Attachment Efficiency Research Articles

Kinetic analysis of cell decay during the filling process: Application to lot size determination in manufacturing systems for human induced pluripotent and mesenchymal stem cells

Scale-up of a cell-manufacturing system involves an extension of the filling process time because of the increased number of vials to be filled with the cells suspended in a cryopreservation solution. As the solution includes cryoprotective agents such as dimethyl sulfoxide that is toxic to cells, a longer processing time has an adverse impact on the quality of cell products. Therefore, understanding the related cell-decay kinetics is a major challenge for the development of robust and scalable cell-manufacturing systems. In this study, a new set of performance indices were defined to quantify the state and potential of cells suspended in a cryopreservation solution, i.e., the survival ratio of cells suspended in cryopreservation solution was provided for the cell state estimation, and the recovery ratio of cells after freezing and thawing, the attachment efficiency of cells after seeding, and the specific growth rate of surviving cells were provided for the cell potential estimation. Using these indices, the time-dependencies of cell viability variation in human induced pluripotent (hiPSCs) and human mesenchymal stem cells (hMSCs) suspended in a cryopreservation solution were investigated. Based on the obtained kinetic data, strategies for developing and controlling cell-manufacturing systems were discussed.

Transport of polymer stabilized nano-scale zero-valent iron in porous media

This study presents a set of laboratory-scale transport experiments and numerical simulations evaluating carboxymethyl cellulose (CMC) polymer stabilized nano-scale zero-valent iron (nZVI) transport. The experiments, performed in a glass-walled two-dimensional (2D) porous medium system, were conducted to identify the effects of water specific discharge and CMC concentration on nZVI transport and to produce data for model validation. The transport and movement of a tracer lissamine green B® (LGB) dye, CMC, and CMC-nZVI were evaluated through analysis of the breakthrough curves (BTCs) at the outlets, the time-lapsed images of the plume, and retained nZVI in the sandbox. The CMC mass recovery was >95% when injected alone and about 65% when the CMC-nZVI mixture was used. However, the mean residence time of CMC was significantly higher than that of LGB. Of significance for field implementation, viscous fingering was observed in water displacement of previously injected CMC and CMC-nZVI. The mass recovery of nZVI was lower (<50%) than CMC recovery due to attachment onto sand grain surfaces. Consecutive CMC-nZVI injections showed higher nZVI recovery in the second injection, a factor to be considered in field trials with successive CMC-nZVI injections.Transport of LGB, CMC, and nZVI were modeled using a flow and transport model considering LGB and CMC as solutes, and nZVI as a colloid, with variable solution viscosity due to changes in CMC concentrations. The simulation results matched the experimental observations and provided estimates of transport parameters, including attachment efficiency, that can be used to predict CMC stabilized nZVI transport in similar porous media, although the extent of viscous fingering may be underpredicted. The experimental and simulation results indicated that increasing specific discharge had a greater effect on decreasing CMC-nZVI attachment efficiency (corresponding to greater possible travel distances in the field) than increasing CMC concentration.

Different transport behaviors of Bacillus subtilis cells and spores in saturated porous media: Implications for contamination risks associated with bacterial sporulation in aquifer

In this study, we compared the transport characteristics of Bacillus subtilis cells and their spores in saturated porous media. Both the bacteria cells and spores were readily transported under lower ionic strengths (IS) (1mM), featuring high mass recovery rates (>75%). The bacteria cells recovery rate declined to 58.05% when the IS increased to 100mM; but the spores recovery rate remained at 74.54%. Spores appear to be more mobile than bacterial cells over wide range of IS. Surface properties and breakthrough curves analysis of the two bio-particles (cells and spores) indicated that attachment, straining and blocking mechanisms contribute differently to deposition processes. The extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory further indicated that the particles attachment may primarily occurred at the secondary energy minimum. More important, XDLVO calculations showed that the dominant forces changed with IS, altering the efficiency of particles attachment. The electric double layer repulsion mainly controlled particle deposition at 1mM. In contrast, the van der Waals attraction and Lewis acid-base interactions dominated particle deposition at 100mM. This study showed that spores are more mobile than bacteria cells in subsurface environment. Given that spores form under harsh environmental conditions, we must consider spore formation and transport when groundwater microbial contamination occurs.

Influence of Biochar on Deposition and Release of Clay Colloids in Saturated Porous Media.

