scholarly journals Towards a scaled-up T cell-mediated cytotoxicity assay in 3D cell culture using microscopy

2019 ◽  
Author(s):  
Elinor Gottschalk ◽  
Eric Czech ◽  
Bulent Arman Aksoy ◽  
Pinar Aksoy ◽  
Jeff Hammerbacher
Keyword(s):  
Cell Culture ◽  
T Cells ◽  
T Cell ◽  
Cytotoxic T Cells ◽  
Three Dimensional ◽  
Collagen Gel ◽  
3D Cell Culture ◽  
3D Scaffold ◽  
Tumor Spheroids ◽  
Drug Induced

AbstractThree-dimensional (3D) cell culture systems with tumor spheroids are being adopted for research on the antitumor activity of drug treatments and cytotoxic T cells. Analysis of the cytotoxic effect on 3D tumor cultures within a 3D scaffold, such as collagen, is challenging. Image-based approaches often use confocal microscopy, which greatly limits the sample size of tumor spheroids that can be assayed. We explored a system where tumor spheroids growing in a collagen gel within a microfluidics chip can be treated with drugs or co-cultured with T cells. We attempted to adapt the system to measure the death of cells in the tumor spheroids directly in the microfluidics chip via automated widefield fluorescence microscopy. We were able to successfully measure drug-induced cytotoxicity in tumor spheroids, but had difficulties extending the system to measure T cell-mediated tumor killing.Abstract Figure

scholarly journals The Combined Effects of Co-Culture and Substrate Mechanics on 3D Tumor Spheroid Formation within Microgels Prepared via Flow-Focusing Microfluidic Fabrication

Pharmaceutics ◽  
2018 ◽  
Vol 10 (4) ◽  
pp. 229 ◽  
Author(s):  
Dongjin Lee ◽  
Chaenyung Cha
Keyword(s):  
Mechanical Properties ◽  
Cell Culture ◽  
Microfluidic Device ◽  
3D Cell Culture ◽  
Breast Adenocarcinoma ◽  
Adherent Cell ◽  
Flow Focusing ◽  
Spheroid Formation ◽  
3D Scaffold ◽  
Tumor Spheroids

Tumor spheroids are considered a valuable three dimensional (3D) tissue model to study various aspects of tumor physiology for biomedical applications such as tissue engineering and drug screening as well as basic scientific endeavors, as several cell types can efficiently form spheroids by themselves in both suspension and adherent cell cultures. However, it is more desirable to utilize a 3D scaffold with tunable properties to create more physiologically relevant tumor spheroids as well as optimize their formation. In this study, bioactive spherical microgels supporting 3D cell culture are fabricated by a flow-focusing microfluidic device. Uniform-sized aqueous droplets of gel precursor solution dispersed with cells generated by the microfluidic device are photocrosslinked to fabricate cell-laden microgels. Their mechanical properties are controlled by the concentration of gel-forming polymer. Using breast adenocarcinoma cells, MCF-7, the effect of mechanical properties of microgels on their proliferation and the eventual spheroid formation was explored. Furthermore, the tumor cells are co-cultured with macrophages of fibroblasts, which are known to play a prominent role in tumor physiology, within the microgels to explore their role in spheroid formation. Taken together, the results from this study provide the design strategy for creating tumor spheroids utilizing mechanically-tunable microgels as 3D cell culture platform.

scholarly journals A reliable and affordable 3D tumor spheroid model for natural product drug discovery: A case study of curcumin

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Loh Teng Hern Tan ◽  
Liang Ee Low ◽  
Siah Ying Tang ◽  
Wei Hsum Yap ◽  
Lay Hong Chuah ◽  
...  
Keyword(s):  
Cell Culture ◽  
Drug Discovery ◽  
Three Dimensional ◽  
3D Cell Culture ◽  
Automated Image Analysis ◽  
Tumor Spheroids ◽  
Culture Methods ◽  
In Vivo Models

Three-dimensional cell culture methods revolutionize the field of anticancer drug discovery, forming an important link-bridge between conventional in vitro and in vivo models and conferring significant clinical and biological relevant data. The current work presents an affordable yet reproducible method of generating homogenous 3D tumor spheroids. Also, a new open source software is adapted to perform an automated image analysis of 3D tumor spheroids and subsequently generate a list of morphological parameters of which could be utilized to determine the response of these spheroids toward treatments. Our data showed that this work could serve as a reliable 3D cell culture platform for preclinical cytotoxicity testing of natural products prior to the expensive and time-consuming animal models

scholarly journals Closing the system: production of viral antigen-presenting dendritic cells eliciting specific CD8+ T cell activation in fluorinated ethylene propylene cell culture bags

