Oksana Sirenko, Senior Scientist, Molecular Devices and Evan Cromwell, President & CEO, Protein Fluidics, explain the benefits of automated organoid assays with microfluidics and 3D imaging.
Microfluidic flowchip technology combined with three-dimensional (3D) imaging is a powerful duo poised to change the future of drug discovery and development. The integration of combined technologies into one workflow has demonstrated efficiency in the life sciences field and created opportunities to outperform traditional techniques in scientific research.
Also known as lab-on-a-chip (LOC), microfluidics gained popularity in the early 1990s for its demonstrated success manipulating small volumes of fluid to control chemical, biological, and physical processes1. Over the last three decades, advancements in microfluidics have allowed scientists to maximise throughput and increase productivity while also ensuring the highest levels of accuracy and reproducibility in their drug discovery research by enabling assays with complex cell-based models like organoids where it is uniquely suited to recapitulating the in vivomicroenvironment.
However, two-dimensional (2D) cell-based screening still accounts for most assays. Traditional high-throughput drug screening in oncology for example, routinely relies on 2D cell models which inadequately recapitulates the physiologic context of cancer 4. Therefore, methods for producing and screening 3D organoids like tumor organoids for high throughput is essential to cancer drug discovery.
3D systems portend to have more in vivo relevance and perform as a more predictive tool for the success or failure of a drug screening campaign2. Common advanced cellular models that researchers rely on include spheroids, tumoroids, structured co-cultures, and multicellular organoids3, which are found in many areas of the drug development pipeline, from Target ID through to preclinical safety assessment. More common are phenotypic high-throughput screens using complex models like tumor organoids4, as they are more physiologically relevant than their biochemical counterparts, yet they can be both difficult to handle as well as to image5.
Here, we introduce a new method that’s advancing the possibilities of drug discovery using fluidics to automate 3D cell culture and cell analysis, allowing a hands-free process for media exchanges and treatments while simplifying and improving workflow when compared to conventional cell culture.
Minimal handling of complex 3D samples for maximum results
Manual treatment, staining, and processing of spheroids and organoids is typically labor-intensive as caution is practiced heavily in order to decrease the risk of disrupting or destroying the samples. Furthermore, manual media changes and spheroid treatment may lead to compound, drug, and staining concentration inconsistencies in each well across a microplate, generating unreliable or inconsistent results.
Yet 3D cell-based assay workflows aren’t without their own challenges. Growing 2D cells and plating them on flat surfaces is a mature process that has been developed over many decades. It is also amenable to high throughput as cells can be suspended and dispensed into high-density plates robustly and with a high degree of reproducibility. On the other hand, 3D cell structures can take weeks to mature and are more delicate once they do, making manual manipulation during standard processes such as media exchange tricky, leading to high probability of spheroid damage. While these steps can be automated, the equipment required has traditionally been expensive and designed for higher throughputs.
The automated organoid assay workflow
To alleviate workflow challenges that arise when performing high-content 3D imaging, Molecular Devices and Protein Fluidics partnered to further standardise protocols and improve reproducibility of phenotypic and functional assays, elevating researchers’ confidence in adopting 3D assays for screening programs. The collaboration used automated applications of a 3D cell-based assay system to perform complex protocols with spheroids and organoids, and analyzed the novel biological assays using high content 3D imaging. The automated in situ supernatant sampling and compound exchange of microfluidics allows in parallel measurement of secreted factors and correlation of those to phenotypic changes elucidated by organoid imaging, resulting in efficient synergy between automated culture and processing, along with automated imaging, recording and analysis.
On the automated culture and processing side, Protein Fluidics’ novel microfluidic-based technology exchanges up to 95% of the media without drying the cells or disrupting the spheroid while manual media changes usually follow the process of removing on 50% of the media in order to minimise spheroid degradation. And, without a direct fluid flow over the cells, spheroids remain uninterrupted in place. With Molecular Devices’ imaging solutions, flowchip plates can be analysed directly in the imager through its specially designed flowchip holder that conforms to the SLAS/ANSI microplate standards. Researchers do not have to transfer their spheroids onto a plate that is suitable for imaging, removing an extra step in the workflow that often leads to destruction or loss of spheroids. Image acquisition is fast and easy, as the spheroids are all positioned in the same location housed in the sample wells.
