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Microfluidic Method for Primary Neuron Culture and Analysis

Figure 1: Rat primary cortical neurons were seeded and cultured on the microfluidic plate for 21 days, and the medium was replenished every day until Day 5. After Day 5, the medium was replenished every two days. The phase contrast images were captured every three days under 200x magnification (A); Each image was then analyzed using CellProfiler software, and the total length of the neurites in each image was measured and normalized to the seeding density of the cells in the image. Error bars represent standard deviation of the pixel counts across five tracked positions (B). Image: EMD Millipore

Dissecting neuron function, while crucially important for understanding normal and pathological neurological processes, requires measuring the responses of live cells to external stimuli, such as temperature, chemical signals, electrical signals, cell-cell and cell-matrix contact. Because of the inherent difficulties in performing perturbation analyses inside living organisms, there has been a longstanding drive towards developing methodologies for in vitroanalysis of neurons.

With recent advancements in microdevices specially designed for primary neuron culture, clinically relevant in vitro models are more accessible than ever before. These culture models not only allow neuroscientists easy access to individual neurons for electrophysiological stimulation and pharmacological manipulations, but they are also compatible with high-resolution microscopic analysis.

However, still there are major challenges in keeping primary neuron cultures stable and viable for long-term experiments. Primary neurons are well documented for being very sensitive to microenvironment cues, such as temperature, pH value, osmolarity, oxygen availability, nutrient availability, cell-cell communication and extracellular matrix coating. Although advances in the commercial space have allowed consistent supplies of cryopreserved primary neurons, little is known about how microenvironment parameters affect the stabilization of primary neurons in culture.

Recently, microfluidic technology has been applied to primary neuron culture to help understand how changes in microenvironment can affect different parts of neurons. For example, microfluidics can isolate neuron soma from axons, enabling spatially restricted studies of injury and exposure to changes in pH, neurotoxins and cell-cell communication. Microfluidic neuronal culture devices offer the potential for higher reproducibility and experimental flexibility. Widespread adoption of microfluidics for neuronal culture has been limited, however, by the complexity of the devices and the need for specialized training.

Here, we demonstrate use of a commercially available microfluidic platform (CellASIC ONIX Platform, EMD Millipore) to optimize the growth of rat primary cortical neurons. The platform requires minimal user training and no prior microfluidic experience, and it enables users to program automated changes to culture conditions without interrupting live cell analysis experiments.

The microfluidic chamber recreates the physiologic mass transport condition for optimized cell health. Four upstream fluidic channels allow controlled exposure of the cells to different solutions during live cell analysis. Each microfluidic plate contains four parallel chambers, which are centralized under a single viewing window. Flows can be controlled using an external pneumatic manifold connected to a control system, without perturbing the microscope stage. The plate can also be cultured in a standard incubator using a dedicated gravity-driven flow channel. An integrated microincubator system delivers temperature and gas control to the microfluidic chambers.

In the following experiments, primary neurons were successfully cultured on the microfluidic platform for 21 days. Time-lapse microscopy and image analysis were then used to evaluate the health of colonies in real time. This was achieved by measuring the total length of neurites and comparing the microfluidic cell culture model to colonies cultivated in standard dish culture. Characterization of cultured cells in the microfluidic platform was accomplished through immunostaining of the neuron marker, microtubule-associated protein 2 (MAP2), and the astrocyte marker, glial fibrillary acidic protein (GFAP).

Results and discussion
After carefully recovering the rat primary cortical neurons from a frozen vial, the cells were seeded and the cultures kept growing in the microfluidic plate for up to 21 days. The growth of neurites forming from the same colonies was also tracked every three days by live cell analysis (Figure 1A).The optimum seeding density on the microfluidic plate was only 1.5 x105 cells per cm2, suggesting that only a very small amount of neural cells is required for sustaining healthy primary cortical neuron cultures in a microfluidic system.

To evaluate the growth rate of the neurites cultured in the microfluidic plate, CellProfiler software was used to quantify the total length of the neurites in each image for five positions. As shown in Figure 1B, the neurite outgrowth peaked at Day 15. These observations were consistent with the live cell images (Figure 1A) and were conserved throughout different rounds of the culture experiments.

To further characterize the cells cultured on the microfluidic device, the neuron marker, MAP2, and the astrocyte marker, GFAP, were identified by automated immunocytochemistry using the microfluidic platform. The plate was set up with the staining reagents in the inlet wells and then attached to the control system via the manifold. The automated immunocytochemistry protocol was run and analysis completed in four hours.

In conclusion, the CellASIC ONIX microfluidic culture platform allowed primary neurons to thrive up to 15 days in a controlled, perfusion environment. The platform enables live-cell viewing and image analysis with a standard microscopy setup, allowing dynamic cellular processes to be monitored in real time. It also offers a superior platform for visualization when compared to the plasticware used for conventional static culture.

With the use of this microfluidic platform, primary neuron cultures can be sufficiently stabilized to enable novel investigations, such as interrogating the dynamics of neurotransmitter transporter activity or defining neurite navigation by substrate patterning. This research can be achieved with little to no specialized expertise or dedicated personnel for platform operation, therefore presenting significant advantages over traditional microfluidic cell culture systems.


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