A novel method for assessing adherent single-cell stiffness in tension: design and testing of a substrate-based live cell functional imaging device

Various micro-devices have been used to assess single cell mechanical properties. Here, we designed and implemented a novel, mechanically actuated, two dimensional cell culture system that enables a measure of cell stiffness based on quantitative functional imaging of cell-substrate interaction. Based on parametric finite element design analysis, we fabricated a soft (5 kPa) polydimethylsiloxane (PDMS) cell substrate coated with collagen-I and fluorescent micro-beads, thus providing a favorable terrain for cell adhesion and for substrate deformation quantification, respectively. We employed a real-time tracking system that analyzes high magnification images of living cells under stretch, and compensates for gross substrate motions by dynamic adjustment of the microscope stage. Digital image correlation (DIC) was used to quantify substrate deformation beneath and surrounding the cell, leading to an estimate of cell stiffness based upon the ability of the cell to resist the applied substrate deformation. Sensitivity of the system was tested using chemical treatments to both “soften” and “stiffen” the cell cytoskeleton with either 0.5 μg/ml Cytochalasin-D or 3% Glutaraldehyde, respectively. Results indicate that untreated osteosarcoma cells (SAOS-2) exhibit a 1.5 ± 0.7% difference in strain from an applied target substrate strain of 8%. Compared to untreated cells, those treated with Cyochalasin-D passively followed the substrate (0.5 ± 0.5%, p < 0.001), whereas Glutaraldehyde enhanced cellular stiffness and the ability to resist the substrate deformation (2.9 ± 1.6%, p < 0.001). Nano-indentation testing showed differences in cell stiffness based on culture treatment, consistent with DIC findings. Our results indicate that mechanics and image analysis approaches do hold promise as a method to quantitatively assess tensile cell constitutive properties.     

A microperfused incubator for tissue mimetic 3D cultures

High density, three-dimensional (3D) cultures present physical similarities to in vivo tissue and are invaluable tools for pre-clinical therapeutic discoveries and development of tissue engineered constructs. Unfortunately, the use of dense cultures is hindered by intra-culture transport limits allowing just a few layer thick cultures for reproducible studies. In order to overcome diffusion limits in intra-culture nutrient and gas availability, a simple scalable microfluidic perfusion platform was developed and validated. A novel perfusion approach maintained laminar flow of nutrients through the culture to meet metabolic need, while removing depleted medium and catabolites. Velocity distributions and 3D flow patterns were measured using microscopic particle image velocimetry. The effectiveness of forced convection laminar perfusion was confirmed by culturing 700 μm thick neural-astrocytic (1:1) constructs at cell density approaching that of the brain (50,000 cells/mm³). At the optimized flow rate of the nutrient medium, the culture viability reached 90% through the full construct thickness at 2 days of perfusion while unperfused controls exhibited widespread cell death. The membrane aerated perfusion platform was integrated within a miniature, imaging accessible enclosure enabling temperature and gas control of the culture environment. Temperature measurements demonstrated fast feedback response to environmental changes resulting in the maintenance of the physiological temperature within 37 ± 0.2°C. Reproducible culturing of tissue equivalents within dynamically controlled environments will provide higher fidelity to in vivo function in an in vitro accessible format for cell-based assays and regenerative medicine.     

A Method to Replicate the Microstructure of Heart Tissue <Emphasis Type="Italic">In Vitro</Emphasis> Using DTMRI-Based Cell Micropatterning

