Time-lapse microscopic observation of non-dividing cells in cultured human osteosarcoma MG-63 cell line

Abstract

Cancer stem cells resemble normal tissue-specific stem cells in many aspects, such as self-renewal and plasticity. Like their non-malignant counterparts, cancer stem cells are suggested to exhibit a relative quiescence. The established cancer cell lines reportedly harbor slow-proliferating cells that are positive for some cancer stem cells markers. However, the fate of these cells and their progeny remains unknown. We used time-lapse microscopy and the contrast-based segmentation algorithm to identify and monitor actively dividing and non-dividing cells in human osteosarcoma MG-63 cell line. Within the monitored field of view the non-dividing cells were represented by three cells that never divided, and one cell that attempted to divide, but failed cytokinesis, and later, after significantly prolonged division, produced the progeny with enlarged segmented nuclei, thus pointing to a possible mitotic catastrophe. Together, these cells initially constituted about 6.2% of the total number of seeded cells, yet only 0.02% of all cells at the end of the observation period when cells became confluent. Non-dividing cells were characterized by rounded shape, dark nuclei, random cytoplasmic streaming and subtle oscillatory movement, however, they did not migrate and rarely formed cell-cell contacts as compared to actively dividing cells. Our data indicate that the observed non-dividing MG-63 cells do not have a growth advantage over other cells and, therefore, they do not contribute to the cancer stem cells pool.

Time-Lapse Microscopy

“…Early reports demonstrated the feasibility of TLM for comparative studies of cultured cells [6-8] and for monitoring living blood and lymph cells [1], cell division [9, 10] and reaction of cells to varying contents of electrolytes in perfusion chambers [11]. TLM was helpful to decode the process of multinucleation in the developing skeletal muscles [12] [36]; primordial germ cells, a migratory cell population that will eventually give rise to the gametes [37][38][39]; the migration route of progenitor cells in cell cultures obtained from live chicken embryos [40,41]; microglial cells [42][43][44]; olfactory cells from schizophrenia patients [45]; neurons [46]; chemokines that drive migration of megakaryocytes from the proliferative osteoblastic niche within the bone marrow to the capillary-rich vascular niche, which is an essential step for platelet production [47]; migration of osteoclasts toward bone surfaces [48]; motility of cultured endothelial cells to study remodeling of their intercellular junctions [49]; generation of a complete polarized epithelial monolayer by the epithelial cells of mammary gland [17]; movement of cancer cells that were cultured under hypoxic conditions [50] and treated with salinomycin [24]; individual cell motility in fibroblastoid L929 cells [51]; human osteosarcoma MG-63 cells [52]; B35 neuroblastoma cells transiently expressing GFP and C6 glioma cells after staining with Hoechst 33258…”

“…TLM allows investigators to visualize and characterize cell-cell contacts [46,52,[64][65][66][67][68][69][70][71]. The most interesting reports are concerned with the contacts between the various types of stem/ progenitor cells as well as the tumor-environment cell interactions: the importance of proper cell-cell contacts level for their correct positioning and cell polarity during organogenesis [39], glial-neuronal interactions [72][73][74], interactions between microglia and brain tumors [75], between astrocytes and neural progenitor cells [42], between mesenchymal stem cells and human myoblasts [76], dendritic cells [77], endothelial cells [78], cancer cells [79], extracellular matrix molecules [80], between erythroblastic islands in bone marrow [81], between neural progenitor cells [62,[82][83][84], between neural cells and hematopoietic stem cells that migrate to the central nervous system [85], hematopoietic stem cells and stromal cells [20], endothelial progenitor cells and cardiac myocytes [86], between induced pluripotent cells during the early reprogramming phase [53], vesicle traffic through intercellular bridges between prostate cancer cells [87] and synaptic contacts [88][89][90][91][92][93][94].…”

“…Cell division and cell death can be well investigated with TLM [50,52,[95][96][97]. Division and growth of both labeled [96,[98][99][100] and non-labeled [101,102] cells in culture [52,95,98,[103][104][105] and tissue slices [106], including monitoring of a single cell [95,99,[107][108][109][110][111][112], can be observed and assessed with TLM. The fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can effectively label individual G1, S/G2/M and G1/S-transition phase nuclei as red, green and yellow, respectively, to visualize the real-time cell cycle transitions in living mammalian cells [113][114][115][116].…”

“…The up-to-date portable live cell culture monitor (CytoSMART Technologies, Eindhoven, The Netherlands) works within the regular CO 2 incubator. The culture flask (T-flask, Petri dish, wells or any other transparent vessel) is positioned onto the lens of the device; the field of view is chosen by the investigator, and the cell growth and migration can be monitored and analyzed in the real-time mode by accessing the cloud [52].…”

Continuous Live-Cell Culture Monitoring by Compact Lensless LED Microscopes

“…Applying live-cell imaging to monitor cell dynamics requires highly equipped microscopes, which are extremely expensive [1]. Nevertheless, real-time in situ analysis of cell cultures is highly demanded by both research academia and industry in order to gain more insights into cell growth, including the cell dynamic behavior and morphology [2]. Therefore, miniaturized microscopes, which enable lensless real-time imaging of cells inside a conventional CO2 cell culture incubator, are a promising inexpensive alternative.…”