In-vitro analysis

For many studies, which are not possible to try with test persons, in vitro test methods become more and more important.

For cosmetic products, which are often used over a long period of time, dermatological testing for safety is an essential prerequisite for high application safety and customer satisfaction. By determining and evaluating reliable and reproducible biological endpoints by means of histological and biochemical analyzes, a statement about the effects of a product on regenerative biological processes of the skin can be made.

The 3D skin model is particularly suitable for such an examination as it forms the natural histological sequence of epidermal cell layers and dermal structures as known from human epidermis. Therefore the stratum basal, the basal keratinocyte layer, is located directly on a living dermis equivalent (fibroblasts) and is followed histologically by the stratum spinosum and the stratum granulosum, which are then finally covered by the hardskin layers of the stratum corneum.
Disorders of regular epidermal keratinization (orthokeratosis), caused by various stimulus effects of physical (mechanical, UV-A / B) or chemical (SLS) nature, can be analyzed and evaluated quantitatively and qualitatively.

In in vitro studies, we investigate:

  • Integrity of skin barrier / regeneration after injury,
  • UVA / B damage,
  • Product compatibility.

 

We examine:

  • Epidermal differentiation,
  • Skin barrier of relevant structures,
  • Vitality,
  • Integrity of the Dermo-Epidermal Zone (DEJ),

We analyze:
  • Morphologically-histological structures after special stains,
  • Immunofluorescence staining of proteins involved in the differentiation of the corneocytes,
  • Quantification of Interleukins (e.g., IL-1α, Il-6) by Enzyme Linked Immunoabsorbent Assay (ELISA),
  • Further methods in development.

Histological analysis

Histology is the science of biological tissues (histos = tissue & logos = teaching). With the aid of histology, fine-tissue-morphological changes can be visualized on the cellular level by specific staining and examined accordingly. The most common staining of histology and histopathology is H & E staining. In this case, nucleic acids are intensively colored dark blue, cytoplasm and connective tissue pink-red. The image below shows an H & E staining of a healthy model (A) and of a UVB-exposed, damaged model (B).

invitro1  invitro2
Hematoxilin and Eosin (H & E) staining of an ultra-thin section A) untreated model B) UVB-irradiated model (250 mJ / cm2) after 48 h. Closed arrows: sunburned cells; Open arrows hydropic cells (massive fluid accumulation). *: Typical Dyskeratosis (keratinization disorder) after UV exposure.

 The model has all the physiological layers (stratum basale, spinosum, granulosum and corneum) of native human skin as well as a regular keratinization on (A). Figure B shows a model 48 h after UVB exposure (250 mJ / cm2).
The typical features of a sunburn are clearly recognizable. Numerous so-called "sun burned cells", hydropic cells, which contain more fluid and have a less strongly colored nucleus (open arrows), as well as typical dyskeratosis.
Additionally, isolated ulcerated keratinocytes can be seen. Using this model, the protective function (sun protection) and / or the regenerative potential of a formulation or individual substances can be examined and evaluated at the cellular level.

 

Analysis of cell division rate

The cell division rate (proliferation) is an important indicator of the vitality or "fitness" of a tissue. In the human epidermis, only the keratinocytes of the stratum basale (basal keratinocytes) are capable of cell division. These compensate for the corneocytes lost as a result of the permanent process of scaling, thus providing for the homeostasis of the tissue and preventing a cellular thinning of the epidermis.

invitro3  invitro4
Ultra thin section of a full skin model. A) Bright field B) Fluorescence. Blue = cell nuclei, red: dividing cells.

 
Using the Click-It EdU® technology, the basal keratinocytes of the epidermis and the fibroblasts of the dermis can be specifically labeled during cell division (mitosis) in the skin model (A + B). On the basis of this, it can be determined whether a substance or formulation influences the cell division behavior according to a defined topical or systemic application and treatment time.
Carrying out an untreated control allows the percentage quantification of the cell division rate. Examples of increased cell division activity: wound healing, inflammatory processes.

Integrity of the skin barrier

Another important parameter for a healthy skin is an intact skin barrier, which on the one hand prevents excessive loss of water, on the other hand an effective protective shield against pathogens, environmental influences and (chemical) noxious substances.

invitro5  invitro6
Ultra thin section of a full skin model. A) Treatment with sterile PBS B) 45-minute treatment with 1% SDS.
Blue = cell nuclei, green: Lucifer yellow coloration.

Individual substances, complete formulations or chemical noxes can influence the integrity of the skin barrier. This can result in increased water loss (increased evaporation) and the ingestion of harmful agents from the environment (passively) into the skin. In the full-skin model, the Lucifer Yellow coloration can be used to determine whether a product affects the skin barrier. Sterile water or PBS (phosphate buffered saline solution) have no effect on the integrity of barrier (A). The topically applied dye remains completely in the topmost layers of the skin (stratum corneum).
However, a 45 minute treatment with the surfactant SDS (1%) leads to damage to the skin barrier (B). The dye penetrates the deeper skin layers (dermis). This method can also be used to determine whether a product can restore its barrier function after injury

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