Figure 5.

Pretreatment of mesenchymal stem cells enhances chemotaxis toward gradients of GCM. (A): Schematic of multilayer microfluidic device used to generate gradients of GCM. The red layer represents the cell loading areas. Light blue highlights the narrow channels where linear gradients form. Green represents control layers used to operate the device. (Ai): Phase contrast image of hAMSCs moving through narrow channels. (Aii): Channels filled with Alexa Fluor 594 fluorescent dye, allowing visualization of gradients in the device. (B): Quantification of hAMSC chemotaxis measured by speed and persistence. Cells migrated toward 100% GCM or the negative control, SFM. Movement toward the higher concentration was considered positive (n ≥ 60 cells, mean ± SEM, ∗, p < .05, Mann-Whitney rank sum test). (C): Schematic of 8-µm-pore Transwell chamber used to analyze sensitivity to gradient. (D, E): Quantification of hAMSC chemotaxis measured by numbers of cells migrating through the membrane. Values are normalized and expressed as the fold change (n = 27 fields, mean ± SEM, ∗, p < .05, Mann-Whitney rank sum test). (D): SFM, NCCM, or GCM served as the chemoattractant. (E): hAMSCs were subjected to overnight GCM pretreatment. In the Ctrl condition, hAMSCs were exposed to SFM overnight. Abbreviations: Ctrl, control; GCM, glioma-conditioned medium; hAMSC, human adipocyte-derived mesenchymal stem cell; NCCM, noncancerous astrocyte-conditioned medium; SFM, serum-free medium.