An Improved Transwell Design for Microelectrode Ion-Flux Measurements

The microelectrode ion flux estimation (MIFE) is a powerful, non-invasive electrophysiological method for cellular membrane transport studies. Usually, the MIFE measurements are performed in a tissue culture dish or directly with tissues (roots, parts of the plants, and cell tissues). Here, we present a transwell system that allows for MIFE measurements on a cell monolayer. We introduce a measurement window in the transwell insert membrane, which provides direct access for the cells to the media in the upper and lower compartment of the transwell system and allows direct cell-to-cell contact coculture. Three-dimensional multiphoton lithography (MPL) was used to construct a 3D grid structure for cell support in the measurement window. The optimal polymer grid constant was found for implementation in transwell MIFE measurements. We showed that human umbilical vein endothelial cells (HUVECs) efficiently grow and maintain their physiological response on top of the polymer structures.

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Development of three-dimensional articular cartilage construct using silica nano-patterned substrate

10.1101/472332 ◽

2018 ◽

Author(s):

In-Su Park ◽

Ye Ji Choi ◽

Bo Ram Song ◽

Hyo-Sop Kim ◽

Sang-Hyug Park ◽

...

Keyword(s):

Stem Cells ◽

Particle Size ◽

Culture System ◽

Cell Monolayer ◽

Three Dimensional ◽

3D Culture ◽

Cell Contact ◽

Spheroid Culture ◽

Cell Substrate ◽

Cell Cell

AbstractCurrent strategies for cartilage cell therapy are mostly based on the use of autologous chondrocytes or mesenchymal stem cells (MSCs). However, these cells have limitations of a small number of cells available and of low chondrogenic ability, respectively. Many studies now suggest that fetal stem cells are more plastic than adult stem cells and can therefore more efficiently differentiate into target tissues. This study introduces, efficiency chondrogenic differentiation of fetal cartilage-derived progenitor cells (FCPCs) to adult cells can be achieved using a three-dimensional (3D) spheroid culture method based on silica nanopatterning techniques. In evaluating the issue of silica nano-particle size (Diameter of 300, 750, 1200 nm), each particle size was coated into the well of a 6-well tissue culture plate. FCPCs (2 x 105 cells/well in 6-well plate) were seeded in each well with chondrogenic medium. In this studys, the 300 nm substrate that formed multi-spheroids and the 1200 nm substrate that showed spreading were due to the cell-cell adhesion force(via N-cadherin) and cell-substrate(via Integrin) force, the 750 nm substrate that formed the mass-aggregation can be interpreted as the result of cell monolayer formation through cell-substrate force followed by cell-cell contact force contraction. We conclude that our 3D spheroid culture system contributes to an optimization for efficient differentiation of FCPC, offers insight into the mechanism of efficient differentiation of engineered 3D culture system, and has promise for wide applications in regeneration medicine and drug discovery fields.

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Cell-surface plasminogen activation causes a retraction of in vitro cultured human umbilical vein endothelial cell monolayer

Blood ◽

10.1182/blood.v83.4.994.bloodjournal834994 ◽

1994 ◽

Vol 83 (4) ◽

pp. 994-1005

Author(s):

G Conforti ◽

C Dominguez-Jimenez ◽

E Ronne ◽

G Hoyer-Hansen ◽

E Dejana

Keyword(s):

