Correlation between in vitro expansion-related cell stiffening and differentiation potential of human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) are an attractive cell source for tissue regeneration, given their self-renewal and multilineage potential. However, they are present in only small percentages in human bone marrow, and are generally propagated in vitro prior to downstream use. Previous work has shown that hMSC propagation can lead to alterations in cell behavior and differentiation potency, yet optimization of differentiation based on starting cell elastic modulus is an area still under investigation. To further advance the knowledge in this field, hMSCs were cultured and routinely passaged on tissue-culture polystyrene to investigate the correlation between cell stiffening and differentiation potency during in vitro aging. Local cell elastic modulus was measured at every passage using atomic force microscopy indentation. At each passage, cells were induced to differentiate down myogenic and osteogenic paths. Cells induced to differentiate, as well as undifferentiated cells were assessed for gene and protein expression using quantitative polymerase chain reaction and immunofluorescent staining, respectively, for osteogenic and myogenic markers. Myogenic and osteogenic cell potential are highly reliant on the elastic modulus of the starting cell population (of undifferentiated cells), and this potential appears to peak when the innate cell elastic modulus is close to that of differentiated tissue. However, the latent expression of the same markers in undifferentiated cells also appears to undergo a correlative relationship with cell elastic modulus, indicating some endogenous effects of cell elastic modulus and gene/protein expression. Overall, this study correlates age-related changes with regards to innate cell stiffening and gene/protein expression in commercial hMSCs, providing some guidance as to maintenance and future use of hMSCs in future tissue engineering applications.

Improving fluorescence imaging of biological cells on biomedical polymers.

Immunofluorescence imaging on polymeric biomaterials is often inhibited by autofluorescence and other optical phenomena. This often limits the analysis that can be performed on cells that are in contact with these materials. This study outlines a method that will quench these inhibitive optical phenomena on a variety of polymeric materials, including poly(glycerol sebacate), poly(urethane), poly(L-lactide-co-ε-caprolactone), and poly(lactic acid-co-glycolic acid). The method uses a simple material treatment method utilizing Sudan Black B (SB), which is commonly used as an autofluorescence quenching molecule in tissue histology, but has not yet been used in biomaterials analysis. The quenching mechanism in the selected polymers is investigated using attenuated total reflectance Fourier transform infrared spectroscoy, ultraviolet-visible light absorbance and fluorescence analysis, and scanning electron microscopyobservation of the material morphology prior to and after SB treatment. The results point to SB eliminating the inhibitive light phenomena of these materials by two methods: (i) chemical interaction between SB and the polymer molecules and (ii) physical interaction whereby SB forms a physical barrier that can absorb scattered light and quench autofluorescence interference during fluorescence microscopy. The studies show that the treatment of polymers with SB is robust across the polymers tested, in both porous and non-porous formats. The method does not interfere with immunofluorescent imaging of fluorescently labeled biological cells cultured on these polymers. This quick, simple, and affordable method enables a variety of analyses to be conducted that may otherwise have been impractical or impossible.