Published on 12 January 2026
This application note is based on the work of Nergishan İyisan, Oliver Hausdörfer, Chen Wang, Lukas Hiendlmeier, Philipp Harder, Bernhard Wolfrum, and Berna Özkale Edelmann, from the Technical University of Munich, who introduced the 3D-PRESS platform for mechanoactivation of single stem cells encapsulated in alginate microgels (Mechanoactivation of Single Stem Cells in Microgels Using a 3D‐Printed Stimulation Device, Small Methods, 2024). The study demonstrates how precisely controlled hydrostatic pressure can be applied to hundreds of individually encapsulated mesenchymal stem cells in parallel, enabling single-cell–resolved mechanotransduction studies in a true 3D environment. By combining microfluidic encapsulation, a transparent 3D-printed pressure chamber, and Elveflow’s OB1 pressure control technology, the platform enables stable, reproducible mechanical stimulation under live-cell imaging conditions, providing a powerful tool to monitor mechanosensitive downstream signaling events such as nuclear Yes-associated protein (YAP) translocation.
Microgels provide a tunable 3D microenvironment for single cells, enabling precise control over biochemical and biomechanical cues essential for stem-cell behavior[1],[2]. While these systems successfully mimic static aspects of native tissues, dynamic mechanical forces are also critical regulators of stem-cell function and therapeutic potential[1],[3],[4]. However, current approaches rarely achieve mechanical stimulation at single-cell resolution within a true 3D culture environment. To address this gap, the team developed 3D-PRESS, a 3D-printed pressure chamber that applies:
The platform integrates a custom-designed, transparent 3D-printed pressure chamber with the Elveflow OB1 MK3+ Pressure Controller and MPS Pressure Sensor, allowing precise, leak-free, and biocompatible hydrostatic pressure application up to 400 kPa (4 bar). The team first uses pressure-induced Ca²⁺ signaling as a rapid, live indicator that hydrostatic stimulation is sensed by singly encapsulated cells. The team then evaluates downstream mechanotransductive signaling through YAP nuclear translocation as a mechanosensitive transcriptional readout. In RGD (a cell adhesion sequence that binds to integrins) presenting microgels, integrin engagement provides a defined biochemical context for force transmission, linking applied pressure to YAP activation. Together, these readouts connect controlled hydrostatic loading to integrin-dependent signaling at the single-cell level. This configuration supports real-time mechanotransduction readouts, including pressure-induced intracellular Ca²⁺ signaling as an early indicator of mechanoactivation. This integration provides a versatile approach for single-cell mechanotransduction studies by combining:
The 3D-PRESS system (Figure 1a) integrates microfluidic single-cell encapsulation with pneumatic actuation. Individual Mesenchymal Stem Cells (MSCs) are encapsulated in monodisperse alginate microgels using a two-inlet microfluidic device, where the aqueous phase (alginate, crosslinker, and cells) meets the oil phase containing acetic acid to generate single-cell droplets collected at the outlet (Figure 1b). These microgels are then transferred into a transparent, 3D-printed pressure chamber featuring a bayonet-lock lid and O-ring seal to ensure secure, leak-free operation (Figure 1c).
Overall, the experimental setup incorporates (Figure 1d):
The Elveflow OB1 MK3+ provides precisely regulated air pressure to the chamber, monitored via a MPS sensor. The device can be positioned directly on a stage-top incubator, enabling real-time live-cell imaging during pressure application, as shown in the complete setup photographs (Figure 1e). The entire setup allows in situ observation of hundreds of individually encapsulated cells during mechanical stimulation, while maintaining full biocompatibility and sterility.
Figure 1. (a) Schematic of the 3D-PRESS platform showing single MSCs encapsulated in alginate microgels and stimulated by cyclic hydrostatic pressure. (b) Microfluidic encapsulation using a two-inlet device producing single-cell alginate microgels. (c) CAD design of the pressure chamber with and without the lid, featuring a bayonet lock and O-ring seal. (d) Experimental setup with the 3D-PRESS chip, Elveflow OB1 pressure controller, and MPS pressure sensor. (e) Photographs of the complete setup, 3D-PRESS chip positioned on the microscope stage.
