The in vivo > ex vivo > in vitro Approach for Your Innovative Claim Support
In addition to standard in vitro methods including cell culture techniques and organotypic skin models, we offer an innovative approach that allows us to combine in vivo treatment via ex vivo sampling with a large range of available in vitro analyses. The obtained samples (tissue and tissue fluids) can be subjected to biochemical tests such as ELISA tests, immunohistological, histological and molecular tests:
in vivo Topical Treatment
ex vivo Samples (complete viable epidermis with intact basal cell layer plus interstitial fluid within the blister)
in vitro Analyses (biochemistry, cell biology, immunology, molecular biology, histology)
Sampling via punch biopsies provides epidermal and dermal material but requires a local anesthetization and leaves scars. The suction blister method, initially developed by Kiistala et al. in 1968, is a valuable alternative that is considered to be non-invasive.
Approved by the Ethics Committee in a general vote, we have established this method as a standard trial that allows us to obtain epidermal material for subsequent in vitro analyses.
As the name implies, blisters are artificially induced. Custom-made plexi glass chambers with round openings are attached to the respective test areas and connected to a vacuum pump:
Within approximately 2 hours, the epidermis is slowly detached from the underlying dermis. The induced blister fills with fluids from the surrounding tissue (interstitial fluid):
The procedure is painless – causing itching at worst – and the wounds heal without forming scars within 7 to 10 days. However, some subjects develop hyperpigmentation on the former wound that can last for some months or longer.
After removal of the chambers, the blister fluid (interstitial fluid) can be extracted with a sterile syringe and the blister roof carefully removed using sterile instruments.
For the in vitro analyses, a large variety of methods such as immunohistological stainings and a continuously extended list of commercially available analysis kits are available.
Parameters that can be estimated using in vitro enzyme-linked immunosorbent assays (ELISA), immunohistological or other methods:
- Structural proteins: pro-collagen I as a measure for the de novo synthesis of collagen I, fibrillin and elastin (ELISA)
- Extracellular matrix: hyaluronic acid, MMPs such as Matrix Metalloproteinase-1 (ELISA)
- Oxidative stress: 8-isoprostane as a marker for lipid peroxidation, carbonyl proteins as a marker for protein oxidation (ELISA)
- Cytokines: e.g. IL-6, IL-8 and TNF-α (ELISA)
- UV-induced DNA damage: immunohistological detection of DNA damage induced by UV light (thymine dimers, 8-oxo-guanine)
- Differential gene expression via Real Time Quantitative PCR
- Stem cell activation: number of p63-positive epidermal stem cells by immunohistological analysis, colony forming efficiency by cultivation of epidermal stem cells (colony forming assay)
Please contact us for further information, consultation or a detailed proposal.