148 A NOVEL METHOD FOR EXPANDING NORMAL HUMAN UROTHELIAL CELLS WITH PROGENITOR PHENOTYPE

Abstract

You have accessJournal of UrologyStem Cell Research1 Apr 2010148 A NOVEL METHOD FOR EXPANDING NORMAL HUMAN UROTHELIAL CELLS WITH PROGENITOR PHENOTYPE Monica Liebert, Xu Cheng, J. Stuart Wolf, Cheryl T. Lee, Stephen Feinberg, Cynthia Marcelo, and John Park Monica LiebertMonica Liebert More articles by this author , Xu ChengXu Cheng More articles by this author , J. Stuart WolfJ. Stuart Wolf More articles by this author , Cheryl T. LeeCheryl T. Lee More articles by this author , Stephen FeinbergStephen Feinberg More articles by this author , Cynthia MarceloCynthia Marcelo More articles by this author , and John ParkJohn Park More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.201AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Current techniques of culturing normal urothelial cells for tissue engineering and biologic studies are limited by slow growth rate, sensitivity to low cell density, and senescence. The objective of this study was to evaluate a novel method for rapid expansion of normal human urothelial cells in culture. METHODS Normal human urothelial cells were cultured in a low calcium, serum-free medium (Epi-Life Medium, Invitrogen), originating from ureteral explants obtained under an IRB approved protocol. Cells were expanded as adherent cultures. At the first pass into a T25 culture flask, some adherent cell cultures were fed with high medium volume (25-35 ml), which was changed every day to collect supernatant cells, while the remaining adherent cell cultures were fed with 5-7 ml of medium and passed when confluent. Cells were harvested, counted, and analyzed by flow cytometry for size and expression using a panel of antibodies: mouse IgG (negative control); CD44v6, α6 and β4 integrins and CD71 (putative stem cell markers); and a differentiation marker, W6/32 (HLA Class I). RESULTS The supernatant cells were smaller than the cells from parental adherent cultures. Over 5 days, supernatant culture yielded 3.1 x 106 cells, while 4 x 105 cells were obtained from adherent culture. Supernatant culture cells successfully seeded additional adherent cultures and differentiate normally. Flow cytometric results are shown in Table 1. Mean % positive by flow cytometry Cell type CD44v6 α6 CD71 β4 W6/32 Supernatant cells 67bright 50 24 16 6 Parental adherent 10dim 95 56 92 96 CONCLUSIONS The cells from the supernatant method showed intense CD44v6 expression, one putative marker for urothelial stem cells, while lower in α6 and β4 integrins and CD71 expression, other proposed urothelial stem cell markers. Supernatant cells showed less differentiation based on lower expression of W6/32. These data suggest that cell growth using the supernatant method may capture progenitor/transit amplifying cells. Supernatant culture generated over 7-fold more cells than adherent cultures over 5 days. The rapid expansion of cell numbers makes this method attractive for tissue engineering and other biologic studies. Ann Arbor, MI© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e60-e61 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Monica Liebert More articles by this author Xu Cheng More articles by this author J. Stuart Wolf More articles by this author Cheryl T. Lee More articles by this author Stephen Feinberg More articles by this author Cynthia Marcelo More articles by this author John Park More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...