Chemically defined xeno- and serum-free cell culture medium to grow human adipose stem cells

Adipose tissue is an abundant source of stem cells. However, liposuction cannot yield cell quantities sufficient for direct applications in regenerative medicine. Therefore, the development of GMP-compliant ex vivo expansion protocols is required to ensure the production of a “cell drug” that is safe, reproducible, and cost-effective. Thus, we developed our own basal defined xeno- and serum-free cell culture medium (UrSuppe), specifically formulated to grow human adipose stem cells (hASCs). With this medium, we can directly culture the stromal vascular fraction (SVF) cells in defined cell culture conditions to obtain hASCs. Cells proliferate while remaining undifferentiated, as shown by Flow Cytometry (FACS), Quantitative Reverse Transcription PCR (RT-qPCR) assays, and their secretion products. Using the UrSuppe cell culture medium, maximum cell densities between 0.51 and 0.80 × 105 cells/cm2 (=2.55–4.00 × 105 cells/mL) were obtained. As the expansion of hASCs represents only the first step in a cell therapeutic protocol or further basic research studies, we formulated two chemically defined media to differentiate the expanded hASCs in white or beige/brown adipocytes. These new media could help translate research projects into the clinical application of hASCs and study ex vivo the biology in healthy and dysfunctional states of adipocytes and their precursors. Following the cell culture system developers’ practice and obvious reasons related to the formulas’ patentability, the defined media’s composition will not be disclosed in this study

An approach towards a GMP compliant in-vitro expansion of human adipose stem cells for autologous therapies

Human Adipose Tissue Stem Cells (hASCs) are a valuable source of cells for clinical applications (e.g., treatment of acute myocardial infarction and inflammatory diseases), especially in the field of regenerative medicine. However, for autologous (patient-specific) and allogeneic (off-the-shelf) hASC-based therapies, in-vitro expansion is necessary prior to the clinical application in order to achieve the required cell numbers. Safe, reproducible and economic in-vitro expansion of hASCs for autologous therapies is more problematic because the cell material changes for each treatment. Moreover, cell material is normally isolated from non-healthy or older patients, which further complicates successful in-vitro expansion. Hence, the goal of this study was to perform cell expansion studies with hASCs isolated from two different patients/donors (i.e., different ages and health statuses) under xeno- and serum-free conditions in static, planar (2D) and dynamically mixed (3D) cultivation systems. Our primary aim was I) to compare donor variability under in-vitro conditions and II) to develop and establish an unstructured, segregated growth model as a proof-of-concept study. Maximum cell densities of between 0.49 and 0.65 × 105 hASCs/cm2 were achieved for both donors in 2D and 3D cultivation systems. Cell growth under static and dynamically mixed conditions was comparable, which demonstrated that hydrodynamic stresses (P/V = 0.63 W/m3, τnt = 4.96 × 10−3 Pa) acting at Ns1u (49 rpm for 10 g/L) did not negatively affect cell growth, even under serum-free conditions. However, donor-dependent differences in the cell size were found, which resulted in significantly different maximum cell densities for each of the two donors. In both cases, stemness was well maintained under static 2D and dynamic 3D conditions, as long as the cells were not hyperconfluent. The optimal point for cell harvesting was identified as between cell densities of 0.41 and 0.56 × 105 hASCs/cm2 (end of exponential growth phase). The growth model delivered reliable predictions for cell growth, substrate consumption and metabolite production in both types of cultivation systems. Therefore, the model can be used as a basis for future investigations in order to develop a robust MC-based hASC production process for autologous therapies

Activation of the S100A7/RAGE Pathway by IGF-1 Contributes to Angiogenesis in Breast Cancer

