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標題: | 利用聲學鑷子建立三維多細胞球團培養系統 Developments of Three-dimensional Culture Systems Multicellular Spheroid Using Acoustic Tweezer |
作者: | Mao-Wei Huang 黃茂維 |
指導教授: | 王兆麟 |
關鍵字: | 超音波,聲學鑷子,聲輻射力,三維細胞培養,細胞球團, Ultrasound,Acoustic tweezer,Acoustic radiation force,Three-dimensional cell culture,Multicellular spheroid, |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | 研究目的:
開發一利用聲學鑷子使細胞聚集成球團培養的系統,使用35 mm的培養盤進行培養,研究在不同超音波頻率的條件下,細胞球團的大小、間距以及生成時間。 背景簡介: 相對於二維細胞培養,三維細胞培養更能完整的保存細胞本身之性質且更有利於細胞分化,並在藥物檢測及組織培養等方面具有良好的反應。二維細胞培養方式由於細胞與培養盤直接接觸,細胞沿著塑膠平面生長,這樣的生長環境可能改變細胞本來的分化特性,造成體外細胞培養的研究難以在生物體中有相同的結果,又或使細胞失去原來的特性;而三維的細胞培養方式,能模擬出近似細胞實際生長的立體環境,並且能讓細胞在增值、分化的過程更貼近真實情況。現行研究中有許多三維細胞培養的方式,其中有讓細胞貼附於微載體、凝膠或生物支架上等使細胞不再只是在二維平面的環境下生長;此外,還有利用力學或化學方式使細胞不貼附培養盤讓細胞彼此貼附並形成球團的培養方式。本實驗利用自製的超音波探頭產生駐波,運用聲學鑷子原理使培養盤中的細胞成團聚集並以陣列方式排列,使用不同頻率的超音波探頭,可以改變細胞球團的大小以及細胞聚集成團所需的生成時間。透過這樣的方式不僅可以在單位面積的培養環境中產生更多的細胞球團,亦可能增加細胞球團的生成效率。 材料與方法: 本研究於35 mm培養盤中使用小鼠纖維母細胞(L929)為實驗素材,使用四顆匹配良好相同中心頻率的超音波探頭,於培養盤的四周施打超音波,實驗使用的超音波探頭其中心頻率有1 MHz、1.5 MHz、3 MHz此三種。超音波系統是由訊號產生器輸出正弦波的電訊號,透過功率放大器進行訊號放大,能量在輸入到超音波探頭前會先經過電功率計與電功率感測器,量測系統的FWD、RFL、VSWR,最後再經過匹配電路傳到超音波探頭。四顆分別匹配好且相同頻率的超音波探頭使用串並聯的方式連接,並將輸入電功率與輸出聲功率校正曲線相近之探頭放置於培養盤對側。將細胞培養盤放置於設計的夾具上固定,施打的超音波能量會經過夾具再傳遞到培養盤中。透過改變訊號產生器所輸出的電壓,來改變最後輸入到培養盤的聲功率,藉此觀察不同能量下細胞的聚集效果;透過使用不同頻率的超音波探頭,能改變細胞球團的大小、細胞球團之間的間距以及細胞球團的生成時間。細胞聚集的結果由顯微鏡觀察並拍攝記錄,細胞球團的大小與球團之間的距離使用ImageJ進行影像分析。細胞培養方面,先使用幾丁聚醣(Chitosan)對35 mm細胞培養盤進行表面處理避免細胞貼附培養盤,實驗分為一般培養的對照組與使用超音波使細胞聚集的超音波組,使用Alamar Blue進行螢光檢定作為細胞活性比較的依據,檢測並比較實驗對照組與超音波組的細胞球團在培養三天之後的代謝效率。 結果: 在1MHz的超音波環境置中,細胞球團的間距約為717 ± 10 µm,細胞球團的大小約為23,461 ± 7,887 µm2;而在1.5 MHz的超音波環境置中,細胞球團的間距約為514 ± 36 µm,細胞球團的大小約為7,132 ± 3,078 µm2;而在3 MHz的超音波環境置中,細胞球團的間距約為241 ± 10 µm,細胞球團的大小約為1,805 ± 1,105 µm2。Alamar Blue的檢測結果中,對照組與超音波組所測得的相對螢光值分別為9,039 RFU以及9,593 RFU。 結論: 利用聲學鑷子的原理可使細胞聚集成球團,以此方法所形成的細胞球團彼此之間的距離為所施打超音波的半倍波長,而使用高頻率超音波探頭時,由於波長較短,超音波聲場中波節與波節之間的距離較短,所形成的壓力節點較小;壓力節點變小意味著所能聚集的細胞也跟著變少,所產生的細胞球團因而變小,較小的細胞球團生成所需要的時間也較短。實驗上主要遇到兩大問題導致實驗結果不穩定,首先是四個探頭的能量校正要達到平衡,再者細胞球團的大小受細胞分佈影響,若無法讓細胞平均分佈在聲場節點,所形成的細胞球團大小不能一致。細胞活性的檢測結果顯示超音波組略高於對照組,但未達顯著差異。 Objective: To develop an ultrasound acoustic tweezer system to aggregate the cells into multicellular spheroids, and to determine the effect of frequency of this system on the spheroids formation. Summary of background data: The three-dimensional cell culture systems are reported to be more efficient than two-dimensional cell culture systems. The three-dimensional cell culture systems have good responses on drug testing and tissue culture especially on stem cell and cancer cell culture. In two-dimensional culture systems, the cells are directly adhered to the culture dish and grow along the plastic surface of the culture dish so that the cells may change the properties of the differentiation. As a result, the cultured cells sometimes become difficult to plant back into the organism or lose the original cell characteristics. In three-dimensional culture systems, one can accurately simulate the environment where the cells reside in, keep the cells in reproduction and make them differentiate in vitro. Currently, many three-dimensional culture systems are being researched, including