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.

The capability to generate induced pluripotent stem cell (iPSC) lines, in tandem with CRISPR-Cas9 DNA editing, offers great promise to understand the underlying genetic mechanisms of human disease. The low efficiency of available methods for homogeneous expansion of singularized CRISPR-transfected iPSCs necessitates the coculture of transfected cells in mixed populations and/or on feeder layers. Consequently, edited cells must be purified using labor-intensive screening and selection, culminating in inefficient editing. Here, we provide a xeno-free method for single-cell cloning of CRISPRed iPSCs achieving a clonal survival of up to 70% within 7-10 days. This is accomplished through improved viability of the transfected cells, paralleled with provision of an enriched environment for the robust establishment and proliferation of singularized iPSC clones. Enhanced cell survival was accompanied by a high transfection efficiency exceeding 97%, and editing efficiencies of 50%-65% for NHEJ and 10% for HDR, indicative of the method\'s utility in stem cell disease modeling.

Genetically-modified monoclonal cell lines are currently used for monoclonal antibody (mAbs) production and drug development. The isolation of single transformed cells is the main hindrance in the generation of monoclonal lines. Although the conventional limiting dilution method is time-consuming, laborious, and skill-intensive, high-end approaches such as fluorescence-activated cell sorting (FACS) are less accessible to general laboratories. Here, we report a bench-top approach for isolating single Chinese hamster ovary (CHO) cells using an adapted version of a simple microwell-based microfluidic (MBM) device previously reported by our group. After loading the cell suspension to the device, the electrostatically trapped cells can be viewed under a microscope and transferred using a micropipette for further clone establishment. Compared to the conventional method, the invented approach provided a 4.7-fold increase in the number of single cells isolated per round of cell loading and demonstrated a 1.9-fold decrease in total performing time. Additionally, the percentage of correct single-cell identifications was significantly improved, especially in novice testers, suggesting a reduced skill barrier in performing the task. This novel approach could serve as a simple, affordable, efficient, and less skill-intensive alternative to the conventional single-cell isolation for monoclonal cell line establishment.

Autoantibodies targeting brain antigens can mediate a wide range of neurological symptoms ranging from epileptic seizures to psychosis to dementia. Although earlier experimental work indicated that autoantibodies can be directly pathogenic, detailed studies on disease mechanisms, biophysical autoantibody properties, and target interactions were hampered by the availability of human material and the paucity of monospecific disease-related autoantibodies. The emerging generation of patient-derived monoclonal autoantibodies (mAbs) provides a novel platform for the detailed characterization of immunobiology and autoantibody pathogenicity in vitro and in animal models. This Feature Review focuses on recent advances in mAb generation and discusses their potential as powerful scientific tools for high-resolution imaging, antigenic target identification, atomic-level structural analyses, and the development of antibody-selective immunotherapies.

Tumor cell heterogeneity mediated drug resistance has been recognized as the stumbling block of cancer treatment. Elucidating the cytotoxicity of anticancer drugs at single-cell level in a high-throughput way is thus of great value for developing precision therapy. However, current techniques suffer from limitations in dynamically characterizing the responses of thousands of single cells or cell clones presented to multiple drug conditions.
We developed a new microfluidics-based \"SMART\" platform that is Simple to operate, able to generate a Massive single-cell array and Multiplex drug concentrations, capable of keeping cells Alive, Retainable and Trackable in the microchambers. These features are achieved by integrating a Microfluidic chamber Array (4320 units) and a six-Concentration gradient generator (MAC), which enables highly efficient analysis of leukemia drug effects on single cells and cell clones in a high-throughput way.
A simple procedure produces 6 on-chip drug gradients to treat more than 3000 single cells or single-cell derived clones and thus allows an efficient and precise analysis of cell heterogeneity. The statistic results reveal that Imatinib (Ima) and Resveratrol (Res) combination treatment on single cells or clones is much more efficient than Ima or Res single drug treatment, indicated by the markedly reduced half maximal inhibitory concentration (IC50). Additionally, single-cell derived clones demonstrate a higher IC50 in each drug treatment compared to single cells. Moreover, primary cells isolated from two leukemia patients are also found with apparent heterogeneity upon drug treatment on MAC.
This microfluidics-based \"SMART\" platform allows high-throughput single-cell capture and culture, dynamic drug-gradient treatment and cell response monitoring, which represents a new approach to efficiently investigate anticancer drug effects and should benefit drug discovery for leukemia and other cancers.

