A Principal component analysis to detect cancer cell line aggressiveness

In this paper, we propose the use of Principal Component Analysis (PCA) as a new post-processing method for the detection of breast and bone cancer cell lines cultured in vitro using a microwave biosensor. MDA-MB-231 and MCF7 breast cancer cell lines and SaOS-2 and 143B osteosarcoma cell lines were characterized using a circular patch resonator in the 1 MHz – 3 GHz frequency range. The return loss of each cancer cell line was analyzed, and the differences among each other were determined through Principal Component Analysis according to a protocol previously proposed mainly for electrocardiogram processing and X-ray photoelectron spectroscopy. Our results showed that the four cancer cell lines analyzed exhibited peculiar dielectric properties when compared to each other and the growth medium, confirming that PCA could be employed as an alternative methodology to analyze microwave characterization of cancer cell lines which, in turn, may be deeply exploited as a tool for the detection of cancer cells in healthy tissues

Development and validation of a device for in vitro uniaxial cell substrate deformation with real-time strain control

Substrate deformation affects the behavior of many cell types, as for example bone, skeletal muscle and endothelial cells. Nowadays, in vitro tests are widely employed to study the mechanotransduction induced by substrate deformation. The aim of in vitro systems is to properly reproduce the mechanical stimuli sensed by the tissue in the cellular microenvironment. An accurate strain measurement and control is therefore necessary to ensure the cell sensing the proper strain for the entire treatment. Different types of in vitro systems are commercially available or custom made designed; however, none of these devices performs a real-time measurement of the induced strains. In this study, we proposed a uniaxial strain device for in vitro cell stimulation with an innovative real-time strain control. The system was designed to induce sinusoidal waveform stimulation in a huge range of amplitude and frequency, to three silicone chambers stretched by a linear actuator. The real-time strain measurement and control algorithm is based on an optical tracking method implemented in LabView 2015, and it is able adapting the input amplitude to the linear motor, if necessary, hanging the stimulation signal for about 120 ms. A validation of the strain values measured during the real-time tracking algorithm was carried out through a comparison with digital image correlation (DIC) technique. We investigated the influence of number of reference points and image size on the algorithm accuracy. Experimental results showed that the tracking algorithm allowed for a real-time measurement of the membrane longitudinal strains with a relative error of 0.3%, on average, in comparison to the strains measured with DIC in post-processing analysis. We showed a high homogeneity of the strain pattern on the entire chamber base for different stimulation conditions. Finally, as proof of concept, we employed the uniaxial strain device to induce substrate deformation on human Osteosarcoma cell line (SaOS-2). Experimental results showed a consistent cells’ change in shape in response to the mechanical strain

Measuring the maximum power of an ex vivo engineered muscle tissue with isovelocity shortening technique

The final aim of muscle tissue engineering (TE) is to create a new tissue able to restore the functionality of impaired muscles once transplanted in the site of injury. Therefore, functional contractile properties close to that of healthy muscles are desirable to allow for a good compatibility and a proper functional contribution. Since skeletal muscles deal with locomotion during their normal activity, an accurate measurement of ex vivo muscle engineered tissues' isotonic properties is crucial. In this paper, we devised an experimental system to measure the mechanical power generated by an ex vivo muscle engineered tissue, the X-MET, based on the isovelocity contraction technique. The X-MET is developed without the use of any scaffolds, so that its mechanical properties are not affected by endogenous components. Our experiments allowed for delimiting the ranges of shortening and shortening velocity for which the tissue is able to generate and maintain power for the entire stimulation, which is the condition that better reproduces muscle physiological activity. Then, we measured the power generated by the X-MET and fit the experimental results to the Hill's equation usually employed for modeling the force-velocity relationship of skeletal muscles. The use of this model yielded to the measurement of maximum power and maximum shortening velocity. Results revealed that most of the isotonic properties were consistent with that proposed in the literature for slow-twitch muscles; in particular, the X-METs were able to generate a maximum power of 2.08± 0.78 W/kg and had a maximum shortening velocity of 1.84 ± 0.57 L₀/s, on average

Remodeled eX vivo muscle engineered tissue improves heart function after chronic myocardial ischemia

