3D Spheroid co-culture platform using biocompatible polymers for disease modeling and drug evaluation = 질병 모델링과 약물 평가를 위한 생체적합 고분자 기반 3D 스페로이드 공배양 플랫폼

국문 초록 (Abstract)

3 차원(3D) 세포 배양 시스템은 기존의 이차원(2D) 배양법이 갖는 한계를 보완하고, 실제 생체조직에서 관찰되는 복잡한 질병 기전과 약물 반응을 보다 충실하게 재현할 수 있는 필수 도구로 부상하고 있다. 이러한 3D 플랫폼은 세포–세포 및 세포–기질 간 상호작용, 미세환경 내의 농도 기울기, 그리고 다세포 구조 등 2D 배양으로는 재현하기 어려운 조직 고유의 특성을 재현하는 데 유리하다. 본 논문에서는 생체적합성 고분자를 이용한 3D 스페로이드 공동배양(co-culture) 플랫폼의 개발 및 적용에 관한 세 가지 연속적 연구를 제시한다. 우선, 유방암 세포 스페로이드를 콜라겐 기질에 삽입하여 전이 과정에서의 이동성을 평가하는 “전이성 이동성 분석”을 확립하였다. 이는 세포의 침윤 능력과 약물 민감도를 보다 정밀하게 측정할 수 있어, 기존 독성 중심 평가에 한정된 항암제 스크리닝을 개선하는 데 기여한다. 다음으로는, 염증성 질환 모델을 구축하기 위해 폴리카프로락톤(polycaprolactone) 기반의 나노섬유성 마이크로웰을 이용한 3D 활막 증식(synovial hyperplasia) 모델을 수립하였다. 여기서 환자 유래 활막 섬유아세포(fibroblast-like synoviocytes)와 내피세포(endothelial cells)가 공동으로 스페로이드를 형성함으로써 병리적 혈관신생과 관절 염증을 모사하였으며, 이를 통해 여러 종류의 질병조절항류마티스약(disease-modifying antirheumatic drugs, DMARDs)을 평가하였다. 마지막으로, 활성산소종(reactive oxygen species, ROS)에 감응하는 페로센(ferrocene) 나노입자를 제작하여 류마티스 관절염을 대상으로 설파살라진(sulfasalazine)을 표적 전달하는 시스템을 개발하였다. 이 나노입자는 염증 부위에서 선택적으로 약물을 방출하여 치료 효과를 현저히 높였고, 이는 3D 활막 증식 모델과 콜라겐 유도 관절염(collagen-induced arthritis) 마우스 모델을 통해 확인되었다. 본 연구에서 제시된 일련의 결과들은 고분자 지지체, 질환 특이적 공동배양 전략, 그리고 자극 감응형 약물 전달체를 융합함으로써 기존 in vitro 195 시스템의 한계를 극복하는 방법을 보여준다. 또한 기전 기반의 신약개발과 환자맞춤형 치료 전략 수립에 있어 3D 플랫폼이 갖는 잠재적 가치를 강조하며, 실험실 연구에서 임상적 적용으로 이어지는 전이 연구의 가교 역할을 할 것으로 기대된다

다국어 초록 (Multilingual Abstract)

Three-dimensional (3D) culture systems have emerged as essential tools for modeling complex disease processes and evaluating therapeutic agents under physiologically relevant conditions. Unlike traditional two-dimensional (2D) cell cultures, 3D platforms can capture key features of native tissues, including cell–cell and cell–matrix interactions, microenvironmental gradients, and multicellular architecture. This dissertation explores the development and application of 3D spheroid co-culture platforms using biocompatible polymers, focusing on three sequential studies.
First, a metastatic migration assay was established by embedding breast cancer spheroids in a collagen matrix. This model allowed for more accurate assessments of cell invasiveness and drug sensitivity, advancing anti-cancer drug screening beyond conventional toxicity measurements. Second, the work transitioned to inflammatory disease by constructing a 3D synovial hyperplasia model on polycaprolactone-based nanofibrous microwells. Patient-derived fibroblast-like synoviocytes and endothelial cells formed stable spheroids mimicking pathologic angiogenesis and joint inflammation, enabling the testing of multiple disease-modifying antirheumatic drugs. Third, reactive oxygen species (ROS)-sensitive ferrocene nanoparticles were developed for targeted sulfasalazine delivery in rheumatoid arthritis. Their site-specific release significantly enhanced therapeutic effects, as verified in a 3D hyperplasia model and a collagen-induced arthritis mouse model.
Collectively, these studies illustrate how integrating polymeric scaffolds, disease-relevant co-culture strategies, and responsive drug carriers can overcome limitations of existing in vitro systems. The findings highlight the potential of engineered 3D platforms to facilitate mechanism-based drug discovery, guide personalized therapy, and bridge the translational gap between laboratory research and clinical intervention.

