Mesenchymal Stem Cell Sheet Centrifuge-Assisted Layering Augments Pro-Regenerative Cytokine Production

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.

Umbilical cord-derived mesenchymal stem cell sheets transplanted subcutaneously enhance cell retention and survival more than dissociated stem cell injections

“…Previous reports consistently indicate that hUC-MSCs secrete various therapeutic cytokines in vitro including HGF and TGF-β1 [8,23,24,[50][51][52][53]. However, cytokine secretion after in vivo hUC-MSC transplantation has not been reported.…”

“…Most MSC sheet applications have used bone marrow-or adipose tissue-derived MSC sheets; UC-MSCs are more recently reported in MSC sheets. hUC-MSC sheets have been characterized in vitro by observing cellular microstructures, ECM, cell-cell junctions, cell-ECM junctions, layered sheet constructs, and various cytokine and chemokine production [49][50][51][52][53]. Further, hUC-MSC sheet interactions with tissue culture surfaces have been investigated in vitro [8].…”