Search Results (80)

The potential of water-soluble membrane proteins (wsMPs) has not been fully realized. In this article, we exploit the nearly identical functionality of wsMPs with their membrane-bound counterparts and show that we can create water-soluble membrane proteins that incorporate into the plasma membranes of cells and alter their fate. As a proof of concept, we demonstrate the functional properties of water-soluble engineered pore-forming proteins, K⁺ ionic channels (MthK), and constitutively active GPCRs—among them frizzled receptors—both in vitro and in vivo. We call this method in vivo deployment of recombinant viable MPs, iDRIVE. Furthermore, we demonstrate that our strategy mediates the unidirectional insertion of MPs into the plasma membrane, and through constitutively active receptors, we present evidence for similar signaling pathway activation between small molecules and our water-soluble proteins using model phenotypes and molecular signaling assays. We present three examples where wsMPs are functional in dictating cellular fate, both in vitro and in vivo. Lastly, we show the induction of similar differential methylation via the activation of the Wnt signaling pathway using the conventional small molecule agonist, CHIR99021, or our wsFrizzled receptors (iDRIVE-FZD) in human embryonic kidney (HEK 293) embryoid spheroids (ESs). Additionally, we show that Wnt activation via wsFrizzled receptors results in even more biologically relevant epigenetic changes than via the small molecule CHIR99021. Future work will employ iDRIVE to differentiate stem cells in the production of research and clinically relevant organoids. Full article

The growing demand for alternative meat products is accelerating research into reproductive cell sources for cell-based meat processes, also called cultured meat. Porcine umbilical cord tissue is recognized as an advantageous source of mesenchymal stem cells (MSCs). However, effective decontamination must be achieved without compromising tissue integrity and cell recovery. In this study, we evaluated the decontamination of porcine umbilical cords using plasma-functionalized liquid (PFL) generated by a microwave-driven plasma source. It was applied alone and in combination with ultrasound, with the combined approach demonstrating superior performance. Specifically, the ultrasound–PFL combination treatment reduced the initial microbial load of individual tissue samples, ranging from 4.08 to 7.41 log₁₀ CFU/g, approaching the limit of detection of the applied microbiological assays. Statistical analysis indicated a significant contribution of both PFL and ultrasound to microbial reduction, while mesenchymal stem cell yields (5.4 × 10⁵ cells/g tissue) and cell viability (84%) remained comparable to antibiotic-rinsed controls. Recovered cells retained functional capacity, as demonstrated by successful 3D spheroid formation. These results highlight ultrasound-assisted PFL rinsing as an efficient, long-lasting, and antibiotic-free decontamination strategy without compromising tissue compatibility. This study thereby extends the application of plasma-functionalized liquids and demonstrates the feasibility of sustainable stem cell sourcing. It offers opportunities in cultured meat bioprocessing. Full article

Direct metal laser sintering (DMLS) is an advanced powder bed fusion (PBF) technology widely utilized in the medical device and aerospace sectors for the production of intricate and high-value components. The powdered metal materials used in the DMLS process can be expensive, and it is uncommon for a single build to exhaust an entire batch of powder. As a result, the un-melted powder characterized by differences in particle size and morphology compared to fresh virgin powder is recommended to be recycled for use in subsequent builds. This comprehensive review delves into the essential role that powder quality plays in the realm of DMLS with a particular focus on effective and sustainable powder recycling strategies. In this study, the effects of recycling stainless steel powder, specifically used in the DMLS process, are rigorously investigated in relation to the quality of the finished components. This paper monitors critical powder material characteristics, including particle size, particle morphology, and the overall bulk chemical composition throughout the recycling workflow. Furthermore, this review brings to light significant challenges associated with the recycling of stainless steel powders, such as the need to maintain consistency in particle size and shape, manage contamination risks, and mitigate the degradation effects that can arise from repeated usage, including wear, fragmentation, and oxidation of the particles. In addition, this paper explores a variety of recycling techniques aimed at rejuvenating powder quality. These techniques, including sieving, blending, and plasma spheroidization, are emphasized for their vital role in restoring the integrity of recycled powders and facilitating their reuse in innovative and efficient manufacturing processes. Full article

