12 research outputs found
preliminary study of inkjet printed sensors for monitoring cell cultures
Abstract An extremely promising methodology able to obtain feedbacks from cell cultures is represented by the direct integration within culture substrates of specific sensitive elements capable to provide information related to cell adhesion, migration, differentiation and growth. At present, the most common materials used in the implementation of sensors monitoring 2D cell culture are noble metals. However, printed electronics allow instead an innovative approach, from both sensor realization technique and utilization of sensitive materials. This project aims to develop and test 2D ink-jet printed sensors, focusing on biocompatible substrates and conductive inks. Both biocompatibility and printability of two different sensor designs were evaluated, followed by electronic measurements that estimate fibroblast adhesion. Preliminary findings show a good biocompatibility of the Kapton® substrate coupled with PEDOT:PSS ink. This solution allowed us to correlate cell adhesion with an increase of impedance module, in agreement with the optical observation. On-going works rely on the evaluation of different materials used for both substrates and inks, addressing the possibility to monitor cardiomyocyte activity
Human iPSC-Derived 3D Hepatic Organoids in a Miniaturized Dynamic Culture System
The process of identifying and approving a new drug is a time-consuming and expensive procedure. One of the biggest issues to overcome is the risk of hepatotoxicity, which is one of the main reasons for drug withdrawal from the market. While animal models are the gold standard in preclinical drug testing, the translation of results into therapeutic intervention is often ambiguous due to interspecies differences in hepatic metabolism. The discovery of human induced pluripotent stem cells (hiPSCs) and their derivatives has opened new possibilities for drug testing. We used mesenchymal stem cells and hepatocytes both derived from hiPSCs, together with endothelial cells, to miniaturize the process of generating hepatic organoids. These organoids were then cultivated in vitro using both static and dynamic cultures. Additionally, we tested spheroids solely composed by induced hepatocytes. By miniaturizing the system, we demonstrated the possibility of maintaining the organoids, but not the spheroids, in culture for up to 1 week. This timeframe may be sufficient to carry out a hypothetical pharmacological test or screening. In conclusion, we propose that the hiPSCderived liver organoid model could complement or, in the near future, replace the pharmacological and toxicological tests conducted on animals
Preparation and assessment of high performance gelatin-based hydrogels for tissue engineering
Generation and characterization of human cardiomyocytes derived from pluripotent stem cells
In vitro development has been widely studied using murine ESCs (mESCs), whose differentiation procedure in culture implies the initial
leukemia inhibitory factor (LIF) removal and the formation of cellular aggregates using the “hanging drop” method. These three dimensional
(3D) structures, called embryoid bodies (EBs), replicate in vitro the different stages of murine embryonic development. Around differentiation
day (dd) 8, clusters of spontaneously beating cells appear in culture; these cells express several transcriptional and structural cardiac markers
and were therefore classified generically as cardiomyocytes. Since their isolation, human ESCs have shown different culturing needs from
the murine counterpart, and their behavior revealed also minor differentiation plasticity. Cardiac differentiation is the most glaring example
of this statement: only a modest proportion of EBs derived from either hESC or hiPSC contains contracting cells. This occurrence leads to
the setup of different methods aimed to increase cardiac differentiation during in vitro development of pluripotent stem cells. While some
of these procedures retain an initial 3D EB formation, others start from a confluent monolayer. However, CMs differentiated in vitro vary
considerably from cells isolated from a mature human heart, because of the absence of humoral factors and organized mechanical and
electrical stress. In general, many of the features of hPSC-CMs are reminiscent of normal fetal cells. hPSC-CMs are spontaneously beating
cells co-expressing atrial-, ventricular-, and nodal- markers, with unorganized sarcomeres, immature mitochondria, and an expression
profile different from adult CMs. The CMs that arise during early hESC or hiPSC in vitro differentiation exhibit spontaneous AP, with a
relatively depolarized resting membrane potential, probably due to the temporary absence of the inward rectifier potassium current. The
expression of the ion channels and, consequently, the ionic currents will undergo developmental maturation over time, as assessed by
modifications in current density and property. hPSC-CMs immaturity is also reflected in their excitation–contraction machinery, lacking
clear T-tubuli, disorganized sarcomeric striations, and immature Ca2+ handling.
