A comparison of methods to assess cell mechanical properties.

The mechanical properties of cells influence their cellular and subcellular functions, including cell adhesion, migration, polarization, and differentiation, as well as organelle organization and trafficking inside the cytoplasm. Yet reported values of cell stiffness and viscosity vary substantially, which suggests differences in how the results of different methods are obtained or analyzed by different groups. To address this issue and illustrate the complementarity of certain approaches, here we present, analyze, and critically compare measurements obtained by means of some of the most widely used methods for cell mechanics: atomic force microscopy, magnetic twisting cytometry, particle-tracking microrheology, parallel-plate rheometry, cell monolayer rheology, and optical stretching. These measurements highlight how elastic and viscous moduli of MCF-7 breast cancer cells can vary 1,000-fold and 100-fold, respectively. We discuss the sources of these variations, including the level of applied mechanical stress, the rate of deformation, the geometry of the probe, the location probed in the cell, and the extracellular microenvironment.

Nanoreactors based on DNAzyme-functionalized magnetic nanoparticles activated by magnetic field.

A new biomimetic nanoreactor design, MaBiDz, is presented based on a copolymer brush in combination with superparamagnetic nanoparticles. This cellular nanoreactor features two species of magnetic particles, each functionalized with two components of a binary deoxyribozyme system. In the presence of a target mRNA analyte and a magnetic field, the nanoreactor is assembled to form a biocompartment enclosed by the polymeric brush that enables catalytic function of the binary deoxyribozyme with enhanced kinetics. MaBiDz was demonstrated here as a cellular sensor for rapid detection and imaging of a target mRNA biomarker for metastatic breast cancer, and its function shows potential to be expanded as a biomimetic organelle that can downregulate the activity of a target mRNA biomarker.

Magnetic Field-Activated Sensing of mRNA in Living Cells.

Detection of specific mRNA in living cells has attracted significant attention in the past decade. Probes that can be easily delivered into cells and activated at the desired time can contribute to understanding translation, trafficking and degradation of mRNA. Here we report a new strategy termed magnetic field-activated binary deoxyribozyme (MaBiDZ) sensor that enables both efficient delivery and temporal control of mRNA sensing by magnetic field. MaBiDZ uses two species of magnetic beads conjugated with different components of a multicomponent deoxyribozyme (DZ) sensor. The DZ sensor is activated only in the presence of a specific target mRNA and when a magnetic field is applied. Here we demonstrate that MaBiDZ sensor can be internalized in live MCF-7 breast cancer cells and activated by a magnetic field to fluorescently report the presence of specific mRNA, which are cancer biomarkers.

An Enzyme-based 1:2 Demultiplexer Interfaced with an Electrochemical Actuator.

An enzyme-based 1:2 demultiplexer is designed in a flow system composed of three cells where each one is modified with a different enzyme: hexokinase, glucose dehydrogenase and glucose-6-phosphate dehydrogenase. The Input signal activating the biocatalytic cascade is represented by glucose, while the Address signal represented by ATP is responsible for directing the Input signal to one of the output channels, depending on the logic value of the Address. The biomolecular 1:2 demultiplexer is extended to include two electrochemical actuators releasing entrapped DNA molecules in the active output channel. The modular design of the system allows for easy exchange and extension of the functional elements. The present demultiplexer can be easily integrated in various biomolecular logic systems, including different logic gates based on the enzyme- or DNA-based reactions, as well as containing different chemical actuators, for example, with a biomolecular release function.

AFM study shows prominent physical changes in elasticity and pericellular layer in human acute leukemic cells due to inadequate cell-cell communication.

Biomechanical properties of single cells in vitro or ex vivo and their pericellular interfaces have recently attracted a lot of attention as a potential biophysical (and possibly prognostic) marker of various diseases and cell abnormalities. At the same time, the influence of the cell environment on the biomechanical properties of cells is not well studied. Here we use atomic force microscopy to demonstrate that cell-cell communication can have a profound effect on both cell elasticity and its pericellular coat. A human pre-B p190BCR/ABL acute lymphoblastic leukemia cell line (ALL3) was used in this study. Assuming that cell-cell communication is inversely proportional to the distance between cells, we study ALL3 cells in vitro growing at different cell densities. ALL3 cells demonstrate a clear density dependent behavior. These cells grow very well if started at a relatively high cell density (HD, >2 × 105 cells ml-1) and are poised to grow at low cell density (LD, <1 × 104 cells ml-1). Here we observe ∼6× increase in the elastic (Young's) modulus of the cell body and ∼3.6× decrease in the pericellular brush length of LD cells compared to HD ALL3 cells. The difference observed in the elastic modulus is much larger than typically reported for pathologically transformed cells. Thus, cell-cell communication must be taken into account when studying biomechanics of cells, in particular, correlating cell phenotype and its biophysical properties.

