1313180 九、發明說明： 【發明所屬之技術領域】 本發明是有關於一種奈米級幾丁聚醣的製造方法，特 別是一種適用於細胞培養基材之奈米級幾丁聚醣的製造方 法0 【先前技術】 • 動物細胞的培養應用在許多疫苗及蛋白質基因工程方 面，已行之有年了。根據動物細胞的生長型態。大致可將 動物細胞分成三類。第一類細胞是需在懸浮狀態下生長， 如淋巴細胞。第二類細胞是需貼附於物體的表面上才能生 長，一般稱之為「貼附型」細胞，此類的細胞包含纖維母 細胞、上皮細胞及内皮細胞等等。第三類細胞則是介於第 一類細胞和第二類細胞間，可在懸浮狀態下成長也可以貼 附在物體的表面上成長。 φ 其中’第一類細胞因為需要貼附在物體的表面上才能 成長。故其所貼附的細胞培養基材的性質不僅會對細胞吸 附的情开> 造成影響，也會對細胞生長的情形造成影響。 因為細胞膜一般都是帶負電，所以在帶正電的材料上 其吸附性會比在中性的材料上來得好。因為幾丁聚聽的價 格低廉，且為帶正電荷的材料，故近來有人以幾丁聚醋來 作細胞培養基材。但因所用幾丁聚醣的粒徑比較大，在次 微米等級以上，所以能讓細胞吸附的表面積有限，並且對 細胞活性的促進效果不夠好。 1313180 【發明内容】 本發明的目的之-是在提供一種適用於細胞培養基材 之奈米級幾丁聚骑的製造方法。以此方法製造出的奈米級 幾丁聚醣的粒徑較習知次微米級的幾丁聚醋的粒徑小，使 其具有較大的表面積可供細胞吸附，而且具有較佳之促進 細胞活性的效果。 本發明的另一目的是在提供一種奈米級幾丁聚醣的製 造方法。利用溶膠凝膠法製造奈米級幾丁聚聽 方法簡易，並且適於進行量產。 表仏 =本發明之上述目的，提出一種奈米級幾丁聚醣的 “方法。首先，將幾丁聚醣溶解於弱酸水溶液中， f、幾丁聚醋水溶液。之後，加人驗性水溶液於幾丁聚_水 命液’調整幾丁聚醣水溶液的pH值在約6 :㈣幾丁聚一以形成奈米級幾丁聚= 根據本發明之上述目的，提出一種適用 材之奈米級幾丁聚_製造方法。其製造M、、養基 幾丁聚酶膠體溶液乾燥，以得到奈米級幾丁聚=將上述的 將奈米級幾丁聚醣塗佈於細胞培養容器内表面…之後， 實施例，乾燥奈米級幾丁聚醣膠體溶液 亡、。在較佳 法，細胞培養容器為培養皿或搖瓶。 / 冷來乾燥 由上述可知，本發明利用溶膠凝膠法來 丁聚醣’其製程相當簡易。製造出的奈米級奈米級幾 、戌丁聚聰所作 1313180 成細胞培養基材’對細胞不具有毒性。因為奈米級幾丁聚 醣的粒徑小，故相較於習知次微米級奈米幾丁聚醣，可供 細胞吸附的表面積更大，且細胞更加容易吸附於其表面 上。此外’奈米級幾丁聚醣還可增加細胞的活性及促進細 胞增生。 【實施方式】1313180 IX. Description of the Invention: [Technical Field] The present invention relates to a method for producing a nano-scale chitosan, and more particularly to a method for producing a nano-scale chitosan suitable for a cell culture substrate 0 [Prior Art] • The cultivation of animal cells has been used in many vaccine and protein genetic engineering for many years. According to the growth pattern of animal cells. Animal cells can be roughly classified into three categories. The first type of cells are required to grow in suspension, such as lymphocytes. The second type of cells need to be attached to the surface of an object to grow, and are generally referred to as "attached" cells. Such cells include fibroblasts, epithelial cells, and endothelial cells. The third type of cells are between the first type of cells and the second type of cells, which can grow in suspension or attach to the surface of the object to grow. φ where 'the first type of cells can grow because they need to be attached to the surface of the object. Therefore, the nature of the cell culture substrate to which it is attached not only affects the cell's absorption, but also affects the cell growth. Because cell membranes are generally negatively charged, they are more adsorptive on positively charged materials than on neutral materials. Because the price of chitosan is low and it is a positively charged material, some people have recently used a few diced vinegar as a cell culture substrate. However, since the chitosan used has a relatively large particle size and is above the sub-micron level, the surface area for cell adsorption is limited, and the effect on cell activity is not sufficiently good. 1313180 SUMMARY OF THE INVENTION An object of the present invention is to provide a method for producing a nano-sized chitosan suitable for use in