In the following two examples, we demonstrate a microfluidics system for automation of complex cell protocols with 3D samples, and compatibility with high-content imaging and fast fluorescence kinetic read-outs. Scientists studied the long-term 3D cell-based toxicity by performing automated assays with spheroids, organoids, and microtissues using a novel microfluidic-based Pu·MA System, and high-content 3D imaging using ImageXpress Micro Confocal High-Content Imaging System. This was an ideal application workflow for studying long-term toxicity, oncology therapeutics, secretion of growth factors by single spheroid or organoid, as well as efficient processing of samples for metabolic profiling.
In the first application, scientists automated a 3D assay protocol with cancer spheroids formed from HCT116 colon cancer cells to access the efficacy of selected anti-cancer drugs. The flowchip plate containing spheroids was loaded into a Pu·MA System that was then placed into a conventional cell incubator allowing prolonged culture of live cells. The system enabled a hands-free cell culture process using automated compound additions, media exchanges, staining and processing. Various media or components were simply pre-loaded into the chip using multiple reservoirs for different solutions which could be moved automatically in the microfluidics chip, at set time points, using pneumatic pressure. Specifically, 48-hour cell culture protocol was performed automatically, which included two media exchanges with re-addition of compounds, then addition of viability dyes, and a final wash of spheroids from staining reagents. At that point spheroids were ready for imaging.
Because the flowchips have standard plate dimensions and are not connected to external tubes, they are compatible with various imaging instruments and plate readers, and were easily taken out of the device to be imaged for various cell viability markers. Confocal imaging delivered 3D resolution of spheroid structures and complex analysis, allowing measurement of different compounds’ impact on the size, volume, and integrity of spheroids, as well the number of total, viable and affected cells. Advanced image analysis was made possible using high-content imaging tools that allowed characterisation of nuclei, mitochondria, viability and apoptotic markers. The system can be used for multiple applications, including processing of patient-derived tissues, testing sensitivity of patient cells to drugs ex-vivo, and allow personalising an approach for patient treatment.
In a second application, the flowchip system was used for the functional evaluation of calcium oscillations in neurospheroids upon treatment with neuro-active compounds. Neural 3D cell spheroids (microBrain 3D, Stemonix) were placed into the chip and incubated with various known neuromodulator compounds. While challenging to manually add and remove different solutions when working with 3D spheroids or organoids, the automated microfluidics process allows careful and precise exchange of media or compounds without disturbing micro-tissues. It also allowed for addition or washes of different compounds, and observation of changes in response and dose-responses in the single spheroid.
Neural network activity was assessed via Ca2+oscillations using calcium sensitive dyes and fast kinetic fluorescence imaging. The Pu·MA System was used for single or multiple additions of compounds to the neurospheroids. It automatically added, washed, or exchanged different compounds on the sample, allowing researchers to evaluate concentration-responses and complex kinetics of compound effects. Kinetic fluorescence patterns were recorded directly in the flowchip plates and demonstrated modulation in neural network activity of the spheroids. Observed changes in the calcium oscillations activity were consistent with the mechanism of action of the compounds.
Increasing lab efficiency with hands-free automation
Many researchers and scientists have long implemented various screening systems to eliminate repetitive manual labor and accelerate their lead generation process5. The novel automated 3D organoid assay system provides a way to automate complex assays using special microfluidic flowchip. What the system provides is hands-free functionality that allows researchers to initiate an automatic 3D cell culture and imaging workflow, and then walk away during the process while the system does the work.
Further, the flowchip contains an organoid sample well, which is connected to multiple reservoirs that can hold numerous assay reagents. Different solutions can then be transferred to the samples sequentially using pneumatic pressures to allow multiple processing steps such as media exchange, sample staining with fluorescent dyes, wash steps, among others to be performed automatically. In contrast to traditional organ-on-chip devices, assay plates in this system can be easily taken in and out from the device for reading on-various instruments, or additional manipulations.
Such advanced automated technology has helped to increase the throughput of device production, improved rapid prototyping efforts, and enabled researchers to enhance the complexity and sophistication of experiments that can be performed on a microfluidic chip.
