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

Materials ◽  
2019 ◽  
Vol 12 (20) ◽  
pp. 3308 ◽  
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.

scholarly journals Artificial Tumor Microenvironments in Neuroblastoma

Cancers ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1629
Author(s):  
Colin H. Quinn ◽  
Andee M. Beierle ◽  
Elizabeth A. Beierle
Keyword(s):  
Extracellular Matrix ◽  
Three Dimensional ◽  
Therapeutic Interventions ◽  
3D Bioprinting ◽  
Culture Studies ◽  
In Vivo Models ◽  
Novel Treatments ◽  
Tumor Microenvironments ◽  
Novel Approach

In the quest to advance neuroblastoma therapeutics, there is a need to have a deeper understanding of the tumor microenvironment (TME). From extracellular matrix proteins to tumor associated macrophages, the TME is a robust and diverse network functioning in symbiosis with the solid tumor. Herein, we review the major components of the TME including the extracellular matrix, cytokines, immune cells, and vasculature that support a more aggressive neuroblastoma phenotype and encumber current therapeutic interventions. Contemporary treatments for neuroblastoma are the result of traditional two-dimensional culture studies and in vivo models that have been translated to clinical trials. These pre-clinical studies are costly, time consuming, and neglect the study of cofounding factors such as the contributions of the TME. Three-dimensional (3D) bioprinting has become a novel approach to studying adult cancers and is just now incorporating portions of the TME and advancing to study pediatric solid. We review the methods of 3D bioprinting, how researchers have included TME pieces into the prints, and highlight present studies using neuroblastoma. Ultimately, incorporating the elements of the TME that affect neuroblastoma responses to therapy will improve the development of innovative and novel treatments. The use of 3D bioprinting to achieve this aim will prove useful in developing optimal therapies for children with neuroblastoma.

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

2019 ◽  
Vol 9 (1) ◽  
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.

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

Chemosensors ◽  
2019 ◽  
Vol 7 (1) ◽  
pp. 6 ◽  
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.

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

2018 ◽  
Author(s):  
Madoka Imura ◽  
Ryota Sakiyama ◽  
Koji Yamamoto ◽  
Yusuke Morita ◽  
Eiji Nakamachi
Keyword(s):  
Pc12 Cells ◽  
Three Dimensional ◽  
Collagen Gel ◽  
3D Cell Culture ◽  
Cyclic Stretch ◽  
Uniform Strain ◽  
Enhancement Effect ◽  
Environmental Stimulation ◽  
Axonal Extension

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.

scholarly journals Lipodendriplexes mediated enhanced gene delivery: a cellular to pre-clinical investigation

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Imran Tariq ◽  
Muhammad Yasir Ali ◽  
Muhammad Farhan Sohail ◽  
Muhammad Umair Amin ◽  
Sajid Ali ◽  
...  
Keyword(s):  
Cell Culture ◽  
Gene Delivery ◽  
Cell Viability ◽  
3D Cell Culture ◽  
Serum Biochemistry ◽  
Cellular Protein ◽  
Ros Generation ◽  
Gfp Expression

AbstractClinical success of effective gene therapy is mainly hampered by the insufficiency of safe and efficient internalization of a transgene to the targeted cellular site. Therefore, the development of a safe and efficient nanocarrier system is one of the fundamental challenges to transfer the therapeutic genes to the diseased cells. Polyamidoamine (PAMAM) dendrimer has been used as an efficient non-viral gene vector (dendriplexes) but the toxicity and unusual biodistribution induced by the terminal amino groups (–NH2) limit its in vivo applications. Hence, a state of the art lipid modification with PAMAM based gene carrier (lipodendriplexes) was planned to investigate theirs in vitro (2D and 3D cell culture) and in vivo behaviour. In vitro pDNA transfection, lactate dehydrogenase (LDH) release, reactive oxygen species (ROS) generation, cellular protein contents, live/dead staining and apoptosis were studied in 2D cell culture of HEK-293 cells while GFP transfection, 3D cell viability and live/dead staining of spheroids were performed in its 3D cell culture. Acute toxicity studies including organ to body index ratio, hematological parameters, serum biochemistry, histopathological profiles and in vivo transgene expression were assessed in female BALB/c mice. The results suggested that, in comparison to dendriplexes the lipodendriplexes exhibited significant improvement of pDNA transfection (p < 0.001) with lower LDH release (p < 0.01) and ROS generation (p < 0.05). A substantially higher cellular protein content (p < 0.01) and cell viability were also observed in 2D culture. A strong GFP expression with an improved cell viability profile (p < 0.05) was indicated in lipodendriplexes treated 3D spheroids. In vivo archives showed the superiority of lipid-modified nanocarrier system, depicted a significant increase in green fluorescent protein (GFP) expression in the lungs (p < 0.01), heart (p < 0.001), liver (p < 0.001) and kidneys (p < 0.001) with improved serum biochemistry and hematological profile as compared to unmodified dendriplexes. No tissue necrosis was evident in the animal groups treated with lipid-shielded molecules. Therefore, a non-covalent conjugation of lipids with PAMAM based carrier system could be considered as a promising approach for an efficient and biocompatible gene delivery system.

