Abstract 9: Temperature-responsive Cell Delivery Biopolymers for Cardiac Tissue Engineering

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Brisa Pena ◽  
Valentina Martinelli ◽  
Susanna Bosi ◽  
Carmen Sucharov ◽  
Mark Jeong ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Body Temperature ◽  
Cell Viability ◽  
Cultured Cells ◽  
Ventricular Myocytes ◽  
3D Culture ◽  
Polymeric Matrix ◽  
Neonatal Rat ◽  
Cell Delivery

Background: Advances in cell therapy and material science have made tissue engineering a promising strategy for heart regeneration. We developed an injectable biomimetic reverse thermal gel (RTG) that is liquid at room temperature but gel-like at body temperature, with the ultimate goal of being able to serve as a vehicle for cell-based delivery (liquid) to targeted tissue areas (gel-phase at 37°C). In this study we tested the suitability of this biomimetic RTG on cell viability. Methods and results: We tested different biomimetic RTG systems with and without the chemical incorporation of lysine. In vitro 3D culture experiments were performed with neonatal rat ventricular myocytes (NRVM) by mixing 3x104 cells with 50 μl of polymeric solution and allowing gel formation at 37°C. The cultured cells were incubated for 21 days. For controls we used NRVMs plated on 2D traditional gelatin coated dishes. We found that the 3D polymeric matrix induces rapid coordinated contraction with improved functionality when compared with standard 2D-cultured NRVM. By immunostaining for the morphology of the sarcomere (alpha-actinin) and DAPI, we also observed that the 3D polymeric matrix stimulates cells to spread and form 3D syncytia. Conclusion: These proof-of-concept results demonstrate long-term cell viability in this unique biomimetic system and therefore provide feasibility of a polymeric cell delivery system that permits reversible liquid-to-gel transition at body temperature. These results offer potential for a tissue engineering approach to cardiac regeneration.

scholarly journals A Perfusion Bioreactor for Longitudinal Monitoring of Bioengineered Liver Constructs

Nanomaterials ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 275
Author(s):  
Lisa Sassi ◽  
Omolola Ajayi ◽  
Sara Campinoti ◽  
Dipa Natarajan ◽  
Claire McQuitty ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Liver Disease ◽  
Cell Viability ◽  
3D Culture ◽  
Culture Conditions ◽  
Disease Models ◽  
Non Invasive ◽  
And Function

In the field of in vitro liver disease models, decellularised organ scaffolds maintain the original biomechanical and biological properties of the extracellular matrix and are established supports for in vitro cell culture. However, tissue engineering approaches based on whole organ decellularized scaffolds are hampered by the scarcity of appropriate bioreactors that provide controlled 3D culture conditions. Novel specific bioreactors are needed to support long-term culture of bioengineered constructs allowing non-invasive longitudinal monitoring. Here, we designed and validated a specific bioreactor for long-term 3D culture of whole liver constructs. Whole liver scaffolds were generated by perfusion decellularisation of rat livers. Scaffolds were seeded with Luc+HepG2 and primary human hepatocytes and cultured in static or dynamic conditions using the custom-made bioreactor. The bioreactor included a syringe pump, for continuous unidirectional flow, and a circuit built to allow non-invasive monitoring of culture parameters and media sampling. The bioreactor allowed non-invasive analysis of cell viability, distribution, and function of Luc+HepG2-bioengineered livers cultured for up to 11 days. Constructs cultured in dynamic conditions in the bioreactor showed significantly higher cell viability, measured with bioluminescence, distribution, and functionality (determined by albumin production and expression of CYP enzymes) in comparison to static culture conditions. Finally, our bioreactor supports primary human hepatocyte viability and function for up to 30 days, when seeded in the whole liver scaffolds. Overall, our novel bioreactor is capable of supporting cell survival and metabolism and is suitable for liver tissue engineering for the development of 3D liver disease models.

Abstract 308: Chronic Aspirin Treatment Possibly Poses a Risk to Ischemic Heart via Impairment of Cardiac Proteasome Functions

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Chunjiang Tan ◽  
Wenlie Chen ◽  
Yanbing Wu ◽  
Jiumao Lin ◽  
Ruhui Lin ◽  
...  
Keyword(s):  
Cardiac Function ◽  
Cell Viability ◽  
Cultured Cells ◽  
Ventricular Myocytes ◽  
Cell Model ◽  
Selective Inhibition ◽  
Ischemic Heart ◽  
Saline Control ◽  
Normal Male

