Modifications of previously reported methods for obtaining purified conjunctival mast cells and primary HCECs in monodispersed suspension were used in these studies.
8 Briefly, human conjunctival tissue was obtained with prior consent from organ/tissue donors (8–10 sets of tissue per experiment were obtained through the Lion’s Eye Bank of Wisconsin, Madison, WI, and the Kansas Eye Bank and Cornea Research Center, Wichita, KS, and were approved by the University of Wisconsin Human Subjects Committee). Upper and lower bulbar conjunctiva aseptically collected within 8 hours after death (average time, 4.5 hours) were transported in corneal preservation medium (Dexsol; Chiron Ophthalmics, Irvine, CA) and were stored at 4°C for up to 5 days. Eight to 10 sets of tissue weighing 4 to 5 g were used per experiment. Hyaluronidase and collagenase were used to digest tissue. The digestion process (30 minutes at 37°C on a rotating shaker) was first performed at a low concentration of enzymes (2 digests at 200 U/g in 10 mL final volume), followed by tissue digestion at a high concentration of enzymes (3–6 digests at 2000 U/g in 10 mL final volume). Each digest was followed by washing of the enzyme-treated tissue (with TGCM) over a 100-μm nylon mesh filter to collect freed cells. After the digestion procedure, the freed cells were pelleted, pooled, resuspended in TG, and layered over a single-density gradient (Percoll [Sigma Chemical]; 1.041 g/mL) and centrifuged (500
g, 20 minutes). The resultant top cell layer (epithelial cells) was harvested, washed, and resuspended in media (EpiLife; Cascade Biological) without hydrocortisone, at a concentration of 1 × 10
6 cells/mL, and was transferred to fibronectin/collagen (FNC Coating Mix; AthenaES)–coated 24-well plates (0.5 mL/well) for culture at 37°C. Media were changed every 48 hours until confluence. Primary HCECs were passaged (10 passages) using trypsin-EDTA and a serum-free defined trypsin inhibitor. The mast cell–enriched pelleted cells were washed in TG, resuspended to a concentration of 1 × 10
6 cells/mL in mast cell culture medium, and transferred to a 24-well plate (0.5 mL/well) for an equilibration period (up to 72 hours at 37°C). After the equilibration period, the cell suspension was removed from the plate, layered over a double-density gradient (Percoll [Sigma Chemical]; 1.08 g/mL layered over 1.123 g/mL), and centrifuged (500
g, 20 minutes). Purified conjunctival mast cell suspensions (>90%) harvested from the interface between the densities were washed and allowed to equilibrate in mast cell culture medium for 24 hours at 37°C before experimentation. Differentiation of other cells was obtained using Wright-stained cytospins.