Abstract 359: The Production of Acellular Cardiac Extracellular Matrix from P3 Murine Heart

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Stefan M Kren ◽  
Daniel J Garry ◽  
Mary G Garry
Keyword(s):  
Extracellular Matrix ◽  
Cardiac Tissue ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Culture Media ◽  
Heart Tissue ◽  
Cell Nuclei ◽  
Dapi Staining ◽  
Neonatal Heart ◽  
The Matrix

Introduction: Understanding the role of extracellular matrix (ECM) in the creation of the cellular microenvironment during tissue formation and regeneration could be vital in extending this capability to injured adult tissue. To date the only confirmed mammalian heart tissue regeneration/regrowth has been in neonatal murine heart, with the regenerative capacity ceasing after day P3. By focusing on ECM from P3 heart tissue we hope to elucidate its contribution to regenerative plasticity and transfer this capacity to injured adult myocardium. Hypothesis: Detergent decellularization protocols used in adult tissues can be modified to function on a neonatal scale, serving to remove the cellular components of the neonatal heart, leaving structurally intact ECM to serve as a scaffold for the generation of cardiac tissue equivalents. Materials and Methods: Murine P3 hearts were perfused with 1% SDS in water at 20mm Hg for 12 hrs. Following detergent decellularization, perfusion with water, 1% Triton X-100, PBS and culture media restored biocompatibility to the isolated ECM. P1 and P7 primary cardiomyocytes expressing the mCherry red fluorescent protein reporter under control of the alpha myosin promoter were isolated by enzymatic disassociation and cultured in the heart matrix in a perfusion based bioreactor. Results: The decellularized ECM demonstrated removal of 97% of native DNA when compared to control by pico-green dsDNA binding assay. Histologic analysis demonstrated an absence of cell nuclei by H & E and DAPI staining. The preservation of the matrix structure and the maintenance of matrix immunoreactivity (collagen IV) were also demonstrated histologically. Following infusion of P1 or P7 mCherry positive cells, contractile behavior of the recellularized heart constructs was observed, and markers of cardiac linage (alpha-actinin in mCherry positive cells) were present. Conclusions: Neonatal heart matrix can be effectively decellularized. With appropriate modification of perfusion parameters, pediatric ECM structure can be preserved. This isolated matrix can serve as a scaffold for growth and maintenance of immature and mature cardiomyocytes, supporting continued contractility of cultured cells.

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

Micromachines ◽  
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.

A bioartificial rat heart tissue: Perfusion decellularization and characterization

2019 ◽  
Vol 42 (12) ◽  
pp. 757-764 ◽  
Author(s):  
Busra Ozlu ◽  
Mert Ergin ◽  
Sevcan Budak ◽  
Selcuk Tunali ◽  
Nuh Yildirim ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Three Dimensional ◽  
Tissue Perfusion ◽  
Heart Tissue ◽  
Cell Nuclei ◽  
Tissue Sections ◽  
The Past ◽  
Significant Difference ◽  
Structural Preservation

Despite remarkable advancement in the past decades, heart-related defects are still prone to progress irreversibly and can eventually lead to heart failure. A personalized extracellular matrix–based bioartificial heart created by allografts/xenografts emerges as an alternative as it can retain the original three-dimensional architecture combined with a preserved natural heart extracellular matrix. This study aimed at developing a procedure for decellularizing heart tissue harvested from rats and evaluating decellularization efficiency in terms of residual nuclear content and structural properties. Tissue sections showed no or little visible cell nuclei in decellularized heart, whereas the native heart showed dense cellularity. In addition, there was no significant variation in the alignment of muscle fibers upon decellularization. Furthermore, no significant difference was detected between native and decellularized hearts in terms of fiber diameter. Our findings demonstrate that fiber alignment and diameter can serve as additional parameters in the characterization of biological heart scaffolds as these provide valuable input for evaluating structural preservation of decellularized heart. The bioartificial scaffold formed here can be functionalized with patient’s own material and utilized in regenerative engineering.

scholarly journals Studies of fibronectin matrices in living cells with fluoresceinated gelatin.

