Liver Tissue Engineering Through Three-Dimensional Hepatocyte Culture

通过三维肝细胞培养进行肝组织工程

• 批准号: 8678906
• 负责人: TAMMY T CHANG
• 金额: $ 15.18万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8678906
• 关键词: Advisory Committees; Award; Basic Science; Biocompatible; Biological Assay; Biology; Biomedical Engineering; Biomedical Research; Bioreactors; California; Cause of Death; Cell Communication; Cell Culture Techniques; Cell Shape; Cells; Clinical; Coculture Techniques; Collaborations; Collagen; Complex; Cytoskeletal Gene; Cytoskeleton; Data; Development; Discipline; Disease; Doctor of Medicine; Doctor of Philosophy; Dominant-Negative Mutation; Drug Implants; Elasticity; Engineering; Engraftment; Environment; Extracellular Matrix; Fellowship; Fellowship Program; Fibroblasts; Freedom; Funding; Gastroenterology; Gel; Gene Expression; Goals; Hepatic; Hepatocyte; Hepatology; Hospital Referrals; Implant; Institution; Integrins; Investigation; K-Series Research Career Programs; Knowledge; Laboratories; Lentivirus Vector; Liver; Liver diseases; Mediating; Mentors; Metabolic; Molecular; Myosin ATPase; Normal Cell; Operative Surgical Procedures; Organ; Organ Donor; Organ Transplantation; Organoids; PTK2 gene; Pathologist; Pathology; Pathway interactions; Patients; Phenotype; Plastics; Play; Postdoctoral Fellow; Proteins; Recruitment Activity; Reliance; Replacement Therapy; Research; Research Personnel; Research Training; Residencies; Resources; Ribose; Role; San Francisco; Science; Scientist; Shapes; Signal Transduction; Solid; Staging; Stromal Cells; Structure; Surgeon; Synthetic Genes; System; Techniques; Technology; Testing; Therapeutic; Therapeutic Studies; Time; Tissue Engineering; Tissues; Training Activity; Transplantation; Universities; Up-Regulation; Vascularization; Work; base; blebbistatin; career; career development; clinical application; crosslink; culture plates; gene function; implantation; improved; inhibitor/antagonist; liver transplantation; medical schools; member; minimally invasive; monolayer; novel strategies; paracrine; polyacrylamide gels; prevent; professor; programs; research and development; response; rho; scaffold; shear stress; small molecule; stellate cell; success

DESCRIPTION (provided by applicant): My goal in seeking a K08 Career Development Award is to become an independent investigator and leader in the field of liver tissue engineering. I have demonstrated a sustained commitment to become a clinician- scientist, having completed an M.D.-Ph.D. combined degree at Harvard Medical School, General Surgery Residency at the University of California, San Francisco (UCSF), and two years of post-doctoral research investigating three-dimensional (3D) culture as a novel approach to tissue engineering. I completed a clinical fellowship in Minimally Invasive Surgery and have been recruited to UCSF as Assistant Professor of Surgery with 75% protected research time. As a surgeon-scientist, my long-term career goal is to tissue engineer a functional liver unit for therapeutic implantation, preferably by minimally invasive techniques. This award will help me achieve that goal by supporting development of my independent research program based on my post- doctoral work on 3D hepatocyte culture in solid-body rotational bioreactors. Liver transplantation is currently the only treatment for patients with end-stage liver disease (ESLD), which is the 12th leading cause of death by disease in the U.S. The shortage of donor organs remains a major treatment limitation. Alternatives such as hepatocyte transplantation have shown promise in treating metabolic liver disorders, but low engraftment efficiency and poor long-term efficacy are barriers to broader clinical application. Another strategy is ex vivo tissue engineering of a functional livr unit that can be implanted. However, progress thus far towards building 3D tissue structure with biocompatible