Additionally, minimal sample is needed when performing analysis in small volume experiments using microfluidic solutions – an attractive benefit for many scientists dealing with limited samples or low abundant analytes. The small size of the individual fluidic circuit components in microfluidic devices provides an arsenal of experimental advantages, which makes microfluidic solutions desirable. For example, small components enable researchers to perform experiments which are not possible or practical to execute with more traditional methods. When analyzing the biological measurement of fluidic components at the cellular scale, microfluidic circuits provides exceptional liquid handling capabilities, allowing manipulation of single cells and even single molecules in vitro6. The automated microfluidic solution combined with high-content 3D imaging significantly helps researchers save time on tedious manual labor and materials, and fundamentally leads to higher precision measurements.
Expected advancements with newly standardised, automated workflow
Every day, scientists around the globe make important discoveries and breakthroughs in understanding illness. However, converting such discoveries into improvements in human health can be difficult because the drug development journey is exhaustive, costs too much, and fails far too often, driving a need for more advanced in vitro screening technologies to increase biological relevance and reduce costs associated with experimentation. This represents a challenge, but also a huge opportunity for next generation technology to automate and change workflows that lead to breakthroughs and improve human health.
Technology including the rapid sample processing and precise control of fluids in an assay has made microfluidics an attractive application to replace traditional experimental approaches7. Rather than a true LOC, most experiments still involve putting a small microfluidic device on a sizable instrument, such as a fluorescence microscope, or interfacing it with macroscale control architecture. This situation is changing, however, and some truly portable and automated systems are beginning to be realized and, more importantly, commercialised.
Nevertheless, combining microfluidic systems to perform assays with 3D cell-based structures with automated confocal imaging offers a powerful tool to researchers looking to increase assay complexity, improve the efficiency of their phenotypic assays, and obtain improved biological data from their precious samples. And it is especially beneficial to scientists using limited samples of starting material, like patient-derived samples, patient-derived organoids research, translational research, preclinical studies, or patient response to specific drugs. Optimal for multiplexed drug screening, assay development, and disease modelling, the future potential of pairing microfluidics with 3D imaging into a single step is essential for research advancement.
Figure 1: Schematic layout of the workflow
Figure 2: Confocal images and analysis of tumour spheroids stained for assessment of drug effects.
Figure 3: Kinetic fluorescent recording or calcium oscillations measured by FLIPR Penta High-Throughput Cellular Screening System
Volume 21, Issue 4 – Fall 2020
Main image credit: ThisIsEngineering RAEng
Oksana Sirenko, Senior Scientist, Molecular DevicesOksana Sirenko, PhD is a Senior Scientist at Molecular Devices specialising in complex cell-based model development for research and compound screening. With 10+ years of industry experience, she explores use of 3D cell models for cancer, neurotoxicity, angiogenesis and other complex biological process studies. Sirenko has authored 30 scientific papers.
Evan Cromwell, President & CEO, Protein FluidicsEvan F Cromwell, PhD, President and CEO, Protein Fluidics, is an entrepreneur with 30 years of executive and technical experience in commercialising assay systems. Prior to founding Protein Fluidics, he served as Research Director at Molecular Devices. He holds a BS from Caltech and a PhD from UC Berkeley. He has 40+ papers and 16 patents.
References:
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- Gupta N, Liu J, Patel B, et al. Microfluidics-based 3D cell culture models: Utility in novel drug discovery and delivery research. July 5, 2016. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5689508/ (accessed July 20, 2020).
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- Johnston 2019: Stanton JK, Close DA, Johnston PA.High Content Screening Characterization of Head and Neck Squamous Cell Carinoma Multicellular Tumor Spheroid Cultures Generated in 384-Well Ultra-Low Attachment Plates to Screen for Better Cancer Drug Leads. ADDT, 2019, 17, 17-36.
- Sirenko 2015:Sirenko O, Mitlo T, Hesley J, Luke S, Owens W, Cromwell EF. High-Content Assays for Characterizing the Viability and Morphology of 3D Cancer Spheroid Cultures. ADDT, 2015, 13, 402-414
- Hou S, Tiriac H, Sridharan BP, et al. Advanced Development of Primary Pancreatic Organoid Tumor Models for High-Throughput Phenotypic Drug Screening.SLAS Discovery, 2018, 23, 574-584.
- Hamilton, E. How Close Are We to End-to-End Automated Drug Discovery? The Science Times. May 19, 2020 https://www.sciencetimes.com/articles/25060/20200319/close-end-automated-drug-discovery.htm (accessed July 20, 2020)
