DNA nanotechnology is a rapidly growing field that provides exciting tools for biomedical applications. Targeting lysosomal functions with nanomaterials, such as DNA nanostructures (DNs), represents a rational and systematic way to control cell functionality. Here we present a versatile DNA nanostructure-based platform that can modulate a number of cellular functions depending on the concentration and surface decoration of the nanostructure. Utilizing different peptides for surface functionalization of DNs, we were able to rationally modulate lysosomal activity, which in turn translated into the control of cellular function, ranging from changes in cell morphology to modulation of immune signaling and cell death. Low concentrations of decalysine peptide-coated DNs induced lysosomal acidification, altering the metabolic activity of susceptible cells. In contrast, DNs coated with an aurein-bearing peptide promoted lysosomal alkalization, triggering STING activation. High concentrations of decalysine peptide-coated DNs caused lysosomal swelling, loss of cell-cell contacts, and morphological changes without inducing cell death. Conversely, high concentrations of aurein-coated DNs led to lysosomal rupture and mitochondrial damage, resulting in significant cytotoxicity. Our study holds promise for the rational design of a new generation of versatile DNA-based nanoplatforms that can be used in various biomedical applications, like the development of combinatorial anti-cancer platforms, efficient systems for endolysosomal escape, and nanoplatforms modulating lysosomal pH.

• Keywords
• DNA nanotechnology, Interferon, Lysosomal rupture, Nanotechnology, bio/nano interactions, lysosome interference,
• Publication type
• Journal Article MeSH

The regulatory requirements in cell processing, in the choice of a biomaterial scaffold and in quality control analysis, have to be followed in the clinical application of tissue-engineered grafts. Confirmation of sterility during quality control studies requires prolonged storage of the cell-based construct. After storage, preservation of the functional properties of the cells is an important prerequisite if the cells are to be used for cell-based tissue therapies. The study presented here shows the generation of 3D constructs based on Wharton's jelly multipotent mesenchymal stromal cells (WJ-MSCs) and the clinically-acceptable HyaloFast® scaffold, and the effect of two- and six-day hypothermic storage of 3D cell-based constructs on the functional properties of populated cells. To study the viability, growth, gene expression, and paracrine secretion of WJ-MSCs within the scaffolds before and after storage, xeno-free culture conditions, metabolic, qPCR, and multiplex assays were applied. The WJ-MSCs adhered and proliferated within the 3D HyaloFast®. Our results show different viability of the cells after the 3D constructs have been stored under mild (25 °C) or strong (4 °C) hypothermia. At 4 °C, the significant decrease of metabolic activity of WJ-MSCs was detected after 2 days of storage, with almost complete cell loss after 6 days. In mild hypothermia (25 °C) the decrease in metabolic activity was less remarkable, confirming the suitability of these conditions for cell preservation in 3D environment. The significant changes were detected in gene expression and in the paracrine secretion profile after 2 and 6 days of storage at 25 °C. The results presented in this study are important for the rapid transfer of tissue engineering approaches into clinical applications.

• Keywords
• Hyalofast®, Wharton’s jelly, hypothermic storage, multipotent mesenchymal stromal cells, paracrine activity,
• Publication type
• Journal Article MeSH

Lysosome-activated apoptosis represents an alternative method of overcoming tumor resistance compared to traditional forms of treatment. Pulsed magnetic fields open a new avenue for controlled and targeted initiation of lysosomal permeabilization in cancer cells via mechanical actuation of magnetic nanomaterials. In this study we used a noninvasive tool; namely, a benchtop pulsed magnetic system, which enabled remote activation of apoptosis in liver cancer cells. The magnetic system we designed represents a platform that can be used in a wide range of biomedical applications. We show that liver cancer cells can be loaded with superparamagnetic iron oxide nanoparticles (SPIONs). SPIONs retained in lysosomal compartments can be effectively actuated with a high intensity (up to 8 T), short pulse width (~15 µs), pulsed magnetic field (PMF), resulting in lysosomal membrane permeabilization (LMP) in cancer cells. We revealed that SPION-loaded lysosomes undergo LMP by assessing an increase in the cytosolic activity of the lysosomal cathepsin B. The extent of cell death induced by LMP correlated with the accumulation of reactive oxygen species in cells. LMP was achieved for estimated forces of 700 pN and higher. Furthermore, we validated our approach on a three-dimensional cellular culture model to be able to mimic in vivo conditions. Overall, our results show that PMF treatment of SPION-loaded lysosomes can be utilized as a noninvasive tool to remotely induce apoptosis.

