Classification of analytics, sensorics, and bioanalytics with polyelectrolyte multilayer capsules

Polyelectrolyte multilayer (PEM) capsules, constructed by LbL (layer-by-layer)-adsorbing polymers on sacrificial templates, have become important carriers due to multifunctionality of materials adsorbed on their surface or encapsulated into their interior. They have been also been used broadly used as analytical tools. Chronologically and traditionally, chemical analytics has been developed first, which has long been synonymous with all analytics. But it is not the only development. To the best of our knowledge, a summary of all advances including their classification is not available to date. Here, we classify analytics, sensorics, and biosensorics functionalities implemented with polyelectrolyte multilayer capsules and coated particles according to the respective stimuli and application areas. In this classification, three distinct categories are identified: (I) chemical analytics (pH; K+, Na+, and Pb2+ ion; oxygen; and hydrogen peroxide sensors and chemical sensing with surface-enhanced Raman scattering (SERS)); (II) physical sensorics (temperature, mechanical properties and forces, and osmotic pressure); and (III) biosensorics and bioanalytics (fluorescence, glucose, urea, and protease biosensing and theranostics). In addition to this classification, we discuss also principles of detection using the above-mentioned stimuli. These application areas are expected to grow further, but the classification provided here should help (a) to realize the wealth of already available analytical and bioanalytical tools developed with capsules using inputs of chemical, physical, and biological stimuli and (b) to position future developments in their respective fields according to employed stimuli and application areas

AFM analysis enables differentiation between apoptosis, necroptosis, and ferroptosis in murine cancer cells

Regulated cell death (RCD) has a fundamental role in development, pathology, and tissue homeostasis. In order to understand the RCD mechanisms, it is essential to follow these processes in real time. Here, atomic force microscopy (AFM) is applied to morphologically and mechanically characterize four RCD modalities (intrinsic and extrinsic apoptosis, necroptosis, and ferroptosis) in murine tumor cell lines. The nano-topographical analysis revealed a distinct surface morphology in case of necroptosis, ∼ 200 nm membrane disruptions are observed. Using mechanical measurements, it is possible to detect the early onset of RCD. Combined elasticity and microrheology analysis allowed for a clear distinction between apoptotic and regulated necrotic cell death. Finally, immunofluorescence analysis of the cytoskeleton structure during the RCD processes confirm the measured mechanical changes. The results of this study not only demonstrate the possibility of early real-time cell death detection but also reveal important differences in the cytoskeletal dynamics between multiple RCD modalities

Identification and analysis of key parameters for the ossification on particle functionalized composites hydrogel materials

Developing materials for tissue engineering and studying the mechanisms of cell adhesion is a complex and multifactor process that needs analysis using physical chemistry and biology. The major challenge is the labor-intensive data mining as well as requirements of the number of advanced techniques. For example, hydrogel-based biomaterials with cell-binding sites, tunable mechanical properties and complex architectures have emerged as a powerful tool to control cell adhesion and proliferation for tissue engineering. Composite hydrogels could be used for bone tissue regeneration, but they exhibit poor ossification properties. In current work, we have designed new osteoinductive gellan gum hydrogels by a thermal annealing approach and consequently functionalized them with Ca/Mg carbonates submicron particles. Determination of key parameters, which influence a successful hydroxyapatite generation, were done via the principal component analysis of 18 parameters (Young’s modulus of the hydrogel and particles, particles size and mass) and cell behaviour at various time points (like viability, numbers of the cells, rate of alkaline phosphatase production and cells area) obtained by characterizing such composite hydrogel. It is determined that the particles size and concentration of calcium ions have a dominant effect on the hydroxyapatite formation, because of providing local areas with a high Young’s modulus in a hydrogel – a desirable property for cell adhesion. The presented here detailed analysis allows identifying hydrogels for cell growth applications, while on the other hand, material properties can be predicted, and their overall number can be minimized leading to efficient optimization of bone reconstruction and other cell growth applications

Carbon nanotubes transform soft gellan gum hydrogels into hybrid organic–inorganic coatings with excellent cell growth capability

Carbone nanotubes (CNTs) possess distinct properties, for example, hardness, which is very complementary to biologically relevant soft polymeric and protein materials. Combining CNTs with bio-interfaces leads to obtaining new materials with advanced properties. In this work, we have designed novel organic-inorganic hybrid coatings by combining CNTs with gellan gum (GG) hydrogels. The surface topography of the samples is investigated using scanning electron microscopy and atomic force microscopy. Mechanical properties of synthesized hybrid materials are both assessed at the macro-scale and mapped at the nanoscale. A clear correlation between the CNT concentration and the hardness of the coatings is revealed. Cell culture studies show that effective cell growth is achieved at the CNT concentration of 15 mg/mL. The presented materials can open new perspectives for hybrid bio-interfaces and can serve as a platform for advanced cell culture

