Snd450

Neurons were cultured in microfluidic chambers (SND450, Xona Microfluidics) as described previously (Prots et al., 2018 (link)). In brief, 80,000 MN progenitors and 10,000 astrocytes (primary human cerebellar astrocytes, HA-c, ScienCell Research Laboratories) were seeded on the soma side, and 20,000 astrocytes on the axonal side. Cells located in the soma side were lentivirally infected 2 days post-seeding at a multiplicity of infection of 1, with an incubation time of 48 h, followed by a complete medium change. Mito-DsRed-positive mitochondria were recorded on day 30 after differentiation when the axons had projected through the 450 μm-long microgrooves reaching the axonal side. Microfluidic chambers were used to analyze morphology of neuritic mitochondria (Figure 1), colocalization of Lysotracker with mitochondria (Figure 2), axonal density of mitochondria (Figure 3), and mitochondrial axonal transport (Figure 3).

Primary cortical neurons were extracted using a previously described procedure [22 (link)]. Embryonic mice were taken on day E18. The cerebral cortex was dissected, and the meninges were stripped and digested with trypsin. The neurons were then inoculated in Petri dishes coated with polylysine. After 4 h, serum-free 96% nerve medium containing B27, glutamine (0.5 mM, Thermo Fisher Scientific), penicillin (100 IU/mL), and streptomycin (100 mg/mL, Thermo Fisher Scientific) was replaced. To separate axons from neurons, a microfluidic device (SND450, Xona Microfluidics) was used [17 (link)]. The microfluidic device was placed on the Petri dish coated with polylysine in advance, and neurons were inoculated at the density of 6 × 10⁵ neurons per well. The other steps were the same as mentioned earlier. The medium was changed every alternate day. The concentrations of IL-1α and TNF-β used to culture neurons in an inflammatory environment were 50 ng/mL and 200 ng/mL, respectively. Lentivirus infection was used for the process of overexpression or knockdown in neurons. The amount of virus was calculated according to the optimal multiplicity of infection (MOI), and the serum-free medium mixed with virus and polybrene was incubated with cells for 6 h. The medium was then changed to normal medium, and the follow-up experiment was performed 48 h later.

HPSC-dNeurons from SPG11 patients and controls were grown in microfluidic chambers (SND450, Xona Microfluidics, Temecula; Fig. 3C) as described before (11 (link)). The grooves in the microfluidic chambers enabled the axons to grow parallel and in unidirectional (Fig. 3C). A total of 60 000 NPCs were plated on the soma side and cells were cultured for 15 days in NDM. Axonal-like processes passing through the grooves were visualized using a Zeiss inverted fluorescent microscope (Zeiss). At least 20 cells per NPC line were imaged for analysis. Neurites of individual cells, starting from the end of the groove, were traced using NeuronJ (ImageJ; sbweb.nih.gov/ij/) to calculate neurite length and number of branching points.

Xona microfluidic devices (Xona Microfluidics, Cat#SND450) were sterilised and plasma bonded to glass coverslips (24 × 40 mm; Menzel Glaser) using a plasma cleaner (PDC-32G-2, Harrick Plasma). After bonding, the devices were coated with 15 µg/mL poly-L-ornithine (Sigma) and 10 µg/mL laminin (Sigma). HDF51i-509 NPCs were seeded in both panels of a microfluidic device at a density of 8 × 10⁴ cells per panel in BrainPhys™ complete neural differentiation media to initiate differentiation. The media in the wells were replenished every 2–3 days and the differentiation was continued for up to 21 days.
Microfluidic chambers were infected as described previously (Sundaramoorthy et al. 2020a ) with sarbecoviruses or RABV at a MOI 1 at day 20–21 of differentiation. Briefly, media from the panel to be infected was removed and the appropriate volume of viral inoculum in BrainPhys™ neural medium required to infect at MOI 1 was added. A unidirectional flow of media was strictly maintained by a higher volume of media in the non-infected panel (200 µL) and a lower volume in the infected panel (100 µL). Sarbecovirus- or RABV-infected microfluidic chambers were incubated at 37 °C and 5% CO₂ for 24 h.

Compartmentalized axon chambers: For separating the neuronal cell bodies and axons, cortical and DRG neurons were cultured in xona microfluidic devices [28 (link)] (XONA Microfluidics, Cat#SND450) mounted on glass coverslips (24 x 40 mm; Menzel Glaser) coated with poly-L-ornithine (Sigma-Aldrich). Approximately 10 μL of cell suspension containing 120,000 cells were added to the cell body panel. The chambers were then incubated at 37°C for 10 min to allow attachment of neurons. Then 200 μL of neuronal culture media was added to the top and bottom wells of cell body panel and 150 μL of media was added to the wells in the axon panel. Approximately half the volume of media in each well was replaced with fresh media every two days and the higher volume of media on the cell body panel was maintained.
Transynaptic microfluidic model: To model synaptically connected ex-vivo neuronal cultures, cortical neurons were extracted as above and seeded on to both the panels of microfluidic device (SND450, XONA microfluidics), each with 10 μL of cell suspension containing 120,000 cells.