Engineering thick cell sheets by electrochemical desorption of oligopeptides on membrane substrates

Abstract

We developed a gold-coated membrane substrate modified with an oligopeptide layer that can be used to grow and subsequently detach a thick cell sheet through an electrochemical reaction. The oligopeptide CCRRGDWLC was designed to contain a cell adhesive domain (RGD) in the center and cysteine residues at both terminals. Cysteine contains a thiol group that forms a gold–thiolate bond on a gold surface. Cells attached to gold-coated membrane substrates via the oligopeptide layer were readily and noninvasively detached by applying a negative electrical potential to cleave the gold–thiolate bond. Because of the effective oxygen supply, fibroblasts vigorously grew on the membrane substrate and the thickness of the cell sheets was ∼60 μm at 14 days of culture, which was 2.9-fold greater than that of cells grown on a conventional culture dish. The cell sheets were detached after 7 min of electrical potential application. Using this approach, five layers of cell sheets were stacked sequentially with thicknesses reaching >200 μm. This approach was also beneficial for rapidly and readily transplanting cell sheets. Grafted cell sheets secreted collagen and remained at the transplanted site for at least 2 months after transplantation. This simple electrochemical cell sheet engineering technology is a promising tool for tissue engineering and regenerative medicine applications.

Wavelength‐Dependent Stiffening of Hydrogel Matrices via Redshifted [2+2] Photocycloadditions

“…Thus, our photoresponsive gel design can potentially be employed in “cell sheet” engineering for tissue regeneration. Critically, we can induce the detachment by using a very mild trigger, which is visible light, compared to thermal49 or electrical50 induction.…”

Expansion of bone marrow-derived human mesenchymal stem/stromal cells (hMSCs) using a two-phase liquid/liquid system

“…enzymes, chelators), magnetic or even stimuli‐responsive (e.g. thermo‐responsive, pH‐responsive, electro‐responsive, photo‐responsive).…”

“…However, despite having some advantages over enzymatic treatments, these methods can be complicated, not necessarily scalable and potentially not of economic value. For example, electrochemically‐induced detachment can achieve cell detachment within minutes; however, it requires the elaborate fabrication of sensor features and often costly specialised coatings . Another example of efficient cell detachment was from photo‐sensitive substrates resulting in >90% viable cells detached .…”

“…For example, electrochemically-induced detachment can achieve cell detachment within minutes; however, it requires the elaborate fabrication of sensor features and often costly specialised coatings. 16,21 Another example of efficient cell detachment was from photo-sensitive substrates resulting in >90% viable cells detached. 21,22 Despite their potential, this method can induce an irreversible modification of the substrate that impedes recycling of the material which can be expensive as it typically requires the synthesis of complicated substrates.…”

Optically transparent electrodes to study living cells: a mini review

“…In connection with the development of technologies in tissue engineering, there is an increase in interest in research in this area, as evidenced by studies on the electrochemical adhesion and desorption of various types of cells (Jiang, Ferrigno, Mrksich & Whitesides, 2003, Inaba, Khademhosseini, Suzuki & Fukuda, 2009, Sun, Jiang & Jiang, 2011, Enomoto et al, 2016, Kobayashi et al, 2019.…”