Cytoplasmic Viscosity Research Articles

Optical trapping of lipid bodies reveals increased cytoplasm viscosity in Candida tropicalis post antimicrobial photodynamic therapy

Abstract Antimicrobial photodynamic therapy (aPDT) is a common treatment for large cell colonies, but its effectiveness is typically assessed through colony-forming unit counting, which lacks microscopic details about cell death. This study monitors the trap stiffness of optically trapped lipid bodies of Candida tropicalis of approximately 1 μm of radius following aPDT treatment. Methylene blue served as the photosensitizer at 10 μM concentration, with a lethal light dose of 60 J cm-². The results revealed a significant increase in viscosity after aPDT treatment. Additionally, image analysis confirmed substantial morphological changes indicative of cell death. These findings demonstrate the potential of optical tweezers as a non-invasive tool for assessing cellular health by providing both functional (viscosity) and morphological data on the response to aPDT.

Robust trigger wave speed in Xenopus cytoplasmic extracts

Self-regenerating trigger waves can spread rapidly through the crowded cytoplasm without diminishing in amplitude or speed, providing consistent, reliable, long-range communication. The macromolecular concentration of the cytoplasm varies in response to physiological and environmental fluctuations, raising the question of how or if trigger waves can robustly operate in the face of such fluctuations. Using Xenopus extracts, we find that mitotic and apoptotic trigger wave speeds are remarkably invariant. We derive a model that accounts for this robustness and for the eventual slowing at extremely high and low cytoplasmic concentrations. The model implies that the positive and negative effects of cytoplasmic concentration (increased reactant concentration vs. increased viscosity) are nearly precisely balanced. Accordingly, artificially maintaining a constant cytoplasmic viscosity during dilution abrogates this robustness. The robustness in trigger wave speeds may contribute to the reliability of the extremely rapid embryonic cell cycle.

Age-specific and compartment-dependent changes in mitochondrial homeostasis and cytoplasmic viscosity in mouse peripheral neurons.

Mitochondria are dynamic bioenergetic hubs that become compromised with age. In neurons, declining mitochondrial axonal transport has been associated with reduced cellular health. However, it is still unclear to what extent the decline of mitochondrial transport and function observed during ageing are coupled, and if somal and axonal mitochondria display compartment-specific features that make them more susceptible to the ageing process. It is also not known whether the biophysical state of the cytoplasm, thought to affect many cellular functions, changes with age to impact mitochondrial trafficking and homeostasis. Focusing on the mouse peripheral nervous system, we show that age-dependent decline in mitochondrial trafficking is accompanied by reduction of mitochondrial membrane potential and intramitochondrial viscosity, but not calcium buffering, in both somal and axonal mitochondria. Intriguingly, we observe a specific increase in cytoplasmic viscosity in the neuronal cell body, where mitochondria are most polarised, which correlates with decreased cytoplasmic diffusiveness. Increasing cytoplasmic crowding in the somatic compartment of DRG neurons grown in microfluidic chambers reduces mitochondrial axonal trafficking, suggesting a mechanistic link between the regulation of cytoplasmic viscosity and mitochondrial dynamics. Our work provides a reference for studying the relationship between neuronal mitochondrial homeostasis and the viscoelasticity of the cytoplasm in a compartment-dependent manner during ageing.

Mechanics of spindle orientation in human mitotic cells is determined by pulling forces on astral microtubules and clustering of cortical dynein

The forces that orient the spindle in human cells remain poorly understood due to a lack of direct mechanical measurements in mammalian systems. We use magnetic tweezers to measure the force on human mitotic spindles. Combining the spindle's measured resistance to rotation, the speed at which it rotates after laser ablating astral microtubules, and estimates of the number of ablated microtubules reveals that each microtubule contacting the cell cortex is subject to ∼5 pN of pulling force, suggesting that each is pulled on by an individual dynein motor. We find that the concentration of dynein at the cell cortex and extent of dynein clustering are key determinants of the spindle's resistance to rotation, with little contribution from cytoplasmic viscosity, which we explain using a biophysically based mathematical model. This work reveals how pulling forces on astral microtubules determine the mechanics of spindle orientation and demonstrates the central role of cortical dynein clustering.

