**4.2.1 Neural network by μCP**

26 Advances in Unconventional Lithography

In Fig.10, We show that neurons cultured on the PEI- and PLL-coated surfaces adhered to and extended neurites along the grid-shape patterns, whereas neurons cultured on the LNcoated coverslips clustered into clumps of cells. In addition, we found that the neurons on the PEI and PLL-coated grids survived for more than 2 weeks in serum-free conditions,

Fig. 10. Images obtained using the phase contrast microscope, showing cells cultured on PEI, PLL and LN polymeric lms at different time points. (a, d, and g) Representative images of neurons cultured on PEI grid patterns at 24 h, 7 days, and 14 days, respectively, show that neurons adhere and grow accurately along the PEI grids at differently time points. (b, e, and h) Representative images of neurons cultured on PLL grid patterns at 24 h, 7 days, and 14 days, respectively. (c, f, and i) Representative images of neurons cultured on LN grid patterns at 24 h, 7 days, and 14 days, respectively. The images (c and f) show that the neurons on the LNgrid patterns often accumulate at the cross points of the grids. The image (i) shows that most neurons disappear after 14 days in culture and only small areas of the

**4.2 Specific neural network with two relative neurons, such as dopaminergic neurons** 

There is a closely relationship in the respect of function and structure between dopaminergic neurons in the substantial nigra and GABAergic neurons in the striatum. As well-known,

neural cells exist. (a–c) Bar = 100 μm; (d–i) bar = 50 μm.

**and GABAergic neurons** 

whereas most neurons cultured on the LN-coated grids died after 1 week[13].

**4.1.2 Cells viability** 

Different kinds of neural network by μCP were established with neuron from the striatum, dopaminergic neurons from the substantial nigra and both of them co-culture. The conditions of neuronal adhesion on different pattern figures were observed using several techniques, including immunocytochemical staining, transmission electron microscope and scanning electron microscope. Using immunocytochemical staining, transmission electron microscope, we identified the types of neural cells and observed some neurosynapse-like structures near the neuronal soma on PEI-coated coverslips. These ndings indicate that PEI is a suitable surface for establishing a functional neuronal network in vitro.

### **4.2.1.1 Investigation of neural cell types and neurite elongation along the grid-like patterns by immunocytochemical staining and SEM**

PEI, PLL and LN were used to produce grid-shape patterns on glass coverslips by microcontact printing. GABAergic neurons and medium spiny neuron from the rat striatum, dopaminergic neurons from the rat substantial nigra and both of them co-culture were researched separately on the different polymers coated surface. The viability and morphology of these neurons under serum-free culture conditions were observed using uorescent microscopy in Fig. 11, Fig. 13, Fig. 14. After 7 days in culture, we found that the neural cell bodies on the PEI patterns were located mostly at the cross-points of the grid, whereas neurites extended along the line of the grid-like patterns. More continuous and integrated neural network was achieved finally. On the PLL-coated coverslips, the neural patterns appeared to be integrated. But several cells clustered at the cross-points of the grid disappeared gradually after the media was replaced. In contrast, cells cultured on the LN-coated grids were generally clustered into clumps and cannot form satisfied patterns. Different sizes of PEI pattern were produced by microcontact printing. In Fig. 12, compared with 50μm, 100μm, 200μm pattern sizes, we found few difference early. After 7 days or 14 days culture, most neural cells on 200μm size grew well and were seldom found to overlap each other, unlike those on 50μm size clustered into clumps at the cross-points of grid and disappeared gradually. Identified with immunocytochemical staining, we found that neural cells from the rat substantial nigra were TH positive, synaptic vesicle protein were synaptophysin positive in Fig. 13 and cells from the rat striatum were GABA positive or DARPP-32 positive in Fig. 11, Fig. 12, Fig. 14. SEM show neurons outgrowth on PEI-coated patterns and validate the findings by immunocytochemical staining in Fig. 15, Fig. 16.

