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On-Demand Webinar Available: Cell Freezing Technologies and Disposable Bioreactors Towards a USP Process
Develop a Fully-Closed USP Process: Use Cell Freezing in Bags and SU Bioreactors
  • Recorded on May 22, 2014
  • Duration: 50 minutes
  • A single-cell and feeder-free culture system for monkey embryonic stem cells. 24505480

    Primate pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold great potential for research and application in regenerative medicine and drug discovery. To maximize primate PSC potential, a practical system is required for generating desired functional cells and reproducible differentiation techniques. Much progress regarding their culture systems has been reported to date; however, better methods would still be required for their practical use, particularly in industrial and clinical fields. Here we report a new single-cell and feeder-free culture system for primate PSCs, the key feature of which is an originally formulated serum-free medium containing FGF and activin. In this culture system, cynomolgus monkey ESCs can be passaged many times by single-cell dissociation with traditional trypsin treatment and can be propagated with a high proliferation rate as a monolayer without any feeder cells; further, typical PSC properties and genomic stability can be retained. In addition, it has been demonstrated that monkey ESCs maintained in the culture system can be used for various experiments such as in vitro differentiation and gene manipulation. Thus, compared with the conventional culture system, monkey ESCs grown in the aforementioned culture system can serve as a cell source with the following practical advantages: simple, stable, and easy cell maintenance; gene manipulation; cryopreservation; and desired differentiation. We propose that this culture system can serve as a reliable platform to prepare primate PSCs useful for future research and application.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
  • Human umbilical cord mesenchymal stromal cells exhibit immature nucleus pulposus cell phenotype in a laminin-rich pseudo-three-dimensional culture system. 24405888

    Cell supplementation to the herniated or degenerated intervertebral disc (IVD) is a potential strategy to promote tissue regeneration and slow disc pathology. Human umbilical cord mesenchymal stromal cells (HUCMSCs) - originating from the Wharton's jelly - remain an attractive candidate for such endeavors with their ability to differentiate into multiple lineages. Previously, mesenchymal stem cells (MSCs) have been studied as a potential source for disc tissue regeneration. However, no studies have demonstrated that MSCs can regenerate matrix with unique characteristics matching that of immature nucleus pulposus (NP) tissues of the IVD. In our prior work, immature NP cells were found to express specific laminin isoforms and laminin-binding receptors that may serve as phenotypic markers for evaluating MSC differentiation to NP-like cells. The goal of this study is to evaluate these markers and matrix synthesis for HUCMSCs cultured in a laminin-rich pseudo-three-dimensional culture system.HUCMSCs were seeded on top of Transwell inserts pre-coated with Matrigel™, which contained mainly laminin-111. Cells were cultured under hypoxia environment with three differentiation conditions: NP differentiation media (containing 2.5% Matrigel™ solution to provide for a pseudo-three-dimensional laminin culture system) with no serum, or the same media supplemented with either insulin-like growth factor-1 (IGF-1) or transforming growth factor-β1 (TGF-β1). Cell clustering behavior, matrix production and the expression of NP-specific laminin and laminin-receptors were evaluated at days 1, 7, 13 and 21 of culture.Data show that a pseudo-three-dimensional culture condition (laminin-1 rich) promoted HUCMSC differentiation under no serum conditions. Starting at day 1, HUCMSCs demonstrated a cell clustering morphology similar to that of immature NP cells in situ and that observed for primary immature NP cells within the similar laminin-rich culture system (prior study). Differentiated HUCMSCs under all conditions were found to contain glycosaminoglycan, expressed extracellular matrix proteins of collagen II and laminin α5, and laminin receptors (integrin α3 and β4 subunits). However, neither growth factor treatment generated distinct differences in NP-like phenotype for HUCMSC as compared with no-serum conditions.HUCMSCs have the potential to differentiate into cells sharing features with immature NP cells in a laminin-rich culture environment and may be useful for IVD cellular therapy.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
  • The dynamin-related GTPase Opa1 is required for glucose-stimulated ATP production in pancreatic beta cells. 21551073

