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Andy Wen Loong Liew and Yilei Zhang

bricate vascularized tissue constructs made of stacked sheets consisting of cardiac and ECs were layered on
cell sheet layers [66] . EC monoculture sheets were top of a collagen construct containing microchannels
stacked with fibroblast monoculture sheets in multiple and cultured in a bioreactor. ECs within the cell sheets
different configurations to study the effect of EC posi- were seen migrating through the collagen ECM to
tioning within the tissue construct on vascularization. form vascular networks which were connected to the
After 3 days in culture, in vitro vascular networks pre-fabricated perfusable microchannels, thus allow-
were formed within the cell sheet stacks, and these ing medium perfusion throughout the layered tissue
pre-vascularized constructs showed enhanced vascu- construct. As more sheets were stacked on the layered
logenesis upon implantation in vivo. Subsequent re- construct (12 sheets, more than 100 μm in thickness),
ports managed to control the alignment of endothelial ECs continued to form new vessels and connect with
networks through cell-cell interactions with surround- pre-existing microvessels to form an interconnected
ing focally oriented fibroblast sheets [67] . In another vascular network. Using a similar method, Sekine et
study, a thick (30 cell sheets, close to 1 mm thick) al. demonstrated in a study that stacked layers of cell
myocardial tissue stack with an interconnected, per- sheets composed of neonatal rat cardiomyocytes and
fusable vascular network was fabricated using cell ECs improved cardiac function when implanted into
sheet technology in tandem with in vivo vasculariza- infarcted rat hearts with increasing EC density, and
tion achieved by subcutaneous poly-surgery (up to 10 showed higher capillary density and inosculation [70] .
cycles) implantation into nude rats [68] . This approach Overall, cell sheet technology offers a unique me-
may not be feasible for clinical translation as it would thod for the vascularization of tissue constructs with
require the patient to undergo repetitive surgical pro- distinct advantages. Firstly, the ECM material is depo-
cedures. A technique able to vascularize thick cell sited naturally by the cells themselves, thus negating
sheet stacks in vitro would negate the need for the need to fabricate a biodegradable scaffold which
poly-surgery. Sakaguchi et al. proposed a strategy for may require the use of cytotoxic chemicals. Secondly,
thick cardiac tissue vascularization in vitro using cell the high cell-density and homogeneous distribution
sheet technology in combination with a perfusion bio- achieved in a cell sheet leads to higher regenerative
[4]
reactor and microfluidics [4,69] (Figure 7). Stacks of cell function , and the method of cell sheet harvesting

Figure 7. Cell sheet technology combined with a collagen based perfusion bioreactor for the preservation of cell viability by the
vascularization of 3D tissues. (Adopted from Sakaguchi et al. [69] )
International Journal of Bioprinting (2017)–Volume 3, Issue 1 13