scholarly journals Human Serum for Culture of Articular Chondrocytes

2005 ◽  
Vol 14 (7) ◽  
pp. 469-479 ◽  
Author(s):  
Tommi Tallheden ◽  
Josefine Van Der Lee ◽  
Camilla Brantsing ◽  
Jan-Eric Månsson ◽  
Eva Sjögren-Jansson ◽  
...  
Keyword(s):  
Growth Factor ◽  
Human Serum ◽  
Articular Chondrocytes ◽  
Autologous Serum ◽  
Foreign Protein ◽  
Articular Chondrocyte ◽  
Cell Therapies ◽  
High Proliferation Rate ◽  
Protein Contamination

In the field of cell and tissue engineering, culture expansion of human cells in monolayer plays an important part. Traditionally, cell cultures have been supplemented with serum to support attachment and proliferation, but serum is a potential source of foreign protein contamination and viral protein transmission. In this study, we evaluated the use of human serum for experimental human articular chondrocyte expansion and to develop a method for preparation of large volumes of high-quality human serum from healthy blood donors. Human autologous serum contained high levels of epidermal-derived growth factor and platelet-derived growth factor-AB and supported proliferation up to 7 times higher than FCS in primary chondrocyte cultures. By letting the coagulation take place in a commercially available transfusion bag overnight, up to 250 ml of growth factor-rich human serum could be obtained from one donor. The allogenic human serum supported high proliferation rate without loosing expression of cartilage-specific genes. The expanded chondrocytes were able to redifferentiate and form cartilage matrix in comparable amounts to autologous serums. In conclusion, the transfusion bags allow preparation of large volumes of growth factor-rich human serum with the capacity to support in vitro cell expansion. The data further indicate that by controlling the coagulation process there are possibilities of optimizing the release of growth factors for other emerging cell therapies.

scholarly journals Calcium Input Potentiates the Transforming Growth Factor (TGF)-β1-dependent Signaling to Promote the Export of Inorganic Pyrophosphate by Articular Chondrocyte

2011 ◽  
Vol 286 (22) ◽  
pp. 19215-19228 ◽  
Author(s):  
Frederic Cailotto ◽  
Pascal Reboul ◽  
Sylvie Sebillaud ◽  
Patrick Netter ◽  
Jean-Yves Jouzeau ◽  
...  
Keyword(s):  
Growth Factor ◽  
Promoter Activity ◽  
Transforming Growth Factor ◽  
Articular Chondrocytes ◽  
Specific Protein ◽  
Dependent Manner ◽  
Articular Chondrocyte ◽  
Experimental Conditions ◽  
Inorganic Pyrophosphate ◽  
Tgf Β1

Transforming growth factor (TGF)-β1 stimulates extracellular PPi (ePPi) generation and promotes chondrocalcinosis, which also occurs secondary to hyperparathyroidism-induced hypercalcemia. We previously demonstrated that ANK was up-regulated by TGF-β1 activation of ERK1/2 and Ca2+-dependent protein kinase C (PKCα). Thus, we investigated mechanisms by which calcium could affect ePPi metabolism, especially its main regulating proteins ANK and PC-1 (plasma cell membrane glycoprotein-1). We stimulated articular chondrocytes with TGF-β1 under extracellular (eCa2+) or cytosolic Ca2+ (cCa2+) modulations. We studied ANK, PC-1 expression (quantitative RT-PCR, Western blotting), ePPi levels (radiometric assay), and cCa2+ input (fluorescent probe). Voltage-operated Ca2+-channels (VOC) and signaling pathways involved were investigated with selective inhibitors. Finally, Ank promoter activity was evaluated (gene reporter). TGF-β1 elevated cCa2+ and ePPi levels (by up-regulating Ank and PC-1 mRNA/proteins) in an eCa2+ dose-dependent manner. TGF-β1 effects were suppressed by cCa2+ chelation or L- and T-VOC blockade while being mostly reproduced by ionomycin. In the same experimental conditions, the activation of Ras, the phosphorylation of ERK1/2 and PKCα, and the stimulation of Ank promoter activity were affected similarly. Activation of SP1 (specific protein 1) and ELK-1 (Ets-like protein-1) transcription factors supported the regulatory role of Ca2+. SP1 or ELK-1 overexpression or blockade experiments demonstrated a major contribution of ELK-1, which acted synergistically with SP1 to activate Ank promoter in response to TGF-β1. TGF-β1 promotes input of eCa2+ through opening of L- and T-VOCs, to potentiate ERK1/2 and PKCα signaling cascades, resulting in an enhanced activation of Ank promoter and ePPi production in chondrocyte.

scholarly journals Effect of human serum and some of its components on neutrophil adherence and migration across an epithelium.

