以微流體技術開發懸浮式細胞培養系統及其應用

Developing a microfluidic-based hanging-drop culture system for cells interaction and application

黃書葦(Shu-Wei Huang)

指導教授 ： 林峯輝

共同指導教授 ： 孫瑞昇(Jui-Sheng Sun)

國立臺灣大學/工學院/醫學工程學研究所/博士(2020年)

https://doi.org/10.6342/NTU202001890

摘要

在過去的十年中，已經有許多微流體技術與懸浮液滴相結合用於細胞培養。這些設備中的一個常見問題是將細胞懸浮液集中引入進口，這將導致每個微孔中的細胞數量不規則。而且，在球體形成過程中液滴的不穩定性仍然是未解決的難題。在這項研究中，我們設計了一種基於微流體的懸浮液滴培養系統，該系統採用錐形管設計，可以增加液滴的穩定性，同時提高液體交換的速度。錐形管周圍有一個環。環可以容納細胞，使我們能夠在灌注之前播種足夠數量的細胞。此外，在細胞培養期間，細胞周圍的機械力相對較小，以防止細胞分化並維持表型。作為我們懸掛系統設計的結果，細胞被設計為聚集在液滴的底部。它增加了觀察活動和實驗分析的便利性。我們使用替加環素和棕櫚酸丙酮酸脂作為聯合治療模型，以評估該微流體芯片中藥物遞送的效果。根據結果，可以根據需要以不同濃度輸送藥物。在最後，我們使用幹細胞評估此培養系統對於再生醫學的應用。本研究透過幹細胞的細胞表面標記評估培養前後細胞的型態，以及此系統是否能維持幹細胞可分化為其它組織的特性。根據試驗結果，透過微流道系統培養的幹細胞，不但能大量培養，且維持其細胞型態之外，更能夠保有其分化成其他組織的特性。因此，該微流體芯片可以用作代表體內生理狀況的體外平台，並且在未來可以用於再生醫學。

關鍵字

微流體 ； 懸浮液滴 ； 三維細胞培養 ； 細胞治療 ； 再生醫學

並列摘要

There have been many microfluid technologies combine with hanging-drop for cell culture gotten developed in the past decade. A common problem within these devices is the cell suspension is centralized introduced at the inlet would cause the number of cells in each microwell not regularize. Also, the instability of droplets during the spheroid formation remain an unsolved ordeal. In this study, we designed a microfluidic-based hanging-drop culture system with the design of taper-tube that can increase the stability of droplets meanwhile enhance the rate of liquid exchange. And a ring is surrounding the taper-tube. The ring can hold the cells to enable us to seed the adequate amount of cells before perfusion. Moreover, during the period of cell culture, the mechanical force around the cell is relatively small to prevent cells from differentiate and maintain the phenotype. As the result of our hanging system design, cells are designed to accumulate at the bottom of the droplet. It enhances convenience for observation activities and analysis of experiments. In this study, we used tigecycline and pyrvinium pamoate as a combined therapy model to evaluate the effect of drug delivery in this microfluid chip. According to the results, drugs can be delivered in different concentrations as we need. At last part, we focus on the application for regenerative medicine.. We check the cell markers on the WJ-MSCs cultured in the microfluidic hanging drop chip. According the result, it showed the same distribution before culture and after culture, and demonstrating that the chip maintained the original stem cell phenotype. Thus, this microfluid chip can be used as an in vitro platform representing in vivo physiological conditions, and in regenerative therapy.

並列關鍵字

Microfluid ； Hanging drop ； 3D culture ； Cell therapy ； Regenerative Medicine.

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