One-step delivery of a functional multi-layered cell sheet using a thermally expandable hydrogel with controlled presentation of cell adhesive proteins.

In this study, we developed a new system enabling rapid delivery of a multi-layered cell sheet by combining layer-by-layer (LBL) coating of a cell membrane and surface engineered thermally expandable hydrogel. Human dermal fibroblasts were LBL-coated with fibronectin (FN) and gelatin to form a multi-layered cell sheet in a single seeding step via spontaneous 3D cell-cell interactions. FN was covalently immobilized onto the surface of a Tetronic®-based hydrogel at two different concentrations (1 and 5 µg ml-1) for stable adhesion of the multi-layered cell sheet, followed by polydopamine coating. In both conditions, a multi-layered cell sheet was stably formed. Then, the cell sheet on the hydrogel modified with 1 µg ml-1 FN rapidly detached (>90% efficiency) in response to the expansion of the hydrogel when temperature changed from 37 °C to 4 °C, while the other group had a reduced detachment due to excessive cell-hydrogel interaction. The multi-layered cell sheet was evident in cell-extracellular matrix and cell-cell junction formation, and bFGF was continuously secreted over 7 days of in vitro culture. The multi-layered transplanted to the mouse subcutaneous tissue also exhibited evidence of vascular ingrowth, which collectively suggest that the delivery system maintaining cellular functions is applicable for regenerative medicine.

Spatially Assembled Bilayer Cell Sheets of Stem Cells and Endothelial Cells Using Thermosensitive Hydrogels for Therapeutic Angiogenesis.

Although the coculture of multiple cell types has been widely employed in regenerative medicine, in vivo transplantation of cocultured cells while maintaining the hierarchical structure remains challenging. Here, a spatially assembled bilayer cell sheet of human mesenchymal stem cells and human umbilical vein endothelial cells on a thermally expandable hydrogel containing fibronectin is prepared and its effect on in vitro proangiogenic functions and in vivo ischemic injury is investigated. The expansion of hydrogels in response to a temperature change from 37 to 4 °C allows rapid harvest and delivery of the bilayer cell sheet to two different targets (an in vitro model glass surface and in vivo tissue). The in vitro study confirms that the bilayer sheet significantly increases proangiogenic functions such as the release of nitric oxide and expression of vascular endothelial cell genes. In addition, transplantation of the cell sheet from the hydrogels into a hindlimb ischemia mice model demonstrates significant retardation of necrosis particularly in the group transplated with the bilayer sheet. Collectively, the bilayer cell sheet is readily transferrable from the thermally expandable hydrogel and represents an alternative approach for recovery from ischemic injury, potentially via improved cell-cell communication.

Facile Cell Sheet Harvest and Translocation Mediated by a Thermally Expandable Hydrogel with Controlled Cell Adhesion.

Facile cell sheet translocation system is developed based on a thermally expandable hydrogel with modular cell adhesion favorable for both robust cell sheet formation and harvest. Efficient translocation is achieved at moderate cell-substrate interaction, which can be tuned by two-step reactions of mussel-inspired coating.

Mussel adhesive protein inspired coatings on temperature-responsive hydrogels for cell sheet engineering.

