Microfluidic Optical Stretcher Research Articles

Actin and microtubule networks contribute differently to cell response for small and large strains

Cytoskeletal filaments provide cells with mechanical stability and organization. The main key players are actin filaments and microtubules governing a cell’s response to mechanical stimuli. We investigated the specific influences of these crucial components by deforming MCF-7 epithelial cells at small (≤5% deformation) and large strains (>5% deformation). To understand specific contributions of actin filaments and microtubules, we systematically studied cellular responses after treatment with cytoskeleton influencing drugs. Quantification with the microfluidic optical stretcher allowed capturing the relative deformation and relaxation of cells under different conditions. We separated distinctive deformational and relaxational contributions to cell mechanics for actin and microtubule networks for two orders of magnitude of drug dosages. Disrupting actin filaments via latrunculin A, for instance, revealed a strain-independent softening. Stabilizing these filaments by treatment with jasplakinolide yielded cell softening for small strains but showed no significant change at large strains. In contrast, cells treated with nocodazole to disrupt microtubules displayed a softening at large strains but remained unchanged at small strains. Stabilizing microtubules within the cells via paclitaxel revealed no significant changes for deformations at small strains, but concentration-dependent impact at large strains. This suggests that for suspended cells, the actin cortex is probed at small strains, while at larger strains; the whole cell is probed with a significant contribution from the microtubules.

Mechanical mismatch between Ras transformed and untransformed epithelial cells.

The organization of the actin cytoskeleton plays a key role in regulating cell mechanics. It is fundamentally altered during transformation, affecting how cells interact with their environment. We investigated mechanical properties of cells expressing constitutively active, oncogenic Ras (RasV12) in adherent and suspended states. To do this, we utilized atomic force microscopy and a microfluidic optical stretcher. We found that adherent cells stiffen and suspended cells soften with the expression of constitutively active Ras. The effect on adherent cells was reversed when contractility was inhibited with the ROCK inhibitor Y-27632, resulting in softer RasV12 cells. Our findings suggest that increased ROCK activity as a result of Ras has opposite effects on suspended and adhered cells. Our results also establish the importance of the activation of ROCK by Ras and its effect on cell mechanics.

Biophysical Approach to Mechanisms of Cancer Prevention and Treatment with Green Tea Catechins.

Green tea catechin and green tea extract are now recognized as non-toxic cancer preventives for humans. We first review our brief historical development of green tea cancer prevention. Based on exciting evidence that green tea catechin, (−)-epigallocatechin gallate (EGCG) in drinking water inhibited lung metastasis of B16 melanoma cells, we and other researchers have studied the inhibitory mechanisms of metastasis with green tea catechins using biomechanical tools, atomic force microscopy (AFM) and microfluidic optical stretcher. Specifically, determination of biophysical properties of cancer cells, low cell stiffness, and high deformability in relation to migration, along with biophysical effects, were studied by treatment with green tea catechins. The study with AFM revealed that low average values of Young’s moduli, indicating low cell stiffness, are closely associated with strong potential of cell migration and metastasis for various cancer cells. It is important to note that treatments with EGCG and green tea extract elevated the average values of Young’s moduli resulting in increased stiffness (large elasticity) of melanomas and various cancer cells. We discuss here the biophysical basis of multifunctions of green tea catechins and green tea extract leading to beneficial effects for cancer prevention and treatment.

All-silica microfluidic optical stretcher with acoustophoretic prefocusing

Acoustophoresis is a widely reported and used technique for microparticle manipulation and separation. In the study described here, acustophoresis is employed to prefocus the flow (i.e., focusing occurring upstream of the analysis region) in a microfluidic chip intended for optical trapping and stretching. The whole microchip is made of silica with optical waveguides integrated by femtosecond laser writing. The acoustic force is produced by driving an external piezoelectric ceramic attached underneath the microchip at the chip resonance frequency. Thanks to an efficient excitation of acoustic waves in both water and glass, acoustophoretic focusing is observed along the channel length (>40 mm) and it is successfully demonstrated both with polystyrene beads, swollen red blood cell, and cells from mouse fibroblast cellular lines (L929). Moreover, by comparing results of cell stretching measurements, we demonstrate that acoustic waves do not alter the optical deformability of the cells and that the acoustic prefocusing results in a considerable enhancement of throughput in optical stretching experiments.

