Cancer cells can utilize different invasion strategies to overcome physical arrest during confined migration through tissues with small pores. Cancer cell plasticity allows switches between different migration modes and transitions between single-cell and collective migration. The biophysical parameters that guide these decisions are poorly understood. In this work, we investigated the link between cell deformability and migration efficacy in constrictions of two mesenchymal cancer cell-types with similar invasion strategies: HT1080 fibrosarcoma cells and MV3 melanoma cells. To this end, we designed microfluidic platforms for (1) high-throughput cell deformability measurements and (2) migration through a variety of confining geometries. We measured different deformabilities for HT1080 and MV3 cells and correlated this with their migration efficacy through confinements. However, higher deformability and improved squeezing ability did not impact path selection at junctions of channels of different widths. Our findings show that cell deformability correlates with better squeezing abilities through confinements, but minimally impacts confinement directionality.

The intestinal mucus layer serves as a critical first line of defense against external agents, functioning as a barrier to the absorption of drugs, food, and pathogens. While numerous in vitro studies have explored the role of mucus in preventing particle penetration, the effects of flowing luminal material, dislodging of mucus because of induced shear rate by lumen material and interfacial phenomena remain poorly understood. This study introduces a microfluidic approach to simulate the interaction between flowing luminal material and the mucus layer. The approach successfully measures both particle penetration into the mucus layer and the rate of mucus dislodgement by flowing luminal material. A biosimilar mucus model (BSM) and Hank's Balanced Salt Solution (HBSS) were employed as mimics of human intestinal mucus and luminal fluid, respectively. To investigate the effect of viscosity on the particle penetration pattern, two variants of the mucus model were used: BSM-1, representing a low-viscosity mucus model, and BSM-2, representing a high-viscosity mucus model. The velocity fields in the mucus and luminal material were extracted by tracking fluorescent particles. The results revealed significant differences between BSM-1 and BSM-2, attributed to their rheological properties. These findings were further confirmed through an assessment of the viscoelastic properties of the BSM models. The study utilized COMSOL Multiphysics for numerical simulations, successfully predicting experimental outcomes by solving fluid flow equations. Physicochemical characterizations of BSM and HBSS were performed to link the experimental results with numerical simulations, including flow sweep tests, the application of the power-law model for viscosity, and measurements of mucus density and wettability. This study proposes a microfluidic platform for examining mucus dislodgement and particle penetration in both low- and high-viscosity mucus models. The findings offer valuable insights into the intestinal mucus barrier's response to shear stress. The validated numerical approach and physicochemical characterizations provide a foundation for future studies on mucus dislodgement rates and penetration in more complex intestinal geometries and diverse flow conditions.

Collagen networks form the structural backbone of the extracellular matrix in both healthy and cancerous tissues, exhibiting nonlinear mechanical properties that crucially regulate tissue mechanics and cell behavior. Here, we investigate how the presence of invasive breast cancer cells (MDA-MB-231) influences the polymerization kinetics and mechanics of collagen networks using bulk shear rheology and rheo-confocal microscopy. We show that embedded cancer cells delay the onset of collagen polymerization due to volume exclusion effects. During polymerization, the cells (at 4% volume fraction) cause an unexpected time-dependent softening of the network. We show that this softening effect arises from active remodeling via adhesion and contractility rather than from proteolytic degradation. At higher cell volume fractions, the dominant effect of the cells shifts to volume exclusion, causing a two-fold reduction of network stiffness. Additionally, we demonstrate that cancer cells suppress the characteristic stress-stiffening response of collagen. This effect (partially) disappears when cell adhesion and contractility are inhibited, and it is absent when the cells are replaced by passive hydrogel particles. These findings provide new insights into how active inclusions modify the mechanics of fibrous networks, contributing to a better understanding of the role of cells in the mechanics of healthy and diseased tissues like invasive tumors. Statement of significance: Understanding how cells influence tissue mechanics is crucial to unravel disease progression. While fibroblasts are known to stiffen tissues, the role of invasive cancer cells is less clear. Using collagen-based tissue models, we reveal that cancer cells unexpectedly soften the collagen matrix and disrupt its stress-stiffening response. By comparing active cells to passive particles and selectively blocking cell functions, we show that volume exclusion, adhesion, and contractility each play distinct roles in shaping tissue mechanics. This work sheds light on the physical impact of cancer cells on their environment, advancing our understanding on how cells dynamically alter the mechanical properties of tissues.

