Sabine Maurer

In Vitro Investigations of Tissue-Engineered Multilayered Urothelium Established from Bladder Washings

OBJECTIVE Human urothelial cells (HUCs) are commonly isolated from native urothelium requiring open or endoscopic surgery. The aim of this study was to raise primary monolayer cultures of HUCs from bladder washings, to generate multilayered urothelial sheets in vitro, to characterise the sheets immunologically, and to prove their viability. METHODS Irrigation fluids were taken from 29 adult patients. Isolated cells were cultured in serum-free keratinocyte medium. Confluent monolayer cultures were stratified, and evolved cell sheets were harvested after 10-16 d. Pancytokeratins and cytokeratin 20 (CK20) in the stratified cultures and the detached sheets were immunologically detected. To exclude the presence of mesenchymal cells, antibodies against fibroblast surface antigen and smooth muscle alpha-actin were used. In addition, expression of p63 and uroplakin III was investigated. The viability of the detached cell sheets was proven by establishing explant cultures of small sheet sections. RESULTS Confluent primary HUC cultures were established in 55.2% of the collected bladder washings between days 15-20. Multilayered urothelium developed in 62.5% of the monolayers. Histology revealed stratified cell layers similar to native urothelium. Both stratified cultures and detached sheets stained 100% positive for pancytokeratins and partially for CK20, indicating differentiation into superficial cells. No positive staining was observed with the mesenchymal markers used. p63 was expressed partially. Uroplakin III expression was not observed. Cell sheet viability was confirmed by rapid cell outgrowth in explant cultures. CONCLUSIONS Isolation of HUCs from bladder washings is a minimally invasive approach to establish primary urothelial cultures for creating autologous multilayered urothelial sheets.

In vitro stratifiziertes Urothelium und seine Bedeutung für die rekonstruktive Urologie

ZusammenfassungIm Bereich „tissue engineering“ innerhalb der rekonstruktiven Urologie stehen zwei Ziele im Vordergrund: Die Bereitstellung geeigneten Ersatzgewebes für die rekonstruktive Chirurgie und die damit verbundenen grundlegenden Forschungsarbeiten zum Verständnis der Komplexität und physiologischen Funktionalität der beteiligten Gewebe. Die langjährigen experimentellen Annäherungen an das Übergangsepithel des Harntraktes führten zur routinemäßigen Primärkultur humaner urothelialer Zellen und zur Möglichkeit, diese Zellen zur Besiedlung von biodegradierbaren Trägermaterialien zu verwenden.In den jüngsten Forschungsarbeiten steht nicht mehr nur die Anwendung von Urothelzellen in Form von Einzelzellsuspensionen im Vordergrund. Vielmehr werden die isolierten und propagierten Zellen in vitro stratifiziert, um dadurch mehrlagige Konstrukte zur Übertragung auf geeignete Trägermatrices zur Verfügung stellen zu können und den Aufbau eines funktionellen Urothelgewebes zu beschleunigen. Dieser Ansatz führte in Tiermodellen zu vielversprechenden Ergebnissen. Die klinische Anwendung der in vitro etablierten Urothelkonstrukte steht bislang noch aus. Dieser Beitrag gibt eine Übersicht über die aktuellen Strategien zur Entwicklung von Urothelkonstrukten.AbstractThere are two main objectives regarding tissue engineering in reconstructive urology: (1) to provide the surgeon with autologous tissue for urogenital reconstructive purposes and (2) to create the framework for experimental investigations to better understand the structure and function of the tissues involved.In the last years urothelial cell culture has become a routine laboratory technique. There is sufficient cellular output after isolation and propagation to seed cells as single cell suspensions on biodegradable matrices for the construction of cell-matrix implants. In recent publications attention was directed toward using established primary cell cultures for in vitro stratification of multilayered urothelial sheets. Urothelial sheets have been used quite successfully for covering acellular matrices for bladder augmentation in dog and minipig models. However, up to now there has been no clinical application in humans of urothelial sheets generated in vitro. Here we review facts about the different strategies for generating multilayered urothelial sheets.There are two main objectives regarding tissue engineering in reconstructive urology: (1) to provide the surgeon with autologous tissue for urogenital reconstructive purposes and (2) to create the framework for experimental investigations to better understand the structure and function of the tissues involved. In the last years urothelial cell culture has become a routine laboratory technique. There is sufficient cellular output after isolation and propagation to seed cells as single cell suspensions on biodegradable matrices for the construction of cell-matrix implants. In recent publications attention was directed toward using established primary cell cultures for in vitro stratification of multilayered urothelial sheets. Urothelial sheets have been used quite successfully for covering acellular matrices for bladder augmentation in dog and minipig models. However, up to now there has been no clinical application in humans of urothelial sheets generated in vitro. Here we review facts about the different strategies for generating multilayered urothelial sheets.

