受損神經在修復過程中容易產生神經瘤 (neuroma) 與神經疤 (neural scar)阻礙神經傳導。近期研究中，幹細胞已被證實對於神經修復具有潛在治療效果。本實驗室先前研究發現，將脂肪中分離出之脂肪幹細胞 (adipose derived stem cells, ASCs) 種植在甲殼素塗佈之表面，其表面塗層能促使細胞形成細胞球並分化為神經譜系細胞 (neural linage cells, NLCs)。但對於如何控制形成單一尺寸大小的細胞球，仍具有其挑戰性。因此，本篇論文的研究目的是製造出能夠形成單一尺寸細胞球的細胞晶片，並評估在各種控制條件下形成的神經球特性。我們使用3D Printing技術與微影加工技術 (BioMEMS) 製作具有不同幾何形狀與大小的母模，並以聚二甲基矽氧烷 (PDMS) 將其轉印下來形成細胞晶片，然後以甲殼素塗佈其表面並將 ASCs 種植於晶片中形成神經球。為確認 ASCs 分化成 NLCs 的最佳條件環境，本實驗以光學顯微鏡觀察於不同幾何形狀、深度和直徑所形成的細胞球大小，並以聚合酶連鎖反應 (Polymerase chain reaction) 及螢光免疫染色標定神經特定標誌，包括 Nestin、NeuN、GFAP，來觀察細胞球表現神經特定基因和蛋白的程度。由相位差影像結果可得知，培養於由 BioMEMS 構成之直徑0.5µm 高度0.4µm模板上之細胞球，可為細胞球培養微環境之最佳參數條件並形成單一細胞球於孔洞中。在基因及蛋白表現上，在細胞為數量3.6×105顆，其Nestin、NeuN、GFAP 表現量高於其他細胞密度組別。因此，我們成功地優化神經球培養條件並依此研發能夠有效形成單一大小且均一性高之神經球的細胞晶片。期望未來此晶片能應用於神經修復，以解決目前臨床所遇問題。
Neuroma and neuron scar usually follow nerve injury and hinder nerve regeneration. Recently, stem cell therapy has been proved to be a potential treatment for nerve repair. In our lab, we are able to isolate adipose derived stem cells (ASC) and seed the cells on chitosan-coated surfaces to form spheres which causing the cells undergo transdifferentiation into neural linage cells (NLCs). However, it remains challenging to form spheres with single size which then makes it impossible to observe the optimized condition to transdifferentiate ASCs. The study aims to fabricate biochips that are able to form single size spheres and then evaluate the characteristic of neural spheres formed in various condition. We shaped polydimethylsiloxane (PDMS) biochips with different geometries, heights and diameters by 3D printer and lithography processing technology and coated with chitosan. ASCs were seeded in the wells to form neurosphere. To aware the optimized condition to transdifferentiate ASCs into NLCs, light microscope was used to observe the regulation of well geometry, depth and diameter in sphere size. And attempts to change the seeding density to adjust the sphere size. In addition, RT-PCR and immunofluorescent staining were applied to evaluate expression of neural marker for spheres in different condition. Images took by light microscope indicated the possibility of forming spheres with uniform size, and the sphere size could be regulated by the microenvironmental condition and cell seeding density. In the expression of neural markers, seeding cell number 3.6×105, BioMEMS D=0.5, H=0.4 group has higher Nestin, NeuN, and GFAP gene and protein expression level as compared to other groups. In summary, we successfully optimized culture conditions for forming neurospheres and developed a cell biochip which can form single sphere with uniform size in a well. We expect this cell biochip can be used in promoting nerve regeneration.

Neuroma and neuron scar usually follow nerve injury and hinder nerve regeneration. Recently, stem cell therapy has been proved to be a potential treatment for nerve repair. In our lab, we are able to isolate adipose derived stem cells (ASC) and seed the cells on chitosan-coated surfaces to form spheres which causing the cells undergo transdifferentiation into neural linage cells (NLCs). However, it remains challenging to form spheres with single size which then makes it impossible to observe the optimized condition to transdifferentiate ASCs. The study aims to fabricate biochips that are able to form single size spheres and then evaluate the characteristic of neural spheres formed in various condition. We shaped polydimethylsiloxane (PDMS) biochips with different geometries, heights and diameters by 3D printer and lithography processing technology and coated with chitosan. ASCs were seeded in the wells to form neurosphere. To aware the optimized condition to transdifferentiate ASCs into NLCs, light microscope was used to observe the regulation of well geometry, depth and diameter in sphere size. And attempts to change the seeding density to adjust the sphere size. In addition, RT-PCR and immunofluorescent staining were applied to evaluate expression of neural marker for spheres in different condition. Images took by light microscope indicated the possibility of forming spheres with uniform size, and the sphere size could be regulated by the microenvironmental condition and cell seeding density. In the expression of neural markers, seeding cell number 3.6×105, BioMEMS D=0.5, H=0.4 group has higher Nestin, NeuN, and GFAP gene and protein expression level as compared to other groups. In summary, we successfully optimized culture conditions for forming neurospheres and developed a cell biochip which can form single sphere with uniform size in a well. We expect this cell biochip can be used in promoting nerve regeneration.

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