Single cell organization and cell cycle characterization of DNA stained multicellular tumor spheroids

AbstractMulticellular tumor spheroids (MCTSs) can serve as in vitro models for solid tumors and have become widely used in basic cancer research and drug screening applications. The major challenges when studying MCTSs by optical microscopy are imaging and analysis due to light scattering within the 3-dimensional structure. Herein, we used an ultrasound-based MCTS culture platform, where A498 renal carcinoma MCTSs were cultured, DAPI stained, optically cleared and imaged, to connect nuclear segmentation to biological information at the single cell level. We show that DNA-content analysis can be used to classify the cell cycle state as a function of position within the MCTSs. We also used nuclear volumetric characterization to show that cells were more densely organized and perpendicularly aligned to the MCTS radius in MCTSs cultured for 96 h compared to 24 h. The method presented herein can in principle be used with any stochiometric DNA staining protocol and nuclear segmentation strategy. Since it is based on a single counter stain a large part of the fluorescence spectrum is free for other probes, allowing measurements that correlate cell cycle state and nuclear organization with e.g., protein expression or drug distribution within MCTSs.

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Multicellular tumor spheroids as in vitro models for studying tumor responses to anticancer therapies

Animal Biotechnology ◽

10.1016/b978-0-12-811710-1.00011-2 ◽

2020 ◽

pp. 251-268

Author(s):

Suchit Khanna ◽

Ankit Chauhan ◽

Anant Narayan Bhatt ◽

Bilikere Srinivasa Rao Dwarakanath

Keyword(s):

In Vitro Models ◽

Multicellular Tumor Spheroids ◽

Tumor Spheroids

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Encapsulated multicellular tumor spheroids as a novel in vitro model to study small size tumors

Hemijska industrija ◽

10.2298/hemind0312585m ◽

2003 ◽

Vol 57 (12) ◽

pp. 585-588 ◽

Cited By ~ 4

Author(s):

Elena Markvicheva ◽

Lina Bezdetnaya ◽

Artur Bartkowiak ◽

Annie Marc ◽

Jean-Louis Gorgen ◽

...

Keyword(s):

Tumor Cells ◽

Tumor Biology ◽

Cell Encapsulation ◽

Large Cell ◽

In Vitro Models ◽

Multicellular Tumor Spheroids ◽

Tumor Spheroids ◽

Vitro Model

Presently multicellular tumor spheroids (MTS) are being widely used in various aspects of tumor biology, including studies in biology and photodynamic therapy. The cellular organization of spheroids allows the recreation of in vivo small tumors much better than all common two-dimensional in vitro models. The cell encapsulation method could be proposed as a novel technique to quickly and easily prepare a large number of spheroids with narrow size distribution within a desirable diameter range. Moreover, the proposed technique for spheroid generation using encapsulated growing tumor cells could provide entirely new avenues to develop a novel spheroid co-culture model (for instance, the in vitro co-cultvation of tumor cells and monocytes, or epithelial cells, or fibroblasts etc). The current research was aimed at developing a simple and reliable method to encapsulate tumor cells and to cultivate them in vitro. In order to generate spheroids, MCF-7 cells were encapsulated and cultivated in 200 ml T-flasks in a 5% CO2 atmosphere at 37?C for 4-5 weeks. The cell proliferation was easily observed using a light microscope. The cells grew in aggregates increasing in size with time. The cell growth resulted in the formation of large cell clusters (spheroids) which filled the whole microcapsule volume in 4-5 weeks.

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Semiautomatic Growth Analysis of Multicellular Tumor Spheroids

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057111419501 ◽

2011 ◽

Vol 16 (9) ◽

pp. 1119-1124 ◽

Cited By ~ 25

Author(s):

Bjoern Rodday ◽

Franziska Hirschhaeuser ◽

Stefan Walenta ◽

Wolfgang Mueller-Klieser

Keyword(s):

