scholarly journals A 3D Cell Death Assay to Quantitatively Determine Ferroptosis in Spheroids

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 703 ◽  
Author(s):  
Robin Demuynck ◽  
Iuliia Efimova ◽  
Abraham Lin ◽  
Heidi Declercq ◽  
Dmitri V. Krysko
Keyword(s):  
Cell Death ◽  
Three Dimensional ◽  
Drug Efficacy ◽  
Cost Effective ◽  
3D Cultures ◽  
Cell Death Assay ◽  
Tumour Spheroids ◽  
A Cell ◽  
Triton X

The failure of drug efficacy in clinical trials remains a big issue in cancer research. This is largely due to the limitations of two-dimensional (2D) cell cultures, the most used tool in drug screening. Nowadays, three-dimensional (3D) cultures, including spheroids, are acknowledged to be a better model of the in vivo environment, but detailed cell death assays for 3D cultures (including those for ferroptosis) are scarce. In this work, we show that a new cell death analysis method, named 3D Cell Death Assay (3DELTA), can efficiently determine different cell death types including ferroptosis and quantitatively assess cell death in tumour spheroids. Our method uses Sytox dyes as a cell death marker and Triton X-100, which efficiently permeabilizes all cells in spheroids, was used to establish 100% cell death. After optimization of Sytox concentration, Triton X-100 concentration and timing, we showed that the 3DELTA method was able to detect signals from all cells without the need to disaggregate spheroids. Moreover, in this work we demonstrated that 2D experiments cannot be extrapolated to 3D cultures as 3D cultures are less sensitive to cell death induction. In conclusion, 3DELTA is a more cost-effective way to identify and measure cell death type in 3D cultures, including spheroids.

scholarly journals Cortical Spheroid Model for Studying the Effects of Ischemic Brain Injury

2021 ◽  
Author(s):  
Rachel M McLaughlin ◽  
Amanda Laguna ◽  
Ilayda Top ◽  
Christien Hernadez ◽  
Liane L Livi ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Cost Effective ◽  
Glucose Deprivation ◽  
In Vitro Models ◽  
3D Architecture ◽  
A Cell ◽  
Antioxidant Agent

Stroke is a devastating neurological disorder and a leading cause of death and long-term disability. Despite many decades of research, there are still very few therapeutic options for patients suffering from stroke or its consequences. This is partially due to the limitations of current research models, including traditional in vitro models which lack the three-dimensional (3D) architecture and cellular make-up of the in vivo brain. 3D spheroids derived from primary postnatal rat cortex provide an in vivo-relevant model containing a similar cellular composition to the native cortex and a cell-synthesized extracellular matrix. These spheroids are cost-effective, highly reproducible, and can be produced in a high-throughput manner, making this model an ideal candidate for screening potential therapeutics. To study the cellular and molecular mechanisms of stroke in this model, spheroids were deprived of glucose, oxygen, or both oxygen and glucose for 24 hours. Both oxygen and oxygen-glucose deprived spheroids demonstrated many of the hallmarks of stroke, including a decrease in metabolism, an increase in neural dysfunction, and an increase in reactive astrocytes. Pretreatment of spheroids with the antioxidant agent N-acetylcysteine (NAC) mitigated the decrease in ATP seen after 24 hours of oxygen-glucose deprivation. Together, these results show the utility of our 3D cortical spheroid model for studying ischemic injury and its potential for screening stroke therapeutics.

scholarly journals Pre-Clinical In Vitro Models Used in Cancer Research: Results of a Worldwide Survey

Cancers ◽  
2021 ◽  
Vol 13 (23) ◽  
pp. 6033
Author(s):  
Sarai Martinez-Pacheco ◽  
Lorraine O’Driscoll
Keyword(s):  
Cancer Research ◽  
Three Dimensional ◽  
3D Cell Culture ◽  
Cost Effective ◽  
Cell Types ◽  
3D Models ◽  
In Vitro Models ◽  
3D Cultures