scaffolds has been hindered because the slow degradation rates of these materials prevent normal cell-cell and cell-to- extracellular matrix (ECM) interactions. My post-doctoral research demonstrated that hepatocytes cultured in Rotating Wall Vessel (RWV) bioreactors, which provide minimal shear stress with maximal 3D spatial freedom, produced self-aggregated spheroids with optimal metabolic and synthetic gene expression and function as compared to those cultured in 2D monolayers. This proposal builds upon those findings and aims to define the role of matrix and stromal cells in maintaining hepatocyte functions and to determine the underlying mechanisms. The research is driven by the hypotheses that 1) 3D cell shape and matrix rigidity are interconnected factors that together modulate hepatocyte function through cytoskeletal tension mediated by the Rho-ROCK and integrin signal cascades, and 2) 3D co-culture of hepatocytes with stellate cells will generate organoids demonstrating optimal scaffold matrix rigidity, ECM composition, and paracrine crosstalk for maintaining hepatocyte functions. Specifically, this proposal will 1) Determine the effect of matrix rigidity on hepatocyt function and the underlying molecular mechanisms and 2) Characterize the matrix rigidity, ECM composition, and paracrine crosstalk of hepatocyte-stellate cell 3D aggregates and the underlying mechanisms required to maintain hepatocyte specific functions. The results of this study are expected to advance the field of tissue engineering by increasing our understanding of hepatocyte cellular response to matrix stiffness and 3D stromal cell interactions. This research will provide the necessary preliminary data for me to initiate an R01-funded study of liver tissue engineering. Importantly, other training activities will be utilized to increase my knowledge in various disciplines important to my career goal of becoming an independent investigator in liver tissue engineering. The research and training will be carried out at UCSF, a top-ranked tertiary referral hospital and internationally-known institution for biomedical research with an incredibly rich environment for scientific investigation and collaboration. I will pursue didactic coursework in extracellular matrix scaffolds and bioengineering through the UCSF Biomedical Sciences Graduate Program. I will attend seminars offered through UCSF's Hepatology Fellowship Program in the Division of Gastroenterology and study liver pathology with a world-renowned liver pathologist. My primary mentor is Dr. Hobart Harris, who is Chief of the Division of General Surgery and an accomplished surgeon-scientist with an extramurally-funded basic science laboratory. Dr. Harris is a well- suited scientific mentor with extensive research background in primary hepatocyte biology and an invaluable overall career mentor in guiding me through the challenges of becoming a surgeon-scientist. The proposed project is interdisciplinary and my co-mentor Dr. Valerie Weaver will provide expertise in extracellular matrix, cytoskeletal, and Rho-ROCK/integrin signaling manipulation. My other co-mentor, Dr. Holger Willenbring, will provide additional expertise in primary hepatocyte and stellate cell culture and functional assays. In addition, I will be working with Dr. Jacquelyn Maher, who is a member of my advisory committee and director of the UCSF Liver Center. Dr. Maher and the UCSF Liver Center will be important resources to support the isolation and analysis of primary hepatocytes and stellate cells proposed in this research. With this mentoring team and UCSF's rich research environment, success of my proposed research and career development are very high. Support through the K08 Career Development Award will be invaluable in helping me achieve my long-term career goal of becoming an independent surgeon-scientist with a research focus in liver tissue engineering.

