Characterisation of Chinese Hamster Ovary (CHO) cells at the single cell level

Biopharmaceuticals are a class of biological macromolecules that include antibodies and antibody derivatives, generally produced from cultured mammalian cells line via secretion directly into the media. Manufacturing requires the generation of Chinese hamster ovary (CHO) clonal cell lines capable of expressing the biopharmaceutical product at commercially relevant quantities with desirable product quality. The isolation of cell clones based on random single cell deposition via fluorescence activated cell sorting (FACS) provides a heterogeneous panel of expressers. We hypothesize that the application of FACS to provide an additional sorting step based on cell characteristics that correlate with productivity, product quality or cell growth attributes could lead to the isolation of higher producing cell lines with enhanced product quality attributes. A panel of 20 cell lines expressing a model recombinant monoclonal antibody were characterised in terms of growth, productivity, and intracellular recombinant protein and mRNA amounts. Assays were also developed to investigate cell attributes and organelle content using the ImageStream instrument, an imaging flow cytometer, which enables the investigation of cellular characteristics that correlate with cell productivity at the single cell level. Characterisation revealed the cell lines exhibited a range of values for productivity, growth, and intracellular (IC) antibody mRNA and protein expression, ideal for further ImageStream characterisation. Western blot and qRT-PCR analysis demonstrated that final titre correlated with both IC heavy chain (HC) protein and mRNA amounts (Pearson Correlation Coefficient (R) = 0.70 and R = 0.80, respectively). To assess productivity at the single cell level, assays multiplexing IC HC protein and mRNA with organelles, such as mitochondria, endoplasmic reticulum and Golgi apparatus, were therefore developed. ImageStream quantification of HC mRNA and protein amounts also showed correlations between titre and IC HC protein and mRNA (R = 0.84 and R = 0.79, respectively), confirming results from western blots and qRT-PCR analysis. A cell attribute that correlates with specific productivity has been found, and current work is investigating whether this cell attribute could be used during cell sorting for the isolation of more productive clones. Future experiments will also look at cell attributes that could lead to improved product quality. The developed assays are expected to allow a greater understanding of the intracellular mechanisms underlying productivity and product quality in CHO cells. Moreover, outcomes from this study have the potential to not only integrate into the cell line development clonal selection process, shortening timelines and reducing cost and resource requirements, but also inform host cell engineering projects with the potential for the development of an improved CHO host

Application of Imaging Flow Cytometry for the Characterization of Intracellular Attributes in Chinese Hamster Ovary Cell Lines at the Single Cell Level

Biopharmaceutical manufacturing using Chinese hamster ovary (CHO) cells requires the generation of high-producing clonal cell lines. During cell line development, cell cloning using fluorescence activated cell sorting (FACS) has the potential to combine isolation of single cells with sorting based on specific cellular attributes that correlate with productivity and/or growth, identifying cell lines with desirable phenotypes for manufacturing. This study describes the application of imaging flow cytometry (IFC) to characterize recombinant cell lines at the single cell level to identify cell attributes predictive of productivity. IFC assays to quantify organelle content, and recombinant heavy (HC) and light (LC) chain polypeptide and mRNA amounts in single cells were developed. The assays were then validated against orthogonal standard flow cytometry, western blot and qRT-PCR methods. We describe how these IFC assays may be used in cell line development and show how cellular properties can be correlated with productivity at the single cell level, allowing the isolation of such cells during the cloning process. Our analysis found HC polypeptide and mRNA to be predictive of productivity early in the culture, however specific organelle content did not show any correlation with productivity

Definition and Functional Profiling of HIV-1-Specific T-Cell Responses

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Distal lung epithelial progenitor cell function declines with age.

Tissue stem cell exhaustion is a key hallmark of aging, and in this study, we characterised its manifestation in the distal lung. We compared the lungs of 3- and 22-month old mice. We examined the gross morphological changes in these lungs, the density and function of epithelial progenitor populations and the epithelial gene expression profile. Bronchioles became smaller in their cross-sectional area and diameter. Using long-term EdU incorporation analysis and immunohistochemistry, we found that bronchiolar cell density remained stable with aging, but inferred rates of bronchiolar club progenitor cell self-renewal and differentiation were reduced, indicative of an overall slowdown in cellular turnover. Alveolar Type II progenitor cell density and self-renewal were maintained per unit tissue area with aging, but rates of inferred differentiation into Type I cells, and indeed overall density of Type I cells was reduced. Microarray analysis revealed age-related changes in multiple genes, including some with roles in proliferation and differentiation, and in IGF and TGFβ signalling pathways. By characterising how lung stem cell dynamics change with aging, this study will elucidate how they contribute to age-related loss of pulmonary function, and pathogenesis of common age-related pulmonary diseases

Annexin-1 modulates T-cell activation and differentiation

Annexin-1 is an anti-inflammatory protein that plays an important homeostatic role in innate immunity; however, its potential actions in the modulation of adaptive immunity have never been explored. Although inactive by itself, addition of annexin-1 to stimulated T cells augmented anti-CD3/CD28-mediated CD25 and CD69 expression and cell proliferation. This effect was paralleled by increased nuclear factor-κB (NF-κB), nuclear factor of activated T cells (NFATs), and activator protein-1 (AP-1) activation and preceded by a rapid T-cell receptor (TCR)–induced externalization of the annexin-1 receptor. Interestingly, differentiation of naive T cells in the presence of annexin-1 increased skewing in Th1 cells; in the collagen-induced arthritis model, treatment of mice with annexin-1 during the immunization phase exacerbated signs and symptoms at disease onset. Consistent with these findings, blood CD4(+) cells from patients with rheumatoid arthritis showed a marked up-regulation of annexin-1 expression. Together these results demonstrate that annexin-1 is a molecular “tuner” of TCR signaling and suggest this protein might represent a new target for the development of drugs directed to pathologies where an unbalanced Th1/Th2 response or an aberrant activation of T cells is the major etiologic factor