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Cardiovascular Research Mar 2021Succinate accumulates several-fold in the ischaemic heart and is then rapidly oxidized upon reperfusion, contributing to reactive oxygen species production by...
AIMS
Succinate accumulates several-fold in the ischaemic heart and is then rapidly oxidized upon reperfusion, contributing to reactive oxygen species production by mitochondria. In addition, a significant amount of the accumulated succinate is released from the heart into the circulation at reperfusion, potentially activating the G-protein-coupled succinate receptor (SUCNR1). However, the factors that determine the proportion of succinate oxidation or release, and the mechanism of this release, are not known.
METHODS AND RESULTS
To address these questions, we assessed the fate of accumulated succinate upon reperfusion of anoxic cardiomyocytes, and of the ischaemic heart both ex vivo and in vivo. The release of accumulated succinate was selective and was enhanced by acidification of the intracellular milieu. Furthermore, pharmacological inhibition, or haploinsufficiency of the monocarboxylate transporter 1 (MCT1) significantly decreased succinate efflux from the reperfused heart.
CONCLUSION
Succinate release upon reperfusion of the ischaemic heart is mediated by MCT1 and is facilitated by the acidification of the myocardium during ischaemia. These findings will allow the signalling interaction between succinate released from reperfused ischaemic myocardium and SUCNR1 to be explored.
Topics: Animals; Cells, Cultured; Disease Models, Animal; Female; Isolated Heart Preparation; Male; Metabolome; Mice, Inbred C57BL; Mice, Knockout; Mitochondria, Heart; Monocarboxylic Acid Transporters; Myocardial Infarction; Myocardial Reperfusion; Myocardial Reperfusion Injury; Myocytes, Cardiac; Oxidation-Reduction; Rats; Reactive Oxygen Species; Receptors, G-Protein-Coupled; Succinic Acid; Sus scrofa; Symporters; Time Factors; Mice
PubMed: 32766828
DOI: 10.1093/cvr/cvaa148 -
BMC Biology Nov 2023Cerebral organoids are three-dimensional in vitro cultured brains that mimic the function and structure of the human brain. One of the major challenges for cerebral...
BACKGROUND
Cerebral organoids are three-dimensional in vitro cultured brains that mimic the function and structure of the human brain. One of the major challenges for cerebral organoids is the lack of functional vasculature. Without perfusable vessels, oxygen and nutrient supplies may be insufficient for long-term culture, hindering the investigation of the neurovascular interactions. Recently, several strategies for the vascularization of human cerebral organoids have been reported. However, the generalizable trends and variability among different strategies are unclear due to the lack of a comprehensive characterization and comparison of these vascularization strategies. In this study, we aimed to explore the effect of different vascularization strategies on the nervous system and vasculature in human cerebral organoids.
RESULTS
We integrated single-cell RNA sequencing data of multiple vascularized and vascular organoids and fetal brains from publicly available datasets and assessed the protocol-dependent and culture-day-dependent effects on the cell composition and transcriptomic profiles in neuronal and vascular cells. We revealed the similarities and uniqueness of multiple vascularization strategies and demonstrated the transcriptomic effects of vascular induction on neuronal and mesodermal-like cell populations. Moreover, our data suggested that the interaction between neurons and mesodermal-like cell populations is important for the cerebrovascular-specific profile of endothelial-like cells.
CONCLUSIONS
This study highlights the current challenges to vascularization strategies in human cerebral organoids and offers a benchmark for the future fabrication of vascularized organoids.
Topics: Humans; Single-Cell Gene Expression Analysis; Organoids; Endothelial Cells; Brain
PubMed: 37940920
DOI: 10.1186/s12915-023-01711-1 -
Cell Reports Methods Sep 2022In this study, we report static and perfused models of human myocardial-microvascular interaction. In static culture, we observe distinct regulation of electrophysiology...
