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In vitro culture of cortical neurons in 3D Rodrigo Lozano1,2, Brianna C. Thompson1, Kerry J. Gilmore1, 2 1 Mario Romero-Ortega , Gordon G. Wallace 1IPRI,

University of Wollongong and 2University of Texas at Arlington Email: rl137@uowmail.edu.au

Cortical neurons in 2D

Abstract Two dimensional (2D) neuronal cell cultures on glass or plastic substrates do not completely mimic or resemble the in vivo microenvironment of neuronal tissues.

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To address these limitations, a 3D in vitro cell culture system has been reconstructed . We assembled a 3D neural cell culture system using primary mouse cortical neurons encapsulated in a biologically-derived hydrogel matrix (collagen).

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Figure 3. (a)-(c) Cortical neurons after 7 days on glass coverslip (b)-(c) Cell staining with B-III tubulin (red) for cortical neurons, GFAP for glial cells (green) and DAPI for cell nuclei(blue). d) Image (50X) of Neurons (B-III tubulin channel). e) Metamorph image showing nucleus of different cells and respective neurite outgrowths. f) Overlay image of the nuclear segmentation, cell body and neurite outgrowths on the original image.

We present the cell morphology and analysis of neuronal length in both 2D and 3D culture environments. The 3D cell culture enhanced differentiation in terms of neurite length and number of neurites per cell, and typical 3D cell morphology.

Cortical neurons in 3D

We plan to further develop this co-culture system towards application in neurobiological studies and tissue engineering.

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Methods and Materials •Cortical neurons were collected from embryonic mice between 15 – 19 days ( E15-E19), Fig 1.

Figure 4. (a)-(b).Cortical neurons in collagen, after 10 days of encapsulation. (b) Cell staining same as figure 3(b)-(c). (c)-(d) Images showing the neurite tracing of different cells. (e) 2D –projected confocal image of neuronal network in 3D. (f) Fiji(imageJ) 3D viewer of neuronal network.

•2D culture of the cortical neurons was done on Poly-DLysine(PDL) coated glass coverslips at 2500 cell/cm 2 , Fig 2. •3D culture of the cortical neurons was done by encapsulating cells in collagen at 400 x 106 cell/ml.

Results There was a statistically significance difference in the average number of processes per cell and in the average neuronal processes outgrowth between the 2D and 3D environments.

•Cortical Neurons were encapsulated in collagen with B27 added at 2% of the final gel concentration. •2D image analysis was done using the neuron tracing module from Metamorph (Molecular Devices software). •3D image analysis was done using Fiji(Image J) software. •The total neurite outgrowth was calculated from the 2D and 3D data as well as the number of processes from each cell.

Average neuronal process outgrowth per cell

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Number of Processes

Average number of Processes per cell

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Figure 5. Comparison of average neurite length per cell(right) and number of neurites in 2D and 3D (left). * indicates a statistically significant between 2D and 3D (p≤ 0.025) by Students t-test.

Conclusion

Figure 1. Embryonic mouse (E15E19) dissection process.

Culturing cortical neurons in a 3D state that mimics the tissue environment enables more cell to cell interaction to occur, changing the cell shape (morphology), increasing the number of processes and length of neurites per cell. Therefore 3D cell culture more accurately mimics in vivo neuronal networks and should be used for future applications in neurobiological and tissue engineering.

Figure 2. Schematic diagram of cell culture in 2D and 3D environment.

Acknowledgment and References •ARC Laureate Fellowship(Prof. Gordon Wallace) for funding this research. •Consejo Nacional de Ciencia y Tecnología (CONACYT) Mexico for their financial support. 1. 2. 3. 4.

Figure 2 was reproduce from Baker B M , and Chen C S J Cell Sci 2012;125:3015-3024. Shih-Feng Lan, Binil Starly. Alginate based 3D hydrogels as an in vitro co culture model platform for the toxicity Screening of new chemical entities. Toxicology and applied Pharmacology.(2011). Brian M. Gillette, Ninna S Rossen, Nikkan Das, Debra Leong, Meixing Wang, Arushi Dugar, Samuel K. Sia. Engineering extracellular matrix structure in 3D multiphase tissues. Biomaterial. 2011. Brian M. Gillette, Jacob A. Jensen, Meixin Wang, Jason Tchao, and Samuel K. Sia. Dynamic Hydrogels: Switching of 3D Microenvironments Using Two-Component Naturally Derived Extracellular Matrices, Advance materials, 2010 Junmin Zhu and Roger E Marchant. Design properties of hydrogel tissue engineering scaffolds. Expert Revision Medical Devices. 2011.


Rodrigo lozano