Although the potential application of biochar in soil remediation has been recognized, the effect of biochar on the transport of clay colloids, and accordingly the fate of colloid-associated contaminants, is unclear to date. This study conducted saturated column experiments to systematically examine transport of clay colloids in biochar-amended sand porous media in different electrolytes at different ionic strengths. The obtained breakthrough curves were simulated by the convection-diffusion equation, which included a first-order deposition and release terms. The deposition mechanisms were interpreted by calculating Derjaguin-Landau-Verwey-Overbeek interaction energies. A linear relationship between the simulated deposition rate or the attachment efficiency and the fraction of biochar was observed ( ≥ 0.91), indicating more favorable deposition in biochar than in sand. The interaction energy calculations show that the greater deposition in biochar occurs because the half-tube-like cavities on the biochar surfaces favor deposition in secondary minima and the nanoscale physical and chemical heterogeneities on the biochar surfaces increase deposition in primary minima. The deposited clay colloids in NaCl can be released by reduction of ionic strength, whereas the presence of a bivalent cation (Ca) results in irreversible deposition due to the formation of cation bridging between the colloids and biochar surfaces. The deposition and release of clay colloids on or from biochar surfaces not only change their mobilizations in the soil but also influence the efficiency of the biochar for removal of pollutants. Therefore, the influence of biochar on clay colloid transport must be considered before application of the biochar in soil remediation.

Measuring Nanoparticle Attachment Efficiency in Complex Systems.

As process-based environmental fate and transport models for engineered nanoparticles are developed, there is a need for relevant and reliable measures of nanoparticle behavior. The affinity of nanoparticles for various surfaces (α) is one such measure. Measurements of the affinity of nanoparticles obtained by flowing particles through a porous medium are constrained by the types of materials or exposure scenarios that can be configured into such column studies. Utilizing glass beads and kaolinite as model collector surfaces, we evaluate a previously developed mixing method for measuring nanoparticle attachment to environmental surfaces, and validate this method with an equivalent static column system over a range of organic matter concentrations and ionic strengths. We found that, while both impacted heteroaggregation rates in a predictable manner when varied individually, neither dominated when both parameters were varied. The theory behind observed nanoparticle heteroaggregation rates (αβB) to background particles in mixed systems is also experimentally validated, demonstrating both collision frequency (β) and background particle concentration (B) to be independent for use in fate modeling. We further examined the effects of collector particle composition (kaolinite vs glass beads) and nanoparticle surface chemistry (PVP, citrate, or humic acid) on α, and found a strong dependence on both.

Effects of temperature on aggregation kinetics of graphene oxide in aqueous solutions

Laboratory experiments were conducted to determine the effects of temperature (6, 25, and 40°C) on GO aggregation kinetics under different combinations of ionic strength, cation type, humic acid (HA) concentration by monitoring GO hydrodynamic radii and attachment efficiencies. The results showed that, without HA, temperature increase promoted GO aggregation in both monovalent (Na+ and K+) and divalent (Ca2+) solutions. This phenomenon might be caused by multiple processes including enhanced collision frequency, enhanced cation dehydration, and reduced electrostatic repulsion. The presence of HA introduced steric repulsion forces that enhanced GO stability and temperature showed different effects GO aggregation kinetics in monovalent and divalent electrolytes. In monovalent electrolytes, cold temperature diminished the steric repulsion of HA-coated GO. As a result, the fastest increasing rate of GO hydrodynamic radius and the smallest critical coagulation concentration value appeared at the lowest temperature (6°C). Conversely, in divalent electrolyte solutions with HA, high temperature favored GO aggregation, probably because the interactions between Ca2+ and HA increased with temperature resulting in lower HA coating on GO. Findings of this work emphasized the importance of temperature as well as solution chemistry on the stability and fate of GO nanoparticles in aquatic environment.

