A multi-channel microfluidic platform based on human flavin-containing monooxygenase 3 for personalised medicine.

Human flavin-containing monooxygenase 3 (FMO3) is a drug-metabolizing enzyme (DME) which is known to be highly polymorphic. Some of its polymorphic variants are associated with inter-individual differences that contribute to drug response. In order to measure these differences, the implementation of a quick and efficient in vitro assay is highly desirable. To this end, in this work a microfluidic immobilized enzyme reactor (µ-IMER) was developed with four separate serpentines where FMO3 and its two common polymorphic variants (V257M and E158K) were covalently immobilized via glutaraldehyde cross-linking in the presence of a polylysine coating. Computational fluid dynamics simulations were performed to calculate the selected substrate retention time in serpentines with different surface areas at various flow rates. The oxidation of tamoxifen, an anti-breast cancer drug, was used as a model reaction to characterize the new device in terms of available surface area for immobilization, channel coating, and applied flow rate. The highest amount of product was obtained when applying a 10 µL min-1 flow rate on polylysine-coated serpentines with a surface area of 90 mm2 each. Moreover, these conditions were used to test the device as a multi-enzymatic platform by simultaneously assessing the conversion of tamoxifen by FMO3 and its two polymorphic variants immobilized on different serpentines of the same chip. The results obtained demonstrate that the differences observed in the conversion of tamoxifen within the chip are similar to those already published (E158K > WT > V257M). Therefore, this microfluidic platform provides a feasible option for fabricating devices for personalised medicine.

Development of a Paper-based Hematocrit Test and a Lateral Flow Assay to Detect Critical Fibrinogen Concentrations Using a Bottom-Up Pyramid Workflow Approach.

Fibrinogen is a coagulation factor in human blood and the first one to reach critical levels in major bleeding. Hypofibrinogenemia (a too low fibrinogen concentration in blood) poses great challenges to first responders, clinicians, and healthcare providers since it represents a risk factor for exsanguination and massive transfusion requirements. Thus, the rapid assessment of the fibrinogen concentration at the point of care has gained considerable importance in preventing and managing major blood loss. However, in whole blood measurements, hematocrit variations affect the amount (volume fraction) of plasma that passes the detection zone. In an attempt to accurately determine realistic critical levels of fibrinogen (<1.5 mg/mL) in patients needing immediate treatment and medical interventions, we have developed novel diagnostic systems capable of estimating hematocrit and critical fibrinogen concentrations. A lateral flow assay (LFA) for the detection of fibrinogen has been developed by establishing a workflow employing rapid characterization methods to streamline LFA development. The integration of two detection lines enables (i) the identification of fibrinogen (first line) present in the sample and (ii) the determination of the clinically critical fibrinogen concentrations below 1.5 mg/mL (second line). Furthermore, the paper-based separation of blood cells from plasma provides a semiquantitative estimate of the hematocrit by analyzing the fractions. Initial validation of the point-of-care (PoC) hematocrit test revealed good comparability to a standard laboratory method. The developed diagnostic systems have the ability to accelerate decision-making in cases with major bleeding.

Application of a Biomimetic Nanoparticle-Based Mock Virus to Determine SARS-CoV-2 Neutralizing Antibody Levels in Blood Samples Using a Lateral Flow Assay.

The presence of neutralizing antibodies against SARS-CoV-2 in blood, acquired through previous infection or vaccination, is known to prevent the (re)occurrence of outbreaks unless the virus mutates. Therefore, the measurement of neutralizing antibodies constitutes an indispensable tool in assessing an individual's and a population's immunity against SARS-CoV-2. For this reason, we have developed an innovative lateral flow assay (LFA) capable of detecting blood-derived neutralizing antibodies using a biomimetic SARS-CoV-2 mock virus system. Here, functionalized gold nanoparticles (AuNPs) featuring the trimeric spike (S) protein at its surface imitate the virus's structure and are applied to monitor the presence and efficacy of neutralizing antibodies in blood samples. The detection principle relies on the interaction between mock virus and the immobilized angiotensin-converting enzyme 2 (ACE2) receptor, which is inhibited when neutralizing antibodies are present. To further enhance the sensitivity of our competitive assay and identify low titers of neutralizing antibodies, an additional mixing pad is embedded into the device to increase the interaction time between mock virus and neutralizing antibodies. The developed LFA is benchmarked against the WHO International Standard (21/338) and demonstrated reliable quantification of neutralizing antibodies that inhibit ACE2 binding events down to a detection limit of an antibody titer of 59 IU/mL. Additional validation using whole blood and plasma samples showed reproducible results and good comparability to a laboratory-based reference test, thus highlighting its applicability for point-of-care testing.

Using Rapid Prototyping to Develop a Cell-Based Platform with Electrical Impedance Sensor Membranes for In Vitro RPMI2650 Nasal Nanotoxicology Monitoring.

