Study on Optimal Parameter and Target for Pulsed-Field Ablation of Atrial Fibrillation

Pulsed-field ablation (PFA) had potential advantages in atrial fibrillation ablation, and we aim to confirm the optimal parameter and target of PFA for atrial fibrillation. Two ablation modes in vitro of single-cell system (ablation in electrode cup) and monolayer cell system (ablation in inserts with electrode tips) were established to perform PFA for myocardial cell H9C2 and smooth muscle cell A7r5. Ablation effect, calcium ion influx, the expression of Cx45, and surface morphological change were observed. Three Bama minipigs were used to verify the in vivo ablation effect of PFA. In monolayer cell system, H9C2 was significantly sensitive to PFA compared with A7r5, with shrinking of the whole monolayer. The ablation effect of bidirectional pulse was weaker than that of the two mono-polar pulses. Expressed Cx45 proteins were increased in H9C2 but decreased in A7r5 cells. Bidirectional PFA performed on Bama minipigs was able to effectively block electrical activity from the pulmonary vein to the atrium with week muscle contraction, not generating pulmonary vein stenosis. Bidirectional PFA was able to significantly ablate myocardial cells, maintain cell–cell connection, and reduce muscle contraction, which was a kind of optimized PFA strategy for atrial fibrillation.

The Role of Organoids as a Novel Platform for Modeling of Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) is a chronic relapsing-remitting immune-mediated disorder affecting the gut. It is common in Westernized regions and is increasing in incidence in developing countries. At a molecular level, intrinsic deficiencies in epithelial integrity, mucosal barrier function, and mechanisms of immune response and resolution contribute to the development of IBD. Traditionally two platforms have been utilized for disease modeling of IBD;in-vitromonolayer cell culture andin-vivoanimal models. Both models have limitations, including cost, lack of representative cell types, lack of complexity of cellular interactions in a living organism, and xenogeneity. Organoids, three-dimensional cellular structures which recapitulate the basic architecture and functional processes of the organ of origin, hold potential as a third platform with which to investigate the pathogenesis and molecular defects which give rise to IBD. Organoids retain the genetic and transcriptomic profile of the tissue of origin over time and unlike monolayer cell culture can be induced to differentiate into most adult intestinal cell types. They may be used to model intestinal host-microbe interactions occurring at the mucosal barrier, are amenable to genetic manipulation and can be co-cultured with other cell lines of interest. Bioengineering approaches may be applied to render a more faithful representation of the intestinal epithelial niche. In this review, we outline the concept of intestinal organoids, discuss the advantages and disadvantages of the platform comparative to alternative models, and describe the translational applications of organoids in IBD.

Superparamagnetic Iron Oxide Nanoparticles Carrying Chemotherapeutics Improve Drug Efficacy in Monolayer and Spheroid Cell Culture by Enabling Active Accumulation

Cytotoxic and cytostatic chemotherapeutics act by attacking rapidly dividing tumor cells, predominantly affecting malignant tissue and to a certain degree preserving healthy cells. Nonetheless, severe side effects are caused as quickly proliferating healthy cells such as hematopoietic precursors and mucous membranes are impaired as well. This limits the administered dose and eventually allows tumor cells to escape treatment. In order to increase intratumoral drug concentration and simultaneously reduce systemic side effects, nanoparticles have come into focus as drug carriers. The functionalization of superparamagnetic iron oxide nanoparticles (SPIONs) with chemotherapeutics such as mitoxantrone (MTO) enables targeted drug transport by using magnetic forces. Here, we investigate SPIONs consisting of individual iron oxide cores of 10 nm in diameter and a total hydrodynamic diameter of 53 ± 0.8 nm as a transporting system for MTO. Comparing the killing efficacy in monolayer cell culture and multicellular tumor spheroids of HT-29 cells, we show that spheroids tolerate considerably higher doses of nanoparticle-loaded MTO. Therefore, dose predictions from conventional monolayer cell cultures are often misleading for in vivo applications. This was true for both soluble and nanoparticle-bound MTO. Using flow chambers mimicking in vivo blood flow, we furthermore demonstrate that SPIONs can magnetically accumulate MTO. We conclude that SPIONs can function as an effective delivery platform to increase local drug concentrations, thereby potentially overcoming chemotherapy resistance of cells.

The human arthritic hip joint is a source of mesenchymal progenitor cells (MPCs) with extensive multipotent differentiation potential

