201229240 六、發明說明： 【發明所屬之技術領域】 本發明係關於一種用於控制細胞貼附性之方法，特別 係關於一種藉由调整培養基之pH值來控制細胞貼附性之方 法。 【先前技術】 細胞培養之技術係將細胞自有機體中取出，並將其培 養於模擬有機體之生長條下’使之繁殖、生存、生長之技 • 術，藉由此種技術，係可進行藥物療效、組織工程、细胞 1$選等方面之研究。因此，隨著生醫技術之發展，細胞择 養技術之重要性亦日趨增加。 σ 一般而言，依據動物細胞生長型態可將動物細胞分成 三類，懸浮型細胞（如血液細胞、淋巴組織細胞、造血幹細 胞）、貼附型細胞（如：間質幹細胞、上皮細胞）及介於該兩 種類型間之細胞’其中貼附型細胞因需貼附於物體上方能 生長，因此，若要將其與物體表面分離，則需經過特別之 處理。然，常見之處理程序通常過為繁雜，抑或於分離之 • 過程中，會破壞細胞之結構，進而造成其死亡，影響後續 之研究。 對此，Yamada等人已研發出—種可讓細胞順利脫附之 方法，該方法係利用以溫度應答型高分子聚N_異丙烯丙烯 醯胺所接枝之表面可於37艽培養環境下呈現疏水性而讓細 胞貼附於其上，但當培養溫度降至溫度應答型高分子材料 之最低臨界時，會使之轉變為親水性而使該些細胞能夠順 利至表面分離。然，於該方法中，細胞之貼附或脫附主要 係受不同 '皿度的效應所调控’因此，會有細胞於培養過程 201229240 中容易受溫度震盪過大的效應影響而導致其死亡之缺點。 因此，實有必要開發出另一種可有效使細胞自貼附表面脫 附之方法。 【發明内容】 有鑒於此，本發明之目的係開發出一種可有效使細胞 自貼附表面脫附之方法，其主要係應用酸鹼應答型高分子 於不同酸鹼環境下之表面電性不同之特性而完成。 為達上述目的，本發明係提供一種用於控制細胞貼附 性之方法，其包含：提供一基材，其中該基材之表面上係 具有一酸鹼應答型高分子；於該基材之表面上培養細胞； 及調整培養該細胞用之培養基之pH值，以控制該細胞之貼 附性。 於一較佳實施態樣中，該基材為細胞培養皿、細胞培 養盤或細胞培養瓶。 於一較佳實施態樣中，該酸鹼應答型高分子之酸離係 數(pKa)為6.0〜8.0。更佳地，該酸鹼應答型高分子之酸離係 數為6.4〜7.2。 於一較佳實施態樣中，該酸鹼應答型高分子為具胺基 之高分子。更佳地，該具胺基之高分子為幾丁聚醣、聚離 胺酸、聚丙烯胺、或聚丙烯醯胺。（由於 於一較佳實施態樣中，使該基材之表面上具有一酸驗 應答型高分子之方式係包含下述步驟：將該酸鹼應答型高 分子溶解於弱酸水溶液中，以得一高分子溶液；將該高分 子溶液塗佈至一基材上，得一經塗佈之基材；乾燥該經塗 佈之基材，得一經乾燥之基材；以驗性水溶液中和該經乾 燥之基材，得經中和之基材；及以水清洗該經中和之基材 並乾燥。 201229240 中，該弱酸水落液之ph值係介於 3門5〜6.5之間。更佳地’該弱酸水溶液之pH值介於4〜6之 間。 於-較=施態樣中，該培養基之pH值為65〜8卜 養基中’該PH值之調整係透過更換該培 行。於-較佳實施態樣中’該培養之步驟係於培養箱中進 於-較佳實施態樣中’該pH值之 養箱中之二氧化碳之分壓來完成。 ，、曰由文交忒坧 於：較，實施態樣中，該pH值之調整係透過該培養箱 中之—乳化板及4培養基中之碳酸氫納平衡來完成。< 於-較佳實施態樣中’該_係包含貼附或脫附。 由上可知，本發明主要係利用酸驗應答型高分子 =酸驗環境下之表面電性不同之特性，來調控細胞對其之 進而可藉由改變培養基之阳值，輕易地將細胞自 酸鹼應答型高分子上脫附。 、肥目 【實施方式】 $發明係提供-種可有效使細胞自貼附表面脫附之方 表主要係制酸驗應答型高分子 表面電性不同之特性而完成。 鐵A下之 法，=言提提r用於控制細胞貼附性之方 酸驗應二高材之表面上係具有-培養該細胞用之表面上培#細胞；及調整 於-較佳*% 以控制仙胞之貼附性。 更佳為6.ti 7祕中，鱗養基之ΡΗ值為6.5〜8.5， 201229240 ▲ 明中，該基材可為此領域具有通常知識者所熟 中所稱之「酸驗應答型高分子」係指於弱酸環 境下係帶正電荷，於弱鹼環境下係不帶電之高分子。一般 而言’該餐應答变高分子之酸離係數較佳4 6力〜8 〇，更 佳為6_4 7.2。於-較佳實施態樣中，該酸驗應答型高分子 包含具胺基之高分子（諸如：幾丁聚酿、聚離胺酸、聚丙稀 胺、或聚丙烯醯胺），但並不限於此。 於本發明中，使该基材之表面上具有_酸驗應答型高 分子之方式係包含下述步驟：將該酸鹼應答型高分子溶解 於弱酸水溶液中，以得一高分子溶液；將該高分子溶液塗 佈至一基^上，得一經塗佈之基材；乾燥該經塗佈之基材， 知一經乾燥之基材；以驗性水溶液中和該經乾燥之基材， 得經中和之基材；及以水清洗該經中和之基材並乾燥。前 述之弱酸水溶液及鹼性水溶液均為本領域技術人員所熟知 的，舉例而言，弱酸水溶液可為乙酸水溶液、曱酸水溶液、 檸檬酸水溶液、或填酸水溶液，但並不限於此，而驗性水 溶液可為氫氧化鈉水溶液、氫氧化鉀水溶液、或氳氧化鋰 水溶液，但並不限於此。而該弱酸水溶液之pH值較佳係介 於3.5〜6_5之間，更佳係介於4〜6之間。 本發明所用之細胞可為任何種類之哺乳類細胞，包含 人類之上皮性肺癌細胞(human lung carcinoma，Η1299)、老 鼠之胚胎纖維母細胞(mouse embryonic fibroblast，3Τ3)或牛 之角膜間質細胞(bovine keratocyte/fibroblast)，但並不限於 此。 於一較佳實施態樣中，該pH值之調整係透過更換該培 養基來完成。 201229240 於另^父佳實施態樣中，該培養之步驟係於培養箱中 進行。而該pH值之調整係透過該培養箱中之 礙及該 培養基中之碳酸氫鈉平衡來完成。較佳地，兮二 之 分壓為.30.00%。更佳地，該二氧化碳一之分壓為 0.10〜15.00%。此外，不難理解的是，於本發明中，亦可藉 由改變該培養箱中之二氧化碳之分壓來調整該。201229240 VI. Description of the Invention: [Technical Field] The present invention relates to a method for controlling cell adhesion, and more particularly to a method for controlling cell adhesion by adjusting the pH of a medium. [Prior Art] The technique of cell culture removes cells from an organism and cultures them under the growth bar of a simulated organism to make them propagate, survive, and grow. With this technique, drugs can be used. Efficacy, tissue engineering, cell 1$ selection and other aspects of research. Therefore, with the development of biomedical technology, the importance of cell selection technology is increasing. σ In general, animal cells can be divided into three types according to the growth pattern of animal cells, such as suspension