Gonçalves Lobato de Almeida, Maria Teresa
(2021)
Design and fabrication of a device for three-dimensional neuronal development.
PhD thesis, University of Nottingham.
Abstract
The assessment of axonal growth improves our knowledge of neurological disorders, such as Alzheimer’s disease (AD). Currently, axonal growth is being assessed with compartmentalised two-dimensional (2D) cell culture devices; where is possible to study and treat the neuron cell counterparts (i.e. axons) individually. However, 2D cell culture models fail in recreating the traits displayed as in vivo environment. Therefore, a novel three-dimensional (3D) compartmentalised cell culture device has been developed to allow the assessment of axonal growth in three dimensions within a more relevant cell environment.
The main feature of the new 3D compartmentalised prototype device consists of a physical barrier (i.e. membrane), which facilitates the isolation of axonal cellular material, by allowing only the passage of axons between the two compartments. The materials and manufacturing techniques were selected to achieve the design requirements: having such as transparency, biocompatibility and low-cost. The design of the device consists of sealing two polydimethylsiloxane (PDMS) blocks to a polycarbonate membrane. The PDMS blocks, manufactured using soft-lithography, are sealed to a polycarbonate membrane with silicone double adhesive transfer tape. As each PDMS block incorporates one compartment, the membrane allows the passage of axons between compartments without their cell bodies.
Matrigel and Agarose are two natural hydrogels that mimic the extracellular matrix (ECM) environment. Primary cortical neurons were mixed in pre-gel solutions and platted in one compartment of the new device. The neurons started to develop with their axons crossing to the other compartment. A comparison between neuron morphology and cell survival in 2D and 3D environments were achieved for up to 7 days in vitro (DIV). The results show that neurons growing in Matrigel form a pyramidal shape, which is closer to the morphology found in vivo. While Agarose neuron cultures, presented more rounded neurons with a shorter axons. This confirms that neuron development is different in hydrogel (a 3D environment) than in media (a 2D environment). In addition, it was found that using a high plating density (3.75 x 106 cells/ml) of cells in a lower concentration of Matrigel (5 mg/ml) is the optimal solution for cell survival.
Matrigel was also shown to be the best host for culturing neurons in 3D, since they presented a similar cell survival to the 2D neuron cultures. Neurons laden Matrigel showed 79% cell survival at 7 DIV, whereas neurons laden Agarose shown over 50% cell survival, and for 2D neuron cultures this was 75%.
The new 3D compartmentalised device was characterised and validated by fluidic isolation. Isolating axons from their cell bodies and keeping neurons alive and healthy during the time of culture. The fluidic isolation is achieved by applying a volume difference between two compartments, which prevents the passage of a molecule from the lower volume compartment to the higher volume compartment. The length of time a molecule is isolated in one compartment was determined by the fluorescein diffusion profile for two different hydrogel types tested: Agarose and Matrigel. Results showed that the new device was able to isolate the fluorescein in one compartment for 25 minutes in Agarose and for 8 minutes in Matrigel.
The device was validated showing 3D axonal isolation. Neurons were cultured in Matrigel, platted in the new 3D device and the axonal growth was assessed in three dimensions after 2 DIV. The new device provides the ability to conduct fast biological experiments. As this 3D device is manufactured using rapid prototyping techniques, it is simple and cost-effective to be used in most biological laboratories. Finally, the proof of concept was successfully achieved, a new device provides 3D axonal isolation, which provides more realistic experimental results.
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