Isolating Cranial Neural Folds From a Chick Embryo for a Neural Crest Cell Culture

Take a chick embryo supported within a filter paper frame and position it under a dissecting microscope.

Remove the vitelline membrane, a protective coating surrounding the embryo.

Identify the midbrain neural folds located posterior to the optic lobes and anterior to the cardiac crescent, the embryonic precursor of the mature heart.

The neural folds contain premigratory neural crest cells, which are multipotent stem cells that transform into migratory cells during development.

These migratory cells differentiate into neuronal and non-neuronal cells.

Excise the dorsal-most aspect of the neural folds and transfer the tissue to a culture dish containing buffer.

Next, place coverslips coated with an adhesion protein in culture plate wells containing media.

Transfer the neural folds onto the coated coverslips. Incubate to facilitate tissue attachment.

During culturing, migratory neural crest cells emerge from the neural folds onto the coated surface, establishing a neural crest cell culture.

Transfer an embryo to a clean dish containing Ringer's Pen-Strep solution. Gently swish the embryo back and forth to clear away any yolk that obscures the view, and exchange the Ringer's Pen-Strep solution or transfer it to a fresh dish if it becomes cloudy. Position the embryo dorsal and frame-side up under a dissecting microscope. Leave the embryo on the paper frame to keep it stretched and held in place.

Then, remove the vitelline membrane using forceps to expose the neural folds. Include tissue caudal to the expanding optic vesicles and rostral to the hindbrain, where rhombomere constrictions are just beginning to appear. Using spring scissors, carefully excise the midbrain neural folds. Take care to excise the dorsalmost aspect of the neural fold with minimal contamination of the neural tube and non-neural ectoderm, and transfer the neural folds to a clean dish containing Ringer's Pen-Strep solution using a P20 pipettor or a sterile glass Pasteur pipette rinsed with yolky Ringer's Pen-Strep solution.

Store collected folds on ice while dissecting additional folds. After removing the culture dish from the incubator, use a pipettor or Pasteur pipette to remove the fibronectin solution from coverslips dish or well. After rinsing the fibronectin-coated substrate with Ringer's Pen-Strep solution, add an appropriate volume of complete culture media to the dish or well.

To block the plastic and prevent the tissue from sticking, use a P20 or P200 pipettor, and rinse the pipette tip with yolky Ringer's Pen-Strep solution. Then, transfer the isolated neural folds towards the center of the fibronectin-coated coverslip, taking care to transfer as little Ringer's Pen-Strep solution as possible. After allowing the explants to settle for 10 to 15 minutes, place them into a humidified chamber at 38 degrees Celsius by slowly and carefully carrying the culture dish with plated neural folds.