Application of Soft Lithography and Micro-Fabrication on Neurobiology 29

(a–c) co-culture neurons growing on LN grid patterns. (a) Immunostaining with anti-TH (red uorescence) labelled dopaminergic neurons. (b) Immunostaining with anti-DARPP-32 (green uorescence) labeled medium spiny neuron from the striatum(arrow). (c) Merged image of (a) and (b), showing TH positive neurons on the LN-coated patterns actually adhere to the cluster formed by medium spiny neuron. (d–f) co-culture neurons growing on PEI grid patterns. (d) Immunostaining with anti-TH (red uorescence) labelled dopaminergic neurons(arrows). (e) Immunostaining with anti-DARPP-32 (green uorescence) labeled medium spiny neuron from the striatum(arrow). (f) Merged image of (d) and (e) showing that DARPP32 positive neurons cultured on the PEI-coated patterns form a continuous and integrated neural network, and two TH positive neurons adhere to the cross-points of grid. (g–i) co-culture neurons growing on PLL grid patterns. (g) Immunostaining with anti-TH (red uorescence) labeled dopaminergic neurons(arrow). (h) Immunostaining with anti-DARPP-32 (green uorescence) labeled medium spiny neuron from the striatum(arrows). (i) Merged image of (g) and (h)showing only two DARPP32 positive neurons adhere to the cross-points of grid. TH positive neurons

Fig. 14. Immunofluorescent image of anti-TH labelled dopaminergic neurons from the substantial nigra and anti-DARPP32 labeled medium spiny neuron from the striatum coculture for 7days on different substrates, the nuclei of neurons were stained with Hochest

were not restricted by the grid pattern. bar=100μm

x200

(a) on LN grid patterns (b) on PEI 100μm grid patterns (c) on PLL 100μm grid patterns bar=50μm Fig. 11. Immunofluorescent image of anti-GABA (green fluorescence) +anti-MAP2 (red fluorescence) labelled striatal neurons cultured for 7 days on different substrates, the nuclei of neurons were stained with Hochest x400

(a)on 50μm patterns (b) on 100μm patterns (c) on 200 μm patterns bar=50μm

Fig. 12. Immunofluorescent image of anti-GABA(green fluorescence)+anti-MAP2(red fluorescence) labeled striatal neurons cultured for 7 days on different sizes of PEI patterns, the nuclei of neurons were stained with Hochest x400

(a) on LN grid patterns (b) on PEI 100μm grid patterns (c) on LN+PEI bar=20μm

Fig. 13. Immunofluorescent image of anti-TH(red fluorescence) labeled dopaminergic neurons from the substantial nigra and anti-synaptophysin(green fluorescence) labeled synaptic vesicle protein cultured for 7 days on different substrates. x200

(a) on LN grid patterns (b) on PEI 100μm grid patterns (c) on PLL 100μm grid patterns bar=50μm Fig. 11. Immunofluorescent image of anti-GABA (green fluorescence) +anti-MAP2 (red fluorescence) labelled striatal neurons cultured for 7 days on different substrates, the nuclei

(a)on 50μm patterns (b) on 100μm patterns (c) on 200 μm patterns bar=50μm

(a) on LN grid patterns (b) on PEI 100μm grid patterns (c) on LN+PEI bar=20μm

synaptic vesicle protein cultured for 7 days on different substrates. x200

Fig. 13. Immunofluorescent image of anti-TH(red fluorescence) labeled dopaminergic neurons from the substantial nigra and anti-synaptophysin(green fluorescence) labeled

the nuclei of neurons were stained with Hochest x400

**TH+Syn**

Fig. 12. Immunofluorescent image of anti-GABA(green fluorescence)+anti-MAP2(red fluorescence) labeled striatal neurons cultured for 7 days on different sizes of PEI patterns,