Myosin II Activity Softens Cells in Suspension

The cellular cytoskeleton is crucial for many cellular functions such as cell motility and wound healing, as well as other processes that require shape change or force generation. Actin is one cytoskeleton component that regulates cell mechanics. Important properties driving this regulation include the amount of actin, its level of cross-linking, and its coordination with the activity of specific molecular motors like myosin. While studies investigating the contribution of myosin activity to cell mechanics have been performed on cells attached to a substrate, we investigated mechanical properties of cells in suspension. To do this, we used multiple probes for cell mechanics including a microfluidic optical stretcher, a microfluidic microcirculation mimetic, and real-time deformability cytometry. We found that nonadherent blood cells, cells arrested in mitosis, and naturally adherent cells brought into suspension, stiffen and become more solidlike upon myosin inhibition across multiple timescales (milliseconds to minutes). Our results hold across several pharmacological and genetic perturbations targeting myosin. Our findings suggest that myosin II activity contributes to increased whole-cell compliance and fluidity. This finding is contrary to what has been reported for cells attached to a substrate, which stiffen via active myosin driven prestress. Our results establish the importance of myosin II as an active component in modulating suspended cell mechanics, with a functional role distinctly different from that for substrate-adhered cells.

Unique Mechanical Properties of Cell Nuclei Regulated by Chromatin

Nuclear mechanics and structure could affect gene regulation and gene expression. Chromatin, a major component of cell nuclei, could play an important role in maintaining nuclear integrity and their mechanical properties. Previous studies on nuclear mechanical properties have focused largely on the role of the nuclear lamina, using techniques such as AFM and micropipette aspiration. In this work, we explicitly address the contributions of chromatin to nuclear rheology after isolation from the cell using a microfluidic optical stretcher. We find that isolated nuclei swell in volume under uni-axial stress and exhibit significant softening with increased nuclear size, which can be described by a filtration model for the nuclear membrane and a cortical chromatin model, respectively. In addition, changes to the state of chromatin condensation via histone modifications or chromatin remodeling processes (ATP, topoisomerase II) can strongly impact nuclear morphology and compliance. Moreover, isolated nuclear mechanics is also sensitive to ionic conditions: nuclei stiffen with increasing ionic strength of the buffer and exhibit a transition from stretch to contraction in the presence of multivalent ions (only). Finally, we find that in contrast to other studies suggesting a high refractive index of cell nuclei compared to the cytoplasm, the refractive index of isolated cell nuclei of a variety of cell types can be lower than the refractive index of the cells. The presented work establishes a quantitative link between nuclear mechanical properties and the compaction state of chromatin, which can be modulated by a change in nuclear volume, chromatin remodeling or electrochemical environment.

Impact of heating on passive and active biomechanics of suspended cells.

A cell is a complex material whose mechanical properties are essential for its normal functions. Heating can have a dramatic effect on these mechanical properties, similar to its impact on the dynamics of artificial polymer networks. We investigated such mechanical changes by the use of a microfluidic optical stretcher, which allowed us to probe cell mechanics when the cells were subjected to different heating conditions at different time scales. We find that HL60/S4 myeloid precursor cells become mechanically more compliant and fluid-like when subjected to either a sudden laser-induced temperature increase or prolonged exposure to higher ambient temperature. Above a critical temperature of 52 ± 1°C, we observed active cell contraction, which was strongly correlated with calcium influx through temperature-sensitive transient receptor potential vanilloid 2 (TRPV2) ion channels, followed by a subsequent expansion in cell volume. The change from passive to active cellular response can be effectively described by a mechanical model incorporating both active stress and viscoelastic components. Our work highlights the role of TRPV2 in regulating the thermomechanical response of cells. It also offers insights into how cortical tension and osmotic pressure govern cell mechanics and regulate cell-shape changes in response to heat and mechanical stress.

Evaluation of single‐cell biomechanics as potential marker for oral squamous cell carcinomas: a pilot study

Early detection of oral cancer is a major health issue. The objective of this pilot study was to analyze the deformability of healthy and cancer cells using a microfluidic optical stretcher (OS). Different cancer cell lines, primary oral cancer cells, and their healthy counterparts were cultivated and characterized, respectively. A measurable deformation of the cells along the optical axis was detected, caused by surface stress, which is optically induced by the laser power. All cells revealed a viscoelastic extension behavior and showed a characteristic deformation response under laser light exposure. The CAL-27/-33 cells exhibited the highest relative deformation. All other cells achieved similar values, but on a lower level. The cytoskeleton reacts sensitively of changing environmental conditions, which may be influenced by growth behavior of the cancer specimens. Nevertheless, the statistical analysis showed significant differences between healthy and cancer cells. Generally, malignant and benign cells showed significantly different mechanical behavior. Cancer-related changes influence the composition of the cytoskeleton and thus affect the deformability, but this effect may be superimposed by cell cultivation conditions or cell doubling time. These influences had to be substituted by brush biopsies to minimize confounders in pursuing investigations.

