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 dilution. Although widely utilized, these methods present both technical and biological challenges, and technologies such as the CellRaft 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 dilution, 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 dilution is seen as a lower-cost option, it is time-consuming, lacks proof of monoclonality, requires sample preparation, and drives a tremendous amount of nonbiodegradable recalcitrant plastic waste. Furthermore, two rounds are minimally recommended to be able to approach monoclonality 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 dilution, 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: CellRaft Technology workflow. The advantage of the CellRaft 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 CellRaft Technology
The use of CellRaft 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 System 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 monoclonality, and as the saying goes “a picture is worth a thousand words”. The CellRaft AIR can image an entire CellRaft array in as little as 6 minutes in brightfield, providing a saved image of every single CellRaft 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 CellRaft 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, CellRaft 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, CellRaft 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.
As drug discovery and disease modeling become increasingly sophisticated, 3D cell culture has emerged as a powerful alternative to traditional 2D assays and animal models. In a recent webinar Dr. Allysa Stern showcased how researchers are using the CERO 3D Incubator and Bioreactor to streamline development of organoid models that are scalable, reproducible, and biologically relevant.
Despite decades of optimization in drug development, the failure rate for candidate therapeutics remains high, often due to poor translation from animal models to human biology. That’s why researchers are shifting toward more predictive, human-relevant systems like organoids derived from induced pluripotent stem cells (iPSCs). These models better capture tissue-specific physiology and patient diversity, offering an edge in everything from disease modeling to toxicity screening.
The CERO 3D is a benchtop bioreactor system that enables gentle, dynamic suspension culture of spheroids and organoids. Designed for ease of use, it supports up to four independently controlled tubes, allowing researchers to optimize protocols in parallel. Unlike traditional bioreactors, the CERO 3D requires no impellers or external fluidics. Instead, it uses integrated baffles to create a low-shear, homogenized environment ideal for 3D culture.
Cell culture is the backbone of modern biomedical research, drug development, and regenerative medicine. Traditionally, cells are grown in static environments—like petri dishes or flasks—where stationary media can lead to inconsistent results. Today, researchers are adopting shear flow systems that use dynamic media circulation to mimic the natural forces and nutrient dynamics cells experience in vivo. Here’s why shear flow outperforms static cultures, with a focus on its revolutionary impact on cell culture media:
Physiological Relevance: Media Flow Activates Mechanobiology
Cells like endothelial or bone cells evolved to respond to fluid shear stress. Shear flow systems replicate these forces via media movement, activating critical pathways.
Continuous Fresh Media Supply: Eliminating Feast-or-Famine Cycles
Static cultures rely on manual media changes, creating peaks and crashes in nutrient availability. Shear flow systems ensure steady, automated media replenishment, optimizing cell health and reproducibility.
Constant Waste Removal: Cleaner Media, Healthier Cells
Static media allows metabolic waste (e.g., lactate, ammonia) to accumulate, poisoning cells. Shear flow continuously flushes byproducts, mirroring in vivo clearance mechanisms.
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.
