Genomics of Vero cells: Understanding this cell line and its virus-host interactions for improved vaccine production

The Vero cell line is the most used continuous cell line for viral vaccine manufacturing with more than 40 years of accumulated experience in the vaccine industry. Additionally, the Vero cell line has shown high affinity for infection by MERS-CoV, SARS-CoV and recently SARS-CoV-2, emerging as an important discovery and screening tool to support the global research and development efforts in this COVID-19 pandemic. Furthermore, Vero cells anchorage-dependent use renders scaling-up challenging and operations very labor intensive which affects cost effectiveness. Thus, efforts to adapt Vero cells to suspension cultures have been invested but hurdles such as the long doubling time and low cell viability remain to be addressed. However, the lack of a reference genome for the Vero cell line has limited our understanding of host-virus interactions underlying such affinity of the Vero cell towards key emerging pathogens, and more importantly our ability to re-design high-yield vaccine production processes using Vero genome editing. In this study, we present an annotated highly contiguous 2.9 Gb assembly of the Vero cell genome. Please click Download on the upper right corner to see the full abstract

전기 습윤 기반 SELEX 플랫폼 구상 및 1단계 성능 특성 분석

학위논문 (석사)-- 서울대학교 대학원 : 공과대학 기계항공공학부, 2018. 8. Junghoon LEE.압타머는 3차원 구조의 짧은 DNA, RNA 또는 펩타이드로 종래의 항체로는 진단이 어려운 분자나 단백질에도 높은 결합력과 특이성을 보인다. 이 화학적 항체는 기능적으로 기존의 항체와 유사할 뿐만 아니라 유연한 구조, 작은 크기, 높은 특이성 및 안정성 등의 장점을 가지고 있어, 질병의 변천에 대응할 수 있고 미지의 진료 기법을 개척하는 첨단 기술로 부상하고 있다. 이러한 압타머는 지수적 농축에 의한 리간드의 계통 진화(Systematic Evolution of Ligands by Exponential Enrichment, SELEX)라는 체외 분류 및 증폭 기법으로 생산되며, 수 회의 복합적인 체외 화학 반응이 수반된다. 또 제조 공정 중 지속적인 인력에 의한 오염의 위험이 있으며, 긴 제조 시간과 시약 낭비 등의 문제점이 있어 진단 및 치료 분야에서 압타머의 광범위한 활용을 저해하는 장벽으로 자리잡고 있다. 따라서 다양한 목표 물질에 적용 가능한 고속∙고효율∙전자동 미세유체기반 SELEX 플랫폼이 요구된다. 본 논문은 새로운 EWOD (Electrowetting-on-dielectric) 디지털 미세유체 플랫폼 기반 자동화 SELEX 시스템을 제시한다. 최신 미세 전자 기계 시스템 (Microelectromechanical systems, MEMS) 제작 기술을 활용하여 SELEX 과정에 요구되는 가열, 혼합 및 분리 등 다양한 통합 기능을 EWOD 플랫폼 상에 구현한다. 본 EWOD 플랫폼은 SELEX 전 과정이 하나의 물방울 내에서 진행될 수 있도록 디자인 되었고, 물방울은 여러 반응 부로 옮겨지면서 기존의 SELEX 과정과 같은 반응 조건을 거치게 된다. 이러한 물방울의 움직임은 물방울과 유전체의 양단에 인가된 전압으로 물방울의 표면과 접촉각을 변화시키는 전기적 인터페이스에 의해 구현된다. 본 연구는 총 3 단계로 구성되며, 그중 두 단계가 본 석사 과정 내에 진행되었다. 1 단계에서 SELEX를 위한 EWOD 디지털 미세 유체 플랫폼이 제작되었고, 2 단계에서 SELEX 과정의 정밀한 열 제어를 위한 고성능 RTD (Resistance thermal detector) 히터/센서 시스템의 설계 및 성능 분석이 진행되었다. 3 단계에서는 개발된 플랫폼의 생물학적 실험 및 SELEX 전 단계 검증이 실행될 것이다. 본 시스템의 구상과 1차 성능 특성 분석 단계에서 저항가열과 RTD 원리를 기반으로 한 고성능의 가열/센싱 시스템을 개발하였다. Indium tin oxide (ITO)를 다양한 물질로 도핑하여 전기적인 특성을 변환하였다. 문헌 상 기록된 값에 비해 수 천에서 수 만 배 높은 저항온도계수를 포함하는 등 독특한 성질을 가진 센서를 개발하였다.Aptamers are short DNA, RNA, or peptide 3D-shaped structures with the ability to target molecules and proteins for which antibodies are not well suited, with high specificity and affinity. Not only are these chemical antibodies functionally similar to traditional antibodies, but they also present various advantages such as, their flexible structure, small size, high specificity and stability, which make aptamers a cutting edge way to reach previously unexplored areas of therapeutics and to create treatments that can keep up with the 21st century diseases. These aptamers are developed through an in vitro selection and amplification process named Systematic Evolution of Ligands by Exponential Enrichment (SELEX). This in vitro combination of chemical reactions that synthetically isolates aptamers is performed over several rounds. Nevertheless, some issues such as time consumption, loss of reagents, risks of cross contamination due to constant human intervention during the SELEX process are impeding the widespread application of aptamers in diagnostics and therapy. Thus, there is a need for a microfluidic SELEX platform more rapid, highly efficient, completely automatic and applicable to a