1238851 玖、發明說明： 【發明所屬之技術領域】 ’特別是有關於一種用於 本發明係有關於一種生物反應器 組織細胞培養之生物反應器。 【先前技術】 a趨ΐί來或隨著生物科技的進步，組織細胞體外培養的技術 氣一1? 了提供組織細胞於體外生長時能有較佳的養分及 乳肢父換模式，目前已有許多不同類型的生物反應器 (:咖叫及相關的培養裝置被開發出來，期能在較短時間 3率=的培養效率在體外培養大量的組織細胞，供應蛋白 二==、組織工程、藥物筛選、生物適應性測試及細胞治 療寺不同應用領域的需求。 利用生物反應器進行體外組織細胞培養的過程中，養分運 =亂體交換料開發設計生物反應器時何或缺的兩個基 辟及在養分運輸方面’可採用授掉、搖晃、灌注、旋 ::體床寻方式使養分均勾分佈於培養槽體中，而在氣體交 :’如何在封閉隔絕外來污染的無菌培養系統，使氣相的 二體二液相培養液，同時使溶於液相的氣體溶出反應器槽 體’以達成氣體傳輸交換的目的目丨θ “时a 铷又㈣目的則疋在目前開發相關的生物 反應态過程中亟需克服的問題。 一般的生物發酵槽體，可使用氣泡進入培養液中，直接藉 二在，中所產生的氣液面，來進行氣體的： 血且織細胞培養系統中’由於培養液内含大量的蛋白質 =二，泡式_氣方式進行氣體交換，於培養液面將產 里的水泡堆積，一方面阻礙了氣體交換也易造成系統的 1238851 污染。 體:此，目前在組織細胞體外培養的系統通常只使用培養槽 之的曝氣方式（via surface aerati〇n)，係藉培養液面愈空氣 培行溶氧及氣體交換，再湘攪拌的方式將氧氣帶至 對於7冑位。此方式僅有培養液面可作為氣體交換的表面， 心Γ應小量組織細胞所需的氧量尚堪夠用，若要進行組織細 ，的大量培養則會受到㈣。此外，由於培養“需旬卜界* 礼保持相通’遂系統中會預留-些開口（如旋鬆的瓶口）使空氣 ，出’也因此各種微生物及污染源往往易從旋鬆的瓶口污染势 養槽。 σ 目前亦有使用人工氣體交換膜如sillc〇n gas exchange embrane，此氣體父換膜可製備成平面或中空纖維管的型式以 與培養液接觸進行氣體交換，例如美國專利第5,658,797、 6’〇〇1’585與6，G8G，581號所揭露者。然其氣體交換效率及抗 性仍顯不足。 【發明内容】 、有鑑於此，本發明之目的係揭露一種蛋殼朱物反應器，其 為-價格低廉、易於取得之天然生物容器，且具有高的氣液交 換通透率以及抗菌阻絕效果。 為了達成上述目的，本發明提供一種生物反應器，包括： -蛋殼’作為該反應||之容器，用以容納—培養液且該蛋殼 至少包括-開口；以及-流體混合裝置，其一端穿過該開口伸 入該蛋殼内，用以浸入一樣品於該培養液中。 生物體早已發展出-套相當完美的體外繁殖培養系統_ 蛋，例如-顆雞蛋僅憑藉本身的結構與養分，在體外環境下， 1238851 即可提供大量的氣體交換及代 、及代謝循％所需的所有物質，使此一 糸、4於數週之内可成長出大體5q公克的複雜組織。 。。本發明擬以仿生科技的概念，以蛋殼作為生物反應器的容 二：t入液體授拌循環系統’期利用殼膜高通透氣體及阻絕 从生物侵入的特性，於濟外真 '體外長期培養組織細胞時，提供組織細 月l所需的大量交換氣體與密閉的生長空間。 A 了 述目的’本發明另提供—種生物反應器，包括： 二Uu生物反應益單π ’其中每一生物反應器單元至少包括— 成以及机體合裝置’該蛋殼係作為該反應器之容器，用 :容納-培養液，且該蛋殼至少包括_開口，該流體混合裝置 :、一端係穿過該開口伸人該蛋殼内，用以浸人—樣品於該培養 液中，以及一連接管路’連接該等流體混合裝置。 為讓本發明之上述目的、特徵及優點能更明顯易懂，下文 特牛-較佳實施例’並配合所附圖式，作詳細說明如下： 【實施方式】 實施例 兑在製作此蛋设生物反應器前，係先對所選定的材料·蛋進行 則處理序，首先，提供_完整無裂痕的蛋，其可擇自例如 禽鳥類的蛋如鴨蛋'雞蛋、鵝蛋或駝鳥蛋，本實施例係選擇以 鴨蛋作為製作生物反應器10的材料。 以下係對蛋殼結構12作-詳細制，蛋殼12共由三個主 要層次所構成，最内部為—内層膜（inner sheu membrane)，是 厚度大體為15微米的非纖維薄葉（糖蛋白外套（mande)圍繞蛋 白質中心所組成），具有阻隔微生物侵入的功能，該内層膜外側 1238851 為一外層膜，其厚度為300微米，外層膜具有極強的韌性與通 乳性，其氣孔可交換氣體及阻隔微生物，此兩層膜除在氣室部 位外皆緊密相合。 '殼膜的最外層則是以碳酸鈣組成的無機蛋殼，碳酸鈣晶體 在蛋殼的大部分區域緊密聚集。每顆蛋平均約有1〇,〇〇〇個小 孔，平均孔徑為1 7微米，係作為氣體交換的管道。1238851 发明 Description of the invention: [Technical field to which the invention belongs] 'In particular, it relates to a bioreactor used in the present invention for a bioreactor for tissue cell culture. [Previous technology] A trend or with the advancement of biotechnology, the technology of in vitro culture of tissue cells provides a better nutrient and paternal replacement mode for tissue cells when grown in vitro. At present, there are already Many different types of bioreactors have been developed (eg, coffee and related culture devices), and a large number of tissue cells can be cultured in vitro in a short period of time. Screening, biocompatibility testing, and cell therapy needs in different application areas. In the process of in vitro tissue cell culture using bioreactors, nutrient transport = two basic elements that are missing when developing and designing bioreactors. In terms of nutrient transport, 'can be used to distribute, shake, infuse, spin :: body bed search to distribute nutrients uniformly in the culture tank, and in the gas exchange:' How to seal and isolate the externally contaminated sterile culture system The gas-phase two-body two-liquid culture solution is made, and the gas dissolved in the liquid phase is dissolved out of the reactor tank at the same time to achieve the purpose of gas transmission and exchange 丨 θ "时 a 铷 又 ㈣ The purpose is to solve the problems that need to be overcome in the current development of related biological reaction states. In general biological fermentation tanks, air bubbles can be used to enter the culture medium, and the gas-liquid surface produced by the two-phase can be used to carry out gas. : In the blood and tissue culture system, 'because the culture medium contains a large amount of protein = two, gas exchange is performed by the bubble-gas method, and the vesicles in the culture liquid are accumulated, on the one hand, it hinders the gas exchange and is easy to cause System 1238851 is contaminated. Body: Therefore, the current system of in vitro culture of tissue cells usually only uses the aeration method of the culture tank (via surface aeration), which is based on the cultivation of dissolved oxygen and gas exchange through the culture surface. The method of stirring is used to bring oxygen to the 7 胄 position. In this method, only the culture liquid surface can be used as a surface for gas exchange. The amount of oxygen required for a small amount of tissue cells is