The patient had a 4 month history of hematuria prior to removal of the tumor.

Metastases to the bone marrow were discovered later.

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with Ca++/Mg++ free Dulbeccos phosphate-buffered saline (D-PBS) or 0.25% (w/v) Trypsin - 0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
3. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
   Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
4. Add 2.0 to 3.0 mL of complete growth medium and aspirate cells by gently pipetting
5. Resuspend the cell pellet in fresh growth medium. Add appropriate aliquots of the cell suspension to new culture vessels.
6. Incubate cultures at 37°C. Subculture every 6 to 8 days.

Medium Renewal: 2 to 3 times per week Protocol: Volumes used in this protocol are for 75 cm² flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.

OToole CHuman bladder cancer lines: HLA Class I and Class II antigen expression and susceptibility to cytostatic and cytotoxic effects in vitroIn: OToole CIn vitro models for cancer researchvol. IVBoca Raton, FLCRC Presspp. 103-125.

Fogh J. Cultivation, characterization, and identification of human tumor cells with emphasis on kidney, testis, and bladder tumors. Natl. Cancer Inst. Monogr. 49: 5-9, 1978. PubMed: 571047

Bellet D, et al. Malignant transformation of nontrophoblastic cells is associated with the expression of chorionic gonadotropin beta genes normally transcribed in trophoblastic cells. Cancer Res. 57: 516-523, 1997. PubMed: 9012484

Nayak SK, et al. A cell line from an anaplastic transitional cell carcinoma of human urinary bladder. Br. J. Cancer 35: 142-151, 1977. PubMed: 836756