Biophysical differences between chronic myelogenous leukemic quiescent and proliferating stem/progenitor cells.

The treatment of chronic myeloid leukemia (CML), a clonal myeloproliferative disorder has improved recently, but most patients have not yet been cured. Some patients develop resistance to the available tyrosine kinase treatments. Persistence of residual quiescent CML stem cells (LSCs) that later resume proliferation is another common cause of recurrence or relapse of CML. Eradication of quiescent LSCs is a promising approach to prevent recurrence of CML. Here we report on new biophysical differences between quiescent and proliferating CD34+ LSCs, and speculate how this information could be of use to eradicate quiescent LSCs. Using AFM measurements on cells collected from four untreated CML patients, substantial differences are observed between quiescent and proliferating cells in the elastic modulus, pericellular brush length and its grafting density at the single cell level. The higher pericellular brush densities of quiescent LSCs are common for all samples. The significance of these observations is discussed.

Diffusion of Oligonucleotides from within Iron-Cross-Linked, Polyelectrolyte-Modified Alginate Beads: A Model System for Drug Release.

An analytical model to describe diffusion of oligonucleotides from stable hydrogel beads is developed and experimentally verified. The synthesized alginate beads are Fe(3+) -cross-linked and polyelectrolyte-doped for uniformity and stability at physiological pH. Data on diffusion of oligonucleotides from inside the beads provide physical insights into the volume nature of the immobilization of a fraction of oligonucleotides due to polyelectrolyte cross-linking, that is, the absence of a surface-layer barrier in this case. Furthermore, the results suggest a new simple approach to measuring the diffusion coefficient of mobile oligonucleotide molecules inside hydrogels. The considered alginate beads provide a model for a well-defined component in drug-release systems and for the oligonucleotide-release transduction steps in drug-delivering and biocomputing applications. This is illustrated by destabilizing the beads with citrate, which induces full oligonucleotide release with nondiffusional kinetics.

Towards early detection of cervical cancer: Fractal dimension of AFM images of human cervical epithelial cells at different stages of progression to cancer.

We used AFM HarmoniX modality to analyse the surface of individual human cervical epithelial cells at three stages of progression to cancer, normal, immortal (pre-malignant) and carcinoma cells. Primary cells from 6 normal strains, 6 cancer, and 6 immortalized lines (derived by plasmid DNA-HPV-16 transfection of cells from 6 healthy individuals) were tested. This cell model allowed for good control of the cell phenotype down to the single cell level, which is impractical to attain in clinical screening tests (ex-vivo). AFM maps of physical (nonspecific) adhesion are collected on fixed dried cells. We show that a surface parameter called fractal dimension can be used to segregate normal from both immortal pre-malignant and malignant cells with sensitivity and specificity of more than 99%. The reported method of analysis can be directly applied to cells collected in liquid cytology screening tests and identified as abnormal with regular optical methods to increase sensitivity. FROM THE CLINICAL EDITOR: Despite cervical smear screening, sometimes it is very difficult to differentiate cancers cells from pre-malignant cells. By using AFM to analyze the surface properties of human cervical epithelial cells, the authors were able to accurately identify normal from abnormal cells. This method could augment existing protocols to increase diagnostic accuracy.

If cell mechanics can be described by elastic modulus: study of different models and probes used in indentation experiments.

Here we investigated the question whether cells, being highly heterogeneous objects, could be described with the elastic modulus (effective Young's modulus) in a self-consistent way. We performed a comparative analysis of the elastic modulus derived from the indentation data obtained with atomic force microscopy (AFM) on human cervical epithelial cells (both normal and cancerous). Both sharp (cone) and dull (2500-nm radius sphere) AFM probes were used. The indentation data were processed through different elastic models. The cell was approximated as a homogeneous elastic medium that had either 1), smooth hemispherical boundary (Hertz/Sneddon models) or 2), the boundary covered with a layer of glycocalyx and membrane protrusions ("brush" models). Consistency of these approximations was investigated. Specifically, we tested the independence of the elastic modulus of the indentation depth, which is assumed in these models. We demonstrated that only one model showed consistency in treating cells as a homogeneous elastic medium, namely, the brush model, when processing the indentation data collected with the dull AFM probe. The elastic modulus demonstrated strong depth dependence in all models: Hertz/Sneddon models (no brush taken into account), and when the brush model was applied to the data collected with sharp conical probes. We conclude that it is possible to describe the elastic properties of the cell body by means of an effective elastic modulus, used in a self-consistent way, when using the brush model to analyze data collected with a dull AFM probe. The nature of these results is discussed.

