Biology subjects

Stepien, E.

Publications and source records attributed to Stepien, E..

4 recordsLinked to original sources

bioRxiv2025

Small extracellular vesicles reflect senescence progression in human bone marrow-derived mesenchymal stem cells during hollow fiber bioreactor culture

Prolonged three-dimensional culture exposes stem cells to sustained microenvironmental and mechanical stresses that can promote aging- and senescence-associated phenotypic alterations. This study examined how long-term expansion of human bone marrow-derived mesenchymal stem cells (BMSCs) in a hollow fiber bioreactor (HFB) influences cellular senescence and the molecular composition of secreted small extracellular vesicles (sEVs). During extended HFB culture, BMSCs exhibited progressive morphological flattening and cytoskeletal disorganization, accompanied by increased senescence-associated {beta}-galactosidase activity and immunophenotypic remodeling characterized by reduced fluorescence intensity and spatial redistribution of canonical MSC markers, consistent with a stress-adapted, early senescence-associated cellular state. In parallel, sEVs were collected longitudinally over 40 days and characterized by nanoparticle tracking analysis, immunoblotting, and quantitative proteomics. While vesicle size, marker expression, and yield remained stable throughout culture, proteomic profiling revealed pronounced, phase-dependent remodeling of sEV cargo, including coordinated alterations in oxidative stress-related processes, lysosomal and extracellular matrix-associated pathways, and relative depletion of cytoskeletal and translational components. Notably, these vesicular signatures closely mirrored senescence-associated changes observed at the cellular level. The strong correspondence between cellular phenotypes and sEV proteomic profiles establishes vesicle analysis as a convergent and noninvasive readout of BMSC aging, enabling sensitive monitoring of senescence progression while reducing reliance on parallel, labor-intensive cellular assays. Collectively, these findings indicate that prolonged HFB culture promotes a controlled, stress-associated senescence program in BMSCs and position sEV proteomic profiling as a robust approach for assessing stem cell aging dynamics during long-term three-dimensional bioreactor culture.

molecular biology↗

bioRxiv2025

Fresh Paraformaldehyde Preserves Thrombus Biochemistry - An ATR-FTIR and PCA Investigation of Pulmonary Artery Thrombi

BackgroundBiochemical analyses of pulmonary embolism derived thrombi depend critically on fixative quality. ObjectiveTo quantify the impact of paraformaldehyde (PFA) shelf life on thrombus molecular integrity using attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and time-of-flight secondary ion mass spectroscopy (SIMS) combined with principal component analysis. MethodsTen pulmonary artery thrombi were fixed in either aged PFA (6 months; n = 6) or freshly prepared 4% PFA (n = 4); an in-vitro plasma clot control received the same fresh PFA. Triplicate ATR-FTIR spectra (400-4000 cm-1) were collected and vector-normalized to the Amide I band. ToF-SIMS measurements were performed using a 30 keV Bi primary ion beam Fixative chemistry was assessed by the carbonyl absorbance at 1700 cm-1. ResultsFreshly fixed thrombi displayed narrow Amide I (~1650 cm-1) and Amide II (~1540 cm-1) bands with a higher signal-to-noise ratio (SNR; median 41.8) than aged-PFA samples (18.2; P < .01). Carbonyl absorbance at 1700 cm-1 was markedly higher in fresh PFA. Aged PFA introduces chemical variances of biological samples in low-mass molecule fragments. PCA showed clear separation of fresh versus aged specimens and alignment of fresh thrombi with plasma controls across spectral windows. ConclusionsPFA solutions older than three months markedly deteriorate thrombus biochemical fidelity. ATR-FTIR and ToF-SIMS offer a rapid quality-control assay prior to molecular analyses. Key pointsO_LIPFA stored [≥]3 months loses >70% of reactive aldehydes, yielding broad Amide envelopes, attenuated lipid bands and a two fold SNR drop. C_LIO_LIAged PFA introduces chemical variances of biological samples in low-mass molecule fragments. C_LIO_LIPCA of fingerprint, protein or lipid windows cleanly separates aged from fresh fixations (PC1 up to 80% variance). C_LIO_LIA rapid QC workflow (carbonyl absorbance [≥]0.25 a.u. plus PCA verification) safeguards molecular integrity irrespective of patient age, DVT history or clinical severity. C_LIO_LIAdopting this workflow will harmonise multi centre clot biorepositories and enhance the reproducibility of proteomic, lipidomic and imaging studies. C_LI

biophysics↗

bioRxiv2022

3D melanoma spheroid model for the development of positronium biomarker

It was recently demonstrated that newly invented positronium imaging may be used for improving cancer diagnostics by providing additional information about tissue pathology with respect to the standardized uptake value currently available in positron emission tomography (PET). Positronium imaging utilizes properties of a positronium atoms, which are built from the electron and positron produced in the body during PET examinations. We hypothesized whether positronium imaging would be sensitive to in vitro discrimination of tumour-like three-dimensional structures (spheroids) build of melanoma cell lines with different cancer activity and biological properties. The lifetime of ortho-Positronium (o-Ps) was evaluated in melanoma spheroids from two cell lines (WM266-4 and WM115) differing in the stage of malignancy. Additionally, we considered such parameters: as cell size, proliferation rate and malignancy to evaluate their relationship with o-Ps lifetime. We demonstrate the pilot results for the o-Ps lifetime measurement in extracellular matrix free spheroids. With the statistical significance of two standard deviations, we demonstrated that the higher the degree of malignancy and the rate of proliferation of neoplastic cells the shorter the lifetime of ortho-positronium. In particular we observed following indications encouraging further research: (i) WM266-4 spheroids characterized with higher proliferation rate and malignancy showed shorter o-Ps lifetime compared to WM115 spheroids characterized by lower growth rate, (ii) Both cell lines showed a decrease in the lifetime of o-Ps after spheroid generation in 8th day comparing to 4th day in culture and the mean o-Ps lifetime is longer for spheroids formed from WM115 cells than these from WM266-4 cells, regardless spheroid age. The results of these study revealed that positronium is a promising biomarker that may be applied in PET diagnostics for the assessment of the degree of cancer malignancy.

biophysics↗

bioRxiv2021

Ectosome effect on endothelial monolayers in hyperglycemic and normoglycemic conditions

Extracellular vesicles, namely those larger ones - Ectosomes (Ect), are thought to be important cell-to-cell communication medium. Ect are considered as a potential therapeutic for type-1 and type-2 diabetes mellitus. Ect can be internalized by endothelial cells and, owing to their cargo, they modulate targeted cell behavior. Under hyperglycemic conditions (HGC), endothelial cells changed their properties and became stiffer and less mobile which causes endothelial dysfunction and abnormalities in micro- and macrovascular systems. The aim of this study was to find whether Ect restore mobility and motility of macrovascular endothelial cells under HGC. Uptake of Ect, cell morphology, cytoskeleton organization and membrane stiffness (by atomic force microscopy) were analyzed after the exposure to isolated Ect. To find which cellular pathways were deregulated by HGC and whether Ect could potentially restore gene expression profile, transcriptome analysis was done. We observed that endothelial cells internalized more Ect under normoglycemic conditions (NGC) then HGC. Hyperglycemic cells (HG) were bigger and showed the stiffer surface with denser actin cytoskeleton in comparison to normoglycemic cells. Number of metabolic pathways was influenced under HGC, especially those related to intracellular transport, metabolism and cellular component organization and Ect did not restore HGC impaired cell signaling. Ectosomes cannot reverse this harmful effect of hyperglycemia in endothelial cells, which can have clinical implication in use Ect as therapeutic target in diabetes treatment.

cell biology↗