利用飛秒雷射加工技術再塗覆斥細胞聚合層玻璃基材表面上動態控制神經突觸的生長

Abstract

仿生裝置的研發已成為細胞行為研究上不可或缺的技術之一，我們的研究目的是對於在細胞培養基板上建構一個活體細胞網絡所提供一種新的研究方法。由於細胞相關的仿生裝置在控制活體細胞網路是非常重要的。我們展示使用飛秒雷射引導神經突觸生長至任意方向的方法，此項新穎的方法是藉由飛秒雷射控制基材表面對於神經元與神經突觸的親和性。這個基材表面塗覆上具有斥細胞性質的聚合層。飛秒雷射燒蝕後，利用poly-L-Lys聚合層將原本斥細胞表面轉變為親細胞性。相似的結果顯示，被飛秒雷射燒蝕後塗覆poly-L-Lys的表面轉變成較為斥細胞的玻璃表面。基於這些結果，我們建立了一項動態控制神經突觸的實驗。我們在斥細胞表面上劃出兩條塗覆poly-L-Lys平行的親細胞性通道。PC12黏附在此通道上後並將其神經元往此線形兩邊伸長。神經元的生長形式有兩種：原來的與新劃上的通道。結果顯示神經突觸往原本通道與新劃上通道的機率是近乎一樣的。

當通道再度被飛秒雷射燒蝕過後，多數的細胞被引導至沒有燒蝕過的通道。從結果來看，我們成功利用飛秒雷射改變基材表面對細胞的親和性來控制神經突觸生長至任意的方向。對於未來神經網路的研究是一個重要且不可或缺的突破。此類的裝置對於研究神經相關的功能非常重要，如信號傳遞與記憶。這個神經相關的仿生裝置被預期可應用在毒物學與藥物學檢測。

Construction of biomimetic devices have become one of the most important technique for studying of cellular behaviour. We are aiming at providing a new method to construct a neuron network on a culture substrate toward cell-based devices. Here we show a novel method to guide neurite growth to arbitrary direction by using femtosecond laser ablation of surface polymer for controlling the affinity of the surface to neurons and neurites. The substrate surface was layered with cytophobic poly-(2-methacryloyloxyethylphosphorylcholine). The femtosecond laser ablates the polymer to fabricate cytophilic lines covered with poly-L-lysine (poly-L-Lys) in the cytophobic surface. Similarly, the poly-L-Lys surface was partly removed and converted to relatively cytophobic glass surface by femtosecond laser ablation. Based on these results, we conducted the experiment for neurite guidance. Cytophilic channels covered with poly-L-Lys was fabricated in the MPC polymer surface. The neurites elongated along the initial channel and later branched channel. The probability of the elongation along the original channel and the branched channel was almost same.

When the original channel was ablated by femtosecond laser, most neurites were guided to the non-ablated branched channel. Consequently, we succeeded to control the neurite growth to arbitrary channels. This result is important to constrict neuron circuit on a substrate toward the development of neuron network devices.