Cells In Microfluidic Systems Research Articles

Single-cell characterization of deformation and dynamics of mesenchymal stem cells in microfluidic systems: A computational study.

Understanding the homing dynamics of individual mesenchymal stem cells (MSCs) in physiologically relevant microenvironments is crucial for improving the efficacy of MSC-based therapies for therapeutic and targeting purposes. This study investigates the passive homing behavior of individual MSCs in micropores that mimic interendothelial clefts through predictive computational simulations informed by previous microfluidic experiments. Initially, we quantified the size-dependent behavior of MSCs in micropores and elucidated the underlying mechanisms. Subsequently, we analyzed the shape deformation and traversal dynamics of each MSC. In addition, we conducted a systematic investigation to understand how the mechanical properties of MSCs impact their traversal process. We considered geometric and mechanical parameters, such as reduced cell volume, cell-to-nucleus diameter ratio, and cytoskeletal prestress states. Furthermore, we quantified the changes in the MSC traversal process and identified the quantitative limits in their response to variations in micropore length. Taken together, the computational results indicate the complex dynamic behavior of individual MSCs in the confined microflow. This finding offers an objective way to evaluate the homing ability of MSCs in an interendothelial-slit-like microenvironment.

How to Perform a Microfluidic Cultivation Experiment-A Guideline to Success.

As a result of the steadily ongoing development of microfluidic cultivation (MC) devices, a plethora of setups is used in biological laboratories for the cultivation and analysis of different organisms. Because of their biocompatibility and ease of fabrication, polydimethylsiloxane (PDMS)-glass-based devices are most prominent. Especially the successful and reproducible cultivation of cells in microfluidic systems, ranging from bacteria over algae and fungi to mammalians, is a fundamental step for further quantitative biological analysis. In combination with live-cell imaging, MC devices allow the cultivation of small cell clusters (or even single cells) under defined environmental conditions and with high spatio-temporal resolution. Yet, most setups in use are custom made and only few standardised setups are available, making trouble-free application and inter-laboratory transfer tricky. Therefore, we provide a guideline to overcome the most frequently occurring challenges during a MC experiment to allow untrained users to learn the application of continuous-flow-based MC devices. By giving a concise overview of the respective workflow, we give the reader a general understanding of the whole procedure and its most common pitfalls. Additionally, we complement the listing of challenges with solutions to overcome these hurdles. On selected case studies, covering successful and reproducible growth of cells in MC devices, we demonstrate detailed solutions to solve occurring challenges as a blueprint for further troubleshooting. Since developer and end-user of MC devices are often different persons, we believe that our guideline will help to enhance a broader applicability of MC in the field of life science and eventually promote the ongoing advancement of MC.

Simulation of hypoxia of myocardial cells in microfluidic systems

The paper presents a newly designed microfluidic system that allows simulation of myocardial hypoxia by biochemical method. The geometry of the microsystem was designed in such a way, that quantitative fluorescent measurements using a spectrofluorometric plate reader was possible. Biochemical simulation of hypoxia was carried out using potent mitochondrial oxidative phosphorylation uncoupler—Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP). Two cardiac cell lines were used in the study—rat cardiomyoblasts (H9C2) and human cardiomyocytes. The effectiveness of biochemical simulation of hypoxia was studied using two fluorescent dyes: carbocyanine iodide (JC-1) and Fluo-4. Changes in the mitochondrial membrane potential and concentration of intracellular calcium ions were tested. The major novelty of this research was the applying the microfluidic system to create hypoxia conditions for cardiac cells using the biochemical approach. In further studies, the presented hypoxia model could be used to develop new methods of treatment of ischemic heart disease for example in cell therapy based on stem cells.

