Magnetic Fluid Hyperthermia as Treatment Option for Pancreatic Cancer Cells and Pancreatic Cancer Organoids.

INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) is a cancer with a meager prognosis due to its chemotherapy resistance. A new treatment method may be magnetic fluid hyperthermia (MFH). Magnetoliposomes (ML), consisting of superparamagnetic iron oxide nanoparticles (SPION) stabilized with a phospholipid-bilayer, are exposed to an alternating magnetic field (AMF) to generate heat. To optimize this therapy, we investigated the effects of MFH on human PDAC cell lines and 3D organoid cultures. MATERIAL AND METHODS: ML cytotoxicity was tested on Mia PaCa-2 and PANC-1 cells and on PDAC 3D organoid cultures, generated from resected tissue of patients. The MFH was achieved by AMF application with an amplitude of 40-47 kA/m and a frequency of 270 kHz. The MFH effect on the cell viability of the cell lines and the organoid cultures was investigated at two different time points. Clonogenic assays evaluated the impairment of colony formation. Altering ML set-ups addressed differences arising from intra- vs extracellular ML locations. RESULTS: Mia PaCa-2 and PANC-1 cells showed no cytotoxic effects at ML concentrations up to 300 µg(Fe)/mL and 225 µg(Fe)/mL, respectively. ML at a concentration of 225 µg(Fe)/mL were also non-toxic for PDAC organoid cultures. MFH treatment using exclusively extracellular ML presented the highest impact on cell viability. Clonogenic assays demonstrated remarkable impairment as long-term outcome in MFH-treated PDAC cell lines. Additionally, we successfully treated PDAC organoids with extracellular ML-derived MFH, resulting in notably reduced cell viabilities 2h and 24 h post treatment. Still, PDAC organoids seem to partly recover from MFH after 24 h as opposed to conventional 2D-cultures. CONCLUSION: Treatment with MFH strongly diminished pancreatic cancer cell viability in vitro, making it a promising treatment strategy. As organoids resemble the more advanced in vivo conditions better than conventional 2D cell lines, our organoid model holds great potential for further investigations.

Choosing the Right Differentiation Medium to Develop Mucociliary Phenotype of Primary Nasal Epithelial Cells In Vitro.

In vitro differentiation of airway epithelium is of interest for respiratory tissue engineering and studying airway diseases. Both applications benefit from the use of primary cells to maintain a mucociliated phenotype and thus physiological functionality. Complex differentiation procedures often lack standardization and reproducibility. To alleviate these shortfalls, we compared differentiation behavior of human nasal epithelial cells in four differentiation media. Cells were differentiated at the air-liquid interface (ALI) on collagen-coated inserts. Mucociliary differentiation status after five weeks was analyzed by electron microscopy, histology and immunohistochemistry. The amount of ciliation was estimated and growth factor concentrations were evaluated using ELISA. We found that retinoic-acid-supplemented mixture of DMEM and Airway Epithelial Cell Growth Medium gave most promising results to obtain ciliated and mucus producing nasal epithelium in vitro. We discovered the balance between retinoic acid (RA), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and fibroblast growth factor ß (FGF-ß) to be relevant for differentiation. We could show that low VEGF, EGF and FGF-ß concentrations in medium correspond to absent ciliation in specific donors. Therefore, our results may in future facilitate donor selection and non-invasive monitoring of ALI cultures and by this contribute to improved standardization of epithelial in vitro culture.

Combining Bulk Temperature and Nanoheating Enables Advanced Magnetic Fluid Hyperthermia Efficacy on Pancreatic Tumor Cells.

Many efforts are made worldwide to establish magnetic fluid hyperthermia (MFH) as a treatment for organ-confined tumors. However, translation to clinical application hardly succeeds as it still lacks of understanding the mechanisms determining MFH cytotoxic effects. Here, we investigate the intracellular MFH efficacy with respect to different parameters and assess the intracellular cytotoxic effects in detail. For this, MiaPaCa-2 human pancreatic tumor cells and L929 murine fibroblasts were loaded with iron-oxide magnetic nanoparticles (MNP) and exposed to MFH for either 30 min or 90 min. The resulting cytotoxic effects were assessed via clonogenic assay. Our results demonstrate that cell damage depends not only on the obvious parameters bulk temperature and duration of treatment, but most importantly on cell type and thermal energy deposited per cell during MFH treatment. Tumor cell death of 95% was achieved by depositing an intracellular total thermal energy with about 50% margin to damage of healthy cells. This is attributed to combined intracellular nanoheating and extracellular bulk heating. Tumor cell damage of up to 86% was observed for MFH treatment without perceptible bulk temperature rise. Effective heating decreased by up to 65% after MNP were internalized inside cells.

Bioengineered vascular constructs as living models for in vitro cardiovascular research.