In this study, we report static and perfused models of human myocardial-microvascular interaction. In static culture, we observe distinct regulation of electrophysiology of human induced pluripotent stem cell derived-cardiomyocytes (hiPSC-CMs) in co-culture with human cardiac microvascular endothelial cells (hCMVECs) and human left ventricular fibroblasts (hLVFBs), including modification of beating rate, action potential, calcium handling, and pro-arrhythmic substrate. Within a heart-on-a-chip model, we subject this three-dimensional (3D) co-culture to microfluidic perfusion and vasculogenic growth factors to induce spontaneous assembly of perfusable myocardial microvasculature. Live imaging of red blood cells within myocardial microvasculature reveals pulsatile flow generated by beating hiPSC-CMs. This study therefore demonstrates a functionally vascularized model of human myocardium with widespread potential applications in basic and translational research.
Topics: Humans; Endothelial Cells; Induced Pluripotent Stem Cells; Myocardium; Myocytes, Cardiac; Coculture Techniques
PubMed: 36160044
DOI: 10.1016/j.crmeth.2022.100280 -
Cancers Jun 2021Conventional static culture fails to replicate the physiological conditions that exist in vivo. Recent advances in biomedical engineering have resulted in the creation... (Review)
Review
Conventional static culture fails to replicate the physiological conditions that exist in vivo. Recent advances in biomedical engineering have resulted in the creation of novel dynamic culturing systems that permit the recapitulation of normal physiological processes ex vivo. Whilst the physiological benefit for its use in the culture of two-dimensional cellular monolayer has been validated, its role in the context of primary human tissue culture has yet to be determined. This systematic review identified 22 articles that combined dynamic physiological culture techniques with primary human tissue culture. The most frequent method described (55%) utilised dynamic perfusion culture. A diverse range of primary human tissue was successfully cultured. The median duration of successful ex vivo culture of primary human tissue for all articles was eight days; however, a wide range was noted (5 h-60 days). Six articles (27%) reported successful culture of primary human tissue for greater than 20 days. This review illustrates the physiological benefit of combining dynamic culture with primary human tissue culture in both long-term culture success rates and preservation of native functionality of the tissue ex vivo. Further research efforts should focus on developing precise biochemical sensors that would allow for real-time monitoring and automated self-regulation of the culture system in order to maintain homeostasis. Combining these techniques allows the creation of an accurate system that can be used to gain a greater understanding of human physiology.
PubMed: 34201273
DOI: 10.3390/cancers13122870 -
American Journal of Physiology.... Sep 2022Physiologically relevant and broadly applicable liver cell culture platforms are of great importance in both drug development and disease modeling. Organ-on-a-chip... (Review)
Review
Physiologically relevant and broadly applicable liver cell culture platforms are of great importance in both drug development and disease modeling. Organ-on-a-chip systems offer a promising alternative to conventional, static two-dimensional (2-D) cultures, providing much-needed cues such as perfusion, shear stress, and three-dimensional (3-D) cell-cell communication. However, such devices cover a broad range of complexity both in manufacture and in implementation. In this review, we summarize the key features of the human liver that should be reflected in a physiologically relevant liver-on-a-chip model. We also discuss different material properties of importance in producing liver-on-a-chip devices and summarize recent and current progress in the field, highlighting different types of devices at different levels of complexity.
