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Fabrication of multi-well platform for efficient myogenic commitment of C2C12 cells via electrical stimulation : 근육 조직 전구 세포의 분화 촉진을 위한 전기 자극 멀티웰 플랫폼의 제작

DC Field Value Language
dc.contributor.advisor황석연-
dc.contributor.author송주현-
dc.date.accessioned2017-07-19T05:54:51Z-
dc.date.available2017-07-19T05:54:51Z-
dc.date.issued2015-02-
dc.identifier.other000000026291-
dc.identifier.urihttps://hdl.handle.net/10371/129374-
dc.description학위논문 (석사)-- 서울대학교 대학원 : 화학생물공학부, 2015. 2. 황석연.-
dc.description.abstractTo engineer tissue-like structures, cells are required to organize themselves into three-dimensional networks that mimic the native tissue microarchitecture. Here, we present agarose-based multi-well platform incorporated with electrical stimulation (ES) to build skeletal muscle-like structures in a facile and highly reproducible fashion. Our observations showed that ES improved myogenic differentiation of C2C12 cells in two-dimensional monolayer culture and increased the diameter of the resulting myotubes. Furthermore, C2C12 skeletal muscle cells were encapsulated in collagen/matrigel scaffolds in 3D manner, and electrical stimulation was applied for efficient myogenic commitment. ES also facilitated the formation of free-standing 3D muscle tissues with or without mechanical stretch at both ends of the gel structure. Consequently, we confirmed the transcriptional upregulations of myogenic related genes in the ES group compared to non-stimulated control group in our multi-well 3D culture platform. Interestingly, the cell-encapsulated gel consisted solely of matrigel did not give admirable myotube formation-
dc.description.abstracthowever, collagen showed upregulated myosin heavy chain, muscle regulatory factor expression and prominent effect in building myotubular structures. Given the robust fabrication, engineered muscle tissues in multi-well platform may find their use in high-throughput biological studies drug screenings.-
dc.description.tableofcontentsCONTENTS

ABSTRACT..........................................................................1

CONTENTS..........................................................................3

LIST OF FIGURES AND TABLES..............................................5

PART 1. INTRODUCTION

1.1 Overview ...................................................................6

1.2 Electrical stimulation control of myogenic differentiation ..........7

PART 2. TWO-DIMENSIONAL CULTURE AND EVALUATION OF

ELECTRICAL STIMULATION

2.1 Introduction ................................................................. 13

2.2 Materials and methods

2.2.1 Cell culture .......................................................... 14

2.2.2 Electrical stimulation .............................................. 14

2.2.3 Viability analysis ....................................................15

2.2.4 Proliferation quantification ........................................15

2.2.5 Quantitative gene analysis ...........................................16

2.2.6 Immunofluorescence staining .....................................16

2.2.7 Statistical analysis ..................................................17

3

2.3 Results .........................................................................19

2.4 Discussion .....................................................................27

PART 3. THREE-DIMENSIONAL CULTURE: DESIGN OF 3D

AGAROSE-BASED BULTI-WELL PLATFORM AND ITS APPLICATION

3.1 Introduction ..................................................................28

3.2 Materials and Methods

3.2.1 Design of multi-well mold and making agarose-based multi-
well ......................................................................................... 29

3.2.2 3D culture of C2C12 cells .........................................30

3.2.3 Viability analysis ...................................................31

3.2.4 Contraction measurement on agarose-based multi-well ......31

3.2.5 Quantitative analysis ...............................................32

3.2.6 Immunohistochemistry analysis of fabricated muscle tissue .33

3.2.7 Statistical analysis ..................................................33

3.3 Results .........................................................................34

3.4 Discussion ....................................................................40

PART 4. CONCLUSION ...........................................................43

References............................................................................49

국문초록 ........................................................................... 58

4

List of Figures and tables

Figure 1. Schematic illustration of Electrical stimulation study..............18

Figure 2. C2C12 cell proliferation and viability under electrical stimulation.......21

Figure 3. Application of ES on 2D monolayer C2C12 and EdU staining...22

Figure 4. The evaluation of GM or DM cultured C2C12 cell differentiation

through myosin immunostaining ................................................. 24

Figure 5. Real-time PCR analysis after ES for 5 days............................... 26

Figure 6. 3D cultured C2C12 cells underwent ES from day 2 to 7

continuously overnight under 3 different kinds of scaffold.................... 35

Figure 7. Transcriptional regulation analysis comparing 2D culture and three-
dimensional gel-encapsulation culture............................................36

Figure 8. Microscopic image and real-time PCR analysis of C2C12 3D

structure and pinned structure...................................................... 38

Figure 9. Immunohistochemistry of collagen-encapsulated 3D constructs..39

Table 1. Primer list ..................................................................46

Supplementary figure 1 ...........................................................47

Supplementary figure 2, 3 ....................................................... 48		-
dc.formatapplication/pdf-
dc.format.extent1621886 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectC2C12-
dc.subjectelectrical stimulation-
dc.subjectagarose-based multi-well platform-
dc.subjectcollagen-
dc.subjectmatrigel-
dc.subjectencapsulation-
dc.subject.ddc660-
dc.titleFabrication of multi-well platform for efficient myogenic commitment of C2C12 cells via electrical stimulation-
dc.title.alternative근육 조직 전구 세포의 분화 촉진을 위한 전기 자극 멀티웰 플랫폼의 제작-
dc.typeThesis-
dc.contributor.AlternativeAuthorJoo-hyun Song-
dc.description.degreeMaster-
dc.citation.pages58-
dc.contributor.affiliation공과대학 화학생물공학부-
dc.date.awarded2015-02-
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