Papers by Author: Teruo Okano

Abstract: Rapid recovery of cell sheets is considered important to maintain the biological function and viability of recovered cell sheets. To accelerate required culture substrate hydrophilic/ hydrophobic structural changes in response to culture temperature alteration, poly(2-hydroxyethyl methacrylate) (PHEMA) and poly(N-isopropylacrylamide) (PIPAAm) were grafted successively onto tissue culture polystyrene (TCPS) dishes by electron beam irradiation. Analysis by attenuated total reflection-Fourier transform infrared revealed that PHEMA and PIPAAm were successfully grafted to surfaces of TCPS dish. PIPAAm-PHEMA-grafted TCPS (PIPAAm-PHEMA-TCPS) dishes were compared with PIPAAm-grafted TCPS dishes for cell sheet detachment experiments. Approximately 75 min was required to completely detach cell sheets from PIPAAm-TCPS dish, compared to only 13 min to detach cell sheets from PIPAAm-PHEMA-TCPS dish, which is successively grafted with PHEMA and PIPAAm. PHEMA is a well-known as a high hygroscopic polymer. In the case of PIPAAm-PHEMA-TCPS dish, PHEMA layer acted as a water pool to accelerate the hydration of PIPAAm layer due to the effective and simultaneous water supply to PIPAAm layer, resulting in rapid hydration of grafted PIPAAm molecules and detachment of cell sheet compare to PIPAAm-TCPS dish without PHEMA layer.

Abstract: In this work, we developed a novel patterned co-culture method with thermo-responsive poly(N-isopropylacrylamide) (PIPAAm) and poly(N-ρ-vinylbenzyl-Ο-β-D-galactopyranosyl-(1→ 4)-D-gluconamide) (PVLA) inducing active hepatocyte attachment. Patterned graft of PIPAAm onto PS dishes was carried out by electron beam irradiation using cover-glass as a photomask. PVLA was only coated onto PIPAAm-ungrafted domain because of hydrated hydrophilic property of PIPAAm at below the LCST. Analysis by attenuated total reflection-Fourier transform infrared and electron spectroscopy for chemical analysis revealed that PIPAAm and PVLA were successfully grafted and coated on surfaces of PS dishes. PIPAAm-grafted surface exhibited decreasing contact angle by changing temperature from 37 to 20°C, while PVLA-coated PS and non-treated PS had negligible contact angle changes with temperature alternation. Atomic force microscopy (AFM) results showed that PIPAAm-grafted and PVLA-coated PS had smoother surfaces than that of ungrafted PS dishes. After culture for 12 hours, hepatocytes were well attached on PVLA-coated domain. Hepatocytes adherent on PIPAAm-grafted domain were detached by decreasing temperature. And then, fibroblasts were seeded onto PIPAAm pattern-grafted domain. Fibroblasts were only attached and spread onto PIPAAm-grafted domain. Co-cultured hepatocytes showed better differentiated function of albumin expression compared to homotypic hepatocyte culture

Abstract: In order to realize true regenerative medicine, we have developed a novel technology for the reconstruction of tissues and organs by utilizing intelligent materials including temperature-responsive polymers. We developed temperature-responsive culture surfaces, on which temperature-responsive polymers are covalently immobilized. Cells are cultured on the surfaces at 37°C, and harvested as transplantable cell sheets by reducing temperature to 20°C. With these cell sheets we regenerate various kinds of tissues such as cornea and heart.

Abstract: In order to avoid several complications resulted from biodegradable scaffolds or single cell injection, we have developed “cell sheet engineering”. Our concept is novel tissue reconstruction not from single cells but from cell sheets. In order to prepare cell sheets, temperature-responsive culture dishes are utilized. Since temperature-responsive polymers are covalently grafted on the dishes, various types of cells adhere and proliferate on them at 37°C, but are spontaneously detached only by reducing temperature below 32°C without any need for proteolytic enzyme. All the confluent cells are noninvasively harvested as a single contiguous cell sheets with intact cell-cell junctions and deposited extracellular matrix. We have utilized these harvested cell sheets for various tissue reconstructions including ocular surfaces, periodontal ligament, cardiac patches as well as bladder.