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研究生：	沈彥平 Shen, Yen-Ping
論文名稱：	利用viscous fingering pattern在AIM微流體晶片產生光滑與環形之血管 Generation of a smooth and circular vessel lumen using viscous fingering pattern in AIM microfluidic chips
指導教授：	涂庭源 Tu, Ting-Yuan
學位類別：	碩士 Master
系所名稱：	工學院 - 生物醫學工程學系 Department of BioMedical Engineering
論文出版年：	2020
畢業學年度：	108
語文別：	英文
論文頁數：	48
中文關鍵詞：	viscous finger pattering 、血管、人類臍靜脈內皮細胞、通透係數
外文關鍵詞：	viscous finger patterning, HUVECs, permeability coefficient
相關次數：	點閱：136 下載：0
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在人體中血管遍布全身，許多病症的轉移都與此有很大的關聯。同樣地，養分以及藥物的傳遞也是透過血管輸送至身體的各個部位。因此，了解血管的運行機制對於病症的研究來說愈來愈重要。為了瞭解血管的機制，近年來許多研究發展出了一套能輕易在體外觀察人體微環境的模擬平台。有許多研究皆透過體外微流體系統製造出血管。但是，在多流道的微流道中血管研究仍有更多的發展空間。因此，我們希望在現成的Aim Biochip上製造出尺寸最佳化後的血管並且進行通透性的測試。
在本實驗中，我們展示了在多通道的微流道系統中利用纖維蛋白(fibrin)填充中間的流道，利用第一型膠原蛋白(collagen type I)灌注在兩側流道。通過viscous finger patterning(VFP)的技術將未聚合的膠原蛋白由被動式幫浦(passive pump)擠出而形成中空的管狀結構。在中空結構內側接種人類臍靜脈內皮細胞(Human Umbilical Vein Endothelial Cells, HUVECs)來形成血管。結果顯示出，通過改變環境溫度、被動式幫浦的壓力、第一型膠原蛋白的聚合時間以及濃度來製造出不同尺寸的管徑。透過細胞的接種能再進一步擴張血管的尺寸。最後，藉由加入螢光標記物(分子量為70kDa之FITC-dextran)於具備VFP結構與沒有VFP結構之血管可比較血管的通透性。結果顯示出有VFP結構的血管之通透性大幅降低。比起沒有VFP結構的血管，我們透過VFP技術所製造出的血管更接近人體中的微血管結構。

Blood vessels were ubiquitous in the human body. Nutrients and drugs rely on them to be delivered to all corners of the body. Nonetheless, they are also implicated in many diseases. Therefore, understanding the operating mechanism of blood vessels is important for the study of various diseases. In order to understand the mechanism of blood vessels, various microfluidic systems have been used to create microvasculature-on-a-chip that recapitulates the vascular microenvironment in vitro.. However, the current microvasculature-on-a-chip suffers from complex process in microfluidic chip and hard to control accurate flow in channel. Therefore, we would like to create an optimized blood vessel on the commercial AIM 3D Cell Culture Chips to control flow volume at entrance and exit reservoir and conduct permeability tests. The chips consist of three micro-channels whereby the hydrogel channel in the middle is flanked by 2 media channels on the side.. Using viscous finger patterning (VFP) technique, the unpolymerized collagen in the media channels was extruded by the less viscous phosphate-buffered saline (PBS) through passive pumping to form hollow tubular structures. The results showed that different diameters of tubes are produced by varying the ambient temperature, the pressure of the passive pump, the polymerization time and the concentration of the collagen type I. Subsequently, human umbilical vein endothelial cells (HUVECs) were seeded and cultured inside the hollow structure to form blood vessels. The diameter of the tubular strictures would further expand after the formation of blood vessels. In order to determine the permeability of blood vessels that are grown within the VFP-created tubular structures, the blood vessels were perfused with fluorescent markers (FITC-dextran with a molecular weight of 70 kDa). The results showed that, as compared to the vessel without VFP, the permeability of blood vessels within the VFP-created structure was greatly reduced. Compared to blood vessels without VFP structure, we demonstrated the optimized circular blood vessels with a great barrier in AIM biochip through VFP technology with Extracellular matrix around the vessels. And it made blood vessel be closer to the microvascular structure in the human body.