a cell culture substrate. The particle size of the nano-scale chitosan produced by this method is smaller than that of the conventional sub-micron-sized chitosan, so that it has a large surface area for cell adsorption, and has a better promoting cell. The effect of activity. Another object of the present invention is to provide a method for producing a nano-scale chitosan. The method of making nanometer chitosan by the sol-gel method is simple and suitable for mass production. Table 仏 = the above object of the present invention, a "method of nano-scale chitosan is proposed. First, the chitosan is dissolved in a weak acid aqueous solution, f, a small amount of poly-acetic acid aqueous solution. After that, an artificial aqueous solution is added. Adjusting the pH value of the aqueous solution of chitosan in the chitosan water solution to about 6: (4) forming a nano-single polymerization to form a nano-sized polybutadiene = according to the above object of the present invention, a nanometer of suitable material is proposed. A method for producing a polybutanization method, which comprises preparing a M, a nutrient chitin polymerase colloidal solution to obtain a nanometer chitosan = applying the above-mentioned nanoscale chitosan to a cell culture vessel After the surface, in the embodiment, the dried nano-chitosan colloidal solution is dead. In a preferred method, the cell culture vessel is a petri dish or a shake flask. / Cold drying. As described above, the present invention utilizes a sol-gel method. The process of butyl glycan is quite simple. The nano-scale nano-prepared and the 1313180 cell culture substrate made by 聚丁聚聪 are not toxic to cells. Because the particle size of nano-scale chitosan is small, Compared to the conventional submicron nanobutadiene , Greater surface area for cell attachment, and the cells on the surface thereof is more readily adsorbed. Further 'nanoscale chitosan may also increase the activity of the cells and promote cell proliferation. [Embodiment
本發明中’是以溶膠凝膠法製造奈米級幾丁聚醣，其 製造步驟如下。首先，以弱酸水溶液，溶解幾丁聚醣形 成幾丁聚醣水溶液。在較佳實施例中，弱酸水溶液為醋酸、 乳酸或壓克力酸的水溶液，弱酸在幾丁聚醣水溶液中的重 里百刀/辰度約為0.5〜5%。之後，加入鹼性水溶液於幾丁聚 醣水溶液，調整其pH值在約6 5到8之間，經由麟使幾 丁聚釀進行轉縮合職，㈣成奈綠幾T聚醣膠體溶 貫施例中，鹼性水溶液為氫氧化鈉水溶液或氨 水’鹼性水溶液的重量百分濃度約為〇 5〜5%。此外，在上 =的奈米級幾丁聚卿體溶液中可以選擇性的加入保存 '。在較佳實施例中’保存劑可為仙此 jg neonate。最後’乾燥奈米級幾丁聚醣膠 奈米級幾丁聚醣。 于」 米，八=方法所得奈米級幾丁聚酶之粒徑約為4〇〜15〇奈 醋酸^347360。分子量之;収其流動相為〇·3Μ :之’ .2Μ醋酸鈉；管柱型號為τ 速為 〇.7mL/min。 wwx_L，肌 1313180 在較佳實施例中，乾燥奈米級幾丁聚醣膠體溶液是採 ^ /東乾燥法。乾燥所得的奈米級幾丁聚醣膠可塗佈在如 料皿和搖瓶等細胞培養容器的内表面上，以作為細胞培 養基材之用。 在實施例中，先在不鏽鋼槽中置入1980克的水與20 克的幾丁聚醣。再加入333克18 %的乳酸水溶液來溶解幾 聚醣以开^成幾丁聚醣水溶液。其中乳酸在幾丁聚醣水 ·/谷液中的重量百分濃度約為2.5%。之後，以濾網濾掉不要 的雜質。將過據後的幾丁聚醣水溶液置入不输鋼槽，分兩 加氫氧化鈉水溶液，加入的氫氧化鈉水溶液的重量百 辰度、力為1 ·5 /〇。第一次加入氫氧化納水溶液，以調整幾 丁聚醣水溶液的pH值至接近7,經由攪拌使幾丁聚醣水解 縮β以形成幾丁聚醣膠體溶液。此時，幾丁聚醣膠體已 初步成形。第二次加入氫氧化納水溶液，調整幾丁聚聽膠 體溶液的ΡΗ值在約7到8的範圍心然後經由授掉，使幾 • 了聚膽膠體成形為最終的結構。最後，離心幾丁聚膽膠體 溶液’取得幾丁聚膽之膠體。再以冷来乾燥 膠體，得到奈米級幾丁聚酷。 ^ 本發明所得到的奈米級幾丁聚醣相當適於作為細胞培 養基材，因為其粒徑大小適合細胞吸附於其上，並且奈米 級幾丁聚醋本身可供細胞食用，提供細胞生長所許需$養 分。此外，與習知次微米級幾丁聚醣相比，有更大的表面 積可供細胞吸附。 