Cranial Neural Crest Cells Three-Dimensional In Vitro Differentiation Protocol for Multiplexed Assay

We're interested in understanding how certain decision is regulated during development. Organoid models and single-cellomics technologies are allowing us to ask question that were not possible before. The usage of in vitro organoid models allows us to generate greater amount of data, especially for transient cell populations, such as the neural crest.

The significant variability model outputs remains a major experimental challenge. We have demonstrated that cranial crest cells re-express prepotency genes to expand the differentiation potential. To begin, prepare a fresh collagenase solution at a concentration of two milligrams per milliliter in the DMEM knockout medium.

Aspirate the ESC medium from the well plate containing 70 to 80%confluent mESC. Now, gently add one milliliter of PBS to the side of the well. Rock the plate to ensure even washing.

Pipette out the PBS and replace it with two milliliters of collagenase solution. Incubate at 37 degrees Celsius for 30 to 45 minutes. Check the plate under a light microscope at 10X magnification after 20 minutes of incubation, and then every five minutes.

When the colonies show rolled-up edges, tap vigorously on the plate side to detach the colonies. Using a five milliliter serological pipette, collect the detached colonies and transfer them to a 15 milliliter conical tube. Centrifuge the colonies at 16 G for three minutes at room temperature.

Aspirate as much medium as possible from the conical tube without disturbing the colonies at the bottom. Next, add one milliliter of 0.05%trypsin solution to the pellet and incubate. With a P1000 and P200 micropipettes, pipette the suspension up and down vigorously to dissociate the colonies, then add two milliliters of mESC culture medium to block the trypsin.

Centrifuge the tube at 160 G for three minutes at room temperature. Pipette out the supernatant, then add one milliliter of CNCC differentiation medium. Count the cells with an automated cell counting device or under a microscope, then dilute cells in CNCC differentiation medium to achieve a concentration of 3, 000 live cells per 50 microliters.

With a P200 micropipette, seed 50 microliters of the cell suspension into each well of a non-TC treated U-bottomed 96-well plate. Top up each well to 200 microliters with CNCC differentiation medium and incubate. Observe the plate containing 24 hour culture of differentiated cells under a light microscope to ensure small clusters with clear borders are visible at the bottom of each well.

Put the plate back into the incubator overnight. On day two, slowly remove 100 microliters of medium from each well. Then, use a P200 micropipette with its tip cut three to four millimeters from the end to aspirate the neurospheres along with the remaining medium.

Transfer the neurospheres and remaining medium into a non-TC treated flat-bottomed 96-well plate. Verify the transfer under a light microscope. Top each well to 200 microliters of prewarmed CNCC differentiation medium.

On day four, aspirate 100 microliters of medium from each well. Replace it with 100 microliters of prewarmed CNCC differentiation medium, and then incubate again. On days five and six, check the neurospheres'attachment under a light microscope.

Ensure that lighter cells delaminating from the neurospheres start surrounding its main body. Prepare CNCC maintenance medium as per the composition given. Filter bovine serum albumin through a 0.22 micrometer filter before adding it to the medium.

Once growth factors are added, store the medium at four degrees Celsius for up to three weeks, ensuring it is protected from light. Add 100 microliters of fibronectin solution into each well of a non-TC treated 96-well plate. While the plate is resting, aspirate as much CNCC differentiation medium as possible from the wells of the non-TC treated flat-bottomed 96-well plate.

Replace the medium with 50 microliters of Accutase. Incubate at 37 degrees Celsius for five minutes. Next, add 100 microliters of CNCC maintenance medium into each well to quench the Accutase.

Remove fibronectin from the coated wells of the receiving plate. then filter the detached post-migratory CNCC through a 40 micrometer filter into the wells of the receiving plate. At the desired time point, use a cut tip P200 micropipette to transfer neurospheres from the 96-well plate into a DNA low binding two milliliter tube.

After letting neurospheres settle in the tube for three minutes at room temperature, pipette out as much medium as possible, then rinse with one milliliter of cold PBS. To prepare mounting chambers, place three layers of double-sided transparent fiberless tape on a microscope slide. With a razor, cut a three millimeter by eight millimeter window in the double-sided tape.

Under a stereoscope, with a P200 cut tip, carefully pipette the neurospheres in the clearing agent into the mounting chamber. Use magnifications between 2X and 4X to provide a field of view of the whole chamber and identify the neurospheres. Place a cover slip on the chamber surface and lightly press its sides to adhere it.

Neurosphere cultures in non-tissue plates displayed uniform attachments starting on day five, whereas Petri dish cultures showed variable attachment and fusion of neurospheres, particularly evident by day four. Neurospheres'size variability was significantly reduced in 96-well plate cultures compared to Petri dish cultures on days four and seven. The diameter of neurospheres in 96-well plates ranged from 139 micrometers to 295 micrometers on day four and 383 micrometers to 552 micrometers on day seven, whereas Petri dish cultures showed a wider range.

Neurosphere growth was exponential in terms of cell numbers, increasing from an average of 1, 082 cells on day two to 48, 352 cells on day nine. Diameter growth was linear, increasing from an average of 136 micrometers on day two to 570 micrometers on day nine. Immunofluorescence analysis confirmed that neurospheres and delaminating cells express CNCC markers AP2 alpha and SOX9 and EMT marker TWIST1 on days nine and 13.

PAX7 was only present in the neurospheres.