Immunotherapy has revolutionized cancer treatment, yet clinical success remains limited, with only a fraction of patients responding. Tumor heterogeneity and patient-specificity hinder response prediction, emphasizing the need for human-based models that accurately reproduce tumor-immune interactions. Conventional preclinical platforms, such as murine models, lack the human-specific HLA-TCR complexity, limiting their ability to accurately evaluate immunotherapy responses. To address this, we established a patient-specific pipeline for precision immunotherapy in renal cell carcinoma (RCC) and bladder cancer (BC). Tumor and adjacent tissues were used to generate patient-derived tumor organoids (PDTOs) and patient-derived cells (PDCs) for immunopeptidome profiling. Using our in-house microfluidic platform, PeptiCHIP, we identified tumor-associated HLA-I peptides as potential T cell targets. Their immunogenicity was evaluated using peptide-expanded, HLA-matched peripheral blood mononuclear cells (PBMCs), revealing peptides capable of inducing antigen-specific CD8+ T cell activation and cytotoxicity against the patient's tumor. This study integrates PDCs, immunopeptidomics, and functional immune assays to design and test personalized cancer immunotherapies. By recreating patient-specific tumor-immune interactions ex vivo, our platform enables the discovery of therapeutic targets, the evaluation of immune responses, and validation in a patient-specific context. This approach demonstrates the feasibility of bridging a major gap in translational immunotherapy research and supports the development of personalized cancer immunotherapy strategies.

Extracellular vesicles (EVs) are increasingly recognized as promising biomarkers and mediators of intercellular communication, yet their cell type-selective quantitation remains challenging within current workflows, which typically require multi-step EV isolation followed by orthogonal characterization. This study evaluates electrochemical impedance spectroscopy (EIS) as an in situ, label-free strategy for cell type-selective EV quantitation. A microfabricated chip comprising interdigitated gold electrodes was developed and functionalized with Protein A/G and antibodies targeting hepatocyte (HepPar 1), erythrocyte (CD235ab), or cardiomyocyte (CD172a) protein markers. For EV quantitation, the EIS spectra were fitted to a modified Randles circuit to determine changes in the resistive element after EV binding (ΔR/Rantibody). The optimized sensor design enabled cell type-selective capture of targeted EV populations with chip-to-chip repeatability of 5.5-16.4 % (n=6 each) across all sample types. A correlation analysis (Pearson r) indicated positive association of EIS signal (DR/Rantibody) with total EV protein (r=0.959; p<0.001) and total particle concentrations (r=0.966; p<0.001), and inverse association with vesicle size (r=-0.707; p=0.003). For EVs of the same origin (featuring similar size), the EIS signal was most sensitive to their normalized protein content (µg per 109 particles). Finally, EIS analysis of CD172a(+)-EVs secreted during human induced pluripotent stem cells (hiPSC)-to-cardiomyocyte differentiation revealed differentiation-associated changes not detectable using non-selective EV metrics, such as total particle or EV protein content. Collectively, these results identify immunoaffinity-coupled EIS as a promising strategy for cell type-selective EV quantitation, suggesting its potential for non-invasive monitoring of dynamic cellular processes in vitro.

ABSTRACT Antimicrobial resistance (AMR), largely triggered by bacterial biofilms, is a major threat to global health. Bacteria within biofilms exhibit up to 1000‐fold higher AMR than in their planktonic form. Standard biofilm analysis methods rely on static well‐plate assays and destructive endpoints. Therefore, new methods enabling dynamic culture conditions and real‐time measurements are urgently needed to better understand biofilm evolution and eradication. This study presents BioEISense, a microfluidic biofilm sensing platform, which incorporates a flow‐through biofilm culture channel made from off‐stoichiometric thiol‐ene and polydimethylsiloxane polymers (bottom and cover, respectively). In addition, the BioEISense has integrated gold sensors for noninvasive monitoring of biofilm formation by electrochemical impedance spectroscopy (EIS) in real‐time. The performance of BioEISense is first optimized in terms of electrode design, bacterial concentration, and culture media, using EIS. The EIS results are additionally validated by measuring biofilm thickness using confocal microscopy. Finally, the feasibility of BioEISense for screening of the efficacy of antibiofilm agents is evaluated using both biofilm prevention and eradication assays. Together, our findings highlight the great competence of microfluidic biofilm assays for real‐time, noninvasive monitoring of biofilm growth and degradation, with a view to discovering new antibiofilm agents to combat AMR.