為了讓讀者能清楚瞭解奈米級幾丁聚醣在細胞培養基 1313180 材上的應用，以及本伞 聚醣在細胞培養基材知的次微米級幾丁 幾丁聚糖的製造參數1 上兩者的差異。因此，藉由調整 照。以下將就粒徑分_、 了—組次微米級幾丁聚酷作對 活性和細胞計數，分別作::毒性、細胞生長情形、細胞 粒徑分析 第1A-1B圖分別甚+ , 丁聚酶粒#大小料_ 與習知次微米級幾 範圍約為40到15〇 + 級幾丁聚膽粒徑大小的分佈 級# τ _ *未，平均粒徑約為77.8奈米。次微米 、 ";徑大小的分佈範圍約為200到900夺米，直 中在500奈米，有最大的分佈。 不十-細胞每料 細胞毒性測試是根據ASTM F813-83的規範。首先， 將L929老鼠纖維母細胞培養於直徑6公分的細胞培養皿 中Μ細胞生長達到占滿整個細胞培養皿時，分別將已消 母過的奈米級幾丁聚醣及次微米級幾丁聚醣的塊材置於培 養皿的中央後，再將細胞培養一天。最後，用濃度2。/〇的 結晶紫對細胞作染色。因為死細胞無法染色，故可藉由觀 察染色的範圍來判定細胞毒性。 第2 A-2C圖分別是是控制組、奈米級幾丁聚醣與習知 次微米級幾丁聚醣對細胞毒性測試的結果。在第2A圖中的 控制纟且，不加入任何幾丁聚醣，其目的是用來和另兩組有 C§) 9 1313180 加幾丁聚_的作對照。 第2B-2C圖中，幾丁聚醣塊材的置入，使得位於細胞 坧養皿中間的細胞因為被壓死而無法染色，故中間無紫 色。若置入細胞培養m中的材料具有毒性，細胞染色的情 形會以置入的材料為中心，顏色會一圈圈的向外呈現漸層 的改變，愈往外顏色愈深，愈靠近内顏色愈淺。顯示愈靠 近材料的細胞，愈易受材料毒性的影響，細胞死亡的情形 也愈嚴重。無論是第2B圖中的奈米級幾丁聚餹或是第2C 圖中的次微米級幾丁聚醣，皆無上述一圈圈色變的情形。 故奈米級幾丁聚醣和次微米級幾丁聚醣對細胞皆無毒性上 的顧慮’故可以繼續作後面其他與細胞培養基材有關的測 試。 細胞生長情形 分別將已消毒過的奈米級幾丁聚醣與次微米級幾丁聚 醋置入培養皿’作為培養基材。再將L929老鼠纖維母細胞 培養於其中。之後，用掃瞄式電子顯微鏡觀察細胞第一天 和第二天的增生情形和細胞幾何型態的變化。 第3 A-3B圖分別是奈米級幾丁聚醣所培養的細胞用掃 描式電子顯微鏡拍攝得到的第一天和第二天照片。第 4A-4B圖分別是習知次微米級幾丁聚醣所培養的細胞用掃 描式電子顯微鏡拍攝得到的第一天和第二天照片。在第 3A-3B圖中，以奈米級幾丁聚醣所培養的細胞，在經過兩 天後，細胞延展開來，從原先一個個單獨分離的圓球型態， 1313180 成長為纺錘狀，且細胞和細胞之間彼此連接。由此顯示， 以奈米級幾丁聚醣為培養基材，細胞成長相當良好。 反觀，在第4A-4B圖中，以次微米級幾丁聚醣所培養 的細胞，細胞不僅沒有延展攤平成紡錘狀，部分細胞還開 始出現壞死崩解的現象。 細胞活性 細胞活性是用MTT法來量測。藉由MTT與細胞的粒 腺體作用所生成的Formazan的量來判定細胞的活性。 Formazan的量可利用其對波長570奈米的光吸收值來測 定。將奈米級幾丁聚醣和次微米級幾丁聚醣分別置入培養 m中培養細胞。在測定細胞活性的一開始，將培養皿中的 培養液吸除後，以構酸鹽緩衝液（phosphate buffered saline，PBS)沖洗數次。之後，加入1毫升含MTT之新鮮 培養液（0.5毫克MTT/毫升），置於37°C之培養箱内反應三 個小時，讓細胞與MTT充分作用。將未反應之培養液移除， 添加入500微升二甲基亞颯（dimethyl sulfoxide，DMSO)溶 解細胞生成的Formazan，利用震盪混合器震盪使其完全溶 解。最後’取出200微升溶有formazan之二甲基亞礙’利 用酵素免疫分析儀（ELISA ’ enzyme-linked immunosorbent assay)下偵測其對波長570奈米的光的吸收值’所用的參考 波長為650奈米。 表1是對奈米級幾丁聚醣與習知次微米級幾丁聚醣培 養的細胞對波長570奈米的光的吸收值。表中’奈米級幾 11 1313180 丁聚醣所培養的細胞的吸收值較高。顯示，相較於習知次 微米級幾丁聚聽’奈米級幾丁聚醣更能 性。 0.356 ----— 0.243 增加量 0.131 0.087 表1 第一天 奈米級幾丁聚醣 0.225 次微米級幾丁聚醣 0.156 第二天 細胞計數 於奈米級幾丁聚骑和次微米級幾丁聚驗所培養的⑽ 老鼠纖維母細胞中，分別加入適當量的胰蛋白酵素 ㈣㈣。反應3分鐘之後，取1G毫升細胞懸浮液與ι〇毫 升trypan blue等體積混合均勾於i 5毫升小離心管中。 取約10毫升上述的混合液加在血球計數盤上方凹槽 内，再蓋上蓋玻片，^⑽倍的顯微鏡下觀察。活的細胞 不會被染色’死的細胞則會被染絲L計數血球計數盤 上四個大方格之細胞總數’若細胞位於血球計數盤的線 上’則只計數上線與右線之細胞（或計數下線與左線之細 胞）。將四個大方格之細胞總數除以4，乘以稀釋倍數2, 釋倍數2是因為細胞懸浮液與—等體積混外最 後除以方格的體積，得到每毫升中細胞懸浮液之細胞數。 表2是對奈米級幾丁聚醣與習知次微米級幾丁聚聽择 養的細胞作細胞計數的結果。其中，以奈米級幾丁聚膽诗 養的細胞數目有明顯的增加，且增加的速度高於習知次微 * 12 1313180 幾丁聚醣有助於細胞的 米級幾丁聚醣。由此可知，奈米級 増生。 表2 (細胞單位濃度為4x1 〇3個。 --- --—~—— 第一天 第二天 增加量 奈米級幾丁聚醣 8.7 ------_ 一 18 9.3 f欠微米級幾丁聚醣 8 14.2 6.2In the present invention, a nano-scale chitosan is produced by a sol-gel method, and the production steps are as follows. First, an aqueous solution of chitosan is formed by dissolving chitosan in a weak acid aqueous solution. In a preferred embodiment, the aqueous weak acid solution is an aqueous solution of acetic acid, lactic acid or acrylic acid, and the weight of the weak acid in the aqueous solution of chitosan is about 0.5 to 5%. After that, an alkaline aqueous solution is added to the aqueous solution of chitosan, and the pH value is adjusted to be between about 65 and 8. The