Geodiamolides are depsipeptides previously isolated from marine sponges that are able to disrupt cytoskeleton microfilaments, inhibit cell migration and invasion, and reverse the malignant phenotype of human breast cancer cell lines to polarized spheroid-like structures. Such cytotoxicity to different cellular targets in breast cancer cells suggests that these molecules might also act in other cancer types such as non-melanoma skin cancer (NMSC), one of the cancer types with high incidence worldwide. Thus, the goal of this work was to study the effects of the marine sponge depsipeptides Geodiamolide A and H (Geo A and Geo H) in human squamous cell carcinoma (A431, NMSC) in order to investigate their effects on cell proliferation and cell death. While no significant statistical difference was observed after Geo H treatment, an expressive dose-dependent reduction in A431 cell viability (IC₅₀ of 368 nM, MTT assay; p < 0.05) and proliferation pattern (real-time cell analysis assay) was shown after 48 h exposure with Geo A. The cell proliferation blockade was confirmed after 24 h of Geo A treatment at 500 nM, with a 46% (p < 0.0001) reduction in the total number of cells (cell counting) and G2/M phase cell cycle arrest. Other cytotoxic evidence such as DNA fragmentation, phosphatidylserine exposure (flow cytometry), and time-dependent plasma membrane damage (Trypan Blue) suggested cell death by apoptosis. Therefore, Geo A showed both cytostatic and cytotoxic effects on A431 cells. Taken together, these data point out Geo A as a promising therapeutic molecule for NMSC treatment and is the first depsipeptide (marine or terrestrial), to our knowledge, to target this type of cancer cell. Full article

Introduction: A 3D endothelial spheroid model expressing mosaic gain-of-function KRAS mutations was established to further understand the molecular changes associated with sporadic brain arteriovenous malformations (AVMs). Methods: Repellent 96-well U-bottom plates were seeded with human cerebral microvascular endothelial cells and resultant spheroids transduced with recombinant adeno-associated virus expressing KRAS^G12V. Spheroids were monitored using live-cell imaging for extended culture periods. Results: In the early growth period, KRAS^G12V expression increased spheroid growth rates and enhanced spheroid sprouting on gel matrices consistent with known AVM characteristics. With extended culture, novel endothelial characteristics were observed. KRAS^G12V-expressing spheroids displayed dynamic blebbing associated with the formation of rounded, hypertrophic cells disposed to engage in spheroid escape. These cells displayed reduced cell–cell adherence with rapid plasma membrane blebbing characteristic of amoeboid-like migration and mesenchymal-to-amoeboid transition. Spheroid growth and blebbing were reversed with MEK and mTOR inhibitors; Rho/ROCK inhibition specifically targeted the blebbing phenotype. Conclusions: Endothelial spheroids expressing KRAS^G12V exhibit characteristic features associated with abnormal vessel development in brain AVMs as well as novel phenotypes not previously observed in 2D monolayers. The ability to extend culture periods in this simple 3D model may allow further phenotypic exploration of important AVM driver mutations. Full article

Despite its ultrahigh theoretical capacity, silicon anodes for lithium-ion batteries suffer from severe capacity decay caused by over 300% volume changes during cycling. While Si–Ge alloying and spherical nanostructuring have been demonstrated to improve ionic/electronic transport and mechanical resilience, scalable synthesis of homogeneous, sub-150 nm SiGe nanospheres from low-cost precursors remains challenging. Here, we report a hybrid plasma-spraying physical vapor deposition (PS-PVD) process that directly converts metallurgical-grade Si and Ge powders into phase-pure Si_0.8Ge_0.2 nanospheres (<100 nm) at a continuous rate of 1 g min⁻¹. The co-condensation mechanism during formation was elucidated through molecular dynamics (MD) simulations, which revealed a process initiated by inhomogeneous nucleation and followed by uniform cluster growth and spheroidization. Multiscale characterization confirmed the spherical morphology, compositional uniformity, and crystalline structure of the produced Si_0.8Ge_0.2 nanoparticles. The resulting anodes exhibited a stable capacity of ~1500 mAh g⁻¹ at 0.1C over 100 cycles (>80% retention) and a Coulombic efficiency of ~98%. This approach bridges the gap between high-performance design and industrial manufacturability, offering a practical route to next-generation anodes for electric vehicles. Full article

