570 Biowissenschaften, Biologie
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Tissue constructs of physiologically relevant scale require a vascular system to maintain cell viability. However, in vitro vascularization of engineered tissues is still a major challenge. Successful approaches are based on a feeder layer (FL) to support vascularization. Here, we investigated whether the supporting effect on the self‐assembled formation of prevascular‐like structures by microvascular endothelial cells (mvECs) originates from the FL itself or from its extracellular matrix (ECM). Therefore, we compared the influence of ECM, either derived from adipose‐derived stem cells (ASCs) or adipogenically differentiated ASCs, with the classical cell‐based FL. All cell‐derived ECM (cdECM) substrates enabled mvEC growth with high viability. Prevascular‐like structures were visualized by immunofluorescence staining of endothelial surface protein CD31 and could be observed on all cdECM and FL substrates but not on control substrate collagen I. On adipogenically differentiated ECM, longer and higher branched structures could be found compared with stem cell cdECM. An increased concentration of proangiogenic factors was found in cdECM substrates and FL approaches compared with controls. Finally, the expression of proteins associated with tube formation (E‐selectin and thrombomodulin) was confirmed. These results highlight cdECM as promising biomaterial for adipose tissue engineering by inducing the spontaneous formation of prevascular‐like structures by mvECs.
Improvement of a three-layered in vitro skin model for topical application of irritating substances
(2020)
In the field of skin tissue engineering, the development of physiologically relevant in vitro skin models comprising all skin layers, namely epidermis, dermis, and subcutis, is a great challenge. Increasing regulatory requirements and the ban on animal experiments for substance testing demand the development of reliable and in vivo-like test systems, which enable high-throughput screening of substances. However, the reproducibility and applicability of in vitro testing has so far been insufficient due to fibroblast-mediated contraction. To overcome this pitfall, an advanced 3-layered skin model was developed. While the epidermis of standard skin models showed an 80% contraction, the initial epidermal area of our advanced skin models was maintained. The improved barrier function of the advanced models was quantified by an indirect barrier function test and a permeability assay. Histochemical and immunofluorescence staining of the advanced model showed well-defined epidermal layers, a dermal part with distributed human dermal fibroblasts and a subcutis with round-shaped adipocytes. The successful response of these advanced 3-layered models for skin irritation testing demonstrated the suitability as an in vitro model for these clinical tests: only the advanced model classified irritative and non-irritative substances correctly. These results indicate that the advanced set up of the 3-layered in vitro skin model maintains skin barrier function and therefore makes them more suitable for irritation testing.
The extracellular matrix (ECM) naturally surrounds cells in humans, and therefore represents the ideal biomaterial for tissue engineering. ECM from different tissues exhibit different composition and physical characteristics. Thus, ECM provides not only physical support but also contains crucial biochemical signals that influence cell adhesion, morphology, proliferation and differentiation. Next to native ECM from mature tissue, ECM can also be obtained from the in vitro culture of cells. In this study, we aimed to highlight the supporting effect of cell-derived- ECM (cdECM) on adipogenic differentiation. ASCs were seeded on top of cdECM from ASCs (scdECM) or pre-adipocytes (acdECM). The impact of ECM on cellular activity was determined by LDH assay, WST I assay and BrdU assay. A supporting effect of cdECM substrates on adipogenic differentiation was determined by oil red O staining and subsequent quantification. Results revealed no effect of cdECM substrates on cellular activity. Regarding adipogenic differentiation a supporting effect of cdECM substrates was obtained compared to control. With these results, we confirm cdECM as a promising biomaterial for adipose tissue engineering.
Human adipose-derived stem cells (hASCs) have become an important cell source for the use in tissue engineering and other medical applications. Not every biomaterial is suitable for human cell culture and requires surface modifications to enable cell adhesion and proliferation. Our hypothesis is that chemical surface modifications introduced by low-discharge plasma enhance the adhesion and proliferation of hASCs. Polystyrene (PS) surfaces were modified either by ammonia (NH3), carbon dioxide (CO2) or acrylic acid (AAc) plasma. The results show that the initial cell adhesion is significantly higher on all modified surfaces than on unmodified material as evaluated by bright field microscopy, live/dead staining, total DNA amount and scanning electron microscopy. The formation of focal adhesions was well pronounced on the Tissue Culture PS, NH3-, and CO2 plasma modified samples. The number of matured fibrillar adhesions was significantly higher on NH3 plasmamodified surfaces than on all other surfaces. Our study validates the suitability of chemical plasma activation and represents a method to enhance hASCs adhesion and improved cell expansion. All chemical modification promoted hASCs adhesion and can therefore be used for the modification of different scaffold materials whereby NH3-plasma modified surfaces resulted in the best outcome concerning hASCs adhesion and proliferation.