个人描述(申请者提供)：我的目标是成为一名肝组织工程领域的独立研究员和领导者。我一直致力于成为一名临床科学家，在哈佛医学院获得医学博士学位，在加州大学旧金山分校(UCSF)担任普通外科住院医师，并从事了两年的博士后研究，研究将三维(3D)培养作为组织工程的一种新方法。我完成了微创外科的临床研究，并被加州大学旧金山分校录用为外科助理教授，有75%的研究时间受到保护。作为一名外科医生兼科学家，我的长期职业目标是通过组织工程设计出一种用于治疗性植入的功能性肝脏单元，最好是通过微创技术。这个奖项将帮助我实现这一目标，支持我的独立研究项目的发展，该项目基于我在固体旋转生物反应器中进行3D肝细胞培养的博士后工作。肝移植目前是终末期肝病(ESLD)患者的唯一治疗方法，在美国，终末期肝病是第12大疾病死亡原因。供体器官短缺仍然是治疗的主要限制。肝细胞移植等替代疗法在治疗代谢性肝病方面显示出很好的前景，但植入效率低，长期疗效差，阻碍了更广泛的临床应用。另一种策略是体外组织工程的功能性LIVR单位，可以植入。然而，到目前为止，利用生物相容支架构建3D组织结构的进展一直受到阻碍，因为这些材料的缓慢降解速度阻碍了正常的细胞-细胞和细胞-细胞外基质(ECM)的相互作用。我的博士后研究表明，与2D单层培养相比，在旋转壁血管(RWV)生物反应器中培养的肝细胞可以产生最小剪应力和最大3D空间自由度的自聚集球体，具有最佳的代谢和合成基因表达和功能。这项建议建立在这些发现的基础上，旨在确定基质细胞和基质细胞在维持肝细胞功能中的作用，并确定潜在的机制。这项研究基于如下假设：1)3D细胞形状和基质硬度是相互关联的因素，通过Rho-Rock和整合素信号级联介导的细胞骨架张力共同调节肝细胞功能；2)3D肝细胞与星状细胞共培养将产生显示最佳支架基质硬度、ECM组成和旁分泌干扰的有机物质，以维持肝细胞功能。具体地说，这项建议将1)确定基质刚性对肝细胞功能的影响和潜在的分子机制，2)表征基质刚性、ECM组成、肝细胞-星状细胞3D聚集体的旁分泌串扰以及维持肝细胞特定功能所需的潜在机制。这项研究的结果有望通过增加我们对肝细胞对基质僵硬的反应和3D基质细胞相互作用的了解来推动组织工程领域的发展。这项研究将为我启动一项由R01资助的肝脏组织工程研究提供必要的初步数据。重要的是，其他培训活动将被用来增加我在各个学科的知识，这些学科对我成为肝脏组织工程独立研究员的职业目标很重要。研究和培训将在加州大学旧金山分校进行，这是一家顶级的三级转诊医院，也是国际知名的生物医学研究机构，拥有令人难以置信的丰富的科学研究和合作环境。我将通过加州大学旧金山分校生物医学科学研究生项目，攻读细胞外基质支架和生物工程方面的教学课程。我将参加加州大学旧金山分校胃肠病学部的肝病奖学金项目举办的研讨会，并与一位世界知名的肝脏病理学家一起学习肝脏病理学。我的主要导师是霍巴特·哈里斯博士，他是普外科主任，也是一位成就卓著的外科医生兼科学家，拥有一个由外部资金资助的基础科学实验室。哈里斯博士是一位非常合适的科学导师，在初级肝细胞生物学方面拥有广泛的研究背景，也是指导我度过成为外科科学家的挑战的宝贵的整体职业导师。提议的项目是跨学科的，我的共同导师Valerie Weaver博士将提供细胞外基质、细胞骨架和Rho-ROCK/整合素信号处理方面的专业知识。我的另一位合作导师霍尔格·威伦布林博士将在原代肝细胞和星状细胞培养和功能分析方面提供额外的专业知识。此外，我将与Jacquelyn Maher博士合作，他是我的咨询委员会成员和加州大学旧金山分校肝脏中心的主任。Maher博士和加州大学旧金山分校肝脏中心将是支持本研究中提出的原代肝细胞和星状细胞分离和分析的重要资源。有了这个指导团队和加州大学旧金山分校丰富的研究环境，我提出的研究和职业发展的成功率都很高。通过K08职业发展奖的支持将是无价的，帮助我实现我的长期职业目标，成为一名专注于肝脏组织工程研究的独立外科医生兼科学家。