In Vitro Investigation of the Effects of the Hyaluronan-Rich Extracellular Matrix on Neural Crest Cell Migration

This protocol outlines an in vitro migration experiment suitable for the functional analysis of the molecules involved in the in vivo migration of neural crest cells into the hyaluronan-rich extracellular matrix.

Hyaluronic acid is one of the most abundant extracellular matrix in animal model. The importance of hyaluronic acid in embryonic development has been demonstrated. This in vitro model is useful to explore how neural crest cells adhere and migrate within the hyaluronic acid-rich extracellular matrix.

With our technique, we can evaluate the migration and the HA degradation ability of both wholesale population and then individual cells over time. In addition, this technique can be used for drug screening to find compound that accelerate or prevent HA degradation. To begin, dilute the basement membrane matrix with chilled PBS at a 1:50 ratio and keep it on ice.

Coat a 10 centimeter culture plate with 10 milliliters of the diluted matrix solution and incubate the plate at room temperature for one hour. Wash the plate three times with two milliliters of PBS. To culture the O9-1 cells on the matrix coated platem warm the complete embryonic stem or ES cell medium in a water bath at 37 degrees Celsius.

Gently mix the O9-1 cell suspension and count the number of cells using an automated cell counter. Then adjust the cell concentration to 1.1 million cells per milliliter with the complete ES cell medium. Add eight milliliters of prewarmed complete ES cell medium to the matrix coated 10 centimeter culture plate and seed the O9-1 cells at 1.1 million cells per plate.

Incubate at 37 degrees Celsius in a 5%carbon dioxide humidified incubator. The next day, replace the medium with fresh complete ES cell medium prewarmed to 37 degrees Celsius. Replace with fresh medium every two to three days thereafter.

Wash the culture plate with two milliliters of PBS and then add two milliliters of prewarmed 0.25%Trypsin-EDTA. Incubate for five minutes at 37 degrees Celsius. Add two milliliters of prewarmed complete ES cell medium to the culture plate and transfer the dissociated cells to a 15 milliliter conical tube.

Centrifuge the tube at 300g for five minutes and discard the supernatant without disturbing the cell pellet. Add two milliliters of prewarmed complete ES cell medium to the tube and thoroughly resuspend the cells by pipetting. Then seed the cells on a new culture plate at the desired cell density.

Carefully add 50 microliters of undiluted triethoxysilane to a 3.5 centimeter glass bottom dish and incubate for five minutes at room temperature protected from light. Wash the dish three times with two milliliters of distilled water and add 50 microliters of 0.25%glutaraldehyde diluted 100 times in PBS per dish. Incubate at room temperature for 30 minutes then wash four times with two milliliters of PBS and coat the dishes with 300 microliters of collagen type one in 0.2 normal acetic acid at room temperature for one hour.

Again, wash three times with two milliliters of PBS. Add 300 microliters of 200 micrograms per milliliter fluoresceinamine, labeled sodium hyaluronate H2 to each dish and incubate overnight at room temperature. Wash again with two milliliters of PBS three times.

After drying the coated glass bottom dish, attach the two well culture inserts to the dishes and fill the inserts externally with one milliliter of PBS. Seed the O9-1 cells into the wells at 10, 000 cells in 100 microliters of DMEM containing 2%fetal bovine serum or FBS per insert. Culture the cells for two days at 37 degrees Celsius in a 5%carbon dioxide humidified incubator, Capture phase contrast images as the starting time point, using an all-in-one fluorescence microscope.

Remove the inserts carefully from the coated glass bottom dishes with tweezers and gently wash the coated glass bottom dishes with two milliliters of PBS to remove the cells and cell debris. Add two milliliters of fresh DMEM containing 2%FBS into the cultured dishes. Culture the cells for an additional 48 hours at 37 degrees Celsius and 5%carbon dioxide and capture phase contrast images after 24 hours and 48 hours in culture.

Wash the dishes with PBS and fix the cells after 48 hours with one milliliter of 4%paraformaldehyde for 15 minutes at room temperature or overnight at four degrees Celsius. Then wash the dishes three times for five minutes each with one milliliter of fresh PBS. Finally, place a cover slip with the mounting medium for further morphological observation.

Transverse sections of the neural tube of Tmem2 flag embryos at E9.0 are shown in this figure. The sections at the cranial and trunk levels of the neural tube were double labeled for the Tmem2 flag protein and hyaluronan. Tmem2 expression was observed in the neural plate and the border region of the neural tube whereas these sites were devoid of hyaluronan staining.

Double labeling of the neural crest cells for Tmem2 flag and Sox9 is shown here. Transverse sections of the E9.0 neural tube were stained for Tmem2 flag and Sox9. The representative images of Tmem2 depleted and control O9-1 cells cultured on a regular culture dish are shown in this figure.

The expression of Tmem2 in these cells was evaluated by qPCR with GAPDH as an internal control for normalization. Tmem2 depleted and control O9-1 cells were cultured for 48 hours on glass cover slips coated with fluoresceinated hyaluronan. Hyaluronan degrading activity is revealed as dark areas in the fluorescent background.

The level of hyaluronan degradation was also quantitatively compared between Tmem2 depleted and control O9-1 cells. The degradation of substrate-bound hyaluronan at the focal adhesions in O9-1 cells is shown in this figure. O9-1 cells cultured on mixed substrates consisting of Col1/HA were immuno labeled with an anti-vinculin antibody.

The dark spots or streaks represent hyaluronan degradation activity in the FAHA H2 substrate. The sites of hyaluronan degradation and focal adhesions were co-localized on the mixed substrates. Coval coating the glass bottom dishes with HA is a critical step in the protocol because inadequate coating can result in the HA easily removed by mechanical force during adhesion and migration.

To ensure proper coating, we use glutaraldehyde, chemically-immobilized type one collagen to the glass via arming coupling to the aldehyde. It is possible to use extracellular matrix substrate beside type one collagen, but they must have arming groups and there may be limitations in coval coating them on to glass bottom dishes due to their chemical properties. So trial and error may be necessary to determine the best approach.

This lab experiment can be helpful in studying how certain molecule work during the movement of neural crest cells into the HA or HA environment around the neural tube. It could also be a useful way to test drug that could either accelerate or prevent this migration process. It is interesting to know why Tmem2 has both the functions of adhesion over HA and the degradation of HA and why HA need to be degradated at the full cohesion site.

Since HA is the most common substance in the extracellular matrix, figuring out the answer to this question is really important in biology and medicine. This experimental protocol is valuable because it can be applied to various cell types, including skin fibroblasts, epithelial cells, and cancer cells.