design-validation-volumetric-extrusion-bioprinter-for-bioprinting

Design and Validation of a Volumetric-extrusion Bioprinter for Bioprinting of Soluble Basement Membrane Extract for Translational Research

In translational research, 3D in vitro models are essential for advancing scientific understanding and therapeutic development. To this aim, Soluble ...

Politecnico di Milano

Recruitment of leukocytes from blood to tissues is a multi-step process playing a major role in the activation of inflammatory responses. Tethering and rolling of leukocytes along the vessel wall, followed by arrest and transmigration through the endothelium result from chemoattractant-dependent signals, inducing adhesive and migratory events. Shear forces exerted by the blood flow on leukocytes induce rolling via selectin-mediated interactions with endothelial cells and increase the probability of leukocytes to engage their chemokine receptors, facilitating integrin activation and consequent arrest. Flow-derived shear forces generate mechanical stimuli concurring with biochemical signals in the modulation of leukocyte-endothelial cell interactions. In the last few years, a host of in vitro studies have clarified the biochemical adhesion cascade and the role of shear stress in leukocyte extravasation. The limitation of the static environment in Boyden devices has been overcome both by the use of parallel-plate flow chambers and by custom models mimicking the in vivo conditions, along with widespread microfluidic approaches to in vitro modeling. These devices create an in vitro biomimetic environment where the multi-step transmigration process can be imaged and quantified under mechanical and biochemical controlled conditions, including fluid dynamic settings, channel design, materials and surface coatings. This paper reviews the technological solutions recently proposed to model, observe and quantify leukocyte adhesion behavior under shear flow, with a final survey of high-throughput solutions featuring multiple parallel assays as well as thorough and time-saving statistical interpretation of the experimental results.

Microfluidics for in vitro biomimetic shear stress-dependent leukocyte adhesion assays.

Nucleic acid aptamers possess attractive features such as specific molecular recognition, high-affinity binding, and rapid acquisition and replication, which could be feasible components for separating specific cells from other cell types. This study demonstrates that aptamers conjugated to an oligopeptide self-assembled monolayer (SAM) can be used to selectively trap human hepatic cancer cells from cell mixtures containing normal human hepatocytes or human fibroblasts. Molecular dynamics calculations have been performed to understand how the configurations of the aptamers are related to the experimental results of selective cell capture. We further demonstrate that the captured hepatic cancer cells can be detached and collected along with electrochemical desorption of the oligopeptide SAM, and by repeating these catch-and-release processes, target cells can be enriched. This combination of capture with aptamers and detachment with electrochemical reactions is a promising tool in various research fields ranging from basic cancer research to tissue engineering applications.

Catch-and-Release of Target Cells Using Aptamer-Conjugated Electroactive Zwitterionic Oligopeptide SAM.

Label-free approaches to assess cell properties ideally suit the requirements of cell-based therapeutics, since they permit to characterize cells with minimal perturbation and manipulation, at the benefit of sample recovery and re-employment for treatment. For this reason, label-free techniques would find sensible application in adoptive T cell-based immunotherapy. In this work, we describe the label-free and single-cell detection of in vitro activated T lymphocytes in flow through an electrical impedance-based setup. We describe a novel platform featuring 3D free-standing microelectrodes presenting passive upstream and downstream extensions and integrated into microfluidic channels. We employ such device to measure the impedance change associated with T cell activation at electrical frequencies maximizing the difference between non-activated and activated T cells. Finally, we harness the impedance signature of unstimulated T cells to set a boundary separating activated and non-activated clones, so to characterize the selectivity and specificity of the system. In conclusion, the strategy here proposed highlights the possible employment of impedance to assess T cell activation in label-free.

Label-free identification of activated T lymphocytes through tridimensional microsensors on chip.

The in vitro replication of physiological mechanical conditioning through bioreactors plays a crucial role in the development of functional Small-Caliber Tissue-Engineered Blood Vessels. An in silico scaffold-specific model under pulsatile perfusion provided by a bioreactor was implemented using a fluid-structure interaction (FSI) approach for viscoelastic tubular scaffolds (e.g. decellularized swine arteries, DSA). Results of working pressures, circumferential deformations, and wall shear stress on DSA fell within the desired physiological range and indicated the ability of this model to correctly predict the mechanical conditioning acting on the cells-scaffold system. Consequently, the FSI model allowed us to a priori define the stimulation pattern, driving in vitro physiological maturation of scaffolds, especially with viscoelastic properties.