wide range of targets. Inspired by the new advances in MEMS fabrication methods, this thesis presents a novel Electro-Wetting-On-Dielectric (EWOD) digital microfluidic platform with various integrated features (notably heaters, mixers and isolation areas) designed to overcome the obstacles faced in order to create the first EWOD-based SELEX platform. This EWOD platform is designed to carry the whole SELEX process inside a droplet, which is moved from one reaction site to the other while being submitted to the same reaction conditions then the conventional SELEX process. This droplet movement automation is permitted by an electric interface, which alters the droplet wettability –thus altering its contact angle- on a dielectric surface by varying the electrical potential applied. This research project is composed of three major axes, two of which are within the scope of this Master thesis requirement : (1) the design and fabrication of the first EWOD digital microfluidic platform for SELEX implementation, (2) the Phase 1 characterization consisting of the conception and fabrication of a highly performant RTD heater/sensor system for a precise thermal control of the SELEX process. The third axe, Phase 2 characterization, involving the run of biological tests and the whole SELEX process on our developed platform was established as a future final objective. Through the conception and Phase 1 characterization, a highly performant heating/sensing system, based on resistive heating and RTD principles, was developed by altering the electrical properties of ITO with a novel multiple doping method in order to obtain peculiar properties such as a TCR 3 to 4 orders of magnitude higher than documented RTDs.Chapter 1 Introduction 1 1.1 Motivation 1 1.1.1 Challenges 1 1.1.2 Opportunities 3 1.2 Hypothesis 5 1.3 Objectives 6 1.3.1 EWOD Digital Microfluidic platform development 6 1.3.2 Highly performant Resistance Thermal Detector system 7 1.3.3 Adaptation of SELEX process on EWOD platform 7 1.4 Thesis Organization 7 1.5 References 9 Chapter 2 Background and State of the Art 11 2.1 Aptamer 11 2.1.1 Potential 11 2.1.2 Recent progress in Therapeutics 15 2.2 SELEX 15 2.2.1 History of SELEX 15 2.2.2 Conventional SELEX process 16 2.2.3 SELEX processes review 18 2.3 Microfluidic SELEX Implementations 20 2.4 References 24 Chapter 3 EWOD Technology 27 3.1 Theory 27 3.1.1 Static Electrowetting: Lipmann-Young law 30 3.1.2 Dynamic EWOD model 33 3.2 Design considerations for basic EWOD manipulations 37 3.2.1 Current EWOD configurations 37 3.2.2 Droplet dispensing 38 3.2.3 Droplet transportation 42 3.2.4 Droplet merging and splitting 44 3.2.5 Droplet mixing 45 3.3 References 47 Chapter 4 Design and Fabrication of an EWOD-based SELEX platform 53 4.1 Overall system description 53 4.2 Originality 59 4.3 Specific design considerations and fabrication steps 61 4.3.1 Basic EWOD parts 61 4.3.1.1 Overall design 61 4.3.1.2 Mixing electrodes design 62 4.3.1.3 Heating/thermal sensing system design 63 4.3.1.4 Fabrication 65 4.3.2 Separation parts 68 4.3.2.1 Cassie-Wenzel wetting/de-wetting transition 68 4.3.2.2 Design 71 4.3.2.3 Fabrication 73 4.4 References 76 Chapter 5 Platform automation and Phase 1 characterization 81 5.1 Automation with Dropbot system 81 5.2 Heater and sensor calibration 83 5.2.1 Calibration 83 5.2.2 Results 84 5.3 Effects of Diffusion on RTD sensitivity 89 5.3.1 Semiconductor doping for enhanced sensitivity 89 5.3.2 Diffusion principle 91 5.3.3 Gold and chromium diffusion mechanisms in ITO 93 5.4 References 96 Chapter 6 Conclusion and future work 98 6.1 Conclusion 98 6.1.1 Novel bead-less separation method 98 6.1.2 Phase 1 characterization : a highly sensitive RTD sensor. 98 6.1.3 Complete automation 100 6.2 Future work : Phase 2 characterization 100 6.2.1 Biocompatibility test 100 6.2.2 DNA/RNA/target Mixing rate calibration 100 6.2.3 Separation test 101 6.2.4 Overall SELEX process run-test 101 Abstract in Korean 102Maste