Reduction of DNA damage repair efficiency and accumulation of residual damage following chronic UVB-irradiation of HaCaT cells.

Absorption of ultraviolet radiation (UVR) by DNA leads to the predominant formation of cyclobutane pyrimidine dimers (CPD). Since those CPD are responsible for the driver mutations found in skin cancers, their efficient repair is critical. We previously showed that pre-stimulation of fibroblasts with chronic low doses of UVB (CLUV) increases CPD repair efficiency.

Three-Dimensional Model of Hypertrophic Scar Using a Tissue-Engineering Approach.

Following wound healing, skin is replaced by a specialized tissue called scar. Sometime, this scar can become pathologic, called hypertrophic scar, with a high amount of extracellular matrix, capillaries, and myofibroblast persistence. To understand the mechanisms at the origin of the fibrosis is paramount to treat patients, but despite few animal models and in vitro studies using mainly human pathological cells cultured on plastic on monolayer, the treatment of these fibrotic scars remains unsatisfactory.

Hydrodynamic forces influence the gene transcription of mechanosensitive intercellular junction associated genes in corneal endothelial cells.

Mechanicals forces are known to influence cell behavior. In vivo, the corneal endothelium is under the influence of various mechanical forces, such as intraocular pressure (IOP) and fluid flow. In this study, we used a corneal bioreactor to understand the effect of these hydrodynamic forces on the transcription of intercellular junctions associated genes in the corneal endothelium.

[Tridimensional in vitro models of nervous and immune systems in the skin].

The immune system and the sensory nervous system are responsible for perceiving danger under distinct yet complementary forms. In the last few years, neuroimmune interactions have become an important topic of dermatological research for conditions including wound healing, atopic dermatitis and psoriasis. We present here a selection of tridimensional in vitro models that reproduce skin structure and integrate an immune or a sensory function.

Conditioned medium produced by fibroblasts cultured in low oxygen pressure allows the formation of highly structured capillary-like networks in fibrin gels.

Tissue engineering is an emerging and promising concept to replace or cure failing organs, but its clinical translation currently encounters issues due to the inability to quickly produce inexpensive thick tissues, which are necessary for many applications. To circumvent this problem, we postulate that cells secrete the optimal cocktail required to promote angiogenesis when they are placed in physiological conditions where their oxygen supply is reduced. Thus, dermal fibroblasts were cultivated under hypoxia (2% O) to condition their cell culture medium.

Irradiated Human Fibroblasts as a Substitute Feeder Layer to Irradiated Mouse 3T3 for the Culture of Human Corneal Epithelial Cells: Impact on the Stability of the Transcription Factors Sp1 and NFI.

Because of the worldwide shortage of graftable corneas, alternatives to restore visual impairments, such as the production of a functional human cornea by tissue engineering, have emerged. Self-renewal of the corneal epithelium through the maintenance of a sub-population of corneal stem cells is required to maintain the functionality of such a reconstructed cornea. We previously reported an association between stem cell differentiation and the level to which they express the transcription factors Sp1 and NFI.

Development of an innervated tissue-engineered skin with human sensory neurons and Schwann cells differentiated from iPS cells.

Cutaneous innervation is increasingly recognized as a major element of skin physiopathology through the neurogenic inflammation driven by neuropeptides that are sensed by endothelial cells and the immune system. To investigate this process in vitro, models of innervated tissue-engineered skin (TES) were developed, yet exclusively with murine sensory neurons extracted from dorsal root ganglions. In order to build a fully human model of innervated TES, we used induced pluripotent stem cells (iPSC) generated from human skin fibroblasts.

The Tissue-Engineered Human Psoriatic Skin Substitute: A Valuable In Vitro Model to Identify Genes with Altered Expression in Lesional Psoriasis.

Psoriasis is a chronic inflammatory skin disease for which no cure has emerged. Its complex etiology requires the development of an in vitro model representative of the pathology. In this study, we exploited gene profiling analyses on microarray in order to characterize and further optimize the production of a human psoriatic skin model representative of this in vivo skin disease.

Isolation of Human Skin Lymphatic Endothelial Cells and 3D Reconstruction of the Lymphatic Vasculature In Vitro.

