Blood cell analysis is one of the standard clinical tests. Despite the widespread use of exogenous markers for blood cell quantification, label-free optical methods are still of high demand due to their possibility for in vivo application and signal specific to the biochemical state of the cell provided by native fluorophores. Here we report the results of blood cell characterization using label-free fluorescence imaging techniques and flow-cytometry. Autofluorescence parameters of different cell types -white blood cells, red blood cells, erythrophagocytic cells -are assessed and analyzed in terms of molecular heterogeneity and possibilities of differentiation between different cell types in vitro and in vivo.
Extracellular matrix (ECM) provides both structural support and dynamic microenvironment for cells regulating their behavior and fate. As a critical component of stem cell niche ECM maintains stem cells and activates their proliferation and differentiation under specific stimuli. Mesenchymal stem/stromal cells (MSCs) regulate tissue-specific stem cell functions locating in their immediate microenvironment and producing various bioactive factors, including ECM components. We evaluated the ability of MSC-produced ECM to restore stem and progenitor cell microenvironment in vitro and analyzed the possible mechanisms of its effects. Human MSC cell sheets were decellularized by different agents (detergents, enzymes, and apoptosis inductors) to select the optimized combination (CHAPS and DNAse I) based on the conservation of decellularized ECM (dECM) structure and effectiveness of DNA removal. Prepared dECM was non-immunogenic, supported MSC proliferation and formation of larger colonies in colony-forming unit-assay. Decellularized ECM effectively promoted MSC trilineage differentiation (adipogenic, osteogenic, and chondrogenic) compared to plastic or plastic covered by selected ECM components (collagen, fibronectin, laminin). Interestingly, dECM produced by human fibroblasts could not enhance MSC differentiation like MSC-produced dECM, indicating cell-specific functionality of dECM. We demonstrated the significant integrin contribution in dECM-cell interaction by blocking the stimulatory effects of dECM with RGD peptide and suggested the involvement of key intracellular signaling pathways activation (pERK/ERK and pFAK/FAK axes, pYAP/YAP and beta-catenin) in the observed processes based on the results of inhibitory analysis. Taken together, we suppose that MSC-produced dECM may mimic stem cell niche components in vitro and maintain multipotent progenitor cells to insure their effective response to external differentiating stimuli upon activation. The obtained data provide more insights into the possible role of MSC-produced ECM in stem and progenitor cell regulation within their niches. Our results are also useful for the developing of dECM-based cell-free products for regenerative medicine.
Objective Chondrospheres represent a variant of tissue spheroids biofabricated from chondrocytes. They are already being used in clinical trials for cartilage repair; however, their biomechanical properties have not been systematically investigated yet. The aim of our study was to characterize chondrospheres in long-term in vitro culture conditions for morphometric changes, biomechanical integrity, and their fusion and spreading kinetics. Results It has been demonstrated that the increase in chondrospheres secant modulus of elasticity is strongly associated with the synthesis and accumulation of extracellular matrix. Additionally, significant interplay has been found between biomechanical properties of tissue spheroids and their fusion kinetics in contrast to their spreading kinetics. Conclusions Extracellular matrix is one of the main structural determinants of chondrospheres biomechanical properties during chondrogenic maturation in vitro. The estimation of tissue spheroids' physical behavior in vitro prior to operative treatment can be used to predict and potentially control fusogenic self-assembly process after implantation in vivo.
Developing bone scaffolds can greatly improve the patient’s quality of life by accelerating the rehabilitation process. In this paper, we studied the process of composite polycaprolactone supercritical foaming for tissue engineering. The influence of graphene oxide and reduced graphene oxide on the foaming parameters was studied. The structural and mechanical properties were studied. The scaffolds demonstrated mechanical flexibility and endurance. The co-culturing and live/dead tests demonstrated that the obtained scaffolds are biocompatible. Different composite scaffolds induced various surface cell behaviors. The experimental data demonstrate that composite foams are promising candidates for in vivo medical trials.
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