Viscosity-dependent control of protein synthesis and degradation

It has been proposed that the concentration of proteins in the cytoplasm maximizes the speed of important biochemical reactions. Here we have used Xenopus egg extracts, which can be diluted or concentrated to yield a range of cytoplasmic protein concentrations, to test the effect of cytoplasmic concentration on mRNA translation and protein degradation. We find that protein synthesis rates are maximal in ~1x cytoplasm, whereas protein degradation continues to rise to a higher optimal concentration of ~1.8x. We show that this difference in optima can be attributed to a greater sensitivity of translation to cytoplasmic viscosity. The different concentration optima could produce a negative feedback homeostatic system, where increasing the cytoplasmic protein concentration above the 1x physiological level increases the viscosity of the cytoplasm, which selectively inhibits translation and drives the system back toward the 1x set point.

A surge in cytoplasmic viscosity triggers nuclear remodeling required for Dux silencing and pre-implantation embryo development

Embryonic genome activation (EGA) marks the transition from dependence on maternal transcripts to an embryonic transcriptional program. The precise temporal regulation of gene expression, specifically the silencing of the Dux/murine endogenous retrovirus type L (MERVL) program during late 2-cell interphase, is crucial for developmental progression in mouse embryos. How this finely tuned regulation is achieved within this specific window is poorly understood. Here, using particle-tracking microrheology throughout the mouse oocyte-to-embryo transition, we identify a surge in cytoplasmic viscosity specific to late 2-cell interphase brought about by high microtubule and endomembrane density. Importantly, preventing the rise in 2-cell viscosity severely impairs nuclear reorganization, resulting in a persistently open chromatin configuration and failure to silence Dux/MERVL. This, in turn, derails embryo development beyond the 2- and 4-cell stages. Our findings reveal a mechanical role of the cytoplasm in regulating Dux/MERVL repression via nuclear remodeling during a temporally confined period in late 2-cell interphase.

Towards a biomimetic cellular structure and physical morphology with liposome-encapsulated agarose sol systems

The cytoplasm, serving as the primary hub of cellular metabolism, stands as a pivotal cornerstone for the harmonious progression of life. The ideal artificial cell should not only have a biomembrane structure system similar to that of a cell and the function of carrying genetic information, but also should have an intracellular environment. In this pursuit, we employed a method involving the incorporation of glycerol into agarose, resulting in the formation of agarose-glycerol mixed sol (AGs). This dynamic sol exhibited fluidic properties at ambient temperature, closely mimicking the viscosity of authentic cytoplasm. Harnessing the electroformation technique, AGs was encapsulated within liposomes, enabling the efficient creation of artificial cells that closely resembled native cellular dimensions through meticulous parameter adjustments of the alternating current (AC) field. Subsequently, artificial cells harboring AGs were subjected to diverse electrolyte and non-electrolyte solutions, enabling a comprehensive exploration of their deformation phenomena, encompassing both inward and outward budding. This study represents a significant stride forward in addressing one of the most fundamental challenges in the construction of artificial cytoplasm. It is our fervent aspiration that this work shall offer invaluable insights and guidance for future endeavors in the realm of artificial cell construction.

Cytoplasmic viscosity is a potential biomarker for metastatic breast cancer cells.

Cellular microrheology has shown that cancer cells with high metastatic potential are softer compared to non-tumorigenic normal cells. These findings rely on measuring the apparent Young's modulus of whole cells using primarily atomic force microscopy. The present study aims to explore whether alternative mechanical parameters have discriminating features with regard to metastatic potential. Magnetic rotational spectroscopy (MRS) is employed in the examination of mammary epithelial cell lines: MCF-7 and MDA-MB-231, representing low and high metastatic potential, along with normal-like MCF-10A cells. MRS utilizes active micron-sized magnetic wires in a rotating magnetic field to measure the viscosity and elastic modulus of the cytoplasm. All three cell lines display viscoelastic behavior, with cytoplasmic viscosities ranging from 10 to 70 Pa s and elastic moduli from 30 to 80 Pa. It is found that the tumorigenic MCF-7 and MDA-MB-231 cells are softer than the MCF-10A cells, with a twofold decrease in the elastic modulus. To differentiate cells with low and high malignancy however, viscosity emerges as the more discriminating parameter, as MCF-7 exhibits a 5 times higher viscosity as compared to MDA-MB-231. These findings highlight the sensitivity of cytoplasmic viscosity to metastatic activity, suggesting its potential use as a mechanical marker for malignant cancer cells.

Blood Rheology and Hemodynamics.