cells (such as blood cells, lymphoid tissue cells, hematopoietic stem cells), adherent cells (such as mesenchymal stem cells, epithelial cells), and A cell between the two types' in which the attached cell grows because it needs to be attached to the object, and therefore, if it is to be separated from the surface of the object, special treatment is required. Of course, common procedures are often complicated, or in the process of separation, which destroys the structure of the cells, causing them to die, affecting subsequent research. In response, Yamada et al. have developed a method for allowing cells to be successfully desorbed by using a temperature-responsive polymeric poly(N-isopropene acrylamide) grafted surface in a 37 艽 culture environment. Hydrophobicity is applied to allow cells to attach to them, but when the culture temperature is lowered to the lowest critical temperature of the temperature-responsive polymer material, it is converted into hydrophilicity so that the cells can be smoothly separated from the surface. However, in this method, the attachment or detachment of cells is mainly controlled by different effects of the degree of the dish. Therefore, there is a defect that the cells are easily affected by the effect of temperature turbulence in the culture process 201229240. . Therefore, it is necessary to develop another method for effectively desorbing cells from the attached surface. SUMMARY OF THE INVENTION In view of the above, the object of the present invention is to develop a method for effectively desorbing cells from a self-adhesive surface, which mainly uses an acid-base responsive polymer to have different surface electrical properties under different acid-base environments. The characteristics are completed. In order to achieve the above object, the present invention provides a method for controlling cell adhesion, comprising: providing a substrate, wherein the substrate has an acid-base responsive polymer on a surface thereof; The cells are cultured on the surface; and the pH of the medium for culturing the cells is adjusted to control the adhesion of the cells. In a preferred embodiment, the substrate is a cell culture dish, a cell culture tray or a cell culture flask. In a preferred embodiment, the acid-base responsive polymer has an acid separation coefficient (pKa) of from 6.0 to 8.0. More preferably, the acid-base responsive polymer has an acidity coefficient of from 6.4 to 7.2. In a preferred embodiment, the acid-base responsive polymer is a polymer having an amine group. More preferably, the amine-based polymer is chitosan, poly-amino acid, polyacrylamine, or polyacrylamide. (In a preferred embodiment, the method of providing an acid-responsive polymer on the surface of the substrate comprises the step of dissolving the acid-base responsive polymer in a weak acid aqueous solution to obtain a polymer solution; coating the polymer solution onto a substrate to obtain a coated substrate; drying the coated substrate to obtain a dried substrate; neutralizing the solution with an aqueous solution The dried substrate is subjected to a neutralized substrate; and the neutralized substrate is washed with water and dried. In 201229240, the pH value of the weak acid aqueous solution is between 3 and 5 to 6.5. More preferably The pH of the weak acid aqueous solution is between 4 and 6. In the comparison with the application state, the pH of the medium is 65~8, and the pH value is adjusted by replacing the culture line. In the preferred embodiment, the step of culturing is carried out in an incubator in a preferred embodiment in which the partial pressure of carbon dioxide in the pH tank is completed.