Tumor Biology ◽

Three Dimensional ◽

In Vitro Models ◽

Multicellular Tumor Spheroids ◽

Tumor Spheroids ◽

Microtiter Plates ◽

Accuracy And Precision ◽

Ocular Micrometer ◽

Growing Conditions

Multicellular tumor spheroids (MCTS) are routinely employed as three-dimensional in vitro models to study tumor biology. Cultivation of MCTS in spinner flasks provides better growing conditions, especially with regard to the availability of nutrients and oxygen, when compared with microtiter plates. The main endpoint of drug response experiments is spheroid size. It is common practice to analyze spheroid size manually with a microscope and an ocular micrometer. This requires removal of some spheroids from the flask, which entails major limitations such as loss of MCTS and the risk of contamination. With this new approach, the authors present an efficient and highly reproducible method to analyze the size of complete MCTS populations in culture containers with transparent, flat bottoms. MCTS sediments are digitally scanned and spheroid volumes are calculated by computerized image analysis. The equipment includes regular office hardware (personal computer, flatbed scanner) and software (Adobe Photoshop, Microsoft Excel, ImageJ). The accuracy and precision of the method were tested using industrial precision steel beads with known diameter. In summary, in comparison with other methods, this approach provides benefits in terms of semiautomation, noninvasiveness, and low costs.

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Abstract 14391: Transient Cell Cycle Induction in Cardiomyocytes is a Promising Approach to Treat Ischemia Induced Heart Failure

Circulation ◽

10.1161/circ.142.suppl_3.14391 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Riham Abouleisa ◽

Qinghui Ou ◽

Xian-liang Tang ◽

Mitesh Solanki ◽

Yiru Guo ◽

...

Keyword(s):

Cell Cycle ◽

Cardiac Function ◽

Single Cell ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Cardiomyocyte Proliferation ◽

Specific Expression ◽

Control Virus

Rationale: The regenerative capacity of the heart to repair itself after myocardial infarction (MI)is limited. Our previous study showed that ectopic introduction of Cdk1/CyclinB1 andCdk4/CyclinD1 complexes (4F) promotes cardiomyocyte proliferation in vitro and in vivo andimproves cardiac function after MI. However, its clinical application is limited due to the concernsfor tumorigenic potential in other organs. Objectives: To first, identify on a single cell transcriptomic basis the necessary reprogrammingsteps that cardiomyocytes need to undertake to progress through the proliferation processfollowing 4F overexpression, and then, to determine the pre-clinical efficacy of transient andcardiomyocyte specific expression of 4F in improving cardiac function after MI in small and largeanimals. Methods and Results: Temporal bulk and single cell RNAseq of mature hiPS-CMs treated with4F or LacZ control for 24, 48, or 72 h revealed full cell cycle reprogramming in 15% of thecardiomyocyte population which was associated with sarcomere disassembly and metabolicreprogramming. Transient overexpression of 4F specifically in cardiomyocytes was achievedusing non-integrating lentivirus (NIL) driven by TNNT2 (TNNT2-4F-NIL). One week after inductionof ischemia-reperfusion injury in rats or pigs, TNNT2-4F-NIL or control virus was injectedintramyocardially. Compared with controls, rats or pigs treated with TNNT2-4F-NIL showed a 20-30% significant improvement in ejection fraction and scar size four weeks after treatment, asassessed by echocardiography and histological analysis. Quantification of cardiomyocyteproliferation in pigs using a novel cytokinesis reporter showed that ~10% of the cardiomyocyteswithin the injection site were labelled as daughter cells following injection with TNNT2-4F-NILcompared with ~0.5% background labelling in control groups. Conclusions: We provide the first understanding of the process of forced cardiomyocyteproliferation and advanced the clinical applicability of this approach through minimization ofoncogenic potential of the cell cycle factors using a novel transient and cardiomyocyte-specificviral construct.

