Cell Production Core

为了实现这个PPG的目标，我们需要从小鼠中培养大量的心肌细胞（CM）。 胚胎干（ES）细胞来源，用于亲和纯化质谱（AP-MS）。CM生产将在 从6孔板扩大到大组织烧瓶，然后在生物反应器中悬浮培养。虽然 大规模的细胞生产在一个实验室中是低效的，将我们的努力结合到一个核心实验室中， 增加细胞产量，从而可以有效地实现AP-MS。 具体目标摘要 具体目标1：为计划研究项目提供高质量的细胞生产能力。 细胞生产核心将使研究项目能够生产大量的心脏祖细胞， 来源于工程小鼠ES细胞的CM。通过在悬浮液中扩增细胞， 生产水平至少比培养皿高两个数量级。这些细胞将 提供给蛋白质组学核心，以分析心脏分化的生化复合物。 具体目标2：为负担得起的大批量CM生产开发新的协议。 生物反应器已成功地用于在学术上生产大量的小鼠和人类CM。 和工业实验室，但主要的限制是试剂的成本。典型的生物反应器运行将使用 100升培养基和最先进的方案每次运行的成本可能超过80，000美元。我们将调整我们目前的 协议，使成本下降了70%以上，允许在大量的拟议实验， 细胞系

To achieve the goals of this PPG we will need to grow large quantities of cardiomyocytes (CMs) from mouse embryonic stem (ES) cell sources, for affinity-purification mass spectrometry (AP-MS). CM production will be scaled up from 6-well plates, to large tissue flasks and then suspension culture in a bioreactor. Although large-scale cell production is inefficient in a single lab, combining our efforts into a core laboratory will increase cell production so that the AP-MS can be achieved efficiently. SPECIFIC AIMS SUMMARY Specific Aim 1: To provide high-quality cell production capabilities for program research projects. The cell production core will enable research projects to produce large quantities of cardiac progenitors and CMs derived from engineered mouse ES cells. By expanding cells in suspension, we can attain cell production levels that are at least two orders of magnitude greater than on petri dishes. These cells will be provided to the Proteomics Core to analyze biochemical complexes that underlie cardiac differentiation. Specific Aim 2: To develop new protocols for affordable high volume CM production. Bioreactors have successfully been used to produce large amounts of mouse and human CMs in academic and industrial laboratories, but the main limitation is the cost of reagents. Typical bioreactor runs will use 100 liters of media and state-of-the-art protocols can cost >$80,000 per run. We will adapt our current protocols to bring the cost down by over 70%, allowing for the proposed experiments in a large numbers of cell lines.