The detail provided in this protocol, addresses human skin equivalent generation, which is often technically challenging. Novel human skin equivalent protocol elements, such as vasculature and volumetric imaging are included. The technique enables the straightforward construction of a skin model that more closely matches in-vivo tissue for the study of diseases, aging and personalized medicine.
The most challenging part of this protocol is a transition between submersion and air-liquid interface. It will likely take a few trials to optimize the timing and media levels for consistent results. To begin, add 516 microliters of media to a cold capped tube, and immediately add 375 microliters of cold collagen using the 1000-microliter positive displacement pipette.
Remove the empty pipette tip and switch to the prepared 250-microliter positive displacement pipette to mix. Mix quickly, but gently to prevent excess bubble formation. Keeping the tip in the solution, mix uniformly from different positions of the tube until the solution is of homogenous color, which typically takes about five pipette cycles or 10 seconds.
Immediately disperse 125 microliters of acellular collagen onto the membrane of the 12-well culture insert. To ensure uniform coverage, tilt the plate and use the pipette tip to paint the membrane by gently spreading collagen around. Immediately move the 12-well plate to a 37-degree Celsius cell culture incubator to let it gel for at least 20 minutes.
After the gelation period, take the 12-well plate of acellular collagen out of the incubator. Remove the 1.7 milliliter capped tube from wet ice, then remove the stock collagen from refrigeration and place it on wet ice with the lid open. Add 516 microliters of cooled cell suspension to the cold capped tube.
Use the 1000-microliter pipette to immediately pipette 375 microliters of cold collagen solution directly into the solution inside the capped tube. Expel all collagen from the pipette into the tube and discard the positive displacement pipette tip. Immediately switch to the 250-microliter pipette and mix the collagen solution Once mixed, transfer 250 microliters of cellular collagen solution onto the acellular collagen supports in the 12-well culture insert, then move the 12-well plate to a 37 degrees Celsius cell culture incubator.
After the 30 minute gel time, gently tilt the plate to assess the gelation and make sure that the collagen has solidified. Add 500 microliters and 1000 microliters of blend media to the upper chamber and lower chamber of the insert, respectively. Ensure that the collagen gel is submerged, adding more media if necessary.
Place the well plate in the cell culture incubator for overnight incubation. On submersion day seven, use a manual pipette to collect and discard media from the bottom and top chamber of each construct well. To collect media stuck directly under the permeable membrane, place the pipette tip under the membrane, knocking the insert out of place temporarily.
Add one milliliter of human skin equivalent or HSE media supplemented with 5%FPS to the lower chamber of each well and 200 microliters of cell suspension to the top chamber of each well. Seed the keratinocytes directly onto the dermal construct surface and allow them to settle for two hours in the incubator. Two hours after seeding the keratinocytes, carefully add 300 microliters of HSE media, supplemented with 5%FPS to the top chamber of each construct well.
After loading the media, place the construct back into the incubator. Using a manual pipette, lift each construct to their air-liquid interface, or ALI, by removing media waste from the upper chamber only. Try to get as close to the epidermal layer as possible without touching or damaging it.
Tilt the plates slightly at different angles to collect the media, then add approximately two milliliters of sterile water to the surrounding wells in the plate to maintain consistent humidity. Check the plate a few hours later to make sure the keratinocytes are still at the ALI. Remove any media in the upper chamber, keeping track of how much media is removed from each well.
The epidermal layers should look hydrated, not dry, but there should not be media pooled on top of the construct. To prepare the construct for immunofluorescent staining, turn an insert upside down and place it over its well on the well plate. Stabilize the insert with one hand while using fine tip forceps or a precision knife to cut about half of the circumference of the membrane.
Cut as close to the plastic housing as possible. Using the fine tip forceps, grab the edge of the cut membrane flap and gently peel the porous membrane off the insert as well as the VHSE construct. If the construct gets stuck on the side of the chamber, use the fine tip forceps or a small scoopula to move it to the well.
Once the VHSE is in the well, discard any remaining pieces of the insert membrane and keep the culture insert housing in each well to hold the VHSEs in a submerged position during staining. Two days before imaging, prepare polydimethylsiloxane or PDMS. Place any clean mixing vessel on a weighing balance and tare the scale.
Add the base, then add the crosslinker. Stir the solution vigorously for at least four minutes, creating small bubbles. After sufficient mixing, pour the PDMS into a 90 or 100-millimeter Petri dish.
Degas the PDMS in a vacuum chamber until all bubbles disappear and the PDMS is clear. Release the vacuum slowly and remove the PDMS. Place the dish into an oven to cure overnight at 50 to 60 degrees Celsius, making sure that the dish is sitting flat for PDMS to cure evenly.
One day before imaging, use a steel punch or a handheld precision knife to punch or cut out a circular well from the PDMS sheet, around the same size as the VHSE construct. Cut a square patch around the circular well to create a single PDMS well. Using a glass cover slip of a similar size as the PDMS well add cyanoacrylate glue to the bottom surface of the PDMS and smear evenly with a disposable pipette tip.
Center the glass and press it onto the PDMS well while leaving a clear glass window within the punched circle. Let the glue dry overnight before using. Characterization of the epidermis and dermis show appropriate immunofluorescent markers for human skin in the VHSE constructs.
Cytokeratin 10 is an early differentiation keratinocyte marker that usually marks all suprabasal layers in skin equivalents. Involucrin and filaggrin are late differentiation markers in keratinocytes and mark the upper most suprabasal layers in skin equivalents. Clearing the VHSE allowed for straightforward imaging in one setting and eliminated the need to reorient the construct to image the dermis and epidermis separately.
Volumetric images make it possible to generate 3D renderings to map vasculature throughout each construct. Image stacks were loaded into computational software and a custom algorithm was used for 3D rendering and quantification. Skeletonization determined the definitive center of each collagen IV-marked vessel and the resulting data was used to calculate vessel diameter as well as vascular fraction.
Following this procedure, characterization methods such as barrier analysis, scanning electron microscopy, lipid profiles, and others can be performed to understand how vasculature specifically affects epidermal maturation and stability. This protocol enables tissue-relevant and cell type-specific studies in a vascularized skin model. It is particularly suitable for research in aging and personalized medicine.