Tissue Engineered Human Septal Cartilage in a Murine Model

2008 ◽  
Vol 139 (2_suppl) ◽  
pp. P87-P88
Author(s):  
Angela Chang ◽  
Sage August ◽  
Barbara L Schumacher ◽  
Williams Gregory ◽  
Robert L Sah ◽  
...  
Keyword(s):  
Cell Viability ◽  
Murine Model ◽  
Three Dimensional ◽  
Alginate Beads ◽  
Septal Cartilage ◽  
Hematoxylin And Eosin ◽  
Matrix Production ◽  
Nasal Septal Cartilage

Problem Tissue engineering of human nasal septal cartilage represents an alternative technique for creating large quantities of autologous material for use in reconstructive surgery of the head and neck. Septal neocartilage constructs developed in vitro by the alginate method have demonstrated cartilaginous extracellular matrix production, but their biocompatibility and development in vivo remains largely unknown. Methods A murine model was used to examine the behavior of neocartilage constructs in vivo. Chondrocytes collected from donors undergoing septoplasty were expanded in monolayer and suspended in alginate beads for three-dimensional culture in media containing human serum and growth factors. After in vitro incubation for 5 weeks, the neocartilage constructs were implanted subcutaneously in the dorsum of athymic mice for 30 days (n=3). The mice were sacrificed and the constructs were explanted for assessment of cell viability, gross morphology, and histology. Results The mice survived and tolerated the implant well. Infection and extrusion were not observed. Neocartilage constructs maintained their general shape and size, and demonstrated cell viability after implantation. Explanted constructs were firm and opaque, sharing closer semblance to native septal tissue relative to the gelatinous, translucent pre-implant constructs. On hematoxylin and eosin staining, the explanted constructs exhibited distinct morphologies characteristic of native tissue, which were not observed in pre-implant constructs. Conclusion Neocartilage constructs are viable in an in vivo murine model. The morphologic and histologic features of explanted constructs more closely resemble native septal tissue when compared to pre-implant constructs. Significance Septal neocartilage constructs are biocompatible and demonstrate potential for in vivo maturation with eventual clinical application.

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

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

2019 ◽  
Vol 25 (34) ◽  
pp. 3599-3607 ◽  
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.

scholarly journals In Vivo Tomographic Imaging of Near-Infrared Fluorescent Probes

2002 ◽  
Vol 1 (2) ◽  
pp. 153535002002011
Author(s):  
Vasilis Ntziachristos ◽  
Christoph Bremer ◽  
Edward E. Graves ◽  
Jorge Ripoll ◽  
Ralph Weissleder
Keyword(s):  
Gene Expression ◽  
Protein Function ◽  
Near Infrared ◽  
Imaging Technique ◽  
Three Dimensional ◽  
Tomographic Imaging ◽  
Molecular Function ◽  
Fluorescence Reflectance Imaging ◽  
Spatial Congruence

Fluorescence imaging is increasingly used to probe protein function and gene expression in live animals. This technology could enhance the study of pathogenesis, drug development, and therapeutic intervention. In this article, we focus on three-dimensional fluorescence observations using fluorescence-mediated molecular tomography (FMT), a novel imaging technique that can resolve molecular function in deep tissues by reconstructing fluorescent probe distributions in vivo. We have compared FMT findings with conventional fluorescence reflectance imaging (FRI) to study protease function in nude mice with subsurface implanted tumors. This validation of FMT with FRI demonstrated the spatial congruence of fluorochrome activation as determined by the two techniques.

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