Effects of fibrillin mutations on the behavior of heart muscle cells in Marfan syndrome

Marfan syndrome (MFS) is a systemic disorder of connective tissue caused by pathogenic variants in the fibrillin-1 (FBN1) gene. Myocardial dysfunction has been demonstrated in MFS patients and mouse models, but little is known about the intrinsic effect on the cardiomyocytes (CMs). In this study, both induced pluripotent stem cells derived from a MFS-patient and the line with the corrected FBN1 mutation were differentiated to CMs. Several functional analyses are performed on this model to study MFS related cardiomyopathy. Atomic force microscopy revealed that MFS CMs are stiffer compared to corrected CMs. The contraction amplitude of MFS CMs is decreased compared to corrected CMs. Under normal culture conditions, MFS CMs show a lower beat-to-beat variability compared to corrected CMs using multi electrode array. Isoproterenol-induced stress or cyclic strain demonstrates lack of support from the matrix in MFS CMs. This study reports the first cardiac cell culture model for MFS, revealing abnormalities in the behavior of MFS CMs that are related to matrix defects. Based on these results, we postulate that impaired support from the extracellular environment plays a key role in the improper functioning of CMs in MFS

Effects of fibrillin mutations on the behavior of heart muscle cells in Marfan syndrome

Abstract: Marfan syndrome (MFS) is a systemic disorder of connective tissue caused by pathogenic variants in the fibrillin-1 (FBN1) gene. Myocardial dysfunction has been demonstrated in MFS patients and mouse models, but little is known about the intrinsic effect on the cardiomyocytes (CMs). In this study, both induced pluripotent stem cells derived from a MFS-patient and the line with the corrected FBN1 mutation were differentiated to CMs. Several functional analyses are performed on this model to study MFS related cardiomyopathy. Atomic force microscopy revealed that MFS CMs are stiffer compared to corrected CMs. The contraction amplitude of MFS CMs is decreased compared to corrected CMs. Under normal culture conditions, MFS CMs show a lower beat-to-beat variability compared to corrected CMs using multi electrode array. Isoproterenol-induced stress or cyclic strain demonstrates lack of support from the matrix in MFS CMs. This study reports the first cardiac cell culture model for MFS, revealing abnormalities in the behavior of MFS CMs that are related to matrix defects. Based on these results, we postulate that impaired support from the extracellular environment plays a key role in the improper functioning of CMs in MFS

Vaccination with early ferroptotic cancer cells induces efficient antitumor immunity

Background: Immunotherapy represents the future of clinical cancer treatment. The type of cancer cell death determines the antitumor immune response and thereby contributes to the efficacy of anticancer therapy and long-term survival of patients. Induction of immunogenic apoptosis or necroptosis in cancer cells does activate antitumor immunity, but resistance to these cell death modalities is common. Therefore, it is of great importance to find other ways to kill tumor cells. Recently, ferroptosis has been identified as a novel, iron-dependent form of regulated cell death but whether ferroptotic cancer cells are immunogenic is unknown. Methods: Ferroptotic cell death in murine fibrosarcoma MCA205 or glioma GL261 cells was induced by RAS-selective lethal 3 and ferroptosis was analyzed by flow cytometry, atomic force and confocal microscopy. ATP and high-mobility group box 1 (HMGB1) release were detected by luminescence and ELISA assays, respectively. Immunogenicity in vitro was analyzed by coculturing of ferroptotic cancer cells with bone-marrow derived dendritic cells (BMDCs) and rate of phagocytosis and activation/maturation of BMDCs (CD11c(+)CD86(+), CD11c(+)CD40(+), CD11c(+)MHCII(+), IL-6, RNAseq analysis). The tumor prophylactic vaccination model in immune-competent and immune compromised (Rag-2(-/-)) mice was used to analyze ferroptosis immunogenicity. Results: Ferroptosis can be induced in cancer cells by inhibition of glutathione peroxidase 4, as evidenced by confocal and atomic force microscopy and inhibitors' analysis. We demonstrate for the first time that ferroptosis is immunogenic in vitro and in vivo. Early, but not late, ferroptotic cells promote the phenotypic maturation of BMDCs and elicit a vaccination-like effect in immune-competent mice but not in Rag-2(-/-) mice, suggesting that the mechanism of immunogenicity is very tightly regulated by the adaptive immune system and is time dependent. Also, ATP and HMGB1, the best-characterized damage-associated molecular patterns involved in immunogenic cell death, have proven to be passively released along the timeline of ferroptosis and act as immunogenic signal associated with the immunogenicity of early ferroptotic cancer cells. Conclusions: These results pave the way for the development of new therapeutic strategies for cancers based on induction of ferroptosis, and thus broadens the current concept of immunogenic cell death and opens the door for the development of new strategies in cancer immunotherapy

