Cancer Spheroids Embedded in Tissue-Engineered Skin Substitutes: A New Method to Study Tumorigenicity In Vivo.

Tumorigenic assays are used during a clinical translation to detect the transformation potential of cell-based therapies. One of these in vivo assays is based on the separate injection of each cell type to be used in the clinical trial. However, the injection method requires many animals and several months to obtain useful results. In previous studies, we showed the potential of tissue-engineered skin substitutes (TESs) as a model for normal skin in which cancer cells can be included in vitro. Herein, we showed a new method to study tumorigenicity, using cancer spheroids that were embedded in TESs (cTES) and grafted onto athymic mice, and compared it with the commonly used cell injection assay. Tumors developed in both models, cancer cell injection and cTES grafting, but metastases were not detected at the time of sacrifice. Interestingly, the rate of tumor development was faster in cTESs than with the injection method. In conclusion, grafting TESs is a sensitive method to detect tumor cell growth with and could be developed as an alternative test for tumorigenicity.

Speeding up the production of clinical-grade skin substitutes using off-the-shelf decellularized self-assembled dermal matrices.

Patients with deep and extensive wounds need urgent skin coverage to re-establish the cutaneous barrier that prevents life-threatening infections and dehydration. However, the current clinically-available skin substitutes intended for permanent coverage are limited in number, and a trade-off between production time and quality must be made. Here, we report the use of decellularized self-assembled dermal matrices to reduce by half the manufacturing process time of clinical-grade skin substitutes. These decellularized matrices can be stored for over 18 months and recellularized with patients' cells in order to generate skin substitutes that show outstanding histological and mechanical properties in vitro. Once grafted in mice, these substitutes persist over weeks with high graft take, few contraction events, and high stem cell content. These next-generation skin substitutes constitute a substantial advancement in the treatment of major burn patients, combining, for the first time, high functionality, rapid manufacturability and easy handling for surgeons and healthcare practitioners. Future clinical trials will be conducted to assess the advantages of these substitutes over existing treatments. STATEMENT OF SIGNIFICANCE: The number of patients in need for organ transplantation is ever-growing and there is a shortage in tissue and organ donors. In this study, we show for the first time that we can preserve decellularized self-assembled tissues and keep them in storage. Then, in only three weeks we can use them to produce bilayered skin substitutes that have properties very close to those of the native human skin. These findings therefore represent a major step forward in the field of tissue engineering and organ transplantation, paving the way toward a universal off-the-shelf biomaterial for tissue reconstruction and surgery that will be beneficial for many clinicians and patients.

A Newly Developed Chemically Defined Serum-Free Medium Suitable for Human Primary Keratinocyte Culture and Tissue Engineering Applications.

In our experience, keratinocytes cultured in feeder-free conditions and in commercially available defined and serum-free media cannot be as efficiently massively expanded as their counterparts grown in conventional bovine serum-containing medium, nor can they properly form a stratified epidermis in a skin substitute model. We thus tested a new chemically defined serum-free medium, which we developed for massive human primary keratinocyte expansion and skin substitute production. Our medium, named Surge Serum-Free Medium (Surge SFM), was developed to be used alongside a feeder layer. It supports the growth of keratinocytes freshly isolated from a skin biopsy and cryopreserved primary keratinocytes in cultured monolayers over multiple passages. We also show that keratin-19-positive epithelial stem cells are retained through serial passaging in Surge SFM cultures. Transcriptomic analyses suggest that gene expression is similar between keratinocytes cultured with either Surge SFM or the conventional serum-containing medium. Additionally, Surge SFM can be used to produce bilayered self-assembled skin substitutes histologically similar to those produced using serum-containing medium. Furthermore, these substitutes were grafted onto athymic mice and persisted for up to six months. In conclusion, our new chemically defined serum-free keratinocyte culture medium shows great promise for basic research and clinical applications.

Tie-Over Bolster Pressure Dressing Improves Outcomes of Skin Substitutes Xenografts on Athymic Mice.