Cell Surface ◽

Umbilical Vein ◽

Cell Monolayer ◽

Cell Contact ◽

Specific Cell ◽

Human Umbilical Vein ◽

Cell Contacts ◽

Cell Matrix ◽

Circulating Cells ◽

Cell Cell

Vascular endothelium forms a dynamic interface between blood and underlying tissues. Endothelial monolayer integrity is required for controlled vascular permeability and to preclude exposure of subendothelial cell matrix to circulating cells. Recent studies have established that cultured human umbilical vein endothelial cells (ECs) express receptors for plasminogen (plg) and urokinase-like plasminogen activator (uPA). In the present study, we provide evidence that in EC, uPA receptor is present in focal contacts and at cell-cell contact sites. In these cells, addition of plg and uPA to confluent EC generates a retraction of the monolayer that is evidenced by loss of cell-cell contacts and increase in monolayer permeability. The phenomenon is reversible even after 6 hours of plg-uPA treatment. Inhibition of plg-uPA effect is obtained with plasmin inhibitors, as well as reagents that block binding of uPA or plg to the cell surface. The retractive effect of plg-uPA is concomitant to surface activation of plasminogen and to the loss of cell-cell activation of plg can induce EC retraction, possibly by causing proteolysis at specific cell-cell contacts and cell-matrix sites. This process may be important in mediating the passage of metastatic tumor cells through an intact EC monolayer as well as in regulating contacts between circulating cells and endothelium.

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Cell-surface plasminogen activation causes a retraction of in vitro cultured human umbilical vein endothelial cell monolayer

Blood ◽

10.1182/blood.v83.4.994.994 ◽

1994 ◽

Vol 83 (4) ◽

pp. 994-1005 ◽

Cited By ~ 38

Author(s):

G Conforti ◽

C Dominguez-Jimenez ◽

E Ronne ◽

G Hoyer-Hansen ◽

E Dejana

Keyword(s):

Cell Surface ◽

Umbilical Vein ◽

Cell Monolayer ◽

Cell Contact ◽

Specific Cell ◽

Human Umbilical Vein ◽

Cell Contacts ◽

Cell Matrix ◽

Circulating Cells ◽

Cell Cell

Abstract Vascular endothelium forms a dynamic interface between blood and underlying tissues. Endothelial monolayer integrity is required for controlled vascular permeability and to preclude exposure of subendothelial cell matrix to circulating cells. Recent studies have established that cultured human umbilical vein endothelial cells (ECs) express receptors for plasminogen (plg) and urokinase-like plasminogen activator (uPA). In the present study, we provide evidence that in EC, uPA receptor is present in focal contacts and at cell-cell contact sites. In these cells, addition of plg and uPA to confluent EC generates a retraction of the monolayer that is evidenced by loss of cell-cell contacts and increase in monolayer permeability. The phenomenon is reversible even after 6 hours of plg-uPA treatment. Inhibition of plg-uPA effect is obtained with plasmin inhibitors, as well as reagents that block binding of uPA or plg to the cell surface. The retractive effect of plg-uPA is concomitant to surface activation of plasminogen and to the loss of cell-cell activation of plg can induce EC retraction, possibly by causing proteolysis at specific cell-cell contacts and cell-matrix sites. This process may be important in mediating the passage of metastatic tumor cells through an intact EC monolayer as well as in regulating contacts between circulating cells and endothelium.

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In vitro Three Dimensional Scaffold-free Construct of Human Adipose-derived Stem Cells in Coculture with Endothelial Cells and Fibroblasts

Revista de Chimie ◽

10.37358/rc.17.6.5670 ◽

2017 ◽

Vol 68 (6) ◽

pp. 1341-1344

Author(s):

Grigore Berea ◽

Gheorghe Gh. Balan ◽

Vasile Sandru ◽

Paul Dan Sirbu

Keyword(s):

Tissue Engineering ◽

Stem Cells ◽

Endothelial Cells ◽

Single Cell ◽

Cell Line ◽

Bone Repair ◽

Umbilical Vein ◽

Human Fibroblasts ◽

Three Dimensional ◽

Adipose Derived Stem Cells

Complex interactions between stem cells, vascular cells and fibroblasts represent the substrate of building microenvironment-embedded 3D structures that can be grafted or added to bone substitute scaffolds in tissue engineering or clinical bone repair. Human Adipose-derived Stem Cells (hASCs), human umbilical vein endothelial cells (HUVECs) and normal dermal human fibroblasts (NDHF) can be mixed together in three dimensional scaffold free constructs and their behaviour will emphasize their potential use as seeding points in bone tissue engineering. Various combinations of the aforementioned cell lines were compared to single cell line culture in terms of size, viability and cell proliferation. At 5 weeks, viability dropped for single cell line spheroids while addition of NDHF to hASC maintained the viability at the same level at 5 weeks Fibroblasts addition to the 3D construct of stem cells and endothelial cells improves viability and reduces proliferation as a marker of cell differentiation toward osteogenic line.

