Isolation, Culture, and Characterization of Human Fetal Trabecular Meshwork Cells

Abstract

Purpose: To isolate and characterize fetal trabecular meshwork (FTM) cells for study in culture. Cultured adult trabecular meshwork (TM) cells often possess a slower rate of growth and restricted number of population doublings, limiting the ability to perform expanded testing. Methods: Fetal eyes from 24-week gestation abortions were delicately dissected to isolate the developing trabecular meshwork tissue. Three primary cultures were achieved and passaged. Light microscopy was used to compare the FTM cells to two cultured adult TM cell lines. Immunocytochemistry and Western blot analysis were utilized to identify specific protein expression. Results: The FTM cells demonstrated similar microscopic characteristics to adult TM cells, including monolayer formation, cobblestone pattern, and comparable size. FTM cells exhibited faster, more consistent doubling times when compared with adult TM cells. They grew rapidly even after passage 8, whereas their adult counterparts slowed significantly with each successive passage and failed to reach confluence at passages 4 to 5. Immunofluorescent staining was positive for actin, vimentin, fibronectin, laminin, aquaporin-1, CD-44, and myocilin in both FTM and adult TM cells. In both fetal and adult cells, Western blots showed substantial increase in myocilin after exposure to dexamethasone. Conclusions: Characterization by microscopy and immunocytochemistry suggest that FTM cells have properties similar to adult TM cells. Fetal tissues may be a useful source of abundant, rapidly dividing FTM cells for in vitro investigation. The ability to do expanded research in this field may contribute to a better understanding of the molecular mechanisms in glaucoma development.

KEYWORDS:

• Adult trabecular meshwork cells
• fetal trabecular meshwork cells
• glaucoma
• monolayer formation
• population doubling