NPC Neurosphere Assay: An In Vitro Assay to Assess the Migratory Response of Neural Precursor Cells from the Neurosphere

1. NPC Neurosphere Migration Assay

1. Neurosphere Formation
   1. Add 1 mL of 100% Expansion Media into a 35 mm dish with no coating substrate. Incubate dishes for at least 15 min at 37 °C before plating NPCs. Prepare 2–3 dishes to ensure that there will be enough neurospheres for the cell migration assay.
      NOTE: The absence of a coating substrate ensures that NPCs remain suspended in the media, which is essential for neurosphere formation. Coating dishes will prevent neurosphere formation.
   2. Lift, dissociate, and pellet cells. Resuspend cell pellet in 2–5 mL of pre-warmed 100% Expansion Media. Plate 1 million NPCs into each 35 mm dish prepared in section 1.1.1.
   3. Incubate NPCs at 37 °C for 48 to 96 h to allow NPCs to aggregate and form neurospheres. Assess sphere size using a live ruler on a phase-contrast microscope. Wait for most spheres to reach an approximate diameter of 100 µm (± 20 μm) (Figure 1). Smaller spheres will completely disperse and break apart during the migration assay.
2. Preparation of Plates for Neurosphere Migration Assay
   1. Dissolve ECM-mimic gel aliquots into 6 mL of 30% Expansion Media. Once ECM-mimic gel/30% Expansion Media solution is prepared, add vehicles, growth factors, or drugs of interest at desired concentrations.
      NOTE: Vehicle, drug, and growth factor concentrations need to be increased to account for the addition of 200 µL of neurospheres in section 1.3.
   2. Plate 1 mL of the ECM-mimic gel /30% Expansion Media solution (± vehicles, growth factors, or drugs) into one well of a 6-well plate. Make 2–3 wells per experimental condition. Alternatively, 35 mm dishes may be used. Incubate plates for at least 30 min at 37 °C.^.
      NOTE: DO NOT aspirate the ECM-mimic gel/30% Expansion Media solution for this assay. Plating spheres onto an aspirated ECM-Mimic gel will lead to rapid and excess migration.
3. Plating Neurospheres
   1. Collect neurospheres formed in section 1.1 and place them into a conical tube. Wash the 35 mm dishes with 1 mL of 1x PBS to ensure all neurospheres are collected. Spin down the collected neurospheres at 100 x g for 5 min.
   2. Resuspend the neurospheres in 1–3 mL of pre-warmed 30% Expansion Media. If 1 dish of neurospheres is collected, 1 mL of media is used to resuspend spheres. If 2 dishes are collected, 2 mL of media are added to resuspend spheres, etc. Pipette gently and use only a P-1000 to ensure spheres are not broken.
   3. Plate 200 μL of the resuspended neurospheres into the ECM-mimic gel/30% Expansion solution made in section 1.2. Rock plates in all directions to evenly distribute neurospheres. Incubate the plates for 48 h at 37°C.
   4. Remove ECM-mimic gel/30% Expansion Media solution and fix cells in 4% PFA, wash, and keep cells in 1x PBS + 0.05% Sodium Azide.
4. Analysis of Neurospheres
   1. Acquire images of entire neurospheres using phase-contrast settings at 10X. Ensure spheres are not touching each other. Measure average migration using the ImageJ software.
   2. Trace the outer contour of the neurosphere using the freehand line tool. The freehand line can be accessed by right-clicking on the "straight" line icon. Manually trace using a mouse.
   3. Use the measure function to calculate the area of the trace. Ensure that "area" is selected as a read-out in the "Set Measurements" window found under the Analyze tab. See Figure 2 for trace of outer contour in blue.
   4. Trace the inner cell mass of the sphere and measure area. See Figure 2 for trace of inner contour in red. Quantify average migration by subtracting the inner cell mass from the total neurosphere area.
   5. Measure neurospheres that exhibit a densely packed inner cell mass with cells migrating out as a contiguous carpet (Figure 2).
   6. Do not include cells outside of the carpet or detached from the neurosphere for measurement. See Figure 2 for examples of cells (circled in white) that are excluded from the outer carpet measurement. Analyze a minimum of 20 neurospheres for each condition.