-
Military Medical Research May 2022Cerebral ischemia-reperfusion injury (CIRI) refers to a secondary brain injury that can occur when the blood supply to the ischemic brain tissue is restored. However,...
BACKGROUND
Cerebral ischemia-reperfusion injury (CIRI) refers to a secondary brain injury that can occur when the blood supply to the ischemic brain tissue is restored. However, the mechanism underlying such injury remains elusive.
METHODS
The 150 male C57 mice underwent middle cerebral artery occlusion (MCAO) for 1 h and reperfusion for 24 h, Among them, 50 MCAO mice were further treated with Mitochondrial division inhibitor 1 (Mdivi-1) and 50 MCAO mice were further treated with N-acetylcysteine (NAC). SH-SY5Y cells were cultured in a low-glucose culture medium for 4 h under hypoxic conditions and then transferred to normal conditions for 12 h. Then, cerebral blood flow, mitochondrial structure, mitochondrial DNA (mtDNA) copy number, intracellular and mitochondrial reactive oxygen species (ROS), autophagic flux, aggresome and exosome expression profiles, cardiac tissue structure, mitochondrial length and cristae density, mtDNA and ROS content, as well as the expression of Drp1-Ser616/Drp1, RIP1/RIP3, LC3 II/LC3 I, TNF-α, IL-1β, etc., were detected under normal or Drp1 interference conditions.
RESULTS
The mtDNA content, ROS levels, and Drp1-Ser616/Drp1 were elevated by 2.2, 1.7 and 2.7 times after CIRI (P < 0.05). However, the high cytoplasmic LC3 II/I ratio and increased aggregation of p62 could be reversed by 44% and 88% by Drp1 short hairpin RNA (shRNA) (P < 0.05). The low fluorescence intensity of autophagic flux and the increased phosphorylation of RIP3 induced by CIRI could be attenuated by ROS scavenger, NAC (P < 0.05). RIP1/RIP3 inhibitor Necrostatin-1 (Nec-1) restored 75% to a low LC3 II/LC3 I ratio and enhanced 2 times to a high RFP-LC3 after Drp1 activation (P < 0.05). In addition, although CIRI-induced ROS production caused no considerable accumulation of autophagosomes (P > 0.05), it increased the packaging and extracellular secretion of exosomes containing p62 by 4 - 5 times, which could be decreased by Mdivi-1, Drp1 shRNA, and Nec-1 (P < 0.05). Furthermore, TNF-α and IL-1β increased in CIRI-derived exosomes could increase RIP3 phosphorylation in normal or oxygen-glucose deprivation/reoxygenation (OGD/R) conditions (P < 0.05).
CONCLUSIONS
CIRI activated Drp1 and accelerated the p62-mediated formation of autophagosomes while inhibiting the transition of autophagosomes to autolysosomes via the RIP1/RIP3 pathway activation. Undegraded autophagosomes were secreted extracellularly in the form of exosomes, leading to inflammatory cascades that further damaged mitochondria, resulting in excessive ROS generation and the blockage of autophagosome degradation, triggering a vicious cycle.
Topics: Animals; Brain Ischemia; Cerebral Infarction; DNA, Mitochondrial; Exosomes; Glucose; Humans; Inflammation; Male; Mice; RNA, Small Interfering; Reactive Oxygen Species; Reperfusion; Reperfusion Injury; Tumor Necrosis Factor-alpha
PubMed: 35624495
DOI: 10.1186/s40779-022-00383-2 -
STAR Protocols Sep 2020Primary hepatocytes are a vital tool in various biomedical research disciplines, serving as an model for liver physiology. Obtaining high yields of viable primary mouse...
Primary hepatocytes are a vital tool in various biomedical research disciplines, serving as an model for liver physiology. Obtaining high yields of viable primary mouse hepatocytes is technically challenging, limiting their use. Here, we present an improved protocol based on the classic two-step collagenase perfusion technique. The liver is washed by perfusion, hepatocytes are dissociated by collagenase, separated from other cells, and cultured. This protocol was optimized to significantly reduce procedure duration and improve hepatocyte yield and viability.
Topics: Animals; Cell Separation; Cells, Cultured; Collagenases; Hepatocytes; Liver; Mice; Mice, Inbred C57BL; Perfusion; Primary Cell Culture
PubMed: 33111119
DOI: 10.1016/j.xpro.2020.100086 -
Nature Methods Mar 2019Kidney organoids derived from human pluripotent stem cells have glomerular- and tubular-like compartments that are largely avascular and immature in static culture. Here...
Kidney organoids derived from human pluripotent stem cells have glomerular- and tubular-like compartments that are largely avascular and immature in static culture. Here we report an in vitro method for culturing kidney organoids under flow on millifluidic chips, which expands their endogenous pool of endothelial progenitor cells and generates vascular networks with perfusable lumens surrounded by mural cells. We found that vascularized kidney organoids cultured under flow had more mature podocyte and tubular compartments with enhanced cellular polarity and adult gene expression compared with that in static controls. Glomerular vascular development progressed through intermediate stages akin to those involved in the embryonic mammalian kidney's formation of capillary loops abutting foot processes. The association of vessels with these compartments was reduced after disruption of the endogenous VEGF gradient. The ability to induce substantial vascularization and morphological maturation of kidney organoids in vitro under flow opens new avenues for studies of kidney development, disease, and regeneration.
