以培養基設計及饋料策略進行高密度昆蟲細胞培養

Abstract

動物細胞組織培養發展至今已超過40年, 期間所發展之培養方 式種類很多.然而應用於工業生產上往往由於培養基昂貴及培養細胞濃度 低, 而使其產品價格居高不下. 在此我們利用目前廣泛被用於外來蛋白質 生產之昆蟲細胞/桿狀病毒表現系統為研究對象,透過培養基的設計以饋料 式批次培養來使養份充份利用提高細胞濃度. 在本篇文章中, 我 們利用計量模式推算細胞生長時所需之各種主要養份, 包括胺基酸, 葡萄 糖及yeastolate的計量方程式. 再根據計量方程式並配合昆蟲細胞中組 成物含量的分析, 計算而得各養份消耗之計量係數, 這些係數代表了生 成單一細胞所需之各養分量. 藉由這些計量係數, 可推算整個饋料式批 次培養實驗中起始培養基及補充性 培養基的濃度. 起始培養基是以市售 之Grace's培養基為基礎, 針對胺基酸, 葡萄糖及yeastolate的含量利用 計量係數進行改變, 使其能供應細胞由最初之接種濃度 5x10(5)cells/ml 生長至 10(6)cells/ml 所需. 補充性培養基則要同時兼顧提高濃度及 使所有養分溶解, 因此其各組成分濃度決定是以添加 0.8ml補充性培養基 能使100ml培養基中細胞濃度增加 10(6)cells/ml來計算. 所得之補充性 培養基添加方式, 則是以先行預估至下一次添加期間(24小時)內,細胞以 對數生長期之生長速度, 繁殖所增加之細胞數目來決定其培養基添加量. 經過數次饋料式批次培養實驗發現除了氧氣供應外, 由計量方式所計算出 之glutamine與 glutamate因沒有考慮其用於能量及其他非必需性胺基酸 之生成而使其供應量不足. 在經由對整個系統進行表面通氧及額外添加 glutamine後便足以供應細胞生長所需,而使培養細胞濃度達到 1.9x10(7) cells/ml. 在氧氣供應方面, 由實驗中分別對培養系統進行表面 不通氣,表面通入空氣及表面通入氧氣之實驗, 發現在氧氣供應充足時細 胞幾乎不產生乳酸. 由另一實驗設計中可看出, 當整個培養系統初期表面 通入氧氣時, 培養基中乳酸含量相當低. 然而一旦氧氣供應中斷, 則培養 基中乳酸含量立刻升高, 等恢復供氧後才又降低. 由此可發現乳酸可做為 昆蟲細胞培養時培養基中溶氧量的指標. 在glutamine 含量對昆 蟲細胞培養影響的實驗中, 改變培養基中glutmine與glutamate 含量比 例可發現, 細胞之培養密度與glutamine含量成正比. 且培養基中 glutamine的含量不能以glutamate取代. 可知glutamine對於Sf-21細胞之 生長十分重要. Animal cell culture has been studied for more than 40 years. There hasbeen many in vitro cell culture technologies developed during the time, withalmost all of them aiming at lowering the cost of animal cell products. The problem persists; mainly because culture media are expensive and nutrients within are not efficinetly utilized. This leads to cultures of lower celldensities and products, incurring higher purification expenses. It is withinthis context, we propose to achieve high density cell cultures by improving the nutrient utilization efficiency via a rational medium design and feedingstrategy. In animal cell culture domain, insect cell/baculoviurs expressionsystem is becoming one of most widely used methods for the production of heterologous proteins. In this thesis, we used Spodoptera frugiperda Sf-21as the model system for studying high density insect cell cultures. A stoi-chiometric model has been established to study the demand of nutrients, including glucose, 20 amino acid, and yeastolate, for the synthesis of cell mass. The coefficients for individual untrients in the stoichiometric equation governing insect cell growth were determined from the information of cell mass and compositions. Based on the stoichiometric coefficients, theinitial and supplemental medium for fed-batch cell cultures were designed. The experiments began with Sf-21 insect cells inoculated in a spinnerflask using the initial medium, which would provide a starting environment for achieving optimum cell growth. This is subsequent by periodic feeding of supplemental medium designed by utilizing the stoichiometric equation that governs insect cell growth. With this strategy, we have demonstrated that the Sf-21 cell cultures reach cell density in excess of 1.9x10(7) cells/ml. The relationship between lactate production and oxygen in the Sf-21 cell cultures has been studies. We have found that while the lactate produc-tion in insect cell cultures was very low with sufficient oxygen supply; itwould accumulate quickly when oxygen supply was interrupted or insufficient.It is noteworthy that lactate concentration can be used as an indicator of dissolved oxygen concentration in the culture medium. Further experiments have proved that the amount of feeding glutamine was important to Sf-21 cellgrowth, effecting final cell densities. Most importantly, the feeding of glutamine can not be substituted by glutamate, in contrast to most mammaliancell cultures.