200526775 玖、發明說明： 【發明所屬之技術領域】 本發明係關於一具有輔助攪拌元件之細胞培養裝置，該裝置 能降低培養基流動所造成之剪應力對所培養細胞的傷害，同時維 持高效率且均一的培養環境。 【先前技術】 大規模的細胞培養程序已經於近年來被廣泛的開發並使用， 然而，相較於培養細菌、酵母菌與黴菌等擁有強韌的細胞壁及/或 細胞外間質（extra cellular material )、較具彈性的細胞而言，培 養真核細胞、動物細胞、哺乳動物細胞及/或組織的技術較為困難 且複雜，原因在於此類細胞較微生物細胞來的脆弱，並且其生長 所需之養分與氧氣之維持也較為困難而複雜。動物細胞跟微生物 細胞不同的是，動物細胞不能忍受通入空氣或混合性氣體所造成 的劇烈的擾動及/或剪應力；並且，動物細胞不能直接接觸空氣， 多數動物細胞僅能利用溶解於培養基中的氧氣。比起微生物細 胞，動物細胞及哺乳動物細胞更容易被氣體或是於通氣過程中遭 受傷害，因此增加其死亡率。 由於真核細胞及/或原核細胞及/或動物細胞及/或哺乳動物細 胞於實驗室大量培養的需要，生物反應器及培養裝置在研究與生 產細胞以製造活性蛋白質及/或抗體及/或任何細胞副產品的方面 扮演了重要的角色。生物反應器主要之功能在於配合研究需要培 養大量細胞以萃取其衍生之微量活性物質，包含細胞分泌到培養 基中的蛋白質或抗體；生物反應器之另一功能在於培養細胞以大 規模量產細胞或組織生產之活性蛋白質。 200526775 現行應用於大規模培養之生物反應器通f具備—個内部 益，例如.授拌翼（impeller)，使細胞承受非常高的液體煎應力 而造成細胞的傷害或死亡’導致細胞的低存活率及其蛋白質及^或 副產品之產量也隨之降低。同樣的，為使細胞均勻懸浮及/或適: 接觸空氣，而利用他種機械裝置、激烈的氣體運動或強烈的液: 運動之生物反應器也將造成細胞的傷害與妨礙細胞或組織之生 長’進-步造成細胞的副產品’例如：蛋白質，產量之減少。 、錢心㈣了傳統的培養皿與培養瓶外，$有一種用來 細胞與組狀麟係為絲式培輪㈤k kies) : 泛利用於細胞培養已有數年。然而，軸賴式培養瓶具二 料皿或培養瓶所沒有之優點，例如：提供細胞較大的生長^ =面積，然而較無法克服所有的缺點尤其是關於擴大培養規模 方面。這些缺點包含：頂部空間之氣體（gasheadspaee)血大量旋 :拌:L成=大流體剪應力，·由於滾動式培養瓶的高剪應力 =，導致較大的三維結構的組織培養近乎不可能。唯有可以不 或能維持貼附於滾動式培養瓶内壁的細胞才得 持Ά養時間。由此可知，維持長時間於滾動式 =:株養=在:括其高r環 =外，雖錢動式培養瓶的整體面積大於培養皿或培養瓶， ::、、…:㈣供給每個細胞㈣的面積並&定大於培養皿或培養 瓶=提供之面積，特別是在大規模培料。故此高動 =，=?，係描述-種可增進細胞附著二=動 ° 〜、係於滾動式培養瓶的内壁加上—種_狀的通道， 200526775 增加滾動式培養瓶之内表面積以供έ μ 、、、、田胞附者。然而，一般滾動式 培養瓶僅提供約850-1700平方公分少 、 77之面積，故仍需要大量的滾動 式培養瓶才足以達到量產。而自動仆 曰 , 初化、大1的滾動式培養瓶之培 養糸統雖然可節省時間與人力，钬而* ^ t 丁 …、而其操作卻相當昂責及具限制 性。 除了流體剪應力的問題與生县％灶^ 食面積的限制之外，獲得與保持 充分的氧氣供應也是細胞與組織培1 σ胥筏術的中心問題。正如本技 術領域之人士所熟知，原核細胞、直4 具核細胞，包括動物細胞、哺 乳動物細胞、昆蟲細胞、酵母菌魚歡试^ 嫩鹵的生長均有一主要的速率 限制步驟（rate-limiting step)，即惫友 乳乳質置傳遞（0Xygen mass transfer) ° 除了某些進行發酵作用的真核微生物（如酵母菌）之外，對於多 數的原核細胞與真核細胞，氧氣的代謝對於細胞的代謝功能是相 當重要的，尤其s針對哺乳動物細胞與動物細胞之培養技術，氧 氣的供應對於細胞快速分裂的早期階段又㈣的重要。當細㈣ 浮時’對氧氣的需求量最大’而當細胞聚集或是分化後，氧需求 量就會降低。有些哺乳動物細胞與動物細胞為附著依存 (—age-dependent )，另外有些哺乳動物細胞與動物細胞則可 於液體環境中懸浮生長，即非附著依存（—。哪independent)， 然而所有的細胞均需生長於充分的溶氧環境中。在細胞培養的後 期’不論是附著依存或是非㈣依存的細胞，每單位體積的細胞 數量會大量增加，因而再度需要大量的氧f量傳遞以提供足夠氧 氣。 ，可以利用機器攪拌以及 然而，如前所述，搜拌和 基本上’至少對於附著生長的細胞 通入氣體的方式來供應其需求的氧氣， 200526775 通氣的動作均會傷害細胞，因而減低其存活率及整體細胞及/或組 織培養的的效率與產量。此外，直接通入氣體到細胞及組織培養 基中會導致氣泡產生，同樣不利於細胞存活。 為解決細胞培養時氧氣提供的問題而發明的裝置，例如：Wolf et al.所擁有的美國專利第5，153，131號，係關於一種沒有攪拌翼 (blades)的生物反應槽（bioreactor vessel)。此生物反應槽係將 空氣由輸氣通道通入，經由一支持板元件（support plate member) 穿過屏幕（screen)，並通過炎於反應槽外箱（housing)兩側間的 一扁盤狀之滲透膜，利用外箱兩側之間的氧濃度梯度，使氧氣可 以擴散通過滲透膜而到達培養室。 然而，Wolf的生物反應槽具有許多缺點，尤其是，氧氣經由 扁盤狀之滲透膜擴散的速率成為培養室規模的主要限制因素，另 外一個缺點是扁盤狀之滲透膜設計成可伸縮以具有攪拌功能，此 點卻可能造成細胞的死亡。利用攪拌功能以使空氣均勻分布於培 養基中是相當重要的，然而，攪拌的同時也將增加培養室内部的 剪應力，如前所述可能造成對細胞的傷害。因此，當設計生物反 應器或培養槽時，如何提供足夠的氣體交換以維持培養較大的細 胞結構，係相當重要並且為一實際的限制。 為了解決前述缺點的裝置，利用氣體可滲透材料所製造的反 應器例如·· Schwarzet al.所擁有的美國專利第5,702,941號，名稱 為”可透氣的生物反應器及其使用方法”，係關於一種可水平轉動 的容器，並且其容器壁係部分採用透氣材料，以期達到直接透過 此透氣材料與培養基進行氣體交換之目的。 然而，Schwarz所揭露之裝置的規模仍然受到限制，原因在於 氣體交換需依據透氣面積的大小來決定，Schwarz雖然強調當裝置 200526775 的表面積增加，培養基的體積量亦相對增加，而於其說明書中所 揭露之較佳實施例之規模限制於直徑1至6英吋，寬度則介於1/4 至1英吋間，這樣的大小並不合適生長具有三維空間的細胞集結 物與組織及/或任何大量規模生產。 同樣地，Falkenbergetal.所擁有的美國專利第5,449,617號， 標題為”用於細胞培養之培養容器”，係關於一水平旋轉之裝置， 此培養容器藉由一透析膜將内部區分為細胞培養腔室與營養液儲 存槽，透氣材料用在本容器壁使得細胞培養室可進行氣體交換， 不過營養液儲存槽也並非完全充滿營養液，而是兩個腔室的培養 液上方都維持大量的氣體。然而，Falkenberg容器並未設計用以減 少細胞培養室内部的擾動，相反的，為了保持透析膜的潮濕，攪 拌是必要的步驟；此外，Falkenberg並未提及使用此容器於培養細 胞集結物或是任何類型的組織。 為解決氧氣供應的問題之發明還有，例如：Liau所擁有的美 國專利第5,766,949號，標題為”培養附著依存之單層細胞的方法 與裝置”，係描述一種上下震盪培養基以增加氧氣提供的培養系 統0 然而Liau的發明具有很多缺點，其中之一就是其裝置的複雜 性。Liau的系統包括兩個外接的儲存槽以及一個含有一系列垂直 基材平盤（vertical substrate plates )的分離式培養室，另外還需要 數個蠕動式幫浦推動培養液由一個儲存槽流經培養室再流到另一 個儲存槽，最後再流回到第一個儲存槽。因為Liaii的裝置複雜而 且有一部份為培養室外部的設備，例如··外部管線、儲存槽與幫 浦，所以極有可能將外界的污染源導入内部，另外，因為裝置的 設備龐大，滅菌工作不易進行又耗費人力。 200526775 廍明另、r個缺點是培麵流經系_所造成的流體剪 r^^bs干擾亚且移動生長於基材平盤上的細胞，因而減低 率。另外’垂直基材平盤的設計並不利於細胞附著， 口^厂、法即刻㈣的細胞會掉落並堆積在平盤的底端，這些 細i取後大多會死亡，因此降低細胞的存活率以及蛋白質產量， 而此系統必須-再地重新開始’相當沒有效率而且不理想、。再者， 二：此:、，复雜性，收集任何分泌的蛋白質或是細胞產物都很 秫煩且耗#。最後，當生長液低於細胞生㈣基許盤時，細胞 將曰暴路於工氣中’即，直接接觸氣體環境，因此將導致細胞死 亡。細胞與組織培養技術對於生物科技研究、藥物研究、患者護 理與學術研究都相當的重要。 為了克服習知技術之缺陷與限制並且補足其不便之處，台灣 專利申請㈣921G1625號提供-種細胞培養|置，培養基與細胞 生長於其中的載體於不同腔室内進行相對運動，提供一種可用來 ，養細胞及/或組織以及收集其產物之可靠、簡易、便宜且可以用 凡即丟與有效率的方法與裝置。更具體而言，係教導與揭露一種 新穎的方法與裝置可以有效的培養欲培養之細胞，例如：原核細 胞、真核細胞、動物細胞與哺乳動物細胞，可以持續提供氧氣和 養分卻不會讓任何細胞接觸空氣，因此可以降低細胞的傷害甚至 細胞死亡率。此外，台灣專利申請案第92101625號之方法與裝置 係可減少污染，避免細胞於提供氣體時直接受到剪應力的傷害， 而了以預防氣泡與氣體對細胞的負面影響。該跋置亦可為自動 或手動操作，再者，此裝置係提供一更簡單、更便利之方法生產 與收集細胞產物，例如··蛋白質，及/或抗體，及/或任何細胞及/ 或組織之產物。 200526775 然而，上述之台灣專利申請案第92101625號中，為了提昇培 養基内之養分的分散效率’或使外加之特定養分能有效分布以提 仏更仏的細胞生長環境，則利用增高細胞載體與培養基的相對運 動速度來達成此養分均勻分布、移除代謝廢棄物及氧氣充分供應 標；在此情況下，隨著流速增加，伴隨產生的剪應力也相應 提高’此提高的剪應力對於脆弱的細胞，尤其是無細胞壁保護的 動物細：’其影響將更加明顯；因此，為了保護脆弱的動物細胞 不受到剪應力的傷害’必需使培養基與細胞載體相對流速降低， =在低流速的情形下，上培養裝置中心的溫度將較培養裝置外緣 ^低而產生溫度梯度於整體培養環境中，並且養分分布、代謝廢 ，物移除及氧氣供應都受會受到影響，這㈣題將使細胞生長速 -文到抑制。因此，本發明提出一種輔助裝置以解決上述問題。 【發明内容】 署处ί發明係提供-種具有辅助勝元件之細胞培養裝置，該裝 维：“rc動所造成之剪應力對所培養細胞的傷害，同時 維捋间效率且均一的培養環境。 署，明之目的係提供—種具有輔助㈣元件之細胞培養裝 附之腔室’該腔室包含—可允許細胞生長及/或貼 ，帛—腔室’係與第一腔室互通;及一辅助攪拌元件。 氣體胞培養裝置中的輔助㈣元件，具有混合液體' 播刀月刀及/或細胞、降低系統穩定 =平衡之“可加速平衡_ m 效分散葡萄糖、生長因子等。 又/合虱值有 及/或實施態樣料—射改變通過氣體 定。具體而：，二或狀恶之隔板，該隔板可為靜態或動態固 之隔板儀可Α — Γ '可改，通過氣體及/或液體流動方向或狀態 W 、一、有導流葉片或導流槽之隔板，該隔板可靜態固 200526775 定於第一或第二腔室中，或者，該隔板可動態固定於第一或第二 腔室中，其中動態固定之隔板並可進一步藉由氣體、流體或機械 動力移動或轉動而達到攪拌之效果。 本發明之輔助攪拌元件的一較佳實施態樣係為一具有導流 葉片之隔板，該隔板可為靜態或動態固定。前述具有導流葉片之 隔板，其葉片型態包含，但不限於，直線放射狀、曲線螺旋放射 狀、圓孤放射狀或平行板狀。 前述輔助攪拌元件的一較佳實施態樣係為一具有導流葉片 之隔板，該隔板係靜態固定於第二腔室底部，藉由該具有導流葉 片隔板之導流與擾動，達到穩定與均勻系統之目的。 修 前述輔助攪拌元件的另一較佳實施態樣係為一具有導流葉 片之隔板，該隔板係動態固定於第二腔室，藉由氣體、液體或與 該隔板連接之機械攪拌元件移動或轉動該隔板，達到穩定與均勻 系統之目的。 本發明之輔助攪拌元件的另一較佳實施態樣係為一具有導 流槽之隔板，該隔板可為靜態或動態固定。前述具有導流槽之隔 板，其中導流槽之型態包含，但不限於，螺旋狀之溝槽、平板放 射狀斜槽、錐面放射狀溝槽、直線放射狀溝槽、螺旋線放射狀溝 φ 槽。 前述輔助攪拌元件的另一較佳實施態樣係為一具有導流槽 之隔板，該隔板係靜態固定於第一腔室之載體底部，藉由導流槽 使流經之液體產生渦流，進而達到攪拌之效果。或者，前述之隔 板亦可靜態固定於第二腔室中。 本發明之具有輔助攪拌元件之細胞培養裝置，其中該輔助攪 拌元件的另一實施態樣進一步包含一空氣管路，該空氣管路係可 將氣體注入腔室液體中，進而產生擾動以達到攪拌並提供氣體之 效果。 12 200526775 μ本ι明之具有輔助攪拌元件之細胞培養裝置的一較佳實施 怨樣&含·一第一腔室，該腔室包含一可允許細胞生長及/或 貼附^載體，一第二腔室，係與第一腔室互通；一輔助授拌元件， 係靜態或動態固定於第二腔室。 本發明之具有輔助攪拌元件之細胞培養裝置的一較佳實施 態樣，包含：一第一腔室，該腔室包含一可允許細胞生長及/或 貼附之載體,·-第二腔室，係與第一腔室互通；一輔助授掉元件， 係靜態固定於第一腔室之載體底部。 本發明之具有輔助攪拌元件之細胞培養裝置的一較佳實施 態樣，包含：-第-腔室，該腔室包含一可允許細胞生長及，或 φ 貼附=載體；一第二腔室，係與第一腔室互通；一辅助攪拌元件， 係動怨固定於第二腔室；一渦輪葉片，係與輔助攪拌元件連結； 一進氣官路，係可通入氣體推動渦輪葉片；及一排氣管路，係排 出多於氣體至大氣中。 本發明之具有輔助攪拌元件之細胞培養裝置的另一較佳實 施態樣，包含：一第一腔室，該腔室包含一可允許細胞生長及/ 或貼附之載體；一第二腔室，係與第一腔室互通；一輔助攪拌元 件，係動態固定於第二腔室；一機械攪拌元件，係可直接或間接 帶動辅助攪拌元件。 · 在本發明中，”靜態固定”係指固定之隔板不會因氣體、流體 或機械力量而產生移動或轉動。，，動態固定，，係指固定之隔板可因 氣體、流體或機械力量等外加因素而產生移動或轉動。 在本發明之具有輔助攪拌元件之細胞培養裝置中，其中第一 腔室、第二腔室、細胞載體之配置與材料皆可參考台灣專利申請 案第92101625號之說明。 本發明係利用具有特定形狀的輔助攪拌元件將所流經的液 體藉由葉瓣或溝槽的引導，進而產生足夠的渦流以分散液體中的 13 200526775 養刀生長因子以及降低溫度、pH值、溶氧值的平衡時間，有 利整體培養環境的穩定。 【實施方式】 ^ 本發明係提供一種具有輔助攪拌元件之細胞培養裝置，該 扃置已第一腔至，該腔室包含一可允許細胞生長及/或貼 附之載體卜第二腔室，係與第„_腔室互通；及—輔助攪掉元件、。 藉由輔助授拌元件的輔助，能迅速的將培養基整體溫度加以均衡 而有利於細胞生長。 本發明之輔助_元件可為—具有導流葉片之隔板，藉由細 胞生長所必需通入的氣體直接驅動而產生旋轉或是移動，造成強 制攪拌…方面提供充足的溶氧予細胞生長之用，—方面，亦直 接產生擾拌的效果達到培養基對流的作用，而無須額外的電力驅 使推動授拌葉。或者’藉由該導流葉片引導流體產生渦流或擾 動’達到均於混合之目的。 此外，細胞初始於載體内的分布亦決定培養的成果。本發明 ^輔助授拌元件亦可為-具有導流槽之隔板，利用特定形式的導 ，槽引導細胞方面可以使細胞均句分散而不致於堆積於局 邛仏成降低有效培養體積利用率的負面影響：細胞堆積於局部 鲁 會造成載體㈣局部細胞錢過冑，養分無法有㈣透以提供細 ^生長所A而抑制總細胞數的成長，並可能造成蛋白質或是病 :等產此釋放的缺點；另一方面，藉由導流槽提昇細胞於載體内 部的移動距離，增加細胞與載體的接觸機率，可提昇細胞固定化 (Immobilization)的效率。 "或者，本發明進一步可利用通入的氣體產生 微細氣泡加以擾 拌坧養基，其中產生的氣泡不會直接接觸培養的細胞，並可藉由 程式控制氣體通人時間與速率’錢泡不會持續產生泡泳 14 200526775 (Foam) 〇 本發明之實施例係藉由圖式來進—步闇述，下列實施例僅 用於=明本發明，並非用於限制本發明之_請專利範圍。 第圖係為本發明之一實施態樣，細胞培養裝置⑽包含第 腔至101，第二腔室102,位於第一腔室1〇1中的細胞載體⑻， ^位於，二腔室1G2之㈣葉刚；其中第—腔室⑻係裝設於 f :02中，亚且第二腔室102係環繞第一腔室10卜及授 "茱HM係裝設於第二腔室1()2底部。使用此細胞培養裝置⑽ 時，該細胞培養裝置⑽彳利用外加之空氣廢縮機1〇5配合闕件 i〇6、1〇7推動空氣交替進入第一腔室1〇1或第二腔冑1〇2中；藉 由在第-腔室ΗΠ肖第二腔室102中交替切換的氣體，可使培^ 基在復於第-腔室1()1與第二腔室1G2中。當氣體推動培養基在 兩腔室間流動時’攪拌葉104之延伸葉片能引導由第二腔室1〇2 流向第-腔室ιοί或由第一腔室101流向第二腔室1〇2之培養基 產生渦流’達到混合㈣之絲，敎並均勾整個培養環境。土 一第一圖至第五圖係為前述攪拌葉之數種態樣。第二圖之 葉200中，攪拌葉2〇〇之中心具有一圓弧頂面2〇1，葉片2〇2為 直線放射狀，葉片數大於或#於2組，其中葉片2()2與水平面成 一夾角θ，θ介於20度至90度之間，並且葉片2〇2之長度[與 二腔室102(參考第一圖）内徑之比值較佳為1/2至1/8。 。 第二圖係為攪拌葉之另一態樣。攪拌葉3〇〇之中心具有一圓 弧頂面301，葉片302為圓弧狀，葉片數大於或等於2組，每— 組葉片具有-曲率半徑£ ’並且每組葉片可分別具有不同之: 半徑。 第四圖係為攪拌葉之另一態樣。攪拌葉4〇〇之中心具有一。 弧頂面401，葉片402為曲線螺旋放射狀，葉片數大於或等於^ 組，其中每一葉片不限翻轉一圈。 、 15 200526775 第五圖係為攪拌葉之另一態樣。攪拌葉500之葉片501採平 板狀形式，每一板狀葉片501與水平面成一夾角Θ，此夾角Θ介於 20度之90度之間，且每一組板狀葉片大於1片，同時不限一組。 第六圖係為本發明之具有輔助攪拌元件之細胞培養裝置之 另一實施態樣。細胞培養裝置600包含第一腔室601，第二腔室 602，位於第一腔室601中的細胞載體603，及空氣管路604與通 氣環605 ;其中第一腔室601係裝設於第二腔室602中，及第二 腔室602係環繞第一腔室601。使用此細胞培養裝置600時，該 細胞培養裝置600可利用外加之空氣壓縮機606配合閥件607、 608將空氣交替注入第一腔室601與第二腔室602中，使液面與 載體603產生相對運動。同時，以電腦程式控制閥609進行週期 性的開啟與關閉，將空氣透過空氣管路604輸送至通氣環605， 將氣體注入不含載體之第二腔室602中，進而產生培養基之擾動 以達到攪拌之效果，並可避免氣泡對載體内細胞的傷害。 第七圖係為本發明之具有輔助攪拌元件之細胞培養裝置之 另一實施態樣。細胞培養裝置700包含第一腔室701，第二腔室 702，位於第一腔室701中的細胞載體703，攪拌葉704，渦輪葉 片705，空氣進氣管路706與排氣管路707 ;其中第一腔室701 係裝設於第二腔室702中，及第二腔室702係環繞第一腔室70卜 使用此細胞培養裝置700時，該細胞培養裝置700可利用外加之 空氣壓縮機708配合閥件709、710將空氣交替注入第一腔室701 與第二腔室702中，使液面與載體703產生相對運動。同時，以 電腦程式控制閥711進行週期性的開啟與關閉，將空氣經由空氣 進氣管路706注入並推動渦輪葉片705,進而帶動旋轉攪拌葉 704，而達到攪拌的效果，其中多餘的空氣則經由排氣管路707 排至大氣中。 第八圖係為前述渦輪葉片705之一實施態樣，氣體係由進氣 16 200526775 口 712進人’推動渴輪葉片713產生旋轉，多餘的线則由排 口 714 4非出。 f九圖係為本發明之具有輔助攪拌元件之細胞培養裝置之 另-實施態樣。在細胞培養裝置_中，包含第一腔室9〇1，第 二腔，902,位於第一腔室9〇1之細胞載體9〇3及導流隔板9〇4; …中第&至9〇1係與第二腔室902連結，且第一腔室9〇ι與第 二腔室9G2可以貼合’或合為一體，導流隔板9()4設置於細胞載 體903下方，藉由隔板9〇4中的導流槽（圖未顯示）引導流體產生 渦流，有效提供培養基之混合並提高細胞固定化效率。 第十圖至第十二圖係為前述導流隔板之數種實施態樣。第十 鲁 圖係為具有螺旋狀溝槽1001之導流隔板1〇〇〇，溝槽1〇〇1與水平 面具有一介於20度至90度之間的傾斜角，同時此溝槽1〇〇1之 螺旋圈數大於一圈。 第十一圖係為具有平板放射斜槽11〇1之導流隔板11〇〇，該 導流隔板1100上刻劃有一組以上之傾斜溝槽11〇1，同時該傾斜 槽1101與水平面具有一介於20度至90度之間的夾角。 第十二圖係為一錐面導流隔板12〇〇，該導流隔板12〇〇上刻 劃有一組以上之放射狀傾斜溝槽12〇1，同時該傾斜槽12〇1與水 平面具有-介於2G度至9G度之間的夾角。此—錐面導流隔板籲 1200可防止軋泡堆積於隔板下方而產生無效區域（心以z〇〇n)，並 可將氣泡引導至隔板外源使其不致衝擊上方培養於載體内部的 細胞。 第十三圖係為本發明之具有輔助攪拌元件之細胞培養裝置 之另一貫施態樣。在細胞培養裝置丨3〇〇中，包含第一腔室1301， 第二腔室1302,位於第一腔室13〇1之細胞載體13〇3及導流隔板 1304 ;其中第一腔室13〇1係裝設於第二腔室13〇2中，並且第二 腔室1302係環繞第一腔室13〇1;導流隔板13〇4係設置於細胞載 17 200526775 體1303下方。前述導流隔板1304係可為第十圖至第十二圖所示 之導流隔板。使用此細胞培養裝置1300時，該細胞培養裝置1300 可利用外加之空氣壓縮機1305配合閥件1306、1307推動空氣交 替進入第一腔室1301或第二腔室1302中；藉由在第一腔室1301 與第二腔室1302中交替切換的氣體，可使培養基往復於第一腔 室1301與第二腔室1302中。藉由隔板1304中的導流槽（圖未顯 示）能引導出入第一腔室1301與第二腔室1302之培養基產生渦 流，有效提供培養基之混合並提高細胞固定化效率。 第十四圖係為本發明之具有輔助攪拌元件之細胞培養裝置 之另一實施態樣。在細胞培養裝置1400中，包含第一腔室1401， 第二腔室1402,位於第一腔室1401之細胞載體1403及圓環狀導 流隔板1404;其中第一腔室1401係裝設於第二腔室1402中，並 且第二腔室1402係環繞第一腔室1401 ;該圓環狀導流隔板1404 係設置於第二腔室1402之下方，並且該環狀導流隔板1404可同 時裝設一組以上，藉由此圓環狀隔板1404可有效提供培養基之 混合與細胞固定化的均勻度。使用此細胞培養裝置1400時，該 細胞培養裝置1400可利用外加之空氣壓縮機1405配合閥件 1406、1407推動空氣交替進入第一腔室1401或第二腔室1402 中；藉由在第一腔室1401與第二腔室1402中交替切換的氣體， 可使培養基往復於第一腔室1401與第二腔室1402中。藉由隔板 1404中的導流槽（圖未顯示），能使被氣體推動之培養基產生渦 流，提高培養基在進入第一腔室1401前的混合均勻度，有效提 供培養基之混合並提高細胞固定化效率。 第十五圖與第十六圖係為前述圓環狀導流隔板的實施態樣 之一。第十五圖之圓環狀導流隔板1500上刻劃有至少一條之放 射狀直線導流槽1501，該導流槽與水平面具有一介於20度至90 度之夾角。 18 200526775 第十六圖係為圓環狀導流隔板之另一實施態樣。該圓環狀導 流隔板1600上刻劃有至少一條之放射狀螺旋線導流槽腦，其 中每-螺旋線導流槽16G1之曲率半徑可相同也可不同，並且該 導流t 1601與水平面具有一介於2〇度至9〇度之夾角。 第^七圖係為本發明之具有輔助授拌元件之細胞培養裝置 =另-實施態樣。在細胞培養裝置！中，包含第一腔室測， 第二腔室17G2,位於第__腔室丨期之細胞載體丨期及位於第二 腔至1702之授拌葉1704;其中第一腔室17〇1係裝設於第二腔室 1702中，並且第二腔室17〇2係環繞第一腔室ΐ7〇ι;擾摔葉！綱 係設置於第二腔室底部，第二腔室下方裝設有一機械授拌設備，鲁 利用馬達1705帶動轉轴⑽進而驅動祕葉携 攪拌混合之效益。 7 實驗實施例1 以本發明之具有輔助攪拌元件之細胞培養裝置進行實驗 (參考第六圖）。在本實驗中，分別測量不含任何輔助授摔元件 之細胞培養裝置及具有第六圖之辅助授拌元件之細胞培養裝置 岡系，pH值平衡¥間。第十八圖係為系統值與時間關係 :’八中線(A)為不含任何輔助裝置之細胞培養裝置，線⑻為 第六圖的具有辅助㈣元件之細胞培養裝置，通氣時間為1〇 # 睹’ f(C)為第六圖的具有輔助授拌元件之細胞培養裝置，通氣 二’、’、W秒。由第十八圖之PH反應時間可明顯發現：不含任 言^借^件的情形下，很難達到穩定的混合時間：而藉由進氣 °又、辅助，則可將混合時間縮短至3分鐘以内。 實驗實施例2 二本發明之具有輔助授摔元件之細胞培養裝置進行實驗（參 與養获圖）在本實驗中’分別測量不含任何輔助授拌元件之細胞 。凌置及具有第一圖之輔助攪拌元件之細胞培養裝置的系統 19 200526775 pH值平衡時間。第十九圖係為系 (A)為第一圖之細胞培養裝置 值/、守間關係圖，其中線 含任何輔助元件之細胞培養，置【值與時間關係，線(B)係為不 時間可明顯發現··不含任何辅助L的與二關係。由p H的反應 其他實施態樣 ，所有本說明書所揭露之特徵皆可以任何形式與其他方法合 併使用。本說明書中所揭露之特徵可以具有相同、㈣或相似目 的的特徵所取代。HI此，除了明確強調的部分之外，所有本說明 書中揭露的特徵僅為眾多相等或相似特徵中的一個實施例。例 如，本說明書中細胞培養裝置之第—腔室、第二腔室配置位置及 培養基在兩腔室間進行相互運動之操作方式，皆可參考台灣專利 申請案第92101625號之說明全文，並結合本發明之輔助元件， 併入本發明中。 根據本說明書所揭露的内容’任何一位熟習本技術領域的人 士可輕易明確瞭解本發明之重要特色，在不脫離本發明之精神 與範圍下，係可對本發明做不同的改變與修飾使其符合不同的 使用目的與情況。因此’其他實施態樣亦包含在下列申請專利 範圍内。 20 200526775 【圖式簡單說明】 第一圖係為具攪拌葉之細胞培養裝置示意圖。 第二圖係為直線放射狀攪拌葉示意圖。 第三圖係為圓弧放射狀攪拌葉示意圖。 第四圖係為曲線螺旋放射狀攪拌葉示意圖。 第五圖係為平行板狀攪拌葉示意圖。 第六圖係為具空氣管路之細胞培養裝置示意圖。 第七圖係為氣體帶動攪拌葉之細胞培養裝置示意圖。 第八圖係為渦輪葉片元件示意圖。 第九圖係為具導流隔板之細胞培養裝置示意圖。 第十圖係為螺旋溝槽導流隔板示意圖。 第十一圖係為平板放射狀斜槽導流隔板示意圖。 第十二圖係為錐面放射狀溝槽導流隔板示意圖。 第十三圖係為具導流隔板之細胞培養裝置示意圖。 第十四圖係為具圓環狀導流隔板之細胞培養裝置示意圖。 第十五圖係為放射狀直線槽圓環狀之導流隔板示意圖。 第十六圖係為放射狀螺旋線槽圓環狀之導流隔板示意圖。 第十七圖係為具機械攪拌裝置之細胞培養裝置示意圖。 第十八圖係為比較本發明第六圖及未含輔助攪拌元件之培 養裝置之pH值與時間關係圖。 第十九圖係為比較本發明第一圖及未含輔助攪拌元件之培 養裝置之pH值與時間關係圖。 200526775 【主要元件符號對照說明】 100_—細胞培養裝置 7〇〇…細胞培養裝置 101…第一腔室 7〇1…第一腔室 102…第二腔室 702…第二腔室 103---細胞載體 7〇3…細胞載體 104…攪拌葉 704…攪拌葉 105…空氣壓縮機 705…渦輪葉片 106、107---閥件 706…空氣管路 2〇〇…攪拌葉 707…排氣管路 201 —固弧頂面 708-—空氣壓縮機 202…葉片 709、710、711 …閥件 300…攪拌葉 712—進氣口 301 —圓弧頂面 713…渦輪葉片 302…葉片 714…排氣口 400…攪拌葉 900-—細胞培養裝置 401 — S] 5瓜頂面 9〇1…第一腔室 402…葉片 902…第二腔室 500—攪拌葉 903…細胞載體 501 —葉片 904 —導流隔板 6〇〇…細胞培養裝置 1000 —導流隔板 601…第一腔室 1001—-溝槽 602…第二腔室 1100 —導流隔板 603…細胞載體 1101…溝槽 604…空氣管路 1200 —導流隔板 605…通氣環 1201…溝槽 606…空氣壓縮機 1300—-細胞培養裝置 607 ' 608 > 609——閥件 1301…第一腔室 200526775 1302…第二腔室 1501…溝槽 1303…細胞載體 1600…圓環隔板 1304…導流隔板 1601…溝槽 1305…空氣壓縮機 1700…細胞培養裝置 1306、1307…閥件 1701…第一腔室 1400…細胞培養裝置 1702…第二腔室 1401…第一腔室 1703…細胞載體 1402…第二腔室 1704…攪拌葉 1403…細胞載體 1705…馬達 1404 —導流隔板 1706---轉軸 1405—-空氣壓縮機 1707—-空氣壓縮機 1406、1407…閥件 1708、1709…閥件 1500 —圓壤隔板 23200526775 发明 Description of the invention: [Technical field to which the invention belongs] The present invention relates to a cell culture device with an auxiliary stirring element, which can reduce the damage of the shear stress caused by the flow of the culture medium on the cultured cells, while maintaining high efficiency and Uniform cultivation environment. [Previous technology] Large-scale cell culture programs have been widely developed and used in recent years. However, compared with the culture of bacteria, yeast and mold, they have a strong cell wall and / or extracellular material (extra cellular material). ), For more flexible cells, the technology of culturing eukaryotic cells, animal cells, mammalian cells and / or tissues is more difficult and complicated, because such cells are more fragile than microbial cells, and their growth needs The maintenance of nutrients and oxygen is also difficult and complicated. Animal cells are different from microbial cells in