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Conductive GelMA–Collagen–AgNW Blended Hydrogel for Smart Actuator

Polymers ◽

10.3390/polym13081217 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1217

Author(s):

Jang Ho Ha ◽

Jae Hyun Lim ◽

Ji Woon Kim ◽

Hyeon-Yeol Cho ◽

Seok Geun Jo ◽

...

Keyword(s):

Mechanical Properties ◽

Umbilical Vein ◽

Three Dimensional ◽

Silver Nanowire ◽

Structural Deformation ◽

Rheological Analysis ◽

Sem Image ◽

Smart Actuator ◽

Umbilical Vein Endothelial Cells ◽

Electrical Stimuli

Blended hydrogels play an important role in enhancing the properties (e.g., mechanical properties and conductivity) of hydrogels. In this study, we generated a conductive blended hydrogel, which was achieved by mixing gelatin methacrylate (GelMA) with collagen, and silver nanowire (AgNW). The ratio of GelMA, collagen and AgNW was optimized and was subsequently gelated by ultraviolet light (UV) and heat. The scanning electron microscope (SEM) image of the conductive blended hydrogels showed that collagen and AgNW were present in the GelMA hydrogel. Additionally, rheological analysis indicated that the mechanical properties of the conductive GelMA–collagen–AgNW blended hydrogels improved. Biocompatibility analysis confirmed that the human umbilical vein endothelial cells (HUVECs) encapsulated within the three-dimensional (3D), conductive blended hydrogels were highly viable. Furthermore, we confirmed that the molecule in the conductive blended hydrogel was released by electrical stimuli-mediated structural deformation. Therefore, this conductive GelMA–collagen–AgNW blended hydrogel could be potentially used as a smart actuator for drug delivery applications.

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Acoustic Cell Patterning in Hydrogel for Three-Dimensional Cell Network Formation

Micromachines ◽

10.3390/mi12010003 ◽

2020 ◽

Vol 12 (1) ◽

pp. 3

Author(s):

Kyo-in Koo ◽

Andreas Lenshof ◽

Le Thi Huong ◽

Thomas Laurell

Keyword(s):

Network Formation ◽

Umbilical Vein ◽

Three Dimensional ◽

Extrusion Process ◽

Fibroblast Cells ◽

Glass Capillary ◽

Cell Network ◽

Significant Difference ◽

The One ◽

Umbilical Vein Endothelial Cells

In the field of engineered organ and drug development, three-dimensional network-structured tissue has been a long-sought goal. This paper presents a direct hydrogel extrusion process exposed to an ultrasound standing wave that aligns fibroblast cells to form a network structure. The frequency-shifted (2 MHz to 4 MHz) ultrasound actuation of a 400-micrometer square-shaped glass capillary that was continuously perfused by fibroblast cells suspended in sodium alginate generated a hydrogel string, with the fibroblasts aligned in single or quadruple streams. In the transition from the one-cell stream to the four-cell streams, the aligned fibroblast cells were continuously interconnected in the form of a branch and a junction. The ultrasound-exposed fibroblast cells displayed over 95% viability up to day 10 in culture medium without any significant difference from the unexposed fibroblast cells. This acoustofluidic method will be further applied to create a vascularized network by replacing fibroblast cells with human umbilical vein endothelial cells.

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Cell-to-cell contact influences proliferative marker expression and apoptosis in MIN6 cells grown in islet-like structures

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00424.2004 ◽

2005 ◽

Vol 288 (3) ◽

pp. E502-E509 ◽

Cited By ~ 40

Author(s):

Melanie J. Luther ◽

Emma Davies ◽

Dany Muller ◽

Moira Harrison ◽

Adrian J. Bone ◽

...