忒坧 较: In comparison, in the embodiment, the pH is adjusted through the emulsified plate and the 4 medium in the incubator. The sodium bicarbonate balance is completed. < In the preferred embodiment, the _ system comprises attachment or desorption. From the above, the present invention mainly utilizes an acid-test response type polymer = acid test environment. The characteristics of the surface electrical properties are different to regulate the cells, and the cells can be easily desorbed from the acid-base response type polymer by changing the positive value of the medium. The method for effectively desorbing the cells from the attached surface is mainly performed by the characteristics of the surface electrical properties of the acid-reacting polymer. The method of iron A is used to control cell adhesion. The acidity test on the surface of the two high-grade materials has the surface-cultured cells for culturing the cells; and is adjusted to - preferably *% to control the adhesion of the celestial cells. More preferably 6.ti 7 secret In the medium, the ΡΗ value of the nucleus is 6.5~8.5, 201229240 ▲ In the middle of the Ming Dynasty, the substrate can be called “acid-test response polymer” as commonly known in the field. a polymer with a positive charge that is not charged in a weak alkaline environment. Generally speaking, the meal should be The acid-off coefficient of the polymer is preferably 4 6 〜8 〇, more preferably 6 _ 7.2. In the preferred embodiment, the acid-responsive polymer contains an amine-based polymer (such as: However, the present invention is not limited thereto. In the present invention, the method of providing the acid-responsive type polymer on the surface of the substrate includes The following steps: dissolving the acid-base responsive polymer in a weak acid aqueous solution to obtain a polymer solution; applying the polymer solution to a substrate to obtain a coated substrate; drying the coated substrate a substrate of the cloth, a dried substrate; neutralizing the dried substrate with an aqueous solution to obtain a neutralized substrate; and washing the neutralized substrate with water and drying. The aqueous solution and the alkaline aqueous solution are well known to those skilled in the art. For example, the weak acid aqueous solution may be an aqueous acetic acid solution, an aqueous citric acid solution, an aqueous citric acid solution, or an aqueous acid solution, but is not limited thereto, and the aqueous solution may be It is an aqueous solution of sodium hydroxide, an aqueous solution of potassium hydroxide, Or an aqueous solution of lithium niobate, but is not limited thereto. Preferably, the pH of the aqueous weak acid solution is between 3.5 and 6 and 5, more preferably between 4 and 6. The cells used in the present invention may be any kind of mammalian cells, including human lung carcinoma cells (Η1299), mouse embryonic fibroblasts (3Τ3) or bovine corneal interstitial cells (bovine). Keratocyte/fibroblast), but not limited to this. In a preferred embodiment, the adjustment of the pH is accomplished by replacing the medium. 