Thomas Gervais

On‐chip 3D cancer models are important tools to investigate treatment response and tumor biology. With their increasing adoption, there is a need for non‐destructive methods that enable the sequential monitoring of cell death and tumor response over time. This study presents quantifying cell‐free DNA (cfDNA) as a proxy for cell death for on‐chip 3D sarcoma models. qPCR analysis of media cfDNA from spheroids and micro‐dissected tumor explants (MDTs) generated from SK‐LMS‐1 and MCA‐205 cell lines cultured on microfluidic chips showed a correlation between baseline cfDNA concentration and on‐chip tumor burden. Furthermore, cfDNA levels increased in a dose‐dependent manner following cytotoxic treatments, including staurosporine and hydrogen peroxide. Single‐dose radiation therapy also raised cfDNA levels in media from MCA‐205 but not SK‐LMS‐1 MDTs, in accordance with their 2D culture response. Finally, cfDNA release within the first days of culture was shown to reflect patient‐derived MDT survival, addressing a critical unmet need for ex vivo culture. This study suggests that media cfDNA quantification could be used as a reliable, non‐disruptive, and rapidly quantifiable measure of cell death for on‐chip 3D models, which facilitates more robust use of 3D model systems for pre‐clinical assessment of treatment‐induced cell death and biological response.

Background: Hypoxia influences the biology and response of tumors. Current methods to study hypoxia are low-throughput or use artificial induction, limiting the translational potential of findings. We previously described a user-friendly microfluidic chip to rapidly generate 240 large spheroids naturally harboring a hypoxic core. However, we had yet to validate the clinical relevance of these hypoxic-core (HyCo) spheroids, their use in understanding treatment resistance and their applications in targeted therapy testing. Methods: HyCo and small normoxic spheroids were generated on-chip from commercial and patient-derived soft-tissue sarcoma (STS) cell lines. Presence of hypoxia was validated through in silico modelling, immunofluorescence staining and RTqPCR. Whole-genome RNA sequencing was used to establish HyCo gene expression profile, compared to clinically-derived datasets, and used to identify target genes in hypoxic STSs. Apoptosis staining and clonogenic assay were used to evaluate HyCo therapy resistance to chemotherapy and radiotherapy. Matrigel invasion assay was used to quantify HyCo invasiveness and aggressivity. Diethyl-pythiDC was used to demonstrate the efficacy of P4HA1-inhibition on reducing HyCo invasiveness. Results: Large STSs spheroids generated on-chip naturally exhibit a diffusion-driven hypoxic core in only a few days. These HyCo spheroids recapitulate clinical hallmarks of hypoxia in STS: upregulation of hypoxia-related genes, resistance to chemotherapy/radiotherapy, and increased invasiveness. Our results suggest evidence of treatment resistance driven by hypoxia-induced paracrine signaling in STS. We identified P4HA1 gene as both upregulated in HyCo and prognostic in clinical STS and demonstrated that P4HA1-inhibition using diethyl-pythiDC reduced HyCo invasiveness. Conclusions: Our results demonstrate HyCo spheroids as efficient translational tools to integrate hypoxia in cancer research and drug development. We uncovered evidence of hypoxia-mediated chemoresistance in normoxic regions and successfully identified P4HA1-inhibition as a viable strategy for the treatment of hypoxic STSs, warranting further exploration.

