Center Loex Of Luniversite Laval
Center Loex Of Luniversite Laval
Gibot L.,University Paul Sabatier |
Galbraith T.,Center Loex Of Luniversite Laval |
Bourland J.,Center Loex Of Luniversite Laval |
Rogic A.,Mount Sinai School of Medicine |
And 2 more authors.
Nature Protocols | Year: 2017
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. Fibroblasts are then cultured to produce connective tissue rich in extracellular matrix (stage 2), onto which LECs are seeded to form a network (stage 3). After stacking of tissue layers and tissue maturation at the air-liquid interface (stage 4), the 3D construct containing the lymphatic microvascular network can be analyzed by microscopy (stage 5). Lymphatic vasculature generated by this approach exhibits the major cellular and ultrastructural features of native in vivo human dermal lymphatic microvasculature and is stable over many weeks. The protocol for generating a 3D construct takes 6 weeks to complete, and it requires experience in cell culture techniques. The system described here offers a unique opportunity to study the mechanisms underlying lymphatic vessel formation, remodeling and function in a human cell context. © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
Leroy M.,Laval University |
Leroy M.,Center Loex Of Luniversite Laval |
Labbe J.-F.,Laval University |
Ouellet M.,Laval University |
And 5 more authors.
Acta Biomaterialia | Year: 2014
Research in the field of bioengineered skin substitutes is motivated by the need to find new dressings for people affected by skin injuries (burns, diabetic ulcers), and to develop adequate skin models to test new formulations developed in vitro. Thanks to advances in tissue engineering, it is now possible to produce human skin substitutes without any exogenous material, using the self-assembly method developed by the Laboratoire d'Organogénèse Expérimentale. These human skin substitutes consist of a dermis and a stratified epidermis (stratum corneum and living epidermis). Raman microspectroscopy has been used to characterize and compare the molecular organization of skin substitutes with normal human skin. Our results confirm that the stratum corneum is a layer rich in lipids which are well ordered (trans conformers) in both substitutes and normal human skin. The amount of lipids decreases and more gauche conformers appear in the living epidermis in both cases. However, the results also show that there are fewer lipids in the substitutes and that the lipids are more organized in the normal human skin. Concerning the secondary structure of proteins and protein content, the data show that they are similar in the substitutes and in the normal human skin. In fact, the epidermis is rich in α-keratin, whereas the dermis contains mainly type I collagen. © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Dubois Declercq S.,Center Loex Of Luniversite Laval |
Dubois Declercq S.,Laval University |
Pouliot R.,Center Loex Of Luniversite Laval |
Pouliot R.,Laval University
The Scientific World Journal | Year: 2013
Psoriasis is a chronic, proliferative, and inflammatory skin disease affecting 2-3% of the population and is characterized by red plaques with white scales. Psoriasis is a disease that can affect many aspects of professional and social life. Currently, several treatments are available to help control psoriasis such as methotrexate, ciclosporin, and oral retinoids. However, the available treatments are only able to relieve the symptoms and lives of individuals. The discovery of new immunological factors and a better understanding of psoriasis have turned to the use of immunological pathways and could develop new biological drugs against specific immunological elements that cause psoriasis. Biological drugs are less toxic to the body and more effective than traditional therapies. Thus, they should improve the quality of life of patients with psoriasis. This review describes new psoriasis treatments, which are on the market or currently in clinical trials that are being used to treat moderate-to-severe plaque psoriasis. In addition, this paper describes the characteristics and mechanisms in detail. In general, biological drugs are well tolerated and appear to be an effective alternative to conventional therapies. However, their effectiveness and long-term side effects need to be further researched. © 2013 Sarah Dubois Declercq and Roxane Pouliot.
Moulin V.J.,Center Loex Of Luniversite Laval |
Moulin V.J.,University of Québec |
Moulin V.J.,Laval University
Methods in Molecular Biology | Year: 2013
Skin fibrosis is involved in several pathologies as hypertrophic scar or scleroderma. The determination of the mechanisms at the origin of these problems is however difficult due to the low number of in vivo models. To bypass this absence of animal models, studies typically use human pathological cells cultured in a monolayer way on plastic. However, cell behavior is different according to the fact that cells are on plastic or embedded in matrix. Using a tissue engineering method, we have developed new in vitro models to study these pathologies of the skin. Human pathological cells are used to reconstitute a three dimensional fibrotic tissue comprising the dermal and the epidermal parts of the skin. This method is called the selfassembly approach and is based on the cell capacity to reconstitute in vitro their own environment as in vivo. In this chapter, protocols generating reconstructed pathological skin using this approach are detailed. The methods include extraction and culture of human skin keratinocytes and fibroblasts from very small cutaneous biopsies. In addition, a description of the protocols for the production of fibrotic dermal sheets can be found to obtain a model of fibrotic dermis that can be associated or not with a fully differentiated epidermis. © Springer Science+Business Media, LLC 2013.
Giasson C.J.,University of Montréal |
Giasson C.J.,Center Loex Of Luniversite Laval |
Deschambeault A.,Center Loex Of Luniversite Laval |
Carrier P.,Center Loex Of Luniversite Laval |
And 2 more authors.