Analysis of multiple physical parameters for mechanical phenotyping of living cells

Since the cytoskeleton is known to regulate many cell functions, an increasing amount of effort to characterize cells by their mechanical properties has occured. Despite the structural complexity and dynamics of the multicomponent cytoskeleton, mechanical measurements on single cells are often fit to simple models with two to three parameters, and those parameters are recorded and reported. However, different simple models are likely needed to capture the distinct mechanical cell states, and additional parameters may be needed to capture the ability of cells to actively deform. Our new approach is to capture a much larger set of possibly redundant parameters from cells' mechanical measurement using multiple rheological models as well as dynamic deformation and image data. Principal component analysis and network-based approaches are used to group parameters to reduce redundancies and develop robust biomechanical phenotyping. Network representation of parameters allows for visual exploration of cells' complex mechanical system, and highlights unexpected connections between parameters. To demonstrate that our biomechanical phenotyping approach can detect subtle mechanical differences, we used a Microfluidic Optical Cell Stretcher to mechanically stretch circulating human breast tumor cells bearing genetically-engineered alterations in c-src tyrosine kinase activation, which is known to influence reattachment and invasion during metastasis.

Calcium imaging in the optical stretcher

The Microfluidic Optical Stretcher (MOS) has previously been shown to be a versatile tool to measure mechanical properties of single suspended cells. In this study we combine optical stretching and fluorescent calcium imaging. A cell line transfected with a heat sensitive cation channel was used as a model system to show the versatility of the setup. The cells were loaded with the Ca(2+) dye Fluo-4 and imaged with confocal laser scanning microscopy while being stretched. During optical stretching heat is transferred to the cell causing a pronounced Ca(2+) influx through the cation channel. The technique opens new perspectives for investigating the role of Ca(2+) in regulating cell mechanical behavior.

Detection of Plasmodium falciparum-infected red blood cells by optical stretching

We present the application of a microfluidic optical cell stretcher to measure the elasticity of malaria-infected red blood cells. The measurements confirm an increase in host cell rigidity during the maturation of the parasite Plasmodium falciparum. The device combines the selectivity and sensitivity of single-cell elasticity measurements with a throughput that is higher than conventional single-cell techniques. The method has potential to detect early stages of infection with excellent sensitivity and high speed.

The regulatory role of cell mechanics for migration of differentiating myeloid cells

Migration of cells is important for tissue maintenance, immune response, and often altered in disease. While biochemical aspects, including cell adhesion, have been studied in detail, much less is known about the role of the mechanical properties of cells. Previous measurement methods rely on contact with artificial surfaces, which can convolute the results. Here, we used a non-contact, microfluidic optical stretcher to study cell mechanics, isolated from other parameters, in the context of tissue infiltration by acute promyelocytic leukemia (APL) cells, which occurs during differentiation therapy with retinoic acid. Compliance measurements of APL cells reveal a significant softening during differentiation, with the mechanical properties of differentiated cells resembling those of normal neutrophils. To interfere with the migratory ability acquired with the softening, differentiated APL cells were exposed to paclitaxel, which stabilizes microtubules. This treatment does not alter compliance but reduces cell relaxation after cessation of mechanical stress six-fold, congruent with a significant reduction of motility. Our observations imply that the dynamical remodeling of cell shape required for tissue infiltration can be frustrated by stiffening the microtubular system. This link between the cytoskeleton, cell mechanics, and motility suggests treatment options for pathologies relying on migration of cells, notably cancer metastasis.

Oral Cancer Diagnosis by Mechanical Phenotyping

Oral squamous cell carcinomas are among the 10 most common cancers and have a 50% lethality rate after 5 years. Despite easy access to the oral cavity for cancer screening, the main limitations to successful treatment are uncertain prognostic criteria for (pre-)malignant lesions. Identifying a functional cellular marker may represent a significant improvement for diagnosis and treatment. Toward this goal, mechanical phenotyping of individual cells is a novel approach to detect cytoskeletal changes, which are diagnostic for malignant change. The compliance of cells from cell lines and primary samples of healthy donors and cancer patients was measured using a microfluidic optical stretcher. Cancer cells showed significantly different mechanical behavior, with a higher mean deformability and increased variance. Cancer cells (n approximately 30 cells measured from each patient) were on average 3.5 times more compliant than those of healthy donors [D(normal) = (4.43 +/- 0.68) 10(-3) Pa(-1); D(cancer) = (15.8 +/- 1.5) 10(-3) Pa(-1); P < 0.01]. The diagnosis results of the patient samples were confirmed by standard histopathology. The generality of these findings was supported by measurements of two normal and four cancer oral epithelial cell lines. Our results indicate that mechanical phenotyping is a sensible, label-free approach for classifying cancer cells to enable broad screening of suspicious lesions in the oral cavity. It could in principle be applied to any cancer to aid conventional diagnostic procedures.

