Isolation of primary open-angle glaucomatous trabecular meshwork cells from whole eye tissue.
OBJECTIVE
Isolation and culture of human trabecular meshwork (TM) cells from primary open-angle glaucomatous (POAG) tissue has proven difficult. The objective of this study was to directly compare the utility of two different isolation methods to obtain viable human TM cells from POAG whole eye tissue.


METHODS
Using a blunt dissection technique, human TM tissue was obtained from four pairs of donor eyes (67, 77, 81 and 82 years) with a documented history of POAG. TM tissue from one eye was explanted into tissue culture. TM from the contralateral eye was digested with a collagenase mixture and seeded onto culture plates.


RESULTS
Primary cell isolates were obtained from all donors with both techniques. However, only cells obtained using the digestion method (3 of 4 TMs) could be passaged for expansion and freeze-downs (3 x 107 second passage cells/donor). None of the cells obtained from explanted TMs could be passaged. Cells from successful isolations were of uniform size, possessed typical TM morphology and had doubling times < 48 hours.


CONCLUSIONS
These results demonstrate a clear advantage to digesting the extracellular matrix of glaucomatous TM tissue to obtain sufficient numbers of healthy cells for use in experiments. In contrast to cells obtained from explants, cells liberated from POAG TM tissue by digestion appear indistinguishable morphologically and behaviorally from "normal" TM cells.
Stamer
D W
DW
Departments of Ophthalmology and Pharmacology, The University of Arizona, Tucson, Arizona 85719, USA. dstamer@eyes.arizona.edu
Roberts
B C
BC
Epstein
D L
DL
Allingham
R R
RR
D000368:Aged; D000369:Aged, 80 and over; D018929:Cell Culture Techniques / Q000379:methods; D002469:Cell Separation / Q000379:methods; D017364:Collagenases / Q000494:pharmacology; D005902:Glaucoma, Open-Angle / Q000473:pathology*; D006801:Humans; D014129:Trabecular Meshwork / Q000187:drug effects / Q000473:pathology*
D017364:Collagenases