Agilent BioTek MultiFlo FX Multimode Dispenser

The establishment of in vitro models that mimic tumor invasion as part of the metastatic process is a critical part of oncology research. The need to incorporate multiple cell types, long-term kinetic analysis, methods to allow 3D invasion into the surrounding matrix, and appropriate detection and analysis, has made it difficult to create new models. In this application note, learn more about the procedure that meets these needs through the inclusion of advanced imaging capabilities allowing for the capture of multiple images throughout a range of z-heights, using brightfield and fluorescence channels in a kinetic fashion.

Establishment of in vitro models that mimic tumor invasion as part of the metastatic process, are a critical part of oncology research. The need to incorporate multiple cell types, long-term kinetic analysis, methods to allow 3D invasion into the surrounding matrix, and appropriate detection and analysis, has made it difficult to create new models. The procedure described here meets these needs through inclusion of advanced imaging capabilities allowing for capture of multiple images throughout a range of z-heights, using brightfield and fluorescence channels in a kinetic fashion. Final processed images, following stitching and z-projection, enable accurate cellular analysis to discern the invasive capabilities of 3D cellular structures over time.

In this application note, BioTek demonstrates the use of a novel, automated tool to create consistent and reproducible scratch wounds in cell monolayers formed on the bottom of a microplate well. This application note highlights the use of a single cancer cell model as well as a co-culture of fibroblasts and cancer cells; each plated in collagen-coated microplate wells to more closely simulate in vivo microenvironments and facilitate cell migration.

Media exchanges with unattached cell models can be daunting if performed by hand, even when performed on a single plate. Multichannel pipettes must remove and dispense media at an extremely slow rate, and care must be taken to keep pipette tips away from the actual spheroids. Here, BioTek shows that through incorporation of the AMX Media Exchange Module on the MultiFlo FX, risk of accidental spheroid removal from wells is eliminated. Media is automatically removed and replaced with fresh media only, or fresh media containing treatment concentrations.

Metastasis is the main cause of death in cancer patients and one of the most complex biological processes in human diseases. The development of therapies designed to forestall the metastatic activity of tumors has been met with multiple challenges. In this poster, BioTek demonstrate a combined procedure for the generation of 3D spheroidal tumoroid structures, creation of a suitable invasion matrix, automated kinetic image-based monitoring of tumor invasion, and cellular analysis of captured z-stacked images.

Culturing cells in 3D provides a more in vivo-like environment, allowing cells to maintain high viability when cultured for extended time periods. To maintain the highest levels of viability within untreated cells and ensure that observed effects are solely from treatment, media exchanges and re-dosing are necessary throughout the experiment, particularly in vitro tests lasting weeks. Through incorporation of the AMX™ Automated Media Exchange module on the MultiFlo™ FX Multi-Mode Dispenser, risk of accidental spheroid removal from wells is eliminated. Spent media is automatically removed and replaced with fresh media only, or fresh media containing treatment concentrations.

Interest has intensified recently in the use of 3D models in life sciences research, to improve the understanding of basic cellular processes and reduce drug attrition rate. In the past, however, 3D models have proven difficult to reliably cultivate long enough to perform multiple dosing studies as well as to monitor chronic toxicity, long-term kinetic analyses, and stem cell differentiation. With a focus on four key applications, this application eBook speaks to these challenges - demonstrating robust workflows for high-throughput spheroid assembly and long-term quantitative analysis.

The wound healing or "scratch" assay is one of the most highly used in vitro methods to monitor and quantify collective cell migration. The current standard involves manual wound creation, which yields low reproducibility between wounds, high variability within generated data, and possible false conclusions regarding test molecules. Using an automated wound creation tool, in addition to kinetic image capture and analysis, repeatable wounds and robust and repeatable results are easily attained.

3D cell culture methods encourage cell-cell and cell-matrix interactions and also promote more physiological cell morphologies and behaviors compared to cells grown on flat 2D surfaces. Due to the need to generate the most in-vivo like data during in vitro drug discovery of toxicology testing, 3D spheroids are often used in long-term experimental procedures, and in microplate format to increase throughput. Since these procedures may extend to weeks, media exchange and dosing steps are critically important. When incorporating unattached spheroids, care must be taken not to evacuate or damage the spheroid suspended in the culture media.

Three dimensional (3D) spheroidal cell models have become a mainstay in life science research due to the ability to mimic in vivo-like environments. Performing media exchanges and washes with spheroids in cell repellent microplates can be problematic due to the risk of accidental spheroid removal. By incorporating a novel peristaltic pump-based tool, these procedures can be carried out in a controlled manner that eliminates spheroid disruption and removal, enabling long-term 3D experimental procedures requiring multiple media exchanges.

Chris Laucius, Ph.D., Imaging and Microscopy Specialist at BioTek Instruments, explains how the plate washers and imagers from BioTek Instruments can help with every step of your workflow: from gentle media exchange, sample prep, image acquisition all the way to data analysis.

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