Direct Electrochemical DNA Sensor based on a new redox oligomer modified with ferrocene and carboxylic acid: Application to the detection of Mycobacterium tuberculosis mutant strain

A new redox oligomer “oligo-methoxy-phenyl-acetonitrile” (Fc-acid-OMPA) modified with ferrocene groups and carboxylic acids as functional groups in side chains, has been synthesized. The redox indicator has been used for monitoring the electrochemical behavior. The functional groups give the flexibility for direct covalent attachment of biomolecules. The electrochemical properties of the redox oligomer film deposited on gold electrodes have been studied by cyclic voltammetry (CV), which showed a rate of electron transfer of 6.43 s−1. The oligomer has been studied as a transducer for electrochemical DNA sensing and for this purpose the acid functional group of Fc-acid-OMPA was attached with the DNA probe of hepatitis C bearing amino group in 5′ position through amid link. The efficiency of DNA attachment on the oligomer film has been analyzed by X-Ray Photoelectron Spectrometry (XPS) and FT-IR spectroscopy. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) have been used to analyze the biosensor construction and DNA detection. A wide linear range of detection from 1 fM to 100 pM have been demonstrated with a limit of detection (LOD) of 0.2 fM. The biosensor has showed an appreciated sensitivity to PCR samples of genomic DNA from Mycobacterium tuberculosis, and has been able to detect a single mutation which confers resistance of M. tuberculosis to rifampicin drug.

Simulating Multiwalled Carbon Nanotube Transport in Surface Water Systems Using the Water Quality Analysis Simulation Program (WASP).

Under the Toxic Substances Control Act (TSCA), the Environmental Protection Agency (EPA) is required to perform new chemical reviews of nanomaterials identified in premanufacture notices. However, environmental fate models developed for traditional contaminants are limited in their ability to simulate nanomaterials' environmental behavior by incomplete understanding and representation of the processes governing nanomaterial distribution in the environment and by scarce empirical data quantifying the interaction of nanomaterials with environmental surfaces. In this study, the well-known Water Quality Analysis Simulation Program (WASP) was updated to incorporate particle collision rate and particle attachment efficiency to simulate multiwalled carbon nanotube (MWCNT) fate and transport in surface waters. Heteroaggregation attachment efficiencies (αhet) values derived from sediment attachment studies are used to parametrize WASP for simulation of MWCNTs transport in Brier Creek, a coastal plain river located in central eastern Georgia, and a tributary to the Savannah River. Simulations using a constant MWCNT load of 0.1 kg d-1 in the uppermost Brier Creek water segment showed that MWCNTs were present predominantly in the Brier Creek water column, while downstream MWCNT surface and deep sediment concentrations exhibited a general increase with time and distance from the source, suggesting that MWCNT releases could have increasing ecological impacts in the benthic region over long time frames.

A robust upscaling of the effective particle deposition rate in porous media

In the upscaling from pore to continuum (Darcy) scale, reaction and deposition phenomena at the solid–liquid interface of a porous medium have to be represented by macroscopic reaction source terms. The effective rates can be computed, in the case of periodic media, from three-dimensional microscopic simulations of the periodic cell. Several computational and semi-analytical models have been studied in the field of colloid filtration to describe this problem. They typically rely on effective deposition rates defined by complex fitting procedures, neglecting the advection-diffusion interplay, the pore-scale flow complexity, and assuming slow reactions (or large Péclet numbers). Therefore, when these rates are inserted into general macroscopic transport equations, they can lead to several conceptual inconsistencies and significant errors. To study more accurately the dependence of deposition on the flow parameters, in this work we advocate a clear distinction between the surface processes (that altogether defines the so-called attachment efficiency), and the pore-scale processes. With this approach, valid when colloidal particles are small enough, we study Brownian and gravity-driven deposition on a face-centred cubic (FCC) arrangement of spherical grains, and define a robust upscaling based on a linear effective reaction rate. The case of partial deposition, defined by an attachment probability, is studied and the limit of perfect sink is retrieved as a particular case. We introduce a novel upscaling approach and a particularly convenient computational setup that allows the direct computation of the asymptotic stationary value of effective rates. This allows to drastically reduce the computational domain down to the scale of the single repeating periodic unit. The savings are ever more noticeable in the case of higher Péclet numbers, when larger physical times are needed to reach the asymptotic regime and thus, equivalently, much larger computational domain and simulation time would be needed in a traditional setup. We show how this new definition of deposition rate is more robust and extendable to the whole range of Péclet numbers; it also is consistent with the classical heat and mass transfer literature.

Effects of Cd(II) on the stability of humic acid-coated nano-TiO2 particles in aquatic environments.