Studies of lymphangiogenesis and lymphatic endothelial biology in vitro require pure cultures of lymphatic endothelial cells and 3D vascular constructs, which closely resemble native human lymphatic vasculature. We describe a method for the isolation of human dermal microvascular lymphatic endothelial cells and generation of a 3D lymphatic capillary network. The lymphatic vascular construct is generated by coculturing primary lymphatic endothelial cells and fibroblasts in their native matrix, without the use of synthetic scaffolds or exogenous factors.

Contribution of and Organotypic 3D Models to Understanding the Role of Macrophages and Neutrophils in the Pathogenesis of Psoriasis.

Psoriasis, a common chronic immune-mediated skin disease, is histologically characterized by a rapid keratinocyte turnover and differentiation defects. Key insights favor the idea that T cells are not the only key actors involved in the inflammatory process. Innate immune cells, more precisely neutrophils and macrophages, provide specific signals involved in the initiation and the maintenance of the pathogenesis.

A 3D-psoriatic skin model for dermatological testing: The impact of culture conditions.

Background: Inadequate representation of the human tissue environment during a preclinical screen can result in inaccurate predictions of compound effects. Consequently, pharmaceutical investigators are searching for preclinical models that closely resemble original tissue for predicting clinical outcomes.

Methods: The current research aims to compare the impact of using serum-free medium instead of complete culture medium during the last step of psoriatic skin substitute reconstruction.

Tissue-engineered 3D human lymphatic microvascular network for in vitro studies of lymphangiogenesis.

This protocol describes a unique in vitro method for the generation of a 3D human lymphatic network within native connective tissue devoid of any exogenous material such as scaffolds or growth factors. In this five-stage protocol, human lymphatic endothelial cells (LECs) cocultured with dermal fibroblasts spontaneously organize into a stable 3D lymphatic capillary network. Stage 1 involves the isolation of primary fibroblasts and LECs from human skin.

Urothelial cell expansion and differentiation are improved by exposure to hypoxia.

Cells obtained from a patient's biopsy have to be expanded after extraction to produce autologous tissues, but standard cell culture conditions often limit their growth or lifespan and could induce early and inadequate cell differentiation. Moreover, it has previously been reported that the air-liquid interface, that induces maturation of the urothelium, stimulated inadequate differentiation associated with aberrant keratin-14 expression. The aim of this study was to test the benefits of hypoxia during expansion of urothelial cells and maturation of the bladder epithelium in the context of tissue engineering.

Remodeling of Fibroblast-Derived Vascular Scaffolds Implanted for 6 Months in Rats.

There is a clinical need for tissue-engineered small-diameter (<6 mm) vascular grafts since clinical applications are halted by the limited suitability of autologous or synthetic grafts. This study uses the self-assembly approach to produce a fibroblast-derived decellularized vascular scaffold (FDVS) that can be available off-the-shelf. Briefly, extracellular matrix scaffolds were produced using human dermal fibroblasts sheets rolled around a mandrel, maintained in culture to allow for the formation of cohesive and three-dimensional tubular constructs, and decellularized by immersion in deionized water.

Biotechnological Management of Skin Burn Injuries: Challenges and Perspectives in Wound Healing and Sensory Recovery.

Many wound management protocols have been developed to improve wound healing after burn with the primordial aim to restore the barrier function of the skin and also provide a better esthetic outcome. Autologous skin grafts remain the gold standard in the treatment of skin burn, but this treatment has its limitation especially for patients presenting limited donor sites due to extensive burn areas. Deep burn injuries also alter the integrity of skin-sensitive innervation and have an impact on patient's quality of life by compromising perceptions of touch, temperature, and pain.

Tissue-engineered human psoriatic skin supplemented with cytokines as an in vitro model to study plaque psoriasis.

Aim: Psoriasis is a chronic inflammatory skin disease. To study its complex etiology, a psoriatic skin substitute model supplemented with a cytokine cocktail has been used.

Materials & Methods: Reconstructed psoriatic skin substitutes were supplemented with a cocktail of four cytokines: TNF-α, IL-1α, IL-6 and IL-17A, to monitor their impact on gene expression by DNA microarray.