Topics: Cells, Cultured; Fibroblasts; Human Umbilical Vein Endothelial Cells; Humans; In Vitro Techniques; Kidney; Lab-On-A-Chip Devices; Organ Culture Techniques; Organoids; Printing, Three-Dimensional; Tissue Engineering
PubMed: 30742039
DOI: 10.1038/s41592-019-0325-y -
Nature Protocols Feb 2015Hepatic stellate cells (HSCs) have been identified as the main fibrogenic cell type in the liver. Hence, efforts to understand hepatic fibrogenesis and to develop...
Hepatic stellate cells (HSCs) have been identified as the main fibrogenic cell type in the liver. Hence, efforts to understand hepatic fibrogenesis and to develop treatment strategies have focused on this cell type. HSC isolation, originally developed in rats, has subsequently been adapted to mice, thus allowing the study of fibrogenesis by genetic approaches in transgenic mice. However, mouse HSC isolation is commonly hampered by low yield and purity. Here we present an easy-to-perform protocol for high-purity and high-yield isolation of quiescent and activated HSCs in mice, based on retrograde pronase-collagenase perfusion of the liver and subsequent density-gradient centrifugation. We describe an optional add-on protocol for ultrapure HSC isolation from normal and fibrotic livers via subsequent flow cytometric sorting, thus providing a validated method to determine gene expression changes during HSC activation devoid of cell culture artifacts or contamination with other cells. The described isolation procedure takes ∼4 h to complete.
Topics: Animals; Centrifugation, Density Gradient; Collagenases; Cytological Techniques; Flow Cytometry; Hepatic Stellate Cells; Liver; Liver Cirrhosis; Mice, Transgenic; Perfusion
PubMed: 25612230
DOI: 10.1038/nprot.2015.017 -
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 -
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 -
PloS One 2020A lack of perfusion has been one of the most significant obstacles for three-dimensional culture systems of organoids and embryonic tissues. Here, we developed a simple...
A lack of perfusion has been one of the most significant obstacles for three-dimensional culture systems of organoids and embryonic tissues. Here, we developed a simple and reliable method to implement a perfusable capillary network in vitro. The method employed the self-organization of endothelial cells to generate a capillary network and a static pressure difference for culture medium circulation, which can be easily introduced to standard biological laboratories and enables long-term cultivation of vascular structures. Using this culture system, we perfused the lumen of the self-organized capillary network and observed a flow-induced vascular remodeling process, cell shape changes, and collective cell migration. We also observed an increase in cell proliferation around the self-organized vasculature induced by flow, indicating functional perfusion of the culture medium. We also reconstructed extravasation of tumor and inflammatory cells, and circulation inside spheroids including endothelial cells and human lung fibroblasts. In conclusion, this system is a promising tool to elucidate the mechanisms of various biological processes related to vascular flow.
Topics: Animals; Cell Culture Techniques; Cells, Cultured; Fibroblasts; Human Umbilical Vein Endothelial Cells; Humans; Mice; Perfusion; Tissue Engineering
PubMed: 33112918
DOI: 10.1371/journal.pone.0240552 -
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 -
Biotechnology Advances 2018The manufacturing of recombinant protein is traditionally divided in two main steps: upstream (cell culture and synthesis of the target protein) and downstream... (Review)
Review
The manufacturing of recombinant protein is traditionally divided in two main steps: upstream (cell culture and synthesis of the target protein) and downstream (purification and formulation of the protein into a drug substance or drug product). Today, cost pressure, market uncertainty and market growth, challenge the existing manufacturing technologies. Leaders in the field are active in designing the process of the future and continuous manufacturing is recurrently mentioned as a potential solution to address some of the current limitations. This review focuses on the application of continuous processing to the first step of the manufacturing process. Enabling technologies and operation modes are described in the first part. In the second part, recent advances in the field that have the potential to support its successful future development are critically discussed.
Topics: Animals; Batch Cell Culture Techniques; Bioreactors; CHO Cells; Cricetinae; Cricetulus; Perfusion; Recombinant Proteins
PubMed: 29738813
DOI: 10.1016/j.biotechadv.2018.04.011 -
Journal of Visualized Experiments : JoVE Nov 2022The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and molecular...
The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and molecular functional experiments (e.g., contraction, electrophysiology, calcium cycling, etc.) on cardiomyocytes are the gold standard for establishing mechanism(s) of disease. The mouse is the species of choice for functional experiments and the described technique is specifically for the isolation of mouse cardiomyocytes. Previous methods requiring a Langendorff apparatus require high levels of training and precision for aortic cannulation, often resulting in ischemia. The field is shifting toward Langendorff-free isolation methods that are simple, are reproducible, and yield viable myocytes for physiological data acquisition and culture. These methods greatly diminish ischemia time compared to aortic cannulation and result in reliably obtained cardiomyocytes. Our adaptation to the Langendorff-free method includes an initial perfusion with ice-cold clearing solution, use of a stabilizing platform that ensures a steady needle during perfusion, and additional digestion steps to ensure reliably obtained cardiomyocytes for use in functional measurements and culture. This method is simple and quick to perform and requires little technical skill.
Topics: Mice; Animals; Myocytes, Cardiac; Cardiac Electrophysiology; Calcium; Perfusion; Acclimatization
PubMed: 36440883
DOI: 10.3791/63056