1227736 \ 九、發明說明： 【發明所屬之技術領域】 本舍明係關於一種用於生化科技領域的胞膜電穿孔 (electroporation)基因轉殖模擬方法及其應用的裝置，更詳而 έ之，係指一種實驗過程簡單快速，能縮短實驗時效及提高效 能，且，能以平面式依被治療部位運用於活體外（in_vitr〇)治療， 而可避免對治療部位直接或間接傷害。 【先前技術】 基因轉殖技術是一種以基因治療方式，其藉具有特殊作用 的基因（DNA序列）由載體（Vector)或外力推送進入活細胞 中（Deliver)，外來的DNA序列藉由細胞本身的生理反應可以 轉錄RNA，RNA又被轉譯成蛋白或酵素使基因表現 (Expression)。在基因轉殖技術中將基因投遞（Deliver)的方 法，概分為兩類··病毒法及非病毒法，其中與本發明有關者為參 非病骨法中的電脈衝胞膜穿孔法（Electroporation )，其不需要 任何載體便可以將DNA序列或藥物傳遞進入細胞，藉由電脈衝 電流的刺激使細胞膜上的微小孔洞張開，讓細胞膜外界物質（藥 物）有機會擴散進入細胞體内，並使DNA帶有負電荷藉由外界 電場的作用而加速移動，增加進入細胞的機會。 再者’習知電脈衝胞膜穿孔法對被處理細胞的數量有一定 6 1227736 的&準’低於該數量標準則無法順利進行，且，被治療的細胞 ^養不易’往往在長時間培養至接近實驗的數量標準前已壞 死而必須花時間重新培養。且，即便細胞順利培養成功，但 疋’因為實驗過程基因轉殖、檢測等皆必須分開進行，是以細 胞須被多次刮除移至實驗區才能進行，其間步驟的繁瑣可想而 知。除此，習知整體設備大成本高對從事基礎研究人員實非經 濟0 【發明内容】 本發明之主要目的，在於提供一種用於生化科技領域的胞膜電穿孔基因 轉殖柄擬方法，進言之，係提供—種將麵電穿孔基目轉殖技術在模組化的 轉殖基板上進行，並直接由螢光顯微鏡觀察轉殖效果，藉以縮減實驗步驟， 減:Λ、、田胞培養基、疆、血清及抗生素的使用量，降低研究成本、設備，提 供更方便、更便宜的研究方法，以提昇實驗的效率。 本發明一種用於生化科技領域的胞膜電穿孔基因轉殖模擬φ 方法係提供-電脈衝產生系統以電腦虛擬儀控單元將波形輸出至 數位/類比轉換卡，產生類比的脈衝信號及精準的顧脈衝訊號，來控制脈衝 的幅度、維持日夺間（DuratlonTime)、頻率及脈衝次數，並藉由驅動電路單元 ^大脈衝信號’以提供足夠的電流供應基因轉殖反應之所需，使脈衝波形不 受負載影響而改變。 依據上述的系統，其中產生類比的脈衝維持時間可自約〇1至 7 1227736 以下，而所提供的工作電壓可在lov以下，以產生高電場在n〇〇〇v/cm。 本發明一種用於生化科技領域的胞膜電穿孔基因轉殖模擬 方法再提供一基因轉殖相容系統，使細胞可在具有生物相容性 的環境下生長，該系統係於基板表面上以微加工方式佈局設計出可電極 的金屬微電極圖型（Pattern)，而製作出基因轉殖基板（Electroporati()nchip)， 並容置於容納有培養基的反應槽内，以提供作為培養細胞的環境。 本發明一種用於生化科技領域的胞膜電穿孔基因轉殖模擬 方法又提供一表面處理系統係將帶負電不會被細胞吸收的分子 物質如：蛋白質、Poly-D-Lysine等溶液塗佈（Coating)於前述 基因轉殖相容系統中基板的表面，以改變轉殖基板表面的介面 特性，以使得細胞更容易緊密貼附於基板表面，不會剝落或位 移0 本發明一種用於生化科技領域的胞膜電穿孔基因轉殖模擬 方法，在表面處理系統後提供一繼代培養系統係將原本在培養皿中鲁 大量培養的細胞移到基因轉殖相容系統的反應槽中繼續培養，以細胞懸 浮、細胞計數及轉殖基板培養等步驟，使繼續培養的細胞貼附在轉殖基板表 面，倣繼代培養。 本發明一種用於生化科技領域的胞膜電穿孔基因轉殖模擬 方法，在繼代培養系統後提供一實驗系統將在基因轉殖相容系 統内反應槽所培養的細胞，以固定DN A質量及培養基體積，將 12277361227736 \ IX. Description of the invention: [Technical field to which the invention belongs] The present invention relates to a method for simulating the cell membrane electroporation gene transfer and its application in the field of biochemical technology. It refers to an experimental process that is simple and fast, can shorten the experimental time and increase the efficiency, and can be used for in-vitre treatment in a flat manner according to the treated part, and can avoid direct or indirect damage to the treated part. [Previous technology] Gene transfer technology is a gene therapy method, which uses special genes (DNA sequences) to be pushed into vectors (Vector) or external forces into living cells (Deliver), and the foreign DNA sequences are transferred by the cell itself. The physiological response can transcribe RNA, which is then translated into proteins or enzymes for gene expression (Expression). Methods of gene delivery in gene transfer technology can be roughly divided into two types: viral methods and non-viral methods. Among them, the person related to the present invention is the electric pulsed cell membrane perforation method in the non-disease bone method. ), Which can pass DNA sequences or drugs into the cell without any carrier, and the tiny holes in the cell membrane are opened by the stimulation of the electric pulse current, so that the external material (drug) of the cell membrane can diffuse into the cell, and Making DNA with a negative charge accelerates the movement by the external electric field, increasing the chance of entering the cell. Furthermore, 'the conventional electric pulsed cell membrane perforation method has a certain number of treated cells 6 1227736 & quasi' cannot be performed smoothly below this number standard, and the treated cells are not easy to maintain 'often over a long period of time It has been necrotic before being cultured close to the experimental quantity standard and must take time to reculture. Moreover, even if the cells are successfully cultured successfully, because of the experimental process, gene transfer and detection must be performed separately, and the cells must be scraped and moved to the experimental area for multiple times. The tedious steps can be imagined. In addition, it is not economical for the basic researchers to understand the large cost of the overall equipment. [Summary of the invention] The main purpose of the present invention is to provide a method for the transduction of cell membrane electroporation genes in the field of biochemical technology. In other words, we provide a method of transfection of electroporation basal cells on a modular transgenic substrate, and observe the transfection effect directly with a fluorescent microscope, thereby reducing the experimental steps, reducing: Λ ,, cell culture medium The use of serum, antibiotics, serum, and antibiotics reduces research costs and equipment, and provides more convenient and cheaper research methods to improve the efficiency of experiments. The invention provides a cell membrane electroporation gene transfer simulation simulation method method used in the field of biochemical technology. The electrical pulse generation system uses a computer virtual instrument control unit to output a waveform to a digital / analog conversion card to generate analog pulse signals and accurate The pulse signal is used to control the amplitude of the pulse, maintain the DuratlonTime, frequency, and the number of pulses, and the driving