1980 ◽  
Vol 87 (1) ◽  
pp. 14-22 ◽  
Author(s):  
P Hsieh ◽  
R Segal ◽  
L B Chen
Keyword(s):  
Extracellular Matrix ◽  
Chick Embryo ◽  
Cell Surface ◽  
Cell Lines ◽  
Cultured Cells ◽  
Living Cells ◽  
Low Concentration ◽  
Established Cell Lines ◽  
The Matrix ◽  
Established Cell

We have used fluorescein isosthiocyanate-conjugated gelatin (FITC-gelatin) (1 mg/ml) to localize cell surface fibronectin in unfixed live cells in cultures. FITC-gelatin stains the fibronectin matrix on primary cultures of rat and chick embryo fibroblasts as well as untransformed, established cell lines. In live cultured cells, fibronectin in many areas of the extracellular matrix is inaccessible to antibody and cannot be visualized by immunofluorescence staining. In contrast, fibronectin in these areas is fully stainable by FITC-gelatin. At a low concentration (20 micrograms/ml), FITC-gelatin stains the fibronectin matrix of primary cultured cells but not of "untransformed" established cell lines. SEM can detect only the matrix stainable with the low concentration of FITC-gelatin, such as that expressed by primary chick embryo fibroblasts. The binding of fibronectin to the extracellular matrix is very stable and FITC-gelatin remained bound to the matrix for at least 10 d in culture. Radioiodinated gelatin has been used to quantitate the level of cell surface fibronectin in living normal and transformed cells. FITC-gelatin appears to be a useful probe for studying the fibronectin of living cells in culture.

scholarly journals Changes in the components of extracellular matrix and in growth properties of cultured aortic smooth muscle cells upon ascorbate feeding.

1982 ◽  
Vol 92 (2) ◽  
pp. 462-470 ◽  
Author(s):  
E Schwartz ◽  
R S Bienkowski ◽  
B Coltoff-Schiller ◽  
S Goldfischer ◽  
O O Blumenfeld
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Time Course ◽  
Cultured Cells ◽  
Growth Parameters ◽  
Muscle Cells ◽  
Cellular Protein ◽  
Collagen Accumulation ◽  
The Matrix

Culture conditions can modify the composition of the extracellular matrix of cultured calf aortas smooth muscle cells. In the absence of ascorbate the major components of the matrix are microfibrillar proteins; deposition of collagen occurs upon ascorbate supplementation and, with increased time of exposure of cells to ascorbate, collagen becomes the dominant protein of the extracellular matrix (greater than 80%). Collagen accumulation follows a sigmoidal time-course, suggesting that it is a cooperative phenomenon. Covalent crosslinks are not required for collagen accumulation in the matrix. Microfibrillar proteins and increased amounts of proteoglycans and fibronectin accumulate concurrently with collagen but elastin deposition was not observed either with or without ascorbate feeding. Addition of ascorbate leads to a general stimulation of incorporation of [14C]proline into cellular protein and to changes in cell growth parameters and morphology: cell-doubling time decreases from 62 to 47 h and plating efficiency increases approximately fourfold. We conclude that the composition of the extracellular matrix assembled by cultured cells is subject to experimental manipulation and that changes in endogenously deposited matrix may have significant effects on cellular functions.