Stimuli-responsive materials have been a major subject of investigation for cell sheet technology in regenerative medicine and pharmaceutical research. Most materials and processes, however, have shortcomings, such as a relatively long operation time, varied detachment efficiency, and potential toxicity. In this study, we develop an effective cell sheet translocation protocol using a cell adhesive and temperature-responsive hydrogel. Cell adhesion on the hydrogel was modulated with a bio-inspired coating of polydopamine (PD), the amount of which was tuned to increase as a function of coating time (30-120 min). This PD-coated hydrogel promotes the adhesion of human dermal fibroblasts (HDFBs), mediated by serum protein adsorption, and allows for the formation of a cell sheet (confluent cell monolayer) following culture. Hydrogel cell sheets with 30 min of PD coating (PD30) were readily translocated to a target surface (glass) within 10 min of thermal expansion (induced by changing the temperature from 37 to 4 °C). Under these conditions, the translocation efficiency and cell viability were greater than 90%. However, hydrogel cell sheets with >60 min PD coating remained almost completely attached, while the surfaces exhibited significantly lower cell viability (<50%), suggesting that the regulation of PD coating is a major consideration for translocating cell sheets to their target. Furthermore, the PD30 hydrogel-cultured cell sheet was applied in vivo to the subcutaneous tissue of the mouse model, and thermal expansion was induced by dropping 4 °C saline solution onto the hydrogel, at which point the hydrogel expanded and the cell sheet successfully translocated to the tissue within approximately 20 min. These translocated cell sheets exhibited stable retention over the following 7 days post-transplantation. Together, this shows that the cell adhesive hydrogels developed here could be effectively utilized as a rapid cell delivery tool for use in regenerative medicine.

Construction of 3-D Cellular Multi-Layers with Extracellular Matrix Assembly Using Magnetic Nanoparticles.

Construction of 3-dimensional (3-D) engineered tissue is increasingly being investigated for use in drug discovery and regenerative medicine. Here, we developed multi-layered 3-D cellular assembly by using magnetic nanoparticles (MNP) isolated from Magnetospirillum sp. AMB-1 magnetotactic bacteria. Magnetized human dermal fibroblasts (HDFBs) were prepared by treatment with the MNP, induced to form 3-D assembly under a magnetic field. Analyses including LIVE/DEAD assay, transmission electron microscopy revealed that the MNP were internalized via clathrin-mediated endocytosis without cytotoxicity. The magnetized HDFBs could build 3-D structure as a function of seeding density. When the highest seeding density (5 × 105 cells/mm2 was used, the thickness of assembly was 41.90 ± 1.69 µm, with approximately 9.3 ± 1.6 cell layers being formed. Immunofluorescence staining confirmed homogeneous distribution of ECM and junction proteins throughout the 3-D assembly. Real-time PCR analysis showed decrease in expression levels of collagen types I and IV but increase in that of connexin 43 in the 3-D assembly compared with the 2-D culture. Finally, we demonstrated that the discernible layers can be formed hierarchically by serial assembly. In conclusion, our study showed that a multi-layered structure can be easily prepared using magnetically-assisted cellular assembly with highlighting cell-cell and cell-ECM communication.

Delivery of a Cell Patch of Cocultured Endothelial Cells and Smooth Muscle Cells Using Thermoresponsive Hydrogels for Enhanced Angiogenesis.

Cell-based therapy has been studied as an attractive strategy for therapeutic angiogenesis. However, obtaining a stable vascular structure remains a challenge due to the poor interaction of transplanted cells with native tissue and the difficulty in selecting the optimal cell source. In this study, we developed a cell patch of cocultured human umbilical vein endothelial cells (HUVECs) and smooth muscle cells (SMCs) using thermosensitive hydrogels for regeneration of mature vasculatures. In vitro characterization of HUVECs in the cocultured group revealed the formation of a mesh-like morphology over 5 days of culture. Vascular endothelial growth factor expression was also upregulated in the cocultured group compared with HUVECs only. The cell patch seeded with HUVECs, SMCs, or both cell type was prepared on the synthetic thermosensitive and cell interactive hydrogels, and readily detached from the hydrogel within 10 min by expansion of the hydrogel when the temperature was decreased to 4°C. We then investigated the therapeutic effect of the cell patch using a hind limb ischemic model of an athymic mouse. Overall, the group that received a cell patch of cocultured HUVECs and SMCs had a significantly retarded rate of necrosis with a significant increase in the number of arterioles and capillaries for 4 weeks compared with the groups transplanted with only HUVECs or SMCs. Dual staining of smooth muscle alpha actin and human nuclear antigen showed that the implanted cell patch was partially involved in vessel formation. In summary, the simple transplantation of a cocultured cell patch using a hydrogel system could enhance therapeutic angiogenesis through the regeneration of matured vascular structures.