Please use this identifier to cite or link to this item:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/74286
Title: | 使用超音波評估細胞貼附之可行性研究 The Feasibility Study of Assessment of Cell Adhesion using Ultrasound |
Authors: | Yen-Ting Chou 周彥廷 |
Advisor: | 王兆麟(Jaw-Lin Wang) |
Keyword: | 細胞貼附,超音波非破壞性,初始峰值,生醫材料, Cell adhesion,Ultrasonic nondestructive testing,First peak,Biomaterial, |
Publication Year : | 2019 |
Degree: | 碩士 |
Abstract: | 目的:使用超音波進行非破壞性的細胞貼附量測。
背景簡介:細胞貼附指的是細胞黏附於其他細胞或是細胞外基質的能力,對於行為調控、訊號傳遞以及生命週期等細胞常見的現象來說,細胞貼附是重要的因子。在生醫材料的領域中,細胞貼附現象為探討材料生物相容性的重要指標之一。在體外實驗中,細胞貼附分為初始接觸、胞體扁平化以及完全擴張等三個階段。當前對於細胞貼附的觀測,光學顯微鏡為最普遍使用的儀器,而探究更精準的細胞動態變化則有QCM-D和ECIS。隨著生醫材料的發展,培養細胞的環境日趨多元,許多具有細胞表面依附性的材料較難透過前述儀器進行貼附觀測。因此,本研究希冀使用已用於多種材料測試的超音波,驗證其對細胞進行非破壞性量測的可行性,最終克服非透明材料對細胞貼附觀測所帶來的限制。 材料與方法:本研究使用長形載玻片作為細胞貼附與超音波訊號傳導的介質,兩端分別放置訊號觸發與接收探頭,玻片中段以O型環圈出細胞培養區域。此設置可同時進行細胞貼附的顯微觀測和訊號偵測,並使用2.25 MHz以及5 MHz兩種訊號源頻率。控制變因除了頻率的差異,玻片分為有FN塗層及無FN塗層,使試樣間的細胞貼附速率產生差異。在1mm厚度的玻片中,超音波會以體波的形式傳遞,且因空氣與玻片聲阻抗有差別,訊號會以不斷反射的方式進行傳遞。當訊號傳遞至細胞培養區域,由於細胞—玻璃介質層的聲阻抗作用,部分訊號會產生反射,而部分訊號會因透射繼續進入培養液中傳遞。接收訊號的分析以介質層反射波為主,擷取前端接收訊號並將固定時間區域內的最大峰值稱為初始峰值,並以該值的峰對峰值變化及訊號接收時間差量化細胞貼附於體外環境的三個階段。 結果:經由多種細胞以及培養液濃度的嘗試,最終選定老鼠肌纖維母細胞C2C12作為試樣,試樣濃度為每毫升50萬顆細胞。實驗結果顯示,初始峰值之峰對峰值會隨著細胞貼附產生一致性的變化趨勢,而接收時間差則無顯著的變化。分析實驗數據後進行以下假設:一、當細胞與玻片初始接觸時,幾乎無法產生具有聲阻抗特性的介質層,使得訊號振幅強度較為穩定。二、細胞進行胞體扁平化的過程中,動態變化使接觸面積快速增加而形成另一種介質層,造成透射係數增加,所量測的反射訊號強度也隨之降低。2.25 MHz的組別有60%的降幅,5 MHz的組別則有40%的降幅。三、當細胞完全擴張處於安定的狀態時,訊號強度的變化幅度再度進入穩定期。實驗結果大致符合假設,而2.25 MHz的組別則因細胞完全擴張後產生的第二介質層,導致訊號強度回升,因此推論使用5 MHz或是更高頻率的訊號源能有更真實、穩定的表現。 結論:以目前實驗成果所示,使用超音波進行細胞貼附偵測已具有初步的可能,也提供細胞與訊號傳導介質間容納其他生物基質的擴充性,預期能進一步應用於測量細胞及不同生醫材料的貼附。若再將其他種類的波形如表面波、蘭姆波作為量測訊號,或許能進行更精確的量測及更廣泛的運用。 關鍵字:細胞貼附、超音波非破壞性、初始峰值、生醫材料 Objective: To apply ultrasonic nondestructive testing for cell adhesion measurement. Summary of background: Cell adhesion is a complex process, involving both physical and chemical interaction between cells and their surrounding environment. It plays a key role in regulating cell behaviors, such as proliferation and differentiation. From a biomaterial development point of view, the ability to quantify cell adhesion is also important in providing crucial information relating to the interaction between cells and the biomaterials. The process of static in vitro cell adhesion is classified by three stages: initial attachment, flattening of the cell body, and fully spreading of the cell. Available cell adhesion measurement tools, such as ECIS and QCM-D, are difficult to accommodate biomaterials between and measure the surface of the cells. In this study, we present a method to quantify cell/substrate adhesion using ultrasound with the possibility to accommodate biomaterial substrates like hydrogel between cells and base plate. Method: We used 5 MHz and 2.25 MHz ultrasonic transducers in a transmitter-receiver mode. A glass slide is used as the base plate to seed cells in designated area (surround by an O-ring). Optional bio-substrate (such as FN coating) can be applied to the glass slide before cell seeding. In the case of 1 mm thick slide, bulk model ultrasound is propagated between the transmitter and the receiver. When ultrasound passes through the seeding area, part of acoustic signals will be reflected because of the cell-glass interference, and part of them will leak into the medium. Therefore, the reflected signals are affected by the cell/glass coverslip interface. We measure the arrival time and peak-to-peak value of the first arrival signals, called first peak, to quantify cell adhesion. In both frequencies, we speculate different cell adhesion states will affect the amount of energy leaked into the seeding area. Thus by measuring the receiving amplitude, we can quantify the cell adhesion. Result: To validate our measurement setup, we seeded C2C12 cells onto the seeding area and measured changes in receiving amplitude. After seeding, cells started to attach to the glass substrate. During initial attachment of the cell body to its substrate, the intensity of receiving amplitude is stable. Then, during the flattening and spreading of the cell body, the dynamic change caused the contact area to rapidly increase, changing the acoustic impedance and decreasing the intensity by 60% in 2.25 MHz group and 40% in 5 MHz group. After fully spreading, the cells are in a stable state. The change in the receiving amplitude gets into a stable phase again. However, due to the second boundary, the intensity of 2.25 MHz group rises rather than staying stable. The results validate our assumption that different states of cell adhesion will affect the receiving amplitude. To sum up, it is inferred that employing a signal of 5 MHz or higher frequency can have a more reliable and stable result. Conclusion: In this study, we present an ultrasonic method to measure cell adhesion. This approach offers the possibility to accommodate additional bio-substrate between cells and the base plate. By measuring the changes of receiving amplitude as the cells start to attach to the glass coverslip, the proposed approach has been validated. The method can be further applied to measure cell adhesion with different bio-substrates. Key word: Cell adhesion, Ultrasonic nondestructive testing, First peak, Biomaterial |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/74286 |
DOI: | 10.6342/NTU201903351 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 醫學工程學研究所 |
Files in This Item:
File | Size | Format | |
---|---|---|---|
ntu-108-1.pdf Restricted Access | 2.78 MB | Adobe PDF |
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.