IntroductionReliable techniques for separating the different layers in human skin are not widely available.

The aim of this work was to develop an appropriate methodology for separating the different layers of human skin biopsy samples (dermis and epidermis) to enable quantification of concentrations of test material within each layer.

In order to achieve this, the use of two different techniques (cryosectioning and heat separation) was assessed in order to separate the different layers of human skin biopsy samples. The more complicated cryosectioning technique was used to try and determine a concentration gradient in the skin.

Study DesignHuman SkinOne large piece of human skin was supplied from a single donor and was provided as full thickness skin, without adipose tissue. The skin was stored at <-10°C (nominally -20°C) until use.

On the morning of dosing, the skin sample was removed from frozen storage and allowed to reach room temperature. The full thickness human skin sample was washed with lukewarm water and placed in an incubator set to ca 32°C on to a firm board prior to dermal application (Figure 1).

The test substance was administered as a single dermal application at a nominal dose volume of 50 µL per 2 cm diameter (equivalent to 16 µL/cm2 skin). Dermal applications were applied using a suitable pipette. The required dose was spread evenly within a marked area (Figure 2). The actual dose administered was determined by weighing the pipette before and after dose administration.

At 2 and 6 hours post-dose, the treated area of skin was washed with a mild soap solution (Liquid Ivory) and rinsed with warm water. The application area was then dried.

Skin biopsies of ca 3 mm diameter were taken from a central area of the dose site (Figure 3 and Figure 4).

Figure 1. Human skin placed in an incubator set to ca 32°C.

Figure 2. Human skin attached to a firm board following dermal application.

Figure 3. Collection of the 3 mm human skin biopsy sample.

Figure 4. Human skin 3 mm biopsy sample.

Comparison of Procedures Used to Separate the Different Layers of Human Skin Biopsy SamplesClaire A Valentine1*, Georgina Adjin-Tettey1, Neil Ibbotson1, David T Akroyd1, John S Kendrick1 and Vibeke H Sunesen21Covance Laboratories Ltd, Harrogate, UK; 2LEO Pharm A/S, Ballerup, Denmark

Presented at the Joint DMDG/GMP Open Meeting 2016

CryosectioningMethodsInitially, attempts were made to separate human skin biopsy samples using cryosectioning techniques. It was noted that the biopsy sections were very small and difficult to handle. An embedding fluid was required in order to collect the sections for analysis by cryosectioning. Biopsies from control human skin were embedded in OCT (Optimum Cutting Temperature) and sectioned at 30 µm. The samples were analysed to see if the OCT embedding fluid had an effect on the bioanalysis determinations. The mass spectrometer response of the two stock solutions (one containing OCT and the other without OCT) were compared and the results demonstrated that the presence of OCT did not affect quantification. As such, OCT was used as an embedding fluid to surround each skin biopsy for this study.

Following test substance administration and sample collection, the biopsy samples were mounted onto card and placed into a cryovial. The cryovial was placed in liquid nitrogen for approximately 3-4 minutes until frozen. The frozen samples were embedded in OCT (Optimum Cutting Temperature) reagent, before being mounted on a cryomicrotome and sectioned at a nominal thickness of 30 µm (Figure 5). At selected intervals, 5 µm sections were taken for Hematoxylin and Eosin (H&E) staining to confirm morphology of the sectioned tissues.

Figure 5. The embedded human skin biopsy sample being sectioned at a nominal thickness of 30 µm on a cryomicrotome.

ResultsOn review of the biopsy samples obtained, a “mushroom effect” was noted on each biopsy where the edges of the epidermis surrounded the dermis at the top of the biopsy. Figure 6 is an enlargement of a skin biopsy which clearly shows the “mushroom effect”. This effect is thought to be caused by the pressure applied when obtaining the skin biopsy. The result of this “mushroom effect” following cryosectioning can be seen in Figure 7 where H&E staining clearly shows the epidermis surrounding the dermis layer.

Although successful sections were obtained at different levels through human skin, it was difficult to separate them into the individual layers, i.e., dermis and epidermis.

Figure 6. An enlargement showing the “mushroom effect” of the skin biopsy sample.

Figure 7. Representative micrograph showing the dermis and epidermis following cryosectioning and H&E staining, 2 hours post-application (x40 objective).

Heat SeparationMethodsSeparation of the different layers of human skin utilising heat separation was employed. The human skin was prepared and the test substance was administered as a single dermal application. Biopsies were collected at 2 and 6 hours post-application and each biopsy was transferred to an individual Eppendorf tube. The Eppendorf tubes were placed in a water bath at ca 60°C (Figure 8). After 2 minutes, the biopsies were removed from the water bath and the epidermis was carefully separated from the dermis using forceps (Figure 9 and Figure 10). The different layers of the skin were placed in individual Eppendorf tubes and stored frozen at <-10°C (nominally -20°C) prior to analysis for test material content.

Figure 8. An Eppendorf tube, containing the human skin biopsy sample, being placed in a water bath at ca 60°C.

Figure 9. The epidermis of a human skin biopsy sample being removed from the dermis using forceps.

Figure 10. Separation of the epidermis from the dermis of a human skin biopsy sample.

ResultsConcentrations of test material in the dermis and epidermis following heat separation are presented in Figure 11. The results show that maximal skin concentrations of test substance were observed in the epidermis at 2 and 6 hours post-application compared to the dermis. Although concentrations were lower in the dermis, this indicated that the test substance had penetrated the stratum cornea and epidermis. At 6 hours, concentrations of test substance were higher in both the dermis and epidermis compared to 2 hours post-application. This suggested that test material continued to penetrate through the skin to the dermis layer over time.

Figure 11. Concentrations of the epidermis and dermis following heat separation.

ConclusionsQuantification of the test substance within the separate layers of the skin (dermis and epidermis) was not achieved using cryosectioning techniques.

Using the heat separation technique, it was possible to separate the epidermis from the dermis and analyse them separately. This technique proved to be a robust and more reliable method of separating the different layers of human skin in a small biopsy.

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Individual Skin Biopsy Samples

Dermis -2 hour

Dermis - 6 hour

Epidermis 2 hour

Epidermis - 6 hour

AcknowledgementsThe authors would like to thank Beverley Matharu (Department of Histology, Covance) and Ranbir Mannu (Department of Bioanalytical Services, Covance) for their valuable contributions to this project.