Valve interstitial cell culture: Production of mature type I collagen and precise detection

2017 ◽  
Vol 80 (8) ◽  
pp. 936-942 ◽  
Author(s):  
Jana Liskova ◽  
Daniel Hadraba ◽  
Elena Filova ◽  
Miroslav Konarik ◽  
Jan Pirk ◽  
...  
Keyword(s):  
Cell Culture ◽  
Type I Collagen ◽  
Interstitial Cell ◽  
Type I ◽  
Mature Type ◽  
Culture Production

scholarly journals Regulation of valve interstitial cell homeostasis by mechanical deformation: implications for heart valve disease and surgical repair

2017 ◽  
Vol 14 (135) ◽  
pp. 20170580 ◽  
Author(s):  
Salma Ayoub ◽  
Chung-Hao Lee ◽  
Kathryn H. Driesbaugh ◽  
Wanda Anselmo ◽  
Connor T. Hughes ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Interstitial Cell ◽  
Smooth Muscle Actin ◽  
Heart Valve Disease ◽  
Type I ◽  
Tissue Remodelling ◽  
Cell Responses ◽  
Stress States

Mechanical stress is one of the major aetiological factors underlying soft-tissue remodelling, especially for the mitral valve (MV). It has been hypothesized that altered MV tissue stress states lead to deviations from cellular homeostasis, resulting in subsequent cellular activation and extracellular matrix (ECM) remodelling. However, a quantitative link between alterations in the organ-level in vivo state and in vitro- based mechanobiology studies has yet to be made. We thus developed an integrated experimental–computational approach to elucidate MV tissue and interstitial cell responses to varying tissue strain levels. Comprehensive results at different length scales revealed that normal responses are observed only within a defined range of tissue deformations, whereas deformations outside of this range lead to hypo- and hyper-synthetic responses, evidenced by changes in α-smooth muscle actin, type I collagen, and other ECM and cell adhesion molecule regulation. We identified MV interstitial cell deformation as a key player in leaflet tissue homeostatic regulation and, as such, used it as the metric that makes the critical link between in vitro responses to simulated equivalent in vivo behaviour. Results indicated that cell responses have a delimited range of in vivo deformations that maintain a homeostatic response, suggesting that deviations from this range may lead to deleterious tissue remodelling and failure.

Exposure of mouse preosteoblasts to pulsed electromagnetic fields reduces the amount of mature, type I collagen in the extracellular matrix

2006 ◽  
Vol 24 (2) ◽  
pp. 242-253 ◽  
Author(s):  
Yoshitada Sakai ◽  
Thomas E. Patterson ◽  
Michael O. Ibiwoye ◽  
Ronald J. Midura ◽  
Maciej Zborowski ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Electromagnetic Fields ◽  
Type I Collagen ◽  
Type I ◽  
Pulsed Electromagnetic Fields ◽  
Mature Type ◽  
Pulsed Electromagnetic

scholarly journals Effects of ECM Proteins and Cationic Polymers on the Adhesion and Proliferation of Rat Islet Cells

2008 ◽  
Vol 2 (1) ◽  
pp. 133-137 ◽  
Author(s):  
Guoping Chen ◽  
Naoki Kawazoe ◽  
Tetsuya Tateishi
Keyword(s):  
Cell Culture ◽  
Type Iv Collagen ◽  
Type I Collagen ◽  
Islet Cells ◽  
Type Ii Collagen ◽  
Type I ◽  
Cationic Polymers ◽  
Type Iv ◽  
Ecm Proteins ◽  
Cationic Polyelectrolytes

The effects of extracellular matrix (ECM) proteins and cationic polymers on the adhesion and proliferation of rat islet cells, RIN-5F cells, were investigated. ECM proteins of laminin, fibronectin, vitronectin, type I collagen, type II collagen, and type IV collagen, and cationic polyelectrolytes of poly(L-lysine) and poly(allylamine) were coated on the wells of polystyrene cell culture plates. Their effects on the adhesion and proliferation of RIN-5F in serum-free and serum mediums were compared. The cell number on the laminin-coated surface was the highest among the coated surfaces. Laminin promoted cell adhesion more strongly than did the other ECM proteins and cationic polyelectrolytes. Vitronectin, type IV collagen, and poly(L-lysine) showed moderate effects, but type I collagen and type II collagen did not have any effects on adhesion and proliferation compared with the uncoated polystyrene cell culture plate. Fibronectin promoted cell adhesion but not cell proliferation. Cationic poly(allylamine) had an inhibitory effect in serum-free medium and for longterm culture in serum medium. The ECM proteins of laminin, vitronectin, and type IV collagen, and cationic poly(Llysine) will be useful for the surface modification and construction of biomaterials and scaffolds for islet cell culture and tissue engineering.