A biocatalytic cascade with several output signals--towards biosensors with different levels of confidence.

The biocatalytic cascade based on enzyme-catalyzed reactions activated by several biomolecular input signals and producing output signal after each reaction step was developed as an example of a logically reversible information processing system. The model system was designed to mimic the operation of concatenated AND logic gates with optically readable output signals generated at each step of the logic operation. Implications include concurrent bioanalyses and data interpretation for medical diagnostics.

Ultrabright fluorescent mesoporous silica nanoparticles for prescreening of cervical cancer.

We report on the first functional use of recently introduced ultrabright fluorescent mesoporous silica nanoparticles, which are functionalized with folic acid, to distinguish cancerous and precancerous cervical epithelial cells from normal cells. The high brightness of the particles is advantageous for fast and reliable identification of both precancerous and cancerous cells. Normal and cancer cells were isolated from three healthy women and three cancer patients. Three precancerous cell lines were derived by immortalization of primary cultures of normal cells with human papillomavirus type-16 (HPV-16) DNA. We observed substantially different particle internalization by normal and cancerous/precancerous cells after a short incubation time of 15 minutes. Compared to HPV-DNA and cell pathology tests, which are currently used for prescreening of cervical cancer, we demonstrated that the specificity of our method was similar (94-95%), whereas its sensitivity was significantly better (95-97%) than the sensitivity of those currently used tests (30-80%). FROM THE CLINICAL EDITOR: This team of investigators reports on the development of a new screening test for cervical cancer using ultrabright fluorescent mesoporous silica nanoparticles functionalized with folic acid, enabling significantly better sensitivity (95-97% vs. 30-80%) and maintained specificity (94-95%) compared with current clinical tests. This test should find a way to clinical use in the near future.

Quantitative study of the elastic modulus of loosely attached cells in AFM indentation experiments.

When measuring the elastic (Young's) modulus of cells using AFM, good attachment of cells to a substrate is paramount. However, many cells cannot be firmly attached to many substrates. A loosely attached cell is more compliant under indenting. It may result in artificially low elastic modulus when analyzed with the elasticity models assuming firm attachment. Here we suggest an AFM-based method/model that can be applied to extract the correct Young's modulus of cells loosely attached to a substrate. The method is verified by using primary breast epithelial cancer cells (MCF-7) at passage 4. At this passage, approximately one-half of cells develop enough adhesion with the substrate to be firmly attached to the substrate. These cells look well spread. The other one-half of cells do not develop sufficient adhesion, and are loosely attached to the substrate. These cells look spherical. When processing the AFM indentation data, a straightforward use of the Hertz model results in a substantial difference of the Young's modulus between these two types of cells. If we use the model presented here, we see no statistical difference between the values of the Young's modulus of both poorly attached (round) and firmly attached (close to flat) cells. In addition, the presented model allows obtaining parameters of the brush surrounding the cells. The cellular brush observed is also statistically identical for both types of cells. The method described here can be applied to study mechanics of many other types of cells loosely attached to substrates, e.g., blood cells, some stem cells, cancerous cells, etc.

Method for quantitative measurements of the elastic modulus of biological cells in AFM indentation experiments.

Here we overview and further develop a quantitative method to measure mechanics of biological cells in indentation experiments, which is based on the use of atomic force microscopy (AFM). We demonstrate how the elastic modulus of the cell body should be measured when the cellular brush is taken into account. The brush is an essential inelastic part of the cell, which surrounds all eukaryotic (the brush is mostly microvilli and glycocalyx) and gram-negative prokaryotic cells (the brush is polysaccharides). The other main feature of the described method is the use of a relatively dull AFM probe to stay in the linear stress-strain regime. In particular, we show that the elastic modulus (aka the Young's modulus) of cells is independent of the indentation depth up to 10-20% deformation for the eukaryotic cells studied here. Besides the elastic modulus, the method presented allows obtaining the parameters of cellular brush, such as the effective length and grafting density of the brush. Although the method is demonstrated on eukaryotic cells, it is directly applicable for all types of cells, and even non-biological soft materials surrounded by either a brush or any field of long-range forces.