Cell-penetrating peptides offer a promising strategy for intracellular delivery; however, non-specific uptake and off-target cytotoxicity limit their clinical utility. To address these limitations, a cold atmospheric plasma-responsive delivery platform was developed in which the membrane activity of a peptide was transiently suppressed upon complexation with a DNA-based nanostructure. Upon localized plasma exposure, DNA masking was disrupted, restoring the biological functions of the peptides. Transmission electron microscopy revealed that the synthesized DNA nanoflower structures were approximately 150–250 nm in size. Structural and functional analyses confirmed that the system remained inert under physiological conditions and was rapidly activated by plasma treatment. Fluorescence recovery, cellular uptake assays, and cytotoxicity measurements demonstrated that the peptide activity could be precisely controlled in both monolayer and three-dimensional spheroid models. This externally activatable nanomaterial-based system enables the spatial and temporal regulation of peptide function without requiring biochemical triggers or permanent chemical modifications. This platform provides a modular strategy for the development of potential peptide therapeutics that require precise control of activation in complex biological environments. Full article

Multiple myeloma is a hematologic malignancy characterized by the clonal proliferation of plasma cells within the bone marrow. The tumor microenvironment plays a crucial role in multiple myeloma pathogenesis, progression, and drug resistance. Traditional two-dimensional cell culture models have been instrumental in multiple myeloma research. However, they fail to recapitulate the complex in vivo bone marrow microenvironment, leading to limited predictive value for clinical outcomes. Three-dimensional cell culture models emerged as more physiologically relevant systems, offering enhanced insights into multiple myeloma biology. Scaffold-based systems (e.g., hydrogels, collagen, and Matrigel), scaffold-free spheroids, and bioprinted models have been developed to simulate the bone marrow microenvironment, incorporating key components like mesenchymal stromal cells, osteoblasts, endothelial cells, and immune cells. These models enable the functional assessment of cell adhesion-mediated drug resistance, cytokine signaling networks, and hypoxia-induced adaptations, which are often lost in 2D cultures. Moreover, 3D platforms demonstrated improved predictive value in preclinical drug screening, facilitating the evaluation of novel agents and combination therapies in a setting that better mimics the in vivo tumor context. Hence, 3D cultures represent a pivotal step toward bridging the gap between basic myeloma research and translational applications, supporting the development of more effective and patient-specific therapies. Full article

This work evaluates the effectiveness of in situ and ex situ passivation methods for mitigating the pyrophoricity of uranium–6 wt.% niobium spherical powders produced via the hydride–dehydride process coupled with plasma spheroidization. Oxide layer thickness was characterized using STEM/EDX, and pyrophoricity was assessed by a UN-recommended test method, which involves directly dropping the powders in the air. In situ passivation, performed by introducing flowing oxygen during spheroidization, produced oxide layers ranging from tens to hundreds of nanometers but resulted in inconsistent pyrophoricity mitigation at lower oxygen flow rates. Ex situ passivation, achieved by slow oxygen exposure over several months, formed uniform oxide layers of approximately 20 nm and consistently mitigated pyrophoricity. Despite requiring higher bulk oxygen content, in situ passivation enables faster processing and control of oxygen, while ex situ passivation achieves superior oxide uniformity with lower oxygen incorporation. These findings highlight the trade-offs between passivation methods and provide a foundation for improving the safety and scalability of reactive metal powder production. Full article