The stability of nanoparticles (NPs) in aquatic environments is important to evaluate their adverse effects on aquatic ecosystems and human health. Nanoparticle stability is known to be influenced by coexisting ions and dissolved organic matter. This study was designed to investigate the effects of coexisting low-level Cd(II) on the stability of humic acid-coated nano-TiO2 (HA-TiO2) particles in aquatic environments by measuring their aggregation kinetics through time-resolved dynamic light scattering (DLS) and monitoring suspended HA-TiO2 concentrations via optical absorbance changes over time. The particles exhibited aggregation behavior consistent with the classic Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. The results showed that Cd(II) concentration, pH, and ionic strength had various effects on the aggregation kinetics of the HA-TiO2 NPs. The HA-TiO2 particles aggregated faster as the Cd(II) concentration increased whereas the stability of the nanoparticles increased as the solution pH increased or ionic strength decreased regardless of the Cd(II) concentration. At the fixed pH and ionic strength conditions, the addition of Cd(II) promoted aggregation of nanoparticles, leading to higher attachment efficiencies. The enhanced aggregation of the HA-TiO2 NPs in the presence of coexisting cadmium ions in aqueous solutions indicated that the fate and transport of nanoparticles could be greatly affected by heavy metals in aquatic environments.

Anammox biomass carrying efficiency of polyethylene non-woven sheets as a carrier material

ABSTRACTTo access the effects of the surface modification and fabric structure of polyethylene (PE) non-woven fabric sheets on retaining the attachment efficiency of anammox biomass, three different non-woven sheets were prepared and inserted in an anammox reactor. The hydrophobic surface modification with 10% KMnO4 and gelatin did not improve the attachment efficiency of the anammox biomass on the surface of the PE non-woven fibers. Densely packed PE-755 having the highest specific surface area to volume ratio (SA/V) (755) retained 221.4 mg biomass per unit sheet, whereas PE-181 having the lowest SA/V (181) retained only 66.4 mg biomass per unit. Accordingly, the volumetric anammox activity of non-woven sheet PE-755 was the highest among the three PE non-woven sheets because of the strong positive relationship between the specific anammox activity and biomass amount (R = 0.835, P < .01). The specific surface area to volume ratio (cm2 cm−3) as well as the bulk density should be considered as important parameters for the selection of non-woven biocarriers for anammox biomass.

The effect of polarity in the electrospinning process on PCL/chitosan nanofibres' structure, properties and efficiency of surface modification

The aim of this research was to study the effect of charge polarity applied to the spinning nozzle on the structure and properties of polycaprolactone/chitosan (PCL/CHT) blends, in particular the efficiency of further surface modification by chondroitin sulphate (CS). The observed differences in the morphology and properties of fibres formed at different polarities were interpreted in terms of molecular interactions occurring in the system. FTIR results indicate stronger PCL-chitosan interactions at negative polarity, resulting in lower PCL crystallinity and crystal size distribution determined by DSC, as well as lower wettability. The charge polarity influences PCL/CHT fibre morphology and tailors some of their properties, e.g. wettability, mechanical properties and the efficiency of surface modification. Better efficiency of CS attachment was observed at negative polarity using atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS) is most probably related to higher chitosan content at the fibres' surface being attracted by the negative external potential.

Numerical study of hydrophobic micron particle’s impaction on liquid surface

In this study, a simulation method is established for the impaction of micron particles on liquid surfaces, by which the processes of two impaction modes (submergence and oscillation) are studied. The submergence is found to go through three stages, each of which shows different characteristics of particle velocity and gas–liquid interface variance. The dominant forces of the early and late times of the submergence mode are hydrodynamic force and surface tension, respectively, the accumulated work of which is in the same order. The lost particle kinetic energy is converted to the surface energy of the interfaces, the internal energy and the kinetic energy of fluids. The primary part of the oscillation is the first cycle, and the characteristics of its sinking process are similar to that of the submergence. In the reverting stage, the particle rising velocity increases first and then decreases, and the cavity retracts until the gas–liquid interface flattens. The dominant forces of the early and late times of the reverting stage are surface tension and hydrodynamic force, respectively. The positive accumulated work of surface tension on the particle is considerably limited due to the large contact angle hysteresis at the early times of the reverting stage. The negative accumulated work of the hydrodynamic force on the particle at the late times causes a fast decrease in particle kinetic energy, which leads to particle floating on the gas–liquid interface. The results are helpful in understanding the mechanism of micron particle impaction and developing the prediction method of attachment efficiency.