A portable high-resolution microscope based on combination of fiber-optic array and pre-amplification lens

A portable high-resolution microscope based on combination of fiber-optic array (FOA) and pre-amplification lens, which can overcome the magnification limit of FOA, was developed. By employing the amplification lens at the small-end of the FOA, the resolution was improved from 4.4 μm to 1.0 μm over a field of view (FOV) of 140 μm × 100 μm without using any algorithm processing, and the distortion only slightly increases from 0.13% to 0.19%. The distortion is still lower than that of normal lens-based microscopy. Moreover, the combination methods of the amplification lens and FOA were investigated, which including only FOA, the pre-amplification lens at the small-end of the FOA (termed preAL-FOA) and the post-amplification lens at the big-end of FOA (termed postAL-FOA). The result proved the best resolution was achieved by using the preAL-FOA This microscopy is suitable for real-time monitoring of cells in microfluidic systems.

Maxwell’s Mixing Equation Revisited: Characteristic Impedance Equations for Ellipsoidal Cells

We derived a series of, to our knowledge, new analytic expressions for the characteristic features of the impedance spectra of suspensions of homogeneous and single-shell spherical, spheroidal, and ellipsoidal objects, e.g., biological cells of the general ellipsoidal shape. In the derivation, we combined the Maxwell-Wagner mixing equation with our expression for the Clausius-Mossotti factor that had been originally derived to describe AC-electrokinetic effects such as dielectrophoresis, electrorotation, and electroorientation. The influential radius model was employed because it allows for a separation of the geometric and electric problems. For shelled objects, a special axial longitudinal element approach leads to a resistor-capacitor model, which can be used to simplify the mixing equation. Characteristic equations were derived for the plateau levels, peak heights, and characteristic frequencies of the impedance as well as the complex specific conductivities and permittivities of suspensions of axially and randomly oriented homogeneous and single-shell ellipsoidal objects. For membrane-covered spherical objects, most of the limiting cases are identical to—or improved with respect to—the known solutions given by researchers in the field. The characteristic equations were found to be quite precise (largest deviations typically <5% with respect to the full model) when tested with parameters relevant to biological cells. They can be used for the differentiation of orientation and the electric properties of cell suspensions or in the analysis of single cells in microfluidic systems.

Siphon-driven microfluidic passive pump with a yarn flow resistance controller.

Precise control of media delivery to cells in microfluidic systems in a simple and efficient manner is a challenge for a number of cell-based applications. Conventional syringe pumps can deliver culture media into microfluidic devices at precisely controlled flow rates, but they are bulky and require a power source. On the other hand, passive microflow-generating systems cannot maintain continuous, controllable and long-term delivery of media. We have developed an on-chip microflow control technology that combines flow rate control and passive, long-term delivery of media to microwell tissue culture chambers. Here, a passive flow is initiated using the siphon effect and a yarn flow resistor is used to regulate the flow rate in the microchannel. Using the yarn flow resistor, the medium flow rate into the microfluidic cell culture system is made adjustable to a few hundred microliters per hour. To evaluate the effects of controlled flow on microfluidic cell culture properties (feasibility test), we measured the cell alignment and cytoskeletal arrangement of endothelial cells cultured in a microwell array inside the microfluidic channel.

Generation of 3D functional microvascular networks with human mesenchymal stem cells in microfluidic systems.

The generation of functional microvascular networks is critical for the development of advanced in vitro models to replicate pathophysiological conditions. Mural cells provide structural support to blood vessels and secrete biomolecules contributing to vessel stability and functionality. We investigated the role played by two endothelium-related molecules, angiopoietin (Ang-1) and transforming growth factor (TGF-β1), on bone marrow-derived human mesenchymal stem cell (BM-hMSC) phenotypic transition toward a mural cell lineage, both in monoculture and in direct contact with human endothelial cells (ECs), within 3D fibrin gels in microfluidic devices. We demonstrated that the effect of these molecules is dependent on direct heterotypic cell-cell contact. Moreover, we found a significant increase in the amount of α-smooth muscle actin in microvascular networks with added VEGF and TGF-β1 or VEGF and Ang-1 compared to networks with added VEGF alone. However, the addition of TGF-β1 generated a non-interconnected microvasculature, while Ang-1 promoted functional networks, confirmed by microsphere perfusion and permeability measurements. The presence of mural cell-like BM-hMSCs coupled with the addition of Ang-1 increased the number of network branches and reduced mean vessel diameter compared to EC only vasculature. This system has promising applications in the development of advanced in vitro models to study complex biological phenomena involving functional and perfusable microvascular networks.

