臺灣博碩士論文加值系統

本研究使用光薄片顯微術觀測在微流道裝置中的球體細胞 (cell spheroids)。光薄片顯微術具有光學切片的能力、和共軛焦顯微術比較起來有較低的光毒性以及光漂白性，這些特性對於研究細胞球體是有極有幫助的。
球體細胞為一個三維的細胞聚集體(3-D aggregates)，這種球體細胞比較貼近真實的組織，這種培養方法使細胞提供了較多的生物資訊，可以改進在體外生物醫學研究以及藥物測試。相比之下，傳統的細胞培養方法是二維的，只有單一層面，並不能夠提供於組織所產生的幾何、生化及機械力的影響因素。本研究的實驗使用一個簡單的微流道裝置做為細胞的共同培養(co-culture)的腔體，使用的細胞為肝癌細胞(HepG2)和人類臍靜脈內皮細胞(HUVEC)。我們用光薄片顯微術觀察細胞球體的生長，我們能夠使用光薄片顯微術觀測細胞至少四天，而且在培養細胞的過程中，細胞的培養液是持續置換的。使用光薄片顯微術，我們可以定量地測量細胞的體積，我們發現了細胞的體積一天天地增長。此外，我們觀測到在HepG2裡面的HUVEC有順序地排列成一個圓形的結構，這或許為HUVEC形成管狀結構的徵兆，我們稱之為pre tube formation。
結合微流道和光薄片顯微術我們可以長時間的研究細胞球體的細胞與細胞間的交互作用，這項研究或許可以成為研究腫瘤血管新生及藥物測試的一種新的模型，為新模型的開發鋪一條嶄新的路。

This study demonstrates the possibility of using selective plane illumination microscopy (SPIM) to observe live tumor spheroid inside a microfluidic device . SPIM provides optical sectioning capability, low photo toxicity, and low photo-bleaching effect which are useful for studying large specimen like 3-D cell spheroids.
Culture of cells as three-dimensional (3-D) aggregates, such as cancer spheroids, can improve in vitro tests for basic biomedical research and drug testing. In comparison, conventional two dimensional monolayer cultures do not provide the geometrical, biochemical and mechanical factors created in real tissues. We used a simple microfluidic device for the formation and co-culture spheroids of liver carcinoma cells ( HepG2) and human umbilical vein endothelial cells (HUVEC) using gravity driven cell aggregation, and used selective plane illumination microscopy (SPIM) to study the growth of a single spheroid inside the microfluidic device. We could culture the 3-D spheroids inside the device for more than 4 days while exchanging the culture medium. We could measure cell spheroid volume quantitatively and we found out spheroid grow up day by day. We observed that HUVEC inside HepG2 tumor spheroid form approximate circular structure similar to pre tube formation.
Combining microfluidics and SPIM we could study the cell-cell interactions in 3-D spheroids for a long period of time. This study in co-culture spheroids may pave a way to develop new models for in vitro drug testing and tumor angiogenesis.

Contents
Signature Page
Thesis Approval Form
Acknowledgments.............................. i
Chinese Abstract ............................ iii
English Abstract ............................ iv
Table of Contents ........................... v
List of Figures ............................. vii

Chapter 1 Introduction ...................... 1

1.1 Motivation and research background ...... 1
1.2 Selective plane illumination microscopy (SPIM) ...................................... 2
1.3 Compare SPIM with confocal microscopy ... 4
1.4 Literature review ....................... 7
1.5 Light sheet information and optical resolution .................................. 9

Chapter 2 Experimental setup and specimen preparation ................................. 12

2.1 SPIM system ............................. 12
2.1.1 Setup configuration ................... 12
2.1.2 Setup laser source .................... 17
2.1.3 System control ........................ 20
2.1.2 System resolution ..................... 23
2.2 Microfluidic device ..................... 26
2.2.1 Definition of microfluidic device...... 26
2.2.2 Poly-dimethlsioxane ................... 26
2.2.3Fabrication ............................ 28
2.3 Cell culture ............................ 29
2.3.1 Cell culture .......................... 29
2.3.2 Spheroid formation and culture ........ 30
2.4 Image process............................ 31

Chapter 3 Result ............................ 32

3.1 Cell spheroid growth up ................. 32
3.2 HUVEC arrange a pre tube formation, a circular structure ................................... 34

Chapter 4 Conclusion and discussion ......... 39

Future work ................................. 40

Reference ................................... 41



List of Figures
Fig. 1.1 (A) Typical microscopy (B) Selective plane illumination microscopy ......................3
Fig. 1.2. Schematic of selective plane illumination microscopy....................................3
Fig. 1.3 Principle of confocal microscopy.....5
Fig. 1.4 Comparison of light sheet microscopy with confocal microscopy ...................................6
Fig. 1.5 Schematic of multidirectional selective plane illumination microscopy ......................8
Fig. 1.6 Schematic of inverted selective plane illumination microscopy....................................8
Fig. 1.7 The schematic of a Gaussian beam focused by a cylindrical lens .............................9
Fig. 1.8 PSF schematics and theoretical functions.....................................10
Fig. 1.9 Defining resolutions as the ability to distinguish two Airy disks .............................. 11
Fig. 1.10 The numerical aperture of a microscope objective.................................... 11
Fig. 2.1 The selective plane illumination microscope setup used in this work.............................13
Fig. 2.2 Transmission spectra of 470 nm and 545 nm excitation filter ............................14
Fig. 2.3 The detail information of Leica 20x water immersion objective lens ...............................15
Fig. 2.4 Transmission spectra of 535 nm and 600 nm emission filter........................................16
Fig. 2.5 The overview of the super continuum laser system ..............................................17
Fig. 2.6 The rear panel controls .............18
Fig. 2.7 The principle of operation of white light laser.........................................18
Fig. 2.8 The parameter of HyperTerminal to control laser.........................................20
Fig. 2.9 The correlation of q value and laser power in blue and green light...............................20
Fig. 2.10 Flow chart of SPIM system LABVIEW program ..............................................22
Fig. 2.11 Three images using maximum intensity projection and curve fitting function....................24
Fig. 2.12 The statistics of point spread functions FWHM of agarose-bead cubes on x, y and z axis..........................................25
Fig. 2.13 (A)The microfluidic device we used contain two layer, one is medium
channel and another is cell culture chamber. (B) Device after assembly ...............................29
Fig. 2.14 The de-convolution parameter we used on Huygens Essential software............................30
Fig. 3.1 The time laps experiment of HepG2 cells inside the microfluidic device,
the image is captured by a normal microscopy ..............................................32
Fig. 3.2 The HepG2 spheroid images take by SPIM..........................................33
Fig. 3.3 Schematic of high threshold value in Imaris software......................................34
Fig. 3.4 Schematic of low threshold value in Imaris software......................................34
Fig. 3.5 The merged images of HepG2 and HUVEC co-culture in 3-D at (A) day2 (B) day3 and (C) day4 (D) The statistic of spheroid volume using Imaris software.........35
Fig. 3.6 The co-culture of HepG2 and HUVEC in 3-D and surface plot .................................36
Fig. 3.7 The white dash line shows the pre tube formation of HUVEC ........................................37
Fig. 3.8 We observe the pre tube formation again, some of HUVEC attach to each other very close.........37
Fig. 3.9 We observe the pre tube formation again at day2 ..............................................38
Fig. 3.10 We observe the pre tube formation again on different plane at day3 ......................38
Fig. 3.11 (A) The pre tube formation we observed again on day2 and (B) day3 ............................39

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