The hydrogel–cell constructs were stained using a LIVE/DEAD viability/cytotoxicity kit (Molecular Probes) following the vendor’s protocol. Live cells were stained with green fluorescence by intracellular esterase-catalyzed hydrolysis of Calcein AM, and dead cells were stained red by ethidium homodimer-1 penetrated through the damaged membranes and bound with nucleic acids. After being stained for 30–45 min at 37 °C, the stained hydrogel–cell construct was retrieved and mounted in a Cellview culture dish (Greiner) and imaged on a Leica TCS SP2 confocal microscope. Calcein was excited at 488 nm and observed with the FITC filter (518–542 nm) while ethidium homodimer-1 was excited at 543 nm and observed with the TEXAS RED filter (625–665 nm). Confocal Z-stack images of encapsulated rMSC over the depth of 80 µm (8 consecutive 10 µm slices) were overlaid.
Cellview culture dish
Manufactured by Greiner
Sourced in Germany
The Cellview culture dish is a laboratory equipment designed for cell culture applications. It provides a sterile, controlled environment for the growth and maintenance of cells. The dish features a flat, transparent surface for direct observation and analysis of cell cultures.
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2 protocols using cellview culture dish
1
Live/Dead Staining of Hydrogel-Cell Constructs
2
Osteogenic Differentiation of MSCA-1+ Cells
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The DMEM-cultured MSCA-1+ cell fractions were seeded at a density of 4 × 104 cells and the MC-cultured cell fraction was seeded at a density of 6 × 104 cells per CELLview culture dish with integrated glass bottom (Greiner Bio-One, Frickenhausen, Germany). For all Raman analyses, CELLview culture dishes were precoated with MesenCult-XF attachment substrate for better cell adherence. Preliminary tests revealed low cell adherence with partial cell detachment without precoating (Figure 1 ) particularly for osteogenically induced cells. For osteogenic differentiation, MSCA-1+ cell fractions were treated with osteogenic medium (ob, DMEM/F12 containing 10% FCS, 10 mM β-glycerophosphate, 100 µM L-ascorbic acid 2-phosphate, and 4 µm dexamethasone, Sigma-Aldrich) for 20 days.
Xeno-free (MC-) cultured MSCA-1+/‒ cell fractions were osteogenically induced using the MesenCult osteogenic medium (ob), containing MesenCult MSC basal medium, 5% osteogenic stimulatory supplement, and 3.5 mM ß-glycerophosphate, as described before [2 (link)] for the same time period as DMEM-cultured cells. For both, serum-free and serum-containing culture conditions, untreated and undifferentiated controls (co) were simultaneously cultured.
Xeno-free (MC-) cultured MSCA-1+/‒ cell fractions were osteogenically induced using the MesenCult osteogenic medium (ob), containing MesenCult MSC basal medium, 5% osteogenic stimulatory supplement, and 3.5 mM ß-glycerophosphate, as described before [2 (link)] for the same time period as DMEM-cultured cells. For both, serum-free and serum-containing culture conditions, untreated and undifferentiated controls (co) were simultaneously cultured.
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