Development of Stretch Stimulation Device for Three-Dimensional Culture of PC12 Cells

Enhancement of nerve axonal extension by using the extracellular environmental stimulation were reported. In this study, we focused on the stretch stimulation, and developed a 3D cell culture system to mimic the in vivo extracellular matrices and investigated the fundamental mechanism of axonal extension enhancement. Firstly, we fabricated the stretch stimulation device. The rat phenocromocytoma cells (PC12), the nerve-like cells, embedded in the collagen gel were poured into the stretch chamber. It was set in the stretch stimulation device, which could load the strain to the collagen gel. Secondly, we determined the structure of the stretch chamber to implement the uniform strain distribution in the culture region. Using the finite element (FE) analyses, we confirmed that the uniform strain is assigned in a region of 2.7 × 3.0 × 0.5 mm in the culture region, which is the candidate for the observation region. Thirdly, PC12 cells axonal extension under uniaxial cyclic stretch stimulation (4% strain, 1 Hz) of 24 hours was carried out. After 96 hours’ culture, we observed the 3D morphology of PC12 cells by the multiphoton excitation fluorescence microscope (MPM). Finally, we confirmed the availability of our stretch stimulation device and the enhancement effect of axonal extension.

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Mammary gland 3D cell culture systems in farm animals

Veterinary Research ◽

10.1186/s13567-021-00947-5 ◽

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.

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iTRAQ-Based Quantitative Proteomic Comparison of 2D and 3D Adipocyte Cell Models Co-cultured with Macrophages Using Online 2D-nanoLC-ESI-MS/MS

Scientific Reports ◽

10.1038/s41598-019-53196-0 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Sun Young Lee ◽

Sung Bum Park ◽

Young Eun Kim ◽

Hee Min Yoo ◽

Jongki Hong ◽

...

Keyword(s):

Metabolic Disorders ◽

Cultured Cells ◽

Three Dimensional ◽

3D Cell Culture ◽

Proteomic Profiling ◽

Esi Ms ◽

Mitochondrial Fatty Acid ◽

2D And 3D

AbstractThe demand for novel three-dimensional (3D) cell culture models of adipose tissue has been increasing, and proteomic investigations are important for determining the underlying causes of obesity, type II diabetes, and metabolic disorders. In this study, we performed global quantitative proteomic profiling of three 3D-cultured 3T3-L1 cells (preadipocytes, adipocytes and co-cultured adipocytes with macrophages) and their 2D-cultured counterparts using 2D-nanoLC-ESI-MS/MS with iTRAQ labelling. A total of 2,885 shared proteins from six types of adipose cells were identified and quantified in four replicates. Among them, 48 proteins involved in carbohydrate metabolism (e.g., PDHα, MDH1/2, FH) and the mitochondrial fatty acid beta oxidation pathway (e.g., VLCAD, ACADM, ECHDC1, ALDH6A1) were relatively up-regulated in the 3D co-culture model compared to those in 2D and 3D mono-cultured cells. Conversely, 12 proteins implicated in cellular component organisation (e.g., ANXA1, ANXA2) and the cell cycle (e.g., MCM family proteins) were down-regulated. These quantitative assessments showed that the 3D co-culture system of adipocytes and macrophages led to the development of insulin resistance, thereby providing a promising in vitro obesity model that is more equivalent to the in vivo conditions with respect to the mechanisms underpinning metabolic syndromes and the effect of new medical treatments for metabolic disorders.

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Impedance Study of Dopamine Effects after Application on 2D and 3D Neuroblastoma Cell Cultures Developed on a 3D-Printed Well

Chemosensors ◽

10.3390/chemosensors7010006 ◽

2019 ◽

Vol 7 (1) ◽

pp. 6 ◽

Cited By ~ 2

Author(s):

Georgia Paivana ◽

Theofylaktos Apostolou ◽

Sophie Mavrikou ◽

Dimitris Barmpakos ◽

Grigoris Kaltsas ◽

...

Keyword(s):

Cell Cultures ◽

Immobilized Cells ◽

Neuroblastoma Cell ◽

Three Dimensional ◽

3D Cell Culture ◽

Impedance Analysis ◽

Neural Cells ◽

Opposite Behavior ◽

2D And 3D

In this work, the assessment of the interactions of a bioactive substance applied to immobilized cells in either a two-dimensional (2D) or three-dimensional (3D) arrangement mimicking in vivo tissue conditions is presented. In particular, dopamine (DA) was selected as a stimulant for the implementation of an impedance analysis with a specific type of neural cells (murine neuroblastoma). The aim of this study was the extraction of calibration curves at various frequencies with different known dopamine concentrations for the description of the behavior of dopamine applied to 2D and 3D cell cultures. The results present the evaluation of the mean impedance value for each immobilization technique in each frequency. The differential responses showed the importance of the impedance when frequency is applied in both 2D and 3D immobilization cases. More specifically, in 2D immobilization matrix impedance shows higher values in comparison with the 3D cell culture. Additionally, in the 3D case, the impedance decreases with increasing concentration, while in the 2D case, an opposite behavior was observed.

