Module 1: Skin absorption experiments

a) Skin preparation and permeation experiments

In vitro permeation experiments with human skin require careful handling of the
tissue to guarantee reproducible results. In this lab course the handling of human
skin tissue is demonstrated and the proposed methods are practiced hands on by the
participants:
First, excised human skin obtained from abdominal plastic surgery is prepared for
storage. In this procedure, the subcutaneous fatty tissue is separated from the outer
skin layers, namely the epidermis and the dermis by a scalpel.
Second, the epidermis is separated from the underlying dermis by a heat-separation
technique. The separated epidermis will be used for subsequent permeation
experiments. The epidermis sheets are transferred to static Franz diffusion cells to
conduct permeation experiments.
All critical points of each method as well as the obtained results are highlighted and
discussed with the participants.

Skin preparation

Franz diffusion cell,

Image courtesy Mr. Puetz,
Saarland University

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Module 1: Skin absorption experiments

b) Skin segmentation and penetration experiments
Tape stripping is an established technique to investigate the pharmacokinetic
response to dermally applied formulations. It is a non-invasive technique which is
widely applied in vivo. Drug levels within the stratum corneum are assessed by
subsequent sampling of corneocyte layers with adhesive film and quantifying the
drug concentration in each layer. Results are often reported as drug concentration
skin depth profiles or cumulative amounts in the stratum corneum. By obtaining these
data for several time points during the absorption and elimination phase of a topically
applied drug dermato-pharmacokinetic parameters can be calculated. Especially for
locally acting drugs the pharmacodynamic response can often be predicted from
these pharmacokinetic parameters. Tape stripping has also been used to evaluate
the bioequivalence of topically applied drugs.
In this lab-course we will introduce tape stripping as an in vitro sampling technique of
human stratum corneum. It is imperative that tape stripping is performed under highly
standardized conditions. Participants will learn techniques how to control skin
hydration during incubation, how to standardize sampling and how to quantify the
amount of corneocytes removed by each tape. We will evaluate and discuss
experimental results obtained in the workshop.

Tape-stripping technique by Wagner et al.

(Wagner H et al., Pharm Res 24 (2000) 224-230),
Image courtesy Mr. Puetz, Saarland University

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Module 2: In vitro skin imaging

a) Raman-CLSM
Confocal Raman microscopy is a technology with growing impact in dermal research.
The possibility of label-free and non-destructive follow-up of substances inside the
skin facilitates its use in penetration and permeation studies.
After all attendees received a theoretical introduction in Raman spectroscopy, the lab
course will demonstrate basic aspects of successful hard- and software operation.
During the module all participants will deal with spectral data acquisition of different
specimen, including the preparation of skin as biological sample for spectroscopic
analysis. A demonstration on mathematical processing of recorded raw spectra and
the use of chemometric approaches for conversion of a spectral data set into
chemically selective colour images will close up the session.

CH 2

Conversion of individual Raman peaks (left side) into chemically selective colour images of a two
component system (right side).

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Module 2: In vitro skin imaging

b) Multiphoton-LSM (Particle Penetration and Analysis)
Laser scanning microscopy (confocal and multiphoton microscopy) is an important
non-invasive, non-destructive method for tracking dermal penetration of
nanoparticles. However, imaging techniques are mainly qualitative methods. In
response, we developed in our lab an experimental approach for semiquantitation of
confocal/multiphoton images. In this module, both the basic principles of the
technique in addition to image analysis are demonstrated. The participants will be
trained on handling the microscope, imaging model gold nanoparticles in skin
sections, followed by image analysis.

A schematic presentation of the experimental approach used for semiquantitation of the penetration of
gold nanoparticles in a longitudinal skin section imaged by multiphoton microscopy (Labouta H. I et al.,
Int J Pharm 413 (2011) 279-282).

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Module 3: In vivo skin imaging

This module is organized in cooperation with JenLab GmbH using respective imaging
techniques from the company such as the MPTflex.

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