以明膠覆膜-幾丁聚醣球作為未分化的間質幹細胞在生物反應器內增生的細胞載體

Abstract

間質幹細胞為多功能性幹細胞(Multipotent stem cell)，具有多能分化及免疫調節的特性，常用來作為組織工程或探討免疫途徑、機制的主角。由於間質幹細胞在研究的需求量愈來愈大，與一般平面培養所能增生的細胞數差異甚大，故科學家期望能發展出大量增生的系統來作細胞培養。 本研究利用pH值敏感型水膠幾丁聚醣球作為間質幹細胞在生物反應器內增生的細胞載體，間質幹細胞取自於四周大成鼠的四肢。幾丁聚醣具有良好的生物相容性，因其對pH值敏感的特性，已被廣泛地應用於藥物釋放及組織工程上。然而，幾丁聚醣為細胞不易貼附的材料，故利用明膠在幾丁聚醣球上覆膜，加強細胞貼附性。除了細胞載體外，本研究將細胞置入過程功能封閉式生物反應器系統(Functionally-closed process bioreactor system)作培養。此生物反應器以灌流(perfused system)的方式使細胞生長於液體流動的環境中，帶走細胞新陳代謝的廢物並帶進充足的養分，而不間斷地供應細胞培養液更減少更換培養液所造成汙染的機會。 本研究結果顯示，利用明膠覆膜-幾丁聚醣球作為間質幹細胞增生的細胞載體，其直徑大小約為 1.5 mm。由FITC接枝明膠，並以共軛焦顯微鏡觀察及BCA蛋白質的測定得知明膠均勻地覆膜於幾丁聚醣球。以WST-1作增生分析，培養於生物反應器中的細胞有顯著的增長，其速率略大於在 96 well中培養，以Live/Dead staining及SEM觀察亦可得到相同的結論。另外，以SEM觀察可發現細胞在幾丁聚醣球上的生長型態與在平面培養下的形態(morphology)不同，在平面下培養，細胞呈紡錘狀，大小約為 100 μm，而在幾丁聚醣球上培養，細胞亦會有觸角的展開，但細胞體較為圓滾，大小約為 20 μm。培養至第三天後，將細胞收集下來以流式細胞儀作細胞標記的分析，並針對特定的細胞標記以共軛焦顯微鏡觀察。與在平面上培養的細胞標記相比較，除了CD45不表現外，CD29，CD44，CD54，CD90，CD106均有表現，顯示間質幹細胞並未在此增生的系統中分化，並維持間質幹細胞的型態(phenotype)，證明以這套系統增生大量未分化的間質幹細胞的可行性。

Mesenchymal stem cell is a multipotent stem cell. It has the ability to differentiate to diverse tissues and immunomodulatory properties. It has been widely investigated in tissue engineering and immune pathways and mechanism recently. Duo to its growing demand, it is expected to develop a system to proliferate mesenchymal stem cell. In this study, chitosan was used as cell carrier of mesenchymal stem cell proliferating in bioreactor. Mesenchymal stem cell was isolated from four limbs of four-week rat. Chitosan has good biocompatibility and it is been widely investigated for drug delivery and tissue engineering due to its pH-sensitive characteristics. However, chitosan is a hydrophobic material and that makes it difficult for cells to attach on. Therefore, gelatin-coated chitosan beads was developed in this study in order to enhance cell adhesion. Furthermore, functionally-closed process bioreactor system was set up for the cell proliferation. It contains a disposable tube, a pump, and fresh medium. This is a perfused system which provides a circulating environment for cells to proliferate. Adequate nutrients and oxygen can be taken into the system by pump and the wastes that cell produced can also be taken out. The system continuously provides fresh medium without manual operation and that can reduce the possibility of contamination. Gelatin-coated chitosan beads was investigated in this study. Its diameter was about 1.5 mm. The amount of gelatin coated on chitosan beads was evaluated by confocal and BCA assay and the results showed that gelatin was uniformly coated on chitosan beads. The results of proliferation rate showed that mesenchymal stem cell cultured in bioreactor had significant growth, and was slightly faster than cultured in 96 well. The results from Live/Dead staining and SEM observation were also corresponded to the proliferation rate. From the SEM observation, it was discovered that the cell morphology was different between cultured in bioreactor and in dish. Mesenchymal stem cell cultured in dish is 100 μm spindle-like cell, while it is 20 μm round cell with filopodia in bioreactor. After 3-day culture in bioreactor, cells were evaluated by flow cytometry to determine whether its phenotype changed or not, and were also observed by confocal microscopy. Cell was negative in CD45 and positive in CD29, CD44, CD54, CD90, CD106 both cultured in dish and in bioreactor. It represented that the phenotype was remain after mesenchymal stem cell was cultured in this system. The results showed that it is a possible way to proliferate mesenchymal stem cell in functionally-closed process bioreactor system with gelatin-coated chitosan beads as cell carrier.