chitosan is used to carry out the transcondensation operation, and (4) the Nai green green T-polysaccharide colloid is applied. In the example, the alkaline aqueous solution is an aqueous sodium hydroxide solution or an aqueous ammonia aqueous alkaline solution having a concentration of about 5% to 5% by weight. In addition, it can be selectively added to the upper-side nano-single poly-stain solution. In a preferred embodiment, the preservative can be a jg neonate. Finally 'drying the nano-scale chitosan gum nano-scale chitosan. The particle size of the nano-sized chitosan obtained by the method of rice, eight = method is about 4 〇 15 15 〇 acetic acid ^ 347360. The molecular weight; the mobile phase is 〇·3Μ: ’.2Μ sodium acetate; the column type is τ speed is 〇.7mL/min. wwx_L, Muscle 1313180 In a preferred embodiment, the dried nano-chitosan colloidal solution is a dry/dry method. The dried nano-grade chitosan gum can be applied to the inner surface of a cell culture vessel such as a dish and a shake flask to serve as a cell culture substrate. In the examples, 1980 grams of water and 20 grams of chitosan were placed in a stainless steel tank. Further, 333 g of an 18% aqueous lactic acid solution was added to dissolve the chitosan to open an aqueous solution of chitosan. The concentration by weight of lactic acid in chitosan water/valley solution is about 2.5%. After that, filter the unwanted impurities with a strainer. The treated aqueous solution of chitosan was placed in a non-steel trough, and an aqueous solution of sodium hydroxide was added in two portions. The weight of the aqueous sodium hydroxide solution added was 1. 5 /〇. The aqueous sodium hydroxide solution was added for the first time to adjust the pH of the aqueous solution of chitosan to near 7, and the chitosan was hydrolyzed by stirring to form a chitosan colloidal solution. At this point, the chitosan colloid has been initially formed. A second time, an aqueous solution of sodium hydroxide was added, and the enthalpy of the chitosan colloidal solution was adjusted to be in the range of about 7 to 8 and then transferred to form a final structure. Finally, the chitosan colloidal solution was centrifuged to obtain a colloid of chitin. Then dry the colloids with cold to get the nano-scale. The nano-scale chitosan obtained by the present invention is quite suitable as a cell culture substrate because its particle size is suitable for cells to adsorb thereon, and the nano-sized chitosan itself can be used for cell consumption, providing cells. The nutrient is required for growth. In addition, there is a larger surface area available for cell adsorption than conventional submicron chitosan. In order to let the reader clearly understand the application of nano-scale chitosan in cell culture medium 1313180, and the manufacturing parameters of sub-micron chitosan in the cell culture medium, The difference. Therefore, by adjusting the photo. In the