Andrea Pavesi

Controlled electromechanical cell stimulation on-a-chip

Stem cell research has yielded promising advances in regenerative medicine, but standard assays generally lack the ability to combine different cell stimulations with rapid sample processing and precise fluid control. In this work, we describe the design and fabrication of a micro-scale cell stimulator capable of simultaneously providing mechanical, electrical, and biochemical stimulation, and subsequently extracting detailed morphological and gene-expression analysis on the cellular response. This micro-device offers the opportunity to overcome previous limitations and recreate critical elements of the in vivo microenvironment in order to investigate cellular responses to three different stimulations. The platform was validated in experiments using human bone marrow mesenchymal stem cells. These experiments demonstrated the ability for inducing changes in cell morphology, cytoskeletal fiber orientation and changes in gene expression under physiological stimuli. This novel bioengineering approach can be readily applied to various studies, especially in the fields of stem cell biology and regenerative medicine.

Engineering a 3D microfluidic culture platform for tumor-treating field application

The limitations of current cancer therapies highlight the urgent need for a more effective therapeutic strategy. One promising approach uses an alternating electric field; however, the mechanisms involved in the disruption of the cancer cell cycle as well as the potential adverse effects on non-cancerous cells must be clarified. In this study, we present a novel microfluidic device with embedded electrodes that enables the application of an alternating electric field therapy to cancer cells in a 3D extracellular matrix. To demonstrate the potential of our system to aid in designing and testing new therapeutic approaches, cancer cells and cancer cell aggregates were cultured individually or co-cultured with endothelial cells. The metastatic potential of the cancer cells was reduced after electric field treatment. Moreover, the proliferation rate of the treated cancer cells was lower compared with that of the untreated cells, whereas the morphologies and proliferative capacities of the endothelial cells were not significantly affected. These results demonstrate that our novel system can be used to rapidly screen the effect of an alternating electric field on cancer and normal cells within an in vivo-like microenvironment with the potential to optimize treatment protocols and evaluate synergies between tumor-treating field treatment and chemotherapy.

Monophasic and biphasic electrical stimulation induces a precardiac differentiation in progenitor cells isolated from human heart.

Electrical stimulation (ES) of cells has been shown to induce a variety of responses, such as cytoskeleton rearrangements, migration, proliferation, and differentiation. In this study, we have investigated whether monophasic and biphasic pulsed ES could exert any effect on the proliferation and differentiation of human cardiac progenitor cells (hCPCs) isolated from human heart fragments. Cells were cultured under continuous exposure to monophasic or biphasic ES with fixed cycles for 1 or 3 days. Results indicate that neither stimulation protocol affected cell viability, while the cell shape became more elongated and reoriented more perpendicular to the electric field direction. Moreover, the biphasic ES clearly induced the upregulation of early cardiac transcription factors, MEF2D, GATA-4, and Nkx2.5, as well as the de novo expression of the late cardiac sarcomeric proteins, troponin T, cardiac alpha actinin, and SERCA 2a. Both treatments increased the expression of connexin 43 and its relocation to the cell membrane, but biphasic ES was faster and more effective. Finally, when hCPCs were exposed to both monophasic and biphasic ES, they expressed de novo the mRNA of the voltage-dependent calcium channel Cav 3.1(α1G) subunit, which is peculiar of the developing heart. Taken together, these results show that ES alone is able to set the conditions for early differentiation of adult hCPCs toward a cardiac phenotype.

Electrical conditioning of adipose‐derived stem cells in a multi‐chamber culture platform

In tissue engineering, several factors play key roles in providing adequate stimuli for cells differentiation, in particular biochemical and physical stimuli, which try to mimic the physiological microenvironments. Since electrical stimuli are important in the developing heart, we have developed an easy‐to‐use, cost‐effective cell culture platform, able to provide controlled electrical stimulation aimed at investigating the influence of the electric field in the stem cell differentiation process. This bioreactor consists of an electrical stimulator and 12 independent, petri‐like culture chambers and a 3‐D computational model was used to characterize the distribution and the intensity of the electric field generated in the cell culture volume. We explored the effects of monophasic and biphasic square wave pulse stimulation on a mouse adipose‐derived stem cell line (m17.ASC) comparing cell viability, proliferation, protein, and gene expression. Both monophasic (8 V, 2 ms, 1 Hz) and biphasic (+4 V, 1 ms and −4 V, 1 ms; 1 Hz) stimulation were compatible with cell survival and proliferation. Biphasic stimulation induced the expression of Connexin 43, which was found to localize also at the cell membrane, which is its recognized functional mediating intercellular electrical coupling. Electrically stimulated cells showed an induced transcriptional profile more closely related to that of neonatal cadiomyocytes, particularly for biphasic stimulation. The developed platform thus allowed to set‐up precise conditions to drive adult stem cells toward a myocardial phenotype solely by physical stimuli, in the absence of exogenously added expensive bioactive molecules, and can thus represent a valuable tool for translational applications for heart tissue engineering and regeneration. Biotechnol. Bioeng. 2014;111: 1452–1463.

Advances in microfluidics in combating infectious diseases.

Abstract One of the important pursuits in science and engineering research today is to develop low-cost and user-friendly technologies to improve the health of people. Over the past decade, research efforts in microfluidics have been made to develop methods that can facilitate low-cost diagnosis of infectious diseases, especially in resource-poor settings. Here, we provide an overview of the recent advances in microfluidic devices for point-of-care (POC) diagnostics for infectious diseases and emphasis is placed on malaria, sepsis and AIDS/HIV. Other infectious diseases such as SARS, tuberculosis, and dengue are also briefly discussed. These infectious diseases are chosen as they contribute the most to disability-adjusted life-years (DALYs) lost according to the World Health Organization (WHO). The current state of research in this area is evaluated and projection toward future applications and accompanying challenges are also discussed.