Enniatins (ENNs) are emerging Fusarium mycotoxins detected in food and feed. Despite their widespread occurrence, their toxicity remains poorly understood; thus, advanced in vitro systems that can mimic human physiology are of interest. We evaluated the cytotoxic and genotoxic effects of ENN A1 and ENN B1 exposure on differentiated (DIFF) and undifferentiated (UND) HepaRG liver cells cultured as 2D monolayers and 3D spheroids. Cytotoxicity, assessed by ATP-based luminescence, revealed a time-dependent decrease in inhibitory concentration 50 (IC₅₀) values between 24 h and 48 h across all models. In DIFF HepaRG cells, ENN A1 IC₅₀ values in 3D spheroids decreased from 14.4–18.2 µM at 24 h to 2.2–3.0 µM at 48 h, reaching values comparable to those measured in 2D DIFF cells at 48 h (2.2–2.6 µM), while no IC₅₀ could be determined in 2D at 24 h. For ENN B1, a pronounced time-dependent toxicity was observed, with IC₅₀ values in 3D DIFF spheroids decreasing from 4.1–6.6 µM at 24 h to 1.3–1.6 µM at 48 h, remaining lower than those measured in 2D DIFF cells at 48 h (2.4–3.0 µM). ENN A1 primarily induced apoptotic responses, whereas both ENN A1 and B1 were associated with necrotic responses, and ENN B1 induced a transient and limited autophagic signal, suggesting a minor role for autophagy. To further characterize cellular responses to ENN exposure, spheroids cultured in microfluidic chips were sectioned, and proliferation (Ki67), DNA damage (γH2AX), and apoptosis (cleaved caspase-3) was assessed. Immunostaining revealed no proliferative response, whereas significant DNA damage was detected, particularly in DIFF spheroids. At low, sub-cytotoxic concentrations (~5 µM, 24 h), ENN A1 induced significant DNA damage, as shown by increased γH2AX levels, while cytotoxic effects were only observed at higher concentrations (IC₅₀ ~ 18 µM, 24 h), supporting a potential genotoxic effect independent of cytotoxicity. Despite the structural similarities between ENN A1 and ENN B1, our results highlighted distinct cell death pathways between the two analogues. Both ENNs were detected throughout spheroids without evidence of peripheral restriction, although a homogeneous functional test could not be conclusively demonstrated. Overall, the 3D HepaRG spheroid model proved to be a more physiologically relevant system, offering differential sensitivity, as well as enhanced mechanistic insight, compared to 2D cultures.

Lateral flow immunoassays (LFIAs) are widely used for point‐of‐care diagnostics, but their development is challenged by the complexity and variability of patient samples. In particular, LFIAs often exhibit reduced sensitivity and specificity when used with patient samples, compared to their performance with analyte‐spiked idealized matrices. Patient samples are inherently complex, with variations in physical and biochemical properties between patients. This complexity has consequences for the performance of LFIAs, and can result in non‐specific binding on the test line, discoloration of the nitrocellulose membrane, and incomplete sample flow along the test strip. To address these challenges, a magnetically retrievable platinum nanoreporter (termed Pt@Fe ₃ O ₄ ) is developed for LFIAs. Leveraging the magnetic properties of the Fe ₃ O ₄ core, magnetic separation is utilized to enable the purification and concentration of target antigens from complex human matrices, including serum, saliva, and even stool samples. This also eliminates assay inconsistencies caused by inter‐sample variability. Further, the suitability of Pt@Fe ₃ O ₄ nanoreporters has been explored for use as detection probes in LFIAs. Signal enhancement is demonstrated by the utilization of the magnetic and enzyme‐mimicking activity of the nanoreporter, resulting in a marked improvement in sensitivity, as evidenced by a 2‐ to 4‐fold decrease in the visual limit of detection.

Microdissected tumor tissue explants (MDTs) represent promising ex vivo models for oncology research but remain limited by poor preservation methods and limited viability once resected. We systematically evaluated cryopreservation as a solution to this problem by combining strategies across two ovarian cancer models, comparing slow-freeze and vitrification while integrating antioxidants, temperature modulation, and pre-cryopreservation culture. Vitrification consistently preserved morphology and reduced apoptosis more effectively than slow-freeze, though proliferation outcomes varied by cell line. Supplementation with a defined antioxidant mix (A3) improved proliferation but required careful dosing to avoid toxicity. Pre-cooling enhanced recovery depending on the cell line, while a short resting period prior to freezing further improved survival independent of culture platform. By combining optimized parameters, our protocol preserved up to 98% (49F) and 69% (TOV112D) of proliferative capacity relative to fresh controls. These findings establish vitrification, when rationally optimized, as a reproducible and high-yield strategy for MDT cryopreservation over traditional slow-freezing methods, enabling more reliable use of explant models in personalized medicine and research.

