3D tissue formation by stacking detachable cell sheets formed on nanofiber mesh

Area of Science:

• Biomaterials Engineering
• Tissue Engineering
• Cell Biology

Background:

• Developing methods for constructing complex 3D tissues is crucial for regenerative medicine.
• Existing tissue engineering techniques face challenges in cell manipulation and large-scale tissue assembly.
• Aligned nanofiber scaffolds offer potential for guiding cell behavior and tissue organization.

Purpose of the Study:

• To develop a novel layer-by-layer approach for assembling 3D tissue using cell sheets.
• To investigate the formation and characteristics of cell sheets cultured on aligned nanofiber meshes.
• To demonstrate the potential of this method for creating vascularized and aligned muscle tissues.

Main Methods:

• Aligned polycaprolactone (PCL) nanofiber meshes were fabricated using electrospinning.
• Human umbilical vein endothelial cells (HUVECs), dermal fibroblasts, and skeletal muscle cells (C2C12) were cultured on the meshes to form cell sheets.
• Cell sheets were collected and stacked layer-by-layer using nested frames to create 3D constructs.
• Cell alignment, differentiation, and network formation were analyzed using microscopy and marker expression (CD31).

Main Results:

• Confluent cell sheets (≥ 3x3 cm²) of HUVECs, fibroblasts, and C2C12 cells were successfully formed on nanofiber meshes.
• C2C12 cells aligned with nanofibers and differentiated into aligned myotubes, forming 3-layered tissues (∼50 µm).
• Sandwiching HUVEC sheets with fibroblast sheets resulted in vascularized 3D tissue with extensive endothelial networks (CD31+).

Conclusions:

• Layer-by-layer stacking of cell sheets on aligned nanofiber meshes is a viable method for 3D tissue assembly.
• This approach facilitates the creation of organized, multi-layered tissues, including aligned muscle and vascularized structures.
• The technique offers advantages in cell sheet manipulation and stacking, with broad applications in tissue engineering.