Reconfigurable microfluidic integration of a dual-beam laser trap with biomedical applications

A dual-beam fiber laser trap, termed the optical stretcher when used to deform objects, has been combined with a capillary-based microfluidic system in order to serially trap and deform biological cells. The design allows for control over the size and position of the trap relative to the flow channel. Data is recorded using video phase contrast microscopy and is subsequently analyzed using a custom edge fitting routine. This setup has been regularly used with measuring rates of 50-100 cells/h. One such experiment is presented to compare the distribution of deformability found within a normal epithelial cell line to that of a cancerous one. In general, this microfluidic optical stretcher can be used for the characterization of cells by their viscoelastic signature. Possible applications include the cytological diagnosis of cancer and the gentle and marker-free sorting of stem cells from heterogeneous populations for therapeutic cell-based approaches in regenerative medicine.

Optical Deformability as an Inherent Cell Marker for Testing Malignant Transformation and Metastatic Competence

The relationship between the mechanical properties of cells and their molecular architecture has been the focus of extensive research for decades. The cytoskeleton, an internal polymer network, in particular determines a cell's mechanical strength and morphology. This cytoskeleton evolves during the normal differentiation of cells, is involved in many cellular functions, and is characteristically altered in many diseases, including cancer. Here we examine this hypothesized link between function and elasticity, enabling the distinction between different cells, by using a microfluidic optical stretcher, a two-beam laser trap optimized to serially deform single suspended cells by optically induced surface forces. In contrast to previous cell elasticity measurement techniques, statistically relevant numbers of single cells can be measured in rapid succession through microfluidic delivery, without any modification or contact. We find that optical deformability is sensitive enough to monitor the subtle changes during the progression of mouse fibroblasts and human breast epithelial cells from normal to cancerous and even metastatic state. The surprisingly low numbers of cells required for this distinction reflect the tight regulation of the cytoskeleton by the cell. This suggests using optical deformability as an inherent cell marker for basic cell biological investigation and diagnosis of disease.

Characterizing single suspended cells by optorheology

The measurement of the mechanical properties of individual cells has received much attention in recent years. In this paper we describe the application of optically induced forces with an optical stretcher to perform step-stress experiments on individual suspended fibroblasts. The conversion from creep-compliance to frequency-dependent complex shear modulus reveals characteristic viscoelastic signatures of the underlying cytoskeleton and its dynamic molecular properties. Both normal and cancerous fibroblasts display a single stress relaxation time in the observed time and frequency space that can be related to the transient binding of actin crosslinking proteins. In addition, shear modulus and steady-state viscosity of the shell-like actin cortex as the main module resisting small deformations are extracted. These values in combination with insight into the cells’ architecture are used to explain their different deformability. This difference can then be exploited to distinguish normal from cancerous cells. The nature of the optical stretcher as an optical trap allows easy incorporation in a microfluidic system with automatic trapping and alignment of the cells, and thus a high measurement throughput. This carries the potential for using the microfluidic optical stretcher to investigate cellular processes involving the cytoskeleton and to diagnose diseases related to cytoskeletal alterations.

Deformability‐based flow cytometry

Elasticity of cells is determined by their cytoskeleton. Changes in cellular function are reflected in the amount of cytoskeletal proteins and their associated networks. Drastic examples are diseases such as cancer, in which the altered cytoskeleton is even diagnostic. This connection between cellular function and cytoskeletal mechanical properties suggests using the deformability of cells as a novel inherent cell marker. The optical stretcher is a new laser tool capable of measuring cellular deformability. A unique feature of this deformation technique is its potential for high throughput, with the incorporation of a microfluidic delivery of cells. Rudimentary implementation of the microfluidic optical stretcher has been used to measure optical deformability of several normal and cancerous cell types. A drastic difference has been seen between the response of red blood cells and polymorphonuclear cells for a given optically induced stress. MCF-10, MCF-7, and modMCF-7 cells were also measured, showing that while cancer cells stretched significantly more (five times) than normal cells, optical deformability could even be used to distinguish metastatic cancer cells from nonmetastatic cancer cells. This trimodal distribution was apparent after measuring a mere 83 cells, which shows optical deformability to be a highly regulated cell marker. Preliminary work suggests a deformability-based cell sorter similar to current fluorescence-based flow cytometry without the need for specific labeling. This could be used for the diagnosis of all diseases, and the investigation of all cellular processes, that affect the cytoskeleton.