Keyword(s):

Cellular Proliferation ◽

Kinase Inhibitors ◽

Three Dimensional ◽

Cell Aggregation ◽

Cell Interactions ◽

Cell Phenotype ◽

Cell Contact ◽

Min6 Cells ◽

Primary Mouse ◽

E Cadherin

Cell-to-cell interactions play an important role in the development and maintenance of the β-cell phenotype. Here, we have investigated whether E-cadherin plays a role in regulating the growth of insulin-secreting MIN6 cells configured as three-dimensional islet-like clusters (pseudoislets). Pseudoislets form by cell aggregation rather than by proliferation from individual cells and attain the size of primary mouse islets after ∼7 days of maintenance in culture. E-cadherin is known to mediate homotypic cell adhesion between β-cells and has also been implicated in a number of cellular processes, including proliferation, apoptosis, and differentiation. E-cadherin and its associated intracellular elements, α- and β-catenin, were upregulated in MIN6 pseudoislets. Pseudoislet formation was associated with an increased expression of cyclin-dependent kinase inhibitors and a concomitant downregulation of Ki67, suggesting an overall reduction in cellular proliferation. However, measurements of 5-bromo-2′-deoxyuridine incorporation revealed that there were no differences in the rate of MIN6 cell proliferation whether they were configured as monolayers or as pseudoislets, which is likely to be a result of their being a transformed cell line. Cells within pseudoislets were not necrotic, but apoptosis appeared to be upregulated in the islet-like structures. However, no differential expression of Fas and FasL was detected in monolayers and pseudoislets. These results suggest that cell-to-cell interactions within islet-like structures may initiate antiproliferative and proapoptotic signals.

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Effect of aminaphtone on in vitro vascular permeability and capillary-like maintenance

Phlebology The Journal of Venous Disease ◽

10.1177/0268355517737662 ◽

2017 ◽

Vol 33 (9) ◽

pp. 592-599 ◽

Cited By ~ 1

Author(s):

Francesca Felice ◽

Ester Belardinelli ◽

Alessandro Frullini ◽

Tatiana Santoni ◽

Egidio Imbalzano ◽

...

Keyword(s):

Endothelial Cells ◽

Chronic Venous Insufficiency ◽

Venous Insufficiency ◽

Umbilical Vein ◽

Three Dimensional ◽

Endothelial Permeability ◽

Human Umbilical Vein ◽

Pre Treatment ◽

Umbilical Vein Endothelial Cells

Objectives Aminaphtone, a naphtohydrochinone used in the treatment of capillary disorders, may affect oedema in chronic venous insufficiency. Aim of study is to investigate the effect of aminaphtone on vascular endothelial permeability in vitro and its effects on three-dimensional capillary-like structures formed by human umbilical vein endothelial cells. Method Human umbilical vein endothelial cells were treated with 50 ng/ml VEGF for 2 h and aminaphtone for 6 h. Permeability assay, VE-cadherin expression and Matrigel assay were performed. Results VEGF-induced permeability was significantly decreased by aminaphtone in a range concentration of 1–20 µg/ml. Aminaphtone restored VE-cadherin expression. Finally, 6 h pre-treatment with aminaphtone significantly preserved capillary-like structures formed by human umbilical vein endothelial cells on Matrigel up to 48 h compared to untreated cells. Conclusions Aminaphtone significantly protects endothelium permeability and stabilises endothelial cells organised in capillary-like structures, modulating VE-cadherin expression. These data might explain the clinical benefit of aminaphtone on chronic venous insufficiency.

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Bioinspired nano-ordered liquid membrane for precise molecular separation

10.21203/rs.3.rs-87445/v1 ◽

2020 ◽

Author(s):

Haozhen Dou ◽

Mi Xu ◽

Baoyu Wang ◽

Zhen Zhang ◽

Guobin Wen ◽

...

