scholarly journals Functional scaffold-free 3-D cardiac microtissues: a novel model for the investigation of heart cells

2012 ◽  
Vol 302 (10) ◽  
pp. H2031-H2042 ◽  
Author(s):  
B. R. Desroches ◽  
P. Zhang ◽  
B.-R. Choi ◽  
M. E. King ◽  
A. E. Maldonado ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cardiac Fibroblasts ◽  
Neonatal Rat ◽  
Resting Membrane Potential ◽  
Heart Cells ◽  
Cell Type ◽  
Adenoviral Gene Transfer ◽  
Culture Model ◽  
Cell Type Specific ◽  
Novel Scaffold

To bridge the gap between two-dimensional cell culture and tissue, various three-dimensional (3-D) cell culture approaches have been developed for the investigation of cardiac myocytes (CMs) and cardiac fibroblasts (CFs). However, several limitations still exist. This study was designed to develop a cardiac 3-D culture model with a scaffold-free technology that can easily and inexpensively generate large numbers of microtissues with cellular distribution and functional behavior similar to cardiac tissue. Using micromolded nonadhesive agarose hydrogels containing 822 concave recesses (800 μm deep × 400 μm wide), we demonstrated that neonatal rat ventricular CMs and CFs alone or in combination self-assembled into viable (Live/Dead stain) spherical-shaped microtissues. Importantly, when seeded simultaneously or sequentially, CMs and CFs self-sorted to be interspersed, reminiscent of their myocardial distribution, as shown by cell type-specific CellTracker or antibody labeling. Microelectrode recordings and optical mapping revealed characteristic triangular action potentials (APs) with a resting membrane potential of −66 ± 7 mV ( n = 4) in spontaneously contracting CM microtissues. Under pacing, optically mapped AP duration at 90% repolarization and conduction velocity were 100 ± 30 ms and 18.0 ± 1.9 cm/s, respectively ( n = 5 each). The presence of CFs led to a twofold AP prolongation in heterogenous microtissues (CM-to-CF ratio of 1:1). Importantly, Ba2+-sensitive inward rectifier K+ currents and Ca2+-handling proteins, including sarco(endo)plasmic reticulum Ca2+-ATPase 2a, were detected in CM-containing microtissues. Furthermore, cell type-specific adenoviral gene transfer was achieved, with no impact on microtissue formation or cell viability. In conclusion, we developed a novel scaffold-free cardiac 3-D culture model with several advancements for the investigation of CM and CF function and cross-regulation.

Abstract 349: Functional Scaffold-Free 3D Cardiac Microtissues: A Novel Model for the Investigation of Heart Cells

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Bethany Desroches ◽  
Peng Zhang ◽  
Bum-Rak Choi ◽  
Michelle E King ◽  
Angel E Maldonado ◽  
...  
Keyword(s):  
Cell Culture ◽  
Gene Transfer ◽  
Action Potentials ◽  
Neonatal Rat ◽  
Calcium Handling ◽  
Specific Gene ◽  
Heart Cells ◽  
Cell Type ◽  
Cell Type Specific ◽  
Novel Scaffold

To bridge the gap between 2D cell culture and tissue, various 3D cell culture approaches have been developed for the investigation of cardiac myocytes (CM) and fibroblasts (CF), but limitations still exist. The goal of this study was to develop a scaffold-free cardiac culture that could easily and inexpensively create many uniform cardiac microtissues that mimic the cellular distribution and functional behavior of CM and CF in tissue. Using micromolded non-adhesive agarose hydrogels containing 822 concave recesses (800 λm deep x 400 λm wide), we demonstrate that neonatal rat ventricular CM and CF, when seeded alone or in combination, self-assembled into viable (Live/Dead® stain) spherical-shaped microtissues. Importantly, when CM and CF were seeded simultaneously or sequentially, they self-sorted to be highly interspersed, reminiscent of their distribution in ventricular tissue, as shown by cell type-specific CellTracker TM or antibody labeling. Depending on their cellular composition, microtissues expressed extracellular matrix and calcium handling proteins (including SERCA2a), featured typical I K1 and I Ca-L current densities and IV relationships, and formed functional cell-cell connections (as evidenced by spontaneous action potentials, contractions and connexin 43 expression). Microelectrode recordings and optical mapping showed characteristic triangular action potentials (AP) with a resting membrane potential of -66±7 mV (n=4) in spontaneously contracting CM microtissues. Under pacing, optically mapped AP duration at 90% repolarization and conduction velocity were 100±30 ms and 18.0±1.9 cm/s, respectively (n=5 each). The presence of CF led to a prolongation of both AP and calcium transients in CM:CF microtissues (1:1). Furthermore, cell-type-specific adenoviral gene transfer was achieved, with no impact on microtissue formation or cell viability. In conclusion, we have developed a novel scaffold-free cardiac 3D culture model, in which more than 800 homotypic (CM or CF) or heterotypic (CM:CF) microtissues can be formed in a 6-well with ease, analyzed functionally and subjected to cell-type-specific gene transfer, paving the way for future in vitro investigations of CM and CF behavior and their interactions in a 3D environment.

