Alessandro Zambon

High-efficiency cellular reprogramming with microfluidics

We report that the efficiency of reprogramming human somatic cells to induced pluripotent stem cells (hiPSCs) can be dramatically improved in a microfluidic environment. Microliter-volume confinement resulted in a 50-fold increase in efficiency over traditional reprogramming by delivery of synthetic mRNAs encoding transcription factors. In these small volumes, extracellular components of the TGF-β and other signaling pathways exhibited temporal regulation that appears critical to acquisition of pluripotency. The high quality and purity of the resulting hiPSCs (μ-hiPSCs) allowed direct differentiation into functional hepatocyte- and cardiomyocyte-like cells in the same platform without additional expansion.

Microfluidic-driven viral infection on cell cultures: Theoretical and experimental study.

Advanced cell culture systems creating a controlled and predictable microenvironment together with computational modeling may be useful tools to optimize the efficiency of cell infections. In this paper, we will present a phenomenological study of a virus-host infection system, and the development of a multilayered microfluidic platform used to accurately tune the virus delivery from a diffusive-limited regime to a convective-dominated regime. Mathematical models predicted the convective-diffusive regimes developed within the system itself and determined the dominating mass transport phenomena. Adenoviral vectors carrying the enhanced green fluorescent protein (EGFP) transgene were used at different multiplicities of infection (MOI) to infect multiple cell types, both in standard static and in perfused conditions. Our results validate the mathematical models and demonstrate how the infection processes through perfusion via microfluidic platform led to an enhancement of adenoviral infection efficiency even at low MOIs. This was particularly evident at the longer time points, since the establishment of steady-state condition guaranteed a constant viral concentration close to cells, thus strengthening the efficiency of infection. Finally, we introduced the concept of effective MOI, a more appropriate variable for microfluidic infections that considers the number of adenoviruses in solution per cell at a certain time.

High Temporal Resolution Detection of Patient-Specific Glucose Uptake from Human ex Vivo Adipose Tissue On-Chip.

Human tissue in vitro models on-chip are highly desirable to dissect the complexity of a physio-pathological in vivo response because of their advantages compared to traditional static culture systems in terms of high control of microenvironmental conditions, including accurate perturbations and high temporal resolution analyses of medium outflow. Human adipose tissue (hAT) is a key player in metabolic disorders, such as Type 2 Diabetes Mellitus (T2DM). It is involved in the overall energy homeostasis not only as passive energy storage but also as an important metabolic regulator. Here, we aim at developing a large scale microfluidic platform for generating high temporal resolution of glucose uptake profiles, and consequently insulin sensitivity, under physio-pathological stimulations in ex vivo adipose tissues from nondiabetic and T2DM individuals. A multiscale mathematical model that integrates fluid dynamics and an intracellular insulin signaling pathway description was used for assisting microfluidic design in order to maximize measurement accuracy of tissue metabolic activity in response to perturbations. An automated microfluidic injection system was included on-chip for performing precise dynamic biochemical stimulations. The temporal evolution of culture conditions could be monitored for days, before and after perturbation, measuring glucose concentration in the outflow with high temporal resolution. As a proof of concept for detection of insulin resistance, we measured insulin-dependent glucose uptake by hAT from nondiabetic and T2DM subjects, mimicking the postprandial response. The system presented thus represents an important tool in dissecting the role of single tissues, such as hAT, in the complex interwoven picture of metabolic diseases.

Determination of glucose flux in live myoblasts by microfluidic nanosensing and mathematical modeling

Glucose is the main energy source for cells in an organism and its blood concentration is tightly regulated in healthy individuals. However, impaired blood glucose control has been found in diseases such as metabolic syndrome and diabetes, and anomalous glucose utilization in cancer tissues. Dissecting the dynamics of the different phenomena involved in glucose handling (extracellular mass transport, membrane diffusion, and intracellular phosphorylation) is very relevant to identify which mechanisms are disrupted under disease conditions. In this work, we developed an effective methodology for quantitatively analyzing these phenomena in living cells. A measurement of steady-state glucose uptake is, by itself, insufficient to determine the dynamics of intracellular glucose. For this purpose, we integrated two types of measurements: cytosolic glucose concentration at the single-cell level, obtained using a cytosolic FRET nanosensor, and cell population glucose uptake, obtained without perturbing culture conditions using a microfluidic perfusion system. Microfluidics enabled accurate temporal stimulation of cells through cyclic pulses of glucose concentration at defined flow rates. We found that both, glucose uptake and phosphorylation, are linearly dependent on glucose concentration in the physiological range. Mathematical modeling enabled precise determination of the kinetic constants of membrane transport (0.27 s(-1)) and intracellular phosphorylation (2.01 s(-1)).

Flow biosensing and sampling in indirect electrochemical detection.

