In vitro culture Muller cell model to study the role of inverted internal limiting membrane flap technique in macular hole closure.

Inverted internal limiting membrane (ILM) flap may constitute a scaffold for Muller cells whose migration and proliferation on its surface begin the process of macular hole closure. The goal of the study was to establish an in vitro model of the interaction between ILM and the Muller cells. Vitrectomy with inverted ILM flap was performed in 23 patients due to a full-thickness macular hole (FTMH). After dissection of the inverted flap, the area of ILM peeling was extended and material was collected for cell culture experiments. Muller cells cultured on adherent cell plates showed significantly better growth than on suspension plates. Our results reveal that the presence of the ILM can overcome the growth inhibitory effect of the non-adhesive surface. Moreover, the ILM appears to be the optimal growth surface under normoxia conditions mimicking the microenvironment after vitrectomy and hypoxia which is natural state for Muller cells. The closure rate of FTMH was 100%. Our study revealed that in non-adhesive culture conditions patient derived ILM constitutes an optimal growth surface for Muller cells. We have demonstrated that the ILM effectively stimulates attachment, proliferation, and survival of Müller cells in conditions of normoxia which is the case after vitrectomy. The results strongly advocate for the use of inverted ILM flap method in macular hole closure surgeries.

ELISPOT assays provide reproducible results among different laboratories for T-cell immune monitoring--even in hands of ELISPOT-inexperienced investigators.

Measurements of antibodies in bodily fluids (e.g., by ELISA) have provided robust and reproducible results for decades and such assays have been validated for monitoring of B-cell immunity. In contrast, measuring T-cell immunity has proven to be a challenge due to the need to test live cells in functional assays ex vivo. Several previous efforts looking into the reproducibility of ex vivo T-cell assays between different laboratories, or even within the same laboratory, have provided rather discouraging results. The hypothesis we tested in this study is that those poor results are due to the lack of assay and data analysis standardization, rather than the inherent complexity of T-cell assays. In this study, 11 laboratories across Europe and the United States were provided identical reagents and were asked to follow the same protocol while testing aliquots of the same three cryopreserved peripheral blood mononuclear cells (PBMC) in an interferon-gamma (IFNgamma) ELISPOT assay measuring the antigen-specific T-cell response to a CMV peptide. All individuals performing the assays were ELISPOT novices. At their first attempt, while three of these individuals failed with the basic logistics of the trial, eight detected the peptide-specific CD8+ T-cells in frequencies approximating the values established by the Reference Laboratory. The data show that ELISPOT assays provide reproducible results among different laboratories when the assay procedure and data analysis is standardized. Since ELISPOT assays have been qualified and validated for regulated studies, they are ideal candidates for robust and reproducible monitoring of T-cell activity in vivo.