FleXert: A Soft, Actuatable Multiwell Plate Insert for Cell Culture under Stretch.

Porous multiwell plate inserts are widely used in biomedical research to study transport processes or to culture cells/tissues at the air-liquid interface. These inserts are made of rigid materials and used under static culture conditions, which are unrepresentative of biological microenvironments. Here, we present FleXert, a soft, actuatable cell culture insert that interfaces with six-well plates. It is made of polydimethylsiloxane (PDMS) and comprises a porous PDMS membrane as cell/tissue support. FleXerts can be pneumatically actuated using a standard syringe pump, imparting tensile strains of up to 30%. A wide range of actuation patterns can be achieved by varying the air pressure and pumping rate. Facile surface functionalization of FleXert's porous PDMS membrane with fibronectin enables adhesion of human dermal fibroblasts and strains developing on FleXert's membrane are successfully transduced to the cell layer. 3D tissue models, such as fibroblast-laden collagen gels, can also be anchored to PDMS following polydopamine coating. Furthermore, collagen-coated FleXert membranes support the establishment of a human skin model, demonstrating the material's excellent biocompatibility required for tissue engineering. In contrast to existing technologies, FleXerts do not require costly fabrication equipment or custom-built culture chambers, making them a versatile and low-cost solution for tissue engineering and biological barrier penetration studies under physiological strain. This paper is an extensive toolkit for multidisciplinary mechanobiology studies, including detailed instructions for a wide variety of methods such as device fabrication, theoretical modeling, cell culture, and image analysis techniques.

3D Bioprinting: The Emergence of Programmable Biodesign.

Until recently, bioprinting was largely limited to highly interdisciplinary research teams, as the process requires significant input from specialists in the fields of materials science, engineering, and cell biology. With the advent of commercially available high-performance bioprinters, the field has become accessible to a wider range of research groups, who can now buy the hardware off the shelf instead of having to build it from scratch. As a result, bioprinting has rapidly expanded to address a wide array of research foci, which include organotypic in vitro models, complex engineered tissues, and even bioprinted microbial systems. Moreover, in the early days, the range of suitable bioinks was limited. Now, there is a plethora of viable options to suit many cell phenotypes. This rapidly evolving dynamic environment creates endless opportunities for scientists to design and construct highly complex biological systems. However, this scientific diversity presents its own set of challenges, such as defining standardized protocols for characterizing bioprinted structures, which is essential for eventual organ replacement. In this progress report, the current state-of-the-art in the field of bioprinting is discussed, with a special emphasis on recent hardware developments, bioprinting for regenerative medicine, and late-breaking nontraditional topics.

Glycosylated superparamagnetic nanoparticle gradients for osteochondral tissue engineering.

In developmental biology, gradients of bioactive signals direct the formation of structural transitions in tissue that are key to physiological function. Failure to reproduce these native features in an in vitro setting can severely limit the success of bioengineered tissue constructs. In this report, we introduce a facile and rapid platform that uses magnetic field alignment of glycosylated superparamagnetic iron oxide nanoparticles, pre-loaded with growth factors, to pattern biochemical gradients into a range of biomaterial systems. Gradients of bone morphogenetic protein 2 in agarose hydrogels were used to spatially direct the osteogenesis of human mesenchymal stem cells and generate robust osteochondral tissue constructs exhibiting a clear mineral transition from bone to cartilage. Interestingly, the smooth gradients in growth factor concentration gave rise to biologically-relevant, emergent structural features, including a tidemark transition demarcating mineralized and non-mineralized tissue and an osteochondral interface rich in hypertrophic chondrocytes. This platform technology offers great versatility and provides an exciting new opportunity for overcoming a range of interfacial tissue engineering challenges.

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells.

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

Suitability of human and mammalian cells of different origin for the assessment of genotoxicity of metal and polymeric engineered nanoparticles.