201229240 In the other embodiment, the cultivation step is carried out in an incubator. The pH adjustment is accomplished by the interaction in the incubator and the sodium bicarbonate balance in the medium. Preferably, the partial pressure of the second is .30.00%. More preferably, the partial pressure of the carbon dioxide is 0.10 to 15.00%. Further, it is not difficult to understand that in the present invention, this can also be adjusted by changing the partial pressure of carbon dioxide in the incubator.
於培養基之制方面，並無任何特概制，可為任何 市售之培養基’由於其為此領域具有通常知識者所熟知 的’於此不再贅述。較佳地，該培養基中係含有Q()i〜5〇〇〇% 之血清。更佳地’該培養基中係含有〇 1〇〜2〇〇〇%之血清。 此外，當可理解的是，本發明所提供之方法不僅可用 來控制細胞之貼附性，亦可用來控制細胞之脫附性，其完 全取決使用者之需要而變。 / 本發明之技術特徵已具體敘述於發明說明中，其他各 項之材料與配方係屬於習知技藝，本領域熟知該項技藝者 當可輕易實施本發明。以下將藉由實施例的方式例示:發 明之特徵與優點。 _ 實施例1:表面上具有一酸鹼應答型高分子的基材之製備 材料· 1. 幾丁聚醣：購自Sigma-aldrich公司，商品名稱為 Chitosan。 2. 乙酸：購自Sigma-aldrich公司，商品名稱為Acetic Acid。 3. 24孔盤：購自Corning公司’商品名稱為]yiultiwell Culture Plate 。 4. 氫氧化鈉：購自Sigma-aldrich公司，商品名稱為Sodium Hydroxide。 201229240 製備方法： 將0.5克之酸離係數6.5之幾丁聚醣溶於濃度為1%、 pH值為4之乙酸水溶液中，得一高分子溶液。將之 高分子溶液加至培養細胞用之聚苯乙烯24孔盤（24_wdi plate)，此時，該高分子溶液即會塗佈於該μ孔盤上。接# 將該經塗佈之24孔盤置於60°C之烘箱内，乾燥24小時者 使溶液蒸乾。蒸乾後’加入1M之氫氧化鈉水溶液，進行中 和。中和後’以水清洗經中和之24孔盤並該乾燥之，即得 表面上具有一酸鹼應答型高分子的基材。 實施例2:培養基之配製 材料： 1·碳酸氫鈉：購自Sigma-aldrich公司，商品名稱為Sodium Bicarbonate ° 2. DMEM:購自 Invitrogen 公司，商品名稱為 Dulbecco，s Modified Eagle Medium。 3. 鹽酸：購自Sigma_aldrich公司，商品名稱為Hydrogen Chloride 〇 配製方法： 依據表1所示，在含有10%FBS之DMEM培養基内分 別添加600 mg/L至3600 mg/L之碳酸氫鈉後，利用1M氫 氧化鈉與1M之鹽酸滴定，調整培養基之pH值為7.40後， 放入含有分壓為5%之二氧化碳培養箱，經過一小時的平 衡，即可分別得到pH值為6.99至7.65的細胞培養液。 201229240 表1 : 組別 碳酸氮納浪度 (mg/L) PH值（誤差值） 培養基樣品1 600 6·99(〇·〇6、 培養基樣品2 1200 7.2〇(〇加 培養基樣品3 1800 ----, 7-36(0.05) 培養基樣品4 2400 7.48(〇.〇5、 培養基樣品5 3000 ----—-/ 7.57(0.04、 培養基樣品6 3600 --, 7.65(0.04) 實施例3:貼附實驗測試 將上皮性肺癌細胞株(H1299)、胚胎纖維母細胞(3T3)、 及牛角膜間質細胞(keratocyte/fibroblast)以實施例二之培養 基樣品1至培養基樣品6，培養於實施例1所製得之24孔 盤’以未經幾丁聚醣塗佈之24孔盤作為對照組。每孔細胞 數量為2xl04 ’於分壓為5%之二氧化碳、37°C之培養環境 下’培養24小時’利用倒立式相位差顯微鏡觀察細胞，結 果如第一圖所示。進一步移除培養液，並使用CyQuant® 鲁 Assay(購自 Invitrogen’ 商品名 CyQuant® Proliferation Assay) 測試細胞的貼附數量’其結果如第二圖所示。 第一圖A至第一圖c係分別顯示實施例3上皮性肺癌 細胞株（H1299)、胚胎纖維母細胞（3T3)及角膜間質細胞 (keratocyte/fibroblast)之顯微鏡觀察結果。第一圖A至第一 圖C之結果顯示’於塗佈幾丁聚醣之24孔盤中，細胞對孔 盤之貼附性會隨著培養基之pH值增加而減少，其貼附之臨 界點大約介於培養基樣品3及培養基樣品4之間，亦即pH 值為7.36至7.48之間。第二圖A至第二圖c係分別顯示實 201229240 施例3上皮性肺癌細胞株、胚胎纖維母細胞及角臈間質細 胞之細胞貼附量之量化結果’其顯現之結果同於第一 A圖 至第一 C圖之觀察結果。相較於本發明’作為對照組之未 塗佈幾丁聚醣之24孔盤’並未觀察到相同之現象。以上即 證實，本發明係可藉由調整培養基之PH值來控制細胞貼附 性。 實施例4 :脫附實驗-1/更換培養基 根據實施例3之結果’篩選合適之pH值的培養基，用 於進行脫附實驗測試。本實施例係使用培養基樣品2作為 貼附培養基。將上皮性肺癌細胞株、胚胎纖維母細胞、及 角膜間質細胞，培養於實施例1所製得之24孔盤，以未塗 佈幾丁聚醣之24孔盤作為對照組’每孔細胞數量為2χ1〇4， 於5%二氧化碳、37°C培養環境下，培養24小時，將該貼 附培養基移除後，改加入培養基樣品6，再於5%二氧化碳、 37。(：之培養環境下’培養4小時’期間以倒立式顯微鏡养 配礦時攝影系統觀察，並於培養4小時後，以 Assay測試其細胞脫附之數量’結果如第三圖所示。⑽ 第三圖A係顯示加人培養基樣品6後，㈣ 於不同時間下之顯微鏡觀察結果。由第三圖A之結可= 現，培養於幾丁聚醣上之角膜間質細胞於加入捭^ 6，於5%二氧化碳、37Ϊ下培養4小時後，原：貼 胞會慢慢聚集成團’至4小時時，整個細胞團塊會自孔 表面上脫附。 盟 第三圖B係顯示以培養基樣品6培養4小時 間質細胞之細胞脫附量之量化結果。第三圖3之結月膘 90%之細胞會從孔盤上脫附，而在對照組中，則無°觀察^此 種現象。 201229240 第二圖C係顯不加入培養基樣品6後，纖維母細胞於 不同時間下之觀察結果。第三圖D係顯示加入培養基樣品6 後’上皮性肺癌細胞於不同時間下之觀察結果。由第三圖c 及第三圖D之結果亦可發現同於第三圖B之現象。由上即 證實，本發明係可透過調整培養細胞用之培養基之pH值來 控制該細胞之貼附性。 