circuit unit ^ large pulse signal 'to provide sufficient current to supply the gene transgenic reaction required to make the pulse The waveform is not changed by the load. According to the above-mentioned system, the analog pulse sustaining time can be from about 0.01 to 7 1227736 and the working voltage provided can be below lov to generate a high electric field at 2000 v / cm. The invention provides a method for simulating transfection of cell membrane electroporation genes in the field of biochemical technology, and further provides a transfection-compatible system for cells to grow in a biocompatible environment. The system is arranged on the surface of a substrate to The micro-processing method is used to design an electrode-capable metal micro-electrode pattern, and a gene transfer substrate (Electroporati () nchip) is produced, which is housed in a reaction tank containing a culture medium, so as to provide a culture cell. surroundings. The invention provides a method for simulating the transfection of cell membrane electroporation genes in the field of biochemical technology, and provides a surface treatment system for coating negatively charged molecules such as proteins, Poly-D-Lysine and other solutions ( Coating) on the surface of the substrate in the aforementioned transgenic compatible system to change the interface characteristics of the surface of the transgenic substrate so that the cells can more easily adhere to the substrate surface without peeling or displacement. The present invention is used for biochemical technology In the field of cell membrane electroporation gene transfection simulation methods, a secondary culture system is provided after the surface treatment system. Cells originally cultured in large numbers in a petri dish are transferred to the reaction tank of the gene transfection compatible system for continued cultivation. With the steps of cell suspension, cell counting, and transfection substrate culture, cells that continue to be cultured are adhered to the surface of the transfection substrate, imitating subculture. The invention provides a method for simulating transfection of cell membrane electroporation gene in the field of biochemical science and technology. After the subculture system, an experimental system is provided to fix cells in a reaction tank in a transgenic compatible system to fix DNA quality. And medium volume, will be 1227736
Plasmid DNA輸送入該細胞内進行電脈衝胞膜穿孔實驗。 本發明一種用於生化科技領域的胞膜電穿孔基因轉殖模擬 方法’在貫驗系統後再提供一檢測系統來觀察轉殖基因結果。 檢測系統可採用基因轉殖成功的細胞所製造出螢光蛋白Gfp，經過 一定時間培養後，提高GFP的濃度，藉螢光物質吸收短波長的光能，放射出 長波長的光之特性，並利用反射式汞燈光源螢光顯微鏡搭配適當的濾鏡組， 便可在螢光顯微鏡下觀察轉殖結果，計算綠色螢光細胞的數量以反映轉殖的 程度。 本發明另一特徵，在於提供一種使用於活體外治療的胞膜 電穿孔基因轉殖基板，該轉質基板具有微電極圖型的金屬層， 在低工作電壓下可以直接貼於治療部位進行治療。 【實施方式】 本發明用於生化科技領域的胞膜電穿孔（如血叩〇加丨〇11)基因轉殖轉 基板模擬方法，係包括：電脈衝產生系統、基因轉殖相容系統、表面 處理系統、繼代培養系統及實驗系統，以及檢測系統等作業系統， 其中： 5亥電脈衝產生系統（Electricpulsegenerator)是以電腦虛擬儀控程 式（LabVIEW)將波形輸出至數位/類比轉換卡，產生類比的脈衝信號產生 精準的電壓脈衝訊號，控制脈衝的幅度、維持時間（Durati〇n Time)、頻率 9 1227736 及脈衝次數，藉由外部電路放大脈衝信號，並且提供足夠的電流供應基因轉 殖轉殖基板反應之所需，使脈衝波形不受負載影響而改變。其詳細可分兩部 份進一步說明： (a) 控制元件部分 個人電腦採用Intel Pentium II 450MHz微處理器，128MB DRAM，虛 擬儀控軟體採用美國National Instruments™ Lab VIEW 5.1，是一種圖形化的 程式語言（Graphical Programming Language)，提供程式設計者圖形化的發 展環境，快速建立儀器所需的軟體及人性化的人機介面（UserInterface)，脈 衝參數的控制由軟體介面輸入，包括：電壓、脈衝維持時間（Durati〇n Time)、 脈衝次數，脈衝間隔時間。 (b ) 驅動電路設計元件部分 請配合第觀之，脈細動電路9G_貞喊混合共賴(c麵^Plasmid DNA was delivered into the cells for electrical pulsed membrane perforation experiments. The present invention provides a method for simulating transfection of a cell membrane electroporation gene used in the field of biochemical technology, and provides a detection system to observe the result of the transfection gene after the system is tested. The detection system can use the fluorescent protein Gfp produced by cells that have been successfully transgenic. After a certain period of culture, the concentration of GFP can be increased. The fluorescent substance can absorb short-wavelength light energy and emit long-wavelength light. Using a reflection-type mercury light source fluorescent microscope with an appropriate filter set, the transfection results can be observed under a fluorescent microscope, and the number of green fluorescent cells can be calculated to reflect the degree of transfection. Another feature of the present invention is to provide a cell membrane electroporation gene transgenic substrate for in vitro treatment. The transgenic substrate has a metal layer with a microelectrode pattern, and can be directly attached to a treatment site for treatment at a low working voltage. . [Embodiment] The method for simulating gene transfection and transfection substrate of cell membrane electroporation (such as blood 叩 〇plus〇〇11) used in the field of biochemical technology according to the present invention includes: an electric pulse generation system, a gene translocation compatible system, and a surface Operating system such as processing system, subculture system and experiment system, and detection system. Among them: Electric pulse generation system (Electricpulsegenerator) uses a computer virtual instrument control program (LabVIEW) to output waveforms to a digital / analog conversion card to generate The