Background: Breast cancer (BC) mortality is increased among obese and diabetic patients. Both obesity and diabetes are associated with dysregulation of both the IGF-1R and the RAGE (Receptor for Advanced Glycation End Products) pathways, which contribute to complications of these disorders. The alarmin S100A7, signaling through the receptor RAGE, prompts angiogenesis, inflammation, and BC progression. Methods: We performed bioinformatic analysis of BC gene expression datasets from published studies. We then used Estrogen Receptor (ER)-positive BC cells, CRISPR-mediated IGF-1R KO BC cells, and isogenic S100A7-transduced BC cells to investigate the role of IGF-1/IGF-1R in the regulation of S100A7 expression and tumor angiogenesis. To this aim, we also used gene silencing and pharmacological inhibitors, and we performed gene expression and promoter studies, western blotting analysis, ChIP and ELISA assays, endothelial cell proliferation and tube formation assay. Results: S100A7 expression correlates with worse prognostic outcomes in human BCs. In BC cells, the IGF-1/IGF-1R signaling engages STAT3 activation and its recruitment to the S100A7 promoter toward S100A7 increase. In human vascular endothelial cells, S100A7 activates RAGE signaling and prompts angiogenic effects. Conclusions: In ER-positive BCs the IGF-1 dependent activation of the S100A7/RAGE signaling in adjacent endothelial cells may serve as a previously unidentified angiocrine effector. Targeting S100A7 may pave the way for a better control of BC, particularly in conditions of unopposed activation of the IGF-1/IGF-1R axis

Human adipose stem cells (hASCs) grown on biodegradable microcarriers in serum- and xeno-free medium preserve their undifferentiated status

Human adipose stem cells (hASCs) are promising candidates for cell-based therapies, but they need to be efficiently expanded in vitro as they cannot be harvested in sufficient quantities. Recently, dynamic bioreactor systems operated with microcarriers achieved considerable high cell densities. Thus, they are a viable alternative to static planar cultivation systems to obtain high numbers of clinical-grade hASCs. Nevertheless, the production of considerable biomass in a short time must not be achieved to the detriment of the cells’ quality. To facilitate the scalable expansion of hASC, we have developed a new serum- and xeno-free medium (UrSuppe) and a biodegradable microcarrier (BR44). In this study, we investigated whether the culture of hASCs in defined serum-free conditions on microcarriers (3D) or on planar (2D) cell culture vessels may influence the expression of some marker genes linked with the immature degree or the differentiated status of the cells. Furthermore, we investigated whether the biomaterials, which form our biodegradable MCs, may affect cell behavior and differentiation. The results confirmed that the quality and the undifferentiated status of the hASCs are very well preserved when they grow on BR44 MCs in defined serum-free conditions. Indeed, the ASCs showed a gene expression profile more compatible with an undifferentiated status than the same cells grown under standard planar conditions

Development of a biodegradable microcarrier for the cultivation of human adipose stem cells (hASCs) with a defined xeno- and serum-free medium

Stirred single-use bioreactors in combination with microcarriers (MCs) have established themselves as a technology that has the potential to meet the demands of current and future cell therapeutic markets. However, most of the published processes have been performed using fetal bovine serum (FBS) containing cell culture medium and non-biocompatible MCs. This approach has two significant drawbacks: firstly, the inevitable potential risks associated with the use of FBS for clinical applications; secondly, non-biocompatible MCs have to be removed from the cell suspension before implantation, requiring a step that causes loss of viable cells and adds further costs and complications. This study aimed to develop a new platform based on a chemically defined xeno- and serum-free cell culture medium and biodegradable MC that can support the growth of human adipose stem cells (hASCs) while still preserving their undifferentiated status. A specific combination of components and manufacturing parameters resulted in a MC prototype, called “BR44”, which delivered the desired functionality. MC BR44 allows the hASCs to stick to its surface and grow in a chemically defined xeno- and serum-free medium (UrSuppe). Although the cells’ expansion rate was not as high as with a commercial non-biodegradable standard MC, those cultured on BR44 maintained a better undifferentiated status in both static and dynamic conditions than those cultured on traditional 2D surfaces

Neutralization, effector function and immune imprinting of Omicron variants

Currently circulating SARS-CoV-2 variants have acquired convergent mutations at hot spots in the receptor-binding domain$^{1}$ (RBD) of the spike protein. The effects of these mutations on viral infection and transmission and the efficacy of vaccines and therapies remains poorly understood. Here we demonstrate that recently emerged BQ.1.1 and XBB.1.5 variants bind host ACE2 with high affinity and promote membrane fusion more efficiently than earlier Omicron variants. Structures of the BQ.1.1, XBB.1 and BN.1 RBDs bound to the fragment antigen-binding region of the S309 antibody (the parent antibody for sotrovimab) and human ACE2 explain the preservation of antibody binding through conformational selection, altered ACE2 recognition and immune evasion. We show that sotrovimab binds avidly to all Omicron variants, promotes Fc-dependent effector functions and protects mice challenged with BQ.1.1 and hamsters challenged with XBB.1.5. Vaccine-elicited human plasma antibodies cross-react with and trigger effector functions against current Omicron variants, despite a reduced neutralizing activity, suggesting a mechanism of protection against disease, exemplified by S309. Cross-reactive RBD-directed human memory B cells remained dominant even after two exposures to Omicron spikes, underscoring the role of persistent immune imprinting