Global antimicrobial resistance poses a major threat to human health and is largely driven by bacterial biofilms, which demonstrate significantly greater antibiotic resistance than planktonic bacteria. While most biofilm research targets the development of antibiofilm surfaces, materials that intentionally promote biofilm formation are crucial for creating screening tools to discover new antibiofilm agents. The transition from static to flow-through assay systems is also necessary to increase the methodological readiness of antibiofilm research. This study evaluates the feasibility of an emerging polymer platform, off-stoichiometry thiol-ene (OSTE), in supporting Staphylococcus aureus biofilms. OSTE polymers provide versatile options for rapid prototyping of microfluidic devices, with unique opportunities for on-chip oxygen management. Here, the impacts of OSTE's key materials properties on S. aureus adhesion, biofilm viability, biomass, and metabolic activity are systematically examined in comparison to polystyrene, the current standard in microwell plate-based biofilm assays. Additionally, the composition of the extracellular polymer substances matrix and antimicrobial susceptibility are investigated to determine the most suitable OSTE composition for microfluidic S. aureus biofilm cultures. The results confirm compatibility with S. aureus biofilms, supported by atomic force microscopy analysis of biofilm morphologies under static and microfluidic conditions.

Microfluidic flow reactors functionalized with immobilized human liver microsomes (HLM chips) represent a powerful tool for drug discovery and development by enabling mechanism-based enzyme inhibition studies under flow-through conditions. Additionally, HLM chips may be exploited in streamlined production of human drug metabolites for subsequent microfluidic in vitro organ models or as metabolite standards for drug safety assessment. However, the limited shelf life of the biofunctionalized microreactors generally poses a major barrier to their commercial adaptation in terms of both storage and shipping. The shelf life of the HLM chips in the wetted state is ca. 2-3 weeks only and requires cold storage at 4 degrees C. In this study, we developed a freeze-drying method for lyophilization of HLMs that are readily immobilized inside microfluidic pillar arrays made from off-stoichiometric thiol-ene polymer. The success of lyophilization was evaluated by monitoring the cytochrome P450 and UDP-glucuronosyltransferase enzyme activities of rehydrated HLMs for several months post-freeze-drying. By adapting the freeze-drying protocol, the HLM chips could be stored at room temperature (protected from light and moisture) for at least 9 months (n = 2 independent batches) and up to 16 months at best, with recovered enzyme activities within 60-120% of the non-freeze-dried control chips. This is a major improvement over the cold-storage requirement and the limited shelf life of the non-freeze-dried HLM chips, which can significantly ease the design of experiments, decrease energy consumption during storage, and reduce the shipping costs with a view to commercial adaptation. This study provides proof of concept of the feasibility of freeze-drying to shelf-life extension of human liver microsomal chips by several months.

To study and then harness the tumor-specific T cell dynamics after allogeneic hematopoietic stem cell transplant, we typed the frequency, phenotype, and function of lymphocytes directed against tumor-associated antigens (TAAs) in 39 consecutive transplanted patients, for 1 year after transplant. We showed that TAA-specific T cells circulated in 90% of patients but display a limited effector function associated to an exhaustion phenotype, particularly in the subgroup of patients deemed to relapse, where exhausted stem cell memory T cells accumulated. Accordingly, cancer-specific cytolytic functions were relevant only when the TAA-specific T cell receptors (TCRs) were transferred into healthy, genome-edited T cells. We then exploited trogocytosis and ligandome-on-chip technology to unveil the specificities of tumor-specific TCRs retrieved from the exhausted T cell pool. Overall, we showed that harnessing circulating TAA-specific and exhausted T cells allow to isolate TCRs against TAAs and previously not described acute myeloid leukemia antigens, potentially relevant for T cell–based cancer immunotherapy.

