I am looking at setting up a 3D skin in vitro model (via self-assembly method) without introducing keratinocytes. Is the air-liquid interface necessary in this situation?
Dear Macarena!
Please You look at the protocol:
https://www.jove.com/t/62125/generation-of-self-assembled-vascularized-human-skin-equivalents
Generation of Self-assembled Vascularized Human Skin Equivalents
4. Air-Liquid Interface Day 1 (ALI1) NOTE: ALI is performed the day after step 3.3.Lift each construct to air-liquid interface (ALI) by removing media waste from the upper chamber only. Use a manual pipette to get as close to the epidermal layer as possible without touching or damaging it. Tilt the plate slightly at different angles to collect the media. Remove as much media as possible in this step. Add approximately 2 mL of sterile water to the surrounding wells in the plate to maintain consistent humidity; keep the wells filled with water throughout culture. Check the plate a few h later to make sure the keratinocytes are still at the air-liquid interface. If there is media in the upper chamber remove it. Keep track of how much media is removed for each VHSE well, (The initial volume of upper and lower chambers (1500 µL) - media removed = a good starting point for ALI feeding). NOTE: VHSEs do not necessarily require the same media level for air lift; usually if the VHSEs are seeded together then they need about the same level of media for air lift, but this is not always the case. Adjust the volumes as needed to maintain ALI but ensure that the media levels are not so low that the VHSEs dry out. It is safer to be cautious and remove small media amounts daily until a balance between air lift and hydration has been met.