Nanogenotoxicity is a crucial endpoint in safety testing of nanomaterials as it addresses potential mutagenicity, which has implications for risks of both genetic disease and carcinogenesis. Within the NanoTEST project, we investigated the genotoxic potential of well-characterised nanoparticles (NPs): titanium dioxide (TiO2) NPs of nominal size 20 nm, iron oxide (8 nm) both uncoated (U-Fe3O4) and oleic acid coated (OC-Fe3O4), rhodamine-labelled amorphous silica 25 (Fl-25 SiO2) and 50 nm (Fl-50 SiO) and polylactic glycolic acid polyethylene oxide polymeric NPs - as well as Endorem® as a negative control for detection of strand breaks and oxidised DNA lesions with the alkaline comet assay. Using primary cells and cell lines derived from blood (human lymphocytes and lymphoblastoid TK6 cells), vascular/central nervous system (human endothelial human cerebral endothelial cells), liver (rat hepatocytes and Kupffer cells), kidney (monkey Cos-1 and human HEK293 cells), lung (human bronchial 16HBE14o cells) and placenta (human BeWo b30), we were interested in which in vitro cell model is sufficient to detect positive (genotoxic) and negative (non-genotoxic) responses. All in vitro studies were harmonized, i.e. NPs from the same batch, and identical dispersion protocols (for TiO2 NPs, two dispersions were used), exposure time, concentration range, culture conditions and time-courses were used. The results from the statistical evaluation show that OC-Fe3O4 and TiO2 NPs are genotoxic in the experimental conditions used. When all NPs were included in the analysis, no differences were seen among cell lines - demonstrating the usefulness of the assay in all cells to identify genotoxic and non-genotoxic NPs. The TK6 cells, human lymphocytes, BeWo b30 and kidney cells seem to be the most reliable for detecting a dose-response.

The toxicity, transport and uptake of nanoparticles in the in vitro BeWo b30 placental cell barrier model used within NanoTEST.

Despite the rapid ongoing expansion in the use of nanomaterials, we still know little about their biological interaction and biodistribution within the human body. If medically relevant nanoparticles can cross specific cell barriers they may disseminate through the body beyond the original target and may reach particularly sensitive areas such as the foetus. This study utilised an in vitro barrier model of the placenta to explore toxicity, uptake and transport of iron oxide and silica nanoparticles. The findings indicate that these nanoparticles can transfer extensively across the placental barrier model but physico-chemical characteristics such as surface chemistry impact upon both uptake and transport. Iron oxide cytotoxicity was evident at lower doses and shorter exposure compared with silica and may be of clinical relevance. In vivo correlation of in vitro findings is essential but in vitro models may provide worst case-exposure estimates to help reduce the amount of testing required.

Chlorpyrifos and neurodevelopmental effects: a literature review and expert elicitation on research and policy.

BACKGROUND: Organophosphate pesticides are widely used on food crops grown in the EU. While they have been banned from indoor use in the US for a decade due to adverse health effects, they are still the most prevalent pesticides in the EU, with Chlorpyrifos (CPF) being the most commonly applied. It has been suggested CPF affects neurodevelopment even at levels below toxicity guidelines. Younger individuals may be more susceptible than adults due to biological factors and exposure settings. METHODS: A literature review was undertaken to assess the evidence for CPF contributing to neurodevelopmental disorders in infants and children. Other literature was consulted in order to formulate a causal chain diagram showing the origins, uptake, and neurological effects of animal and human exposure to CPF.The causal chain diagram and a questionnaire were distributed online to scientific experts who had published in relevant areas of research. They were asked to assess their confidence levels on whether CPF does in fact contribute to adverse neurodevelopment outcomes and rate their confidence in the scientific evidence. A second questionnaire queried experts as to which kind of policy action they consider justifiable based on current knowledge. In a special workshop session at the EuroTox congress in Dresden in 2009 the results of both questionnaires were further discussed with invited experts, as a basis for a policy brief with main messages for policy makers and stakeholders. RESULTS: Most experts who responded to the first questionnaire felt that there was already enough evidence to support a ban on indoor uses of CPF in the EU. However, most felt additional research is still required in several areas. The responses from the first questionnaire were used to formulate the second questionnaire addressing the feasibility of government action. In turn, these expert participants were invited to attend a special session at the EuroTox congress in Dresden in 2009. CONCLUSIONS: Some of the evidence that CPF contributes to neurodevelopmental disorders is still disputed among experts, and the overall sense is that further research and public awareness are warranted. There have been campaigns in North America making the potential exposure concerns known, but such information is not widely known in the EU. The ability of government action to produce change is strongly felt in some quarters while others believe better knowledge of consumer use trends would have a greater impact.