The efficacy of skin substitutes is established for the treatment of burn injuries, but its use is not limited to this condition. This technology has the potential to improve the treatment of various conditions by offering highly advanced and personalized treatments. In vivo studies are challenging but essential to move to clinical use in humans. Mice are the most widely used species in preclinical studies, but the main drawback of this model is the limited surface area of the graft in long-term transplantation studies caused by the displacement and the contraction of the graft. We improved the conventional surgical procedures by stabilizing the chamber covering the graft with intramuscular sutures and by adding a tie-over bolster dressing. The current study was therefore performed to compare outcomes of skin grafts between the conventional and optimized skin graft model. Human self-assembled skin substitutes (SASSs) were prepared and grafted to athymic mice either by the conventional method or by the new grafting method. Graft healing and complications were assessed using digital photographs on postoperative days 7, 14, and 21. Similar structure and organization were observed by histological staining. The new grafting method reduced medium and large displacement events by 1.26-fold and medium and large contraction events by 1.8-fold, leading to a 1.6-fold increase in graft surface area compared to skin substitutes grafted with the usual method. This innovation ensures better reproducibility and consistency of skin substitute transplants on mice.

Lysophosphatidic acid enhances collagen deposition and matrix thickening in engineered tissue.

The time needed to produce engineered tissue is critical. A self-assembly approach provided excellent results regarding biological functions and cell differentiation because it closely respected the microenvironment of cells. Nevertheless, the technique was time consuming for producing tissue equivalents with enough extracellular matrix to allow manipulations. Unlike L-arginine supplementation that only increased accumulation of collagen in cell culture supernatant in our model, addition of lysophosphatidic acid, a natural bioactive lipid, did not modify the amount of accumulated collagen in the cell culture supernatant; however, it enhanced the matrix deposition rate without inducing fibroblast hyperproliferation and tissue fibrosis.

An endothelialized urothelial cell-seeded tubular graft for urethral replacement.

INTRODUCTION: Many efforts are used to improve surgical techniques and graft materials for urethral reconstruction. We developed an endothelialized tubular structure for urethral reconstruction. METHODS: Two tubular models were created in vitro. Human fibroblasts were cultured for 4 weeks to form fibroblast sheets. Then, endothelial cells (ECs) were seeded on the fibroblast sheets and wrapped around a tubular support to form a cylinder for the endothelialized tubular urethral model (ET). No ECs were added in the standard tubular model (T). After 21 days of maturation, urothelial cells were seeded into the lumen of both models. Constructs were placed under perfusion in a bioreactor for 1 week. At several times, histology and immunohistochemistry were performed on grafted nude mice to evaluate the impact of ECs on vascularization. RESULTS: Both models produced an extracellular matrix, without exogenous material, and developed a pseudostratified urothelium. Seven days after the graft, mouse red blood cells were present only in the outer layers in T model, but in the full thickness of ET model. After 14 days, erythrocytes were present in both models, but in a greater proportion in ET model. At day 28, both models were well-vascularized, with capillary-like structures in the whole thickness of the tubes. CONCLUSION: Incorporating endothelial cells was associated with an earlier vascularization of the grafts, which could decrease the necrosis of the transplanted tissue. As those models can be elaborated with the patient's cells, this tubular urethral graft would be unique in its autologous property.

Tissue engineering of urinary bladder and urethra: advances from bench to patients.

Urinary tract is subjected to many varieties of pathologies since birth including congenital anomalies, trauma, inflammatory lesions, and malignancy. These diseases necessitate the replacement of involved organs and tissues. Shortage of organ donation, problems of immunosuppression, and complications associated with the use of nonnative tissues have urged clinicians and scientists to investigate new therapies, namely, tissue engineering. Tissue engineering follows principles of cell transplantation, materials science, and engineering. Epithelial and muscle cells can be harvested and used for reconstruction of the engineered grafts. These cells must be delivered in a well-organized and differentiated condition because water-seal epithelium and well-oriented muscle layer are needed for proper function of the substitute tissues. Synthetic or natural scaffolds have been used for engineering lower urinary tract. Harnessing autologous cells to produce their own matrix and form scaffolds is a new strategy for engineering bladder and urethra. This self-assembly technique avoids the biosafety and immunological reactions related to the use of biodegradable scaffolds. Autologous equivalents have already been produced for pigs (bladder) and human (urethra and bladder). The purpose of this paper is to present a review for the existing methods of engineering bladder and urethra and to point toward perspectives for their replacement.