實施例5 :脫附實驗-2/變換二氧化碳分壓 根據實施例3之結果，篩選合適之pH值的培養基，用 $ 於進行脫附實驗測試。本實施例係使用培養基樣品2作為 貼附培養基，將角膜間質細胞培養於實施例1所製得之24 孔盤，以未塗佈幾丁聚醣之24孔盤作為對照組，每孔細胞 數量為2xl04，於5%二氧化碳、37°C培養環境下，培養24 小時後，將細胞盤移至〇%二氧化碳、37°C培養環境，期間 以倒立式顯微鏡搭配曠時攝影系統觀察，並於培養4小時 後，以CyQuant® Assay測試其細胞脫附之數量，並針對脫 附下來之細胞進行LIVE/DEAD cell assay(購自Invitrogen， 商品名稱為 LIVE/DEAD® Cell Viability Assays)，結果如第 鲁 五圖所示。 第四圖A係顯示實施例5角膜間質細胞之顯微鏡觀察 結果。該結果顯示，培養於幾丁聚醣上之角膜間質細胞於 轉換至0%二氧化碳、37°C之培養環境，培養4小時後，原 本貼附之細胞亦會慢慢聚集成團，至4小時時，整個細胞 團塊會自孔盤上脫附。第四圖B係顯示實施例5角膜間質 細胞之細胞脫附量之量化結果。其結果發現約90%之細胞 會從孔盤上脫附，而於對照組中，則未觀察到相同之現象。 此外，第五圖係顯示顯示脫附所得之角膜間質細胞之 LIVE/DEAD cell assay測試結果。該結果顯示，脫附下來之 201229240 =過;====:該細胞之貼 表丁'上所述’本發明係透過改變 :應?^分子之表面電性，連; 性。精此’可提供一種可輕易將細胞 ：：貼附 法，而脫附所得之細胞仍有極高之存活率。、 .之方 其它實施態樣 本本發明書之特徵係可使用任何方式結合。 特徵可使用相同、相等或相似目的的特 徵取代。因此，除了特別陳述強調處之外，本說明 露之特徵係為-系列相等或相似特徵中的—個實施<。 .此外，依據本說明書揭露之内容，熟悉本技術領域者 係Τ輕易依據本發明之基本特徵，在不脫離本發明之精神 與範圍内，針對不同使用方法與情況作適當改變與修飾， 因此，其它實施態樣亦包含於申請專利範圍中。 【圖式簡單說明】 第一圖Α係顯示實施例3上皮性肺癌細胞株(Η1299) 之顯微鏡觀察結果。 第一圖Β係顯示實施例3胚胎纖維母細胞(3Τ3)之顯微 鏡觀察結果。 第一圖C係顯示實施例3角膜間質細胞 (keratocyte/fibroblast)之顯微鏡觀察結果。 第二圖A係顯示實施例3上皮性肺癌細胞株之細胞貼 附量之量化結果。 12 201229240 第二圖B係顯示實施例3胚胎纖維母細胞之細胞貼附 量之量化結果。 第二圖C係顯示實施例3角膜間質細胞之細胞貼附量 之量化結果。 第三圖A係顯示加入培養基樣品6後，角膜間質細胞 於不同時間下之觀察結果。 第三圖B係顯示以培養基樣品6培養4小時後，角膜 間質細胞之細胞脫附量之量化結果。 第三圖C係顯示加入培養基樣品6後，纖維母細胞於 不同時間下之觀察結果。 第三圖D係顯示加入培養基樣品6後，上皮性肺癌 細胞於不同時間下之觀察結果。 第四圖A係顯示實施例5角膜間質細胞於不同時間下 之顯微鏡觀察結果。 第四圖B係顯示實施例5角膜間質細胞之細胞脫附量 之量化結果。 第五圖係顯示脫附所得之角膜間質細胞之 LIVE/DEAD cell assay 湏1J 試結果。There is no special system for the preparation of the medium, and any commercially available medium 'because it is well known to those of ordinary skill in the art' will not be described herein. Preferably, the medium contains serum of Q()i~5〇〇〇%. More preferably, the medium contains 〇1〇~2〇〇〇% of serum. Moreover, it will be appreciated that the methods provided herein can be used not only to control cell attachment, but also to control cell desorption, which is entirely dependent upon the needs of the user. The technical features of the present invention have been specifically described in the description of the invention, and the materials and formulations of the other items are well-known in the art, and those skilled in the art can easily implement the present invention. The features and advantages of the invention will be exemplified by way of embodiments. _ Example 1: Preparation of a substrate having an acid-base responsive polymer on its surface Material 1. Chitosan: purchased from Sigma-aldrich, trade name Chitosan. 2. Acetic acid: purchased from Sigma-aldrich under the trade name Acetic Acid. 3. 24-well plate: purchased from Corning's product name] yiultiwell Culture Plate. 4. Sodium hydroxide: purchased from Sigma-aldrich under the trade name Sodium Hydroxide. 201229240 Preparation method: 0.5 g of chitosan with an acid separation coefficient of 6.5 is dissolved in an aqueous solution of acetic acid having a concentration of 1% and a pH of 4 to obtain a polymer solution. The polymer solution was applied to a polystyrene 24-well plate (24-wdi plate) for culturing cells, and at this time, the polymer solution was applied to the μ-well plate. # The coated 24-well plate was placed in an oven at 60 ° C and dried for 24 hours to allow the solution to dry. After evaporation to dryness, a 1 M aqueous sodium hydroxide solution was added to carry out neutralization. After the neutralization, the neutralized 24-well disk was washed with water and dried to obtain a substrate having an acid-base responsive polymer on its surface. Example 2: Preparation of medium Materials: 1·Sodium bicarbonate: purchased from Sigma-aldrich, under the trade name Sodium Bicarbonate ° 2. DMEM: purchased from Invitrogen, trade name Dulbecco, s Modified Eagle Medium. 