利用噴墨技術將生物可分解之材料製成圖形來控制細胞培養

Abstract

在本研究過程中，為了達到兼具控制性及選擇性的細胞培養，我們提出了一個簡單且可靠的方法，即為使用噴墨技術製成所需之圖樣，再利用細胞本身所喜好生長環境因素的不同，以準確地控制細胞附著於生物可分解(biodegradable)材料上方。噴墨技術不僅可使材料成本降低，亦可以簡單地製作各式各樣不同的微圖形。 首先，藉由海藻酸鈉和氯化鈣產生交聯，並使用鑄造/溶劑蒸發法(casting / solvent evaporation method)製作出乾的方形基底薄膜，再利用控制液滴(drop-on-demand)的方法，在噴塗速度以及基板加熱溫度分別設定為20mm/s及30℃的情況下，可以在海藻酸鈣薄膜上噴塗各種不同的PLA圖案，例如線、圓形和方形等。本實驗中藉由噴塗不同的圖案提供細胞貼附，以證實可以控制3T3-L1細胞貼附在特定的位置。在實驗中細胞生長的概況分析，如細胞在基材上的貼附、貼附的數量以及細胞貼附的型態等，皆使用螢光顯微鏡與光學顯微鏡觀察。 在先前相關的研究中已經發現3T3細胞喜歡貼附在較硬的基材，故本實驗藉由噴墨技術噴塗一層到五層的PLA圖案，製作出因層數不同而具有硬度差異的PLA圖案，以探討硬度差異對3T3-L1細胞貼附的影響。硬度的計算是先利用AFM分析得到力與位移的曲線圖，再藉由Sneddon model做計算。當PLA層數越多，所得到的楊氏係數就越來越大，細胞貼附的量也就越多。最後，由實驗結果顯示，在細胞培養實驗中，因噴塗五層的PLA其硬度遠大於海藻酸鈣薄膜，可吸引較多的細胞貼附，所以本實驗可以利用基材硬度的不同，控制3T3-L1細胞貼附在特定的位置。

In this research, we present an efficient and reliable approach to accurately control cell attachment positions on biodegradable substrates by inkjets printing technology. Sodium alginate cross-linked with calcium chloride is dried by casting /solvent evaporation method to prepare smooth square films. A drop-on-demand method is used to print various patterns of biodegradable material (PLA), such as circles, squares, and lines, which is a specific marker for 3T3-L1 cells. Cell growth profile, such as cell attachment, density and morphology, on various patterns are examined and studied by confocal fluorescence microscopy and optical microscopy. Smooth PLA pattern on calcium alginate film can be obtained when velocity of moving stage was set to 20 mm/s and the substrate temperature was kept at 30℃. Previous studies showed that 3T3 cells prefer to attach to harder substrates. By this principle, to investigate effects of rigidity variation on cell attachment, multiple layers of PLA were printed on alginate to generate composite substrates with different PLA layer. The Young’s moduli of printed PLA layers on alginate are determined with AFM. Through AFM indentation, force–distance curves can be used to calculate Young’s moduli of composite films from Sneddon model. In the result, significantly more cells were attached to PLA with more printed PLA layers, or composite films with higher Young’s modulus. This rigidity-guided attachment approach can be used to create cell culture on various patterns.