analog pulse signal generates accurate voltage pulse signal, controls the pulse amplitude, duration (DuratiOn Time), frequency 9 1227736 and the number of pulses, amplifies the pulse signal by an external circuit, and provides sufficient current for gene transfer It is necessary for the substrate reaction to make the pulse waveform change without being affected by the load. The details can be further divided into two parts: (a) The control unit part of the personal computer uses Intel Pentium II 450MHz microprocessor, 128MB DRAM, and the virtual instrumentation software uses American National Instruments ™ Lab VIEW 5.1, which is a graphical programming language (Graphical Programming Language), provides a graphical development environment for programmers, quickly builds the software required by the instrument and a user-friendly human-machine interface (UserInterface). The control of pulse parameters is input by the software interface, including: voltage, pulse maintenance time (DuratiOn Time), number of pulses, pulse interval time. (b) Design element of the driving circuit. Please cooperate with the first observation.
Emitter)的汉计’提供負载（基因轉殖轉殖基板）所需之ι〇ν以下電壓。 該脈衝軸轉9G快算放大IC_ (⑽41) %，制減向線性放大 “之又十 <"號輸出缟連接電晶體92的基極（Base) 921，回授輸入端連 接電晶體92的射極（Ejj此er) 922 ’形成負回授電路，並且以回授電阻控制 信號放大倍率，電晶體咖娜41卿接面 電壓準位，由電晶體供給維持貞鮮位所需 93，耐壓砸，最大負載電流7A，採用共射極之電路設計，工作脈衝的輸 出端位於電SB體的射極與負載串聯負回授的電麟計可以確保負載所需的 10V以下的電流。同時可以配 1227736 合後述基因轉殖基板，產生1至l〇〇〇V/cm高電場的環境。（註：本發明上述 的電路设計是以方波形脈衝，實際使用上以任一種電路脈衝皆可） 電晶體射極922串聯的可變電阻（rx) 94是為了電路的阻抗匹配，由 於負載本身具有電阻並聯電容的特性，所以當脈衝結束後，電容會短暫持續 放電使得脈衝變形，為了降低此一效應，負載並聯一個電阻95以消耗電容 儲存之電荷幫助電容放電。由於基因轉殖轉殖基板中有細胞貼附在電極表 面，培養基充滿在電極之間，電性比較不穩定，因此電容大小也不一樣，所 以採用可變電阻的設計以方便調整。 該基因轉殖相容系統係提供細胞可在具有生物相容性的 環境下生長，以微機電製程技術配合機械加工及化學塗佈在基板表面上 佈局設計出電極，而製作出基因轉殖基板，再容置於無菌的容器内。此系統 主要包括：轉殖基板及反應槽，其中轉殖基板的目的是作為承載基礎，同時 具有生物相谷性，使細胞可以生長，採用76x26mm載玻片作為基板。而具 導電金屬層用於建立胞膜穿孔所需之電場，並且具有生物相容性，細胞培養 不會造成不良影響，該基板的表面以金（Gold ; Au)及鈦（Titanium ; Ή) 兩層加工，藉金（Gold ; Au)具有良好的導電率及生物相容性，不容易產 生電化學反應，作為胞膜穿孔的反應電極之金屬層，而以鈦（Titanium ; Ή) 作為金（Gold ; Au)與玻璃基板間的黏著層。另，該反應槽為培養細胞的 位置，為經過處理的無菌槽，可隨胞膜電穿孔的反應範圍決定大小，其内容 納細胞培養時所需的培養基，因此必須有2mm以上的深度（或厚度），本實 施例採用PDMS (Polvdimethvlsiloxane)以機械加工的方式製造作為反應槽 11 1227736 的邊壁，藉其具有化學成分安定，無溶劑揮發，加工容易，材質透明方便實 驗觀察的優點，貼附力佳不易傷害基板與電極，組裝方便。以上詳細製作與 各項製程析述如下： (a) 光罩設計 本發明中元件的隶小線寬採励μιη，依據最小線寬的解析能力選擇低 成本的膠片光罩將基因轉殖基板上設置三個反應區，如第二圖所示，每個反 應區各自獨立，在進行電脈衝的過程中不會發生互相干擾的情況。 (b) 微電極設計 請參閱第三、四圖，本發明採用梳狀交錯結構（interdigitated)的設計， 增加反應區的面積，同時可以在低電壓的條件下產生高電場的環境，具導電 金屬層的圖形主要疋由反應電極、探針接點（Τ〇ικ^ pa(j)及對準記號 (AlignmentMark)所形成；反應區電極的線寬為1〇〇μηι，正、負電極的間 距為励μιη ’探針接點為4000χ4000μηι，正、負接點的間距為6〇〇〇μηι，對 準記號為四個500χ500μπι正方形，間距8000μηι。一片76_Χ26_的轉殖 基板上設計了 3個反應區，反應區之間的間距為6〇〇〇μιη，避免在電脈衝的 過程產生漏電流的現象而互相影響，電極四周有對準記號幫助轉殖基板組裝 時的定位。 (c)製程 本發明轉殖基板製程主要是依微機電製程為基礎，真空蒸鐘的方法沉 積金屬薄膜，微影蝕刻的方式定義金屬電極；配合機械加工的方法封裝；最 12 1227736 後化學塗佈的方式改變轉殖基板表面的性質。其步驟採用電子束蒸鑛機 (E-Beam Evaporator ; EBE)進行高純度金屬層的沉積，分別使用鈦 (Titanium ; Tl)與金（Gold ; Au)兩種金屬作騎材，欽與金都是生物相 容性相當高的材料，不科發生氧化或其他化學反應，因此適合作為生物醫 學研究的導電㈣，配合高真空電子束驗_動目_製程，最符合生物 醫學轉質基板高純度電極製程技術的需求。 為避免電極剝落；先以2A/sec速率蒸鍍100A鈦在載玻片之基板表面， 作為黏著層，翻5A/see速率蒸鍍漏A，將電金·無著制比例鲁 在10 · 1至20 · 1之間，附著效果較佳，詳細處理步驟如下： 1. 以HMDS (Hexamethvldisilazane ;表面黏著劑）蒸發進行表面處理 20min 2. 光阻塗佈，將載玻片之基板至於旋轉塗佈機吸盤中央，在載玻片之 基板中央滴上3ml Shipley 1818光阻液，以2500rpm — 10sec/5000rpm—30sec旋轉塗佈轉殖基板 3·軟烤，水平置於90°C的烘箱30min 鲁 4·曝光’光罩對準載玻片之基板後，設定7 8sec照射 5·顯影’以四倍稀釋的Devei〇per351進行顯影30〜60sec 6·冲洗’去離子水(dei〇nized ; DI water)洗淨載玻片之基板lOmin 7·硬烤’水平置於120°C的烘箱30minEmitter's Han Ji 'provides a voltage below ινν required for the load (gene transgenic substrate). The pulse axis is rotated to 9G and the IC_ (⑽41)% is calculated, and the linear amplification is reduced. “No. Ten” output 缟 is connected to the base 921 of the transistor 92, and the feedback input is connected to the transistor 92 The emitter (Ejj this er) 922 'forms a negative feedback circuit and controls the signal magnification of the feedback resistor. The transistor Gana 41 is connected to the voltage level, and the transistor supplies 93 to maintain the chastity position. Withstand voltage and smash, the maximum load current is 7A. It adopts a common emitter circuit design. The output end of the working pulse is located in the electric SB body with the emitter and the load connected in series. The galvanometer can ensure the current required by the load below 10V. At the same time, it can be equipped with a gene transfer substrate of 1227736, which will be described later, and generate an environment with a high electric field of 1 to 1000V / cm. (Note: The above circuit design of the present invention is a square waveform pulse, and any circuit pulse is used in actual use. Both are possible. The variable resistor (rx) 94 connected in series with the transistor emitter 922 is for the impedance matching of the circuit. Because the load itself has the characteristics of resistance and parallel capacitance, after the pulse ends, the capacitor will continue to discharge for a short time to deform the pulse. drop With this effect, the load is connected in parallel with a resistor 95 to consume the charge stored in the capacitor to help the capacitor discharge. Because the cells in the transgenic substrate are attached to the surface of the electrode, and the medium is filled between the electrodes, the electricity