목차 (Table of Contents)

• Chapter 1. General Introduction 19
• 1.1 Drug Development and Screening 20
• 1.1.1 The High Attrition and Cost of Drug Development 20
• 1.1.2 Preclinical–Clinical Gaps and the Need for Human-Relevant Models 22
• 1.2 Three-Dimensional Cell Culture Systems for Preclinical Drug Screening and Disease Modeling 24
• 1.2.1 Regulatory Momentum and the Rise of 3D Cell Models 24
• 1.2.2 Transition from 2D Monolayers to 3D Cultures 26
• 1.2.3 Cell–Cell and Cell–Matrix Interactions in 3D in vitro system 27
• 1.2.4 Overview of the four principal 3D in vitro platforms 28
• 1.2.5 Advantages of Three-Dimensional Cell Culture 30
• 1.2.6 Challenges Associated with 3D Cultures 33
• 1.2.7 Formation and Characteristics of Spheroids 34
• 1.2.8 The superiority of 3D spheroids in drug development and screening
• 35
• 1.3 Overview of 3D Spheroid Culture Platforms 38
• 1.3.1 Hanging Drop Method 38
• 1.3.2 Low-Adhesion Surfaces and Liquid Overlay 39
• 1.3.3 Dynamic Culture and Bioreactors 40
• 1.3.4 Microwell Arrays and High-Throughput Systems 43
• 1.4 Co-Culture Systems in 3D Models 45
• 1.4.1 Importance of Multicellular Interactions 45
• 1.4.2 Strategies for Co-Culture in 3D in vitro system (Mixed vs. Compartmentalized) 46
• 1.5 Scientific Rationale and Unmet Needs in Cancer Modeling 50
• 1.5.1 Conventional Cancer Models and Limitations 49
• 1.5.2 Key Features and Drug‑Screening Shortcomings of Existing Breast‑Cancer 3D Spheroid Models 52
• 1.6 Scientific Rationale and Unmet Needs in Rheumatoid Arthritis Modeling 54
• 1.6.1 Pathophysiology of RA and Key Cellular Players 54
• 1.6.2 Existing Models of RA: In Vitro and In Vivo 58
• 1.6.3 Need for Human 3D Synovial Models 60
• 1.7 Limitations of Current 3D Culture Techniques and Proposed Co-Culture Platform 63
• 1.7.1 Challenges in Current 3D Culture Techniques 63
• 1.7.2 Limitations of Existing Co-Culture Approaches 66
• 1.8 Advances in Three-Dimensional Disease Models and Targeted Therapeutic Delivery 69
• 1.8.1. 3D Spheroid Co-culture Models of the Tumor Microenvironment 69
• 1.8.2 Three-Dimensional Synovial Microenvironment Platforms for Rheumatoid Arthritis 71
• 1.8.3 Redox-Responsive Nanocarriers for Targeted Inflammatory Disease Therapy 73
• 1.9 The overview of Thesis 75
• Chapter 2. Metastatic Migration Analysis-Based Anti-Cancer Drug Screening Using an 3D Spheroid Model in a Collagen Matrix 77
• 2.1 Introduction 78
• 2.2 Materials and Methods 81
• 2.2.1 Cell culture conditions 81
• 2.2.2 Preparation of agarose gel plate and collagen matrix 81
• 2.2.3 Cell viability assay 82
• 2.2.4 Immunofluorescence staining 82
• 2.2.5 Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) 82
• 2.2.6 Anticancer drug treatment and migration assay 83
• 2.2.7 Scanning electron microscopy (SEM) imaging 83
• 2.2.8 Lactate dehydrogenase(LDH) Assay 83
• 2.2.9 Calculation of correlation efficient 84
• 2.2.10 Statistical analysis 84
• 2.3. Results & Discussion 85
• 2.3.1 Interaction Analysis of ADSCs and Breast Cancer Cells
• (MDA-MB-231 and SK-BR-3) in Collagen Matrix 85
• 2.3.2 3D spheroid system with ADSCs and breast cancer cells to
• mimic human breast cancer microenvironments 88
• 2.3.3. Enhanced migration and invasiveness in 3D spheroid system
• using breast cancer with ADSCs in collagen matrix 92
• 2.3.4 Anticancer drug screening using a 3D breast cancer model based
• on cell migration in collagen matrix 95
• Chapter 3. 3D in vitro synovial hyperplasia model on polycaprolactone-micropatterned nanofibrous microwells for screening disease-modifying anti-rheumatic drugs 100
• 3.1 Introduction 101
• 3.2 Materials and Methods 107
