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.
Imagine working with a rare patient-derived cell samples. In a traditional setup, you might need 1 milliliter of sample (equivalent to about 20 drops) to run an experiment. With a microfluidic system, you could potentially get the same results with just 10 microliters (about one-half of a single drop). This efficiency opens up entirely new research possibilities, especially in fields where sample availability is a limiting factor.
Whether due to rarity, cost, or difficulty in obtaining samples, the ability to conduct meaningful experiments with limited sample volumes has long been a goal in biological and chemical research. In this quest for more efficient and precise laboratory techniques, microfluidic shear flow systems have emerged as game-changing tools. These systems are not only reducing the amount of sample required for experiments but are also proving their versatility across a wide range of sample types.
Traditional experimental setups, including static and non-microfluidic shear flow, often demand significant amounts of material, which can be both costly and impractical. This is especially true when working with precious or limited samples such as rare biological specimens, expensive chemicals, or newly synthesized compounds. Enter microfluidic shear flow systems – an elegant solution that’s revolutionizing how investigators approach assays. These miniaturized platforms are proving invaluable across multiple disciplines by dramatically reducing sample requirements from milliliters down to microliters, while maintaining experimental integrity. This up to 1000-fold reduction in sample volume is particularly crucial when working with substances, such as patient-derived cells or novel pharmaceuticals. By significantly reducing the volume of reagents and samples needed, these systems cut costs and make experiments more sustainable.
Equally as impressive as their sample and reagent reduction ability, is the versatility of microfluidic shear flow systems to adapt to different types of samples.
The ability to work with multiple sample types in the same system allows researchers to explore different experimental conditions without the need for multiple specialized setups. By precisely controlling channel geometries and flow rates, researchers can subject different sample types to well-defined shear conditions, enabling detailed studies of cellular mechanics, protein interactions, and material properties.
By Jessica Hartman, Ph.D.
Toiling in the lab
With a deadline on its way
To the scope I go
On a Saturday
I don’t see a clone
I’ve looked for one that’s right
What misery these data bring,
I’ll have to look all night,
Oh! Single cells, single cells, dilutions aren’t the way
Oh, I wish I had an AIR to pick a clone, to-da-ay!
Single cells, single cells, I need a better way
Use a Raft to grow a clone or be sad this holiday!
Use a Raft to grow a clone or be sad this holiday!
Now it’s getting late
My spirits are so low
My PI will hate
If my cells don’t grow
My eyesight’s getting dim
A clone I cannot see
My chances are so slim
I need 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
Oh! Single cells, single cells, sorters aren’t the way
Oh, I wish I had an AIR to pick a clone, to-da-ay!
Single cells, single cells, I need a better way
Use a Raft to grow a clone or be sad this holiday!
Use a Raft to grow a clone or be sad this holiday!
Oh! Single cells, single cells, dispensers aren’t the way
Oh, I wish I had an AIR to pick a clone, to-da-ay!
Single cells, single cells, I need a better way
Use a Raft to grow a clone or be sad this holiday!
Use a Raft to grow a clone or be sad this holiday!
“Single Cell” vocalist: Virginia Laurie