that animal cells cannot tolerate severe disturbances and / or shear stress caused by air or mixed gas; and animal cells cannot directly contact the air. Most animal cells can only be dissolved in the culture medium. In the oxygen. Compared with microbial cells, animal cells and mammalian cells are more likely to be damaged by the gas or during ventilation, thus increasing their mortality. Due to the need for large-scale culture of eukaryotic cells and / or prokaryotic cells and / or animal cells and / or mammalian cells in the laboratory, bioreactors and culture devices are researching and producing cells to produce active proteins and / or antibodies and / or Any aspect of cellular by-products plays an important role. The main function of the bioreactor is to cooperate with the research needs to cultivate a large number of cells to extract traces of active substances derived from them, including proteins or antibodies secreted by the cells into the culture medium; another function of the bioreactor is to cultivate cells to mass produce cells or Tissue-produced active protein. 200526775 Bioreactors currently used in large-scale culture have one internal benefit, for example. Impellers make the cells subject to very high liquid frying stress and cause injury or death of the cells, resulting in a low survival rate of the cells and a decrease in the production of protein and by-products. Similarly, in order to make the cells uniformly suspended and / or suitable for: exposure to air, the use of other mechanical devices, intense gas movement or strong fluid: moving bioreactors will also cause cell damage and hinder cell or tissue growth 'Progressive by-products of cells', for example: protein, yield reduction. In addition to the traditional petri dishes and culture flasks, Qian Xinyi used a cell and a group of phyllostachys to form a wire-type culture wheel (k kies): It has been widely used in cell culture for several years. However, shaft-type culture flasks have advantages that two dishes or flasks do not have, such as providing a larger growth area of cells, but it is less able to overcome all the shortcomings, especially in terms of expanding the culture scale. These shortcomings include: a large amount of gas in the headspace gas (gasheadspaee): a large amount of fluid shear stress; due to the high shear stress of the rolling culture flask =, the tissue culture of larger three-dimensional structures is almost impossible. Only cells that do not or can remain attached to the inner wall of a rolling culture flask have a culture time. From this, it can be seen that maintaining for a long time in the rolling type =: plant cultivation = in: including its high r ring = outside, although the overall area of the money-moving culture flask is larger than that of a petri dish or culture bottle, :: ,, ...: The area of each cell is not larger than the area provided by the culture dish or flask, especially in large-scale culture. Therefore, high-moving =, = ?, is a description-a type that can promote cell adhesion. Two = moving ° ~, is attached to the inner wall of the rolling culture bottle plus a kind of channel, 200526775 increases the inner surface area of the rolling culture bottle for έ μ, ,,, Tian Fu Attachment. However, the general rolling culture flask only provides an area of about 850-1700 cm 2 and 77, so a large number of rolling culture flasks are still needed to achieve mass production. The automatic servo system, although it can save time and manpower, can be used to save time and manpower, but its operation is quite responsible and restrictive. In addition to the problem of fluid shear stress and the limitation of the food area of the county, obtaining and maintaining sufficient oxygen supply is also the central issue of cell and tissue culture. As is well known to those skilled in the art, prokaryotic cells, straight four nucleated cells, including animal cells, mammalian cells, insect cells, yeast fish, fish, etc. The growth of tender brines has a major rate-limiting step. step), that is, 0Xygen mass transfer ° Except for some eukaryotic microorganisms (such as yeast) that undergo fermentation, for most prokaryotic cells and eukaryotic cells, the metabolism of oxygen to the cells Metabolic function is very important, especially for mammalian and animal cell culture technology. The supply of oxygen is important for the early stages of rapid cell division. When the cell is floating, 'the demand for oxygen is greatest' and when the cells aggregate or differentiate, the demand for oxygen will decrease. Some mammalian cells and animal cells are -age-dependent, and some mammalian cells and animal cells can grow in suspension in a liquid environment, that is, non-attachment dependent. However, all cells are Need to grow in a sufficient dissolved oxygen environment. In the later stage of cell culture, whether it is attachment-dependent or non-dependent cells, the number of cells per unit volume will increase significantly, and therefore a large amount of oxygen f needs to be transferred again to provide sufficient oxygen. You can use machine agitation and, however, as mentioned earlier, the search and mixing basically provides at least the oxygen required for the cells that grow and grow to supply the oxygen they need. 200526775 The aeration action will harm the cells, thus reducing their survival rate. And overall cell and / or tissue culture efficiency and yield. In addition, the direct introduction of gas into cells and tissue culture media will cause air bubbles to be generated, which is also not conducive to cell survival. Device invented to solve the problem of oxygen supply during cell culture, for example: Wolf et al. U.S. Patent No. 5,153,131 is owned by a bioreactor vessel without blades. The biological reaction tank passes air through the gas transmission channel, passes through a screen through a support plate member, and passes through a flat disk shaped between two sides of the housing of the reaction tank. The osmosis membrane uses the oxygen concentration gradient between the two sides of the outer box, so that oxygen can diffuse through the osmosis membrane and reach the cultivation chamber. However, Wolf ’s bioreactor has many disadvantages. In particular, the rate of oxygen diffusion through the flat disk-shaped osmosis membrane has become the main limiting factor for the size of the culture chamber. Another disadvantage is that the flat disk-shaped osmosis membrane is designed to be scalable to have Stirring function, but this may cause cell death. It is important to use the agitation function to uniformly distribute air in the culture medium. However, agitation will also increase the shear stress