The Non-Invasive Cell Sheets Harvest via Thermo-Responsive Surfaces for Corneal Endothelial Cells Transplantation

Abstract

Abstract: : Purpose: To investigate the feasibility for harvesting corneal endothelial cell sheets via temperature modulation of thermo-responsive polymer-grafted surfaces. Methods: Poly-N-isopropylacrylamide (PNIPAAm) can exhibit compact and extended chain conformation to yield hydrophobic and hydrophilic property as the external temperature was above or below its lower critical solution temperature occurred around 32°C in aqueous media. The surface modification of polyethylene (PE) with PNIPAAm was conducted by plasma-activated treatment and UV-light irradiation graft polymerization. Then, the PE-g-PNIPAAm surfaces were obtained. To determine the optimal reaction conditions, surface characterization were performed as follows: the generated peroxides were detected by 1, 1-diphenyl-2-picryhydrzyl (DPPH) method; static water contact angles were measured by sessile drop method; functional groups were learned by attenuated total reflection-Fourier transform infrared (ATR-FTIR). To evaluate cell sheets harvest, the corneal endothelial cells from 3 kg New Zealand adult albino rabbits were cultured on PE-g-PNIPAAm surfaces at 37°C under a humidified atmosphere of 5% CO₂ in air. The ungrafted PE surfaces served as a control. After reaching confluence, the cell sheets detachment via lowering the temperature below 32°C was observed by phase-contrast microscopy. The effects of temperature dependency on cell sheets detachment were discussed. Results: 6*10^-8 mole/cm² of peroxides amount was disclosed at 160 W, 90 s, 200 mtorr of Ar-plasma treatment. 10% (w/w) of aqueous NIPAAm monomer solution was grafted onto PE surface at 100 W, 20 mins UV-light irradiation. The water contact angles of PE-g-PNIPAAm surfaces measured at 37 and 15°C were 65 and 40 respectively while Control surface were 90° and 85°. The PE-g-PNIPAAm surface had an amide absorption band at 1650 cm^-1. The detachment of cell sheets was founded at 15°C incubation for 40 mins. Conclusions: The technology for corneal endothelial cell sheets harvest via termperature modulation of thermo-responsive surfaces was developed. Successful application on corneal endothelial cell transplantation was promising.

Keywords: cornea: endothelium • regeneration • transplantation