3. Hydrochloric acid: purchased from Sigma_aldrich, the trade name is Hydrogen Chloride 〇 Preparation method: According to Table 1, after adding 600 mg/L to 3600 mg/L sodium bicarbonate in DMEM medium containing 10% FBS, After titrating with 1 M sodium hydroxide and 1 M hydrochloric acid, the pH of the medium was adjusted to 7.40, and then placed in a carbon dioxide incubator containing a partial pressure of 5%. After one hour of equilibration, the pH was 6.99 to 7.65, respectively. Cell culture fluid. 201229240 Table 1: Group NbB nanowave (mg/L) PH value (error value) Medium sample 1 600 6·99 (〇·〇6, medium sample 2 1200 7.2〇 (〇 plus medium sample 3 1800 -- --, 7-36 (0.05) Medium sample 4 2400 7.48 (〇.〇5, medium sample 5 3000 ------/ 7.57 (0.04, medium sample 6 3600 --, 7.65 (0.04) Example 3: Attachment test. Epithelial lung cancer cell line (H1299), embryonic fibroblast (3T3), and keratocyte/fibroblast were cultured in the medium sample 1 to medium sample 6 of Example 2, and cultured in the examples. 1 prepared 24-well plate 'with a 24-well plate coated with chitosan as a control group. The number of cells per well was 2xl04 'in a carbon dioxide at a partial pressure of 5%, in a culture environment of 37 ° C' Incubation for 24 hours 'The cells were observed using an inverted phase contrast microscope, and the results are shown in the first figure. The culture solution was further removed, and the cells were attached using CyQuant® Lu Assay (purchased from Invitrogen's brand name CyQuant® Proliferation Assay). The number 'the result is shown in the second figure. The first figure A to the first figure c The microscopic observation results of the epithelial lung cancer cell line (H1299), the embryonic fibroblast (3T3), and the keratocyte/fibroblast of Example 3 were respectively shown. The results of the first panel A to the first panel C show 'in In a 24-well plate coated with chitosan, the adhesion of the cells to the well plate decreases as the pH of the medium increases, and the critical point of attachment is approximately between the medium sample 3 and the medium sample 4. That is, the pH value is between 7.36 and 7.48. The second figure A to the second figure c show the cell attachment amount of the epithelial lung cancer cell line, the embryonic fibroblast, and the keratinocyte interstitial cells of the embodiment 201229240, respectively. The quantified result 'the result of its appearance is the same as that of the first A to the first C. The same is not observed in the 24-well plate of the uncoated chitosan as the control group of the present invention. Phenomenon, it was confirmed that the present invention can control cell adhesion by adjusting the pH value of the medium. Example 4: Desorption experiment-1/replacement medium According to the result of Example 3, 'screening a suitable pH value medium For conducting desorption experiments. In