To begin, remove the FN-silk vials from the minus 80 degrees Celsius freezer, and place them in dry ice for transportation. Bring the vials to a biological safety cabinet, and place them in a micro centrifuge tube rack. Upon thawing, pipette 550 microliters of silk solution into every second well of a 48-well plate.
Place the lid on the plate, and place it inside a sterile box. Carefully transfer the box from the biological safety cabinet, and leave it in ambient conditions overnight. The next day, bring the box containing the plate with the FN-silk membranes and sterile inserts into the biological safety cabinet.
Carefully lift the plate from the box, open the lid of the plate, and visually verify membrane formation by observing cloudiness in the wells. Using sterile tweezers, pick up one insert and guide it down onto the membrane until the handles touch the top of the well. Once all inserts have been lowered, place the lid on the plate, and return the plate to the sterile box.
Allow the membranes to adhere to the inserts for two hours. Next, remove the plate from the box and open the lid. Using sterile tweezers, remove the insert from the well.
Fill the insert with 100 microliters of DMEM/F12 for basal seeding, or 200 microliters for apical seeding. Transfer the insert into an empty well of a 24-well plate, and fill the well with one milliliter of DMEM/F12. After harvesting the desired number of human keratinocytes, using a P200 pipette, aspirate 20 to 50 microliters of cell suspension.
Aim the pipette tip towards the center of the membrane and slowly press the pipette plunger, bringing the droplet into contact with the culture medium inside the insert. Once all membranes are transferred, move the plate in a figure eight pattern to distribute the cells evenly across the membranes. Incubate the culture at 37 degrees Celsius and 5%carbon dioxide.
Using tweezers, lift the inserts out of the 24-well plate, leaving the culture medium in the wells. Then invert the insert so that the basal side faces upwards. Place the inverted insert in the Petri dish.
Aspirate 20 microliters of the resuspended human keratinocyte cell suspension with a P200 pipette. Aim the pipette tip towards the center of the membrane, and slowly press the pipette plunger to form a droplet that falls onto the membrane. Place the lid on the Petri dish.
Transfer the Petri dish and medium-containing 24-well plate to the incubator for 30 minutes. After incubation, bring the 24-well plate and Petri dish into the biological safety cabinet. Using tweezers, pick one insert and invert it so that the apical side of the membrane faces upwards and the basal side downwards.
Using a P200 pipette, add 200 microliters of culture medium inside the insert. Place the insert back into a prefilled well of the 24-well plate. Culture the inserts at 37 degrees Celsius and 5%carbon dioxide.
Keratinocytes cultured on the FN-silk membrane evenly covered the surface and showed cobblestone morphology on day one. Keratinocytes formed a confluent layer by day three, indicating epithelial function, and formed a network of tight junctions indicating they were assuming physiologic epithelial functions. High cell viability was observed across the FN-silk membranes after three days, with no significant difference in viability between the center and periphery.
The FN-silk membrane system supported keratinocyte viability similar to or better than commercial PET membrane systems.