3D-PRESS chambers were SLA-printed directly onto A174-silanized glass slides (76 × 52 × 1 mm) using a Miicraft 50 printer and Medicalprint Mould Clear 2.0 resin. Prints were cleaned in isopropanol, baked at 80 °C, and UV-cured in an Otoflash G171. Then, the protocol involved plasma cleaning the glass-printed parts, sterilization in 70% ethanol, and coating with 10 µg/cm² poly-D-lysine prior to use.
PDMS microfluidic chips (SU-8 3050 master) were fabricated by soft lithography, bonded to plasma-activated glass, hydrophobized with RainX, and sterilized. In addition, D1 ORL UVA MSCs were mixed with CaCO₃ (10 mg/mL) and functionalized alginate. The protocol infused the aqueous phase containing alginate and cells, and the oil phase with surfactant and 0.04% acetic acid, into the two-inlet device using a syringe pump at 1.7 µL/min. Finally, emulsions were collected, demulsified with PFO, and microgels were transferred to culture media.
3D-PRESS devices were connected to an OB1 MK3+ controller through 0.2 µm filters, with pressure monitored via a MPS sensor. Accordingly to the protocole, square-wave profiles (25–400 kPa, 10 Hz) were applied during live imaging. For YAP assays, encapsulated MSCs were stimulated at 200 kPa (0.5 Hz, 30 min) on a 37 °C, 5% CO₂ stage-top incubator and then fixed immediately or after 24 h.
D1 ORL UVA MSCs were cultured in DMEM + 10% FBS + 1% pen/strep. Calcium imaging used 4.5 µM Calbryte 520 AM in isotonic solution. For YAP analysis, cells were fixed, permeabilized, blocked, stained with YAP1 primary and Alexa Fluor 647 secondary antibodies, and counterstained with DAPI.
The OB1 MK3+ Pressure Controller delivers controlled pressure through the medium to each encapsulated single cell. In consequence, this isotropic stimulation enables consistent force transmission across all microgels, ensuring that every cell experiences the same mechanical load.
The 3D-PRESS platform delivers precise and stable hydrostatic pressure profiles that closely follow the programmed square-wave input with minor delay (Figure 2a). MSCs exposed to 25 and 200 kPa for 30 minutes maintained high viability, indicating that moderate pressure loading is well tolerated (Figure 2b). At 400 kPa, viability decreased, demonstrating that very high pressure begins to impose mechanical stress on the cells. Then, live/dead imaging confirmed these trends, with predominantly live cells at lower pressures and a visibly increased number of dead cells at 400 kPa (Figure 2c).
Together, these findings establish that 3D-PRESS supports healthy cell behavior under moderate stimulation while enabling controlled exploration of cellular limits under higher mechanical loads.
Figure 2. (a) Pressure profiles showing the programmed set value (continuous line) and the measured pressure (dotted line) by the inline sensor. (b) Cell viability at t=0 and t=24 hours following stimulation at 25, 200, and 400 kPa. (c) Live (green) and dead (red) fluorescent images of cells in the pressure device under control, 25, 200, and 400 kPa conditions, immediately after stimulation and after 24 hours. Scale bar: 200 µm.
Live-cell calcium imaging was used as an initial qualitative readout to verify whether hydrostatic pressure elicits an immediate mechanosensitive response. In that context, MSCs stimulated at 200 and 400 kPa showed a rapid and pronounced rise in Calbryte fluorescence within seconds, whereas 25 and 50 kPa produced minimal activation in both monolayer and encapsulated format (Figure 3a,b). In parallel, encapsulated MSCs exhibited substantially stronger calcium responses than monolayer cells indicating that 3D culture might cause higher mechanosensitivity (Figure 3c). These findings show that 3D-PRESS triggers rapid mechanosensitive calcium signaling and that encapsulation in microgels markedly alters the magnitude of the cellular calcium response.
