介電泳細胞微陣列微流系統應用於化療藥物之篩檢

Abstract

由於化療對於部份癌症患者之療效有限，因此藉由體外之藥物篩檢以預測化療結果的需求與日俱增。為了提升藥物反應之預測，高通量 (high throughput) 生物檢測工具，如 細胞微陣列 (cellular microarray)，已被用以進行藥物篩檢以預測臨床藥物反應。 本文提出以介電泳（dielectrophoresis）為基礎的細胞微陣列輸流 (perfusion) 系統，進行穩定的藥物篩檢。藉由平面叉環電極（planar interdigitated ring electrode, PIRE）陣列所產生之介電泳力均勻排列細胞微陣列。另外，整合濃度梯度產生器 (concentration gradient generator, CGG) 以及反濃度串擾閥（anti-crosstalk valve, ACV），產生一個無濃度串擾 (concentration crosstalk)的線性藥物濃度梯度，以提供穩定的藥物輸流。藉由一個特別設計的輸流架構提升系統內部的壓力高於常壓進而消除系統內之氣泡，提供無氣泡之細胞培養環境。 實驗結果顯示本文所提出之系統提供一個良好細胞培養的環境以因應藥物篩檢之需。在PIRE上細胞在幾分鐘內被排列佈滿整個PIRE陣列，且被排列之細胞在數量上具有良好的一致性: 對於一個 6 × 6 PIRE陣列，每個PIRE含48 ± 6個細胞;對於一個 1 × 3 PIRE陣列，每個PIRE含92 ± 5個細胞。由細胞存活率測試顯示被排列之細胞在經過24小時的培養後依然健康。藉由細胞存活率測試，進一步將活細胞之螢光強度量化，而細胞存活率重複性(reproducibility of cell viability) 良好（經常態化後，於任一PIRE上之細胞平均螢光強度為 1 ± 0.138）。此外，對於細胞進行24小時之藥物輸流 (24-hr. drug perfusion)後進一步應證：1. 細胞存活率與藥物濃度具有一個良好的關聯性；2. 此系統所得之藥物劑量反應(dose response)與96孔盤(96-well plate) 所得之反應無統計上的差異。此外，在PDMS晶片內消除氣泡的結果顯示消除氣泡所需的時間與氣泡的初始大小為線性關係。 本文所提出的細胞微陣列輸流 (perfusion) 系統將有助於需要一致細胞數量的相關應用，並補充微流輸流系統 (microfluidic perfusion) 於藥物篩檢之相關資訊，以建立與臨床的藥物反應 (in vivo drug response) 之關聯性，至終提高化療之療效。

With low efficiency of chemotherapy for some patients, there is a need for predicting the outcome of a treatment by performing in vitro cellular drug screenings. To predict drug responses, cellular microarray－a powerful tool for high throughput screening－has been used to meet the need. Here, this thesis presents a dielectrophoresis (DEP)-based cellular microarray perfusion system for anticancer drug screenings. A collagen-coated planar interdigitated ring electrode (PIRE) array utilizing positive dielectrophoresis to pattern cells uniformly is put forth. Furthermore, a functional integration of a concentration gradient generator (CGG) and an anti-crosstalk valve (ACV) is presented to generate a linear concentration gradient of drug without unwanted crosstalk of drug concentrations for a stable drug perfusion. Besides, a tailor-made configuration was designed to keep the pressure inside chip higher than ambient pressure which served to provide a bubble-free culture environment. Results suggest that the proposed system provides a viable microfluidic culture environment for drug screenings. Cell patterning on a PIRE showed that cells were patterned within minutes with good uniformity (for a 6×6 PIRE array: 48 ± 6 cells per PIRE; for a 1×3 array: 92 ± 5 cells per PIRE). Cell viability test revealed healthy patterned cells after 24-hour culture. Furthermore, quantification of fluorescence intensity of living cells showed an acceptable reproducibility of cell viability among PIREs (mean normalized intensity per PIRE was 1 ± 0.138). Moreover, observations of patterned cells during 24-hour drug perfusion showed good correlation between cell viability and drug concentration, and no statistical significance in dose responses between the microfluidic system and 96-well plates. Besides, bubble elimination in a PDMS chip showed the relationship between the time for bubble elimination and the associate bubble sizes was linear. The DEP-based cellular microarray perfusion system should benefit applications desiring uniform cellular patterning, and supplement microfluidic perfusion approach for building up a correlation to in vivo drug responses for improving efficacy of chemotherapy.