Keyword(s):

Self Assembly ◽

High Performance ◽

Liquid Membrane ◽

Three Dimensional ◽

Cellular Membrane ◽

Liquid Structure ◽

Long Term Stability ◽

Separation Membranes ◽

Molecular Separation

Abstract Cellular membranes provide ideal archetypes for molecule or ion separations with sub-angstrom scale precision, which are featured with both extremely high permeability and selectivity due to the well-defined membrane protein channels. However, the development of bioinspired membranes with artificial channels for sub-angstrom scale ethylene/ethane (0.416 nm / 0.443 nm) separation remains an uncharted territory and a significant challenge. Herein, a bioinspired nano-ordered liquid membrane is constructed by a facile ion/molecule self-assembly strategy for highly efficient ethylene/ethane separation, which mimics the structure of cellular membrane elegantly and possesses plenty of three-dimensional (3D) nanochannels. The elaborate regulation of non-covalent interactions by optimizing the ion/molecule compositions within membrane confers the nano-ordered liquid structure with interpenetrating and bi-continuous apolar domains and polar domains, which results in the formation of regular carrier wires and enormous 3D interconnected ethylene transport nanochannels. By virtue of these 3D nanochannels, the bioinspired nano-ordered liquid membrane manifests simultaneously super-high selectivity, excellent permeance and long-term stability, which exceeds previously reported ethylene/ethane separation membranes. This methodology in this work for construction of bioinspired membrane with tunable 3D nanochannels through ion/molecule self-assembly will enlighten the design and development of high-performance separation membranes for angstrom/sub-angstrom scale ion or molecule separations.

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Cultivation of hierarchical 3D scaffolds inside a perfusion bioreactor: scaffold design and finite-element analysis of fluid flow

SN Applied Sciences ◽

10.1007/s42452-021-04871-3 ◽

2021 ◽

Vol 3 (12) ◽

Author(s):

Kaylie Sampson ◽

Songmi Koo ◽

Carter Gadola ◽

Anastasiia Vasiukhina ◽

Aditya Singh ◽

...

Keyword(s):

Cell Proliferation ◽

Perfusion Bioreactor ◽

Osteoporotic Bone ◽

Scaffold Design ◽

Culture Dish ◽

Element Analysis ◽

3D Scaffolds ◽

Tissue Culture Dish ◽

Thermally Induced ◽

Bone Nonunion

AbstractThe use of porous 3D scaffolds for the repair of bone nonunion and osteoporotic bone is currently an area of great interest. Using a combination of thermally-induced phase separation (TIPS) and 3D-plotting (3DP), we have generated hierarchical 3DP/TIPS scaffolds made of poly(lactic-co-glycolic acid) (PLGA) and nanohydroxyapatite (nHA). A full factorial design of experiments was conducted, in which the PLGA and nHA compositions were varied between 6‒12% w/v and 10‒40% w/w, respectively, totaling 16 scaffold formulations with an overall porosity ranging between 87%‒93%. These formulations included an optimal scaffold design identified in our previous study. The internal structures of the scaffolds were examined using scanning electron microscopy and microcomputed tomography. Our optimal scaffold was seeded with MC3T3-E1 murine preosteoblastic cells and subjected to cell culture inside a tissue culture dish and a perfusion bioreactor. The results were compared to those of a commercial CellCeram™ scaffold with a composition of 40% β-tricalcium phosphate and 60% hydroxyapatite (β-TCP/HA). Media flow within the macrochannels of 3DP/TIPS scaffolds was modeled in COMSOL software in order to fine tune the wall shear stress. CyQUANT DNA assay was performed to assess cell proliferation. The normalized number of cells for the optimal scaffold was more than twofold that of CellCeram™ scaffold after two weeks of culture inside the bioreactor. Despite the substantial variability in the results, the observed improvement in cell proliferation upon culture inside the perfusion bioreactor (vs. static culture) demonstrated the role of macrochannels in making the 3DP/TIPS scaffolds a promising candidate for scaffold-based tissue engineering.

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