Two-photon polymerization (2PP) is an additive manufacturing technology capable of producing polymeric 3D nano- to mesoscale structures with design flexibility and sub-micron resolution. This study investigates the influence of 2PP printing parameters on the morphology and mechanical properties of solid and porous microstructures fabricated from three commercial resins: IP-Q, IP-S, and IP-polydimethylsiloxane (IP-PDMS). To evaluate micromechanical behavior, micropillar compression tests are conducted using IP-Q, which has not been extensively characterized. Porous structures retained 80–85% of the stiffness of solids for IP-Q and IP-S, and 50% for IP-PDMS. Fourier transform infrared spectroscopy showed degrees of conversion of 38% for IP-Q and 61% for IP-S and IP-PDMS. The optimal printing parameters for IP-Q micropillars were a laser power of 50 mW, slicing distance (s) of 1.2 μm, and hatching distance (h) of 1 μm. These settings correspond to a peak laser intensity of 1.58 × 10⁻¹¹ W cm⁻², a focal spot diameter (dxy) of 3.17 μm, a Rayleigh length (zR) of 10.13 μm, and a voxel overlap (δ) of 0.6. These conditions yielded a Young's modulus of 3.7 GPa and yield strength of 75.21 MPa. Overall, the findings emphasize the challenges of parameter optimization when introducing porosity and comparing materials. The results provide a systematic framework for tailoring 2PP processing to guide biomedical microdevice design.

The dissemination of primary solid tumor cells to distant organs, termed metastasis, is a major cause of cancer-related deaths. Circulating tumor cells (CTCs), which can exist as individual cells or multicellular clusters, travel through the bloodstream. Their isolation from liquid biopsy samples is increasingly recognized as a valuable tool for diagnosis, prognosis, and treatment guidance for cancer patients. Current isolation methods typically rely on biomarkers like epithelial cell adhesion molecule (EpCAM) and utilize technologies such as magnetic beads or microfluidic chips. However, these methods face limitations due to tumor heterogeneity. Furthermore, tumor cells that transfer into CTCs often undergo epithelial-to-mesenchymal transition, gaining invasive characteristics while losing epithelial markers. As a result, these cells are difficult to detect using EpCAM-based methods. Label-free microscale isolation technologies tackle the limitations of biomarker-based methods by leveraging the distinctive physical properties of CTCs, such as their size, electrical charge, viscoelasticity, and deformability that contrast them from normal blood cells. This review evaluates primary label-free isolation methods, including deterministic lateral displacement, microfiltration, acoustophoresis, and dielectrophoresis, and whether they can offer a deeper insight into tumor heterogeneity and the dynamics of cancer progression and treatment. Additionally, it highlights automated platforms for high-throughput CTC isolation and analysis.

Polydimethylsiloxane (PDMS) is one of the materials of choice for the fabrication of microfluidic chips. However, its broad application is constrained by its incompatibility with common organic solvents and the absence of surface anchoring groups for surface functionalization. Current solutions involving bulk-, ex-situ surface-, and in-situ liquid phase modifications are limited and practically demanding. In this work, we present a simple, novel strategy to deposit a metal oxide nano-layer on the inside of bonded PDMS microfluidic channels using atmospheric pressure atomic layer deposition (AP-ALD). Using three important classes of microfluidic experiments, i.e., (i) the production of micron-sized particles, (ii) the cultivation of biological cells, and (iii) the photocatalytic degradation in continuous flow chemistry, we demonstrate that the metal oxide nano-layer offers a higher resistance against organic solvent swelling, higher hydrophilicity, and a higher degree of further functionalization of the wall. We demonstrate the versatility of the approach by not only depositing SiO_x nano-layers, but also TiO_x nano-layers, which in the case of the flow chemistry experiment were further functionalized with gold nanoparticles through the use of AP-ALD. This study demonstrates AP-ALD as a tool to broaden the applicability of PDMS devices.

