4 267 3 3 經濟部中央標隼局員工消費合作社印製 A7 B7 五、發明説明（1 ) 發明範圍 本發明係有關破裂生長於培養皿之細胞的一種方法，其 利用低壓反向噴流製造出流體剪，力量足以破裂細胞但不 至於破壞其内容物。 發明背景 許多生物技術和發酵的程序中需要大量的細胞在生物反 應器中生長，然後使破裂以釋放出所需產物。欲將細胞破 裂可利用機械、化學、生物學或物理學方法來完成。有許 多*己錄偏好機械破裂法，因機械法可避免使用許多額外的 藥劑[清潔劑、酵素或溶質度作用劑（〇sm〇larity effect〇rs)] 及將物理法（如冰涑/融解法）規模化的困難。 許多破裂微生物的機械法已經被發展出來了。這些方法 通常仰賴流體剪及/或壓縮以破裂細胞壁和膜。然而，並 非所有的生物技術和發酵的程序都使用微生物。動物細胞 逐漸成爲普遍選擇的宿主細胞》由於動物的細胞較大，並 有易碎的細胞膜，導致細胞破裂所需輸入的能量要少的多 。許多爲微生物系統設計的市售現成系統並不適合使用於 動物細胞。 轉子/固定子裝置有一圓筒形的轉子在同心固定子内以 高速旋轉可用來使動物細胞破裂。這些裝置在環形部中製 造出陡峭的速度梯度而在液體中產生足夠的剪應力使細胞 破裂。有一種稱爲凱寇夫壓（Chaikoff Press)的類似裝置， 包含一個圓筒和一個直徑較小的活塞。活塞的運動在環形 空間裡的流體中製造了高剪力，導致細胞破裂。此類型裝 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） --],------<-- - - (請先聞讀背面之注項再填寫本頁) 訂 A7 426733 B7 五、發明説明（2 ) ------------文------訂 (請先閲讀背面之注意事項再填寫本頁) 置也稱爲當色（douncer)，並已用來破裂水痘（Varicella)感 染之MRC-5二倍體肺細胞。然而，這些方法僅於實驗室規 模下可行，無法大規模製造。 超音波或聲能可藉由空化作用在液體中製造高剪力區使 細胞破裂。震動音波（〜20kHz)在液體中製造壓力脈衝。 氣泡在低壓區中形成，當進入高壓力區時便崩潰造成高能 震波。當震波由最初空化位置移動時，即產生高剪力，同 時熱能會散逸到液體中。 有一種連續流體聲能裝置已用來使含水痘病毒的MRC-5 二倍體肺細胞破裂。在非常接近震波起源的區域具有足以 破壞水痘感染體的能量。因此，病毒感染體的損失量決定 於每一單位體積液體產生的成核位置數目；而此數目與輸 入液體内的能量有關。然而，這種連續聲波法也造成處理 液體溫度上升5到1 (TC，而増加了病毒的降解率。降低輸 入聲波能量可增加聲波處理後的力價；然而，較少的細胞 破裂在通過濾清器時會造成較大的損失。在聲波處理造成 的感染力價損失和高濾清後力償所需要的細胞破裂程度之 間存有一最佳的能量範圍。 經濟部中央標準局員工消費合作社印製 另一種舊方法，冰凍/融解法，可自培養基中釋放最大 量的輪狀病毒（Rotavirus)。此法難以大量製造。 二重衝擊噴射法爲人所知：將液體噴射衝擊於一板上並 以反向噴射衝擊《此衝擊區製造了一個微小混和區，其剪 力由喷射線速度所控制。這種裝置已被用來破裂微生物細 胞。在商業產品中，微液化器（MICROFLUIDIZER)使用一 __________ _ 5 - 本紙張尺度適用中國國家標準（CNS ) Α4規格（210X297公釐i 426733 A7 B7 經濟部中央標準局員工消費合作社印製 五、發明説明（3 ) 種交互作用室’其中兩個噴流以高達每秒200+公尺的線速 度互相衝擊。其破裂模式根據報告爲空化作用、流體剪力 及衝擊。然而’此裝置不適合使用於動物細胞，因爲不僅 細胞會破裂，其内容物也同樣會損壞。 理想的裝置必須能有效破裂動物細胞並毫無損傷的釋放 細胞内容物。 發明詳述 本發明係有關於一種破裂無細胞壁培養細胞的新方法， 包括將懸浮於懸浮液中的細胞通過—低壓衝擊噴射裝置。 i 此法的優點之一爲利用將培養細胞通過一由衝擊喷射裝 置送出的低壓流體所控制的剪力之方法可將細胞所製造的 產物毫無損傷的釋放出來。因此本發明另一種觀點爲收集 包含於無細胞壁細胞中之細胞產物的方法，包括： a) 於培養基的培養狀態下培養細胞直到產生細胞產物 4 f b) 將細胞懸浮於懸浮液中； c) 將懸浮細胞通過一低壓衝擊噴射裝置使細胞在從大 約每平方英吋5到100磅壓力下破裂並釋放細胞產物 :然後 d) 復原釋放出的細胞產物。 本發明方法可廣範地運用在任何無細胞壁或已去除細胞 壁的細胞上。此法雖然較適於動物細胞，但運用於其他細 胞時也同樣表現良好，如植物或黴菌細胞和細菌球狀細胞 °唯一的要求是這些細胞能夠細胞培養，最好能大規模培 _ -6- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐i —---------^ — (請先閱讀背面之注^項再填寫本頁) 訂 /! 2 6 7 3 3 A7 B7 第85111939號專利申請案 中文說明書修正頁(88年3月） 五、發明説明（ Μ年）曰修正 (請先閲讀背面之注意事項再填寫本頁) 養》適合的動物細胞實例包括' VER〇細胞、CH〇細胞和二 倍體成纖維（fibroblast)細胞，如MRC-5細胞。合適的植物 細胞實例包括菸草屬（NlC0tiana)、矮牽牛花屬（peturda)、 玉蜀黍屬（Zea)、蕓薹屬（Brassica)和雜交混種。細胞株可 予以或不予永生化（immortalized)，而且可培養於固定式或 懸浮式培養基中。並無特殊培養變數不適合本發明方法。 本發明方法只際上可用來復原所有由培養細胞所製造的 產物。產物實例包括：天然發生產物，如蛋白質；多醣 類’重組蛋白貝’包括抗體和酵素；以及病毒。在本發明 較佳的具體實例中，動物細胞在製作疫苗時作為生產病毒 的宿主細胞=因此，本發明包含了收集在動物細胞生長的 病毒的方法，包括： a) 培養病毒感染的動物細胞； b) 將含病毒的動物細胞懸浮於懸浮液中； c) 將懸浮的動物細胞通過低壓衝擊喷射裝置，使細胞 破裂而將病毒釋出； d) 收集釋出的病毒。 經濟部中央橾準局員工消費合作社印装 在 4佳具體例中’將感染了水痘帶狀（Zoster)病毒 (特別是ΟΚΑ型的水痘帶狀病毒）的mrc_5a類二倍體肺細 胞破裂以收集病毒用來製造活毒疫苗，VARIVAX®。 附圖簡述 圖1為可用於本發明方法的一種衝擊噴射裝置的描繪。 主要元件之符號. -7- 本紙張尺度適用中國國家橾準（CNS ) A4規格（210X297公釐） 4#忐171¾¾專利申請案 中文說明書修正頁(88年3月） A7 B7 五'發明説明（ 4a )&修正補充 100 代衣策流， 101 代表噴嘴； 110 代表喷流， 111 代表喷嘴； 120 代表小室； 130 代表輸出水流11 按照一般習慣來培養各種細胞。經過適當的培養期之 後，將細胞從培養基取下（如果其為固定式），懸浮於液體中 (請先閲讀背面之注意事項再填寫本頁) -t 訂 .1 '竦 經濟部中央標準局員工消費合作社印策 -7ίΐ - 本纸張尺度適用中國國家標準（CNS ) A4規格（210X2?7公釐） '426733 經濟部中央標準局員工消費合作社印黎 A7 R7 五、發明説明（5 ) 。懸浮液可與用來培養細胞的相同或類似，或爲安定劑。 針對本發明的目的，懸浮液的成分不受限制。接著將懸浮 細胞通如圖1所示的衝擊噴射裝置。關於圖j，流體剪較佳 由二反向喷流111和1〇1以控制線性速度在12〇小室中衝擊 而產生。此裝置最好以連續模式操作，將輸入的水流劈成 二個噴流100、110，而輸出水流J 30會排出小室外。噴嘴 111及101的配置必須非常相互靠近，如距離小於i英吋， 而更佳爲接近1/8至3/8英叶的距離以便増強最大流體剪力 〇 本方法一嚴謹的觀點爲此裝置在低壓、非空化狀態下操 作，較佳爲低於約每平方英吋15〇磅，而更佳爲低於約每 平方英吋100磅。低操作壓力造成溫和的破裂-細胞在非常 低的壓力破裂，從約每平方英吋5到約1 〇〇磅。這是區別本 法與舊技法的特徵之一。例如市面所提供的衝擊喷射細胞 破裂器’商標名爲微液化器（MICROFLUIDIZER®)，由微 液國際公司（Microfluidics International Corp.