the examples, the culture medium sample 2 was used as the attachment medium. The epithelial lung cancer cell line, the embryonic fibroblast, and the corneal interstitial cells were cultured in the 24-well plate prepared in Example 1, and the uncoated polycondensation was performed. The 24-well plate of sugar was used as the control group. The number of cells per well was 2χ1〇4, cultured in 5% carbon dioxide and 37°C culture environment for 24 hours. After the attachment medium was removed, the medium sample 6 was added. At 5% carbon dioxide, 37. (In the culture environment, 'cultivation for 4 hours' was observed by an inverted microscope to observe the photographic system of the mine, and after 4 hours of culture, the number of cell desorption was measured by Assay'. The results are shown in the third figure. (10) The third panel A shows the results of microscopic observation at different times after adding the culture medium sample 6. (B) The knot of the third graph A can be used to present the corneal interstitial cells cultured on chitosan. 6, after culturing for 4 hours under 5% carbon dioxide and 37 ,, the original: the cells will slowly aggregate into the group '4 hours, the whole cell mass will be detached from the surface of the hole. The quantification result of the amount of cell desorption of the culture medium sample 6 cultured for 4 hours was observed. In the third graph, 90% of the cells of the sputum were desorbed from the well plate, while in the control group, there was no observation. 201229240 The second figure is the observation of fibroblasts at different times after the addition of medium sample 6. The third figure D shows the observation of 'epithelial lung cancer cells' at different times after adding medium sample 6. Result. The knot from the third figure c and the third figure D The phenomenon similar to that of the third graph B can also be found. From the above, it is confirmed that the present invention can control the adhesion of the cells by adjusting the pH of the medium for culturing the cells. Example 5: Desorption experiment-2/ Conversion of Carbon Dioxide Partial Pressure According to the results of Example 3, a suitable pH value of the medium was screened and tested for desorption experiments. In this example, medium sample 2 was used as a patch medium, and corneal stromal cells were cultured in the examples. A 24-well plate prepared by using a 24-well plate with uncoated chitosan as the control group, the number of cells per well was 2xl04, and after culturing for 24 hours in 5% carbon dioxide and 37 ° C culture environment, The cell disk was transferred to a 二氧化碳% carbon dioxide, 37 ° C culture environment, and observed with an inverted microscope with a sputum photographic system, and after 4 hours of culture, the amount of cell detachment was tested with CyQuant® Assay and targeted for desorption. The cells were subjected to LIVE/DEAD cell assay (purchased from Invitrogen, trade name: LIVE/DEAD® Cell Viability Assays), and the results are shown in Figure 5. Figure 4 is a microscope showing the corneal interstitial cells of Example 5. The results showed that the corneal interstitial cells cultured on chitosan were transferred to 0% carbon dioxide, 37 ° C culture environment, and after 