is unstable, so the capacitor The size is also different, so a variable resistance design is used for easy adjustment. The gene transfection compatible system provides cells that can grow in a biocompatible environment. Micro-electromechanical process technology is used in conjunction with mechanical processing and chemical coating. The electrodes are laid out and designed on the surface of the substrate, and the gene transgenic substrate is made, and then placed in a sterile container. This system mainly includes: a transgenic substrate and a reaction tank, wherein the purpose of the transgenic substrate is to serve as a supporting base, and at the same time It is biologically valley-shaped, allowing cells to grow, and uses a 76x26mm glass slide as a substrate. A conductive metal layer is used to establish the electric field required for cell membrane perforation, and is biocompatible, and cell culture will not cause adverse effects. The surface of the substrate is processed with two layers of gold (Gold; Au) and titanium (Titanium; Ή). Electrical conductivity and biocompatibility are not easy to produce electrochemical reactions. As the metal layer of the electrode for the cell membrane perforation, titanium (Titanium; ium) is used as the adhesion layer between gold (Au) and the glass substrate. The reaction tank is the location for culturing cells. It is a treated sterile tank. It can be determined according to the reaction range of cell electroporation. It contains the medium required for cell culture. Therefore, it must have a depth of 2mm or more (or thickness). ), This example uses PDMS (Polvdimethvlsiloxane) to manufacture the side wall of the reaction tank 11 1227736 by mechanical processing, which has the advantages of stable chemical composition, no solvent volatilization, easy processing, transparent material, convenient experimental observation, and adhesion. It is not easy to damage the substrate and electrodes, and it is easy to assemble. The above detailed production and various processes are described as follows: (a) Photomask design The small line width of the element in the present invention is μμη, and the low-cost film photomask is selected based on the minimum line width analysis ability to transfer the gene onto the substrate. Set up three reaction zones, as shown in the second figure, each reaction zone is independent, and mutual interference will not occur during the process of electric pulse. (b) Refer to Figures 3 and 4 for the design of microelectrodes. The present invention adopts a comb-shaped interdigitated design to increase the area of the reaction area, and at the same time can generate a high electric field environment under low voltage conditions, with conductive metals. The pattern of the layer is mainly formed by the reaction electrode, the probe contact (Tóκ ^ pa (j), and the alignment mark (AlignmentMark); the line width of the electrode in the reaction area is 100 μηι, and the distance between the positive and negative electrodes To stimulate μιη 'probe contacts are 4000 × 4000μηι, the spacing between positive and negative contacts is 6000μηι, the alignment marks are four 500 × 500μπι squares, and the spacing is 8000μηι. Three reactions are designed on a 76_ × 26_ transgenic substrate The distance between the reaction zone and the reaction zone is 6,000 μm to avoid mutual influence caused by the phenomenon of leakage current during the electrical pulse process. Alignment marks around the electrodes help the positioning of the transposable substrate during assembly. (C) Process book The invention of the substrate conversion process is mainly based on the micro-electro-mechanical process. The vacuum evaporation method is used to deposit metal thin films and the lithographic etching method is used to define metal electrodes. The method of chemical coating after 1212736 changed the properties of the surface of the transplanted substrate. The steps used an electron beam evaporator (E-Beam Evaporator; EBE) to deposit a high-purity metal layer, using titanium (Titanium; Tl) and Gold (Gold; Au) is used as the riding material. Chin and Au are both highly biocompatible materials. They do not undergo oxidation or other chemical reactions. Therefore, they are suitable as conductive plutonium for biomedical research and are compatible with high vacuum electronics. The beam inspection _ moving head _ process is in line with the requirements of high-purity electrode manufacturing technology for biomedical substrates. To avoid electrode peeling off, 100A titanium is first deposited on the surface of the glass substrate at a rate of 2A / sec as an adhesive layer. Turn over 5A / see to evaporate leakage A, and set the electro-gold / non-deposition ratio between 10 · 1 to 20 · 1. The adhesion effect is better. The detailed processing steps are as follows: 1. Use HMDS (Hexamethvldisilazane; surface adhesive ) Evaporate for surface treatment for 20 min 2. Photoresist coating, place the substrate of the slide on the center of the suction cup of the spin coater, and drip 3ml Shipley 1818 photoresist liquid on the center of the substrate of the slide at 2500rpm — 10sec / 5000rpm — Spin-coated transplanted substrate 30 sec. Soft baking, horizontally placed in an oven at 90 ° C for 30 min. Lu 4 exposure. After the photomask is aligned with the substrate of the slide, set 7 8 sec irradiation 5 development. Dilute four times. Deveiperper 351 is developed for 30 ~ 60sec 6. Rinse 'deionized (DI water) and wash the substrate of the slide 10min 7. Hard bake' horizontally placed in an oven at 120 ° C for 30min
