Josef Skopalik

Autologous adipose tissue-derived stromal vascular fraction cells application in patients with osteoarthritis.

Ahead of Print article withdrawn by publisher

Mesenchymal stromal cell labeling by new uncoated superparamagnetic maghemite nanoparticles in comparison with commercial Resovist – an initial in vitro study

Objective Cell therapies have emerged as a promising approach in medicine. The basis of each therapy is the injection of 1–100×106 cells with regenerative potential into some part of the body. Mesenchymal stromal cells (MSCs) are the most used cell type in the cell therapy nowadays, but no gold standard for the labeling of the MSCs for magnetic resonance imaging (MRI) is available yet. This work evaluates our newly synthesized uncoated superparamagnetic maghemite nanoparticles (surface-active maghemite nanoparticles – SAMNs) as an MRI contrast intracellular probe usable in a clinical 1.5 T MRI system. Methods MSCs from rat and human donors were isolated, and then incubated at different concentrations (10–200 μg/mL) of SAMN maghemite nanoparticles for 48 hours. Viability, proliferation, and nanoparticle uptake efficiency were tested (using fluorescence microscopy, xCELLigence analysis, atomic absorption spectroscopy, and advanced microscopy techniques). Migration capacity, cluster of differentiation markers, effect of nanoparticles on long-term viability, contrast properties in MRI, and cocultivation of labeled cells with myocytes were also studied. Results SAMNs do not affect MSC viability if the concentration does not exceed 100 μg ferumoxide/mL, and this concentration does not alter their cell phenotype and long-term proliferation profile. After 48 hours of incubation, MSCs labeled with SAMNs show more than double the amount of iron per cell compared to Resovist-labeled cells, which correlates well with the better contrast properties of the SAMN cell sample in T2-weighted MRI. SAMN-labeled MSCs display strong adherence and excellent elasticity in a beating myocyte culture for a minimum of 7 days. Conclusion Detailed in vitro tests and phantom tests on ex vivo tissue show that the new SAMNs are efficient MRI contrast agent probes with exclusive intracellular uptake and high biological safety.

Formation of Cell-To-Cell Connection between Bone Marrow Cells and Isolated Rat Cardiomyocytes in a Cocultivation Model

Aims: Limited regenerative potential of cardiomyocytes (CMs) causes irreversible changes in heart tissue during pathological processes. However bone marrow mononuclear cells (BM-MNCs) can migrate to this tissue, incorporate to the area of dead or missing myocytes, and improve the global heart function. The mechanism of BMMSCs’ incorporation and interaction with CMs is not clear. Our aim was to create an in vitro model which would enable to study the interaction of BM-MNCs with CMs and to make a microscopy description of these interactions. Methods and Results: CMs were isolated from adult and newborn rats. BM-MNCs were isolated from bone marrow. BM-MNCs were added to the myocyte culture. Cell-to-cell adherence and Cx43 expression were evaluated by fluorescence microscopy, Ca2+ transients were evaluated in cardiomyocyte-BMC communication under electrical stimulation by fluo-4 fluorescence measurement. Analysis of calcein transport from BM-MNCs to CMs was performed using fluorescence microscopy. Conclusions: The adherence of BM-MNCs to CMs occurred quickly and was stable. Cx43 was detected in contact zones between BM-MNCs and CMs; pairs which displayed Cx43 positivity represented less than 1% from all BM-MNC-cardiomyocyte pairs in the coculture. Conductive structures between CMs and BM-MNCs were formed and verified by imaging calcein transfer and synchronous Ca2+ transients.

Modern Semi-automatic Set-up for Testing Cell Migration with Impact for Therapy of Myocardial Infarction

Ischemic heart disease and resulting acute myocardial infarction (AMI) is one of the main causes of morbidity and mortality in industrial countries. The idea for the modern therapeutic strategy, which should activate the migration of stem/progenitor cells or reduce the migration of inflammatory cells in AMI regions, has emerged in the last 15 years, mainly as a result of physiological observation and post-mortem histology. Published data from direct measurements of cell migration are very limited. We prepared a universal set-up that can be used for the testing of cell migration in AMI micro-environment. Mesenchymal stromal cells (MSCs), the most commonly used stem/progenitor cells in experimental cellular therapy for AMI, were used in the recent set-up tests. The cells, which should be tested for their migration potential, were injected into the starting point in a special micro-chamber on the substrate, and optics of the microscope allowed a time-lapse recording of cells in micrometre resolution every 2 min. Our software tools provided precise 2D and 3D tracking of moving cells and data export for statistical analysis. Set-up should be upgraded to a fully-automatic preclinical screening tool in the future.

