Informe de resultados

13mm2 _{glass coverslips and microfluidic chambers were prepared for culture as described}

in Chapter 3. Glass coverslips were coated with poly-L-lysine (PLL) for glial cells, collagen for primary skeletal muscle cells, or poly-L-lysine/laminin (PLL-L) for C2C12 myoblasts. Microfluidic chambers were filled with PLL-L on the proximal side and laminin/collagen to the distal side (Figure 5.1). Flasks were prepared with either PLL or collagen. All substrates were incubated overnight at room temperature, removed and devices filled with cell-specific medium. All cell culture substrates were allowed to equilibrate for 2 hours prior to addition of cells.

5.2.1.1 Primary glial cells

Spinal glial cells were obtained from the spinal cords of two postnatal day 2 (P2) Sprague-Dawley rats or four P2 C57Bl/6 mice. Neonates were decapitated using sharp scissors. The skin was sterilised using 70% ethanol and pups placed in a clean petri dish with the back side facing up. The skin was opened using sterile large spring-loaded scissors and the spinal cord exposed. The spinal cavity was opened with a single horizontal cut at the base of the tail and the cavity opened using forceps to one side of the spinal cord. Spinal cords were collected in 100µl drop of HBSS in a sterile petri-dish and stripped of meninges. Tissue was trypsinised (0.0125% trypsin) for 5 minutes. Trypsin was removed and tissue triturated in 2ml glial cell medium (Table 8.1) and filtered through 80nm mesh. Cells were pelleted (300 x g, 5 minutes) and plated into 2x prepared PLL coated P25 flasks (Iwaki). Cultures were incubated under standard cell culture

95 conditions for 24 hours followed by a complete medium change with warmed glial cell

medium. Mixed glial cultures were maintained for 2-3 weeks until confluent with weekly ½ medium changes.

Glial cells were passaged by washing the cells once in warmed trypsin/EDTA (refer to Chapter 8.2), followed by incubation in trypsin/EDTA for 5 minutes. Cells were dislodged from flasks by firmly hitting with the heel of the hand. Cells were pelleted (300 x g, 5 minutes) and plated either onto 24x PLL coated glass coverslips (rat) or uncoated coverslips (mouse), or into the proximal chamber of 24x microfluidic devices.

5.2.1.2 Primary skeletal myocytes

Primary myocyte cultures were prepared from the hind limbs of two P2 Sprague-Dawley rats. Neonates were decapitated using sharp scissors. Skin was sterilised in 70% ethanol and hind limbs removed. The feet were severed using scissors and tweezers used to remove skin. Muscle tissue was dissected from hind limb bones and finely minced using spring-loaded scissors, and transferred to 2mL HBSS. Tissue was incubated in 500µl collagenase/dispase (refer to Chapter 8.2) for 40 minutes and mechanically triturated. After trituration, 2ml primary myoblast growth medium (Table 8.1) was added and cells passed through fine gauze. Cells were pelleted (350 x g, 10 minutes), resuspended in 6ml myoblast growth medium and plated into two prepared collagen-coated P25 flasks and grown under standard cell culture conditions. Culture medium was replaced in full after 24 hours and myocytes allowed to grow for a further 4-5 days, or until 70% confluent.

Semi-confluent myoblasts were passaged onto either collagen-coated glass coverslips (density of 50,000 cells per coverslip) or into the distal compartment of microfluidic

96 chambers containing a substrate of laminin/collagen. Primary myoblast growth medium

was removed and cells rinsed 1x (10 seconds) with 1ml warmed trypsin-EDTA. Cells were incubated for 2 minutes with a second 1ml of trypsin-EDTA and dislodged from the flask by hitting the flask firmly with the heel of the hand. Cells were pelleted (350 x g, 10 minutes) and added to coverslips or microfluidic chambers. Passaged myoblasts were allowed to grow for 1 day in primary myoblast medium following passaging, after which they were transferred to neuron initial growth medium (Table 8.1) to promote differentiation.

5.2.1.3 C2C12 myoblasts

C2C12 mouse myoblast cells (American Type Culture Collection (ATCC), Virginia, USA) were maintained at a low passage number (<10) in C2C12 growth medium as recommended by the manufacturers suggestions (Table 8.1). C2C12 myoblasts were transferred onto PLL-L coated coverslips or the distal compartment of microfluidic chambers using the trypsin-EDTA method described above. C2C12 myocytes were differentiated by serum-shock in C2C12 differentiation medium 1 (CDM1) (Table 8.1) for 2 days, and transferred to C2C12 differentiation medium 2 (CDM2) (Table 8.1) for three days thereafter. Cultures were maintained in serum-free neuronal subsequent medium (Table 8.1) for the duration of cultures.

5.2.1.4 Primary rodent motor neurons

Primary spinal motor neurons were obtained from the spinal cords to E14.5 Sprague- Dawley rats or E13.5 C57Bl/6 mice. Time-mated females were killed by CO2, embryos

97 removed and decapitated. Embryo bodies were maintained on ice for the duration of the

dissection to minimise tissue degradation. Spinal cords were removed by placing the bodies in a sterile petri dish with the spinal cord facing up. Using microscopic guidance, tweezers were used to score along both sides of the spinal cord to remove skin. The spinal cord was then removed and transferred to a drop of ice-cold HBSS. Meninges and dorsal root ganglia were removed using fine forceps and cleaned spinal cords transferred to 5ml HBSS. Tissue was trypsinised (0.00625%) for 5 minutes. Trypsin was removed and cells triturated in 2mL HBSS. The cell suspension was pelleted in 3.5% bovine serum albumin (BSA) and enriched for motor neurons in 1.06g/L Optiprep™ gradient for 25 minutes at 400 x g with the brake off. Motor neurons were pelleted at 700 x g and resuspended in 250µl. Cell density was estimated using the trypan blue dye exclusion assay and the volume adjusted to achieve 8x106 cells per ml. 10µl motor neurons (8.5x104 motor neurons) were plated into the proximal compartment of prepared microfluidic chambers (Chapter 2.2.1) or onto prepared 13mm coverslips with glial cells, myocytes or PLL. Plated cells were returned to incubator conditions. Medium was replaced with neuron subsequent medium (Table 8.1) at 2DIV (rat) or 7DIV (mouse) and maintained with weekly ½ medium changes.