1286158 九、發明說明： 【發明所屬之技術領域】 本發明係有關於一種生物反應器，尤其係有關於一種 虹吸式生物反應器’其無需藉由機械動力系統即可進行反 應器中培養液的置換。 【先前技術】 膝關節的軟骨缺損或退化性關節炎常會造成患者膝關 節的疼痛。對年老的病患來說，通常係藉由置換人工關節 來作為治療的手段，而對於年輕的病患來說，一般則係藉 由軟骨鑽洞術(cartilage drilling)或馬赛克形成術來進行治 療。目前最新的技術則係使用骨膜皮瓣及軟骨細胞移植術 進行治療。但前述這些治療的方法皆有其限制，因此運用 組織工程進行膝關節軟骨組織修復的構想便應運而生。 習知運用組織工程製備人工關節軟骨時，必須考量幾 個要素，分別為載體（或稱支架）、細胞、生長調控因子， 以及生物反應器。其中，生物反應器可提供許多重要的功 能’例如用以改善在3D支架（scaffold)上的細胞植入 (seed)、增加物質傳遞(mass transport)效率，以及體外組織 的機械刺激等。因此，將生物反應器應用於人工軟骨組織 的製程中是很重要的事。 對於細胞支架的接種來說，為將分離的細胞散布置於 支架中，首要的目的即建立一個三維的培養系統，此系統 的建立，對於組織生成的進展，將扮演一個重要的角色。 1286158 $般來说’細胞能均勻的接種於支架上，理論上細胞於支 木上生長後亦應呈均勻分布。然而，實際上即使一個小型 的支采（例如，直徑5mm X寬2mm)，欲使高密度的軟骨 細胞(chondrocyte)均勻且有效的分佈於整個支架上 ，仍然 是一個相當大的挑戰。 習知將細胞接種於支架上的方法，係為直接將細胞靜 悲接種於支架上。但研究顯示，此方法的接種效率低、細 胞分布不均、骨架周邊缺乏細胞分布，以及接種的效果會 因操作的方式或操作者的不同而有所差異。為了改善此缺 點’有人提出利用攪拌瓶反應器（spinner flask bi〇react〇r) 來培養細胞，其係藉由將支架置於反應器中，並藉由攪拌 產生對SlL使付懸浮細胞得以流入支架中。相較於上述方 法，此法可顯著地提升靜態接種法的接種效率，以及增加 細胞的分佈均勻度。 然而，利用前述攪拌瓶反應器接種細胞時，由於流體 _ 的對流實際上並無法完全進入支架内部，這將導致整體接 種效率下降以及使得支架表面的細胞密度較高，而導致實 際操作時支架中細胞的分布並不如預期般的均勻。為解決 此一問題’即有人另提出一種將流灌式(perfusion)接種與流 灌式生物反應器整合在一起的方法，此不但能有效地進行 細胞的接種，也能同步進行組織培養。流灌式生物反應器 能將培養液直接灌流並通過已植入細胞之3D支架的孔 洞，藉此即可減少在支架結構周圍與其内部孔洞中之物質 傳遞的限制。因此’此糸統的建立’不但使組織工程的夢 '1286158 程更有效率，也對於組織與反應器之間的處理與轉移的風 險降低。 前述流灌式生物反應器雖可獲得較佳的細胞植入效率 與較高細胞分布均勻性，但生物反應器於培養細胞的過程 中需藉由一機械動力系統提供動力來運輪培養液，此需要 耗費不少的電能。尤其細胞於反應器中進行培養時，二常 需時數天之久，這些所耗費電能的總和是不小的。此外，1286158 IX. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD The present invention relates to a bioreactor, and more particularly to a siphon bioreactor that can perform a culture solution in a reactor without using a mechanical power system. Replacement. [Prior Art] A cartilage defect or degenerative arthritis of the knee joint often causes pain in the knee joint of the patient. For elderly patients, it is usually used as a means of treatment by replacing artificial joints. For younger patients, it is usually done by cartilage drilling or mosaic formation. treatment. The latest technology is currently treated with periosteal flaps and chondrocyte transplantation. However, all of the above methods of treatment have their limitations, so the concept of tissue engineering for the repair of knee cartilage tissue has emerged. Conventional use of tissue engineering to prepare artificial articular cartilage requires consideration of several elements, namely carriers (or scaffolds), cells, growth regulators, and bioreactors. Among them, bioreactors can provide many important functions, such as to improve cell seeding on 3D scaffolds, increase mass transport efficiency, and mechanical stimulation of tissue in vitro. Therefore, it is very important to apply a bioreactor to the process of artificial cartilage tissue. For the inoculation of cell scaffolds, in order to disperse the separated cells in the scaffold, the primary purpose is to establish a three-dimensional culture system, and the establishment of this system will play an important role in the progress of tissue generation. 