結合電穿刺與離心力以評估細胞之粒線體遞送

Abstract

本研究旨在探討處於離心環境下，利用微流道將粒線體遞送至細胞內部之可行性。離心環境和微流控技術在生物醫學領域中具有廣泛應用，如在細胞分離、細胞培養和藥物篩選等方面。近十年來，細胞內粒線體遞送已成為生物醫學領域的熱門議題。

本研究冀望能建立一個完善的實驗平台，在將細胞進行必要的離心步驟時，我們可以透過電穿刺將粒線體傳遞至細胞內部。為了驗證該方法的成功率，我們選定HL-60（人類白血病細胞）進行研究，此細胞在細胞實驗中較為常見。我們預設將在50mL的離心管內利用半導體製程製作微流道與微電極系統(以下簡稱微流道電穿刺系統)。

微流道電穿刺系統的設計和製作過程包括光阻、蝕刻等步驟，以實現高精度的細微結構。將此系統放置在市售離心機中，透過微流道導向細胞，以實現在離心環境下對細胞進行電穿刺。

為驗證實驗方法的有效性，我們調整了不同的參數，如離心機轉速、電訊號頻率等，並比較該方法之成功率。實驗結果發現，在其他參數相同的情況下，使用10Vpp、10kHz、200g、5分鐘之參數可以有最佳的穿刺效果。在後續測試中，我們發現再離心500g、5分鐘，可以有最佳的粒線體遞送效率。

本研究的特點在於，設計一個適合在離心環境下運行的微流道電穿刺系統。未來研究可以進一步優化微流道電穿刺系統和離心條件，以實現更高效、安全的細胞內物質遞送，以求能遞送除粒線體以外之其他物質。

綜上所述，本實驗已建立一個完善的流程與步驟，讓細胞成功在離心環境下完成粒線體遞送，對於在微流道中提高電穿刺的進一步發展具有重要的指導意義。

This study explores the feasibility of delivering mitochondria into cells using microchannels in a centrifugal environment. Centrifugal environments and microfluidic technology have extensive applications in the biomedical field, including cell separation, cell culture, and drug screening. Over the past decade, intracellular mitochondrial delivery has become a prominent topic in biomedical research.

The study aims to establish an experimental platform to deliver mitochondria into cells through electroporation during centrifugation steps. HL-60 (human leukemia cells) were selected for their common use in cell experiments. A microchannel and microelectrode system (microchannel electroporation system) will be created using semiconductor processes within a 50mL centrifuge tube.

The design and fabrication of the microchannel electroporation system involve photolithography and etching to achieve high-precision microstructures. This system is placed in a commercial centrifuge, guiding cells through the microchannels to achieve electroporation in a centrifugal environment.

To validate the method, various parameters such as centrifuge speed and electrical signal frequency were adjusted, and the success rates were compared. The best electroporation results were achieved with parameters of 10Vpp, 10kHz, 200g, and 5 minutes. The highest mitochondrial delivery efficiency was achieved at 500g for 5 minutes.

The study's main feature is the design of a microchannel electroporation system for operation in a centrifugal environment. Future research can optimize the system and conditions to achieve more efficient and safer intracellular substance delivery, including other substances besides mitochondria.

In summary, this experiment establishes a comprehensive procedure for successful mitochondrial delivery in cells within a centrifugal environment, providing important guidance for further development of electroporation efficiency in microchannels.