Expert Tips for Reproducible Neuronal Research

1 Cell culture hacks Multi electrode array assays using iPSC-derived neurons Multi-electrode array (MEA) assays are one of the most powerful tools for measuring neuronal electrical activity. They provide quantitative functional data that can help researchers characterise neuronal models, track subtle cellular changes, and assess the impact of genetic perturbations or drug candidates. However, MEA experiments can be notoriously finicky, with small changes in cell culture conditions dramatically impacting results. bit.bio’s deterministically programmed human iPSC-derived neurons provide a functional and scalable in vitro system for studying neuronal activity and network formation using MEA. We caught up with Luke Foulser, Field Applications Scientist at bit.bio, to understand the nuances of setting up MEA experiments with these cells and gathered his top tips for achieving relevant and reproducible results. density, it’s possible to achieve regular and consistent cell culture success! What is a Multi-Electrode Array (MEA) assay? Multi-electrode array (MEA) technology is a method used to record extracellular electrical signals from neurons.1 It enables researchers to measure key electrophysiological properties such as firing rates, network connectivity, and burst activity in neuronal cultures over time. Unlike patch-clamp techniques, MEA assays are non-invasive and allow for long-term monitoring of neuronal networks in a high-throughput manner. This makes MEA a valuable tool for studying neuronal activity in the context of drug discovery, including disease modelling and neurotoxicity screening. Luke Foulser explains: “MEA is a game-changer for neuronal research, enabling long-term monitoring of neural activity at scale and over long time periods. But with experiments lasting this long, consistency in experimental setup and cell handling is critical. You don’t want to realise four weeks in that an issue on day one compromised your results.” About Luke Foulser, MSc Luke Foulser is a Field Application Scientist at bit.bio, where he supports the adoption of deterministically programmed human iPSC-derived cells in research and drug discovery. Prior to this role, he oversaw cellular phenotyping on the HD-MEA platform at bit.bio, leading extensive functional characterisation of ioGlutamatergic Neurons, ioMotor Neurons, and ioAstrocytes. Luke holds an MSc in neuroscience from King’s College London, and has expertise spanning MEA, calcium imaging, and in vitro stem cell biology. Published 2025 For any questions, contact our experts at technical@bit.bio 2 Cell culture hacks - Multi electrode array assays using iPSC-derived neurons Published 2025 In this guide, we will share expert tips to help researchers optimise MEA experiments using human iPSC-derived neurons. From choosing the right platform to maintaining a stable environment and ensuring proper cell seeding, these best practices will help you obtain consistent and reproducible data. TOP TIP 1 Understand your MEA system Different MEA platforms have varying electrode densities and imaging capabilities. Some systems are designed for finite metrics such as axon tracking, while others are optimised for high-throughput studies. Researchers should evaluate their experimental goals to select the most appropriate system. TOP TIP 2 Set up a consistent environment for your cells To improve experimental reproducibility and reduce contamination risk, we recommend culturing MEA chips inside a sealed chamber with a permeable membrane. We suggest placing the MEA chip containing the culture inside a larger dish (a chamber) which is sealed with a Breathe-Easy® membrane (Figure 1). Adding a small amount of water inside the chamber helps maintain humidity. This simple modification has reduced contamination rates from 50% to just 5% in our labs. The cells are cultured without antibiotics, which leaves them susceptible to contamination. So emphasising good cell culture standards is key: • Regularly spray surfaces with 70% ethanol • Keep workspaces clean and organised • Wear appropriate PPE at all times TOP TIP 3 Plan your plate preparation timeline For the experiment to work with a standard Monday-Friday schedule that many researchers hold – and it is important for a long-term project like this to pick a sustainable work schedule – we recommend starting plate preparation on Thursday so that the plates are ready for cell seeding on Monday, which enables you to continue working Monday through Friday with cell feeding. This schedule works well for us: • Thursday: Begin plate preparation (hydrophilic treatment, ethanol sterilisation, and pre-conditioning in media) • Monday: Seed cells TOP TIP 4 Precision matters on day 0 Mistakes made on Day 0 may not become apparent for weeks, but they can be devastating, especially when using dot spotting. Follow these steps for success with this method. Step 1: Prepare your plates with GeltrexTM Timing matters – Geltrex must be added 1 hour before seeding. Plan for pipetting time – Consider the time it takes to pipette multiple experimental conditions and stagger your Geltrex application accordingly. Example (dot spotting only): If you have three conditions A, B, and C that in total take 10 minutes to pipette, apply Geltrex as follows: • Condition A – 1:00 PM • Condition B – 1:05 PM • Condition C – 1:10 PM This ensures all wells are ready at the right time when seeding. 