Uji Akihito, Matsuo Toshihiko, Uchida Tetsuya, Shimamura Kaoru, Ohtsuki Hiroshi
Department of Ophthalmology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University, Okayama City, Japan.
Artif Organs. 2006 Sep;30(9):695-703. doi: 10.1111/j.1525-1594.2006.00286.x.
Photoelectric dyes, which absorb light and convert photon energy to electric potentials, were shown to stimulate retinal neurons in culture. One of these dyes coupled with polyethylene film surface, as a prototype of retinal prostheses, could induce intracellular calcium elevation in chick embryonic retinal tissues. In this study, we used retinal cells from chick embryos in which no photoreceptor outer segments yet developed, and assessed cell adhesiveness and response to the original and modified types of our retinal prostheses. Extruded-blown film of high density polyethylene was used as the original polyethylene film. Polyethylene film surface was modified either by coupling with type 1 collagen or by crystallization from the melt of the original polyethylene film. A photoelectric dye, 2-[2-[4-(dibutylamino)phenyl]ethenyl]-3-carboxymethylbenzothiazolium bromide, was coupled to these different types of polyethylene films through amide linkage. Retinal cells from chick embryos at the 12-day embryonic stage were cultured on the surface of dye-coupled films. The response of retinal cells to the dye-coupled films was observed by calcium imaging. Cell types, either neurons or glial cells, were detected by immunocytochemical staining and also observed by scanning electron microscopy (SEM). Intracellular calcium elevation was observed in cells, including both neurons and glial cells, on the surface of the original dye-coupled film, collagen-coated dye-coupled film, and dye-coupled recrystallized film, in contrast with no response of cells cultured on the dye-uncoupled plain film. Retinal neurons grew upon colonies of glial cells settling on the surface of all different types of the films. The number of retinal neurons on the collagen-coated dye-coupled film and dye-coupled recrystallized film was significantly larger than the number of neurons on the dye-uncoupled plain film (P = 0.0004, analysis of variance; P < 0.05, Tukey-Kramer test). With SEM, Muller cell-like large cells covered the original dye-coupled film and collagen-coated dye-coupled film, while neuron-like small cells settled directly on the dye-coupled recrystallized film and also formed large colonies on Muller-like cells. Retinal neurons and glial cells were cultured directly on the different types of dye-coupled polyethylene films. Photoelectric dye-coupled polyethylene films could be used as prototypes of retinal prostheses.
光电染料能吸收光线并将光子能量转化为电势，已证实在培养中可刺激视网膜神经元。其中一种与聚乙烯薄膜表面偶联的染料，作为视网膜假体的原型，可诱导鸡胚视网膜组织内的细胞内钙升高。在本研究中，我们使用了尚未发育出光感受器外段的鸡胚视网膜细胞，并评估了细胞黏附性以及对我们原始和改良型视网膜假体的反应。高密度聚乙烯吹塑薄膜用作原始聚乙烯薄膜。聚乙烯薄膜表面通过与I型胶原偶联或通过原始聚乙烯薄膜熔体结晶进行改性。一种光电染料，2-[2-[4-(二丁基氨基)苯基]乙烯基]-3-羧甲基苯并噻唑溴化物，通过酰胺键与这些不同类型的聚乙烯薄膜偶联。将12日龄鸡胚的视网膜细胞培养在染料偶联薄膜表面。通过钙成像观察视网膜细胞对染料偶联薄膜的反应。通过免疫细胞化学染色检测细胞类型（神经元或神经胶质细胞），并通过扫描电子显微镜（SEM）观察。在原始染料偶联薄膜、胶原包被的染料偶联薄膜和染料偶联重结晶薄膜表面的细胞（包括神经元和神经胶质细胞）中观察到细胞内钙升高，相比之下，在未偶联染料的普通薄膜上培养的细胞无反应。视网膜神经元生长在定居于所有不同类型薄膜表面的神经胶质细胞集落上。胶原包被的染料偶联薄膜和染料偶联重结晶薄膜上的视网膜神经元数量显著多于未偶联染料的普通薄膜上的神经元数量（方差分析，P = 0.0004；Tukey-Kramer检验，P < 0.05）。通过SEM观察，穆勒细胞样大细胞覆盖原始染料偶联薄膜和胶原包被的染料偶联薄膜，而神经元样小细胞直接定居在染料偶联重结晶薄膜上，并在穆勒样细胞上形成大集落。视网膜神经元和神经胶质细胞直接培养在不同类型的染料偶联聚乙烯薄膜上。光电染料偶联聚乙烯薄膜可作为视网膜假体的原型。