inside the culture chamber, which may cause damage to the cells as described above. Therefore, when designing a bioreactor or culture tank, how to provide sufficient gas exchange to maintain a large cell structure in culture is very important and a practical limitation. In order to solve the aforementioned disadvantages, a reactor made of a gas-permeable material, such as Schwarzet al. U.S. Patent No. 5,702,941, entitled "Breathable Bioreactor and Method of Use", relates to a horizontally rotatable container, and the wall portion of the container is made of breathable material, in order to achieve direct penetration of the breathable material Purpose of gas exchange with culture medium. However, the scale of the device disclosed by Schwarz is still limited because the gas exchange needs to be determined according to the size of the ventilation area. Although Schwarz emphasizes that when the surface area of the device 200526775 increases, the volume of the culture medium also increases relatively. The scale of the disclosed preferred embodiment is limited to 1 to 6 inches in diameter and 1/4 to 1 inch in width. This size is not suitable for growing cell aggregates and tissues with three-dimensional space and / or any Mass production. Similarly, Falkenbergetal. U.S. Patent No. 5,449,617, entitled "Culture Vessel for Cell Culture", relates to a horizontally rotating device. This culture vessel is divided into a cell culture chamber and a nutrient solution storage tank by a dialysis membrane. The air-permeable material is used on the wall of the container to allow gas exchange in the cell culture chamber, but the nutrient solution storage tank is not completely filled with the nutrient solution, but a large amount of gas is maintained above the culture solution in the two chambers. However, the Falkenberg container is not designed to reduce the disturbance inside the cell culture chamber. On the contrary, stirring is necessary to keep the dialysis membrane moist. In addition, Falkenberg does not mention the use of this container in the culture of cell aggregates or Any type of organization. Another invention to solve the problem of oxygen supply is, for example, US Patent No. 5,766,949 owned by Liau, entitled "Method and Apparatus for Cultivating Dependent Monolayer Cells", which describes a method of shaking the culture medium up and down to increase the supply of oxygen. Cultivation system 0 However, Liau's invention has many disadvantages, one of which is the complexity of its device. Liau's system includes two external storage tanks and a separate culture chamber containing a series of vertical substrate plates. In addition, several peristaltic pumps are required to push the culture medium from one storage tank to the culture. The chamber flows to another storage tank and finally back to the first storage tank. Because Liaii's equipment is complex and some of it is external to the culture room, such as external pipelines, storage tanks, and pumps, it is highly likely that external pollution sources will be introduced into the interior. In addition, because the equipment is large, sterilization is not easy It is labor-intensive. 200526775 Tong Ming Another disadvantage is that the fluid shear r ^^ bs caused by the noodle flow through the system interferes with and moves the cells growing on the flat plate of the substrate, thereby reducing the rate. In addition, the design of the flat plate of the vertical substrate is not conducive to the attachment of cells. The cells immediately after the factory will fall and accumulate at the bottom of the flat plate. Most of these thin cells will die after removal, thus reducing the survival of the cells. Rate and protein yield, and this system has to be-restarted again-quite inefficient and not ideal. Furthermore, two: this: ,,, complexity, collecting any secreted protein or cell product is very annoying and consumes #. Finally, when the growth fluid is lower than the cell growth disk, the cells will rush into the working gas, that is, they will directly contact the gaseous environment, which will lead to cell death. Cell and tissue culture technologies are important for biotechnology research, pharmaceutical research, patient care, and academic research. In order to overcome the shortcomings and limitations of the conventional technology and make up for its inconveniences, Taiwan Patent Application No. 921G1625 provides a kind of cell culture | placement, the medium in which the medium in which the cells grow is moved in different chambers, and provides a kind of Relying on cells and / or tissues and collecting their products are reliable, simple, inexpensive, and can be done with efficient and effective methods and devices. More specifically, it teaches and discloses a novel method and device that can effectively cultivate cells to be cultured, such as prokaryotic cells, eukaryotic cells, animal cells, and mammalian cells, which can continuously provide oxygen and nutrients without letting Any cell that comes into contact with air can therefore reduce cell damage and even cell mortality. In addition, the method and device of Taiwan Patent Application No. 92101625 can reduce pollution, prevent cells from being directly damaged by shear stress when supplying gas, and prevent the negative effects of air bubbles and gas on the cells. The device can also be operated automatically or manually. Furthermore, the device provides a simpler and more convenient way to produce and collect cell products, such as proteins, and / or antibodies, and / or any cells and / or The product of organization. 200526775 However, in the above-mentioned Taiwan Patent Application No. 92101625, in order to improve the efficiency of nutrient dispersion in the culture medium 'or to enable the specific nutrient to be effectively distributed to improve the cell growth environment, the cell carrier and the culture medium are increased. Relative movement speed to achieve this uniform distribution of nutrients, removal of metabolic waste and sufficient supply of oxygen; in this case, as the flow rate increases, the accompanying shear stress will also increase accordingly. , Especially for animals without cell wall protection: 'the effect will be more obvious; therefore, in order to protect fragile animal cells from being damaged by shear stress', the relative flow rate of the medium and the cell carrier must be reduced, = at low flow rates, The temperature of the center of the upper culture device will be lower than the outer edge of the culture device, which will produce a temperature gradient in the overall culture environment, and nutrient distribution, metabolic waste, material removal and oxygen