To develop and subsequently get cancer researchers to use organotypic three-dimensional (3D) models that can recapitulate the complexity of human in vivo tumors in an in vitro setting, it is important to establish what in vitro model(s) researchers are currently using and the reasons why. Thus, we developed a survey on this topic, obtained ethics approval, and circulated it throughout the world. The survey was completed by 101 researchers, across all career stages, in academia, clinical or industry settings. It included 40 questions, many with multiple options. Respondents reported on their field of cancer research; type of cancers studied; use of two-dimensional (2D)/monolayer, 2.5D and/or 3D cultures; if using co-cultures, the cell types(s) they co-culture; if using 3D cultures, whether these involve culturing the cells in a particular way to generate spheroids, or if they use additional supports/scaffolds; techniques used to analyze the 2D/2.5D/3D; and their downstream applications. Most researchers (>66%) only use 2D cultures, mainly due to lack of experience and costs. Despite most cancer researchers currently not using the 3D format, >80% recognize their importance and would like to progress to using 3D models. This suggests an urgent need to standardize reliable, robust, reproducible methods for establishing cost-effective 3D cell culture models and their subsequent characterization.

scholarly journals Dynamics of in vivo ASC speck formation

2017 ◽  
Vol 216 (9) ◽  
pp. 2891-2909 ◽  
Author(s):  
Paola Kuri ◽  
Nicole L. Schieber ◽  
Thomas Thumberger ◽  
Joachim Wittbrodt ◽  
Yannick Schwab ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Death ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Inflammasome Activation ◽  
Caspase Activation ◽  
Endogenous Gene ◽  
Pyrin Domain ◽  
Effector Caspase

Activated danger or pathogen sensors trigger assembly of the inflammasome adaptor ASC into specks, large signaling platforms considered hallmarks of inflammasome activation. Because a lack of in vivo tools has prevented the study of endogenous ASC dynamics, we generated a live ASC reporter through CRISPR/Cas9 tagging of the endogenous gene in zebrafish. We see strong ASC expression in the skin and other epithelia that act as barriers to insult. A toxic stimulus triggered speck formation and rapid pyroptosis in keratinocytes in vivo. Macrophages engulfed and digested that speck-containing, pyroptotic debris. A three-dimensional, ultrastructural reconstruction, based on correlative light and electron microscopy of the in vivo assembled specks revealed a compact network of highly intercrossed filaments, whereas pyrin domain (PYD) or caspase activation and recruitment domain alone formed filamentous aggregates. The effector caspase is recruited through PYD, whose overexpression induced pyroptosis but only after substantial delay. Therefore, formation of a single, compact speck and rapid cell-death induction in vivo requires a full-length ASC.

scholarly journals Transmigration of Trypanosoma cruzi trypomastigotes through 3D cultures resembling a physiological environment

2019 ◽  
Author(s):  
Matías Exequiel Rodríguez ◽  
Mariana Rizzi ◽  
Lucas D. Caeiro ◽  
Yamil E. Masip ◽  
Alina Perrone ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Three Dimensional ◽  
3D Cultures ◽  
Monolayer Cultures ◽  
Host Parasite Interactions ◽  
Virulent Strains ◽  
Host Parasite ◽  
3D Spheroids