systems using carriers, biological stent or hydrogel. In this experiment, a home-made ultrasound system is developed to generate the standing wave. The principle of acoustic tweezers is used to aggregate the cells in the culture dish. By adjusting the output power and the frequency, the size of the multicellular spheroid and the time of multicellular spheroid generation can be tuned. Methods: Mouse fibroblasts (L929) were used to make multicellular spheroids in the 35 mm culture dish. Four well-matched ultrasound probes were placed in four directions of the culture dish. This research used three frequencies of ultrasound probes, i.e. 1 MHz, 1.5 MHz and 3 MHz. Ultrasound system outputted a sine wave electrical signal from a function generator and the signal was amplified by connection to a power amplifier. Before the power input to the ultrasound probe, a RF power meter will measure Forward Power (FWD), Reflect Power (RFL) and Voltage Standing Wave Ratio (VSWR) of the system. The impendence of ultrasound probe was also matched to optimize the power output. The acoustic power efficiency of all probes were measured by the correlation between the input electric power and output acoustic power. The average size of multicellular spheroids and the distance between the multicellular spheroids were analyzed by ImageJ. Alamar Blue fluorescence test is used to analyze cell viability. Result: For the formation of using 1 MHz frequency, the distance between multicellular spheroids is 717±10 µm, and the area of the cellular spheroid is 23,461±7,887 µm2.. For the formation of using 1.5 MHz frequency, the distance between cellular spheroids is about 513±36 µm, and the area of cellular spheroid is 7,132±3,078 µm2. For the formation of using 3 MHz frequency, the distance between cellular spheroids is 241±10 µm, and the area of the cellular spheroid is about 1,805±1,105 µm2. In Alamar Blue fluorescence test, the relative fluorescence unit (RFU) of control group and ultrasound group are 9,039 RFU and 9,593 RFU. Discussion and Conclusion: The distance between multicellular spheroids is half of the wavelength of applied ultrasound frequency. When using the high-frequency ultrasound probes (short wavelength), the nodes in the ultrasound field become closer and smaller. Nodes are where cells aggregated and formed the multicellular spheroids. The area of the node becomes smaller and thus the multicellular spheroid. Limitations in this research should be noted. The size of multicellular spheroid depends on the condition of cellular distribution. If the cells is not evenly distributed, the size of the multicellular spheroid may varies. In conclusion, with four ultrasound probes placed in four directions, the ultrasound system could provide a stable acoustic field to aggregate and align in array. With higher frequency, the aggregation time is shorter, and the area of the multicellular spheroid and the distance between multicellular spheroids become smaller. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77829 |
DOI: | 10.6342/NTU201704162 |
全文授權: | 有償授權 |
顯示於系所單位: | 醫學工程學研究所 |
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