(377e) Precise Tissue Assembly Using Avidin-Biotin Binding System and Optical Tweezers

Introduction: We investigated the feasibility of a new methodology that enables us to organize tissues with precise alignment of cells. This is based on a very quick cell-cell binding using Avidin-Biotin Binding System (ABBS) and a single cell manipulation using an optical tweezers. Materials and Methods: Hep G2 cells, a human hepatoma cell line, were biotinylated with commercially available reagent EZ-Link Sulfo-NHS-LC-Biotin (Pierce). An aliquot of biotinylated-cells was then coated with an avidin protein to prepare avidinylated-cells. To distinguish the two cell fractions, avidinylated- and biotinylated-cells were stained by fluorescence dyes, PKH67 (Sigma) and PKH26 (Sigma), respectively. The optical tweezers consisted of a microscopy (Olympus) and a laser unit (Sigma Koki). Results and Discussion: First, we mixed these cells in a plate and gently shook it for several seconds. Cells immediately formed aggregates due to the strong cell-to-cell bindings mediated by ABBS. Under confocal laser scanning microscopic observation, green-cells and red-cells were arranged like a checkered pattern. These results clearly show that ABBS can aggregate cells in very short time. Second, we tried to assemble cells more precisely using the optical tweezers. We were able to control a single cell with the optical tweezers and to attach an avidinylated- and a biotinylated-cell almost immediately after the contact. After such manipulation, chain-like tissue consisted of red- and green-cells were formed successfully. Although at present these tissues were very small (up to 20 cells), further assembling of these small tissues may lead to the formation of much larger tissues having a precisely-controlled microstructure.