Programmable Manipulation of Adherent Cell Motility Using Biological Breadboards
The dynamic nature of cellular attachment and detachment which regulates cell function must be characterized to understand essential biological phenomena such as embryonic development, inflammatory immune response, wound repair, cancer metastasis, etc. Although extensive efforts have been devoted toward cell motility manipulation for a better understanding cellular dynamics, these efforts have remained in their infancy. A programmable manipulation of adherent-cell motility therefore has remained a challenging task. The goal of this research is to design addressable, multi-functional, and reusable biological platforms, nicknamed biological breadboards (BBBs), to enable the spatiotemporal manipulation of cell motility at cellular and even subcellular levels, offering a new method to analyze cellular dynamics in a quantitative way. In this thesis, this method for cell motility manipulation using BBBs and their applications to quantitative characterizations of single-cellular and intracellular dynamics are summarized.
The BBBs consisting of M×N gold electrodes, identical and independently operated, on Pyrex glass substrate are designed to secure their addressability, thereby achieving a high-degree-of-freedom in programmable manipulation of adherent cell motility. This programmable manipulation of adherent cell motility is implicated by two fundamental mechanisms of cell attachment and detachment. An arginine-glycine-aspartic (RGD) acid-terminated thiol is introduced to achieve those mechanisms by tethering the RGD to the gold electrodes via thiol linker. The first mechanism is solved by a RGD which is a recognition sequence for integrin binding to many extracellular matrix proteins. The RGD-terminated thiol connects a RGD peptide into the gold electrodes using its thiol end which shows a spontaneous chemisorption of R-S-H + Au -> R-S-AU + 1/2H2 where R is a substituent, thereby enhancing cell adhesion on the BBB. The second mechanism depends on a rapid reductive desorption of the thiol-gold self-assembled monolayer under negative bias voltage, R-S-Au + H+ + e- -> R-S-H + Au. This reaction makes the RGD-terminated thiol and even a cell or a part of the cell thereon detached from the gold electrodes. The BBBs are fabricated with microfabrication processes (optical lithography, e-beam evaporation, and lift-off) and their surfaces are modified by two surface-treatments of polyethylene glycol treatment on Pyrex glass and RGD-terminated thiol treatment on gold electrodes. The first is intended to achieve a cell-resistive surface which suppresses cell spreading on them, whereas the second is planned to tether a RGD peptide into gold via thiol linker, thereby providing a cell-adhesive surface.
In the experimental studies with a mouse embryonic fibroblast cell line (NIH 3T3), cellular and subcellular detachments have been made with the fabricated BBBs both to quantitatively characterize cellular and subcellular detachment dynamics and to investigate the addressability, multi-functionality, and reusability of the BBBs, thereby evaluating the BBBs as biological platforms for programmable manipulation of adherent cell motility. In addition, the changes in viscoelastic properties of the detached and retracting thin layer of a NIH 3T3 cell have been investigated.