Seminars in Thrombosis and Hemostasis (STH) celebrates 50 years of publishing in 2024. To celebrate this landmark event, STH is republishing some archival material. This article represents the most highly cited paper ever published in STH. The original abstract follows.Blood is a two-phase suspension of formed elements (i.e., red blood cells [RBCs], white blood cells [WBCs], platelets) suspended in an aqueous solution of organic molecules, proteins, and salts called plasma. The apparent viscosity of blood depends on the existing shear forces (i.e., blood behaves as a non-Newtonian fluid) and is determined by hematocrit, plasma viscosity, RBC aggregation, and the mechanical properties of RBCs. RBCs are highly deformable, and this physical property significantly contributes to aiding blood flow both under bulk flow conditions and in the microcirculation. The tendency of RBCs to undergo reversible aggregation is an important determinant of apparent viscosity because the size of RBC aggregates is inversely proportional to the magnitude of shear forces; the aggregates are dispersed with increasing shear forces, then reform under low-flow or static conditions. RBC aggregation also affects the in vivo fluidity of blood, especially in the low-shear regions of the circulatory system. Blood rheology has been reported to be altered in various physiopathological processes: (1) Alterations of hematocrit significantly contribute to hemorheological variations in diseases and in certain extreme physiological conditions; (2) RBC deformability is sensitive to local and general homeostasis, with RBC deformability affected by alterations of the properties and associations of membrane skeletal proteins, the ratio of RBC membrane surface area to cell volume, cell morphology, and cytoplasmic viscosity. Such alterations may result from genetic disorders or may be induced by such factors as abnormal local tissue metabolism, oxidant stress, and activated leukocytes; and (3) RBC aggregation is mainly determined by plasma protein composition and surface properties of RBCs, with increased plasma concentrations of acute phase reactants in inflammatory disorders a common cause of increased RBC aggregation. In addition, RBC aggregation tendency can be modified by alterations of RBC surface properties because of RBC in vivo aging, oxygen-free radicals, or proteolytic enzymes. Impairment of blood fluidity may significantly affect tissue perfusion and result in functional deteriorations, especially if disease processes also disturb vascular properties.

Mechanisms of Red Blood Cell Splenic Filtration Revealed By Multiscale Modeling and in-Vitro Microfluidic Experiments

Interendothelial slits in the spleen fulfill the major physiological function of continuously filtering red blood cells (RBCs) from the bloodstream to remove abnormal and aged cells. To date, the process of passage of 8 µm RBCs through 0.3-µm wide slits remains enigmatic. Should the slits increase their caliber during RBC passage as sometimes proposed in the literature? Here, we elucidated the mechanisms that govern the passage dynamics or retention of RBCs in slits by combining multiscale modeling, live imaging, and microfluidic experiments on an original device with slits of defined physiological dimensions, including submicron width. We observed that healthy RBCs pass through 0.28-µm wide rigid slits at body temperature. To achieve this tour de force, they must meet two requirements. Geometrically, their surface area-to-volume ratio must be compatible with a shape in two tether-connected equal spheres. Mechanically, they must be able to locally unfold their spectrin cytoskeleton inside the slits. In contrast, activation of the mechanosensitive PIEZO1 channel is not required. The RBC transit time through slits scales with in-slit pressure drop and slit width to the -1 and -3 power, respectively. This transit dynamics is similar to that of a Newtonian fluid in a 2D Poiseuille flow, thus showing that it is controlled by the RBC cytoplasmic viscosity. Altogether, our results clearly show that filtration through submicron-wide slits is possible without further slit opening. Furthermore, our approach addresses the critical need for in-vitro evaluation of splenic clearance of diseased or engineered RBCs for transfusion and drug delivery.

Physical mechanisms of red blood cell splenic filtration.

