✍🏼 Adonary Munoz, José M. Morachis

🏠 NanoCellect Biomedical, San Diego, CA

📑 Biotechnol Lett (2022)

Abstract
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.

How the WOLF was used in this study
The authors used the WOLF Cell Sorter as a gentle, low-pressure microfluidic fluorescence-activated cell sorter to perform single-cell cloning across multiple mammalian cell lines. Cells were prepared at ~1×10⁵ cells/mL, filtered, and then sorted with a sterile single-cell cartridge directly into 96-well plates containing optimized cloning media. The WOLF sorter enabled precise deposition of one cell per well, significantly improving the efficiency of single-cell outgrowth compared to traditional limiting-dilution methods by allowing selection of viable, fluorescently marked or gated cells and reducing wells with multiple cells or no cells at all. This single-cell isolation was validated with beads and used on various adherent and suspension lines, demonstrating higher deposition efficiency and increased survival of single-cell colonies, which is crucial for workflows in cell line development, drug screening, and bioprocessing.