VITRIFICATION OF RECOMBINANT NON-BETA CELLS IN CONSTRUCTS

构建体中重组非 β 细胞的玻璃化

基本信息

批准号：
7350918
负责人：
Ying Song
金额：
$ 9.5万
依托单位：
XYTEX RESEARCH, INC.
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-02-01 至 2010-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7350918
关键词：
3-Dimensional Acute Address Adult AlamarBlue Allogenic American Animals Apoptosis Artificial Endocrine Pancreas Autologous Beta Cell Biological Assay Biological Preservation Biopsy Cell Line Cell Survival Cell model Cell physiology Cells Characteristics Collagen Cryopreservation Cryoprotective Agents Development Diabetes Mellitus Drug Formulations Encapsulated Engineering Freezing Gel Gene Expression Genetic Glucose Goals Grant Hepatocyte Human Ice Immune Implant Individual Injection of therapeutic agent Insulin Insulin-Dependent Diabetes Mellitus Invasive Investigation Islets of Langerhans Maintenance Measures Messenger RNA Metabolic Methods Modeling Molecular Pancreas Patients Phase Physiological Population Primary carcinoma of the liver cells Proteins Protocols documentation Radioimmunoassay Recombinants Relative (related person)Research SCID Mice Source Suspension substance Suspensions Testing Therapeutic Tissue Engineering Tissues Transduction Gene Transgenes Transportation base cellular engineering concept continuous cell line cytotoxic cytotoxicity design diabetic functional outcomes gene therapy genetically modified cells improved in vivo insulin secretion mouse model novel strategies preproinsulin research study three dimensional structure transgene expression

项目摘要

DESCRIPTION (provided by applicant): According to current estimates from the American Diabetes Association, there were approximately 18.2 million diabetics in the U.S. in 2002 (6.3% of the population), with 910,000 to 1,800,000 afflicted with Type I insulin-dependent diabetes. In an envisioned alternative therapeutic approach, adult cells are retrieved by a biopsy from the patient and engineered ex vivo to acquire beta cell characteristics. The cells are then re-implanted in the patient, either without or, preferably, after incorporation in a 3-D substitute for localization, retrievability, and improved maintenance of cell function. It would be most desirable if some of the engineered cells were preserved for later use by the same individual, as needed. A significant hurdle for deployment of such constructs is an effective means for long-term storage and transportation to permit worldwide product distribution. Ice formation within tissue matrices causes matrix damage in three- dimensional structures during "traditional" cryopreservation by freezing. Preliminary studies demonstrated that vitrification is a feasible storage method for tissue-engineered pancreatic substitutes. Effective cryopreservation of cells and tissues requires that they retain their genetic and phenotypic characteristics after thawing. For example, a successful cryopreservation protocol would allow genetically modified cells, prior to preservation, to express the same proteins and at the same levels after thawing. In this proposal, testing of vitrification as a storage method for tissue-engineered pancreas is extended to include genetically engineered pancreatic models (human HepG2 cells). The effects of cryoprotective agent cytotoxicity on gene expression, insulin-secretion, and cell viability will be evaluated using HepG2 cell suspensions. In addition, several new methods of vitrification using molecular ice control will be screened using 3-D constructs and the most effective formulations will be tested in a different gene transduction 3-D model of non-(3 cells (human primary recombinant hepatocytes) to test the efficacy of the optimized cryopreservation method. Finally, a small pilot animal study will test the efficacy of the cryopreservation protocols in vivo. The overall goal of this proposal is to characterize the effect of cryopreservation on the recombinant gene expression, insulin secretion and cell viability from recombinant cells that can be incorporated in pancreatic substitutes design.

描述（申请人提供）：根据美国糖尿病协会目前的估计，2002年美国大约有1820万糖尿病患者（占总人口的6.3%），其中91万至180万患有I型胰岛素依赖型糖尿病。在一种设想的替代治疗方法中，通过对患者进行活检来获取成体细胞，并进行离体改造以获得β细胞特征。然后将细胞重新植入患者体内，无需或优选在掺入 3-D 替代物后，以实现定位、可回收性和改善细胞功能的维持。如果需要保留一些工程细胞以供同一个人以后使用，那将是最理想的。部署此类结构的一个重大障碍是长期储存和运输以允许全球产品分销的有效手段。在通过冷冻进行的“传统”低温保存过程中，组织基质内的冰形成导致三维结构中的基质损伤。初步研究表明，玻璃化冷冻是组织工程胰腺替代品的一种可行的储存方法。细胞和组织的有效冷冻保存要求它们在解冻后保留其遗传和表型特征。例如，成功的冷冻保存方案将允许转基因细胞在保存前表达相同的蛋白质，并在解冻后以相同的水平表达。在该提案中，玻璃化冷冻作为组织工程胰腺储存方法的测试扩展到包括基因工程胰腺模型（人 HepG2 细胞）。将使用 HepG2 细胞悬浮液评估冷冻保护剂细胞毒性对基因表达、胰岛素分泌和细胞活力的影响。此外，将使用 3-D 构建体筛选几种使用分子冰控制的玻璃化新方法，并将在非（3 细胞（人原代重组肝细胞）的不同基因转导 3-D 模型中测试最有效的制剂，以测试优化冷冻保存方法的功效最后，一项小型试点动物研究将测试体内冷冻保存方案的功效。该提案的总体目标是表征冷冻保存对重组基因的影响。重组细胞的表达、胰岛素分泌和细胞活力，可纳入胰腺替代品设计中。