supply will be affected. This problem will cause cell growth Quick-text to suppression. Therefore, the present invention proposes an auxiliary device to solve the above problems. [Summary of the Invention] The Department of Invention provides a cell culture device with auxiliary win element, which is equipped with: "the damage caused by the shear stress caused by rc movement to the cultured cells, and at the same time an efficient and uniform culture environment The purpose of the invention is to provide a chamber containing a cell culture device with auxiliary cells, the chamber contains-allowing cells to grow and / or stick, and the cells are in communication with the first chamber; and An auxiliary stirring element. The auxiliary ㈣ element in the gas cell culture device has a mixture of liquid, sowing knife and / or cells, reducing system stability = balance, and can accelerate the equilibrium_ m to effectively disperse glucose and growth factors. And / or the value of the lice and / or the implementation of the sample-the change in the gas is determined. Specifically, the two or evil-shaped separators, which can be static or dynamic solid separators, can be changed from A to Γ ', through the direction and state of the flow of gas and / or liquid W, one, with diversion Blade or baffle partition, the partition can be statically fixed in the first or second chamber 200526775, or the partition can be dynamically fixed in the first or second chamber, where the dynamically fixed partition The plate can be further moved or rotated by gas, fluid or mechanical power to achieve the effect of stirring. A preferred embodiment of the auxiliary stirring element according to the present invention is a partition plate having a guide vane, and the partition plate can be fixed statically or dynamically. In the aforementioned partition plate with guide vanes, the shape of the blade includes, but is not limited to, straight radial shape, curved spiral radial shape, circular solitary radial shape, or parallel plate shape. A preferred embodiment of the aforementioned auxiliary stirring element is a partition plate with guide vanes, which is statically fixed at the bottom of the second chamber, and through the guide and disturbance of the guide plate with guide vanes, To achieve the purpose of stable and uniform system. Another preferred embodiment of repairing the aforementioned auxiliary stirring element is a partition plate with a guide vane. The partition plate is dynamically fixed in the second chamber, and is stirred by gas, liquid, or mechanically connected to the partition plate. The component moves or rotates the partition to achieve the purpose of stable and uniform system. Another preferred embodiment of the auxiliary stirring element of the present invention is a partition plate having a flow guide groove, and the partition plate can be fixed statically or dynamically. The aforementioned partition plate with a guide groove, wherein the form of the guide groove includes, but is not limited to, a spiral groove, a flat radial inclined groove, a tapered radial groove, a linear radial groove, and a spiral radiation Like groove φ groove. Another preferred embodiment of the aforementioned auxiliary stirring element is a partition plate with a flow guide, which is fixed statically on the bottom of the carrier in the first chamber, and the liquid flowing therethrough is vortexed by the flow guide groove. , And then achieve the effect of stirring. Alternatively, the aforementioned partition may be statically fixed in the second chamber. The cell culture device with auxiliary stirring element of the present invention, wherein another embodiment of the auxiliary stirring element further includes an air line, which can inject gas into the liquid in the chamber, and then generate disturbance to achieve stirring And provide the effect of gas. 12 200526775 A preferred implementation of a cell culture device with auxiliary agitation elements & includes a first chamber containing a cell that allows cells to grow and / or attach to a carrier, a first Two chambers communicate with the first chamber; an auxiliary mixing element is fixed in the second chamber statically or dynamically. A preferred embodiment of the cell culture device with auxiliary stirring element of the present invention includes: a first chamber, the chamber includes a carrier that allows cells to grow and / or attach, and a second chamber Is interconnected with the first chamber; an auxiliary delivery element is statically fixed to the bottom of the carrier of the first chamber. A preferred embodiment of the cell culture device with an auxiliary stirring element of the present invention includes:-a first chamber, the chamber contains a cell that can allow cell growth and, or φ attachment = carrier; a second chamber It is in communication with the first chamber; an auxiliary stirring element is fixed in the second chamber; a turbine blade is connected with the auxiliary stirring element; an intake air path is connected with the gas to push the turbine blade; And an exhaust line, which discharges more than gas into the atmosphere. Another preferred embodiment of the cell culture device with auxiliary stirring elements of the present invention includes: a first chamber, the chamber containing a carrier that allows cells to grow and / or attach; a second chamber It is in communication with the first chamber; an auxiliary stirring element is dynamically fixed in the second chamber; a mechanical stirring element can directly or indirectly drive the auxiliary stirring element. In the present invention, "statically fixed" means that the fixed partition does not move or rotate due to gas, fluid, or mechanical forces. ,, Dynamically fixed, means that the fixed partition can move or rotate due to external factors such as gas, fluid or mechanical force. In the cell culture device with the auxiliary stirring element of the present invention, the configuration and materials of the first chamber, the second chamber, and the cell carrier can refer to the description of Taiwan Patent Application No. 92101625. The present invention uses an auxiliary stirring element with a specific shape to guide the flowed liquid through leaflets or grooves, thereby generating sufficient vortexes to disperse 13 200526775 knife growth factor in the liquid, and reduce temperature, pH, The equilibrium time of the dissolved oxygen value is beneficial to the stability of the overall culture environment. [Embodiment] The present invention provides a cell culture device having an auxiliary stirring element. The device has a first chamber, and the chamber includes a carrier and a second chamber that allow cells