The cell / colony movement index, which is determined by non-invasive, quantitative measurements via the optical flow algorithm, correlates with the epithelial regenerative capacity of oral keratinocytes in cultures of the middle phase, making it a reliable tool for quality control
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 genetically 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 publication to add the ability to determine the threshold cell / colony movement speed required to differentiate inferior oral keratinocyte populations prior to producing one Tissue engineering tissue of the oral mucosa. The interdisciplinary collaboration in which Dr. Izumi and Dr. Hoshikawa, both experts in oral keratinocyte biology, rely on Dr. Sato left 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. As Dr. Izumi noted, “Dynamic image-based analysis, rather than static image analysis like observing colony shape, allowed us to focus on the 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 keratinocytes in the middle phase using the MMS threshold makes this a more robust and predictive quality screening tool to ensure that cells are ready for clinical Application are suitable.
The researchers went one step further in their study, comparing the growth properties of oral p1 keratinocytes exposed to metabolic stress with those under standard conditions: “The creative point in this study was that we had cells in culture under a few bad ones Conditions forced what we called “challenged” protocols to weaken cells, “said Dr. Izumi. “Then we extrapolated the ‘threshold’ in our own culture condition.” The proliferation 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 that are for transfer in 3D culture conditions are suitable. 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 different culture conditions, as reported by Dr. Izumi observes: “Depending on the cell type, the details of the algorithm must be taken into account and adapted. More importantly, the “threshold” has to be determined by different cell types, culture media and culture conditions. ”Since the movement index is correlated with properties that are closely related to cell quality and clinical application, such as proliferation and epithelial regeneration, it can be considered more reliable Serve 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, Izumi notes that it is used even beyond regenerative medicine, for example in cancer research. Ultimately, the team hopes to increase the accuracy of movement speed measurement in the future and to elucidate the molecular relationship underlying the correlation between locomotion and proliferation of oral keratinocytes, which will help determine critical quality characteristics of oral keratinocytes.