，Newton, MA) 出品，報告以每平方英吋2,000磅壓力來破裂動物細胞。 在此高壓下力，有可能發生空化作用且造成破壞》 由於衝擊點的線喷射速度可決定破裂力因此也是本發明 的一個嚴格觀點。適合本方法的較佳線噴射速度約爲每秒 5到50公尺，更佳爲每秒1 〇到30公尺。 本發明方法係專門設計來破裂無細胞壁的細胞，其中以 可控制的方法傳送較低的輸入能量。這點對高壓力裝置來 説很困難：如均化器（homogenizer)或微液化器係設計來傳送 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） ---^--.------V-- (請先聞讀背面之注意事項再填寫本頁) 訂. 426733 經濟部中央標丰局—工消費合作杜印製 A7 B7 五、發明説明（6 ) 每平方英忖高達15,〇〇〇和40,000镑的壓力，個別來説，欲 傳送精確控制的低壓將遠低於其原設計规格，然而本發明 方法卻使用最適於低壓運作之裝置。 本發明的衝擊噴射法顯示可提供適當的細胞破裂，在幾 可忽略感染力價損失的情況下達成高過濾產量β在初步的 實驗中，衝擊噴射提供了較冰凍/融解法更隹的生產量。 本發明方法較目前的細胞破裂法有更多的優點。首先， 本裝置設計簡單且不需要活塞幫浦及冷卻裝置，避免了與 此型零件有關的問題。低壓操作系統有更佳的優點可方便 利用低壓操作幫浦（葉或隔膜）或相對低壓槽來傳送液體。 由於其低操作壓力，在破裂操作期間僅產生可忽略的熱 量（<0.rc)。 本裝置尺寸相當小因此可裝入連接二容器的標準操作管 中。 本衝擊噴射裝置藉著在充分定義之衝擊區中的微混合製 造流體剪來破裂細胞。在使用破裂狀態時不會產生空化作 用。避免了空化破裂法之高剪力在非均勻區造成的破壞。 本衝擊噴射裝置可規模化。固定線速的容積操作率可藉 由増加噴射孔口徑來提昇。 爲原生質或病毒提供一種具高復原率之可控制的流體剪 式破裂法。 本裝置設計衛生並使用市面現有的一貫裝配嘴嘴技術， 而且能直接合併入標準操作裝備内。更甚者，此裝置可適 當的減菌。 -9 - 本紙張尺度適用中國國家標率（CNS ) A4規格（210X297公釐:丨 —^—.------^— % · (請先閲讀背面之注意事項再填寫本頁) 訂 4267 3 3 A7 B7 五、發明説明（7 ) 可避免大於每平方英吋200磅的高壓力操作和其相關問 題。 在較佳的具體實例中’本發明的衝擊噴射細胞破裂法可 將來自動物細胞的水痘帶狀病毒、其它的病毒或細胞内蛋 白質做高產量的復原。在破裂之後，藉由濾清器將細胞碎 片從伴隨的病毒顆粒中分離，並將所得病毒製品凍結直到 進一步的處理製成疫苗。 下列挺出幾個非限制性實例進一步説明本發明a 實例一 以40毫升Ι.Οχ或l.5x PGSE固定劑洗藤、充入内附有感染 水痘病毒之MRC-5細胞的滾筒燒瓶，並從滾筒燒瓶收集以 機械臂從燒瓶上刮下的細胞層。將自滾筒燒瓶除下的細胞 漿收集在容器中，冰;東到-6〇°C。操作前，先測量衝擊噴 射裝置口徑以決定製造所需線速的必需壓力，然後滅菌。 將包含冷凍收集物的容器解凍、集中，並置於連接衝擊噴 射裝置之壓力槽。將壓力槽内容物加壓製造每秒22.5公尺 (l.OxPGSE)或每秒2S.0公尺（1.5xPGSE)的喷射線速度。將 嗔出物移回壓力槽做第二次噴射。 實例二 另一種選擇是_聯二組噴射來使用。然後利用聚丙烯深 度濾過器澄清破裂的細胞。利用溶菌斑試驗測量力價。再 用依榮（Elzone)粒子分析器分析粒子尺寸。 -10- 本紙張尺度適用中國國家標準（CNS ) A4^g· ( 2丨0X297公釐） m^i ^in ^^^1 n^i 1 ^^^1 n * (請先閱讀背面之注意事項再填寫本頁)4 267 3 3 Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs A7 B7 V. Description of the invention (1) Scope of the invention The invention relates to a method for rupturing cells growing in a petri dish, which uses a low-pressure reverse jet to produce a fluid Shears are strong enough to rupture cells but not destroy their contents. BACKGROUND OF THE INVENTION Many biotechnology and fermentation processes require large numbers of cells to grow in a bioreactor and then rupture to release the desired product. Destruction of cells can be accomplished using mechanical, chemical, biological or physical methods. There are many * mechanical preferences for mechanical rupture methods, because mechanical methods can avoid the use of many additional agents [detergents, enzymes or solute effect agents (〇sm〇larity effect〇rs)] and physical methods (such as ice cream / melting) Law) Difficulties in scale. Many mechanical methods for disrupting microorganisms have been developed. These methods often rely on fluid shear and / or compression to rupture cell walls and membranes. However, not all biotechnology and fermentation processes use microorganisms. Animal cells have gradually become the host of choice. Because animal cells are larger and have fragile cell membranes, much less energy is required to cause cell rupture. Many off-the-shelf systems designed for microbial systems are not suitable for use with animal cells. The rotor / fixer device has a cylindrical rotor that rotates at high speed in a concentric fixator and can be used to rupture animal cells. These devices create a steep velocity gradient in the annulus and generate sufficient shear stress in the liquid to rupture the cells. There is a similar device called Chaikoff Press, which contains a cylinder and a smaller diameter piston. The movement of the piston creates high shear forces in the fluid in the annular space, causing the cells to rupture. The size of this type