scholarly journals The shed ectodomain of type XIII collagen associates with the fibrillar fibronectin matrix and may interfere with its assembly in vitro

2005 ◽  
Vol 393 (1) ◽  
pp. 43-50 ◽  
Author(s):  
Marja-Riitta Väisänen ◽  
Timo Väisänen ◽  
Hongmin Tu ◽  
Päivi Pirilä ◽  
Raija Sormunen ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Culture Conditions ◽  
Biologically Active ◽  
Matrix Proteins ◽  
Ectodomain Shedding ◽  
Pericellular Matrix ◽  
Active Molecule ◽  
Type Xiii Collagen ◽  
Cell Culture Conditions

Type XIII collagen is a transmembrane collagen, which is known to exist also as a soluble variant due to ectodomain shedding. Earlier studies with the recombinant ectodomain have shown it to interact in vitro with a number of extracellular matrix proteins, e.g. Fn (fibronectin). In view of its strong binding to Fn, we examined in the present study whether the released soluble ectodomain can bind to the fibrillar Fn matrix under cell-culture conditions and, if so, influence its assembly. In this study, we demonstrate that the type XIII collagen ectodomain of mammalian cells can associate with Fn fibres and may eventually hamper incorporation of the fibrillar Fn meshwork. The association between type XIII collagen and Fn was implicated to be mediated by the C-terminal end of type XIII collagen and the N-terminal end of Fn. The results presented here imply that the shedding of the type XIII collagen ectodomain results in a biologically active molecule capable of remodelling the structure of the pericellular matrix.

scholarly journals Tissue Specific Differentiation of Human Chondrocytes Depends on Cell Microenvironment and Serum Selection

Cells ◽  
2019 ◽  
Vol 8 (8) ◽  
pp. 934 ◽  
Author(s):  
Ecke ◽  
Lutter ◽  
Scholka ◽  
Hansch ◽  
Becker ◽  
...  
Keyword(s):  
Articular Chondrocytes ◽  
Culture Conditions ◽  
Serum Supplement ◽  
3 Dimensional ◽  
The Matrix ◽  
Differentiation Degree ◽  
Overlay Technique ◽  
Fetal Bovine ◽  
Cell Culture Conditions

Therapeutic options to cure osteoarthritis (OA) are not yet available, although cell-based therapies for the treatment of traumatic defects of cartilage have already been developed using, e.g., articular chondrocytes. In order to adapt cell-based therapies to treat OA, appropriate cell culture conditions are necessary. Chondrocytes require a 3-dimensional (3D) environment for redifferentiation after 2-dimensional (2D) expansion. Fetal bovine serum (FBS) is commonly used as a medium supplement, although the usage of a xenogeneic serum could mask the intrinsic behavior of human cells in vitro. The aim of this study was to compare human articular chondrocytes cultivated as monolayers (2D) and the development of microtissues (3D) in the presence of FBS with those cultivated with human serum (HS). Evaluation of the expression of various markers via immunocytochemistry on monolayer cells revealed a higher dedifferentiation degree of chondrocytes cultivated with HS. Scaffold-free microtissues were generated using the agar overlay technique, and their differentiation level was evaluated via histochemistry and immunohistochemistry. Microtissues cultivated in the medium with FBS showed a higher redifferentiation level. This was evidenced by bigger microtissues and a more cartilage-like composition of the matrix with not any/less positivity for cartilage-specific markers in HS versus moderate-to-high positivity in FBS-cultured microtissues. The present study showed that the differentiation degree of chondrocytes depends both on the microenvironment of the cells and the serum type with FBS achieving the best results. However, HS should be preferred for the engineering of cartilage-like microtissues, as it rather enables a "human-based" situation in vitro. Hence, cultivation conditions might be further optimized to gain an even more adequate and donor-independent redifferentiation of chondrocytes in microtissues, e.g., designing a suitable chemically-defined serum supplement.

scholarly journals Defining cell culture conditions to improve human norovirus infectivity assays

2013 ◽  
Vol 67 (4) ◽  
pp. 863-868 ◽  
Author(s):  
T. M. Straub ◽  
J. R. Hutchison ◽  
R. A. Bartholomew ◽  
C. O. Valdez ◽  
N. B. Valentine ◽  
...  
Keyword(s):  
Cell Line ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Qrt Pcr ◽  
Infectivity Assay ◽  
Polymerase Chain ◽  
Single Laboratory ◽  
Cell Culture Conditions ◽  
Human Intestinal Cells