AbstractChaga’ disease, caused by the kinetoplastid parasite Trypanosoma cruzi, presents a variety of chronic clinical manifestations whose determinants are still unknown but probably influenced by the host-parasite interplay established during the first stages of the infection, when bloodstream circulating trypomastigotes disseminate to different organs and tissues. After leaving the blood, trypomastigotes must migrate through tissues to invade cells and establish a chronic infection. How this process occurs remains unexplored. Three-dimensional (3D) cultures are physiologically relevant because mimic the microarchitecture of tissues and provide an environment similar to the encountered in natural infections. In this work, we combined the 3D culture technology with host-pathogen interaction, by studying transmigration of trypomastigotes into 3D spheroids. T. cruzi strains with similar infection dynamics in 2D monolayer cultures but with different in vivo behavior (CL Brener, virulent; SylvioX10 no virulent) presented different infection rates in spheroids (CL Brener ∼40%, SylvioX10 <10%). Confocal microscopy images evidenced that trypomastigotes from CL Brener and other highly virulent strains presented a great ability to transmigrate inside 3D spheroids: as soon as 4 hours post infection parasites were found at 50 µm in depth inside the spheroids. CL Brener trypomastigotes were evenly distributed and systematically observed in the space between cells, suggesting a paracellular route of transmigration to deepen into the spheroids. On the other hand, poor virulent strains presented a weak migratory capacity and remained in the external layers of spheroids (<10µm) with a patch-like distribution pattern. The invasiveness -understood as the ability to transmigrate deep into spheroids- was not a transferable feature between strains, neither by soluble or secreted factors nor by co-cultivation of trypomastigotes from invasive and non-invasive strains. We also studied the transmigration of recent T. cruzi isolates from children that were born congenitally infected, which showed a high migrant phenotype while an isolate form an infected mother (that never transmitted the infection to any of her 3 children) was significantly less migratory. Altogether, our results demonstrate that in a 3D microenvironment each strain presents a characteristic migration pattern and distribution of parasites in the spheroids that can be associated to their in vivo behavior. Certainly, the findings presented here could not have been studied with traditional 2D monolayer cultures.Author SummaryTrypanosoma cruzi is the protozoan parasite that causes Chaga’ disease, also known as American trypanosomiasis. Experimental models of the infection evidence that different strains of the parasite present different virulence in the host, which cannot be always reproduced in 2D monolayer cultures. Three dimensional (3D) cultures can be useful models to study complex host-parasite interactions because they mimic in vitro the microarchitecture of tissues and provide an environment similar to the encountered in natural infections. In particular, spheroids are small 3D aggregates of cells that interact with each other and with the extracellular matrix that they secrete resembling the original microenvironment both functionally and structurally. Spheroids have rarely been employed to explore infectious diseases and host-parasite interactions. In this work we studied how bloodstream trypomastigotes transmigrate through 3D spheroids mimicking the picture encountered by parasites in tissues soon after leaving circulation. We showed that the behavior of T. cruzi trypomastigotes in 3D cultures reflects their in vivo virulence: virulent strains transmigrate deeply into spheroids while non-virulent strains remain in the external layers of spheroids. Besides, this work demonstrates the usefulness of 3D cultures as an accurate in vitro model for the study of host-pathogen interactions that could not be addressed with conventional monolayer cultures.

scholarly journals Pro-apoptotic caspase deficiency reveals a cell-extrinsic mechanism of NK cell regulation

2021 ◽  
Author(s):  
Tayla M. Olsen ◽  
Wei Hong Tan ◽  
Arne C. Knudsen ◽  
Anthony Rongvaux
Keyword(s):  
Cell Death ◽  
Nk Cells ◽  
Nk Cell ◽  
Apoptotic Cell ◽  
Type I ◽  
Dependent Manner ◽  
Apoptosis Pathway ◽  
A Cell

AbstractRegulated cell death is essential for the maintenance of cellular and tissue homeostasis. In the hematopoietic system, genetic defects in apoptotic cell death generally produce the accumulation of immune cells, inflammation and autoimmunity. In contrast, we found that genetic deletion of caspases of the mitochondrial apoptosis pathway reduces natural killer (NK) cell numbers and makes NK cells functionally defective in vivo and in vitro. Caspase deficiency results in constitutive activation of a type I interferon (IFN) response, due to leakage of mitochondrial DNA and activation of the cGAS/STING pathway. The NK cell defect in caspase-deficient mice is independent of the type I IFN response, but the phenotype is partially rescued by cGAS or STING deficiency. Finally, caspase deficiency alters NK cells in a cell-extrinsic manner. Type I IFNs and NK cells are two essential effectors of antiviral immunity, and our results demonstrate that they are both regulated in a caspase-dependent manner. Beyond caspase-deficient animals, our observations may have implications in infections that trigger mitochondrial stress and caspase-dependent cell death.

scholarly journals SIRT1 Promotes Cell Survival under Stress by Deacetylation-Dependent Deactivation of Poly(ADP-Ribose) Polymerase 1