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Development of Three-Dimensional DC Electric Field Stimulation Bio-Reactor for Axonal Outgrowth Enhancement

Volume 3: Biomedical and Biotechnology Engineering ◽

10.1115/imece2018-86637 ◽

2018 ◽

Cited By ~ 1

Author(s):

Shohei Tanaka ◽

Ryota Sakiyama ◽

Koji Yamamoto ◽

Yusuke Morita ◽

Eiji Nakamachi

Keyword(s):

Electric Field ◽

Three Dimensional ◽

3D Culture ◽

Collagen Gel ◽

Control Group ◽

Axonal Outgrowth ◽

The Neural Network ◽

Dc Electric Field ◽

Current Electric Field

Numerous studies of electrical stimulation effects on the nerve regeneration have been carried out. However, there were very few investigations which adopt the 3D culture that mimics the in vivo environment. In this study, we designed and fabricated a new 3D direct current electric field (DCEF) stimulation bio-reactor and investigated the effectiveness on the axonal outgrowth enhancement. We searched an optimum structure using the finite element (FE) analyses to obtain a uniform DCEF in the culture region. A measurement result of DCEF strength showed an agreement with FE results. The rat phenocromocytoma cells (PC12) were disseminated in the collagen gel and 3D culture was performed. We observed the morphologies of cell bodies and neurites using the multiphoton excitation fluorescence microscope (MPM). Both increases in 11.3% of mean axonal length and in 4.2% of axogenesis rate, under the condition of 5.0 mV/mm on 6 hours a day for 4 days, were obtained. Further, there was a tendency of longer connecting distance between cell bodies in the DCEF group than one in the Control group. As a result, we validated the efficacies of our stimulation, both for the axonal extension and the neural network generation, using our newly developed bio-reactor.

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Three-Dimensional In Vitro Staphylococcus aureus Abscess Communities Display Antibiotic Tolerance and Protection from Neutrophil Clearance

Infection and Immunity ◽

10.1128/iai.00293-20 ◽

2020 ◽

Vol 88 (11) ◽

Author(s):

Marloes I. Hofstee ◽

Martijn Riool ◽

Igors Terjajevs ◽

Keith Thompson ◽

Martin J. Stoddart ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Immune Cells ◽

Early Stage ◽

Three Dimensional ◽

Collagen Gel ◽

Maximum Size ◽

Human Neutrophils ◽

Content Type

ABSTRACT Staphylococcus aureus is a prominent human pathogen in bone and soft-tissue infections. Pathophysiology involves abscess formation, which consists of central staphylococcal abscess communities (SACs), surrounded by a fibrin pseudocapsule and infiltrating immune cells. Protection against the ingress of immune cells such as neutrophils, or tolerance to antibiotics, remains largely unknown for SACs and is limited by the lack of availability of in vitro models. We describe a three-dimensional in vitro model of SACs grown in a human plasma-supplemented collagen gel. The in vitro SACs reached their maximum size by 24 h and elaborated a fibrin pseudocapsule, as confirmed by electron and immunofluorescence microscopy. The in vitro SACs tolerated 100× the MIC of gentamicin alone and in combination with rifampin, while planktonic controls and mechanically dispersed SACs were efficiently killed. To simulate a host response, SACs were exposed to differentiated PLB-985 neutrophil-like (dPLB) cells and to primary human neutrophils at an early stage of SAC formation or after maturation at 24 h. Both cell types were unable to clear mature in vitro SACs, but dPLB cells prevented SAC growth upon early exposure before pseudocapsule maturation. Neutrophil exposure after plasmin pretreatment of the SACs resulted in a significant decrease in the number of bacteria within the SACs. The in vitro SAC model mimics key in vivo features, offers a new tool to study host-pathogen interactions and drug efficacy assessment, and has revealed the functionality of the S. aureus pseudocapsule in protecting the bacteria from host phagocytic responses and antibiotics.

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Simple In-House Fabrication of Microwells for Generating Uniform Hepatic Multicellular Cancer Aggregates and Discovering Novel Therapeutics

Materials ◽

10.3390/ma12203308 ◽

2019 ◽

Vol 12 (20) ◽

pp. 3308 ◽

Cited By ~ 2

Author(s):

Chiao-Yi Chiu ◽

Ying-Chi Chen ◽

Kuang-Wei Wu ◽

Wen-Chien Hsu ◽

Hong-Ping Lin ◽

...