Due to advances in additive manufacturing and prototyping, affordable and rapid microfluidic sensor-integrated assays can be fabricated using additive manufacturing, xurography and electrode shadow masking to create versatile platform technologies aimed toward qualitative assessment of acute cytotoxic or cytolytic events using stand-alone biochip platforms in the context of environmental risk assessment. In the current study, we established a nasal mucosa biosensing platform using RPMI2650 mucosa cells inside a membrane-integrated impedance-sensing biochip using exclusively rapid prototyping technologies. In a final proof-of-concept, we applied this biosensing platform to create human cell models of nasal mucosa for monitoring the acute cytotoxic effect of zinc oxide reference nanoparticles. Our data generated with the biochip platform successfully monitored the acute toxicity and cytolytic activity of 6 mM zinc oxide nanoparticles, which was non-invasively monitored as a negative impedance slope on nasal epithelial models, demonstrating the feasibility of rapid prototyping technologies such as additive manufacturing and xurography for cell-based platform development.

Breaking the Third Wall: Implementing 3D-Printing Technics to Expand the Complexity and Abilities of Multi-Organ-on-a-Chip Devices.

The understanding that systemic context and tissue crosstalk are essential keys for bridging the gap between in vitro models and in vivo conditions led to a growing effort in the last decade to develop advanced multi-organ-on-a-chip devices. However, many of the proposed devices have failed to implement the means to allow for conditions tailored to each organ individually, a crucial aspect in cell functionality. Here, we present two 3D-print-based fabrication methods for a generic multi-organ-on-a-chip device: One with a PDMS microfluidic core unit and one based on 3D-printed units. The device was designed for culturing different tissues in separate compartments by integrating individual pairs of inlets and outlets, thus enabling tissue-specific perfusion rates that facilitate the generation of individual tissue-adapted perfusion profiles. The device allowed tissue crosstalk using microchannel configuration and permeable membranes used as barriers between individual cell culture compartments. Computational fluid dynamics (CFD) simulation confirmed the capability to generate significant differences in shear stress between the two individual culture compartments, each with a selective shear force. In addition, we provide preliminary findings that indicate the feasibility for biological compatibility for cell culture and long-term incubation in 3D-printed wells. Finally, we offer a cost-effective, accessible protocol enabling the design and fabrication of advanced multi-organ-on-a-chip devices.

A Microfluidic Multisize Spheroid Array for Multiparametric Screening of Anticancer Drugs and Blood-Brain Barrier Transport Properties.

Physiological-relevant in vitro tissue models with their promise of better predictability have the potential to improve drug screening outcomes in preclinical studies. Despite the advances of spheroid models in pharmaceutical screening applications, variations in spheroid size and consequential altered cell responses often lead to nonreproducible and unpredictable results. Here, a microfluidic multisize spheroid array is established and characterized using liver, lung, colon, and skin cells as well as a triple-culture model of the blood-brain barrier (BBB) to assess the effects of spheroid size on (a) anticancer drug toxicity and (b) compound penetration across an advanced BBB model. The reproducible on-chip generation of 360 spheroids of five dimensions on a well-plate format using an integrated microlens technology is demonstrated. While spheroid size-related IC50 values vary up to 160% using the anticancer drugs cisplatin (CIS) or doxorubicin (DOX), reduced CIS:DOX drug dose combinations eliminate all lung microtumors independent of their sizes. A further application includes optimizing cell seeding ratios and size-dependent compound uptake studies in a perfused BBB model. Generally, smaller BBB-spheroids reveal an 80% higher compound penetration than larger spheroids while verifying the BBB opening effect of mannitol and a spheroid size-related modulation on paracellular transport properties.

Cytotoxicity, Retention, and Anti-inflammatory Effects of a CeO2 Nanoparticle-Based Supramolecular Complex in a 3D Liver Cell Culture Model.

Both cerium oxide (CeOx) nanoparticles and mefenamic acid (MFA) are known anti-inflammatory agents with hepatoprotective properties and are therefore prescribed for one of the major diseases in the world, nonalcoholic fatty liver disease (NAFLD). To study the potential cytotoxicity and anti-inflammatory effects as well as drug retention of a potential therapeutic CeOx/MFA supramolecular complex, a well-standardized hepatic (HepG2) spheroid model was used. Results showed that the highest cytotoxicity for the CeOx/MFA supramolecular complex was found at 50 µg/mL, while effective doses of 0.1 and 1 µg/mL yielded a significant decrease of TNF-α and IL-8 secretion. Time-resolved analysis of HepG2 spheroids revealed a spatiotemporal distribution of the supramolecular complex and limited clearance from the internal microtissue over a period of 8 days in cultivation. In summary, our results point at rapid uptake, distribution, and biostability of the supramolecular complex within the HepG2 liver spheroid model as well as a significant anti-inflammatory response at noncytotoxic levels.