Cell-type specific trafficking of expressed mutant COMP in a cell culture model for PSACH

2004 ◽  
Vol 23 (7) ◽  
pp. 433-444 ◽  
Author(s):  
Tung-Ling L. Chen ◽  
Jeff W. Stevens ◽  
William G. Cole ◽  
Jacqueline T. Hecht ◽  
Barbara M. Vertel
Keyword(s):  
Cell Culture ◽  
Cell Type ◽  
Cell Culture Model ◽  
Culture Model ◽  
A Cell ◽  
Cell Type Specific

scholarly journals Osteogenic differentiation of human mesenchymal stromal cells and fibroblasts differs depending on tissue origin and replicative senescence

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Vera Grotheer ◽  
Nadine Skrynecki ◽  
Lisa Oezel ◽  
Jan Grassmann ◽  
Joachim Windolf ◽  
...  
Keyword(s):  
Cell Culture ◽  
Osteogenic Differentiation ◽  
Stromal Cells ◽  
Replicative Senescence ◽  
Cell Type ◽  
Differentiation Potential ◽  
Culture Passage ◽  
Cell Type Specific ◽  
Cell Culture Passage ◽  
Tissue Origin

AbstractThe need for an autologous cell source for bone tissue engineering and medical applications has led researchers to explore multipotent mesenchymal stromal cells (MSC), which show stem cell plasticity, in various human tissues. However, MSC with different tissue origins vary in their biological properties and their capability for osteogenic differentiation. Furthermore, MSC-based therapies require large-scale ex vivo expansion, accompanied by cell type-specific replicative senescence, which affects osteogenic differentiation. To elucidate cell type-specific differences in the osteogenic differentiation potential and replicative senescence, we analysed the impact of BMP and TGF-β signaling in adipose-derived stromal cells (ASC), fibroblasts (FB), and dental pulp stromal cells (DSC). We used inhibitors of BMP and TGF-β signaling, such as SB431542, dorsomorphin and/or a supplemental addition of BMP-2. The expression of high-affinity binding receptors for BMP-2 and calcium deposition with alizarin red S were evaluated to assess osteogenic differentiation potential. Our study demonstrated that TGF-β signaling inhibits osteogenic differentiation of ASC, DSC and FB in the early cell culture passages. Moreover, DSC had the best osteogenic differentiation potential and an activation of BMP signaling with BMP-2 could further enhance this capacity. This phenomenon is likely due to an increased expression of activin receptor-like kinase-3 and -6. However, in DSC with replicative senescence (in cell culture passage 10), osteogenic differentiation sharply decreased, and the simultaneous use of BMP-2 and SB431542 did not result in further improvement of this process. In comparison, ASC retain a similar osteogenic differentiation potential regardless of whether they were in the early (cell culture passage 3) or later (cell culture passage 10) stages. Our study elucidated that ASC, DSC, and FB vary functionally in their osteogenic differentiation, depending on their tissue origin and replicative senescence. Therefore, our study provides important insights for cell-based therapies to optimize prospective bone tissue engineering strategies.