sufficient. A large number of cultures will suffer. In addition, due to the cultivation of "need to communicate with the world, the etiquette must be kept in common", some openings (such as the loose bottle mouth) will be reserved for air, and therefore These microorganisms and pollution sources tend to contaminate potential tanks from unscrewed bottles. Σ At present, artificial gas exchange membranes such as sillcon gas exchange embrane are also used. This gas-exchange membrane can be prepared as a flat or hollow fiber tube. Contact with the culture medium for gas exchange, such as disclosed in U.S. Patent Nos. 5,658,797, 6'00001'585 and 6, G8G, 581. However, its gas exchange efficiency and resistance are still insufficient. [Summary of the Invention], there are In view of this, the object of the present invention is to disclose an eggshell and chubby reactor, which is a natural biological container that is inexpensive and easy to obtain, and has a high gas-liquid exchange permeability and an antibacterial barrier effect. In order to achieve the above object, The present invention provides a bioreactor, comprising:-an eggshell 'as a container for the reaction || for containing a culture liquid and the eggshell at least includes-an opening; and-a fluid mixing device, one end of which extends through the opening It is inserted into the egg shell for immersing a sample in the culture solution. Organisms have already developed a set of quite perfect in vitro reproduction and culture systems _ eggs, for example-eggs rely on their structure and nutrients alone. In an in vitro environment, 1238851 can provide a large amount of gas exchange and generation, and metabolic cycle. All the materials needed make it possible to grow a complex structure of approximately 5q grams in a few weeks. . . The present invention intends to use the concept of bionic technology and take eggshell as the bioreactor's container two: t-into the liquid mixing circulation system in the 'phase' uses the shell membrane high-pass air-permeable body and the characteristics of blocking the invasion from organisms, and it is used in the outside world. When culturing tissue cells for a long period of time, it provides a large amount of exchange gas and closed growth space required for tissue fine month l. A The stated purpose 'The present invention also provides a bioreactor, including: two Uu biological reaction benefits π' wherein each bioreactor unit includes at least-a body and a body combination device 'the eggshell system as the reactor The container is used to hold the culture medium, and the egg shell at least includes an opening. The fluid mixing device: one end is inserted into the egg shell through the opening, so as to immerse the human in the sample in the culture medium. And a connection line 'connects the fluid mixing devices. In order to make the above-mentioned objects, features, and advantages of the present invention more comprehensible, the following is a detailed description of the preferred embodiment of the present invention and the accompanying drawings: [Embodiment] The embodiment is to make this egg device. Before the bioreactor, the selected materials and eggs are processed first. First, complete and crack-free eggs are provided, which can be selected from, for example, eggs of poultry and birds such as duck eggs, goose eggs, or ostrich eggs. In the embodiment, duck eggs are selected as materials for manufacturing the bioreactor 10. The following is a detailed description of the egg shell structure 12. The egg shell 12 is composed of three main layers. The inner part is the inner sheu membrane, which is a non-fibrous thin leaf (glycoprotein coat) with a thickness of approximately 15 microns. (Mande) is composed around the center of the protein), and has the function of blocking the invasion of microorganisms. The outer side of the inner film 1238851 is an outer film with a thickness of 300 microns. The outer film has strong toughness and milk permeability, and its pores can exchange gas. As well as blocking microorganisms, the two layers are tightly connected except at the air chamber. 'The outermost layer of the shell membrane is an inorganic egg shell composed of calcium carbonate, and calcium carbonate crystals are tightly packed in most areas of the egg shell. Each egg has about 10,000 pores on average, with an average pore size of 17 microns, and is used as a gas exchange duct.
