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Three-dimensional (3D) cell cultures were created with the use of fur keratin associated proteins (F-KAPs) as scaffolds. The procedure of preparation F-KAP involves combinations of chemical activation and enzymatic digestion. The best result in porosity and heterogeneity of F-KAP surface was received during pepsin digestion. The F-KAP had a stable structure, no changes were observed after heat treatment, shaking and washing. The 0.15-0.5 mm fraction had positive effect for formation of 3D scaffolds and cell culturing. Living rat mesenchymal cells on the F-KAP with no abnormal morphology were observed by SEM during 32 days of cell culturing.

Sylwia Dabrowska,1 Andrea Del Fattore,2 Elzbieta Karnas,3,4 Malgorzata Frontczak-Baniewicz,5 Hanna Kozlowska,6 Maurizio Muraca,7 Miroslaw Janowski,1,8 Barbara Lukomska1 1NeuroRepair Department, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland; 2Multifactorial Disease and Complex Phenotype Research Area, Bambino Gesù Children’s Hospital, IRCCS, Rome, Italy; 3Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland; 4Malopolska Centre of Biotechnology, Krakow, Poland; 5Electron Microscopy Platform, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland; 6Laboratory of Advanced Microscopy Techniques, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland; 7Department of Women’s and Children’s Health, University of Padua, Padua, Italy; 8Russel H. Morgan Department of Radiology and Radiological Science, Division of MR Research, The Johns Hopkins University School of Medicine, Baltimore, MD, USA Background: Mesenchymal stem cells have been shown therapeutic in various neurological disorders. Recent studies support the notion that the predominant mechanism by which MSCs act is through the release of extracellular vesicles (EVs). EVs seem to have similar therapeutic activity as their cellular counterparts and may represent an interesting alternative standalone therapy for various diseases. The aim of the study was to optimize the method of EV imaging to better understand therapeutic effects mediated by EVs.Methods: The fluorescent lipophilic stain PKH26 and superparamagnetic iron oxide nanoparticles conjugated with rhodamine (Molday ION Rhodamine B™) were used for the labeling of vesicles in human bone marrow MSCs (hBM-MSCs). The entire cycle from intracellular vesicles to EVs followed by their uptake by hBM-MSCs has been studied. The identity of vesicles has been proven by antibodies against: anti-CD9, -CD63, and -CD81 (tetraspanins). NanoSight particle tracking analysis (NTA), high-resolution flow cytometric analysis, transmission electron microscopy (TEM), ELYRA PS.1 super-resolution microscopy, and magnetic resonance imaging (MRI) were used for the characterization of vesicles.Results: The PKH26 and Molday ION were exclusively localized in intracellular vesicles positively stained for EV markers: CD9, CD63, and CD81. The isolated EVs represent heterogeneous population of various sizes as confirmed by NTA. The TEM and MRI were capable to show successful labeling of EVs using ION. Co-culture of EVs with hBM-MSCs revealed their uptake by cells in vitro, as visualized by the co-localization of PKH26 or Molday ION with tetraspanins inside hBM-MSCs.Conclusion: PKH26 and Molday ION seem to be biocompatible with EVs, and the labeling did not interfere with the capability of EVs to re-enter hBM-MSCs during co-culture in vitro. Magnetic properties of IONs provide an additional advantage for the imaging of EV using TEM and MRI. Keywords: mesenchymal stem cells, extracellular vesicles, cell tracking, fluorescent dye, iron oxide, MRI