Effect of Pore Size of Honeycomb-Patterned Polymer Film on Spontaneous Formation of 2D Micronetworks by Coculture of Human Umbilical Vein Endothelial Cells and Mesenchymal Stem Cells.

The geometrical control of micronetwork structures ( µ NSs) formed by endothelial cells is an important topic in tissue engineering, cell-based assays, and fundamental biological studies. In this study, µ NSs are formed using human umbilical vein endothelial cells (HUVECs) by the coculture of HUVECs and human mesenchymal stem cells (MSCs) confined in a honeycomb-patterned poly-l-lactic acid film (honeycomb film (HCF)), which is a novel cell culture scaffold. The HCF is produced using the breath figure method, which uses condensed water droplets as pore templates. The confinement of the HUVECs and MSCs in the HCF along with the application of centrifugal force results in µ NS formation when the pore size is more than 20  µ m. Furthermore, µ NS development is geometrically restricted by the hexagonally packed and connected pores in the horizontal direction of the HCF. Network density is also controlled by changing the seeding density of the HUVECs and MSCs. The threshold pore size indicates that µ NSs can be formed spontaneously by using an HCF with a perfectly uniform porous structure. This result provides an important design guideline for the structure of porous cell culture scaffolds by applying a blood vessel model in vitro.

Effect of separation distance on the growth and differentiation of mouse embryoid bodies in micropatterned cultures.

Embryoid body (EB) culture has been widely used for in vitro differentiation of embryonic stem (ES) cells. Micropatterning of cultures is a promising technique for regulating EB development, because it allows for controlling the EB size and the distance between neighboring EBs. In this study, we examined the relationship of EB separation distance to their growth and differentiation using a micropatterned chip. The basic chip design consisted of 91 gelatin spots (300 µm in diameter) in a hexagonal arrangement on a glass substrate that served as the cell adhesion area; the region without gelatin spots was modified with polyethylene glycol to create the non-adhesion area. Two similar chips were fabricated with distances between gelatin spots of 500 and 1500 µm. Mouse ES cells adhered on the gelatin spots and then proliferated to form EBs. When the EB-EB distance was at 1500 µm, their size and the expression of developmental gene markers were almost the same for all EBs on the chip. This indicated that interference between neighboring EBs was avoided. In contrast, when the EB-EB distance was at 500 µm, the size of EBs located in the inside region of the chip was smaller than that in the outside region. Additionally, in the inside region, hepatic differentiation of EB cells was increased over cardiac and vascular differentiation. These results indicate that the distance between EBs is an important factor in the regulation of their growth and differentiation.