接著，利用潤濕的紙巾輕輕擦拭蛋殼12上的髒汙，切勿 使用刷子刷洗，以避免具有延緩微生物侵入功能的角皮層被除 去。之後，進行一開口 14的製作，開口 14可由如鑽鑿或研磨 的方式形成，本實施例係以電動鑽鑿的方式對蛋殼12的徑向 鑽馨-孔洞’以形成此—開π 14，開口 14的直徑大體可為 1.5〜2·0厘米，在此為17厘米。續利用真空馬達抽除蛋殼u 内原有的内容物如蛋白、蛋黃繫帶等物質。 接著，浸泡空蛋殼12於去離子水中大 達1·5小時’再以大量的去離子水沖洗大體2〜3二欠，此處沖 2次，取後以濃度為01mM，ρΗ值為7 4，體積1〇毫升的ρ] 緩衝溶液浸潤備用。另過程中需以1QKGY的^射線消毒滅菌 以上即完成一製作生物反應器1〇的前置工作。Next, use a moistened paper towel to gently wipe the dirt on the egg shell 12, and do not use a brush to avoid the corneal layer that has the function of delaying the invasion of microorganisms. After that, an opening 14 is made. The opening 14 can be formed by drilling or grinding. In this embodiment, the radial drilling of the egg shell 12 is performed by electric drilling. The diameter of the opening 14 may be generally 1.5 to 2.0 cm, and is 17 cm here. Continue to use the vacuum motor to extract the original contents such as protein, egg yolk and other substances in the egg shell u. Next, soak the empty eggshell 12 in deionized water for about 1.5 hours', and then rinse roughly 2 to 3 times with a large amount of deionized water. Here, rinse twice, and take it at a concentration of 01mM and a ρΗ value of 7 4. Infiltrate the buffer solution with a volume of 10 ml. In the other process, it is necessary to sterilize and sterilize with 1QKGY ray. The above-mentioned pre-work of making a bioreactor 10 is completed.
接下來，請參閱第i目，說明本發明之一實施例，生物 應為之製作。首先’提供—已具有開口 14的蛋殼η，作為 物反應器1〇的容器。續植入-流體混合裝置16至蛋殼容器 中’机體此合裳置16的結構敘述如下，該裝置的製作 為玻璃、塑膠或金屬，本實施例選定塑膠為該裝置的 料，其直徑大體可介於U〜U厘米，此處為Μ厘米。裝置 部具備-容置空間18，其外壁結構製作成包含_無孔、 一多孔隙或網狀22結構兩部分。 “ 9 1238851 =流體混合裝置16時，一培養樣品24已固定於該裝 =工間18中’而包圍樣品24所在區域的外壁結構係為 二隙或網狀22的部分，以利後續培養液養分的進出交換， β的樣24可為一貼附性的動物或植物細胞例如軟骨 '肌 肉，幹細胞等’本實施例所選用的培養樣品為軟骨。上述用來 固定樣品24的方法與支架材料料載於後。樣品_可為縣 洋性的動物或植物細胞例如細g或大腸桿菌等，而懸浮性㈣ 可由流體混合裝置16的頂部注人，經過該農置進人整個 槽體。 ° 接著’由流體混合裝置16的頂部注入一培養液％，培養 液26可包括組織細胞培養液、蛋白液、蛋黃液、純水、 糖水溶液、蔗糖水溶液、果糖水溶液、纽切水、林格氏液 或含離子之水溶液等，此處選用D祕似。，s改良的Eagle，s於 養基（DMEM)作為本試驗培養溶液。 口 之後’塗佈-壓克力骨水泥28於流體混合裝置16刚 12之間的空隙’以固定、黏合流體混合装置16與蛋殼η，其 中壓克力骨水;尼28係由甲基丙烯酸醋聚合物所構成。固定Next, referring to item i, an embodiment of the present invention will be described, and the organism should be made for it. First, the eggshell η, which has an opening 14, is provided as a container for the bioreactor 10. Subsequent implantation-fluid mixing device 16 into the egg shell container The structure of the body and the combination device 16 is described below. The device is made of glass, plastic or metal. In this embodiment, plastic is selected as the material of the device, and its diameter Generally it can be between U ~ U cm, here is M cm. The device section is provided with an accommodating space 18, and the outer wall structure thereof is made up of two parts including a non-porous, a multi-porous, or a net 22 structure. "9 1238851 = fluid mixing device 16, a culture sample 24 has been fixed in the equipment = workshop 18 ', and the outer wall structure surrounding the area where the sample 24 is located is a two-gap or net-shaped portion 22 to facilitate subsequent culture fluids The nutrient in and out exchange, β sample 24 may be an adherent animal or plant cell such as cartilage 'muscle, stem cells, etc.' The culture sample selected in this embodiment is cartilage. The above method for fixing sample 24 and the scaffold material The sample is contained in the sample. The sample can be an animal or plant cell of the prefecture, such as fine g or E. coli, etc., while the suspended maggot can be injected from the top of the fluid mixing device 16, and then enter the entire tank through the farm. Next, a