Fastest growth.
Biggest colonies.
Healthiest clones.
Prettiest morphology.
Relying on these commonly used, albeit biased, superlatives to make decisions about which cells are seemingly the “best” can ignore true biological phenotypes. As the story below highlights, the best clone may ultimately be the one that breaks the mold of these canonical criteria.
During a recent visit to a large pharma company that uses the CellRaft AIR System for cell line development, a dynamic user of the system shared a remarkable story about how the unique ability of the CellRaft AIR systemAn all-in-one platform for imaging, identifying, and isolating viable single cells, colonies, or organoids using CellRaft Arrays and software-guided selection with CellRaft Cytometry. More to support the viability of all cells, not just the strongest cells, allowed her to finally generate a long-awaited clone.
500 Clones, 0 Knockouts
In this experiment, the researcher was asked to make a CRISPR knockout of a gene in a human bladder carcinoma cell line. Using both FACS and the CellRaftA microscale polystyrene growth surface within an array used to spatially segregate cells/colonies while maintaining shared media access, enabling imaging over time and targeted isolation. More AIR in parallel, she performed five independent transductions and screened more than 500 clones without identifying a single homozygous knockout clone. As she laughingly admitted, she was picking “the best clones” that were growing robustly and assumed that the failure to generate a knockout clone was due to a technical issue such as electroporation efficiency or construct design.
Biological Breakthrough
After numerous failed attempts, she began to suspect that it could be a true biological phenotype and not a technical limitation. She noticed that many of the clones that survived on the CellRaft ArrayConsumables containing thousands of microwells (containing CellRafts) for spatial segregation, imaging, and isolation of single cells, monoclonal colonies, or 3D cultures. More were small and growing very slowly. In desperation, she isolated the slow-growing clones using the CellRaftA microscale polystyrene growth surface within an array used to spatially segregate cells/colonies while maintaining shared media access, enabling imaging over time and targeted isolation. More AIR, and remarkably, those “less beautiful” clones were her long-awaited triple homozygous knockouts. In contrast, none of the clones generated by FACS were knocked out for the gene of interest, likely because the loss of the gene conferred a growth disadvantage, and the cells could not survive after the stress of single-cell sorting.
Sharing the Knowledge
Amazingly, she was able to use this knowledge to accelerate not only her own research but also that of her colleagues at a sister site. When she learned that another group was making point mutation edits to the same gene in a different cell line, she advised them to leverage the CellRaftA microscale polystyrene growth surface within an array used to spatially segregate cells/colonies while maintaining shared media access, enabling imaging over time and targeted isolation. More Cytometry software to select CellRafts containing slow-growing, small colonies. Using this information, the group was able to generate their desired clonal cell line in ONE round of screening, not five, in only two months as compared to the year she spent uncovering the phenomenology of the gene.
From Zero to Hero
As she told me “I was a sorting devotee, but I am converted to the CellRaftA microscale polystyrene growth surface within an array used to spatially segregate cells/colonies while maintaining shared media access, enabling imaging over time and targeted isolation. More. Here, I have pretty much every well growing because we already picked the cells that expanded from the raft. With flow, we are just putting cells in a well and hoping. I’m not even turning on the cytometer anymore because there’s no point; it takes too long and isn’t going to work. The CellRaftA microscale polystyrene growth surface within an array used to spatially segregate cells/colonies while maintaining shared media access, enabling imaging over time and targeted isolation. More AIR is really improving our success rate, because we need to understand the biology, and we can use the (CellRaftA microscale polystyrene growth surface within an array used to spatially segregate cells/colonies while maintaining shared media access, enabling imaging over time and targeted isolation. More) AIR to aid in the process.”
In an increasingly fast-paced scientific landscape where “-est” is everything, this real-world experience acutely highlights the importance of allowing biology to drive discovery, not superlatives.
Click below to learn how tools like the CellRaftA microscale polystyrene growth surface within an array used to spatially segregate cells/colonies while maintaining shared media access, enabling imaging over time and targeted isolation. More AIR can make that goal easily attainable.
