This article details the methodology for assessing the migratory response of neural precursor cells (NPCs) from neurospheres. It outlines the preparation, culture conditions, and analysis techniques necessary for studying NPC migration.
Neurospheres are non-adherent spherical aggregates of neural precursor cells or NPCs.
When plated under specific adherent culture conditions, NPCs may spontaneously migrate out from the neurosphere.
To assess the migratory response of NPCs, first, prepare an NPC suspension of desired density in a suitable media.
Next, seed the cell suspension into a media-containing culture plate and incubate.
The specific growth factors in the media support the proliferation of cells. Additionally, the absence of a coating substrate keeps the cells suspended in media, facilitating them to form 3D cell aggregates called neurospheres.
On attaining the desired size, collect the neurospheres.
Centrifuge and resuspend the neurosphere pellet in the diluted media.
Then, plate the neurosphere suspension into an extracellular matrix-coated plate containing diluted media and incubate.
During culture, the neurospheres adhere to the extracellular matrix.
Over time, due to the constituents of the extracellular matrix, a few NPCs begin to migrate away from the neurosphere core.
Now, remove the media and fix the cells.
Capture phase-contrast images of the neurospheres exhibiting cell migration.
Using suitable software, measure the area of the total neurosphere and the inner cell mass.
Finally, subtract the inner cell mass area from the total neurosphere area to calculate the average NPC migration from the neurosphere.
To form the neurosphere, pipette 1 milliliter of 100% expansion media into a 35-millimeter dish without any coating substrate. Then, add one million neural precursor cells in each of these plates and incubate the plate at 37 degrees Celsius for 48 to 96 hours.
To prepare the plate for neurosphere migration, dissolve ECM-mimic gel aliquots in 6 milliliters of 30% expansion medium. Add 1 milliliter of ECM-mimic gel with 30% expansion medium in a single well of a 6-well plate. Then, incubate the plate at 37 degrees Celsius for 30 minutes.
To plate the neurospheres use a pipette to collect the cells in the medium from the 35-millimeter dish and transfer them to a conical tube. Then, spin the tube at 100 x g to pellet the neurospheres. Then, resuspend the pellet in 1 to 3 milliliters of prewarmed 30% expansion medium.
Next, pipette 200 microlitres of the resuspended neurospheres into the 6-well plate containing the ECM-mimic gel with 30% expansion medium and rock the plate. Then, incubate the plate at 37 degrees Celsius for 48 hours. Aspirate the ECM-mimic gel with 30% expansion medium and then fix the cells for 30 minutes using 4% paraformaldehyde.
To analyze the neurospheres, capture images using phase-contrast settings at 10X magnification. Use the NIH ImageJ software to measure the average migration of the neurospheres. On ImageJ, use the freehand line tool to trace the outer contour of the neurosphere.
Then, use measure function to calculate the trace area and trace the inner cell mass of the sphere. Subtract the inner cell mass from the total neurosphere area to quantify the average migration.
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