culture medium is injected from the top of the fluid mixing device 16. The culture medium 26 may include a tissue cell culture solution, a protein solution, an egg yolk solution, pure water, an aqueous sugar solution, an aqueous sucrose solution, an aqueous fructose solution, a Nucleus water, and Ringer's. Solution or ion-containing aqueous solution, etc., D is chosen here, s modified Eagle, s medicinal solution (DMEM) as the culture solution for this test. After the mouth 'coated-acrylic bone cement 28 mixed with fluid The gap between the devices 16 and 12 is used to fix and bond the fluid mixing device 16 and the egg shell η, in which acrylic bone water is used; Ni 28 is composed of methacrylic acid vinegar polymer. Fixed
後的流體混合裝置16’其多孔隙或網狀22結構的外 於蛋殼殼體12内。 ’'I 最後’覆盍一頂蓋30於流體混合裂置16的頂部，以 整個培養系統10。生物反應器10更包括—攪拌裝置例如為： 力搜拌裝置、機械授拌裝置或流體㈣裂置，#中該磁力授掉 裝置例如是以-外加磁力如慢速磁_機帶動—磁叾3 拌方式’該機械授拌裝置例如是以一外加機械力帶動 ： 的攪拌方式，而該流體攪拌裝置例如是為利用導管導入一 〇 流體的授拌方式。上述磁石34或旋轉器可接於:體 1238851 16的底部 置。 而本實施例所選用的攪拌裝署 夏係為一磁力攪拌裝 溶氧量之測定 請參照第1圖與第2圖，說明試驗組 液％中，氧氣(〇2)與二氧化碳(c〇2)的溶解量 ' =培養 本發明的蛋殼反應器，對昭 1 ^且係私 竣反雁哭〆 對…且C則為以石蠟封閉蛋殼表面的蛋 j應^。弟i圖為隨時間（天數）變化，氧氣溶解量（毫蛋 的"乂圖(試驗組Ε νί·對照組〇,由圖 明(試驗組一6所溶解的氧氣量均 應斤:解的減量高出大體U〜h5毫克/升，為—較佳的蛋殼反 第2圖為隨時間（天數）變化，二氧化碳溶解量（毫克/升）的 比較圖（試驗組Ev& «組由圖中可看出，隨時間變化， 本發明（試驗組E)培養液26所溶解的二氧化碳量均較對照組c 所溶解的二氧化碳量降低大體25〜5〇毫克/升，再次驗證本發明 的蛋殼反應器係為一較佳的蛋殼反應器。 酸鹼值平衡測定 _ 請參閱表1，係比較試驗組E與對照組（Cl與c2)其培養液 酸鹼值的差異，並同時作溶氧量（D〇,毫克/升）、溫度（τ，攝氏）、 乳酉夂（lactate，L，毫克/升）以及葡萄糖（giuc〇se，G，毫克/升）的測 試。本試驗所選用的試驗組E為本發明製備的蛋殼反應器，而 對照組分別為以石蝶將蛋殼所有表面封閉的蛋殼反應器c 1及 市售的稅拌式反應器（Spinner flask) C2。 反應條件如下，分別於反應器中注入體積50毫升，pH值 Ϊ238851 為8·9的偏鹼性DMEM細胞培養 «仆z山、曲— 罝入改/皿攝氏32度 礼化妷遍度為5%的組織細胞培養箱達48小時。 DO T L G PH DMEM 8.9 25.0 172 3,823 8.85 C! 4.7 *' .·? ;.ϋ 二'··*',：：· II 3,791 8.43 c2 5.9 32.7 175 3,785 7.76 E 5.9 32.7 172 3,794 7·81 表1 由表1巾可看出，該試驗組E(即本發明）的蛋殼反應器可 、且4、’、田胞培養箱中的二氧化碳順利進出，達到平衡培 鹼值的作用。 氧氣傳輸速率測試 一 +本測試方法係根據HuaZha〇等人提出的試驗模式，其利用 葡萄糖水解酵素水解葡萄糖成為葡萄糖醛酸，過程中由於需要 氧氣的參與，因此，可推算出各反應器之最大氧氣傳輸速率 值’反應式如下； -The rear fluid mixing device 16 'has a porous or mesh 22 structure outside the egg shell casing 12. '' I Finally ', a cover 30 is placed on top of the fluid mixing crack 16 to complete the culture system 10. The bioreactor 10 further includes a stirring device such as: a force search and mixing device, a mechanical mixing device, or a fluid cracking device. The magnetic force dropping device in # is, for example, an externally-applied magnetic force such as a slow magnetic machine. 3 Stirring method 'The mechanical stirring device is, for example, a stirring method driven by an external mechanical force, and the fluid stirring device is, for example, a stirring method for introducing a fluid through a duct. The magnet 34 or the rotator can be connected to the bottom of the body 1238851 16. In the example, the stirring system used in this example is a magnetic stirring device to measure the dissolved oxygen content. Please refer to Figure 1 and Figure 2 to explain the oxygen content of the test group, oxygen (02) and carbon dioxide (c0). The amount of dissolution '= the eggshell reactor of the present invention is cultivated, and it is a pair of anti-cryptophores, and C is the egg j that seals the surface of the eggshell with paraffin. The figure i shows the change with time (days), the amount of dissolved oxygen (in milligrams) (test group Ε νί · control group 0, as shown in the figure (the amount of dissolved oxygen in test group 6 should be: The weight loss is higher than the general U ~ h5 mg / L, which is—the better egg shell. The second figure is a comparison chart of the