An Optimized O9-1/Hydrogel System for Studying Mechanical Signals in Neural Crest Cells

Detailed step-by-step protocols are described here for studying mechanical signals in vitro using multipotent O9-1 neural crest cells and polyacrylamide hydrogels of varying stiffness.

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The method aids in the study of the mechanical signaling of the neural crest cells and its interaction the with chemical signals. It can also aid in the molecular and genetic mechanisms of the neural crest development in diseases. Transfer the glass cover slips is the most challenging therefore transfer them slowly and attentively to prevent breaking and dropping them.

Demonstrating the procedure will be me and Dr.Sherry Zhao, a postdoctoral fellow from my laboratory. Begin by placing the desired number of glass cover slips onto a piece of laboratory wipe. Then sterilize each cover slip by passing it back and forth through the flame of the alcohol burner.

When the cover slips cool down, cover the 12 millimeter cover slip with approximately 200 microliters and 25 millimeter cover slip with 800 microliters of 0.1 molar sodium hydroxide. After five minutes, aspirate sodium hydroxide and allow the cover slips to air dry for five minutes to form an even film. Once the cover slips are dried, add approximately 18 microliters of aminopropyl triethoxysilane or APTS on a 12 millimeter cover slip and 150 microliters of APTS on a 25 millimeter cover slip.

Aspirate access APTS to allow the residual APTS to dry for five minutes. Later, rinse the cover slips thrice by submerging them in sterile deionized water for five minutes each time. Move the washed cover slips to a fresh Petri dish with the reactive side facing up.

And treat the cover slips by soaking in enough 0.5%glutaraldehyde for 30 minutes. After aspirating the glutaraldehyde, rinse the cover slips once with deionized water for three minutes and air dry the cover slips with the reactive side up. To prepare the siliconized cover slips, place the equal number of cover slips as that aminosilane coated cover slips in a Petri dish lined with para film.

Then, treat the 12 millimeter cover slips with 40 microliters and 25 millimeter cover slips with 150 microliters of dichloromethane silane or DCMS for five minutes. Wash the cover slips with sterile deionized water once for one minute before placing the reactive cover slips face up on a laboratory wipe. To prepare the hydrogels, pipette freshly prepared acrylamide gel solution onto the dried aminosilane coated cover slips.

Using curved tweezers, immediately transfer the DCMS treated cover slip on top of the gel solution with the treated side touching the gel solution thus sandwiching change the gel solution between two cover slips. Once the gel is polymerized, separate the DCMS treated cover slip with curved tweezers, leaving the gel attached to the aminosilane coated cover slip. Next, submerge the 12 millimeter cover slip with the attached hydrogel in a predetermined 4-well or 24-well plate covered with 500 microliters of sterile PBS or deionized water and the 25 millimeter cover slip in a 6-well plate covered with two milliliters of sterile PBS or deionized water for 30 minutes.

After removing excess acrylamide solution, store the hydrogels in sterile PBS or deionized water at room temperature for 30 minutes. Later, aspirate PBS or deionized water from the well plate before adding Sulfo-succinimydyl or Sulfo-SUNPAH solution to cover the gel entirely. And place the gels with the solution under a 15 watt, 365 nanometer ultraviolet light uncovered for 10 minutes.

Collect as much of the Sulfo-SANPAH per solution as possible by tilting the plate. And then wash the gel two to three times with 15 millimolar HEPES. Add 15 milligrams per milliliter Collagen 1 diluted in 0.2%acetic acid to each well containing the hydrogel.

And incubate the gels overnight at 37 degrees Celsius and 5%carbon dioxide. The next day, perform the wash as described in the manuscript before incubating the hydrogels with PBS containing 10%horse serum and 5%FBS at 37 Celsius and 5%carbon dioxide. After two hours of incubation, add 500 microliters of sterile filtered DMEM with 10%FBS and 1%penicillin streptomycin to each well and store the gels at 37 degrees Celsius and 5%carbon dioxide.

When the cell culture is ready, plate approximately 1.5 times 10 to the fourth 09-1 cells per centimeter squared in the bazell medium. Use tweezers to transport the cover slips to a new plate to minimize false signals from the cells grown directly onto the plate. Then, fix the cells using 500 microliters of 4%paraformaldehyde for 10 minutes before treating the cells with 500 microliters of 0.1%Triton X-100 at room temperature.

After 15 minutes, walk the cells with 250 microliters of 10%donkey serum. Stain the cells for F-actin with felodipine at a dilution of 1 to 400 in 250 microliters of 10%donkey serum, followed by incubation with DAPI for 10 minutes. Wash the cells with PBS for two minutes before adding three to four drops of mounting medium to each well.

On a fluorescence microscope, capture the images of at least three random frames per hydrogel sample producing individual and merged channels. In the stiffness assessment of the hydrogel through atomic force microscopy or AFM, the slope of the force curve was gentle for soft hydrogels as the required force from the AFM probe was less. However, on stiff hydrogels, the generated slope was much steeper.

The AFM measurements were then used to calculate the average stiffness of hydrogels from Young's elastic modulus in kilopascals. The growth characteristics of P19 and 09-1 cells were observed in one kilopascal and 40 kilopascals hydrogels of control and modified gel systems. The 09-1 cells grown on the original gel systems resulted in a higher number of dead cells.

In contrast the 09-1 cells grown on the modified gel systems exhibited healthy cell growth and sufficient attachment to the hydrogels substrate with little cell deck indicated by minimal round cells. The P19 cells plated on both hydrogel systems displayed an excess of round floating cells and a lack of cell substrate attachments. The compatibility of neural crest cells or NCCs with the modified hydrogel system was assessed by immunofluorescent staining for AP-2.

A significant increase in AP-2 expression in 09-1 cells plated on the modified hydrogel system was observed. Additionally, the 09-1 cells exhibited different morphologies based on the low or high stiffness hydrogels through varying amounts of the stress fibers. The anti-vinculin expression in 09-1 cells grown on the 40 kilopascals hydrogel was higher than those grown on the one kilopascal hydrogel reflecting that the cells grown on softer substrates form minimal focal addition complexes than the stiff substrate.

Appropriate waiting time is crucial for the polymerization of the hydrogels as acrylamides are toxic to the cells. Submerge the hydrogels in PBS or deionized water for at least 30 minutes to ensure better results.

Journal (Developmental Biology)

Journal (Developmental Biology)

An Optimized O9-1/Hydrogel System for Studying Mechanical Signals in Neural Crest Cells

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