Metabolic

The cell / colony movement index of oral keratinocytes predicts the epithelial regenerative capacity

PICTURE: The optical flow (OF) algorithm was applied to image segmentation and analysis of oral first passage keratinocyte cultures (p1) to assess total cell / colony growth and speed of movement. The mean … view More

Photo credit: Niigata University

Niigata, Japan – A comprehensive study of cell and colony movement provides new insights into the proliferative and epithelial regenerative abilities of human primary oral keratinocyte cultures with implications for the quality control of engineered cells used in regenerative medicine. Dr. Kenji Izumi and his colleagues, Dr. Emi Hoshikawa and Dr. Taisuke Sato, modified the optical flow (OF) protocol originally presented in their 2019 paper to add the ability to determine the threshold cell / colony movement speed required to differentiate inferior oral keratinocyte populations prior to the Fabrication of a tissue engineering tissue construct of the oral mucosa. The interdisciplinary collaboration was crucial for the success of this study. Dr. Izumi and Dr. Hoshikawa, both experts in oral keratinocyte biology, relied on Dr. Sato to improve the protocol procedure of the OF algorithm implementation. Oral mucosal keratinocytes are currently used in regenerative dentistry and extraoral regenerative medicine, and differentiating cell health is critical to making high quality products for cell-based therapies. While previous studies have non-invasively characterized the optimal metabolic activities of grafts produced by tissue engineering of the oral mucosa, the non-invasive measurements for oral first passage keratinocytes (p1) have not yet emerged as a tool for quality control of cells. The researchers first determined the specific spatiotemporal growth pattern of p1 cells and allowed them to make correlative estimates that reduced the time of photomicrography from 24 hours to 4 hours, resulting in fewer image analyzes. The cell / colony proliferation and speed of movement of each frame were then assessed by applying the OF algorithm with image segmentation. Like Dr. Izumi noted, “Dynamic image-based analysis, rather than static image analysis like observing colony shape, allowed us to focus on cell / colony movement of oral keratinocytes in our cell culture system where cells do not form densely. Packed colonies.” Using this approach, mean movement speed (MMS), a marker of the ability to move, was assessed. As expected, this analysis confirmed their previous finding that the MMS of oral keratinocytes correlates positively with proliferative capacity. Surprisingly, the team found that this was not the only cellular feature associated with MMS, as histological examination of the cells showed that the epithelial regeneration potential correlates with the speed of movement. The ability to assess both the proliferative capacity and epithelial regenerative capacity of oral mid-phase keratinocytes using the MMS threshold makes this a more robust and predictive quality screening tool to ensure that cells are ready for clinical use are suitable.

The researchers took their investigation a step further, comparing the growth properties of oral p1 keratinocytes exposed to metabolic stress to those under standard conditions: “The creative point in this study was that we could see cells in culture under a few poor conditions forced what we call “challenged” protocols to weaken cells, “said Dr. Izumi. “Then we extrapolated the ‘threshold’ into our own cultural state.” The proliferative potential of cells grown under different conditions was compared and the population doubling times were calculated to determine the MMS threshold, which is indicative of cells suitable for transfer under 3D culture conditions. In their specific culture system, a movement index of less than 40 μm / h reflected cell damage induced by metabolic challenges and sub-par cell colonies. However, these results have wider application, with the movement index serving as a threshold to determine the quality of cultured cells under various culture conditions, as reported by Dr. Izumi observes: “Depending on the cell type, the details of the algorithm have to be taken into account and adapted. More importantly, the “threshold” must be determined by different cell types, culture media and culture conditions. “Since the movement index correlates with properties closely related to cell quality and clinical application, such as proliferation and epithelial regeneration, it can be used as a reliable indicator for quality control. “Because the non-invasive and quantitative monitoring of cells enables multiple observations over time, it is useful for quality control of cells in regenerative medicine and contributes to the cell manufacturing industry.” However, Dr. Izumi notes that it can even be used beyond regenerative medicine, for example in cancer research. Ultimately, the team hopes to increase the accuracy of the measurement of the speed of movement in the future and to elucidate the molecular relationship underlying the correlation between locomotion and proliferation of oral keratinocytes is what contributes to the determination of critical quality features of oral keratinocytes.

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References: https://doi.org/10.1177/2041731419881528

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