微流體整合晶片應用於單一細胞定位與藥物測試

An Integrated Single-Cell Microfluidic Chip for Single Cell Positioning and Drug Testing

陳俐瑾(Li-Ching Chen)

指導教授 ： 曾繁根 ； 楊重熙

國立清華大學/原子科學院/工程與系統科學系/碩士(2014年)

摘要

近年來，由於醫學及生物學的發展，細胞行為的探討，不再侷限細胞群體的研究，而個體化細胞篩選、定位及單顆細胞間的變異性等議題日趨重要，單顆細胞個體的細胞形態、生物意義，以及細胞間訊息的聯繫，甚至是單顆細胞於環境中扮演的角色等等帶給整體環境的影響等研究亦逐漸興起。為了瞭解單顆細胞的行為，單顆細胞定位、培養和實時檢測，以及細胞間的交互作用已成為今日重要的技術。
我們提出了一個整合性的微流體晶片，提供一簡單的技術來實時檢測單顆細胞個體的行為以及細胞間的交互作用。我們設計了一個獨特的晶片結合捕捉細胞陷阱結構以及可控制流體的真空閥門，並整合於微流道晶片上。晶片中，為了精確捕捉到單顆細胞，我們設計了僅能容納單顆細胞大小的結構陷阱，並提供細胞適當的培養環境讓細胞於晶片中亦能表現出正常的細胞行為。此外，本研究不僅針對單顆細胞進行觀測，甚至我們希望能即時觀測細胞間的訊息傳遞，觀察其細胞行為以及其化學分泌物質。因此，我們利用閥門適當控制流體運行方式，藉由閥門的開關不僅能將晶片分隔成兩個獨立細胞培養系統，用於培養兩單顆細胞，並利用閥門亦能打開通路來傳輸外界藥物等刺激，或者打開兩獨立細胞培養系統間閥門，觀測細胞通訊所產生之信號以及交互影響，進而推斷單顆細胞個體於環境所扮演的角色。
本研究中，為了確定此微流道晶片捕捉細胞之效果與真空閥門系統的運作及封閉性，還有將其發展成一個具有生物意義的設計。我們對晶片設計了四個階段一系列的實驗進行探討。第一個實驗，我們通入不同顏色的墨水，當作兩種不同細胞類型的懸浮液或培養液，模擬後期的細胞實驗。墨水實驗結果顯示，真空閥門的控制十分精確，更能
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利用閥門的開關控制流體的方向。第二個實驗，將染有螢光的細胞通入晶片中，確定此系統中設計的陷阱結構能準確抓取細胞，定位於晶片中的位置。第三個實驗，我們將熱敏感性微脂體(Thermosensitive liposome)模擬為細胞，利用溫度變化模擬給予刺激，使其釋放出包覆的螢光染劑，進而影響到第二個細胞的表現，此實驗不僅可確定晶片設計的完整度，更表示此晶片可觀測到細胞間交互作用的情形，具有後期更多生物研究上的意義。第四個實驗，我們再次運用微指體包覆致毒性藥物，利用溫度上升使得其釋放藥物，給予第二個細胞刺激，再藉由螢光染色去觀察細胞受刺激後其活性改變情形，再次確認此平台於往後實驗的可行性。由以上這些實驗結果可預期，後期研究能持續朝細胞交互作用觀測實驗方面進行深入探討。

關鍵字

微流體晶片 ； 單一細胞定位

並列摘要

The interactions between cells and cells play essential roles in cell biology. Nowadays reports showed that studying cells to cells interactions is an important research to understand cell behaviors such as variation or tumor progression and metastasis, which cannot be fully revealed by traditional methods based on the ensemble average behaviors of large population of cells incubated in petri dish. Thus single cell positioning, incubation and observation have become an important technique to analyze single cell behaviors. In this study, we proposed an unique chip combining micro snare to capture single cells and controllable valves to manipulate fluid operation, for facilitating the study of single cell-cell interactions.
In this research, a process consisting mechanical traps and valves was presented for studying single-cell interactions. Firstly, two single-cells were captured independently by the 15μm x 15μm snare which was designed as the diameter of the cell. Afterward, the two single-cells underwent perfusion culture. After both cells adhered to the bottom of the channel, cell A was stimulated by some chemicals and released some substance. Then the substance released by cell A stimulates cell B by opening the path between them. Finally, the substance released by cell B passed into the detection part to examine the variation of cell B.
We used simple technique to fabricate our chip, including microfluidic channel, controllable valve and cell trap. Moreover, the valves could be manipulated on-off by a vacuum pump controlled via Lab-view software (National Instruments). The testing of the four series valves exhibited the precise operation and control of the valves. Certain fluid was
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injected into two different directions to ensure the valves could switch the pathway between two single-cell culturing systems. In this way, the two single-cells could be cultured under independent medium route avoiding mutual mixture and could also be affected by each other by opening the valve between two systems.
After single cell trapped inside the snare, the streamline will not go through the snare center since the increase of the flow resistance and the rest of cells will pass by without accumulation inside the trap. Hela cells dyed with Calcein AM Fluorescent were loaded into the chip and the single-cell was captured by the trap. Discovering captured cells simultaneous in our unique chip is feasible. Finally, we simulated thermosensitive liposome as one of single-cell (cell A), and induced liposome releasing some fluorescent dye to affect another real single-cell (cell B). The fluorescent change showed that the second single-cell apparently received the signal released by liposome from the cell A channel. Thus, the way as the third testing, Doxorubicin was encapsulated by liposome. Heating up the temperature made the liposome release Doxorubicin to stimulate another single-cell. Finally, we examined the stimulated single cell viability by Calcein. The above four serial experiments not only prove the completeness of the chip but reveal its biological feasibility. We could further use this special design to undergo cell-to-cell mutual interactions in the future. It is a prospective platform for cell-cell interaction research, which can also combine with biological or electrochemical detection someday.

參考文獻

[32] Sarah Brown and David R. Khan, “The Treatment of Breast Cancer Using Liposome Technology” Journal of Drug Delivery, 2012

[33] Juan Lao and JuliaMadani and Teresa Puértolas, “Liposomal Doxorubicin in the Treatment of Breast Cancer Patients”, Journal of Drug Delivery, 2013.

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