A novel cell culture methodology is described in which diffusion tensor magnetic resonance imaging (DTMRI) and cell micropatterning are combined to fabricate cell monolayers that replicate realistic cross-sectional tissue structure. As a proof-of-principle, neonatal rat ventricular myocyte (NRVM) monolayers were cultured to replicate the tissue microstructure of murine ventricular cross-sections. Specifically, DTMRI-measured in-plane cardiac fiber directions were converted into soft-lithography photomasks. Silicone stamps fabricated from the photomasks deposit fibronectin patterns to guide local cellular alignment. Fibronectin patterns consisted of a matrix of 190 μm² subregions, each comprised of parallel lines 11–20 μm-wide, spaced 2–8.5 μm apart, and angled to match local DTMRI-measured fiber directions. Within 6 days of culture, NRVMs established confluent, electrically coupled monolayers, and for 18 μm-wide, 5 μm-spaced lines, directions of cell alignment in subregions microscopically replicated DTMRI-measurements with a local error of 7.2 ± 4.1°. By adjusting fibronectin line widths and spacings, cell elongation, gap junctional membrane distribution, and local cellular disarray were altered without changing the dominant directions of cell alignment in individual subregions. Changes in the anisotropy of electrical propagation were assessed by optically mapping membrane potentials. This novel methodology is expected to enable systematic studies of intramural structure–function relationships in both healthy and structurally remodeled hearts.     

Design of Multifunctional Nanomedical Systems

Multifunctional nanoparticles hold great promise for drug/gene delivery and simultaneous diagnostics and therapeutics (“theragnostics”) including use of core materials that provide in vivo imaging and opportunities for externally modulated therapeutic interventions. Multilayered nanoparticles can act as nanomedical systems with on-board molecular programming done through the chemistry of highly specialized layers to accomplish complex and potentially decision-making tasks. The targeting process itself is a multi-step process consisting of initial cell recognition through cell surface receptors, cell entry through the membrane in a manner to prevent undesired alterations of the nanomedical system, re-targeting to the appropriate sub-region of the cell where the therapeutic package can be localized, and potentially control of that therapeutic process through feedback systems using molecular biosensors. This paper describes a bionanoengineering design process in which sophisticated nanomedical platform systems can be designed for diagnosis and treatment of disease. The feasibility of most of these subsystems has been demonstrated, but the full integration of these interacting sub-components remains a challenge for the field. Specific examples of sub-components developed for specific applications are described.     

Efficacy of ginkgolic acids against Cryptosporidium andersoni in cell culture

Cryptosporidium is a worldwide waterborne parasite and the treatment is a severe problem in immunocompromised patients. In this study, we used the in vitro culture system to evaluate the anti-Cryptosporidium activity of ginkgolic acids (GAs), nitazoxanide (NTZ), garlicin (GAR), and artemether (ART). The growth of Cryptosporidium andersoni in HCT-8 cells was determined by real-time PCR assay. When exposed to 5.00 μg/ml GAs or 10.00 μg/ml NTZ for 48 h, the number of C. andersoni in cultures was on a very low lever, but the number of parasites did not significantly decrease when exposed to GAR and ART. Our results indicate that GAs could be a potential drug for the treatment of cryptosporidiosis.     

Perfluorocarbon Nanoemulsions for Quantitative Molecular Imaging and Targeted Therapeutics

A broad array of nanomaterials is available for use as contrast agents for molecular imaging and drug delivery. Due to the lack of endogenous background signal in vivo and the high NMR sensitivity of the ¹⁹F atom, liquid perfluorocarbon nanoemulsions make ideal agents for cellular and magnetic resonance molecular imaging. The perfluorocarbon core material is surrounded by a lipid monolayer which can be functionalized with a variety of agents including targeting ligands, imaging agents and drugs either individually or in combination. Multiple copies of targeting ligands (∼20–40 monoclonal antibodies or 200–400 small molecule ligands) serve to enhance avidity through multivalent interactions while the composition of the particle’s perfluorocarbon core results in high local concentrations of ¹⁹F. Additionally, lipophilic drugs contained within molecularly targeted nanoemulsions can result in contact facilitated drug delivery to target cells. Ultimately, the dual use of perfluorocarbon nanoparticles for both site targeted drug delivery and molecular imaging may provide both imaging of disease states as well as conclusive evidence that drug delivery is localized to the area of interest. This review will focus on liquid perfluorocarbon nanoparticles as ¹⁹F molecular imaging agents and for targeted drug delivery in cancer and cardiovascular disease.     

