Suga Mika, Kii Hiroaki, Niikura Keiichi, Kiyota Yasujiro, Furue Miho K
Laboratory of Stem Cell Cultures, National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, Japan; Stem Cell Evaluation Technology Research Association, Tokyo, Japan; Microscope Solution Business Unit, Nikon Corporation, Tokyo, Japan.
Laboratory of Stem Cell Cultures, National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, Japan; Stem Cell Evaluation Technology Research Association, Tokyo, Japan; Microscope Solution Business Unit, Nikon Corporation, Tokyo, Japan
Stem Cells Transl Med. 2015 Jul;4(7):720-30. doi: 10.5966/sctm.2014-0242. Epub 2015 May 13.
: Cell growth is an important criterion for determining healthy cell conditions. When somatic cells or cancer cells are dissociated into single cells for passaging, the cell numbers can be counted at each passage, providing information on cell growth as an indicator of the health conditions of these cells. In the case of human pluripotent stem cells (hPSCs), because the cells are usually dissociated into cell clumps of ∼50-100 cells for passaging, cell counting is time-consuming. In the present study, using a time-lapse imaging system, we developed a method to determine the growth of hPSCs from nonlabeled live cell phase-contrast images without damaging these cells. Next, the hPSC colony areas and number of nuclei were determined and used to derive equations to calculate the cell number in hPSC colonies, which were assessed on time-lapse images acquired using a culture observation system. The relationships between the colony areas and nuclei numbers were linear, although the equation coefficients were dependent on the cell line used, colony size, colony morphology, and culture conditions. When the culture conditions became improper, the change in cell growth conditions could be detected by analysis of the phase-contrast images. This method provided real-time information on colony growth and cell growth rates without using treatments that can damage cells and could be useful for basic research on hPSCs and cell processing for hPSC-based therapy.
This is the first study to use a noninvasive method using images to systemically determine the growth of human pluripotent stem cells (hPSCs) without damaging or wasting cells. This method would be useful for quality control during cell culture of clinical hPSCs.
细胞生长是确定健康细胞状态的重要标准。当体细胞或癌细胞解离为单细胞进行传代时，每次传代时可对细胞数量进行计数，从而提供细胞生长信息，作为这些细胞健康状况的指标。对于人类多能干细胞（hPSC），由于细胞传代时通常解离为约50 - 100个细胞的细胞团，细胞计数很耗时。在本研究中，我们使用延时成像系统开发了一种方法，可从未标记的活细胞相差图像中确定hPSC的生长情况，且不会对这些细胞造成损伤。接下来，确定hPSC集落面积和细胞核数量，并用于推导计算hPSC集落中细胞数量的方程，这些方程在使用培养观察系统获取的延时图像上进行评估。集落面积与细胞核数量之间的关系呈线性，尽管方程系数取决于所使用的细胞系、集落大小、集落形态和培养条件。当培养条件变得不合适时，通过分析相差图像可检测到细胞生长条件的变化。该方法无需使用可能损伤细胞的处理方式即可提供集落生长和细胞生长速率的实时信息，对hPSC的基础研究和基于hPSC的治疗的细胞处理可能有用。
这是第一项使用非侵入性图像方法系统地确定人类多能干细胞（hPSC）生长情况而不损伤或浪费细胞的研究。该方法在临床hPSC细胞培养的质量控制中可能有用。