: The adult heart displays poor reparative capacities after injury. Cell transplantation and tissue engineering approaches have emerged as possible therapeutic options. Several stem cell populations have been largely used to treat the infarcted myocardium. Nevertheless, transplanted cells displayed limited ability to establish functional connections with the host cardiomyocytes. In this study, we provide a new experimental tool, named 3D eX vivo muscle engineered tissue (X-MET), to define the contribution of mechanical stimuli in triggering functional remodeling and to rescue cardiac ischemia. We revealed that mechanical stimuli trigger a functional remodeling of the 3D skeletal muscle system toward a cardiac muscle-like structure. This was supported by molecular and functional analyses, demonstrating that remodeled X-MET expresses relevant markers of functional cardiomyocytes, compared to unstimulated and to 2D- skeletal muscle culture system. Interestingly, transplanted remodeled X-MET preserved heart function in a murine model of chronic myocardial ischemia and increased survival of transplanted injured mice. X-MET implantation resulted in repression of pro-inflammatory cytokines, induction of anti-inflammatory cytokines, and reduction in collagen deposition. Altogether, our findings indicate that biomechanical stimulation induced a cardiac functional remodeling of X-MET, which showed promising seminal results as a therapeutic product for the development of novel strategies for regenerative medicine

Determination of a Measurement Procedure for the Study of Cells’ Dielectric Properties through Descriptive Statistic

This paper presents a measurement procedure for analyzing the dielectric properties of cells using descriptive statistics. The study focuses on four cancer cell lines (MDA-MB-231 and MCF-7 breast cancer, SaOS-2, and 143B osteosarcoma) and DMEM culture medium, utilizing the Lorentzian fit model of the return-loss function. The measurements are performed using a circular patch resonator with a 40 mm diameter, powered by a miniVNA operating in the frequency range of 1 MHz to 3 GHz. Eight specimens are prepared for each group to ensure reliability, and the return loss is recorded ten times for each specimen. Various statistical parameters are calculated and evaluated, including the average value, standard deviation, coefficient of variation, and relative error between the average and the first values. The results demonstrate that one single acquisition highly represents the entire set of ten data points, especially for the resonant frequency, with an accuracy error lower than 0.05%. These findings have significant implications for the methodological approach to detecting cells’ dielectric properties, as they substantially reduce time and preserve the specimens without compromising the accuracy of the experimental results

Development and mechanical validation of an in vitro system for bone cell vibration loading

Vibration loading, both low magnitude and high magnitude at high frequency, has been demonstrated to have an anabolic effect on bone cells. The study of the mechanotransduction, the process by which mechanical loadings are detected by cells and converted in a chemical signal, is made accessible through the use of in vitro loading system. The aim of an in vitro loading system is to recreate the forces acting in the cell microenvironment. The goal of this study was to develop and mechanically validate a vibration loading system able to engender sinusoidal vertical vibration at different combinations of magnitude (0.3 g, 1 g, and 3 g) and frequency (30 Hz, 60 Hz and 90 Hz). A system like this can be therefore employed to study cell response to high and low magnitudes at high frequencies, thus providing a comprehensive evaluation of bone cell mechanotransduction. The mechanical validation, that is the characterization of the right loading input to the system to obtain the desired stimulation on cell culture, was performed in two different methods: open-loop and closed-loop mode. The results obtained in the open-loop mode showed a good intra-day repeatability of the measurements with values of index of dispersion always lower than 0.6%. While in the closed-loop mode a systematic search was implemented to reach the optimal amplitude stimulation. The vibration signals acquired on long-term test following the systematic search showed a good stability with index of dispersion always lower than 1%. Following the mechanical validation, the system was used to stimulate osteoblast like cells (Saos-2) with vibration loading of nine combinations of magnitude and frequency and the cell proliferation was studied 24h after the treatment by cell counting. Our preliminary results showed that no alterations in the proliferation were induced by 90 Hz vibration loading. On the other hand, small modulations in the proliferation were reported for lower stimulation frequency, being statistically significant when using 0.3 g of amplitude at 30 Hz

Measuring the X-MET’s maximum power: a preliminary study

The measurement of the mechanical properties is a crucial point for new engineered muscle tissues. The final aim is to implant these tissues to substitute or restore the functionality of impaired muscles, so that functional properties as close as possible to the healthy native muscles are required. We developed an engineered skeletal muscle tissue, X-MET, whose strong point is to be created without any endogenous component. This construct is able to contract spontaneously as well as to respond to electrical stimulation. In this work, we developed an experimental system to measure for the first time, the power developed by the X-MET. The power was measured by applying the isovelocity contraction technique. This technique has never been applied on muscle engineered tissues so far, so the aim of this work was to find out the optimal stimulation parameters. Once determined the range of displacement and velocity of shortening for which the X-MET was able to develop power, we proceeded looking at the optimal parameters allowing the production of its maximum power. Preliminary tests showed that the X-MET generates the optimal power when stimulated to shorten 3% of its ideal length at a speed of 0.2 L0/s