Cardiovascular diseases represent the most common cause of morbidity and mortality worldwide. In this review, we explore the potential of bioengineered vascular constructs as living models for in vitro cardiovascular research to advance the current knowledge of pathophysiological processes and support the development of clinical therapies. Bioengineered vascular constructs capable of recapitulating the cellular and mechanical environment of native vessels represent a valuable platform to study cellular interactions and signaling cascades, test drugs and medical devices under (patho)physiological conditions, with the additional potential benefit of reducing the number of animals required for preclinical testing.

Targeted mutagenesis of zebrafish antithrombin III triggers disseminated intravascular coagulation and thrombosis, revealing insight into function.

Pathologic blood clotting is a leading cause of morbidity and mortality in the developed world, underlying deep vein thrombosis, myocardial infarction, and stroke. Genetic predisposition to thrombosis is still poorly understood, and we hypothesize that there are many additional risk alleles and modifying factors remaining to be discovered. Mammalian models have contributed to our understanding of thrombosis, but are low throughput and costly. We have turned to the zebrafish, a tool for high-throughput genetic analysis. Using zinc finger nucleases, we show that disruption of the zebrafish antithrombin III (at3) locus results in spontaneous venous thrombosis in larvae. Although homozygous mutants survive into early adulthood, they eventually succumb to massive intracardiac thrombosis. Characterization of null fish revealed disseminated intravascular coagulation in larvae secondary to unopposed thrombin activity and fibrinogen consumption, which could be rescued by both human and zebrafish at3 complementary DNAs. Mutation of the human AT3-reactive center loop abolished the ability to rescue, but the heparin-binding site was dispensable. These results demonstrate overall conservation of AT3 function in zebrafish, but reveal developmental variances in the ability to tolerate excessive clot formation. The accessibility of early zebrafish development will provide unique methods for dissection of the underlying mechanisms of thrombosis.

A low-volume tester for the thrombogenic potential of mechanical heart valve prostheses.

BACKGROUND AND AIM OF THE STUDY: During the development of a mechanical heart valve prosthesis, many studies are conducted to guarantee its correct function. Currently, investigations into the thrombogenic potential of a valve after its replacement are conducted with expensive and time-consuming chronic animal trials. Hence, the study aim was to develop and test an alternative system to resolve such thrombogenic issues. METHODS: The Thrombosis Tester of the Helmholtz Institute Aachen (THIA II) has a reasonably small priming volume (220-270 ml) that allows analysis of the thrombogenic potential of two valves, using one human blood bottle. RESULTS: Hydrodynamic evaluation demonstrated an absolutely stable physiological pressure and flow progression at the aortic and pulmonary positions. A sinus geometry of the human aortic root is implemented downstream of the valve in order to guarantee physiological leaflet motion. The tester remained absolutely thrombus-free during several tests carried out with minimally anticoagulated porcine blood, while the valves showed reproducible thrombus formation in reasonable locations. Tests with fully heparinized porcine blood showed that a soft silicon fixture for the valve could reduce hemolysis in the THIA II. CONCLUSION: This in-vitro test protocol can enable the optimization of a valve design during the early stages of its research and development. The system can provide a unique and suitable supplement to animal trials for testing thrombogenic performance, under constant and reproducible boundary conditions, including considerable physiological and pathological circumstances such as the influence of valve position (aortic, pulmonic), and a comparison of different valve types.

Multi-slice computed tomography: A tool for non-invasive temperature measurement?

PURPOSE: To investigate the potential of multi-slice computed tomography (MSCT) as a tool for non-invasive temperature measurement. MATERIALS AND METHODS: Samples of water, 0.9% saline, sunflower oil and dilutions of (1:32, 1:64, 1:128) contrast agent (Iopromid 370, BayerSchering Pharma, Berlin) were heated in a plexiglass phantom. In a first set-up, samples of 0.9% saline solution were scanned at defined temperatures (25 degrees -75 degrees C; 5 degrees C intervals) using a clinical CT scanner. Scan parameters (tube current-time product, tube voltage, collimation, slice thickness) were systematically varied. In a second set-up samples of the different fluids (water, sunflower oil, contrast agent dilutions) were scanned using the following scan protocol: 250 mAs, 140 kV, 1.2 mm collimation, 9.6 mm slice thickness. CT numbers were measured in reconstructed axial images at the different temperatures. A regression analysis was performed to investigate the relationship between temperature and CT number. RESULTS: Standard deviation of measured CT numbers decreased with increasing tube current-time product, increasing tube voltage, thicker collimation and higher slice thickness. Regression analysis showed an inverse relationship between temperature and CT number for all fluids with regression coefficients of -0.471 (0.9% saline), 0.447 (water), -0.679 (sunflower oil), -0.420 (contrast agent 1:32), -0.414 (contrast agent 1:64) and -0.441 (contrast agent 1:128), respectively. CONCLUSION: Multi-slice computed tomography can depict thermal density expansion of different fluids. Based on these results the implementation of a temperature discrimination of several degrees C at a high spatial resolution is achievable.

Thermoablation of malignant kidney tumors using magnetic nanoparticles: an in vivo feasibility study in a rabbit model.

