An engineered approach to stem cell culture: automating the decision process for real-time adaptive subculture of stem cells.
An engineered approach to stem cell culture: automating the decision process for real-time adaptive subculture of stem cells.
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DOI：
10.1371/journal.pone.0027672
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Campbell PG
中科院分区：
文献类型：
--
作者：
Ker DF;Weiss LE;Junkers SN;Chen M;Yin Z;Sandbothe MF;Huh SI;Eom S;Bise R;Osuna-Highley E;Kanade T;Campbell PG
当前的细胞培养实践依赖于人类操作者，并且仍然是费力和高度主观的，导致很大的变化和不一致的结果，特别是当使用细胞融合度的视觉评估来确定细胞传代培养的适当时间时。尽管用机器人系统自动化细胞培养的努力正在进行中，但大多数这样的系统仍然需要人工干预来决定何时进行传代培养。因此，有必要准确客观地确定细胞传代的合适时间。维持细胞多能性同时最大限度地提高细胞产量的最佳干细胞培养对于高效、经济的干细胞治疗尤其重要。为了实现这一目标，我们开发了一个基于计算机视觉的实时系统，该系统监测细胞融合程度，精度为0.791±0.031，召回率为0.559±0.043。该系统由一台自动相衬延时显微镜和一台服务器组成。多个培养皿依次成像，数据上传到执行计算机视觉处理的服务器，预测细胞何时将超过预定义的最佳细胞融合阈值，并提供基于web的远程细胞培养监控界面。人工操作员也会在达到该阈值前4小时通过短信和电子邮件收到通知，并在达到该阈值后立即收到通知。该系统已成功用于指导范例干细胞群体C2C12细胞的扩增。计算机定向和人定向对照传代培养需要3次连续培养才能达到5000万个C2C12细胞的理论目标细胞产量，并且在成肌分化和成骨分化方面没有差异。这种基于视觉的自动化系统具有潜在的潜力，可用于继代培养、细胞培养优化和质量保证/质量控制的自适应实时控制，并且可以与当前和正在开发的机器人细胞培养系统集成以实现完全自动化。
Current cell culture practices are dependent upon human operators and remain laborious and highly subjective, resulting in large variations and inconsistent outcomes, especially when using visual assessments of cell confluency to determine the appropriate time to subculture cells. Although efforts to automate cell culture with robotic systems are underway, the majority of such systems still require human intervention to determine when to subculture. Thus, it is necessary to accurately and objectively determine the appropriate time for cell passaging. Optimal stem cell culturing that maintains cell pluripotency while maximizing cell yields will be especially important for efficient, cost-effective stem cell-based therapies. Toward this goal we developed a real-time computer vision-based system that monitors the degree of cell confluency with a precision of 0.791±0.031 and recall of 0.559±0.043. The system consists of an automated phase-contrast time-lapse microscope and a server. Multiple dishes are sequentially imaged and the data is uploaded to the server that performs computer vision processing, predicts when cells will exceed a pre-defined threshold for optimal cell confluency, and provides a Web-based interface for remote cell culture monitoring. Human operators are also notified via text messaging and e-mail 4 hours prior to reaching this threshold and immediately upon reaching this threshold. This system was successfully used to direct the expansion of a paradigm stem cell population, C2C12 cells. Computer-directed and human-directed control subcultures required 3 serial cultures to achieve the theoretical target cell yield of 50 million C2C12 cells and showed no difference for myogenic and osteogenic differentiation. This automated vision-based system has potential as a tool toward adaptive real-time control of subculturing, cell culture optimization and quality assurance/quality control, and it could be integrated with current and developing robotic cell cultures systems to achieve complete automation.
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影响因子：
4.8
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通讯作者：
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