Estimation of the physiological mechanical conditioning in vascular tissue engineering by a predictive fluid-structure interaction approach.

Selected adsorption efficiency of a molecule in solution in a microchannel is strongly influenced by the convective/diffusive mass transport phenomena that supply the target molecule to the adsorption surface. In a standard microchannel with a rectangular cross section, laminar flow regime limits the fluid mixing, thus suggesting that mass transport conditions can be improved by the introduction of herringbone-like structures. Tuning of these geometrical patterns increases the concentration gradient of the target molecule at the adsorption surface. A computational fluid dynamic (CFD) study was performed to evaluate the relation between the geometrical herringbone patterns and the concentration gradient improvement in a 14 mm long microchannel. The results show that the inhomogeneity of the concentration gradient can provide an improved and localized adsorption under specific geometrical features, which can be tuned in order to adapt the adsorption pattern to the specific assay requirements.

Herringbone-like hydrodynamic structures in microchannels: A CFD model to evaluate the enhancement of surface binding.

The liver is organized in hexagonal functional units - termed lobules - characterized by a rather peculiar blood microcirculation, due to the presence of a tangled network of capillaries - termed sinusoids. A better understanding of the hemodynamics that governs liver microcirculation is relevant to clinical and biological studies aimed at improving our management of liver diseases and transplantation. Herein, we built a CFD model of a 3D sinusoidal network, based on in vivo images of a physiological mouse liver obtained with a 2-photon microscope. The CFD model was developed with Fluent 16.0 (ANSYS Inc., Canonsburg, PA), particular care was taken in imposing the correct boundary conditions representing a physiological state. To account for the remaining branches of the sinusoids, a lumped parameter model was used to prescribe the correct pressure at each outlet. The effect of an adhered cell on local hemodynamics is also investigated for different occlusion degrees. The model here proposed accurately reproduces the fluid dynamics in a portion of the sinusoidal network in mouse liver. Mean velocities and mass flow rates are in agreement with literature values from in vivo measurements. Our approach provides details on local phenomena, hardly described by other computational studies, either focused on the macroscopic hepatic vasculature or based on homogeneous porous medium model.

Microcirculation in the murine liver: a computational fluid dynamic model based on 3D reconstruction from in vivo microscopy.

Encapsulating molecules into red blood cells (RBCs) is a challenging topic for drug delivery in clinical practice, allowing to prolong the residence time in the body and to avoid toxic residuals. Fluidic shear stress is able to temporary open the membrane pores of RBCs, thus allowing for the diffusion of a drug in solution with the cells. In this paper, both a computational and an experimental approach were used to investigate the mechanism of shear-induced encapsulation in a microchannel. By means of a computational fluid dynamic model of a cell suspension, it was possible to calculate an encapsulation index that accounts for the effective shear acting on the cells, their distribution in the cross section of the microchannel and their velocity. The computational model was then validated with micro-PIV measurements on a RBCs suspension. Finally, experimental tests with a microfluidic channel showed that, by choosing the proper concentration and fluid flow rate, it is possible to successfully encapsulate a test molecule (FITC-Dextran, 40 kDa) into human RBCs. Cytofluorimetric analysis and confocal microscopy were used to assess the RBCs physiological shape preservation and confirm the presence of fluorescent molecules inside the cells.

Shear-Induced Encapsulation into Red Blood Cells: A New Microfluidic Approach to Drug Delivery.

Aerosol and pollutants, in form of particulates 5-8 μm in main size face every day our respiratory system as natural suspension in air or forced to be inhaled as a coadjutant in a medical therapy for respiratory diseases. This inhalation happens in children to elderly, women and men, healthy or sick and disable people. In this paper we analyzed the inhalation of aerosol in conditions assimilable to the thermal therapy. We use a computational fluid dynamic 3D model to compute and visualize the trajectories of aerosol (3-7-10-25 µm) down to the sixth generation of bronchi, in a steady and dynamic condition (7 µm) set as breath cycle at rest. Results, compared to a set of milestone experimental studies published in literature, allow the comprehension of particles behavior during the inhalation from mouth to bronchi sixth generation, the visualization of jet at larynx constriction and vortices, in an averaged characteristic rigorous geometrical model including tracheal rings. Results on trajectories and deposition show the importance of the including transient physiological breath cycle on aerosol deposition analyses. Numerical and graphical results, may enable the design of medical devices and protocols to make the inhalations more effective in all the users' population.