Reorientation of microfluidic channel enables versatile dielectrophoretic platforms for cell manipulations

Dielectrophoresis is a versatile tool for the sorting, immobilization, and characterization of cells in microfluidic systems. The performance of dielectrophoretic systems strongly relies on the configuration of microelectrodes, which produce a nonuniform electric field. However, once fabricated, the microelectrodes cannot be reconfigured to change the characteristics of the system. Here, we show that the reorientation of the microfluidic channel with respect to the microelectrodes can be readily utilized to alter the characteristics of the system. This enables us to change the location and density of immobilized viable cells across the channel, release viable cells along customized numbers of streams within the channel, change the deflection pattern of nonviable cells along the channel, and improve the sorting of viable and nonviable cells in terms of flow throughput and efficiency of the system. We demonstrate that the reorientation of the microfluidic channel is an effective tool to create versatile dielectrophoretic platforms using the same microelectrode design.

Modelling single- and tandem-bubble dynamics between two parallel plates for biomedical applications

Carefully timed tandem microbubbles have been shown to produce directional and targeted membrane poration of individual cells in microfluidic systems, which could be of use in ultrasound-mediated drug and gene delivery. This study aims at contributing to the understanding of the mechanisms at play in such an interaction. The dynamics of single and tandem microbubbles between two parallel plates is studied numerically and analytically. Comparisons are then made between the numerical results and the available experimental results. Numerically, assuming a potential flow, a three-dimensional boundary element method (BEM) is used to describe complex bubble deformations, jet formation, and bubble splitting. Analytically, compressibility and viscous boundary layer effects along the channel walls, neglected in the BEM model, are considered while shape of the bubble is not considered. Comparisons show that energy losses modify the bubble dynamics when the two approaches use identical initial conditions. The initial conditions in the boundary element method can be adjusted to recover the bubble period and maximum bubble volume when in an infinite medium. Using the same conditions enables the method to recover the full dynamics of single and tandem bubbles, including large deformations and fast re-entering jet formation. This method can be used as a design tool for future tandem-bubble sonoporation experiments.

Dielectrophoresis has broad applicability to marker-free isolation of tumor cells from blood by microfluidic systems

The number of circulating tumor cells (CTCs) found in blood is known to be a prognostic marker for recurrence of primary tumors, however, most current methods for isolating CTCs rely on cell surface markers that are not universally expressed by CTCs. Dielectrophoresis (DEP) can discriminate and manipulate cancer cells in microfluidic systems and has been proposed as a molecular marker-independent approach for isolating CTCs from blood. To investigate the potential applicability of DEP to different cancer types, the dielectric and density properties of the NCI-60 panel of tumor cell types have been measured by dielectrophoretic field-flow fractionation (DEP-FFF) and compared with like properties of the subpopulations of normal peripheral blood cells. We show that all of the NCI-60 cell types, regardless of tissue of origin, exhibit dielectric properties that facilitate their isolation from blood by DEP. Cell types derived from solid tumors that grew in adherent cultures exhibited dielectric properties that were strikingly different from those of peripheral blood cell subpopulations while leukemia-derived lines that grew in non-adherent cultures exhibited dielectric properties that were closer to those of peripheral blood cell types. Our results suggest that DEP methods have wide applicability for the surface-marker independent isolation of viable CTCs from blood as well as for the concentration of leukemia cells from blood.

