Biophysical stimulation for in vitro engineering of functional cardiac tissues

2017 ◽  
Vol 131 (13) ◽  
pp. 1393-1404 ◽  
Author(s):  
Anastasia Korolj ◽  
Erika Yan Wang ◽  
Robert A. Civitarese ◽  
Milica Radisic
Keyword(s):  
Cardiac Tissue ◽  
Culture Media ◽  
Culture Conditions ◽  
Lineage Specification ◽  
Cardiac Tissues ◽  
Cardiac Lineage ◽  
And Function ◽  
Tissue Models

Engineering functional cardiac tissues remains an ongoing significant challenge due to the complexity of the native environment. However, our growing understanding of key parameters of the in vivo cardiac microenvironment and our ability to replicate those parameters in vitro are resulting in the development of increasingly sophisticated models of engineered cardiac tissues (ECT). This review examines some of the most relevant parameters that may be applied in culture leading to higher fidelity cardiac tissue models. These include the biochemical composition of culture media and cardiac lineage specification, co-culture conditions, electrical and mechanical stimulation, and the application of hydrogels, various biomaterials, and scaffolds. The review will also summarize some of the recent functional human tissue models that have been developed for in vivo and in vitro applications. Ultimately, the creation of sophisticated ECT that replicate native structure and function will be instrumental in advancing cell-based therapeutics and in providing advanced models for drug discovery and testing.

scholarly journals Optimization of Co-Culture Conditions for a Human Vascularized Adipose Tissue Model

2020 ◽  
Vol 7 (3) ◽  
pp. 114 ◽  
Author(s):  
Feipeng Yang ◽  
Ronald N. Cohen ◽  
Eric M. Brey
Keyword(s):  
Adipose Tissue ◽  
Endothelial Cell ◽  
Metabolic Diseases ◽  
Culture Media ◽  
Culture Conditions ◽  
Extensive Evaluation ◽  
Proliferation And Differentiation ◽  
And Function ◽  
Tissue Models

In vitro adipose tissue models can be used to provide insight into fundamental aspects of adipose physiology. These systems may serve as replacements for animal models, which are often poor predictors of obesity and metabolic diseases in humans. Adipose tissue consists of a rich vasculature that is essential to its function. However, the study of endothelial cell–adipocyte interactions has been challenging due to differences in culture conditions required for the survival and function of each cell type. To address this issue, we performed an extensive evaluation of the cell culture media composition to identify the conditions optimal for the co-culture of endothelial cells and adipocytes. The effects of individual media factors on cell survival, proliferation, and differentiation were systematically explored. Several media factors were determined to disrupt the co-culture system. Optimized culture conditions were identified and used to generate a vascularized human adipose microtissue. An interconnected vascular network was established within an adipose micro-tissue, and the networks were anastomosed with perfused channels to form a functional network. In conclusion, media conditions were identified that enabled endothelial cell–adipocyte co-culture and were used to support the formation of a vascularized adipose tissue within a microfluidic device.

Abstract 37: Nrip Deficiency Leads to Dilated Cardiomyopathy

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Show-Li Chen
Keyword(s):  
Calcium Transient ◽  
Receptor Interaction ◽  
Cell Width ◽  
Interacting Protein ◽  
Cardiac Tissues ◽  
Calmodulin Binding ◽  
Calcineurin Phosphatase ◽  
And Function