of paper is applicable to the Chinese National Standard (CNS) A4 (210X297 mm)-], ------ <---(Please read the notes on the back before filling this page) Order A7 426733 B7 V. Description of Invention (2) ------------ Article ---- Order (please read the precautions on the back before filling this page) (Douncer) and has been used to rupture MRC-5 diploid lung cells infected with Varicella. However, these methods are only feasible at the laboratory scale and cannot be manufactured on a large scale. Ultrasound or acoustic energy can cause cells to rupture by creating high shear regions in the liquid through cavitation. Vibratory sound waves (~ 20kHz) create pressure pulses in liquids. Bubbles form in the low-pressure zone, and when they enter the high-pressure zone, they collapse and cause high-energy shock waves. When the shock wave moves from the initial cavitation position, a high shear force is generated, and at the same time, the heat energy is dissipated into the liquid. One continuous fluid acoustic energy device has been used to rupture the MRC-5 diploid lung cells of hydropox virus. It has enough energy to destroy chickenpox infections in a region very close to the origin of the shock wave. Therefore, the amount of virus infection is determined by the number of nucleation sites generated per unit volume of liquid; this number is related to the energy input into the liquid. However, this continuous sonication method also caused a 5 to 1 ° C increase in the temperature of the treatment fluid, which increased the degradation rate of the virus. Reducing the input sonic energy can increase the force value after sonication; however, fewer cell ruptures pass through the filter Larger losses will be caused when cleaning. There is an optimal energy range between the loss of infectious power caused by sonication and the degree of cell rupture required for high filtration after compensation. Employees' Cooperatives, Central Standards Bureau, Ministry of Economic Affairs Print another old method, the freeze / thaw method, which can release the maximum amount of Rotavirus from the culture medium. This method is difficult to manufacture in large quantities. The double impact spray method is known: spraying liquid on a plate The impact zone is reversed. This impact zone creates a micro-mixing zone whose shear force is controlled by the linear velocity of the jet. This device has been used to rupture microbial cells. In commercial products, micro-liquifiers (MICROFLUIDIZER) Use 1 __________ _ 5-This paper size applies to Chinese National Standards (CNS) Α4 specifications (210X297 mmi 426733 A7 B7 Employees of the Central Standards Bureau of the Ministry of Economic Affairs Cooperative printed 5. Invention description (3) Interaction chamber 'Two jets impact each other at a linear velocity of up to 200+ meters per second. Their rupture modes are reported as cavitation, fluid shear and impact. However, 'This device is not suitable for use on animal cells, because not only the cells will rupture, but their contents will also be damaged. The ideal device must be able to effectively rupture animal cells and release the cell contents without damage. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to A new method of rupturing cell-free wall culture cells, including passing cells suspended in suspension through a low-pressure impingement jet device. One of the advantages of this method is the use of controlled culture cells through a low-pressure fluid sent from the impulse jet device. The method of shearing force can release the products produced by cells without damage. Therefore, another aspect of the present invention is a method for collecting cell products contained in cell-free cells, including: a) culturing in a culture state of a medium Cells until cell product 4 fb) Suspension of cells in suspension; c) Suspension of cells Ejecting means through a low pressure impactor disrupt the cells at about 5-100 lbs per inch square cell and release the pressure from the product: and d) recovery of the released cell product. The method of the present invention can be widely applied to any cell wall without or without cell wall removal. Although this method is more suitable for animal cells, it also performs well when applied to other cells, such as plant or mold cells and bacterial spheroid cells. The only requirement is that these cells can be cell cultured, preferably large-scale culture. -6 -This paper size applies to China National Standard (CNS) A4 specification (210X297 mmi —--------- ^ — (Please read the note on the back before filling this page) Order /! 2 6 7 3 3 A7 B7 Chinese Patent Application No. 85111939 Revised page (March 88) V. Description of the invention (year M) Revised (please read the precautions on the back before filling this page) Examples of suitable animal cells Including 'VERO cells, CH0 cells and diploid fibroblast cells such as MRC-5 cells. Examples of suitable plant cells include Nicotiana, peturda, Zea ), Brassica and hybrids. The cell line can be immortalized or not, and can be cultured in fixed or suspended medium. No special culture variables are not suitable for the method of the present invention. The present invention Method Can be used to restore all products made by cultured cells. Examples of products include: naturally occurring products such as proteins; polysaccharides 'recombinant protein shells' including antibodies and enzymes; and viruses. In a preferred embodiment of the present invention, animals Cells serve as host cells for virus production when vaccines are made = Therefore, the present invention includes a method for collecting virus grown in animal cells, including: a) culturing virus-infected animal cells; b) suspending virus-containing animal cells in suspension C) Pass the suspended animal cells through a low-pressure shock jet device to rupture the cells to release the virus; d) Collect the released virus. The Consumer Cooperatives of the Central Government Bureau of the Ministry of Economic Affairs of the People's Republic of China printed in 4 specific examples' mrc_5a type diploid lung cells infected with varicella zoster virus (especially OVZ type varicella zoster virus) will be ruptured to collect The virus is used to make live vaccines, VARIVAX®. Brief Description of the Drawings Figure 1 is a depiction of an impact injection device that can be used in the method of the present invention. Symbols of main components. -7- This paper size is applicable to China National Standard (CNS) A4 specification (210X297 mm) 4 # 忐 171¾¾ Revised page of Chinese specification of patent application (March 88) A7 B7 Five 'invention description ( 4a) & amended and supplemented 100 generations of clothing flow, 101 for nozzles; 110 for sprays, 111 for nozzles; 120 for small chambers; 130 for output water streams 11 According to general habits, various cells are cultured. After an appropriate incubation period, remove the cells from the culture medium (if they are stationary) and suspend them in the liquid (please read the precautions on the back before filling this page)-t.1 '竦 Central Standards Bureau, Ministry of Economic Affairs Employee Consumption Cooperative Seal-7-7-This paper size applies to the Chinese National Standard (CNS) A4 specification (210X2? 