Analysis of Gene Expression and Morphology of P19 Cells Cultured in an Apatite-Fiber Scaffold

2012 ◽  
Vol 529-530 ◽  
pp. 370-373 ◽  
Author(s):  
Hide Ishii ◽  
Yuya Mukai ◽  
Mamoru Aizawa ◽  
Nobuyuki Kanzawa
Keyword(s):  
Heart Failure ◽  
Tissue Engineering ◽  
Cardiac Tissue ◽  
Cultured Cells ◽  
Three Dimensional ◽  
Heart Tissue ◽  
Cardiac Tissue Engineering ◽  
Cardiac Differentiation ◽  
Embryonal Carcinoma Cell ◽  
Cells Cultured

Heart disease is the second most common cause of mortality in Japan. Most cases of late stage heart failure can only be effectively treated by a heart transplant. Cardiac tissue engineering is emerging both as a new approach for improving the treatment of heart failure and for developing new cardiac drugs. Apatite-fiber scaffold (AFS) was originally designed as a substitute material for bone. AFS contains two sizes of pores and is appropriate for the three dimensional proliferation and differentiation of osteoblasts. To establish engineered heart tissue, a pluripotent embryonal carcinoma cell line, P19.CL6, was cultured in AFS. P19.CL6 cells seeded into AFS proliferated well. Generally, cardiac differentiation of P19.CL6 cells is induced by treating suspension-cultured cells with dimethyl sulfoxide (DMSO), after which the cells form spheroids. However, our results showed that P19.CL6 cells cultured in AFS differentiated into myocytes without forming spheroidal aggregates, and could be cultured for at least one month. Thus, we conclude that AFS is a good candidate as a scaffold for cardiac tissue engineering.

scholarly journals The role of intermolecular disulfide bonding in deposition of GP140 in the extracellular matrix.

1984 ◽  
Vol 99 (1) ◽  
pp. 105-114 ◽  
Author(s):  
W G Carter
Keyword(s):  
Extracellular Matrix ◽  
Culture Media ◽  
Molecular Forms ◽  
Cell Protein ◽  
Cell Contact ◽  
Relative Molecular Mass ◽  
Disulfide Bonding ◽  
Sequence Of Events ◽  
The Matrix ◽  
Matrix Precipitation

Human WI-38 fibroblasts in cultures synthesized at least three molecular forms of the major, extracellular matrix glycoprotein (GP), GP140: (a) cytoplasmic GP140 (1.2 ng of GP140/micrograms of cell protein) was detergent-soluble, underglycosylated, and possessed detectable levels of intermolecular disulfide bonding; (b) matrix GP140 (3.6 ng of GP140/micrograms of cell protein) was detergent-insoluble, more highly glycosylated and polymerized by intermolecular disulfide bonding, and co-distributed in the extracellular matrix with fibronectin; and (c) released GP140 (2 ng of GP140/micrograms of cell protein per 24 h) was recovered in the conditioned culture media and lacked intermolecular disulfide bonding. Cytoplasmic GP140 was the immediate biosynthetic precursor of the matrix form of GP140. In addition, various human adult and fetal tissues contained a form of GP140 that resembled the fibroblast matrix GP140 in the degree of intermolecular disulfide bonding, relative molecular mass, and immunological reactivity. Analysis of the sequence of events in assembly of GP140 and fibronectin in the extracellular matrix detected the following: (a) fibronectin was first to appear in the extracellular matrix; (b) GP140 accumulated in the cytoplasm, then deposited in the extracellular matrix and co-aligned with the established fibronectin; and (c) maturation of the extracellular matrix proceeded by continued intermolecular disulfide bonding. To evaluate possible roles for intermolecular disulfide bonding in cell interactions, a unique assay system was utilized based on the ability of labeled cells to incorporate radioactive matrix components into a biotinylated exogenous matrix. Precipitation of the biotinylated matrix from extracts of the cultures using avidin indicated: (a) disulfide bonding of radioactive GP140 and fibronectin into the exogenous biotinylated matrix required cell contact with the matrix. The newly deposited GP140 and fibronectin derived from the cells and not from GP140 and fibronectin present in the conditioned culture media. (b) Pro-alpha 1 and Pro-alpha 2 procollagens, present in the culture media, bound to the exogenous matrix in a noncovalent manner and were independent of cell contact. (c) SV40 transformed cells (WI-38 VA13) synthesized released form GP140 but did not deposit GP140 into the biotinylated matrix.

