Abstract
Surface plasmon resonance (SPR) biosensors have been recognized as a useful tool and widely used for real-time dynamic analysis of molecular binding affinity because of its high sensitivity to the change of the refractive index of tested objects. The conventional methods in molecular biology to evaluate cell differentiation require cell lysis or fixation, which make investigation in live cells difficult. In addition, a certain amount of cells are needed in order to obtain adequate protein or messenger ribonucleic acid for various assays. To overcome this limitation, we developed a unique SPR-based biosensing apparatus for real-time detection of cell differentiation in live cells according to the differences of optical properties of the cell surface caused by specific antigen-antibody binding. In this study, we reported the application of this SPR-based system to evaluate the osteogenic differentiation of mesenchymal stem cells (MSCs). OB-cadherin expression, which is up-regulated during osteogenic differentiation, was targeted under our SPR system by conjugating antibodies against OB-cadherin on the surface of the object. A linear relationship between the duration of osteogenic induction and the difference in refractive angle shift with very high correlation coefficient was observed. To sum up, the SPR system and the protocol reported in this study can rapidly and accurately define osteogenic maturation of MSCs in a live cell and label-free manner with no need of cell breakage. This SPR biosensor will facilitate future advances in a vast array of fields in biomedical research and medical diagnosis.
Highlights
Dramatic progress in the biological understanding and the potential clinical use of mesenchymal stem cells (MSCs) has been made in recent years
The OB-cadherin protein expression level was measured by a semi-quantitative assay in which the relative amount of immunoreactive bands on Western blot film was quantified as arbitrary units using a computerized software program (LabWorks 4.6; UVP Inc., Cambridge, UK) and was normalized to the intensity of SaOS2 (Fig. 2C)
Since the expression of the membrane molecule OB-cadherin is up-regulated during maturation of osteoblasts, we used it as a marker to analyze the extent of differentiation by quantifying it under Surface plasmon resonance (SPR) system
Summary
Dramatic progress in the biological understanding and the potential clinical use of mesenchymal stem cells (MSCs) has been made in recent years. MSCs have been initially identified in bone marrow stroma as non-hematopoietic stem cells which are capable of differentiation into tissues of mesodermal origin, such as osteoblasts, adipocytes, chondrocytes, tenocytes, and hepatocytes [1,2,3,4,5,6] Due to their multi-lineage differentiation potentials, many pre-clinical studies with tissue engineering approaches are currently under investigation [7,8,9]. To evaluate the maturation of osteogenic differentiation of hMSCs during these processes, histochemical and molecular biological methods such as alkaline phosphatase (ALK-p) staining, von Kossa staining, Western blot, and reverse transcription polymerase chain reaction (RT-PCR), are commonly used [14,15,16]. The conventional methods to detect the extent of osteogenic differentiation require cell lyses or fixation, which causes cell death and makes continuous analysis on the same cell impossible
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