Limitations imposed by non-physiological cell culture practices: characterization and solutions

非生理细胞培养实践带来的限制：表征和解决方案

• 批准号: RGPIN-2020-05274
• 负责人: Stuart, Jeffrey
• 金额: $ 3.06万
• 依托单位: Brock University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=740192
• 关键词: Limitations; imposed; non; physiological; cell

Each year thousands of original research papers report results of studies performed on cultured mammalian cells to better understand fundamental biology processes at the level of individual molecules. Almost all of these studies use commercial media that look nothing like human plasma (in which cells are bathed inside the body) and fail to regulate oxygen at the levels found in the body. The failure to mimic normal body conditions when working with human cells affects the reliability of the data we glean from experiments. Data from cell culture is critical not only to understanding normal biology, but also mechanisms of disease. If cells are grown in media with little resemblance to blood and behave differently than in the body, their use as experimental models is compromised. Just this year (2019) there has been attention given to developing cell culture media with compositions very similar to blood plasma, and we have begun to use these. Unfortunately, we have already gathered enough data to know that virtually all of our cell culture results are different in this new media compared to traditional cell culture media upon which the foundation of cell biology has been built. This Discovery Grant proposal focus on identifying and characterizing these differences in the behaviour of cells growing in new media that resemble human blood vs the old commercial media that bear little resemblance to blood. Ultimately, this will contribute to our ability to do better experiments that are more likely to accurately model reproduce in vivo biology and thus to allow better drugs to be developed. We propose investigating gene expression, metabolism, free radical production, and growth under the old conditions and in the new media. Once we have characterized problems with the old cell culture approaches, we will work to develop solutions that help us maintain the right conditions when working with cells in culture. For example, we will investigate the use of a synthetic hemoglobin to aid in oxygen delivery to cells. We will also investigate how to prevent changes in media composition over the course of long experiments. We will continue to develop our existing tools that allow us to visualize mitochondrial form and function inside of cells. Mitochondria play important roles in energy and metabolism, so their health and function are important. This work will be done mainly by MSc and PhD students who will work with me in my research laboratory to gather data and develop new tools. They will attend international conferences to share their discoveries and inventions with other scientists. Undergraduate BSc students and grade 12 students will also work with us to learn laboratory and research skills. Through these opportunities, all these students will progress toward highly skilled professions like medicine and allied health, technical positions in hospitals, universities, and government institutions, industrial or academic research, and teaching.

每年，数以千计的原创研究论文报告了在培养的哺乳动物细胞上进行的研究结果，以更好地在单个分子水平上了解基本的生物学过程。几乎所有这些研究都使用了看起来与人类血浆(细胞在体内沐浴)完全不同的商业介质，并且未能将氧气调节到体内发现的水平。在使用人体细胞工作时，无法模拟正常的身体条件，这影响了我们从实验中收集的数据的可靠性。来自细胞培养的数据不仅对了解正常生物学至关重要，而且对疾病的机制也至关重要。如果细胞在与血液几乎没有相似之处的培养液中生长，并且在体内的表现不同，它们作为实验模型的使用就会受到影响。就在今年(2019年)，人们开始关注开发成分与血浆非常相似的细胞培养液，我们已经开始使用这些培养液。不幸的是，我们已经收集了足够的数据，知道在这种新的介质中，我们几乎所有的细胞培养结果都不同于传统的细胞培养介质，传统的细胞培养介质是细胞生物学的基础。这项发现拨款提案的重点是识别和描述在类似人类血液的新介质中生长的细胞与在与血液几乎没有相似之处的旧商业介质中生长的细胞行为的这些差异。最终，这将有助于我们有能力进行更好的实验，这些实验更有可能准确地模拟体内复制生物学，从而允许开发更好的药物。我们建议研究基因的表达，代谢，自由基的产生，以及在旧条件下和新媒体中的生长。一旦我们确定了旧的细胞培养方法的问题，我们将努力开发解决方案，帮助我们在处理培养中的细胞时保持正确的条件。例如，我们将研究使用合成的血红蛋白来帮助向细胞输送氧气。我们还将研究如何在长期的实验过程中防止媒体成分的变化。我们将继续开发现有的工具，使我们能够可视化细胞内线粒体的形态和功能。线粒体在能量和代谢中起着重要的作用，因此它们的健康和功能非常重要。这项工作将主要由硕士和博士生完成，他们将在我的研究实验室与我一起收集数据并开发新工具。他们将参加国际会议，与其他科学家分享他们的发现和发明。本科生和12年级的学生也将与我们一起学习实验室和研究技能。通过这些机会，所有这些学生都将进入高技能的职业，如医药和专职卫生，医院、大学和政府机构的技术职位，工业或学术研究和教学。