Preparation of arrays of cell spheroids and spheroid-monolayer cocultures within a microfluidic device.

This study describes a novel method for generation of an array of three-dimensional (3D) multicellular spheroids within a microchannel in patterned cultures containing one or multiple cell types. This method uses a unique property of a cross-linked albumin coated surface in which the surface can be switched from non-adhesive to cell adhesive upon electrostatic adsorption of a polycation. Introduction of a solution containing albumin and a cross-linking agent into a microchannel with an array of microwells caused the entire surface, with the exception of the interior of the microwells, to become coated with the cross-linked albumin layer. Cells that were seeded within the microchannel did not adhere to the surface of the microchannel and became entrapped in the microwells. HepG2 cells seeded in the microwells formed 3D spheroids with controlled sizes and shapes depending upon the dimensions of the microwells. When the albumin coated surface was subsequently exposed to an aqueous solution containing poly(ethyleneimine) (PEI), adhesion of secondary cells, fibroblasts, occurred in the regions surrounding the arrayed spheroids. This coculture system can be coupled with spatially controlled fluids such as gradients and focused flow generators for various biological and tissue engineering applications.

Cell micropatterning inside a microchannel and assays under a stable concentration gradient.

We describe the use of a microfluidic device to micropattern cells in a microchannel and investigated the behavior of these cells under a concentration gradient. The microfluidic device consisted of 3 parts: a branched channel for generating a stable concentration gradient, a main channel for culturing cells, and 2 side channels that flowed into the main channel. The main channel was coated with a cross-linked albumin that was initially cell-repellent but that could become cell-adherent by electrostatic adsorption of a polycation. A sheath flow stream, which was generated by introducing a polycation solution from the branched channel and a buffer solution from the 2 side channels, was used to change a specific region in the main channel from cell-repellent to cell-adhesive. In this way, cells attached to the central region along the main channel. The remaining surface was subsequently changed to cell-adhesive, thereby facilitating cell migration from a fixed location under a concentration gradient. We demonstrated that with this device, the gradient generator could be used to conduct simultaneous cytotoxic assays with anticancer agents; further, by combining this device with cell micropatterning, migration assays under a concentration gradient of biological factors could be conducted.

Engineering of capillary-like structures in tissue constructs by electrochemical detachment of cells.

A major challenge in the development of functional thick tissues is the formation of vascular networks for oxygen and nutrient supply throughout the engineered tissue constructs. This study describes an electrochemical approach for fabrication of capillary-like structures, precisely aligned within micrometer distances, whose internal surfaces are covered with vascular endothelial cells. In this approach, an oligopeptide containing a cell adhesion domain (RGD) in the center and cysteine residues at both ends was designed. Cysteine has a thiol group that adsorbs onto a gold surface via a gold-thiolate bond. The cells attached to the gold surface via the oligopeptide were readily and noninvasively detached by applying a negative electrical potential and cleaving the gold-thiolate bond. This approach was applicable not only for a flat surface but also for various configurations, including cylindrical structures. By applying this approach to thin gold rods aligned in a spatially controlled manner in a perfusion culture device, human umbilical vein endothelial cells (HUVECs) were transferred onto the internal surface of capillary structures in collagen gel. In the subsequent perfusion culture, the HUVECs grew into the collagen gel and formed luminal structures, thereby forming vascular networks in vitro.