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Jean-Philippe Bastien ◽  
Natalie Fekete ◽  
Ariane V. Beland ◽  
Marie-Paule Lachambre ◽  
Veronique Laforte ◽  
...  
Keyword(s):  
Cell Culture ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Viral Antigen ◽  
Cell Activation ◽  
Cytotoxic T Cells ◽  
Surface Marker ◽  
Fluorinated Ethylene Propylene

Abstract Background A major obstacle to anti-viral and -tumor cell vaccination and T cell immunotherapy is the ability to produce dendritic cells (DCs) in a suitable clinical setting. It is imperative to develop closed cell culture systems to accelerate the translation of promising DC-based cell therapy products to the clinic. The objective of this study was to investigate whether viral antigen-loaded monocyte-derived DCs (Mo-DCs) capable of eliciting specific T cell activation can be manufactured in fluorinated ethylene propylene (FEP) bags. Methods Mo-DCs were generated through a protocol applying cytokine cocktails combined with lipopolysaccharide or with a CMV viral peptide antigen in conventional tissue culture polystyrene (TCPS) or FEP culture vessels. Research-scale (< 10 mL) FEP bags were implemented to increase R&D throughput. DC surface marker profiles, cytokine production, and ability to activate antigen-specific cytotoxic T cells were characterized. Results Monocyte differentiation into Mo-DCs led to the loss of CD14 expression with concomitant upregulation of CD80, CD83 and CD86. Significantly increased levels of IL-10 and IL-12 were observed after maturation on day 9. Antigen-pulsed Mo-DCs activated antigen-responsive CD8+ cytotoxic T cells. No significant differences in surface marker expression or tetramer-specific T cell activating potency of Mo-DCs were observed between TCPS and FEP culture vessels. Conclusions Our findings demonstrate that viral antigen-loaded Mo-DCs produced in downscaled FEP bags can elicit specific T cell responses. In view of the dire clinical need for closed system DC manufacturing, FEP bags represent an attractive option to accelerate the translation of promising emerging DC-based immunotherapies.

scholarly journals Trinity of Three-Dimensional (3D) Scaffold, Vibration, and 3D Printing on Cell Culture Application: A Systematic Review and Indicating Future Direction

2018 ◽  
Vol 5 (3) ◽  
pp. 57 ◽  
Author(s):  
Haobo Yuan ◽  
Ke Xing ◽  
Hung-Yao Hsu
Keyword(s):  
Cell Culture ◽  
3D Printing ◽  
Rapid Development ◽  
Three Dimensional ◽  
3D Cell Culture ◽  
3D Scaffold ◽  
Cell Scaffold ◽  
3D Environments ◽  
Culturing Conditions ◽  
Future Direction

Cell culture and cell scaffold engineering have previously developed in two directions. First can be ‘static into dynamic’, with proven effects that dynamic cultures have benefits over static ones. Researches in this direction have used several mechanical means, like external vibrators or shakers, to approximate the dynamic environments in real tissue, though such approaches could only partly address the issue. Second, can be ‘2D into 3D’, that is, artificially created three-dimensional (3D) passive (also called ‘static’) scaffolds have been utilized for 3D cell culture, helping external culturing conditions mimic real tissue 3D environments in a better way as compared with traditional two-dimensional (2D) culturing. In terms of the fabrication of 3D scaffolds, 3D printing (3DP) has witnessed its high popularity in recent years with ascending applicability, and this tendency might continue to grow along with the rapid development in scaffold engineering. In this review, we first introduce cell culturing, then focus 3D cell culture scaffold, vibration stimulation for dynamic culture, and 3DP technologies fabricating 3D scaffold. Potential interconnection of these realms will be analyzed, as well as the limitations of current 3D scaffold and vibration mechanisms. In the recommendation part, further discussion on future scaffold engineering regarding 3D vibratory scaffold will be addressed, indicating 3DP as a positive bridging technology for future scaffold with integrated and localized vibratory functions.