Keyword(s):

Liver Cancer ◽

Cell Viability ◽

Near Infrared ◽

Hollow Spheres ◽

Three Dimensional ◽

3D Cell Culture ◽

Therapeutic Interventions ◽

Therapeutic Modalities ◽

Photothermal Treatment

Three-dimensional (3D) cell culture models have become powerful tools because they better simulate the in vivo pathophysiological microenvironment than traditional two-dimensional (2D) monolayer cultures. Tumor cells cultured in a 3D system as multicellular cancer aggregates (MCAs) recapitulate several critical in vivo characteristics that enable the study of biological functions and drug discovery. The microwell, in particular, has emerged as a revolutionary technology in the generation of MCAs as it provides geometrically defined microstructures for culturing size-controlled MCAs amenable for various downstream functional assays. This paper presents a simple and economical microwell fabrication methodology that can be conveniently incorporated into a conventional laboratory setting and used for the discovery of therapeutic interventions for liver cancer. The microwells were 400–700 µm in diameter, and hepatic MCAs (Huh-7 cells) were cultured in them for up to 5 days, over which time they grew to 250–520 µm with good viability and shape. The integrability of the microwell fabrication with a high-throughput workflow was demonstrated using a standard 96-well plate for proof-of-concept drug screening. The IC50 of doxorubicin was determined to be 9.3 µM under 2D conditions and 42.8 µM under 3D conditions. The application of photothermal treatment was demonstrated by optimizing concanavalin A-FITC conjugated silica-carbon hollow spheres (SCHSs) at a concentration of 500:200 µg/mL after a 2 h incubation to best bind with MCAs. Based on this concentration, which was appropriate for further photothermal treatment, the relative cell viability was assessed through exposure to a 3 W/cm2 near-infrared laser for 20 min. The relative fluorescence intensity showed an eight-fold reduction in cell viability, confirming the feasibility of using photothermal treatment as a potential therapeutic intervention. The proposed microwell integration is envisioned to serve as a simple in-house technique for the generation of MCAs useful for discovering therapeutic modalities for liver cancer treatment.

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Culture Models for Studying Thyroid Biology and Disorders

ISRN Endocrinology ◽

10.5402/2011/275782 ◽

2011 ◽

Vol 2011 ◽

pp. 1-9 ◽

Cited By ~ 10

Author(s):

Shuji Toda ◽

Shigehisa Aoki ◽

Kazuyoshi Uchihashi ◽

Aki Matsunobu ◽

Mihoko Yamamoto ◽

...

Keyword(s):

Thyroid Tissue ◽

Three Dimensional ◽

Collagen Gel ◽

Cell Types ◽

Experimental Models ◽

C Cells ◽

Culture Systems ◽

Culture Models ◽

Normal Polarity

The thyroid is composed of thyroid follicles supported by extracellular matrix, capillary network, and stromal cell types such as fibroblasts. The follicles consist of thyrocytes and C cells. In this microenvironment, thyrocytes are highly integrated in their specific structural and functional polarization, but monolayer and floating cultures cannot allow thyrocytes to organize the follicles with such polarity. In contrast, three-dimensional (3-D) collagen gel culture enables thyrocytes to form 3-D follicles with normal polarity. However, these systems never reconstruct the follicles consisting of both thyrocytes and C cells. Thyroid tissue-organotypic culture retains 3-D follicles with both thyrocytes and C cells. To create more appropriate experimental models, we here characterize four culture systems above and then introduce the models for studying thyroid biology and disorders. Finally, we propose a new approach to the cell type-specific culture systems on the basis of in vivo microenvironments of various cell types.

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

Progress in Drug Discovery & Biomedical Science ◽

10.36877/pddbs.a0000017 ◽

2019 ◽

Vol 2 (1) ◽

Cited By ~ 4

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

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

10.1101/842039 ◽

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

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Application of Three-dimensional (3D) Tumor Cell Culture Systems and Mechanism of Drug Resistance

Current Pharmaceutical Design ◽

10.2174/1381612825666191014163923 ◽

2019 ◽

Vol 25 (34) ◽

pp. 3599-3607 ◽

Cited By ~ 3

Author(s):

Adeeb Shehzad ◽

Vijaya Ravinayagam ◽

Hamad AlRumaih ◽

Meneerah Aljafary ◽

Dana Almohazey ◽

...

Keyword(s):

Cell Culture ◽

Cancer Cells ◽

Anticancer Drugs ◽

Three Dimensional ◽

3D Culture ◽

3D Cell Culture ◽

Cancer Therapeutics ◽

In Vivo Model

: The in-vitro experimental model for the development of cancer therapeutics has always been challenging. Recently, the scientific revolution has improved cell culturing techniques by applying three dimensional (3D) culture system, which provides a similar physiologically relevant in-vivo model for studying various diseases including cancer. In particular, cancer cells exhibiting in-vivo behavior in a model of 3D cell culture is a more accurate cell culture model to test the effectiveness of anticancer drugs or characterization of cancer cells in comparison with two dimensional (2D) monolayer. This study underpins various factors that cause resistance to anticancer drugs in forms of spheroids in 3D in-vitro cell culture and also outlines key challenges and possible solutions for the future development of these systems.

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