Yokohama National University, Faculty of Engineering
(Bio Microsystem lab)
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Surface research

 

Fabrication of Thick Cell Sheet via Electrochemical Reactions on Membrane Culture Substrate
Objective

　We developed a method for noninvasively harvesting cells, cell sheets, and spheroids from a culture surface along with the electrochemical desorption of a self-assembled monolayer (SAM). Cells were attached on a gold culture surface on which Arg-Gly-Asp (RGD) containing peptides were covalently bonded via the SAM of an alkanethiol. The application of ?1.0 V (vs Ag/AgCl) caused the reductive desorption of the SAM, resulting in the detachment of the cells. By this method, more than 90% of the cells were detached within 5 min. Two-dimensional (2D) cell sheets could also be detached from the gold surface in the same manner. The detached cell sheets consisted of viable cells that could easily attach to other cell sheets in succession and form a multilayered cell sheet. Moreover, by combining this approach with photolithography and micro-contact printing (μCP) technologies, we fabricated a chip system in which hepatocytes formed spheroids of a uniform diameter at the density of 280/cm2 and could be harvested by the application of a negative potential. This cell manipulation technology could potentially be a useful tool for the fabrication and assembly of building blocks such as cell sheets and spheroids for regenerative medicine and tissue engineering applications.
Results
　A commercially available membrane substrate (cell culture insert, pore size, 400 nm; pore density, 100 × 106 pores/cm2, BD Falcon) was coated with a few nm layer of chromium and a ~40 nm layer of gold. In this system, an improved supply of oxygen and nutrient can be expected.
　The oligopeptide, CCRRGDWLC, was designed to contain an arginine-glycine-aspartate (RGD) domain in the center and cysteine at both ends. Because cysteine contains a thiol group, the oligopeptide was chemically adsorbed onto a gold layer on a membrane substrate via the formation of a gold-thiolate bond. Fibroblasts seeded on the membrane were grown to form a thick cell layer. Then, by applying a negative potential, the gold-thiolate bonds were reductively cleaved, and the cell sheet was detached along with the desorption of the peptide (Figure 1, 2).
　Because of the oxygen supply through the membrane, cells grew and formed a 50-µm-thick cell sheet after 14 days of culture; this sheet is significantly thicker than the sheets formed in a conventional culture dish. The proposed approach was further employed to stack detached cell sheets to obtain approximately 200 µm thick multilayered sheets (Figure 3).


Fig. 1 Cell detachment via Electrochemical reactions


Fig.2 Image of detached cell sheet


Fig.3 Multilayered cell sheet (5 layers)


[Reference]
N. Mochizuki, T. Kakegawa, T. Osaki, N. Sadr, NN. Kachouie, H. Suzuki, J. Fukuda*, Tissue Engineering Based on Electrochemical Desorption of an RGD-Containing Oligopeptide, Journal of Tissue Engineering and Regenerative Medicine (IF=3.53), (2012)
J. Enomoto, N. Mochizuki, K. Ebisawa, T. Osaki, T. Kageyama, D. Myasnikova, T. Nittami, J. Fukuda, Engineering thick cell sheets by electrochemical desorption of oligopeptides on membrane substrates, Regenerative Therapy, 3, pp24-21 (2016)

 

 

 
● Vascular
● Liver
● Hair
● Pacnreas
● Bone
● Lab Chip/ MEMS
● Surface modification
● Microbe
 
 
 
 

 
Fukuda Lab, Faculty of Engineering, Yokohama National University