This protocol describes a straightforward method to cultivate three-dimensional reconstituted human epidermis in a reproducible and robust manner. Additionally, it characterizes the structure-function relationship of the epidermal barrier model. The biological responses of the reconstituted human epidermis upon proinflammatory stimuli are also presented.
In vitro or constructed human epidermis model were first developed in the nineties with a primary goal of developing alternative testing methods to animal experimentation. Those 3D epidermal models are now commonly used to test both the safety and efficacy of cosmetic ingredients. Whereas it can be purchased from several tissue suppliers, being able to reconstitute a 3D model in a laboratory gives more flexibility to the end point of interest.
There are several research articles describing briefly the reconstitution protocol of a 3D epidermal model, but there was, to date, no video available showing the critical steps of the reconstitution process. This is the reason behind the publication of this video paper. The preparation of in-house reconstructed human epidermis allows for a lot of freedom in the culturing process, such as changes to the culture medium composition, pro-inflammatory or oxidative stress triggers, silencing of genes of interest, and the inhibition or stimulation of certain biological processes.
To begin, thaw a vial with 1 million cryo-preserved normal human epidermal keratinocytes, or NHEKs, in a water bath at 37 degrees Celsius by submerging part of the vial in water. Incubate the vial for one to two minutes in the water bath, until only a small sliver of ice is visible. Resuspend the cells very carefully by pipetting up and down two to three times.
Transfer the cell suspension into two T75 flasks containing a total of 15 milliliters of pre-warned thawing medium, resulting in a seeding density of 6.7 times 10 to the fourth cells per centimeter squared. Pre-fill 24 well plates with 1.5 milliliters of submerged medium, ideally using a dispenser pipette. After culturing the cells to 80%confluency, they are ready for seeding in inserts for the cultivation of reconstructed human epidermis, or RHEs.
Remove the basal medium from the T75 flasks with the NHEKs. Rinse the cells by adding five milliliters of pre-warmed PBS to each T75 flask, then remove the PBS from the flasks. Add two to three milliliters of pre-warmed 0.05%Trypsin-EDTA to each flask, making sure that the trypsin solution is equally distributed on the cell culture area of the flask.
Place the flasks in the cell culture incubator for four minutes, then check whether the cells detach using the microscope at a 10 X magnification. Rap the flask to help the cells release from the surface of the flask. Once all the cells are detached, add an equal volume of pre-warmed trypsin inhibitor to each T75 flask, and transfer the cell suspension from the flasks to a centrifuge tube.
Rinse the flasks with five milliliters of pre-warmed PBS and transfer it to the centrifuge tube containing the cell suspension. Centrifuge the harvested cells at 400 times G for five minutes. Carefully discard most of the supernatant leaving approximately 100 to 200 microliters in the tube.
Gently flick the tube with your fingers to carefully loosen the pellet. Gently resuspend the pellet of cells in a low volume of submerged medium, pipetting up and down 5 to 10 times to ensure a uniform cell suspension. Start with a low volume to avoid the formation of cell aggregates, and add up to one milliliter of submerged medium per initial T75 flask.
Count the cells in the suspension using the trypan blue exclusion method. Dilute the cell suspension with additional submerged medium to reach a concentration of 3.525 times 10 to the fifth cells per milliliter, using the first equation provided in the text manuscript. Perform a second cell count of the diluted solution and use the second equation in the text manuscript to calculate the cell suspension volume to be seeded into the culture insert.
Hang the 24 well culture inserts in the highest position of the carrier plate, and transfer the carrier plate to the 24 well plate pre-filled with submerged medium. Add the determined volume of the cell suspension to each insert, taking care not to touch the insert. After seeding, incubate the 24 well plates for 10 to 15 minutes at room temperature to overcome an edge effect.
Do not move the plates during this time. Transfer the plates to the cell culture incubator, and leave them in submerged conditions for three days. After a three-day incubation in the cell culture incubator, expose the cells that have adhered to the membrane surface to the air liquid interface, or ALI, by removing the submerged medium from the apical compartment with an aspiration system and a glass Pasteur pipette.
Fill new 24 well plates with 1.5 milliliters of fresh pre-warmed ALI medium, and transfer the carrier plates with the culture inserts to the new multi-well plates. Transfer the multi-well plates back to the cell culture incubator. Refresh the ALI medium every two to three days, for 14 days.
Keratinocytes in 2D culture display a traditional morphology with a consistent polygonal shape. After 15 days at ALI, the reconstructed human epidermis forms a fully stratified tissue, which is indicated by its four main epidermal layers. Ultrastructural analysis of the reconstructed human epidermis at different time points in the reconstitution protocol reveals the cornification process, with an increased number of cornea site layers over time.
Keratinocytes in the epidermis show different protein expression profiles according to their differentiation stage. Involucrin expression appears more predominantly in the SG layer, whereas the expression of filaggrin and loricrin is located in the upper layers. Keratin-10 expression was found in all the viable layers except for the SB layer.
The reconstructed human epidermis displays functional desmosomal junctions, as indicated by the expression of Desmoglein-1 in the intercellular space of the viable epidermal layers. The barrier properties of the reconstructed epidermis model were investigated by assessing the tissue viability upon topical treatment with a known barrier disrupter, and by assessing the tissue integrity. The tissue integrity was determined after 15 days by measuring the TEER with a voltmeter.
Responsiveness of the epidermis to Lipopolysaccharide and Tumor Necrosis Factor alpha was investigated. Both LPS and TNF alpha treatments were non-toxic. relative to the Triton X-100 control.
The release of Interleukin-1 alpha and Interleukin-8 in the RHE medium was quantified using IL lysis. LPS treatment resulted in a statistically significant up-regulation in the release of Interleukin-1 alpha and Interleukin-8. TNF alpha treatment resulted in a statistically significant up-regulation in only the release of Interleukin-1 alpha.
However, a clear tendency of increased Interleukin-8 levels was also observed upon stimulation with TNF alpha. Following this protocol, TEER can be measured as a value for barrier integrity. The viability or cytotoxicity can be measured by an MTT or LDH assay, for example.
The supernatant can be used to measure the secretion of cytokines or other proteins. Furthermore, the tissue can be used to study the expression of proteins or genes of interest.
