Engineering Tissues without the Use of a Synthetic Scaffold: A Twenty-Year History of the Self-Assembly Method

Twenty years ago, Dr. François A. Auger, the founder of the Laboratory of Experimental Organogenesis (LOEX), introduced the self-assembly technique. This innovative technique relies on the ability of dermal fibroblasts to produce and assemble their own extracellular matrix, differing from all other tissue-engineering techniques that use preformed synthetic scaffolds. Nevertheless, the use of the self-assembly technique was limited for a long time due to its main drawbacks: time and cost. Recent scientific breakthroughs have addressed these limitations. New protocol modifications that aim at increasing the rate of extracellular matrix formation have been proposed to reduce the production costs and laboratory handling time of engineered tissues. Moreover, the introduction of vascularization strategies in vitro permits the formation of capillary-like networks within reconstructed tissues. These optimization strategies enable the large-scale production of inexpensive native-like substitutes using the self-assembly technique. These substitutes can be used to reconstruct three-dimensional models free of exogenous materials for clinical and fundamental applications.

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

Dermatology Research and Practice ◽

10.1155/2016/5397319 ◽

2016 ◽

Vol 2016 ◽

pp. 1-9 ◽

Cited By ~ 3

Author(s):

Stéphane Chabaud ◽

Stéphane Bolduc

Keyword(s):

Self Assembly ◽

Standard Technique ◽

Dermal Fibroblasts ◽

The Self ◽

Cell Behaviour ◽

Assembly Method ◽

The Matrix ◽

Assembly Technique ◽

Fundamental Research ◽

Tissue Reorganization

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. It also avoids the use of animals, which gave sometimes nontranslatable results. Among the various techniques existing, the self-assembly method allows production of a near native skin, free of exogenous material. After cultivating human dermal fibroblasts in the presence of ascorbate during two weeks, a reseeding of these cells takes place after elevation of the resulting stroma on a permeable ring and culture pursued for another two weeks. This protocol induces a clear realignment of matrix fibres and cells parallel to the horizon. The thickness of this stretched reconstructed tissue is reduced compared to the stroma produced by the standard technique. Cell count is also reduced. In conclusion, a new, easy, and inexpensive method to produce aligned tissue free of exogenous material could be used for fundamental research applications in dermatology.

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Biophysical and biochemical properties of Deup1 self-assemblies: a potential driver for deuterosome formation during multiciliogenesis

Biology Open ◽

10.1242/bio.056432 ◽

2021 ◽

Vol 10 (3) ◽

pp. bio056432 ◽

Cited By ~ 1

Author(s):

Shohei Yamamoto ◽

Ryoichi Yabuki ◽

Daiju Kitagawa

Keyword(s):

Self Assembly ◽

Large Scale ◽

Molecular Mechanisms ◽

Coiled Coil ◽

Biochemical Properties ◽

The Self ◽

Centrosomal Protein ◽

Biochemical Analyses ◽

Stable Structures

ABSTRACTThe deuterosome is a non-membranous organelle involved in large-scale centriole amplification during multiciliogenesis. Deuterosomes are specifically assembled during the process of multiciliogenesis. However, the molecular mechanisms underlying deuterosome formation are poorly understood. In this study, we investigated the molecular properties of deuterosome protein 1 (Deup1), an essential protein involved in deuterosome assembly. We found that Deup1 has the ability to self-assemble into macromolecular condensates both in vitro and in cells. The Deup1-containing structures formed in multiciliogenesis and the Deup1 condensates self-assembled in vitro showed low turnover of Deup1, suggesting that Deup1 forms highly stable structures. Our biochemical analyses revealed that an increase of the concentration of Deup1 and a crowded molecular environment both facilitate Deup1 self-assembly. The self-assembly of Deup1 relies on its N-terminal region, which contains multiple coiled coil domains. Using an optogenetic approach, we demonstrated that self-assembly and the C-terminal half of Deup1 were sufficient to spatially compartmentalize centrosomal protein 152 (Cep152) and polo like kinase 4 (Plk4), master components for centriole biogenesis, in the cytoplasm. Collectively, the present data suggest that Deup1 forms the structural core of the deuterosome through self-assembly into stable macromolecular condensates.This article has an associated First Person interview with the first author of the paper.