RNA interference (RNAi) is a powerful and rapidly developing technology that enables precise silencing of genes of interest. However, the clinical development of RNAi is hampered by the limited cellular uptake and stability of the transferred molecules. Electroporation (EP) is an efficient and versatile technique for the transfer of both RNA and DNA. Although the mechanism of electrotransfer of small nucleic acids has been studied previously, too little is known about the potential effects of significantly larger pDNA on this process. Here we present a fundamental study of the mechanism of electrotransfer of oligonucleotides and siRNA that occur independently and simultaneously with pDNA by employing confocal fluorescence microscopy. In contrast to the conditional understanding of the mechanism, we have shown that the electrotransfer of oligonucleotides and siRNA is driven by both electrophoretic forces and diffusion after EP, followed by subsequent entry into the nucleus within 5 min after treatment. The study also revealed that the efficiency of siRNA electrotransfer decreases in response to an increase in pDNA concentration. Overall, the study provides new insights into the mechanism of electrotransfer of small nucleic acids which may have broader implications for the future application of RNAi-based strategies.

Epithelial-to-mesenchymal transitions (EMT) and unjamming transitions provide two distinct pathways for cancer cells to become invasive, but it is still unclear to what extent these pathways are connected. Here, we addressed this question by performing 3D spheroid invasion assays on epithelial-like (A549) and mesenchymal-like (MV3) cancer cell lines in collagen-based hydrogels, where we varied both the invasive character of the cells and matrix porosity. We found that the onset time of invasion was correlated with the matrix porosity and vimentin levels, while the spheroid expansion rate correlated with MMP1 levels. Spheroids displayed solid-like (non-invasive) states in small-pore hydrogels and fluid-like (strand-based) or gas-like (disseminating cells) states in large-pore hydrogels or for mesenchymal-like cells. Our findings are consistent with different unjamming states as a function of cell motility and matrix confinement predicted in recent models for cancer invasion, but show that cell motility and matrix confinement are coupled via EMT-related matrix degradation.

The growth and invasion of solid tumors are associated with changes in their viscoelastic properties, influenced by both internal cellular factors and physical forces in the tumor microenvironment. Due to the lack of a comprehensive investigation of tumor tissue viscoelasticity, the relationship between such physical properties and cancer malignancy remains poorly understood. Here, the viscoelastic properties of breast cancer spheroids, 3D (in vitro) tumor models, are studied in relation to their metastatic potentials by imposing controlled, dynamic compression within a microfluidic constriction, and subsequently monitoring the relaxation of the imposed deformation. By adopting a modified Maxwell model to extract viscoelastic properties from the compression data, the benign (MCF-10A) spheroids are found to have higher bulk elastic modulus and viscosity compared to malignant spheroids (MCF-7 and MDA-MB-231). The relaxation is characterized by two timescales, captured by a double exponential fitting function, which reveals a similar fast rebound for MCF-7 and MCF-10A. Both the malignant spheroids exhibit similar long-term relaxation and display residual deformation. However, they differ significantly in morphology, particularly in intercellular movements. These differences between malignant spheroids are demonstrated to be linked to their cytoskeletal organization, by microscopic imaging of F-actin within the spheroids, together with cell-cell adhesion strength.

Tissue surface tension influences cell sorting and tissue fusion. Earlier mechanical studies suggest that multicellular spheroids actively reinforce their surface tension with applied force. Here we study this open question through high-throughput microfluidic micropipette aspiration measurements on cell spheroids to identify the role of force duration and spheroid deformability. In particular, we aspirate spheroid protrusions of mice fibroblast NIH3T3 and human embryonic HEK293T homogeneous cell spheroids into micron-sized capillaries for different pressures and monitor their viscoelastic creep behavior. We find that larger spheroid deformations lead to faster cellular retraction once the pressure is released, regardless of the applied force. Additionally, less deformable NIH3T3 cell spheroids with an increased expression level of alpha-smooth muscle actin, a cytoskeletal protein upregulating cellular contractility, also demonstrate slower cellular retraction after pressure release for smaller spheroid deformations. Moreover, HEK293T cell spheroids only display cellular retraction at larger pressures with larger spheroid deformations, despite an additional increase in viscosity at these larger pressures. These new insights demonstrate that spheroid viscoelasticity is deformation-dependent and challenge whether surface tension truly reinforces at larger aspiration pressures.