Identification of HLA class I ligands from the tumour surface (ligandome or immunopeptidome) is essential for designing T-cell mediated cancer therapeutic approaches. However, the sensitivity of the process for isolating MHC-I restricted tumor-specific peptides has been the major limiting factor for reliable tumor antigen characterization, making clear the need for technical improvement. Here, we describe our work from the fabrication and development of a novel microfluidic-based chip (PeptiCHIP) and to its use to identify and characterize tumor-speficic ligands on clinically relevant human organoids. Specifically, we assessed the potential of immobilizing a pan-HLA antibody on solid surfaces via well-characterized streptavidin-biotin chemistry, overcoming the limitations of the cross-linking chemistry used to prepare the affinity matrix with the desired antibodies in the immunopeptidomics workflow. Furthermore, to address the restrictions related to the handling and the limited availability of tumour samples, we further developed the concept towards the implementation of a microfluidic through-flow system. Thus, the biotinylated pan-HLA antibody was immobilized on streptavidin-functionalized surfaces, and immune-affinity purification (IP) was carried out on customized microfluidic pillar arrays made of thiol-ene polymer. Compared to the standard methods reported in the field, our methodology drastically reduces the handling, the amount of antibody and the time required for peptide isolation. In this work, we carefully examined the specificity and robustness of our customized technology for immunopeptidomics workflows. We tested this novel platform by immunopurifying HLA-I complexes from as few as 106 cells both in a widely studied B-cell line and in patients-derived ex vivo cell cultures. After the final elution in mild acid, HLA-I-presented peptides were identified by tandem mass spectrometry and further investigated by in vitro methods. These results highlight the potential to exploit microfluidics-based strategies in immunopeptidomics platforms and in personalized immunopeptidome analysis from cells isolated from individual tumour biopsies to design tailored cancer therapeutic vaccines.

Identification of HLA class I ligands from the tumor surface (ligandome or immunopeptidome) is essential for designing T-cell mediated cancer therapeutic approaches. However, the sensitivity of the process for isolating MHC-I restricted tumor-specific peptides has been the major limiting factor for reliable tumor antigen characterization, making clear the need for technical improvement. Here, we describe our work from the fabrication and development of a microfluidic-based chip (PeptiCHIP) and its use to identify and characterize tumor-specific ligands on clinically relevant human samples. Specifically, we assessed the potential of immobilizing a pan-HLA antibody on solid surfaces via well-characterized streptavidin-biotin chemistry, overcoming the limitations of the cross-linking chemistry used to prepare the affinity matrix with the desired antibodies in the immunopeptidomics workflow. Furthermore, to address the restrictions related to the handling and the limited availability of tumor samples, we further developed the concept toward the implementation of a microfluidic through-flow system. Thus, the biotinylated pan-HLA antibody was immobilized on streptavidin-functionalized surfaces, and immune-affinity purification (IP) was carried out on customized microfluidic pillar arrays made of thiol-ene polymer. Compared to the standard methods reported in the field, our methodology reduces the amount of antibody and the time required for peptide isolation. In this work, we carefully examined the specificity and robustness of our customized technology for immunopeptidomics workflows. We tested this platform by immunopurifying HLA-I complexes from 1 × 106 cells both in a widely studied B-cell line and in patients-derived ex vivo cell cultures, instead of 5 × 108 cells as required in the current technology. After the final elution in mild acid, HLA-I-presented peptides were identified by tandem mass spectrometry and further investigated by in vitro methods. These results highlight the potential to exploit microfluidics-based strategies in immunopeptidomics platforms and in personalized immunopeptidome analysis from cells isolated from individual tumor biopsies to design tailored cancer therapeutic vaccines. Moreover, the possibility to integrate multiple identical units on a single chip further improves the throughput and multiplexing of these assays with a view to clinical needs.

