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.

A new workflow combining magnetic cell separation and impedance-based cell dispensing for gentle, simple and reliable cloning of specific CD8+ T cells.

Reverse immunology has open the door to innovative cancer immunotherapy strategies such as immunogenic antigen-based vaccination and transgenic T cell receptor (TCR)-based adoptive cell transfer. This approach enables the identification of immunogenic tumor specific antigen derived peptides. One of the major challenges is the rapid selection of antigen-specific CD8+ T cell clones. Thus, IFNγ-producing CD8+ T cells magnetic sorting combined with limiting dilution cloning approach represents the most common method of specific T cell cloning. However, during plate setup several wells will not contain T cells whereas others will contain mixed population of T cells. In this case, a re-cloning step is required which make limiting dilution based cloning a laborious, inefficient, expensive and a time-consuming method. To address these obstacles, here we present a novel 2-step workflow combining simple, affordable and gentle magnetic cell separation followed by single cell isolation using a device called DispenCell-S1. We aimed to compare this new workflow with the traditional limiting dilution method using in vitro generated antigen-specific CD8+ T cells. Herein, we reported the reliability of DispenCell-S1 method and its efficiency in T cell clones isolation.

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.

Impedance-Based Single-Cell Pipetting.

Many biological methods are based on single-cell isolation. In single-cell line development, the gold standard involves the dilution of cells by means of a pipet. This process is time-consuming as it is repeated over several weeks to ensure clonality. Here, we report the modeling, designing, and testing of a disposable pipet tip integrating a cell sensor based on the Coulter principle. We investigate, test, and discuss the effects of design parameters on the sensor performances with an analytical model. We also describe a system that enables the dispensing of single cells using an instrumented pipet coupled with the sensing tip. Most importantly, this system allows the recording of an impedance trace to be used as proof of single-cell isolation. We assess the performances of the system with beads and cells. Finally, we show that the electrical detection has no effect on cell viability.

Traceable Impedance-Based Dispensing and Cloning of Living Single Cells.

Single-cell cloning is essential in stem cell biology, cancer research, and biotechnology. Regulatory agencies now require an indisputable proof of clonality that current technologies do not readily provide. Here, we report a one-step cloning method using an engineered pipet combined with an impedance-based sensing tip. This technology permits the efficient and traceable isolation of living cells, stem cells, and cancer stem cells that can be individually expanded in culture and transplanted.

[Doctor Charles Marx 1903-1946]. / Docteur Charles Marx 1903-1946.

Charles Marx (1903-1946) an outstanding Luxembourg physician was a communist. He had very well succeeded in his medical studies in Paris starting as an interne (1929), to become "chef de clinique chirurgicale", then a "Lauréat de l'Académie de Médecine" and finally a member of the "Comité de l'Association française de Chirurgie" (1935). He founded his own hospital (50 beds) in Ettelbruck. There, in 1940, he helped two interned French airmen, to escape to France. Charles Marx escaped himself, with his family, just before the German invasion. He briefly directed four French hospitals, in Nevers and in Quillan. In July he founded the first resistance group of the "Armée secrète", which in May 1943 was followed by a first maquis in the western Pyrenees. In June 43, he was condemned to death "in absentia" in Montpellier by the Nazis. In February 1944 Charles Marx was appointed medical commander of the FFI and chief health manager of the Resistance in the oriental Pyrenees. In September 1944 he took part in the liberation of Lyon. In October, being the delegate of the medical resistance council, he was named attaché to the Health Minister and was charged to organize French-American military surgical structures. End of July he took up the management of the Ettelbruck hospital. In November 1945 he was nominated minister of Public Health in Luxembourg. In June 1946 Charles Marx and his Rumanian wife lost their lives in a car accident.

[Dogs and people]. / Des chiens et des hommes.

A neuropsychiatrist remembers all the dogs he met in his life. Ten of them, with different characters, their own ways to express affectionate wishes by continuous body language. Not only patients are helped by animals who are their friends but so are doctors.