In vitro stratified urothelium and its relevance in reconstructive urology

ZusammenfassungIm Bereich „tissue engineering“ innerhalb der rekonstruktiven Urologie stehen zwei Ziele im Vordergrund: Die Bereitstellung geeigneten Ersatzgewebes für die rekonstruktive Chirurgie und die damit verbundenen grundlegenden Forschungsarbeiten zum Verständnis der Komplexität und physiologischen Funktionalität der beteiligten Gewebe. Die langjährigen experimentellen Annäherungen an das Übergangsepithel des Harntraktes führten zur routinemäßigen Primärkultur humaner urothelialer Zellen und zur Möglichkeit, diese Zellen zur Besiedlung von biodegradierbaren Trägermaterialien zu verwenden.In den jüngsten Forschungsarbeiten steht nicht mehr nur die Anwendung von Urothelzellen in Form von Einzelzellsuspensionen im Vordergrund. Vielmehr werden die isolierten und propagierten Zellen in vitro stratifiziert, um dadurch mehrlagige Konstrukte zur Übertragung auf geeignete Trägermatrices zur Verfügung stellen zu können und den Aufbau eines funktionellen Urothelgewebes zu beschleunigen. Dieser Ansatz führte in Tiermodellen zu vielversprechenden Ergebnissen. Die klinische Anwendung der in vitro etablierten Urothelkonstrukte steht bislang noch aus. Dieser Beitrag gibt eine Übersicht über die aktuellen Strategien zur Entwicklung von Urothelkonstrukten.AbstractThere are two main objectives regarding tissue engineering in reconstructive urology: (1) to provide the surgeon with autologous tissue for urogenital reconstructive purposes and (2) to create the framework for experimental investigations to better understand the structure and function of the tissues involved.In the last years urothelial cell culture has become a routine laboratory technique. There is sufficient cellular output after isolation and propagation to seed cells as single cell suspensions on biodegradable matrices for the construction of cell-matrix implants. In recent publications attention was directed toward using established primary cell cultures for in vitro stratification of multilayered urothelial sheets. Urothelial sheets have been used quite successfully for covering acellular matrices for bladder augmentation in dog and minipig models. However, up to now there has been no clinical application in humans of urothelial sheets generated in vitro. Here we review facts about the different strategies for generating multilayered urothelial sheets.There are two main objectives regarding tissue engineering in reconstructive urology: (1) to provide the surgeon with autologous tissue for urogenital reconstructive purposes and (2) to create the framework for experimental investigations to better understand the structure and function of the tissues involved. In the last years urothelial cell culture has become a routine laboratory technique. There is sufficient cellular output after isolation and propagation to seed cells as single cell suspensions on biodegradable matrices for the construction of cell-matrix implants. In recent publications attention was directed toward using established primary cell cultures for in vitro stratification of multilayered urothelial sheets. Urothelial sheets have been used quite successfully for covering acellular matrices for bladder augmentation in dog and minipig models. However, up to now there has been no clinical application in humans of urothelial sheets generated in vitro. Here we review facts about the different strategies for generating multilayered urothelial sheets.

From tissue engineering to regenerative medicine in urology — The potential and the pitfalls ☆

Tissue engineering is a promising technique for the development of biological substitutes that can restore, maintain, or improve tissue function. The creation of human tissue-engineered products, generated of autologous somatic cells or adult stem cells with or without seeding of biocompatible matrices is a vision to resolve the lack of tissues and organs for transplantation and to offer new options for reconstructive surgery. Tissue engineering in urology aims at the reconstruction of the urinary tract by creating anatomically and functionally equal tissue. It is a rapidly evolving field in basic research and the transfer into the clinic has yet to be realized. Necessary steps from bench to bed are the proof of principle in animal models and the proof of concept in clinical trials following good manufacturing practice and ethical and legal requirements for human tissue-engineered products. Up to now, obstacles still occur in the neovascularization of implants and ingrowth of nerves in vivo. Moreover the harvesting of mesenchymal stem cells out of bone marrow as well as the explant of urothelial cells yet demands rather invasive surgery to achieve a successful outcome. Thus, other cell sources and harvesting techniques like placenta and adipose tissue for mesenchymal stem cells and bladder irrigation for urothelial cells require closer investigation.