Abstract Knowledge about naturally presented HLA class I ligands from the tumor surface (the ligandome or immunopeptidome) is essential for designing T-cell mediated cancer therapeutic approaches. Indeed, the generation of specific anti-tumor CD8+ T cells relies on the recognition of tumor-antigens in the HLA-I complex. However, the sensitivity of the process for isolating the tumor antigens have been the major limiting factor for reliable HLA-I peptide characterization, making clear the need for technical improvement.Here, we assessed the potential of immobilizing a pan-HLA antibody on solid surfaces via well-characterized streptavidin-biotin chemistry, overcoming the limitations of the cross-linking chemistry used to prepare the affinity matrix with the desired antibodies in the immunopeptidomic workflow. Furthermore, to address the restrictions related to the handling and the limited availability of tumor samples, we further developed the concept towards the implementation of a microfluidic through-flow system.Thus, the biotinylated pan-HLA antibody was immobilized on streptavidin-functionalized surfaces, and immune-affinity purification (IP) was carried out on customized microfluidic pillar arrays made of thiol-ene polymer. Compared to the standard methods reported in the field, our methodology drastically reduces the handling, the amount of antibody and the time required for peptide isolation.In this work, we carefully examined the specificity and robustness of our customized technology for immunopeptidomics workflows. We tested this novel platform by immunopurifying HLA-I complexes from as few as 106 cells both in a widely studied B-cell line and in patients-derived ex vivo cell cultures. After the final elution in mild acid, HLA-I-presented peptides were identified by tandem mass spectrometry and further investigated by in vitro methods. These results highlight the potential to exploit microfluidics-based strategies in immunopeptidomic platforms and in personalized immunopeptidome analysis from cells isolated from individual tumor biopsies to design tailored cancer therapeutic vaccines. Citation Format: Sara Feola, Markus Happala, Karita Peltonen, Cristian Capasso, Gabriella Antignani, Antonio Federico, Vilja Pietiäinen, Jacopo Chiaro, Michaela Feodoroff, Antti Ranniko, Manlio Fusciello, Satu Koskela, Jukka Partanen, Firas Hamdan, Sari Tähkä, Erkko Ylösmäki, Dario Greco, Mikaela Grönholm, Rui M M Branca, Janne Lehtiö, Tiina Siikanen, Vincenzo Cerullo. PeptiCHIP: A novel microfluidic-based chip platform for tumor antigen landscape identification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1897.

The superfamily of hepatic cytochrome P450 (CYP) enzymes is responsible for the intrinsic clearance of the majority of therapeutic drugs in humans. However, the kinetics of drug clearance via CYPs varies significantly among individuals due to both genetic and external factors, and the enzyme amount and function are also largely impacted by many liver diseases. In this study, we developed a new methodology, based on digital microfluidics (DMF), for rapid determination of individual alterations in CYP activity from human-derived liver samples in biopsy-scale. The assay employs human liver microsomes (HLMs), immobilized on magnetic beads to facilitate determination of the activity of microsomal CYP enzymes in a parallelized system at physiological temperature. The thermal control is achieved with the help of a custom-designed, inkjet-printed microheater array modularly integrated with the DMF platform. The CYP activities are determined with the help of prefluorescent, enzyme-selective model compounds by quantifying the respective fluorescent metabolites based on optical readout in situ. The selectivity and sensitivity of the assay was established for four different CYP model reactions, and the diagnostic concept was validated by determining the interindividual variation in one of the four model reaction activities, that is, ethoxyresorufin O-deethylation (CYP1A1/2), between five donors. Overall, the developed protocol consumes only about 15 μg microsomal protein per assay. It is thus technically adaptable to screening of individual differences in CYP enzyme function from biopsy-scale liver samples in an automated fashion, so as to support tailoring of medical therapies, for example, in the context of liver disease diagnosis.