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Human Organ-Specific 3D Cancer Models Produced by the Stromal Self-Assembly Method of Tissue Engineering for the Study of Solid Tumors

BioMed Research International ◽

10.1155/2020/6051210 ◽

2020 ◽

Vol 2020 ◽

pp. 1-23 ◽

Cited By ~ 1

Author(s):

Vincent Roy ◽

Brice Magne ◽

Maude Vaillancourt-Audet ◽

Mathieu Blais ◽

Stéphane Chabaud ◽

...

Keyword(s):

Tissue Engineering ◽

Tumor Microenvironment ◽

Self Assembly ◽

The Self ◽

Human Organ ◽

Assembly Method ◽

Cancer Models ◽

Organ Specific

Cancer research has considerably progressed with the improvement of in vitro study models, helping to understand the key role of the tumor microenvironment in cancer development and progression. Over the last few years, complex 3D human cell culture systems have gained much popularity over in vivo models, as they accurately mimic the tumor microenvironment and allow high-throughput drug screening. Of particular interest, in vitrohuman 3D tissue constructs, produced by the self-assembly method of tissue engineering, have been successfully used to model the tumor microenvironment and now represent a very promising approach to further develop diverse cancer models. In this review, we describe the importance of the tumor microenvironment and present the existing in vitro cancer models generated through the self-assembly method of tissue engineering. Lastly, we highlight the relevance of this approach to mimic various and complex tumors, including basal cell carcinoma, cutaneous neurofibroma, skin melanoma, bladder cancer, and uveal melanoma.

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An Innovative Vegetative Propagation System for Large-scale Production of Globe Artichoke Transplants. Part II. Propagation System Validation

HortTechnology ◽

10.21273/horttech.15.4.0817 ◽

2005 ◽

Vol 15 (4) ◽

pp. 817-819 ◽

Cited By ~ 8

Author(s):

Mariateresa Cardarelli ◽

Youssef Rouphael ◽

Francesco Saccardo ◽

Giuseppe Colla

Keyword(s):

In Vitro Propagation ◽

Large Scale ◽

Central Italy ◽

Production Costs ◽

Production Cycle ◽

Scale Production ◽

Globe Artichoke ◽

Large Scale Production ◽

Propagation Technique

Research was conducted at the University of Tuscia (central Italy) to validate the propagation system for globe artichoke (Cynara cardunculus var. scolymus) described in a previous paper for a 1-year production cycle. The resulting globe artichoke plants were used in a 2-year field trial to investigate the field response of plantlets obtained with our propagation technique in comparison with plantlets produced by in vitro propagation and by offshoots harvested in commercial fields. The total number of artichoke plantlets obtained with our propagation system was 62.7 plantlets/m2 per year. In the first year, the globe artichoke production (bud number and fresh bud weight) was higher in plants obtained with our propagation system and by micropropagation than in those obtained from offshoots harvested in commercial fields. The production cost of plantlets obtained with our propagation technique was 52% lower than those of the micropropagated plantlets. This could lead to a significant reduction of production costs for artichoke growers, while preserving the advantages of in vitro propagation (disease-free plants and plant uniformity).

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Specialized Living Wound Dressing Based on the Self-Assembly Approach of Tissue Engineering

Journal of Functional Biomaterials ◽

10.3390/jfb9030053 ◽

2018 ◽

Vol 9 (3) ◽

pp. 53 ◽

Cited By ~ 6

Author(s):

Laurence Cantin-Warren ◽

Rina Guignard ◽

Sergio Cortez Ghio ◽

Danielle Larouche ◽

François Auger ◽

...

Keyword(s):