amount of carbon dioxide dissolved (mg / L) with time (days). (Test group Ev & «Group by It can be seen in the figure that with time, the amount of carbon dioxide dissolved in the culture solution 26 of the present invention (test group E) is reduced by approximately 25 to 50 mg / liter compared with the amount of carbon dioxide dissolved in the control group c. The eggshell reactor is a better eggshell reactor. PH-base balance determination_ Please refer to Table 1 to compare the difference in the pH value of the culture medium between the test group E and the control group (Cl and c2), and at the same time Tests for dissolved oxygen (D0, mg / L), temperature (τ, Celsius), lactate (L, mg, L), and glucose (giucose, G, mg / L). This test The selected test group E is the eggshell reactor prepared by the present invention, and the control group is Egg shell reactor c 1 with all shells closed and commercially available spinner flask C 2. The reaction conditions are as follows. The reactor is filled with a volume of 50 ml, and the pH value Ϊ 238851 is 8 · 9 partial alkali. DMEM cell culture «仆 z 山 、 曲 — 罝 入 改 / dish 32 ° C polite, 5% tissue cell incubator for 48 hours. DO TLG PH DMEM 8.9 25.0 172 3,823 8.85 C! 4.7 * ' . ·?; .Ϋ Two '·· *', :: · II 3,791 8.43 c2 5.9 32.7 175 3,785 7.76 E 5.9 32.7 172 3,794 7 · 81 Table 1 As can be seen from Table 1, the test group E (that (Invention) The eggshell reactor can, and the carbon dioxide in the cell culture incubator smoothly enters and exits to achieve the effect of equilibrium cultivation. Oxygen transmission rate test 1+ This test method is based on the test proposed by HuaZha〇 et al. Mode, which uses glucose hydrolysis enzymes to hydrolyze glucose to glucuronic acid. Since oxygen is required in the process, the maximum oxygen transmission rate value of each reactor can be calculated as follows:-
GluCose^H^D_gluc()n()ilact〇ne. h2oGluCose ^ H ^ D_gluc () n () ilact〇ne. H2o
Gluconic acid 反應中的氧氣進出量與酵素濃度直接影響到反應速率， 2反應速率又與產物-葡萄糖醛酸的量成一正比關係，遂藉由滴 疋樣本中葡萄糖醛酸的量，即可推算出氧氣進出反應器 _ 〇 一 試驗步驟如下所述，首先，注入50毫升的葡萄糖水溶浪 12 1238851 於忒驗組E與對照組（Ci與Gy的反應器令，其中試驗組E係指 本發明的蛋殼反應器，對照組分別為以石蠟將蛋殼所有表面封 =的蛋殼反應器Ci及市售的攪拌式反應器Q。之後，每隔5 2鐘自各反應器取出1亳升的樣本，並加人過量的氫氧化納以 終止反應，靜置20分鐘後，用鹽酸滴定出葡萄糖醛酸的含量。 續以葡萄糖醛酸的含量對反應時間作圖，所得直線的斜率 ^上0.5即為氧氣的傳輸速率。重複操作上述反應步驟於不同 /辰度的酵素環境時各反應器即可獲得一氧氣傳輸速率的極大The amount of oxygen in and out of the Gluconic acid reaction directly affects the reaction rate with the enzyme concentration. 2 The reaction rate is directly proportional to the amount of the product-glucuronic acid. Then, the amount of glucuronic acid in the titan sample can be calculated. Oxygen in and out of the reactor_〇 One test procedure is as follows. First, inject 50 ml of glucose water soluble wave 12 1238851 into test group E and control group (reactor order of Ci and Gy, where test group E refers to the The eggshell reactor and the control group were the eggshell reactor Ci and the commercially available stirred reactor Q with all surfaces of the eggshell sealed with paraffin. Then, a 1 liter sample was taken from each reactor every 52 minutes. , And added an excess of sodium hydroxide to stop the reaction, and after standing for 20 minutes, the content of glucuronic acid was titrated with hydrochloric acid. Continued to plot the content of glucuronic acid against the reaction time, the slope of the straight line obtained is above 0.5. Is the oxygen transmission rate. When the above reaction steps are repeated in different enzyme environments, each reactor can obtain a maximum oxygen transmission rate.
