3D cell sheet structure augments mesenchymal stem cell cytokine production

Mesenchymal stem cells (MSCs) secrete paracrine factors that play crucial roles during tissue regeneration. An increasing body of evidence suggests that this paracrine function is enhanced by MSC cultivation in three-dimensional (3D) tissue-like microenvironments. Toward this end, this study explored scaffold-free cell sheet technology as a new 3D platform. MSCs cultivated on temperature-responsive culture dishes to a confluent 2D monolayer were harvested by temperature reduction from 37 to 20 °C that induces a surface wettability transition from hydrophobic to hydrophilic. Release of culture-adherent tension induced spontaneous cell sheet contraction, reducing the diameter 2.4-fold, and increasing the thickness 8.0-fold to render a 3D tissue-like construct with a 36% increase in tissue volume.

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FIG: Microscopic cell morphology influences macroscopic tissue structure. Macroscopic image and microscopic cell morphology of hUC-MSC 2D monolayers seeded onto an insert membrane, and contracted 3D cell sheets following temperature-detachment and placement on an insert membrane. In both groups, hUC-MSCs were seeded at 41,580 cells/cm2 initial cell densities. Macroscopic images of a (a) 2D monolayer (white dashed circle) on an insert membrane (red dashed circle, 24 mm diameter) and a (b) 3D cell sheet (white dashed circle) on an insert membrane, placed in the center of tissue-culture plastic dishes (35-mm diameter) for imaging. Morphology of hUC-MSCs in a 2D monolayer at (c) × 10 and (e) × 20 magnification, and in a 3D cell sheet at (d) × 10 and (f) × 20 magnification, observed using phase-contrast microscopy. Scale bars = 1 cm in (a) and (b). Scale bars = 200 μm in (c) through (f).

This 2D-to-3D transition reorganized MSC actin cytoskeleton from aligned to multidirectional, corresponding to a cell morphological change from elongated in 2D monolayers to rounded in 3D cell sheets. 3D culture increased MSC gene expression of cell interaction proteins, β-catenin, integrin β1, and connexin 43, and of pro-tissue regenerative cytokines, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and interleukin-10 (IL-10), and increased VEGF secretion per MSC 2.1-fold relative to 2D cultures. Together, these findings demonstrate that MSC therapeutic potency can be enhanced by 3D cell sheet tissue structure.

Bou-Ghannam, S., Kim, K., Grainger, D.W. et al. 3D cell sheet structure augments mesenchymal stem cell cytokine production. Sci Rep 11,8170 (2021). https://doi.org/10.1038/s41598-021-87571-7

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