Abstract
A focal advantage of cell sheet technology has been as a scaffold-free three-dimensional (3D) cell delivery platform capable of sustained cell engraftment, survival, and reparative function. Recent evidence demonstrates that the intrinsic cell sheet 3D tissue-like microenvironment stimulates mesenchymal stem cell (MSC) paracrine factor production. In this capacity, cell sheets not only function as 3D cell delivery platforms, but also prime MSC therapeutic paracrine capacity. This study introduces a "cell sheet multilayering by centrifugation" strategy to non-invasively augment MSC paracrine factor production. Cell sheets fabricated by temperature-mediated harvest were first centrifuged as single layers using optimized conditions of rotational speed and time. Centrifugation enhanced cell physical and biochemical interactions related to intercellular communication and matrix interactions within the single cell sheet, upregulating MSC gene expression of connexin 43, integrin β1, and laminin α5. Single cell sheet centrifugation triggered MSC functional enhancement, secreting higher concentrations of pro-regenerative cytokines vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and interleukin-10 (IL-10). Subsequent cell sheet stacking, and centrifugation generated cohesive, bilayer MSC sheets within 2 h, which could not be accomplished within 24 h by conventional layering methods. Conventional layering led to H1F-1α upregulation and increased cell death, indicating a hypoxic thickness limitation to this approach. Comparing centrifuged single and bilayer cell sheets revealed that layering increased VEGF production 10-fold, attributed to intercellular interactions at the layered sheet interface. The "MSC sheet multilayering by centrifugation" strategy described herein generates a 3D MSC-delivery platform with boosted therapeutic factor production capacity.