• Keywords
• apoptosis, lysosomal death pathways, lysosomal membrane permeabilization, magnetic nanoparticles, pulsed magnetic field,
• Publication type
• Journal Article MeSH

Cryopreservation is the only established method to provide long-term storage and fast availability of cellular product for therapeutic applications. The overwhelming majority of cryopreservation media contain toxic concentrations of dimethyl sulfoxide (DMSO) limiting the possibility for the direct administration of cryopreserved cells to the patients. Here, we propose a novel approach for nontoxic xeno-free cryopreservation of human multipotent mesenchymal stromal cells (MSCs) aimed at ensuring high viability, ready-to-use availability, and localized delivery of the cell-based graft into damaged tissues. For MSC cryopreservation, we applied sucrose pretreatment procedure and xeno-free cryoprotective medium containing human platelet-poor blood plasma (PPP), sucrose, and nontoxic concentration of DMSO. Using the combination of PPP, 0.2 M sucrose, and 1% DMSO, the recovery rate of cryopreserved MSCs reached 73% of the values obtained for noncryopreserved cells. Moreover, the presence of PPP in the cryoprotective medium provided the possibility to create a ready-to-use 3D hydrogel for the localized delivery and additional support of MSCs in vivo. In a proof-of-concept study, we assessed the regenerative capacity of cryopreserved MSCs in a full-thickness wound model in mice. The positive impact of MSCs within 3D gel on wound healing rates was confirmed by morphometric and histological examinations. Our results demonstrate the possibility to apply cryopreserved cells immediately after thawing using a cryoprotective medium as the vehicle solution.

• Publication type
• Journal Article MeSH

The transplantation of Wharton's jelly derived mesenchymal stromal cells (WJ-MSCs) possesses therapeutic potential for the treatment of a spinal cord injury (SCI). Generally, the main effect of MSCs is mediated by their paracrine potential. Therefore, application of WJ-MSC derived conditioned media (CM) is an acknowledged approach for how to bypass the limited survival of transplanted cells. In this study, we compared the effect of human WJ-MSCs and their CM in the treatment of SCI in rats. WJ-MSCs and their CM were intrathecally transplanted in the three consecutive weeks following the induction of a balloon compression lesion. Behavioral analyses were carried out up to 9 weeks after the SCI and revealed significant improvement after the treatment with WJ-MSCs and CM, compared to the saline control. Both WJ-MSCs and CM treatment resulted in a higher amount of spared gray and white matter and enhanced expression of genes related to axonal growth. However, only the CM treatment further improved axonal sprouting and reduced the number of reactive astrocytes in the lesion area. On the other hand, WJ-MSCs enhanced the expression of inflammatory and chemotactic markers in plasma, which indicates a systemic immunological response to xenogeneic cell transplantation. Our results confirmed that WJ-MSC derived CM offer an alternative to direct stem cell transplantation for the treatment of SCI.

• Keywords
• Wharton’s jelly, cell secretome, cell therapy, conditioned medium, mesenchymal stem cells, spinal cord injury,
• MeSH
• Cytokines blood   MeSH
• Rats MeSH
• Culture Media, Conditioned pharmacology   MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Mesenchymal Stem Cells cytology   MeSH
• Spinal Cord Injuries blood   physiopathology   therapy   MeSH
• Rats, Wistar MeSH
• Mesenchymal Stem Cell Transplantation * methods   MeSH
• Wharton Jelly cytology   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cytokines MeSH
• Culture Media, Conditioned MeSH