Lacrimal glands were dissected from adult Sabra rats and divided by pincers to 0.3–0.4 mm fragments. Animals were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The “raft” culture technique
8 was applied: Between three and six tissue fragments were suspended on filter paper (Hogla-Kimberly Ltd., Ramle, Israel) within 4-cm center-well organ culture plates (model 353037; Falcon-BD Biosciences, Bedford, MA). DMEM medium (Beit Haemek Industries, Kibbutz Beit-Haemek, Israel) was in contact with the filter paper from below, with 10% fetal calf serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 1% glutamine, and 10 mM HEPES buffer to maintain pH at 7.0. The tissue fragments were thus at the interface between liquid media from below and air above. Plates were maintained in an incubator at 37°C and under 5% CO
2. Viability of the tissue was assessed by the tetrazolium dye 3-(4,5-dfimethylthiazol-2-yl)-2,5-diphyenyltetrazolium bromide mitochondrial assay (MTT) adapted for organ culture as described by Connelly et al.
9 The MTT dye, reduced by active mitochondria and other cellular enzymes, forms a dark purple precipitate in viable areas within the tissue fragments. Fragments assayed two hours after excision served as a positive control, showing that living cells are present throughout the specimen at this time
(Fig. 1A) . Freshly excised fragments maintained for 2 hours in distilled water to cause cellular death served as a negative control, and, indeed, no MTT staining was seen
(Fig. 1B) . After 7 to 10 days in culture, viability of the tissue was largely maintained.
Figures 1C and 1D exemplify the range of MTT staining observed at this time. Whereas viability was reduced to a certain degree in some fragments
(Fig 1C) , large viable regions could be identified in all cases and staining was often present throughout the tissue
(Fig. 1D) .