Muscle-Cell-Based 'Living Diodes'

Muscle Cell-Based ‘Living Diodes’ Uryan Isik Can1, Neerajha Nagarajan1, Dervis Can Vural3, and Pinar Zorlutuna*1

1Aerospace and Mechanical Engineering Department, University of Notre Dame, Notre Dame, IN 46556, USA 2Department of Physics, University of Notre Dame, Notre Dame, IN 46556, USA

In recent years, there has been tremendous progress in materials science and electronics engineering, in particularly the development of stretchable, flexible electronics that are optimized for interfacing with soft materials.[1, 2] The next step is to expand the domain of such devices to functional biological structures by augmenting, engineering or re-designing “computational tissues”, such that part of an organ can act as a programmable arrangement of logic gates or an interface to an external device. Current approaches exploring the use of cell-based elements in creating biocircuits primarily rely on genetic manipulation of the cell, as well as introducing chemicals and other biomolecules to achieve certain functions. Currently, there is no cell- based signal-processing device that operates solely on external electrical triggers. Here, we design, fabricate and characterize a new type of diode that is made entirely of living cells through micropatterned co-cultures of electrically excitable cardiac muscle cells (CMs, i.e. cardiomyocytes) and non-excitable cardiac fibroblasts (CFs). Non-excitable cells can electrically couple to excitable cells through cell-cell junctions and relay signals passively up to a certain distance, though they cannot amplify or propagate external signals directly coming to their membranes. In contrast, excitable cells can initiate, amplify and propagate signals in response to external stimuli through their specialized membrane channels. Our muscle cell-based diode (MCD) design confines these two cell types in a rectangular pattern using a novel, self-forming micropatterning approach, where one side consists of electrically excitable cells and the other side consists of purely non-excitable cells. This configuration allows the signal initiated on the excitable side to pass to the non-excitable side, whereas, it is not possible to pass any signals in the other direction since the cells on the non-excitable side are not able to initiate any action potentials (AP). As such, we find that the controlled arrangement of excitable and non-excitable cell types can be used to transduce electrical signals unidirectionally, essentially achieving a diode function, as we have shown through the characterization of the electrical response of the MCDs using microelectrode arrays (MEA). Biocomputing is a recent field that emerged around the idea of applying biomolecular systems to information processing.[3-5] Initially limited to chemical reactions that behave as single logic-gates,[6, 7] this technology has moved towards using reaction networks of cell-derived biomacromolecules (such as enzyme complexes) to instantiate multiple logic gates.[8, 9] More recently, genetically modified cells have been used to perform rudimentary computational tasks.[10] Current state-of-the-art biocomputing primarily involves single cells (either bacteria[11-13] or mammalian[14-16]), and information processing is done at the gene or protein level rather than between cell groups. Specifically, information is processed chemically through differential gene and protein expression, in the end controlling the rate of production of certain enzymes.[17] In addition to chemical signals (which naturally tend to be slow), some cells, such as the CMs, also respond to electrical signals. CMs are excitable cells and can receive an electrical input both internally, through their gap junctions that connect to other cells, and externally, through their voltage-gated ion channels. In contrast, CFs are non-excitable cells and can only receive electrical signals through their gap junctions. Therefore, an electrical signal can be initiated from a CM and transferred to both CMs and CFs. However, such signals cannot be initiated from a CF. The degree of CM – CF coupling is relevant to a number of pathological conditions and has been investigated extensively.[18-25] Although CFs cannot initiate an action potential like muscle cells, they can still propagate the electrical signal passively, for a limited distance.[18, 21] For cultured rat neonatal ventricular CMs, it was shown that electrical activity could be conducted over a distance of up to 300 µm via CF inserts, causing insert-length dependent delays in wave propagation.[19] This result was reproduced by comparative studies with gap junction deficient cells as controls, confirming the need for gap junctions in electrical conduction.[26] First, we tested the unidirectional signal transduction potential of specific patterns of these two cell types using a computational model. Our two variable model was b

…(Full text truncated)…