Capillary Networks for Bio-Artificial Three-Dimensional Tissues Fabricated Using Cell Sheet Based Tissue Engineering

Abstract

One of the most important challenges facing researchers in the field of regenerative medicine is to develop methods to introduce vascular networks into bioengineered tissues. Although cell scaffolds that slowly release angiogenic factors can promote post-transplantation angiogenesis, they cannot be used to construct thick tissues because of the time required for sufficient vascular network formation. Recently, the co-culture of graft tissue with vascular cells before transplantation has attracted attention as a way of promoting capillary angiogenesis. Although the co-cultured vascular cells can directly contribute to blood vessel formation within the tissue, a key objective that needs to be met is the construction of a continuous circulatory structure. Previously described strategies to reconstruct blood vessels include the culture of endothelial cells in a scaffold that contains microchannels or within the original vascular framework after decellularization of an entire organ. The technique, as developed by authors, involves the progressive stacking of three-layered cell sheets onto a vascular bed to induce the formation of a capillary network within the cell sheets. This approach enables the construction of thick, functional tissue of high cell density that can be transplanted by anastomosing its artery and vein (provided by the vascular bed) with host blood vessels.

Keywords: bioreactor; cell sheet technology; regenerative medicine; tissue culture; tissue engineering; vascular bed; vascularization.

Figure 1

Approaches to myocardial tissue engineering. (A) Dissociated cells are poured into a pre-made, highly porous scaffold. The scaffold undergoes biodegradation, and extracellular matrix (ECM) fills the intercellular space to create a 3D tissue. (B) A mixture of dissociated cells and biodegradable polymers is poured into a mold, and the molecules are polymerized. Since the gel is poured into a mold, tissue of any shape can be constructed. (C) Cells suspended in a polymer solution are injected directly into injured/defective tissue using a syringe. This approach is similar to that used for cell infusion therapy. (D) Cells are actively removed from human or xenobiotic tissues and organs to leave only the ECM component, which serves as a scaffold for the seeding of dissociated cells. Since the 3D structure of the original tissue is preserved, the entire organ can be reconstructed. (E) A 3D printer uses a mixture of cells and polymer gel as bio-ink to assemble 3D tissues in a bottom-up fashion. (F) Cell sheets harvested from temperature-responsive culture surfaces are layered. The cell sheets adhere to each other via the ECM that they produce, yielding a 3D tissue that does not contain a biodegradable scaffold. The schematic diagram was drawn with the graphic design software, Adobe Illustrator.

Figure 2

Generation of a cell sheet on a temperature-responsive surface. The temperature-responsive dish allows cultured cells to be harvested as an intact, confluent sheet simply by lowering the temperature (culture in a CO₂ incubator set at 20 °C). This technique prevents the need for enzymatic harvesting. The schematic diagram was drawn with the graphic design software, Adobe Illustrator.

Figure 3

Technologies available for the induction of capillaries in bioengineered tissue. (A) A microfluidic system is used to introduce artificial capillaries (microchannels) within the tissues that are perfused with fluid. (B) A microfluidic system based on the creation of a scaffold that imitates a 3D microcirculatory structure. Vascular endothelial cells are seeded on the luminal side of the scaffold and perfusion culture is performed. (C) A whole organ is decellularized and vascular endothelial cells are seeded into the original vascular structure and subjected to perfusion culture so as to reproduce a circulatory system. (D) Engineered tissue is implanted onto well-vascularized host tissue to induce the growth of capillaries into the graft. The host tissue is supplied by blood vessels that are suitable for anastomosis onto other blood vessels, enabling later transplantation of the vascularized graft to the desired site. The schematic diagram was drawn with the graphic design software, Adobe Illustrator.

Figure 4

Construction of thick cardiac tissue grafts with blood vessels by the sequential implantation of multiple cardiomyocyte sheets containing vascular endothelial cells. (A) A three-layered cardiac cell sheet containing endothelial cells is transplanted onto a vascular bed. One day later, after a capillary network has formed in the cardiac tissue graft, a new three-layered cardiac cell sheet containing endothelial cells is transplanted on top of the original cardiac cell sheet. Thick myocardial tissue with a capillary network is constructed by repeating this procedure multiple times. (B) Fabrication of cardiac tissue suitable for ectopic transplantation. Three-layered cardiomyocyte sheets containing endothelial cells are repeatedly grafted onto existing large vessels in vivo to create pulsatile cardiac tissue supplied by an artery and vein. (C) A vascularized cardiac tissue graft is fabricated by the repeated implantation of three-layered cardiomyocyte sheets onto the femoral muscle of a rat. The cardiac tissue graft and accompanying femoral blood vessels are then excised and implanted into a region of the neck by anastomosis of the femoral blood vessels with neck vessels. Pulsation of the graft resumes immediately after vascular anastomosis. Reproduced with permission, Copyright 2006 Federation of American Societies for Experimental Biology. The schematic diagram was drawn with the graphic design software, Adobe Illustrator.

Figure 5

Tissue perfusion culture system and stacking of cell sheets on a vascular bed. The schematic diagram was drawn with the graphic design software, Adobe Illustrator.