Spheroid Assembly in Microwells of Defined Geometry for Quantitative Assessment of Visco-Capillary Velocity and Shape Control

Efremov, Y. M.; Makarova, E. Y.; Koteneva, P. I.; Golubchikov, D. O.; Yanbarisov, R. M.; Vassilevski, Y. V.; Kosheleva, N. V.; Timashev, P. S.

doi:10.1101/2025.09.15.676122

bioRxiv · 10.1101/2025.09.15.676122

Spheroid Assembly in Microwells of Defined Geometry for Quantitative Assessment of Visco-Capillary Velocity and Shape Control

Efremov, Y. M.·Makarova, E. Y.·Koteneva, P. I.·Golubchikov, D. O.·Yanbarisov, R. M.·Vassilevski, Y. V.·Kosheleva, N. V.·Timashev, P. S.

View original source ↗Export metadata ↓

Abstract

Three-dimensional (3D) cell spheroids are widely used as in vitro tissue models, yet quantitative understanding of their morphogenesis remains limited. We present an integrated experimental-computational framework to analyze, model, and modulate the compaction of cell aggregates in agarose microwells of defined geometries. Custom 3D-printed stamps produced circular, square, and triangular microwells of equal cross-sectional area. Time-lapse imaging combined with AI-based segmentation enabled tracking of spheroid morphology, with circularity and projected area serving as quantitative descriptors of compaction. The process followed predictable exponential kinetics, with mesenchymal (HDF) spheroids compacting faster than epithelial (ARPE-19) ones. Computational fluid dynamics (CFD) simulations modeled spheroid rounding as a visco-capillary-driven process, where the extracted visco-capillary velocity unified experimental and simulated dynamics. Mechanical measurements by atomic force microscopy and compression confirmed that differences in surface tension predominantly governed the observed kinetics. Pharmacological modulation of cytoskeletal tension revealed that inhibition of contractility markedly altered spheroid formation dynamics, enabling the generation of stable, non-spherical aggregates. Using this principle as a shape-engineering strategy, we produced aggregates with distinct geometries (brick-like, prismatic, and star-shaped), characterized by an increased surface-to-volume ratio compared to conventional spheroids. Limitations of the approach include the use of pharmacological cytoskeletal modulation and constraints in geometric fidelity arising from printing resolution, agarose casting, cell filling, and intrinsic smoothing of sharp features during cell aggregation. Collectively, this work establishes a geometry-controlled platform for quantitative analysis of spheroid formation and mechanical behavior, and provides a versatile framework for designing cell aggregates with defined shapes.

Source connections

Published article ↗

Explore related subjects

biophysics Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Efremov, Y. M., Makarova, E. Y., Koteneva, P. I., Golubchikov, D. O., Yanbarisov, R. M., Vassilevski, Y. V., Kosheleva, N. V., Timashev, P. S.. 2025-09-17. Spheroid Assembly in Microwells of Defined Geometry for Quantitative Assessment of Visco-Capillary Velocity and Shape Control. https://doi.org/10.1101/2025.09.15.676122

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

A quantum state of mitochondria in the living cell

The high energy-efficiency of life is hard to understand only with classical physics. Many efforts have been made to study its mechanism based on quantum mechanics; the progress is nevertheless slow due to lack of experimental evidence with living cells. Here, combining experiments on cells, tissues and mitochondria with a theoretical model, we demonstrate a quantum state of mitochondria, which can be employed to modulate ATP production in living cells. We found an anomalous 71.0-THz oscillation mode only in living cells and tissues, which is highly determined by intact structure of mitochondria, and cannot be assigned to any specific molecules. Based on experimental data, a quantum model of light-matter coupling was introduced to trace the origin of this mode. Our calculations suggest a quantum superposition state of functional mitochondrion that forms by the coupling of light and lipid CH2 bonds in functional cristae, and induces a splitting of the intrinsic CH2 vibration mode of 87 THz to two levels at 71 THz and 103 THz, respectively. The former can be observed only in living cells and tissues; whereas the latter falls in the range (90-110 THz) of biomolecular and water vibrations, thus indistinguishable. Additional experiments revealed this mitochondrial quantum state able to serve as an efficient channel to modulate ATP production. Our findings provide a quantum mechanics view for understanding living cells, and it will be interesting to further explore whether such quantum state could act as a channel for energy metabolism, and even information transmission in life.

biophysics↗

bioRxiv2026

Mechanistical and structural basis of Kv channel inhibition by 4 aminopyridine

Inhibition of Kv channels by 4-aminopyridine (4AP) improves motor function in multiple sclerosis by enhancing neuronal excitability. The mechanism of inhibition and the structural basis of 4AP binding to Kv channels remain unclear. Here, we determined the structure of the Shaker V369I-I372L-S376T (ILT) mutant bound to 4AP at 3.3 [A], demonstrating that 4AP binds to the closed state of the channel. This structure is inconsistent with an open channel block mechanism. Electrophysiology experiments show that 4AP binds even when intracellular pore access is constitutively blocked, suggesting that 4AP enters the pore through membrane-facing fenestrations. MD simulations and mutational analysis agree with the proposed fenestration pathway and suggest that 4AP binds in its neutral form. These results support a mechanism where 4AP binds to a partially activated closed state that prevents complete activation of Kv channels, explaining its pharmacological activity.

biophysics↗

bioRxiv2026

Substrate binding reorganizes the energetic landscape of Plasmodium falciparum hexose transporter PfHT1

Malaria parasites depend on the Plasmodium falciparum hexose transporter PfHT1 for sugar uptake, yet how substrate binding reshapes transporter energetics and kinetics of sugar transport remains poorly understood. Here, we investigate how glucose reorganizes the conformational landscape, transition pathways, and residue interaction network underlying membrane transport. Using over 800 s of adaptive molecular dynamics simulations combined with Markov state models, transition-path theory, residue-contact analysis, and graph attention learning, we reconstruct the apo and glucose-bound conformational cycles. We show that glucose selectively stabilizes productive outward-facing, occluded, and inward-facing conformations, reshapes transition kinetics, and channels reactive flux through the occluded state. We identify TM7b helix cracking as a local structural transition coupled to extracellular-gate closure and substrate progression, providing a flexible connection between the binding pocket and global alternating access. Experimental testing of mechanistically critical residues validated their functional importance in PfHT1-dependent sugar utilization. Together, these results show how substrate binding reorganizes the energetic, kinetic, and interaction landscape of membrane transport and establish a transferable framework for studying transporter mechanisms.

biophysics↗