The development of an innovative embedded sensor for the optical measurement of ex-vivo engineered muscle tissue contractility

Tissue engineering is a multidisciplinary approach focused on the development of innovative bioartificial substitutes for damaged organs and tissues. For skeletal muscle, the measurement of contractile capability represents a crucial aspect for tissue replacement, drug screening and personalized medicine. To date, the measurement of engineered muscle tissues is rather invasive and not continuous. In this context, we proposed an innovative sensor for the continuous monitoring of engineered-muscle-tissue contractility through an embedded technique. The sensor is based on the calibrated deflection of one of the engineered tissue's supporting pins, whose movements are measured using a noninvasive optical method. The sensor was calibrated to return force values through the use of a step linear motor and a micro-force transducer. Experimental results showed that the embedded sensor did not alter the correct maturation of the engineered muscle tissue. Finally, as proof of concept, we demonstrated the ability of the sensor to capture alterations in the force contractility of the engineered muscle tissues subjected to serum deprivation

Electric field distribution analysis for the design of an electrode system in a 3D neuromuscular junction microfluidic device

Electrical stimulation (ES) highly influences the cellular microenvironment, affecting cell migration, proliferation and differentiation. It also plays a crucial role in tissue engineering to improve the biomechanical properties of the constructs and regenerate the damaged tissues. However, the effects of the ES on the neuromuscular junction (NMJ) are still not fully analyzed. In this context, the development of a specialized microfluidic device combined with an ad-hoc electrical stimulation can allow a better investigation of the NMJ functionality. To this aim, we performed an analysis of the electric field distribution in a 3D neuromuscular junction microfluidic device for the design of several electrode systems. At first, we designed and modeled the 3D microfluidic device in order to promote the formation of the NMJ between neuronal cells and the muscle engineered tissue. Subsequently, with the aim of identifying the optimal electrode configuration able to properly stimulate the neurites, thus enhancing the formation of the NMJ, we performed different simulation tests of the electric field distribution, by varying the electrode type, size, position and applied voltage. Our results revealed that all the tested configurations did not induce an electric field dangerous for the cell vitality. Among these configurations, the one with cylindrical pin of 0.3 mm of radius, placed in the internal position of the neuronal chambers, allowed to obtain the highest electrical field in the zone comprising the neurites

A prospective cohort analysis of the prevalence and predictive factors of delayed discharge after laparoscopic cholecystectomy in Italy: the DeDiLaCo Study

Background: The concept of early discharge ≤24 hours after Laparoscopic Cholecystectomy (LC) is still doubted in Italy. This prospective multicentre study aims to analyze the prevalence of patients undergoing elective LC who experienced a delayed discharge >24 hours in an extensive Italian national database and identify potential limiting factors of early discharge after LC. Methods: This is a prospective observational multicentre study performed from January 1, 2021 to December 31, 2021 by 90 Italian surgical units. Results: A total of 4664 patients were included in the study. Clinical reasons were found only for 850 patients (37.7%) discharged >24 hours after LC. After excluding patients with nonclinical reasons for delayed discharge >24 hours, 2 groups based on the length of hospitalization were created: the Early group (≤24 h; 2414 patients, 73.9%) and the Delayed group (>24 h; 850 patients, 26.1%). At the multivariate analysis, ASA III class ( P <0.0001), Charlson's Comorbidity Index (P=0.001), history of choledocholithiasis (P=0.03), presence of peritoneal adhesions (P<0.0001), operative time >60 min (P<0.0001), drain placement (P<0.0001), pain ( P =0.001), postoperative vomiting (P=0.001) and complications (P<0.0001) were independent predictors of delayed discharge >24 hours. Conclusions: The majority of delayed discharges >24 hours after LC in our study were unrelated to the surgery itself. ASA class >II, advanced comorbidity, the presence of peritoneal adhesions, prolonged operative time, and placement of abdominal drainage were intraoperative variables independently associated with failure of early discharge