Myocardial slices come to age: an intermediate complexity in vitro cardiac model for translational research

Abstract Although past decades have witnessed significant reductions in mortality of heart failure together with advances in our understanding of its cellular, molecular, and whole-heart features, a lot of basic cardiac research still fails to translate into clinical practice. In this review we examine myocardial slices, a novel model in the translational arena. Myocardial slices are living ultra-thin sections of heart tissue. Slices maintain the myocardium’s native function (contractility, electrophysiology) and structure (multicellularity, extracellular matrix) and can be prepared from animal and human tissue. The discussion begins with the history and current advances in the model, the different interlaboratory methods of preparation and their potential impact on results. We then contextualize slices’ advantages and limitations by comparing it with other cardiac models. Recently, sophisticated methods have enabled slices to be cultured chronically in vitro while preserving the functional and structural phenotype. This is more timely now than ever where chronic physiologically relevant in vitro platforms for assessment of therapeutic strategies are urgently needed. We interrogate the technological developments that have permitted this, their limitations, and future directions. Finally, we look into the general obstacles faced by the translational field, and how implementation of research systems utilizing slices could help in resolving these.

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Sitotoksik Analizlerin Su Kalitesi Değerlendirmeleri Üzerine Etkinliğinin Belirlenmesi

Turkish Journal of Agriculture - Food Science and Technology ◽

10.24925/turjaf.v8i2.478-483.3221 ◽

2020 ◽

Vol 8 (2) ◽

pp. 478

Author(s):

Nuray Emin ◽

Ekrem Mutlu ◽

Ayşegül Emin Güzel

Keyword(s):

Water Quality ◽

Water Samples ◽

Heart Tissue ◽

In Vitro Cytotoxicity ◽

Elemental Content ◽

Tissue Samples ◽

Thin Sections ◽

Cytotoxicity Studies

In the studies carried out by us, IC50 value of water was determined by using mesenchymal stem cells and endothelial cell lines in in vitro cultures and positive and negative effect doses on the cells were determined. Depending on the results obtained, the effects on the whole organism, if necessary, can be examined in vivo on subjects. For this purpose, young wistar rats are divided into experimental groups (experimental and control) and their water needs are met with water samples obtained from the study area for 3 to 6 months. Blood and urine samples were taken from the subjects in each group at certain time points and the changes were recorded by analyzing the routine biochemistry and hemogram. In addition, the subjects in the sampling are sacrificed at monthly intervals and general examination of endogenous tissues are performed and liver, kidney and heart tissue samples are taken for histological and chemical analyzes. Some of the tissue samples are homogenized using a microwave and changes in elemental content are determined by ICP_OES. The other part is fixed in 10% formaldehyde and then 5 µm thin sections are examined histopathologically according to freeze sectioning method. These studies showed that although the known methods used in water analysis are very valuable, the water quality study carried out by cytotoxic analysis method provides reliability in terms of observing the direct effects of water quality on living tissue. In the present study, in vitro cytotoxicity studies for water samples taken from İncesu Basin are given as examples because of their striking results. Based on the obtained results, it is suggested that in vitro cytotoxicity tests should be added to routine water quality analyzes.

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Probing the ultrastructure of the mitotic and meiotic spindle in eggs: An expeditious approach based on semi-thin (0.25 μm) serial sections

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100076408 ◽

1983 ◽

Vol 41 ◽

pp. 552-555

Author(s):

Conly L. Rieder ◽

S. Bowser ◽

R. Nowogrodzki ◽

K. Ross ◽

G. Sluder

Keyword(s):

Small Sample Size ◽

Small Sample ◽

Meiotic Spindle ◽

Serial Sections ◽

Mitotic Apparatus ◽

Thin Sections ◽

Cell Cycles ◽

Ultrastructural Studies

Eggs have long been a favorite material for studying the mechanism of karyokinesis in-vivo and in-vitro. They can be obtained in great numbers and, when fertilized, divide synchronously over many cell cycles. However, they are not considered to be a practical system for ultrastructural studies on the mitotic apparatus (MA) for several reasons, the most obvious of which is that sectioning them is a formidable task: over 1000 ultra-thin sections need to be cut from a single 80-100 μm diameter egg and of these sections only a small percentage will contain the area or structure of interest. Thus it is difficult and time consuming to obtain reliable ultrastructural data concerning the MA of eggs; and when it is obtained it is necessarily based on a small sample size.We have recently developed a procedure which will facilitate many studies concerned with the ultrastructure of the MA in eggs. It is based on the availability of biological HVEM's and on the observation that 0.25 μm thick serial sections can be screened at high resolution for content (after mounting on slot grids and staining with uranyl and lead) by phase contrast light microscopy (LM; Figs 1-2).

