scholarly journals Pak1 regulates branching morphogenesis in 3D MDCK cell culture by a PIX and β1-integrin-dependent mechanism

2010 ◽  
Vol 299 (1) ◽  
pp. C21-C32 ◽  
Author(s):  
Michael P. Hunter ◽  
Mirjam M. Zegers
Keyword(s):  
Cell Culture ◽  
Mdck Cell ◽  
Three Dimensional ◽  
3D Culture ◽  
Branching Morphogenesis ◽  
Dominant Negative ◽  
Β1 Integrin ◽  
Blocking Antibody ◽  
Important Regulator ◽  
P21 Activated Kinase

Branching morphogenesis is a fundamental process in the development of the kidney. This process gives rise to a network of ducts, which form the collecting system. Defective branching can lead to a multitude of kidney disorders including agenesis and reduced nephron number. The formation of branching tubules involves changes in cell shape, cell motility, and reorganization of the cytoskeleton. However, the exact intracellular mechanisms involved are far from understood. We have used the three-dimensional (3D) Madin-Darby canine kidney (MDCK) cell culture system to study how p21-activated kinase 1 (Pak1), which is an important regulator of the cytoskeleton, modulates branching. Our data reveal that Pak1 plays a crucial role in regulating branching morphogenesis. Expression of a dominant-negative Pak1 mutant (DN-Pak1) in MDCK cysts resulted in the spontaneous formation of extensions and branching tubules. Cellular contractility and levels of phosphorylated myosin light chain (pMLC) were increased in DN-Pak1 cells in collagen. Expression of a DN-Pak1 mutant that does not bind to PIX (DN-Pak1-ΔPIX) failed to form extensions in collagen and did not have increased contractility. This shows that the DN-Pak1 mutant requires PIX binding to generate extensions and increased contractility in 3D culture. Furthermore, a β1-integrin function-blocking antibody (AIIB2) inhibited the formation of branches and blocked the increased contractility in DN-Pak1 cysts. Taken together, our work shows that DN-Pak1-induced branching morphogenesis requires PIX binding and β1-integrin signaling.

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 Factors to consider when interrogating 3D culture models with plate readers or automated microscopes

2021 ◽  
Author(s):  
Terry Riss ◽  
O. Joseph Trask
Keyword(s):  
Cell Culture ◽  
Three Dimensional ◽  
3D Culture ◽  
Fluorescent Imaging ◽  
Culture Models ◽  
Assay Methods ◽  
Diffusion Rates ◽  
Cell Culture Models ◽  
Dimensional Cell ◽  
Cells Cultured

AbstractAlong with the increased use of more physiologically relevant three-dimensional cell culture models comes the responsibility of researchers to validate new assay methods that measure events in structures that are physically larger and more complex compared to monolayers of cells. It should not be assumed that assays designed using monolayers of cells will work for cells cultured as larger three-dimensional masses. The size and barriers for penetration of molecules through the layers of cells result in a different microenvironment for the cells in the outer layer compared to the center of three-dimensional structures. Diffusion rates for nutrients and oxygen may limit metabolic activity which is often measured as a marker for cell viability. For assays that lyse cells, the penetration of reagents to achieve uniform cell lysis must be considered. For live cell fluorescent imaging assays, the diffusion of fluorescent probes and penetration of photons of light for probe excitation and fluorescent emission must be considered. This review will provide an overview of factors to consider when implementing assays to interrogate three dimensional cell culture models.

A rhabdomyosarcoma hydrogel model to unveil cell-extracellular matrix interactions

2021 ◽  
Author(s):  
Mattia Saggioro ◽  
Stefania D'Agostino ◽  
Anna Gallo ◽  
Sara Crotti ◽  
Sara D'Aronco ◽  
...  
Keyword(s):  
Cell Culture ◽  
Extracellular Matrix ◽  
Three Dimensional ◽  
3D Culture ◽  
3D Cell Culture ◽  
Culture Systems ◽  
Matrix Interactions ◽  
In Vitro Systems ◽  
Extracellular Matrix Interactions

Three-dimensional (3D) culture systems are progressively getting attention given their potential in overcoming limitations of the classical 2D in vitro systems. Among different supports for 3D cell culture, hydrogels (HGs)...

scholarly journals Identification and evaluation of an appropriate housekeeping gene for real-time gene profiling of hepatocellular carcinoma cells cultured in three-dimensional scaffold

