Culturing and Manipulation of O9-1 Neural Crest Cells

O9-1 is a multipotent mouse neural crest cell line. Here we describe detailed step-by-step protocols for culturing O9-1 cells, differentiating O9-1 cells into specific cell types, and genetically manipulating O9-1 cells by using siRNA-mediated knockdown or CRISPR-Cas9 genome editing.

The O9-1 cell line is a very useful tool for studying neural crest cells in vitro. It has a great potential for use in a wide range of application in various use of research. O9-1 cells have obvious advantages, such as easy access, and quickly obtaining sufficient cell numbers for experiments, making it a powerful method complimentary to in vitro studies of neural crest cells.

To begin, prepare basal media for O9-1 cell culture, according to the text protocol. Then, filter the basal media, and wrap it in foil to protect it from the light. After this, collect conditioned basal media from an activated STO cells according to the text protocol.

First, thaw an aliquot of basement membrane matrix on ice for two hours. Then, dilute the basement membrane matrix with filtered sterilized DMEM with 10%FBS to a final concentration of 0.5mg/ml. Coat the plate with the basement membrane matrix at room temperature for one hour.

Then, aspirate the basement membrane matrix while tilting the plate. Dry the plates at 30 degrees Celsius for 30 minutes. When adding the basement membrane matrix to the plate, make sure the solution covered the well evenly to maintain cell characteristics and avoid unnecessary variation in between experiments.

Recover O9-1 cells by placing a cryo-vial into a 37 degree Celsius water-bath. Slowly swirl the vial until the solution completely turns to liquid. After this, transfer the cell solution from the cryo-vial to a 15ml centrifuge tube.

Add five volumes of DMEM with 10%FBS to the tube to re-suspend the cells. Centrifuge the cells for three minutes at 180 Gs.Then, aspirate the supernatent, taking care not to disturb the pellet. Next, re-suspend the pellet in conditioned basal media supplemented with LIF and BFGF.

Seed the cells in a six well plate, and agitate the plate to seed the cells evenly. When agitating the plate, do so horizontally and vertically, as swirling the plate will cause the cells to concentrate towards the center. And allow those cells to attach and grow in a standard cell culture incubator overnight.

Ensure the cells are attached before changing the media. When the O9-1 cells reach 80%confluency, thaw the basement membrane matrix and prepare a basement membrane matrix coated plate as described previously. Then add basal media supplemented with LIF and BFGF to the membrane matrix.

Gently rinse the wells with 2ml of DPBS twice. Then, dissociate the cells with 1ml of 0.05%trypsin EDTA at 37 degrees Celsius for three minutes. Neutralize the trypsin with an equal amount of DMEM with 10%FBS by repeatedly pipetting the liquid over the whole surface of the well.

Then, transfer the cell solution to a centrifuge tube. And centrifuge for three minutes at 180 Gs.Aspirate the supernatent without disturbing the pellet of cells. Next, resuspend the cell pellet by gently pippetting conditioned basal media supplemented with LIF and BFGF.

Seed the cells to the coated plate as previously described. Then, allow the cells to attach and grow in a standard cell culture incubator. First, prepare the 2X freezing media according to the text protocol.

Then, filter-sterilize the media. Rinse the walls of the plate with DPBS twice, pipetting gently to avoid losing cells. Next dissociate the cells with 0.05%trypsin EDTA at 37 degrees Celsius for three minutes.

Neutralize the trypsin with an equal amount of 10%FBS and DMEM. Transfer the contents of the plate to a centrifuge tube, and centrifuge for three minutes at 180 Gs.Then, aspirate the supernatent and add 2ml of PBS to resuspend the pellet. Centrifuge the cell solution once more and aspirate the supernatent.

Adjust the amount of basal media in the tube as needed, and add an equal amount of 2X freezing media. Next, transfer the cells to the labeled cryo vials. Place the cryo vials inside a polystyrene box with a lid to achieve a slow cooling rate at 80 degrees Celsius for at least 24 hours.

To perform SIRNA knockdown in O9-1 cells, recover and seed the cells. Dilute liposomes in an appropriate volume of serum-free media. Then, dilute SIRNA in serum-free media according to the text protocol.

After this, add the diluted SIRNA to the diluted liposomes, according to the manufacturer's instructions. Mix the solution by pipetting, and incubate for five minutes at room temperature. Next, add an appropriate volume of SIRNA lipid complex to the cells.

Then, incubate the cells for 24 hours in a standard cell culture incubator. O9-1 cells can differentiate into different cell types under specific differentiation culture conditions. To differentiate O9-1 cells into osteoplasts, prepare osteogenic differentiation media according to the text protocol.

Finally, detect the osteoplast marker osteocalcin in differentiated osteoplasts by immuno staining. To differentiate O9-1 cells into smooth muscle cells, culture them under differentiation media according to the text protocol, and evaluate the differentiation by smooth-muscle actin immuno staining. In this protocol, both knock out and knock down experiment to study Yap loss of function in O9-1 cells were performed.

Under smooth muscle cell differentiation conditions, most wild type O9-1 cells gave rise to smooth muscle actin positive smooth muscle cells. Yap null cells however generally failed to differentiate into SMA positive smooth muscle cells. This indicates that Yap plays a critical role in the differentiation of neural crest cells into smooth muscle cells.

While attempting this procedure, it's important to remember to handle the O9-1 cell line gently and carefully during the entire process. Make sure to dilute and use the basal membrane matrix properly, and use 0.05%trypsin for cell dissociation. Don't forget that during the preparation for O9-1 cell culture, working with mitomycin c can be extremely hazardous, and precautions such as wearing a lab coat and gloves should always be taken while performing this procedure.