Modifying the Secretome of Mesenchymal Stem Cells Prolongs the Regenerative Treatment Window for Encephalopathy of Prematurity.

Clinical treatment options to combat Encephalopathy of Prematurity (EoP) are still lacking. We, and others, have proposed (intranasal) mesenchymal stem cells (MSCs) as a potent therapeutic strategy to boost white matter repair in the injured preterm brain. Using a double-hit mouse model of diffuse white matter injury, we previously showed that the efficacy of MSC treatment was time dependent, with a significant decrease in functional and histological improvements after the postponement of cell administration. In this follow-up study, we aimed to investigate the mechanisms underlying this loss of therapeutic efficacy. Additionally, we optimized the regenerative potential of MSCs by means of genetic engineering with the transient hypersecretion of beneficial factors, in order to prolong the treatment window. Though the cerebral expression of known chemoattractants was stable over time, the migration of MSCs to the injured brain was partially impaired. Moreover, using a primary oligodendrocyte (OL) culture, we showed that the rescue of injured OLs was reduced after delayed MSC coculture. Cocultures of modified MSCs, hypersecreting IGF1, LIF, IL11, or IL10, with primary microglia and OLs, revealed a superior treatment efficacy over naïve MSCs. Additionally, we showed that the delayed intranasal administration of IGF1-, LIF-, or IL11-hypersecreting MSCs, improved myelination and the functional outcome in EoP mice. In conclusion, the impaired migration and regenerative capacity of intranasally applied MSCs likely underlie the observed loss of efficacy after delayed treatment. The intranasal administration of IGF1-, LIF-, or IL11-hypersecreting MSCs, is a promising optimization strategy to prolong the window for effective MSC treatment in preterm infants with EoP.

Polymer film-based microwell array platform for long-term culture and research of human bronchial organoids.

The culture of lung organoids relies on drops of basement membrane matrices. This comes with limitations, for example, concerning the microscopic monitoring and imaging of the organoids in the drops. Also, the culture technique is not easily compatible with micromanipulations of the organoids. In this study, we investigated the feasibility of the culture of human bronchial organoids in defined x-, y- and z-positions in a polymer film-based microwell array platform. The circular microwells have thin round/U-bottoms. For this, single cells are first precultured in drops of basement membrane extract (BME). After they form cell clusters or premature organoids, the preformed structures are then transferred into the microwells in a solution of 50% BME in medium. There, the structures can be cultured toward differentiated and mature organoids for several weeks. The organoids were characterized by bright-field microscopy for size growth and luminal fusion over time, by scanning electron microscopy for overall morphology, by transmission electron microscopy for the existence of microvilli and cilia, by video microscopy for beating cilia and swirling fluid, by live-cell imaging, by fluorescence microscopy for the expression of cell-specific markers and for proliferating and apoptotic cells, and by ATP measurement for extended cell viability. Finally, we demonstrated the eased micromanipulation of the organoids in the microwells by the example of their microinjection.

Polystyrene Pocket Lithography: Sculpting Plastic with Light.

Tissue-culture-ware polystyrene is the gold standard for in vitro cell culture. While microengineering techniques can create advanced cell microenvironments in polystyrene, they require specialized equipment and reagents, which hinder their accessibility for most biological researchers. An economical and easily accessible method is developed and validated for fabricating microstructures directly in polystyrene with sizes approaching subcellular dimensions while requiring minimal processing time. The process involves deep ultraviolet irradiation through a shadow mask or ink pattern using inexpensive, handheld devices followed by selective chemical development with common reagents to generate micropatterns with depths/heights between 5 and 10 µm, which can be used to guide cell behavior. The remarkable straightforwardness of the process enables this class of microengineering techniques to be broadly accessible to diverse research communities.

Thin fluorinated polymer film microcavity arrays for 3D cell culture and label-free automated feature extraction.

There is an increasing need for automated label-free morphometric analysis using brightfield microscopy images of 3D cell culture systems. This requires automated feature detection which can be achieved by improving the image contrast, e.g. by reducing the refractive index mismatch in the light path. Here, a novel microcavity platform fabricated using microthermoforming of thin fluorinated ethylene-propylene (FEP) films which match the refractive index of cell culture medium and provide a homogenous background signal intensity is described. FEP is chemically inert, mechanically stable and has been used as a substrate for light sheet microscopy. The microcavities promote formation of mouse embryonic stem cell (mESC) aggregates, which show axial elongation and germ layer specification similar to embryonic development. A label-free feature extraction pipeline based on a machine-learning plugin for FIJI is used to extract morphometric features from time-lapse imaging in a highly robust and reproducible manner. Lastly, the pipeline is utilized for testing the effect of the drug Latrunculin A on the mESC aggregates, highlighting the platform's potential for high-content screening (HCS) in drug discovery. This new microengineered tool is an important step towards label-free imaging of free-floating stem cell aggregates and paves the way for high-content drug testing and translational studies.

Serum enhanced cytokine responses of macrophages to silica and iron oxide particles and nanomaterials: a comparison of serum to lung lining fluid and albumin dispersions.