Abstract 98: Embryonic Extracellular Matrix and Cell Fate Determination

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Stefan M Kren ◽  
Christopher S Chapman ◽  
Andrew Loza ◽  
Daniel J Garry ◽  
Mary G Garry
Keyword(s):  
Extracellular Matrix ◽  
Cell Fate ◽  
Cellular Proliferation ◽  
Cultured Cells ◽  
Culture Media ◽  
Fluorescent Microscopy ◽  
Embryonic Stem ◽  
Vascular Perfusion ◽  
Murine Embryos

Background - The determination of cell fate during development is governed by intrinsic factors, but also by interaction with the milieu in which they reside. The extracellular matrix (ECM) from rapidly developing tissue should form a rich signaling environment for cellular proliferation and differentiation. Hypothesis - Murine embryonic ECM can be prepared by detergent decellularization that is morphologically preserved, biocompatible for cell culture, and at E13.5 substantial enough to permit vascular catheterization and recellularization by perfusion. Methods and Results - To test the contribution of embryonic extracellular matrix (ECM) to the determination of cell fate, we undertook isolation of ECM from developing murine embryos. Triton X-100 and SDS detergent decellularization were used to isolate ECM from E10.5 and E13.5 embryos. Acellularity was confirmed by pico-green DNA assay (98.7 % ± 0.95 of DNA removed compared to control), as well as the lack of visible nuclei by H & E and DAPI histology. The matrix scaffolds were washed thoroughly with PBS and culture media to return them to a biocompatible state. Murine embryonic stem cells (mESC) modified to express EGFP were cultured on the exterior or the interior of the ECM scaffolds. mESCs seeded on the exterior of the E10.5 scaffolds or perfused through the E13.5 umbilical vasculature were highly adherent and proliferative during the 17 day culture period as evidenced by fluorescent microscopy. Perfused mESCs exhibited engrafted in the heart, liver, and vascular conduit E13.5 matrix 2 days post-infusion. Histology confirmed the attachment and morphologic alteration of the cultured cells on the exterior of the E10.5 ECM and presence of the perfused cells in the E13.5 embryo matrix interior. Conclusion - Biocompatible, acellular morphologically preserved embryonic ECM can be extracted from E10.5 and E13.5 murine embryos. By E13.5 the structural integrity of the acellular matrix can sustain vascular perfusion for delivery of mESCs to internal organoid structures. These ECM preparations support the proliferation and maintenance of mESCs externally and internally.

scholarly journals Molecular assembly, secretion, and matrix deposition of type VI collagen.

1986 ◽  
Vol 102 (3) ◽  
pp. 703-710 ◽  
Author(s):  
E Engvall ◽  
H Hessle ◽  
G Klier
Keyword(s):  
Extracellular Matrix ◽  
Culture Media ◽  
Molecular Assembly ◽  
Collagen Molecule ◽  
Type Vi Collagen ◽  
Cultured Fibroblasts ◽  
Intracellular Pool ◽  
Matrix Deposition ◽  
The Matrix ◽  
Tissue Form

Monoclonal antibodies reactive with the tissue form of type VI collagen were used to isolate the type VI collagen polypeptides from cultured fibroblasts and muscle cells. Two [35S]methionine-labeled polypeptides of 260 and 140 kD were found intracellularly, in the medium, and in the extracellular matrix of metabolically labeled cells. These polypeptides were disulfide cross-linked into very large complexes. The 260- and 140-kD polypeptides were intimately associated and could not be separated from each other by reduction without denaturation. In the absence of ascorbic acid, both polypeptides accumulated inside the cell, and their amounts in the medium and in the matrix were decreased. These results suggest that both the 260- and the 140-kD polypeptides are integral parts of the type VI collagen molecule. Examination of type VI collagen isolated from the intracellular pool by electron microscopy after rotary shadowing revealed structures corresponding to different stages of assembly of type VI collagen. Based on these images, a sequence for the intracellular assembly of type VI collagen could be discerned. Type VI collagen monomers are approximately 125 nm long and are composed of two globules separated by a thin strand. The monomers assemble into dimers and tetramers by lateral association. Only tetramers were present in culture media, whereas both tetramers and multimers were found in extracellular matrix extracts. The multimers appeared to have assembled from tetramers by end-to-end association into filaments that had prominent knobs and a periodicity of approximately 110 nm. These results show that, unlike other collagens, type VI collagen is assembled into tetramers before it is secreted from the cells, and they also suggest an extracellular aggregation mechanism that appears to be unique to this collagen.