4 hours of culture, the originally attached cells will slowly aggregate. At 4 hours, the entire cell mass will desorb from the well plate. Figure 4B shows the quantitative results of the cell desorption of the corneal interstitial cells of Example 5. The results showed that about 90% of the cells would The well plate was desorbed, whereas in the control group, the same phenomenon was not observed. In addition, the fifth figure shows the results of the LIVE/DEAD cell assay showing the corneal interstitial cells obtained by desorption. The results show that the desorbed 201229240 = over; ====: the cell is labeled as described above. The invention is modified by the surface electrical properties of the molecule. This can provide a cell-attachment method, and the cells obtained by desorption still have a very high survival rate. Further aspects of the present invention The features of the present invention can be combined in any manner. Features can be replaced with features of the same, equal or similar purpose. Therefore, in addition to the special statement emphasis, the features of this description are - implementations of - series equal or similar features. In addition, according to the disclosure of the present specification, those skilled in the art can easily make appropriate changes and modifications to different methods and situations without departing from the spirit and scope of the present invention. Other implementation aspects are also included in the scope of the patent application. BRIEF DESCRIPTION OF THE DRAWINGS The first figure shows the results of microscopic observation of the epithelial lung cancer cell line (Η1299) of Example 3. The first panel shows the results of microscopic observation of the embryonic fibroblasts (3Τ3) of Example 3. The first panel C shows the results of microscopic observation of the corneal interstitial cells (keratocyte/fibroblast) of Example 3. Fig. 2A shows the results of quantification of the amount of cell attachment of the epithelial lung cancer cell line of Example 3. 12 201229240 The second panel B shows the quantitative results of the cell attachment amount of the embryonic fibroblasts of Example 3. Fig. 2C shows the results of quantification of the amount of cell attachment of the corneal interstitial cells of Example 3. The third panel A shows the observation of corneal interstitial cells at different times after the addition of the medium sample 6. The third panel B shows the quantified results of the amount of cell desorption of corneal interstitial cells after 4 hours of culture in the culture medium sample 6. The third panel, C, shows the observation of fibroblasts at different times after the addition of the medium sample 6. The third panel D shows the observation of epithelial lung cancer cells at different times after the addition of the medium sample 6. Figure 4A shows the results of microscopic observation of the corneal interstitial cells of Example 5 at different times. Fig. 4B is a graph showing the results of quantification of the amount of cell desorption of the corneal interstitial cells of Example 5. The fifth panel shows the LIVE/DEAD cell assay 湏1J test results of debranching corneal interstitial cells.
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