Electrotransfer of nucleic acids and proteins has become crucial in biotechnology for gene augmentation and genome editing. This review explores the applications of electrotransfer in both ex vivo and in vivo scenarios, emphasizing biomedical uses. We provide insights into completed clinical trials and successful instances of nucleic acid and protein electrotransfer into therapeutically relevant cells such as immune cells and stem and progenitor cells. In addition, we delve into emerging areas of electrotransfer where nanotechnology and deep learning techniques overcome the limitations of traditional electroporation.

The Poisson's ratio and elastic modulus are two parameters determining the elastic behavior of biomaterials. While the effects of elastic modulus on the cell response is widely studied, very little is known regarding the effects of the Poisson's ratio. The micro-architecture of meta-biomaterials determines not only the Poisson's ratio but also several other parameters that also influence cell response, such as porosity, pore size, and effective elastic modulus. It is, therefore, very challenging to isolate the effects of the Poisson's ratio from those of other micro-architectural parameters. Here, we computationally design meta-biomaterials with controlled Poisson's ratios, ranging between -0.74 and +0.74, while maintaining consistent porosity, pore size, and effective elastic modulus. The 3D meta-biomaterials were additively manufactured at the micro-scale using two-photon polymerization (2PP), and were mechanically evaluated at the meso‑scale. The response of murine preosteoblasts to these meta-biomaterials was then studied using in vitro cell culture models. Meta-biomaterials with positive Poisson's ratios resulted in higher metabolic activity than those with negative values. The cells could attach and infiltrate all meta-biomaterials from the bottom to the top, fully covering the scaffolds after 17 days of culture. Interestingly, the meta-biomaterials exhibited different cell-induced deformations (e.g., shrinkage or local bending) as observed via scanning electron microscopy. The outcomes of osteogenic differentiation (i.e., Runx2 immunofluorescent staining) and matrix mineralization (i.e., Alizarin red staining) assays indicated the significant potential impact of these meta-biomaterials in the field of bone tissue engineering, paving the way for the development of advanced bone meta-implants. Statement of significance: We studied the influence of Poisson's ratio on bone cell response in meta-biomaterials. While elastic modulus effects are well-studied, the impact of Poisson's ratio, especially negative values found in architected biomaterials, remains largely unexplored. The complexity arises from intertwined micro-architectural parameters, such as porosity and elastic modulus, making it challenging to isolate the Poisson's ratio. To overcome this limitation, this study employed rational computational design to create meta-biomaterials with controlled Poisson's ratios, alongside consistent effective elastic modulus, porosity, and pore size. The study reveals that two-photon polymerized 3D meta-biomaterials with positive Poisson's ratios displayed higher metabolic activity, while all the developed meta-biomaterials supported osteogenic differentiation of preosteoblasts as well as matrix mineralization. The outcomes pave the way for the development of advanced 3D bone tissue models and meta-implants.

Within the tumor microenvironment (TME), cancer cells use mechanotransduction pathways to convert biophysical forces to biochemical signals. However, the underlying mechanisms and functional significance of these pathways remain largely unclear. The upregulation of mechanosensitive pathways from biophysical forces such as interstitial flow (IF), leads to the activation of various cytokines, including transforming growth factor-β (TGF-β). TGF-β promotes in part via a Smad-dependent signaling pathway the epithelial–mesenchymal transition (EMT) in cancer cells. The latter process is linked to increased cancer cell motility and invasion. Current research models have limited ability to investigate the combined effects of biophysical forces (such as IF) and cytokines (TGF-β) in a 3D microenvironment. We used a 3D-matrix based microfluidic platform to demonstrate the potentiating effect of IF on exogenous TGF-β induced upregulation of the Smad-signaling activity and the expression of mesenchymal marker vimentin in A549 lung cancer spheroids. To monitor this, we used stably integrated fluorescent based reporters into the A549 cancer cell genome. Our results demonstrate that IF enhances exogenous TGF-β induced Smad-signaling activity in lung cancer spheroids embedded in a matrix microenvironment. In addition, we observed an increased cell motility for A549 spheroids when exposed to IF and TGF-β. Our 3D-microfluidic model integrated with real-time imaging provides a powerful tool for investigating cancer cell signaling and motility associated with invasion characteristics in a physiologically relevant TME.