This work presents a new approach for the fabrication of 316L/Al₂O₃ composites, based on a combination of spray granulation, radio frequency (RF) plasma spheroidization and spark plasma sintering (SPS). Initially, a suspension containing 316L and alumina powders is formulated by precisely adjusting the pH and selecting an appropriate dispersant, thereby ensuring homogeneous dispersion of the constituents. The spray granulation process then produces granules with controlled size and morphology. RF plasma spheroidization, carried out using a TekSphero-40 system, is investigated by varying parameters such as the power, gas flow rates, injection position and feed rate, in order to optimize the formation of spherical and dense particles. The analysis reveals a marked sensitivity to heat transfer from the plasma to the particles, with a tendency for fine particles to segregate, which underscores the necessity for precise control of the processing conditions. Finally, SPS densification, performed under a constant pressure and a rigorously controlled thermal cycle, yields composites with excellent density and hardness characteristics. This study thus demonstrates that the proposed hybrid process offers an optimal synergy between a uniform distribution of alumina and a controlled microstructure, opening up promising avenues for the design of high-performance composite materials for demanding applications. Full article

Standard 2D cultures inadequately mimic the natural microenvironment of mesenchymal stromal cells (MSCs), compromising their properties. This study investigated the impact of 3D cultures in spheroids, alginate microspheres (AMSs), and blood plasma scaffolds on human-adipose-derived MSC behavior. The cell morphology, viability/apoptosis (6-CFDA/Annexin-Cy3.18), actin filament development (phalloidin-FITC), and metabolic activity (Alamar Blue) were assessed on the 3rd day of the generated 3D construct cultures. The abilities for adipogenic and osteogenic differentiation were evaluated after 21 days of culture in media with inducers by Nile Red and Alizarin Red staining, respectively. The 3D culture supported closer-to-physiological cell interactions and morphology and resulted in F-actin reduction compared with the 2D culture. While the metabolic activity was elevated in the scaffolds, it was significantly reduced in the spheroids and AMSs, which reflected natural-like quiescence. The differentiation was maintained across all the 3D constructs. These findings highlight the essential influence of 3D construct design on MSC function, underscoring its potential for advancing both in vitro models and cell-based therapies. Full article

Background/Objectives: Cold atmospheric plasma (CAP) has shown a strong anticancer effect on a variety of tumors, presenting a new approach for the effective treatment of oral squamous cell carcinoma (OSCC), one of the most prevalent malignant neoplasms with a high mortality rate. Here, we aimed to comprehensively investigate the antitumor potential of two approaches of CAP treatment on both two-dimensional and three-dimensional OSCC cell line models, as well as to analyze whether plasma treatment enhances the sensitivity of OSCC to chemotherapy. Methods: An in-house designed plasma needle, with helium as a working gas, was used to treat the SCC-25 cell line directly or indirectly via plasma-treated medium (PTM). The antitumor effect of CAP was assessed by measuring cell viability, apoptosis, adhesion, and migration. In addition, the combined effect of PTM and cisplatin was analyzed in SCC-25 tumor spheroids, as a more complex and reliable in vitro model. Results: Both plasma treatments showed time-dependent antitumor effects affecting their viability, adhesion, and migration. The rate of apoptosis was higher after incubation with PTM and is mediated by the intrinsic pathway. By utilizing the 3D spheroid carcinoma model, we confirmed the antitumor potential of CAP and additionally demonstrated an increased chemosensitivity of PTM-treated carcinoma cells. Conclusions: The results of our study illustrate a promising avenue for the application of CAP as a therapeutic option for OSCC, either as a standalone treatment or in combination with cisplatin. Full article

