy number, and integration site analysis. Significant diversity in cell growth, protein productivity, product quality attributes, and copy number was observed between the subclones, despite stability of the parent clone over time. The diversity in protein productivity and quality of the subclones were reproduced across time and production scales, suggesting that the resulting population post sub-cloning originating from a single cell is stable but with unique properties. Overall, this work demonstrates that the characteristics of isolated subclones are not predictive of a clonally derived parental clone. Consequently, the analysis of subclones may not be an effective approach to demonstrate clonal origin of a cell bank. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:613-623, 2018.

SUBMITTER: Tharmalingam T

PROVIDER: S-EPMC6099272 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Characterization of phenotypic and genotypic diversity in subclones derived from a clonal cell line.

Tharmalingam Tharmala T Barkhordarian Hedieh H Tejeda Nicole N Daris Kristi K Yaghmour Sam S Yam Pheng P Lu Fang F Goudar Chetan C Munro Trent T Stevens Jennitte J

Biotechnology progress 20180501 3

Regulatory guidelines require the sponsors to provide assurance of clonality of the production cell line, and when such evidence is not available, additional studies are typically required to further ensure consistent long-term manufacturing of the product. One potential approach to provide such assurance of clonal derivation of a production cell line is to characterize subclones generated from the original cell line and assess their phenotypic and genotypic similarity with the hypothesis that c ...[more]

PMID: 29882350

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Project description:Declines in genetic diversity within a species can affect the stability and functioning of populations. The conservation of genetic diversity is thus a priority, especially for threatened or endangered species. The importance of genetic variation, however, is dependent on the degree to which it translates into phenotypic variation for traits that affect individual performance and ecological processes. This is especially important for predominantly clonal species, as no single clone is likely to maximise all aspects of performance. Here we show that intraspecific genotypic diversity as measured using microsatellites is a strong predictor of phenotypic variation in morphological traits and shoot productivity of the threatened, predominantly clonal seagrass Posidonia australis, on the east coast of Australia. Biomass and surface area variation was most strongly predicted by genotypic richness, while variation in leaf chemistry (phenolics and nitrogen) was unrelated to genotypic richness. Genotypic richness did not predict tissue loss to herbivores or epiphyte load, however we did find that increased herbivore damage was positively correlated with allelic richness. Although there was no clear relationship between higher primary productivity and genotypic richness, variation in shoot productivity within a meadow was significantly greater in more genotypically diverse meadows. The proportion of phenotypic variation explained by environmental conditions varied among different genotypes, and there was generally no variation in phenotypic traits among genotypes present in the same meadows. Our results show that genotypic richness as measured through the use of presumably neutral DNA markers does covary with phenotypic variation in functionally relevant traits such as leaf morphology and shoot productivity. The remarkably long lifespan of individual Posidonia plants suggests that plasticity within genotypes has played an important role in the longevity of the species. However, the strong link between genotypic and phenotypic variation suggests that a range of genotypes is still the best case scenario for adaptation to and recovery from predicted environmental change.

Project description:To sum up our results, we managed to isolate a reproducible SP cell fraction of human gastric carcinoma cell lines for the first time. Those cells differed from the rest of the population regarding morphology and biological behaviour. They had the ability to build a population resembling the basic tumor population and therefore performed differenziation and asymmetric cell division. SP cells expressed potential stem cell markers like musashi 1 and Cd133 and also showed a high expression of genes that encode for stem cell properties. The so identified phenotypic and genetic factors showed a variable expression in tumor samples of patient series and might be applicable as prognostic factors in the diagnosis of gastric carcinoma. Aims: The side population (SP) of tumor cell lines shares characteristics with tumor stem cells. In this study we phenotypically and genotypically characterized the SP of gastric cancer cell lines. Methods: The SP was obtained from MKN45- and AGS-gastric cancer cells using Hoechst 33342 staining and fluorescence-activated cell sorting (FACS). SP cells were subsequently studied morphologically (cytology, immunocytochemistry), on the transcriptional level (gene array) and in cell culture (recultivation assays). Genes differentially expressed in the SP cells were finally searched by immunohistochemistry in neoplastic and non-neoplastic gastric tissue from gastric cancer patients. Results: The SP was reproducibly obtained from gastric cancer cell lines. The SP cells were smaller and rounder then non-SP cells. SP cells self-renewed in re-cultivation experiments and differentiated into SP- and non-SP cells. Re-cultivated SP- and non-SP cells showed distinct phenotypes in culture regarding cell shape and colony-formation. SP cells had increased levels of the stem cell markers CD133 and Musashi 1. Transcriptional analyses demonstrated that SP cells express genes that encode for stem cell properties like FZD7, HEY1, SMO and ADAM17. Finally we detected the transcripts of these genes in tissue samples from patients with gastric cancer. Conclusions: Human gastric cancer cell lines enclose a phenotypically and genotypically distinct cell population with tumor stem cell features. Phenotypical characteristics of this distinct cell population are also present in gastric cancer tissue.

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Characterization of phenotypic and genotypic diversity in subclones derived from a clonal cell line.

Publications

Characterization of phenotypic and genotypic diversity in subclones derived from a clonal cell line.

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