Polycationic carriers promise low cost and scalable gene therapy treatments, however inefficient intracellular unpacking of the genetic cargo has limited transfection efficiency. Charge-reversing polycations, which transition from cationic to neutral or negative charge, can offer targeted intracellular DNA release. We describe a new class of charge-reversing polycation which undergoes a cationic-to-neutral conversion by a reaction with cellular nucleophiles. The deionization reaction is relatively slow with primary amines, and much faster with thiols. In mammalian cells, the intracellular environment has elevated concentrations of amino acids (∼10×) and the thiol glutathione (∼1000×). We propose this allows for decationization of the polymeric carrier slowly in the extracellular space and then rapidly in the intracellular milleu for DNA release. We demonstrate that in a lipopolyplex formulation this leads to both improved transfection and reduced cytotoxicity when compared to a non-responsive polycationic control.

Cell spheroids are in vitro multicellular model systems that mimic the crowded micro-environment of biological tissues. Their mechanical characterization can provide valuable insights in how single-cell mechanics and cell-cell interactions control tissue mechanics and self-organization. However, most measurement techniques are limited to probing one spheroid at a time, require specialized equipment and are difficult to handle. Here, we developed a microfluidic chip that follows the concept of glass capillary micropipette aspiration in order to quantify the viscoelastic behavior of spheroids in an easy-to-handle, more high-throughput manner. Spheroids are loaded in parallel pockets via a gentle flow, after which spheroid tongues are aspirated into adjacent aspiration channels using hydrostatic pressure. After each experiment, the spheroids are easily removed from the chip by reversing the pressure and new spheroids can be injected. The presence of multiple pockets with a uniform aspiration pressure, combined with the ease to conduct successive experiments, allows for a high throughput of tens of spheroids per day. We demonstrate that the chip provides accurate deformation data when working at different aspiration pressures. Lastly, we measure the viscoelastic properties of spheroids made of different cell lines and show how these are consistent with previous studies using established experimental techniques. In summary, our chip provides a high-throughput way to measure the viscoelastic deformation behavior of cell spheroids, in order to mechanophenotype different tissue types and examine the link between cell-intrinsic properties and overall tissue behavior.

In the research of cancer cell invasion and metastasis, recreation of physiologically relevant and faithful three-dimensional (3D) tumor models that recapitulate spatial architecture, spatiotemporal control of cell communication and signaling pathways, and integration of extracellular cues remains an open challenge. Here, a programmable multifunctional 3D cancer cell invasion microbuckets-hydrogel (Mb-H) platform is developed by integrating various function-variable microbuckets and extracellular matrix (ECM)-like hydrogels. Based on this Mb-H micro platform, the aggregation of multi-cancer cells is well controlled to form cancer cell spheroids, and the guiding relationship of single-cell migration and collective cell migration during the epithelial-mesenchymal transition (EMT) of cancer cell invasion are demonstrated. By programming and precisely assembling multiple functions in one system, the Mb-H platform with spatial-temporal controlled release of cytokine transforming growth factor beta (TGF-β) and various functionalized Mb-H platforms with intelligent adjustment of cell-matrix interactions are engineered to coordinate the 3D invasive migration of cancer cell spheroids. This programmable and adaptable 3D cancer cell invasion micro platform takes a new step toward mimicking the dynamically changing (localized) tumor microenvironment and exhibits wide potential applications in cancer research, bio-fabrication, cell signaling, and drug screening.