Then the possibility to obtain human CMs in a culture dish is a powerful technique that will allow identification of new drugs in
pharmacological studies as well as the identification of new causative genes in modeling genetic pathologies. Nevertheless, newly differentiated
human CMs show a low degree of maturation that must be considered and, eventually, overcome by physical, mechanical or cultural stimuli
Preparation and properties of high performance gelatin-based hydrogels with chitosan or hydroxyethyl cellulose for tissue engineering applications
High performance gelatin-based biocompatible hybrid hydrogels are developed using functionalized polyethylene glycol as a cross-linker in presence of chitosan or hydroxyethyl cellulose. Tensile test shows robust and tunable mechanical properties and reveals non-linear and J-shaped stress-strain curves similar to those found for native extracellular matrix. Degradation study demonstrates that the mass loss and change in mechanical properties are dependent on hydrogel composition and cross-linking density. Structural features of the hydrogels are confirmed by infrared spectroscopy. A preliminary biological evaluation is carried out using rat myoblasts and human fibroblasts cell lines. The results show that all hydrogels allow cell adhesion and proliferation during four days culture, hence, they might have a great potential for use in the biomedical applications
MEMS force microactuator with displacement sensing for mechanobiology experiments
This paper presents a Micro Electro-Mechanical System (MEMS) that performs electrostatic force actuation and capacitive microdisplacement sensing in the same chip. By driving the actuator with a given voltage, a known force can be applied to a microsample under test by using a silicon probe tip, while the obtained displacement is measured. This allows to extract the mechanical properties of the microsample entirely on chip, and to derive its force-displacement curve without external equipment. The proposed device is intended for mechanobiology experiments, where the microsample is made of biological tissues or cells. The device generates a force in the order of few micronewtons and a maximum displacement of 1.8 ÎĽm can be measured
Ink-jet printed stretchable sensors for cell monitoring under mechanical stimuli: A feasibility study
Impedance-based sensors represent a promising tool for cell monitoring to improve current invasive biological assays. A novel research field is represented by measurements performed in dynamic conditions, monitoring cells (e.g., myocytes) for which the mechanical stimulus plays an important role for promoting maturation. In this picture, we applied printed and stretchable electronics principles, developing a system able to evaluate cells adhesion during substrate cyclic strain. Cytocompatible and stretchable sensors were ink-jet printed using carbon-based ink on crosslinked poly(\u3f5-caprolactone) electrospun mats. Moreover, a customized stretching device was produced, with a complete user interface to control testing condition, validated in order to correlate impedance changes with myoblasts - i.e., myocytes precursors - adhesion. Overall system sensitivity was evaluated using three different cell concentrations and DAPI imaging assay was performed to confirm myoblast adhesion. Preliminary results showed the possibility to correlate an average increase of impedance magnitude of 1k\u3c9 every 15,000 cells/cm2 seeded, suggesting the possibility to discriminate between different cell concentrations, with a sensitivity of 80m\u3c9/(cells/cm2). In conclusion, the present system might be generalized in the development of future applications, including the differentiation process of cardiac myocytes with the aid of mechanical stimuli
Ink-jet printed stretchable sensors for cell monitoring under mechanical stimuli: A feasibility study
Impedance-based sensors represent a promising tool for cell monitoring to improve current invasive biological assays. A novel research field is represented by measurements performed in dynamic conditions, monitoring cells (e.g., myocytes) for which the mechanical stimulus plays an important role for promoting maturation. In this picture, we applied printed and stretchable electronics principles, developing a system able to evaluate cells adhesion during substrate cyclic strain. Cytocompatible and stretchable sensors were ink-jet printed using carbon-based ink on crosslinked poly(ϵ-caprolactone) electrospun mats. Moreover, a customized stretching device was produced, with a complete user interface to control testing condition, validated in order to correlate impedance changes with myoblasts - i.e., myocytes precursors - adhesion. Overall system sensitivity was evaluated using three different cell concentrations and DAPI imaging assay was performed to confirm myoblast adhesion. Preliminary results showed the possibility to correlate an average increase of impedance magnitude of 1kω every 15,000 cells/cm2 seeded, suggesting the possibility to discriminate between different cell concentrations, with a sensitivity of 80mω/(cells/cm2). In conclusion, the present system might be generalized in the development of future applications, including the differentiation process of cardiac myocytes with the aid of mechanical stimuli
Carbon on poly(epsilon-caprolactone) (PCL) ink-jet printed sensor for monitoring cell cultures of myoblasts
Nowadays techniques for sensitive non-invasive, real-time monitoring of cell differentiation and maturation are highly demanded. In light of this, the development of electrochemical printed sensors impedance-based could represent a promising tool. In the present work, we developed 2D ink-jet printed sensors for myoblasts adhesion monitoring, using carbon-based ink on a substrate consisting in non-woven electrospun mats made in crosslinked poly(\uce\ub5-caprolactone) (PCL). First of all, sensors printability was optimized and the biocompatibility tested. In order to determine the possibility to employ the prepared systems as scaffolds for dynamic cellular cultures, the mechanical response of the PCL scaffold was evaluated through the application of cyclic deformation tests. After that, electrical characterization of ink and substrate was performed, followed by electrochemical impedance-based measurements to evaluate myoblasts adhesion. Biocompatibility assessment showed good results for both carbon and PCL. Mechanical tests findings suggested that a training of 50 cycles and a proper value of strain should be applied before the cell seeding, in order to ensure a subsequent controlled strain amplitude. The sensorized scaffold allowed us to correlate cell adhesion with an increase of impedance module, in agreement with biocompatibility testing. Thus, this first preliminary testing suggested that this non-invasive impedance spectroscopy-based measurement system can be used for sensitive monitoring of cells adhesion, in static and moreover, as suggested from mechanical characterization, in dynamic conditions