值，請參照第4圖，說明試驗組E與對照組（Ci與其氧氣傳 輸速率（OTR)的差異。 ” 第4圖的橫座標為酵素濃度，縱座標為氧氣傳輸速率（莫耳 /分鐘.升）’由圖中之試驗結果可看出’試驗組e(即本發明、)的 反應器較其餘㊉組對照組⑹與C2)均為佳，試驗，組E的氧氣傳 輸速率為1.12毫莫耳/分鐘，對照組C2的氧氣傳輸速率為〇 89 毫莫耳/分鐘，而對照组C|的效果最差，該幾乎無法進出此 一反應系統。 貼附性細胞培養測試Value, please refer to Fig. 4 to explain the difference between test group E and the control group (Ci and its oxygen transmission rate (OTR). "The abscissa of Fig. 4 is the enzyme concentration, and the ordinate is the oxygen transmission rate (mol / min. L) 'From the test results in the figure, it can be seen that the reactor of the test group e (that is, the present invention) is better than the rest of the ㊉ group control group ⑹ and C2). In the test, the oxygen transmission rate of group E was 1.12 millimeters. Mole / minute, the oxygen transmission rate of the control group C2 is 089 millimoles / minute, while the control group C | has the worst effect, and it is almost impossible to enter and exit this reaction system.
在進行貼附性細胞培養前，須先製作一生物性支架以〈 該貼附性細胞的附著生長，太岬私^ Μ + 、 1 ^本5式驗即選定以開環聚合方式製」 得到的PLGA高分子（分子量奸擔 1 丁 里根據 gel permeatic chromatography 測定為 20〇,〇〇〇)，祚焱 & ；作為此一生物支架的材料 以下說明該生物性支架的製作程序。 首先，置入塊狀的PLGA其八工μ ^ 。刀子於粉碎機中粉碎，並使」 通過60〜80孔目的師網’過篩结果^^權 巾"果可獲得粒徑大體介於177〜2ί 微米的高分子顆粒，之後，添加可奋丨 加了創造孔洞結構的水溶性材5 13 1238851 例如為粒徑大體250微米的氣化鈉顆粒於高分子顆粒中。 _ 入plga高分子顆粒與氣化鈉顆粒以重量比1〇:9〇均勻混 合，隨後置入該混合顆粒於一下端接有抽氣裝置，直徑為7毫 米的圓形過濾模器並壓實之’此時倒入有機溶劑丙“ 粒中浸潤，接著，打開抽氣閥門以產生—負壓抽去多餘:溶 劑’使表面溶融態的高分子顆粒相互黏結。待高分子顆粒相互 ㈣後’由過渡器上方倒入大量的去離子水，同時打開抽氣閥 抽以帶走大部分水分，益洗出内部的氣化納顆粒，進而促 使高分子顆粒固化。 固化後的高分子顆粒自濾器中取出，置於裝有去離子水的 大燒杯中浸泡，在室溫下每六小時換水一次達一個工作天，以 徹底洗出内部殘留的溶劑或鹽粒，最後，再置於攝氏度的 真空供箱中加熱乾燥—天，即可得到—孔徑大體介於⑼〜⑽ 微米，孔隙率大體為9〇v〇l%，孔洞相互連通的多孔性基材，作 為本试驗之生物性支架使用。 製備完成的基材續以手術刀裁切為直徑7毫米，高度2毫 米的圓片狀材料，並以濃度為75%的酒精浸泡消毒6小時，再 用大里無菌的phosphate buffered saline(PBS)溶液置換酒精。 接下來，進行培養細胞接種至多孔隙基材的步驟，本試驗馨 的培養細胞係選自大白鼠大腿骨的軟骨組織。首先，以顯微器 材取出剛新生-週的大白鼠軟骨組織’之後’以滅過菌的組織 剪剪碎並分離關節軟骨，碎片大小以2〇〜4〇孔目的篩網控制在 大體400〜800微米之間。 績收集碎片於15毫升的離心管中，並倒入1〇毫升的 DMEM洛液沖洗三次，沖洗後的碎片，待吸除pBS後，加入濃 度為1毫克/PBS毫升，體肖5毫升的膠原蛋白(c〇Uagenase， 14 1238851 COL) ’共同置於攝氏t 又的i口養相中達24小時。游離的軟骨 細胞以細胞計數盤計數（大體個），並接種於該plga多 孔隙基材上’而完成此一接種步驟。 來置入攻已凡成上述接種步驟的多孔隙基材於蛋殼 反應器中培養兩週，並配合每週更換2次培養液。之後，間隔 不同的培養時間，自反應器中取出材料製成試片，先以PBS沖 洗，再浸於4%福馬林（formaHn)的pBS溶液中固定續以石堪 軟組織包埋切片的方式切片，最後以蘇木紫伊紅（he)、膠原蛋 白免疫木色及沙黃_0(safranin_〇)染色，觀察組織型態、膠原蛋 白種類及分析軟骨細胞間質。 觀察試驗結果可發現’載體孔洞中已長出許多新生的軟骨 組織’且其細胞型態與體内組織相同，另以沙黃-〇染色法染 色，可觀察新生軟骨的細胞間質富含醣胺素 (glyC〇a_glycan)，而若以膠原蛋白免疫染色，則可證實新生 ^織中所合成的勝原蛋白種類為第二型的膠原蛋白，與關節軟 月組成相同。根據本試驗的組織檢體，本發明的蛋殼反應器， :可維持細胞正常的生長型態外’亦可於體外培養貼附性細胞 % ’分泌合成體内組織細胞的間質。 懸浮性細胞培養測試 本試驗所選用的懸浮性細胞為大腸桿菌，試驗組e為轉 =製備的蛋殼反應器，對照組分別為以石墩將蛋殼所有表面封 閉的蛋殼反應器c,及市售的㈣式反應器（响⑽祕）c2。 :驗步驟如下，首先，於各反應器中加入_ %毫升的谇 ，液’並同時注入濃度為1〇6細胞數/毫升的菌液，隨後置於攝 P度惶溫的培養箱中進行—天的培養，初期每2小時取樣 15 1238851 一次，之後，每隔1小時取樣，每次取樣丨毫升。 當菌液漠度為1〇9細胞數/毫升時，其於波長奈米的吸 光測定值A 1 ’藉此可利用線性内插推算各反應器中的細胞濃 度，培養結果如第5圖所示。帛5圖的橫座標為培養時間（小 時），縱座標為吸光值（OD)，纟圖中可看出，試驗组e(即本發 明）的反應器與對照組〇2的㈣式反應器對懸浮性細胞的培養 效果相當’不具統計上的差別，而對照組q㈣的蛋殼反應 器，則因無法提供充足的氧氣，致使内部微生物到後期時幾乎 已停止生長。 此外’本發明亦可透過複數個生物反應器的串接，同時培 養更為大量的細胞組織’以供生化產業上的需求，該連接管^ 的材質可與流體混合裝S 16相同，例如為玻璃或塑膠，在此 使用塑膠材質的管路，而該連接管路上，更可設置複數個幫 浦’以產生壓力差推動流體。 相較於目前眾多的人工生物反應器，本發明揭示一種最完 美的生化反應m可提供充足的培#養分、適度的氣體交 換以及阻止微生物侵入的天然屏障。 雖然本發明已以較佳實施例揭露如上，然其並非用以限定 本發明Μ壬何熟習此技藝I，在不脫離本發明之精神和範圍 内曰可作更動與潤飾，因此本發明之保護範圍當視後附之申 請專利範圍所界定者為準。 16 1238851 【圖式簡單說明】 第1圖係為本發明蛋殼反應器之剖面示意圖。 第2圖係為本發明的反應器與以石蠟封閉蛋殼表面的反應 器之溶氧量比較圖。 第3圖係為本發明的反應器與以石蠟封閉蛋殼表面的反應 器之溶解二氧化碳量比較圖。 第4圖係為發明的反應器與以石蠟封閉蛋殼表面的反應器 及市售的攪拌式反應器之氧氣傳輸速率比較圖。Before performing the adherent cell culture, a biological scaffold must be made to adhere to the growth of the adherent cells. Tai Chishui Private ^ M +, 1 ^ This is selected by the formula 5 in the open-loop polymerization method. PLGA polymer (molecular weight: 1 butyl, measured by gel permeatic chromatography: 20,000), 祚 焱 &; as the material of this biological scaffold, the procedure for making the biological scaffold will be described below. First, a block-shaped PLGA is placed in its octave μ ^. The knife is pulverized in a pulverizer, and the result of sieving through a mesh of 60 to 80 meshes is obtained ^ Right towel " Polymer particles with a particle size of approximately 177 ~ 2 µm