scholarly journals Extracellular matrix micropatterning technology for whole cell cryogenic electron microscopy studies

2019 ◽  
Author(s):  
Leeya Engel ◽  
Guido Gaietta ◽  
Liam P. Dow ◽  
Mark F. Swift ◽  
Gaspard Pardon ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Culture ◽  
Extracellular Matrix ◽  
Strain Energy ◽  
Control Cell ◽  
Traction Force ◽  
Culture Technique ◽  
Force Transmission ◽  
Energy States

ABSTRACTCryogenic electron tomography is the highest resolution tool available for structural analysis of macromolecular organization inside cells. Micropatterning of extracellular matrix (ECM) proteins is an established in vitro cell culture technique used to control cell shape. Recent traction force microscopy studies have shown correlation between cell morphology and the regulation of force transmission. However, it remains unknown how cells sustain increased strain energy states and localized stresses at the supramolecular level. Here, we report a technology to enable direct observation of mesoscale organization in epithelial cells under morphological modulation, using a maskless protein photopatterning method to confine cells to ECM micropatterns on electron microscopy substrates. These micropatterned cell culture substrates can be used in mechanobiology research to correlate changes in nanometer-scale organization at cell-cell and cell-ECM contacts to strain energy states and traction stress distribution in the cell.

Development of an in vitro Model for the Study of the Response of Equine Tendon Fibroblasts to Injury and Medication

1997 ◽  
Vol 10 (01) ◽  
pp. 6-11 ◽  
Author(s):  
R. F. Rosenbusch ◽  
L. C. Booth ◽  
L. A. Dahlgren
Keyword(s):  
Electron Microscopy ◽  
Extracellular Matrix ◽  
Light Microscopy ◽  
Light And Electron Microscopy ◽  
Tendon Healing ◽  
Matrix Proteins ◽  
Isolated Cells ◽  
Superficial Digital Flexor Tendon ◽  
Vitro Model

SummaryEquine tendon fibroblasts were isolated from explants of superficial digital flexor tendon, subcultured and maintained in monolayers. The cells were characterized by light microscopy, electron microscopy and radiolabel studies for proteoglycan production. Two predominant cell morphologies were identified. The cells dedifferentiated toward a more spindle shape with repeated subcultures. Equine tendon fibroblasts were successfully cryopreserved and subsequently subcultured. The ability to produce proteoglycan was preserved.The isolated cells were identified as fibroblasts, based on their characteristic shape by light microscopy and ultrastructure and the active production of extracellular matrix proteins. Abundant rough endoplasmic reticulum and the production of extracellular matrix products demonstrated active protein production and export. Proteoglycans were measurable via liquid scintillation counting in both the cell-associated fraction and free in the supernatant. This model is currently being utilized to study the effects of polysulfated glycosaminoglycan on tendon healing. Future uses include studying the effects of other pharmaceuticals, such as hyaluronic acid, on tendon healing.A model was developed for in vitro investigations into tendon healing. Fibroblasts were isolated from equine superficial digital flexor tendons and maintained in monolayer culture. The tenocytes were characterized via light and electron microscopy. Proteoglycan production was measured, using radio-label techniques. The fibroblasts were cryopreserved and subsequently subcultured. The cells maintained their capacity for proteoglycan production, following repeated subculturing and cryopreservation.

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 The LINC complex transmits integrin-dependent tension to the nuclear lamina and represses epidermal differentiation

2020 ◽  
Author(s):  
Emma Carley ◽  
Rachel K. Stewart ◽  
Abigail Zieman ◽  
Iman Jalilian ◽  
Diane. E. King ◽  
...  
Keyword(s):  
Cell Fate ◽  
Control Cell ◽  
Nuclear Lamina ◽  
Epidermal Differentiation ◽  
Keratinocyte Differentiation ◽  
Linc Complex ◽  
Force Transmission ◽  
Cell Fate Decisions

AbstractWhile the mechanisms by which chemical signals control cell fate have been well studied, how mechanical inputs impact cell fate decisions are not well understood. Here, using the well-defined system of keratinocyte differentiation in the skin, we examine whether and how direct force transmission to the nucleus regulates epidermal cell fate. Using a molecular biosensor, we find that tension on the nucleus through Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes requires integrin engagement in undifferentiated epidermal stem cells, and is released during differentiation concomitant with decreased tension on A-type lamins. LINC complex ablation in mice reveals that LINC complexes are required to repress epidermal differentiation in vivo and in vitro and influence accessibility of epidermal differentiation genes, suggesting that force transduction from engaged integrins to the nucleus plays a role in maintaining keratinocyte progenitors. This work reveals a direct mechanotransduction pathway capable of relaying adhesion-specific signals to regulate cell fate.

scholarly journals Surface Characteristics and Cell Adhesion Behaviors of the Anodized Biomedical Stainless Steel

2020 ◽  
Vol 10 (18) ◽  
pp. 6275
Author(s):  
Heng-Jui Hsu ◽  
Chia-Yu Wu ◽  
Bai-Hung Huang ◽  
Chi-Hsun Tsai ◽  
Takashi Saito ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Culture ◽  
Stainless Steel ◽  
Cell Adhesion ◽  
Oxide Layer ◽  
Photoelectron Spectroscopy ◽  
X Ray ◽  
In Vitro Cell Culture ◽  
Cell Culture Assay

In this study, an electrochemical anodizing method was applied as surface modification of the 316L biomedical stainless steel (BSS). The surface properties, microstructural characteristics, and biocompatibility responses of the anodized 316L BSS specimens were elucidated through scanning electron microscopy, X-ray photoelectron spectroscopy, X-ray diffractometry, transmission electron microscopy, and in vitro cell culture assay. Analytical results revealed that the oxide layer of dichromium trioxide (Cr2O3) was formed on the modified 316L BSS specimens after the different anodization modifications. Moreover, a dual porous (micro/nanoporous) topography can also be discovered on the surface of the modified 316L BSS specimens. The microstructure of the anodized oxide layer was composed of amorphous austenite phase and nano-Cr2O3. Furthermore, in vitro cell culture assay also demonstrated that the osteoblast-like cells (MG-63) on the anodized 316L BSS specimens were completely adhered and covered as compared with the unmodified 316L BSS specimen. As a result, the anodized 316L BSS with a dual porous (micro/nanoporous) oxide layer has great potential to induce cell adhesion and promote bone formation.