The potential hazard to humans exposed to nanomaterials such as silica and iron oxide was investigated using an in vitro macrophage cell culture system. Amorphous silica and iron oxide particles and nanomaterials (NMs) were dispersed in cell culture medium supplemented with either bovine serum albumin (BSA), lung lining fluid (LLF) or serum, in order to mimic the body fluids encountered during different routes of exposure in the body. End points investigated included macrophage viability and cytokine production. Silica NMs and particles (50 and 200 nm, respectively) were unmodified (plain) or aminated (NH2 ). Iron oxide NMs and particles, Fe3 O4 45 nm and Fe2 O3 280 nm were also used in this study. Silica particles and NMs induced a dose-dependent increase in cytotoxicity as measured by lactate dehydrogenase (LDH) release. Serum enhanced silica-induced interleukin (IL)-6, IL-10, IL-1ß and MCP-1 release, whereas albumin partially inhibited MCP-1 release. Aminated silica, 50 nm was more potent than the 200-nm particles at inducing monocyte chemoattractant protein-1 (MCP-1) production when dispersed in medium or LLF, suggesting a size specific effect for these particles and this cytokine. Iron oxide particles were relatively inert compared with the silica particles and NMs; however, serum and albumin did affect cytokine release in some treatments. In conclusion, the data suggests that serum, compared with medium, BSA and LLF is very potent at enhancing macrophage responses to silica and iron oxide particles and NMs. Size was only influential in LLF for a limited number of parameters, whereas surface chemistry was not of consequence in this in vitro macrophage system.

Cytotoxicity and cytokine release in rat hepatocytes, C3A cells and macrophages exposed to gold nanoparticles--effect of biological dispersion media or corona.

The study aim was to investigate how gold nanoparticles (NPs) of different sizes (20 and 100 nm) influence primary hepatocytes, the hepatocyte cell line C3A and macrophage cytokine responses when dispersed in lung or blood relevant fluids. Gold Au NPs induced cytotoxicity in primary hepatocytes at the highest dose of 66 µg/cm2, this effect was modified by the dispersant, the effect was greater with lung lining fluid (LLF). Release of interleukin (IL)-6, Monocyte chemoattractant protein-1 (MCP-1) and IL-1ß was enhanced by the Au NPs and the effects were influenced by the particle size and dispersant. In medium, the smallest particle size was most effective at inducing IL-6 release, while in LLF the largest particles were most effective at inducing IL-6 release. Both 20 nm and 100 nm particles enhanced MCP-1 and IL-1ß in the presence of LLF. The Au particles had no cytotoxic effects nor did they stimulate the release of cytokines in the C3A hepatoma cell line. The Au NPs had no significant impact on macrophage viability. Particles induced IL-6 and TNF-α release. LLF and serum reduced the IL-6 response while albumin enhanced the TNF-α response compared to medium dispersed Au NPs. The Au NPs did not impact on MCP-1 release, but this cytokine was enhanced by albumin and serum, while it was depressed by LLF. The macrophage responses were lower than those evoked in primary hepatocytes. In conclusion, when assessing the cytotoxic and pro-inflammatory responses induced by Au NPs, the response is influenced by the dispersant, with different dispersants having different effects in different cell types.

Investigating the relationship between nanomaterial hazard and physicochemical properties: Informing the exploitation of nanomaterials within therapeutic and diagnostic applications.

Nanomaterials (NMs) have the potential to improve the treatment and diagnosis of disease as they are suitable candidates for a number of diagnostic and therapeutic applications. On entering the body via a variety of exposure routes, and during their translocation to secondary target sites it is inevitable that NMs interact with biological molecules, such as proteins. These interactions may influence the behaviour and toxicity of NMs following exposure. As the surface of NMs is what interacts with cells and tissues it is necessary to identify the influence of NM surface properties on their toxicity, and determine how this is influenced by the route of exposure, and physico-chemical characteristics of NMs. The term protein corona is used to describe the coating of the NM surface with protein. The protein corona is a dynamic and complex structure whose composition is dictated by the biological medium and the physico-chemical properties of NMs (such as their size, composition, hydrophobicity and charge) as this influences protein binding specificity and affinity. Depending on the route of exposure (e.g. inhalation or injection) NMs will encounter different proteins. We have observed that i) the composition of protein corona of NMs is likely to be dictated by their route of entry, ii) the translocation of NMs to secondary target sites may influence the composition of the protein corona (i.e. they encounter different proteins on their transport in the body) so that the composition of the protein corona evolves over time, iii) the physico-chemical characteristics of NMs dictate the composition of the protein corona, and the toxicity of NMs and iv) NMs can affect secondary target sites that vary according to delivery route and corona composition following exposure. These findings, and evidence from the wider literature has therefore led us to hypothesise that NM toxicity is dictated by the exposure route due to the acquisition of a surface coating (protein corona) that is determined by the route of entry and physico-chemical properties of the NM. This information can be exploited within the intelligent design of NMs in the future (e.g. to control protein adsorption and the subsequent cellular response), and be used to improve the design of toxicology investigations (e.g. to inform how NMs should be dispersed within in vitro experiments to more accurately reflect in vivo conditions).

Phytotoxicity of silver nanoparticles to Lemna minor L.

The use of silver nanoparticles (AgNPs) in commercial products has increased significantly in recent years. Although there has been some attempt to determine the toxic effects of AgNPs, there is little information on aquatic plants which have a vital role in ecosystems. This study reports the use of Lemna minor L. clone St to investigate the phytotoxicity of AgNPs under modified OECD test conditions. AgNPs were synthesised, characterised and subsequently presented to the L. minor. Results showed that inhibition of plant growth was evident after exposure to small (~ 20 nm) and larger (~100 nm) AgNPs at low concentrations (5 µg L⁻¹) and this effect became more acute with a longer exposure time. There was a linear dose-response relationship after 14 d exposure. Using predicted environmental concentrations for wastewaters it was found that AgNPs may pose a significant potential risk to the environment.