Miniaturization in biological analyses has several advantages, such as sample volume reduction and fast response time. The integration of miniaturized biosensors within lab-on-a-chip setups under flow conditions is highly desirable, not only because it simplifies process handling but also because measurements become more robust and operator-independent. In this work, we study the integration of flow amperometric biosensors within a microfluidic platform when analyte concentration is indirectly measured. As a case study, we used a platinum miniaturized glucose biosensor, where glucose is enzymatically converted to [Formula: see text] that is oxidized at the electrode. The experimental results produced are strongly coupled to a theoretical analysis of fluid dynamic conditions affecting the electrochemical response of the sensor. We verified that the choice of the inlet flow rate is a critical parameter in flow biosensors, because it affects both glucose and [Formula: see text] transport, to and from the electrode. We identify optimal flow rate conditions for accurate sensing at high time resolution. A dimensionless theoretical analysis allows the extension of the results to other sensing systems according to fluid dynamic similarity principles. Furthermore, we developed a microfluidic design that connects a sampling unit to the biosensor, in order to decouple the sampling flow rate from that of the actual measurement.

In Situ Raman Analysis of CO2—Assisted Drying of Fruit-Slices

This work explores the feasibility of applying in situ Raman spectroscopy for the online monitoring of the supercritical carbon dioxide (SC-CO2) drying of fruits. Specifically, we investigate two types of fruits: mango and persimmon. The drying experiments were carried out inside an optical accessible vessel at 10 MPa and 313 K. The Raman spectra reveal: (i) the reduction of the water from the fruit slice and (ii) the change of the fruit matrix structure during the drying process. Two different Raman excitation wavelengths were compared: 532 nm and 785 nm. With respect to the quality of the obtained spectra, the 532 nm excitation wavelength was superior due to a higher signal-to-noise ratio and due to a resonant excitation scheme of the carotenoid molecules. It was found that the absorption of CO2 into the fruit matrix enhances the extraction of water, which was expressed by the obtained drying kinetic curve.

Straightforward Micropatterning of Oligonucleotides in Microfluidics by Novel Spin-On ZrO2 Surfaces

DNA biochip assays often require immobilization of bioactive molecules on solid surfaces. A simple biofunctionalization protocol and precise spatial binding represent the two major challenges in order to obtain localized region specific biopatterns into lab-on-a-chip (LOC) systems. In this work, a simple strategy to anchor oligonucleotides on microstructured areas and integrate the biomolecules patterns within microfluidic channels is reported. A photosensitive ZrO2 system is proposed as an advanced platform and versatile interface for specific positioning and oriented immobilization of phosphorylated DNA. ZrO2 sol-gel structures were easily produced on fused silica by direct UV lithography, allowing a simple and fast patterning process with different geometries. A thermal treatment at 800 °C was performed to crystallize the structures and maximize the affinity of DNA to ZrO2. Fluorescent DNA strands were selectively immobilized on the crystalline patterns inside polydimethylsiloxane (PDMS) microchannels, allowing high specificity and rapid hybridization kinetics. Hybridization tests confirmed the correct probe anchoring and the bioactivity retention, while denaturation experiments demonstrated the possibility of regenerating the surface.

Enzymatic, physicochemical, nutritional and phytochemical profile changes of apple (Golden Delicious L.) juice under supercritical carbon dioxide and long-term cold storage

The impact of supercritical carbon dioxide (SCCD) (10-60 MPa/45 °C/30 min) and subsequent 10 weeks storage at 4 °C on polyphenol oxidase (PPO), peroxidase (POD) activities, phenolic profile, vitamin C, sugars, physicochemical properties of cloudy apple juices was investigated. No significant changes in sugars and total polyphenols were observed, whereas significant degradation (≈28%) of vitamin C and individual polyphenols (≈18%) was noted after SCCD treatment. After 4 weeks storage only 34% of vitamin C was retained and no vitamin C was detected after this time. Ten weeks of storage caused hydrolysis of sucrose in 15%, whereas degradation of individual polyphenols ranged from 43 to 50% depending on the pressure applied. The highest pressure was applied the highest retention of polyphenols was observed. The lightness of juice significantly increased by 15% after SCCD and decreased during storage. Moreover, the synergistic effect of both enzymes with chlorogenic acid and catechol was found.

Microbial inactivation efficiency of supercritical CO2 drying process

ABSTRACT Conventional drying of spices, as hot air treatment, often needs an additional downstream inactivation step to decrease the microbial load of the dried product and improve its microbial safety and microbial quality. In this regard, the present work explored the possibility to dry and decontaminate food in a single step using supercritical carbon dioxide (scCO2) as a drying agent. A case study was focused on the drying of herbs and the antimicrobial effects were evaluated on the naturally present microbiota. For this purpose, experiments were carried out on coriander leaves using a high pressure vessel at 10 MPa, at two different temperatures (40 and 50°C) with drying time of 0 and 150 min to establish the influence of each parameter on the microbial inactivation. Yeasts and molds appeared to be the least resistant to scCO2 as they could never be detected after the treatment (<2 log CFU/g). Mesophilic bacteria were also significantly reduced, up to 4 log CFU/g, but remained above the limit of quantification. The quality of the dried product was comparable with the quality of air-dried samples in terms of phenolic constituents. Overall, the results indicated that scCO2 drying was a promising green drying technique combining both drying and microbial inactivation in a single step with a relevant impact on safety and costs.

Erratum to: Simvastatin Rapidly and Reversibly Inhibits Insulin Secretion in Intact Single-Islet Cultures.

In the original publication, a statement of equal contribution was not included. The text should read as: Valentina Scattolini and Camilla Luni contributed equally to the manuscript. Open Access. This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/ by-nc/4.0/), which permits any noncommercial use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.