A novel cell culture technique for electron microscopy

1993 ◽  
Vol 26 (6) ◽  
pp. 517-522
Author(s):  
Fen Wang ◽  
Lindsay B. Ledford ◽  
Jonathan F. Head ◽  
Robert L. Elliott
Keyword(s):  
Electron Microscopy ◽  
Cell Culture ◽  
Culture Technique ◽  
Cell Culture Technique

scholarly journals Isolated Hepatocytes versus Hepatocyte Spheroids: In Vitro Culture of Rat Hepatocytes

2005 ◽  
Vol 14 (6) ◽  
pp. 397-401 ◽  
Author(s):  
Giovanni Ambrosino ◽  
Stefano M. M. Basso ◽  
Sergio Varotto ◽  
Enrico Zardi ◽  
Antonio Picardi ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Functional Activity ◽  
Statistical Significance ◽  
Isolated Hepatocytes ◽  
Culture Technique ◽  
Cell Contact ◽  
Close Link ◽  
Nonparenchymal Cells ◽  
Hepatocyte Spheroids

The use of hepatocytes that express liver-specific functions to develop an artificial liver is promising. Unfortunately, the loss of specialized liver functions (dedifferentiation) is still a major problem. Different techniques, such as collagen entrapment, spherical multicellular aggregates (spheroids), and coculture of hepatocytes with extracellular matrix, have been used to improve the performance of hepatocytes in culture. The aim of this study was to compare two different models of hepatocyte isolation in culture: isolated hepatocytes (G1) and hepatocyte spheroids (60% hepatocytes, 40% nonparenchymal cells, and extracellular matrix) (G2). To test functional activity of hepatocytes, both synthetic and metabolic, production of albumin and benzodiazepine transformation into metabolites was tested. G2 showed a high albumin secretion, while a decrease after 15 days of culture in G1 was noted. Diazepam metabolites were higher in G2 than in G1 in all samples, but had statistical significance at days 14 and 21 (p < 0.01). The glycogen content, after 30 days of culture, was very low in G1 (14.2 ± 4.4%), while in G2 it was 72.1 ± 2.6% (p < 0.01). Our study confirms the effectiveness of a culture technique with extracellular matrix and nonparenchymal cells. Maintenance of a prolonged functional activity has been related to restoration of cell polarity and close cell-to-cell contact. We showed that isolated hepatocytes maintain their functional activity for a period significantly reduced, when compared to the hepatocyte spheroids. We confirmed the role of extracellular matrix as a crucial component to promote hepatocyte homeostasis, and the close link between cellular architecture and tissue-specific functions.

Electron Microscopy of Virus in L1210V Cells

1973 ◽  
Vol 31 ◽  
pp. 586-587
Author(s):  
Pearl L. Chen ◽  
Nurul H. Sarkar ◽  
Dan H. Moore
Keyword(s):  
Electron Microscopy ◽  
Cell Culture ◽  
Cancer Research ◽  
Female Mouse ◽  
Double Dose ◽  
Cancer Research Institute ◽  
Lymphatic Leukemia ◽  
Research Institute

Two cell culture lines established in vitro, L1210VA and L1210VB, were obtained from Dr. Dorris J. Hutchison, Sloan- Kettering Cancer Research Institute, Rye, N. Y. They were maintained in Earl'-Eagle' medium containing a double dose of glutamine.The original lymphatic leukemia L1210 was induced by Law et al in a DBA/2 female mouse. In these cells grown intraperitoneally in DBA mice the presence of intracytoplasmic A particles and their incorporation into budding virions, as well as extracellular particles with the morphology of leukemiasarcoma viruses have been described. Since the intracytoplasmic A particles are related to the development of B particles it was our interest to investigate whether or not the cells produce B particles.

Cultured smooth muscle approach in the study of hypertension

1992 ◽  
Vol 70 (4) ◽  
pp. 573-579 ◽  
Author(s):  
Stephen C. Pang ◽  
Shannon L. Venance
Keyword(s):  
Cell Culture ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Spontaneously Hypertensive Rat ◽  
Marked Change ◽  
Culture Technique ◽  
Mesenteric Arteries ◽  
Experimental Hypertension ◽  
In Vitro Techniques

The systemic vasculature is known to undergo marked change in both human and experimental hypertension. The in vitro study of individual cellular components from the blood vessel wall and the regulation of their intracellular biochemical processes will aid in developing an understanding of the pathogenesis of hypertension. Vascular smooth muscle cells derived from the aorta and mesenteric arteries of normotensive and hypertensive rats can be successfully maintained in culture, providing a system free of confounding variables such as blood pressure. To assist in fully understanding the pathophysiology of hypertension, this cell culture model can be used to examine interactions between receptor and ligand, the transduction of an associated signal, characterization of subsequent intracellular responses and ultimately, quantification of a physiological and functional consequence of these events, for example, proliferation. The application of in vitro techniques to hypertension research will continue to contribute new knowledge to increase our understanding of the mechanisms behind the hypertensive disease process.Key words: experimental hypertension, spontaneously hypertensive rat, vascular smooth muscle, aorta, mesenteric arteries, cell culture technique.

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