3D Electrophysiological Measurements on Cells Embedded within Fiber‐Reinforced Matrigel

Abstract

of the matrix. [9] Mechanical properties such as the stiffness of the 3D surrounding environment are known to affect differentiation of certain cells, [10] and are believed to be critical for neuronal maturation and neurotransmission. [9] More discrete and higher resolved structures, such as those made with additive manufacturing (AM) [11] and electrojetting technology, [12] are promising to provide reproducible conditions. Electrojetting uses electrostatic forces to fabricate monodispersed, nanomicro particles in a simple, versatile, and cost-effective method for drug delivery and tissue engineering applications. [13] Electrospinning allows processability for polymer solutions and polymer melts. [14] In addition, polymer melts can be directly written using a programmable path in a technique known as melt electrowriting (MEW). [15] The precise placement of low-micrometer diameter fibers that are stackable using additive manufacturing principles are achievable using MEW (Figure 1a,b). The advantages of MEW include solvent-free processing and avoidance of the chaotic fiber deposition often seen in electrospinning. Bioprinting approaches for cell hierarchy have been reinforced with extruded, stiffer structures [16] that reinforce the bioink and aid with in vitro handling [17] and extend a processing window for such hydrogels. [16,17] The reinforcing of matrices and bioinks with much smaller, low-micrometer scale fibers on matrices has recently been of particular interest. [18,19] Using MEW, [15] well-ordered, small diameter fibers can be distributed throughout a matrix in low volume fractions and provide significant increase in overall mechanical properties. [18][19][20] Furthermore, the mechanics of MEW fiber-reinforced hydrogels can be modulated further with sinusoidal direct-writing of the fibers. [18] Since the fiber diameter made using MEW can be readily changed with the nozzle mass flow rate and/or the collection speed, the diameter of the printed fiber can be significantly altered. [21] Therefore, MEW reinforcement of matrices (such as Matrigel shown here) have the potential to regulate the environment of cells through both mechanotransductive [22] or haptotaptic [23] cues.An experimentally designed in vitro 3D structure for electrophysiological studies requires a relevant receptor model, with the inhibitory glycine receptor (GlyR) used in this study. The GlyR is a pentameric, ligand gated ion channel, which belongs to the superfamily of Cys-loop receptors. [24] Upon binding of 2D electrophysiology is often used to determine the electrical properties of neurons. In the brain however, neurons form extensive 3D networks. Thus, performing electrophysiology in a 3D environment provides a closer situation to the physiological condition and serves as a useful tool for various applications in the field of neuroscience. In this study, 3D electrophysiology is established within a fiber-reinforced matrix to enable fast readouts from transfected cells, which are often used as model systems for 2D electrophysiology. Usin...

Tumor Cells Develop Defined Cellular Phenotypes After 3D-Bioprinting in Different Bioinks

“…Matrigel, the current gold standard for a rich physiological ECM, but not classical bioink as it lacks shape fidelity and long-term stability, has been used in the field of biofabrication for some time. However, in published studies, Matrigel was used basically as a coating material for 3D printed scaffolds, as the physiological component of a printable matrix mixture with hydrogels like alginate, gelatin or agarose or it was printed into predefined molds (e.g., [38,39,40,41,42]). Here, we developed a method to print pure Matrigel, which, although revealing the poorest printability in comparison with the other inks, was printable with the tumor cells and was able to keep a 3D structure over the culturing period of 14 days without dissolving in the medium.…”

Polymers for Melt Electrowriting

“…Finally, to provide 3D neuronal cell culture models with a weak matrix microenvironment closer to the native tissue, MEW fabricated scaffolds were embedded into Matrigel. [38,41] Those reinforced scaffolds within the Matrigel improved the mechanical properties and therefore the handling of the delicate constructs. [38,41] Another approach to mimic native tissue and its composition was demonstrated by using orthogonal microfiber scaffolds embedded into compressed collagen to design an artificial cornea.…”

Layer-By-Layer: The Case for 3D Bioprinting Neurons to Create Patient-Specific Epilepsy Models

“…MEW is more reproducible than typical electrospinning which can often have chaotic fibre deposition. Schaefer et al (2019) developed a 3D prototype neural model of MEW PCL seeded with fibroblast embedded Matrigel, and demonstrated that the MEW PCL did not interfere with whole cell patch clamp recordings [142]. Neurons are more vulnerable to environmental stressors than fibroblasts, in 2D neuron electrophysiological behaviour was found not be affected when seeded on PCL electrospun fibres [44].…”