Innovations in the flotation of fine and coarse particles

Research on the mechanisms of particle-bubble interaction has provided valuable information on how to improve the flotation of fine (<20 µm) and coarse particles (>100 µm) with novel flotation machines which provide higher collision and attachment efficiencies of fine particles with bubbles and lower detachment of the coarse particles. Also, new grinding methods and technologies have reduced energy consumption in mining and produced better mineral liberation and therefore flotation performance.

In vitro and in vivo evaluation of a small-caliber coaxial electrospun vascular graft loaded with heparin and VEGF

IntroductionTo date, clinically available expanded polytetrafluoro-ethylene (ePTFE) vascular grafts are suboptimal for reconstructing small caliber (D < 6 mm) arteries, owing to thrombosis in early and restenosis in late stage. Our aim in this preliminary study was to fabricate a nano-fibrous vascular graft which was biofunctionalized with VEGF165 and heparin. The short term performance was evaluated both in vitro and in vivo. MethodFour-mm caliber grafts were prepared by the coaxial-elctrospun technique, which consisted of poly(l-lactide-co-caprolactone) [P(LLA-CL)] collagen and elastin. Heparin and endothelial cell growth factor-165 (VEGF165) were encapsulated in the core of the fibrous. Controlled release of the heparin and VEGF165 were evaluated for 28 days. Endothelial cells were cultured on the electrospun grafts or ePTFE grafts as controls. The cellular adhesion, proliferation and morphology were examined. Electrospun or ePTFE grafts were randomly implanted into a rabbit infrarenal aortic replacement model (n = 30) for 28 days without any antiplatelet therapy. At the termination, all grafts were examined by Doppler ultrasound and then evaluated with histology and scanning electron microscopy. ResultsThe cumulative release amount of heparin (6.93 ± 1.03 mg) and VEGF165 (22.17 ± 5.56 μg) during 28 days were measured. Endothelial cells cultured on electrospun grafts showed significantly higher attachment efficiency and proliferation compared to the ePTFE ones (P < 0.001). At 2 h more ECs had attached to the P(LLA-CL)/Collagen/Elatin grafts (83.26 ± 8.02%) compared to P(LLA-CL) (67.07 ± 4.16%) and ePTFE (46.87 ± 8.85%). ECs proliferated faster on VEGF loaded grafts (O.D = 2.9 ± 1.2, n = 12) compared to ePTFE (O.D = 1.7 ± 1.0, n = 12). The patency was significantly higher in electrospun grafts (86.6%) than ePTFE grafts (40.0%) (P = 0.021). Correspondingly, the microscope images of electrospun implants showed little thrombus when compared with the ePTFE implants. ConclusionBiofunctionalized electrospun graft showed surgical properties, hemocompatibility and higher short-term patency compared with the ePTFE grafts. Despite good early performances, profound study should be designed for long-term evaluation.

Effect of magnesium on the osteogenesis of normal human osteoblasts.

Biomaterials containing magnesium are used for implants and bone regeneration. However, mechanisms underlying the biologic effects of magnesium are still largely unknown and have not been examined on normal human osteoblasts. This study was designed to test the effect of supplemented Mg2+ concentrations between 0.5mM and 16mM on the osteogenic behaviors of normal human primary osteoblasts. Human primary osteoblasts were cultured in the groups with various concentrations of supplemented magnesium for various time intervals. Cell proliferation was measured using crystal violet staining. Degree of Alkaline Phosphatase (ALP) activity was measured by fluorometric assay. Expression of osteocalcin was measured by immunosorbent assay. Mineralization of cultures was determined by Alizarin Red S staining. Results showed that initial cell attachment efficiency was not affected by supplemented Mg2+ (P>0.05). At 21 days, proliferation rates increased in groups containing 0.5mM-4mM supplemented Mg2+ and decreased in groups of supplemented 8mM and 16mM Mg2+. ALP activity and osteocalcin expression were upregulated in groups of supplemented Mg2+ between 0.5mM-2.0mM (P<0.05), but downregulated in groups with supplemented Mg2+ concentrations of 4mM and above (P<0.05). Cultures with 1mM and 2mM supplemented Mg2+ showed upregulated mineralization activity compared to the control (P<0.05), but downregulated in groups with supplemented Mg2+ concentrations of 4mM and above (P<0.05). The present study based on an experimental design demonstrated the impact of 2mM supplemented Mg2+ on induced-proliferation and differentiation of normal human osteoblasts.

