The research objective of my research team is to understand tissue development and the regeneration. This goal is particularly important in the stem cell research today. We hope to understand the principles, how a organ form, and this knowledge is not only help us to understand the nature, but also can be applied to help regenerative medicine.
The challenge we face is to understand the molecular and cellular process involved in this morphogenetic processes. So therefore, we need to have a explant culture system to analyze molecular and cellular processes carefully because it's not possible to do this in vivo. To fill in this knowledge gap, scientists have developed different ways of organoid cultures.
In the organoid culture, we will be able to tape collective cell movement to see how cells interact with each other. We are also able to manipulate the cells so they will express different molecules to test the function of the molecules. The unique approach we use is the chicken skin explant culture.
Chicken skin is used because they are easily accessible from the egg and they form distinct feather, but arranged in hexagonal pattern as a readout for tissue patterns. There are three ways we do the culture as you will see. One is how the explant culture allow us to take the movies or deliver molecules to perturb the system.
Second, to study the contribution of epicilia or mesenchyme, we can take them apart and put them back. Third, to further challenge the ability of each cells, we dissociate them into the single cells and test how they come back to reorganize into the tissue patterns. Through these approaches, we have been able to gain new understanding toward the tissue patterning.
To start, incubate fertilized chicken eggs in a humidified incubator at 38 degrees Celsius. Then place a stage 28 to 32 chicken embryo in a 60 millimeter Petri dish filled with HBSS. After dissecting out the dorsal embryo skin and head, gently pull the limb buds to reposition the ventral side of the body downwards.
Now along the two sides of the embryo body, make an anterior to posterior incision. Grip the limb buds longitudinally with a pair of forceps to stabilize the body. Use a watchmaker's forceps to carefully peel the skin from the neck down to the tail region.
Use sharp scissors to delicately remove any remaining subcutaneous tissues still attached to the peeled skin and smooth the skin edges. Next, add two milliliters of supplemented DMEM to each well of a six-well dish. After adding antibiotics to the wells, place a sterile tissue culture insert inside the well, ensuring the medium surrounds the exterior of the culture insert.
Then move the skin onto a spatula while immersed in HBSS to prevent creasing and folding. Once done, transfer the excised skin to a dish containing HBSS. Slowly slide the skin off the spatula onto the culture insert without folding.
Using a 200-microliter pipette, remove any excess HBSS from the insert. Finally, incubate the skin explant cultures at 37 degrees Celsius with a blend of 5%carbon dioxide and 95%air. Replace the medium every two days.
The feather primordia developed into short feather buds after two days in culture and long feather buds developed after four days in culture. The dermal plaque codes were more mature in the midline than at the sides. To begin, incubate fertilized chicken eggs in a humidified incubator.
Next, prepare two times CMF saline with 0.25%EDTA buffer. Then peel the skin off the chicken embryos in HBSS solution and incubate them in two times CMF-EDTA solution on ice for 15 to 20 minutes. Use a pair of watchmakers forceps to carefully separate the epithelium and mesenchyme.
Then transfer the isolated epithelium and mesenchyme to a single clean dish containing HBSS. For recombination, place the mesenchyme on a Petri dish containing HBSS. Then place the epithelium on it, aligning it along the mesenchyme's anterior-posterior axis.
Alternatively, rotate the epithelium by 90 degrees from the mesenchymal anterior-posterior axis. Next, transfer the recombined skins to the culture inserts in six-well culture dishes. Remove any excess HBSS surrounding the recombined skin.
After adding DMEM containing 10%FBS to the exterior well of the insert, pipette a thin layer of DMEM into the insert chamber, ensuring the explant remains semi-hydrated. Finally, place the skin recombinants in an incubator at 37 degrees in an environment of 5%carbon dioxide and 95%air. Monitor the phenotypic changes in the initial skin development phases using time-lapse photography with a mounted dissecting microscope.
New plaque codes appeared shortly after recombination and developed into short feather buds two days post recombination. Long feather buds developed in four days. When the epithelium was rotated by 90 degrees, the orientation of the new buds was determined by the epithelium.
To start, incubate fertilized chicken eggs in a humidified incubator at 38 degrees Celsius. Now dissect stage 30 to 33 chicken embryo dorsal skins in HBSS, then incubate the skins in two times calcium and magnesium-free saline with 0.25%EDTA for 15 to 20 minutes. Carefully separate the epithelium and the mesenchyme using watchmaker's forceps and move the separated material to HBSS placed on ice.
Collect the separated mesenchyme into a 15-milliliter centrifuge tube and add two milliliters of 0.1%collagenase trypsin made in PBS. Then incubate the solution in a water bath. Next, use a Pasteur pipet to disperse the skin mesenchyme into a single cell suspension.
Then add eight milliliters of DMEM containing 10%fetal bovine serum to halt digestion. Centrifuge the cells at 233G for five minutes, then resuspend the pellet in culture medium. Next, place the cell culture insert in a well of a six-well dish.
Drop a 10-microliter droplet of the mesenchymal cells onto the insert. In the well outside of the insert, pipette two milliliters of DMEM with 10%fetal bovine serum. Incubate the plated cells at 37 degrees in an incubator set with 5%carbon dioxide and 95%air for one hour.
Using a one milliliter pipette, transfer the intact epithelium on top of the plated mesenchymal droplet. Flatten the intact epithelium over the mesenchyme to form the reconstituted skin explant. Leave a thin layer of medium on the insert to keep the skin explant semi-wet, providing an air liquid interface.
Incubate the reconstituted skin explant at 37 degrees in an incubator with 5%carbon dioxide and 95%air environment. The ex vivo organ cultures appear homogeneous at first. Short feather buds formed after two to three days in culture and long feather buds formed after four to five days in culture.
