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
- 1Leibniz University Hannover
Abstract
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.
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BibTeX
@misc{hussein2026direct,
title = {{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}},
author = {N. Hussein and C. Miller and Y. Roger and L. Venturini and M. Stadler and M. Haertle and A. Lavrentieva and N. Haase and S. Rudorf and A. Hoffmann and D. Egger},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.10.758039},
url = {https://www.biorxiv.org/content/10.64898/2026.10.10.758039v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - 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
AU - N. Hussein
AU - C. Miller
AU - Y. Roger
AU - L. Venturini
AU - M. Stadler
AU - M. Haertle
AU - A. Lavrentieva
AU - N. Haase
AU - S. Rudorf
AU - A. Hoffmann
AU - D. Egger
PY - 2026
DA - 2026/10/10
PB - bioRxiv
DO - 10.64898/2026.10.10.758039
UR - https://www.biorxiv.org/content/10.64898/2026.10.10.758039v1
AB - 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.
ER -