Two-Photon Polymerization 3D-Printing of Micro-scale Neuronal Cell Culture Devices

We are broadly interested in the relationship between structure and functions in the brain, particularly at the level of micro circuits. We often employ reduced experimental preparations such as dissociated neuron cell culture because they are a precious model to investigate electrical activity, synaptic transmission, and overall the emergence of collective states. The method we propose is ideal for rapid fabrication of microscale polymeric devices.

This can be immediately used for the experimental study of modular neural networks in a dish, and it do not requires a high level of technical expertise in soft photolithography. This method and our simple proof of concept allow the designing of modular neural networks where we can define the structure and control the flow of biological signals. Drug screening and investigation of pathological conditions immediately benefit from these sophisticated in vitro neuronal networks.

To begin, launch the design software desktop application from the top menu bar, select new, and then choose part a 3D representation of a single design component. To establish the top view of the sketch, press control and five. From the side panel, select sketch, and choose corner rectangle.

Click on one of the edges of the rectangle and on parameters panel, set the length to 100 units and set the length of the perpendicular edge to 50 units. Drag over the rectangle and select it while holding the left mouse button. From the add relation menu, select fix to constrain the relations among lines.

After creating a new small overlapping rectangle from the side panel, select features and choose extruded base. Set the depth of the large rectangle to five in the smaller one to 10 units. Following these basic principles, more complex objects can be designed.

Finally, from the file menu, save the part in STL format. To begin, generate and process the computer assisted design file in STL format. Position the glass substrate on the holder.

Using an electronic multimeter, confirm that the indium tin oxide or ITO coated side of the glass substrate is oriented upwards, and tape the glass thoroughly. Under a chemical fume hood, apply a drop of IPS Photoresist at the center of the glass substrate and insert the holder into the 3D printer with the resin drop facing the objective. Through the software file menu, load the GWL files, select approach sample, and then find interface.

Select start job to initiate printing, and when done, press exchange holder. Gently remove the substrate with the printed part, and under a fume hood, incubate the glass substrate in PGMEA followed by isopropanol. To cure the air dried printed part, expose it to 365 to 405 nanometer UV light for five to 20 minutes.

Under a laminar flow hood, gently remove the printed part from the glass substrate and place it on a two microliter drop of resin in a Petri dish. After five minutes of UV exposure, cap the dish and incubate at 80 degrees Celsius for 30 minutes. To begin, print a 3D mold using two photon polymerization and mount it to a Petri dish, which is now referred to as a mold.

For fabrication of the device, incubate the mold with 10 microliters of hydrophobing agent for seven minutes, and rinse it with 70%ethanol, followed by deionized water two times. Gently add previously prepared PDMS mixture onto the mold until the intended final height of the device is reached. Incubate the covered mold in a preheated 80 degree Celsius oven for 18 minutes.

Then gently detach the cured PDMS block from the mold under the laminar flow hood and submerge it in isopropanol for 10 minutes inside a glass Petri dish. Under a stereo microscope, using an ophthalmic stab knife, carefully cut out the central square section and then along the edges of the device. Then submerge the device in ethanol for 30 minutes.

Under a laminar flow hood, transfer the device into a new Petri dish and let it dry. To begin, obtain a 3D printed mold and fabricate the polydimethylsiloxane or PDMS device. To sterilize the micro electrode array or MEA, submerge it in 70%ethanol for 30 minutes, and then rinse it with sterile deionized water three times.

Using sterile fine tweezers, mount the PDMS device onto the inner area of a MEA under a stereo microscope. Align one side of the PDMS device to the center of the inner MEA area. Under a fume hood, add one milliliter of 0.1%polyethylenimine solution to MEA and incubate it at 37 degrees Celsius overnight.

After rinsing the MEA five times with deionized water, add one milliliter of the cell culture medium and incubate the MEA at 37 degrees Celsius. Next, seed the MEA with one milliliter of culture containing 1.8 million neuronal cells isolated from rat pup's brain and incubate. To begin, fabricate a PDMS device from a 3D printed mold.

Enculture neuronal cells in the MEA mounted to the PDMS. Gently mount the MEA inside the head stage of a multichannel electronic amplifier placed inside a dry 37 degree Celsius and incubate for 10 minutes. Launch the experimenter software, set the sampling rate to 25 kilohertz, and press start data acquisition to initiate data acquisition.

In the data display panel, the raw traces of extracellularly recorded activity for each channel appear. Go to the stimulator panel of the software, choose three pairs of neighboring micro electrodes that are active during network wide bursts, and select a biphasic pulse waveform. Set the peak amplitudes to 800 millivolts and the pulse duration to 200 microseconds.

To achieve the bipolar configuration, select and set the values of the neighboring electrodes. Finally, set the recorder to 300 milliseconds before, and 1000 milliseconds after the stimulation, and press start data acquisition to initiate recording. Live imaging revealed that fluorescently tagged neurites showed outgrowth from source to target in a microchannel of the PDMS device.

Electrophysiology analysis indicated that the evoked response of the source and the target in the modular neuronal networks are asymmetric, whereas the control without PDMS did not show this pattern.