另’於基板以含有鹽酸（Hydrochloric Acid; HC1)的鈦蝕刻液，在70°C 13 1227736 對__為論see，树級會_觸彡__，金的抗 刻力強可作為敍的侧罩幕，以值溫切槽隔水加触刻劍，並且以電磁 授拌器擾動溶液，使溶液均勻穩定麵刻速率。㈣完成後用光阻去除液 CRemoveO (Acetone) (is〇m〇]) ^ 並且以m_ (deiGnized;去離子水）徹底清除表面的殘留物，避免造成 細胞生長的障礙，以上詳細處理步驟如下： •以TPX (4-methylpentene ;鐵弗龍）塑膠染色架承載轉殖基板 2. 放入金侧液並且用電磁授拌器、以300rpm_溶液60sec 3. 以DI Water (deioni就去離子水）、冲洗轉殖基板i〇min 4. 將欽餘刻液以怪溫水槽加熱至7〇〇c 5. 連同染色架將轉殖基板放入鈦餘刻液並且用電磁獅器以綱啊 攪:動溶液15sec 6.以DI Water沖洗轉殖基板i〇min 7·以光阻去除液浸泡轉殖基板5min 8·以DI Water (deionized ;去離子水）沖洗轉殖基板i〇min 9.用N2吹乾轉殖基板 該表面處理系統，主要目的使貼壁性的細胞更容易緊密貼附於轉 殖基板表面，使得實驗操作過程中細胞不會剝落或位移；表面處理是將分子 量70,000〜150,000的Poly-D-Lysine塗覆在轉殖基板表面，改變轉殖基板表面 14 1227736 的介面特性，藉以達到細胞貼附的目的；轉殖基板表面處理的過程必須在無 菌操作台内進行，避免轉殖基板受到外界環境的污染，Poly-D-Lysine的塗佈 可以增加表面正電荷離子的數量，增進細胞貼附的機會。濃度l〇〇ppm的水 /谷液1 ml可以處理25cm2的面積。Poly-D-Lysine塗佈完成的表面為親水性介 面，同時表面帶正電荷。 另’ Poly-cpLysine對細胞具有毒性，當表面處理完成後，必須將殘留 在轉殖基板上的Poly-D-Lysine溶液沖洗乾淨，否則會影響細胞的生長塗佈 (Coating) Poly_D-Lysine的方法採用滴入法，其步驟如下· · 1·將滅卤並乾燥完成之轉殖基板置於無菌的9cm塑膠培養皿； 2.於反應槽中滴入50|nl的P〇ly-D-Lysine水溶液； 3·將培養皿連同轉殖基板置於37°C的恆溫箱24小時； 4. 完成後將反應槽的P〇ly-D-Lysine水溶液吸乾； 5. 以PBS或無鹵的MQ water徹底清洗反應槽兩次； 6·培養皿置於無菌操作台以紫外光燈連續照射8小時以上滅菌並且自 然乾燥。 該繼代培養系統，係將原本在培養皿中大量培養的細胞移到基因 轉殖相容系統的反應槽中繼續培養。本實施例所使用的細胞為293T (腎 臟癌細胞的一種），是一種纖維母細胞（fiberbrast)，具有貼壁的特性（註： 人造細胞、表皮細胞等亦可）。首先，細胞必須放置於37〇〇〖1溫無菌培養箱 15 1227736 中維持生長’並且通以5%的c〇2氣體維持培養基的酸驗值72〜74。以 Dulb_'s modified Eagle's medium培養基提供維持細胞的生長與活性所需 的養分，培養基添加了 1〇% (v/v)胎牛血清（脑也牆獅編 serum^BS). lOunit/mlM^S^CPenicillin), 1 ΟΟμ/ml^streptomycin)- 及4mM毅胺酿胺（L_ghitamine)。 在培養皿中大量培養的細胞必須先在培養皿或Flask中培養到足夠的 數量後（約七到八分滿，正常情況一個9cm的培養皿可以得到1〇7 cdls)， 將細胞從培養皿表面取下，分散到轉殖基板上培養，一個9cm培養皿的細 胞數里足夠供應650個轉殖基板使用。繼代培養分為三個步驟：細胞懸浮、 細胞計數及轉殖基板培養，其中轉殖基板培養（Culture 〇n Chip)是將固定 數量的細胞放在反應槽的轉殖基板内，使細胞附著在轉殖基板或電極表面 上。根據細胞濃度計算的結果，加入適量的培養基調整細胞濃度到1〇5 cells/ml;稀釋細胞濃度時，最好以多次稀釋的方式，每次稀釋倍數不要超過 10倍，避免發生不均勻的情況。取稀釋後的細胞樣本兄…注入反應槽的轉 殖基板，繼續在無菌培養箱中培養。使每個進行實驗的反應槽中約有5〇〇〇 顆細胞。懸浮的細胞在新的反應槽中，30分鐘内會沉澱到反應槽底部，4〜6 小時便會完全貼附在轉殖基板表面，18小時後細胞完全穩定就可以提供電 胞膜穿孔的實驗。 該實驗系統，係將在基因轉殖相容系統内反應槽所培養的 細胞’以固定DNA質量及培養基體積，將Plasniid DNA輸送入 細胞内進行電脈衝胞膜穿孔實驗。本實施例以轉殖基板代替傳統 16 1227736 cuvette作為反應器，對293T細胞進行電脈衝胞膜穿孔，將PlasmidDNA輸送 到細胞内。Electroporation必須在無血清的環境下進行，避免血清内複雜的 成分影響實驗結果，過程中必須固定DNA的質量，確保轉殖的結果不受^^人 濃度的影響，反應時培養基的體積必須固定，以確保每一片轉殖基板的阻抗 值及電容值相同。詳細步驟如下： 1. 用37°C無血清的培養基清洗反應區的細胞兩次 2. 用無血清的培養基將PlasmidDNA稀釋，使DNA濃度到達80pg/ml 3·每個反應槽加入20μ1 DNA試劑 4·轉殖基板靜置於37°C無菌培養箱中1〇〜Ι5ηώι 5.設定電脈衝的參數 6·以探棒輕壓在電極接點（Pad)觸發脈衝 7·反應完成後立刻加入25ml培養基於培養皿，使培養基完全覆蓋細胞 8·放置在37°C無菌培養箱中繼續培養 該檢測系統，係用於觀察本發明轉殖基因的結果，即由本發明 轉殖成功的細胞會以pEGFP-Nl (螢光表現）Vector為模板，而遺傳密碼子 679〜1398會被轉譯綠色螢光蛋白（GFP)，使得細胞膜内充滿螢光蛋白物質， 藉以確認細胞的基因轉殖成效。依螢光物質吸收短波長的光能，放射出長波 長的光，EGFP的激發（Excitation)峰值波長為4〇〇nm (UV)與475nm (藍 光），發射（Emission)峰值波長為507nm (綠光），轉殖成功的細胞，利用 17 1227736 反射式汞燈光源螢光顯微鏡搭配適當的濾鏡組，便可判讀轉殖細胞的數目。 其中，EGFP為GFP強度35倍。因而，基因轉殖成功的細胞會開始製造螢光 蛋白GFP，經過24小時的培養後，GFp的濃度提高，便可以產生在螢光顯微 鏡下觀察轉殖結果，計算綠色螢光細胞的數量以反映轉殖的程度。 本發明的實施例中採用Olympus BX40螢光顯微鏡，搭配濾光鏡組 U-MWIB ( Excitation 460〜490nm; Emission 515nm〜），影像擷取系統是 pixera 600CL Cooled CCD配合桌上型電腦，即時擷取螢光影像，pixera 600(χ Cooled CCD 系統採用 1/2” IT-CCD (1.5 mega pixel)，最大解析度 2776\2074卩丨\6卜快門速度1/1000〜60 86(：。 上述實驗系統與檢驗系統可便捷看出胞膜電穿孔的實際所產生的成 效，請配合第五圖至第七圖參閱，其中第五圖所示由上而下係細胞數量 為：5000 cells/well、2500 cells/well、500 cells/well ;細胞密 度：177 cells/mm2、88.5 cells/ mm2、17.7 cells/mm2 ’ 容置於轉殖 基板反應區内的細胞影像，而第六、七圖係分別以7.2V及lv 工作電壓做胞膜電穿孔後轉殖基板上的細胞培養影像，從第 六、七圖可發現細胞在基因轉殖後培養24小時，GFP DNA轉 錄為RN A，RN A再轉譯為蛋白，使螢光蛋白可在螢光顯微鏡 的激發下表現。且，第七圖中小電壓（IV)細胞轉殖機率較第六 圖高電壓（7.5V)更增加。是以，本發明可依照不同細胞轉殖所 須條件，以最佳化的轉殖機率進行實際的實驗作業上。 另，請參閱第八圖為以本發明之胞膜電穿孔基因轉殖模擬 1227736 方法依照上述各系統與步驟以0.1V及0.05V不同工作電壓，對 不同細胞進行胞膜電穿孔後於轉殖基板上的細胞培養影像，其 中：A為人體肝細胞（Chang liver cells ); B為人體臍靜脈血管 内皮細胞（Primary human umbilical vein endothelial cells ; HUVEC ) ; C 為人體分化型肝細胞（Human hepatocellular carcinoma cell line ; Hep-G2 ) ; D 為肝癌細胞（Human hepatocellular carcinoma cell line ； Huh-7 cells ) ° 是以，可知本 發明的胞膜電穿孔基因轉殖模擬方法可在不同工作電壓下對不# 同細胞進行實驗，可減少對稀有細胞的取得，餅可使得整個實 驗過程在同一個平台上進行，免除細胞培養、刮除、轉殖等繁 複的作業程序。歸納以上所述，本發明所提供一種胞膜電穿孔基因轉 殖（electroporation)的平台，結合活細胞與電極電路於轉殖基板上，經胞 膜電穿孔基因轉殖所需之電場脈衝，並使轉殖後的細胞可在勞光顯微鏡下 觀察，Ifg減實驗步驟’減少細胞、培養基、DNA、血清及抗生素的使用量， 降低研究成本；降低胞膜電穿孔基因轉殖的工作電壓同時減少設備成· 本，對於從事胞膜電穿孔基因轉殖基礎研究的人員，本發明將可提供更 方便、更便宜的研究方法，以提昇實驗的效率。 另，本發明除上述提供醫師臨床上的實驗外，該模擬方法中 的基因轉殖基板實際運用上亦可採用高分子材料，製作成軟質(或硬 貝）’並以上述製程將金在其表面上製出具微電極的導電層，其電極圖型的電 極線寬可為以下，正、負電極的間距可為觸叫以下，電極形狀以 19 1227736 幾何形皆可。藉以可》/ ^ > Γ在低工作電壓10V以下直接貼於欲治療的部位，進行胞 膜電穿孔的轉。易言之，本發明具有如下優點： 1·實驗過賴單而且快速可在同—平台上完成。 2. 適用於各種細胞的臨床實驗。 3. 可以低工作電義於活體外治療，降低胞膜電穿孔基因轉殖 (electroporation)對治療部位或附近細胞及神經的損傷。 综上所述，本發明一種用於生化科技領域的胞膜電穿孔旯 因轉殖模擬方法確具首創新穎，合乎產業利用 ^ 、 丨隹以上所 揭露者，僅為本發明之較佳實施例而已，自不< 明之專利範圍，因此，依照本發明所做之等效辦 >、定化或修飾 仍屬本發明所涵蓋之範圍。 +犯以此限定本發 者， 【圖式簡單說明】 第一圖係本發明一種用於生化科技領域的胞膜電穿 模擬方法之驅動電路圖。 因 第二圖係本發明一種用於生化科技領域的胞膜電 模擬方法之轉殖基板上的三個反應區。 第三圖係本發明一種用於生化科技領域的胞膜電 模擬方法之電極佈局光罩圖。 第四圖係本發明一種用於生化科技領域的胞膜電 穿 穿 穿 孔基 因轉 轉 扎基 轉 20 1227736 模擬方法之電極線寬、正負極間距及電場有效區域圖。 第五圖：是容置於轉殖基板反應區的細胞培養影像，由上而下 詳細資料如下： 細胞數量為：5000 cells/well、2500 cells/well、500 cells/well ； 細胞密度：177 cells/mm2、88.5 cells/ mm2、17.7 cells/mm 〇 第六圖：以7.2V工作電壓做胞膜電穿孔後轉殖基板上的細胞培 養影像。 