7 mm) '426733 Employee Consumption Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs, Employees Cooperative, India Lei A7 R7 5. Description of the invention (5). The suspension may be the same as or similar to that used to culture the cells, or may be a stabilizer. For the purposes of the present invention, the composition of the suspension is not limited. The suspended cells were then passed through an impact jet as shown in FIG. Regarding Figure j, the fluid shear is preferably generated by two reverse jets 111 and 101 at a controlled linear velocity to impinge in the 120 cell. The device is preferably operated in continuous mode, splitting the incoming water stream into two jets 100, 110, and the outgoing water stream J 30 is discharged out of the small room. The arrangement of the nozzles 111 and 101 must be very close to each other, such as a distance less than i inches, and more preferably a distance of 1/8 to 3/8 inches in order to strengthen the maximum fluid shear. A rigorous viewpoint of this method is this device Operating in a low pressure, non-cavitated state, preferably below about 150 pounds per square inch, and more preferably below about 100 pounds per square inch. Low operating pressure causes mild rupture-cells rupture at very low pressure, from about 5 to about 100 pounds per square inch. This is one of the characteristics that distinguishes this method from old techniques. For example, the commercially available impact jet cell disruptor 'brand name is MICROFLUIDIZER®, produced by Microfluidics International Corp. (Newton, MA) and reported to rupture at a pressure of 2,000 pounds per square inch. Animal cells. Under this high pressure, cavitation may occur and damage may occur. Since the line ejection speed at the impact point can determine the breaking force, it is also a strict point of the present invention. A preferred linear spray speed suitable for this method is about 5 to 50 meters per second, and more preferably 10 to 30 meters per second. The method of the present invention is specifically designed to rupture cell-free cells in which a lower input energy is delivered in a controlled manner. This is very difficult for high-pressure devices: such as a homogenizer or micro-liquefier designed to transmit the paper size to the Chinese National Standard (CNS) A4 (210X297 mm) --- ^-. ------ V-- (Please read the precautions on the back before filling out this page) Order. 426733 Central Standards Bureau, Ministry of Economic Affairs—Industrial and Consumer Cooperation Du printed A7 B7 5. Description of Invention (6) per square With pressures of up to 15,000 pounds and 40,000 pounds, individually, the low pressure to be accurately controlled will be much lower than its original design specifications, but the method of the present invention uses the device most suitable for low pressure operation. The impact spray method of the present invention has been shown to provide appropriate cell rupture and achieve high filtration yields with negligible loss of infectious power. In preliminary experiments, the impact spray provided a greater throughput than the freeze / thaw method. . The method of the invention has more advantages than the current cell rupture