The possibilities of Cell-IQ phase-contrast microscopy for a continuous real-time observation of multipotent mesenchymal stromal cell culture

摘要

Continuous monitoring of multipotent mesenchymal stromal cells (MMSCs) is a promising tool that could be used in cellular biology, environmental research and biotechnology to study in vitro real-time morphology and behavioural response of stem cells alone, as well at contacts with other cells and for controlling a sustainable production of scaffolds for tissue engineering. The in vitro processes of human adipose-derived MMSCs (hAMMSCs) morphology, motility, cell division, and secretion were studied by means of Cell-IQ v2 MLF (CM? Technologies Oy, Finland) integrated phase-contrast microscopic platform for a continuous real-time surveillance imaging of living cells. 70 μL suspension (5×10 4 viable karyocytes) of the cells was applied into the center of the wells of 12-well plastic plates, and cells were allowed to adhere in a moist chamber for 120 min. Nonadherent cells were washed, and the wells were carefully filled with 1.5 mL of a nutrient medium DMEM/F12 (1 : 1) without osteogenic additions. Cells were cultured for 14 days at 100% humidity in a 5% carbondioxide atmosphere at 37°C until a monolayer formation. Digital images of cell culture growth were captured every 45 min. The cells were positively stained with alizarin red (osteoblasts), alcian blue (chondrocytes), or oil red (adipocytes). More than 95% of attached cells expressed CD73, CD90, and CD105 markers, mainly. Thus, the cells corresponded to the morphological criteria of MMSCs. The Cell-IQ system has allowed establishing 182 μm/h linear velocity of free (until the cell contacts) motility of spindle or fibroblast-like cells. Maximum number of cells achieved 136 cells per field of view; 13-24 % of cells divided each 1-3 h until a monolayer was formed. Chemokine cooperation between hAMMSCs and poor macrophages intermixture was proposed. Cell-IQ could be useful for in vitro real-time imaging of cell subpopulations and/or their response to biodegradable scaffolds and/or (micro)environmental factors.