DNA Intracellular Delivery into 3T3 Cell Line Using Fluorescence Magnetic Ferumoxide Nanoparticles

Gene delivery is a widespread strategy in current experimental medicine. In this work, we report a method for low-toxic intracellular DNA vector delivery and post transfection localisation of this vector in mouse embryonic fibroblast cell lines. The surface of modified ferumoxide nanoparticles conjugated with Rhoda-mine B isothiocyanate (FeNV-Rh) was modified with linear polyethyleneimine and medium molecular weight chitosan to increase Accelerated Sensor of Action Potentials DNA vector adhesion. The size of the FeNV-Rh/DNA transfection complex was studied using dynamic light scattering (DLS) and scanning electron microscopy (SEM) techniques. The transfection complex internalisation of plasmid expression and FeNV-Rh, and stability of rhodamine fluorescence in intracellular space were observed at time periods 6, 12, 24 and 48 h post transfection. Results showed high transfection complex intracellular biocompatibility—cell viability after Rh-MNP labelling was higher than 97% 24 h after transfection, and higher than 95% after the next 24 h. Selective FeNV-Rh localisation in the lysosomes was quantified. More than 82% of nanoparticles were localised in the lysosomes 12 h post transfection and 94% of lysosomes had a significant and long-term deposit of nanoparticles. DNA vector expression was visible in >65% of the cells and precise protein localisation on the cell membrane was confirmed using confocal microscopy.

Three-dimensional fluorescence lifetime imaging in confocal microscopy of living cells

Fluorescence lifetime imaging (FLIM) is a modern optical method which increases the potential of standard microscopy. This paper shows the possibilities of extended fluorescence lifetime evaluation and imaging in studying three-dimensional structures such as compartments of living cells with different fluorescence lifetimes. The method for quasi-FLIM image calculation is presented and image processing steps useful for biological experiments are suggested. The method was tested on isolated cardiomyocyte cells (CMs) and rat bone marrow stromal cells (MSCs) labelled with SPIO-rhodamine nanoparticles and stained with standard fluorescent dyes. We proved it is possible to use an exponential decrease of fluorescence in time and lifetime parameters for pseudo-colour 3D image mapping of living cells and their compartments that is not a standard function of confocal microscopes.

Stromal Vascular Fraction Cells of Adipose and Connective Tissue in People with Osteoarthritis: A Case Control Prospective Multi-Centric Non-Randomized Study

Objective: Stromal vascular fraction (SVF), containing high amount of stem cells and other regenerative cells, can be easily obtained from loose connective tissue that is associated with adipose tissue. Here we evaluated safety and clinical efficacy of freshly isolated autologous SVF cells in a case control prospective multi-centric non-randomized study in patients with grade 2-4 degenerative osteoarthritis. Methods: A total of 1128 patients underwent standard liposuction under local anesthesia and SVF cells were isolated and prepared for application into 1-4 large joints. A total of 1856 joints, mainly knee and hip joints, were treated with a single dose of SVF cells. 1114 patients were followed for 12.1-54.3 months (median 17.2 months) for safety and efficacy. Modified KOOS/HOOS Clinical Score was used to evaluate clinical effect and was based on pain, non-steroid analgesic usage, limping, extent of joint movement, and joint stiffness evaluation before and at 3,6 and 12 months after the treatment. Results: No serious side effects, systemic infection or cancer was associated with SVF cell therapy. Most patients gradually improved 3-12 months after the treatment. At least 75% Score improvement was noticed in 63% of patients and at least 50% Score improvement was documented in 91% of patients 12 months after SVF cell therapy. Obesity and higher grade of OA were associated with slower healing. Conclusion: Here we report a novel and promising treatment approach for patients with degenerative osteoarthritis that is safe, cost-effective, and relying only on autologous cells.

Characterization of cells migration through cardiac tissue using advanced microscopy techniques and matlab simulation

Mesenchymal stromal cells (MSC) and neutrophils (NP) migration are important factors of the postinfarcted hearts remodeling. These both types of the cells can migrate through cardiac extracellular matrix to the central ischemic region. The quantitative description of MSC and NP migration through collagen matrix is important aim of modern bio-medicine. NP and MSC migration through peri-infarct zone was simulated in a custom-made microphantom: two chambers (bottom 10×20 mm) connected by a collagen tunnel. Bottom of the system was constructed from glass plate, compatible with confocal microscopy. System was heated at 37°C in 5-21 % O2 environment. The first chamber was starting point of migrating MSC and NP. The second chamber included living or apoptotic myocytes (model of central infarcted). Monitoring of migrating cells was performed on the confocal laser scanning microscope Leica. Chemotaxis movement of MSC through collagen tunnel between two chambers was approved. Speed was significantly modulated by collagen fiber orientation and hypoxic condition. The speed constants of cell motility were quantified by originally-made microphantom Matlab utility and basic equations for cell motility was proposed, usable for future creating of in-silico simulator of real cell invasivity in patients.