可應用於微組織之微流體系統開發－以質傳為基礎之生物反應器

Development of a Microfluidic System for Microtissue Applications - Bioreactors Based on Mass Transport

吳東翰(Tung-Han Wu)

指導教授 ： 許聿翔

國立臺灣大學/工學院/應用力學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201803287

摘要

疾病晶片是未來醫療保健個人化醫療領域的優先研究領域之一。在主要的慢性疾病中，癌症是繼心血管疾病之後的第二大死因。這是由於其高度異質性，準確而有效的診斷及治療仍是目前巨大的挑戰。因此，開發腫瘤晶片的方法在該領域引起了廣泛關注。透過使用晶片上的腫瘤系統進行藥物篩選或遺傳篩檢，我們可以根據篩查結果為不同患者設計個人化的藥物治療。
在本研究團隊之前的研究中，設計了兩種微流體系統，透過不同的流動模式來產生兩種不同的培養環境：(1)營養剝奪，透過擴散流體提供緩慢的營養供應，使得養分不及供應細胞的代謝(2)對流支配，透過孔隙間的對流提供養分完全補充，達到快速供給養分。兩者之間的養分供給時間相差兩個數量級，而在本研究中，我們透過微流體技術來展示對營養剝奪與完全營養補充對腫瘤發展的貢獻研究。設計長的微流體通道以產生擴散特性的微腫瘤腔室，並且透過兩種方法控制營養剝奪的程度。一種是控制細胞的初始注膠濃度，另一種是控制營養供應的灌注速率從兩個側通道擴散，不同的程度壓力驅動下，可造成腔室內養分替換的快慢，以研究微腫瘤的生長。我們的實驗結果表明，較低的初始細胞濃度，具有均勻的生長速率，可以在1mm×2mm的腔室中發展連續性的微腫瘤。
微腫瘤的生長可以透過營養剝奪的程度來調節。這表示營養缺乏可能在微腫瘤的一開始發展起重要作用。應用該模型系統，可以針對不同類型的腫瘤細胞進行優化。因此，我們可以提供個人化系統來開發患者異質性微腫瘤，以研究患者之間腫瘤的差異性並提供有效的治療方法。
在另一項研究中我們嘗試讓微血管連接到微腫瘤組織中以提供腫瘤大量的血氧供給。腫瘤剛形成時，癌細胞之間靠擴散作用就可以吸收周遭環境養分，排除代謝所產之廢物；但是腫瘤生長一旦超過100μm擴散極限，細胞之間的擴散作用就不再管用，開始養分供應不足，進而產生缺氧狀態，必需誘發血管新生的機制來供應癌細胞生長所需養分。否則腫瘤中心會因擴散的限制導致氧氣導入的困難而形成過度缺氧(<2%)，因為代謝物的累積會逐漸酸化，最後導致細胞壞死。
本論文，我們設計了兩種微流體系統在這項研究中，透過不同階段的培養對象來產生兩種不同的微環境，兩者在模擬的驗證下皆是營養剝奪，透過擴散流提供緩慢的營養支持。我們成功的開發了大尺度的血管網絡與腫瘤組織，證明血管網絡受到側旁的纖維母細胞腔室誘導血管生成芽。此平台能提供完整的生理與工程條件，相信血管仿生癌組織也能此生物反應器上實現。
所有的微流體平台皆搭配有限元素模擬，針對不同厚度的微流體與驅動壓力做計算作分析，探討微流體在流道內的的流速、腔室的壓力梯度以及腔室內的速度分布，最後作時間相依的濃度分析。結過表明，我們成功開發可應用於微組織之微流體系統，透過質傳方式建構生物的微環境。最後藉由流體週轉時間實驗驗證模擬質傳時間的相符程度。這種新開發的系統，突破以往腫瘤與血管組織在體外培養毫米尺寸的限制，這種3-D的組織不僅提高人體相容性，在未來，可以在臨床應用之前進行患者腫瘤模型或不同的癌細胞類型用於個人化醫學研究。