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Neurofilaments and Paired Helical Filaments

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100120357 ◽

1985 ◽

Vol 43 ◽

pp. 740-743

Author(s):

J. Metuzals

Keyword(s):

Animal Model ◽

Posttranslational Modifications ◽

Posttranslational Modification ◽

Giant Axon ◽

Paired Helical Filaments ◽

Squid Giant Axon ◽

In Vitro Experiments ◽

Thin Sections ◽

Structural Relationships

It has been demonstrated that the neurofibrillary tangles in biopsies of Alzheimer patients, composed of typical paired helical filaments (PHF), consist also of typical neurofilaments (NF) and 15nm wide filaments. Close structural relationships, and even continuity between NF and PHF, have been observed. In this paper, such relationships are investigated from the standpoint that the PHF are formed through posttranslational modifications of NF. To investigate the validity of the posttranslational modification hypothesis of PHF formation, we have identified in thin sections from frontal lobe biopsies of Alzheimer patients all existing conformations of NF and PHF and ordered these conformations in a hypothetical sequence. However, only experiments with animal model preparations will prove or disprove the validity of the interpretations of static structural observations made on patients. For this purpose, the results of in vitro experiments with the squid giant axon preparations are compared with those obtained from human patients. This approach is essential in discovering etiological factors of Alzheimer's disease and its early diagnosis.

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Observations of Frozen-Hydrated Microtubules

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100181270 ◽

1990 ◽

Vol 48 (1) ◽

pp. 504-505

Author(s):

D. Chrétien ◽

D. Job ◽

R.H. Wade

Keyword(s):

Fourier Transforms ◽

Structural Complexity ◽

Image Contrast ◽

Phase Behaviour ◽

Experimental Conditions ◽

Thin Sections ◽

Surface Lattice ◽

Cilia And Flagella ◽

Outstanding Question

Microtubules are filamentary structures found in the cytoplasm of eukaryotic cells, where, together with actin and intermediate filaments, they form the components of the cytoskeleton. They have many functions and show various levels of structural complexity as witnessed by the singlet, doublet and triplet structures involved in the architecture of centrioles, basal bodies, cilia and flagella. The accepted microtubule model consists of a 25 nm diameter hollow tube with a wall made up of 13 paraxial protofilaments (pf). Each pf is a string of aligned tubulin dimers. Some results have suggested that the pfs follow a superhelix. To understand how microtubules function in the cell an accurate model of the surface lattice is one of the requirements. For example the 9x2 architecture of the axoneme will depend on the organisation of its component microtubules. We should also note that microtubules with different numbers of pfs have been observed in thin sections of cellular and of in-vitro material. An outstanding question is how does the surface lattice adjust to these different pf numbers?We have been using cryo-electron microscopy of frozen-hydrated samples to study in-vitro assembled microtubules. The experimental conditions are described in detail in this reference. The results obtained in conjunction with thin sections of similar specimens and with axoneme outer doublet fragments have already allowed us to characterise the image contrast of 13, 14 and 15 pf microtubules on the basis of the measured image widths, of the the image contrast symmetry and of the amplitude and phase behaviour along the equator in the computed Fourier transforms. The contrast variations along individual microtubule images can be interpreted in terms of the geometry of the microtubule surface lattice. We can extend these results and make some reasonable predictions about the probable surface lattices in the case of other pf numbers, see Table 1. Figure 1 shows observed images with which these predictions can be compared.

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Studies on Thrombolysis with Streptokinase

Thrombosis and Haemostasis ◽

10.1055/s-0038-1654310 ◽

1971 ◽

Vol 25 (02) ◽

pp. 354-378 ◽

Cited By ~ 5

Author(s):

R Gottlob ◽

L Stockinger ◽

U Pötting ◽

G Schattenmann

Keyword(s):

Electron Microscopy ◽

Cross Section ◽

Whole Blood ◽

Jugular Vein ◽

Thin Sections ◽

The Third ◽

Morphological Findings ◽

Blood Clots ◽

Arteries And Veins

SummaryIn vitro whole blood clots of various ages, experimental thrombi produced in the jugular vein of rabbits and human thrombi from arteries and veins were examined in semi-thin sections and by means of electron microscopy.In all types of clots examined a typical course of retraction was found. Retraction starts with a dense excentrical focus which grows into a densification ring. After 24 hours the entire clot becomes almost homogeneously dense; later a secondary swelling sets in.Shortly after coagulation the erythrocytes on the rim of the clot are bi-concave discs. They then assume the shape of crenate spheres, turn into smooth spheres and finally become indented ghosts which have lost the largest part of their contents. In the inner zone, which makes up the bulk of the clot, we observed bi-concave discs prior to retraction. After retraction we see no crenations but irregularly shaped erythrocytes. Once the secondary swelling sets in, the cross-section becomes polygonal and later spherical. After extensive hemolysis we observe the “retiform thrombus” made up of ghosts.Experimental and clinical thrombi present the same morphology but are differentiated from in vitro clots by: earlier hemolysis, immigration of leukocytes, formation of a rim layer consisting of fibrin and thrombocytes, and the symptoms of organization. Such symptoms of organization which definitely will prevent lysis with streptokinase were found relatively late in experimental and clinical thrombi. Capillary buds and capillary loops were never found in clinical thrombi prior to the third month.The morphological findings agree with earlier physical and enzymatic investigations. The observation that phenomena of reorganization occur relatively late and frequently only in the rim areas of large thrombi explains why lytic therapy is possible in some of the chronic obliterations.

