輕量化高分子微載體對細胞貼附及細胞產物生產之影響

Application of light weight PLGA microcarrier to adhesive cell suspension culture

廖慧帆(Hui-fan Liao)

指導教授 ： 王鐘毅

大同大學/工程學院/生物工程學系所/碩士(2008年)

摘要

以哺乳類動物細胞生產重組蛋白，獲得之蛋白結構與醣修飾與人體內的結構較相像，但產率遠比以微生物表現系統低，產率偏低的問題可藉由選殖較好的細胞株來改善。中國倉鼠卵巢細胞是最早用於生產重組醣蛋白藥物的細胞株，基因重組人類巨噬細胞刺激因子 (recombinant human macrophage colony-stimulating factor, M-CSF) 能刺激巨噬細胞增生與分化，是目前應用於血液疾病相當重要的蛋白質藥物。本實驗使用基因重組之中國倉鼠卵巢細胞株作為實驗用細胞株。
首先使用PLGA配合二次乳化法製備具有多孔洞之輕量化微載體，將細胞接種於含有微載體之培養基中，以旋轉角瓶配合低速攪拌器進行細胞培養，微載體之多孔洞結構可避免細胞受到攪拌所產生的剪切力影響，使細胞在懸浮的環境中增殖、分化及產生目標產物。
本實驗以乳化法所製備之實心微載體粒徑約為117.8±15.0 μm ，1 % NH4HCO3-PLGA微載體粒徑為375.8±78.5 μm，5 % NH4HCO3-PLGA粒徑為442.4±25.8μm，兩者孔洞大小均介於17~18μm。而發泡劑濃度越高，所製備之微載體粒徑越大，孔洞越多，相對密度較低。對照組為Ctyodex 3商品化微載體。商品化微載體細胞培養到達第四天即到最高密度，隨即引發大量細胞凋亡。以乳化法製備之實心微載體由於其表面平滑，細胞貼附在其表面，在spinner flask中生長的情況不佳。添加1 %發泡劑 NH4HCO3所製備之微載體所培養之細胞密度生長到達第12天才到最高，但其最終細胞數量仍可達到與商品化微載體相仿。添加5% 發泡劑之PLGA微載體細胞生長至第十六天才到達最高密度，其細胞生長速率及細胞密度均不如1% 發泡劑所製備之微載體良好。
以M-CSF ELISA kit 測定細胞所產生之M-CSF產量，1 % NH4HCO3-PLGA在細胞生長及M-CSF生產量上有較佳的表現，在100 ml spinner flask 中所測得的M-CSF含量為5.7 μg，約為12盤T-75細胞的產量。
本研究結果顯示，以1 % NH4HCO3-PLGA配合spinner flask在及時監控細胞密度和培養程序上都非常簡便，且確實可有效增加細胞密度及細胞產物的含量，具有發展為生產細胞產物系統的潛力。

關鍵字

中國倉鼠卵巢細胞 ； 細胞培養 ； 微載體 ； 巨噬細胞刺激因子

並列摘要

Glycosylation and post-translational modification of recombinant proteins are the unique feature of mammalian cell system. However, the productivity of recombinant protein by mammalian cells is much lower than that of microorganisms. Chinese hamster ovary cell line is the most popular mammalian host for the commercial production of therapeutic proteins. We used Chinese hamster ovary cells (5/9 M alpha 3-18, BCRC 60185, CHO cells) to express macrophage colony-stimulating factor (M-CSF), which can stimulate macrophage proliferaction, differenciation and survival.
Cells were inoculated into culture system containing light weight porous microspheres, which were made of biodegradable polymer, PLGA. The support of PLGA microspheres reduced cell damage resulting from the shear force.
The particle size of dense PLGA particles was 117.8±15.0 μm；1% NH4HCO3-PLGA microcarrier was about 375.8±78.5 μm；5 % NH4HCO3-PLGA particle size was 442.4±25.8μm。The pore size of 1 % NH4HCO3-PLGA and 5 % NH4HCO3-PLGA was between 17~18μm。
Cytodex 3 is a commercial cell culture microcarrier, which was cross-linked dextran coated with denatured collagen. After cell culture with Cytodex 3 for 4 days, the cell density reached maximum, and the onset of cell death was observed. The cell culture with PLGA microcarriers, made of 1% NH4HCO3 and PLGA, the cells grew till day 13. The final density of cells was similar to that cultured with Cytodex 3. The 5 % NH4HCO3-PLGA microcarriers showed the massive cell death up to day 17, but the cell growth rate and cell density were both smaller to that with 1% PLGA microcarriers. The yield of M-CSF is higher for cell culture with 1 % NH4HCO3-PLGA microcarrier. The 1% NH4HCO3-PLGA microcarrier was the best in cell density and M-CSF production.

並列關鍵字

CHO cell ； cell culture ； microcarrier ； M-CSF

參考文獻

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