Mesenchymal stem cells: multilayer polyelectrolyte microcapsules uptake, toxicity and influence upon functional properties
K. V. Lepik1, V. S. Sergeev1, A. R. Muslimov1, D. S. Romanyuk1, R. T. Mikhelashvili1, I. S. Moiseev1, E. V. Popova2,I. L. Radchenko2, A. D. Vilesov1, G. B. Sukhorukov2, 3, B. V. Afanasyev1
1 The First St. Petersburg State I. Pavlov Medical University, St. Petersburg, Russia.
2 Peter the Great St. Petersburg Polytechnic University, RASA-center, Russia,
3 Queen Mary University of London, UK
Multilayer polyelectrolyte microcapsules are gathering an increasing interest as novel mean for drug delivery, a diagnostic and investigational tool. The main advantages of these microcapsules are their well-controlled size and shape, finely tuned wall thickness, and variable wall compositions enabling controlled release of their content via magnetic field, light and/or ultrasound exposure. Ease of surface modification allows further functionalization of the microcapsules. At this point, mesenchymal stem cells (MSCs) represent a popular cell model in the wide range of clinical and scientific trials. Currently, there is a lack of data concerning efficiency of microcapsules uptake by the MSCs, and their influence on functional properties of these cells.
Materials and methods
Bone marrow-derived MSCs were isolated using a standard operating procedure. The cells harvested from the second and third passages were used for further experiments. The study included two main sections: in the first arm, a suspension of magnetic PAH/PSS FITC-labeled microcapsules (3-5 μm in size) were added to the surface-adhered MSCs at the cell/particle ratios of 1:1, 1:5, 1:10, 1:20. In the second arm, the same preparations of microcapsules were added to the MSC suspensions at the same ratios. After 24 h of incubation, the uptake rates were tested by means of confocal microscopy and flow cytometry. Cell morphology and viability was tested by differential counting in haemocytometer using trypan blue and propidium iodide vital dyes. Differentiation capacity of the cells was tested using standard staining for osteogenic and adipogenic differentiation. Magnetic separation of MSCs bound with magnetic-labeled microcapsules was tested using midiMACS cell separation system. Adhesive properties of MSCs were tested as follows: MSC associated with microcapsules at the ratios of 1:5; 1:10; 1:20 were seeded into the cultural flasks at the concentrations of 20x104/cm2 under standard conditions. After 24-h incubation, the culture medium was removed, adherent cells were detached with trypsine/EDTA. Both suspension and adherent cell fraction were counted by means of haemocytometer and normalized agains a control group (without microcapsules).
The cell-capsule association rates correlate with numbers of added capsules and the method applied. Average proportion of adherent cells associated with microparticles is, respectively, 8% (at 1:1 cell/capsule ratio); 18% (1:3 ratio) 32% (1:10 ratio). Similar testing of MSC/particle contacts in suspension phase yielded more impressive results: 90% at the 1:1 ratio; 98% (1:3 ratio); 99% (1:10 ratio). Single cells are able to capture up to 30 microcapsules (at 1:20 cell/capsule ratio). Further increase of the microcapsule concentrations do not lead to significant enhance of their uptake by the cells. A lesser part of capsules are only associated with membrane, but not internalized by cells. Following internalization, the microcapsules are dispersed in cytoplasm, mainly, in the perinuclear compartment. Mean percentage of viable cells after 24 h of incubation of MSCs with microcapsules, normalized for the control group results, was 90% (at the 1:5 cell/capsule ratio); 85% (1:10); 83% (1:20 ratio), 5% (1:100 ratio). The cells, associated with microcapsules can be isolated with high efficiency by routine magnet separation methods. The ability of MSCs for osteogenic and adipogenic differentiation was not significantly affected by the microcapsules uptake. Conclusion. Adhesion assay has shown that adherent fraction of the culture-plated cells treated with capsules was significantly decreased in dose-dependent manner, i. e., average percentage of adherent cells was 85% (1:5 ratio); 64% (1:10 ratio); and 38% (1:20 ratio), in comparison with 85% adherence in untreated control group.
Polyelectrolyte microcapsules can be internalized by MSCs at a high efficiency. Cell suspension provides optimal conditions for effective capsule uptake. Polyelectrolyte microcapsules show very mild toxicity and only minimally influence cell functions under the cell:capsule ratio of<1:10. A significant decrease in cell viability was observed only in experiments with very high (1:100+) cell/capsule ratio. The ability of MSCs for osteogenic and adipogenic differentiation was not impaired by the microcapsules uptake. Adherent fraction of the plated capsule-exposed cells was significantly decreased in a dose-dependent manner. Ingestion of microcapsules at high amounts causes inhibition of MSCs adherence Polyelectrolyte microcapsules may be considered a promising tool for diagnostics, therapeutic interventions and investigational approaches, due to potential ability of controlled cell delivery using external magnetic fields.
Adherence, differentiation, polyelectrolyte microcapsules, internalization, mesenchymal stem cells