Biohybridized Neural Interface Microsystems

Living Neuro-Prosthetic Interfaces

The Cullen Lab blends neural tissue engineering techniques and micro-electrical technology to create “biohybrid” interface microsystems. These living biohybridized neural-electrical interface microsystems are being investigated for functional integration with the nervous system. With this design, host axonal ingrowth and synaptic integration may occur with the living component, potentially exploiting a more natural integration with the non-organic interface.  In the case of driving a prosthetic limb, the neural interface may occur with the peripheral nervous system rather than placing a device into the otherwise non-injured brain or spinal cord.  In addition, this neural interface occurs at the point of final motor output and primary sensory input, thus leveraging the exquisite processing power of the brain and spinal cord rather than straining to decipher it.  However, peripheral nerve axonal require a living target for innervation, hence the necessity of our living biohybrid neural interfaces.  Overall, these tissue engineered 3-D neural interfce microsystems may significantly advance regeneration or device-based deficit mitigation in the nervous system.

biohybridized

 

Biohybridized neural interface microsystems formed around 3-D electrodes create unique microsystems that are powerful platforms for enhancing the interface with the nervous system.  (a) Cells can be grown in vitro on electro-conductive microfibers and encapsulated with hydrogel or (b) the microfibers can be incorporated with the axonal constructs prior to transplantation.

neurons and azons

 

Controlled neuronal adhesion to conductive polymer microfibers.  Confocal reconstructions of neuronal cultures plated on microfibers immunolabeled at 7 days in vitro for MAP-2 (green) and tau (fiber locations denoted by dashed lines). By controlling the relative electrostatic surface charge of the microfiber and the substrate, adhesion to the microfiber was increased.  (a) Low-density adhesion on the microfibers resulted in the axonal projections to the substrate.  (b) Robust neuronal adhesion resulted in neuronal somata and axonal containment on the microfiber. Scale bar = 200 µm.

 

Neurons

 

Neuronal encapsulation on microfibers.  For future transplantation, removal from culture while maintaining neuronal network integrity and viability is necessary.  In order to demonstrate this using hydrogel encapsulation, neurons were plated on collagen-coated conductive polymer microfibers and, at 6-9 days in vitro, encapsulated using 0.5-1.0% agarose.  (a-c) Representative fluorescent confocal reconstructions of encapsulated neuronal cultures on microfibers stained to discriminate live cells (green) from the nuclei of dead cells (red) (scale bar = 200 µm). (a) The encapsulation process did not reduce the cell density or the cell viability versus non-encapsulated controls.  Increased magnification of regions of interest from showing (b) a cluster of neuronal somata and (c) a neurite-rich segment following encapsulation (scale bars = 50 µm).

 

Advanced Microsystems In Vitro

These techniques are also useful to enhance the capabilities of investigational platforms in vitro.  Here, interfacing novel 3-D neural cellular constructs with micro-fluidic and/or micro-electrical systems has created biohybridized platforms, providing unprecendented 3-D microenvironmental control and allowing non–invasive probing and manipulation of cultured neural cells. This effort was selected as one of the Highlights of Neural Engineering entitled “Microfluidic Engineered High Cell Density 3-D Neural Cultures”. Currently, the Cullen Lab is applying these 3-D neural cellular constructs as powerful investigational platforms for the study of basic neurobiology, network neurophysiology, injury/disease mechanisms, pharmacological screening, or test-beds for cell replacement therapies.

advanced microsystems

 

Biohybridized neural interfaces. (a-b) Concept: 3-D neural cell cultures formed around 3-D micro-towers to create biohybridized neural interface microsystems. (c-d) Live neurons (green fluorescent protein; GFP+) forming 3-D networks around micro-towers in culture. (c) At 1 DIV, neurons are homogenously distributed throughout the matrix (>500 µm thick); (d) However, by 13 DIV, there was increased cell density around the towers and the culture thickness had decreased to approximately 300 µm. (e) Live (green) and dead (red) neural cells at 23 DIV, demonstrating viable neural networks adhering to the micro-towers.  (f-g) Immunocytochemistry labeling astrocytes (GFAP+, red) with GFP+ neurons. Neurons and astrocytes were in intimate contact with each other and the micro-towers in the bottom 300 mm of the cultures. (h) In some cases, neural cells and processes had coalesced into tracts spanning neural populations on micro-towers (top 200 µm shown here). With micro-electrical capability, these microsystems may serve as powerful in vitro platforms to continuously monitor the electrical activity and dynamics of “small world” neuronal networks in 3-D matrices.

 

 

Contact Information

D. Kacy Cullen, Ph.D
105E Hayden Hall, 3320 Smith Walk
Philadelphia, PA 19104
dkacy@mail.med.upenn.edu

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News and Events

  • The Cullen Lab was awarded a one-year pilot grant through the Penn Medicine Neuroscience Center to explore "Neuromodulation of Brain Circuits Using Transplantable Micro-Tissue Engineered Neural Networks."
  • James Harris, Ph.D., was awarded an NIH NRSA Postdoctoral Fellowship on the "Brain Injury Training Grant" through Penn's Center for Brain Injury & Repair. Dr. Harris' project is on "Mechanisms of Biophysical Responses and Neurodegeneration following Concomitant Blast and Inertial Traumatic Brain Injury."
  • Dr. Cullen gave a lecture at the American Society for Peripheral Nerve Annual Meeting in Kauai, Hawaii in January. The talk was titled "Tissue Engineered Grafts Accelerate Peripheral Nerve Repair By Direct Axon-Induced Axon Regeneration." 2013
  • Laura Struzyna was a Poster Award Finalist at the Tissue Engineering and Regenerative Medicine International Society Annual Meeting in Atlanta, Georgia in November. Her poster was titled "Tissue Engineered Grafts Accelerate Peripheral Nerve Repair By Direct Axon-Induced Axon Regeneration."
  • Dr. Cullen gave an Invited Lecture at the Department of Biomedical Engineering at the Georgia Institute of Technology in May. He presented "Tissue Engineering Strategies to Restore Neural Circuitry and Create Living Scaffolds for Targeted Axonal Regeneration."
  • Dr. Cullen and collaborator Dr. Douglas Smith were awarded funding from the Department of Defense through the Rutgers - Cleveland Clinic Consortium of the Armed Forces Inst. of Regenerative Medicine. This project will investigate the efficacy of novel tissue engineered constructs encased in custom-built nerve guidance tubes to promote nerve regeneration following major peripheral nerve injury.
  • Bioengineering Ph.D. Candidate, Laura Struzyna, was selected for a prestigious 3-year Graduate Research Fellowship through the National Science Foundation based on her proposal for "Tissue Engineered Constructs for Neural Regeneration."

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