2021 ◽  
Author(s):  
Anjana Kaveri Badekila ◽  
Praveen Rai ◽  
Sudarshan Kini
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Culture ◽  
Real Time ◽  
Internal Control ◽  
18S Rrna ◽  
Three Dimensional ◽  
Melting Curve ◽  
3D Culture ◽  
Gene Profiling ◽  
3D Microenvironment

Abstract Assessing an optimal reference gene as an internal control for target gene normalization is important during quantitative real time polymerase chain reaction (RT-qPCR) of three-dimensional cell culture. Especially, gene profiling of cancer cells under a complex 3D microenvironment in a polymer scaffold provides a deeper understanding of recapitulation of in vivo tumors. In this aspect, expression of six housekeeping genes (HKG’s): glyceraldehyde-3-phosphodehydrogenase (GAPDH), β-actin (ACTB), beta-2-microglobulin (B2M), 18S ribosomal RNA (18S rRNA), peptidyl-propyl-isomerase A (PPIA), and ribosomal protein L13 (RPL-13)) during the monolayer culture (two-dimensional), and alginate-carboxymethylcellulose scaffold based three-dimensional (3D) cell culture conditioned up to 21 days was analyzed for hepatocellular carcinoma (Huh-7) cell line. The real-time gene expression using RT-qPCR of HCC spheroids in 3D culture were analyzed by determining the primer efficiency, melting curve and quantification cycle analysis of the selected candidate HKG’s. Further, RT-qPCR data were validated using analysis softwares i.e., geNorm and NormFinder for statistical significance. The study indicated RPL-13, 18S rRNA and B2M to be stable among selected referral HKG candidates and considered them as potential internal controls during varying cell culture conditions.

Hydroxyapatite Nanoparticle-Crosslinked Peptide Hydrogels for Three-Dimensional Culture and Differentiation of MC3T3-E1 Osteoblasts

2019 ◽  
Vol 15 (12) ◽  
pp. 2351-2362
Author(s):  
Yingjie Xu ◽  
Xin Wu ◽  
Shuyi Wang ◽  
Changzhou Yang ◽  
Ying Li ◽  
...  
Keyword(s):  
Cell Culture ◽  
Bone Marrow ◽  
Mechanical Stability ◽  
Bone Marrow Cells ◽  
Three Dimensional ◽  
3D Culture ◽  
Self Assembling ◽  
Hybrid Hydrogels ◽  
Peptide Hydrogels ◽  
Marrow Cells

Hydrogels have been widely used to mimic the biochemical and mechanical environments of native extracellular matrices for cell culture and tissue engineering. Among them, self-assembling peptide hydrogels are of special interest thanks to their great biocompatibility, designability and convenient preparation procedures. In pioneering studies, self-assembling peptide hydrogels have been used for the culture of bone marrow cells. However, the low mechanical stability of peptide hydrogels seems to be a drawback for these applications, as bone marrow cells prefer hard substrates for osteogenic differentiation. In this work, we explored the use of hydroxyapatite (HAP)-peptide hybrid hydrogels for three-dimensional (3D) culture and differentiation of osteogenic MC3T3-E1 cells. We used HAP nanoparticles as crosslinkers to increase the mechanical stability of peptide hydrogels. Meanwhile, HAP provided unique chemical cues to promote the differentiation of osteoblasts. A phosphate group was introduced to the self-assembling peptide so that the peptide fibers could bind to HAP nanoparticles specifically and strongly. Rheological characterization indicated that the hybrid hydrogels were mechanically more stable than the hydrogels containing only peptides and can be used for long term cell culture. Moreover, the hydrogels were biocompatible and showed very low cytotoxicity. The favorable mechanical properties of the hybrid hydrogels and the chemical properties of HAP synergistically supported the differentiation of MC3T3-E1 cells. Based on these characterizations, we believe that these hybrid hydrogels can potentially be used as scaffolds for cartilage and bone regeneration in the future.

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 Biomaterials for Three-Dimensional Cell Culture: From Applications in Oncology to Nanotechnology

Nanomaterials ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 481
Author(s):  
Tarek Saydé ◽  
Omar El Hamoui ◽  
Bruno Alies ◽  
Karen Gaudin ◽  
Gaëtane Lespes ◽  
...  
Keyword(s):  
Cell Culture ◽  
Complex Structure ◽  
Tumor Development ◽  
Three Dimensional ◽  
3D Culture ◽  
Supramolecular Assembly ◽  
Hydrogel Scaffold ◽  
Scale Characterization ◽  
Dimensional Cell

