To optimise the culture conditions for human Wharton’s jelly-derived mesenchymal stem cells (hWJ-MSCs) intended for clinical use, we investigated ten different properties of these cells cultured under 21% (atmospheric) and 5% (physiological normoxia) oxygen concentrations. The obtained results indicate that 5% O₂ has beneficial effects on the proliferation rate, clonogenicity, and slowdown of senescence of hWJ-MSCs; however, the oxygen level did not have an influence on the cell morphology, immunophenotype, or neuroprotective effect of the hWJ-MSCs. Nonetheless, the potential to differentiate into adipocytes, osteocytes, and chondrocytes was comparable under both oxygen conditions. However, spontaneous differentiation of hWJ-MSCs into neuronal lineages was observed and enhanced under atmospheric oxygen conditions. The cells relied more on mitochondrial respiration than glycolysis, regardless of the oxygen conditions. Based on these results, we can conclude that hWJ-MSCs could be effectively cultured and prepared under both oxygen conditions for cell-based therapy. However, the 5% oxygen level seemed to create a more balanced and appropriate environment for hWJ-MSCs.

Tuning stem cells microenvironment in vitro may influence their regenerative properties. In this study Wharton's Jelly-derived mesenchymal stem cells (WJ-MSCs) were encapsulated in 3D hydrogels derived from human fibrin (FB) or platelet lysate (PL) and the oxygen level was adjusted to physiological normoxia (5% O₂). The influence of the type of the scaffold and physiological normoxia conditions was tested on the WJ-MSCs' survivability, proliferation, migratory potential, the level of expression of selected trophic factors, cytokines, and neural markers. Encapsulated WJ-MSCs revealed high survivability, stable proliferation rate, and ability to migrate out of the hydrogel and the up-regulated expression of all tested factors, as well as the increased expression of neural differentiation markers. Physiological normoxia stimulated proliferation of encapsulated WJ-MSCs and significantly enhanced their neuronal, but not glial, differentiation. Ex vivo studies with indirect co-culture of organotypic hippocampal slices and cell-hydrogel bio-constructs revealed strong neuroprotective effect of WJ-MSCs against neuronal death in the CA1 region of the rat hippocampus. This effect was potentiated further by FB scaffolds under 5% O₂ conditions. Our results indicating significant effect of oxygen and 3D cytoarchitecture suggest the urgent need for further optimization of the microenvironmental conditions to improve therapeutical competence of the WJ-MSCs population.

Arrays of human umbilical cord blood-neural stem cells have been patterned in high density at single cell resolution. Pre-patterns of adhesive molecules, i.e. fibronectin and poly-L-lysine, have been produced on anti-adhesive poly (ethylene) oxide films deposited by plasma-enhanced chemical vapour deposition, which prevents cell adsorption. The structures consisted of adhesive squares and lines with 10μm lateral dimensions, which correspond approximately to the size of one cell nucleus, separated by 10μm anti-adhesive gap. The stem cells cultured on these platforms redistribute their cytoplasm on the permitted areas. Spherical cells were deposited on the square patterns in a single cell mode, while on the lines they spread longitudinally; the extent of elongation being dependent on the specific (fibronectin) or non-specific (poly-L-lysine) attachment biomolecule. The cell patterns were retained up to 12 days, which will be useful for recording statistical data of individual chronic responses to chemical, physical or physiologically relevant stimuli.

Stroke poses a considerable challenge for regenerative medicine due to the complex and multidimensional specificity of the central nervous system, and cell therapy is one of the currently considered treatments. The aim of this study was to determine the pro-regenerative outcome after transplantation of preconditioned and scaffold-encapsulated mesenchymal stem/stromal cells isolated from Wharton’s Jelly (WJ-MSCs) in an experimental rat model of brain injury. For this purpose, WJ-MSCs cultured at different oxygen concentrations (21% O₂ or 5% O₂) were transplanted into the injured rat brain in saline or hydrogel scaffolds derived from human platelet lysate or fibrinogen. Using magnetic resonance imaging, we observed the signal of labelled WJ-MSCs at injection site at different time-points after transplantation. By diffusion-weighted imaging we detected the signal at the lesion site only after WJ-MSCs transplantation. Furthermore, cell transplantation resulted in a significant decrease in the extent of brain damage and dynamic change in the expression of investigated rat trophic factors (BDNF, GDNF, VEGF-A) in the brain and cerebrospinal fluid. The highest increase in this expression was observed after transplantation of physioxia-preconditioned and scaffold-encapsulated cells. The results demonstrated the regenerative effect of WJ-MSCs, which was enhanced by their transplantation within human protein-based hydrogel scaffolds in the rat model of brain injury.

Piezoelectric materials, due to their ability to generate an electric charge in response to mechanical deformation, are becoming increasingly attractive in the engineering of bone and neural tissues. This manuscript reports the effects of the addition of nanohydroxyapatite (nHA), introduction of gold nanoparticles (AuNPs) via sonochemical coating, and collector rotation speed on the formation of electroactive phases and biological properties in electrospun nanofiber scaffolds consisting of poly(vinylidene fluoride) (PVDF). FTIR, WAXS, DSC, and SEM results indicate that introduction of nHA increases the content of electroactive phases and fiber alignment. The collector rotational speed increases not only the fiber alignment but also the content of electroactive phases in PVDF and PVDF/nHA fibers. Increased fiber orientation and introduction of each of additives resulted in increased SFE and water uptake. In vitro tests conducted on MG-63 and hiPSC-NSC cells showed increased adhesion and cell proliferation. The results indicate that PVDF-based composites with nHA and AuNPs are promising candidates for the development of advanced scaffolds for bone and neural tissue engineering applications, combining electrical functionality and biological activity to support tissue regeneration.

Despite ongoing debate surrounding mesenchymal stem/stromal cells (MSCs), the number of clinical trials involving MSCs has continued to increase due to their strong therapeutic potential. These cells exhibit immunosuppressive and neuroprotective properties, making them a promising approach for treating neurodegenerative diseases. Given the growing interest in stem cell-based therapies, it is essential to establish specific protocols for manufacturing adipose-derived MSCs. This study outlines the process of obtaining an ATMP cell product from ASCs, evaluating cell quality, isolation efficiency, and optimal conditions for cryopreservation, preparation, and transport. Cells were isolated from fresh, cryopreserved, and 4°C-stored adipose tissue to evaluate how different storage conditions affect cell quality. In addition, cell viability in different transport solutions over time was examined. To evaluate the therapeutic potential of ASCs, cells were cultured in cerebrospinal fluid (CSF) and neural gene and protein expression, as well as the secretome profile, were analysed. Our findings show that isolating ASCs immediately after liposuction yields the highest viable cell count. Adipose tissue can be stored at 4 °C for up to 48 h without affecting cell population doubling time, but cryopreservation negatively impacts ASCs' clonogenicity. For optimal long-term preservation, a medium with 5 % platelet lysate and 10 % DMSO is recommended. Additionally, we found that 0.9 % NaCl is the best transport medium for maintaining cell viability. CSF exposure induced neural markers (NG2, A2B5, Nestin, β-III-tubulin, S-100-β) and morphological changes, suggesting neural-like differentiation. These findings contribute to the development of protocols for MSC therapy in neurodegenerative diseases.