following, the activity and cell counts will be determined for the particle size distribution, the sub-micron-scale, and the following:: toxicity, cell growth, cell size analysis, Figure 1A-1B, respectively, +, polymerase The size of the granules and the conventional submicron range is about 40 to 15 〇 + the distribution of the size of the chitosan particle size # τ _ * No, the average particle size is about 77.8 nm. The sub-micron, " diameter size distribution range is about 200 to 900 meters, and the straight line is 500 nm, with the largest distribution. Not ten-cell per cytotoxicity tests are in accordance with ASTM F813-83. First, the L929 mouse fibroblasts were cultured in a 6 cm diameter cell culture dish. When the cells were grown to occupy the entire cell culture dish, the nano-severe chitosan and the sub-micron-sized chitin were respectively removed. After the block of the glycan is placed in the center of the culture dish, the cells are cultured for one day. Finally, use concentration 2. / 〇 Crystal violet stains cells. Since dead cells cannot be stained, cytotoxicity can be determined by observing the extent of staining. The second A-2C plot is the result of a cytotoxicity test of the control group, the nano-scale chitosan and the conventional sub-micron chitosan. In the control in Fig. 2A, no chitosan is added, and the purpose is to compare with the other two groups of C§) 9 1313180 plus chitosan. In Fig. 2B-2C, the chitosan block was placed so that the cells located in the middle of the cell culture dish could not be stained because they were crushed, so there was no purple color in the middle. If the material placed in the cell culture m is toxic, the cell staining will be centered on the placed material, and the color will gradually change outwards in a circle. The deeper the color, the closer the color is. shallow. The closer the cells are to the material, the more susceptible they are to the toxicity of the material, and the more severe the cell death. Neither the nano-sized chitosan in Figure 2B nor the sub-micron-sized chitosan in Figure 2C have the above-described one-circle color change. Therefore, the nano-scale chitosan and the sub-micron-sized chitosan have no toxicity concerns on the cells, so that other tests related to the cell culture substrate can be continued. Cell growth conditions The sterilized nano-scale chitosan and the sub-micron-sized chitosan were separately placed in a petri dish as a culture substrate. L929 mouse fibroblasts were then cultured therein. Thereafter, the proliferation and cell geometry of the cells on the first and second days of the cells were observed by a scanning electron microscope. Fig. 3A-3B is a photograph of the first day and the second day of the cells cultured by the nano-chitosan using a scanning electron microscope. Fig. 4A-4B are photographs of the first day and the second day taken by a conventional electron microscopic chitosan cultured by a scanning