scholarly journals Cobalt-containing Calcium Phosphate Induces Resorption of Biomineralized Collagen by Human Osteoclasts

2020 ◽  
Author(s):  
Daniel de Melo Pereira ◽  
Matthias Schumacher ◽  
Pamela Habibović
Keyword(s):  
Cell Culture ◽  
Calcium Phosphate ◽  
Calcium Release ◽  
Type I Collagen ◽  
Cell Culture Supernatant ◽  
Type I ◽  
Cobalt Ions ◽  
Collagen Membranes ◽  
Human Osteoclasts ◽  
Osteoclast Resorption

Abstract Background: Biomineralized collagen, consisting of fibrillary type-I collagen with embedded hydroxyapatite mineral, is a bone-mimicking material with potential application as a bone graft substitute. Despite the chemical and structural similarity with bone extracellular matrix, no evidence exists so far that biomineralized collagen can be resorbed by osteoclasts. The aim of the current study was to induce resorption of biomineralized collagen by osteoclasts by a two-fold modification: increasing the calcium phosphate content and introducing cobalt ions (Co2+), which have been previous shown to stimulate resorptive activity of osteoclasts.Methods: To this end, we produced biomineralized collagen membranes and coated them with a cobalt-containing calcium phosphate (CoCaP). Human osteoclasts, derived from CD14+ monocytes from peripheral blood, were differentiated directly on the membranes. Their morphology was assessed by laser confocal microscopy and their capacity for resorption observed by scanning electron microscopy (SEM), as well as indirectly quantified by calcium release into cell culture supernatant. Results: The CoCaP coating increased the mineral content of the membranes by 4 wt.% and their elastic modulus from 1 to 10 MPa. The coated membranes showed a sustained Co2+ release of about 7 nM per 2 days. In contrast to uncoated membranes, on CoCaP-coated biomineralized collagen membranes, osteoclasts sporadically formed actin rings, and caused resorption lacunae to form, as observed by SEM and confirmed by increase in Ca2+ concentration in cell culture medium. The effect of the CoCaP layer on osteoclast function is thought to be mainly caused by the increase of membrane stiffness, although the effect of Co2+, which was released in very low amounts, cannot be fully excluded.Conclusions: This work shows the potential of this relatively simple approach to induce osteoclast resorption of biomineralized collagen, despite the fact that the extent of osteoclast resorption was limited, and the method needs further optimization,. Moreover, the coating method is suitable for incorporating bioactive ions of interest into biomineralized collagen, which is typically not possible using the common biomineralization methods, such as polymer-induced liquid precursor method.

scholarly journals SAT0293 EXOSOMES DERIVED FROM PLASMA OF SYSTEMIC SCLEROSIS (SSC) PATIENTS AND FROM SSC CULTURED FIBROBLASTS CONTAIN PRO-FIBROTIC MIRNA SIGNATURES AND COULD INDUCE MYOFIBROBLAST DIFFERENTIATION IN VITRO

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1091.2-1092
Author(s):  
C. Corallo ◽  
M. Cutolo ◽  
S. Soldano ◽  
E. Selvi ◽  
F. Bellisai ◽  
...  
Keyword(s):  
Cell Culture ◽  
Systemic Sclerosis ◽  
Real Time Pcr ◽  
Type I Collagen ◽  
Type I ◽  
Myofibroblast Differentiation ◽  
Plasma Samples ◽  
Fibroblast Cultures ◽  
Control Plasma