The objective of this study was to assess the technical feasibility of CT-guided magnetic thermoablation for the treatment of malignant kidney tumors in a VX2 tumor rabbit model. VX2 tumors were implanted into the kidneys of five rabbits and allowed to grow for 2 weeks. After preinterventional CT perfusion imaging, CT-guided injection of superparamagnetic iron oxide particles (300 microl) was performed, followed by exposure of the animals to an alternating electromagnetic field for 15 min (approximately 0.32 kA/m). Then animals underwent CT perfusion imaging again. Afterward, animals were sacrificed and kidneys were dissected for macroscopic and histological evaluation. Changes in perfusion before and after exposure to the alternating magnetic field were analyzed. In one animal no tumor growth could be detected so the animal was used for optimization of the ablation procedure including injection technique and peri-interventional cross-sectional imaging (CT, MRI). After image-guided intratumoral injection of ferrofluids, the depiction of nanoparticle distribution by CT correlated well with macroscopic evaluation of the dissected kidneys. MRI was limited due to severe susceptibility artefacts. Postinterventional CT perfusion imaging revealed a perfusion deficiency around the ferrofluid deposits. Histological workup showed different zones of thermal damage adjacent to the ferrofluid deposits. In conclusion, CT-guided magnetic thermoablation of malignant kidney tumors is technically feasible in an animal model and results in a perfusion deficiency indicating tumor necrosis as depicted by CT perfusion imaging and shown in histological evaluation.

Robot-based methodology for a kinematic and kinetic analysis of unconstrained, but reproducible upper extremity movement.

Although arm movements play an important role in everyday life, there is still a lack of procedures for the analysis of upper extremity movement. The main problems for standardizing the procedure are the variety of arm movements and the difficult assessment of external hand forces. The first problem requires the predefinition of motions, and the second one is the prerequisite for calculation of net joint forces and torques arising during motion. A new methodology for measuring external forces during prespecified, reproducible upper extremity movement has been introduced and validated. A robot-arm has been used to define the motion and 6 degrees of freedom (DoF) force sensor has been attached to it for acquiring the external loads acting on the arm. Additionally, force feedback has been used to help keeping external loads constant. Intra-individual reproducibility of joint angles was estimated by using correlation coefficients to compare a goal-directed movement with robot-guided task. Inter-individual reproducibility has been evaluated by using the mean standard deviation of joint angles for both types of movement. The results showed that both inter- and intra-individual reproducibility have significantly improved by using the robot. Also, the effectiveness of using force feedback for keeping a constant external load has been shown. This makes it possible to estimate net joint forces and torques which are important biomechanical information in motion analysis.

Noninvasive inductive stent heating: alternative approach to prevent instent restenosis?

RATIONALE AND OBJECTIVES: To test noninvasive inductive heating of implanted vascular stents as an alternative approach for reduction or prevention of neointimal hyperplasia. METHODS: Calorimetric pretests were performed to get an orientation on the different parameters of influence for inductive heating of stents. The field strength was set to a maximum of 90 kA/m within a frequency range from 80 kHz to 320 kHz. The electromagnetic field was emitted by a custom-made water-cooled copper winding antenna. A flow model for stent heating was set up to assess the increase in temperature of an expanded 316L stainless steel stent with typical coronary stent dimensions of 3.5 mm diameter and 14.5 mm in length, and in a second setup with 4.5 mm diameter and 13 mm in length, respectively. The stent was located in a bioartificial artery, simulated by a fibrinogen matrix with a defined number of vital cells. The system was exposed to a pulsating perfusion and to an electromagnetic field of 200 kHz over a period of 20 minutes and in a second setup to an electromagnetic field of 300 kHz and increasing intensity up to maximum power-output. Afterward, the artificial vessel was sliced and examined by fluorescence microscopy to evaluate the number and location of damaged cells. RESULTS: The calorimetric tests show an exponential correlation of energy uptake in the stent with an increase in frequency and a constant generator output. At a frequency of 80 kHz, the power uptake accounts for 0.1 W (250 kHz 1.0 W; 320 kHz 1.9 W, respectively). The flow tests confirmed feasibility to elevate the stent temperature from 37 degrees C body temperature to 44 degrees C at 200 kHz within 55 seconds. The temperature increase of the fluid passing the heated vessel region was only marginal (maximum of 0.5 degrees C). Cell necrosis after 20 minutes of treatment was not observed. In a second set-up with 4.5 mm stent diameter, a frequency of 300 kHz and with maximum power output, the stent temperature was increased to 80 degrees C and there was extensive necrosis area around the stent. Treatment time and stent temperature were optimized in further tests. CONCLUSION: Selective noninvasive energy transfer to coronary stainless steel stents by inductive heating is possible within a wide range of power. By thermal conduction, vital cells close to the stent struts can be affected. The frequency of 200 kHz turned out to be favorable. There is still room for further optimization of energy dosage with regard to material and stent design, to induce controlled cell death. The method has potential to serve as an alternative approach for prevention of instent restenosis.