2009 ◽  
Vol 29 (15) ◽  
pp. 4116-4129 ◽  
Author(s):  
Senthilkumar B. Rajamohan ◽  
Vinodkumar B. Pillai ◽  
Madhu Gupta ◽  
Nagalingam R. Sundaresan ◽  
Konstantin G. Birukov ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Cell Survival ◽  
Cross Talk ◽  
Gene Promoter ◽  
Stress Conditions ◽  
Transcriptional Level ◽  
A Cell

ABSTRACT Poly(ADP-ribose) polymerase 1 (PARP1) and SIRT1 deacetylase are two NAD-dependent enzymes which play major roles in the decision of a cell to live or to die in a stress situation. Because of the dependence of both enzymes on NAD, cross talk between them has been suggested. Here, we show that PARP1 is acetylated after stress of cardiomyocytes, resulting in the activation of PARP1, which is independent of DNA damage. SIRT1 physically binds to and deacetylates PARP1. Increased acetylation of PARP1 was also detected in hearts of SIRT1−/− mice, compared to that detected in the hearts of SIRT1+/+ mice, confirming a role of SIRT1 in regulating the PARP1 acetylation in vivo. SIRT1-dependent deacetylation blocks PARP1 activity, and it protects cells from PARP1-mediated cell death. We also show that SIRT1 negatively regulates the activity of the PARP1 gene promoter, thus suggesting that the deacetylase controls the PARP1 activity at the transcriptional level as well. These data demonstrate that the activity of PARP1 is under the control of SIRT1, which is necessary for survival of cells under stress conditions.

scholarly journals Multi-Organs-on-Chips: Towards Long-Term Biomedical Investigations

Molecules ◽  
2019 ◽  
Vol 24 (4) ◽  
pp. 675 ◽  
Author(s):  
Yi Zhao ◽  
Ranjith Kankala ◽  
Shi-Bin Wang ◽  
Ai-Zheng Chen
Keyword(s):  
Real Time ◽  
Three Dimensional ◽  
Drug Efficacy ◽  
Dynamic Responses ◽  
Physical Parameters ◽  
Real Time Monitoring ◽  
Multiple Organs ◽  
On Chip

With advantageous features such as minimizing the cost, time, and sample size requirements, organ-on-a-chip (OOC) systems have garnered enormous interest from researchers for their ability for real-time monitoring of physical parameters by mimicking the in vivo microenvironment and the precise responses of xenobiotics, i.e., drug efficacy and toxicity over conventional two-dimensional (2D) and three-dimensional (3D) cell cultures, as well as animal models. Recent advancements of OOC systems have evidenced the fabrication of ‘multi-organ-on-chip’ (MOC) models, which connect separated organ chambers together to resemble an ideal pharmacokinetic and pharmacodynamic (PK-PD) model for monitoring the complex interactions between multiple organs and the resultant dynamic responses of multiple organs to pharmaceutical compounds. Numerous varieties of MOC systems have been proposed, mainly focusing on the construction of these multi-organ models, while there are only few studies on how to realize continual, automated, and stable testing, which still remains a significant challenge in the development process of MOCs. Herein, this review emphasizes the recent advancements in realizing long-term testing of MOCs to promote their capability for real-time monitoring of multi-organ interactions and chronic cellular reactions more accurately and steadily over the available chip models. Efforts in this field are still ongoing for better performance in the assessment of preclinical attributes for a new chemical entity. Further, we give a brief overview on the various biomedical applications of long-term testing in MOCs, including several proposed applications and their potential utilization in the future. Finally, we summarize with perspectives.

scholarly journals Extensive transcriptional and chromatin changes underlie astrocyte maturation in vivo and in culture

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Michael Lattke ◽  
Robert Goldstone ◽  
James K. Ellis ◽  
Stefan Boeing ◽  
Jerónimo Jurado-Arjona ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Cell Culture Model ◽  
Functional Maturation ◽  
Culture Model ◽  
Mature Astrocyte ◽  
Transcriptional Changes ◽  
A Cell ◽  
Brain Homeostasis

