Standard cancer lab models lack many attributes of the in-vivo cancer microenvironment. Oxygen levels for example are not commonly controlled in 2D cell-culture well plate experiments. However, low O2 (hypoxia) in particular is common in cancerous tissue due to high proliferation rates of cancer cells and inadequate vasculature. Hypoxia is also associated with cancer recurrence and drug resistance. We report a microfluidic system capable of exposing a 2D cell-culture to a dissolved oxygen gradient ranging from hypoxia (<1 mg/L) to hyperoxia (40 mg/L) for over 30 minutes, measurable in real-time using an integrated sensor film. The film incorporates a photostable, non-cytotoxic oxygen-sensitive fluorescent dye, which exhibits a linear response and high contrast (I0 /I100 = 12) within the range of interest, was integrated onto glass substrates as a cell culture substrate. To demonstrate the applicability of the platform, Ishikawa cancer cells were cultured on the platform and exposed to linear cross-stream oxygen gradients. The platform provides a valuable tool for the culture of cancer cells in an in-vivo like microenvironment and will enable more accurate screening of new anti-cancer drugs.

The life expectancy of patients with high-grade glioma (HGG) has not improved in decades. One of the crucial tools to enable future improvement is advanced models that faithfully recapitulate the tumour microenvironment; they can be used for high-throughput screening that in future may enable accurate personalised drug screens. Currently, advanced models are crucial for identifying and understanding potential new targets, assessing new chemotherapeutic compounds or other treatment modalities. Recently, various methodologies have come into use that have allowed the validation of complex models—namely, spheroids, tumouroids, hydrogel-embedded cultures (matrix-supported) and advanced bioengineered cultures assembled with bioprinting and microfluidics. This review is designed to present the state of advanced models of HGG, whilst focusing as much as is possible on the paediatric form of the disease. The reality remains, however, that paediatric HGG (pHGG) models are years behind those of adult HGG. Our goal is to bring this to light in the hope that pGBM models can be improved upon.

This work introduces casein microstructures with surface features as a biodegradable biomedical platform technology for enhancing tissue-engineering applications. An optimized fabrication process is presented to reduce the hydrophobicity of intermediate polydimethylsiloxane (PDMS) molds and to transfer high-resolution regular and biomimetic features onto the surface of casein devices. Four different cross-linking reagents, glutaraldehyde, formaldehyde, citric acid and transglutaminase (TG) were investigated to increase the degradation time of casein and their influence on swelling and biocompatibility of the films was studied. TG was found to be the only cross-linker to effectively increase the degradation time and show reduced film swelling after immersion into media, while remaining compatible with cell-culture. The maximum expansion of the films cross-linked via TG was 33% after 24 hours of immersion in cell-culture media. C2C12 cells were successfully cultured on the patterned films for up to 72 hours. The patterned biodegradable casein substrates presented here have promising applications in stem-cell engineering, regenerative medicine, and implantable devices.

Mechanical forces created by the extracellular environment regulate biochemical signals that modulate the inter-related cellular phenotypes of morphology, proliferation, and migration. A stiff microenvironment induces glioblastoma (GBM) cells to develop prominent actin stress fibres, take on a spread morphology and adopt trapezoid shapes, when cultured in 2D, which are phenotypes characteristic of a mesenchymal cell program. The mesenchymal subtype is the most aggressive among the molecular GBM subtypes. Recurrent GBM have been reported to transition to mesenchymal. We therefore sought to test the hypothesis that stiffer microenvironments—such as those found in different brain anatomical structures and induced following treatment—contribute to the expression of markers characterising the mesenchymal subtype. We cultured primary patient-derived cell lines that reflect the three common GBM subtypes (mesenchymal, proneural and classical) on polyacrylamide (PA) hydrogels with controlled stiffnesses spanning the healthy and pathological tissue range. We then assessed the canonical mesenchymal markers Connective Tissue Growth Factor (CTGF) and yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ) expression, via immunofluorescence. Replating techniques and drug-mediated manipulation of the actin cytoskeleton were utilised to ascertain the response of the cells to differing mechanical environments. We demonstrate that CTGF is induced rapidly following adhesion to a rigid substrate and is independent of actin filament formation. Collectively, our data suggest that microenvironmental rigidity can stimulate expression of mesenchymal-associated molecules in GBM.

Cancer cells resist some anti-cancer drugs in a hypoxic environment, which is often present in-vivo due to high proliferation rates and inadequate vasculature in dense tumour cores. Oxygen control and measurement are therefore valuable tools in culturing cancer cells in an in-vivo-like microenvironment. We report on the polyvinylpyrrolidone (PVP) treatment of polystyrene (PS)/platinum(II) octaethylporphyrin ketone (PtOEPK) fluorescent oxygen sensor films. This treatment improves cell attachment and subsequent long-term cell culture compared to native PS/PtOEPK sensor films by decreasing the water contact angle of the films to 19 o , whilst sensor sensitivity to oxygen remains high (I 0 /I 100 = 12).

Mechanical forces created by the extracellular environment regulate biochemical signals that modulate the inter-related cellular phenotypes of morphology, proliferation, and migration. A rigid microenvironment induces glioblastoma (GBM) cells to develop prominent actin stress fibres, take on a spread morphology and adopt trapezoid cells shapes when cultured in 2D, which are phenotypes characteristic of a mesenchymal cell program. The mesenchymal subtype is the most aggressive among the molecular GBM subtypes and recurrent GBM of other subtypes have been reported to transition to mesenchymal. We therefore sought to test the hypothesis that rigid microenvironments - such as those found in different brain anatomical structures and induced following treatment - contribute to the expression of markers characterising the mesenchymal subtype. We cultured primary patient-derived cell lines that reflect the three common GBM subtypes (mesenchymal, proneural and classic) on polyacrylamide (PA) hydrogels with controlled stiffnesses spanning the healthy and pathological tissue range. We then assessed the canonical mesenchymal marker Connective Tissue Growth Factor (CTGF) and yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ) expression, via immunofluorescence. Replating techniques and drug-mediated manipulation of the actin cytoskeleton were utilised to ascertain the response of the cells to differing mechanical environments. We demonstrate that CTGF is induced rapidly following adhesion to a rigid substrate and is independent of actin filament formation. Collectively, our data suggest that microenvironmental rigidity may stimulate the GBM mesenchymal signalling program.

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