Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

The overall goal of this procedure is to build up a three dimensional full thickness skin equivalent, which resembles natural skin and to generate precise and reproducible wounds under sterile conditions using an automated wounding device. This is accomplished by first embedding primary human dermal fibroblasts in a collagen one hydrogel. Next primary human epidermal keratinocytes are seated on the top of the fibronectin coated hydrogel.

Then the model is cultured under submerged conditions, and then under an air liquid interface, a three-dimensional full thickness skin equivalent is formed, composed of a dermal component and a multi-layered epidermis. Finally, a standardized wound is generated in a sterile environment. Ultimately, immunohistochemical staining and microscopy is used to assess the histological structure of the wounded full thickness skin equivalent.

The main advantage of this technique of existing methods, like animal models, for example, is that the full thickness skin equivalent is composed of primary human cells. So together with the automated wounding device, we can generate standardized wounds, So the automated wounding device can provide insight into skin wound healing processes in humanized environment. It can also be applied to our systems such as bone tissue engineering.

This method can help answer key questions of wound healing, such as biological processes, underlying skin repair, and the cellular crosstalk between the epidermal and dermal component. To begin dissolve collagen with 0.1%acetic acid to a final concentration of six milligrams per milliliter. Prepare gel neutralization solution by mixing 232.5 milliliters of two X dmm, 7.5 milliliters of FCS, 7.5, milliliters of three molar hees, and 2.5 milliliters of five milligrams per milliliter.

Chondroitin sulfate place inserts into the wells of a 24 well plate detach human dermal fibroblasts or HDFS and centrifuge the cell suspensions for five minutes. At 270 G, count the cells and centrifuge an Eloqua of 1, 200, 000 HDF for five minutes. At 270 G, remove the snat and use four milliliters of cooled gel neutralization solution to carefully resuspend the HDF without causing air bubbles.

Then resuspend the solution with eight milliliters of collagen solution. Next, use a multi-step pipette and add 500 microliters of collagen cell mixture to each insert. In the 24 well plate incubate the gels for 20 minutes at 37 degrees Celsius and 5%carbon dioxide to allow the gels to solidify.

Then use two milliliters of DMM plus 10%FCS per well. To submerge the gels, incubate the gels for 24 hours. Use ultrapure water to dissolve human fibronectin protein to a final concentration of 50 micrograms per milliliter.

After incubating the dermal equivalent gels for 24 hours, remove the medium. Use 25 microliters of fibronectin solution to cover each dermal equivalent and incubate the gels for 30 minutes. In the meantime, resuspend the human epidermal, keratinocytes or HKS with kgm two plus 5%FCS to a final concentration of 1 million cells per liter seed, 100, 000 heeks on top of each gel.

Then incubate gels for 45 minutes to allow cells to adhere using kgm two ready plus 5%FCS submersed the cells and continue the incubation. After incubating for two to three days, replace the medium with 2%FCS followed by medium replacement without FCS, two to three days later on. Day seven, completely remove the medium.

Use sterile forceps to place each insert into one well of a six well plate. Then add 1.5 milliliters of airlift medium per well. Taking care not to wet the surface of the FTSC.

Change the medium every two to three days for an additional 14 days. Prepare the automated wounding device by using 70%ethanol and ultraviolet light to disinfect the working area using sterile forceps. Place skin equivalents into designated areas of the autoclave sample carrier plate.

Place a sample carrier plate into the socket below the drilling head. Use the control software to set the wounding parameters as follows. Spin velocity 15, 000 RPM penetration, 1.5 millimeters.

Propulsion 100 hertz. Transfer the wounding parameters to the automated wounding device. Begin the wounding procedure when complete.

Remove the sample carrier plate from the socket and use sterile forceps to transfer the wounded FTSE back into the insert used for culturing. Incubate the wounded FTSE until experimental evaluation. Use the models for his histological analysis at any time.

The isolated HDFS and HKS were characterized by immunohistochemical staining before using them for FTSE. The HDF are positive for menton, a marker for fibroblasts primary HEK highly express early differentiation protein CYTOKERATIN 14, but nearly no late keratinocyte differentiation protein CYTOKERATIN 10. The FTSE were cultured for seven days under submerged medium conditions, followed by 14 days at the air liquid interface.

During this process, the FTSC contracts significantly within 21 days. The HEK are proliferating by changing the medium level in such a way that the surface of the models is exposed to air. And by increasing calcium concentration, the HKS are stimulated to differentiate into a multicellular epidermis that is composed of several vital cell layers of keratinocytes.

In different states of D differentiation and atrium cornea. Comparing the hematin and eosin staining of FTSE with native human skin, it becomes obvious that FTSE mimic histological architecture of the skin. The HDF in the collagen one hydrogel can be stained with primary antibodies against vimentin.

In addition, keratinocytes form an epidermal layer composed of cytokeratin 14 positive cells and the late differentiation marker. CYTOKERATIN 10 is expressed in the super basal layers. Furthermore, the St.Stratum cornium is positive for RIN After its development.

This technique paved the way for researchers in the field of dermatology to explore cutaneous wound healing and a standardized in vitro setup that closely mimics the human skin.