Biology subjects

Stridfeldt, F.

Publications and source records attributed to Stridfeldt, F..

4 recordsLinked to original sources

bioRxiv2025

LoAR-Exo and HiAR-Exo: One-step label-free isolation of extracellular vesicles using inertial microfluidic devices

Extracellular vesicles (EVs) are lipid membrane-bound nanoscale (20 nm - 1000 nm) objects shed by all cells. As EVs play an important role in intercellular signaling, these have emerged as promising biomarkers for many diseases including cancer and neurodegenerative disorders. A major bottleneck in research into EVs and their smooth translation to clinic as disease biomarkers is a lack of access to affordable and user-friendly technologies to quickly isolate EVs from complex biological and clinical samples with acceptable purity and yield. Here, we report two different designs of inertial microfluidic devices (e.g. LoAR-Exo and HiAR-Exo) that can isolate EVs from different kinds of biological samples in a single label-free step using a spiral microchannel design with different aspect ratios. LoAR-Exo has a height-to-width aspect ratio [~] 1, while HiAR-Exo has a height-to-width aspect ratio of 2.5, optimized by COMSOL simulations. We tested both designs by separating polystyrene particles of size < 1 m from a heterogeneous mixture. We then benchmarked the performance of the microfluidic chips against ultracentrifugation and a precipitation kit by isolating EVs from the cell culture-conditioned media (CCM) of MDA-MB-231 cells. We also compared the microchip with SEC by using the CCM from H1975/OR cells. The size distribution of EVs isolated by the microfluidic chip was comparable with ultracentrifugation, precipitation and SEC. In summary, both devices isolated EVs from as little as 1 mL of sample volume using a label-free technique in a continuous manner and without any user intervention. Both microfluidic platforms offer a simple, efficient, and scalable alternative to conventional methods for EV isolation.

bioengineering↗

bioRxiv2024

Surface adhesion and membrane fluctuations influence the elastic modulus of extracellular vesicles

Elastic properties of nanoscale extracellular vesicles (EVs) are believed to influence their cellular interactions, thus having a profound implication in intercellular communication. Yet, an accurate quantification of the elasticity of such small lipid vesicles is difficult even with AFM-based nanoindentation experiments as it crucially depends on the reliability of the theoretical interpretation of such measurements. Here we describe a complete method composed of theoretical framework, experimental procedure, and appropriate statistical approach for an accurate determination of bending modulus and effective elastic modulus of EVs. Further, we experimentally demonstrate that the quantification of EVs by the elastic modulus from AFM-based force spectroscopy measurement is marred by the interplay of their compositionally inhomogeneous fluid membrane with the adhesion forces from the substrate and thermal effects - two exquisite phenomena that could thus far only be theoretically predicted. The effects result in a large spreading of elastic modulus even for a single EV. Our unified model is then applied to genetically engineered classes of EVs to understand how the alterations in tetraspanin expression may influence their elastic modulus.

biophysics↗

bioRxiv2022

Analyses of single extracellular vesicles from non-small lung cancer cells to reveal effects by Epidermal growth factor inhibitor treatments

Precision cancer medicine have changed the treatment landscape of non-small cell lung cancer (NSCLC) as illustrated by tyrosine kinase inhibitors (TKIs) towards mutated Epidermal growth factor receptor (EGFR). Yet, responses to such TKIs e.g., erlotinib and osimertinib among patients are heterogenous and there is a need for non-invasive blood-based analytics to follow treatment response and reveal resistance to improve patients treatment outcome. Recently, extracellular vesicles (EVs) have been identified as an important source of tumor biomarkers promising to revolutionize liquid biopsy-based diagnosis of cancer. However, high heterogeneity has been a major bottleneck. The pathological signature is often hidden in the differential expression of membrane proteins in a subset of EVs which are difficult to identify with bulk techniques. Using a fluorescence-based approach, we for the first time demonstrate that the single-EV technique can be used to monitor the treatment response of targeted cancer therapies such as TKIs towards EGFR. To test the hypothesis, we analyzed the membrane proteins of native EVs extracted from EGFR-mutant NSCLC cell line, both prior and post treatment with EGFR-TKIs erlotinib or osimertinib. The selected cell line being refractory to erlotinib and responsive to osimertinib makes it a suitable model system. The expression level of five surface proteins; two common tetraspanins (CD9, CD81) and three markers of specific interest in lung cancer (EGFR, PD-L1, HER2) were studied. The data suggest that in contrast to erlotinib, the osimertinib treatment increases the population of PD-L1, EGFR and HER2 positive EVs while the expression level per EV decreases for all the three markers. The PD-L1 and HER2 expressing EV population seems to increase by several fold because of osimertinib treatment. The observations agree with the previous reports performed on cellular level indicating the biomarker potential of EVs for liquid-biopsy based monitoring of targeted cancer treatments. HighlightsO_LIMembrane protein analyses of single EVs may reveal distinct differences when lung cancer cells are refractory vs responsive under different EGFR-TKI treatments. C_LIO_LIComparison of 1st generation erlotinib and 3rd generation osimertinib shows clear signature on the expression of PD-L1, EGFR, HER2 on single EVs C_LIO_LIColocalization showed a change in common marker combinations before after treatment. C_LIO_LIPD-L1 expression per vesicle decreases while the number of PD-L1 positive EVs increases as a result of osimertinib treatment, indicating that such signature may not be detectable under bulk analysis C_LI

bioengineering↗

bioRxiv2021

Multiparametric Profiling of Single Nanoscale Extracellular Vesicles by Combined Atomic Force and Fluorescence Microscopy: Correlation and Heterogeneity in Their Molecular and Biophysical Features

Being a key player in intercellular communications, nanoscale extracellular vesicles (EVs) offer unique opportunities for both diagnostics and therapeutics. However, their cellular origin and functional identity remain elusive due to the high heterogeneity in their molecular and physical features. Here, for the first time, multiple EV parameters involving membrane protein composition, size and mechanical properties on single small EVs (sEVs) are simultaneously studied by combined fluorescence and atomic force microscopy. Furthermore, their correlation and heterogeneity in different cellular sources are investigated. The study, performed on sEVs derived from Human Embryonic Kidney 293, Cord Blood Mesenchymal Stromal and Human Acute Monocytic Leukemia cell lines, identifies both common and cell line-specific sEV subpopulations bearing distinct distributions of the common tetraspanins (CD9, CD63 and CD81) and biophysical properties. Although the tetraspanin abundances of individual sEVs are independent of their sizes, the expression levels of CD9 and CD63 are strongly correlated. A sEV population co-expressing all the three tetraspanins in relatively high abundance, however, having on average diameters <100 nm and relatively low Young moduli, is also found in all cell lines. Such a multiparametric approach is expected to provide new insights regarding EV biology and functions, potentially deciphering unsolved questions in this field.

bioengineering↗