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Yonathan Garfias, Mariana A. Garcia-Mejia, Jessica Nieves, Carlos Estrada, Concepcion Agundis; Apoptosis Effect Of Frozen Denuded Amniotic Membrane Supernatant Is Restricted To Lymphoid Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):512.
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© ARVO (1962-2015); The Authors (2016-present)
Amniotic Membrane is the innermost layer of the placenta which is in contact with the fetus. Although its efficacy in clinical ophthalmology has been well demonstrated as a co-adyuvant in ocular cell surface therapy, the mechanism of action of this tissue remains partially unknown. The aim of this study was at determining the effect of the supernatant (SN) of frozen amniotic membrane on different cell types.
Frozen amniotic membrane was thawed at room temperature; the epithelium was enzimatically removed. The amnion without epithelium (dAM) was incubated with 10% FBS complemented medium for 24 h; the SN was collected and kept at -800C until use. Cell cultures were performed with primary cells: Peripheral Blood Mononuclear Cells (PBMC), limbal fibroblasts and limbal epithelial cells; and stable cell lines: Jurkat cells, U937 monocytic cells and HEK epithelial cells. Different cell types were cultured in conditioned medium, which was prepared from 0 to 50% of SN in fresh complete medium for 24 h. Inhibitory Concentration 50 (IC50) for each cell type was calculated in a quantal-dose response curve. Finally, the conditioned medium was used to determine its effect on the proliferation of antiCD3/CD28 stimulated PBMC. Cell viability was measured by CFSE, while cell death was analyzed by propidium iodide incorporation; all analyses were performed by flow cytometry.
When cells were cultured in the presence of increasing concentrations of SN, primary lymphoid cells and lymphoid stable cell lines were more susceptible to apoptosis with an IC50 of conditioned medium at 16% of SN, compared to epithelial cells either primary or stable cells with an IC50 up to 40% of SN. When PBMC were cultured in the presence of proteinase K and heat treated SN, apoptosis and cell proliferation percentages were not statistically different from those percentages of cells cultured with non-treated SN.
Taken together these results indicate that there are soluble proteinase-K and heat stable apoptosis inducing factors, which effect is restricted to lymphoid cells. This could explain in part the success of amniotic membrane transplantation in inflammatory ocular cell surface diseases.
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