關鍵字

生物反應器 ； 微流體 ； 營養剝奪 ； 腫瘤晶片 ； 微腫瘤 ； 血管生成 ； 微組織

並列摘要

Disease-on-a-chip is one of the leading research fields in personalized medicine for future health care. Among the major chronical diseases, cancer is the second leading cause of death next to cardiovascular disease. It is due to its high heterogeneity, and identification of an effective treatment sets a big challenge to cure cancer. Hence, the method to develop tumor-on-a-chip has drawn much attention in this field. By using a tumor-on-a-chip system for drug screening or genetic screening, we can design personalized medical treatment for different patients according to the screening results.
In this study, two kinds of microfluidic systems to generate two different culture environments through generating different flow patterns are designed: (1) Nutrition-deprived condition, which offers a slow nutrient support through diffusive flow, (2) Convection-dominated condition, which offers a rapid nutrient support through interstitial convective flow to achieve a rapid nutrient supply. In this thesis, different initial cell numbers and nutrition supply to investigate the growth of a microtumor are studied and compared. A long microfluidic channel is designed to create a diffusion dominant microtumor chamber, and the level of nutrition deprivation is controlled by two methods. One is to control the initial loading concentration of cells, and the other is to control the perfusion rate of the nutrition supply diffuses from two side channels. Our experimental finding suggests that a lower initial cell concentration can developed into continuous microtumors across the 1 mm by 2 mm microchamber with a uniform growth rate.
The growth of microtumors can be regulated by the level of nutrition deprivation. It suggests that nutrition deprivation could play an important role on the initial development of a microtumor. Applying this model system, it could potentially be optimized for the tumor growth among different kinds of tumor cells. Thus, we can provide a personalized system to develop patient specific microtumors to study the heterogeneity of tumors between patients and to identify an effective treatment.
In the second study we attempt to connect microvasculature into microtumor tissue to mimic nutrition and oxygen supply of tumor. Once the tumor is formed, cancer cells can directly uptake surrounding nutritions or exclude the metabolic waste by the diffusion between the cells; however, once the tumor grows beyond the diffusion limit of 100 μm, the diffusion between the cells is insufficient, resulting in hypoxic conditions. Thus, tumors can tirger angiogenic process to create an environment for tumor growth. Otherwise, the tumor is anoxia (<2%) due to limited oxygen induction, and acidification due to reprogramming of metabolic procss, it eventually creates apoptosis and necrosis.
We further design two kinds of microfluidic systems to generate two different microenvironments through different stages of tissue development. Both are nutritionally deprived and provide slow nutritional support through the diffusion transport. We successfully develop a large-scale vascular network and microtumor. It was shown that the angiogenic process is induced by the adjacent fibroblast chamber. In the second study, we successfully developed a large-scale microtumor next to developed vasculature. This platform can provide complete physiological and engineering conditions, and it is believed that vascular biomimetic cancer can be realized on this bioreactor.
All microfluidic platforms are verified with finite element analysis. The microfluidic and driving pressures of different chamber height are analyzed. The flow velocity of the microfluid in the microchannel, the pressure gradient of the chamber and the velocity distribution in the chamber also are discussed. Finally, time-dependent concentration analysis is performed. The results show that we successfully developed microfluidic systems that can be applied to microtissues. These newly developed systems break through the limitations of the mm size of microtumor and blood vessels in lab-on-a-chip systems. The 3-D tissue not only improves human compatibility, and in the future, patient-specific tumor models or different cancer cell types also can be applied for personalized health before clinical applications.

並列關鍵字

Bioreactor ； microfluidics ； nutrition-deprivation ； Tumor-on-a-Chip ； microtumor ； angiogenesis ； microtissue

參考文獻

[1] N. Kashaninejad et al., "Organ-tumor-on-a-chip for chemosensitivity assay: a critical review," Micromachines, vol. 7, no. 8, p. 130, 2016.

[2] A. Annibaldi and C. Widmann, "Glucose metabolism in cancer cells," Current Opinion in Clinical Nutrition & Metabolic Care, vol. 13, no. 4, pp. 466-470, 2010.

[3] X. Zhang, Y. Lin, and R. J. Gillies, "Tumor pH and its measurement," Journal of nuclear medicine: official publication, Society of Nuclear Medicine, vol. 51, no. 8, p. 1167, 2010.

[4] T. Van Dyke and T. Jacks, "Cancer modeling in the modern era: progress and challenges," Cell, vol. 108, no. 2, pp. 135-144, 2002.

[5] I. Vuletic et al., "Validation of bevacizumab therapy effect on colon cancer subtypes by using whole body imaging in mice," Molecular Imaging and Biology, vol. 19, no. 6, pp. 847-856, 2017.