Spinal Cord Neuronal Network Formation in a 3D Printed Reinforced Matrix—A Model System to Study Disease Mechanisms

Abstract

3D cell cultures allow a better mimicry of the biological and mechanical environment of cells in vivo compared to 2D cultures. However, 3D cell cultures have been challenging for ultrasoft tissues such as the brain. The present study uses a microfiber reinforcement approach combining mouse primary spinal cord neurons in Matrigel with melt electrowritten (MEW) frames. Within these 3D constructs, neuronal network development is followed for 21 days in vitro. To evaluate neuronal development in 3D constructs, the maturation of inhibitory glycinergic synapses is analyzed using protein expression, the complex mechanical properties by assessing nonlinearity, conditioning, and stress relaxation, and calcium imaging as readouts. Following adaptation to the 3D matrix-frame, mature inhibitory synapse formation is faster than in 2D demonstrated by a steep increase in glycine receptor expression between days 3 and 10. The 3D expression pattern of marker proteins at the inhibitory synapse and the mechanical properties resemble the situation in native spinal cord tissue. Moreover, 3D spinal cord neuronal networks exhibit intensive neuronal activity after 14 days in culture. The spinal cord cell culture model using ultrasoft matrix reinforced by MEW fibers provides a promising tool to study and understand biomechanical mechanisms in health and disease.

The Impact of Including Carbonyl Iron Particles on the Melt Electrowriting Process

“…[ 12–14 ] The resulting MEW‐processed scaffolds are often used to guide cell growth in tissue engineering applications [ 15,16 ] or to reinforce hydrogels providing mechanical stability and improved handling. [ 12,17 ]…”

“…[12][13][14] The resulting MEW-processed scaffolds are often used to guide cell growth in tissue engineering applications [15,16] or to reinforce hydrogels providing mechanical stability and improved handling. [12,17] After poly(ɛ-caprolactone) (PCL), PVDF is the most commonly MEW-processed polymer [18,19] but has not yet been combined with filler particles. While particles of hydroxyapatite (HAp), [20] strontium-substituted bioactive glass (SrBG), [21] bioactive milk proteins, lactoferrin, whey protein, [22] or reduced graphene oxide (rGO) [23] have been combined with PCL, particles with magnetic capacity such as carbonyl iron (CI) have not previously been studied for MEW.…”

Biocompatible Synthetic Polymers for Tissue Engineering Purposes

“…147 In comparison with hollow conduits, more complex designs yield improved nerve repair. 142 Among the synthetic polymers used, PCL is one of the best picks for nerve TE because it is flexible and can be 3D printed to give such complex structures 141,148,149 In our opinion, the combination of these properties with the availability in the market at a relatively good price as well as the previous regulatory approval in medical devices make PCL-based materials such as copolymers (P(DLLA-co-CL)) the most reasonable synthetic polymers of choice for nerve TE. Copolymerization with D,Llactide helps accelerate the otherwise slow hydrolytic degradation of PCL.…”

Multifaceted analysis of nanotoxicity using primary cultured neurons

“…The primary neuronal culture method has been established since the 1970s, and many findings have been reported using cultured neurons [15,16]. With the development of biomaterials and cell preparative technology, various culture methods have been established for multiple studies [13,17,18]. Although many studies on neurotoxicity and neuronal cell death have been reported using these primary culture methods [16,[19][20][21], there is a need to optimize those protocols to up-to-date cytotoxicity assessment methods and to evaluate the safety of nanomaterials.…”