In Vitro Study of Extracellular Vesicles Migration in Cartilage-Derived Osteoarthritis Samples Using Real-Time Quantitative Multimodal Nonlinear Optics Imaging

Abstract

Mesenchymal stromal cells (MSCs)-derived extracellular vesicles (EVs) are promising therapeutic nano-carriers for the treatment of osteoarthritis (OA). The assessment of their uptake in tissues is mandatory but, to date, available technology does not allow to track and quantify incorporation in real-time. To fill this knowledge gap, the present study was intended to develop an innovative technology to determine kinetics of fluorescent MSC-EV uptake by means of time-lapse quantitative microscopy techniques. Adipose-derived mesenchymal stromal cells (ASCs)-EVs were fluorescently labeled and tracked during their uptake into chondrocytes micromasses or cartilage explants, both derived from OA patients. Immunofluorescence and time-lapse coherent anti-Stokes Raman scattering, second harmonic generation and two-photon excited fluorescence were used to follow and quantify incorporation. EVs penetration appeared quickly after few minutes and reached 30–40 μm depth after 5 h in both explants and micromasses. In explants, uptake was slightly faster, with EVs signal overlapping both extracellular matrix and chondrocytes, whereas in micromasses a more homogenous diffusion was observed. The finding of this study demonstrates that this innovative technology is a powerful tool to monitor EVs migration in tissues characterized by a complex extracellular network, and to obtain data resembling in vivo conditions.

Adipose-Derived Mesenchymal Stromal Cells Treated with Interleukin 1 Beta Produced Chondro-Protective Vesicles Able to Fast Penetrate in Cartilage

“…In this regard, it has been observed that the EVs derived from ASCs have the ability to fast penetrate for several micrometers into the cartilage, arriving predominantly at the matrix, but also reaching the cells. Herein presented results confirmed the quick EVs penetration along 30–40 μm depth already observed after a 5 h time-lapse in both micromasses and cartilage explants with the same technology [ 13 ], and further extended both intensity and spatial distribution measurements up to 16 h. Moreover, the observation of two diffusion kinetics of EVs suggests an active and steady initial process, followed by a more passive and slower one, which could be linked both to the differences in EVs penetration in the collagen-rich areas, but also to the progressive achievement of saturation, considering the 16 h as the identified time point when the signal reached a plateau. Finally, the highest EVs prevalence in ECM than in cells observed in experiments conducted on cartilage explants was not previously observed in micromasses [ 13 ], where a more homogenous EVs diffusion was reported.…”

“…Herein presented results confirmed the quick EVs penetration along 30–40 μm depth already observed after a 5 h time-lapse in both micromasses and cartilage explants with the same technology [ 13 ], and further extended both intensity and spatial distribution measurements up to 16 h. Moreover, the observation of two diffusion kinetics of EVs suggests an active and steady initial process, followed by a more passive and slower one, which could be linked both to the differences in EVs penetration in the collagen-rich areas, but also to the progressive achievement of saturation, considering the 16 h as the identified time point when the signal reached a plateau. Finally, the highest EVs prevalence in ECM than in cells observed in experiments conducted on cartilage explants was not previously observed in micromasses [ 13 ], where a more homogenous EVs diffusion was reported. These data evidencing differences in EVs kinetics between the two 3D in vitro models might be due to the different cell to matrix ratios and local homogeneity, both higher in micromasses, and suggest the use of cartilage specimens for kinetic studies being more representative of the real cartilage physiology.…”

“…The multimodal microscope allowed to collect images with Coherent Anti-Stokes Raman Scattering (CARS) for the cellular lipidic structures combined with Second Harmonic Generation (SHG) techniques for the collagen of the extracellular matrix [ 19 ] and Two-Photon Excitation Fluorescence (TPEF) for the EVs [ 13 ].…”

“…The depth of EV penetration during the time-lapse together with the average occupied area and volume were computed using a custom-made ImageJ plugins processing the 3D images as already described [ 13 ].…”

“…In this specific context, it is essential to analyze also the EV interaction with the tissue and the cells resident therein to better understand their real therapeutic potential. Recently, the ability of ASC-EVs to penetrate in chondrocyte micromasses and cartilage explants from osteoarthritic patients was shown through a novel, real-time, microscopy technique [ 13 ].…”