• 3.2.1 Rheumatoid patient cell isolation and culture 107
• 3.2.2 Fabrication method of the PCL-micropatterned nanofibrous microwells 107
• 3.2.3 Rheumatoid patient cell seeding on the 3D macro-patterned scaffold 108
• 3.2.4 Cell viability test 109
• 3.2.5 In Vitro Cell Proliferation Assessment in 3D Spheroids 109
• 3.2.6 Immunofluorescence staining 109
• 3.2.7 Real-time quantitative polymerase chain reaction (RT-qPCR) 110
• 3.2.8 Lactate dehydrogenase(LDH) Assay 110
• 3.2.9 ROS Quantitative assay DCFDA/H2DCFDA assay 111
• 3.2.10 Reactive oxygen species (ROS) imaging 111
• 3.2.11 Enzyme-linked immunosorbent assay (ELISA) assay 112
• 3.2.12 Fibroblast-like synoviocytes (FLS) activation 112
• 3.2.13 Dot blot assay using human inflammatory cytokine array 112
• 3.2.14 Scanning electron microscopy (SEM) imaging 113
• 3.2.15 DMARDs treatment and Assessment of drug contribution by patient
• 113
• 3.3 Results and Discussion 115
• 3.3.1. Optimization of 3D in vitro RA model in PCL-micropatterned nanofibrous microwells 115
• 3.3.2 Study on rheumatoid disease modeling and development of DMARDs screening platform to inhibit abnormal proliferation of FLSs 119
• 3.3.3 Comprehensive assessment of immune interactions and ROS level
• in a 3D in vitro RA model 122
• 3.3.4. Evaluating the inflammatory environment and responses of DMARDs in the 3D in vitro RA model 126
• 3.3.5 Assessment of the drug responsiveness of DMARDs using patient-derived FLSs on a 3D in vitro RA model. 130
• Chapter 4. Development of ROS-Sensitive Sulfasalazine-Loaded Ferrocene Nanoparticles and Evaluation of Their Antirheumatic Effects in a 3D Synovial Hyperplasia Model 134
• 4.1. Introduction 135
• 4.2 Materials and Methods 140
• 4.2.1 Synthesis of Ferrocene Polymers (FcPs) 140
• 4.2.2 Characterization of FcPs 140
• 4.2.3 Fabrication and Characterization of the Ferrocene Nanoparticles (FcNPs) 141
• 4.2.4 ROS-Sensitive Properties of the FcNPs 141
• 4.2.5 Characterization of Sulfasalazine-Loaded Ferrocene Nanoparticles (SSZ@FcNPs) 142
• 4.2.6 ROS-Sensitive Drug Release of SSZ@FcNPs 143
• 4.2.7 Fabrication and Characterization of Enriched FcNPs and SSZ@FcNPs
• 4.2.8 Isolation and Culture of Rheumatoid Patient Cells 143
• 4.2.9 Fabrication Method of PCL-Micropatterned Nanofibrous Microwell3 144
• 4.2.10 In Vitro Immune Response Test Using Macrophages 145
• 4.2.11 Immunofluorescence Staining 146
• 4.2.12 Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) 146
• 4.2.13 Western Blot Assay 146
• 4.2.14 Lactate Dehydrogenase (LDH) Assay 147
• 4.2.15 ROS Quantitative Assay (DCFDA/H2DCFDA Assay) 148
• 4.2.16 Fibroblast-like Synoviocytes (FLS) Activation 148
• 4.2.17 Drug Treatment on 3D Spheroid In Vitro Model 148
• 4.2.18 Dot Blot Assay Using Human Inflammatory Cytokine Array 148
• 4.2.19 Animals and Arthritis Induction 149
• 4.2.20 Arthritis Scoring 149
• 4.2.21 Flow Cytometric Analysis 150
• 4.2.22 Enzyme-Linked Immunosorbent Assay (ELISA) 150
• 4.2.23 Histological Examination 151
• 4.2.24 Serum Test 151
• 4.3 Results and Discussion 152
• 4.3.1 Synthesis and Characterization of ROS-Sensitive FcPs for Targeted Drug Delivery 153
• 4.3.2 Optimization and Characterization of FcNPs for Targeted SSZ Delivery. 159
• 4.3.3 Stability Evaluation of Enriched SSZ@FcNPs for Enhanced In Vivo SSZ Delivery 163
• 4.3.4 Effectiveness of SSZ@FcNPs on In Vitro 3D Synovial Hyperplasia Model 163
• 4.3.5 Enhanced Inhibition of NF-κB Signaling Pathway in Rheumatoid Arthritis through SSZ@FcNPs 168
• 4.3.6 SSZ@FcNPs Effects in Collagen-Induced Arthritis (CIA) Mouse Model 174
• 4.3.7 Immune Modulation of SSZ@FcNPs During CIA Development 178