electron microscope. In Fig. 3A-3B, the cells cultured with nano-scale chitosan, after two days, the cells are extended, and the spherical shape separated from the original one, 1313180 grows into a spindle shape. And the cells and cells are connected to each other. This shows that with nano-scale chitosan as the culture substrate, the cell growth is quite good. On the other hand, in the 4A-4B figure, the cells cultured with the submicron-sized chitosan not only did not spread to form a spindle shape, but some cells also began to show necrosis and disintegration. Cellular activity Cell viability is measured by the MTT method. The activity of the cells was determined by the amount of Formazan produced by the action of MTT and the granulosa of the cells. The amount of Formazan can be measured using its light absorption value of 570 nm. The cells in which the nano-scale chitosan and the sub-micron-sized chitosan were separately placed in the culture m were cultured. At the beginning of the measurement of the cell activity, the culture solution in the culture dish was aspirated, and then washed several times with phosphate buffered saline (PBS). Thereafter, 1 ml of fresh culture medium containing MTT (0.5 mg MTT/ml) was added and placed in an incubator at 37 ° C for three hours to allow the cells to fully function with MTT. The unreacted culture solution was removed, and 500 μl of dimethyl sulfoxide (DMSO)-dissolved Formazan was added and shaken to complete dissolution by an oscillating mixer. Finally, 'take out 200 microliters of dimethyl sulphate dissolved in formazan' using a ELISA 'enzyme-linked immunosorbent assay to detect the absorption of light at a wavelength of 570 nm' is used as the reference wavelength. 650 nm. Table 1 shows the absorption of light at a wavelength of 570 nm for cells cultured with nano-scale chitosan and conventional sub-micron chitosan. The cells cultured in the table of 'nano grade 11 1313180 chitosan have higher absorption values. It is shown that it is more energy-efficient than the conventional sub-micron chitosan nano- chitosan. 0.356 ----- 0.243 increase 0.131 0.087 Table 1 The first day of the nano-scale chitosan 0.225 micron-scale chitosan 0.156 The second day of cell count in the nano-scale chit and sub-micron In the (10) mouse fibroblasts cultured in Dingju, the appropriate amount of trypsin (4) was added (4). After 3 minutes of reaction, 1 g of the cell suspension was mixed with ι 〇 trypan blue in equal volume and hooked into a 5 ml microcentrifuge tube. Approximately 10 ml of the above mixture was added to the groove above the hemocytometer disk, and then covered with a cover glass, and observed under a microscope (10) times. Live cells will not be stained. 