The splenic interendothelial slits fulfill the essential function of continuously filtering red blood cells (RBCs) from the bloodstream to eliminate abnormal and aged cells. To date, the process by which 8 [Formula: see text]m RBCs pass through 0.3 [Formula: see text]m-wide slits remains enigmatic. Does the slit caliber increase during RBC passage as sometimes suggested? Here, we elucidated the mechanisms that govern the RBC retention or passage dynamics in slits by combining multiscale modeling, live imaging, and microfluidic experiments on an original device with submicron-wide physiologically calibrated slits. We observed that healthy RBCs pass through 0.28 [Formula: see text]m-wide rigid slits at 37 °C. To achieve this feat, they must meet two requirements. Geometrically, their surface area-to-volume ratio must be compatible with a shape in two tether-connected equal spheres. Mechanically, the cells with a low surface area-to-volume ratio (28% of RBCs in a 0.4 [Formula: see text]m-wide slit) must locally unfold their spectrin cytoskeleton inside the slit. In contrast, activation of the mechanosensitive PIEZO1 channel is not required. The RBC transit time through the slits follows a [Formula: see text]1 and [Formula: see text]3 power law with in-slit pressure drop and slip width, respectively. This law is similar to that of a Newtonian fluid in a two-dimensional Poiseuille flow, showing that the dynamics of RBCs is controlled by their cytoplasmic viscosity. Altogether, our results show that filtration through submicron-wide slits is possible without further slit opening. Furthermore, our approach addresses the critical need for in vitro evaluation of splenic clearance of diseased or engineered RBCs for transfusion and drug delivery.

Release of Arabidopsis seed dormancy by cold atmospheric plasma relies on cytoplasmic glass transition

When mature Arabidopsis thaliana seeds are dormant, their germination is prevented in apparently favorable conditions. This primary dormancy can be released during seed dry storage through a process called after-ripening whose duration can last several months. To reduce this delay, cold atmospheric plasmas (CAPs) can be used as sources of reactive oxygen species (ROS) capable of inducing heterogeneous chemical reactions. While CAP are known to stimulate the germination of various seed species, the relationship between CAP treatments and the amorphous solid state of dry seeds remains unexplored. Here, we demonstrate that seed dormancy can be alleviated using a cold plasma of ambient air and that this alleviation can be amplified for seeds with high water-content (typically 30%DW) or seeds heated at 60 °C during plasma treatment. Differential scanning micro-calorimetry shows that these characteristics control the glassy/rubbery state of the seed cytoplasm. This technique indicates also that a glass transition to the rubbery state strengthens the CAP effects to alleviate seed dormancy. We propose that lower cytoplasmic viscosity can promote the oxidative signaling induced by CAP which, in turn, improves the germination process.

AFM-Based Poroelastic@Membrane Analysis of Cells and its Opportunities for Translational Medicine.

Cell mechanics is an emerging field of research for translational medicine. Here, the cell is modeled as poroelastic cytoplasm wrapped by tensile membrane (poroelastic@membrane model) and is characterized by the atomic force microscopy (AFM). The parameters of cytoskeleton network modulus EC , cytoplasmic apparent viscosity ηC , and cytoplasmic diffusion coefficient DC are used to describe the mechanical behavior of cytoplasm, and membrane tension γ is used to evaluate the cell membrane. Poroelastic@membrane analysis of breast cells and urothelial cells show that non-cancer cells and cancer cells have different distribution regions and distribution trends in the four-dimensional space composed of EC , ηC . From non-cancer to cancer cells, there is often a trend of γ, EC , ηC decreases and DC increases. Patients with urothelial carcinoma at different malignant stages can be distinguished at high sensitivity and specificity by analyzing the urothelial cells from tissue or urine. However, sampling directly from tumor tissues is an invasive method, may lead to undesirable consequences. Thus, AFM-based poroelastic@membrane analysis of urothelial cells from urine may provide a non-invasive and no-bio-label method to detecting urothelial carcinoma.

Vast heterogeneity in cytoplasmic diffusion rates revealed by nanorheology and Doppelgänger simulations

The cytoplasm is a complex, crowded, actively driven environment whose biophysical characteristics modulate critical cellular processes such as cytoskeletal dynamics, phase separation, and stem cell fate. Little is known about the variance in these cytoplasmic properties. Here, we employed particle-tracking nanorheology on genetically encoded multimeric 40 nm nanoparticles (GEMs) to measure diffusion within the cytoplasm of individual fission yeast (Schizosaccharomyces pombe) cellscells. We found that the apparent diffusion coefficients of individual GEM particles varied over a 400-fold range, while the differences in average particle diffusivity among individual cells spanned a 10-fold range. To determine the origin of this heterogeneity, we developed a Doppelgänger simulation approach that uses stochastic simulations of GEM diffusion that replicate the experimental statistics on a particle-by-particle basis, such that each experimental track and cell had a one-to-one correspondence with their simulated counterpart. These simulations showed that the large intra- and inter-cellular variations in diffusivity could not be explained by experimental variability but could only be reproduced with stochastic models that assume a wide intra- and inter-cellular variation in cytoplasmic viscosity. The simulation combining intra- and inter-cellular variation in viscosity also predicted weak nonergodicity in GEM diffusion, consistent with the experimental data. To probe the origin of this variation, we found that the variance in GEM diffusivity was largely independent of factors such as temperature, the actin and microtubule cytoskeletons, cell-cyle stage, and spatial locations, but was magnified by hyperosmotic shocks. Taken together, our results provide a striking demonstration that the cytoplasm is not “well-mixed” but represents a highly heterogeneous environment in which subcellular components at the 40 nm size scale experience dramatically different effective viscosities within an individual cell, as well as in different cells in a genetically identical population. These findings carry significant implications for the origins and regulation of biological noise at cellular and subcellular levels.