第七圖：以IV工作電壓做胞膜電穿孔後轉殖基板上的細胞培養 影像。 第八圖：為以本發明之胞膜電穿孔基因轉殖模擬方法以0.1V 及0.05V工作電壓對人體肝細胞、人體臍靜脈血管内皮 細胞、人體分化型肝細胞、肝癌細胞，進行胞膜電穿孔 後於轉殖基板上的細胞培養影像。 【主要元件符號說明】 脈衝驅動電路 90 放大 IC (OP741) 91 電晶體 92 基極（Base) 921 射極（Emitter) 922 電晶體開關 93 可變電阻（Rx) 94 電阻 95 21In addition, on the substrate, a titanium etching solution containing hydrochloric acid (HC1) is used at 70 ° C 13 1227736. For the __ see, the tree level meeting _ touches __, the strong resistance to gold can be used as Syria The side cover screen is cut with water at the value of temperature and added a touch sword, and the solution is disturbed by an electromagnetic stirrer to make the solution uniform and stable face engraving rate. ㈣After completion, use photoresist removal solution CRemoveO (Acetone) (is〇m〇)) ^ and use m_ (deiGnized; deionized water) to completely remove the residue on the surface to avoid obstacles to cell growth. The above detailed processing steps are as follows: • Use TPX (4-methylpentene; Teflon) plastic dyeing rack to carry the transplanted substrate 2. Put the gold side solution and use an electromagnetic stirrer at 300rpm_ solution for 60sec 3. Use DI Water (deioni is deionized water) Rinse the transgenic substrate i0min 4. Heat the Qinyu etching solution to 700 ℃ with a strange warm water tank 5. Put the transgenic substrate into the Titanium etching solution together with the dyeing stand and stir it with a magnetic lion device: Run the solution for 15sec 6. Rinse the transgenic substrate i0min with DI Water 7. Soak the transgenic substrate 5min with photoresist removal solution 8. Rinse the transgenic substrate imin with DI Water (deionized) 9. Use N2 The main purpose of this surface treatment system is to dry the transplanted substrate. The main purpose is to make adherent cells more easily adhere to the surface of the transposed substrate, so that the cells will not peel or move during the experimental operation. The surface treatment is to make the molecular weight of 70,000 ~ 150,000. Poly-D-Lysine is coated on the surface of the transgenic substrate. The interface characteristics of the transfection substrate surface 14 1227736, so as to achieve the purpose of cell attachment; the surface treatment process of the transfection substrate must be performed in a sterile operation table to prevent the transfection substrate from being polluted by the external environment. Poly-D-Lysine coating Cloth can increase the number of positively charged ions on the surface and increase the chance of cell attachment. 1 ml of 100 ppm water / valley solution can handle an area of 25 cm2. Poly-D-Lysine coated surface is hydrophilic and the surface is positively charged. In addition, Poly-cpLysine is toxic to cells. After the surface treatment is completed, the Poly-D-Lysine solution remaining on the transplanted substrate must be washed away, otherwise the growth of the cells will be affected. (Coating) Poly_D-Lysine The drop-in method is used. The steps are as follows: 1. Place the halogen-free and dried transgenic substrate on a sterile 9 cm plastic petri dish; 2. Drop 50 nL of Polly-D-Lysine into the reaction tank. Aqueous solution; 3. Place the petri dish with the transplanted substrate in a 37 ° C incubator for 24 hours; 4. Dry the reaction solution ’s Poly-D-Lysine aqueous solution after completion; 5. Use PBS or halogen-free MQ The reaction tank was washed thoroughly with water twice; 6. The petri dish was placed on a sterile operation table and irradiated with UV light for more than 8 hours to sterilize and dry naturally. In this subculture system, cells originally cultured in large numbers in a petri dish are transferred to a reaction tank of a gene transfection compatible system and cultured. The cell used in this embodiment is 293T (a type of kidney cancer cell), which is a type of fiberbrast, and has the characteristics of adherence (note: artificial cells, epidermal cells, etc.) may also be used. First, the cells must be placed in a 37 ° C 1-temperature sterile incubator 15 1227736 to maintain growth 'and the acid test value of the culture medium maintained at 72 to 74 with 5% CO2 gas. Dulb_'s modified Eagle's medium provides nutrients necessary to maintain cell growth and viability. The medium is supplemented with 10% (v / v) fetal bovine serum (brain wall lion series serum ^ BS). LOunit / mlM ^ S ^ CPenicillin ), 100 μ / ml ^ streptomycin)-and 4mM L_ghitamine. A large number of cells cultured in a petri dish must first be cultured in a Petri dish or Flask to a sufficient number (approximately seven to eight full, a 9cm petri dish can normally obtain 107 cdls). The surface was removed, and the cells were dispersed and cultured on a transfection substrate. A cell of 9 cm was enough to supply 650 transfection substrates. Subculture is divided into three steps: cell suspension, cell counting, and transfection substrate culture. Among them, Culture On Chip is a method in which a fixed number of cells are placed in the transfection substrate of a reaction tank to allow the cells to attach. On the substrate or electrode surface. According to the results of cell concentration calculation, add an appropriate amount of medium