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Nicole Qiaozhi Lu, Michael Peter Grant, Jennifer Elisseeff; Isolation of Goblet Cells from Conjunctival Epithelium by Density-Gradient Centrifugation. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1441.
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Primary conjunctival epithelial cells were subject to Percoll® density-gradient centrifugation, in order to isolate goblet cells for the treatment of conjunctival diseases.
Rabbit conjunctiva tissue was digested with dispase II and trypsin/EDTA to obtain single cell suspension. Six densities (1.03 - 1.08 g/mL) were prepared by adjusting Percoll with 10× PBS solution and H2O. The discontinuous gradients were created by layering 2 mL of each Percoll solution in a centrifuge tube, with decreasing density. The cell suspension was added on top of the gradients, and centrifuged at 400×g for 20 min. Fractions from each interface were collected and washed with PBS. Primary cells without centrifugation were used as control. To identify cell phenotypes, qRT-PCR (comparative ΔΔCT; gapdh as the reference) was used to analyze gene expression (ck4, non-goblet cell epithelium; muc5ac, goblet cells; abcg2, stem/progenitor cells). Flow cytometry experiments were also carried out to quantify the percent of goblet cells in the fractions with CK4 and MUC5AC labeling.
After centrifugation, cell suspensions were fractionated into 6 layers (one on each interface), and they were labeled as L1-6 from top to bottom. The qRT-PCR values were interpreted as muc5ac/ck4 ratio. With increasing density, the relative expression of muc5ac/ck4 decreased, and L1 had a higher ratio than control. With respect to abcg2, L2 had the highest expression level, while both L1 and L2 expressed more abcg2 transcripts than control. Flow cytometry quantification further implicated that the MUC5AC-positive population became smaller from L1 to L6. When compared to control (15%), L1-L3 contained more MUC5AC-positive cells (18-20%). At the same time the CK4-positive population became bigger from L1 to L6. Therefore, with both results combined, L1 and L2 could be considered to contain the majority of goblet cells and more stem cells than other layers and the control.
Goblet cell-rich population can be separated from the primary conjunctival cell mixture, by simple centrifugation with discontinuous Percoll gradients. Both goblet and stem cells were enriched in the top 2 layers after centrifugation. Potentially, this isolation method could be applied to increase the mucin production and proliferation capacity of harvested cells before transplantation, which may improve outcomes in conjunctival reconstruction.
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