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

为了更好地理解单个分子水平上的基本生物学过程，每年都有数千篇原创研究论文报告在培养的哺乳动物细胞上进行的研究结果。几乎所有这些研究都使用商业媒介，这些媒介看起来一点也不像人体血浆（细胞浸泡在体内），也无法调节体内的氧气水平。在处理人体细胞时，无法模拟正常的身体状况会影响我们从实验中收集到的数据的可靠性。细胞培养的数据不仅对理解正常生物学，而且对疾病的机制也至关重要。如果细胞生长在与血液几乎没有相似之处的培养基中，并且表现与在体内不同，那么它们作为实验模型的用途就会受到损害。就在今年（2019年），人们开始关注开发与血浆成分非常相似的细胞培养基，我们已经开始使用这些培养基。不幸的是，我们已经收集了足够的数据，知道几乎所有的细胞培养结果在这种新介质中与传统的细胞培养介质相比都是不同的，而传统的细胞培养介质是细胞生物学的基础。这项发现基金提案的重点是识别和描述细胞在类似人类血液的新媒体中与在与血液几乎没有相似之处的旧商业媒体中生长的行为差异。最终，这将有助于我们进行更好的实验，更有可能准确地模拟体内繁殖生物学，从而允许开发更好的药物。我们建议在旧条件和新介质下研究基因表达、代谢、自由基产生和生长。一旦我们确定了旧细胞培养方法的问题，我们将努力开发解决方案，帮助我们在培养细胞时保持正确的条件。例如，我们将研究使用合成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实验过程中防止介质组成的变化。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式和功能。线粒体在能量和代谢中起着重要的作用，因此其健康和功能非常重要。这项工作将主要由硕士和博士研究生完成，他们将与我一起在我的研究实验室收集数据并开发新的工具。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。本科理学士学生和12年级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职业，如医学和联合健康，医院、大学和政府机构的技术职位，工业或学术研究和教学。

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.