to adjust the cell concentration to 105 cells / ml; when diluting the cell concentration, it is best to use multiple dilutions, and the dilution multiple should not exceed 10 times each time to avoid unevenness. Happening. Take the diluted cell sample ... and inject it into the transfer substrate of the reaction tank, and continue to culture in a sterile incubator. Make approximately 5000 cells in each reaction tank where the experiment is performed. The suspended cells will settle to the bottom of the reaction tank within 30 minutes, and will be completely adhered to the surface of the transplantation substrate within 4 to 6 hours. After 18 hours, the cells are completely stable and can provide an electromembrane perforation experiment. . In this experimental system, cells cultivated in a reaction tank in a transgenic compatible system are used to fix the quality of the DNA and the volume of the culture medium, and transport Plasniid DNA into the cells for electrical pulsed cell membrane perforation experiments. In this embodiment, a transgenic substrate is used instead of the conventional 16 1227736 cuvette as a reactor, and electric pulse cell membrane perforation is performed on 293T cells to deliver PlasmidDNA into the cells. Electroporation must be performed in a serum-free environment to avoid complex components in the serum from affecting the experimental results. The quality of the DNA must be fixed during the process to ensure that the results of transfection are not affected by human concentration. The volume of the medium must be fixed during the reaction. To ensure that the impedance value and capacitance value of each transplanted substrate are the same. The detailed steps are as follows: 1. Wash the cells in the reaction zone twice with 37 ° C serum-free medium 2. Dilute PlasmidDNA with serum-free medium so that the DNA concentration reaches 80 pg / ml 3. Add 20 μ1 DNA reagent to each reaction tank 4 · The transgenic substrate is placed in a sterile incubator at 37 ° C for 10 ～ Ι5η. 5. Set the parameters of the electric pulse. 6. Press the probe lightly on the electrode pad (Pad) to trigger the pulse. 7. Add 25ml culture immediately after the reaction is completed. Based on a petri dish, the medium is completely covered with cells. 8 · Placed in a 37 ° C sterile incubator to continue culturing the detection system, which is used to observe the results of the transgenic genes of the present invention. The Nl (fluorescent expression) Vector is used as a template, and the genetic codons 679 to 1398 are translated into green fluorescent protein (GFP), so that the cell membrane is filled with fluorescent protein material, thereby confirming the cell's gene transfer effect. The fluorescent substance absorbs short-wavelength light energy and emits long-wavelength light. The excitation peak wavelength of EGFP is 400nm (UV) and 475nm (blue light), and the peak wavelength of Emission is 507nm (green Light), successfully transfected cells, using 17 1227736 reflective mercury lamp light source fluorescence microscope with appropriate filter set, you can determine the number of transfected cells. Among them, EGFP is 35 times stronger than GFP. Therefore, cells successfully transgenic will begin to produce fluorescent protein GFP. After 24 hours of culture, the concentration of GFp increases, and the result of transfection can be observed under a fluorescent microscope, and the number of green fluorescent cells can be calculated to reflect Degree of transplantation. In the embodiment of the present invention, an Olympus BX40 fluorescence microscope is used, with a filter set U-MWIB (Excitation 460 ~ 490nm; Emission 515nm ~). The image capture system is a pixera 600CL Cooled CCD combined with a desktop computer for instant capture. Fluorescence image, pixera 600 (χ Cooled CCD system uses 1/2 ”IT-CCD (1.5 mega pixel), maximum resolution 2776 \ 2074 卩 丨 \ 6b shutter speed 1/1000 ~ 60 86 (: the above experimental system And the test system can easily see the actual effect of cell membrane electroporation. Please refer to Figures 5 to 7, where the number of cells in the top-down line shown in Figure 5 is 5000 cells / well, 2500. cells / well, 500 cells / well; cell density: 177 cells / mm2, 88.5 cells / mm2, 17.7 cells / mm2 'image of cells contained in the transfection substrate reaction zone, and the sixth and seventh images are respectively 7.2 V and lv operating voltages were used to perform cell culture images on the transplanted substrate after cell membrane electroporation. From the sixth and seventh images, it can be found that cells were cultured for 24 hours after gene transfection. Fluorescent protein The performance of the cells under the small voltage (IV) in the seventh chart is higher than that in the high voltage (7.5V) in the sixth chart. Therefore, the present invention can be optimized according to the conditions required for cell transplantation. The actual translocation probability will be used for actual experimental operations. In addition, please refer to the eighth figure for the cell membrane electroporation gene translocation simulation 1227736 method of the present invention in