CRISPR & Cell Engineering
CRISPR technology enables quick and easy modification of any gene in a given cell type. In a typical CRISPR workflow, target cells are transfected with a plasmid containing both the CRISPR editing construct and a fluorescent reporter, enabling easy identification of successful transfections. Edited cells are then isolated for clonal expansion. Single cell isolation has traditionally been done with two approaches, both of which have inherent drawbacks.
Limiting dilution can be extremely inefficient and costly for clonal cell isolation, particularly when transfection efficiency is low. In the event of low transfection rates, it is nearly impossible to isolate clones via limiting dilution. Moreover, single cell isolation efficiency is highly variable with limiting dilutions, resulting in many empty wells or multiple cells per well.
Fluorescence-Activated Cell Sorters (FACS), while faster and higher throughput than limiting dilutions, operate at high system pressure (up to 70 psi) that can easily damage or kill the sorted cells. Induced pluripotent stem (iPS) cells, a commonly used cell type for CRISPR cloning, are particularly sensitive to the high pressure and have low survival rates following isolation. Additionally, FACS is difficult to operate and prone to contamination.
For CRISPR workflows, cells are transfected with a CRISPR construct and fluorescent reporter, then cultured during a recovery period. Following recovery, fluorescently-labeled transfected cells are identified and dispensed with Namocell’s Single Cell Dispenser. After clonal expansion, colonies are analyzed via PCR to identify which clones contain the desired genotype.
- Gentle sorting (< 2 psi) preserves cell viability
- Higher clonality, better outgrowth
- Compact size fits in tissue culture hood
- Sterile and disposable cell cartridge
- 96-well plate in 1 min
- 384-well plate in 6 min
- Quick set up in under 3 min
- No calibration, no maintenance
Namocell Single Cell Dispensers are highly efficient and consistent at dispensing single cells. With dispensing efficiency (percentage of dispensed wells with single cells) of 80-90% for CHO cells compared to ~30% using manual pipetting.
Clonal growth of mammalian cells sorted using the Namocell Single Cell Dispenser alongside both limiting dilution (LDC) and FACS. Cells sorted with the Namocell showed higher cloning efficiencies than LDC and FACS.
Generating Clonal GFP Cell Lines
Single Cell Dispensing Efficiency
Single Cell Isolation with Minimal Input
Protocols & Resources
Unlabeled iPS Cell Prep Protocol
Practical Tips for Single Cell Isolation
Webinar: Fast, Gentle and Easy Single Cell Isolation
Webinar: Chemical Cytoprotection for Stress-Free Use of hPSC’s
High-throughput and automation advances for accelerating single-cell cloning, monoclonality and early phase clone screening steps in mammalian cell line development for biologics production. Tejwani V, et al. Biotechnol Prog. 2021 Nov;37(6):e3208. doi: 10.1002/btpr.3208. Epub 2021 Sep 20. PMID: 34478248.
Efficient and safe single-cell cloning of human pluripotent stem cells using the CEPT cocktail. Tristan, C.A., et al. Nat Protoc 18, 58–80 (2023). https://doi.org/10.1038/s41596-022-00753-z
Integrating Micro and Nano Technologies for Cell Engineering and Analysis: Toward the Next Generation of Cell Therapy Workflows Prithvijit Mukherjee, et al. ACS Nano 2022 16 (10), 15653-15680. DOI: 10.1021/acsnano.2c05494
Age-Related Low Bone Mineral Density in C57BL/6 Mice Is Reflective of Aberrant Bone Morphogenetic Protein-2 Signaling Observed in Human Patients Diagnosed with Osteoporosis. Halloran D, et al. Int J Mol Sci. 2022 Sep 23;23(19):11205. doi: 10.3390/ijms231911205. PMID: 36232525; PMCID: PMC9570292.
Coexpression network and trans-activation analyses of maize reproductive phasiRNA loci. Zhan, J., et al. (2023), Plant J, 113: 160-173. https://doi.org/10.1111/tpj.16045
Identification of Inhibitors of Tubulin Polymerization Using a CRISPR-Edited Cell Line with Endogenous Fluorescent Tagging of β-Tubulin and Histone H1. Khachatryan H, et al. Biomolecules. 2023; 13(2):249. https://doi.org/10.3390/biom13020249
A versatile polypharmacology platform promotes cytoprotection and viability of human pluripotent and differentiated cells. Chen, Y., et al. Nat Methods 18, 528–541 (2021). https://doi.org/10.1038/s41592-021-01126-2
What Scientists are Saying
Here at Cytovance, we love our Namocell. This machine has increased our plating capacity and efficiency nearly four-fold. The accuracy of Namocell’s 384-well plating coupled with the easy changes between samples has been a game changer for our workflow.
Namocell Hana single cell dispenser provides a great solution for gentle single cell cloning using sterile disposable cartridges. The system is easy to use and quick to initialize. Hana combines the advantages of FACS and microfluidic cell dispensing to enable high assurance of monoclonality with high throughput and great cell viability.
We’ve been using the Namo to do single cell sorting for genomics/ transcriptomics applications as well as for colony selection for tissue culture. We’re overall very satisfied with our results and find the setup to be very easy, as well as standard maintenance. After sterility protocols (which can take an hour), being able to immediately jump to sorting in less than ten minutes has saved us time and precious samples.
The Hana has had a positive impact on our workflow. We are now able, with a high degree of confidence, to generate clonal populations of cells with reduced cytotoxicity (compared to FACS) while simultaneously saving hands-on time due to not needing to immediately screen for single cells after manual limited dilutions. In many cell lines, we see a significantly higher efficiency of clonality from plate to plate compared to our previous methods.
The instrument works very well for our purposes of single-cell dispensing, primarily gene-edited lines. Easy to use; the setup and dispensing time is very short. It has the benefits of a FACS machine (binning by forward/side scatter), with lower pressure to help increase cell survival.
Namocell’s disposable cartridges have allowed us to increase the throughput of the validation of our CRISPR engineered cell lines since we can sort a full plate in less than 3 minutes, and can move to the next plate without the hassle of cleaning. This outstanding product is paired with Namocell’s exceptional customer service that has been responsive and attentive to our needs. While evaluating different FACS machines, we simply did not find a rival that combined Namocell’s simplicity, performance and value, all while occupying a small footprint.
Our stem cell core facility was looking for ways to speed up our human induced pluripotent cell cloning to generate clonal cell lines and to analyze our gene editing efficiencies in single cells. Our criteria for an instrument for single cell dispensing was 1) sterility, 2) gentle on cells, 3) ease of use, and 4) reasonable cost. The Namocell fitted our criteria perfectly. We are able to reduce the time and effort for generating clonal CRISPR-engineered iPSC lines significantly. The instrument requires very little maintenance and users do not need to be highly trained with the main requirement being good aseptic technique. We are excited to use the Namocell potentially for other single cell applications.