shows images of the microfabricated PDMS co-culture device. The scanning electron micrographs show arrays of 15 µm wide axon-guiding microchannels separated from one another by approximately 60 µm distance. The inset in shows the overall device (22 mm by 22 mm) placed inside a conventional 6-well polystyrene culture plate. Axon-guiding channel having width of 30 µm with 30 µm distance between channels and 15 µm with 60–500 µm distances have been tested to find the optimal dimension that maximizes the channel area between the compartments (i.e. maximum channel width and minimum distance between channels) for high density axon growth into the axon/glia compartment while maintaining a stable fluidic seal. Too wide of a channel caused the PDMS channel to sag and blocked the channel. Too short distance between channels resulted in insufficient contact area between the PDMS structure and the substrate and prevented a tight fluidic seal. Experimental results showed that axon-guiding channels with 15 µm width and 60 µm distance provided maximum opening between the two compartments while maintaining a robust fluidic seal between the PDMS device and the substrate throughout the culture period of four weeks without leakage. Without the oxygen plasma treatment, the fluidic seal between the device and the PDL or MatrigelTM coated substrate broke after several hours, and the hydrophobic property of the PDMS caused bubbles to be trapped inside the compartment while loading cells or changing out the culture media. The 30 µm wide channels with 30 µm distances provided more than two folds of opening area between compartments, but the short distance between the channels deteriorated the adhesion of the PDMS device to the substrate, resulting in a fluidic seal failure after several hours. Forty to sixty PDMS microfluidic co-culture platform devices have been routinely fabricated and tested simultaneously in one cell culture run that typically last for 4–5 weeks.
Figure 3 Scanning electron micrographs (SEMs) and an optical photograph of the PDMS co-culture device showing (a) bottom side of the axon-guiding channel arrays before bonding (Inset: Device filled with color dye for visualization and placed inside a conventional (more ...)
3.2. Fluidic isolation
The efficiency of fluidic isolation was tested on both the circular design and the square design by creating a minute fluidic level difference between the reservoirs that resulted in difference in hydrostatic pressure. Initially, color dye (red and blue) was mixed with PBS and loaded into each reservoir. Fluidic level differences of 130, 400, 650, and 1000 µm were achieved by creating volume differences of 5, 15, 25, 40 µl between reservoirs, respectively. Successful fluidic isolation between the compartments was achieved with as small as 400 µm fluidic level difference that was maintained for over 70 hours in both the circular and the square design culture platform (). Fluidic pressure from the soma compartment (red color, higher fluidic level) to the axon/glia compartment (blue color, lower fluidic level) through the 2.5 µm high microchannel array counteracted diffusion. For more accurate test, the axon/glia compartment and the soma compartment were filled with fluorescent dye (FITC) and PBS respectively, with the soma compartment having a slightly higher fluidic level. With a fluidic level difference of 400 µm between the two compartments, FITC was confined to the axon/glia compartment as shown in where a sharp boundary can be seen between the compartment and the axon-guiding microchannel arrays.
Figure 4 Fluidic isolation in the (a) circular design and (b) square design neuron culture platform. Dotted white lines in the center fluorescence images delineate compartment boundaries. Green fluorescent part indicates the axon/glia compartment while the black (more ...)
3.3. Cell loading
To facilitate high density of axons to cross into the axon/glia compartment through the axon-guiding microchannels, neurons loaded into the soma compartment of the microfluidic co-culture platform have to be positioned close to the entrance of the microchannels during the initial cell loading process. Neuron cells were loaded into the soma compartment to characterize and compare the distances between the loaded cells and the entrance of the axon-guiding microchannels in the two different microdevice designs. The areal cell density was same in both designs (500 cells/mm2). We first tested the square design neuron culture platform. In our initial experiments, 120 µl/reservoir of additional culture media was added to each reservoir immediately after loading the neurons to the soma compartment. This cell loading protocol, however, resulted in many of the cells inside the soma compartment washing out to the outlet reservoir due to the rather strong culture media flow from one reservoir to the other reservoir caused by the addition of culture media. To prevent such washing out of cells, a short incubation step was added before loading culture media since neurons loaded inside the compartment attach to the substrate after a while and their locations remain relatively stable. Incubation times of 10–60 minutes were tested to optimize the incubation step. Sufficient adhesion of the cells to the substrate was achieved after 30 minutes of incubation that prevented cell wash-out while maintaining good cell viability. Longer incubation times (more than 30 minutes) provided stronger adhesion of the cells but with a drop in cell viability. Neurons incubated for 60 minutes before adding culture media showed slight aggregation inside the soma compartment after 24 hours, and most of them were observed dead at DIV 4. Short incubation times (less than 30 minutes) resulted in many cells still being washed out to the other side of the reservoir while adding the culture media.
