Primary neuron culture and analysis facilitated by the CellASIC® ONIX Microfluidic Platform

Abstract

Perturbation analysis of live neurons is crucial for fully understanding the nervous system and requires spatiotemporal microenvironment control of cultured neurons. Although advances in microfluidics have enabled such studies, most microfluidic analyses of neurons have required prohibitive commitment of resources. We have demonstrated a novel, optimized microfluidic platform for long-term culture of primary neurons to monitor dynamic cellular processes in real time. The platform enables users to program automated changes to culture conditions without interrupting live cell analysis experiments. In addition, the microfluidic plates offer a superior platform for visualization when compared to plasticware used for conventional static culture. Finally, the platform requires little to no specialized expertise or dedicated personnel for operation, thereby presenting signifcant advantages over traditional microfluidic cell culture systems.

Introduction

Dissecting the functions of neurons, 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 cellmatrix contact1. Because of the diffculties inherent in performing this sort of perturbation analysis of live neurons inside living organisms, there has been a longstanding drive towards developing methodologies for in vitro analysis of neurons2. With recent advancements in microdevices specially designed for primary neuron culture, clinically relevant in vitro models are more accessible than ever before3. These culture models not only allow neuroscientists easy access to individual neurons for electrophysiological stimulation and pharmacological manipulations, but are also compatible with high-resolution microscopic analysis3,4.

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 temperature5, pH value6, osmolarity7, oxygen availability8, nutrient availability9, cell-cell communication10 and extracellular matrix coating10-12. Although advances in the commercial space have allowed consistent supplies of cryopreserved primary neurons, little is known about how microenvironment parameters and their dynamics 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 injury13 and exposure to changes in pH14 , neurotoxins15 and cell-cell communication16. Microfluidic neuronal culture devices offer the potential for higher reproducibility and experimental flexibility; however, widespread adoption of microfluidics for neuronal culture has been limited by requirements for device production/development by dedicated personnel and specialized training for platform operation.

Here, we demonstrate the use of a commercially available microfluidic platform, which requires minimal user training and no prior microfluidic experience, to optimize the growth of rat primary cortical neurons. Based on past experience and recent literature11,12,17, we have established a protocol for cultivating dissociated primary neurons on a dual-coated matrix layer in this microfluidic platform.

We have previously described this technology for perfusion-based microenvironment control for longterm, live cell microscopy18. The microfluidic chamber recreates the physiologic mass transport condition for optimized cell health (Figure 1). 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.

After successfully culturing primary neurons on the platform for 21 days, we used time-lapse microscopy and image analysis to evaluate the health of colonies in real time. This was done by measuring the total length of neurites and comparing our 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 (MAP219)and the astrocyte marker, glial fbrillary acidic protein (GFAP20).

Materials and Methods

Cell culture, reagents, and automated immunocytochemistry materials
Rat primary cortical neurons were purchased from Life Technologies. Cells were maintained in Neurobasal® medium containing 0.5 mM GlutaMAX™ supplement and 2% B27 supplement (all from Life Technologies) at 5% CO2 at 37 °C after proper thawing procedures as described in the manufacturer’s protocols. Mouse anti-MAP2, anti-GFAP, Alexa Fluor® 488 goat antimouse IgG (H+L), Alexa Fluor® 594 goat anti-rabbit IgG (H+L), and Hoechst 33342 were all purchased from Life Technologies. 4% paraformaldehyde solution was purchased from Affymetrix Inc., Triton® X-100 and bovine serum albumin (BSA) were both purchased from Sigma. Microfluidic perfusion cell culture and automated immunocytochemistry was performed using the CellASIC® ONIX Platform, consisting of the CellASIC® ONIX System (Cat. No. EV262), CellASIC® ONIX Microincubator Controller (Cat. No. MIC230) and CellASIC® ONIX M04S Microfluidic Plates (Cat. No. M04S-03- 5PK).

