Cell-based assays

We offer a portfolio of patented reporter skin models (EP 2 041 172) for the detection of typical cell stress responses such as inflammation, sensitization or stress induced by defective protein folding in the endoplasmic reticulum (ER stress). These in vivo reconstituted epidermis and full-thickness skin models with intact skin barrier are based on immortalized primary keratinocytes, the use of which guarantees a high donor-independent reproducibility. The activation of a specific cellular stress signaling pathway in these immortalized primary keratinocytes can be read out quickly and easily via a reporter protein that has been stably integrated into the genome using a reporter construct. In this way, both skin-damaging and skin-regenerating or skin-protecting substance effects can be specifically identified and evaluated: 

• Reporter skin models for the detection of anti-/inflammatory effects:

are reconstructed epidermis and full-thickness skin models based on NF-kB reporter keratinocytes to evaluate the inflammation-inducing or -inhibiting potency of substances via activation of the NF-B signaling pathway.

As a central mediator of inflammatory reactions, the transcription factor NF-kB induces the expression of various pro-inflammatory genes and is involved in the regulation of inflammasomes. 

• Reporter skin models for the detection of sensitizing and anti-/oxidative effects: 

Are reconstructed epidermis and full-thickness skin models based on Nrf2 reporter keratinocytes to assess the sensitizing/oxidative sstress-inducing potential of substances via activation of the Nrf2 pathway. 

The transcription factor NRf2 plays a crucial role as a master regulator in maintaining the cellular redox balance in the adaptive response to oxidative stress, which can lead to sensitization to the s-stress-inducing substance.

• Reporter skin models for the detection of ER stress-inducing effects:

are reconstructed epidermis and full-thickness skin models based on stress sensor keratinocytes to evaluate the ER stress-inducing potential of substances via activation of specific transcription factors that are significantly involved in cell stress. ER stress is caused by the accumulation of misfolded proteins in the endoplasmic reticulum (ER).

Challenge / Your challenges

In order to guarantee the consumer safety of new chemical substances - as ingredients in cosmetics, cleaning agents, clothing, biocides, pesticides or as industrial chemicals - their safety for humans must be proven before they are approved for the market. In vitro tissue models, for example of the skin, are ideally suited to replace animal testing.

Our approach / Our development

With our decades of experience in the development and targeted genetic modification of human cell lines, the establishment of human reconstituted 3D skin models and adherence to the highest quality standards, we establish specific reporter skin models that are suitable for reliable testing of substances. Our reporter keratinocytes form a multilayered epidermis with full physiological skin barrier function in vitro and show a unique correspondence of characteristic differentiation markers of the models with native human skin.

Advantages / unique selling points

Reporter skin models are suitable for obtaining precise results immediately and on living tissue when testing different substances. By genetically modifying the skin cells, we can differentiate between the most important cell stress responses such as inflammation, sensitization or oxidative oxidative stress or ER stress using different reporter skin models. Whether a chemical substance damages human skin cells is visualized by means of a reporter protein secreted into the culture medium, which has been activated via the respective cellular signaling cascade, and can thus be read out quickly, easily and quantitatively. To do this, it is sufficient to remove a few microliters of the culture medium and add a suitable substrate. 

Our patented reporter skin models 

• are donor-independent reproducible through the use of immortalized primary cells and are available at any time, 
• allow an investigation and comparison of substances in systemic and topical application, 
• allow multiple sampling due to a reporter protein secreted into the culture medium and are thus suitable for studies of the temporal course of signaling pathway activation,
• enable more realistic exposure scenarios, as the substance to be tested can be applied directly to the skin barrier, which also enables the testing of water-insoluble, viscous or (semi-)solid substances and the evaluation of mixtures of substances with different skin penetration properties, 
• allow simultaneous investigation of the skin stress potential via the activation of various cellular stress signaling pathways, cytotoxicity and skin penetration of a substance in the same model,
• can be cultivated without media additives of animal origin, and 
• allow studies with repeated substance application.

Applications / areas of use

Our reporter skin models are suitable as a consistent solution for avoiding and reducing animal testing, for risk assessment of chemical substances and products that come into contact with the skin as well as for biomedical research in order to 

• efficacy studies in the preclinical development of pharmaceuticals, for example to test substances with anti-inflammatory or skin-soothing effects, 
• to investigate skin-damaging effects of chemical substances as ingredients of cosmetics, cleaning agents, clothing, biocides, pesticides or as industrial chemicals and
• evaluate the skin-damaging potential of formulated chemical substances and products such as cosmetics, care and cleaning products, etc. 

Range of services / Our services

We offer a wide range of services related to our reporter skin models, which we carry out in modern laboratories on behalf of and in close cooperation with and for our customers:

• Conducting studies as well as substance and product screenings, 
• Development of innovative reporter skin models including

o Isolation of primary cells

o Production of genetically modified cell lines (stable and transient)

o Reporter cell lines with different intracellular and secreted reporters

o 3-D tissue reconstitution

o Phenotypic and functional characterization of models

• Shipping of reporter skin models

Methods of analysis

Our reporter skin models enable the investigation of skin-damaging effects of chemical substances and products coming into contact with the skin on the activation of molecular signaling cascades associated with skin stress and the tissue architecture. The analyses can be performed on epidermis models, on the entire full skin model as well as separately in dermis and epidermis. 

We offer the following analytical methods:

• Analysis and quantification of secreted reporter proteins in the culture medium as a measure of cell stress responses
• Vitality measurements, for example using the MTT test (colorimetric test), alamarBlueTM . .
• Multiplex analysis for the detection and quantification of secreted proteins such as cytokines, chemokines, growth factors and antimicrobial peptides,
• Histological and immunohistochemical staining of tissue thin sections,
• Protein analyses using Western blot and
• RNA and DNA analyses using PCR or qPCR and sequencing

Several non-clinical tests are running at Fraunhofer IGB to support R&D projects that investigate biological parameters of samples using cell-based assays.

Examples are:

• Testing of bioactivity, cytotoxicity and immunogenicity of compounds using immune receptor-based assays
• Screening of TLR agonists/antagonists
• Testing of antimicrobial properties of substances or surfaces
• Detection of pyrogens and microbial residues (pathogen-associated microbial patterns, PAMPs)
• Detection of viruses

• Novel screening assay for antimycotics
• Cell-based test system for the detection of pyrogenic residues
• Virus-protection assay (Antiviral Assay, AVA)
• The Tissue Culture Infectious Dose50 (TCID50) and the Plaque Assay