Tissue engineering in urothelium regeneration

The development of therapeutic treatments to regenerate urothelium, manufacture tissue equivalents or neourethras for in-vivo application is a significant challenge in the field of tissue engineering. Many studies have focused on urethral defects that, in most cases, inadequately address current therapies. This article reviews the primary tissue engineering strategies aimed at the clinical requirements for urothelium regeneration while concentrating on promising investigations in the use of grafts, cellular preparations, as well as seeded or unseeded natural and synthetic materials. Despite significant progress being made in the development of scaffolds and matrices, buccal mucosa transplants have not been replaced. Recently, graft tissues appear to have an advantage over the use of matrices. These therapies depend on cell isolation and propagation in vitro that require, not only substantial laboratory resources, but also subsequent surgical implant procedures. The choice of the correct cell source is crucial when determining an in-vivo application because of the risks of tissue changes and abnormalities that may result in donor site morbidity. Addressing an appropriately-designed animal model and relevant regulatory issues is of fundamental importance for the principal investigators when a therapy using cellular components has been developed for clinical use.

„Tissue engineering“ und Stammzellforschung in der Urologie für den rekonstruktiven bzw. regenerativen Therapieansatz

ZusammenfassungNachdem sich die rekonstruktive Urologie in den letzten Jahren neuen Gebieten gewidmet hat, konnten im Bereich „tissue engineering“, nach herausragenden Ergebnissen in der Grundlagenforschung, erste Erfolge in klinischen Bereichen verzeichnet werden. Auch die Stammzellenforschung eröffnet weitere Bereiche bezüglich regenerativer Ansatzpunkte.Durch die enge Kooperation der Urologie mit verschiedensten Disziplinen der Universität Tübingen werden Fragestellungen mit engem klinischem Bezug bearbeitet. Die Regeneration des äußeren Sphinkters bedarf funktionell integrierter Muskelzellen. Darüber hinaus wird zur Rekonstruktion der Harnröhre Urothel benötigt, um nicht nur die Invasivität, sondern auch die Möglichkeit der Restriktur weiter zu verringern.Nachdem mesenchymale Stammzellen (MSC) erfolgreich zu Muskelzellen differenziert und appliziert werden konnten, müssen für den klinischen Einsatz, entsprechend der gesetzlichen Vorgaben, die Vorraussetzungen geschaffen werden. Neben der Entwicklung entsprechender Nährmedien und Verifizierung im Tiermodell, wurden aus humanem Hodengewebe omnipotente Stammzellen gewonnen und in Zellen aller drei Keimblätter differenziert.Zur Herstellung eines mehrlagigen Urothels wurde zur Gewinnung autologer Urothelzellen durch die „Blasenwaschung“ die Invasivität weiter vermindert. Entsprechende Spül- und Nährmedien verbessern die Ausbeute, die mit der entsprechenden Stratifizierung das mehrlagige Urothel entstehen lässt. Darüber hinaus wird im Großtiermodell die Applikation von Urothelzellen endoskopisch mit entsprechenden Trägersubstanzen zur Minimierung der Invasivität erfolgreich untersucht.In der Urologie werden im Bereich „tissue engineering“ und in der Stammzellforschung durch die enge Kooperation mit anderen Disziplinen in kürzerer Zeit und mit nahem klinischem Bezug neue therapeutische Ansatzmöglichkeiten für die rekonstruktive und regenerative Urologie eröffnet.AbstractWith the involvement of clinical reconstructive urology in the field of tissue engineering, outstanding results have been achieved in basic research as well as in some clinics. Stem cell research has even opened up possibilities for regenerative aspects. In close cooperation with various disciplines, the Department of Urology at the University of Tübingen investigates different clinical aspects with regard to reconstructive and regenerative urology. The regeneration of the external urethral sphincter requires functionally integrated muscle cells. In addition stricture reconstruction with multilayer urothelium should become less invasive and the re-stricture rate reduced.After the application of differentiating stem cells was proven, the clinical setting needed to be set for legal issues. In addition to the specification of culture media and verification in the animal model, the possibility to harvest omnipotent stem cells out of human testis and to differentiate those into the three germ layers was demonstrated.With the reduced invasiveness of harvesting the urothelium cells by a bladder wash using specific culture fluids, the cell culture was significantly improved enabling successful creation of urothelium by stratification. In addition urothelial cells in a matrix are further improved for endoscopic application.The close cooperation of different disciplines shortens the time to develop therapeutic approaches with a close clinical relationship in reconstructive and regenerative urology.With the involvement of clinical reconstructive urology in the field of tissue engineering, outstanding results have been achieved in basic research as well as in some clinics. Stem cell research has even opened up possibilities for regenerative aspects. In close cooperation with various disciplines, the Department of Urology at the University of Tübingen investigates different clinical aspects with regard to reconstructive and regenerative urology. The regeneration of the external urethral sphincter requires functionally integrated muscle cells. In addition stricture reconstruction with multilayer urothelium should become less invasive and the re-stricture rate reduced. After the application of differentiating stem cells was proven, the clinical setting needed to be set for legal issues. In addition to the specification of culture media and verification in the animal model, the possibility to harvest omnipotent stem cells out of human testis and to differentiate those into the three germ layers was demonstrated. With the reduced invasiveness of harvesting the urothelium cells by a bladder wash using specific culture fluids, the cell culture was significantly improved enabling successful creation of urothelium by stratification. In addition urothelial cells in a matrix are further improved for endoscopic application. The close cooperation of different disciplines shortens the time to develop therapeutic approaches with a close clinical relationship in reconstructive and regenerative urology.