Norepinephrine and ANG II stimulate secretion of TGF-beta by neonatal rat cardiac fibroblasts in vitro

1995 ◽  
Vol 268 (4) ◽  
pp. C910-C917 ◽  
Author(s):  
S. A. Fisher ◽  
M. Absher
Keyword(s):  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Cardiac Fibroblasts ◽  
Neonatal Rat ◽  
Heart Cells ◽  
Ang Ii ◽  
Tgf Beta ◽  
Hypertrophic Growth ◽  
Beta 2

Transforming growth factor-beta (TGF-beta) is a ubiquitous growth-regulating protein that is capable of influencing the growth and function of heart cells in vitro. To better understand the role TGF-beta might play as a paracrine mediator of cardiac hypertrophy, the expression, secretion, and growth effects of TGF-beta were examined. Neonatal cardiac fibroblasts in vitro secreted latent TGF-beta 1 and TGF-beta 2 as high as 15 ng/10(6) cells. Angiotensin II (ANG II) and norepinephrine (NE) each augmented up to threefold the expression and secretion of latent TGF-beta 1 and TGF-beta 2 and also induced a shift in isoform predominance from beta 1 to beta 2. Each agent individually produced hypertrophic growth of neonatal cardiocytes and hyperplastic growth of cardiac fibroblasts. Paradoxically, the combination of NE and ANG II at intermediate and high concentrations resulted in less TGF-beta secretion (compared with either agent alone) and in hypertrophic growth of fibroblasts. These results suggest that the growth-promoting effects of ANG II and NE may in part be mediated via a paracrine stimulation of TGF-beta secretion.

scholarly journals The Cell Type–Specific Functions of miR-21 in Cardiovascular Diseases

2020 ◽  
Vol 11 ◽  
Author(s):  
Beibei Dai ◽  
Feng Wang ◽  
Xiang Nie ◽  
Hengzhi Du ◽  
Yanru Zhao ◽  
...  
Keyword(s):  
Cardiovascular Diseases ◽  
Vascular System ◽  
Cardiac Fibroblasts ◽  
Pressure Overload ◽  
Cell Type ◽  
Physiological Processes ◽  
A Cell ◽  
Cell Type Specific ◽  
Underlying Mechanisms ◽  
Endothelial Nitric Oxide

Cardiovascular diseases are one of the prime reasons for disability and death worldwide. Diseases and conditions, such as hypoxia, pressure overload, infection, and hyperglycemia, might initiate cardiac remodeling and dysfunction by inducing hypertrophy or apoptosis in cardiomyocytes and by promoting proliferation in cardiac fibroblasts. In the vascular system, injuries decrease the endothelial nitric oxide levels and affect the phenotype of vascular smooth muscle cells. Understanding the underlying mechanisms will be helpful for the development of a precise therapeutic approach. Various microRNAs are involved in mediating multiple pathological and physiological processes in the heart. A cardiac enriched microRNA, miR-21, which is essential for cardiac homeostasis, has been demonstrated to act as a cell–cell messenger with diverse functions. This review describes the cell type–specific functions of miR-21 in different cardiovascular diseases and its prospects in clinical therapy.

scholarly journals The First Nonmammalian Pegivirus Demonstrates Efficient In Vitro Replication and High Lymphotropism

2020 ◽  
Vol 94 (20) ◽  
Author(s):  
Zhen Wu ◽  
Yuanyuan Wu ◽  
Wei Zhang ◽  
Andres Merits ◽  
Peter Simmonds ◽  
...  
Keyword(s):  
Cell Culture ◽  
Animal Experiments ◽  
Clinical Manifestations ◽  
Viral Loads ◽  
Culture Model ◽  
Route Of Transmission ◽  
Replication Systems ◽  
Goose Parvovirus ◽  
Cell Type Specific