to grow and / or attach. The system communicates with the first chamber; and-the auxiliary stirring-off element. With the assistance of the auxiliary mixing element, the overall temperature of the medium can be quickly balanced to facilitate cell growth. The auxiliary element of the present invention may be- The baffle with diversion blades is directly driven by the gas necessary for cell growth to rotate or move, resulting in forced agitation .... On the one hand, it provides sufficient dissolved oxygen for cell growth. On the other hand, it also directly generates disturbance. The mixing effect achieves the effect of convection of the culture medium without the need for additional power to drive the mixing leaves. Or, 'the fluid is guided by the guide vanes to generate vortices or disturbances' to achieve the purpose of mixing. In addition, the cells originally in the carrier The distribution also determines the results of cultivation. The auxiliary mixing element of the present invention can also be a baffle with a guide groove, which uses a specific form of guide to guide the cells. It can make the cells evenly dispersed without accumulating in the local area and reduce the negative impact of the effective culture volume utilization rate: the accumulation of cells in the local area will cause the carrier, the local cell will be too expensive, and the nutrients cannot be thoroughly penetrated to provide fine growth It inhibits the growth of the total number of cells, and may cause protein or disease: the disadvantages of such release; on the other hand, the flow distance of the cell inside the carrier is increased by the flow channel, and the probability of contact between the cell and the carrier is increased. , Can improve the efficiency of cell immobilization (Immobilization). Alternatively, the present invention can further generate micro-bubbles using the gas introduced to disturb the culture medium, wherein the generated bubbles will not directly contact the cultured cells, and can be borrowed. Controlling the time and rate of gas passage by program 'money bubble will not continue to produce bubble swimming 14 200526775 (Foam) 〇 The embodiment of the present invention is carried out by means of diagrams-step by step, the following examples are only used for = The invention is not intended to limit the scope of the present invention. Please refer to the patent. The figure is an embodiment of the present invention. The cell culture device includes the first cavity to the 101st, and the second The chamber 102 is a cell carrier 位于 located in the first chamber 101, and 位于 is located in the second chamber 1G2, Ye Gang; where the first chamber ⑻ is installed in f: 02, and the second chamber The 102 series surrounds the first chamber 10 and the "Ju Zhu HM" is installed at the bottom of the second chamber 1 () 2. When using this cell culture device ⑽, the cell culture device ⑽ 彳 uses an external air shrinking machine 105 cooperates with the files 106 and 107 to push the air into the first chamber 101 or the second chamber 102 alternately; by alternately in the first chamber 102 and the second chamber 102 The switched gas can make the culture medium in the first chamber 1 () 1 and the second chamber 1G2. When the gas pushes the medium to flow between the two chambers, the extension blade of the stirring blade 104 can guide the The medium flowing from the two chambers 102 to the first chamber or the medium flowing from the first chamber 101 to the second chamber 102 generates a vortex 'to reach the mixed silk, and the whole culture environment is hooked. The first to fifth pictures of soil are several aspects of the aforementioned stirring leaves. In the leaf 200 of the second figure, the center of the stirring leaf 200 has an arc top surface 201, and the blades 202 are linear and radial. The number of the blades is greater than or # in two groups, among which the blades 2 () 2 and The horizontal plane forms an included angle θ, which is between 20 degrees and 90 degrees, and the ratio of the length of the blade 2 [to the inner diameter of the two chambers 102 (refer to the first figure) is preferably 1/2 to 1/8. . The second picture is another aspect of the stirring blade. The center of the stirring blade 300 has an arc top surface 301, the blade 302 is arc-shaped, the number of blades is greater than or equal to 2 groups, each group of blades has a radius of curvature of £ 'and each group of blades can have different differences: radius. The fourth picture is another aspect of the stirring blade. The center of the stirring blade 400 has one. The top surface 401 of the arc, and the blades 402 are curved and spiral radial, and the number of blades is greater than or equal to ^ groups, where each blade is not limited to one turn. , 15 200526775 The fifth picture is another aspect of the stirring blade. The blade 501 of the stirring blade 500 is in the form of a flat plate. Each plate-shaped blade 501 forms an included angle Θ with the horizontal plane, and the included angle Θ is between 90 degrees and 20 degrees. One group. The sixth figure is another embodiment of the cell culture device having the auxiliary stirring element of the present invention. The cell culture device 600 includes a first chamber 601, a second chamber 602, a cell carrier 603 located in the first chamber 601, and an air pipe 604 and a ventilation ring 605. The first chamber 601 is installed in the first chamber 601. The two chambers 602 and the second chamber 602 surround the first chamber 601. When using this cell culture device 600, the cell culture device 600 can use an external air compressor 606 in cooperation with valves 607 and 608 to inject air into the first chamber 601 and the second chamber 602 alternately, so that the liquid surface and the carrier 603 Generate relative motion. At the same time, the computer program control valve 609 is used to open and close periodically. The air is sent to the ventilation ring 605 through the air pipe 604, and the gas is injected into the second chamber 602 without the carrier. The effect of stirring can avoid the air bubbles from damaging the cells in the carrier. The seventh figure is another embodiment of the cell culture apparatus having the auxiliary stirring element of the present invention. The cell culture device 700 includes a first chamber 701, a second chamber 702, a cell carrier 703 located in the first chamber 701, a stirring blade 704, a turbine blade 705, an air intake pipe 706 and an exhaust pipe 707; The first chamber 701 is installed in the second chamber 702, and the second chamber 702 surrounds the first chamber 70. When the cell culture device 700 is used, the cell culture