'Dead cells will be stained with silk. Counting the total number of cells in the four large squares on the blood cell count disk. If the cells are on the line of the blood cell count disk, then only the cells on the upper line and the right line are counted. Or count the cells below the line and the left line). Divide the total number of cells in the four large squares by 4, multiply by the dilution factor of 2, and the factor of 2 is because the cell suspension is mixed with the same volume and finally divided by the volume of the square to obtain the cells of the cell suspension per ml. number. Table 2 shows the results of cell counting of cells of the nano-scale chitosan and the conventional sub-micron chitosan. Among them, the number of cells raised in nanometer-sized chitosan poetry has a significant increase, and the rate of increase is higher than that of the conventional sub-micro-* 12 1313180 chitosan which contributes to the cell-grade chitosan. It can be seen that the nano-level is alive. Table 2 (cell unit concentration is 4x1 〇3. --- ---~ - The second day of the first day increases the amount of nano-butanose 8.7 ------ _ 18 9.3 f under micron Chitosan 8 14.2 6.2
由上述本發明較佳實施例可知，應用奈米級幾丁聚釀 作細胞培養基材’具有下列優點。 (1) 奈米級幾丁聚醣對細胞不具有毒性。 (2) 奈米級幾丁聚醣更能增加細胞的活性。 (3) 奈米級幾丁聚醣有助於細胞的增生。 雖然本發明已以一較佳實施例揭露如上，然其並非用 • 卩限定本發明，任何熟習此技藝者，在不脫離本發明之精 神和範圍内，當可作各種之更動與潤飾，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。 【圖式簡單說明】 為讓本發明之上述和其他目的、特徵、優點與實施例 能更明顯易懂，所附圖式之詳細說明如下： 第IA-Ιβ圖分別是奈米級幾丁聚醣幾丁聚醣與習知次 微米級幾丁聚醣粒徑大小統計圖。 C§) 13 1313180 第2A-2C圖分別是控制組、奈米級幾丁聚醣與習知次 微米級幾丁聚醣對細胞毒性測試的結果。 第3 A-3B圖分別是奈米級幾丁聚醣所培養的細胞用掃 描式電子顯微鏡拍攝得到的第一天和第二天照片。 第4A-4B圖分別是習知次微米級幾丁聚醣所培養的細 胞用掃描式電子顯微鏡拍攝得到的第一天和第二天照片。As apparent from the above preferred embodiment of the present invention, the use of a nano-sized chitosan cell culture substrate has the following advantages. (1) Nano-scale chitosan is not toxic to cells. (2) Nano-scale chitosan can increase the activity of cells. (3) Nano-scale chitosan helps cell proliferation. Although the present invention has been described above in terms of a preferred embodiment, it is not intended to limit the invention, and various modifications and changes may be made without departing from the spirit and scope of the invention. The scope of the invention is defined by the scope of the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS In order to make the above and other objects, features, advantages and embodiments of the present invention more obvious, the detailed description of the drawings is as follows: The IA-Ιβ diagram is a nano-scale Statistical diagram of the particle size of sugar chitosan and conventional submicron chitosan. C§) 13 1313180 Figure 2A-2C shows the results of the cytotoxicity test of the control group, the nano-scale chitosan and the conventional sub-micron chitosan. Fig. 3A-3B is a photograph of the first day and the second day of the cells cultured by the nano-chitosan using a scanning electron microscope. Figures 4A-4B are photographs of the first and second days taken by scanning electron microscopy of cells cultured with conventional submicron chitosan, respectively.
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