3 Figure 1. Recommended culture hood layout for sample preparation including the closed culturing chamber (plastic tray with petri dish of water, sealed with Breathe-Easy® membrane) to avoid contamination while maintaining the cells. Cell culture hacks - Multi electrode array assays using iPSC-derived neurons Published 2025 Step 2: Start thawing cells at the right time Thawing starts 30 minutes after Geltrex application. Example: If Geltrex is added at 1:00 PM, start thawing cells at 1:30 PM so the cells are ready when the plate is prepared. Be methodical with multiple cell types. • Example: If you are handling three different cell types, prepare three separate thawing containers and label them clearly. • Use a timer to ensure each vial thaws for the same duration. • Work in parallel, while one vial is thawing, prepare the next. • Example: If it takes 2 minutes to thaw a single vial, and you have 3 vials, your total thawing process should take no more than 6-7 minutes. Step 3: Count and plate cells methodically Count cells accurately. Pipette with precision. We advise a culture hood layout as shown in Figure 1 for efficiency. This step is a critical point of failure – if the Geltrex dries out, or is not removed enough, or if the cells are perturbed in the process, you may not find out for weeks. This can be very costly, so get it right – prepare. TOP TIP 5 Use controls to ensure your seeding protocol works Use a dummy plate to confirm: • Proper attachment of all cell types • Even distribution across the measurement area • No clustering or disproportionate spread • Check cell health For systems with transparent plates, brightfield microscopy can be used to check cell distribution. For non-transparent plates, prepare a control plate for staining and imaging. 4 REFERENCES 1. Spira ME, Hai A. Multi-electrode array technologies for neuroscience and cardiology. Nat. Nanotechnol. 2013 Feb;8(2):83-94. https://doi.org/10.1038/ nnano.2012.265 Cell culture hacks - Multi electrode array assays using iPSC-derived neurons Published 2025 BB/B/CCMEN/V1 TOP TIP 6 Choose the right seeding method Different platforms may allow for varied seeding methods, such as dot-spot plating (small 10 μL droplets forming ‘comets’) or full-well seeding. Researchers should determine the optimal method based on their study goals. TOP TIP 7 Optimise cell density and ratios MEA is highly sensitive to changes in cell type, ratios and densities. We recommend optimising these for your specific study. Even small variations can impact network synchrony and spike patterns. TOP TIP 8 Wait at least 4 hours post-feeding to record your cells (and be consistent) Cell activity fluctuates after media changes, so standardise your recording schedule: • Wait at least 4 hours post-feeding before recording • In most cases, we recommend feeding at 9:00 AM, followed by recording at 1:00 PM • Recordings should be taken on days 7, 14, 21, 28, 35, 42, and 49 (once a week for 49 days) TOP TIP 9 Time your drug dosing wisely MEA is highly sensitive to changes in cell type, ratios and densities. We recommend starting with our preferred ratios but adjusting as needed for your specific study. Even small variations can impact network synchrony and spike patterns. TOP TIP 10 Long-term consistency is key MEA experiments can last 54 days or longer, requiring careful handling and consistency: • Minimise plate disturbances to avoid network disruption • Stick to a Monday/Wednesday/Friday feeding schedule for consistency • Assign cell maintenance to a single person to reduce variability Summary MEA is a powerful tool for neuronal electrophysiology, but its sensitivity requires careful planning and execution. From selecting the right system to optimising plating and maintaining long-term consistency, each step matters. At bit.bio, we have extensively optimised human iPSC-derived neurons for MEA experiments. Our cells, protocols, and expertise help researchers obtain reliable, reproducible data faster. Further reading The top tips in this blog have been used successfully in the following protocol: Tri-culture of Glutamatergic Neurons, GABAergic Neurons and astrocytes for MEA assays The tri-culture results, including functional network formation and modulation of network activity by ioGABAergic Neurons, were presented in a WEBINAR and can be explored in detail HERE.