Bladder tissue was obtained from 18-80 y/o IC/BPS patients and non-IC/BPS controls via cystoscopically-guided biopsy under an IRB-approved protocol. Tissues were placed in urothelial cell media (UCM) reconstituted with growth supplements and antibiotics, incubated at 37° C, and cut using a scalpel into four 1x1 mm2 pieces. Three pieces were placed in a 60 mm petri dish and subsequently covered with a glass coverslip anchored in the center with vacuum grease. The fourth piece was placed into one chamber of an 8-chamber plate and anchored in place with a sterile silver bead. Reconstituted UCM was added to sufficiently cover all tissue pieces and changed every 3-4 days.
Eleven IC/BPS and three control samples were obtained for culture. IC/BPS samples (7/11;63.6%) and control samples (3/3;100%) grew successfully using this method. Cell growth and/or migration from the originally plated tissue pieces were present in as little as three days with increased cell numbers present over the subsequent weeks. We confirmed that the cells observed were UC via immunohistochemical (IHC) staining for pan cytokeratin [AE1/AE3]: a marker characteristic for human UC (Figure 1A). We then confirmed the presence of proliferating cells in culture through IHC staining for two markers characteristic of epithelial progenitor cells: a nuclear marker, P63, and a cell surface marker, CD44 (Figure 1B-1C).
Figure 1: Immunohistochemical staining of interstitial cystitis/bladder pain syndrome patient urothelial cells (UC) Panel A: UC Immunostained using Pain Cytokeratin [AE1/AE3], a marker characteristic for human urothelial cells. Panel B: UC Immunostained with P63, a nuclear marker characteristic for human epithelial progenitor cells. Panel C: UC Immunostained with CD44, a cell-surface marker characteristic for human epithelial progenitor cells.
The evaluation of UC growth characteristics in IC/BPS is a useful tool to further define the underlying pathophysiology of the disease. While published UC culture methods typically start with a larger piece of explant tissue, the approach described here can be performed with tissue from a single cold-cup biopsy, enabling the evaluation of large numbers of individual patient samples across the broad spectrum of IC/BPS phenotypes.
Presented by: Tyler Overholt, MD1
Co-Authors: Jeffrey Schachar, MD,2 Trang Simon, BS,3 Robert Evans, MD,1,2 Catherine Matthews, MD,1,2 Andre Plair, MD,2 Whitney Smith, MD,2 Gopal Badlani, MD,1,2 Stephen Walker, MD1,2,3
1. Wake Forest Baptist Health Department of Urology, 2. Wake Forest Baptist Health Female Pelvic Medicine and Reconstructive Surgery, 3.Wake Forest Institute for Regenerative Medicine
Written by: Bilal Farhan, MD, Assistant Professor, Division of Urology, University of Texas, Medical Branch, Texas; @BilalfarhanMD, at the Society of Urodynamics, Female Pelvic Medicine & Urogenital Reconstruction Winter Meeting, SUFU 2020, February 25 - February 29, 2020, Scottsdale, Arizona
Presented by: Tyler Overholt, MD1
Co-Authors: Jeffrey Schachar, MD,2 Trang Simon, BS,3 Robert Evans, MD,1,2 Catherine Matthews, MD,1,2 Andre Plair, MD,2 Whitney Smith, MD,2 Gopal Badlani, MD,1,2 Stephen Walker, MD1,2,3
1. Wake Forest Baptist Health Department of Urology, 2. Wake Forest Baptist Health Female Pelvic Medicine and Reconstructive Surgery, 3.Wake Forest Institute for Regenerative Medicine
Written by: Bilal Farhan, MD, Assistant Professor, Division of Urology, University of Texas, Medical Branch, Texas; @BilalfarhanMD, at the Society of Urodynamics, Female Pelvic Medicine & Urogenital Reconstruction Winter Meeting, SUFU 2020, February 25 - February 29, 2020, Scottsdale, Arizona