Previously, we demonstrate a gene, nuclear receptor interaction protein (NRIP, also named DCAF6 or IQWD1) as a Ca2+- dependent calmodulin binding protein that can activate calcineurin phosphatase activity. Here, we found that α-actinin-2 (ACTN2), is one of NRIP-interacting proteins from the yeast two-hybrid system using NRIP as a prey. We further confirmed the direct bound between NRIP and ACTN2 using in vitro protein-protein interaction and in vivo co-immunoprecipitation assays. To further map the binding domain of each protein, the results showed the IQ domain of NRIP responsible for ACTN2 binding, and EF hand motif of ACTN2 responsible for NRIP bound. Due to ACTN2 is a biomarker of muscular Z-disc complex; we found the co-localization of NRIP and ACTN2 in cardiac tissues by immunofluorescence assays. Taken together, NRIP is a novel ACTN2-interacting protein. To investigate insights into in vivo function of NRIP, we generated conventional NRIP-null mice. The H&E staining results are shown in the hearts of NRIP KO mice are enlarged and dilated and the cell width of NRIP KO cardiomyocyte is increased. The EM of NRIP KO heart muscles reveal the reduction of I-band width and extension length of Z-disc in sarcomere structure; and the echocardiography shows the diminished fractional shortening in heart functions. Additionally, the calcium transient and sarcomere contraction length in cardiomyocytes of NRIP KO is weaker and shorter than wt; respectively. In conclusion, NRIP is a novel Z-disc protein and has function for maintenance of sarcomere integrity structure and function for calcium transient and muscle contraction.

scholarly journals Bioreactors as physiologicallike in vitro models

2020 ◽  
Author(s):  
Sara Mantero ◽  
Federica Boschetti
Keyword(s):  
Culture Conditions ◽  
In Vitro Models ◽  
In Vivo Models ◽  
In Vitro Development ◽  
Culture Cells ◽  
Vitro Development ◽  
Tissue Models ◽  
Mechanical Stretching

Bioreactors are powerful tools for in vitro development of engineered substitutes through controlled biological, physical, and mechanical culture conditions: bioreactor technology allows a closer in vitro replication of native tissues. One of bioreactors applications is the design of in vitro 3D tissue models as a bridge between 2D and in vivo models, allowing the application of 3R (replacement, reduction, refinement) principle. To this aim, bioreactors can be used to culture cells seeded on engineered scaffolds under in vivo-like conditions. Another key use of bioreactors is for perfusion decellularization of tissues and organs to be used as scaffolds. This contribution describes a dynamic stretching. bioreactor, imposing a mechanical stretching to the cultured constructs, allowing the development of skeletal muscle engineered constructs, and a decellularization bioreactor, designed for decellularization of blood vessels.

scholarly journals Versatile electrical stimulator for providing cardiac-like electrical impulses in vitro

2020 ◽  
Author(s):  
Stefano Gabetti ◽  
Giovanni Putame ◽  
Federica Montrone ◽  
Giuseppe Isu ◽  
Anna Marsano ◽  
...  
Keyword(s):  
Cardiac Tissue ◽  
Animal Experiments ◽  
Cost Effective ◽  
Culture Conditions ◽  
Animal Testing ◽  
Electrical Stimulator ◽  
Electrical Impulses ◽  
Reliable Methods

In the perspective of reliable methods alternative to in vivo animal testing for cardiac tissue engineering (CTE) research, the versatile electrical stimulator ELETTRA has been developed. ELETTRA delivers controlled and stable cardiac-like electrical impulses, and it can be coupled to already existing bioreactors for providing in vitro combined biomimetic culture conditions. Designed to be cost-effective and easy to use, this device could contribute to the reduction and replacement of in vivo animal experiments in CTE.

scholarly journals BOARD INVITED REVIEW: Post-transfer consequences of in vitro-produced embryos in cattle

2019 ◽  
Vol 97 (6) ◽  
pp. 2555-2568 ◽  
Author(s):  
Alan D Ealy ◽  
Lydia K Wooldridge ◽  
Sarah R McCoski
Keyword(s):  
Production Systems ◽  
Culture Media ◽  
Methylation Status ◽  
Culture Conditions ◽  
Retention Rates ◽  
Binucleate Cell ◽  
Low Oxygen ◽  
Embryo Production

