Use of iodixanol self-generated density gradients to enrich for viable urothelial cells from nonneurogenic and neurogenic bladder tissue.
Suspensions of viable urothelial cells (UC) isolated from patient bladder biopsies often contain considerable amounts of extraneous materials comprised of cellular debris, dead and dying UC, and red blood cells. We have consistently observed an inversely proportional relationship between UC attachment efficiency and the amount of extraneous materials in the suspension; viable UC cell attachment efficiency decreases as the amount of extraneous materials in the cell suspension increases. Processing the initial cell isolate to reduce the amount of extraneous materials can enrich for viable UC capable of attaching and proliferating in ex vivo cultures. In this report, we describe the isolation of an enriched population of viable UC from nonneurogenic and neurogenic bladder tissue biopsies using iodixanol self-generated density gradients (OptiPrep), and characterization by trypan blue exclusion, fluorescence-activated cell sorting, immunofluorescence, and growth kinetics.
Bruce
Andrew T
AT
Bioprocess Research and Assay Development, Tengion Inc., Winston-Salem, NC, USA.
Sangha
Namrata
N
Richmond
Allison
A
Johnson
Kenny
K
Jones
Sarah
S
Spencer
Thomas
T
Ludlow
John W
JW
D001706:Biopsy; D002469:Cell Separation; D002470:Cell Survival; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D003287:Contrast Media / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods; D044968:Regenerative Medicine; D023822:Tissue Engineering / Q000379:methods; D014283:Triiodobenzoic Acids / Q000737:chemistry / Q000494:pharmacology*; D014343:Trypan Blue / Q000494:pharmacology; D001743:Urinary Bladder / Q000473:pathology*; D001750:Urinary Bladder, Neurogenic / Q000473:pathology*; D019459:Urothelium / Q000473:pathology*
D003287:Contrast Media; D014283:Triiodobenzoic Acids; C044834:iodixanol; D014343:Trypan Blue