Two major challenges in tissue engineering are mimicking the native cell-cell arrangements of tissues and maintaining viability of three-dimension (3D) tissues thicker than 300 µm. Cell printing and prevascularization of engineered tissues are promising approaches to meet these challenges. However, the printing technologies used in biofabrication must balance the competing parameters of resolution, speed, and volume, which limit the resolution of thicker 3D structures. We suggest that high-resolution conformal printing techniques can be used to print 2D patterns of vascular cells onto biopaper substrates which can then be stacked to form a thicker tissue construct. Towards this end we created 1 cm × 1 cm × 300 µm biopapers to be used as the transferable, stackable substrate for cell printing. 3.6% w/v poly-lactide-co-glycolide was dissolved in chloroform and poured into molds filled with NaCl crystals. The salt was removed with DI water and the scaffolds were dried and loaded with a Collagen Type I or Matrigel. SEM of the biopapers showed extensive porosity and gel loading throughout. Biological laser printing (BioLP) was used to deposit human umbilical vein endothelial cells (HUVEC) in a simple intersecting pattern to the surface of the biopapers. The cells differentiated and stretched to form networks preserving the printed pattern. In a separate experiment to demonstrate "stackability," individual biopapers were randomly seeded with HUVECs and cultured for 1 day. The mechanically stable and viable biopapers were then stacked and cultured for 4 days. Three-dimensional confocal microscopy showed cell infiltration and survival in the compound multilayer constructs. These results demonstrate the feasibility of stackable "biopapers" as a scaffold to build 3D vascularized tissues with a 2D cell-printing technique.
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