scholarly journals Liver Progenitor Cells Develop Cholangiocyte-Type Epithelial Polarity in Three-dimensional Culture

2007 ◽  
Vol 18 (4) ◽  
pp. 1472-1479 ◽  
Author(s):  
Naoki Tanimizu ◽  
Atsushi Miyajima ◽  
Keith E. Mostov
Keyword(s):  
Bile Duct ◽  
Molecular Mechanism ◽  
Basolateral Membrane ◽  
Three Dimensional ◽  
3D Culture ◽  
Cytokeratin 19 ◽  
Epithelial Polarity ◽  
Extracellular Matrix Components ◽  
Duct Development

Cholangiocytes are cellular components of the bile duct system of the liver, which originate from hepatoblasts during embryonic liver development. Although several transcription factors and signaling molecules have been implicated in bile duct development, its molecular mechanism has not been studied in detail. Here, we applied a three-dimensional (3D) culture technique to a liver progenitor cell line, HPPL, to establish an in vitro culture system in which HPPL acquire differentiated cholangiocyte characteristics. When HPPL were grown in a gel containing Matrigel, which contains extracellular matrix components of basement membrane, HPPL developed apicobasal polarity and formed cysts, which had luminal space inside. In the cysts, F-actin bundles and atypical protein kinase C were at the apical membrane, E-cadherin was localized at the lateral membrane, and β-catenin and integrin α6 were located at the basolateral membrane. HPPL in cysts expressed cholangiocyte markers, including cytokeratin 19, integrin β4, and aquaporin-1, but not a hepatocyte marker, albumin. Furthermore, HPPL transported rhodamine 123, a substrate for multidrug resistance gene products, from the basal side to the central lumen. These data indicate that HPPL develop cholangiocyte-type epithelial polarity in 3D culture. Phosphatidylinositol 3-kinase signaling was essential for proliferation and survival of HPPL in culture, whereas laminin-1 was a crucial component of Matrigel for inducing epithelial polarization of HPPL. Because HPPL cysts display structural and functional similarities with bile ducts, the 3D culture of HPPL recapitulates in vivo cholangiocyte differentiation and is useful to study the molecular mechanism of bile duct development in vitro.

scholarly journals Crosstalk between invadopodia and the extracellular matrix

2020 ◽  
Author(s):  
Shinji Iizuka ◽  
Ronald P. Leon ◽  
Kyle P. Gribbin ◽  
Ying Zhang ◽  
Jose Navarro ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Type I Collagen ◽  
Complex Structure ◽  
Three Dimensional ◽  
3D Culture ◽  
Model Systems ◽  
Type I ◽  
Extracellular Matrix Components

ABSTRACTThe scaffold protein Tks5α is required for invadopodia-mediated cancer invasion both in vitro and in vivo. We have previously also revealed a role for Tks5 in tumor cell growth using three-dimensional (3D) culture model systems and mouse transplantation experiments. Here we use both 3D and high-density fibrillar collagen (HDFC) culture to demonstrate that native type I collagen, but not a form lacking the telopeptides, stimulated Tks5-dependent growth, which was dependent on the DDR collagen receptors. We used microenvironmental microarray (MEMA) technology to determine that laminin, collagen I, fibronectin and tropoelastin also stimulated invadopodia formation. A Tks5α-specific monoclonal antibody revealed its expression both on microtubules and at invadopodia. High- and super-resolution microscopy of cells in and on collagen was then used to place Tks5α at the base of invadopodia, separated from much of the actin and cortactin, but coincident with both matrix metalloprotease and cathepsin proteolytic activity. Inhibition of the Src family kinases, cathepsins or metalloproteases all reduced invadopodia length but each had distinct effects on Tks5α localization. These studies highlight the crosstalk between invadopodia and extracellular matrix components, and reveal the invadopodium to be a spatially complex structure.