1286158 $Generally speaking, cells can be uniformly inoculated on the scaffold. In theory, the cells should be evenly distributed after growing on the branches. However, in fact, even a small branch (for example, 5 mm in diameter X and 2 mm in width) is a considerable challenge to uniformly and efficiently distribute high-density chondrocytes throughout the stent. It is conventional to inoculate cells on a scaffold by directly inoculating the cells on the scaffold. However, studies have shown that this method has low inoculation efficiency, uneven cell distribution, lack of cell distribution around the skeleton, and the effect of inoculation varies depending on the mode of operation or the operator. In order to improve this disadvantage, it has been proposed to use a spinner flask (spinner flask bi〇react〇r) to culture cells by placing the stent in a reactor and by stirring to cause the suspension cells to flow into the S1L. In the bracket. Compared with the above method, this method can significantly improve the inoculation efficiency of the static inoculation method and increase the uniformity of cell distribution. However, when the cells are inoculated with the aforementioned stirred bottle reactor, since the convection of the fluid_ does not actually enter the inside of the stent completely, this will result in a decrease in the overall inoculation efficiency and a higher cell density on the surface of the stent, resulting in the actual operation of the stent. The distribution of cells was not as uniform as expected. In order to solve this problem, a method of integrating perfusion inoculation with a flow-through bioreactor has been proposed, which not only enables efficient cell inoculation but also simultaneous tissue culture. The flow-through bioreactor can directly perfuse the culture fluid through the pores of the 3D scaffold that has been implanted into the cell, thereby reducing the restriction of material transfer around the scaffold structure and its internal pores. Therefore, the establishment of this system not only makes the organizational engineering dream more efficient, but also reduces the risk of processing and transfer between the organization and the reactor. Although the above-mentioned flow-through bioreactor can obtain better cell implantation efficiency and higher cell distribution uniformity, the bioreactor needs to be powered by a mechanical power system to transport the culture solution in the process of culturing the cells. This requires a lot of power. Especially when cells are cultured in a reactor, it usually takes several days, and the sum of these consumed electric energy is not small. In addition,
於細胞培養的過程中如遇到無預期的停電時，先前的細胞 培養工作即有可能功齡一匱。 【發明内容】 為解決前述習知技術之問題，本發明之目的即在於提 供-種虹吸式生物反應器’其無需任何機械動力系統來運 达培養液，僅需藉由重力即可執行反應財培養液的置換。 、為達成本發明之目的，根據本發明所指出之一種虹吸 式生物反應器，包含： -用以裝載騎狀培輪儲存容器、；以及 -經由-管路與該培養液儲存容器連通的反應腔室， 献應腔室内部能容納一用以供細胞 之支架，且其設置高度低_培錢儲縣 其中，該反應腔室的下部連接一於垂㈣ 之虹吸管，且該虹吸管於垂直面上 反應腔室的頂點。 呵^不回於该 位於本發明虹吸式生物反應器之培養液儲存容器中的 1286158 培養液，可藉由重力經由管路流至其内裝載有支架之反應 腔室中。當流入反應腔室中之培養液的液面高度高於虹吸 管的最高點時，位於虹吸管内的培養液即會受重力影響， 順著虹吸管流出，藉此即可於反應腔室内形成虹吸， 將反應腔室中之培養液導出，使支架暴露於空氣中，並得 以充分與氧氣制。之後，重力會魏錢再次充滿反應 腔室，而使支架再度浸潰於培養射，朗下—個培養液 流出的循環。 本發明虹吸式生物反應器中，培養基自培養液儲存容 器流過反應腔室的流祕產生—類似f知流灌式生物反應 器的流灌效應。本發明虹吸式生物反應器的系統中無需任 何電源的供應’僅需藉由重力及虹吸效應即可輕易的置換 反應腔室内的培養基。 、 本發明將藉由參考下列的實施方式做進—步的說明， 在此所述之實财式並秘制本發明前面所_之内容。 