Differences in interaction of graphene/graphene oxide with bacterial and mammalian cell membranes.

Graphene, a single layer, hexagonally packed two-dimensional carbon sheet is an attractive candidate for diverse applications including antibacterial potential and drug delivery. One of the knowledge gaps in biomedical application of graphene is the interaction of these materials with the cells. To address this, we investigated the interaction between graphene materials (graphene and graphene oxide) and plasma membranes of cells (bacterial and mammalian cells). The interactions of four of the most abundant phospholipids in bacteria and mammalian plasma membranes with graphene materials were studied using density functional theory (DFT) at the atomic level. The calculations showed that the mammalian phospholipids have stronger bonding to each other compared to bacterial phospholipids. When the graphene/graphene oxide sheet is approaching the phospholipid pairs, the bacterial pairs exhibit less repulsive interactions, thereby a more stable system with the sheets was found. We also assembled bacterial and mammalian phospholipids into liposomes. We further observed that the bacterial liposomes and cells let the graphene flakes penetrate the membrane. The differential scanning calorimetry measurements of liposomes revealed that the bacterial liposomes have the lowest heat capacity; this strengthens the theoretical predictions of weaker interaction between the bacterial phospholipids compared to the mammalian phospholipids. We further demonstrated that graphene oxide could be internalized into the mammalian liposomes without disrupting the membrane integrity. The results suggest that the weak bonding among bacteria phospholipids and less repulsive force when graphene materials approach, result in graphene materials interacting differently with the bacteria compared to mammalian cells.

Direct On-Chip Differentiation of Intestinal Tubules from Induced Pluripotent Stem Cells.

Intestinal organoids have emerged as the new paradigm for modelling the healthy and diseased intestine with patient-relevant properties. In this study, we show directed differentiation of induced pluripotent stem cells towards intestinal-like phenotype within a microfluidic device. iPSCs are cultured against a gel in microfluidic chips of the OrganoPlate, in which they undergo stepwise differentiation. Cells form a tubular structure, lose their stem cell markers and start expressing mature intestinal markers, including markers for Paneth cells, enterocytes and neuroendocrine cells. Tubes develop barrier properties as confirmed by transepithelial electrical resistance (TEER). Lastly, we show that tubules respond to pro-inflammatory cytokine triggers. The whole procedure for differentiation lasts 14 days, making it an efficient process to make patient-specific organoid tubules. We anticipate the usage of the platform for disease modelling and drug candidate screening.

Development of a Gut-On-A-Chip Model for High Throughput Disease Modeling and Drug Discovery.

A common bottleneck in any drug development process is finding sufficiently accurate models that capture key aspects of disease development and progression. Conventional drug screening models often rely on simple 2D culture systems that fail to recapitulate the complexity of the organ situation. In this study, we show the application of a robust high throughput 3D gut-on-a-chip model for investigating hallmarks of inflammatory bowel disease (IBD). Using the OrganoPlate platform, we subjected enterocyte-like cells to an immune-relevant inflammatory trigger in order to recapitulate key events of IBD and to further investigate the suitability of this model for compound discovery and target validation activities. The induction of inflammatory conditions caused a loss of barrier function of the intestinal epithelium and its activation by increased cytokine production, two events observed in IBD physiopathology. More importantly, anti-inflammatory compound exposure prevented the loss of barrier function and the increased cytokine release. Furthermore, knockdown of key inflammatory regulators RELA and MYD88 through on-chip adenoviral shRNA transduction alleviated IBD phenotype by decreasing cytokine production. In summary, we demonstrate the routine use of a gut-on-a-chip platform for disease-specific aspects modeling. The approach can be used for larger scale disease modeling, target validation and drug discovery purposes.

Membrane-free culture and real-time barrier integrity assessment of perfused intestinal epithelium tubes.

In vitro models that better reflect in vivo epithelial barrier (patho-)physiology are urgently required to predict adverse drug effects. Here we introduce extracellular matrix-supported intestinal tubules in perfused microfluidic devices, exhibiting tissue polarization and transporter expression. Forty leak-tight tubules are cultured in parallel on a single plate and their response to pharmacological stimuli is recorded over 125 h using automated imaging techniques. A study comprising 357 gut tubes is performed, of which 93% are leak tight before exposure. EC50-time curves could be extracted that provide insight into both concentration and exposure time response. Full compatibility with standard equipment and user-friendly operation make this Organ-on-a-Chip platform readily applicable in routine laboratories.Efforts to determine the effects of drugs on epithelial barriers could benefit from better in vitro models. Here the authors develop a microfluidic device supporting the growth and function of extracellular matrix-supported intestinal tubules, and evaluate the effect of staurosporine and acetylsalicylic acid on barrier integrity.

Fusogenic Liposomes as Nanocarriers for the Delivery of Intracellular Proteins.

