Xeno-Free In Vitro Cultivation and Osteogenic Differentiation of hAD-MSCs on Resorbable 3D Printed RESOMER®.

The development of alloplastic resorbable materials can revolutionize the field of implantation technology in regenerative medicine. Additional opportunities to colonize the three-dimensionally (3D) printed constructs with the patient's own cells prior to implantation can improve the regeneration process but requires optimization of cultivation protocols. Human platelet lysate (hPL) has already proven to be a suitable replacement for fetal calf serum (FCS) in 2D and 3D cell cultures. In this study, we investigated the in vitro biocompatibility of the printed RESOMER® Filament LG D1.75 materials as well as the osteogenic differentiation of human mesenchymal stem cells (hMSCs) cultivated on 3D printed constructs under the influence of different medium supplements (FCS, human serum (HS) and hPL). Additionally, the in vitro degradation of the material was studied over six months. We demonstrated that LG D1.75 is biocompatible and has no in vitro cytotoxic effects on hMSCs. Furthermore, hMSCs grown on the constructs could be differentiated into osteoblasts, especially supported by supplementation with hPL. Over six months under physiological in vitro conditions, a distinct degradation was observed, which, however, had no influence on the biocompatibility of the material. Thus, the overall suitability of the material LG D1.75 to produce 3D printed, resorbable bone implants and the promising use of hPL in the xeno-free cultivation of human MSCs on such implants for autologous transplantation have been demonstrated.

Comparative Analysis of Mesenchymal Stem Cell Cultivation in Fetal Calf Serum, Human Serum, and Platelet Lysate in 2D and 3D Systems.

In vitro two-dimensional (2D) and three-dimensional (3D) cultivation of mammalian cells requires supplementation with serum. Mesenchymal stem cells (MSCs) are widely used in clinical trials for bioregenerative medicine and in most cases, in vitro expansion and differentiation of these cells are required before application. Optimized expansion and differentiation protocols play a key role in the treatment outcome. 3D cell cultivation systems are more comparable to in vivo conditions and can provide both, more physiological MSC expansion and a better understanding of intercellular and cell-matrix interactions. Xeno-free cultivation conditions minimize risks of immune response after implantation. Human platelet lysate (hPL) appears to be a valuable alternative to widely used fetal calf serum (FCS) since no ethical issues are associated with its harvest, it contains a high concentration of growth factors and cytokines and it can be produced from expired platelet concentrate. In this study, we analyzed and compared proliferation, as well as osteogenic and chondrogenic differentiation of human adipose tissue-derived MSCs (hAD-MSC) using three different supplements: FCS, human serum (HS), and hPL in 2D. Furthermore, online monitoring of osteogenic differentiation under the influence of different supplements was performed in 2D. hPL-cultivated MSCs exhibited a higher proliferation and differentiation rate compared to HS- or FCS-cultivated cells. We demonstrated a fast and successful chondrogenic differentiation in the 2D system with the addition of hPL. Additionally, FCS, HS, and hPL were used to formulate Gelatin-methacryloyl (GelMA) hydrogels in order to evaluate the influence of the different supplements on the cell spreading and proliferation of cells growing in 3D culture. In addition, the hydrogel constructs were cultivated in media supplemented with three different supplements. In comparison to FCS and HS, the addition of hPL to GelMA hydrogels during the encapsulation of hAD-MSCs resulted in enhanced cell spreading and proliferation. This effect was promoted even further by cultivating the hydrogel constructs in hPL-supplemented media.

Gelatin-Methacryloyl (GelMA) Formulated with Human Platelet Lysate Supports Mesenchymal Stem Cell Proliferation and Differentiation and Enhances the Hydrogel's Mechanical Properties.