Computational fluid dynamic models as tools to predict aerosol distribution in tracheobronchial airways.

Cell seeding on 3D scaffolds is a very delicate step in tissue engineering applications, influencing the outcome of the subsequent culture phase, and determining the results of the entire experiment. Thus, it is crucial to maximize its efficiency. To this purpose, a detailed study of the influence of the geometry of the scaffold fibers on dynamic seeding efficiency is presented. 3D printing technology was used to realize polylactic acid porous scaffolds, formed by fibers with a non-circular cross-sectional geometry, named multilobed to highlight the presence of niches and ridges. An oscillating perfusion bioreactor was used to perform bidirectional dynamic seeding of MG63 cells. The fiber shape influences the fluid dynamic parameters of the flow, affecting values of fluid velocity and wall shear stress. The path followed by cells through the scaffold fibers is also affected and results in a larger number of adhered cells in multilobed scaffolds compared to scaffolds with standard pseudo cylindrical fibers. Geometrical and fluid dynamic features can also have an influence on the morphology of adhered cells. The obtained results suggest that the reciprocal influence of geometrical and fluid dynamic features and their combined effect on cell trajectories should be considered to improve the dynamic seeding efficiency when designing scaffold architecture.

Improving cell seeding efficiency through modification of fiber geometry in 3D printed scaffolds.

Cancer is one of the most life-threatening diseases worldwide. Despite the huge efforts, the failure rate of therapies remains high due to cells heterogeneity, so physiologically relevant models are strictly necessary. Bioprinting is a technology able to form highly complex 3D tissue models and enables the creation of large-scale constructs. In cancer research, Matrigel is the most widely used matrix, but it is hardly bioprinted pure, without the use of any other bioink as reinforcement. Its complex rheological behavior makes the control with a standard bioprinting process nearly impossible. In this work, we present a customized bioprinting strategy to produce pure Matrigel scaffolds with good shape fidelity. To this aim, we realized a custom-made volumetric dispensing system and performed printability evaluations. To determine optimal printing parameters, we analyzed fibers spreading ratio on simple serpentines. After identifying an optimal flow rate of 86.68 ± 5.77 µL/min and a printing speed of 10 mm/min, we moved forward to evaluate printing accuracy, structural integrity and other key parameters on single and multi-layer grids. Results demonstrated that Matrigel was able to maintain its structure in both simple and complex designs, as well as in single and multilayer structures, even if it does not possess high mechanical strength. In conclusion, the use of volumetric dispensing allowed printing pure Matrigel constructs with a certain degree of shape fidelity on both single and multiple layers.

Bioprinting of Matrigel Scaffolds for Cancer Research.

Drug discovery is an expensive and lengthy process. Among the different phases, drug discovery and preclinical trials play an important role as only 5-10 of all drugs that begin preclinical tests proceed to clinical trials. Indeed, current high-throughput screening technologies are very expensive, as they are unable to dispense small liquid volumes in an accurate and quick way. Moreover, despite being simple and fast, drug screening assays are usually performed under static conditions, thus failing to recapitulate tissue-specific architecture and biomechanical cues present  even in the case of 3D models. On the contrary, microfluidics might offer a more rapid and cost-effective alternative. Although considered incompatible with high-throughput systems for years, technological advancements have demonstrated how this gap is rapidly reducing. In this Review, we want to further outline the role of microfluidics in high-throughput drug screening applications by looking at the multiple strategies for cell seeding, compartmentalization, continuous flow, stimuli administration (e.g., drug gradients or shear stresses), and single-cell analyses.

The impact of microfluidics in high-throughput drug-screening applications.

