Lavrentieva, A. | bioRxiv Science

bioRxiv2026

Direct 3D isolation in gelatin methacryloyl microbeads versus conventional 2D isolation and culture: a trade-off between mesenchymal stromal cell purity and niche-associated transcriptional profiles

Bone-marrow mesenchymal stromal cell (MSC) isolation typically couples plastic-adherence selection to planar expansion. Therefore, the recovered cells may reflect culture adaptation as much as native stromal biology. Direct isolation within a three-dimensional matrix could reduce planar selection, but its effects on cellular recovery, composition and MSC transcriptional profiles remain insufficiently resolved. We therefore developed a gelatin methacryloyl (GelMA) workflow progressing from bulk hydrogels to medium-surrounded GelMA microbeads (GMBs) and compared 3D-derived cells with conventional 2D cultures. Qualitative live/dead staining with calcein acetoxymethyl ester (CAM) and propidium iodide (PI) and 5-ethynyl-2'-deoxyuridine (EdU) imaging established cellular activity in both GelMA formats. In bulk GelMA cultures from four human donors, CD73-, CD90- and CD105-associated flow-cytometric readouts did not increase from day 7 to day 21. At day 21, the combined marker readout was numerically higher in donor-matched 2D cultures than in bulk 3D cultures, which retained heterogeneous marker-defined populations. GMBs enabled cell recovery and flow-cytometric characterization and motivated an exploratory one-donor single-cell RNA sequencing (scRNA-seq) analysis. The final dataset contained 37,784 cells across four samples and 23 annotated populations. MSC-focused re-embedding resolved 13,175 cells into two 3D and four 2D clusters. A pooled comparison identified 4,840 differentially expressed genes (DEGs, false discovery rate [FDR]<0.01, |log2FC|[≥]1), indicating a broad method-associated expression shift. The 3D C1 cluster combined progenitor-, extracellular-matrix-, adhesion-, cytokine-, hypoxia/glycolysis- and predicted-interaction-associated programmes, whereas 2D clusters showed mitochondrial, cytoskeletal/mechanoadaptive and proliferative programmes. Thus, 2D culture yielded a more strongly represented and compositionally purer MSC compartment, while 3D isolation contained rare MSC clusters with niche-associated transcriptional profiles within a heterogeneous marrow environment. These findings support a testable native-associated tendency for 3D, but donor replication, matched recovery and direct native-reference mapping are required before inferring greater in-vivo similarity or functional superiority.

cell biology↗

bioRxiv2026

Overcoming oxygen impermeability in PDMS-free organ-on-a-chip microfluidics with nanoporous thermoplastic

Oxygen availability is a critical yet all-too-often overlooked variable in organ-on-a-chip (OoC) systems. PDMS-based microfluidics remain the most common approach to facilitating oxygen equilibration with the incubator environment, but the materials tendency to ad- and absorb small hydrophobic molecules can pose significant concerns for pharmacological and toxicological studies. Yet there remains a lack of alternative gas-exchange materials feasible for OoC integration, even as the use of thermoplastic microfluidics in particular has otherwise proliferated. Here, we present commercially available track-etched nanoporous polycarbonate (50 nm pores, 1.18% porosity, [~]0.1 {euro}/cm2) as a practical alternative to polydimethylsiloxane (PDMS) for gas exchange in OoC. We show that nanoporous polycarbonate provides a thermoplastic material with an oxygen permeability of 3290 {+/-} 240 fs mol / kg, over an order of magnitude higher than PDMS. We demonstrate integration into existing lamination-based thermoplastic microfluidic fabrication workflows with sustained leak-free operation well above physiologically relevant pressures. We find that nanoporous polycarbonate does not compromise cell viability, but that high water vapor permeance necessitates a high-humidity environment around the device - though thickness-normalized water vapor permeability is notably similar to PDMS. We validate the OoC application with Caco-2 intestinal epithelial cells by monitoring oxygen levels during the critical cell attachment phase, with nanoporous polycarbonate allowing for maintenance of stable oxygen tension, in stark contrast to severe hypoxia in nonporous controls within 30 minutes. We further show that this uncontrolled hypoxia correlates with a time-delayed increase in cellular hypoxia inducible factor-1 reporter expression. Overall, our findings position nanoporous polycarbonate as a low-cost, mechanically robust, and fabrication-friendly alternative that can bring controlled oxygen availability to PDMS-free microfluidics and OoC.

bioengineering↗