摘要	i
Abstract	iii
致謝	v
Contents	vi
Figure contents	viii
List of Abbreviations	xiv
Chapter 1.	Introduction	1
1.1	Background	1
1.2	Research aims	5
Chapter 2.	Materials and Methods	6
2.1	Experimental workflow	6
2.2	Cell culture	6
2.3	Microfluidic chip	6
2.4	Hydrogels preparation	8
2.5	Device operation for the formation of lumen with/without VFP structure	9
2.6	HUVECs seeding in the microfluidic channels	9
2.7	Measurement of lumen within the collagen diameter	11
2.8	Fluorescent staining and imaging	11
2.9	Permeability coefficients measurement	12
2.10	Statistical Analysis	12
Chapter 3.	Results and discussion	13
3.1	The effect of temperature for lumen within the collagen formation	13
3.2	The effect of PDF for the lumen within the collagen formation	16
3.3	The effect of PT for the lumen within the collagen formation	18
3.4	The effect of CC for the lumen within the collagen formation	20
3.5	Formation of vessels in VFP lumen within the collagen	23
3.6	Diffusional permeability coefficient	26
Chapter 4.	Conclusion	30
Chapter 5.	Supplementary	31
Chapter 6.	Reference	45
 
Figure contents
Figure 1. Figures and schematic illustration of the AIM chips and the viscous fingering pattern (VFP) processes in the channels. (a) Image of an AIM Biochip, each chip comprises three independent sites for three experiments. Scale bar: 5mm. (b) Phase contrast image of the three channels and the trapezoidal micro-pillars that are designed to contain unpolymerized hydrogel within the hydrogel channel in AIM chips. Scale bar: 200 µm. (c) Schematic illustration of an AIM chip, with two media channels (blue) and one gel channel (red). (d) The cross-sectional view (i-i’) of the media channel depicting the formation of a lumen within the collagen using VFP technique and subsequent seeding of HUVECs in the lumen. (e) The cross-sectional view (ii-ii’) of the lumen within the collagen and vessel.	4
Figure 2. Comparison of the lumen within the collagen formed at 25℃ or 37℃, where pressure driven flow (PDF) at 12 Pa, polymerization time (PT) at 3.5 min and collagen concentration (CC) at 3 mg/ml. (a) Morphology of the lumen within the collagen at different locations of the media channel, i.e. front (1.6-2.8 mm), middle (4.8-6 mm) and back (8-8.8 mm) position. (b) Quantification of the average lumen within the collagen diameter at different locations in media channel polymerized at 25℃ and 37℃. Scale bar: 200 µm.	15
Figure 3. Comparison of the lumen within the collagen formed at different PDF of 18 Pa and 12 Pa, where the temperature set at 25℃, PT at 3.5 min and CC at 3 mg/ml. (a) Morphology of the lumen within  the collagen formed at different locations of the media channel. (b) Quantification of the average lumen within the collagen diameter at different locations in media channel polymerized at PDF of 18 Pa and 12 Pa. Scale bar: 200 µm.	17
Figure 4. Comparison of the lumen within the collagen formed at different PT, i.e. 2.5, 3 and 3.5 min, where the temperature set at 25℃, PDF at 12 Pa and CC at 3 mg/ml. (a) Morphology of the lumen within the collagen formed at different locations of the media channel at different PT. (b) Quantification of the average lumen within the collagen diameter at different locations in media channel polymerized at PT of 2.5, 3 and 3.5 min. Scale bar: 200 µm.	19
Figure 5. Comparison of the lumen within the collagen formed at different CC at 2, 2.5 and 3 mg/ml, where the temperature set at 25℃, PDF at 12 Pa and PT at 2.5 min. (a) Morphology of the lumen within the collagen formed at different locations of the media channel at different CC. (b) Quantification of the average lumen within the collagen diameter at different locations in media channel polymerized at CC of 2, 2.5 and 3 mg/ml. (c) Top- and cross-sectional views of the fluorescent labeled lumen within the collagen. Scale bar: 200 µm	22
Figure 6. Process of HUVECs seeding and the vessel formation in two days in the lumen within the collagen formed at 25℃, PDF at 12 Pa, PT at 2.5 min and CC at 3 mg/ml. (a) Images of HUVECs seeding and formation of vessel in two days at different locations of the device, i.e. front (1.2-2 mm), middle (4.8-5.6 mm) and rear position (8.4-9.2 mm) in media channel. (b) Quantification of the average lumen diameter over time at different location in media channel. Scale bar:100 µm.	24
Figure 7. Immunofluorescence images of the vessels formed with- or without VFP lumen. (a) Comparison of the vessels formed without VFP method (cells grew on engineering plastics) or with VFP method (cell grew on collagen). (b) 3D rendering of the HUVECs vessel formed without or with- collagen. Scale bar:50 µm.	26
Figure 8. Measurement of permeability in vessels formed in the device without- or with VFP lumen. (a) Fluorescent images of the 70 kDa FITC-Dextran incubated in the lumen and observed at time 0, 5, 15 and 75 min without or with VFP lumen. Scale bar: 500 µm. (b) Intensity maps of the FITC-Dextran observed without or with VFP collagen at 0, 5, 15 and 75 min. (c) Permeability coefficient calculated from the fluorescent time lapse images in without- (n=4) and with VFP lumen (n=3).	27