Downsizing surface-enhanced Raman spectroscopy (SERS) within microfluidic devices has opened interesting perspectives for the development of low-cost and portable (bio)sensors for the optical analysis of liquid samples. Despite the research efforts, SERS-fluidic devices still rely either on the use of expensive bulky set-ups or on polymeric devices giving spurious background signals fabricated via expensive manufacturing processes. Here, polymeric platforms integrating fluidics and optics were fabricated with versatile designs allowing easy coupling with fiber-based Raman systems. For the first time, anti-fouling photocurable perfluoropolyether (PFPE) was explored for high-throughput SERS-integrating chip fabrication via replica molding of negative stamps obtained through standard and advanced fabrication processes. The PFPE devices comprised networks of channels for fluid handling and for optical fiber housing with multiple orientations. Embedded microfeatures were used to control the relative positioning of the fibers, thus guaranteeing the highest signal delivering and collection. The feasibility of PFPE devices as fiber-based SERS fluidic platforms was demonstrated through the straightforward acquisition of Raman-SERS spectra of a mixture of gold nanoparticles as SERS substrates with rhodamine 6G (Rh6G) at decreasing concentrations. In the presence of high-performing gold nanostars, the Rh6G signal was detectable at dilutions down to the nanomolar level even without tight focusing and working at low laser power-a key aspect for analyte detection in real-world biomedical and environmental applications.

Fiber-Based SERS-Fluidic Polymeric Platforms for Improved Optical Analysis of Liquids.

Red Blood Cells (RBCs) are a promising drug delivery system candidate for many drugs. Using autologous cells helps to overcome biocompatibility issues, while microfluidics allows accurate control of the intracellular delivery of molecules through fluidic shear stress. With the ultimate goal of exploiting this delivery technique for clinical applications, we investigate how the chemical/rheological characteristics of the suspension and the properties of the RBCs in different animals influence the delivery mechanism. As regard the suspension of RBC, we study the effects induced by the hematocrit and by the presence of proteins such as albumin (Bovine Serum Albumin-BSA). Regarding the cellular properties of RBCs, we aim to investigate the exportability of the technique to the RBC of the most used animal models and identify the most suitable one. The presence of BSA implies a more significant variability of the intracellular delivery. However, 70 ÷ 94% of the cells have successfully encapsulated the probe molecule. Regarding the effect of hematocrit, however, the implementation of the experiment is more challenging due to the increase in viscosity and the easier sedimentation at low flow rates. Evaluation of intracellular delivery in the RBCs of various animal samples has instead led to the proposal of the mouse as the most suitable model for preclinical studies on this particular delivery approach.

A Microfluidic Approach for Intracellular Delivery into Red Blood Cells: A Deeper Understanding of the Role of Chemical/Rheological Properties of the Cellular Suspension.

Article	Year
Microfluidics for in vitro biomimetic shear stress-dependent leukocyte adhesion assays. Journal of biomechanics\| PubMed ID: 23200903	2013
Catch-and-Release of Target Cells Using Aptamer-Conjugated Electroactive Zwitterionic Oligopeptide SAM. Scientific reports\| PubMed ID: 28266533	2017
Label-free identification of activated T lymphocytes through tridimensional microsensors on chip. Biosensors & bioelectronics\| PubMed ID: 28284079	2017
Estimation of the physiological mechanical conditioning in vascular tissue engineering by a predictive fluid-structure interaction approach. Computer methods in biomechanics and biomedical engineering\| PubMed ID: 28569086	2017
Herringbone-like hydrodynamic structures in microchannels: A CFD model to evaluate the enhancement of surface binding. Medical engineering & physics\| PubMed ID: 28802780	2017
Microcirculation in the murine liver: a computational fluid dynamic model based on 3D reconstruction from in vivo microscopy. Journal of biomechanics\| PubMed ID: 28917579	2017
Shear-Induced Encapsulation into Red Blood Cells: A New Microfluidic Approach to Drug Delivery. Annals of biomedical engineering\| PubMed ID: 31410713	2020
Computational fluid dynamic models as tools to predict aerosol distribution in tracheobronchial airways. Scientific reports\| PubMed ID: 33441807	2021
Improving cell seeding efficiency through modification of fiber geometry in 3D printed scaffolds. Biofabrication\| PubMed ID: 33578401	2021
Bioprinting of Matrigel Scaffolds for Cancer Research. Polymers\| PubMed ID: 34205767	2021
The impact of microfluidics in high-throughput drug-screening applications. Biomicrofluidics\| PubMed ID: 35646223	2022
Fiber-Based SERS-Fluidic Polymeric Platforms for Improved Optical Analysis of Liquids. Bioengineering (Basel, Switzerland)\| PubMed ID: 37370607	2023
A Microfluidic Approach for Intracellular Delivery into Red Blood Cells: A Deeper Understanding of the Role of Chemical/Rheological Properties of the Cellular Suspension. Annals of biomedical engineering\| PubMed ID: 39971860	2025

Elena Bianchi

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