    Previous studies using in vitro cell culture systems have shown the role of the dynamin-related GTPase Opa1 in apoptosis prevention and mitochondrial DNA (mtDNA) maintenance. However, it remains to be tested whether these functions of Opa1 are physiologically important in vivo in mammals. Here, using the Cre-loxP system, we deleted mouse Opa1 in pancreatic beta cells, in which glucose-stimulated ATP production in mitochondria plays a key role in insulin secretion. Beta cells lacking Opa1 maintained normal copy numbers of mtDNA; however, the amount and activity of electron transport chain complex IV were significantly decreased, leading to impaired glucose-stimulated ATP production and insulin secretion. In addition, in Opa1-null beta cells, cell proliferation was impaired, whereas apoptosis was not promoted. Consequently, mice lacking Opa1 in beta cells develop hyperglycemia. The data suggest that the function of Opa1 in the maintenance of the electron transport chain is physiologically relevant in beta cells.
    Document Type:
    Reference
    Product Catalog Number:
    AB932
    Product Catalog Name:
    Anti-Glucagon Antibody
  • Cyclin D3 accumulation and activity integrate and rank the comitogenic pathways of thyrotropin and insulin in thyrocytes in primary culture. 10602491

    The proliferation of most normal cells depends on the synergistic interaction of several growth factors and hormones, but the cell cycle basis for this combined requirement remains largely uncharacterized. We have addressed the question of the requirement for insulin/IGF-1 also observed in many cell culture systems in the physiologically relevant system of primary cultures of dog thyroid epithelial cells stimulated by TSH, which exerts its mitogenic activity only via cAMP. The induction of cyclin A and cdc2, the phosphorylation of cdk2, the nuclear translocation of cdk4 and the assembly of cyclin D3-cdk4 complexes required the synergy of TSH and insulin. Cyclin D3 (the most abundant cyclin D) was necessary for the proliferation stimulated by TSH in the presence of insulin as shown by microinjection of a neutralizing antibody. Cyclin D3 accumulation and activity were differentially regulated by insulin and TSH, which points out this cyclin as an integrator that ranks these comitogenic pathways as supportive and activatory, respectively. Paradoxically TSH alone strongly repressed cyclin D3 accumulation. This inhibition was overridden by insulin, which markedly stimulated cyclin D3 mRNA and protein accumulation, but failed to assemble cyclin D3-cdk4 complexes in the absence of TSH. TSH unmasked the DCS-22 epitope of cyclin D3 and assembled cyclin D3-cdk4 in the presence of insulin. These data demonstrate that cyclin D synthesis and cyclin D-cdk assembly can be dissociated and complementarily regulated by different agents and signalling pathways.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
  • Apoptosis-like cell death induction and aberrant fibroblast properties in human incisional hernia fascia. 21641387

    Incisional hernia often occurs following laparotomy and can be a source of serious problems. Although there is evidence that a biological cause may underlie its development, the mechanistic link between the local tissue microenvironment and tissue rupture is lacking. In this study, we used matched tissue-based and in vitro primary cell culture systems to examine the possible involvement of fascia fibroblasts in incisional hernia pathogenesis. Fascia biopsies were collected at surgery from incisional hernia patients and non-incisional hernia controls. Tissue samples were analyzed by histology and immunoblotting methods. Fascia primary fibroblast cultures were assessed at morphological, ultrastructural, and functional levels. We document tissue and fibroblast loss coupled to caspase-3 activation and induction of apoptosis-like cell-death mechanisms in incisional hernia fascia. Alterations in cytoskeleton organization and solubility were also observed. Incisional hernia fibroblasts showed a consistent phenotype throughout early passages in vitro, which was characterized by significantly enhanced cell proliferation and migration, reduced adhesion, and altered cytoskeleton properties, as compared to non-incisional hernia fibroblasts. Moreover, incisional hernia fibroblasts displayed morphological and ultrastructural alterations compatible with autophagic processes or lysosomal dysfunction, together with enhanced sensitivity to proapoptotic challenges. Overall, these data suggest an ongoing complex interplay of cell death induction, aberrant fibroblast function, and tissue loss in incisional hernia fascia, which may significantly contribute to altered matrix maintenance and tissue rupture in vivo.
    Document Type:
    Reference
    Product Catalog Number:
    CBL171
    Product Catalog Name:
    Anti-Actin Antibody, smooth muscle, clone ASM-1
  • Endothelial cell responses towards low-fouling surfaces bearing rGD in a three-dimensional environment. 21679704