Direct delivery of proteins and peptides into living mammalian cells has been accomplished using phospholipid liposomes as carrier particles. Such liposomes are usually taken up via endocytosis where the main part of their cargo is degraded in lysosomes before reaching its destination. Here, fusogenic liposomes, a newly developed molecular carrier system, were used for protein delivery. When such liposomes were loaded with water-soluble proteins and brought into contact with mammalian cells, the liposomal membrane efficiently fused with the cellular plasma membrane delivering the liposomal content to the cytoplasm without degradation. To explore the key factors of proteofection processes, the complex formation of fusogenic liposomes and proteins of interest and the size and zeta potential of the formed fusogenic proteoliposoms were monitored. Intracellular protein delivery was analyzed using fluorescence microscopy and flow cytometry. Proteins such as EGFP, Dendra2, and R-phycoerythrin or peptides such as LifeAct-FITC and NTF2-AlexaFluor488 were successfully incorporated into mammalian cells with high efficiency. Moreover, correct functionality and faithful transport to binding sites were also proven for the imported proteins.

Development of a rapid screen for the endodermal differentiation potential of human pluripotent stem cell lines.

A challenge facing the human pluripotent stem cell (hPSC) field is the variability observed in differentiation potential of hPSCs. Variability can lead to time consuming and costly optimisation to yield the cell type of interest. This is especially relevant for the differentiation of hPSCs towards the endodermal lineages. Endodermal cells have the potential to yield promising new knowledge and therapies for diseases affecting multiple organ systems, including lung, thymus, intestine, pancreas and liver, as well as applications in regenerative medicine and toxicology. Providing a means to rapidly, cheaply and efficiently assess the differentiation potential of multiple hPSCs is of great interest. To this end, we have developed a rapid small molecule based screen to assess the endodermal potential (EP) of hPSCs, based solely on definitive endoderm (DE) morphology. This drastically reduces the cost and time to identify lines suitable for use in deriving endodermal lineages. We demonstrate the efficacy of this screen using 10 different hPSCs, including 4 human embryonic stem cell lines (hESCs) and 6 human induced pluripotent stem cell lines (hiPSCs). The screen clearly revealed lines amenable to endodermal differentiation, and only lines that passed our morphological assessment were capable of further differentiation to hepatocyte like cells (HLCs).

A bioanalytical assay to distinguish cellular uptake routes for liposomes.

Lipid-based nanoparticles are frequently used for drug or DNA delivery into mammalian cells. However it is difficult to determine whether such particles are taken up via endocytosis or fusion to the plasma membrane. Here, we propose a simple and reliable analytical method to do so based on the unique spectral properties of the fluorescent tracer BODIPY FL. At high local concentrations, this dye displays an additional red-shifted emission peak that is absent at low concentrations. In dye-loaded liposomes taken up by endocytosis, the local dye concentration did not significantly change upon internalization. Accordingly, unchanged fluorescence spectra were detected. When cells were incubated with liposomes able to fuse with the plasma membrane of mammalian cells, a reduction of local dye concentration and much weaker emission in the red-shifted peak were observed. The ratio of intensities in both fluorescence channels was shown to be a reliable indicator of the cellular uptake mechanism.

Small-molecule-driven hepatocyte differentiation of human pluripotent stem cells.

The differentiation of pluripotent stem cells to hepatocytes is well established, yet current methods suffer from several drawbacks. These include a lack of definition and reproducibility, which in part stems from continued reliance on recombinant growth factors. This has remained a stumbling block for the translation of the technology into industry and the clinic for reasons associated with cost and quality. We have devised a growth-factor-free protocol that relies on small molecules to differentiate human pluripotent stem cells toward a hepatic phenotype. The procedure can efficiently direct both human embryonic stem cells and induced pluripotent stem cells to hepatocyte-like cells. The final population of cells demonstrates marker expression at the transcriptional and protein levels, as well as key hepatic functions such as serum protein production, glycogen storage, and cytochrome P450 activity.

Plasma membrane functionalization using highly fusogenic immune activator liposomes.

Cell surface functionalization and target molecule incorporation into living cell membranes without functional damage represent major biotechnological challenges. One possible way to achieve these goals is to induce cell membrane fusion with an artificial membrane containing molecules equipped with reactive groups or ligands. In this work we developed a carrier system to incorporate lipopolysaccharide (LPS), an immune cell activating molecule from Gram-negative bacteria, into mammalian membranes. LPS is not present in untreated mammalian cells which hence are not detectable by the immune system. Here, we demonstrate the successful incorporation of LPS into fusogenic liposomes (FLs) and subsequent incorporation into mammalian plasma membranes using these FLs. Additionally, the presence of LPS in cell membranes was probed by the addition of non-activated macrophages. A high concentration of LPS in the plasma membrane of immortalized fibroblasts activated the immune cells, which in turn started to eliminate LPS-exhibiting cells. Our method for cellular membrane functionalization is a promising tool for biomedical applications and could provide the basis for specific cell targeting approaches.