Tissue engineering and stem cell research in urology for a reconstructive or regenerative treatment approach

ZusammenfassungNachdem sich die rekonstruktive Urologie in den letzten Jahren neuen Gebieten gewidmet hat, konnten im Bereich „tissue engineering“, nach herausragenden Ergebnissen in der Grundlagenforschung, erste Erfolge in klinischen Bereichen verzeichnet werden. Auch die Stammzellenforschung eröffnet weitere Bereiche bezüglich regenerativer Ansatzpunkte.Durch die enge Kooperation der Urologie mit verschiedensten Disziplinen der Universität Tübingen werden Fragestellungen mit engem klinischem Bezug bearbeitet. Die Regeneration des äußeren Sphinkters bedarf funktionell integrierter Muskelzellen. Darüber hinaus wird zur Rekonstruktion der Harnröhre Urothel benötigt, um nicht nur die Invasivität, sondern auch die Möglichkeit der Restriktur weiter zu verringern.Nachdem mesenchymale Stammzellen (MSC) erfolgreich zu Muskelzellen differenziert und appliziert werden konnten, müssen für den klinischen Einsatz, entsprechend der gesetzlichen Vorgaben, die Vorraussetzungen geschaffen werden. Neben der Entwicklung entsprechender Nährmedien und Verifizierung im Tiermodell, wurden aus humanem Hodengewebe omnipotente Stammzellen gewonnen und in Zellen aller drei Keimblätter differenziert.Zur Herstellung eines mehrlagigen Urothels wurde zur Gewinnung autologer Urothelzellen durch die „Blasenwaschung“ die Invasivität weiter vermindert. Entsprechende Spül- und Nährmedien verbessern die Ausbeute, die mit der entsprechenden Stratifizierung das mehrlagige Urothel entstehen lässt. Darüber hinaus wird im Großtiermodell die Applikation von Urothelzellen endoskopisch mit entsprechenden Trägersubstanzen zur Minimierung der Invasivität erfolgreich untersucht.In der Urologie werden im Bereich „tissue engineering“ und in der Stammzellforschung durch die enge Kooperation mit anderen Disziplinen in kürzerer Zeit und mit nahem klinischem Bezug neue therapeutische Ansatzmöglichkeiten für die rekonstruktive und regenerative Urologie eröffnet.AbstractWith the involvement of clinical reconstructive urology in the field of tissue engineering, outstanding results have been achieved in basic research as well as in some clinics. Stem cell research has even opened up possibilities for regenerative aspects. In close cooperation with various disciplines, the Department of Urology at the University of Tübingen investigates different clinical aspects with regard to reconstructive and regenerative urology. The regeneration of the external urethral sphincter requires functionally integrated muscle cells. In addition stricture reconstruction with multilayer urothelium should become less invasive and the re-stricture rate reduced.After the application of differentiating stem cells was proven, the clinical setting needed to be set for legal issues. In addition to the specification of culture media and verification in the animal model, the possibility to harvest omnipotent stem cells out of human testis and to differentiate those into the three germ layers was demonstrated.With the reduced invasiveness of harvesting the urothelium cells by a bladder wash using specific culture fluids, the cell culture was significantly improved enabling successful creation of urothelium by stratification. In addition urothelial cells in a matrix are further improved for endoscopic application.The close cooperation of different disciplines shortens the time to develop therapeutic approaches with a close clinical relationship in reconstructive and regenerative urology.With the involvement of clinical reconstructive urology in the field of tissue engineering, outstanding results have been achieved in basic research as well as in some clinics. Stem cell research has even opened up possibilities for regenerative aspects. In close cooperation with various disciplines, the Department of Urology at the University of Tübingen investigates different clinical aspects with regard to reconstructive and regenerative urology. The regeneration of the external urethral sphincter requires functionally integrated muscle cells. In addition stricture reconstruction with multilayer urothelium should become less invasive and the re-stricture rate reduced. After the application of differentiating stem cells was proven, the clinical setting needed to be set for legal issues. In addition to the specification of culture media and verification in the animal model, the possibility to harvest omnipotent stem cells out of human testis and to differentiate those into the three germ layers was demonstrated. With the reduced invasiveness of harvesting the urothelium cells by a bladder wash using specific culture fluids, the cell culture was significantly improved enabling successful creation of urothelium by stratification. In addition urothelial cells in a matrix are further improved for endoscopic application. The close cooperation of different disciplines shortens the time to develop therapeutic approaches with a close clinical relationship in reconstructive and regenerative urology.