This study investigates the use of industrial-grade polydimethylsiloxane (PDMS) for mass production of microfluidic chips through Liquid Silicone Rubber Injection Molding (LSR-IM) technologies. Injection molding of PDMS is ubiquitous in the automotive and aerospace industries. Yet, it is not currently applied to large-scale production of microfluidic devices. Here, we manufactured PDMS microfluidics devices using industrial IM systems and measured the values of several physicochemical properties of PDMS important in microfluidics. For the two grades of injection-moldable PDMS studied, properties were found close to those of the standard Sylgard 184 PDMS, except Young’s modulus, which varied from 66% to 250% of the Sylgard 184 value and the absorption of small molecules, which was lower or equal to that within Sylgard 184. Yet, the main benefits of LSR-IM production lie in its systematic process, resulting in an increase in reproducibility of both PDMS surface and bulk properties by decreasing the variance within and between production batches of several variables: Young’s modulus (30-fold), oxygen permeation (10-fold), mean fluorescence intensity of Nile Red (5-fold). Emphasis on biocompatibility of LSR-IM was evaluated by culturing complex and sensitive 3D biology models (tumor spheroids and explants) in injection-molded PDMS devices. We observed no significant differences in cell proliferation relative to samples cultured on conventional Sylgard 184 PDMS devices. Overall, these findings open a direct path from benchtop prototyping to industrialization of PDMS-based microfluidic devices for applications in healthcare.

Open-space microfluidics allows the precise spatiotemporal control of cell stimuli on adjacent cell patches on the same petri dish. It is used here to monitor the frequency-dependent expression of key genes in the NOTCH pathway.

We describe the first microfluidic device for in vitro testing of brachytherapy (BT), with applications in translational cancer research. Our PDMS-made BT-on-chip system allows highly precise manual insertion of clinical BT seeds, reliable dose calculation using standard clinically-used TG-43 formalism and easy culture of naturally hypoxic spheroids in less than 3 days, thereby increasing the translational potential of the device. As the BT-on-chip platform is designed to be versatile, we showcase three different gold-standard post-irradiation bioassays and recapitulate, for the first time on-chip, key clinical observations such as dose rate effect and hypoxia-induced radioresistance. Our results suggest that BT-on-chip can be used to safely and efficiently integrate BT and radiotherapy to translational research and drug development pipelines, without expensive equipment or complex workflows.

The use of microfluidic devices in the field of plasma-liquid interaction can unlock unique possibilities to investigate the effects of plasma-generated reactive species for environmental and biomedical applications. So far, very little simulation work has been performed on microfluidic devices in contact with a plasma source. We report on the modelling and computational simulation of physical and chemical processes taking place in a novel plasma-microfluidic platform. The main production and transport pathways of reactive species both in plasma and liquid are modelled by a novel modelling approach that combines 0D chemical kinetics and 2D transport mechanisms. This combined approach, applicable to systems where the transport of chemical species occurs in unidirectional flows at high Péclet numbers, decreases calculation times considerably compared to regular 2D simulations. It takes advantage of the low computational time of the 0D reaction models while providing spatial information through multiple plug-flow simulations to yield a quasi-2D model. The gas and liquid flow profiles are simulated entirely in 2D, together with the chemical reactions and transport of key chemical species. The model correctly predicts increased transport of hydrogen peroxide into the liquid when the microfluidic opening is placed inside the plasma effluent region, as opposed to inside the plasma region itself. Furthermore, the modelled hydrogen peroxide production and transport in the microfluidic liquid differs by less than 50% compared with experimental results. To explain this discrepancy, the limits of the 0D–2D combined approach are discussed.

We present a corollary to Washburn's equation in capillary dynamics. We show that, during capillary filling, in cases where flow path decreases with time, an accelerating capillary flow or reverse-Washburn flow regime occurs. We provide a description of this phenomenon following Washburn's classic analysis and characterize a “reverse-Washburn” capillary flow regime in both inertial and viscous regimes. This regime is observed and characterized in experiments and numerical simulations of recently discovered self-coalescence flows, opening the door to engineering devices with naturally accelerating capillary inflows.