Figure 3. (a) Fluorescence images showing intracellular calcium changes in monolayer MSCs at 50, 200, and 400 kPa, captured at t = 0, 5 s, 10 s, and 5 min. (b) Fluorescence images of encapsulated MSCs at 50, 200, and 400 kPa, captured at t = 0, 5 s, 10 s, and 5 min. Red indicates Rhodamine B–functionalized microgels. Green indicates Calbryte staining. Scale bar: 100 µm. (c) Maximum Calbryte fluorescence intensity in monolayer (n = 30) and encapsulated (n = 10) MSCs across all pressure values. ***p < 0.001
The experimental timeline shows that encapsulated MSCs were stimulated at 200 kPa for 30 minutes and fixed either immediately or after 24 hours to assess YAP translocation (Figure 4a). Quantification demonstrated that pressure stimulation significantly increased the YAP nuclear-to-cytoplasmic ratio in RGD+ microgels, with higher values observed at both timepoints compared to their unstimulated controls (Figure 4b). Here, RGD microgels refer to alginate matrix functionalized with the integrin-binding RGD peptide, providing cell adhesion sites for cell–matrix attachment. Notably, RGD-presenting microgels showed the greatest increase, indicating ligand-dependent sensitivity to applied pressure. Representative fluorescence images confirm these trends, displaying clear YAP nuclear enrichment after stimulation in both microgel types, particularly at 24 hours (Figure 4c).
Figure 4. (a) Experimental timeline. Cells were stimulated for 30 min at 200 kPa and fixed either immediately (t = 0 h) or after 24 h (t = 24 h). (b) YAP nucleus-to-cytoplasm ratios at t = 0 h and t = 24 h for single cells in RGD-absent (RGD–) and RGD-presenting (RGD+) microgels. Plain bars show unstimulated controls; striped bars show samples stimulated at 200 kPa. (c) Fluorescence images of single MSCs in RGD– and RGD+ microgels at t = 24 h, with and without 200 kPa stimulation. DAPI (blue) marks nuclei, YAP (green) shows intracellular localization, and Rhodamine B–labeled alginate (red) denotes microgels.
The 3D-PRESS platform enables precise and reproducible mechanical stimulation of hundreds of individually encapsulated stem cells, supported by stable pressure regulation from the OB1 MK3+ system. According to the experimental data, cell viability remained high after stimulation, while encapsulated MSCs showed markedly stronger calcium responses compared to 2D cultures. In addition, pressure-induced YAP nuclear translocation occurred only in RGD-presenting microgels, confirming integrin-dependent mechanotransduction at the single-cell level.
Building on these findings, a recently published work (Hydrostatic Pressure Induces Osteogenic Differentiation of Single Stem Cells in 3D Viscoelastic Microgels, Small Science, 2025) from the same group demonstrated that cyclic hydrostatic pressure applied to RGD-functionalized microgels can drive osteogenic differentiation of single MSCs solely through mechanical cue.
Overall, this integrated approach provides a controlled and biocompatible method for studying single-cell mechanosensitivity and mechanically directed lineage specification in 3D microenvironments, where defined mechanical regimes can be systematically applied to precondition stem cells for downstream therapeutic use.
This work was supported by the ONE MUNICH Project, Munich Multiscale Biofabrication, funded by the Federal Ministry of Education and Research (BMBF) and the Free State of Bavaria under the Excellence Strategy of the Federal Government and the Länder. The authors also acknowledge funding from the Bavarian High-Tech Agenda.
Written and reviewed by Nergishan İyisan, Berna Özkale Edelmann and Louise Fournier, PhD. For more content about microfluidics, you can have a look here.
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