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.

Immunoreactivity of p63 in monolayered and in vitro stratified human urothelial cell cultures compared with native urothelial tissue.

OBJECTIVE: To investigate the immunoreactivity of p63 in monolayered and stratified human urothelial cell cultures and in normal urothelial tissues to assess the differentiation status of in vitro stratified urothelial constructs. METHODS: p63 expression was detected immunohistochemically in native normal human bladder, ureter, and renal pelvis tissues and immunocytochemically in monolayered urothelial cell cultures and urothelial constructs stratified in vitro. Additionally, expression of pancytokeratin, cytokeratin 20 (CK20), uroplakin III, and fibroblast surface antigen was investigated. RESULTS: In native tissues, urothelial cell layers showed the most intensive p63 staining in the basal cells; the superficial umbrella cells were predominantly negative. Monolayered urothelial cell cultures revealed reduced p63 expression with ongoing culture passages. In vitro stratified urothelial constructs exhibited p63 expression similar to that of native urothelium. CK20-reactive cells were absent in the monolayered cultures but present in the stratified cell cultures and in the urothelial constructs. In native urothelium, only superficial cells stained positive for CK20. Uroplakin III was not present in either monolayered urothelial cell cultures or stratified urothelial constructs. Cultured cells were always positive for pancytokeratin and negative for fibroblast surface antigen. CONCLUSIONS: p63 is a new biomarker for differentiation and stratification of urothelium created in vitro. For proposed clinical applications of in vitro stratified urothelium in reconstructive urology, urothelial constructs should exhibit expression of significant marker proteins similar to that of native urothelium. Our results show such similarity of expression for pancytokeratin, p63, and CK20, an encouraging possibility for confirming the functionality of tissue-engineered urothelia after clinical application.

Cytotoxic effects of treosulfan on prostate cancer cell lines.

OBJECTIVE: Despite various therapeutical options in metastatic prostate cancer, the lack of a curative approach motivates further investigations. Treosulfan is an alkylating agent that has proven its indication in the treatment of e.g. ovarian carcinoma. This study focused on the objective of evaluating the effect of in vitro intoxication of human prostate carcinoma cell lines with treosulfan. MATERIALS AND METHODS: Human prostate cancer cell lines LNCaP, DU145 and PC3 were treated with treosulfan concentrations from 0.5-500 microM for up to six days. Analysis of cell viability was performed using colorimetric WST-1 assay. Control data were obtained from identical cell lines cultivated without treosulfan. RESULTS: Incubation with treosulfan inhibited cell viability and led to cell death in all cell lines in a dose- and time-dependent manner. After one day, viability of LNCaP, DU145 and PC3 cells was constantly reduced with a dose rate of at least 10 microM (p < 0.001), 10 microM (p < 0.0001) and 100 microM (p < 0.0001) treosulfan, respectively. Minimum dose rates leading to death of nearly all LNCaP, DU145 and PC3 cells were 250 microM, 100 microM and 200 microM treosulfan, respectively. CONCLUSION: The results demonstrate a sensitivity of prostate carcinoma cells to the cytotoxic activity of treosulfan. Therefore, treosulfan might be a promising compound for novel treatment protocols for prostate cancer.

Investigations of urothelial cells seeded on commercially available small intestine submucosa.

OBJECTIVES: To examine adherence and viability of human urothelial cells seeded on commercially available small intestine submucosa (SIS) specimens under serum-free conditions. MATERIALS AND METHODS: Before seeding, SIS was either washed with incubation medium or coated with collagen A, fibronectin, or pronectin. A possible influence of SIS itself on the viability of urothelial cells was analysed with conditioned cell culture medium obtained by incubation of SIS for 24hours. In addition, untreated SIS and a setting without SIS were used as controls. Viability of urothelial cells was analysed with the WST-1 assay until day 9. Histology of seeded and unseeded SIS specimens was investigated after Papanicolaou staining. To demonstrate urothelial cell adherence on SIS, immunohistology was performed with a mixture of monoclonal AE1 and AE3 anticytokeratin antibodies. RESULTS: Urothelial cells seeded on SIS revealed no measurable cell viability. SIS-conditioned cell culture medium was cytotoxic for urothelial cells after 24 hours. Histology only demonstrated cell nuclei and no cytoplasm both in seeded and unseeded SIS specimens, thus indicating porcine DNA. Expression of the cell type-specific marker proteins AE1/AE3 could not be demonstrated. CONCLUSION: Since the commercially available SIS specimens used contained porcine DNA residues and demonstrated cytotoxic effects on urothelial cells, SIS is not suitable for in vitro construction of urothelial cell-matrix implants.