Self Assembly ◽

Wound Dressing ◽

Reference Method ◽

The Self ◽

Wound Dressings ◽

Skin Substitute ◽

Seeding Density ◽

Skin Substitutes ◽

Assembly Method

There is a high incidence of failure and recurrence for chronic skin wounds following conventional therapies. To promote healing, the use of skin substitutes containing living cells as wound dressings has been proposed. The aim of this study was to produce a scaffold-free cell-based bilayered tissue-engineered skin substitute (TES) containing living fibroblasts and keratinocytes suitable for use as wound dressing, while considering production time, handling effort during the manufacturing process, and stability of the final product. The self-assembly method, which relies on the ability of mesenchymal cells to secrete and organize connective tissue sheet sustaining keratinocyte growth, was used to produce TESs. Three fibroblast-seeding densities were tested to produce tissue sheets. At day 17, keratinocytes were added onto 1 or 3 (reference method) stacked tissue sheets. Four days later, TESs were subjected either to 4, 10, or 17 days of culture at the air–liquid interface (A/L). All resulting TESs were comparable in terms of their histological aspect, protein expression profile and contractile behavior in vitro. However, signs of extracellular matrix (ECM) digestion that progressed over culture time were noted in TESs produced with only one fibroblast-derived tissue sheet. With lower fibroblast density, the ECM of TESs was almost completely digested after 10 days A/L and lost histological integrity after grafting in athymic mice. Increasing the fibroblast seeding density 5 to 10 times solved this problem. We conclude that the proposed method allows for a 25-day production of a living TES, which retains its histological characteristics in vitro for at least two weeks.

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Behaviour of Endothelial Cells in a TridimensionalIn VitroEnvironment

BioMed Research International ◽

10.1155/2015/630461 ◽

2015 ◽

Vol 2015 ◽

pp. 1-9 ◽

Cited By ~ 8

Author(s):

Raif Eren Ayata ◽

Stéphane Chabaud ◽

Michèle Auger ◽

Roxane Pouliot

Keyword(s):

Endothelial Cells ◽

Self Assembly ◽

The Self ◽

Biochemical Tests ◽

Area Of Interest ◽

Assembly Method ◽

Engineering Models ◽

Prevascularized Tissue ◽

Biological Environment

Angiogenesis is a fundamental process in healing, tumor growth, and a variety of medical conditions. For this reason,in vitroangiogenesis is an area of interest for researchers. Additionally,in vitroangiogenesis is important for the survival of prevascularized tissue-engineering models. The aim of this study was to observe the self-tubular organization behaviour of endothelial cells in the self-assembly method. In this study, bilayered and dermal substitutes were prepared using the self-assembly method. Histological, immunostaining, and biochemical tests were performed. The behavioural dynamics of endothelial cells in this biological environment of supportive cells were observed, as were the steps of thein vitroangiogenic cascade with self-organizing capillary-like structures formation. The epidermal component of the substitutes was seen to promote network expansion and density. It also increased the quantity of angiogenic factors (VEGF and Ang-1) without increasing the proinflammatory factor (IL-8). In addition, the increased MMP activity contributed to matrix degradation, which facilitated capillary formation.

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Lentiviral Vectors for Delivery of Gene-Editing Systems Based on CRISPR/Cas: Current State and Perspectives

Viruses ◽

10.3390/v13071288 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1288

Author(s):

Wendy Dong ◽

Boris Kantor

Keyword(s):

Large Scale ◽

Lentiviral Vector ◽

Clinical Applications ◽

Scale Production ◽

Base Editing ◽

Epigenome Editing ◽

Vector Systems ◽

Dividing Cells

CRISPR/Cas technology has revolutionized the fields of the genome- and epigenome-editing by supplying unparalleled control over genomic sequences and expression. Lentiviral vector (LV) systems are one of the main delivery vehicles for the CRISPR/Cas systems due to (i) its ability to carry bulky and complex transgenes and (ii) sustain robust and long-term expression in a broad range of dividing and non-dividing cells in vitro and in vivo. It is thus reasonable that substantial effort has been allocated towards the development of the improved and optimized LV systems for effective and accurate gene-to-cell transfer of CRISPR/Cas tools. The main effort on that end has been put towards the improvement and optimization of the vector’s expression, development of integrase-deficient lentiviral vector (IDLV), aiming to minimize the risk of oncogenicity, toxicity, and pathogenicity, and enhancing manufacturing protocols for clinical applications required large-scale production. In this review, we will devote attention to (i) the basic biology of lentiviruses, and (ii) recent advances in the development of safer and more efficient CRISPR/Cas vector systems towards their use in preclinical and clinical applications. In addition, we will discuss in detail the recent progress in the repurposing of CRISPR/Cas systems related to base-editing and prime-editing applications.

