Spheroid culture has gained increasing popularity, arising as a promising tool for regenerative medicine applications. Importantly, spheroids may present advantages over single-cell suspensions in cell-based therapies (CT). Unfortunately, most growth media used for spheroid culture contain animal origin-components, such as fetal bovine serum (FBS). The presence of FBS compromises the safety of CT and presents economic and ethical constraints. SCC (supplement for cell culture) is a novel xeno-free (XF) industrial cell culture supplement, derived from well-controlled pooled human plasma and processed under good manufacturing practice rules. Here, we developed a XF SCC-based formulation for 2D-culture of outgrowth endothelial cells (OEC), and then used it for generating co-culture spheroids of OEC and mesenchymal stem cells (MSC). XF MSC-OEC spheroids were characterized in detail and compared to spheroids cultured in FBS-supplemented medium. XF spheroids presented comparable integrity, size and morphology as the reference culture. The use of both media resulted in spheroids with similar structure, abundant extracellular matrix deposition and specific patterns of OEC distribution and organization. Notably, XF spheroids presented significantly enhanced angiogenic potential, both in vitro (fibrin sprouting assay) and in vivo (CAM assay). These findings are particularly promising in the context of potential therapeutic applications.

Highlights Epifluorescence microscopy and image processing enable single-cell expression analysis. Escherichia coli biofilm heterogeneity increased during biofilm development. Fluorescence heterogeneity was correlated with spatial heterogeneity. 2 AbstractBioprocesses based on surface-associated microorganisms are emerging in environmental and industrial areas owing to the physiological specificities and heterogeneities of biofilm cells. This study describes a simple and accurate method for evaluating recombinant protein expression at a single-cell scale during Escherichia coli biofilm development. The model recombinant protein used was enhanced Green Fluorescent Protein (eGFP), as its intrinsic fluorescence allows the quantification of expression at both population and single-cell levels. Specific cell fluorescence intensity sharply increased during the first 4 days of biofilm cultivation and thereafter decreased abruptly to reach a low-level plateau until the end of the experiment. During biofilm development, the population became increasingly heterogeneous with regard to eGFP expression. Three distinct biofilm types were observed along the experimental time: one with a homogeneous population (days 3-5), the second with a moderately heterogeneous population (days 6-8) and the third with a strongly heterogeneous population (days 9-11). Observation of E. coli biofilms by confocal laser scanning microscopy demonstrated marked spatial heterogeneity, with the cells actively producing eGFP restricted to the top layer of the biofilm. The proposed methodology allows a fine analysis of the recombinant protein expression within E. coli biofilms, and it may be used for optimizing the processing conditions. Keywords: Biofilm; Escherichia coli; recombinant protein expression; fluorescence microscopy; single-cell scale; spatial heterogeneity Introduction 3The Gram-negative bacterium Escherichia coli is one of the most commonly used recombinant protein production hosts 1 due to its ability to grow rapidly and to high densities on inexpensive substrates, its well-known genetics and the availability of various systems for gene expression.2 Several bacteria such as E. coli naturally grow in a community attached to a substratum and not in liquid cultures. The biocatalytic potential of these bacterial communities, termed biofilms, can be attributed to their high cell density; the former feature is widely used for wastewater treatment 3 and also for the production of industrial chemicals such as ethanol, butanol and lactic acid. 4,5 Recombinant protein production in biofilms has been mostly studied in the context of waste biodegradation 6,7 ; however, this strategy could also be advantageous in other processes such as the biosynthesis of pharmaceutical intermediates 8 and catalysts for the food industry. In fact, using a recombinant Aspergillus niger strain, which contained a gene encoding the glucoamylase-GFP fusion protein, Talabardon and Yang 9 showed that higher amounts of GFP (Green Fluorescent Protein) and glucoamylase...

Angiogenesis, the formation of blood vessels from pre-existing ones, is a key event in pathology, including cancer progression, but also in homeostasis and regeneration. As the phenotype of endothelial cells (ECs) is continuously regulated by local biomechanical forces, studying endothelial behaviour in altered gravity might contribute to new insights towards angiogenesis modulation. This study aimed at characterizing EC behaviour after hypergravity exposure (more than 1g), with special focus on cytoskeleton architecture and capillary-like structure formation. Herein, human umbilical vein ECs (HUVECs) were cultured under two-dimensional and three-dimensional conditions at 3g and 10g for 4 and 16 h inside the large diameter centrifuge at the European Space Research and Technology Centre (ESTEC) of the European Space Agency. Although no significant tendency regarding cytoskeleton organization was observed for cells exposed to high g's, a slight loss of the perinuclear localization of b-tubulin was observed for cells exposed to 3g with less pronounced peripheral bodies of actin when compared with 1g control cells. Additionally, hypergravity exposure decreased the assembly of HUVECs into capillary-like structures, with a 10g level significantly reducing their organization capacity. In conclusion, short-term hypergravity seems to affect EC phenotype and their angiogenic potential in a time and g-level-dependent manner.