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Live-cell time-lapse imaging and single-cell tracking of in vitro cultured neural stem cells – Tools for analyzing dynamics of cell cycle, migration, and lineage selection

Methods ◽

10.1016/j.ymeth.2017.10.003 ◽

2018 ◽

Vol 133 ◽

pp. 81-90 ◽

Cited By ~ 11

Author(s):

Katja M. Piltti ◽

Brian J. Cummings ◽

Krystal Carta ◽

Ayla Manughian-Peter ◽

Colleen L. Worne ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Neural Stem Cells ◽

Single Cell ◽

Cell Tracking ◽

Time Lapse ◽

Live Cell ◽

Time Lapse Imaging

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Effects of T-lymphocytes and interferon-γ on different stages of development of multicellular tumor spheroids in vitro

Cytology and Genetics ◽

10.3103/s0095452714050065 ◽

2014 ◽

Vol 48 (5) ◽

pp. 285-292 ◽

Cited By ~ 1

Author(s):

O. Perepelytsina ◽

T. Gergeliuk ◽

M. Sydorenko

Keyword(s):

T Lymphocytes ◽

Interferon Γ ◽

Multicellular Tumor Spheroids ◽

Tumor Spheroids ◽

Stages Of Development

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Folic acid-conjugated soybean protein-based nanoparticles mediate efficient antitumor ability in vitro

Journal of Biomaterials Applications ◽

10.1177/0885328216679571 ◽

2016 ◽

Vol 31 (6) ◽

pp. 832-843 ◽

Cited By ~ 13

Author(s):

Weijing Yao ◽

Qian Zha ◽

Xu Cheng ◽

Xin Wang ◽

Jun Wang ◽

...

Keyword(s):

Folic Acid ◽

Soy Protein ◽

Soybean Protein ◽

Soy Protein Isolate ◽

Three Dimensional ◽

Multicellular Tumor Spheroids ◽

Tumor Spheroids ◽

Solution Ph ◽

Protein Nanoparticles

In this study, soy protein isolate was hydrolyzed by compound enzymes to give aqueous soy protein with low molecular weights. Folic acid modified and free soy protein nanoparticles were successfully prepared by a desolvation method as target-specific drug delivery, respectively. Ultraviolet spectrophotometry demonstrated that folic acid was successfully grafted onto soy protein. The shape and size of folic acid modified soy protein nanoparticles were detected by transmission electron microscopy, scanning electron microscope, and dynamic light scattering. In addition, a series of characteristics including kinetic stability, pH stability, and time stability were also performed. Doxorubicin was successfully loaded into folic acid modified soy protein nanoparticles, and the encapsulation and loading efficiencies were 96.7% and 23%, respectively. Doxorubicin-loaded folic acid modified soy protein nanoparticles exhibited faster drug release rate than soy protein nanoparticles in PBS solution (pH = 5). The tumor penetration and antitumor experiments were done using three-dimensional multicellular tumor spheroids as the in vitro model. The results proved that folic acid modified soy protein nanoparticles display higher penetration and accumulation than soy protein nanoparticles, therefore possessing efficient growth inhibitory ability against multicellular tumor spheroids.

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Vascularized Microfluidics and the Blood–Endothelium Interface

Micromachines ◽

10.3390/mi11010018 ◽

2019 ◽

Vol 11 (1) ◽

pp. 18 ◽

Cited By ~ 4

Author(s):

Christopher A. Hesh ◽

Yongzhi Qiu ◽

Wilbur A. Lam

Keyword(s):

In Vitro Models ◽

Biological Information ◽

Microvascular Endothelium ◽

Basic Principles ◽

Peripheral Tissues ◽

Bidirectional Communication ◽

In Vivo Animal Models ◽

Complex Phenomena

The microvasculature is the primary conduit through which the human body transmits oxygen, nutrients, and other biological information to its peripheral tissues. It does this through bidirectional communication between the blood, consisting of plasma and non-adherent cells, and the microvascular endothelium. Current understanding of this blood–endothelium interface has been predominantly derived from a combination of reductionist two-dimensional in vitro models and biologically complex in vivo animal models, both of which recapitulate the human microvasculature to varying but limited degrees. In an effort to address these limitations, vascularized microfluidics have become a platform of increasing importance as a consequence of their ability to isolate biologically complex phenomena while also recapitulating biochemical and biophysical behaviors known to be important to the function of the blood–endothelium interface. In this review, we discuss the basic principles of vascularized microfluidic fabrication, the contribution this platform has made to our understanding of the blood–endothelium interface in both homeostasis and disease, the limitations and challenges of these vascularized microfluidics for studying this interface, and how these inform future directions.

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