Abstract In vitro embryo production (IVP) in cattle has gained worldwide interest in recent years, but the efficiency of using IVP embryos for calf production is far from optimal. This review will examine the pregnancy retention rates of IVP embryos and explore causes for pregnancy failures. Based on work completed over the past 25 yr, only 27% of cattle receiving IVP embryos will produce a live calf. Approximately 60% of these pregnancies fail during the first 6 wk of gestation. When compared with embryos generated by superovulation, pregnancy rates are 10% to 40% lower for cattle carrying IVP embryos, exemplifying that IVP embryos are consistently less competent than in vivo-generated embryos. Several abnormalities have been observed in the morphology of IVP conceptuses. After transfer, IVP embryos are less likely to undergo conceptus elongation, have reduced embryonic disk diameter, and have compromised yolk sac development. Marginal binucleate cell development, cotyledon development, and placental vascularization have also been documented, and these abnormalities are associated with altered fetal growth trajectories. Additionally, in vitro culture conditions increase the risk of large offspring syndrome. Further work is needed to decipher how the embryo culture environment alters post-transfer embryo development and survival. The risk of these neonatal disorders has been reduced by the use of serum-free synthetic oviductal fluid media formations and culture in low oxygen tension. However, alterations are still evident in IVP oocyte and embryo transcript abundances, timing of embryonic cleavage events and blastulation, incidence of aneuploidy, and embryonic methylation status. The inclusion of oviductal and uterine-derived embryokines in culture media is being examined as one way to improve the competency of IVP embryos. To conclude, the evidence presented herein clearly shows that bovine IVP systems still must be refined to make it an economical technology in cattle production systems. However, the current shortcomings do not negate its current value for certain embryo production needs and for investigating early embryonic development in cattle.

scholarly journals Myofibrils in Cardiomyocytes Tend to Assemble Along the Maximal Principle Stress Directions

2017 ◽  
Vol 139 (12) ◽  
Author(s):  
Hongyan Yuan ◽  
Bahador Marzban ◽  
Kevin Kit Parker
Keyword(s):  
Rational Design ◽  
Spatial Organization ◽  
Cardiac Tissue ◽  
Contractile Function ◽  
Focal Adhesions ◽  
Cardiac Tissues ◽  
Mechanics Model ◽  
Stress Directions

The mechanisms underlying the spatial organization of self-assembled myofibrils in cardiac tissues remain incompletely understood. By modeling cells as elastic solids under active cytoskeletal contraction, we found a good correlation between the predicted maximal principal stress directions and the in vitro myofibril orientations in individual cardiomyocytes. This implies that actomyosin fibers tend to assemble along the maximal tensile stress (MTS) directions. By considering the dynamics of focal adhesion and myofibril formation in the model, we showed that different patterns of myofibril organizations in mature versus immature cardiomyocytes can be explained as the consequence of the different levels of force-dependent remodeling of focal adhesions. Further, we applied the mechanics model to cell pairs and showed that the myofibril organizations can be regulated by a combination of multiple factors including cell shape, cell–substrate adhesions, and cell–cell adhesions. This mechanics model can guide the rational design in cardiac tissue engineering where recapitulating in vivo myofibril organizations is crucial to the contractile function of the heart.

262. Towards derivation of primordial germ cells from murine embryonic stem cells

2005 ◽  
Vol 17 (9) ◽  
pp. 106
Author(s):  
J. C. Young ◽  
V. L. Dias ◽  
M. Holland ◽  
K. L. Loveland
Keyword(s):  
Culture Media ◽  
Es Cells ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Lineage Specification ◽  
Signalling Molecules

The process of primordial germ cell (PGC) specification begins at the earliest stages of murine embryogenesis. The mechanisms underlying this process are the strong instructive cues generated by the extra-embryonic ectoderm, which, via ligand-receptor signalling in the visceral endoderm, activate pathways in the proximal epiblast to induce the PGC phenotype. We have subjected murine embryonic stem (ES) cells to similar cues in order to drive PGC lineage specification in vitro. ES cells were differentiated as aggregates (embryoid bodies (EBs)), a process that is thought to recapitulate the early stages of embryogenesis by providing an environment conducive to the spontaneous emergence of multiple cell lineages. To date, we have shown that EBs can also support the spontaneous emergence of cells expressing PGC markers. Expression analysis was performed on EBs from 1 to 30 days in culture. PGC markers, including nanog, dazl, fragilis, stella and SSEA1, are expressed in undifferentiated ES cells, but rapidly become undetectable in EBs as the constituent ES cells undergo differentiation. The spontaneous emergence of cells expressing these markers occurred only following long-term EB culture. This indicates a lag in the signalling normally required for PGC specification. In vivo, the lack of BMP4, its receptor (ALK-2) or downstream signalling molecules (Smad 1 and 5), results in the absence of PGCs in embryos. Therefore, in order to enhance PGC specification in our in vitro system, we have added BMP4 into the culture media. Under these conditions, the emergence of cells expressing PGC markers occurs at both an apparently higher efficiency and in a shorter time period. This suggests that BMP4 response pathways are present within the EB context and, when activated, can direct PGC specification. Thus, by recapitulating an in vivo physical and biochemical environment, we are able to direct PGC lineage specification in vitro.