can be obtained, and then added Kefen丨 A water-soluble material 5 13 1238851 is added to create a pore structure. For example, sodium carbonate particles with a particle size of approximately 250 microns are added to the polymer particles. _ The plga polymer particles and sodium gas particles are uniformly mixed in a weight ratio of 10:90, and then the mixed particles are placed at the bottom and connected to a circular filter mold with a suction device with a diameter of 7 mm and compacted. "At this time, pour the organic solvent C into the particles to infiltrate. Then, open the suction valve to generate-negative pressure to remove the excess: the solvent 'makes the polymer particles on the surface melted to adhere to each other. Wait until the polymer particles are mixed with each other' Pour a large amount of deionized water from the top of the transition device, and open the air extraction valve to take away most of the water, which will wash out the internal vaporized sodium particles, and then promote the polymer particles to solidify. Remove it, place it in a large beaker filled with deionized water, and change the water every six hours at room temperature for a working day to thoroughly wash out the residual solvent or salt particles. Finally, place it in a vacuum of Celsius The heating and drying in the supply box—day, you can get—porous substrate with a pore size generally between ⑼ ~ 微米 microns, a porosity of 90 vol%, and pores interconnected with each other, used as the biological scaffold in this test. . The prepared substrate was further cut with a scalpel into a disc-shaped material with a diameter of 7 mm and a height of 2 mm, and was soaked and sterilized with 75% alcohol for 6 hours, and then sterilized with phosphate buffered saline (PBS) solution. Replace the alcohol. Next, the step of inoculating the cultured cells on the porous substrate is performed. The cultured cell line of this test is selected from the cartilage tissue of the femur of the rat. First, the newly-wounded cartilage tissue of the rat is removed with a microscope. 'After', the sterilized tissue was cut and shredded to separate and separate the articular cartilage, and the size of the debris was controlled between approximately 400 and 800 microns with a mesh of 20 to 40 holes. Collect the debris in a 15 ml centrifuge tube. Pour 10 ml of DMEM solution and rinse three times. After washing the pBS, add collagen (co. Uagenase, 14 1238851 COL) at a concentration of 1 mg / PBS ml and 5 ml body weight. Co-placed in a nutrient phase at celsius for 24 hours. Free chondrocytes are counted on a cell counter (generally) and seeded on the plga porous substrate to complete this inoculation step. The perforated substrate that has already completed the above-mentioned inoculation step is cultured in an eggshell reactor for two weeks, and the culture solution is changed twice a week. After that, the material is taken out of the reactor to make a test. The slices were washed with PBS, then immersed in 4% formalin (formaHn) pBS solution, fixed and then sliced with Shikan soft tissue embedded sections, and finally hematoxylin (he) and collagen were used to immunize the wood. And safranin_〇 (safranin_〇) staining, observe the tissue type, collagen type and analysis of chondrocyte interstitial. Observation test results can be found that 'the carrier holes have grown a lot of new cartilage tissue' and its cell type It is the same as in vivo tissues. In addition, it is stained with sand yellow-0 staining method, and the interstitial tissue of newly cartilage can be observed to be rich in glycosaminoglycan (glyC〇a_glycan). The type of the synthesized protamine is type II collagen, which has the same composition as the joint soft moon. According to the tissue specimen of this test, the eggshell reactor of the present invention can maintain the normal growth pattern of the cells' and can also culture adherent cells in vitro to secrete and synthesize