of neurons were stained with Hochest x400

(a–c) co-culture neurons growing on LN grid patterns. (a) Immunostaining with anti-TH (red uorescence) labelled dopaminergic neurons. (b) Immunostaining with anti-DARPP-32 (green uorescence) labeled medium spiny neuron from the striatum(arrow). (c) Merged image of (a) and (b), showing TH positive neurons on the LN-coated patterns actually adhere to the cluster formed by medium spiny neuron. (d–f) co-culture neurons growing on PEI grid patterns. (d) Immunostaining with anti-TH (red uorescence) labelled dopaminergic neurons(arrows). (e) Immunostaining with anti-DARPP-32 (green uorescence) labeled medium spiny neuron from the striatum(arrow). (f) Merged image of (d) and (e) showing that DARPP32 positive neurons cultured on the PEI-coated patterns form a continuous and integrated neural network, and two TH positive neurons adhere to the cross-points of grid. (g–i) co-culture neurons growing on PLL grid patterns. (g) Immunostaining with anti-TH (red uorescence) labeled dopaminergic neurons(arrow). (h) Immunostaining with anti-DARPP-32 (green uorescence) labeled medium spiny neuron from the striatum(arrows). (i) Merged image of (g) and (h)showing only two DARPP32 positive neurons adhere to the cross-points of grid. TH positive neurons were not restricted by the grid pattern. bar=100μm

Fig. 14. Immunofluorescent image of anti-TH labelled dopaminergic neurons from the substantial nigra and anti-DARPP32 labeled medium spiny neuron from the striatum coculture for 7days on different substrates, the nuclei of neurons were stained with Hochest x200

Application of Soft Lithography and Micro-Fabrication on Neurobiology 31

**4.2.1.2 Examination of synaptic formations by immunocytochemical staining and TEM**  To further understand functional activities of neurons on the grid pattern, we observed the microstructure of the neuronal cell body, neuritis extension and processes of the synapse formation by using laser confocal microscope after 7 days in culture. It is found that several neural cell bodies aggregated on the cross point of the grid pattern (Fig.17.b), which neurites extended along the lines clearly by cytoskeletal proteins MAP2 and Synaptophysin double immunocytochemical staining of patterned neurons on PEI(Fig.17.a). Some processes can even gather into a bundle, span the distance between two cross-points and make connection with another neuron. Synaptophysin was a kind of granular protein scattered around the cell bodies and neurites, The neurites from neuron at the cross-point seemed to communicate with those at the lines which express a large number of synaptic vesicle

(a) neurites extended along the lines and synaptic vesicle protein around

**4.2.2 Neural network by microfluidic technique** 

(c) synapse connection between two neurons at the grid pattern bar=25μm

(b) neural cell bodies at the cross-point and synaptic vesicle protein scattering around them

be electrical synapse structure due to double-membrane structure adjacent closely.

Fig. 17. Immunofluorescent image of anti-MAP2(red fluorescence) labeled neurons and antisynaptophysin(green fluorescence) labeled synaptic vesicle protein cultured for 7 days on

We chose PEI group which can construct more clearly neural network and continued to observe intercellular ultrastructure under transmission electron microscope (Figure18). Two periphery of neurites thicken show high electron density to form a synaptic contact (Red border). Width of synaptic cleft was measured to 30 ~ 50nm. Figure18(a) shows clear synaptic vesicles. Figure18(b) shows the synaptic cleft is relatively narrow and suspected to

In this experiment,, we made a big progress on microfluidic technique by re-designing the parameters and enhancing the photoresist coating thickness of the Cr template. After 7 days in culture, poly-l-lysine and laminin+polyethyleneimine were found to be formed more complete and clearer flow patterns by the application of microfluidic technique. On LN group, neurons were easy to cluster into clumps when channel width was 150μm,almost overshadowed flow pattern itself; On PEI group, even though the flow patterns are more complete, the neurites extend short and cannot constitute a connection between some cells

protein(Fig.17.c).

**MAP2+Syn**

PEI patterns x400

Fig. 15. SEM show neurons outgrowth on PEI-coated patterns. Somata of neuons located on the cross point and neurites extend along the lines

Fig. 16. SEM show that Line of grid were occupied by abound neurites.

Fig. 15. SEM show neurons outgrowth on PEI-coated patterns. Somata of neuons located on

Fig. 16. SEM show that Line of grid were occupied by abound neurites.

the cross point and neurites extend along the lines