热習本發明之技藝者，可做些許之改良與 離本發明之範疇。 1-彳乃+肌 【實施方式】 參閱第一圖，為根據本發明所指出之虹 器的平面:㈣。本發明虹吸式生物反應器10，包= 養液儲存m，以及―經由管路16 * 12連通的反應腔室14。苴由 ，、u養液儲存名 η 至4其中，培養液儲存容器12係斥 儲放大量㈣魏(未_)，其尺寸可_需大小^ 1286158 ，培養液儲存容 器的下‘又有-培養液流出口 18，用以使於 /:儲:容器〗2内的培養液得以經由此培養液流出口、士 =使培養液儲存容器12,便於裝填培 ： ==充:2°’:其上較佳設有-可重複： p ]2 〜為避免培養液流出時於培養液錯存容 生負遷效應，致使培養液不易流出，封口蓋U 12的上部上較佳設有一氣孔(未顯示）， 人18時’外部空氣得以自此氣孔t流 :以平衡培養液儲存容器12 _氣I，以避免產生負 物f °^空氣中可能的污染物質(例如，灰塵、微生 液，培麵齡容1112㈣污染培養 示），科以仰=^又置一空氣過遽裝置或一過濾膜(未顯 J精以濾除空軋中的污染物質。 口 24與-氣體置有一培養液流入 拉m 下部則設有—廢液排放口 28。 ^：：：；：：^ 反ϋ形虹吸总3〇。"；另廢液排放口 28上連通有一 可仗及虛1 I虹吸管3〇位於垂直面上的最高點， 計，々但14内所欲保持之培養液的液面高度來設 ^取祕的高度需不高於反應腔室14的頂點。 計成器12與反應腔室14可依需要設 5 11刀離的形式，此於本發明中並沒有特 1286158 別$限制，但培養液儲存容器12的設置的高度需高於反應 腔至14，以使培養液儲存容器I)内的培養液得以藉由重 力’經由管路16流至反應腔室14内。 反應腔室14的尺寸與形狀，於本發明中並沒有特別的 限制，其可依所需培養之支架大小來設計。為使用以培養 細胞的支架可易於放入反應腔室14内，反應腔室14的上 部上可進一步開設一支架放入口 32,藉此便於支架的放 入。且支架放入口 32上較佳設有一可重複開關之上蓋34， ’以避免外部污染物質經由支架放入口 32進入反應腔室14 内。另外’為避免空氣中的污染物質(例如，灰塵、微生物… 等）自氣體流入口 26流入反應腔室14内，氣體流入口 26 其上較佳係進一步設置一過濾裝置或一過濾膜(未顯示）， 藉以濾除空氣中的污染物質。此外，為避免空氣中的污染 物質經由虹吸管30的末端進入反應腔室14内，較佳為於 虹吸管30的末端上連接一反U形鵝頸管36。此鵝頸管36 最高點的高度需不高於虹吸管30的最高點。 Α習本發明技術領域之技藝者，經閱讀本發明說明書 後可了解到，本發明培養液儲存容器12上可設複數個流出 口 18 ’藉以同時連接複數個反應腔室14，藉此即可藉由一 種培養液同時做複數種不同支架的零時差培養。 參閱第二至第四圖，為本發明虹吸式生物反應器操作 實施例之連續動作示意圖。本發明虹吸式生物反應器1〇 於使用時，首先係將已預先植入有欲進行培養之細胞的支 架放置於反應腔室14内。另外，將用以培養該細胞之培養 10 1286158 液W預先配製並滅g後置人培#_存容器中，如第 ^圖二=。為避免於培養液4()注人培養液儲存容器Η中 時’培養液先行經由管路16流入反應腔室心，較佳為 於培養液儲存容器12的流出口 18處或於管路㈣進一步 設置一閥門42。 接著，將前述之閥n 42開啟。此時，培養液儲存容器 12内的培養液4G即會受重力作用，由流出口 18通過管路 16流入反應腔室14内。直到位於反應腔室14内的支架38 > 完全浸潰於培魏40巾，藉此即可使支架騎培養，如第 三圖所示。 當培養一段時間後，欲更新反應腔室14内的培養液 40時，僅需藉由將培養液4〇繼續注入至反應腔室μ中， 使培養液40液面超過虹吸管3〇的最高點。此時，超過虹 吸管30最鬲點的培養液4〇會受重力作用而向下流出，藉 此即會於虹吸管30中產生虹吸現象，進而將反應腔室14 内的培養液40完全抽出，經由虹吸管3〇排放掉。反應腔 . 至14内的培養液4〇被抽離時，在其内部會產生負壓效應。 由於氣體較培養液的流動速度為快，因此負壓效應產生 時’空氣會首先經由氣體流入口 26流入反應腔室14中。 此時’支架38會充分暴露於空氣中，藉此即可使細胞增加 與氧氣的接觸量，如第四圖所示。 隨後培養液40會受重力影響，再次自培養液儲存容器 12流至反應腔室14内（如第三圖所示），進行下一個培養循 環。 11 1286158 前述反應腔室14内之培養基的更換速率，可藉由選擇 適當之管路16的内徑大小來控制培養液40流入反應腔室 14内的速率來達成，亦可藉由控制閥門42的開啟或關閉 來達成。此外，前述通入空氣的步驟亦可藉由控制閥門 42，來確保空氣得以先行進入反應腔室14内，或控制支架 暴露於空氣中的時間。In the event of an unexpected power outage during cell culture, the previous cell culture work is likely to be at the same age. SUMMARY OF THE INVENTION In order to solve the problems of the prior art described above, it is an object of the present invention to provide a siphon bioreactor that does not require any mechanical power system to deliver the culture solution, and only needs to perform the reaction by gravity. Replacement of the culture medium. For the purposes of the present invention, a siphonic bioreactor according to the present invention comprises: - a loading tank for loading a riding wheel, and - a reaction via a conduit to the culture fluid storage container a chamber, the interior of the chamber can accommodate a support for cells, and the height of the chamber is low. The lower part of the reaction chamber is connected to a siphon of the vertical (four), and the siphon is in a vertical plane. The apex of the upper reaction chamber. The 1286158 culture solution in the culture solution storage container of the siphon bioreactor of the present invention can be flowed by gravity to a reaction chamber in