高純度螢光激發式罕見細胞分選及細胞培養晶片系統之研究

Study on Fluorescence Activated Micro Cell Sorter with On-Chip Cell Expansion System

王彥翔(Yen-Siang Wang)

指導教授 ： 黃榮山

共同指導教授 ： 趙聖德(Sheng-Der Chao)

國立臺灣大學/工學院/應用力學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.02467

摘要

細胞為構成生物體的最小單位，許多種的細胞均具有研究或是臨床應用上的重要性，但是礙於細胞存在的數量過少而無法更深入研究。以幹細胞為例，幹細胞為身體各組織最原始的細胞，具有分化為各種不同細胞的能力，但是幹細胞在生物體組織中所佔有的比例非常的少，因此，針對幹細胞的研究或是臨床上的應用，必須透過細胞篩選的過程，取得足夠的罕見細胞數量。
本研究之目的在於透過微機電的微製造技術，以微流力及微電子的基礎下，建立一個細胞分選的晶片系統，透過此晶片系統針對罕見細胞作篩選純化的動作，並且在晶片上整合細胞培養的功能，使篩選過後的細胞能夠在不受外界污染的情況下，直接在晶片上作培養的動作，減少細胞運送過程的損失，以利於罕見細胞後續的研究及應用。
本研究的重心為細胞篩選及培養的結果，論文包含了實驗原理、晶片製造與實驗方法及實驗結果三大主軸。在實驗原理的部分，主要闡述微流道及介電泳動的原理，並且包含了測試的結果。晶片製造與實驗方法的部分，詳細記錄了晶片的製造過程，也說明了本實驗微流道、電子操作系統以及光學系統的架設，此外，完整的實驗流程也包含在其中。最後，實驗結果的部分，完整的呈現了本晶片系統在細胞篩選及細胞培養方面所發揮的功能。
本研究所建構之細胞分選及細胞培養系統，在施加電壓10V的狀況下，能夠使細胞樣本之標的細胞純度提昇至90%以上，並且不會對標的細胞產生傷害。此外，也成功的在晶片內培養細胞超過100個小時。本研究之細胞分選晶片具備低成本、高純度以及能夠在晶片內部培養細胞的優點，相信對於未來細胞醫療或是罕見細胞的研究能夠有一定程度的貢獻。

關鍵字

細胞分選 ； 介電泳 ； 水力聚焦 ； 罕見細胞 ； 幹細胞

並列摘要

A new microfabricated platform for rare cells is demonstrated, which provides specific cells with a novel device of multiple functions of cell sorting, cell culturing, and real-time investigation. Due to increasing potential of stem cell in therapy, diagnostics, drug discovery as well as development of its science and technology, few existing techniques can be tailored to be specific for emerging needs of stem cell applications. In primary culture of rare stem cells that account for one out of 103~106 cells, purification and expansion of stem cells requires complicated processes of cell identification, sorting, culture and verification. The present microfabricated platform that provides integration of required functions is especially suitable for purification of stem cells in mixed population of rare cells. The micro device is designed to be disposable for single use and minimal contamination for potential cell therapy. In addition, the single-use device is able to be compatible with the existing microscopes and to be capable of integration with subsequently required cell analysis.
The micro device is simply composed of a polydimethylsiloxane (PDMS) elastomer channel and multiple electrodes deposited on the glass substrate. By using PDMS, a controllable environment for target cells with bio-compatible (hydrophobic), elastic vessel-like channel wall, and transparent (optically transparent down to 300 nm) for microscopic living-cell detection was created. In sorting approach, the dielectrophoresis (DEP) technique with hydrodynamic focusing flow is chosen due to minimal effect to cells and ease of implementation. The moving fluorescent cells tracked under the microscope to be sorted were guided along the designated electrodes by DEP to the specific chambers, while the rest of cells were straight transported through the central flow of the hydrodynamic focusing to the waste chamber. Few amount of the selected cells were required for proliferation on chip to be contamination-free. To facilitate the on-device culture of living cells, the culture condition was additionally controlled by a micro-incubator controller, providing an excellent controlled environment at just 37℃, 90% moisture and 5% CO2 concentration. Several types of human cells chosen for preliminary studies enable the present device to simulate direct applications on clinical trial.
The present system demonstrates the potential for surface marker-specific cell sorting of human bone marrow stem cells simply with the normal, regular procedures of immunostaining techniques by using μ-FACS within the micro device. To evaluate the performance of this on-chip culture system shown, the sorted human bone marrow stem cells had been examined for detailed testing. The cell-sorting performance of rare cells shows 92 % in efficiency. Over one day and several hours of culture, the sorted cells exhibited a well-adhered, healthy morphology comparable to static culture and the surface of untreated glass was almost completely covered by the proliferating cells.
The results demonstrated that the microchamber is a satisfactorily biocompatible microenvironment for the continuous growth of the cells. The multifunctional μ-platform system has been proven to be useful in rare stem cells sorting and cell culturing. The platform offers the potential for the future application on stem cell therapy.

並列關鍵字

cell sorting ； dielectrophoresis ； DEP ； hydrodynamic focusing ； rare cell ； stem cell

參考文獻

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