Although cell adhesion to the substrate is an important factor and the loading method has been optimized for the square design, the chances of axons entering the axon-guiding microchannels and growing into the axon/glia compartment are low unless neurons are positioned close to the inlet of the axon-guiding microchannels during the initial loading process. The location of the cells during the initial cell loading step is heavily influenced by the microfluidic design of the culture compartments and cell loading inlets/outlets. The circular design co-culture platform has one open access compartment in the center for neuron cells. This design allows neuron cells loaded into the center compartment to flow radially toward the axon-guiding channel inlets. This small but sustained flow keeps cells close to the entrance of the axon-guiding channels.
The “cell loading efficiency” in the two different microfluidic co-culture platform designs was analyzed by measuring the average distance of the closest cells from the inlets of the axon-guiding microchannels. Cells loaded into the square design co-culture platform moved smoothly into the soma compartment; however, the average distance of the cells from channel inlets, measured from 263 cells in multiple devices, was 26.0 ± 32.3 µm (means ± SD). In contrast, the circular design co-culture platform showed an average distance of 3.8 ± 11.2 µm, measured from 225 cells in multiple devices, which indicates that the loaded cells are located almost exactly at the inlet of the axon-guiding microchannels (). To see the statistical significance, at a significance level of 1%, we performed a two-sample pooled T-test showing P < 0.001. This shows that the cells are located closer to the inlets of the axon-guiding microchannels when using the circular shape design compared to the square design, increasing the probability of axons growing into the microchannels. Cells loaded into the open soma compartment were naturally positioned close to the channel inlets due to the radial flow pressure resulting from the circular fluidic design, and the fluidic pressure from the added culture media of 120 µl moved the cells even closer to the channel inlets. Although neurons were located at the channel inlets, channel openings were not blocked by the neurons, and axons could successfully pass through the channels into the axon/glia compartment.
Figure 5 Fluorescent images of neuron cells inside the soma compartment at DIV 1. (a) Cells inside the square design device; (b – c) cells inside the circular design device; (d) average distance of the closest cells from the axon-guiding channel inlets (more ...)
The cell loading efficiency was not analyzed for the OLs since they have to attach uniformly on top of the axonal network layer inside the axon/glia compartment rather than being concentrated to the axon-guiding channel outlet area. OL progenitors were loaded to the axon/glia compartment at DIV 14 for the co-culture experiments with a final areal cell density of 400 cells/mm2. A total culture media volume of 10 µl was added through the axon/glia compartment reservoir after aspirating out the excessive culture media inside the reservoir. It is important not to remove the culture media inside the axon/glia compartment during aspiration, since axons inside the axon/glia compartment can be aspirated out with the culture media, causing damages to the axonal network. OL progenitors were uniformly distributed over the axonal network inside the axon/glia compartment in both the circular and the square shaped designs.
3.4. Axon growth
Two different types of substrates, glass coverslips and 6-well polystyrene culture plates, were coated with either PDL or Matrigel™ prior to assembly with the PDMS microfluidic co-culture devices. Substrates coated with Matrigel™ resulted in all axon-guiding microchannels being blocked due to rehydration of the Matrigel™. Therefore, all following experiments were conducted on PDL coated substrates.
The microdevice was designed so that neuron cell bodies are isolated from axons by the height of the axon-guiding microchannels (2.5 µm) that block the cell bodies from moving into the axon/glia compartment. On the other hand, isolation of dendrites from axons is achieved by controlling the length of the axon-guiding channels because both axons and dendrites can grow through the axon-guiding microchannels but dendrites can grow only for a short distance. Microchannels with length ranging from 200 to 800 µm were tested to find the minimum length required for axon/dendrite isolation. Axons and dendrites successfully developed from neurons loaded inside the co-culture platform, and no toxicity or contamination issues were observed throughout four weeks of culture. Axons crossed and filled most of the axon-guiding microchannels by DIV 6 and started to spread out rapidly inside the axon/glia compartment. The microchannel arrays were not only efficient in guiding axons but also successful in physically isolating cell bodies and dendrites from axons. The 2.5 µm high shallow microchannels prevented cell bodies from moving into the axon/glia compartment, and the length of the microchannels kept dendrites from reaching the axon/glia compartment. clearly demonstrates the physical isolation of axons (stained for neurofilament (NF), red) from cell bodies and dendrites (stained for neuron marker, MAP2, green) via the axon-guiding microchannels. A shorter axon-guiding channel has the advantage to form an extensive axonal network inside the axon/glia compartment much earlier compared to longer axon-guiding channels. Axon-guiding channel as short as 200 µm was sufficient to isolate dendrites from growing into the axon/glia compartment. Having a dense axonal network forming inside the axon-glia compartment as early as possible has the advantage of maximizing the co-culture period with OLs, since the overall culture becomes unhealthy after about five weeks of culture inside the co-culture platform.
