Ph.D Thesis

Ph.D StudentMarom Anat
SubjectOptical Probing of Three-Dimensional Engineered
DepartmentDepartment of Biotechnology
Supervisor PROF. Shy Shoham
Full Thesis textFull thesis text - English Version


Planar neural networks and interfaces serve as versatile in vitro models of central nervous system (CNS) physiology, but adaptations of related methods to three dimensions (3D) have met with limited success. In this study we present the process of the development and characterization of a three dimensional (3D) rat cortical neural network culture grown in a transparent hydrogel scaffold. We demonstrate that the dense 3D cultures have high viability rate and cellular composition of roughly 20% neurons. Neurons in these networks develop a multitude of synaptic connections that in mature networks have spine densities similar to those reported in vivo. Because these brain-like cultures provide 3D optical access and can be equipped with probes for optical imaging and stimulation, we term them as “Optonets”.

To explore the electrophysiological activity of the network we perform volumetric functional imaging of the Optonets using two custom-built optical systems. Using these systems, we present for the first time activity patterns recorded from large 3D neural network imaged with a single cell resolution. We characterized the complex spontaneous activity patterns in the 3D Optonets and found that it is changed during network development and included sporadic single-cell activity, bursts and waves.

To gain spatial control over drug administration to the 3D neuronal Optonet, we developed a novel microfluidic chip for site-specific chemical treatment that also allows the optical probing of the changes in networks activity patterns as a result of drug administration. Treatment with Tetrodotoxin (TTX) or Charbachol (Cach) resulted in localized effects on networks’ activity that could be restored following washout of the drug.

Finally, to gain spatial control over the network’s structure, we directed neuronal growth into laser ablated microchannels. Directing neuronal growth can facilitate the creation of 3D modular neuronal networks composed of pieces of cortical tissue or small Optonets embedded in a hydrogel scaffold non permissive to spontaneous growth, and connected by laser-ablated microchannels.

In conclusion, we present and explore a novel Optonet technology that may be applied to various applications in basic neuroscience research as well as medical applications.  The new technology may serve as (i) a tool for studying large and modular neuronal networks in vitro; (ii) may have applications in regenerative medicine for cell replacement or as biological neural interface and (iii) may be a powerful tool for drug screening and serve an effective and low cost device for pharmacological research or diagnostics.