Background: Impaired cardiac proteasome (CP) has been reported in ischemia and heart failure. Recent data highlighted aspirin as an inhibitor of the ubiquitin-proteasome system, however, it’s unclear whether it affects CP functions. Objective: We investigated the influence of aspirin on CP in the rat model of myocardial infarction (MI). Methods and Results: MI, sham or normal male SD rats were injected intraperitoneally with high (300mg/kg), low (5mg/kg) aspirin or saline (control) once a day for seven weeks. Parallel experiments were performed in the hypoxia/reoxygenated cell model of primary human ventricular myocytes incubated with different concentrations of aspirin. Myocardial hypertrophy, cardiac function, cell viability and the functions of 26S, 20S and 19S, including the β1, β2 and β5 subunits of 20S were determined. The activity of 26S, 20S and 19S declined by about 30%, and β5, β2 and β1 by 40%, 20% and 30%, respectively, in the MI rats compared with the non-MI rats ( P <0.05). Compared with the saline-treated MI rats, 26S and 20S in high or low dose aspirin-treated MI rats further decreased by 30% and 20%, and β5 further decreased by 30% and 12%, and β1 by 40% and 30%, respectively, and their lost activity was correlated with compromised cardiac function in the MI rats. The dose-related and selective inhibition of β5 and β1 by aspirin was comparable to their protein expressions in the MI rats and in the cultured cells. However, the CP in the normal settings, or 19S and β2 in all the groups of animals or cultured cells were less affected by aspirin treatment. The impaired CP, dose-dependently enhanced by aspirin, led to the elevation of oxidative and ubiquitinated proteins in the MI rat hearts or in the hypoxia/reoxygenated cells. These aberrant proteins in turn constituted the ultimate causal factor for the cardiac dysfunction and the loss of cell viability. In vitro experiments confirmed that the purified CP from the MI rats or hypoxic cells, but was more susceptible to aspirin treatment, whereas the complexes in the normal settings appeared less affected by aspirin. Conclusions: Chronic aspirin treatment, via the dose-dependent and selective inhibition of CP, enhanced the ischemic CP dysfunction, which constitutes a potential risk factor for the ischemic heart.

scholarly journals A microfluidic-based cell encapsulation platform to achieve high long-term cell viability in photopolymerized PEGNB hydrogel microspheres

2017 ◽  
Vol 5 (1) ◽  
pp. 173-180 ◽  
Author(s):  
Zhongliang Jiang ◽  
Bingzhao Xia ◽  
Ralph McBride ◽  
John Oakey
Keyword(s):  
Tissue Engineering ◽  
Polyethylene Glycol ◽  
Regenerative Medicine ◽  
Cell Viability ◽  
Cell Encapsulation ◽  
Cell Delivery ◽  
Cellular Behavior ◽  
Hydrogel Scaffolds

Cell encapsulation within photopolymerized polyethylene glycol (PEG)-based hydrogel scaffolds has been demonstrated as a robust strategy for cell delivery, tissue engineering, regenerative medicine, and developing in vitro platforms to study cellular behavior and fate.

Abstract 259: KChIP2 is a Key Transcriptional Regulator of Cardiac Excitability Under Normal and Pathogenic Conditions

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Drew M Nassal ◽  
Xiaoping Wan ◽  
Haiyan Liu ◽  
Danielle Maleski ◽  
Angelina Ramirez-Navarro ◽  
...  
Keyword(s):  
Heart Disease ◽  
Ventricular Myocytes ◽  
Direct Interaction ◽  
Neonatal Rat ◽  
K Channel ◽  
Early Event ◽  
Interacting Protein ◽  
Protein Levels ◽  
Cardiac Excitability

