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P050 Intraperitoneally injected 3-dimensional cultured tonsil-derived mesenchymal stem cells in murine colitis model form aggregates with mouse immune cells: A preliminary study

Byeon, J.R.(1)*;Song, E.M.(1);Joo, Y.H.(1);Park, Y.(1);Shim , K.N.(1);Choe, A.R.(1);Tae, C.H.(1);Kim, S.E.(1);Moon, C.M.(1);Jung, H.K.(1);Hong, J.T.(1);Jung, S.A.(1);

(1)Ewha Womans University College of Medicine, Department of Internal Medicine, Seoul, Korea- Republic Of;

Background

3-dimensional (3D) cultured tonsil-derived mesenchymal stem cells (TMSCs) showed higher therapeutic efficacy in murine colitis model than conventional 2D-cultured TMSCs via increased anti-inflammatory cytokine expression. Here, we evaluated the in-vivo localization and formation of 3D-cultured TMSCs using electron microscopy.

Methods

Chronic mouse colitis was induced by oral administration of 1.5% dextran sulfate sodium for 30 days. 3D-cultured TMSCs and 2D-cultured TMSCs was injected intraperitoneally on day 6 and day 16 after inducing colitis in mice. TMSC was 3D-cultured as a spheroid for 24 hours before intraperitoneal injection. On day 31, mice were euthanized and the intraperitoneal localization of 3D-cultured TMSCs was evaluated using immunofluorescence analysis. Moreover, the in-vivo characterization of 3D-cultured TMSCs was evaluated using electron microscopy.

Results

In vitro 3D-cultured TMSC spheroids were measured to have an average size of 182.4 μm on day 1, 169.4 μm on day 2, and 154.1 μm on day 3; however, the size of spheroids gradually decreased after 2 days of culture. In mice treated with 2D-cultured TMSCs, there were no detectable TMSCs at the time of sacrifice. In mice treated with 3D-cultured TMSCs, white variable sized spherical aggregates were observed. These aggregates were attached to peritoneal cavity including omentum and mesentery. In fluorescence microscopy, these aggregates were stained with human nuclear antigen antibody, indicating that the transplanted 3D-cultured TMSCs formed a cluster in the peritoneum even 14 days after intraperitoneal injection. In electron microscopy, these aggregates were covered with mouse lymphocytes. In the inside of spheroids, suspected mesenchymal cell and mesothelial cell were distributed.

Conclusion

Intraperitoneally injected 3D-cultured TMSCs were found as aggregates in the peritoneal cavity of mouse even 14 days after injection. In electron microscopy, these aggregates were covered with mouse lymphocytes. Based on the results of current study, functional studies evaluating the role of TMSC as a cell therapy, including cell-to-cell communication between TMSC and immune cells, are needed.