Flow Analysis of a Porous Polymer-Based Three-Dimensional Cell Culture Device for Drug Screening

2018 ◽  
Author(s):  
Liang Ma ◽  
Lei Gao ◽  
Yichen Luo ◽  
Huayong Yang ◽  
Bin Zhang ◽  
...  
Keyword(s):  
Cell Culture ◽  
Shear Stress ◽  
Three Dimensional ◽  
Flow Simulation ◽  
3D Cell Culture ◽  
Porous Polymer ◽  
Poly Lactic Acid ◽  
Porous Scaffolds ◽  
3D Scaffold

A porous polymer-based three-dimensional (3D) cell culture device has been developed as an in vitro tissue model system for the cytotoxicity of anticancer drug test. The device had two chambers connected in tandem, each loaded with a 3D scaffold made of highly biocompatible poly (lactic acid) (PLA). Hepatoma cells (HepG2) and glioblastoma multiforme (GBM) cancer cells were cultured in the two separate porous scaffolds. A peristaltic pump was adopted to realize a perfusion cell culture. In this study, we focus on cell viability inside the 3D porous scaffolds under flow-induced shear stress effects. A flow simulation was conducted to predict the shear stress based on a realistic representation of the porous structure. The simulation results were correlated to the cell variability measurements at different flow rates. It is shown that the modeling approach presented in this paper can be useful for shear stress predication inside porous scaffolds and the computational fluid dynamics model can be an effective way to optimize the operation parameters of perfused 3D cell culture devices.

A ready-to-use, versatile, multiplex-able three-dimensional scaffold-based immunoassay chip for high throughput hepatotoxicity evaluation

Lab on a Chip ◽  
2015 ◽  
Vol 15 (12) ◽  
pp. 2634-2646 ◽  
Author(s):  
Xiaojun Yan ◽  
Jingyu Wang ◽  
Lu Zhu ◽  
Jonathan Joseph Lowrey ◽  
Yuanyuan Zhang ◽  
...  
Keyword(s):  
Cell Culture ◽  
High Throughput ◽  
Three Dimensional ◽  
3D Cell Culture ◽  
3D Scaffold

A ready-to-use 3D scaffold-based immunoChip combined with a 3D cell culture chip for high throughput drug hepatotoxicity evaluation.

scholarly journals Applications of Biomaterials in 3D Cell Culture and Contributions of 3D Cell Culture to Drug Development and Basic Biomedical Research

2021 ◽  
Vol 22 (5) ◽  
pp. 2491
Author(s):  
Yujin Park ◽  
Kang Moo Huh ◽  
Sun-Woong Kang
Keyword(s):  
Cell Culture ◽  
Three Dimensional ◽  
3D Culture ◽  
3D Cell Culture ◽  
Accurate Method ◽  
New Drugs ◽  
Toxicity Screening ◽  
Cellular Functions ◽  
Toxicity Of Drugs ◽  
External Stimuli

The process of evaluating the efficacy and toxicity of drugs is important in the production of new drugs to treat diseases. Testing in humans is the most accurate method, but there are technical and ethical limitations. To overcome these limitations, various models have been developed in which responses to various external stimuli can be observed to help guide future trials. In particular, three-dimensional (3D) cell culture has a great advantage in simulating the physical and biological functions of tissues in the human body. This article reviews the biomaterials currently used to improve cellular functions in 3D culture and the contributions of 3D culture to cancer research, stem cell culture and drug and toxicity screening.

scholarly journals Mammary gland 3D cell culture systems in farm animals

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Laurence Finot ◽  
Eric Chanat ◽  
Frederic Dessauge
Keyword(s):  
Cell Culture ◽  
Mammary Gland ◽  
Bacterial Infections ◽  
Three Dimensional ◽  
3D Cell Culture ◽  
Farm Animals ◽  
Culture Systems ◽  
Cell Culture Systems

AbstractIn vivo study of tissue or organ biology in mammals is very complex and progress is slowed by poor accessibility of samples and ethical concerns. Fortunately, however, advances in stem cell identification and culture have made it possible to derive in vitro 3D “tissues” called organoids, these three-dimensional structures partly or fully mimicking the in vivo functioning of organs. The mammary gland produces milk, the source of nutrition for newborn mammals. Milk is synthesized and secreted by the differentiated polarized mammary epithelial cells of the gland. Reconstructing in vitro a mammary-like structure mimicking the functional tissue represents a major challenge in mammary gland biology, especially for farm animals for which specific agronomic questions arise. This would greatly facilitate the study of mammary gland development, milk secretion processes and pathological effects of viral or bacterial infections at the cellular level, all with the objective of improving milk production at the animal level. With this aim, various 3D cell culture models have been developed such as mammospheres and, more recently, efforts to develop organoids in vitro have been considerable. Researchers are now starting to draw inspiration from other fields, such as bioengineering, to generate organoids that would be more physiologically relevant. In this chapter, we will discuss 3D cell culture systems as organoids and their relevance for agronomic research.