Three-dimensional systems for in vitro follicular culture: overview of alginate-based matrices

2014 ◽  
Vol 26 (7) ◽  
pp. 915 ◽  
Author(s):  
Ivina R. Brito ◽  
Isadora M. T. Lima ◽  
Min Xu ◽  
Lonnie D. Shea ◽  
Teresa K. Woodruff ◽  
...  
Keyword(s):  
Ovarian Follicle ◽  
Three Dimensional ◽  
3D Culture ◽  
Culture Conditions ◽  
Physical Interaction ◽  
Oocyte Growth ◽  
Follicle Culture ◽  
Culture Systems ◽  
Extracellular Matrix Components

The in vitro culture of ovarian follicles has provided critical insight into the biology of the follicle and its enclosed oocyte and the physical interaction and communication between the theca and granulosa cells and the oocyte that is necessary to produce meiotically competent oocytes. Various two-dimensional (2D) and three-dimensional (3D) culture systems have been developed to evaluate the effect of growth factors, hormones, extracellular matrix components and culture conditions on follicle development and oocyte growth and maturation. Among these culture systems, 3D systems make it possible to maintain follicle structure and support communication between the various cell compartments within the follicle. In this review article, we will discuss the three main approaches to ovarian follicle culture: 2D attachment systems, 3D floating systems and 3D encapsulated systems. We will specifically emphasise the development of and advances in alginate-based encapsulated systems for in vitro follicle culture.

3D Culture Modelling: An Emerging Approach for Translational Cancer Research in Sarcomas

2020 ◽  
Vol 27 (29) ◽  
pp. 4778-4788 ◽  
Author(s):  
Victoria Heredia-Soto ◽  
Andrés Redondo ◽  
José Juan Pozo Kreilinger ◽  
Virginia Martínez-Marín ◽  
Alberto Berjón ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Cancer Biology ◽  
Soft Tissues ◽  
Three Dimensional ◽  
3D Culture ◽  
Resistance Mechanisms ◽  
In Vitro Models ◽  
Tumour Spheroids ◽  
Current Therapies

Sarcomas are tumours of mesenchymal origin, which can arise in bone or soft tissues. They are rare but frequently quite aggressive and with a poor outcome. New approaches are needed to characterise these tumours and their resistance mechanisms to current therapies, responsible for tumour recurrence and treatment failure. This review is focused on the potential of three-dimensional (3D) in vitro models, including multicellular tumour spheroids (MCTS) and organoids, and the latest data about their utility for the study on important properties for tumour development. The use of spheroids as a particularly valuable alternative for compound high throughput screening (HTS) in different areas of cancer biology is also discussed, which enables the identification of new therapeutic opportunities in commonly resistant tumours.

scholarly journals Interaction of phosphorylated Rab11-FIP2 with Eps15 regulates apical junction composition

2017 ◽  
Vol 28 (8) ◽  
pp. 1088-1100 ◽  
Author(s):  
Lynne A. Lapierre ◽  
Elizabeth H. Manning ◽  
Kenya M. Mitchell ◽  
Cathy M. Caldwell ◽  
James R. Goldenring
Keyword(s):  
Mdck Cells ◽  
Three Dimensional ◽  
3D Culture ◽  
Cyst Formation ◽  
Epithelial Polarity ◽  
Lateral Membrane ◽  
Low Calcium ◽  
Single Lumen ◽  
Apical Junctions ◽  
Temporal Localization

MARK2 regulates the establishment of polarity in Madin–Darby canine kidney (MDCK) cells in part through phosphorylation of serine 227 of Rab11-FIP2. We identified Eps15 as an interacting partner of phospho-S227-Rab11-FIP2 (pS227-FIP2). During recovery from low calcium, Eps15 localized to the lateral membrane before pS227-FIP2 arrival. Later in recovery, Eps15 and pS227-FIP2 colocalized at the lateral membrane. In MDCK cells expressing the pseudophosphorylated FIP2 mutant FIP2(S227E), during recovery from low calcium, Eps15 was trapped and never localized to the lateral membrane. Mutation of any of the three NPF domains within GFP-FIP2(S227E) rescued Eps15 localization at the lateral membrane and reestablished single-lumen cyst formation in GFP-FIP2(S227E)–expressing cells in three-dimensional (3D) culture. Whereas expression of GFP-FIP2(S227E) induced the loss of E-cadherin and occludin, mutation of any of the NPF domains of GFP-FIP2(S227E) reestablished both proteins at the apical junctions. Knockdown of Eps15 altered the spatial and temporal localization of pS227-FIP2 and also elicited formation of multiple lumens in MDCK 3D cysts. Thus an interaction of Eps15 and pS227-FIP2 at the appropriate time and location in polarizing cells is necessary for proper establishment of epithelial polarity.