device 700 can be compressed by the additional air. The machine 708 cooperates with the valve parts 709 and 710 to inject air into the first chamber 701 and the second chamber 702 alternately, so that the liquid surface and the carrier 703 are relatively moved. At the same time, the computer program control valve 711 is opened and closed periodically, injects air through the air intake pipe 706 and pushes the turbine blades 705, and then drives the rotating stirring blade 704 to achieve the effect of stirring, among which the excess air is It is discharged into the atmosphere through an exhaust pipe 707. The eighth figure is an embodiment of one of the aforementioned turbine blades 705. The air system is driven by the intake air 16 200526775 port 712 'to push the thirsty wheel blade 713 to rotate, and the excess line is discharged from the outlet 714 4. The f-nine figure is another embodiment of the cell culture device with the auxiliary stirring element of the present invention. The cell culture device includes a first chamber 901, a second chamber, 902, a cell carrier 903 and a guide plate 904 located in the first chamber 901; ... To 901 is connected to the second chamber 902, and the first chamber 90m and the second chamber 9G2 can be bonded together or integrated into one, and a deflector 9 () 4 is arranged below the cell carrier 903. The fluid is guided by a guide groove (not shown) in the partition 904 to generate a vortex, which effectively provides the mixing of the culture medium and improves the efficiency of cell immobilization. The tenth to twelfth diagrams are several implementation aspects of the aforementioned deflector. The tenth Lutu is a deflector 100 with a spiral groove 1001, the groove 1001 and the horizontal mask have an inclination angle between 20 degrees and 90 degrees, and the groove 1 The number of spiral turns of 〇1 is greater than one. The eleventh figure is a guide plate 1100 with a flat radial chute 1101. The guide plate 1100 is engraved with more than one set of inclined grooves 1101. At the same time, the inclined groove 1101 and the horizontal plane It has an included angle between 20 degrees and 90 degrees. The twelfth figure is a cone-shaped flow-guiding baffle plate 1200. The flow-guiding baffle plate 1200 is engraved with more than one set of radial inclined grooves 1201. At the same time, the inclined groove 1201 and the horizontal plane Has an included angle between 2G degrees and 9G degrees. This—The cone-shaped diversion baffle 1200 can prevent bubbles from accumulating below the baffle and create an ineffective area (centered by z 00n), and can guide bubbles to the outside of the baffle so that it will not impact on the carrier. Internal cells. The thirteenth figure is another embodiment of the cell culture device with the auxiliary stirring element of the present invention. The cell culture device 300 includes a first chamber 1301, a second chamber 1302, a cell carrier 1330 and a guide plate 1304 located in the first chamber 130, and the first chamber 13 〇1 is installed in the second chamber 1302, and the second chamber 1302 surrounds the first chamber 1301; the deflector 1304 is arranged below the cell carrier 17 200526775 body 1303. The aforementioned guide plate 1304 may be the guide plate shown in the tenth to twelfth figures. When the cell culture device 1300 is used, the cell culture device 1300 can use an external air compressor 1305 to cooperate with valves 1306 and 1307 to push air into the first chamber 1301 or the second chamber 1302 alternately; The gas alternately switched between the chamber 1301 and the second chamber 1302 can cause the culture medium to reciprocate between the first chamber 1301 and the second chamber 1302. The baffle (not shown) in the partition plate 1304 can guide the medium in and out of the first chamber 1301 and the second chamber 1302 to generate vortex, which effectively provides the mixing of the medium and improves the efficiency of cell immobilization. The fourteenth figure is another embodiment of the cell culture device having the auxiliary stirring element of the present invention. The cell culture device 1400 includes a first chamber 1401, a second chamber 1402, a cell carrier 1403 and a ring-shaped guide plate 1404 located in the first chamber 1401, and the first chamber 1401 is installed in In the second chamber 1402, and the second chamber 1402 surrounds the first chamber 1401; the ring-shaped guide plate 1404 is disposed below the second chamber 1402, and the annular guide plate 1404 More than one group can be installed at the same time, so that the annular partition 1404 can effectively provide the uniformity of the mixing of the culture medium and the cell immobilization. When the cell culture device 1400 is used, the cell culture device 1400 can use an external air compressor 1405 to cooperate with valves 1406 and 1407 to push air into the first chamber 1401 or the second chamber 1402 alternately; The gas alternately switched between the chamber 1401 and the second chamber 1402 can cause the culture medium to reciprocate between the first chamber 1401 and the second chamber 1402. The baffle (not shown) in the partition plate 1404 can make the culture medium pushed by the gas generate vortex, improve the uniformity of the mixture of the medium before entering the first chamber 1401, effectively provide the mixture of the medium and improve the cell fixation.化 efficient. The fifteenth and sixteenth figures are one of the embodiments of the aforementioned annular diversion baffle. In the fifteenth figure, at least one radiating linear guide groove 1501 is engraved on the annular guide plate 1500, and the guide groove and the horizontal mask have an included angle between 20 degrees and 90 degrees. 18 200526775 The sixteenth figure is another embodiment of a ring-shaped baffle. At least one radial spiral diversion trough brain is engraved on the annular diversion baffle 1600, wherein the curvature radius of each spiral spiral diversion groove 16G1 may be the same or different, and the diversion t 1601 and The horizontal mask has an angle between 20 degrees and 90 degrees. The seventh figure is a cell culture device with an auxiliary stirring element according to the present invention. In a cell culture device! Including the first chamber measurement, the second chamber 17G2, the cell carrier stage 丨 in the __th stage of the 丨 stage, and the mixing leaf 1704 located in the second chamber to the 1702; the first chamber 1701 is The second chamber 1702 is installed in the second chamber 1702, and the second chamber 1702 surrounds the first chamber ΐ700; disturb the leaves! The outline is set at the bottom of the second chamber, and a mechanical mixing device is installed below the second chamber. Lu uses the motor 1705 to drive the shaft ⑽ to drive the secret leaves to carry the benefits of mixing. 