Generation of Monoclonal Chinese Hamster Ovary Cell Lines Using DISPENCELL-S3.

Single-cell isolation is a key step in the manufacturing of therapeutic proteins, which relies on the development of monoclonal cell lines. It increases production safety and consistency. It also ensures higher manufacturing performances thanks to the selection of the rare clonally derived cell lines with optimal growth and production capacities. DISPENCELL-S3 is a small format single-cell dispenser whose technology is based on impedance spectroscopy. Here, we provide a detailed protocol for generating Chinese hamster ovary (CHO) monoclonal cell lines using DISPENCELL-S3. Production and characterization of an adequate cell sample for single-cell isolation, as well as the optimization of the DISPENCELL-S3 dispensing parameters are described. Monoclonal outgrowth assessment and the use of the recorded impedance signal as evidence of clonality are also outlined.

Traceable impedance-based single-cell pipetting, from a research set-up to a robust and fast automated robot: DispenCell-S1.

Single-cell isolation is a truly transformative tool for the understanding of biological systems. It allows single-cell molecular analyses and considers the heterogeneity of cell populations, which is of particular relevance for the diagnosis and treatment of evolving diseases and for personalized medicine. Single-cell isolation is also a key process in cell line development, where it is used to obtain stable and high producing clonally-derived cell lines, thus contributing to the efficiency, safety and reproducible quality of the drug produced. High producing clonally-derived cell lines are however rare events and their identification is a time-consuming process that requires the screening of thousands of clones. Therefore, there is an unmet need for a device that would allow the fast and efficient isolation of single cells, while preserving their integrity and providing an insurance of their clonality. We proposed earlier an impedance based pipetting technology for isolation of single cells (Bonzon et al., 2020), with initial validations for state-of-the-art stem cell in-vitro and in-vivo assays (Muller et al., 2020). Here, we present the transition from this pioneering technology developed in an academic setting into an automated instrument, called DispenCell-S1, allowing for traceable isolation of single cells. We developed and validated models predicting the performances for 96-well plates single-cell isolation. This resulted in a time of dispense down to 3 min and a plate filling rate up to 96%. Finally, we obtained an impedance signal reliability for proof of single particle isolation of 99% with beads and ranging from 93 to 95% with CHO cells.

Overexpression of transcription factor Foxa1 and target genes remediate therapeutic protein production bottlenecks in Chinese hamster ovary cells.

Despite extensive research conducted to increase protein production from Chinese hamster ovary (CHO) cells, cellular bottlenecks often remain, hindering high yields. In this study, a transcriptomic analysis led to the identification of 32 genes that are consistently upregulated in high producer clones and thus might mediate high productivity. Candidate genes were associated with functions such as signaling, protein folding, cytoskeleton organization, and cell survival. We focused on two engineering targets, Erp27, which binds unfolded proteins and the Erp57 disulfide isomerase in the endoplasmic reticulum, and Foxa1, a pioneering transcription factor involved in organ development. Erp27 moderate overexpression increased production of an easy-to-express antibody, whereas Erp27 and Erp57 co-overexpression increased cell density, viability, and the yield of difficult-to-express proteins. Foxa1 overexpression increased cell density, cell viability, and easy- and difficult-to-express protein yields, whereas it decreased reactive oxygen species late in fed-batch cultures. Foxa1 overexpression upregulated two other candidate genes that increased the production of difficult- and/or easy-to-express proteins, namely Ca3, involved in protecting cells from oxidative stress, and Tagap, involved in signaling and cytoskeleton remodeling. Overall, several genes allowing to overcome CHO cell bottlenecks were identified, including Foxa1, which mediated multiple favorable metabolic changes that improve therapeutic protein yields.

Alu RNA regulates the cellular pool of active ribosomes by targeted delivery of SRP9/14 to 40S subunits.

The human genome contains about 1.5 million Alu elements, which are transcribed into Alu RNAs by RNA polymerase III. Their expression is upregulated following stress and viral infection, and they associate with the SRP9/14 protein dimer in the cytoplasm forming Alu RNPs. Using cell-free translation, we have previously shown that Alu RNPs inhibit polysome formation. Here, we describe the mechanism of Alu RNP-mediated inhibition of translation initiation and demonstrate its effect on translation of cellular and viral RNAs. Both cap-dependent and IRES-mediated initiation is inhibited. Inhibition involves direct binding of SRP9/14 to 40S ribosomal subunits and requires Alu RNA as an assembly factor but its continuous association with 40S subunits is not required for inhibition. Binding of SRP9/14 to 40S prevents 48S complex formation by interfering with the recruitment of mRNA to 40S subunits. In cells, overexpression of Alu RNA decreases translation of reporter mRNAs and this effect is alleviated with a mutation that reduces its affinity for SRP9/14. Alu RNPs also inhibit the translation of cellular mRNAs resuming translation after stress and of viral mRNAs suggesting a role of Alu RNPs in adapting the translational output in response to stress and viral infection.