Significant difficulties remain for determining whether human noroviruses (hNoV) recovered from water, food, and environmental samples are infectious. Three-dimensional (3-D) tissue culture of human intestinal cells has shown promise in developing an infectivity assay, but reproducibility, even within a single laboratory, remains problematic. From the literature and our observations, we hypothesized that the common factors that lead to more reproducible hNoV infectivity in vitro requires that the cell line be (1) of human gastrointestinal origin, (2) expresses apical microvilli, and (3) be a positive secretor cell line. The C2BBe1 cell line, which is a brush-border producing clone of Caco-2, meets these three criteria. When challenged with Genogroup II viruses, we observed a 2 Log10 increase in viral RNA titer. A passage experiment with GII viruses showed evidence of the ability to propagate hNoV by both quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microscopy. In our hands, using 3-D C2BBe1 cells improves reproducibility of the infectivity assay for hNoV, but the assay can still be variable. Two sources of variability include the cells themselves (mixed phenotypes of small and large intestine) and initial titer measurements using qRT-PCR that measures all RNA vs. plaque assays that measure infectious virus.

scholarly journals Direct repression of Nanog and Oct4 by OTX2 modulates contribution of epiblast-derived cells to germline and somatic lineage

Development ◽  
2021 ◽  
Author(s):  
Luca Giovanni Di Giovannantonio ◽  
Dario Acampora ◽  
Daniela Omodei ◽  
Vincenzo Nigro ◽  
Pasquale Barba ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Regulatory Network ◽  
Primordial Germ Cells ◽  
Culture Conditions ◽  
Specific Cell ◽  
Temporal Window ◽  
Pluripotency Gene ◽  
Gene Regulatory ◽  
Cell Culture Conditions

In mammals the pre-gastrula proximal epiblast gives rise to Primordial Germ Cells (PGCs) or somatic precursors in response to BMP4 and WNT signaling. Entry into the germline requires activation of a naïve-like pluripotency gene regulatory network (GRN). Recent work showed that suppression of OTX2 expression in the epiblast by BMP4 allows cells to develop a PGC fate in a precise temporal window. However, the mechanisms by which OTX2 suppresses PGC fate are unknown. Here we show that OTX2 prevents epiblast cells from activating the pluripotency GRN by direct repression of Oct4 and Nanog. Loss of this control during PGC differentiation in vitro causes widespread activation of the pluripotency GRN and deregulated response to LIF, BMP4 and WNT signaling. These abnormalities, in specific cell culture conditions, result in massive germline entry at the expense of somatic mesoderm differentiation. Increased generation of PGCs occurs also in mutant embryos. We propose that the OTX2 repressive control of Oct4 and Nanog is at the basis of the mechanism determining epiblast contribution to germline and somatic lineage.

A Scanning Electron-Microscope Study of the Surface Features of Mammalian Cells In Vitro

1972 ◽  
Vol 11 (1) ◽  
pp. 233-247
Author(s):  
GISELE M. HODGES ◽  
MARJORIE D. MUIR
Keyword(s):  
Cell Surface ◽  
Mammalian Cells ◽  
Physiological State ◽  
Culture Conditions ◽  
Kidney Cells ◽  
Scanning Electron Microscope Study ◽  
Cell Strains ◽  
Scanning Electron ◽  
Morphology Of Surface

The cell surface aspects and the density, distribution and morphology of surface cytoplasmic projections (microprocesses) of HeLa, BHK and baby mouse kidney cells, maintained under different culture conditions, have been compared using scanning electron microscopy. No significant differences were noted in the surface aspects of these cell strains. Comparison of the cell strains, under identical culture conditions, showed variations from one strain to another in the density and morphology of the cell surface microprocesses which may reflect basic physiological differences between the strains. For a given cell strain, variations were observed in response to changes in culture conditions indicative that the presence of surface microprocesses is related to the physiological state of the cell.

