Spheroid-based three-dimensional liver-on-a-chip to investigate hepatocyte-hepatic stellate cell interactions
- 주제(키워드) liver chip , hepatocytes , hepatic stellate cells
- 발행기관 고려대학교 대학원
- 지도교수 이상훈
- 발행년도 2014
- 학위수여년월 2014. 2
- 학위구분 석사
- 학과 일반대학원 바이오융합공학과
- 세부전공 생체의공학 전공
- 원문페이지 57 p
- 실제URI http://www.dcollection.net/handler/korea/000000048986
- 본문언어 영어
- 제출원본 000045786856
초록/요약
We have developed a three-dimensional (3D) liver-on-a-chip to investigate the interaction of hepatocytes and hepatic stellate cells (HSCs) in which primary 3D hepatocyte spheroids and HSCs are co-cultured without direct cell-cell contact. Here, we show that the 3D liver chip offers substantial advantages for the formation and harvesting of spheroids. The most important feature of this liver chip is that it enables continuous flow of medium to the cells through osmotic pumping, and thus requires only minimal handling and no external power source. We also demonstrate that flow assists the formation and long-term maintenance of spheroids. Additionally, we quantitatively and qualitatively investigated the paracrine effects of HSCs, demonstrating that HSCs assist in the maintenance of hepatocyte spheroids and play an important role in the formation of tight cell-cell contacts, thereby improving liver-specific function. Spheroids derived from co-cultures exhibited improved albumin and urea secretion rates compared to mono-cultured spheroids after 9 days. Immunostaining for cytochrome P450 revealed that the enzymatic activity of spheroids co-cultured for 8 days was greater than that of mono-cultured spheroids. These results indicate that this system has the potential for further development as a unique model for studying cellular interactions or as a tool that can be incorporated into other models aimed at creating hepatic structure and prolonging hepatocyte function in culture.
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ABSTRACT
Ⅰ. INTRODUCTION 3
Ⅱ. MATERIALS AND METHODS 8
2. 1. Design and operation of the spheroid-based 3D artificial liver chip 8
2. 2. Fabrication process 13
2. 3. Isolation and culture of primary hepatocytes and HSCs 18
2. 4. Seeding hepatocytes and HSCs in flat and concave chambers 19
2. 5. Cell viability test. 21
2. 6. Scanning electron microscopy 22
2. 7. Immunofluorescence staining 23
2. 8. Functional assessment 24
Ⅲ. RESULTS 25
Ⅳ. DISCUSSION 39
Ⅴ. CONCLUSIONS 44
Ⅵ. REFERENCES 45