7 Experimental Example 1 An experiment was performed with a cell culture device having an auxiliary stirring element according to the present invention (refer to FIG. 6). In this experiment, a cell culture device without any auxiliary teaching element and a cell culture device with the auxiliary teaching element of Fig. 6 were respectively measured. The pH value was balanced between ¥. The eighteenth figure is the relationship between system value and time: 'Eight center line (A) is a cell culture device without any auxiliary device, and the line ⑻ is a cell culture device with auxiliary ㈣ element in the sixth figure, and the ventilation time is 1 〇 # See 'f (C) is the cell culture device with auxiliary feeding element in the sixth figure, aeration two', ', W seconds. From the pH reaction time in the eighteenth figure, it can be clearly found that it is difficult to achieve a stable mixing time without any words ^ borrowed ^: With the air intake and auxiliary, the mixing time can be shortened to Within 3 minutes. Experimental Example 2 The cell culture device of the present invention having an auxiliary feeding element is subjected to an experiment (see the diagram of harvesting). In this experiment, cells that do not contain any auxiliary feeding element are measured separately. Ling Chi and cell culture system with auxiliary stirring element shown in the first figure 19 200526775 pH equilibration time. The nineteenth picture is the diagram of the cell culture device value / interval relationship in the first diagram (A), where the line contains any auxiliary elements for cell culture. Set the [value and time relationship, line (B) is not Time can clearly find the relationship between the two without any auxiliary L. From the reaction of p H to other embodiments, all the features disclosed in this specification can be combined with other methods in any form. Features disclosed in this specification may be replaced by features having the same, similar, or similar purpose. HI, except for the part explicitly emphasized, all the features disclosed in this specification are only one embodiment of many equal or similar features. For example, for the first-chamber and second-chamber disposition position of the cell culture device in this specification, and the operation mode of the medium for mutual movement between the two chambers, please refer to the full description of Taiwan Patent Application No. 92101625, combined with The auxiliary element of the present invention is incorporated in the present invention. According to the content disclosed in this specification, 'any person familiar with the technical field can easily and clearly understand the important features of the present invention. Without departing from the spirit and scope of the present invention, different changes and modifications can be made to the present invention. Meet different usage purposes and situations. Therefore, other implementations are also included in the scope of the following patent applications. 20 200526775 [Schematic description] The first diagram is a schematic diagram of a cell culture device with stirring leaves. The second figure is a schematic view of a linear radial stirring blade. The third figure is a schematic diagram of a circular arc stirring blade. The fourth figure is a schematic diagram of a curved spiral radial stirring blade. The fifth figure is a schematic view of a parallel plate-shaped stirring blade. The sixth figure is a schematic diagram of a cell culture device with an air line. The seventh diagram is a schematic diagram of a cell culture device with a gas-driven stirring blade. The eighth diagram is a schematic diagram of a turbine blade element. The ninth figure is a schematic diagram of a cell culture device with a deflector. The tenth figure is a schematic view of a spiral groove guide plate. The eleventh figure is a schematic diagram of a flat radial chute flow guide partition. The twelfth figure is a schematic view of a tapered radial groove guide plate. The thirteenth figure is a schematic diagram of a cell culture device with a deflector. The fourteenth figure is a schematic diagram of a cell culture device with a ring-shaped baffle. The fifteenth figure is a schematic view of a radial straight groove-shaped annular diversion baffle. The sixteenth figure is a schematic view of a circular spiral-shaped groove-shaped annular diversion baffle. Figure 17 is a schematic diagram of a cell culture device with a mechanical stirring device. The eighteenth figure is a graph comparing the pH value and time of the sixth figure of the present invention and the cultivation device without the auxiliary stirring element. The nineteenth figure is a graph comparing the pH value and time of the first figure of the present invention and the cultivation device without the auxiliary stirring element. 200526775 [Comparison of main component symbols] 100_—cell culture device 700… cell culture device 101… first chamber 701… first chamber 102… second chamber 702… second chamber 103 --- Cell carrier 703 ... Cell carrier 104 ... Stirring leaf 704 ... Stirring leaf 105 ... Air compressor 705 ... Turbine blade 106, 107 --- Valve 706 ... Air line 200 ... Stirring leaf 707 ... Exhaust line 201 —Solid arc top surface 708——Air compressor 202… Blades 709, 710, 711… Valve 300… Stirring blade 712—Air inlet 301—Arc top surface 713… Turbine blade 302 ... Blade 714 ... Exhaust port 400 ... Stirring leaf 900 --- Cell culture device 401-S] 5 Top surface of the melon 901 ... First chamber 402 ... Leaf 902 ... Second chamber 500--Stirring leaf 903 ... Cell carrier 501--Leaf 904--Diversion Separator 600… Cell culture device 1000—Deflector 601… First chamber 1001—Groove 602… Second chamber 1100—Deflector 603… Cell carrier 1101… Groove 604… Air tube Road 1200—Baffle 605… Vent ring 1201… Groove 606… Empty Compressor 1300 --- Cell culture device 607 '608 > 609-Valve 1301 ... First chamber 200526775 1302 ... Second chamber 1501 ... Groove 1303 ... Cell carrier 1600 ... Ring plate 1304 ... Drainage compartment Plate 1601 ... Groove 1305 ... Air compressor 1700 ... Cell culture device 1306, 1307 ... Valve 1701 ... First chamber 1400 ... Cell culture device 1702 ... Second chamber 1401 ... First chamber 1703 ... Cell carrier 1402 ... The second chamber 1704 ... agitating blade 1403 ... cell carrier 1705 ... motor 1404-guide plate 1706 --- rotating shaft 1405 --- air compressor 1707 --- air compressors 1406, 1407 ... valves 1708, 1709 ... valves 1500 —Round soil partition 23