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

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 ß1, 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.

Enhancing chondrogenic potential via mesenchymal stem cell sheet multilayering.

Advanced tissue engineering approaches for direct articular cartilage replacement in vivo employ mesenchymal stem cell (MSC) sources, exploiting innate chondrogenic potential to fabricate hyaline-like constructs in vitro within three-dimensional (3D) culture conditions. Cell sheet technology represents one such advanced 3D scaffold-free cell culture platform, and previous work has shown that 3D MSC sheets are capable of in vitro hyaline-like chondrogenic differentiation. The present study aims to build upon this understanding and elucidate the effects of an established cell sheet manipulation technique, cell sheet multilayering, on fabrication of MSC-derived hyaline-like cartilage 3D layered constructs in vitro. To achieve this goal, multilayered MSC sheets are prepared and assessed for structural and biochemical transitions throughout chondrogenesis. Results support MSC multilayering as a means of increasing construct thickness and 3D cellular interactions related to in vitro chondrogenesis, including N-cadherin, connexin 43, and integrin ß-1. Data indicate that increasing construct thickness from 14 µm (1-layer construct) to 25 µm (2-layer construct) increases these cellular interactions and subsequent in vitro MSC chondrogenesis. However, a clear initial thickness threshold (33 µm - 3-layer construct) is evident that decreases the rate and extent of in vitro chondrogenesis, specifically chondrogenic gene expressions (Sox9, aggrecan, type II collagen) and sulfated proteoglycan accumulation in deposited extracellular matrix (ECM). Together, these data support the utility of cell sheet multilayering as a platform for tailoring construct thickness and subsequent MSC chondrogenesis for future articular cartilage regeneration applications.

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. 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, ß-catenin, integrin ß1, 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.

Allogeneic mesenchymal stem cell sheet therapy: A new frontier in drug delivery systems.

The evolution of drug discovery exploded in the early 20th century with the advent of critical scientific advancements in organic chemistry, chemical analysis, and purification. Early drug generations focused largely on symptom control and pain management, effective targets for small-molecule drugs. Recently, the attention in drug discovery has shifted to pursuit of radical cures. Cell therapy presents the ideal attributes of a promising new drug, targeting specific tissues based on chemotactic cues and modulating secretion of instructive regenerative molecules in response to dynamic signaling from disease environments. To actuate the therapeutic potential of cell therapy toward worldwide clinical use, cell delivery methods that can effectively localize and engraft mesenchymal stem cells (MSCs) with high disease-site fidelity and enable dynamic MSC bioactive function are paramount. In this review, we discuss the evolution of cell therapies with a focus on stem cell advantages, as well as the limitations to these therapies. This review aims to introduce cell sheet technology as a breakthrough cell therapy with demonstrated therapeutic success across indications for heart, liver, and kidney tissue regeneration. Opportunities and anticipated clinical impacts of cell sheet technology using MSCs are discussed.

Cell sheet tissue engineering for scaffold-free three-dimensional (3D) tissue reconstruction.

Three-dimensional (3D) reconstruction of highly functional tissues is of great importance in advancing the clinical benefit of tissue engineering and regenerative medicine. In the last quarter century, many studies have found that by engineering a 3D microenvironment that resembles the in vivo tissue condition, cells exhibit behaviors and functions that reflect those of native tissue. Biomaterial scaffolds are a central technology for providing 3D microenvironments in vitro, and, in conjunction with diverse design and cell seeding advents, have produced highly functional and complex 3D tissues. Here, we describe a new approach to creating 3D cell-dense tissue-like constructs without a biomaterial scaffold. Cell sheet technology with cell sheet layering strategies generates highly cell dense, engineered tissue capable of direct crosstalk with the tissue-engraftment surface, in addition to paracrine-mediated signaling. In this chapter, we will introduce methods of reconstructing 3D tissue using cell sheet technology and the advantages of a scaffold-free design.

Stable cell adhesion affects mesenchymal stem cell sheet fabrication: Effects of fetal bovine serum and human platelet lysate.

Cell sheet technology exploits temperature responsive cell culture dishes (TRCDs) as versatile cell harvesting methods to yield contiguous cell monolayers robustly held together by cell-cell junctions, receptors, and endogenous extracellular matrix. More than 15 years of clinical data using autologous-sourced cell sheets demonstrate enhanced therapeutic properties through increased cell retention at target tissue sites. Recently, several preclinical studies have also been reported using mesenchymal stem cell (MSC) sheets in wound healing, cardiac ischemia therapies, and pancreatic regeneration. However, optimized MSC sheet fabrication conditions have not yet been reported. In this study, we identified specific conditions for reliable human MSC sheet fabrication by comparing cell growth media supplements (fetal bovine serum [FBS] and human platelet lysate [hPL]). Human umbilical cord-derived MSCs cultured in FBS and hPL exhibit different actin cytoskeletal structures related to their cell morphologies and adhesion. MSCs cultured in FBS media showed stable cell adhesion on TRCDs with flattened cell shapes and aligned actin cytoskeletal structure. This stable cell adhesion enables production of consistent MSC cell sheets, with controlled cell sheet detachment. Conversely, cell sheet fabrication in hPL media exhibits poor reproducibility being more sensitive to temperature- and culture time-induced release due to weak cell adhesion. These findings suggest that stable MSC adhesion to TRCDs is important to reliable MSC sheet fabrication methods and that MSC growth media supplementation directly affects cell adhesion during culture.