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 Beyond 3D culture models of cancer

2015 ◽  
Vol 7 (283) ◽  
pp. 283ps9-283ps9 ◽  
Author(s):  
Kandice Tanner ◽  
Michael M. Gottesman
Keyword(s):  
In Vitro Culture ◽  
Three Dimensional ◽  
3D Culture ◽  
Drug Efficacy ◽  
Spatiotemporal Evolution ◽  
Culture Models ◽  
Dynamic Interplay

The mechanisms underlying the spatiotemporal evolution of tumor ecosystems present a challenge in evaluating drug efficacy. In this Perspective, we address the use of three-dimensional in vitro culture models to delineate the dynamic interplay between the tumor and the host microenvironment in an effort to attain realistic platforms for assessing pharmaceutical efficacy in patients.

scholarly journals Comparison of Immunosuppressive and Angiogenic Properties of Human Amnion-Derived Mesenchymal Stem Cells between 2D and 3D Culture Systems

2019 ◽  
Vol 2019 ◽  
pp. 1-16 ◽  
Author(s):  
Vitale Miceli ◽  
Mariangela Pampalone ◽  
Serena Vella ◽  
Anna Paola Carreca ◽  
Giandomenico Amico ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Three Dimensional ◽  
3D Culture ◽  
Culture Method ◽  
Paracrine Factors ◽  
Human Amnion ◽  
Tissue Repair And Regeneration ◽  
3D Culture System

The secretion of potential therapeutic factors by mesenchymal stem cells (MSCs) has aroused much interest given the benefits that it can bring in the field of regenerative medicine. Indeed, the in vitro multipotency of these cells and the secretive capacity of both angiogenic and immunomodulatory factors suggest a role in tissue repair and regeneration. However, during culture, MSCs rapidly lose the expression of key transcription factors associated with multipotency and self-renewal, as well as the ability to produce functional paracrine factors. In our study, we show that a three-dimensional (3D) culture method is effective to induce MSC spheroid formation, to maintain the multipotency and to improve the paracrine activity of a specific population of human amnion-derived MSCs (hAMSCs). The regenerative potential of both 3D culture-derived conditioned medium (3D CM) and their exosomes (EXO) was assessed against 2D culture products. In particular, tubulogenesis assays revealed increased capillary maturation in the presence of 3D CM compared with both 2D CM and 2D EXO. Furthermore, 3D CM had a greater effect on inhibition of PBMC proliferation than both 2D CM and 2D EXO. To support this data, hAMSC spheroids kept in our 3D culture system remained viable and multipotent and secreted considerable amounts of both angiogenic and immunosuppressive factors, which were detected at lower levels in 2D cultures. This work reveals the placenta as an important source of MSCs that can be used for eventual clinical applications as cell-free therapies.

scholarly journals Three-Dimensional Spheroids as In Vitro Preclinical Models for Cancer Research

Pharmaceutics ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1186
Author(s):  
Bárbara Pinto ◽  
Ana C. Henriques ◽  
Patrícia M. A. Silva ◽  
Hassan Bousbaa
Keyword(s):  
Cancer Research ◽  
Low Cost ◽  
Three Dimensional ◽  
3D Culture ◽  
Comprehensive Overview ◽  
Culture Techniques ◽  
Drug Candidates ◽  
In Vivo Testing