ABSTRACT Members of the Pegivirus genus, family Flaviviridae, widely infect humans and other mammals, including nonhuman primates, bats, horses, pigs, and rodents, but are not associated with disease. Here, we report a new, genetically distinct pegivirus in goose (Anser cygnoides), the first identified in a nonmammalian host species. Goose pegivirus (GPgV) can be propagated in goslings, embryonated goose eggs, and primary goose embryo fibroblasts, and is thus the first pegivirus that can be efficiently cultured in vitro. Experimental infection of GPgV in goslings via intravenous injection revealed robust replication and high lymphotropism. Analysis of the tissue tropism of GPgV revealed that the spleen and thymus were the organs bearing the highest viral loads. Importantly, GPgV could promote clinical manifestations of goose parvovirus infection, including reduced weight gain and 7% mortality. This finding contrasts with the lack of pathogenicity that is characteristic of previously reported pegiviruses. IMPORTANCE Members of the Pegivirus genus, family Flaviviridae, widely infect humans and other mammals, but are described as causing persistent infection and lacking pathogenicity. The efficiency of in vitro replication systems for pegivirus is poor, thus limiting investigation into viral replication steps. Because of that, the pathogenesis, cellular tropism, route of transmission, biology, and epidemiology of pegiviruses remain largely uncovered. Here, we report a phylogenetically distinct goose pegivirus (GPgV) that should be classified as a new species. GPgV proliferated in cell culture in a species- and cell-type-specific manner. Animal experiments show GPgV lymphotropism and promote goose parvovirus clinical manifestations. This study provides the first cell culture model for pegivirus, opening new possibilities for studies of pegivirus molecular biology. More importantly, our findings stand in contrast to the lack of identified pathogenicity of previously reported pegiviruses, which sheds lights on the pathobiology of pegivirus.

scholarly journals Gq-activated fibroblasts induce cardiomyocyte action potential prolongation and automaticity in a three-dimensional microtissue environment

2017 ◽  
Vol 313 (4) ◽  
pp. H810-H827 ◽  
Author(s):  
C. M. Kofron ◽  
T. Y. Kim ◽  
M. E. King ◽  
A. Xie ◽  
F. Feng ◽  
...  
Keyword(s):  
Action Potential ◽  
Electrical Activity ◽  
Rise Time ◽  
Connexin 43 ◽  
Cardiac Fibroblasts ◽  
Three Dimensional ◽  
Neonatal Rat ◽  
Resting Membrane Potential ◽  
Western Blots ◽  
Cell Cell

Cardiac fibroblasts (CFs) are known to regulate cardiomyocyte (CM) function in vivo and in two-dimensional in vitro cultures. This study examined the effect of CF activation on the regulation of CM electrical activity in a three-dimensional (3-D) microtissue environment. Using a scaffold-free 3-D platform with interspersed neonatal rat ventricular CMs and CFs, Gq-mediated signaling was selectively enhanced in CFs by Gαq adenoviral infection before coseeding with CMs in nonadhesive hydrogels. After 3 days, the microtissues were analyzed by signaling assay, histological staining, quantitative PCR, Western blots, optical mapping with voltage- or Ca2+-sensitive dyes, and microelectrode recordings of CF resting membrane potential (RMPCF). Enhanced Gq signaling in CFs increased microtissue size and profibrotic and prohypertrophic markers. Expression of constitutively active Gαq in CFs prolonged CM action potential duration (by 33%) and rise time (by 31%), prolonged Ca2+ transient duration (by 98%) and rise time (by 65%), and caused abnormal electrical activity based on depolarization-induced automaticity. Constitutive Gq activation in CFs also depolarized RMPCF from –33 to −20 mV and increased connexin 43 and connexin 45 expression. Computational modeling confers that elevated RMPCF and increased cell-cell coupling between CMs and CFs in a 3-D environment could lead to automaticity. In conclusion, our data demonstrate that CF activation alone is capable of altering action potential and Ca2+ transient characteristics of CMs, leading to proarrhythmic electrical activity. Our results also emphasize the importance of a 3-D environment where cell-cell interactions are prevalent, underscoring that CF activation in 3-D tissue plays a significant role in modulating CM electrophysiology and arrhythmias. NEW & NOTEWORTHY In a three-dimensional microtissue model, which lowers baseline activation of cardiac fibroblasts but enables cell-cell, paracrine, and cell-extracellular matrix interactions, we demonstrate that selective cardiac fibroblast activation by enhanced Gq signaling, a pathophysiological trigger in the diseased heart, modulates cardiomyocyte electrical activity, leading to proarrhythmogenic automaticity.

scholarly journals Oligodendrocytes and Microglia Are Selectively Vulnerable to Combined Hypoxia and Hypoglycemia Injury in Vitro

