学位の種別: 課程博士審査委員会委員 : (主査)東京大学教授 石原 一彦, 東京大学教授 吉田 亮, 東京大学准教授 坂田 利弥, 東京大学特任准教授 金野 智浩, 東京大学客員教授(東京医科歯科大学) 塙 隆夫University of Tokyo(東京大学

Gao Botao

高 博韜

English

UTokyo Repository

Cytocompatible and multilayered phospholipid
polymer hydrogel for regulation of cellular
behavior
その他のタイトル 細胞挙動を調節するための細胞適合性リン脂質ポリ
マーハイドロゲル多層膜
学位授与年月日 2015-03-24
URL http://doi.org/10.15083/00008242
 博士論文（要約） 
 
 
 
 
Cytocompatible and multilayered phospholipid 
polymer hydrogel for regulation of cellular 
behavior 
 
（細胞挙動を調節するための細胞適合性リン脂質ポリマー 
ハイドロゲル多層膜） 
 
 
 
 
Gao Botao 
(高 博韜) 
 
 
 
 
 
 
 
 
 
 
 
 The primary objective of this dissertation is to create a defined Three-dimensional (3D) 
cell culture model that provided a multilayered hydrogel platform for the spatial patterning of 
cells, temporal control over soluble molecule diffusion within 3D model, and the ability to 
monitor cellular behavior throughout the culture period. Figure 1 shows overview of this thesis.  
 
Chapter 1 Motivation and design of the thesis 
3D in vitro models span the gap between 2D cell cultures and whole-animal systems. 
These models can replicate some features of the in vivo architecture and allow control of the 
degree of homotypic and heterotypic cell–cell interactions, enabling more accurate quantitative 
studies. However, the application of these microfabrication technologies to elucidate the 
underlying mechanism is still a challenge. 
As shown in Figure 2, the main reason for the slow progress in this area is the complexity 
of the in vivo microenvironment. For example, for more detailed information on indirect 
cell–cell interactions, they should be separated from other parameters and studied following the 
“cue–signal–response” rules in a simplified environment. Another probable reason is that most 
microfabrication technologies are complex and require significant preparation and 
instrumentation not commonly available in biology laboratories.  
To solve these issues, the experimental design should incorporate following aspects:  
a) Suitable biomaterials for 3D cell culture. 
b) Easy availability, low cost and simple manipulation. 
c) Ability of patterning of multiple types of cells within 3D scaffolds with spatially 
localized gradients of soluble molecules. 
d) Control the diffusion of these soluble biomolecules to provide temporal administration 
of cellular stimuli.  
e) Systems to monitor cellular behavior within these environments throughout the 
experiment without sacrificing cells. 
 
  
 
Figure 1 Overview of this thesis. 
 
 According to these aspects of experimental design, the following contributions have been 
done for this project: 
 
Chapter 2 Fabricate of PMBV/PVA multilayer hydrogel 
The contribution of this chapter is developing an easily applicable and generic approach to 
construct PMBV/PVA multilayer hydrogels by using spinning-assisted layer-by-layer (LbL) 
procedures. The LbL assembly is based on the chemical reaction between PMBV and PVA in 
aqueous medium. The thickness of the multilayered hydrogel is regulated by number of gel 
layers. This approach is compatible with existing laboratory techniques and instrumentation. All 
of multilayered hydrogels were built on commonly available 35 mm cell culture dish. A 
commercial centrifuge was transformed into spin-coater. 
 
Chapter 3 Fabrication of precise spatial cellular multilayered phospholipid polymer 
hydrogel 
The contribution of this chapter is development of the multilayered hydrogels that allowed 
the design of cell patterning gels by building 2 cell-laden layers separated by PMBV/PVA 
multilayer hydrogel. These multilayered hydrogels that mimicked stratified structure of human 
 
Figure 2 Motivation of this research to develop an easy available reductionist 3D in vitro 
model for better understanding in vivo environment. 
tissue were stable in cell culture medium and suitable for cell incorporation due to tens of 
micrometer scale thickness. The cells remained alive during the spinning process and 
maintained their metabolic activity for at least 24 h. 
 
Chapter 4 Quantitating distance-dependent, indirect cell-cell interactions with 
PMBV/PVA multilayer hydrogel 
The contribution of this chapter is making use of finely tuned structure and property of 
PMBV/PVA hydrogels to build up a customized reductionist 3D in vitro model for exploring 
the relationship between diffusion distance and indirect cell–cell interactions (soluble factor 
interactions), things that have seldom been investigated experimentally. Due to the presence of 
MPC units in hydrogels, a key feature of this hydrogel system is that it permits the diffusion of 
soluble factors and decouples soluble signals from other signals (cell-cell contact and cell-ECM 
interaction). In a case study, I have used this 3D model for patterning tumor cells and stromal 
cells and quantitatively modulating soluble cell–cell signaling. Cell behavior was evaluated by 
combination of FUCCI fluorescent reporter technology and microscopy. Then I observed clear 
differences in the dynamics of cell cycle progression in HeLa-Fucci cells depending on the 
distance separating them from co-cultured cells.  
 
Chapter 5 Regulation of cells encapsulated in a multilayered hydrogel containing a 
bioactive reagent 
The contribution this chapter is control of cell behavior based on diffusion of bioactive 
reagent that was layer-specific loaded within multilayered hydrogels. This 3D model was 
sandwich constructed, where cell-containing gel layer was separated from PTX reservoir layer 
by a spacing of dual-crosslinked PMBV/AWP hydrogel for controlled PTX diffusion. It was 
also shown that the permeability of dual-crosslinked hydrogel was so low that it worked as a 
diffusion-controlling barrier and barrier effects could be finely controlled by varying thickness 
of diffusion-controlling barrier and PTX concentration. I found that PTX induced weaker 
apoptotic effects of HeLa cells as barrier thickness increased and PTX concentration decreased.  
Finally, these achievements will aid in the elucidation of how cells behave within spatially 
defined microenvironments similar to physiological tissue. It is clear that a great deal of work 
remains to fully understand the underlying mechanism. However, I believe that this work 
provides initial insights that may prove useful in guiding the design of future 3D cell culture 
systems. 


細胞挙動を調節するための細胞適合性リン脂質ポリマーハイドロゲル多層膜

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細胞挙動を調節するための細胞適合性リン脂質ポリマーハイドロゲル多層膜

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