AbstractAstrocytes have essential functions in brain homeostasis that are established late in differentiation, but the mechanisms underlying the functional maturation of astrocytes are not well understood. Here we identify extensive transcriptional changes that occur during murine astrocyte maturation in vivo that are accompanied by chromatin remodelling at enhancer elements. Investigating astrocyte maturation in a cell culture model revealed that in vitro-differentiated astrocytes lack expression of many mature astrocyte-specific genes, including genes for the transcription factors Rorb, Dbx2, Lhx2 and Fezf2. Forced expression of these factors in vitro induces distinct sets of mature astrocyte-specific transcripts. Culturing astrocytes in a three-dimensional matrix containing FGF2 induces expression of Rorb, Dbx2 and Lhx2 and improves astrocyte maturity based on transcriptional and chromatin profiles. Therefore, extrinsic signals orchestrate the expression of multiple intrinsic regulators, which in turn induce in a modular manner the transcriptional and chromatin changes underlying astrocyte maturation.

scholarly journals OXPHOS Promotes Apoptotic Resistance and Persistence in TH17 cells

2021 ◽  
Author(s):  
Hanna S. Hong ◽  
Nneka E. Mbah ◽  
Mengrou Shan ◽  
Kristen Loesel ◽  
Lin Lin ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Death ◽  
Cell Fate ◽  
Apoptotic Cell ◽  
Metabolic Phenotype ◽  
Tumor Studies ◽  
A Cell

AbstractApoptotic cell death is a cell-intrinsic, immune tolerance mechanism that regulates the magnitude and resolution of T cell-mediated responses. Evasion of apoptosis is critical for the generation of memory T cells, as well as autoimmune T cells, and knowledge of the mechanisms that enable resistance to apoptosis will provide insight into ways to modulate their activity during protective and pathogenic responses. IL-17-producing CD4 T cells (TH17s) are long-lived, memory cells. These features enable their role in host defense, chronic inflammatory disorders, and anti-tumor immunity. A growing number of reports now indicate that TH17s in vivo require mitochondrial oxidative phosphorylation (OXPHOS), a metabolic phenotype that is poorly induced in vitro. To elucidate the role of OXPHOS in TH17 processes, we developed a system to polarize TH17s that metabolically resembled their in vivo counterparts. We discovered that directing TH17s to use OXPHOS promotes mitochondrial fitness, glutamine anaplerosis, and an anti-apoptotic phenotype marked by high BCL-XL and low BIM. Through competitive co-transfer experiments and tumor studies, we further revealed how OXPHOS protects TH17s from cell death while enhancing their persistence in the periphery and tumor microenvironment. Together, our work demonstrates a non-classical role of metabolism in regulating TH17 cell fate and highlights the potential for therapies that target OXPHOS in TH17-driven diseases.

scholarly journals Extensive transcriptional and chromatin changes underlie astrocyte maturation in vivo and in culture

2020 ◽  
Author(s):  
Michael Lattke ◽  
Robert Goldstone ◽  
Francois Guillemot
Keyword(s):  
Three Dimensional ◽  
Cell Culture Model ◽  
Culture Model ◽  
Mature Astrocyte ◽  
Transcriptional Changes ◽  
A Cell ◽  
Late Stages ◽  
Brain Homeostasis

SummaryAstrocytes have diverse functions in brain homeostasis. Many of these functions are acquired during late stages of differentiation when astrocytes become fully mature. The mechanisms underlying astrocyte maturation are not well understood. Here we identified extensive transcriptional changes that occur during astrocyte maturation and are accompanied by chromatin remodelling at enhancer elements. Investigating astrocyte maturation in a cell culture model revealed that in vitro-differentiated astrocytes lacked expression of many mature astrocyte-specific genes, including genes for the transcription factors Rorb, Dbx2, Lhx2 and Fezf2. Forced expression of these factors in vitro induced distinct sets of mature astrocytes-specific transcripts. Culturing astrocytes with FGF2 in a three-dimensional gel induced expression of Rorb, Dbx2 and Lhx2 and improved their maturity based on transcriptional and chromatin profiles. Therefore extrinsic signals orchestrate the expression of multiple intrinsic regulators, which in turn induce in a modular manner the transcriptional and chromatin changes underlying astrocyte maturation.

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