Hydrophobic agglomeration kinetics of fine kaolinite particles in aqueous suspensions

ABSTRACTThe hydrophobic agglomeration kinetics of kaolinite has been studied through the in-situ turbidity meter measurement system. The effects of surfactant dosage, stirring rate and particle size on the hydrophobic agglomeration dynamics have been investigated. Appropriate surfactant concentrations provided the strongest hydrophobicity, modest stirring rate offered a higher agglomerates formation rate and a lower disruption rate, and smaller particles had lower energy barrier and higher attachment efficiency. The best hydrophobic agglomeration conditions have been determined and a kinetic model of hydrophobic agglomeration has been proposed by experimental results. The kinetic model has been turned out to be fitted to the dynamic process well and can characterize as well as assess the hydrophobic agglomeration kinetics appropriately.

Role of Humic Acid in Enhancing Dissolved Air Flotation for the Removal of TiO2 Nanoparticles

The particle separation efficiency by flotation sharply decreases or even completely fails when the diameter of dispersed particles falls into the nanoscale. In the present laboratory work, humic acid was used to enhance the removal of TiO2 nanoparticles from suspension in a chemical coagulant-free dissolved air flotation process. Without humic acid, merely 63.8% of TiO2 nanoparticles were removed. For the humic acid-assisted dissolved air flotation, the pH of humic acid solution significantly influenced the removal efficiency: more than 90% of nanoparticles could be separated when the pH of the humic acid stock solution was acidic; however, the basic solutions resulted in rather poor performance. In the acidic solution, the fiberlike humic acid might form colloids through the attraction between hydrophobic moieties. They possibly acted as a fishnet and trapped nanoparticles, leading to the great measured bubble–particle attachment efficiency. In all the effluents, a low residual dissolved organic carbon ...

Heterobifunctional Polyprotein for Efficient Characterization of Mechanically Labile Proteins

Atomic force microscopy (AFM) is a powerful tool for single molecule force spectroscopy, allowing researchers to directly probe the energy landscape of protein folding and unfolding. Despite the power of this technique, it has been hindered by non-specific attachment of protein to the AFM surface and tip. Non-specific attachment allows for simple experimental set up, but results in low attachment efficiency, multiple attachments, incomplete unfolding, and adhesion to the surface which can mask low force unfolding events. Not only does this make data collection incredibly time consuming, it also makes it difficult to test mechanically labile proteins. Despite recent advances in tip and surface functionalization, protein labeling continues to be a major hurdle for many labs. Furthermore, most current labeling protocols require cysteine tag modification which is unsuitable for cysteine containing proteins. To address this issue, we developed a simple and versatile protein cassette utilizing aldehyde tags to facilitate the specific attachment of proteins to both the AFM surface and tip. Combining our labeled protein cassette with newly developed techniques to functionalize the surface and tip resulted in a 75-fold increase in attachment efficiency while greatly reducing the number of incomplete unfolding events and surface adhesion. We have shown the ability to quickly insert new proteins into our cassette and rapidly characterize their response to force. Moreover, we were able to rapidly collect a dynamic force spectrum for α3D, an α-helical protein which unfolds at very low force. Our protein cassette is a powerful tool that not only increases the efficiency of AFM based single molecule force spectroscopy, but also enhances the ability to test mechanically labile proteins.

Mesenchymal Stromal Cells as Active Regulators of Lymphocyte Recruitment to Blood Vascular Endothelial Cells.

Methods are described for analyzing adhesion and migration of isolated lymphocytes on endothelial cell monolayers which have been cocultured with different mesenchymal stromal cells, with or without additional cytokine treatment. The different cells types are grown on opposite sides of 3.0 or 0.4 μm pore filters, depending on whether migration through the whole construct is to be analyzed, or adhesion to the endothelial cells alone. Migration away from the sub-endothelial space and through the stromal layer can also be assessed by culturing mesenchymal stromal cells within a 3-D collagen gel overlaid with endothelial cells. Assays may be "static" or the filter-based constructs can be incorporated into flow chambers so that cell behavior can be directly observed under conditions simulating those in vivo. In general, by choice of method, one can evaluate efficiency of attachment, and ability of cells to migrate across the endothelial monolayer, through the filter and through the stromal cell layer in 2-D or 3-D. Fluorescence microscopic examination of fixed filters can be used, e.g., to ascertain whether lymphocytes are retained by stromal cells. In general, static assays have the higher throughput and greatest ease of use, while the flow-based assays are more physiologically relevant and allow detailed recording of cell behavior in real time.