Molecular Vision | Year: 2014
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. Alternatively, bovine endothelial cells were used. At maturity, sections of stromal equivalents were processed for Masson's trichrome or indirect immunofluorescence using antibodies against pan-, N-, or E-cadherins or α-or β-catenins. Alternatively, stromal equivalents were dissected, to separate the proteins from the epithelium, endothelium, and stroma with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blots of the transferred proteins exposed to these primary antibodies were detected with chemiluminescence. Native corneas were processed similarly. Results: Three or four layers of epithelial cells reminiscent of the native cornea (basal cuboidal and superficial flatter cells) lay over a stromal construct containing fibroblastic cells under which an endothelium is present. Western blots and indirect immunofluorescence revealed that, similarly to the native cornea, the epithelium reacted positively to antibodies against catenins (α and β) and E-cadherin. The endothelium of corneal constructs, whether of human or bovine origin, reacted mildly to catenins and N-cadherin. Conclusions: This collagen-based corneal equivalent simulated the native cornea. Cells from the epithelial and endothelial layers expressed adherens junction proteins, indicating the presence of cell-cell contacts and the existence of polarized morphology of these layers over corneal equivalents. © 2014 Molecular Vision.
Auger F.A.,Center Loex Of Luniversite Laval |
Auger F.A.,Laval University |
Gibot L.,CNRS Institute of Pharmacology and Structural Biology |
Gibot L.,Toulouse 1 University Capitole |
Lacroix D.,Center Loex Of Luniversite Laval
Annual Review of Biomedical Engineering | Year: 2013
Vascularization is one of the great challenges that tissue engineering faces in order to achieve sizeable tissue and organ substitutes that contain living cells. There are instances, such as skin replacement, in which a tissue-engineered substitute does not absolutely need a preexisting vascularization. However, tissue or organ substitutes in which any dimension, such as thickness, exceeds 400 ?mu?m need to be vascularized to ensure cellular survival. Consistent with the wide spectrum of approaches to tissue engineering itself, which vary from acellular synthetic biomaterials to purely biological living constructs, approaches to tissue-engineered vascularization cover numerous techniques. Those techniques range from micropatterns engineered in biomaterials to microvascular networks created by endothelial cells. In this review, we strive to provide a critical overview of the elements that must be considered in the pursuit of this goal and the major approaches that are investigated in hopes of achieving it. Copyright © 2013 by Annual Reviews.
PubMed | Laval University and Center Loex Of Luniversite Laval
Type: | Journal: Scientifica | Year: 2016
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. Postnatal sera also provided thinner stromal compartments than the one obtained using fetal sera, no matter if the fibroblasts from healthy or psoriatic donors were used to reconstruct human skin substitutes.
PubMed | Mount Sinai School of Medicine and Center Loex Of Luniversite Laval
Type: | Journal: Biomaterials | Year: 2015
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. Invitro-generated lymphatic capillaries exhibited the major molecular and ultra-structural features of native, human lymphatic microvasculature: branches in the three dimensions, wide lumen, blind ends, overlapping borders, adherens and tight junctions, anchoring filaments, lack of mural cells, and poorly developed basement membrane. Furthermore, we show that fibroblast-derived VEGF-C and HGF cooperate in the formation of lymphatic vasculature by activating ERK1/2 signaling, and demonstrate distinct functions of HGF/c-Met and VEGF-C/VEGFR-3 in lymphangiogenesis. This lymphatic vascular construct is expected to facilitate studies of lymphangiogenesis invitro and it holds promise as a strategy for regeneration of lymphatic vessels and treatment of lymphatic disorders in various conditions.
PubMed | University of Québec and Center Loex Of Luniversite Laval
Type: Journal Article | Journal: Regenerative medicine | Year: 2016
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.Reconstructed psoriatic skin substitutes were supplemented with a cocktail of four cytokines: TNF-, IL-1, IL-6 and IL-17A, to monitor their impact on gene expression by DNA microarray.Gene profiling analyses identified several deregulated genes reported as being also deregulated in psoriasis skin invivo (S100A12, IL-8, DEFB4A and KYNU). The expression of those genes was dramatically increased compared with basal levels of controls (p<0.005 to<0.05).Psoriatic substitutes supplemented with a cocktail of TNF-, IL-1, IL-6 and IL-17A showed similar transcriptome alterations to those found in psoriasis.
PubMed | Center Loex Of Luniversite Laval
Type: | Journal: Scientific reports | Year: 2015
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. This method is based on mild digestion of tissue followed by gradient density separation allowing to obtain two millions motor neurons over 92% pure from one E14.5 single embryo and more than 30,000 from an adult mouse. These cells can be cultured more than 14 days in vitro at a density of 100,000 cells/cm(2) to maintain optimal viability. Functional astrocytes and microglia and small gamma motor neurons can be purified at the same time. This protocol will be a powerful and reliable method to obtain motor neurons and glia to better understand mechanisms underlying spinal cord diseases.