Introduction: Arrhythmogenesis is the primary cause of death in patients with acquired heart disease, and is the consequence of profound dysregulation of both depolarizing and repolarizing currents. Reduction in expression of the auxiliary subunit K+ channel interacting protein 2 (KChIP2), which regulates the transient outward K+ current (Ito), is a common and early event in many cardiac pathologies. Notably, transcriptional changes observed in heart disease can be elicited through direct KChIP2 silencing without disease signaling, suggesting novel transcriptional capacity for KChIP2. Methods and Results: A miRNA microarray was conducted on neonatal rat ventricular myocytes (NRVM) following in vitro silencing of KChIP2, identifying the miR-34 family as a potential transcriptional target of KChIP2. qPCR confirmed reduction in miR-34b/c when over-expressing KChIP2 and increase following silencing. Luciferase assays conducted on the promoter for miR-34b/c reinforced KChIP2 repression on the promoter, while chromatin immunoprecipitation identified direct interaction of KChIP2 on the promoter. Previous studies show modified expression of KChIP2 can lead to changes in several ion channel subunits. Therefore, we investigated if this was the consequence of KChIP2 regulation via miR-34. Expression of miR-34b/c precursors reduced transcript levels of Nav1.5 and Navβ1, and reduced protein levels for Kv4.3, resulting in loss of INa and Ito. To determine the relevance in disease signaling, NRVMs were exposed to 100 μM phenylephrine for 48 hrs, significantly reducing KChIP2, Nav1.5, Navβ1, and Kv4.3, while elevating miR-34b/c. Maintaining KChIP2 expression by adenovirus or blocking miR-34 activity with antagomirs rescued the changes in channel expression. Consequently, miR-34 inhibition rescued the induction of sustained arrhythmias observed in a 2D culture of myocytes through the maintenance of cardiac excitability. Conclusion: Collectively, these observations identify dysregulation of the KChIP2/miR-34 axis as a nodal event in the development of electrical dysfunction that characterize cardiac pathologies, and further identifies miR-34 as a viable therapeutic target for managing arrhythmogenesis in patients with heart disease.

Abstract 631 ADAM12 Is An Important Regulator Of Myocyte Apoptosis Induced By β-adrenergic Receptor Stimulation

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Hang Xi ◽  
Khadija Rafiq ◽  
Marie Hanscom ◽  
Rachid Seqqat ◽  
Nikolay L Malinin ◽  
...  
Keyword(s):  
Ventricular Myocytes ◽  
Left Ventricular ◽  
Surface Proteins ◽  
Neonatal Rat ◽  
Human Heart Failure ◽  
Activation Mechanisms ◽  
Myocyte Apoptosis ◽  
Protease Deficient

ADAM (A Disintegrin And Metalloprotease)12 is a member of a family of cell surface proteins with protease and cell-binding activities. Recent work showed ADAM12 up-regulation in human heart failure. However, the activation mechanisms of ADAM12 in the heart are obscure. We hypothesized that β-adrenergic receptors (AR) stimulation regulates ADAM12 activation in neonatal rat ventricular myocytes (NRVMs) in-vitro and after injection of isoproterenol (ISO) in-vivo. Wistar rats received a single injection of ISO (5 mg/kg) and were sacrificed 6, 24 and 72 hrs later. In comparison with controls, left ventricular function was impaired in rats 24 hrs after ISO injection and started to improve at 72 hrs. The fraction of myocytes undergoing apoptosis peaked 24 hrs after ISO injection and declined thereafter. ADAM12 protein was reduced in hearts from ISO treated animals at 6 hrs, pointing to a possible increase in ADAM12 proteolytic activity. However, both ADAM12 expression and activation were significantly up-regulated at 24 and 72 hrs after ISO injection. We therefore assessed whether ADAM12 activation was involved in myocyte apoptosis secondary to excess exposure of catecholamine. Acute stimulation with ISO (10 μM, 30 min to 3 hrs) induced accumulation of ADAM12 N-terminal cleavage product in conditioned medium, demonstrating activation of the ADAM metalloprotease activity. However, chronic stimulation with ISO for 24 hrs and 48 hrs significantly increased both ADAM12 expression and secretion. This ISO-induced ADAM12 expression/activation was mediated through β 1 -AR stimulation and was dependent on intracellular calcium elevation and protein kinase C activation. Adenoviral expression of an ADAM12 protease-deficient mutant (ADAM12DeltaMP) blocks β-AR-induced myocyte apoptosis, while transduction of NRVMs with adenovirus harboring ADAM12 significantly increased myocyte apoptosis. These data suggest that ADAM12 is a regulator of myocyte apoptosis induced by β-AR in NRVMs and may play an important autocrine role in mediating the effects of β-AR on myocardial remodeling.

Abstract 356: KChIP2 Mediates Ion Channel Dysregulation by Novel Transcriptional Regulation of miR-34s

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Drew Nassal ◽  
Haiyan Liu ◽  
Xiaoping Wan ◽  
Angelina Ramirez-Navarro ◽  
Eckhard Ficker ◽  
...  
Keyword(s):  
Heart Failure ◽  
Ion Channel ◽  
Transcriptional Control ◽  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Physical Interaction ◽  
Protein Patterns ◽  
Protein Levels ◽  
Disease States

Introduction: Cardiac ion channel dysregulation is a hallmark of heart failure. Consistently, the disease yields dramatic decline in Ito through loss in Kv4 and its auxiliary partner KChIP2. Notably, transcriptional changes in heart failure can be elicited through KChIP2 silencing without disease signaling, suggesting potential transcriptional capacity for KChIP2. Further, disparity between resulting transcript and protein patterns suggests a mechanism compatible with modified miRNA activity. Considering other members of the KChIP family behave as transcriptional repressors, we hypothesize that KChIP2 regulates discrete miRNAs which in turn regulate cardiac excitability. Methods and Results: A miRNA microarray was conducted on neonatal rat ventricular myocytes (NRVM) following in vitro silencing of KChIP2 by siRNA, identifying the miR-34 family as potential transcriptional targets of KChIP2. Regulation, confirmed by quantitative PCR, showed reduction in miR-34a/b/c when over-expressing KChIP2 and increase following silencing. Luciferase assays were performed on the cloned promoter for miR-34b/c which reinforced direct KChIP2 repression on the miR-34b/c promoter. Furthermore, chromatin immunoprecipitation followed by PCR identified physical interaction of KChIP2 to the promoter site. Previous studies show modified expression of KChIP2 can lead to changes in several ion channel subunits. Therefore, we investigated if this was the consequence of KChIP2 regulation via miR-34. miR-34a/b/c precursors were expressed in NRVM which reduced transcript levels of Nav1.5 and Navβ1, and reduced protein levels for Kv4.3. Reflecting these changes, peak INa was reduced following miR precursor treatment. NRVMs were exposed to 100 μM phenylephrine for 48 hrs, significantly reducing KChIP2, Nav1.5, Navβ1, and Kv4.3, while elevating miR-34b/c. Returning KChIP2 expression by adenovirus normalized these changes back towards baseline, implicating the physiologic relevance of this pathway. Conclusion: These observations describe a novel mechanism where KChIP2 regulates a host of cardiac genes through transcriptional control of miRNAs, potentially explaining electrical remodeling observed in disease states where KChIP2 is reduced.

scholarly journals Cardiomyocyte functional screening: interrogating comparative electrophysiology of high-throughput model cell systems

2019 ◽  
Vol 317 (6) ◽  
pp. C1256-C1267 ◽  
Author(s):  
Simon P. Wells ◽  
Helen M. Waddell ◽  
Choon Boon Sim ◽  
Shiang Y. Lim ◽  
Gabriel B. Bernasochi ◽  
...  
Keyword(s):  
Conduction Velocity ◽  
High Throughput ◽  
Microelectrode Array ◽  
Ventricular Myocytes ◽  
Field Potential ◽  
Induced Pluripotent Stem Cell ◽  
Neonatal Rat ◽  
Functional Screening ◽  
Electrophysiological Properties

Cardiac arrhythmias of both atrial and ventricular origin are an important feature of cardiovascular disease. Novel antiarrhythmic therapies are required to overcome current drug limitations related to effectiveness and pro-arrhythmia risk in some contexts. Cardiomyocyte culture models provide a high-throughput platform for screening antiarrhythmic compounds, but comparative information about electrophysiological properties of commonly used types of cardiomyocyte preparations is lacking. Standardization of cultured cardiomyocyte microelectrode array (MEA) experimentation is required for its application as a high-throughput platform for antiarrhythmic drug development. The aim of this study was to directly compare the electrophysiological properties and responses to isoproterenol of three commonly used cardiac cultures. Neonatal rat ventricular myocytes (NRVMs), immortalized atrial HL-1 cells, and custom-generated human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were cultured on microelectrode arrays for 48–120 h. Extracellular field potentials were recorded, and conduction velocity was mapped in the presence/absence of the β-adrenoceptor agonist isoproterenol (1 µM). Field potential amplitude and conduction velocity were greatest in NRVMs and did not differ in cardiomyocytes isolated from male/female hearts. Both NRVMs and hiPSC-CMs exhibited longer field potential durations with rate dependence and were responsive to isoproterenol. In contrast, HL-1 cells exhibited slower conduction and shorter field potential durations and did not respond to 1 µM isoproterenol. This is the first study to compare the intrinsic electrophysiologic properties of cultured cardiomyocyte preparations commonly used for in vitro electrophysiology assessment. These findings offer important comparative data to inform methodological approaches in the use of MEA and other techniques relating to cardiomyocyte functional screening investigations of particular relevance to arrhythmogenesis.

scholarly journals Comparison of the Effect of Sol-Gel and Coprecipitation Routes on the Properties and Behavior of Nanocomposite Chitosan-Bioactive Glass Membranes for Bone Tissue Engineering

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Elke M. F. Lemos ◽  
Sandhra M. Carvalho ◽  
Patrícia S. O. Patrício ◽  
Claudio L. Donnici ◽  
Marivalda M. Pereira
Keyword(s):  
Tissue Engineering ◽  
Cell Viability ◽  
Bioactive Glass ◽  
Bone Regeneration ◽  
Sol Gel ◽  
Composite Films ◽  
Maximum Tensile Stress ◽  
Control Group ◽  
Nanoparticle Dispersion

Recent studies in tissue engineering have highlighted the importance of the development of composite materials based on biodegradable polymers containing bioactive glasses, in particular, composites for high load support and excellent cell viability for potential application in bone regeneration. In this work, hybrid composite films were obtained by combining chitosan with bioactive glass in solution form and in nanoparticle dispersion form obtained by the two different synthesis routes: the sol-gel method and coprecipitation. The bioactive glass served both as a mechanical reinforcing agent and as a triggering agent with high bioactivity. The results ofin vitroassays with simulated body fluid demonstrated the formation of a significant layer of fibrils on the surface of the film, with a typical morphology of carbonated hydroxyapatite, reflecting induction of a favorable bioactivity. Maximum tensile stress increased from 42 to 80 MPa to the sample with 5% wt bioactive glass. In addition, samples containing 5% and 10% wt bioactive glass showed a significant increase in cell viability, 18 and 30% increase compared to the control group. The samples showed significant response, indicating that they could be a potential material for use in bone regeneration through tissue engineering.

scholarly journals 3D Hepatic Organoid-Based Advancements in LIVER Tissue Engineering

2021 ◽  
Vol 8 (11) ◽  
pp. 185
Author(s):  
Amit Panwar ◽  
Prativa Das ◽  
Lay Poh Tan
Keyword(s):  
Tissue Engineering ◽  
Self Assembly ◽  
Liver Tissue ◽  
3D Culture ◽  
Culture Method ◽  
Culture Conditions ◽  
Phenotypic Properties ◽  
Liver Tissue Engineering

Liver-associated diseases and tissue engineering approaches based on in vitro culture of functional Primary human hepatocytes (PHH) had been restricted by the rapid de-differentiation in 2D culture conditions which restricted their usability. It was proven that cells growing in 3D format can better mimic the in vivo microenvironment, and thus help in maintaining metabolic activity, phenotypic properties, and longevity of the in vitro cultures. Again, the culture method and type of cell population are also recognized as important parameters for functional maintenance of primary hepatocytes. Hepatic organoids formed by self-assembly of hepatic cells are microtissues, and were able to show long-term in vitro maintenance of hepato-specific characteristics. Thus, hepatic organoids were recognized as an effective tool for screening potential cures and modeling liver diseases effectively. The current review summarizes the importance of 3D hepatic organoid culture over other conventional 2D and 3D culture models and its applicability in Liver tissue engineering.

scholarly journals Electrically stimulable indium tin oxide plate for long-term in vitro cardiomyocyte culture

2020 ◽  
Author(s):  
Sung-Hwan Moon ◽  
Young-Woo Cho ◽  
Hye-Eun Shim ◽  
Jae-Hak Choi ◽  
Chan-Hee Jung ◽  
...  
Keyword(s):  
Electrical Stimulation ◽  
Tin Oxide ◽  
Indium Tin Oxide ◽  
Ventricular Myocytes ◽  
Culture Conditions ◽  
Neonatal Rat ◽  
Control Group ◽  
Supporting Evidence ◽  
Standard Culture

Abstract Background: We investigated whether electrical stimulation via indium tin oxide (ITO) could enhance the in vitro culture of neonatal rat ventricular myocytes (NRVMs), which are important in vitro models for studying the mechanisms underlying many aspects of cardiology.Methods: Cardiomyocytes were obtained from 1-day-old neonatal rat heart ventricles. To evaluate function of NRVMs cultured on ITO with electrical stimulation, the cell viability, change of cell morphology, immunochemistry using cardiac-specific antibodies, and gene expression were tested.Results: Defined sarcomeric structure, cell enlargement, and increased distribution of NRVMs appeared in the presence of electrical stimulation. These characteristics were absent in NRVMs cultured under standard culture conditions. In addition, the expression levels of cardiomyocyte-specific and ion channel markers were higher in NRVMs seeded on ITO-coated dishes than in the control group at 14 days after seeding. ITO-coated dishes could effectively provide electrical cues to support the in vitro culture of NRVMs.Conclusions: These results provide supporting evidence that electrical stimulation via ITO can be effectively used to maintain culture and enhance function of cardiomyocytes in vitro.

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