A new line for laser-driven light ions acceleration and related TNSA studies

In this paper, we present the status of the line for laser-driven light ions acceleration (L3IA) currently under implementation at the Intense Laser Irradiation Laboratory (ILIL), and we provide an overview of the pilot experimental activity on laser-driven ion acceleration carried out in support of the design of the line. A description of the main components is given, including the laser, the beam transport line, the interaction chamber, and the diagnostics. A review of the main results obtained so far during the pilot experimental activity is also reported, including details of the laser-plasma interaction and ion beam characterization. A brief description of the preliminary results of a dedicated numerical modeling is also provided

Maturation of SARS-CoV-2 Spike-specific memory B cells drives resilience to viral escape

SUMMARYMemory B cells (MBCs) generate rapid antibody responses upon secondary encounter with a pathogen. Here, we investigated the kinetics, avidity and cross-reactivity of serum antibodies and MBCs in 155 SARS-CoV-2 infected and vaccinated individuals over a 16-month timeframe. SARS-CoV-2-specific MBCs and serum antibodies reached steady-state titers with comparable kinetics in infected and vaccinated individuals. Whereas MBCs of infected individuals targeted both pre- and postfusion Spike (S), most vaccine-elicited MBCs were specific for prefusion S, consistent with the use of prefusion-stabilized S in mRNA vaccines. Furthermore, a large fraction of MBCs recognizing postfusion S cross-reacted with human betacoronaviruses. The avidity of MBC-derived and serum antibodies increased over time resulting in enhanced resilience to viral escape by SARS-CoV-2 variants, including Omicron BA.1 and BA.2 sub-lineages, albeit only partially for BA.4 and BA.5 sublineages. Overall, the maturation of high-affinity and broadly-reactive MBCs provides the basis for effective recall responses to future SARS-CoV-2 variants

NURE: An ERC project to study nuclear reactions for neutrinoless double beta decay

Neutrinoless double beta decay (0νββ) is considered the best potential resource to access the absolute neutrino mass scale. Moreover, if observed, it will signal that neutrinos are their own anti-particles (Majorana particles). Presently, this physics case is one of the most important research “beyond Standard Model” and might guide the way towards a Grand Unified Theory of fundamental interactions. Since the 0νββ decay process involves nuclei, its analysis necessarily implies nuclear structure issues. In the NURE project, supported by a Starting Grant of the European Research Council (ERC), nuclear reactions of double charge-exchange (DCE) are used as a tool to extract information on the 0νββ Nuclear Matrix Elements. In DCE reactions and ββ decay indeed the initial and final nuclear states are the same and the transition operators have similar structure. Thus the measurement of the DCE absolute cross-sections can give crucial information on ββ matrix elements. In a wider view, the NUMEN international collaboration plans a major upgrade of the INFN-LNS facilities in the next years in order to increase the experimental production of nuclei of at least two orders of magnitude, thus making feasible a systematic study of all the cases of interest as candidates for 0νββ

Sicilia—silicon carbide detectors for intense luminosity investigations and applications

Silicon carbide (SiC) is a compound semiconductor, which is considered as a possible alternative to silicon for particles and photons detection. Its characteristics make it very promising for the next generation of nuclear and particle physics experiments at high beam luminosity. Silicon Carbide detectors for Intense Luminosity Investigations and Applications (SiCILIA) is a project starting as a collaboration between the Italian National Institute of Nuclear Physics (INFN) and IMM-CNR, aiming at the realization of innovative detection systems based on SiC. In this paper, we discuss the main features of silicon carbide as a material and its potential application in the field of particles and photons detectors, the project structure and the strategies used for the prototype realization, and the first results concerning prototype production and their performance