Electrochemical Manipulation of Living Cells Supported By Polymeric Nanosheets
Authors: Suzuki Jin , Kondo Taro , Nagai Nobuhiro , Nishizawa Matsuhiko , Abe Toshiaki , Kaji Hirokazu 
Published: February 2020

Introduction
Regenerative medicine based on cell sheets has been widely investigated. Developing an effective and minimally invasive cell transplantation process is an important enabling step for such medical treatments. However, cell sheets are fragile and handling them is difficult. Recently, we have investigated the use of biodegradable polymeric nanosheets for introducing cells into the body and found that the flexible nanosheets on which cells are cultured can be aspirated and injected by a syringe needle without significant loss of cell viability. To harvest cell/nanosheet constructs from a substrate, a water soluble sacrificial layer such as polyvinyl alcohol (PVA) was used, however, a drawback in this method is that the timing to detach the cell/nanosheet constructs from the substrate cannot be controlled. In this paper, we use an electrochemical method for harvesting the cell/nanosheet constructs in vitro. Cells are cultured on nanosheets attached on a self-assembled monolayer (SAM) of alkanethiol. The SAM is reductively desorbed from the gold substrate by the application of a negative electrical potential, which detaches the cell/nanosheet constructs from the gold surface in a rapid manner. The harvested cell/nanosheet constructs were transplanted into the subretinal space of rat eyes through syringe needles.
Material and Method A gold layer was patterned by sputter-depositing gold with a patterned silicon mask. The gold surface was modified with 10-carboxy-1-decanethiol, 7-carboxy-1-heptanethiol or L-cysteine by immersing the substrates in any of the three thiol solutions for 30 min at room temperature. The desorption of the SAMs was performed and analyzed electrochemically. Nanosheets were first prepared on PVA-coated substrates by a combination of a spincoating and microcontact printing technique using poly(dimethyl siloxane) (PDMS) stamps. A solution of poly(lactic-co-glycolic acid) (PLGA) was spincoated onto the PDMS stamp and the resulting PLGA layer was transferred onto a PVA-coated substrate. Freestanding nanosheets were obtained by dissolving the PVA sacrificial layer with water and attached on the SAM-modified gold surface. To desorb the nanosheets with SAMs, a voltage of 1.5 V, supplied by an AA-size dry battery, was applied between the gold electrode (cathode) and the Pt wire (anode) in phosphate buffered saline. Retinal pigment epithelium (RPE) cells were cultured on PLGA nanosheets attached on the SAMs for 2 days to form cell/nanosheet constructs. Electrochemical desorption of SAMs with cell/nanosheet constructs was conducted in culture medium. Sprague-Dawley rats (SLC) weighing 250-300 g were used in this study. After the rats were anesthetized with ketamine hydrochloride and xylazine hydrochloride, RPE cell/nanosheet constructs were injected into the subretinal space using a hand-made glass capillary needle.
Result and Discussion Cyclic voltammograms observed in the reductive desorption of SAMs showed a clear dependence of peak potential on the length of the alkyl chain. With longer chains, the peak potential shifted to the negative direction, which has been explained by van der Waals interactions between the alkyl chains. Since the SAM of L-cysteine was reductively desorbed at a lower potential (around -0.7 V vs. Ag/AgCl) than the SAM of the two other thiol molecules and L-cysteine is biocompatible, the SAM of L-cysteine was used for harvesting nanosheets and cell/nanosheet constructs. PLGA nanosheets attached on the SAM of L-cysteine were successively detached from the substrate by gently pipetting within 1 min after applying the reductive potential to the gold electrode. On the other hand, nanosheets on the SAM without applying the voltage did not detach from the substrate. In a similar manner, RPE cell/nanosheet constructs could be detached from the substrate and the live/dead staining showed the cells on the nanosheets are viable.
Finally, we used our system to deliver RPE cell/nanosheet constructs to the subretinal space of a rat eye in vivo. Nanosheets were injected with 2 μL of saline to the subretinal space through the sclera using a glass capillary needle. Optical coherence tomography (OCT) images of the retina taken one week after the injection of the nanosheets showed a shadow of the sheet like structure in the subrentinal space while there was not such shadow in the OCT image of a control. Also, a shadow of the circular nanosheet was observed at the posterior segment of the isolated eye. These results indicate that the nanosheets were successfully injected and spread into the subretinal space of rat eyes.
Conclusion
We reported a method for noninvasively harvesting cell/nanosheet constructs from a culture surface by using the electrochemical desorption of SAM. The harvested RPE/nanosheet constructs could be delivered to the subretinal space using a capillary needle. The harvesting and delivering system of cell/nanosheets constructs could be a useful tool for cell transplantation.