Modeling the interaction of tandem bubbles for biomedical applications

Recent experiments, motivated by ultrasound-mediated drug and gene delivery, have utilized laser-generated tandem microbubbles to produce directional and targeted membrane poration of individual cells in microfluidic systems [Sankin et al., Phys. Rev. Lett. 105, 078101 (2010)]. Two models describing the dynamics of coupled bubbles between parallel plates have been applied to understand these observations. The first approach is based on the Boundary Element Method in both 2D and 3D coordinate systems for bubbles bounded by finite rigid plates. Deformation of the bubble surfaces is taken into account, capturing phenomena such as bubble jetting and fragmentation [Hsiao et al., Ultrasound Med. Biol. 36, 2065-2079 (2010)]. The second approach is semi-analytic, accounts for fluid compressibility and elasticity of the plates, but is limited to spherical bubble pulsation [Hay et al., J. Acoust. Soc. Am. 129, 2477(A) (2011)]. Observations of tandem bubble interaction with adjacent biological cells and their potent...

In Situ Fluorescence Optical Detection Using a Digital Micromirror Device (DMD) for 3D Cell-based Assays

We have developed a fluorescence optical detection system using a digital micromirror device (DMD) for monitoring 3D cell culture matrices in situ. Full 3D imaging with fast scanning speed was implemented by the combined action of a DMD and a motorized stage. Imaging results with fluorescent microbeads measure the minimum axial resolution of the system as <TEX>$6.3{\mu}m$</TEX>, while full 1-mm scanning through 3D alginate-based matrix was demonstrated. For cell imaging, improved images were obtained by removing background fluorescence although the scanning distance was reduced because of low intracellular fluorescence efficiency. The system is expected to be useful to study various dynamics and behaviors of 3-dimensionally cultured cells in microfluidic systems.

Acoustofluidics 20: Applications in acoustic trapping

This part of the Acoustofluidics tutorial series reviews applications in acoustic trapping of micron-sized particles and cells in microfluidic systems. Acoustic trapping enables non-invasive and non-contact immobilisation of cells and particles in microfluidic systems. Acoustic trapping has been used for reducing the time needed to create 3D cell clusters, enhance particle-based bioassays and facilitated interaction studies of both cells and particles. An area that is increasingly interesting is the use of acoustic trapping for enriching low concentration samples and the washing or fractioning of cell populations prior to sensitive detection methods (MALDI-MS, PCR etc.) The main focus of the review is systems where particles can be retained against a flow while applications in which particles are positioned in a stationary fluid will be addressed in part 21 of the Acoustofluidics tutorial series (M. Wiklund, S. Radel and J. J. Hawkes, Lab Chip, 2012, 12, ).

Mathematical analysis of colonial formation of embryonic stem cells in microfluidic system

A fluidic environment affects mechanochemical characteristics of embryonic stem cells (ESCs). Perfusion is recognized as an attractive culture mode of ESCs since the steady fluidic state can enhance ESCs’ controllability, supporting a unique cell culture condition. Cellular membrane motility presents important information about cellular dynamics such as adhesion, spreading, and migration. Thus, an investigation of the perfusion-induced membrane motility is significant to understand the mechanochemical behavior of ESCs in the steady culture state. In this research, we suggest Lfr, the ratio of circumferential membrane unattached to other cells’ to the cell’s circumference, as a new parameter to characterize cells’ shape and motility. Lfr of embryonic stem cells has positive correlations with cellular area (Ar) and free peripheral length (Lf) but a negative correlation with roundness (Rn). We also propose a mathematical model representing ESCs’ membrane motilities and demonstrate their colonical behavior.

Evaluation of cytotoxic effect of 5-fluorouracil on human carcinoma cells in microfluidic system

In this paper, we present cytotoxicity tests performed in a microanalytical hybrid system. The microchambers of cell culture were integrated with a concentration gradient generator (CGG), which created five different concentrations of evaluated agent in a single step. 5-Fluorouracil (5-FU) is one of the most important chemotherapeutic agents for cancer treatment, therefore it was tested in the microsystem. Independent tests of the cytotoxic effect of 5-FU were performed in this microsystem on different cell lines (human lung carcinoma cells – A549 and human colon carcinoma cells – HT-29). We also present the dependence of cell viability at different times of incubation with 5-FU. We proved that 5-FU has a stronger inhibition effect on A549 than on HT-29 cells. Moreover, the results obtained in the microfluidic system were compared with that of a macroscale. It can be concluded that the conditions of cultivation can affect the level of cytotoxicity of drugs, and the microsystem is a good example that could be used in this field of research.

