A Novel Microfluidic Cell Co-culture Platform for the Study of the Molecular Mechanisms of Parkinson's Disease and Other Synucleinopathies

Abstract

Although, the precise molecular mechanisms underlying Parkinson's disease (PD) are still elusive, it is now known that spreading of alpha-synuclein (aSyn) pathology and neuroinflammation are important players in disease progression. Here, we developed a novel microfluidic cell-culture platform for studying the communication between two different cell populations, a process of critical importance not only in PD but also in many biological processes. The integration of micro-valves in the device enabled us to control fluid routing, cellular microenvironments, and to simulate paracrine signaling. As proof of concept, two sets of experiments were designed to show how this platform can be used to investigate specific molecular mechanisms associated with PD. In one experiment, naïve H4 neuroglioma cells were co-cultured with cells expressing aSyn tagged with GFP (aSyn-GFP), to study the release and spreading of the protein. In our experimental set up, we induced the release of the contents of aSyn-GFP producing cells to the medium and monitored the protein's diffusion. In another experiment, H4 cells were co-cultured with N9 microglial cells to assess the interplay between two cell lines in response to environmental stimuli. Here, we observed an increase in the levels of reactive oxygen species in H4 cells cultured in the presence of activated N9 cells, confirming the cross talk between different cell populations. In summary, the platform developed in this study affords novel opportunities for the study of the molecular mechanisms involved in PD and other neurodegenerative diseases.

Keywords: Parkinson's disease; alpha-synuclein; cell culture; co-culture; inflammation; microfluidics; microglia.

Figure 1

Device overview. Image of the microfluidic platform made by stitching several transmitted light microscopy images. The device includes 3 inlets, one main channel served by an outlet and two cell culture chambers with dedicated outlets. The cell culture chambers are connected to each other by three channels. Integrated pneumatic valves (dashed red lines) in a different PDMS layer allow to control fluid flow and to isolate inlets and cameras. A given cell culture chamber can be isolated by activating the valves at the entrance and exit, and the valve serving the central channels. Top right: schematic detail of the microfluidic structure with the cell culture chambers (green), pneumatic channels (red), and channels with a round cross-section (blue). For the mold of the flow layer, culture chambers, inlets and outlets were patterned with SU-8, while the channels with a round-cross section were patterned with AZ 40XT.

Figure 2

Schematic of the flow control mechanism of the device. Valves can be activated independently to route fluid to the left chamber (A) or the right chamber (B). Individual chambers can be isolated from the rest of the platform (C) or both of them can be isolated while allowing diffusion between them (D). Valves are shown in red, channels in blue, and cell culture chambers in green. The direction of the fluid flow is given by the white arrow.

Figure 3

Release of aSyn-GFP by treatment with Triton X-100. (A) Cells producing aSyn-GFP (left) are permeabilized with 0.05% Triton X-100 in PBS and start releasing their contents to the medium in a matter of seconds (right). (B) Transmission microscopy images show that cells treated with Triton X-100 suffer significant morphological changes (left) and that 10 h after opening the central channel the wild type H4 cells are unaffected by Triton X-100 (right). This was also confirmed by viability staining with PI. Fluorescence images acquired with a 300 ms exposure.

Figure 4

Diffusion of released aSyn-GFP. (A) An intensity plot profile of fluorescent signal across the two chambers for several time points shows that after 60 min the concentration of aSyn-GFP is almost the same in both chambers. The line used for data acquisition is shown on the image on the upper right corner (white line). (B) Fluorescence microscopy images taken 1, 20, and 60 min after opening the central channels, showing diffusion of aSyn-GFP from the left to the right chamber. Scale bars are 100 μm, fluorescence images acquired with a 300 ms exposure.

Figure 5

Average fluorescence intensity signal of the H4 cells compared to the background fluorescence measured inside the cell chamber. Fluorescence values of cells were obtained by selecting the areas corresponding to cells in images obtained with an epifluorescence microscope, taken with a 2 s exposure. For the assay with H4 aSyn-GFP (A), the average signal before exposure was calculated counting 124 cells and the average signal 10 h after exposure was calculated over 116 cells; for the assay with H4 GFP (B) the numbers were 42 and 44, respectively. The ratio of cell signal to background fluorescence is 1.053 before exposure to aSyn-GFP and 1.033 after exposure (A) and 1.029 before exposure to GFP and 1.027 after exposure (B). The difference between the background before and after exposure to aSyn-GFP or GFP can be explained by the fact that the fluorescence images of the latter were obtained after washing the device with PBS, which replaced the cell culture medium previously inside the cell chambers.

Figure 6

Fluorescence signal in dead cells. Cells with a higher fluorescence signal from aSyn-GFP, after washing with PBS (left, white arrows) displayed a compromised membrane after PI staining (right). This seems to indicate that aSyn-GFP is able to penetrate and remain inside cells with compromised membranes. Similar results were obtained for H4 wild type cells co-cultured with H4 GFP cells. Fluorescence images were taken with 2 s (left) and 300 ms (right).

Figure 7

H4 cells exhibit higher ROS levels when co-cultured with LPS-activated N9. (A) Plot of the average DHE fluorescence signal of the H4 cells. The signal of cells co-cultured with LPS-activated N9 (32 cells, 77% of the population) is 97% higher than the one emitted from the cells co-cultured with non-activated (vehicle) N9 (74 cells, 78% of the population); ^***p < 0.001 for two-tailed Student's t-test with unpaired data. (B) Fluorescence microscopy image of H4 cells, co-cultured with non-activated LPS, stained with DHE.