以相對親疏水性特性應用於功能性再生醫學研究

Abstract

本研究中利用疏水性材質之特性，以及自動化雷射雕刻技術，並結合兩者之優點，發展自動化且較為簡易之薄膜式細胞陣列製作技術，其中此技術可成功應用製作細胞微陣列，該細胞微陣列隨之被應用於探討細胞與細胞間之交互作用，以及細胞對藥物作用後之生化反應。再者，此疏水性細胞微陣列提供一個適當之細胞培養環境，足以促使毛囊乳頭細胞在一般培養環境下，成功自我聚集形成具功能性之微組織，使其具有在體內環境可成功誘導毛囊之新生，此方法對於大量生產功能性毛囊微組織有很大之效用，對於毛髮再生之研究有很大的幫助。 實驗中首先利用疏水性材質發展一個高效率之細胞轉殖方法，由於疏水性材質對細胞之親和性較弱，間接的使得細胞對實驗中所採用之骨架材質之親和性提升，因此可以達到提高細胞轉殖效率的功效，由於明膠與幾丁聚醣在組織工程上的應用廣泛且兩者皆具有透明之性質，實驗中採用明膠微球體以及幾丁聚醣薄膜分別為立體與平面之模型，在搭配以疏水性材質聚二甲基硅氧烷(PDMS)預先處理之細胞培養環境，成功證實此利用相對親疏水特性的培養方式，的確有助於提升細胞轉殖至組織工程培養骨架基材的效率。 傳統上製作細胞陣列時，通常需要一系列之步驟，其中包含化學性修飾材料表面或是物理性的處理，其中源起於微機電製程之軟蝕刻技術最為被廣泛使用，但是此方法的前置製備過程中，需要較為嚴苛之環境及設備要求，而在本實驗中，利用疏水性材質之特性結合自動化雷射雕刻技術，將製作細胞微陣列母模的製程，成功簡化成單一步驟之雷射雕刻流程，實驗中，將疏水性材質預先製作成搭配實驗所需之厚度後，再將此薄膜送入自動雕刻之雷射平台中，成功在短時間製作出大量的細胞微陣列母模。此細胞微陣列母模隨後在實驗中被應用於探討老鼠腎上腺髓質嗜鉻細胞瘤(PC12 cell)在神經生長激素影響下，轉分化成神經性細胞並生長出似軸突結構之研究，另外，也成功應用於探討藥物對於細胞之移動的相關影響。 最後，此自動化生產之細胞微陣列母模被應用於探討製備具陣列形式之功能性毛囊微組織培養之研究，利用疏水性材質與形成之微凹槽的影響，成功的在市售的聚苯乙烯細胞培養材質上，培養出自我聚集之具毛髮誘導功能之毛囊乳頭細胞微組織，實驗中我們發現，提供適當的孔徑大小以及細胞培養密度，可以有效的形成毛囊乳頭細胞微組織，且此微組織在體內實驗中具有成功誘導毛囊發生之潛力。

Relative hydrophobic culture strategy and robotic laser micromachining were used in this study to develop a simple and automatic process of cell array. This method has been applied in the research of cell-cell interaction and the response of cell to drug treatment. Furthermore, the relative hydrophobic culture strategy and the microwell cell patterning provided a suitable environment for cultivating self-assembled spheroidal dermal papilla microtissues in the commercial polystyrene culture plate. The arrayed functional microtissues of dermal papilla can be applied to the reconstruction of hair for patients with alopecia. In the first experiment, we designed a strategy to improve the efficiency of cell adhesion to the scaffold using a hydrophobic cell culture environment Cells show lower affinity to the surface of PDMS than tissue culture polystyrene (TCPS) plate. When cells were cultured with gelatin microspheres or chitosan films in PDMS-coated plate instead of a normal TCPS plate, there was a significant increase in cell attachment efficiency. The results demonstrate that the method is easy to use and facilitates fast cultivation of cell-scaffold constructs. In the second experiment, we developed a method for fast cell array fabrication using laser sintering and the hydrophobicity of PDMS films (Patterned PDMS-based cell array system, PCAS). This approach can be easily adopted and is cost-effective. We used NIH/3T3 fibroblast cells to demonstrate the feasibility of PCAS. We also used PC-12 cells with poor adhesion ability to demonstrate cell-cell communication. Results showed that the method is very useful for studying cell-cell interaction, cell-substrate interaction or cell migration. Finally, we employed the PCAS to develop a strategy for cultivating dermal papilla (DP) cells to form multiple arrayed spheroidal microtissues for transplantation. By controlling the cell seeding densities, a microwell with arrayed DP spheroidal microtissues was easily formed. Formation of DP microtissues was associated with the overlapping of multilayered cells on microwells and low cell-substrate adhesivity on the PDMS film. A microwell environment enhanced the aggregation of DP cells into spheroidal microtissues on the TCPS culture plate. The aggregation of DP microtissues formed on microwells preserves their hair induction potential for use in follicular cell implantation and is independent of the number of cells in the TCPS plate. Large quantity of DP spheroidal microtissues can be obtained fast and simply by the platform.