1998 ◽  
Vol 18 (5) ◽  
pp. 521-530 ◽  
Author(s):  
Susan A. Lyons ◽  
Helmut Kettenmann
Keyword(s):  
Lactate Dehydrogenase ◽  
Glial Cell ◽  
Glial Cells ◽  
Cell Types ◽  
Neonatal Rat ◽  
Radical Scavenger ◽  
Cell Type ◽  
Hypoxic Conditions ◽  
Cell Type Specific

The major classes of glial cells, namely astrocytes, oligodendrocytes, and microglial cells were compared in parallel for their susceptibility to damage after combined hypoxia and hypoglycemia or hypoxia alone. The three glial cell types were isolated from neonatal rat brains, separated, and incubated in N2/CO2-gassed buffer-containing glucose or glucose substitutes, 2-deoxyglucose or mannitol (both nonmetabolizable sugars). The damage to the cells after 6 hours' exposure was determined at 0, 1, 3, 7 days based on release of lactate dehydrogenase and counting of ethidium bromide–stained dead cells, double-stained with cell-type specific markers. When 2-deoxyglucose replaced glucose during 6 hours of hypoxia, both oligodendrocytes and microglia rarely survived (18% and 12%, respectively). Astroglia initially increased the release of lactate dehydrogenase but maintained 98% to 99% viability. When mannitol, a radical scavenger and osmolarity stabilizer, replaced glucose during 6 hours of hypoxia, oligodendrocytes rarely survived (10%), astroglia survival remained at 99%, but microglia survival increased to 50%. After exposure to 6 and 42 hours, respectively, of hypoxic conditions alone, oligodendrocytes exhibited 10% survival whereas microglia and astroglia were only temporarily stressed and subsequently survived. In conclusion, oligodendrocytes, then microglia, are the most vulnerable glial cell types in response to hypoxia or hypoglycemia conditions, whereas astrocytes from the same preparations recover.

Na+,K+-ATPase as a Target for Treatment of Tissue Fibrosis

2019 ◽  
Vol 26 (3) ◽  
pp. 564-575 ◽  
Author(s):  
Sergei N. Orlov ◽  
Jennifer La ◽  
Larisa V. Smolyaninova ◽  
Nickolai O. Dulin
Keyword(s):  
Cardiac Fibrosis ◽  
Cardiac Fibroblasts ◽  
Lung Fibroblasts ◽  
In Vivo Studies ◽  
Myofibroblast Differentiation ◽  
Cell Type ◽  
Tissue Fibrosis ◽  
Cell Type Specific ◽  
Cox 2

Myofibroblast activation is a critical process in the pathogenesis of tissue fibrosis accounting for 45% of all deaths. No effective therapies are available for the treatment of fibrotic diseases. We focus our mini-review on recent data showing that cardiotonic steroids (CTS) that are known as potent inhibitors of Na+,K+-ATPase affect myofibroblast differentiation in a cell type-specific manner. In cultured human lung fibroblasts (HLF), epithelial cells, and cancer-associated fibroblasts, CTS blocked myofibroblast differentiation triggered by profibrotic cytokine TGF-β. In contrast, in the absence of TGF-β, CTS augmented myofibroblast differentiation of cultured cardiac fibroblasts. The cell type-specific action of CTS in myofibroblast differentiation is consistent with data obtained in in vivo studies. Thus, infusion of ouabain via osmotic mini-pumps attenuated the development of lung fibrosis in bleomycintreated mice, whereas marinobufagenin stimulated renal and cardiac fibrosis in rats with experimental renal injury. In TGF-β-treated HLF, suppression of myofibroblast differentiation by ouabain is mediated by elevation of the [Na+]i/[K+]i ratio and is accompanied by upregulation of cyclooxygenase COX-2 and downregulation of TGF-β receptor TGFBR2. Augmented expression of COX-2 is abolished by inhibition of Na+/Ca2+ exchanger, suggesting a key role of [Ca2+]i-mediated signaling. What is the relative impact in tissue fibrosis of [Na+]i,[K+]iindependent signaling documented in several types of CTS-treated cells? Do the different conformational transitions of Na+,K+-ATPase α1 subunit in the presence of ouabain and marinobufagenin contribute to their distinct involvement in myofibroblast differentiation? Additional experiments should be done to answer these questions and to develop novel pharmacological approaches for the treatment of fibrosis-related disorders.

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