Bioartificial urothelium generated from bladder washings. A future therapeutic option for reconstructive surgery

Reconstructive surgery of lower urinary tract disorders can be limited by a shortage of adequate autologous tissue. Tissue engineering is an option for surgical reconstruction with evolved biological substitutes. Urethral repair with bioartificial urothelial implants can be an innovative method for sustained urothelial regeneration in situ. The needed urothelial cells are commonly isolated from native urothelium requiring surgery.The aim of this study was to establish primary human urothelial cell cultures from bladder washings in serum-free media and to generate urothelial tissue without seeding of matrices in a feeder cell-free system. It could be demonstrated that under these conditions bioartificial urothelium can be developed successfully from bladder washings. Its multilayered cellular structure and the initial differentiation in vitro, similar to native-grown urothelial tissue, are promising with regard to intended clinical application. Current work focuses on establishing cell culture techniques according to legal regulations, terminal differentiation of the urothelial constructs in vitro, and techniques to surgically implant lab-grown bioartificial urothelium.

Bioartifizielles autologes Urothel etabliert aus Spülungen der Harnblase

Reconstructive surgery of lower urinary tract disorders can be limited by a shortage of adequate autologous tissue. Tissue engineering is an option for surgical reconstruction with evolved biological substitutes. Urethral repair with bioartificial urothelial implants can be an innovative method for sustained urothelial regeneration in situ. The needed urothelial cells are commonly isolated from native urothelium requiring surgery.The aim of this study was to establish primary human urothelial cell cultures from bladder washings in serum-free media and to generate urothelial tissue without seeding of matrices in a feeder cell-free system. It could be demonstrated that under these conditions bioartificial urothelium can be developed successfully from bladder washings. Its multilayered cellular structure and the initial differentiation in vitro, similar to native-grown urothelial tissue, are promising with regard to intended clinical application. Current work focuses on establishing cell culture techniques according to legal regulations, terminal differentiation of the urothelial constructs in vitro, and techniques to surgically implant lab-grown bioartificial urothelium.

A Bovine Collagen Type I-Based Biodegradable Matrix as a Carrier for Tissue-Engineered Urothelium

Engineering of lower urinary tract tissue suitable for reconstructive surgery requires biomaterials as cell carriers, particularly in patients for whom autologous grafts are not available. Matrices should support growth, improve mechanical stability, feature excellent biocompatibility, and fully degrade without signs of scarring at the implantation side. In this study, a new bovine collagen type I-based biodegradable non-cross linked matrix was investigated for its suitability as a carrier for porcine and human urothelial cells in vitro. Initial cell adherence, metabolic activity, and proliferation behaviour of cells isolated from tissue biopsies were analyzed. Constructs were characterized immunohistologically in comparison with matrix-free cell sheets established on plastic surface (=controls). Even for high-density seeding, adherence on collagen cell carrier (CCC) was excellent. Metabolic activity and proliferation of stratifying porcine and human urothelial cells cultured on CCC were comparable to that of controls. Immunofluorescence analysis confirmed epithelial phenotype, cell-cell junction formation and ongoing differentiation of the multilayered urothelium on CCC. This study proved CCC as a suitable carrier for urothelial cells for the future aim for urethral reconstruction.