Topics: Cell Communication; Drug Development; Humans; Lab-On-A-Chip Devices; Liver
PubMed: 35819853
DOI: 10.1152/ajpgi.00346.2021 -
Bioengineering (Basel, Switzerland) Jan 2023Hepatocytes are differentiated cells that account for 80% of the hepatic volume and perform all major functions of the liver. In vivo, after an acute insult, adult... (Review)
Review
Hepatocytes are differentiated cells that account for 80% of the hepatic volume and perform all major functions of the liver. In vivo, after an acute insult, adult hepatocytes retain their ability to proliferate and participate in liver regeneration. However, in vitro, prolonged culture and proliferation of viable and functional primary hepatocytes have remained the major and the most challenging goal of hepatocyte-based cell therapies and liver tissue engineering. The first functional cultures of rat primary hepatocytes between two layers of collagen gel, also termed as the "sandwich cultures", were reported in 1989. Since this study, several technical developments including choice of hydrogels, type of microenvironment, growth factors and culture conditions, mono or co-cultures of hepatocytes along with other supporting cell types have evolved for both rat and human primary hepatocytes in recent years. All these improvements have led to a substantial improvement in the number, life-span and hepatic functions of these cells in vitro for several downstream applications. In the current review, we highlight the details, limitations and prospects of different technical strategies being used in primary hepatocyte cultures. We discuss the use of newer biomaterials as scaffolds for efficient culture of primary hepatocytes. We also describe the derivation of mature hepatocytes from other cellular sources such as induced pluripotent stem cells, bone marrow stem cells and 3D liver organoids. Finally, we also explain the use of perfusion-based bioreactor systems and bioengineering strategies to support the long-term function of hepatocytes in 3D conditions.
PubMed: 36829625
DOI: 10.3390/bioengineering10020131 -
Cell Reports Methods Jun 2024Organoids, self-organizing three-dimensional (3D) structures derived from stem cells, offer unique advantages for studying organ development, modeling diseases, and... (Review)
Review
Organoids, self-organizing three-dimensional (3D) structures derived from stem cells, offer unique advantages for studying organ development, modeling diseases, and screening potential therapeutics. However, their translational potential and ability to mimic complex in vivo functions are often hindered by the lack of an integrated vascular network. To address this critical limitation, bioengineering strategies are rapidly advancing to enable efficient vascularization of organoids. These methods encompass co-culturing organoids with various vascular cell types, co-culturing lineage-specific organoids with vascular organoids, co-differentiating stem cells into organ-specific and vascular lineages, using organoid-on-a-chip technology to integrate perfusable vasculature within organoids, and using 3D bioprinting to also create perfusable organoids. This review explores the field of organoid vascularization, examining the biological principles that inform bioengineering approaches. Additionally, this review envisions how the converging disciplines of stem cell biology, biomaterials, and advanced fabrication technologies will propel the creation of increasingly sophisticated organoid models, ultimately accelerating biomedical discoveries and innovations.
Topics: Organoids; Humans; Bioengineering; Animals; Tissue Engineering; Neovascularization, Physiologic; Bioprinting; Coculture Techniques; Stem Cells; Printing, Three-Dimensional; Cell Differentiation
PubMed: 38759654
DOI: 10.1016/j.crmeth.2024.100779 -
Nature Communications Jan 2023The vascularization of engineered tissues and organoids has remained a major unresolved challenge in regenerative medicine. While multiple approaches have been developed...
The vascularization of engineered tissues and organoids has remained a major unresolved challenge in regenerative medicine. While multiple approaches have been developed to vascularize in vitro tissues, it has thus far not been possible to generate sufficiently dense networks of small-scale vessels to perfuse large de novo tissues. Here, we achieve the perfusion of multi-mm tissue constructs by generating networks of synthetic capillary-scale 3D vessels. Our 3D soft microfluidic strategy is uniquely enabled by a 3D-printable 2-photon-polymerizable hydrogel formulation, which allows for precise microvessel printing at scales below the diffusion limit of living tissues. We demonstrate that these large-scale engineered tissues are viable, proliferative and exhibit complex morphogenesis during long-term in-vitro culture, while avoiding hypoxia and necrosis. We show by scRNAseq and immunohistochemistry that neural differentiation is significantly accelerated in perfused neural constructs. Additionally, we illustrate the versatility of this platform by demonstrating long-term perfusion of developing neural and liver tissue. This fully synthetic vascularization platform opens the door to the generation of human tissue models at unprecedented scale and complexity.