Vast heterogeneity in cytoplasmic diffusion rates revealed by nanorheology and Doppelgänger simulations.

The cytoplasm is a complex, crowded, actively-driven environment whose biophysical characteristics modulate critical cellular processes such as cytoskeletal dynamics, phase separation, and stem-cell fate. Little is known about the variance in these cytoplasmic properties. We employed particle-tracking nano-rheology on genetically-encoded multimeric 40-nm nanoparticles (GEMs) to measure diffusion within the cytoplasm of the fission yeast Schizosaccharomyces pombe. We found that the apparent diffusion coefficients of individual GEM particles varied over a 400-fold range, while the average particle diffusivity for each individual cell spanned a 10-fold range. To determine the origin of this heterogeneity, we developed a Doppelgänger Simulation approach that uses stochastic simulations of GEM diffusion that replicate the experimental statistics on a particle-by-particle basis, such that each experimental track and cell had a one-to-one correspondence with their simulated counterpart. These simulations showed that the large intra- and inter-cellular variations in diffusivity could not be explained by experimental variability but could only be reproduced with stochastic models that assume an equally wide intra- and inter-cellular variation in cytoplasmic viscosity. Through experimental perturbations, we found that the variance in diffusivity was largely independent of factors such as temperature, cytoskeleton, cell cycle stage, and spatial locations, but was magnified by hyperosmotic shocks. The model additionally predicts diffusion through a heterogeneous cytoplasm should be weakly non-ergodic at short time scales, consistent with the experimental data. Taken together, our results provide a striking demonstration that the cytoplasm is not “well-mixed” but represents a highly heterogeneous environment in which subcellular components at the 40-nm size-scale experience dramatically different effective viscosities within an individual cell, as well as in different cells in the population. These findings carry significant implications for the origins and regulation of biological noise at cellular and subcellular levels.

Fluorescence and phosphorescence lifetime imaging reveals a significant cell nuclear viscosity and refractive index changes upon DNA damage

Cytoplasmic viscosity is a crucial parameter in determining rates of diffusion-limited reactions. Changes in viscosity are associated with several diseases, whilst nuclear viscosity determines gene integrity, regulation and expression. Yet how drugs including DNA-damaging agents affect viscosity is unknown. We demonstrate the use of a platinum complex, Pt[L]Cl, that localizes efficiently mostly in the nucleus as a probe for nuclear viscosity. The phosphorescence lifetime of Pt[L]Cl is sensitive to viscosity and provides an excellent tool to investigate the impact of DNA damage. We show using Fluorescence Lifetime Imaging (FLIM) that the lifetime of both green and red fluorescent proteins (FP) are also sensitive to changes in cellular viscosity and refractive index. However, Pt[L]Cl proved to be a more sensitive viscosity probe, by virtue of microsecond phosphorescence lifetime versus nanosecond fluorescence lifetime of FP, hence greater sensitivity to bimolecular reactions. DNA damage was inflicted by either a two-photon excitation, one-photon excitation microbeam and X-rays. DNA damage of live cells causes significant increase in the lifetime of either Pt[L]Cl (HeLa cells, 12.5–14.1 µs) or intracellularly expressed mCherry (HEK293 cells, 1.54–1.67 ns), but a decrease in fluorescence lifetime of GFP from 2.65 to 2.29 ns (in V15B cells). These values represent a viscosity change from 8.59 to 20.56 cP as well as significant changes in the refractive index (RI), according to independent calibration. Interestingly DNA damage localized to a submicron region following a laser microbeam induction showed a whole cell viscosity change, with those in the nucleus being greater than the cytoplasm. We also found evidence of a by-stander effect, whereby adjacent un-irradiated cells also showed nuclear viscosity change. Finally, an increase in viscosity following DNA damage was also observed in bacterial cells with an over-expressed mNeonGreen FP, evidenced by the change in its lifetime from 2.8 to 2.4 ns.