Laser microstructured scaffolds for tissue engineering under dynamic cell culture conditions

2020 ◽  
Author(s):  
Ελευθερία Μπαμπαλιάρη
Keyword(s):  
Tissue Engineering ◽  
Cell Culture ◽  
Culture Conditions ◽  
Dynamic Cell ◽  
Cell Culture Conditions

Παρόλο που το περιφερικό νευρικό σύστημα εμφανίζει υψηλότερο ρυθμό αναγέννησης από εκείνο του κεντρικού νευρικού συστήματος μέσω αυθόρμητης αναγέννησης μετά από έναν τραυματισμό, η καθοδηγούμενη αξονική νευρική αναγέννηση και η λειτουργική αποκατάσταση είναι αρκετά σπάνια. Συνεπώς, η ανάπτυξη επιτυχημένων μεθόδων για την καθοδήγηση της νευρικής ανάπτυξης, «in vitro», είναι υψίστης σημασίας. Έχει αναφερθεί λεπτομερώς ότι η τοπογραφία του υποστρώματος επηρεάζει την ανάπτυξη, τον προσανατολισμό και τη διαφοροποίηση των νευρικών κυττάρων. Ωστόσο, η συνδυασμένη δράση της διατμητικής τάσης και της τοπογραφίας του υποστρώματος στην νευρική ανάπτυξη έχει ελάχιστα μελετηθεί, παρόλο που οι διατμητικές τάσεις είναι ευρέως γνωστό ότι διαδραματίζουν καθοριστικό ρόλο στην οργάνωση, ανάπτυξη και λειτουργία των ιστών. Σε αυτή τη μελέτη, ένα σύστημα μικροροών ακριβούς ελεγχόμενης ροής με συγκεκριμένους ειδικά σχεδιασμένους θαλάμους, που ενσωματώνουν μικροδομημένα υποστρώματα λέιζερ, αναπτύχθηκε για να μελετηθεί η συνδυασμένη δράση της διατμητικής τάσης και της τοπογραφίας υποστρώματος στην ανάπτυξη, στον προσανατολισμό, στην επιμήκυνση και στη διαφοροποίηση νευρικών κυττάρων. Πολυμερικά μικροδομημένα υποστρώματα, με ελεγχόμενη γεωμετρία και κανονικότητα μοτίβου, κατασκευάστηκαν με χρήση υπερβραχέων παλμών λέιζερ. Πραγματοποιήθηκε συγκριτική μελέτη μεταξύ στατικών και δυναμικών κυτταρικών καλλιεργειών για να αξιολογηθεί η συνεργατική ή ανταγωνιστική επίδραση της διατμητικής τάσης και της τοπογραφίας στη συμπεριφορά των νευρικών κυττάρων. Τα αποτελέσματα της κυτταρικής καλλιέργειας συμπληρώθηκαν με υπολογιστικές προσομοιώσεις ροής με σκοπό τον ακριβή υπολογισμό των αντίστοιχων τιμών διατμητικής τάσης.

scholarly journals Enhancement Of Dermal Fibroblast Isolation Method

2015 ◽  
Vol 16 (1) ◽  
pp. 65-69
Author(s):  
Milan Zaric ◽  
Ivana Nikolic ◽  
Ivanka Zelen ◽  
Marina Mitrovic ◽  
Zoran Milosavljevic
Keyword(s):  
Dermal Fibroblast ◽  
Petri Dish ◽  
Culture Conditions ◽  
Dermal Fibroblasts ◽  
Enzyme Digestion ◽  
Digestion Method ◽  
Primary Isolation ◽  
Donor Cells ◽  
Cell Culture Conditions

ABSTRACTCultivated fibroblasts have been widely used in a large number of in vitro studies. Although they readily proliferate under cell culture conditions, improvements in methods for their isolation are necessary. Here, we present our modified enzyme digestion method and compare its efficiency with commonly used techniques.Three foreskin samples from young, middle-aged and old donors were used. The classical explant, standard enzyme digestion method with collagenase and our improved enzyme digestion method were compared for efficiency of fibroblast isolation and the time needed to achieve 95% confluence in a 30-mm Petri dish.The explant method was the slowest to achieve fibroblast confluence, especially with the tissues from the older donors (up to 23 days). With the standard enzyme digestion method, the skin tissue was partially digested, but the fibroblasts reached confluence much faster (the younger donor cells needed approximately 7 days to reach confluence). Our modified “mixed” enzyme digestion method was the fastest (the fibroblasts from the younger donors needed up to 5 days to reach confluence).For studies requiring the primary isolation and cultivation of dermal fibroblasts, the best method to achieve this goal is the tissue digestion method with the multiple enzyme solution.