scholarly journals Bifunctional iRGD-anti-CD3 enhances antitumor potency of T cells by facilitating tumor infiltration and T-cell activation

2021 ◽  
Vol 9 (5) ◽  
pp. e001925
Author(s):  
Shujuan Zhou ◽  
Fanyan Meng ◽  
Shiyao Du ◽  
Hanqing Qian ◽  
Naiqing Ding ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Confocal Microscope ◽  
Mechanistic Studies ◽  
Tumor Spheroids ◽  
Antitumor Effects ◽  
Transport Pathway

BackgroundPoor infiltration and limited activation of transferred T cells are fundamental factors impeding the development of adoptive cell immunotherapy in solid tumors. A tumor-penetrating peptide iRGD has been widely used to deliver drugs deep into tumor tissues. CD3-targeting bispecific antibodies represent a promising immunotherapy which recruits and activates T cells.MethodsT-cell penetration was demonstrated in tumor spheroids using confocal microscope, and in xenografted tumors by histology and in vivo real-time fluorescence imaging. Activation and cytotoxicity of T cells were assessed by flow cytometry and confocal microscope. Bioluminescence imaging was used to evaluate in vivo antitumor effects, and transmission electron microscopy was used for mechanistic studies.ResultsWe generated a novel bifunctional agent iRGD-anti-CD3 which could immobilize iRGD on the surface of T cells through CD3 engaging. We found that iRGD-anti-CD3 modification not only facilitated T-cell infiltration in 3D tumor spheroids and xenografted tumor nodules but also induced T-cell activation and cytotoxicity against target cancer cells. T cells modified with iRGD-anti-CD3 significantly inhibited tumor growth and prolonged survival in several xenograft mouse models, which was further enhanced by the combination of programmed cell death protein 1 (PD-1) blockade. Mechanistic studies revealed that iRGD-anti-CD3 initiated a transport pathway called vesiculovacuolar organelles in the endothelial cytoplasm to promote T-cell extravasation.ConclusionAltogether, we show that iRGD-anti-CD3 modification is an innovative and bifunctional strategy to overcome major bottlenecks in adoptive cell therapy. Moreover, we demonstrate that combination with PD-1 blockade can further improve antitumor efficacy of iRGD-anti-CD3-modified T cells.

scholarly journals CD28-B7 interactions allow the induction of CD8+ cytotoxic T lymphocytes in the absence of exogenous help.

1993 ◽  
Vol 177 (6) ◽  
pp. 1791-1796 ◽  
Author(s):  
F A Harding ◽  
J P Allison
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cytotoxic T Cells ◽  
Interleukin 2 ◽  
Cell Receptor ◽  
Histocompatibility Complex ◽  
Necessary And Sufficient ◽  
Additional Antigen

The activation requirements for the generation of CD8+ cytotoxic T cells (CTL) are poorly understood. Here we demonstrate that in the absence of exogenous help, a CD28-B7 interaction is necessary and sufficient for generation of class I major histocompatibility complex-specific CTL. Costimulation is required only during the inductive phase of the response, and not during the effector phase. Transfection of the CD28 counter receptor, B7, into nonstimulatory P815 cells confers the ability to elicit P815-specific CTL, and this response can be inhibited by anti-CD28 Fab or by the chimeric B7-binding protein CTLA4Ig. Anti-CD28 monoclonal antibody (mAb) can provide a costimulatory signal to CD8+ T cells when the costimulatory capacity of splenic stimulators is destroyed by chemical fixation. CD28-mediated signaling provokes the release of interleukin 2 (IL-2) from the CD8+ CTL precursors, as anti-CD28 mAb could be substituted for by the addition of IL-2, and an anti-IL-2 mAb can block the generation of anti-CD28-induced CTL. CD4+ cells are not involved in the costimulatory response in the systems examined. We conclude that CD8+ T cell activation requires two signals: an antigen-specific signal mediated by the T cell receptor, and an additional antigen nonspecific signal provided via a CD28-B7 interaction.

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