which the stent is loaded. When the liquid level of the culture liquid flowing into the reaction chamber is higher than the highest point of the siphon tube, the culture liquid located in the siphon tube is affected by gravity and flows out along the siphon tube, thereby forming a siphon in the reaction chamber, which will The culture medium in the reaction chamber is led out, and the stent is exposed to the air and sufficiently made with oxygen. After that, the gravity will fill the reaction chamber again, and the stent will be immersed again in the culture shot, and the circulation of the culture fluid will flow out. In the siphon bioreactor of the present invention, the flow of the medium from the culture solution storage container through the reaction chamber is generated - similar to the flow irrigation effect of the flow-through bioreactor. The system of the siphon bioreactor of the present invention does not require any supply of power supply. The medium in the reaction chamber can be easily replaced by gravity and siphon effects. The present invention will be described with reference to the following embodiments, and the present invention will be described herein. Those skilled in the art will be able to make some modifications and are within the scope of the invention. 1-彳乃+肌 [Embodiment] Referring to the first figure, the plane of the rainbow indicated in accordance with the present invention: (d). The siphon bioreactor 10 of the present invention, package = nutrient storage m, and "reaction chamber 14" that is in communication via line 16*12.苴,, u nutrient storage name η to 4, the culture fluid storage container 12 is the storage amplification amount (four) Wei (not _), its size can be _ need size ^ 1286158, the lower limit of the culture fluid storage container - there are - The culture solution outlet 18 is configured to allow the culture solution in the container to pass through the culture solution outlet port, and to make the culture solution storage container 12 convenient for loading: ==charge: 2°': Preferably, it is provided with - repeatable: p ] 2 〜 in order to avoid the negative migration effect of the culture solution when the culture solution flows out, the culture solution is not easily discharged, and the upper part of the sealing cover U 12 is preferably provided with a pore ( Not shown), when the person is 18 hours, 'external air can flow from this air hole t: to balance the culture liquid storage container 12 _ gas I to avoid the generation of negative substances f ° ^ possible pollutants in the air (for example, dust, micro-liquid , Pei face age 1112 (four) pollution culture showed), the department to set up an air through the device or a filter membrane (there is no J fine to filter out the pollutants in the empty rolling. Port 24 and - gas set a culture The liquid into the lower part of the pull m is provided with a waste liquid discharge port 28. ^:::;::^ The total of the reversed siphon is 3〇.