Monoclonal cell lines are a powerful tool for biomedical research and drug development. The development and use of monoclonal cell lines have been accelerated due to the advent of breakthrough gene editing technologies such as CRISPR-Cas9. Due to their ease of use and amenability to single cell cloning, there are several well-established and recognizable cell lines that are commonly used to create monoclonal colonies, including the CHO and HEK293 lines. However, primary cells such as induced pluripotent stem cells (iPSCs), or immortalized cancer cell lines have proven more challenging. Historically, monoclonal cell lines have been developed using traditional methods, such as flow cytometry or manual limiting dilutionA statistical cloning approach where cells are diluted to low density to “hopefully” seed one cell per well; commonly used but can be low-efficiency for difficult-to-clone lines. More. Although widely utilized, these methods present both technical and biological challenges, and technologies such as 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 Technology offer several profound advantages over traditional methods. In this blog post, we will provide an overview of available technologies for monoclonal cell line development and how researchers can overcome some of the limitations faced with these technologies.
Traditional Methods for Developing Monoclonal Cell Lines
Monoclonal cell lines can be generated with the incorporation of traditional methods such as limiting dilutionA statistical cloning approach where cells are diluted to low density to “hopefully” seed one cell per well; commonly used but can be low-efficiency for difficult-to-clone lines. More, single cell dispensing, flow cytometry, cell sorting, and cell dispensers. A table comparing these methods can be found in the white paper titled Development of Monoclonal Cell Lines – Available Technologies and Overcoming Challenges. Unfortunately, each of these methods is not without limitations. While limiting dilutionA statistical cloning approach where cells are diluted to low density to “hopefully” seed one cell per well; commonly used but can be low-efficiency for difficult-to-clone lines. More is seen as a lower-cost option, it is time-consuming, lacks proof of monoclonalityEvidence that a colony/line originated from one cell; often supported by time-course imaging and traceability. More, requires sample preparation, and drives a tremendous amount of nonbiodegradable recalcitrant plastic waste. Furthermore, two rounds are minimally recommended to be able to approach monoclonalityEvidence that a colony/line originated from one cell; often supported by time-course imaging and traceability. More and single cell confirmation. The incorporation of laboratory equipment over recent years has improved throughput and reduced the hands-on time that is required with limiting dilutionA statistical cloning approach where cells are diluted to low density to “hopefully” seed one cell per well; commonly used but can be low-efficiency for difficult-to-clone lines. More, but many are expensive to incorporate, require extensive training, and can result in phenotypically perturbed cells that have poor viability and outgrowth.
Figure 1: Current methods for single cell cloning. The majority of technologies available on the market separate a heterogeneous population of cells into single cells within a microwell, either via manual pipetting or fluidics-based segregation.
Overcoming Limitations of Current Technologies
CellRaft Technology is an affordable and modern method for developing monoclonal cell lines that overcomes the limitations of traditional methods. It provides flask-like culture conditions at the resolution of a single cell, with gentle and automated isolation using image-based attributes for function, gene expression, and morphology. Within a single platform, researchers can grow, scan, analyze, and isolate single cell derived monoclonal colonies.
Figure 2: 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 Technology workflow. The advantage of 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 technology is that cells are seeded as a population while maintaining single-cell separation. Cells can grow in situ on individual CellRafts to form discrete colonies that can be identified using image-based software analysis tools. After growth on the array, the monoclonal colony, rather than a single cell, is isolated intact and transferred to a downstream collection plate for continued growth, expansion, and downstream analysis.
Benefits of 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 Technology
The use of 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 Technology offers several advantages over traditional methods for developing monoclonal cell lines including:
Figure 3: Track-and-trace colony growth from a single cell. Three representative CellRafts containing single cells on day 1 and imaged on 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 over a 4-day period until colony formation. This serial imaging allows for precise monitoring of exponential growth and phenotypic characterization, as well as improved viability downstream.
One of the most critical components of cell line development is proof of monoclonalityEvidence that a colony/line originated from one cell; often supported by time-course imaging and traceability. More, and as the saying goes “a picture is worth a thousand words”. 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 image an entire CellRaft arrayConsumables containing thousands of microwells (containing CellRafts) for spatial segregation, imaging, and isolation of single cells, monoclonal colonies, or 3D cultures. More in as little as 6 minutes in brightfield, providing a saved image of every single 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 for impeccable record keeping and the ability to track and trace single cells from seeding to colony formation (Figure 3). In addition, because the single cells are individually segregated within the array but share a contiguous media volume, viability and clonal growth are significantly improved compared to methods that put a single cell in a well. This key feature leads to hundreds to thousands of clonal colonies to choose from, ensuring that the researchers are able to get exactly the phenotypic characteristics they desire, rather than settling for whichever clones survive. Lastly, 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 technology is user-friendly, easy to learn, and requires a very low barrier to entry compared to instrumentation such as flow sorters.
Table 1:
Conclusion
In summary, monoclonal cell line development is an important step in biomedical research that has traditionally relied on time-consuming and labor-intensive techniques or high-cost alternatives that produce low-viability cell populations. However, 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 Technology offers significant advantages over traditional methods due to its affordability, speed, accuracy, and scalability capabilities, as well as its overall improvement of cell viability and clonal growth. For scientists struggling to find an efficient and successful method for growing and recovering high-quality monoclonal cell lines, 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 Technology is a novel solution that will improve overall research productivity and progress.
For a deeper look comparing technologies, download the white paper, Development of Monoclonal Cell Lines – Available Technologies and Overcoming Challenges.
Kap Kumar has over 25 years of experience in the life sciences tools and reagents industry. He started in R&D and product development, where he launched products for cell biology and imaging applications. For the last 15 years, Kap has led strategic marketing, market development, and product management for a variety of companies, including Thermo Fisher Scientific (Life Technologies), Danaher (Beckman Coulter Life Sciences), Cell Signaling Technologies, Nexcelom Biosciences, and Avantor-VWR. Kap has diverse experience managing complex portfolios, including instruments, consumables, and reagents, both in early-stage and mature companies. Kap has a Ph.D. in Cell and Molecular Biology from Kent State University, a post-doctorate from Harvard Medical School, and an MBA from Babson College.
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