Three-dimensional cell culture has revolutionized cellular biology research and opened the door to novel discoveries in terms of cellular behavior and response to microenvironment stimuli. Different types of 3D culture exist today, including hydrogel scaffold-based models, which possess a complex structure mimicking the extracellular matrix. These hydrogels can be made of polymers (natural or synthetic) or low-molecular weight gelators that, via the supramolecular assembly of molecules, allow the production of a reproducible hydrogel with tunable mechanical properties. When cancer cells are grown in this type of hydrogel, they develop into multicellular tumor spheroids (MCTS). Three-dimensional (3D) cancer culture combined with a complex microenvironment that consists of a platform to study tumor development and also to assess the toxicity of physico-chemical entities such as ions, molecules or particles. With the emergence of nanoparticles of different origins and natures, implementing a reproducible in vitro model that consists of a bio-indicator for nano-toxicity assays is inevitable. However, the maneuver process of such a bio-indicator requires the implementation of a repeatable system that undergoes an exhaustive follow-up. Hence, the biggest challenge in this matter is the reproducibility of the MCTS and the associated full-scale characterization of this system’s components.

scholarly journals Identification and Evaluation of an Appropriate Housekeeping Gene for Real-Time Gene Profiling of Hepatocellular Carcinoma Cells Cultured in Three-Dimensional Scaffold

2021 ◽  
Author(s):  
Anjana Kaveri Badekila ◽  
Praveen Rai ◽  
Sudarshan Kini
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Culture ◽  
Real Time ◽  
Internal Control ◽  
18S Rrna ◽  
Three Dimensional ◽  
Melting Curve ◽  
3D Culture ◽  
Gene Profiling ◽  
3D Microenvironment

Abstract Assessing an optimal reference gene as an internal control for target gene normalization is important during quantitative real time polymerase chain reaction (RT-qPCR) of three-dimensional cell culture. Especially, gene profiling of cancer cells under a complex 3D microenvironment in a polymer scaffold provides a deeper understanding of recapitulation of in vivo tumors. In this aspect, expression of six housekeeping genes (HKG’s): glyceraldehyde-3-phosphodehydrogenase (GAPDH), β-actin (ACTB), beta-2-microglobulin (B2M), 18S ribosomal RNA (18S rRNA), peptidyl-propyl-isomerase A (PPIA), and ribosomal protein L13 (RPL-13)) during the monolayer culture (two-dimensional), and alginate-carboxymethylcellulose scaffold based three-dimensional (3D) cell culture conditioned up to 21 days was analysed for hepatocellular carcinoma (Huh-7) cell line. The real-time gene expression using RT-qPCR of HCC spheroids in 3D culture were analyzed by determining the primer efficiency, melting curve and quantification cycle analysis of the selected candidate HKG’s. Further, RT-qPCR data were validated using analysis softwares i.e., geNorm and NormFinder for statistical significance. The study indicated RPL-13, 18S rRNA and B2M to be stable among selected referral HKG candidates and considered them as potential internal controls during varying cell culture conditions.

scholarly journals Two-Dimensional and Three-Dimensional Cell Culture and Their Applications

2021 ◽  
Author(s):  
Sangeeta Ballav ◽  
Ankita Jaywant Deshmukh ◽  
Shafina Siddiqui ◽  
Jyotirmoi Aich ◽  
Soumya Basu
Keyword(s):  
Cell Culture ◽  
Cell Biology ◽  
Low Cost ◽  
Three Dimensional ◽  
3D Culture ◽  
Two Dimensional ◽  
Living Body ◽  
Preclinical Trials ◽  
Dimensional Cell

Cell culture is one of the most important and commonly used in vitro tools to comprehend various aspects of cells or tissues of a living body such as cell biology, tissue morphology, mechanism of diseases, cell signaling, drug action, cancer research and also finds its great importance in preclinical trials of various drugs. There are two major types of cell cultures that are most commonly used- two-dimensional (2D) and three-dimensional culture (3D). The former has been used since the 1900s, owing to its simplicity and low-cost maintenance as it forms a monolayer, while the latter being the advanced version and currently most worked upon. This chapter intends to provide the true meaning and significance to both cultures. It starts by making a clear distinction between the two and proceeds further to discuss their different applications in vitro. The significance of 2D culture is projected through different assays and therapeutic treatment to understand cell motility and treatment of diseases, whereas 3D culture includes different models and spheroid structures consisting of multiple layers of cells, and puts a light on its use in drug discovery and development. The chapter is concluded with a detailed account of the production of therapeutic proteins by the use of cells.

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