Multiple Roles of Alu-Related Noncoding RNAs.

Repetitive Alu and Alu-related elements are present in primates, tree shrews (Scandentia), and rodents and have expanded to 1.3 million copies in the human genome by nonautonomous retrotransposition. Pol III transcription from these elements occurs at low levels under normal conditions but increases transiently after stress, indicating a function of Alu RNAs in cellular stress response. Alu RNAs assemble with cellular proteins into ribonucleoprotein complexes and can be processed into the smaller scAlu RNAs. Alu and Alu-related RNAs play a role in regulating transcription and translation. They provide a source for the biogenesis of miRNAs and, embedded into mRNAs, can be targeted by miRNAs. When present as inverted repeats in mRNAs, they become substrates of the editing enzymes, and their modification causes the nuclear retention of these mRNAs. Certain Alu elements evolved into unique transcription units with specific expression profiles producing RNAs with highly specific cellular functions.

An Alu-like RNA promotes cell differentiation and reduces malignancy of human neuroblastoma cells.

Neuroblastoma (NB) is a pediatric cancer characterized by remarkable cell heterogeneity within the tumor nodules. Here, we demonstrate that the synthesis of a pol III-transcribed noncoding (nc) RNA (NDM29) strongly restricts NB development by promoting cell differentiation, a drop of malignancy processes, and a dramatic reduction of the tumor initiating cell (TIC) fraction in the NB cell population. Notably, the overexpression of NDM29 also confers to malignant NB cells an unpredicted susceptibility to the effects of antiblastic drugs used in NB therapy. Altogether, these results suggest the induction of NDM29 expression as possible treatment to increase cancer cells vulnerability to therapeutics and the measure of its synthesis in NB explants as prognostic factor of this cancer type.

Origin of the photo-cross-linking process in dichromated polyacrylamide under conventional and laser irradiation.

This work is devoted to determining the contribution of amide groups in the photoredox and cross-linking process of dichromated polyacrylamide based on the fate of the photoactive species and of the polymer under conventional and laser irradiation. It was shown that, in parallel to the reduction of chromium(VI) into chromium(V), the cross-linking of the matrix occurred through a complexation reaction around chromium(V) and through formation of covalent bonds between macromolecular chains. A comparison with dichromated poly(vinyl alcohol) was also reported to highlight the role of the chemical structure of the polymeric matrix in the mechanism of hologram formation. Moreover, for the first time it was demonstrated by in situ infrared spectroscopy that the physicochemical modifications undergone by the photosensitive materials were similar for the two modes of irradiation.

A novel non-symbiotic hemoglobin from oak: Roles in root signalling and development?

The cellular and molecular adaptations of non-model woody species to environmental changes are still poorly understood. We have cloned and characterised a novel non-symbiotic hemoglobin from oak roots (QpHb1) which exhibits a specific cellular distribution in the root. The QpHb1 gene is strongly expressed in the protoderm and the protoxylem cells in two Quercus species (Q. petraea and Q. robur) with contrasting adaptive potential to drought and flooding. The constitutive expression of QpHb1 in both oak species in specific root tissues combined with the reported presence of nitric oxide in the same tissues and its potential for protein S-nitrosylation could support a role for non-symbiotic hemoglobins in signalling changes in the root environment and/or in controlling some aspects of root development.

A novel nonsymbiotic hemoglobin from oak: cellular and tissue specificity of gene expression.

This study presents the isolation and characterization of a novel nonsymbiotic Hb gene from sessile oak (Quercus petraea) seedlings, herein designated QpHb1. The cellular and tissue expression of QpHb1 was analysed by Northern blotting and in situ hybridization. The encoded protein was predicted to consist of 161 amino acid residues, and shares 71 and 51% amino acid sequence identity with the Arabidopsis class 1 and 2 nonsymbiotic Hb, respectively. Northern blot analysis revealed that QpHb1 was strongly expressed in roots. Spatial expression analysis of QpHb1 in the root apical region of sessile oak by in situ hybridization indicated that transcripts were mostly abundant in protoxylem cell initials, some cortical cells and the protoderm. In addition, when comparing the expression profile of QpHb1 in sessile and pedunculate oak (Quercus robur), two species with contrasted hypoxia tolerance, the transcript level of QpHb1 rose early in the most flood-tolerant species, pedunculate oak, during root submergence. The spatial-temporal expression of QpHb1 suggests that this gene could participate in perception and signalling during hypoxia.