三維二氧化鈦的孔徑大小對培養肝細胞之影響

Abstract

將不同孔徑大小之PU海綿，利用涵浸二氧化鈦漿料使二氧化鈦吸附於海綿上，再使用高溫燒結法製備出三維多孔之二氧化鈦，接著將大鼠(Rat)肝細胞(Hepatocyte)培養於多孔二氧化鈦試片上，並探討不同孔隙大小的二氧化鈦試片對於培養肝細胞所造成的影響。

本實驗使用X光繞射儀(X-ray diffraction, XRD)、掃描式電子顯微鏡(Scanning electron microscopy, SEM)，分析燒結後之二氧化鈦試片結晶構造以及試片表面、截面形貌。XRD結果顯示在經過1450°C/2小時燒結後，並無殘留除了二氧化鈦以外的成分。在SEM方面，觀察試片表面及截面的孔洞大小與形貌，可以發現燒結後試片的孔洞大小與吸附漿料使用之海綿孔徑成正比，孔徑越大的海綿能製備出孔徑越大的試片。而從SEM圖中也發現，大部分的孔洞都為互相交聯的，但隨著孔徑變小，因表面張力而殘留的不交聯之封閉孔洞也會逐漸變多。

在培養肝細胞的部分，使用細胞存活率分析( MTT assay )與酵素免疫分析法( ELISA )分析細胞存活率與肝細胞功能，以及使用光學顯微鏡觀察細胞型貌。從實驗結果發現，培養三天後的肝細胞，存活率最高的是培養於不含任何試片的二維培養皿中，隨著二氧化鈦試片孔徑越大，存活的細胞數量則越來越少；而在肝細胞功能分析中結果則完全相反，培養於不含試片之二維培養皿中的肝細胞功能最差，而在培養於三維二氧化鈦試片上的肝細胞，隨著孔徑越大，肝細胞的表現也越為優秀，從OM觀察的結果也可發現其細胞型貌的差異。

由本實驗可以得知，用此培養方法在肝細胞的存活率上，二氧化鈦試片表現並不佳，但是在肝細胞功能表現上，其效果是明顯優於傳統培養在二維培養皿中的細胞。

PU foam with different pore size was soaked in titanium dioxide (TiO2) slurry to adsorb TiO2 on the foam. And the foam was sintered to produce three-dimensional (3-D) porous TiO2 specimens. Then rat hepatocytes were cultured on TiO2 specimens. The influence of TiO2 specimen with different pore size on culturing hepatocytes has been studied.

X-ray diffraction (XRD) and scanning electron microscopy (SEM) have been used to analyze crystal structure and morphology of sintered TiO2 specimen. XRD result showed no second phase remained in specimen after sintering at 1450°C for two hours. SEM was used to observe the surface of specimen and pore size and morphology of cross-section. The pore size of sintered specimen depended on the pore size of PU foam and most of the pores were cross-linked. On the other hand, the number of uncross-linked closed pores in the specimen with smaller pores was increased due to the effect of surface tension. In the part of culturing hepatocytes, MTT assay, ELISA and optical microscope (OM) were used to analyze the growth, function and morphology of hepatocytes. The data indicated the highest survival of hepatocytes in the culture dish without TiO2 specimen for three days. TiO2 specimen affected the cell growth in the pore size-dependent manner. However, the function of hepatocytes assessed by its albumin production was better in the culture with TiO2 specimen, specifically in the large size pores. OM indicated the different morphology of hepatocytes in the culture with TiO2 specimen.

In summary, TiO2 specimen affected the cell growth of hepatocytes, while the function of hepatocytes were relatively excellent as compared with traditional 2-D culture dish.