Figure S1. Experimental workflow and parameters involved for the formation of the lumen within the collagen	31
Figure S2. Device operation with the lumen structure and control.	32
Figure S3. The figure showed the collagen both fully covered and didn’t fully cover at outlet.	32
Figure S4. Uniformity of gel boundary in between the gel channel posts after injection of collagen gel into the media channel. (a)collagen gel. (b)fibrin gel. (c)The comparison chart of the collagen and fibrin gel. Scale bar:100 µm	34
Figure S5. Comparison of the lumen within the collagen formed at same PDF, 12 Pa but different volume i.e. (inlet ports: outlet ports(µl)) 90:50, 80:40, and 70:30 (12 Pa), where the temperature set at 25℃, PT at 3.5 min, and CC at 3 mg/ml. (a) Morphology of the lumen within the collagen formed at different locations of the media channel at different volume (b) Quantification of the average lumen within the collagen diameter at different locations in media channel polymerized at PDF of 90:50, 80:40, 70:30. Scale bar:200 µm.	36
Figure S6. Comparison of the lumen within the collagen formed at different stock i.e. 3.59 mg/ml and 3.81 mg/ml, where the temperature set at 25℃, PDF at 12 Pa, PT at 3.5 min, and CC at 3 mg/ml. (a) Morphology of the lumen within the collagen formed at different locations of the media channel. (b) Quantification of the average lumen within the collagen diameter at different locations in media channel polymerized at different stock of 3.59 and 3.81 mg/ml. (c) Quantification of the total average lumen within the collagen diameter polymerized at different stock of 3.59 and 3.81 mg/ml Scale bar:200 µm.	38
Figure S7. Comparison of the lumen within the collagen formed at different stock i.e. 3.59 mg/ml and 3.81 mg/ml, where the temperature set at 25℃, PDF at 12 Pa, PT at 3 min, and CC at 3 mg/ml. (a) Morphology of the lumen within the collagen formed at different locations of the media channel. (b) Quantification of the average lumen within the collagen diameter at different locations in media channel polymerized at different stock of 3.59 and 3.81 mg/ml. (c) Quantification of the total average lumen within the collagen diameter polymerized at different stock of 3.59 and 3.81 mg/ml Scale bar:200 µm.	39
Figure S8. Comparison of the lumen within the collagen formed at different stock i.e. 3.59 mg/ml and 3.81 mg/ml, where the temperature set at 25℃, PDF at 12 Pa, PT at 2.5 min, and CC at 3 mg/ml. (a) Morphology of the lumen within the collagen formed at different locations of the media channel. (b) Quantification of the average lumen within the collagen diameter at different locations in media channel polymerized at different stock of 3.59 and 3.81 mg/ml. (c) Quantification of the total average lumen within the collagen diameter polymerized at different stock of 3.59 and 3.81 mg/ml Scale bar:200 µm.	41
Figure S9. Comparison of the lumen within the collagen formed at CC i.e. 2, 2.5, and 3 mg/ml, where the temperature set at 25℃, PDF at 15 Pa, PT at 3.5 min. (a) Morphology of the lumen within the collagen formed at different locations of the media channel. (b) Quantification of the average lumen within the collagen diameter at different locations in media channel polymerized at different CC at 2, 2.5, and 3 mg/ml. Scale bar:200 µm.	43


                                    

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