scholarly journals Epicardial therapy with atrial appendage micrografts salvages myocardium after infarction

2019 ◽  
Author(s):  
Xie Yanbo ◽  
Milla Lampinen ◽  
Juuso Takala ◽  
Vilbert Sikorski ◽  
Rabah Soliymani ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Extracellular Matrix ◽  
Cardiac Tissue ◽  
Artery Bypass ◽  
Cardiac Regeneration ◽  
Atrial Appendage ◽  
Mortality And Morbidity ◽  
The Matrix ◽  
Clinical Benefits ◽  
Matrix Patch

AbstractIschemic heart disease remains the leading cause of mortality and morbidity worldwide despite improved possibilities in medical care. Alongside interventional therapies, such as coronary artery bypass grafting, adjuvant tissue-engineered and cell-based treatments can provide regenerative improvement. Unfortunately, most of these advanced approaches require multiple lengthy and costly preparation stages without delivering significant clinical benefits.We evaluated the effect of epicardially delivered minute pieces of atrial appendage tissue material, defined as atrial appendage micrografts (AAMs), in mouse myocardial infarction model. An extracellular matrix patch was used to cover and fix the AAMs onto the surface of the infarcted heart. The matrix-covered AAMs salvaged the heart from infarction-induced loss of functional myocardium and attenuated scarring. Site-selective proteomics of injured ischemic and uninjured distal myocardium from AAM-treated and untreated tissue sections revealed an increased expression of several cardiac regeneration-associated proteins (i.e. periostin, transglutaminases and glutathione peroxidases) as well as activation of pathways responsible for angio- and cardiogenesis in relation to AAMs therapy.Epicardial delivery of AAMs encased in an extracellular matrix patch scaffold salvages functional cardiac tissue from ischemic injury and restricts fibrosis after myocardial infarction. Our results support the use of AAMs as tissue-based therapy adjuvants for salvaging the ischemic myocardium.

Extracellular matrix: the gatekeeper of tumor angiogenesis

2019 ◽  
Vol 47 (5) ◽  
pp. 1543-1555 ◽  
Author(s):  
Maurizio Mongiat ◽  
Simone Buraschi ◽  
Eva Andreuzzi ◽  
Thomas Neill ◽  
Renato V. Iozzo
Keyword(s):  
Extracellular Matrix ◽  
Tumor Angiogenesis ◽  
Signaling Cascades ◽  
Cancer Growth ◽  
Cell Behavior ◽  
Metastatic Progression ◽  
Surface Receptors ◽  
The Matrix ◽  
Multiple Signaling

Abstract The extracellular matrix is a network of secreted macromolecules that provides a harmonious meshwork for the growth and homeostatic development of organisms. It conveys multiple signaling cascades affecting specific surface receptors that impact cell behavior. During cancer growth, this bioactive meshwork is remodeled and enriched in newly formed blood vessels, which provide nutrients and oxygen to the growing tumor cells. Remodeling of the tumor microenvironment leads to the formation of bioactive fragments that may have a distinct function from their parent molecules, and the balance among these factors directly influence cell viability and metastatic progression. Indeed, the matrix acts as a gatekeeper by regulating the access of cancer cells to nutrients. Here, we will critically evaluate the role of selected matrix constituents in regulating tumor angiogenesis and provide up-to-date information concerning their primary mechanisms of action.

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