Single-cell selection and cloning is required for multiple bioprocessing and cell engineering workflows. Dispensing efficiency and outgrowth were optimized for multiple common suspension (CHO ES, Expi293F, and Jurkat) and adherent (MCF-7, A549, CHO-K1, and HEK293) cell lines. Single-cell sorting using a low pressure microfluidic cell sorter, the WOLF Cell Sorter, was compared with limiting dilution at 0.5 cells/well to demonstrate the increased efficiency of using flow cytometry selection of cells. In this work, there was an average single cell deposition on Day 0 of 89.1% across all the cell lines tested compared to 41.2% when using limiting dilution. After growth for 14 days, 66.7% of single-cell clones sorted with the WOLF Cell Sorter survived and only 23.8% when using limiting dilution. Using the WOLF Cell Sorter for cell line development results in higher viable single-cell colonies and the ability to select subpopulations of single-cells using multiple parameters.

The baculovirus expression vector system using insect cells as a bioreactor has been used for in vitro expression of recombinant proteins and plays an important role in the fields of biology, agronomy, and medicine. Screening suitable host cell lines is an important part of the construction of insect cell baculovirus expression systems. In previous research, we used a single-cell cloning process with the Papilio xuthus cell line RIRI-PX1 and obtained the monoclonal cell line RIRI-PX1-C31. In this study, we compared the basic biological and recombinant protein expression characteristics of RIRI-PX1-C31 and its parent cell line RIRI-PX1 and found that the expression of recombinant β-galactosidase in RIRI-PX1-C31 was significantly higher than that in the parental cell line. Further serum-free adaptation studies confirmed that RIRI-PX1-C31 can adapt to the growth environment of Express Five Serum-free medium and that its expression level of recombinant β-galactosidase was significantly higher than that before adaptation.

BACKGROUND: Disease-specific human induced pluripotent stem cells (hiPSCs) can be generated directly from individuals with known disease characteristics or alternatively be modified using genome editing approaches to introduce disease causing genetic mutations to study the biological response of those mutations. The genome editing procedure in hiPSCs is still inefficient, particularly when it comes to homology directed repair (HDR) of genetic mutations or targeted transgene insertion in the genome and single cell cloning of edited cells. In addition, genome editing processes also involve additional cellular stresses such as poor cell viability and genetic stability of hiPSCs. Therefore, efficient workflows are desired to increase genome editing application to hiPSC disease models and therapeutic applications.
RESULTS: To this end, we demonstrate an efficient workflow for feeder-free single cell clone generation and expansion in both CRISPR-mediated knock-out (KO) and knock-in (KI) hiPSC lines. Using StemFlex medium and CloneR supplement in conjunction with Matrigel cell culture matrix, we show that cell viability and expansion during single-cell cloning in edited and unedited cells is significantly enhanced. Keeping all factors into account, we have successfully achieved hiPSC single-cell survival and cloning in both edited and unedited cells with rates as maximum as 70% in less than 2 weeks.
CONCLUSIONS: This simplified and efficient workflow will allow for a new level of sophistication in generating hiPSC-based disease models to promote rapid advancement in basic research and also the development of novel cellular therapeutics.

Mammalian cell line development is a multistep process wherein timelines for developing clonal cells to be used as manufacturing cell lines for biologics production can commonly extend to 9 months when no automation or modern molecular technologies are involved in the workflow. Steps in the cell line development workflow involving single-cell cloning, monoclonality assurance, productivity and stability screening are labor, time and resource intensive when performed manually. Introduction of automation and miniaturization in these steps has reduced the required manual labor, shortened timelines from months to weeks, and decreased the resources needed to develop manufacturing cell lines. This review summarizes the advances, benefits, comparisons and shortcomings of different automation platforms available in the market for rapid isolation of desired clonal cell lines for biologics production.

Cell line development (CLD) represents a critical, yet time-consuming, step in the biomanufacturing process as significant resources are devoted to the scale-up and screening of several hundreds to thousands of single-cell clones. Typically, transfected pools are fully recovered from selection and characterized for growth, productivity, and product quality to identify the best pools suitable for single-cell cloning (SCC) using limiting dilution or fluorescence-activated cell sorting (FACS). Here we report the application of the Berkeley Lights Beacon Instrument (BLI) in an early SCC process to accelerate the CLD timeline. Transfected pools were single-cell cloned when viabilities reached greater than 85% or during selection when viabilities were less than 30%. Clones isolated from these accelerated processes exhibited comparable growth, productivity, and product quality to those derived from a standard CLD process and fit into an existing manufacturing platform. With these approaches, up to a 30% reduction in the overall CLD timeline was achieved. Furthermore, early process-derived clones demonstrated equivalent long-term stability compared with standard process-derived clones over 50 population doubling levels (PDLs). Taken together, the data supported early SCC on the BLI as an attractive approach to reducing the standard CLD timeline while still identifying clones with acceptable manufacturability.