Most cancer biologists still rely on conventional two-dimensional (2D) monolayer culture techniques to test in vitro anti-tumor drugs prior to in vivo testing. However, the vast majority of promising preclinical drugs have no or weak efficacy in real patients with tumors, thereby delaying the discovery of successful therapeutics. This is because 2D culture lacks cell–cell contacts and natural tumor microenvironment, important in tumor signaling and drug response, thereby resulting in a reduced malignant phenotype compared to the real tumor. In this sense, three-dimensional (3D) cultures of cancer cells that better recapitulate in vivo cell environments emerged as scientifically accurate and low cost cancer models for preclinical screening and testing of new drug candidates before moving to expensive and time-consuming animal models. Here, we provide a comprehensive overview of 3D tumor systems and highlight the strategies for spheroid construction and evaluation tools of targeted therapies, focusing on their applicability in cancer research. Examples of the applicability of 3D culture for the evaluation of the therapeutic efficacy of nanomedicines are discussed.

scholarly journals Spheroids as a Type of Three-Dimensional Cell Cultures—Examples of Methods of Preparation and the Most Important Application

2020 ◽  
Vol 21 (17) ◽  
pp. 6225 ◽  
Author(s):  
Kamila Białkowska ◽  
Piotr Komorowski ◽  
Maria Bryszewska ◽  
Katarzyna Miłowska
Keyword(s):  
Cell Cultures ◽  
Cell Biology ◽  
Disease Modeling ◽  
Three Dimensional ◽  
3D Culture ◽  
Matrix Interactions ◽  
Vitro Model ◽  
Extracellular Matrix Interactions ◽  
Natural Cell

Cell cultures are very important for testing materials and drugs, and in the examination of cell biology and special cell mechanisms. The most popular models of cell culture are two-dimensional (2D) as monolayers, but this does not mimic the natural cell environment. Cells are mostly deprived of cell–cell and cell–extracellular matrix interactions. A much better in vitro model is three-dimensional (3D) culture. Because many cell lines have the ability to self-assemble, one 3D culturing method is to produce spheroids. There are several systems for culturing cells in spheroids, e.g., hanging drop, scaffolds and hydrogels, and these cultures have their applications in drug and nanoparticles testing, and disease modeling. In this paper we would like to present methods of preparation of spheroids in general and emphasize the most important applications.

scholarly journals VIP17/MAL expression modulates epithelial cyst formation and ciliogenesis

2012 ◽  
Vol 303 (8) ◽  
pp. C862-C871 ◽  
Author(s):  
Vinita Takiar ◽  
Kavita Mistry ◽  
Monica Carmosino ◽  
Nicole Schaeren-Wiemers ◽  
Michael J. Caplan
Keyword(s):  
Epithelial Cells ◽  
Primary Cilium ◽  
Three Dimensional ◽  
3D Culture ◽  
Cyst Formation ◽  
Unknown Etiology ◽  
Development In Vitro ◽  
Renal Cystic Diseases

The polarized organization of epithelial cells is required for vectorial solute transport and may be altered in renal cystic diseases. Vesicle integral protein of 17 kDa (VIP17/MAL) is involved in apical vesicle transport. VIP17/MAL overexpression in vivo results in renal cystogenesis of unknown etiology. Renal cystogenesis can occur as a consequence of defects of the primary cilium. To explore the role of VIP17/MAL in renal cystogenesis and ciliogenesis, we examined the polarization and ciliary morphology of wild-type and VIP17/MAL overexpressing Madin-Darby canine kidney renal epithelial cells grown in two-dimensional (2D) and three-dimensional (3D) cyst culture. VIP17/MAL is apically localized when expressed in cells maintained in 2D and 3D culture. VIP17/MAL overexpressing cells produce more multilumen cysts compared with controls. While the distributions of basolateral markers are not affected, VIP17/MAL expression results in aberrant sorting of the apical marker gp135 to the primary cilium. VIP17/MAL overexpression is also associated with shortened or absent cilia. Immunofluorescence analysis performed on kidney sections from VIP17/MAL transgenic mice also demonstrates fewer and shortened cilia within dilated lumens ( P < 0.01). These studies demonstrate that VIP17/MAL overexpression results in abnormal cilium and cyst development, in vitro and in vivo, suggesting that VIP17/MAL overexpressing mice may develop cysts secondary to a ciliary defect.

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