Origin of Serum Affects Quality of Engineered Tissues Produced by the Self-Assembly Approach.

Despite the emergence of serum-free media for cell culture, the use of serum to supplement the culture media is still essential in order to produce engineered urologic tissues using the self-assembly approach, not only for the stromal compartment but also for the uroepithelium. Effects of sera on thickness of these two compartments were measured and quality of the epithelial differentiation was evaluated. For bladder mucosa substitute reconstruction, the use of postnatal sera failed to produce an adequate uroepithelium whereas the fetal sera supplementation did.

Production of a Self-Aligned Scaffold, Free of Exogenous Material, from Dermal Fibroblasts Using the Self-Assembly Technique.

Many pathologies of skin, especially ageing and cancer, involve modifications in the matrix alignment. Such tissue reorganization could have impact on cell behaviour and/or more global biological processes. Tissue engineering provides accurate study model by mimicking the skin and it allows the construction of versatile tridimensional models using human cells.

Cell-based approach for 3D reconstruction of lymphatic capillaries in vitro reveals distinct functions of HGF and VEGF-C in lymphangiogenesis.

Regeneration of lymphatic vessels is important for treatment of various disorders of lymphatic system and for restoration of lymphatic function after surgery. We have developed a method for generating a human 3D lymphatic vascular construct. In this system, human lymphatic endothelial cells, co-cultured with fibroblasts, spontaneously organized into a stable 3D lymphatic capillary network without the use of any exogenous factors.

High yield extraction of pure spinal motor neurons, astrocytes and microglia from single embryo and adult mouse spinal cord.

Extraction of mouse spinal motor neurons from transgenic mouse embryos recapitulating some aspects of neurodegenerative diseases like amyotrophic lateral sclerosis has met with limited success. Furthermore, extraction and long-term culture of adult mouse spinal motor neurons and glia remain also challenging. We present here a protocol designed to extract and purify high yields of motor neurons and glia from individual spinal cords collected on embryos and adult (5-month-old) normal or transgenic mice.

Demonstration of the direct impact of ketamine on urothelium using a tissue engineered bladder model.

Introduction: Ketamine is a common recreational drug. Severe lower urinary tract symptoms associated with its consumption have been reported, but little is known about the involved mechanisms. The effect of ketamine, which is excreted in urine, was evaluated by its application on an in vitro three-dimensional human tissue-engineered bladder model composed of an urothelium and a submucosa.

Using infrared and Raman microspectroscopies to compare ex vivo involved psoriatic skin with normal human skin.

Psoriasis is a chronic dermatosis that affects around 3% of the world's population. The etiology of this autoimmune pathology is not completely understood. The barrier function of psoriatic skin is known to be strongly altered, but the structural modifications at the origin of this dysfunction are not clear.

In vitro glycation of an endothelialized and innervated tissue-engineered skin to screen anti-AGE molecules.

Glycation is one of the major processes responsible for skin aging through induction of the detrimental formation of advanced glycation end-products (AGEs). We developed an innovative tissue-engineered skin combining both a capillary-like and a nerve networks and designed a protocol to induce continuous AGEs formation by a treatment with glyoxal. We determined the optimal concentration of glyoxal to induce AGEs formation identified by carboxymethyl-lysin expression while keeping their toxic effects low.

Sensory neurons accelerate skin reepithelialization via substance P in an innervated tissue-engineered wound healing model.

Keratinocytes are responsible for reepithelialization and restoration of the epidermal barrier during wound healing. The influence of sensory neurons on this mechanism is not fully understood. We tested whether sensory neurons influence wound closure via the secretion of the neuropeptide substance P (SP) with a new tissue-engineered wound healing model made of an upper-perforated epidermal compartment reconstructed with human keratinocytes expressing green fluorescent protein, stacked over a dermal compartment, innervated or not with sensory neurons.

Adherens junction proteins are expressed in collagen corneal equivalents produced in vitro with human cells.

Purpose: To test whether adherens junction proteins are present in the epithelium and the endothelium of corneal equivalents.

Methods: Corneal cell types were harvested from human eyes and grown separately. Stromal equivalents were constructed by seeding fibroblasts into a collagen gel on which epithelial and endothelial cells were added on each side.