Microfluidic sample manipulation is a key enabler in modern chemical biology research. Both discrete droplet‐based digital microfluidic (DMF) assays and continuous flow in‐channel assays are well established, each featuring unique advantages from the viewpoint of automation and parallelization. However, there are marked differences in the applicable microfabrication materials and methods, which limit the interfacing of DMF sample preparation with in‐channel separation systems, such as the gold standard microchip electrophoresis. Simultaneously, there is an increasing demand for low‐cost and user‐friendly manufacturing techniques to foster the adaptation of microfluidic technology in routine laboratory analyses. This work demonstrates integration of DMF with in‐channel separation systems using only low‐cost and accessible (non‐cleanroom) manufacturing techniques, i.e., inkjet printing of silver for patterning of the driving electrodes and UV curing of off‐stoichiometric thiol–ene (OSTE) polymers both for dielectric coating of the electrode arrays and replica molding of the microchannel network. As a dielectric, OSTE performs similar to Parylene C (a gold standard dielectric in electrowetting), whereas its tunable surface and bulk properties facilitate straightforward bonding of the microchannel with the dielectric layer. In addition, a new chip design that facilitates efficient droplet transfer from the DMF part to the microchannel inlet solely by electrowetting is showcased.

Three-dimensional (3D) printing has recently emerged as a cost-effective alternative for rapid prototyping of microfluidic devices. The feature resolution of stereolithography-based 3D printing is particularly well suited for manufacturing of continuous flow cell culture platforms. Poor cell adhesion or material-induced cell death may, however, limit the introduction of new materials to microfluidic cell culture. In this work, we characterized four commercially available materials commonly used in stereolithography-based 3D printing with respect to long-term (2 month) cell survival on native 3D printed surfaces. Cell proliferation rates, along with material-induced effects on apoptosis and cell survival, were examined in mouse embryonic fibroblasts. Additionally, the feasibility of Dental SG (material with the most favored properties) for culturing of human hepatocytes and human-induced pluripotent stem cells was evaluated. The strength of cell adhesion to Dental SG was further examined over a shear force gradient of 1-89 dyne per cm(2)by using a custom-designed microfluidic shear force assay incorporating a 3D printed, tilted and tapered microchannel sealed with a polydimethylsiloxane lid. According to our results, autoclavation of the devices prior to cell seeding played the most important role in facilitating long-term cell survival on the native 3D printed surfaces with the shear force threshold in the range of 3-8 dyne per cm(2).

A new heated capillary photoionization (CPI) ion source design was developed to photoionize analytes inside a transfer capillary between a gas chromatograph (GC) and a mass spectrometer (MS). The CPI setup included a wide, oval-shaped vacuum-ultraviolet (VUV) transparent magnesium fluoride (MgF2) window to maximize photoionization efficiency and thus sensitivity. The source contained a nitrogen housing around the ionization chamber inlet to avoid undesirable hydrolysis and oxidation reactions with ambient air and to maximize the proportion of formed molecular radical cations of analytes. The feasibility of the ion source was studied by analyzing 18 endogenous steroids in urine as their trimethylsilyl (TMS) derivatives with gas chromatography-tandem mass spectrometry (GC-MS/MS). The method was validated and applied to human urine samples. To our best knowledge, this is the first time that a capillary photoionization ion source has been applied for quantitative analysis of biological samples. The GC-CPI-MS/MS method showed good chromatographic resolution (peak half-widths between 3.1 to 5.3 s), acceptable linearity (coefficient of determination between 0.981 to 0.996), and repeatability (relative standard deviation (RSD%) between 5 to 18%). Limits of detection (LOD) were between 2 to 100 pg mL(-1) and limits of quantitation (LOQ) were between 0.05 to 2 ng mL(-1). In total, 15 steroids were quantified either as a free steroid or glucuronide conjugate from the urine of volunteers. The new CPI source design showed excellent sensitivity for analysis of steroids in complex biological samples. (C) 2019 Elsevier B.V. All rights reserved.

This is the first report on capillary photoionization (CPI) interfacing a liquid chromatograph (LC) and mass spectrometer (MS). A new heated CPI ion source was developed, including a heated transfer capillary, a wide oval-shaped and low-depth ionization chamber with a vacuum ultraviolet (VUV) transparent magnesium fluoride (MgF2) window to increase the photoionization efficiency and thus the sensitivity. As both analytes and eluent are first vaporized and then photoionized inside the CPI ion source between the atmosphere and the vacuum of MS, the ion transfer efficiency into the MS and thus the sensitivity is improved. The effect of the most important operation parameters, the eluent flow rate and temperature of the CPI source, on the signal intensity was studied with selected steroids. The feasibility of LC-CPI-MS/MS for the quantitative analysis of steroids was also studied in terms of linearity, repeatability, and limits of detection. The method showed good quantitative performance and sensitivity down to the low femto-mole level.