1986 ◽  
Vol 102 (5) ◽  
pp. 1868-1877 ◽  
Author(s):  
E B Cramer ◽  
L C Milks ◽  
M J Brontoli ◽  
G K Ojakian ◽  
S D Wright ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Human Serum ◽  
Neutrophil Migration ◽  
Autologous Serum ◽  
Heat Inactivation ◽  
Human Neutrophils ◽  
Epithelial Surface ◽  
Neutrophil Adherence ◽  
And Migration

The effect of human serum and some of its components on the process of transepithelial migration of human neutrophils was investigated in an in vitro system. 10% autologous serum caused an increase in neutrophil adherence to and migration across canine kidney epithelial cells. This increase in neutrophil binding also occurred if the epithelium but not the neutrophils had been preincubated with serum. The binding was lost if the serum was either preabsorbed over the kidney epithelium before use or heat inactivated. Indirect immunofluorescence studies indicated that IgG, IgM, and a component of C3 bound to the epithelial surface, whereas IgA, IgE, or C5a were not detectable. The majority of epithelial cells were immunofluorescent, however epithelial cells with varying degrees of reactivity were also apparent and approximately 5% of the epithelial cells did not bind IgG, IgM, and C3. When epithelia were simultaneously tested for the presence of either IgG, IgM, or C3, and bound neutrophils the few epithelial cells which did not bind IgG or IgM also did not bind C3 or neutrophils. Studies with monoclonal antibodies against Fc and C3 receptors indicate that neutrophil adherence to the epithelial surface was mediated predominately by the receptors for C3b and C3bi. In response to a chemotactic gradient, bound neutrophils were able to detach and migrate across the epithelium. A separate heat-stable factor(s) in serum was able to increase neutrophil migration across the epithelial monolayer. This factor acted independently of the factors which caused the increase in neutrophil binding as the increase in neutrophil migration also occurred under conditions (preabsorption over the kidney epithelium or heat inactivation) that prevented the increase in neutrophil binding. The increase in neutrophil migration may be caused by the permeability-increasing properties of this factor as both serum and heat-inactivated serum lowered the transepithelial electrical resistance an average of 38 and 35%, respectively, in 40 min. Upon removal of serum or heat-inactivated serum, the resistance returned 100 and 81%, respectively, in 5 h.

scholarly journals Ethanol Alters Phenotype and Synthesis Activity of Rat Neonatal Articular Chondrocytes Grown in 2- and 3-Dimensional Culture

Cartilage ◽  
2019 ◽  
pp. 194760351987086
Author(s):  
Natalia Viana Tamiasso ◽  
Carla Maria Osório Silva ◽  
Amanda Maria Sena Reis ◽  
Natália Melo Ocarino ◽  
Rogéria Serakides
Keyword(s):  
Mtt Assay ◽  
Articular Chondrocytes ◽  
Quantitative Polymerase Chain Reaction ◽  
Periodic Acid ◽  
3D Culture ◽  
Articular Chondrocyte ◽  
Periodic Acid Schiff ◽  
3 Dimensional ◽  
Synthesis Activity

Objective We sought to evaluate the effect of different concentrations of ethanol on phenotype and activity of articular chondrocyte synthesis of neonatal rats in 2-dimensional (2D) and 3-dimensional (3D) culture. Methods Chondrocytes were cultured in chondrogenic medium with different concentrations of ethanol: 0.0% v/v (control); 0.05% v/v (8.6 mM); 0.25% v/v (42.9 mM), and 0.5% v/v (85.7 mM). Chondrocytes under 2D culture were subjected to MTT assay, while chondrocytes under 3D culture were processed for paraffin inclusion and stained by periodic acid Schiff (PAS) to evaluate mean chondrocyte diameter and percentages of cells, nucleus, cytoplasm, well-differentiated matrix, and PAS+ areas. The expression of gene transcripts for aggrecan, Sox9, and type II collagen was evaluated by real-time quantitative polymerase chain reaction. Results There was no difference between groups by the MTT assay. PAS staining revealed that chondrocytes treated with 0.5% v/v ethanol had higher percentages of cytoplasm and nuclear areas, but with a reduction in PAS+ matrix area. The mean diameter of chondrocytes was similar between groups. The expression of aggrecan in the group treated with 0.5% v/v ethanol was lower in comparison to that in the control. In the groups treated with 0.25% v/v and 0.5% v/v ethanol, the percentage of differentiated cartilage was lower in comparison with that in the control. The group treated with 0.05% v/v ethanol was similar to the control in all parameters. Conclusions Ethanol acted directly on in vitro cultured articular chondrocytes of newborn rats, altering the chondrocyte phenotype and its synthesis activity, and these effects were dose dependent.

