Human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (hiPSC-RPE) cells suspension have the potential for regenerative treatment. However, practical regenerative applications with hiPSC-RPE cells require the development of simple and cost-effective non-freezing preservation methods. We investigated the effect of non-freezing temperatures on suspended hiPSC-RPE cells in various conditions and analysed mechanisms of cell death, anoikis, Rho GTPases, hypoxia, microtubule destruction, and cell metabolism. Cells stored at 37 °C had the lowest viability due to hypoxia from high cell metabolism and cell deposits, and cells preserved at 4 °C were damaged via microtubule fragility. Cell suspensions at 16 °C were optimal with drastically reduced apoptosis and negligible necrosis. Moreover, surviving cells proliferated and secreted key proteins normally, compared to cells without preservation. hiPSC-RPE cell suspensions were optimally preserved at 16 °C. Temperatures above or below the optimal temperature decreased cell viability significantly yet differentially by mechanisms of cell death, cellular metabolism, microtubule destruction, and oxygen tension, all relevant to cell conditions. Surviving cells are expected to function as grafts where high cell death is often reported. This study provides new insight into various non-freezing temperature effects on hiPSC-RPE cells that are highly relevant to clinical applications and may improve cooperation between laboratories and hospitals.
PURPOSE. Intraepithelial mast cells are observed in giant papillae tissue samples obtained from patients with atopic keratoconjunctivitis (AKC)/vernal keratoconjunctivitis (VKC). We examined the roles of interaction between the conjunctival epithelial cells and mast cells.
METHODS.The interaction between human mast cells and conjunctival epithelial cells (HCjE) was investigated using a coculture model. Protein array analysis, ELISA, and real-time PCR were performed to test the interaction. Tissue samples (n ¼ 6) from giant papillae were resected for therapeutic purposes, and subjected to immunohistological analysis of CCL2 expression. Recombinant CCL2 (10 ng/mL) was reacted with the cultured human mast cells and ultrastructural analysis was performed. A ragweed (RW)-induced mouse experimental allergic conjunctivitis model was used to examine ccl2 mRNA expression and mast cell morphology.RESULTS. Protein array and real-time PCR analyses showed that CCL2 protein/mRNA expression was induced by mast cell-HCjE coculture. Upregulation of CCL2 mRNA was observed in mast cells, whereas in situ CCL2 expression was observed at the conjunctival epithelium of the giant papillae by immunohistochemistry. Ultrastructural analysis showed that recombinant CCL2 treatment induced piecemeal degranulation (PMD) in the mast cells. Ultrastructural analysis of tissues from the giant papillae showed PMD of mast cells within the conjunctival epithelial cells. The RW-induced experimental allergic conjunctivitis model showed increased ccl2 mRNA expression and PMD morphology in the conjunctivae.
CONCLUSIONS. Mast cell-conjunctival epithelial cell interaction induces CCL2 expression and subsequent PMD.Keywords: allergy, conjunctivitis, mast cell, degranulation M ast cell activation and migration within and around the conjunctival epithelium is one of the histopathologic features of severe chronic allergic conjunctivitis, atopic keratoconjunctivitis (AKC), 1 and vernal keratoconjunctivitis (VKC).2 In our study, we investigated possible interactions of mast cells and conjunctival epithelial cells using in vitro coculture models, and we found that CCL2 expression in mast cells was upregulated by coculture. A previous report showed that CCL2 could induce piecemeal degranulation (PMD) in basophils.3 PMD and anaphylactic degranulation are known as two distinct types of mast cell degranulation. 4,5 Anaphylactic degranulation is a degranulation style with antecedent granuleto-granule and/or granule-to-plasma membrane fusions. On the other hand, gradual emptying of cytoplasmic secretory granules in the absence of granule-to-granule or granule-to-plasma membrane fusion events is observed in PMD. Previously, we reported that 34 of 168 mast cells observed in the giant papillae of eight eyes obtained from VKC patients showed PMD, whereas only 28 of the 168 mast cells had the morphology of anaphylactic degranulation. 6 These results suggested that not only anaphylactic degranulation, but also PMD might have some roles in the pathophysiology of AKC and VKC....
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