Dr. Jessica Hartman has a B.S in Biology from the University of Virginia, a Ph.D. from Duke University in Molecular Cancer Biology and postdoctoral training in Biochemistry and Cancer Biology at Baylor College of Medicine and Duke University, respectively. She has previously served in Director-level roles, managing bioscience research and development for biotechnology companies. At Cell Microsystems, Dr. Hartman’s role is to lead the development of new and streamlined workflows using the CellRaft Technology and its associated products.
In the ongoing battle against antibiotic-resistant bacteria, or “superbugs,” scientists are constantly seeking innovative tools to understand the mechanisms of resistance and develop effective treatments. One such groundbreaking technology is shear flow systems, which have emerged as game-changers in microbiological research. By simulating the dynamic conditions of bacteria’s natural environments, such as the human body or food processing, shear flow systems provide unique insights into bacterial behavior and resistance mechanisms that traditional static methods simply cannot achieve. In this blog post, we’ll explore the benefits of using shear flow systems to combat antibiotic resistance and develop next-generation therapies.
Antibiotic resistance is a complex phenomenon influenced by a variety of factors, including genetic mutations, horizontal gene transfer, and the formation of biofilms. Traditional laboratory methods often fail to capture the dynamic nature of these processes, leading to incomplete or misleading results. Shear flow systems address this limitation by providing a more realistic environment for bacterial growth and interaction. Here’s how:
1. Mimicking Real-World Conditions
In the human body, bacteria are rarely in a static state. They are constantly exposed to fluid flow, such as blood circulation or urine flow, which influences their behavior and resistance mechanisms. Shear flow systems can replicate these conditions, allowing researchers to study how bacteria respond to antibiotics under realistic physiological conditions. This is critical for understanding how resistance develops and persists in vivo. This benefit was recently highlighted in a webinar presented by Dr. Katharina Richter, a microbiology researcher from the University of Adelaide in Australia. Using a high-throughput BioFlux Shear Flow System, Dr. Richter and her team were able to test 3 different methods of superbug treatments under physiological conditions.
2. Studying Biofilm Formation and Resistance
Biofilms—structured communities of bacteria encased in a protective matrix—are a major contributor to antibiotic resistance. Biofilms are notoriously difficult to treat because they shield bacteria from antibiotics and the immune system. Shear flow systems enable researchers to study biofilm formation in real time, observing how bacteria adhere to surfaces, form microcolonies, and develop resistance under flow conditions. For example, using a BioFlux, Dr. Richter was able to leverage high-resolution imaging to obtain unprecedented insight into the biofilm killing and prevention efficacy of a copper and diethyldithiocarbamate (DDC) combination nanoparticles¹. The group is currently investigating the use of a Cu-DDC infused gel that can be injected into wounds that are at high risk for chronic infection, such as hernia.
The study of biofilm communities presents unique challenges in experimental design and analysis. While mixed bacterial populations offer fascinating insights into microbial interactions and community dynamics, accurately characterizing and investigating these complex systems has previously required sophisticated and tedious technical approaches. Here, we will discuss advances in technology that have helped to simplify and streamline polymicrobial investigations.
Throughout the last decade, a trend for growing and assaying biofilms under shear flow has gained traction among microbiologists. The rationale for investigating biofilms under flow is multi-dimensional. A recent Cell Microsystems survey asked why microbiologists are using shear flow found that the top responses were:
Of the scientists surveyed, 40% indicated that they use a BioFlux Shear Flow System. This system provides unique advantages over other methods to induce shear stress, such as slide-based parallel plate flow chambers and benchtop rockers.
BioFlux uses microfluidic channels embedded onto the bottom of standard-sized 6, 24, or 48-well plates to create isolated and independent flow chambers where user-defined wall shear stress is initiated and maintained by digitally controlled air pressure. This not only simplifies the setup and experimental processes but also ensures that experiments are contained and do not contaminate the shear flow system.
Although shear flow has been thoroughly demonstrated to improve the biological relevance of biofilm investigations1, an often overlooked aspect of biofilm physiology is the significant role that multicellular aggregates play in biofilm formation2. When investigating Pseudomonas aeruginosa, it has been demonstrated that multicellular aggregates lead to faster biofilm formation and enhanced resistance to antimicrobials and immune responses3. In addition, multispecies microbial aggregates have been shown to dominate single cells in human saliva4. Together, these data show that aggregates are important areas of investigation because they are more likely to represent physiological biofilm formation and growth.