On-chip assessment of human primary cardiac fibroblasts proliferative responses to uniaxial cyclic mechanical strain.

Cardiac cell function is substantially influenced by the nature and intensity of the mechanical loads the cells experience. Cardiac fibroblasts (CFs) are primarily involved in myocardial tissue remodeling: at the onset of specific pathological conditions, CFs activate, proliferate, differentiate, and critically alter the amount of myocardial extra‐cellular matrix with important consequences for myocardial functioning. While cyclic mechanical strain has been shown to increase matrix synthesis of CFs in vitro, the role of mechanical cues in CFs proliferation is unclear. We here developed a multi‐chamber cell straining microdevice for cell cultures under uniform, uniaxial cyclic strain. After careful characterization of the strain field, we extracted human heart‐derived CFs and performed cyclic strain experiments. We subjected cells to 2% or 8% cyclic strain for 24 h or 72 h, using immunofluorescence to investigate markers of cell morphology, cell proliferation (Ki67, EdU, phospho‐Histone‐H3) and subcellular localization of the mechanotransduction‐associated transcription factor YAP. Cell morphology was affected by cyclic strain in terms of cell area, cell and nuclear shape and cellular alignment. We additionally observed a strain intensity‐dependent control of cell growth: a significant proliferation increase occurred at 2% cyclic strain, while time‐dependent effects took place upon 8% cyclic strain. The YAP‐dependent mechano‐transduction pathway was similarly activated in both strain conditions. These results demonstrate a differential effect of cyclic strain intensity on human CFs proliferation control and provide insights into the YAP‐dependent mechano‐sensing machinery of human CFs. Biotechnol. Bioeng. 2016;113: 859–869.

Microfluidic models for adoptive cell-mediated cancer immunotherapies.

Current adoptive T cell therapies have shown promising results in clinical trials but need further development as an effective cancer treatment. Here, we discuss how 3D microfluidic tumour models mimicking the tumour microenvironment could help in testing T cell immunotherapies by assessing engineered T cells and identifying combinatorial therapy to improve therapeutic efficacy. We propose that 3D microfluidic systems can be used to screen different patient-specific treatments, thereby reducing the burden of in vivo testing and facilitating the rapid translation of successful T cell cancer immunotherapies to the clinic.

A pulsatile simulator for the in vitro analysis of the mitral valve with tri-axial papillary muscle displacement.

Purpose We developed a new pulsatile hydrodynamic simulator for the in vitro testing of mitral valve (MV) samples. The required specifications included a 3D positioning system for the papillary muscles (PMs) that is accurate and simple to manage; measurement of the force exerted by the chordae tendineae on the PMs; and the possibility to visually inspect the MV for kinematic analysis. Methods An atrial/ventricular chamber system was developed. The ventricular chamber housed a triaxial actuator system that was aligned to a morphometric Cartesian frame, allowing for PM positioning even while tests are running. Each PM holder had an embedded load cell for force measurement. The atrial chamber was designed so as to permit MV visual inspection, maintaining a non-disturbed flow at the sample inlet. The setup was subjected to trials with fresh porcine MVs. Flow and pressure difference across the MVs and PM forces were measured in different MV configurations, with different PM spatial dislocations. High speed video recordings were acquired. Results The positioning accuracy was assessed. Tests with MVs showed good usability, even by the non-engineering personnel. The effects of PM displacement on valve function (valve competence and PM forces) was consistent with previously published data, thus confirming the general soundness of the design principles. Conclusions The developed simulator is a promising instrument for performing MV in vitro tests in a precise, well-repeatable manner. The ability to completely adjust the PM position while a test is running boosts the simulators potential for detailed investigations of the pathological and surgically treated MV.

Modeling the Blood-Brain Barrier in a 3D triple co-culture microfluidic system.

The need for a blood-brain barrier (BBB) model that accurately mimics the physiological characteristics of the in-vivo situation is well-recognized by researchers in academia and industry. However, there is currently no in-vitro model allowing studies of neuronal growth and/or function influenced by factors from the blood that cross through the BBB. Therefore, we established a 3D triple co-culture microfluidic system using human umbilical vein endothelial cells (HUVEC) together with primary rat astrocytes and neurons. Immunostaining confirmed the successful triple co-culture system consisting of an intact BBB with tight intercellular junctions in the endothelial monolayer. The BBB selective permeability was determined by a fluorescent-based assay using dextrans of different molecular weights. Finally, neuron functionality was demonstrated by calcium imaging.

Using microfluidics to investigate tumor cell extravasation and T-cell immunotherapies

Understanding the mechanism of tumor cell extravasation, cell migration and the role of the immunosystem is crucial in creating targeted and patient-specific cancer therapies. We created an in-vitro microfluidic cell extravasation assay, incorporating a microvascular network and demonstrated its use to study cancer cells extravasation. Separately, we developed an assay for screening T-cell migration and cytotoxicity as a means to evaluate the efficiency of adoptive immunotherapies against cancer. Similar devices using a similar platform can be used to recreate a tumor liver microenvironment, taking in consideration the hypoxic and inflammatory conditions in the liver. These platforms show considerable potential as efficient pre-clinical models for testing the efficiency of cancer drugs and engineered T-cell functionality for personalized medicine.