As my mother, a seasoned scientist and lab veteran who has trained countless students and post-docs, likes to say, “They don’t call it RE-search for nothing. It’s because we RE-do experiments over and over again until it works.” So, it should come as no surprise that she cautioned me to think long and hard about becoming a career researcher.
In the folly of my youth, I didn’t listen, and as mothers tend to be, it turns out she was completely right. Day after day, scientists show up to the lab striving to achieve the impossible. Despite long hours, grueling experiments, and weeks or months of failure, we persevere, hoping for that elusive breakthrough that can impact human health. Yet sometimes, despite our best efforts, flawless experimental designs, and meticulous lab work, our tools fall short, leaving us to RE-peat our labors.
It is our keen understanding of this frustration that drove us to launch our “Clone Challenge”. Our aim was simple: provide struggling scientists access to novel technology that could solve the bottleneck inhibiting their progress. Our first clone challenge winner came to us from Stanford University. We were excited by the complexity of the problem and the opportunity to help.
In their application, the Stanford team explained that they were using a human glioblastoma cell line (the most malignant adult brain cancer type), which must be cultured in suspension as spheroids to prevent differentiation. However, as is often the case, the cell line is not homogenous, and approximately 90% of the population has one type of genetic amplification, while 10% of the population has a different genetic amplification of the same gene. For months, they had been attempting to derive clonal populations of these two genotypes without success, as these cells do not grow well as single cells. Moreover, the screening necessary to confirm the genotypes was labor-intensive, expensive, and not easily scalable.
The team at Stanford provided us with their cell line, and we used 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 to derive monoclonal cell lines from the bulk parental line. Although the initial promise of the clone challenge was five monoclonal cell lines, due to the low frequency (1 in 10) of one of the genotypes, we realized we needed to screen as many as possible. Over the course of about six weeks, we generated clonal cell lines that maintained their ability to grow as spheroids (see below). Upon returning the clones to Stanford, they validated that 26 of the lines were correct, and they were able to obtain isogenic matched pairs with both genotypes after confirmation with their orthogonal methods.
As a scientist who has failed in the lab all too often, the response from the team at Stanford was profoundly gratifying:
“Thank you so much for your help earlier in helping us to get monoclonally expanded cells. We are all very happy with the outcome as it has been quite a challenging cell system for monoclonal expansion. The clones are a great addition to the lab’s expanding cell line models.”
The moral of the story? Sometimes, you just need a little help. This is why I show up every day as a scientist at Cell Microsystems. We develop technology that enables the impossible and eliminates roadblocks. Watching fellow researchers leverage our platform to achieve breakthroughs is incredibly fulfilling. So even though mom may have been right all those years ago, I wouldn’t change a thing about my journey.
A single GBM39 cell on 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 generated a monoclonal population of cells that formed spheroids in culture and demonstrated a specific genotype as measured by fluorescence in situ hybridization (FISH).
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 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 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.