Figure 1. Microfluidic plate for mammalian cell culture. The plate contains four independent flow units (A-D), each with four upstream solution inlets, a gravity flow inlet, a cell inlet, and two waste wells. The culture chamber is surrounded with a microfabricated perfusion barrier. The inlet and outlet channels are 100 µm in width. The barriers are 40 µm tall and 60 µm thick; the spacing between the barriers is 4 µm.

Coating of the microfluidic culture chamber
Coating the chambers of the microfluidic plates prior to loading cells is essential for creating an environment for primary neuronal attachment. In our system, it was found that a double coating of ornithine and laminin was more effective for primary neuron cultures than polyD-lysine, another common coating for neuron culture (unpublished data). Therefore, a coating solution of poly-L- ornithine at 50 µg/mL was prepared in deionized water and a coating solution of laminin at 7 µg/mL was prepared in 1X phosphate-buffered saline (PBS). Once coating solutions were prepared, PBS solution was aspirated from plate Wells 1, 6, 7 and 8 (leaving the inner cutouts with solution in all wells) in a laminar flow hood. Poly-L- ornithine (300 µL of the 50 µg/mL solution) was added to Well 6 and 50 µL to Well 7, thereby breaking the surface tension and ensuring flow.

Plates were incubated for 24 hours in a 37 °C, humidifed environment of 5% CO2 air. The next day, Wells 6, 7 and 8 were aspirated again and 300 µL of deionized water was added to Well 6 and 50 µL to Well 7 to wash away any residual poly-L- ornithine that did not attach to the chambers and channels. Plates were incubated for 4 hours in a 37 °C humidifed environment of 5% CO2 air. For the second layer coating of laminin, Wells 6, 7 and 8 were aspirated (leaving the inner cutouts with solution in all wells) and 300 µL of 7 µg/mL laminin coating solution was added to Well 6 and 50 µL to Well 7. Plates were incubated for 24 hours in a 37 °C humidifed environment of 5% CO2 air. Before seeding the cells, the channels and chambers were rinsed by aspirating well 6, 7, and 8, and adding 300 µL of 1X PBS to Well 6 and 50 µL to Well 7. The plates were then returned to the incubator for 2 hours.

Prior to loading primary neuronal cells, Wells 1, 6, 7, and 8 were aspirated (leaving the inner cutouts with solution in all wells). Cell seeding on microfluidic plates after loading 100 µL of the cell solution (0.5 x 106 cells/mL) to Well 6 and 50 µL to Well 7, the plates were placed on a tilting rack at a 45 degree decline. Plates were incubated in a 37 °C humidifed environment of 5% CO2 air for 3 hours. When cells settled in the chambers, 300 µL of the growth medium (Neurobasal® Medium, 0.5 mM GlutaMAX™ supplement (2.5mL/L), 2% B27 Supplement (20 mL/L), and additional 25 µM L-glutamate for hippocampus neurons) was added to Well 1 and Well 6, and 50 µL of the growth medium to Well 7. Plates were returned to the incubator for long-term culture.

Cell culture maintenance
The growth medium was periodically replenished by aspirating Wells 1, 6, 7 and 8 (leaving liquids in cutouts in all wells) and adding 300 µL of the growth medium to Well 1 and 6, and 50 µL of the growth medium to Well 7. The medium was replenished every day until Day 5. After Day 5, when cells and neurite networks had settled, the medium was replenished every two days.

Image analysis of neurite outgrowth
Images taken from the experiment were aligned using a cross correlation registration algorithm in ImageJ and manually rescaled to make the feld of view and background illumination consistent across all timepoints. An approximate number of neurites was then calculated per image using a custom multi-step pipeline in CellProfiler™ software21,22. First, the soma were identifed by enhancing the dark, circular shaped objects with a morphological reconstruction algorithm and then segmenting using a manual threshold. Next, the neurites were identifed using a series of morphological operations to enhance the long, thin features and then segmenting using a fxed manual threshold. The segmented bodies were then skeletonized and associated with the cell body seed objects. Finally, the total pixel length of neurites in each image was measured.

