Electrophysiological research focuses on the electrical properties of certain living cells (e.g., neurons, cardiac cells). Measuring their electrical activity allows researchers to decode network communication as well as evaluate their health and responses to external stimuli such as chemical insults. To monitor electrophysiological activities non-invasively, cells can be deposited and grown in vitro on a chip where electrodes are then used to measure any changes in their transmembrane or extracellular voltage. For cells that are cultured on a 2D surface, these events can be measured with a biocompatible microelectrode array (MEA). LLNL-developed thin film MEAs are core components of their innovative iCHIP (in vitro Chip-based Human Investigational Platform) systems that can be used to replicate biological systems such as peripheral nervous system (PNS), central nervous system (CNS), cardiac, and the blood–brain barrier (BBB).
However, cells found in biological tissue and organs are arranged in three dimensions, not in a planar configuration, so to mimic physiological conditions, there is a need for recording methods that target in vitro cells cultured in 3D. Currently, electrophysiological activities of cells grown in a 3D matrix are typically measured with single electrodes made of glass or tungsten that are inserted into the matrix after the cells have established themselves. In practice, there are many disadvantages to this traditional approach. Since the electrode is introduced after the maturation of the cell network, damage of the delicate network and supporting structure could occur during the insertion process. Additionally, each electrode is accompanied by bulky support and handling structures, making it extremely difficult to place more than a few into a cell culture well, thus making it challenging to target specific locations within the matrix of cells as well as to evaluate network communication across multiple locations.
To overcome these challenges, LLNL researchers have built upon their innovative MEA technology and developed a novel flexible MEA device that can interrogate in vitro cells cultured in 3D.
To replicate the physiology and functionality of tissues and organs, LLNL has developed an in vitro device that contains 3D MEAs made from flexible polymeric probes with multiple electrodes along the body of each probe. At the end of each probe body is a specially designed hinge that allows the probe to transition from lying flat to a more upright position when actuated and then remains so without additional supports.
To vertically actuate the flexible probes, an approach was developed to flex the probe bodies off the surface with “buckling shanks” and subsequently lift them upright using rigid “lifting shanks”. Their movements are controlled precisely using an actuating apparatus that can simultaneously actuate multiple probes.
Once the probes are actuated and in position, a cell culture well can be mounted, and cell-containing matrix introduced. Having been distributed throughout the volume of the culture well, cells are then able to mature and naturally create a cohesive network around the probes. Their electroactive functions can then be measured and recorded simultaneously using the 3D MEA and compatible instrumentation.
LLNL researchers have been able to fabricate a device for in vitro brain models using flexible 3D MEA to interrogate neurons entrapped in a 3D gel matrix. The researchers were able to use the device to successfully acquire electrophysiological recordings simultaneously in all three dimensions of an in vitro neural network. Moreover, LLNL’s novel flexible 3D MEA technology is not limited to neurons – it is extremely adaptable and can be used to mimic any organ system containing electroactive cells.
Lab researchers develop 3D ‘brain-on-a-chip’ device capable of long-term recording of neural activity (https://www.llnl.gov/news/lab-researchers-develop-3d-brain-chip-device-capable-long-term-recording-neural-activity)
Soscia DA, Lam D, Tooker AC, Enright HA, Triplett M, Karande P, Peters SKG, Sales AP, Wheeler EK, Fischer NO. A flexible 3-dimensional microelectrode array for in vitro brain models. Lab Chip. 2020 Mar 3;20(5):901-911. (https://doi.org/10.1039/c9lc01148j)
Here's How Scientists Replicate Mysterious Organs...on a Chip (https://ipo.llnl.gov/media/578)
Image below of 3D MEA device prior to actuation (A, B) and post-actuation (C). The hinge regions are plastically deformed and allow the probes to stand upright without additional supports.
- LLNL’s 3-dimensional in vitro flexible MEA device allows for the non-invasively interrogation of 3D cultures of cells, which more closely resembles the complex organization of an in vivo organ compared to traditional 2D cultures. The technology could provide more accurate organ models to study disease, chemical insults, or develop new drugs or medical countermeasures to chemical or biological agents.
- The device is non-invasive, biocompatible, relatively easy to fabricate, able to be integrated with existing commercial electrophysiology hardware, and straightforward to use. The flexible probes are robust and can be mechanically actuated without difficulty using equipment that is also commercially available.
- The novel 3D MEA technology allows cells to grow and establish naturally around the device’s probes while they are being studied, which is more favorable than the traditional, possibly damaging, approach of inserting probes into a 3D cell culture, after the cells had established their intricate network.
The device can be used to interrogate electroactive cells (e.g., neurons, cardiac cells) in a 3D culture for research and drug screening applications. It can be used to monitor electrophysiological activities of cells simply mixed with a hydrogel or possibly spheroids/organoids comprised of electroactive cells that have been formed in situ around the probes of the device.
LLNL has filed for patent protection on this technology.
US Application Publication No. 2021/0139828 SYSTEM AND METHOD FOR THREE-DIMENSIONAL IN VITRO FLEXIBLE MICROELECTRODE ARRAY published 5/13/2021 (https://image-ppubs.uspto.gov/dirsearch-public/print/downloadPdf/20210139828)