An Improved Method to Obtain a Soluble Nuclear Fraction from Embryonic Brain Tissue

Giusti, Sebastián; Bogetti, M; Bonafina, Antonela; Plazas, Sara Fiszer de

doi:10.1007/s11064-009-9993-9

Cited by 23 publications

(15 citation statements)

References 20 publications

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“…The ionic detergent SDS is able to solubilize the nuclear and cytoplasmic cell membranes, as well as dissociate DNA from proteins. 34 A high concentration of SDS is more efficient to remove DNA content than a lower concentration of SDS, 35 which is also proved by our results (Fig. 1).…”

supporting

confidence: 80%

Decellularization of Fibroblast Cell Sheets for Natural Extracellular Matrix Scaffold Preparation

Xing

Yates

Tahtinen

et al. 2015

Tissue Engineering Part C: Methods

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The application of cell-derived extracellular matrix (ECM) in tissue engineering has gained increasing interest because it can provide a naturally occurring, complex set of physiologically functional signals for cell growth. The ECM scaffolds produced from decellularized fibroblast cell sheets contain high amounts of ECM substances, such as collagen, elastin, and glycosaminoglycans. They can serve as cell adhesion sites and mechanically strong supports for tissue-engineered constructs. An efficient method that can largely remove cellular materials while maintaining minimal disruption of ECM ultrastructure and content during the decellularization process is critical. In this study, three decellularization methods were investigated: high concentration (0.5 wt%) of sodium dodecyl sulfate (SDS), low concentration (0.05 wt%) of SDS, and freeze-thaw cycling method. They were compared by characterization of ECM preservation, mechanical properties, in vitro immune response, and cell repopulation ability of the resulted ECM scaffolds. The results demonstrated that the high SDS treatment could efficiently remove around 90% of DNA from the cell sheet, but significantly compromised their ECM content and mechanical strength. The elastic and viscous modulus of the ECM decreased around 80% and 62%, respectively, after the high SDS treatment. The freeze-thaw cycling method maintained the ECM structure as well as the mechanical strength, but also preserved a large amount of cellular components in the ECM scaffold. Around 88% of DNA was left in the ECM after the freeze-thaw treatment. In vitro inflammatory tests suggested that the amount of DNA fragments in ECM scaffolds does not cause a significantly different immune response. All three ECM scaffolds showed comparable ability to support in vitro cell repopulation. The ECM scaffolds possess great potential to be selectively used in different tissue engineering applications according to the practical requirement.

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supporting

confidence: 80%

Decellularization of Fibroblast Cell Sheets for Natural Extracellular Matrix Scaffold Preparation

Xing

Yates

Tahtinen

et al. 2015

Tissue Engineering Part C: Methods

180

134

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“…Commonly used detergents include Triton X-100 and CHAPS for thick tissues, or low percentages of SDS (sodium dodecyl sulfate) for whole organ decellularization. These detergents solubilize the cell membrane and disrupt DNA (Meyer et al, 2006; Jones and Wagers, 2008; Giusti et al, 2009; Petersen et al, 2010; Reing et al, 2010; Crapo et al, 2011), though they have negative effects on protein and ultrastructure. Chelating agents, such as EDTA (ethylenediaminetetraacetic acid) and EGTA (ethylene glycol tetraacetic acid), help to dissociate cells from the ECM, while serine protease inhibitors, such as PMSF (phenylmethylsulfonyl fluoride), aprotinin, and leupeptin, prevent ECM damage (Crapo et al, 2011).…”

Section: Decellularization Of Tissues For Ecm-based Scaffold Productionmentioning

confidence: 99%

Native extracellular matrix: a new scaffolding platform for repair of damaged muscle

et al. 2014

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Effective clinical treatments for volumetric muscle loss resulting from traumatic injury or resection of a large amount of muscle mass are not available to date. Tissue engineering may represent an alternative treatment approach. Decellularization of tissues and whole organs is a recently introduced platform technology for creating scaffolding materials for tissue engineering and regenerative medicine. The muscle stem cell niche is composed of a three-dimensional architecture of fibrous proteins, proteoglycans, and glycosaminoglycans, synthesized by the resident cells that form an intricate extracellular matrix (ECM) network in equilibrium with the surrounding cells and growth factors. A consistent body of evidence indicates that ECM proteins regulate stem cell differentiation and renewal and are highly relevant to tissue engineering applications. The ECM also provides a supportive medium for blood or lymphatic vessels and for nerves. Thus, the ECM is the nature's ideal biological scaffold material. ECM-based bioscaffolds can be recellularized to create potentially functional constructs as a regenerative medicine strategy for organ replacement or tissue repopulation. This article reviews current strategies for the repair of damaged muscle using bioscaffolds obtained from animal ECM by decellularization of small intestinal submucosa (SIS), urinary bladder mucosa (UB), and skeletal muscle, and proposes some innovative approaches for the application of such strategies in the clinical setting.

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“…Purified nuclei and pure soluble nuclear fractions derived from them were prepared by an improved method recently developed by our group and described elsewhere (Giusti et al, 2009). Optic lobes of six embryos were pooled to obtain a single sample.…”

Section: Tissue Preparationmentioning

confidence: 99%

Neuroprotection by hypoxic preconditioning involves upregulation of hypoxia‐inducible factor‐1 in a prenatal model of acute hypoxia

Giusti

Plazas

2011

J of Neuroscience Research

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The molecular pathways underlying the neuroprotective effects of preconditioning are promising, potentially drugable targets to promote cell survival. However, these pathways are complex and are not yet fully understood. In this study we have established a paradigm of hypoxic preconditioning based on a chick embryo model of normobaric acute hypoxia previously developed by our group. With this model, we analyzed the role of hypoxia-inducible factor-1α (HIF-1α) stabilization during preconditioning in HIF-1 signaling after the hypoxic injury and in the development of a neuroprotective effect against the insult. To this end, we used a pharmacological approach, based on the in vivo administration of positive (Fe(2+), ascorbate) and negative (CoCl(2)) modulators of the activity of HIF-prolyl hydroxylases (PHDs), the main regulators of HIF-1. We have found that preconditioning has a reinforcing effect on HIF-1 accumulation during the subsequent hypoxic injury. In addition, we have also demonstrated that HIF-1 induction during hypoxic preconditioning is necessary to obtain an enhancement in HIF-1 accumulation and to develop a tolerance against a subsequent hypoxic injury. We provide in vivo evidence that administration of Fe(2+) and ascorbate modulates HIF accumulation, suggesting that PHDs might be targets for neuroprotection in the CNS.

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An Improved Method to Obtain a Soluble Nuclear Fraction from Embryonic Brain Tissue

Cited by 23 publications

References 20 publications

Decellularization of Fibroblast Cell Sheets for Natural Extracellular Matrix Scaffold Preparation

Decellularization of Fibroblast Cell Sheets for Natural Extracellular Matrix Scaffold Preparation

Native extracellular matrix: a new scaffolding platform for repair of damaged muscle

Neuroprotection by hypoxic preconditioning involves upregulation of hypoxia‐inducible factor‐1 in a prenatal model of acute hypoxia

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