Topics: Humans; Microfluidics; Tissue Engineering; Organoids; Hydrogels; Liver; Neovascularization, Pathologic; Printing, Three-Dimensional; Tissue Scaffolds
PubMed: 36635264
DOI: 10.1038/s41467-022-35619-1 -
Perfuse and Reuse: A Low-Cost Three-Dimensional-Printed Perfusion Bioreactor for Tissue Engineering.Tissue Engineering. Part C, Methods Nov 2022This article describes fabrication of a customizable bioreactor, which comprises a perfusion system and coverslip-based tissue culture chamber that allow...
This article describes fabrication of a customizable bioreactor, which comprises a perfusion system and coverslip-based tissue culture chamber that allow centimeter-scale vascularized or otherwise canalized tissue constructs to be maintained in weeks long static and/or perfusion culture at an exceptionally low cost, with intermittent live imaging and media sampling capabilities. The perfusion system includes a reusable polydimethylsiloxane (PDMS) lid generated from a three-dimensional (3D)-printed poly-lactic acid (PLA) mold and several lengths of perfusion tubing. The coverslip tissue culture chamber includes PDMS components built with 3D-printed PLA molds, as well as 3D-printed PLA frames and glass coverslips that house perfusable hydrogel constructs. As proof of concept, we fabricated a vascularized hydrogel construct, which was subjected to static and perfusion tissue culture, as well as flow studies using fluorescent beads and widefield fluorescent microscopy. This system can be readily reproduced, promoting the advancement of tissue engineering and regenerative medicine research.
Topics: Tissue Engineering; Perfusion; Bioreactors; Hydrogels; Polyesters; Printing, Three-Dimensional; Tissue Scaffolds
PubMed: 36094108
DOI: 10.1089/ten.TEC.2022.0139 -
Acta Biomaterialia Jul 2023Pathologies associated with uteroplacental hypoxia, such as preeclampsia are among the leading causes of maternal and perinatal morbidity in the world. Its fundamental...
Pathologies associated with uteroplacental hypoxia, such as preeclampsia are among the leading causes of maternal and perinatal morbidity in the world. Its fundamental mechanisms are yet poorly understood due to a lack of good experimental models. Here we report an in vitro model of the placental barrier, based on co-culture of trophoblasts and endothelial cells against a collagen extracellular matrix in a microfluidic platform. The model yields a functional syncytium with barrier properties, polarization, secretion of relevant extracellular membrane components, thinning of the materno-fetal space, hormone secretion, and transporter function. The model is exposed to low oxygen conditions and perfusion flow is modulated to induce a pathological environment. This results in reduced barrier function, hormone secretion, and microvilli as well as an increased nuclei count, characteristics of preeclamptic placentas. The model is implemented in a titer plate-based microfluidic platform fully amenable to high-throughput screening. We thus believe this model could aid mechanistic understanding of preeclampsia and other placental pathologies associated with hypoxia/ischemia, as well as support future development of effective therapies through target and compound screening campaigns. STATEMENT OF SIGNIFICANCE: The human placenta is a unique organ sustaining fetal growth but is also the source of severe pathologies, such as preeclampsia. Though leading cause of perinatal mortality in the world, preeclampsia remains untreatable due to a lack of relevant in vitro placenta models. To better understand the pathology, we have developed 3D placental barrier models in a microfluidic device. The platform allows parallel culture of 40 perfused physiological miniaturized placental barriers, comprising a differentiated syncytium and endothelium that have been validated for transporter functions. Exposure to a hypoxic and ischemic environment enabled the mimicking of preeclamptic characteristics in high-throughput, which we believe could lead to a better understanding of the pathology as well as support future effective therapies development.
Topics: Pregnancy; Female; Humans; Placenta; Pre-Eclampsia; Endothelial Cells; Hypoxia; Ischemia; Lab-On-A-Chip Devices; Hormones
PubMed: 37116636
DOI: 10.1016/j.actbio.2023.04.033