We have identified the most likely reaction mechanism for oxidizing heptafulvenes to the corresponding tropones by experimental and theoretical investigations. The experimental studies were done by coupling a three-dimensional printed miniaturized reactor with an integrated electrospray ionization needle to a mass spectrometer. Using the experimentally observed ions as a basis, nine alternative reaction pathways were investigated with density functional theory calculations. The lowest energy reaction pathway starts with the formation of an epoxide that is opened upon the addition of a second equivalent of the oxidizing species meta-chloroperoxybenzoic acid. The adduct formed then undergoes a Criegee-like rearrangement to yield a positively charged hemiketal, which on deprotonation dissociates into acetone and tropone. Overall, the reaction mechanism resembles a Hock-like rearrangement.

A simple flow chemistry microreactor with an electrospray ionization tip for real time mass spectrometric reaction monitoring is introduced. The microreactor was fabricated by a laser-based additive manufacturing technique from acid-resistant stainless steel 316L. The functionality of the microreactor was investigated by using an inverse electron demand Diels-Alder and subsequent retro Diels-Alder reaction for testing. Challenges and problems encountered are discussed and improvements proposed. Adsorption of reagents to the rough stainless steel channel walls, short length of the reaction channel, and making a proper ESI tip present challenges, but the microreactor is potentially useful as a disposable device.

We report the development and characterization of digital microfluidic (DMF) immobilized enzyme reactors (IMERs) for studying cytochrome P450 (CYP)-mediated drug metabolism on droplet scale. The on-chip IMERs consist of porous polymer (thiol-ene) monolith plugs prepared in situ by photopolymerization and functionalized with recombinant CYP1A1 isoforms (an important detoxification route for many drugs and other xenobiotics). The DMF devices also incorporate inexpensive, inkjet-printed microheaters for on-demand regio-specific heating of the IMERs to physiological temperature, which is crucial for maintaining the activity of the temperature-sensitive CYP reaction. For on-chip monitoring of the CYP activity, the DMF devices were combined with a commercial well-plate reader, and a custom fluorescence quantification method was developed for detection of the chosen CYP1A1 model activity (ethoxyresorufin-O-deethylation). The reproducibility of the developed assay was examined with the help of ten parallel CYP-IMERs. All CYP-IMERs provided statistically significant difference (in fluorescence response) compared to any of the negative controls (including room-temperature reactions). The average (n = 10) turnover rate was 20.3 ± 9.0 fmol resorufin per minute. Via parallelization, the concept of the droplet-based CYP-IMER developed in this study provides a viable approach to rapid and low-cost prediction of the metabolic clearance of new chemical entities in vitro.

This work describes the interfacing of electrowetting-on-dielectric based digital microfluidic (DMF) sample preparation devices with ambient mass spectrometry (MS) via desorption atmospheric pressure photoionization (DAPPI). The DMF droplet manipulation technique was adopted to facilitate drug distribution and metabolism assays in droplet scale, while ambient mass spectrometry (MS) was exploited for the analysis of dried samples directly on the surface of the DMF device. Although ambient MS is well-established for bio- and forensic analyses directly on surfaces, its interfacing with DMF is scarce and requires careful optimization of the surface-sensitive processes, such as sample precipitation and the subsequent desorption/ionization. These technical challenges were addressed and resolved in this study by making use of the high mechanical, thermal, and chemical stability of SU-8. In our assay design, SU-8 served as the dielectric layer for DMF as well as the substrate material for DAPPI-MS. The feasibility of SU-8 based DMF devices for DAPPI-MS was demonstrated in the analysis of selected pharmaceuticals following on-chip liquid-liquid extraction or an enzymatic dealkylation reaction. The lower limits of detection were in the range of 1–10 pmol per droplet (0.25–1.0 µg/mL) for all pharmaceuticals tested.