Controlled-release Microbeads to Replace Growth Factors in Fetal Bovine Serum

控释微珠替代胎牛血清中的生长因子

• 批准号: 10254493
• 负责人: Jeffrey H Stern
• 金额: $ 22.55万
• 依托单位: STEMCULTURES, LLC
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-20 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10254493
• 关键词: Address; Biological Assay; Bioreactors; Calibration; Cell Culture Techniques; Cell Proliferation; Cells; Characteristics; Clinical Trials; Collaborations; Culture Media; Custom; Development; Enzyme-Linked Immunosorbent Assay; FDA approved; FGF2 gene; Fetal Development; Formulation; Future; Glycolates; Goals; Growth; Growth Factor; Hour; Institutes; Insulin-Like Growth Factor I; Knowledge; Life; MCF7 cell; MDA MB 231; Manuals; Measures; Mesenchymal Stem Cells; Microfluidic Microchips; Microfluidics; Microspheres; Outcome; Phase; Play; Pluripotent Stem Cells; Production; Protocols documentation; Pump; Reproducibility; Series; Serum; Small Business Technology Transfer Research; Stabilizing Agents; Structure of retinal pigment epithelium; Technology; Time; United States National Institutes of Health; base; cell behavior; cell growth; cell type; controlled release; cost effective; feeding; fetal bovine serum; flexibility; human error; improved; manufacturing process; nanoscience; nerve stem cell; new growth; novel; novel strategies; platelet-derived growth factor BB; pressure; scale up; time use; tool

Project Summary / Abstract: Fetal bovine serum (FBS) contains a variety growth factors (GFs) and GF-stabilizing agents that have a potent influence on cell behavior. Lot-to-lot variability of FBS, however, results in fluctuation of initial GF levels. These fluctuations are further complicated by time-dependent decay at rates unique to each GF, resulting in changing GF ratios over time. These three FBS characteristics (variable initial GF levels, time-dependent decay, and varying GF ratios over time) make it difficult to know actual GF levels maintained during the culture period. Knowledge and control of GF levels, however, is needed to systematically formulate media to effectively replace the GFs provided by FBS We propose to manufacture controlled-release microspheres that maintain stable, defined GF levels to facilitate the development of an FBS-free medium. Replacing FBS GFs is a meaningful step toward addressing the FOA PA-18-816 goals of: ‘Better Defining Growth Medium to Improve Reproducibility of Cell Culture’ and ‘supporting technologies and products to expedite development of better serum substitutes’. A challenge to originating serum-free protocols is the rapid degradation of soluble GFs that are typically added to media at the time of use. StemCultures, LLC manufactures controlled-release poly lactic-co-glycolic acid (PLGA) microbeads that slowly release GFs to produce defined and stable GF levels in culture media. This flexible PLGA microbead platform will be used to generate a series of ‘CultureBeads’ that release key FBS GFs to maintain a stable media level over several days. Four FBS GFs will provide proof-of-concept for the controlled- release strategy. The initial four FBS GFs were selected based on: 1) presence in FBS, 2) lability and 3) bioactivity. Each CultureBead replaces one FBS GF, allowing CultureBeads to be combined to provide a stable GF mixture. The second aim advances automated microfluidic manufacturing to facilitate cost-effective current Good Manufacturing Process (cGMP) compliant CultureBeads production. The initial CultureBeads tool kit can be utilized for specific cell types dependent on the selected GFs, including retinal pigment epithelium (RPE) cells, certain mesenchymal stem cells and pluripotent stem cells where there is broad demand. CultureBeads manufacture of RPE has immediate application for an FDA-approved, NIH-sponsored clinical trial. In a future Phase 2 STTR application, the CultureBeads series will be expanded using multiplex analysis to identify additional factors to more completely replace FBS.

项目摘要/摘要： 胎牛血清(FBS)含有多种生长因子(GFS)和GF稳定剂，具有很强的 对细胞行为的影响。然而，FBS的批次到批次的变异性导致初始GF水平的波动。这些 波动因依赖于时间的衰减率而进一步复杂化，衰减率对于每个GF而言都是唯一的，导致变化 随时间变化的GF比率。这三个FBS特征(可变的初始GF水平、随时间变化的衰减和 随着时间的推移，不同的GF比例)使得很难知道在培养期间保持的实际GF水平。 然而，需要对GF级别的知识和控制来系统地制定媒体以有效地替换 FBS提供的GFS我们建议制造保持稳定的控释微球， 定义了GF级别，以促进无FBS介质的开发。更换FBS GFS是有意义的一步 朝向FOA PA-18-816的目标：更好地定义生长介质以提高再生性 《细胞培养》和《支持技术和产品》，加速开发更好的血清 代替者的。 最初的无血清方案面临的一个挑战是可溶性GFS的快速降解，这些GFS通常 在使用时添加到介质中。StemCultures，LLC生产控释聚乳酸-羟基乙酸 酸性(PLGA)微球，缓慢释放GFS，在培养基中产生定义和稳定的GF水平。这 灵活的PLGA微珠平台将用于生成一系列发布密钥FBS GFS的“culturebead” 在几天内保持稳定的媒体水平。四个FBS GF将为受控- 发布策略。最初的四个FBS GF是基于：1)在FBS中的存在，2)不稳定和3) 生物活性。每个CultureBead替换一个FBS GF，允许将CultureBead组合在一起，以提供稳定的 玻璃纤维混合物。第二个目标是推进自动化微流控制造，以促进具有成本效益的电流 符合良好制造工艺(CGMP)的culturebeads生产。最初的CultureBeads工具包可以 根据选定的GFS用于特定的细胞类型，包括视网膜色素上皮(RPE)细胞， 某些有广泛需求的间充质干细胞和多能干细胞。文化珠子 RPE的制造商已经立即申请了FDA批准的、NIH赞助的临床试验。在未来 阶段2 STTR应用程序，将使用多路分析扩展CultureBeads系列，以确定 其他因素，以更完全地取代FBS。