Human mesenchymal stem cell sheets in xeno-free media for possible allogenic applications.

Cell-based therapies are increasingly focused on allogeneic stem cell sources because of several advantages in eliminating donor variability (e.g., aging and disease pathophysiology) affecting stem cell quality and in cell-banked sourcing of healthy donors to enable "off-the-shelf" products. However, allogeneic cell therapy is limited by host patient immunologic competence and inconsistent performance due to cell delivery methods. To address allogeneic cell therapy limitations, this study developed a new allogeneic stem cell sheet using human umbilical cord mesenchymal stem cells (hUC-MSC) that present low antigenicity (i.e., major histocompatibility complex, MHC). Optimal conditions including cell density, passage number, and culture time were examined to fabricate reliable hUC-MSC sheets. MHC II antigens correlated to alloimmune rejection were barely expressed in hUC-MSC sheets compared to other comparator MSC sheets (hBMSC and hADSC). hUC-MSC sheets easily graft spontaneously onto subcutaneous tissue in immune-deficient mice within 10 minutes of placement. No sutures are required to secure sheets to tissue because sheet extracellular matrix (ECM) actively facilitates cell-target tissue adhesion. At 10 days post-transplantation, hUC-MSC sheets remain on ectopic target tissue sites and exhibit new blood vessel formation. Furthermore, implanted hUC-MSC sheets secrete human HGF continuously to the murine target tissue. hUC-MSC sheets described here should provide new insights for improving allogenic cell-based therapies.

Exosomes derived from clinical-grade oral mucosal epithelial cell sheets promote wound healing.

The oral mucosa exhibits unique regenerative properties, sometimes referred to as foetal-like wound healing. Researchers from our institute have used sheets of oral mucosa epithelial cells (OMECs) for regenerative medicine applications including cornea replacement and oesophageal epithelial regeneration for stricture prevention. Here, we have isolated exosomes from clinical-grade production of OMEC sheets from healthy human donors (n = 8), aiming to evaluate the clinical potential of the exosomes to stimulate epithelial regeneration and to improve understanding of the mode-of-action of the cells. Exosomes were isolated from conditioned (cExo) and non-conditioned (ncExo) media. Characterization was performed using Western blot for common exosomal-markers: CD9 and flotillin were positive while annexin V, EpCam and contaminating marker GRP94 were negative. Nanoparticle tracking analysis revealed a diameter of ~120 nm and transmission electron microscopy showed a corresponding size and spherical appearance. Human skin fibroblasts exposed to exosomes showed dose-dependent reduction of proliferation and a considerable increase of growth factor gene expression (HGF, VEGFA, FGF2 and CTGF). The results were similar for both groups, but with a trend towards a larger effect from cExo. To study adhesion, fluorescently labelled exosomes were topically applied to pig oesophageal wound-beds ex vivo and subsequently washed. Positive signal could be detected after as little as 1 min of adhesion, but increased adhesion time produced a stronger signal. Next, labelled exosomes were added to full-thickness skin wounds in rats and signal was detected up to 5 days after application. cExo significantly reduced the wound size at days 6 and 17. In conclusion, exosomes from OMEC sheets showed pro-regenerative effects on skin wound healing. This is the first time that the healing capacity of the oral mucosa is studied from an exosome perspective. These findings might lead to a combinational therapy of cell sheets and exosomes for future patients with early oesophageal cancer.

Evaluation of Multiple Biological Therapies for Ischemic Cardiac Disease.

The development of cell- and gene-based strategies for regenerative medicine offers a therapeutic option for the repair and potential regeneration of damaged cardiac tissue post-myocardial infarction (MI). Human umbilical cord subepithelial cell-derived stem cells (hUC-SECs), human bone marrow-derived mesenchymal stem cells (hBM-MSCs), and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), all derived from human tissue, have been shown to have in vitro and in vivo therapeutic potential. Additionally, S100a1, VEGF165, and stromal-derived factor-1α (SDF-1α) genes all have the potential to improve cardiac function and/or effect adverse remodeling. In this study, we compared the therapeutic potential of hBM-MSCs, hUC-SECs, and hiPSC-CMs along with plasmid-based genes to evaluate the in vivo potential of intramyocardially injected biologics to enhance cardiac function in a mouse MI model. Human cells derived from various tissue types were expanded under hypoxic conditions and injected intramyocardially into mice that had undergone left anterior descending (LAD) artery ligation. Similarly, plasmids were also injected into three groups of mice after LAD ligation. Seven experimental groups were studied in total: (1) control (saline), (2) hBM-MSCs, (3) hiPSC-CMs, (4) hUC-SECs, (5) S100a1 plasmid, (6) VEGF165 plasmid, and (7) SDF-1α plasmid. We evaluated echocardiography, hemodynamic catheterization measurements, and histology at 4 and 12 weeks post-biologic injection. Significant improvement was observed in cardiac function and contractility in hiPSC-CM and S100a1 groups and a significant reduction in left ventricle scar within the hUC-SEC group and a slight improvement in the SDF-1α and VEGF165 groups compared to the control group. These results demonstrate the potential for new biologic therapies to reduce scar burden and improve contractile function.