We developed a localized single-cell electroporation chip to deliver exogenous biomolecules with high efficiency while maintaining high cell viability. In our microfluidic device, the cells are trapped in a microtrap array by flow, after which target molecules are supplied to the device and electrotransferred to the cells under electric pulses. The system provides the ability to monitor the electrotransfer of exogenous biomolecules in real time. We reveal through numerical simulations that localized electroporation is the mechanism of permeabilization in the microtrap array electroporation device. We demonstrate the simplicity and accuracy of this microtrap technology for electroporation by delivery of both small molecules using propidium iodide and large molecules using plasmid DNA for gene expression, illustrating the potential of this minimally invasive method to be widely used for precise intracellular delivery purposes (from bioprocess engineering to therapeutic applications).

A functional vascular system is a prerequisite for bone repair as disturbed angiogenesis often causes non-union. Paracrine factors released from human bone marrow derived mesenchymal stromal cells (BMSCs) have angiogenic effects on endothelial cells. However, whether these paracrine factors participate in blood flow dynamics within bone capillaries remains poorly understood. Here, we used two different microfluidic designs to investigate critical steps during angiogenesis and found pronounced effects of endothelial cell proliferation as well as chemotactic and mechanotactic migration induced by BMSC conditioned medium (CM). The application of BMSC-CM in dynamic cultures demonstrates that bioactive factors in combination with fluidic flow-induced biomechanical signals significantly enhanced endothelial cell migration. Transcriptional analyses of endothelial cells demonstrate the induction of a unique gene expression profile related to tricarboxylic acid cycle and energy metabolism by the combination of BMSC-CM factors and shear stress, which opens an interesting avenue to explore during fracture healing. Our results stress the importance of in vivo - like microenvironments simultaneously including biochemical, biomechanical and oxygen levels when investigating key events during vessel repair. Statement of significance: Our results demonstrate the importance of recapitulating in vivo - like microenvironments when investigating key events during vessel repair. Endothelial cells exhibit enhanced angiogenesis characteristics when simultaneous exposing them to hMSC-CM, mechanical forces and biochemical signals simultaneously. The improved angiogenesis may not only result from the direct effect of growth factors, but also by reprogramming of endothelial cell metabolism. Moreover, with this model we demonstrated a synergistic impact of mechanical forces and biochemical factors on endothelial cell behavior and the expression of genes involved in the TCA cycle and energy metabolism, which opens an interesting new avenue to stimulate angiogenesis during fracture healing.

An early step of metastasis requires a complex and coordinated migration of invasive tumor cells into the surrounding tumor microenvironment (TME), which contains extracellular matrix (ECM). It is being appreciated that 3D matrix-based microfluidic models have an advantage over conventional in vitro and animal models to study tumor progression events. Recent microfluidic models have enabled recapitulation of key mechanobiological features present within the TME to investigate collective cancer cell migration and invasion. Microfluidics also allows for functional interrogation and therapeutic manipulation of specific steps to study the dynamic aspects of tumor progression. In this review, we focus on recent developments in cancer cell migration and how microfluidic strategies have evolved to address the physiological complexities of the TME to visualize migration modes adapted by various tumor cells.

Electroporation has become a powerful tool for nonviral delivery of various biomolecules such as nucleic acids, proteins, and chemotherapeutic drugs to virtually any living cell by exposing the cell membrane to an intense pulsed electric field. Different multiphysics and multiscale models have been developed to describe the phenomenon of electroporation and predict molecular transport through the electroporated membrane. In this paper, we critically examine the existing mechanistic, single-cell models which allow spatially and temporally resolved numerical simulations of electroporation-induced transmembrane transport of small molecules by confronting them with different experimental measurements. Furthermore, we assess whether any of the proposed models is universal enough to describe the associated transmembrane transport in general for all the different pulse parameters and small molecules used in electroporation applications. We show that none of the tested models can be universally applied to the full range of experimental measurements. Even more importantly, we show that none of the models has been compared to sufficient amount of experimental data to confirm the model validity. Finally, we provide guidelines and recommendations on how to design and report experiments that can be used to validate an electroporation model and how to improve the development of mechanistic models.