accordance with the above systems and steps with different operating voltages of 0.1V and 0.05V, Cell culture images on transgenic substrates after cell membrane electroporation of different cells, where: A is human liver cells (Chang liver cells); B is primary human umbilical vein endothelial cells (HUVEC) C is human hepatocellular carcinoma cell line (Hep-G2); D is human hepatocellular carcinoma cell line (Huh-7 cells). The colony simulation method can perform experiments on different cells under different working voltages, which can reduce the acquisition of rare cells. The cake can make the entire experimental process in the same It is performed on the platform, eliminating the complicated operation procedures such as cell culture, scraping and transplantation. To summarize the above, the present invention provides a platform for electroporation of cell membrane electroporation genes, combining live cells and electrode circuits on the substrate for transporation, and the electric field pulses required for gene transfection via cell membrane electroporation, and The transfected cells can be observed under a light microscope. Ifg reduces the experimental steps, which reduces the use of cells, culture media, DNA, serum, and antibiotics, and reduces research costs. At the same time, the working voltage of cell membrane electroporation gene transduction is reduced. Equipment cost. For those who are engaged in basic research of cell membrane electroporation gene transfer, the present invention will provide a more convenient and cheaper research method to improve the efficiency of experiments. In addition, in addition to the above-mentioned clinical experiments provided by the physician, the present invention can also use a polymer material to produce a soft (or hard shell) 'in the practical application of the gene transgenic substrate in the simulation method. A conductive layer with micro-electrodes is fabricated on the surface, and the electrode line width of the electrode pattern can be the following, and the distance between the positive and negative electrodes can be the following, and the electrode shape can be 19 1227736 geometry. Therefore, ^ / ^ > Γ is directly applied to the site to be treated at a low working voltage below 10V, and the membrane electroporation is performed. In other words, the present invention has the following advantages: 1. Experiments can be completed on the same platform quickly. 2. Suitable for clinical experiments of various cells. 3. It can be used for in vitro treatment with low working electricity, which can reduce the damage of cells and nerves at or near the treatment site by electroporation of the cell membrane. To sum up, a method for simulating cell membrane electroporation in the field of biochemical science and technology according to the present invention is indeed the first innovation, which is suitable for industrial use ^, 丨 隹 The above is only a preferred embodiment of the present invention However, since the scope of the patent is not known, the equivalents, modifications, or modifications made in accordance with the present invention are still within the scope of the present invention. + This is to limit the author. [Schematic description] The first diagram is a driving circuit diagram of a method for simulating the electromembrane of a cell membrane used in the biochemical technology field of the present invention. The second diagram is the three reaction zones on the transgenic substrate of a method for cell membrane electrical simulation in the field of biochemical technology. The third diagram is an electrode layout photomask of a method for simulating a cell membrane in the field of biochemical technology according to the present invention. The fourth diagram is a diagram of the cell membrane electrical perforation and perforation in the biochemical science and technology field of the present invention. The electrode line width, positive and negative electrode spacing, and electric field effective area diagram of the simulation method. Fifth image: Cell culture images contained in the transfection substrate reaction zone. Details from top to bottom are as follows: Number of cells: 5000 cells / well, 2500 cells / well, 500 cells / well; Cell density: 177 cells / mm2, 88.5 cells / mm2, 17.7 cells / mm. Figure 6: Cell culture images on the transplanted substrate after electroporation of the cell membrane at a working voltage of 7.2V. Figure 7: Image of cell culture on the transplanted substrate after electroporation of the membrane at the IV operating voltage. Figure 8: The cell membrane of human hepatocytes, human umbilical vein vascular endothelial cells, human differentiated hepatocytes, and liver cancer cells was subjected to the cell membrane by using the method of cell membrane electroporation gene transfection simulation of the present invention at a working voltage of 0.1V and 0.05V Image of cell culture on electrotransformed substrate after electroporation. [Description of main component symbols] Pulse drive circuit 90 Amplification IC (OP741) 91 Transistor 92 Base (Base) 921 Emitter (922) Transistor switch 93 Variable resistor (Rx) 94 Resistor 95 21