Figure 6 Immunocytochemistry images of neurons at two weeks in culture demonstrate that axons grew from the soma compartment into the axon/glia compartment through the arrays of axon-guiding microchannels but dendrites and neuronal soma could not reach into the (more ...)
To compare how the two microfluidic culture platform designs and the different substrates influence axon growth inside the co-culture platform, neurons were cultured in both the circular and the square design assembled on PDL coated polystyrene culture plates and PDL coated glass coverslips. The axon growth efficiency by different conditions was analyzed by the axon coverage ratio (ACR), defined as the percentage of area covered with axons inside the axon/glia compartment. To analyze the ACR by the different device designs, neuron cells were cultured at an areal density of 500 cells/mm2 on top of PDL coated polystyrene culture plates and were fixed at DIV 14. After two weeks of neuron cell culture, the average ACR of the circular design co-culture platform attached on polystyrene culture plate was 51.0 ± 11.8 % (means ± SD), which is statistically significantly higher than that of the square design on the same substrate showing 14.1 ± 4.6 % (, P < 0.0001). Therefore, we concluded that the novel circular co-culture design developed here enables the formation of a denser axonal network layer when compared with the square design.
Figure 7 Axon coverage ratio (ACR) of the axon/glia compartment by (a) different culture platform designs at DIV 14 and by (b) different substrates (polystyrene, glass) at DIV 26 analyzed from 79 images. Neurons cultured on the polystyrene culture plate using (more ...)
The ACR was also affected by the substrate type. Neurons with an areal cell density of 3100 cells/mm2 were cultured inside the circular design culture platforms assembled on PDL coated glass coverslips and PDL coated polystyrene culture plates, respectively. After four weeks of culture, including two weeks of co-culture period, neurons cultured on glass coverslips showed an average ACR of 71.8 ± 7.9%, while the neurons cultured on polystyrene culture plates showed an ACR of 79.9 ± 9.2 % (). Again, to see the statistical significance, at a significance level of 1%, two-sample pooled T-test was performed showing P value of 0.016 and therefore, concluded that neurons form denser axonal network when cultured on polystyrene substrates compared to glass substrates.
In addition, the adhesion of cells to substrates was also different depending on the substrate types. Axons grown on top of polystyrene culture plates were firmly attached to the substrate while many axons cultured on glass coverslips peeled off when detaching the PDMS devices for fixing and staining at the end of the culture periods.
3.5. Co-culture of CNS neurons and oligodendrocyte progenitors
The co-culture capability of the developed platform was tested by plating OL progenitors on top of the axonal network inside the axon/glia compartment that was already formed during the initial two-week culture period. OL progenitors with an areal cell density of 400 cells/mm2 were loaded uniformly on top of the axonal layer inside the axon/glia compartment without any disturbance to the existing axonal network layer. The 2.5 µm high axon-guiding microchannels physically prevented OLs from crossing the axon-guiding channels, and no OLs were observed inside the soma compartment upon OL loading. After loading, neurons and OLs were co-cultured for up to two more weeks and fixed at DIV 26. shows axons (stained for NF, red) and OLs (stained for MBP, green) labelled with fluorescent dyes inside the axon/glia compartment. Myelin basic protein (MBP), stained with green fluorescence, expresses only in mature OLs, thus, the expression of MBP shown in is a clear indication that OLs co-cultured on top of the axonal network successfully developed into mature OLs inside the PDMS microfluidic co-culture platform.
Figure 8 A phase contrast image and immunocytochemistry images of axons and OLs co-cultured inside the axon/glia compartment for two weeks. (a) Phase contrast image of the axon/glia compartment; (b–c) immunostaining of mature OLs grown on top of axonal (more ...)