Fabrication of Scaffold-Free Engineered Cartilage Using Passaged Chondrocytes in the Presence of Insuline Like Growth Factor 1 (IGF-1)

2007 ◽  
Vol 342-343 ◽  
pp. 149-152 ◽  
Author(s):  
Ri Long Jin ◽  
So Ra Park ◽  
Jeong Hwa Son ◽  
Byoung Hyun Min
Keyword(s):  
Growth Factor ◽  
Articular Chondrocytes ◽  
Cartilage Tissue ◽  
Insulin Like Growth Factor ◽  
Free System ◽  
Engineered Cartilage

Two passaged (P2) immature porcine articular chondrocytes were used to fabricate an engineered cartilage tissue in an in vitro scaffold-free system with or without insulin like growth factor 1 (IGF-1). This study shows the possibility of the fabrication of structurally regular neocartilage tissue using passaged chondrocytes in the scaffold-free system with insulin like growth factor-1(IGF-1).

scholarly journals An antibody present in normal human serum inhibits the binding of cytokines to their receptors in an in vitro system

1999 ◽  
Vol 343 (1) ◽  
pp. 125-133 ◽  
Author(s):  
David E. MOSEDALE ◽  
David J. GRAINGER
Keyword(s):  
Growth Factor ◽  
Human Serum ◽  
Transforming Growth Factor ◽  
Extracellular Domain ◽  
Normal Human Serum ◽  
Platelet Derived Growth Factor ◽  
Type Ii ◽  
Serial Transfer ◽  
Normal Human

The presence of active transforming growth factor-β (TGF-β) in serum has not been widely accepted. In particular, although at least five studies have concluded that active TGF-β is present in normal human plasma and serum, assays that use the extracellular domain of the TGF-β type II receptor as a capture agent have given contradictory results. We show that there is an antagonist present in normal human serum which inhibits the binding of active TGF-β to the extracellular domain of the TGF-β type II receptor when it is coated on the well of an ELISA plate. This antagonist activity is due to a pool of immunoglobulins of the G2, D and M classes. Moreover, we show that this same pool of immunoglobulins also recognizes the extracellular domain of the platelet-derived growth factor α-receptor, insulin-like growth factor-1 receptor and interleukin-3 receptor, by serial transfer of serum over the different receptors. In addition, the same immunoglobulin pool inhibits the binding of platelet-derived growth factor-AA to its receptor, in an analogous way to the inhibition of binding of TGF-β to its type II receptor. Circumstantial evidence suggests that the pool of immunoglobulins is recognizing a carbohydrate residue that is attached to the protein when it is synthesized by the mouse myeloma cell line, NSO, in which it is made. If the cytokine receptors are similarly glycosylated in vivo, then the presence of these antibodies in normal human serum may modulate physiological cytokine signalling.

scholarly journals Adrenomedullin and truncated peptide adrenomedullin(22-52) affect chondrocyte response to apoptotis in vitro: downregulation of FAS protects chondrocyte from cell death

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Frédéric Velard ◽  
Aurore Chatron-Colliet ◽  
Dominique Côme ◽  
Marie-Dominique Ah-Kioon ◽  
Hilène Lin ◽  
...  
Keyword(s):  
Cell Death ◽  
Amino Acid ◽  
Fas Ligand ◽  
Articular Chondrocytes ◽  
Hypoxia Inducible Factor ◽  
Adenosine Monophosphate ◽  
Chondrocyte Apoptosis ◽  
Articular Chondrocyte ◽  
Fas L

Abstract Chondrocyte apoptosis may have a pivotal role in the development of osteoarthritis. Interest has increased in the use of anti-apoptotic compounds to protect against osteoarthritis development. In this work, we investigated the effect of adrenomedullin (AM), a 52 amino-acid hormone peptide, and a 31 amino-acid truncated form, AM(22-52), on chondrocyte apoptosis. Bovine articular chondrocytes (BACs) were cultured under hypoxic conditions to mimic cartilage environment and then treated with Fas ligand (Fas-L) to induce apoptosis. The expression of AM and its calcitonin receptor-like receptor (CLR)/receptor activity-modifying protein (RAMP) (receptor/co-receptor) was assessed by immunostaining. We evaluated the effect of AM and AM(22-52) on Fas-L-induced chondrocyte apoptosis. FAS expression was appreciated by RT-qPCR and immunostainings. The expression of hypoxia-inducible factor 1α (HIF-1α), CLR and one co-receptor (RAMP2) was evidenced. With BACs under hypoxia, cyclic adenosine monophosphate production increased dose-dependently with AM stimulation. AM significantly decreased caspase-3 activity (mean 35% decrease; p = 0.03) as a marker of Fas-L-induced apoptosis. Articular chondrocytes treated with AM showed significantly reduced cell death, along with downregulated Fas expression and production, as compared with AM(22-52). AM decreased articular chondrocyte apoptosis by downregulating a Fas receptor. These findings may pave the way for novel therapeutic approaches in osteoarthritis.

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