    This study reveals that it is possible to obtain a specific cell response towards low-fouling carboxymethyl dextran (CMD) surfaces bearing the RGD adhesive peptide in fibrin. To avoid cell sedimentation on surfaces observed in traditional cell culture systems, CMD surfaces bearing RGD were vertically embedded in fibrin containing human umbilical vein endothelial cells (HUVEC) and their effect over cells was investigated. Compared to the CMD surfaces and to CMD layers bearing the negative control RGE, RGD coatings promoted cell adhesion, induced focal contact formation indicated by co-localization of vinculin and actin fibers, and presented a significant effect over HUVEC net growth during the first 24h of the culture, as revealed by Ki67 staining and cell counting. The intracellular localization of caveolin-1 combined with the expression of beta 1 integrins was investigated and the orientation of HUVEC towards and on the RGD surfaces was studied. When compared to the negative controls, HUVEC responded to the RGD surface in fibrin resulting in acceleration of morphological changes. RGD surfaces supported fibrin degradation by HUVEC as revealed by fluorescent fibrin experiments as well as multi-cellular structure formation, vacuolation and lumen formation.Copyright © 2011 Elsevier Inc. All rights reserved.
    Document Type:
    Reference
    Product Catalog Number:
    AB2910
    Product Catalog Name:
    Anti-Mcl-1 Antibody
  • Mammary epithelial-specific disruption of the focal adhesion kinase blocks mammary tumor progression. 18056629

    Elevated expression and activation of the focal adhesion kinase (FAK) occurs in a large proportion of human breast cancers. Although several studies have implicated FAK as an important signaling molecule in cell culture systems, evidence supporting a role for FAK in mammary tumor progression is lacking. To directly assess the role of FAK in this process, we have used the Cre/loxP recombination system to disrupt FAK function in the mammary epithelium of a transgenic model of breast cancer. Using this approach, we demonstrate that FAK expression is required for the transition of premalignant hyperplasias to carcinomas and their subsequent metastases. This dramatic block in tumor progression was further correlated with impaired mammary epithelial proliferation. These observations provide direct evidence that FAK plays a critical role in mammary tumor progression.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
  • Defined carboxy-terminal fragments of insulin-like growth factor (IGF) binding protein-2 exert similar mitogenic activity on cultured rat growth plate chondrocytes as IGF ... 18556354

    The IGF/IGF binding protein (IGFBP) system is an important component in the hormonal regulation of longitudinal growth. Evidence from in vitro studies indicates that IGFBPs may have IGF-independent effects. We analyzed the biological activity of intact IGFBP-2 and defined carboxy-terminal IGFBP-2 fragments isolated from human hemofiltrate in two cell culture systems of the growth plate: rat growth plate chondrocytes in primary culture and the mesenchymal chondrogenic cell line RCJ3.1C5.18. The IGFBP-2 fragments IGFBP-2(167-279), IGFBP-2(167-289), and IGFBP-2(104-289) exerted a strong (2- to 3-fold) mitogenic effect on growth plate chondrocytes, which was comparable with IGF-I in equimolar concentrations (7.8 nm) but was not mediated through the type 1 IGF receptor. In a dose-response experiment, the most effective concentration of IGFBP-2(104-289) for the stimulation of cell proliferation was 10 nm. This biological activity of IGFBP-2 fragments was associated with cell membrane binding, demonstrated by Western blot analysis of fractionated cell lysates and immunohistochemistry. Whereas intact IGFBP-2 did not modulate chondrocyte proliferation, partially reduced (by dithiothreitol) full-length IGFBP-2 stimulated cell proliferation to a comparable extent (3.4-fold) as carboxy-terminal IGFBP-2 fragments. The mitogenic activity of these IGFBP-2 fragments and of partially reduced full-length IGFBP-2 was mediated through the use of the MAPK/ERK 1/2. These data imply a novel role of naturally occurring IGFBP-2 fragments for the endocrine and paracrine/autocrine regulation of longitudinal growth.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple