Extracellular vesicles (EVs) are cell-derived membrane structures exerting major effects in physiological as well as pathological processes by functioning as vehicles for the delivery of biomolecules to their target cells. An increasing number of effects previously attributed to cell-based therapies have been recognized to be actually mediated by EVs derived from the respective cells, suggesting the administration of purified EVs instead of living cells for cell-based therapies. In this review, we focus on the heterogeneity of EVs derived from mesenchymal stem/stromal cells (MSC) and summarize upstream process parameters that crucially affect the resulting therapeutic properties and biological functions. Hereby, we discuss the effects of the cell source, medium composition, 3D culture, bioreactor culture and hypoxia. Furthermore, aspects of the isolation and storage strategies influences EVs are described. Conclusively, optimization of upstream process parameters should focus on controlling MSC-derived EV heterogeneity for specific therapeutic applications. Graphical Abstract

3D printing is increasingly important for the rapid prototyping of advanced and tailor-made cell culture devices. In this context, stereolithography represents a method for the rapid generation of prototypes from photocurable polymers. However, the biocompatibility of commercially available photopolymers is largely unknown. Therefore, we evaluated the cytotoxicity of six polymers, two of them certified as biocompatible according to ISO 10993-5:2009, and we evaluated, if coating with Parylene, an inert polymer widely used in medical applications, might shield cells from the cytotoxic effects of a toxic polymer. In addition, we evaluated the processability, reliability, and consistency of the details printed. Human mesenchymal stem cells (MSCs) were used for cytotoxicity testing as they are widely used and promising for numerous applications in regenerative medicine. MSCs were incubated together with printed photopolymers, and the cytotoxicity was assessed. All photopolymers significantly reduced the viability of MSCs while the officially biocompatible resins displayed minor toxic effects. Further, coating with Parylene completely protected MSCs from toxic effects. In conclusion, none of the tested polymers can be fully recommended for rapid prototyping of cell culture devices. However, coating with Parylene can shield cells from toxic effects and thus might represent a viable option until more compatible materials are available.

Mesenchymal stem cells (MSCs) are of great interest for their use in cell-based therapies due to their multipotent differentiation and immunomodulatory capacities. In consequence of limited numbers following their isolation from the donor tissue, MSCs require extensive expansion performed in traditional 2D cell culture setups to reach adequate amounts for therapeutic use. However, prolonged culture of MSCs in vitro has been shown to decrease their differentiation potential and alter their immunomodulatory properties. For that reason, preservation of these physiological characteristics of MSCs throughout their in vitro culture is essential for improving the efficiency of therapeutic and in vitro modeling applications. With this objective in mind, many studies already investigated certain parameters for enhancing current standard MSC culture protocols with regard to the effects of specific culture media components or culture conditions. Although there is a lot of diversity in the final therapeutic uses of the cells, the primary stage of standard isolation and expansion is imperative. Therefore, we want to review on approaches for optimizing standard MSC culture protocols during this essential primary step of in vitro expansion. The reviewed studies investigate and suggest improvements focused on culture media components (amino acids, ascorbic acid, glucose level, growth factors, lipids, platelet lysate, trace elements, serum, and xenogeneic components) as well as culture conditions and processes (hypoxia, cell seeding, and dissociation during passaging), in order to preserve the MSC phenotype and functionality during the primary phase of in vitro culture.

The use of 3D cell cultures has gained increasing importance in medical and pharmaceutical research. However, the analysis of the culture medium is hardly representative for the culture conditions within a 3D model which hinders the standardization of 3D cultures and translation of results. Therefore, we developed a modular monitoring platform combining a perfusion bioreactor with an integrated minimally invasive sampling system and implemented sensors that enables the online monitoring of culture parameters and medium compounds within 3D cultures. As a proof-of-concept, primary cells as well as cell lines were cultured on a collagen or gelatin methacryloyl (GelMA) hydrogel matrix, while monitoring relevant culture parameters and analytes. Comparing the interstitial fluid of the 3D models versus the corresponding culture medium, we found considerable differences in the concentrations of several analytes. These results clearly demonstrate that analyses of the culture medium only are not relevant for the development of standardized 3D culture processes. The presented bioreactor with an integrated sampling and sensor platform opens new horizons for the development, optimization, and standardization of 3D cultures. Furthermore, this technology holds the potential to reduce animal studies and improve the transferability of pharmaceutical in vitro studies by gaining more relevant results, bridging the gap towards clinical translation.