&quo t;; another waste liquid discharge port 28 is connected with a sputum and virtual 1 I siphon 3 〇 at the highest point on the vertical surface, 々, but the liquid level of the culture liquid to be maintained in 14 is set to The height of the reactor 12 and the reaction chamber 14 may be set to be 5 11 knife-offs as needed, which is not limited to 1286158 in the present invention, but the culture solution is stored. The height of the container 12 is set higher than the reaction chamber to 14 so that the culture liquid in the culture solution storage container I) can flow through the line 16 into the reaction chamber 14 by gravity. The size and shape of the reaction chamber 14 are not particularly limited in the present invention, and can be designed according to the size of the stent to be cultured. The stent for culturing the cells can be easily placed in the reaction chamber 14, and a stent insertion port 32 can be further formed on the upper portion of the reaction chamber 14, thereby facilitating the insertion of the stent. Preferably, the holder inlet 32 is provided with a repeatable switch upper cover 34 to prevent external contaminants from entering the reaction chamber 14 via the holder inlet 32. In addition, in order to prevent airborne pollutants (for example, dust, microorganisms, etc.) from flowing into the reaction chamber 14 from the gas inlet 26, the gas inlet 26 is preferably further provided with a filtering device or a filter membrane (not Display) to filter out pollutants in the air. Further, in order to prevent the pollutants in the air from entering the reaction chamber 14 through the end of the siphon tube 30, it is preferable to connect an inverted U-shaped gooseneck 36 to the end of the siphon tube 30. The height of the highest point of the gooseneck 36 is not higher than the highest point of the siphon 30. After reading the specification of the present invention, it can be understood that a plurality of outlets 18' can be disposed on the culture solution storage container 12 of the present invention, thereby connecting a plurality of reaction chambers 14 at the same time. Zero time difference culture of a plurality of different stents is simultaneously performed by one culture solution. Referring to the second to fourth figures, there is shown a schematic diagram of the continuous operation of the embodiment of the siphon bioreactor according to the present invention. When the siphon bioreactor of the present invention is used, first, a stent which has been previously implanted with cells to be cultured is placed in the reaction chamber 14. In addition, the culture medium 10 1286158 W for cultivating the cells is pre-formulated and sterilized, and then placed in a human culture container, as shown in Fig. 2 =. In order to prevent the culture solution 4 () from being injected into the culture medium storage container, the culture medium first flows into the reaction chamber through the line 16, preferably at the outlet 18 of the culture solution storage container 12 or in the line (4). A valve 42 is further provided. Next, the aforementioned valve n 42 is opened. At this time, the culture solution 4G in the culture solution storage container 12 is subjected to gravity, and flows into the reaction chamber 14 through the line 16 