Optimization of cell culture conditions for production of biologically active proteins

1991 ◽  
Vol 5 (S2) ◽  
pp. 17-34 ◽  
Author(s):  
Shinji Hosoi ◽  
Hiromasa Miyaji ◽  
Mitsuo Satoh ◽  
Tsukasa Kurimoto ◽  
Akira Mihara ◽  
...  
Keyword(s):  
Cell Culture ◽  
Culture Conditions ◽  
Biologically Active ◽  
Cell Culture Conditions

Injection Molding of ProNectin®F Dispersed in Polystyrene for The Fabrication of Plastic Ware Activated Towards Attachment of Mammalian Cells

1993 ◽  
Vol 330 ◽  
Author(s):  
Erwin R. Stedronsky ◽  
Joseph Cappello ◽  
Samuel David ◽  
David M. Donofrio ◽  
Tina Mcarthur ◽  
...  
Keyword(s):  
Injection Molding ◽  
Mammalian Cell ◽  
Mammalian Cells ◽  
De Novo ◽  
Cell Attachment ◽  
Biologically Active ◽  
Polymeric Surfactant ◽  
New Paradigm

ABSTRACTProNectin®F is a recombinant engineered protein polymer of de novo design which incorporates the RGD epitope recognized by mammalian cell integrins. It is biologically active as a cell attachment protein, manifests properties of a planar polymeric surfactant, and is extremely resistant to thermal degradation. ProNectin®F was dispersed onto polystyrene powder, fabricated into plastic ware through injection molding, and the plastic ware was shown to have cell attachment activity. This technology represents a new paradigm for the production of plastic ware useful for mammalian cell culture under serum free conditions; and more generally, for the production of molded devices for use in contact with cells in vitro or in vivo.

Human LTC-IC can be maintained for at least 5 weeks in vitro when interleukin-3 and a single chemokine are combined with O-sulfated heparan sulfates: requirement for optimal binding interactions of heparan sulfate with early-acting cytokines and matrix proteins

Blood ◽  
2000 ◽  
Vol 95 (1) ◽  
pp. 147-155
Author(s):  
Pankaj Gupta ◽  
Theodore R. Oegema ◽  
Joseph J. Brazil ◽  
Arkaduisz Z. Dudek ◽  
Arne Slungaard ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Biologically Active ◽  
Matrix Proteins ◽  
Hematopoietic Progenitor Cell ◽  
Heparin Binding ◽  
Heparan Sulfates ◽  
Kinetics Of ◽  
Better Than

We have shown that stromal O-sulfated heparan sulfate glycosaminoglycans (O-S-GAGs) regulate primitive human hematopoietic progenitor cell (HPC) growth and differentiation by colocalizing heparin-binding cytokines and matrix proteins with HPC in stem cell “niches” in the marrow microenvironment. We now show that long-term culture-initiating cells (LTC-IC) are maintained for 5 weeks in the absence of stroma when O-S-GAGs are added to IL-3 and either MIP-1 or PF4 (LTC-IC maintenance without GAGs, 32 ± 2%; with GAGs, 95 ± 7%; P < .001). When cultured with 5 additional cytokines, O-S-GAGs, IL-3, and MIP-1, LTC-IC expanded 2- to 4-fold at 2 weeks, and 92 ± 8% LTC-IC were maintained at 5 weeks. Similar results were seen when PF4 replaced MIP-1. Although O-S-GAG omission did not affect 2-week expansion, only 20% LTC-IC were maintained for 5 weeks. When O-S-heparin was replaced by completely desulfated-, N-sulfated (O-desulfated), or unmodified heparins, LTC-IC maintenance at week 5 was not better than with cytokines alone. Unmodified- and O-S-heparin, but not desulfated- or N-sulfated heparin, bound to MIP-1, IL-3, PF4, VEGF, thrombospondin, and fibronectin. However, the affinity of heparin for thrombospondin and PF4, and the association and dissociation rates of heparin for PF4, were higher than those of O-S-heparin. We conclude that (i) although cytokines may suffice to induce early expansion, adult human LTC-IC maintenance for longer than 1 month requires O-S-GAGs, and (ii) HPC support may depend not only on the ability of GAGs to bind proteins, but also on optimal affinity and kinetics of interactions that affect presentation of proteins in a biologically active manner to progenitors. (Blood. 2000;95:147-155)

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