Combined Simulation and Experimental Study of Large Deformation of Red Blood Cells in Microfluidic Systems

We investigate the biophysical characteristics of healthy human red blood cells (RBCs) traversing microfluidic channels with cross-sectional areas as small as 2.7×3μm. We combine single RBC optical tweezers and flow experiments with corresponding simulations based on dissipative particle dynamics (DPD), and upon validation of the DPD model, predictive simulations and companion experiments are performed in order to quantify cell deformation and pressure-velocity relationships for different channel sizes and physiologically relevant temperatures. We discuss conditions associated with the shape transitions of RBCs along with the relative effects of membrane and cytosol viscosity, plasma environments, and geometry on flow through microfluidic systems at physiological temperatures. In particular, we identify a cross-sectional area threshold below which the RBC membrane properties begin to dominate its flow behavior at room temperature; at physiological temperatures this effect is less profound.

Controlled deposition of cells in sealed microfluidics using flow velocity boundaries

We present a method for depositing cells in a sealed microfluidic device. The device consists of a poly(dimethylsiloxane) (PDMS) microfluidic network (MFN) sealed with a Si chip. The Si chip has vias and ports that are connected to high-precision motorized pumps. The surfaces of the PDMS MFN are homogeneously coated with fibronectin cell adhesion molecules (CAMs). Flow velocity boundaries are created between vicinal microfluidic structures to prevent or permit deposition of cells in specific regions of the MFN. In narrow flow paths, cells experience a wall shear stress from the fast-moving liquid that overcomes the initial adhesion of the cells with CAMs. Conversely, cells can adhere to CAMs in larger flow paths such as cell chambers inside which the velocity of the liquid and the shear stress are reduced. Interactively changing pumping rates makes the critical velocity (the velocity at which cells deposit in the chamber but not elsewhere) easy to find. The transparent PDMS MFN allows both real-time visualization of the deposition process and cellular assays. We illustrate this method using N9 mouse microglia cells. In one experiment, approximately 75 microglia are deposited per min in a approximately 0.5 microL chamber. The deposited cells remain viable, as assessed from staining and biofunctional assays. This method is simple, reliable, fast, and flexible, and therefore is an attractive technique for depositing cells in microfluidic systems for numerous applications.

Optical tweezers for single cells.

Optical tweezers (OT) have emerged as an essential tool for manipulating single biological cells and performing sophisticated biophysical/biomechanical characterizations. Distinct advantages of using tweezers for these characterizations include non-contact force for cell manipulation, force resolution as accurate as 100aN and amiability to liquid medium environments. Their wide range of applications, such as transporting foreign materials into single cells, delivering cells to specific locations and sorting cells in microfluidic systems, are reviewed in this article. Recent developments of OT for nanomechanical characterization of various biological cells are discussed in terms of both their theoretical and experimental advancements. The future trends of employing OT in single cells, especially in stem cell delivery, tissue engineering and regenerative medicine, are prospected. More importantly, current limitations and future challenges of OT for these new paradigms are also highlighted in this review.

Recent advances in electric analysis of cells in microfluidic systems

Microfluidics offers an ideal platform to integrate cell-based assays with electric measurements. The technological advances in microfluidics, microelectronics, electrochemistry, and electrophysiology have greatly inspired the development of microfluidic/electric devices that work with a low number of cells or single cells. The applications of these microfluidic systems range from the detecting of cell culture density to the probing of cellular functions at the single-cell level. In this review, we introduce the recent advances in the electric analysis of cells on a microfluidic platform, specifically related to the quantification and monitoring of cells in static solution, on-chip patch-clamp measurement, and examination of flowing cells. We also point out future directions and challenges in this field.