Melanoma is an aggressive disease that arises from mutations in the cells that produce the pigment melanin, melanocytes. Melanoma is characterized by a high mortality rate, due to avoidance of applied therapies and metastasis to other organs. The peculiar features of boron neutron capture therapy (BNCT), particularly its cell-level selectivity, make BNCT a promising modality for melanoma treatment. However, appropriate cellular models should be used to study new therapies or improve the efficacy of existing therapies. Spheroids, which have been used for years for in vitro studies of the efficacy of anti-cancer therapies, have many characteristics shared with tumors through which they can increase the accuracy of the cellular response compared to 2D culture in vitro studies and reduce the use of animals for research in the future. To the best of our knowledge, when we started researching the use of spheroids in BNCT in vitro, there was no publication showing such use. Our study aimed to evaluate the efficacy of a 3D cellular model (spheroids) for testing BNCT on melanoma cells. We assessed boronophenylalanine (¹⁰BPA) uptake using inductively coupled plasma mass spectrometry in both spheroids and 2D cultures of melanoma and melanocytes. DNA damage, Ki67 protein expression, and spheroid growth were analyzed. The experimental groups included: (1) IR_B (neutron flux + 50 µg ¹⁰B/mL), (2) IR (neutron flux alone), (3) C_B (no irradiation, 50 µg ¹⁰B/mL), and (4) C (no irradiation and no treatment with boron). The total absorbed doses were estimated to be 2.1–3.1 Gy for IR_B cells and spheroids as well as 8.3–9.4 Gy for IR_B spheroids, while estimated doses for IR cells were 0.5–1.9 Gy. The results indicated that IR_B spheroids might exhibit a reduced diameter. Melanoma cells in the 3D model showed that their DNA damage levels may be higher than those in the 2D model. Moreover, the Ki67 assay revealed differences in the expression of this marker between irradiated melanoma cell lines. In conclusion, preincubation with ¹⁰BPA enhances BNCT efficacy, leading to cell growth inhibition and increased DNA fragmentation. Differences in DNA damage between 2D and 3D models may be due to dissimilarities in cell metabolism caused by a changed cell architecture. Full article

Alumina is the most widely used oxide ceramic, and its applications are widespread in engineering and in biomedical fields. Its spheroidization was performed by a prototypal direct current (DC) thermal plasma, which was designed and installed at ENEA, investigating surface morphology, particle size distribution, crystallinity, spheroidization, and reactivity. Features such as morphology and porosity significantly influence the flowability of the powder on the printer bed and, consequently, the density of the printed parts. It has been reported that spherical powder shape is highly recommended in additive manufacturing (AM) due to its superior flowability compared to other shapes whose interaction between powder particles results in poor flowability. In this paper, the spheroidization process of alumina powders using two different DC plasma powers and two kinds of secondary gas is reported. The average value of the circularity of the powders, after plasma treatment, has always been greater than or equal to 0.8 with the degree of the spheroidization over 90% at high power. The best process parameters of the thermal plasma were properly selected to produce spherical powders suitable for AM applications, and powders with high circularity were successfully obtained. Forming, debinding, and sintering tests were performed to verify the processability and the densification of produced powders, with good results in terms of density (97%). Full article

Lung cancer is the most common type of malignant tumor worldwide. Plasma-activated medium (PAM) is an innovative cancer treatment method that has received considerable scientific attention. The objective of this study is to evaluate the effects of PAM on the anti-tumor characteristics of non-small cell lung cancer (NSCLC) cells in two-dimensional (2D) and three-dimensional (3D) cultures. The effects of PAM treatment on the proliferative and migratory capabilities of A549 cells in 2D and 3D cultures were assessed using MTT, migration, invasion assays, and cell cycle, respectively. The study also investigated the impact of PAM treatment on the changes in the content of intracellular and extracellular reactive species and analyzed protein expression using the Western Blot method. PAM treatment inhibited the viability, migration, and invasion abilities of A549 cells in both 2D and 3D cultures, suppressed the epithelial–mesenchymal transition (EMT) process, and downregulated the expression of the RAS/ERK signaling pathway, which effectively inhibited tumor spheroid formation. Additionally, the effect of PAM on A549 cells was mediated through ROS-induced oxidative reactions, and PAM treatment exhibited greater cytotoxicity in 2D culture compared to 3D culture. As compared to 2D, the 3D cell culture model provides a viable in vitro cell model for studying the mechanisms of PAM treatment in lung cancer. PAM represents an effective new treatment for NSCLC. Full article