The satisfaction that comes from seeing a carefully designed experiment unfold and coalesce into a clue, an answer, or a new tool is what makes research and development so gratifying. But every scientist knows that this process is not always seamless. When the goal is big, like Kite Pharma’s ultimate goal of curing cancer, there are bound to be obstacles.
Kite Pharma, a Gilead Company, develops genetically engineered, autologous CAR T cell therapies for the treatment of cancer. Kite’s mission statement emphasizes how these therapies can be used:
“At Kite, our singular focus is cell therapy to treat and potentially cure cancer. Our goal is to bring the promise of cell therapy to as many eligible patients as possible who may benefit. We believe cell therapy has the ability to change the way cancer is treated. Our industry-leading cell therapy technology uses the power of a patient’s own immune system (their white blood cells) to target and attack their cancer cells.”
At the heart of this mission is the ability to engineer cell lines for target discovery and disease modeling. At Kite, the Flow Cytometry and Cell Engineering Core uses Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) gene editing to develop cell lines for research. Even with expertise and advanced instrumentation at their disposal, the team at Kite found that relying on single cell cloning for cell engineering was causing a bottleneck that was a barrier to efficiency and progress.
The Core at Kite Pharma had been using various techniques for single cell cloning, including 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, high-speed cell sorting, or combinations of bulk sorting and single cell dispensing. They often encountered poor or variable cell outgrowth after isolation of single cells into 96-well plates, resulting in timelines that were not amenable to their fast-paced goals. To alleviate these pain points and accelerate their cell line development pipeline, they leveraged 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® System from Cell Microsystems.
To compare the efficiency of several methods for cell line generation, Kite created CRISPR knockout Raji cells, which are lymphoblast-like, suspension cells derived from B-lymphocytes commonly used to test anti-tumor activity for therapeutic approaches related to B cell malignancies. Kite analyzed various single cell clone generation workflows using 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 as well as traditional cell sorters like the BD FACS Aria Fusion, the SONY SH800 Sorter, and ThermoFisher BigFoot Spectral Cell Sorter.
Concisely stated, cells were transfected with CD19 sgRNAs (Fig. 1), then either sorted directly into 96-well plates using the traditional cell sorters, or bulk sorted to enrich for the CD19PE+ cells and applied to a 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® Array (Fig. 2).
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 to image the CellRaft ArrayConsumables containing thousands of microwells (containing CellRafts) for spatial segregation, imaging, and isolation of single cells, monoclonal colonies, or 3D cultures. More over the course of culture, the system captured an image-based record of single cells as they divide into clonal colonies of cells (Fig. 3A-H). The images shown in Fig. 3A-H are representative of individual CellRafts, with Fig. 3A-D and Fig. 3E-H showing the same 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 over the course of 4 and 5 days, respectively. After isolation, Kite found that colonies isolated from the CellRaft ArrayConsumables containing thousands of microwells (containing CellRafts) for spatial segregation, imaging, and isolation of single cells, monoclonal colonies, or 3D cultures. More grew out in over 60% of wells, compared to only 30% growth from single cells dispensed into 96-well plates using cell sorters.
Ultimately, the team at Kite Pharma implemented 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 in their Core because it provides a greater number of clones due to superior cell growth and expansion, enabling them to shorten their timeline to cell line generation and improve their efficiency. Combined with the ease-of-use, confirmation of monoclonalityEvidence that a colony/line originated from one cell; often supported by time-course imaging and traceability. More, and software-driven clone selection, 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 enables a more researcher-friendly workflow that helps companies achieve their overarching goals.
Dr. Megan Harrison is responsible for demonstrating the power of the CellRaft® AIR System to the scientific community, training new users on CellRaft® Technology, and supporting current users by helping them to develop new workflows and to optimize existing processes. Dr. Harrison has experience as a researcher and leader in industry and academic settings. Most recently, as Principal Scientist for Frequency Therapeutics, she developed and characterized 3D culture models, such as primary tissue-derived organoids and spheroids, which were used for small molecule drug discovery in the regenerative medicine space. Dr. Harrison received post-doctoral training at Yale School of Medicine in the Cell Biology department, where her research focused on endosomal trafficking. She earned her Ph.D. in Pathobiology from The Pennsylvania State University, where her research focused on Paramyxovirus budding. Dr. Harrison holds a B.S. in Pre-Veterinary Medicine and Animal Biotechnology from the University of Delaware, where she first learned to conduct research in avian virology.
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.