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Clinical and immunological effects of mRNA vaccines in malignant diseases

Molecular Cancer ◽

10.1186/s12943-021-01339-1 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Annkristin Heine ◽

Stefan Juranek ◽

Peter Brossart

Keyword(s):

Immune Responses ◽

Messenger Rna ◽

Immune Escape ◽

Special Focus ◽

Potential Efficacy ◽

Protective Antibodies ◽

Technological Developments ◽

Broad Variety ◽

Antigen Loss

AbstractIn vitro-transcribed messenger RNA-based therapeutics represent a relatively novel and highly efficient class of drugs. Several recently published studies emphasize the potential efficacy of mRNA vaccines in treating different types of malignant and infectious diseases where conventional vaccine strategies and platforms fail to elicit protective immune responses. mRNA vaccines have lately raised high interest as potent vaccines against SARS-CoV2. Direct application of mRNA or its electroporation into dendritic cells was shown to induce polyclonal CD4+ and CD8+ mediated antigen-specific T cell responses as well as the production of protective antibodies with the ability to eliminate transformed or infected cells. More importantly, the vaccine composition may include two or more mRNAs coding for different proteins or long peptides. This enables the induction of polyclonal immune responses against a broad variety of epitopes within the encoded antigens that are presented on various MHC complexes, thus avoiding the restriction to a certain HLA molecule or possible immune escape due to antigen-loss. The development and design of mRNA therapies was recently boosted by several critical innovations including the development of technologies for the production and delivery of high quality and stable mRNA. Several technical obstacles such as stability, delivery and immunogenicity were addressed in the past and gradually solved in the recent years.This review will summarize the most recent technological developments and application of mRNA vaccines in clinical trials and discusses the results, challenges and future directions with a special focus on the induced innate and adaptive immune responses.

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Role of interleukins in the pathogenesis of pulmonary fibrosis

Cell Death Discovery ◽

10.1038/s41420-021-00437-9 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Yi Xin She ◽

Qing Yang Yu ◽

Xiao Xiao Tang

Keyword(s):

Pulmonary Fibrosis ◽

Therapeutic Strategy ◽

Future Directions ◽

Regulation Mechanisms ◽

Underlying Mechanisms ◽

Multiple Cells ◽

The Relationship

AbstractInterleukins, a group of cytokines participating in inflammation and immune response, are proved to be involved in the formation and development of pulmonary fibrosis. In this article, we reviewed the relationship between interleukins and pulmonary fibrosis from the clinical, animal, as well as cellular levels, and discussed the underlying mechanisms in vivo and in vitro. Despite the effects of interleukin-targeted treatment on experimental pulmonary fibrosis, clinical applications are lacking and unsatisfactory. We conclude that intervening in one type of interleukins with similar functions in IPF may not be enough to stop the development of fibrosis as it involves a complex network of regulation mechanisms. Intervening interleukins combined with other existing therapy or targeting interleukins affecting multiple cells/with different functions at the same time may be one of the future directions. Furthermore, the intervention time is critical as some interleukins play different roles at different stages. Further elucidation on these aspects would provide new perspectives on both the pathogenesis mechanism, as well as the therapeutic strategy and drug development.

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Recapitulating Cardiac Structure and Function In Vitro from Simple to Complex Engineering

Micromachines ◽

10.3390/mi12040386 ◽

2021 ◽

Vol 12 (4) ◽

pp. 386

Author(s):

Ana Santos ◽

Yongjun Jang ◽

Inwoo Son ◽

Jongseong Kim ◽

Yongdoo Park

Keyword(s):

Tissue Engineering ◽

Extracellular Matrix ◽

Computational Modeling ◽

Cardiac Tissue ◽

Cardiac Development ◽

Heart Tissue ◽

Cardiac Tissue Engineering ◽

Cardiac Cells

Cardiac tissue engineering aims to generate in vivo-like functional tissue for the study of cardiac development, homeostasis, and regeneration. Since the heart is composed of various types of cells and extracellular matrix with a specific microenvironment, the fabrication of cardiac tissue in vitro requires integrating technologies of cardiac cells, biomaterials, fabrication, and computational modeling to model the complexity of heart tissue. Here, we review the recent progress of engineering techniques from simple to complex for fabricating matured cardiac tissue in vitro. Advancements in cardiomyocytes, extracellular matrix, geometry, and computational modeling will be discussed based on a technology perspective and their use for preparation of functional cardiac tissue. Since the heart is a very complex system at multiscale levels, an understanding of each technique and their interactions would be highly beneficial to the development of a fully functional heart in cardiac tissue engineering.

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