method. First, the design of this device is simple and does not require piston pumps and cooling devices, avoiding problems associated with this type of part. Low-pressure operating systems have the advantage that they can facilitate the use of low-pressure operating pumps (lobes or diaphragms) or relatively low-pressure tanks to transfer liquids. Due to its low operating pressure, only negligible heat is generated during the rupture operation (< 0.rc). The unit is quite small and fits into a standard operating tube connecting two containers. The impact jet device ruptures cells by micro-mixing a fluid shear in a well-defined impact zone. No cavitation occurs when using the ruptured state. The damage caused by the high shear force of the cavitation rupture method in the non-uniform area is avoided. The impact injection device can be scaled up. The fixed line speed volume operation rate can be increased by increasing the diameter of the injection hole. Provides a controlled fluid shear rupture method with high recovery for protoplasts or viruses. The device is hygienic in design and uses the existing conventional assembly nozzle technology on the market, and can be directly incorporated into standard operating equipment. What's more, this device can reduce bacteria appropriately. -9-This paper size applies to China National Standards (CNS) A4 specifications (210X297 mm: 丨 — ^ —.------ ^ —% · (Please read the precautions on the back before filling this page) Order 4267 3 3 A7 B7 V. Description of the invention (7) High pressure operation greater than 200 pounds per square inch and related problems can be avoided. In a preferred embodiment, the impact jet cell rupture method of the present invention can be derived from animals Cellular varicella zoster virus, other viruses or intracellular proteins are recovered in high yield. After rupture, the cell debris is separated from the accompanying virus particles by a filter, and the resulting virus preparation is frozen until further processing The following are several non-limiting examples to further illustrate the present invention. Example 1 A rattan was washed with 40 ml of 1.0 × or 1.5x PGSE fixative and filled with a drum containing MRC-5 cells infected with chickenpox virus. Flask and collect the cell layer scraped from the flask with a robotic arm from the roller flask. Collect the cytoplasm removed from the roller flask in a container, ice; east to -60 ° C. Before operation, measure the impact spray Device caliber depends Set the necessary pressure for the line speed required for manufacturing, and then sterilize. Thaw the container containing the frozen collection, concentrate, and place it in a pressure tank connected to the impact spray device. Pressurize the contents of the pressure tank to make 22.5 meters per second (l. OxPGSE) or 2S.0 meters per second (1.5xPGSE) linear velocity of the spray. Move the effluent back to the pressure tank for a second spray. Example 2 Another option is to use the two-group spray. Then use the poly Acrylic depth filter clarifies ruptured cells. Use plaque test to measure force value. Then use Elzone particle analyzer to analyze particle size. -10- This paper size applies Chinese National Standard (CNS) A4 ^ g · (2丨 0X297mm) m ^ i ^ in ^^^ 1 n ^ i 1 ^^^ 1 n * (Please read the precautions on the back before filling this page)
-、1T 經濟部中央標準局員工消費合作社印製 426733-、 1T Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 426733
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