Urothelial Cell Culture: Stratified Urothelial Sheet and Three-Dimensional Growth of Urothelial Structure
Department of Urology, Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, USA.Methods in molecular biology (Clifton, N.J.) (Impact Factor: 1.29). 01/2013; 945:383-99. DOI: 10.1007/978-1-62703-125-7_23
Urothelial cells line the urinary tract, including the renal pelvis, ureters, bladder, superior urethra, and the central ducts of the prostate. They are highly specialized epithelial cell types possessing unique features, imparting important functional roles in the urinary system. They act as a permeability barrier and protect underlying muscle tissues from the caustic effects of urine while also expanding with bladder filling to adjust urine pressures. The multilayered urothelium is typically structured with differentiated, mature surface cells and less mature basal cells. The basal cell layer contains tissue-specific stem cells able to self-renew for the lifetime of the mammal and also produces a pool of maturing cells for tissue homeostasis. Maintaining regenerative basal cells in a culture facilitates urothelial cell growth in vitro. Additionally, epithelial-mesenchymal communication, epithelial-matrix interactions, and cytokines/growth factors are required to maintain the normal structure and function of mature urothelial cells in vitro and to induce stem cell differentiation into urothelial cells. These cultures are useful to study the biology and physiology of the urinary tract, particularly for the development of cell-based tissue engineering strategies in urology. This chapter describes methods for the isolation of urothelial cells and their maintenance in monolayer culture, and methods for the production of multilayer urothelial cell sheets and three-dimensional cocultures of urothelial and mesenchymal cells.
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ABSTRACT: Different pathological conditions such as congenital organ absence, severe organ injuries, end-stage organ failure and malignancy-related organ removal, have few effective therapeutic options a part from a whole organ transplant, that, however, often meets with a serious shortage of suitable donor organs. The purpose of this paper consists in highlighting what the novel tissue engineering approaches might help to solve such problems. EMERGING CONCEPTS: A recent approach in tissue/organ engineering, particularly to build bioartificial airways, is the procedure of decellularizing a whole donor organ to obtain a complex 3D-biomatrix-scaffold mantaining the intrinsic vascular network, that is subsequently recellularized with recipient's autologous organ-specific differentiated cells or/and stem cells, to build a potentially functional biological substitute. Such strategy has been clinically used to replace organ in trachea/broncus tumor patients. In another approach, mainly used to construct a bioartificial urinary bladder tissue, different types of either biodegradable synthetic polymers or naturally-derived matrices or even polymer/biomatrix-composite materials are used as scaffold for either cell-free or autologous cell-seeded tissue engineering procedures. So far, such technique has been mainly used to make an augmentation cystoplasty in patients with end-stage poorly compliant neuropathic bladder or in exstrophic bladder subjects. Intriguing developments in biomaterial science, nanotechnologies, stem cell biology, and further improvements in bioreactor manufactoring will allow to generate, in the near future, tissue engineered organs that, as for structure/function so the native one-like, might represent the optimum solution to replace organs in tumor surgery.European review for medical and pharmacological sciences 03/2013; 17(5):624-31. · 1.21 Impact Factor
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ABSTRACT: Background: We investigated the feasibility of urethral reconstruction using stretched electrospun silk fibroin matrices. Materials and methods: A novel electrospun silk fibroin matrix was prepared. The structure of the material was assessed by scanning electron microscopy and a porosity test. Canine urothelial cells were isolated, expanded, and seeded onto the material for 1 wk to obtain a tissue-engineered graft. The tissue-engineered graft was assessed using hematoxylin and eosin staining and scanning electron microscopy. A dorsal urethral mucosal defect was created in nine female beagle dogs. In the experimental group, tissue-engineered mucosa was used to repair urethra mucosa defects in six dogs. No substitute was used in the three dogs of the control group. Retrograde urethrography was performed at 1, 2, and 6 mo after grafting. The urethral grafts were analyzed grossly and histologically. Results: Scanning electron microscope and a porosity test revealed that the material had a three-dimensional porous structure. Urothelial cells grew on the material and showed good biocompatibility with the stretched silk fibroin matrices. Canines implanted with tissue-engineered mucosa voided without difficulty. Retrograde urethrography revealed no signs of stricture. Histologic staining showed gradual epithelial cell development and stratified epithelial layers at 1, 2, and 6 mo. The canines in the control group showed difficulty in voiding. Retrograde urethrography showed urethra stricture. Histologic staining showed that no or only one layer of epithelial cells developed. A severe inflammatory reaction was also observed in the control group. Conclusions: Stretched electrospun silk fibroin matrices have good biocompatibility with urothelial cells, which could prove to be a potential material for use in urethra reconstruction.Journal of Surgical Research 04/2013; 184(2). DOI:10.1016/j.jss.2013.04.016 · 1.94 Impact Factor
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ABSTRACT: Over the last few decades, both synthetic and natural materials have been utilized to develop bladder substitutes. Most attempts have not been successful because of mechanical, structural, functional, or biocompatibility problems. Bladder acellular matrix (BAM) is obtained by removing cellular components from donor bladders, leaving a tissue matrix consisting of collagen, elastin, fibronectin, glycosaminoglycans (GAGs), proteoglycans, and growth factors. Multiple BAM-based studies now suggest that tissue engineering techniques may provide efficacious alternatives to current methods of bladder augmentation. Efforts to optimize BAM-based scaffolds are ongoing and would be greatly assisted by feasible means of improving scaffold properties and interaction with cells and tissues. Future applications of BAM will likely include cell-seeded grafts with the eventual hope of producing "off the shelf" replacement materials for bladder augmentation.Tissue Engineering Part B Reviews 07/2013; 20(2). DOI:10.1089/ten.TEB.2013.0103 · 4.64 Impact Factor
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