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Abstract 262: Derivation of Duchenne Muscular Dystrophy Disease Specific Cardiomyocytes From Patient Urine

Circulation Research ◽

10.1161/res.113.suppl_1.a262 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Xuan Guan ◽

David L Mack ◽

Claudia M Moreno ◽

Fernando Santana ◽

Charles E Murry ◽

...

Keyword(s):

Stem Cells ◽

Human Urine ◽

Large Scale ◽

Lentiviral Vector ◽

Cellular Reprogramming ◽

Urine Samples ◽

Pcr Analysis ◽

Scale Production ◽

Mechanism Study

Introduction: Human somatic cells can be reprogrammed into primitive stem cells, termed induced pluripotent stem cells (iPSCs). These iPSCs can be extensively expanded in vitro and differentiated into multiple functional cell types, enabling faithful preservation of individual’s genotype and large scale production of disease targeted cellular components. These unique cellular reagents thus hold tremendous potential in disease mechanism study, drugs screening and cell replacement therapy. Due to the genetic mutation of the protein dystrophin, many DMD patients develop fatal cardiomyopathy with no effective treatment. The underlying pathogenesis has not been fully elucidated. Hypothesis: We tested the hypothesis that iPSCs could be generated from DMD patients’ urine samples and differentiated into cardiomyocytes, recapitulating the dystrophic phenotype. Methods: iPSCs generation was achieved by introducing a lentiviral vector expressing Oct4, Sox2, c-Myc and Klf4 into cells derived from patient’s (n=1) and healthy volunteers’ (n=3) urine. Cardiomyocytes were derived by sequentially treating iPSCs with GSK3 inhibitor CHIR99021 and Wnt inhibitor IWP4. Differentiated cardiomyocytes were subjected to calcium imaging, electrophysiology recording, Polymerase Chain Reaction (PCR) analysis, and immunostaining. Results: iPSCs were efficiently generated from human urine samples and further forced to differentiate into contracting cardiomyocytes. PCR analysis and immunostaining confirmed the expression of a panel of cardiac markers. Both normal and patient iPSC derived cardiomyocytes exhibited spontaneous and field stimulated calcium transients (up to 2Hz), as well as action potentials with ventricular-like and nodal-like characteristics. Anti-dystrophin antibodies stained normal iPSC-derived cardiomyocyte membranes but did not react against DMD iPSC-derived cardiomyocytes. Conclusions: Cardiomyocytes can be efficiently generated from human urine, through the cellular reprogramming technology. DMD cardiomyocytes retained the patient’s genetic information and manifested a dystrophin-null phenotype. Functional assessments are underway to determine differences that may exist between genotypes.

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Enhanced Entrapment and Improved in Vitro Controlled Release of N-Acetyl Cysteine in Hybrid PLGA/Lecithin Nanoparticles Prepared Using a Nanoprecipitation/Self-Assembly Method

Journal of Cellular Biochemistry ◽

10.1002/jcb.26070 ◽

2017 ◽

Vol 118 (12) ◽

pp. 4203-4209 ◽

Cited By ~ 10

Author(s):

Parvin Ahmaditabar ◽

Amir A. Momtazi-Borojeni ◽

Ali H. Rezayan ◽

Mahboobeh Mahmoodi ◽

Amirhossein Sahebkar ◽

...

Keyword(s):

Controlled Release ◽

Self Assembly ◽

Assembly Method ◽

Acetyl Cysteine

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Environmentally safe, substrate-independent and repairable nanoporous coatings: large-scale preparation, high transparency and antifouling properties

Journal of Materials Chemistry A ◽

10.1039/c7ta05112c ◽

2017 ◽

Vol 5 (38) ◽

pp. 20277-20288 ◽

Cited By ~ 30

Author(s):

Yong Li ◽

Zhaozhu Zhang ◽

Mengke Wang ◽

Xuehu Men ◽

Qunji Xue

Keyword(s):

Self Assembly ◽

Large Scale ◽

Intrinsic Properties ◽

High Transparency ◽

Assembly Method ◽

Antifouling Coatings ◽

Large Scale Preparation ◽

Environmentally Safe ◽

Scale Preparation ◽

Substrate Independent

Repairable and antifouling coatings were prepared via self-assembly method without destroying the intrinsic properties of substrates, which aims to tackle low transparency and poor durability problems of current coatings.

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