through the outlet port 18. Until the stent 38 > located in the reaction chamber 14 is completely immersed in the Peiwei 40 towel, the stent can be cultured by riding, as shown in Fig. 3. When the culture solution 40 in the reaction chamber 14 is to be renewed after a period of culture, it is only necessary to continuously inject the culture solution 4 into the reaction chamber μ so that the liquid level of the culture solution 40 exceeds the highest point of the siphon 3〇. . At this time, the culture liquid 4 超过 which exceeds the most point of the siphon 30 is flown downward by gravity, whereby a siphon phenomenon is generated in the siphon 30, and the culture liquid 40 in the reaction chamber 14 is completely extracted. The siphon 3 is discharged. Reaction chamber. When the culture solution to 14 is removed, a negative pressure effect is generated inside the reaction solution. Since the gas flows faster than the culture liquid, the negative pressure effect occurs when the air first flows into the reaction chamber 14 via the gas flow inlet 26. At this time, the stent 38 is sufficiently exposed to the air, thereby increasing the amount of contact of the cells with oxygen, as shown in the fourth figure. The culture solution 40 is then subjected to gravity and again flows from the culture solution storage container 12 into the reaction chamber 14 (as shown in the third figure) for the next culture cycle. 11 1286158 The rate of replacement of the culture medium in the reaction chamber 14 can be achieved by selecting the appropriate inner diameter of the conduit 16 to control the rate at which the culture fluid 40 flows into the reaction chamber 14, or by controlling the valve 42. Open or close to achieve. In addition, the aforementioned step of introducing air may also ensure that air is allowed to enter the reaction chamber 14 first by controlling the valve 42, or controlling the time the stent is exposed to the air.
12 6158 【圖式簡單說明】 第— ^ 為根據本發明所指出之虹吸式生物反應器的平面 示意圖； 楚一 一圖為本發明虹吸式生物反應器操作實施例之第一步 . 驟的示意圖； 第二圖為本發明虹吸式生物反應器操作實施例之第二步 驟的示意圖；以及 第四圖為本發明虹吸式生物反應器操作實施例之第三步 • 驟的示意圖。 【主要元件符號說明】 ίο虹吸式生物反應器 12 培養液儲存容器 14 反應腔室 16 管路 18 培養液流出口 φ 20 培養液填充口 22 封口蓋 24 培養液流入口 26 氣體流入口 28 廢液排放口 30 虹吸管 32 支架放入口 34 上蓋 36 鵝頸管 13 1286158 38 支架 40 培養液 42 閥門12 6158 [Simplified description of the drawings] - ^ is a schematic plan view of the siphon bioreactor according to the present invention; Figure 1 is a first step of the operation example of the siphon bioreactor of the present invention. The second diagram is a schematic diagram of the second step of the operation example of the siphon bioreactor of the present invention; and the fourth diagram is a schematic diagram of the third step of the operation example of the siphon bioreactor of the present invention. [Main component symbol description] ίο siphon bioreactor 12 culture fluid storage container 14 reaction chamber 16 pipeline 18 culture fluid outlet φ 20 culture fluid filling port 22 sealing cap 24 culture fluid inlet 26 gas inlet 28 waste liquid Discharge port 30 siphon 32 bracket inlet 34 upper cover 36 gooseneck 13 1286158 38 bracket 40 culture fluid 42 valve