A BODIPY-based fluorescent probe for simultaneous detection of H2O2 and viscosity during the pyroptosis process.

A dual functional BODIPY fluorescent probe was developed for simultaneous detection of H2O2 and viscosity, by collecting fluorescence from 800-1100 nm and 550-750 nm, respectively. Bioimaging based on the probe shows that H2O2 accumulates and cytoplasmic viscosity increases during the palmitic acid (PA)-induced pyroptosis process.

Dependence of diffusion in Escherichia coli cytoplasm on protein size, environmental conditions, and cell growth.

Inside prokaryotic cells, passive translational diffusion typically limits the rates with which cytoplasmic proteins can reach their locations. Diffusion is thus fundamental to most cellular processes, but the understanding of protein mobility in the highly crowded and non-homogeneous environment of a bacterial cell is still limited. Here, we investigated the mobility of a large set of proteins in the cytoplasm of Escherichia coli, by employing fluorescence correlation spectroscopy (FCS) combined with simulations and theoretical modeling. We conclude that cytoplasmic protein mobility could be well described by Brownian diffusion in the confined geometry of the bacterial cell and at the high viscosity imposed by macromolecular crowding. We observed similar size dependence of protein diffusion for the majority of tested proteins, whether native or foreign to E. coli. For the faster-diffusing proteins, this size dependence is well consistent with the Stokes-Einstein relation once taking into account the specific dumbbell shape of protein fusions. Pronounced subdiffusion and hindered mobility are only observed for proteins with extensive interactions within the cytoplasm. Finally, while protein diffusion becomes markedly faster in actively growing cells, at high temperature, or upon treatment with rifampicin, and slower at high osmolarity, all of these perturbations affect proteins of different sizes in the same proportions, which could thus be described as changes of a well-defined cytoplasmic viscosity.

Two‐step nuclear centring by competing microtubule‐ and actin‐based mechanisms in 2‐cell mouse embryos

Microtubules typically promote nuclear centring during early embryonic divisions in centrosome‐containing vertebrates. In acentrosomal mouse zygotes, microtubules also centre male and female pronuclei prior to the first mitosis, this time in concert with actin. How nuclear centring is brought about in subsequent acentrosomal embryonic divisions has not been studied. Here, using time‐lapse imaging in mouse embryos, we find that although nuclei are delivered to the cell centre upon completion of the first mitotic anaphase, the majority do not remain stationary and instead travel all the way to the cortex in a microtubule‐dependent manner. High cytoplasmic viscosity in 2‐cell embryos is associated with non‐diffusive mechanisms involving actin for subsequent nuclear centring when microtubules again exert a negative influence. Thus, following the first mitotic division, pro‐centring actin‐dependent mechanisms work against microtubule‐dependent de‐centring forces. Disrupting the equilibrium of this tug‐of‐war compromises nuclear centring and symmetry of the subsequent division potentially risking embryonic development. This circuitous centring process exposes an embryonic vulnerability imposed by microtubule‐dependent de‐centring forces.

Tank-treading dynamics of red blood cells in shear flow: On the membrane viscosity rheology

In this article, extensive three-dimensional simulations are conducted for tank-treading (TT) red blood cells (RBCs) in shear flow with different cell viscous properties and flow conditions. Apart from recent numerical studies on TT RBCs, this research considers the uncertainty in cytoplasm viscosity, covers a more complete range of shear flow situations of available experiments, and examines the TT behaviors in more details. Key TT characteristics, including the rotation frequency, deformation index, and inclination angle, are compared with available experimental results of similar shear flow conditions. Fairly good simulation-experiment agreements for these parameters can be obtained by adjusting the membrane viscosity values; however, different rheological relationships between the membrane viscosity and the flow shear rate are noted for these comparisons: shear thinning from the TT frequency, Newtonian from the inclination angle, and shear thickening from the cell deformation. Previous studies claimed a shear-thinning membrane viscosity model based on the TT frequency results; however, such a conclusion seems premature from our results and more carefully designed and better controlled investigations are required for the RBC membrane rheology. In addition, our simulation results reveal complicate RBC TT features and such information could be helpful for a better understanding of in vivo and in vitro RBC dynamics.