Three-dimensional (3D) cell culture is a major focus of current research, since cultivation under physiological conditions provides more reliable information about in vivo cell behavior. 3D cell cultures are used in basic research to better understand intercellular and cell-matrix interactions. However, 3D cell culture plays an increasingly important role in the in vitro testing of bioactive substances and tissue engineering. Gelatin-methacryloyl (GelMA) hydrogels of different degrees of functionalization (DoFs) are a versatile tool for 3D cell culture and related applications such as bioprinting. Human platelet lysate (hPL) has already demonstrated positive effects on 2D cell cultures of different cell types and has proven a valuable alternative to fetal calf serum (FCS). Traditionally, all hydrogels are formulated using buffers. In this study, we supplemented GelMA hydrogels of different DoF with hPL during adipose tissue-derived mesenchymal stem cell (AD-MSCs) encapsulation. We studied the effect of hPL supplementation on the spreading, proliferation, and osteogenic differentiation of AD-MSCs. In addition, the influence of hPL on hydrogel properties was also investigated. We demonstrate that the addition of hPL enhanced AD-MSC spreading, proliferation, and osteogenic differentiation in a concentration-dependent manner. Moreover, the addition of hPL also increased GelMA viscosity and stiffness.

The marker choice: Unexpected resolving power of an unexplored CO1 region for layered DNA barcoding approaches.

The potential of DNA barcoding approaches to identify single species and characterize species compositions strongly depends on the marker choice. The prominent "Folmer region", a 648 basepair fragment at the 5' end of the mitochondrial CO1 gene, has been traditionally applied as a universal DNA barcoding region for metazoans. In order to find a suitable marker for biomonitoring odonates (dragonflies and damselflies), we here explore a new region of the CO1 gene (CO1B) for DNA barcoding in 51 populations of 23 dragonfly and damselfly species. We compare the "Folmer region", the mitochondrial ND1 gene (NADH dehydrogenase 1) and the new CO1 region with regard to (i) speed and reproducibility of sequence generation, (ii) levels of homoplasy and (iii) numbers of diagnostic characters for discriminating closely related sister taxa and populations. The performances of the gene regions regarding these criteria were quite different. Both, the amplification of CO1B and ND1 was highly reproducible and CO1B showed the highest potential for discriminating sister taxa at different taxonomic levels. In contrast, the amplification of the "Folmer region" using the universal primers was difficult and the third codon positions of this fragment have experienced nucleotide substitution saturation. Most important, exploring this new barcode region of the CO1 gene identified a higher discriminating power between closely related sister taxa. Together with the design of layered barcode approaches adapted to the specific taxonomic "environment", this new marker will further enhance the discrimination power at the species level.

Direct exposure at the air-liquid interface: evaluation of an in vitro approach for simulating inhalation of airborne substances.

In toxicology, the strategies for testing the hazardous potential of substances are changing as a result of the ongoing progress in the development of in vitro methods and the demand of the authorities to reduce animal testing. Even in the complex field of inhalation toxicology with its high requirements on the technical implementation and cell culture models, the preconditions for using such methods are fulfilled. We here introduce a sophisticated technique that enables the stable and reproducible exposure of cultivated cells to airborne substances at the air-liquid interface by means of the CULTEX(®) Radial Flow System (RFS) module. The feasibility and suitability of the experimental setup is demonstrated by dose-response investigations of mainstream cigarette smoke and particulate matter of four substances in different lung epithelial cell lines. A dose-dependent cytotoxcity of the test substances was verified by applying different exposure times. The high reproducibility of the results indicate the reliability of the presented method and recommend the integration of such in vitro approaches in the field of inhalation toxicology by advancing their regulatory validation.

Importance of material evaluation prior to the construction of devices for in vitro techniques.

The development and validation of new in vitro methods in the field of toxicology have gained more importance in recent years due to stricter guidelines for animal testing, especially in the European Union. Consequently, advances in the construction of technical devices for the exposure of cell or tissue cultures to test substances are necessary. Here, to obtain reliable results, it is important to exclusively use materials that do not interfere with the cell viability. Thus, similar to the biomaterials testing of medical devices which is regulated in the Directive 93/42/EEC, the biocompatibility of the materials has to be verified prior to the construction of such devices. We present here a novel approach for biomaterials testing which allows the quantitative and qualitative assessment of cytotoxicity of material samples. Stainless steel and silicone are often used for laboratory equipment, due to their high chemical, thermal and mechanical resistance. However, our results highlight that some types of silicone may have adverse effects on cultured cells. Moreover, special methods for the surface treatment of metals may also be a critical factor for in vitro devices. Therefore, the testing of all materials coming in contact with cell cultures is highly recommended.