غیر تهاجمی برای کنترل کیفیت فرآیندهای تولید معادل پوست مصنوعی با استفاده از توموگرافی انسجام نوری

The engineering of artificial tissue and organs is widely gaining relevance in biotechnological and biomedical research. Particularly by the law enforced reduction of animal testing, alternative methods need to be developed to simulate and test the effects of chemical substances on human organs. Regarding the branches for pharmaceuticals, chemicals and cosmetics most products need to be tested on penetration of the human skin tissue. As an alternative method to animal testing, skin equivalents (SEs) based on tissue engineering may be used to test the skin barrier function. In the framework of a large scale research project, we built a fully automated production process based on the adaption of manual laboratory protocols to carry out the biological process steps for the manufacturing of SEs. In a first step, keratinocytes are mechanically and enzymatic isolated from human foreskin biopsies. In a second process, the cells are cultivated in MTP format cultivation dishes und undergo a series of passages. In a last process step, the keratinocytes are concentrated and dispensed in 24 well membrane based MTPs. Due to biological fluctuations in cell and tissue growth, the production process faces major challenges to control and process a vast variety in batch duration and timing. This results in a strong impact in the final tissue quality. For quality control in the manual process, histologies of the SEs are prepared followed by staining and microscopic imaging. To overcome this time intensive process, we developed a fully automated optical coherence tomography OCT system. OCT is a non- invasive and non-destructive imaging technique based on the interferometric detection of optical light feedback from the probe tissue in the near infrared light. OCT generates 2D and 3D tomographic images with a micron resolution and a tissue penetration up to 2 mm. We developed custom image processing algorithms, to detect the individual layer structure and corresponding layer thicknesses. The OCT results show a high degree of correlation with the histological findings regarding structure and layer thicknesses. Further, we show in a first study a method to use this OCT layer detection technique as a potential substitution measurement to the ET50 test used for analyzing the in vitro skin barrier function

With the aim of placing greater responsibility on industries when it comes to chemical substances the European Commission released in 2006 the REACH (Registration, Evaluation, Authorization and Restriction)regulation to protect human health through the better and earlier identification of the intrinsic properties of 1. Introduction chemical substances. In many cases public and regulatory requirements force producers to utilize alternative methods to animal testing for analyzing new substances or products regarding their safety. The increasing demand for substituting animal testing in consumer product development has lead to a growing market for in vitro test systems. Here, a significant for irritation, penetration or toxicity tests (i.e. OECD guideline for the testing of chemicals 431: “In vitro Skin Corrosion: Human Skin Model Test”) [1]. SEs are build up by keratinocytes undergoing a differentiation and proliferation process during the air-liquid-interface growth phase. On the basis of specialized growth culture condition, the SEs exhibit the four epidermal layers (fig. 1) achieving one part of the requirements for using it as alternative for skin irritation test.

In this study an alternative method for the process of quality control in the automatic production of SEs is presented. Therefore, we developed an automatic OCT device that generates 2D tomographic high resolution images of the SEs. The OCT measurements strongly correlate with the histological findings in structure and morphology. With the image processing software the layer detection of the membrane, layer of keratinocytes and cornified epidermal layer was realized. Due to morphological chances in the preparation process of the histologies and correlating the right image plane, theAs shown in this first study, the layer detection by OCT of the SEs strongly correlates with the ET50 measurements. To evaluate this finding, further systematic studies of this correlation are initiated. To improve the OCT measurement data consistency, we plan to expand the measurement to an 3D-OCT scan to monitor the entire SE internal structure.