Mechanobiology dynamics in regulated cell death : cells under pressure

Every day, over 50 billion cells die in our body. Luckily, one does not have to worry about this since regulated cell death (RCD) is an essential process to maintain homeostasis in our tissues and bodies. Apoptosis was for a long time seen as the only form of RCD. Since the beginning of 21st century an increasing number of different cellular pathways that lead to RCD have been uncovered, which are referred to as different RCD modalities (e.g., necroptosis, ferroptosis, pyroptosis, NETosis, …). With increasingly broad research performed in the field of RCD, increasing understanding emerged on how these cell death modalities and more importantly malfunctions in these processes are involved in ubiquitous pathologies among which are neurodegenerative diseases, systemic inflammation, and cancer. The omnipresence of RCD processes throughout life underlines the importance of having thorough understanding of what is occurring during these RCD modalities and what differentiates them from each other. Previously research in this field has been focusing on the morphological and biochemical changes during the different RCD modalities. In this work the goal is to explore how mechanobiology is involved in different RCD modalities and how this knowledge can be used for increased insight in the occurring immune reactions towards RCD which will be essential in developing new treatment strategies for diseases such as Alzheimer’s disease and cancer. First, by using a single cell Atomic Force Microscopy (AFM) analysis, it is possible to detect distinct changes in the (visco)elastic properties of cells during four different RCD modalities (intrinsic apoptosis, extrinsic apoptosis, necroptosis and ferroptosis) (1). While AFM provides a high sensitivity single cell data analysis, the low throughput hinders its use as a real diagnostic tool. Therefore, to address this, a high throughput mechano-cytometry method is developed and applied to investigate their correlation with data obtained by AFM. Results from this analysis indicated a clear clustering between viable and dead cancer cells based on differences in mechanical properties (2). Finally, knowledge gained on these changes of the mechanical properties of dying/dead cancer cells was used to gain further understanding in interaction between dead cancer cells and immune cells. From this analysis, it could be concluded that increasing the Young’s modulus of dead cancer cells (using Layer-by-Layer coatings) leads to an increase of their uptake (i.e., efferocytosis) by macrophages (3). The results presented in this work shed a light on the importance of mechanobiological characteristics in essential cellular processes such as cancer cell death. Furthermore, these results provide important future outlook for label-free diagnostic purposes to discriminate different types of RCD and development of new treatment strategies for such diseases as cancer. To obtain the results shown in this work, cells were put “under pressure”, both literally and figuratively

High-throughput mechano-cytometry as a method to detect apoptosis, necroptosis, and ferroptosis

In recent years, the importance of the investigation of regulated cell death (RCD) has significantly increased and different methods are proposed for the detection of RCD including biochemical as well as fluorescence assays. Researchers have shown that early stages of cell death could be detected by using AFM. Although AFM offers a high single-cell resolution and sensitivity, the throughput (<100 cells/h) limits a broad range of biomedical applications of this technique. Here, a microfluidics-based mechanobiology technique, named shear flow deformability cytometry (sDC), is used to investigate and distinguish dying cells from viable cells purely based on their mechanical properties. Three different RCD modalities (i.e., apoptosis, necroptosis, and ferroptosis) are induced in L929sAhFas cells and analysed using sDC. Using machine learning on the extracted parameters, it was possible to predict the dead or viable state with 92% validation accuracy. A significant decrease in elasticity can be noticed for each of these RCD modalities by analysing the deformation of the dying cells. Analysis of morphological characteristics such as cell size and membrane irregularities also indicated significant differences in the RCD induced cells versus control cells. These results highlight the importance of mechanical properties during RCD and the significance of label-free techniques, such as sDC, which can be used to detect regulated cell death and can be further linked with sorting of live and dead cells

Deep learning with digital holographic microscopy discriminates apoptosis and necroptosis

Regulated cell death modalities such as apoptosis and necroptosis play an important role in regulating different cellular processes. Currently, regulated cell death is identified using the golden standard techniques such as fluorescence microscopy and flow cytometry. However, they require fluorescent labels, which are potentially phototoxic. Therefore, there is a need for the development of new label-free methods. In this work, we apply Digital Holographic Microscopy (DHM) coupled with a deep learning algorithm to distinguish between alive, apoptotic and necroptotic cells in murine cancer cells. This method is solely based on label-free quantitative phase images, where the phase delay of light by cells is quantified and is used to calculate their topography. We show that a combination of label-free DHM in a high-throughput set-up (similar to 10,000 cells per condition) can discriminate between apoptosis, necroptosis and alive cells in the L929sAhFas cell line with a precision of over 85%. To the best of our knowledge, this is the first time deep learning in the form of convolutional neural networks is applied to distinguish-with a high accuracy-apoptosis and necroptosis and alive cancer cells from each other in a label-free manner. It is expected that the approach described here will have a profound impact on research in regulated cell death, biomedicine and the field of (cancer) cell biology in general