scholarly journals Engineering Biomaterials to Guide Heart Cells for Matured Cardiac Tissue

Coatings ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 925
Author(s):  
Yongjun Jang ◽  
Yongdoo Park ◽  
Jongseong Kim
Keyword(s):  
Cardiac Tissue ◽  
Structural Integrity ◽  
Cardiac Cells ◽  
Heart Cells ◽  
Cardiac Tissues ◽  
Main Components ◽  
2D And 3D ◽  
Suitable Environment

The extracellular matrix (ECM) is needed to maintain the structural integrity of tissues and to mediate cellular dynamics. Its main components are fibrous proteins and glycosaminoglycans, which provide a suitable environment for biological functions. Thus, biomaterials with ECM-like properties have been extensively developed by modulating their key components and properties. In the field of cardiac tissue engineering, the use of biomaterials offers several advantages in that biophysical and biochemical cues can be designed to mediate cardiac cells, which is critical for maturation and regeneration. This suggests that understanding biomaterials and their use in vivo and in vitro is beneficial in terms of advancing cardiac engineering. The current review provides an overview of both natural and synthetic biomaterials and their use in cardiac engineering. In addition, we focus on different strategies to recapitulate the cardiac tissue in 2D and 3D approaches, which is an important step for the maturation of cardiac tissues toward regeneration of the adult heart.

Myofibrillar degeneration with diphtheria toxin

2020 ◽  
Vol 45 (4) ◽  
pp. 351-357
Author(s):  
Bilge Özerman Edis ◽  
Muhammet Bektaş ◽  
Rüstem Nurten
Keyword(s):  
Protein Synthesis ◽  
Actin Filaments ◽  
Diphtheria Toxin ◽  
Cardiac Tissue ◽  
Culture Conditions ◽  
Bacterial Toxin ◽  
Filamentous Actin ◽  
Cardiac Damage ◽  
Tissue Samples

AbstractObjectivesCardiac damage in patient with diphtheritic myocarditis is reported as the leading cause of mortality. Diphtheria toxin (DTx) is a well-known bacterial toxin inducing various cytotoxic effects. Mainly, catalytic fragment inhibits protein synthesis, induces cytotoxicity, and depolymerizes actin filaments. In this study, we aimed to demonstrate the extent of myofibrillar damage under DTx treatment to porcine cardiac tissue samples.MethodsTissue samples were incubated with DTx for 1–3 h in culture conditions. To analyze whole toxin (both fragments) distribution, conjugation of DTx with FITC was performed. Measurements were carried out with fluorescence spectrophotometer before and after dialysis. Immunofluorescence microscopy was used to show localization of DTx-FITC (15 nM) on cardiac tissue incubated for 2 h. Ultrastructural characterization of cardiac tissue samples treated with DTx (15 or 150 nM) was performed with transmission electron microscopy.ResultsDTx exerts myofibrillar disorganization. Myofilament degeneration, mitochondrial damage, vacuolization, and abundant lipid droplets were determined with 150 nM of DTx treatment.ConclusionsThis finding is an addition to depolymerization of actin filaments as a result of the DTx-actin interactions in in vitro conditions, indicating that myofilament damage can occur with DTx directly besides protein synthesis inhibition. Ultrastructural results support the importance of filamentous actin degeneration at diphtheritic myocarditis.

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