Background: While multiple in vitro studies examined mesenchymal progenitor cells (MPCs) derived from bone marrow or hyaline cartilage, there is little to no data about the presence of MPCs in the joint capsule or the ligamentum capitis femoris (LCF) of the hip joint. Therefore, this in vitro study examined the presence and compared the differentiation potential of MPCs isolated from the bone marrow, arthritic hyaline cartilage, the LCF and full-thickness samples of the anterior joint capsule of the hip joint. Methods: MPCs were isolated and multiplied in adherent monolayer cell culture. Osteogenesis and adipogenesis was induced in monolayer cell cultures for 21 days using a differentiation medium containing specific growth factors, while chondrogenesis in the presence of TGF-ß1 was performed using pellet-culture for 27 days. Control cultures were maintained for comparison over the same duration of time. The differentiation process was analyzed using histological and immunohistochemical stainings as well as semiquantitative RT-PCR for measuring the mean expression levels of tissue-specific genes.Results: This in vitro research showed that the isolated cells from all four donor tissues grew plastic adherent and showed similar adipogenic and osteogenic differentiation capacity as proven by the histological detection of lipid droplets or deposits of extracellular calcium and collagen type I. After 27 days of chondrogenesis proteoglycans accumulated in the differentiated MPC-pellets from all donor tissues. Immunohistochemical staining revealed vast amounts of collagen type II in all differentiated MPC-pellets, except for those from the LCF. Interestingly all differentiated MPCs still showed a clear increase in mean expression of adipogenic, osteogenic and chondrogenic marker genes. In addition the examination of an exemplary donor sample revealed that cells from all four donor tissues were clearly positive for the surface markers CD44, CD73, CD90 and CD105 by flow cytometric analysis.Conclusions: This study proved the presence of MPCs in all four examined donor tissues of the hip joint. No significant differences were observed during osteogenic or adipogenic differentiation depending on the source of MPCs used. Further research is necessary to fully determine the chondrogenic differentiation potential of MPCs isolated from the LCF and capsule tissue of the hip joint.

COMPARATIVE STUDIES OF FELINE VIRAL RHINOTRACHEITIS VIRUS FOR ITS REPLICATION PROPERTIES IN DIFFERENT CELL CULTURES

The results of comparative studies of feline viral rhinotracheitis virus for its culture properties in primary and continuous cell cultures of feline origin (FK, FK (subculture), CrFK, FS, CC-81, FC/Tg) are presented. It was found that viral rhinotracheitis virus replication, irrespective of the route of infection and the culture technique, was consistent and practically equal in susceptible cell cultures. The most pronounced cytopathic effect (more than 75% monolayer degeneration) was observed in all types of cell cultures in 48–72 hours of cultivation. However, the accumulation of feline viral rhinotracheitis virus Grand strain was highest when preliminary adsorption occurred within the specified period of time, monolayer cell cultures were infected with the virus at a dose of 5.5 lg TCID50/ml and roller bottle cultivated, and the рН of the medium was maintained at 7.0–7.4. Single freezing of the virus at a temperature of minus 60 degrees Celsius upon the completion of the cultivation cycle (during 60–72 hours) and its thawing were found to significantly increase the virus titre by 0.5 lg TCID50/ml.

Cystic hygroma of the neck as an alternative sourse of cells for prenatal karyotyping

The results of studies about the possibility of using liquid from the fetal cystic hygroma of the neck for prenatal karyotyping are presented. Aspiration of fetal cystic formations was carry out by five women. A standard method for producing monolayer cell cultures with their own modifications was used for the work. In all cases, the chromosomal pathology of the fetus was confirmed 45,X. There were noted the specific features of the cultivation of various fluid's samples. The reasons that could influence on the change in the cultivation period were analyzed.

In Vitro Replication of Chelonid Herpesvirus 5 in Organotypic Skin Cultures from Hawaiian Green Turtles (Chelonia mydas)

ABSTRACT Fibropapillomatosis (FP) is a tumor disease of marine turtles associated with chelonid herpesvirus 5 (ChHV5), which has historically been refractory to growth in tissue culture. Here we show, for the first time, de novo formation of ChHV5-positive intranuclear inclusions in cultured green turtle cells, which is indicative of active lytic replication of the virus. The minimal requirements to achieve lytic replication in cultured cells included (i) either in vitro cultures of ChHV5-positive tumor biopsy specimens (plugs) or organotypic cultures (rafts) consisting of ChHV5-positive turtle fibroblasts in collagen rafts seeded with turtle keratinocytes and (ii) keratinocyte maturation induced by raising raft or biopsy cultures to the air-liquid interface. Virus growth was confirmed by detailed electron microscopic studies that revealed intranuclear sun-shaped capsid factories, tubules, various stages of capsid formation, nuclear export by budding into the perinuclear space, tegument formation, and envelopment to complete de novo virus production. Membrane synthesis was also observed as a sign of active viral replication. Interestingly, cytoplasmic particles became associated with keratin filaments, a feature not seen in conventional monolayer cell cultures, in which most studies of herpesvirus replication have been performed. Our findings draw a rich and realistic picture of ChHV5 replication in cells derived from its natural host and may be crucial not only to better understand ChHV5 circulation but also to eventually complete Koch's postulates for FP. Moreover, the principles described here may serve as a model for culture of other viruses that are resistant to replication in conventional cell culture. IMPORTANCE A major challenge in virology is the study of viruses that cannot be grown in the laboratory. One example is chelonid herpesvirus 5 (ChHV5), which is associated with fibropapillomatosis, a globally distributed, debilitating, and fatal tumor disease of endangered marine turtles. Pathological examination shows that ChHV5 is shed in skin. Here we show that ChHV5 will grow in vitro if we replicate the complex three-dimensional structure of turtle skin. Moreover, lytic virus growth requires a close interplay between fibroblasts and keratinocytes. Finally, the morphogenesis of herpesviral growth in three-dimensional cultures reveals a far richer, and likely more realistic, array of capsid morphologies than that encountered in traditional monolayer cell cultures. Our findings have applications to other viruses, including those of humans.