Background:Exosomes generated great resonance in the last few years due to their important roles in different biological pathways and diseases, including systemic sclerosis (SSc) (1). They are lipid-like nanovesicles containing biomarkers, such as proteins, lipids, macromolecules and nucleic acids, including microRNA (miRNA) (2). Exosomes are implicated in intercellular communication by fusing and releasing their cargo into the target cells (3).Objectives:In the present study, we evaluated the potential of exosomes deriving from plasma of SSc patients or generating from cultured SSc fibroblasts to drive the fibrotic signaling in the disease.Methods:Exosomes were isolated from plasma of n=10 SSc patients and from n=10 control subjects. Exosomes were also purified from cell culture supernatants of SSc fibroblasts and of control fibroblasts. Exosome size and concentration were assessed by Nanosight Particle Tracking Analysis (NTA) and by transmission electron microscopy (TEM). The content of anti-fibrotic (let-7a, 146a, 200a, 223a) and pro-fibrotic (150, 155) miRNAs was assessed in all the plasma-derived and cell culture-derived exosome populations by semiquantitative real time PCR. Finally, isolated exosomes were used to stimulate control dermal fibroblasts in culture. Gene expressions (COL1A1, ACTA2 and TAGLN) were assessed by quantitative real time PCR (qRT-PCR) and protein levels (type-I-collagen, α-SMA and SM22) by immunofluorescence (IF).Results:Exosomes isolated from SSc plasma samples showed higher concentration (3.3x1010±1.1x1010particles/mL) compared to those isolated from control plasma ones (1.5x1010±0.4x1010particles/mL) (p<0.01). The exosome size did not differ between SSc and control plasma samples and ranged from 50nm to 150nm. Similar results were obtained with exosomes generated from fibroblast cultures: the concentration was higher in SSc fibroblasts (1.1x1010±0.2x1010particles/mL) than in control ones (0.4x1010±0.1x1010particles/mL) (p<0.05) with no significant differences in size distribution. The content of all anti-fibrotic (let-7a, 146a, 200a, 223a) miRNAs was decreased in exosomes coming from both SSc plasma samples and from SSc fibroblasts with respect to control plasma samples (p<0.05) and to control fibroblasts (p<0.05). On the contrary, the pro-fibrotic (150, 155) miRNAs were significantly upregulated in exosomes deriving from SSc plasma samples and from SSc fibroblasts, with respect to control plasma samples (p<0.05) and to control fibroblasts (p<0.05). Finally, only exosomes coming from SSc plasma samples or SSc fibroblast cultures were able to induce pro-fibrotic gene (COL1A1, ACTA2 and TAGLN) and protein (type-I-collagen, α-SMA and SM22) expression in control fibroblasts. No pro-fibrotic induction was seen in presence of exosomes isolated from control plasma samples or control fibroblast cultures.Conclusion:This study demonstrates that plasma from SSc patients contains higher concentration of exosomes compared to plasma from control subjects and SSc-derived exosomes contain specific pro-fibrotic miRNA signatures that can induce myofibroblast differentiationin vitro. These results suggest that exosomes could be fibrotic drivers towards non-affected areasin vivo, and they might represent novel targets for precision medicine treatments in SSc.References:[1]Zhu T, Wang Y, Jin H, Li L. The role of exosome in autoimmune connective tissue disease. Ann Med. 2019 Mar;51(2):101-108.[2]Wermuth PJ, Piera-Velazquez S, Rosenbloom J, et al. Existing and novel biomarkers for precision medicine in systemic sclerosis. Nat Rev Rheumatol. 2018 Jul;14(7):421-432.[3]Colletti M, Galardi A, De Santis M, et al. Exosomes in Systemic Sclerosis: Messengers Between Immune, Vascular and Fibrotic Components? Int J Mol Sci. 2019 Sep 4;20(18). pii: E4337.Disclosure of Interests:Claudio Corallo: None declared, Maurizio Cutolo Grant/research support from: Bristol-Myers Squibb, Actelion, Celgene, Consultant of: Bristol-Myers Squibb, Speakers bureau: Sigma-Alpha, Stefano Soldano: None declared, Enrico Selvi: None declared, Francesca Bellisai: None declared, Nicola Giordano: None declared

scholarly journals Cobalt-containing calcium phosphate induces resorption of biomineralized collagen by human osteoclasts

2021 ◽  
Vol 25 (1) ◽  
Author(s):  
Daniel de Melo Pereira ◽  
Matthias Schumacher ◽  
Pamela Habibovic
Keyword(s):  
Cell Culture ◽  
Calcium Phosphate ◽  
Culture Medium ◽  
Type I Collagen ◽  
Type I ◽  
Cell Culture Medium ◽  
Cobalt Ions ◽  
Collagen Membranes ◽  
Human Osteoclasts ◽  
Osteoclast Resorption

Abstract Background Biomineralized collagen, consisting of fibrillar type-I collagen with embedded hydroxyapatite mineral, is a bone-mimicking material with potential application as a bone graft substitute. Despite the chemical and structural similarity with bone extracellular matrix, no evidence exists so far that biomineralized collagen can be resorbed by osteoclasts. The aim of the current study was to induce resorption of biomineralized collagen by osteoclasts by a two-fold modification: increasing the calcium phosphate content and introducing cobalt ions (Co2+), which have been previously shown to stimulate resorptive activity of osteoclasts. Methods To this end, we produced biomineralized collagen membranes and coated them with a cobalt-containing calcium phosphate (CoCaP). Human osteoclasts, derived from CD14+ monocytes from peripheral blood, were differentiated directly on the membranes. Upon fluorescent staining of nuclei, F-actin and tartrate-resistant alkaline phosphatase, the cells were analyzed by laser confocal microscopy. Their resorption capacity was assessed by scanning electron microscopy (SEM), as well as indirectly quantified by measuring the release of calcium ions into cell culture medium. Results The CoCaP coating increased the mineral content of the membranes by 4 wt.% and their elastic modulus from 1 to 10 MPa. The coated membranes showed a sustained Co2+ release in water of about 7 nM per 2 days. In contrast to uncoated membranes, on CoCaP-coated biomineralized collagen membranes, osteoclasts sporadically formed actin rings, and induced formation of resorption lacunae, as observed by SEM and confirmed by increase in Ca2+ concentration in cell culture medium. The effect of the CoCaP layer on osteoclast function is thought to be mainly caused by the increase of membrane stiffness, although the effect of Co2+, which was released in very low amounts, cannot be fully excluded. Conclusions This work shows the potential of this relatively simple approach to induce osteoclast resorption of biomineralized collagen, although the extent of osteoclast resorption was limited, and the method needs further optimization. Moreover, the coating method is suitable for incorporating bioactive ions of interest into biomineralized collagen, which is typically not possible using the common biomineralization methods, such as polymer-induced liquid precursor method.

