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The best coatings for multi-electrode arrays

The best coatings for multi-electrode arrays

Multi-electrode arrays (MEAs) are grids of small electrodes that allow extracellular recording or stimulation of many cardiac and neural cells neurons simultaneously1. Here, we discuss 2D MEAs designed for in vitro recording of cultured cells or induced pluripotent stem cells (iPSCs), however MEAs can also be designed for in vivo implantation and recording of cell populations within the brain. In vitro MEAs record electrochemical fluctuations in the extracellular field around neurons produced by ion exchange during action potentials2. This non-invasive setup allows for long-term studies of neural network activity with relatively high temporal and spatial resolution and limited disturbance to the system. With the advent of multi-well plates incorporating up to 96 separate MEAs in individual wells, MEAs have become a powerful tool for multiplexing assays aimed at studying the effect of a treatment on the electrophysiological properties of cardiac and neural cells.

One of the key challenges of MEA experiments is being able to detect cellular electrophysiological signals from as many electrodes in the array as possible and being able to maintain those active channels over recording sessions spread over multiple days.In vitro MEA electrodes are typically implanted into a glass base for cell culture1 

. The key to maintaining a high number of  stablelong-term  active channels in your MEA experiments is to nature of many MEA experiments requires a bio-compatibleuse a coating that supports long-term cell adhesion without degradation3. If the coating substrate is degraded, cells will detach from the substrate where the electrode pads are located and will begin growing over on top of each other in clumps4. This effect is particularly problematic for MEAs, since the clumping of cells will reduce the likelihood of an electrophysiologically active cells being on or near an electrode site which require spatial separation of cells to distinguish electrical signals across the network. 

Types of MEA coatings

In vitro MEA electrodes are typically embedded into a glass or polystyrene base which is typically not permissive to the adhesion of the cultured cells1. Therefore, most MEA protocol require an initial coating of the MEA before seeding the cells. Polyornithine and poly-lysine are typical coating for neuronal cells. Both coatings however, contain peptide bonds and are vulnerable to degradation by secreted proteases8. An alternate coating substrate found in many MEA protocols is polyethlyenimine (PEI). It mimics the adhesion-promoting properties of these polypeptides but lack the peptide bonds and are therefore more resistant to degradation. As with all cationic polymer, it needs to be A standard coating found in many MEA protocols is polyethlyenimine (PEI) – supplemented with laminin or another extracellular matrix protein for iPSC cultures - which shows reduced cell clustering compared to polylysine1,5. However, PEI shows diminished neuronal differentiation,  maturation, and electrical responsiveness when compared to the synthetic standard polypeptide substrate poly-dl-ornithine (PDLO)3. Other common coating substrates for MEA iPSC cultures include laminin alone, or poly-ornithine (PLO)PLO used together with laminin, however each of these substrates is susceptible to degradation and cell aggregation in long-term studies4,6,7. 

Polyornithine and poly-dl-ornithine both contain peptide bonds and are vulnerable to degradation by secreted proteases8. Alternate coating substrates (such as PEI) mimic the adhesion-promoting properties of these polypeptides but lack the peptide bonds and are therefore more resistant to degradation. Dendritic polyglycerol amine (dPGA) is another non-peptide polymer that effectively supports long-term cell adhesion for many different neuronal and iPSC cultures. In contrast to PEI, dPGA  supports cell differentiation and maturation at comparable, and sometimes increased levels relative to polypeptide substrates4,8. When used in conjunction with Matrigel as an MEA coating substrate for iPSC derived motor neurons, dPGA supports long-term cultures with minimal clustering, reflected in an increase in number and frequency of spikes recorded4.  


References

  1. Pelkonen, A. et al. Functional Characterization of Human Pluripotent Stem Cell-Derived Models of the Brain with Microelectrode Arrays. Cells 11, 106 (2021).
  2. Ahmadvand, T., Mirsadeghi, S., Shanehsazzadeh, F., Kiani, S. & Fardmanesh, M. A Novel Low-Cost Method for Fabrication of 2D Multi-Electrode Array (MEA) to Evaluate Functionality of Neuronal Cells. Proceedings 60, 51 (2020).
  3. Amin, H. et al. Electrical Responses and Spontaneous Activity of Human iPS-Derived Neuronal Networks Characterized for 3-month Culture with 4096-Electrode Arrays. Front. Neurosci. 10, (2016).
  4. Thiry, L., Clément, J.-P., Haag, R., Kennedy, T. E. & Stifani, S. Optimization of Long-Term Human iPSC-Derived Spinal Motor Neuron Culture Using a Dendritic Polyglycerol Amine-Based Substrate. ASN Neuro 14, 17590914211073381 (2022).
  5. Hales, C. M., Rolston, J. D. & Potter, S. M. How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays (MEAs). JoVE (Journal of Visualized Experiments) e2056 (2010) doi:10.3791/2056.
  6. Kuijlaars, J. et al. Sustained synchronized neuronal network activity in a human astrocyte co-culture system. Sci Rep 6, 36529 (2016).
  7. Thiry, L., Hamel, R., Pluchino, S., Durcan, T. & Stifani, S. Characterization of Human iPSC-derived Spinal Motor Neurons by Single-cell RNA Sequencing. Neuroscience 450, 57–70 (2020).
  8. Clément, J.-P. et al. Dendritic Polyglycerol Amine: An Enhanced Substrate to Support Long-Term Neural Cell Culture. ASN Neuro 14, 17590914211073276 (2022).