Automated immunocytochemistry
All solutions were aspirated from Wells 1 through 8, taking care not to disturb the liquid in the inner cutouts. 350 µL of 1X PBS, 100 µL of 4% paraformaldehyde, 100 µL of 0.1% Triton® X-100 in 1X PBS, 150 µL of the primary antibody solution (10 µg/mL mouse anti-MAP2 antibody, 4 µg/mL mouse anti-GFAP antibody and 1:1000 diluted Hoescht 33342 in 1% BSA solution), 100 µL of the secondary antibody solution (10 µg/mL Alexa Fluor® 488 goat anti- mouse IgG and 10 µg/mL Alexa Fluor® 594 goat anti-rabbit IgG in 1X PBS) were added to Wells 1, 2, 3, 4, and 5, respectively. The plate was sealed to the heater manifold and microincubator controller as described in the manual. The flow program was created using the CellASIC® ONIX FG Software using the parameters in Table 1.

Step Flow valve number Pressure (psi, determines flow rate) Duration (min)
Fix V2 2 24
Wash V1 0.25 8
Permeabilize V3 2 24
Wash V1 0.25 8
Primary antibody V4 2 120
Wash V1 0.25 8
Secondary antibody V5 2 80
Wash V1 0.25 8

Table 1. Flow parameters for automated immunostaining using the microfluidic platform.

Results and Discussion

After carefully recovering the rat primary cortical neurons from a frozen vial, we seeded the cells and kept cultures growing in the microfluidic plate for up to 21 days. We also tracked the growth of neurites forming from the same colonies every 3 days by live cell analysis (Figure 2A).The optimum seeding density on the microfluidic plate was only 1.5 x10 5 cells per cm 2 , suggesting that only a very small amount of neural cells is required for sustaining healthy primary cortical neuron cultures in a microfluidic system.

Microfluidic culture of primary neurons
To evaluate the growth rate of the neurites cultured in the CellASIC® ONIX microfluidic plate, CellProfler™ software was used to quantify the total length of the neurites in each image for 5 positions. As shown in Figure 2B, the neurite outgrowth peaked at Day 15. These observations were consistent with the live cell images (Figure 2A) and were conserved throughout different rounds of the culture experiments.

Figure 2. Neurite outgrowth in the microfluidics-cultured rat primary cortical neurons. 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 2 days. The phase contrast images were captured every 3 days under 200x magnifcation (A); Each image was then analyzed using CellProfler™ 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 5 tracked positions (B).

Immunocytochemistry of microfluidics-cultured primary neurons
To futher characterize the cells cultured on the microfluidic device, the neuron marker, MAP2, and the astrocyte marker, GFAP, were identifed by automated immunocytochemistry using the microfluidic platform. We frst set up the plate with the staining reagents in the inlet wells and attached the plate to the control system via the manifold. We then ran the automated immunocytochemistry protocol and completed the analysis in 4 hours (Figure 3). Figure 3 illustrates the excellent quality of images obtained using microfluidic platform, with many cells all in sharp focus, enabling precise, quantitative image analysis.

Figure 3. Immunocytochemistry of the rat primary cortical neurons cultured, stained and analyzed on the microfluidic platform. The primary cortical neurons were cultured on the device for 19 days, and the neuron marker (MAP2, green) and the astrocyte marker (GFAP, red) were identifed by immunocytochemistry using anti-MAP2 and antiGFAP antibodies. Nuclei were stained with Hoechst 33342 (blue).

Conclusion

We have shown that this novel microfluidic culture platform allowed primary neurons to thrive up to 15 days in a controlled, perfusion environment. The platform enabled live cell viewing and image analysis with a standard microscopy setup. Using laminar fluid exchange and cell loading minimized shear stress, which may alter the behavior and morphologies of neurons caused by media changes and direct pipette-to- cell contact. With the use of this microfluidic platform, primary neuron cultures can be suffciently stabilized to enable novel investigations, such as interrogating the dynamics of neurotransmitter transporter activity or defning neurite navigation by substrate patterning, even by investigators lacking experience in or resources to dedicate to the engineering of microfluidic devices.

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