Immobilization of native type I collagen on polypropylene fabrics as a substrate for HepG2 cell culture

2017 ◽  
Vol 32 (1) ◽  
pp. 93-103 ◽  
Author(s):  
Gongze Peng ◽  
Saina Li ◽  
Qing Peng ◽  
Yang Li ◽  
Jun Weng ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepg2 Cell ◽  
Type I Collagen ◽  
Type I

Type I Collagen-Functionalized Supported Lipid Bilayer as a Cell Culture Platform

2010 ◽  
Vol 11 (5) ◽  
pp. 1231-1240 ◽  
Author(s):  
Chun-Jen Huang ◽  
Nam-Joon Cho ◽  
Chih-Jung Hsu ◽  
Po-Yuan Tseng ◽  
Curtis W. Frank ◽  
...  
Keyword(s):  
Cell Culture ◽  
Lipid Bilayer ◽  
Type I Collagen ◽  
Type I ◽  
Supported Lipid Bilayer ◽  
A Cell

scholarly journals Gelatin-Coated Microfluidic Channels for 3D Microtissue Formation: On-Chip Production and Characterization

Micromachines ◽  
2019 ◽  
Vol 10 (4) ◽  
pp. 265 ◽  
Author(s):  
Gabriele Pitingolo ◽  
Antoine Riaud ◽  
Claudio Nastruzzi ◽  
Valerie Taly
Keyword(s):  
Cell Culture ◽  
Type I Collagen ◽  
3D Cell Culture ◽  
Gelatin Film ◽  
New Drugs ◽  
In Situ Monitoring ◽  
Microfluidic Channels ◽  
Type I ◽  
Gelatin Coating

Traditional two-dimensional (2D) cell culture models are limited in their ability to reproduce human structures and functions. On the contrary, three-dimensional (3D) microtissues have the potential to permit the development of new cell-based assays as advanced in vitro models to test new drugs. Here, we report the use of a dehydrated gelatin film to promote tumor cells aggregation and 3D microtissue formation. The simple and stable gelatin coating represents an alternative to conventional and expensive materials like type I collagen, hyaluronic acid, or matrigel. The gelatin coating is biocompatible with several culture formats including microfluidic chips, as well as standard micro-well plates. It also enables long-term 3D cell culture and in situ monitoring of live/dead assays.

Abstract 155: TGF-β1 Positively Modulates Cell-Metal Interaction in Cardiovascular Applications

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
S Hamed Alavi ◽  
Arash Kheradvar
Keyword(s):  
Cell Culture ◽  
Stainless Steel ◽  
Endothelial Cells ◽  
Type I Collagen ◽  
Cell Attachment ◽  
Proper Time ◽  
Type I ◽  
Stainless Steel Mesh ◽  
Steel Mesh ◽  
Tgf Β1

Introduction: The interaction between a metallic implant and local cells in the cardiovascular system determines the fate of the implant. We have recently developed a hybrid scaffold for tissue engineering of valves, which consists of an extra thin layer of Stainless Steel mesh tightly enclosed by three distinct layers of cells. We have examined the effect of TGF-β1 on degree of cell attachment to the metal mesh. Methods and Results: By using a 3D cell culture method a Stainless Steel mesh with desired size and thickness was enclosed through cultured layers of cells similar to a heart valve leaflet pattern. Human aortic smooth muscle cells and adventitial fibroblast/myofibroblast cells were used to fulfill the role of valvular interstitial cells, and human umbilical vascular endothelial cells were used to cover the construct in place of the valvular endothelial cells. The layers were seeded in sequence on a three dimensional, porous biological scaffold comprised of bovine type I collagen gel mixture. The effect of TGF-β1 supplementation at proper time-steps on the cultured hybrid tissue after two months of cell culture was determined. TGF-β supplementation resulted in significant increase in proliferation, and extra ingrowths into the outer portions of the tissue. DAPI staining of nuclei showed that the number of cells at the surface of the mesh progressively increased due to TGF-β, and eventually formed a thicker tissue around the mesh Conclusions: We have demonstrated the effects of TGF-β1 on cell attachment and tissue development by using a hybrid tissue material that helped us study the direct cell-metal interactions in-vitro.

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