interstitial tissue cells in vivo. Suspension cell culture test The suspension cell used in this test is E. coli. The test group e is the egg shell reactor prepared. The control group is the egg shell reactor c, which covers all the surface of the egg shell with stone pier. And a commercially available tritium reactor (sounding secret) c2. : The test steps are as follows. First, add _% ml of hydrazone solution to each reactor, and simultaneously inject a bacterial solution with a concentration of 106 cells / ml, and then place it in an incubator at a temperature of P degrees. For day culture, 15 1238851 is sampled every 2 hours at the beginning, and then every 1 hour, every milliliter is sampled. When the inoculum of the bacterial solution is 109 cells / ml, the absorbance measurement value A 1 'at the wavelength of nanometer can be used to estimate the cell concentration in each reactor by linear interpolation. The culture results are shown in Figure 5 Show. The horizontal axis of Figure 5 is the culture time (hours) and the vertical axis is the absorbance value (OD). It can be seen in the figure that the reactor of the test group e (that is, the present invention) and the control type 〇 reactor of 02 The effect of culturing suspension cells is quite 'not statistically different, while the eggshell reactor of the control group Q㈣ failed to provide sufficient oxygen, which caused the internal microorganisms to almost stop growing at a later stage. In addition, the present invention can also use a plurality of bioreactors in series to cultivate a larger number of cell tissues at the same time to meet the needs of the biochemical industry. The material of the connection tube ^ can be the same as that of the fluid mixing container S 16, for example, Glass or plastic, plastic pipes are used here, and a plurality of pumps can be set on the connecting pipes to generate a pressure difference to push the fluid. Compared with many artificial bioreactors, the present invention discloses a most perfect biochemical reaction that can provide sufficient nutrients, moderate gas exchange, and a natural barrier to prevent microbial invasion. Although the present invention has been disclosed as above with a preferred embodiment, it is not intended to limit the technical skills of the present invention. It can be modified and retouched without departing from the spirit and scope of the present invention. Therefore, the protection of the present invention The scope shall be determined by the scope of the attached patent application. 16 1238851 [Brief description of the drawings] Figure 1 is a schematic cross-sectional view of the eggshell reactor of the present invention. Fig. 2 is a graph comparing the dissolved oxygen amount of the reactor of the present invention with a reactor in which the surface of an egg shell is closed with paraffin. Fig. 3 is a comparison diagram of the amount of dissolved carbon dioxide between the reactor of the present invention and a reactor in which the surface of an egg shell is closed with paraffin. Fig. 4 is a comparison diagram of the oxygen transmission rate between the inventive reactor, a reactor in which the surface of the egg shell is closed with paraffin, and a commercially available stirred reactor.
第5圖係為發明的反應器與以石壤封閉蛋殼表面的反應器 及市售的授拌式反應器之懸浮性細胞培養比較圖。 【符號說明】 10〜生物反應器； 12〜蛋殼； 14〜開口； 16〜流體混合裝置； 18〜容置空間； 20〜無孔隙之外壁； 22〜多孔隙或網狀外壁； 24〜樣品； 26〜培養液； 28〜壓克力骨水泥； 30〜頂蓋； 32〜慢速磁攪拌機； 34〜磁石。 17Fig. 5 is a comparison diagram of the suspension cell culture between the inventive reactor, the reactor in which the surface of the eggshell is closed with stone soil, and the commercially available mixing reactor. [Symbol description] 10 ~ bioreactor; 12 ~ egg shell; 14 ~ opening; 16 ~ fluid mixing device; 18 ~ receiving space; 20 ~ non-porous outer wall; 22 ~ multi-porous or reticulated outer wall; 24 ~ sample 26 ~ culture fluid; 28 ~ acrylic bone cement; 30 ~ top cover; 32 ~ slow magnetic stirrer; 34 ~ magnet. 17