Microfluidics/CMOS orthogonal capabilities for cell biology

The study of individual cells and cellular networks can greatly benefit from the capabilities of microfabricated devices for the stimulation and the recording of electrical cellular events. In this contribution, we describe the development of a device, which combines capabilities for both electrical and pharmacological cell stimulation, and the subsequent recording of electrical cellular activity. The device combines the unique advantages of integrated circuitry (CMOS technology) for signal processing and microfluidics for drug delivery. Both techniques are ideally suited to study electrogenic mammalian cells, because feature sizes are of the same order as the cell diameter, ∼ 50 μm. Despite these attractive features, we observe a size mismatch between microfluidic devices, with bulky fluidic connections to the outside world, and highly miniaturized CMOS chips. To overcome this problem, we developed a microfluidic flow cell that accommodates a small CMOS chip. We simulated the performances of a flow cell based on a 3-D microfluidic system, and then fabricated the device to experimentally verify the nutrient delivery and localized drug delivery performance. The flow-cell has a constant nutrient flow, and six drug inlets that can individually deliver a drug to the cells. The experimental analysis of the nutrient and drug flow mass transfer properties in the flowcell are in good agreement with our simulations. For an experimental proof-of-principle, we successfully delivered, in a spatially resolved manner, a ‘drug’ to a culture of HL-1 cardiac myocytes.     

Integration of a pump and an electrical sensor into a membrane-based PDMS microbioreactor for cell culture and drug testing

To the extent possible, artificial organs should have characteristics that match those of the in vivo system. To this end, microfabrication techniques allow us to create microenvironments that can help maintain cell organization and functionality in in vitro cultures. We present three new microbioreactors, each of which allows cells to be cultured in a perfused microenvironment similar to that found in vivo. Our microbioreactors use new technology that permits integration onto the chip (35 mm × 20 mm) of an electrical sensor, in addition to one or more pumping systems and associated perfusion circuitry. The monitoring of Caco-2 cell cultures using electrical impedance spectroscopy (EIS) has allowed us to measure the effects of cell growth, cellular barrier formation and the presence of chemical compounds and/or toxins. Specifically, we have investigated the ability of the electrical sensor to maintain appropriate sensitivity and precision. Our results show that the sensor was very sensitive not only to the presence or the absence of the cells, but also to changes in cell state. Our perfused microbioreactors are highly efficient miniaturized tools that are easy to operate. We anticipate that they will offer promising new opportunities in many types of cell culture research, including drug screening and tissue engineering.     

DNA bacterial load in children and adolescents with pneumococcal pneumonia and empyema

The purpose of this investigation was to evaluate a rapid quantitative real-time polymerase chain reaction (PCR) for the direct detection and quantification of pneumococcal DNA bacterial load (DBL) in patients with pneumonia and empyema. DBL and molecular serotype detection was determined by DNA quantification of the pneumolysin (ply) gene and an additional capsular gene by real-time PCR. Plasma or pleural fluid samples from children and adolescents with confirmed pneumococcal pneumonia were analyzed. DBL was correlated with clinical parameters and outcomes. One hundred and sixty-nine patients with pneumococcal pneumonia (145 empyema) had bacterial cultures and real-time PCR assays performed. Among them, 41 (24.3%) had positive results for both, 4 (2.4%) had positive culture alone, and 124 (73.3%) had positive real-time PCR alone. The pleural fluid DBL was lower in patients with prior antibiotics (p = 0.01) and higher in patients with positive culture (p < 0.001). The pleural fluid DBL was positively correlated with serum C-reactive protein (p = 0.009), pleural fluid neutrophils (p < 0.001), and pleural fluid glucose (p < 0.001). The plasma and pleural fluid DBL were higher in patients with ≥8 days of hospital stay (p = 0.002), and the pleural fluid DBL was positively correlated with the number of hours of pleural drainage (p < 0.001). Quantification of pneumococcal DBL by real-time PCR may be helpful for the diagnosis and clinical management of pediatric patients with pneumonia and empyema     

Effect of the antitubulin drug oryzalin on the encystation of Entamoeba invadens

We have recently demonstrated that the antimicrotubule drug oryzalin inhibits the growth of Entamoeba invadens as well as E. histolytica, the former being more resistant to the drug than the latter, and that effective doses of oryzalin are higher for Entamoeba than for the other parasitic protozoa examined thus far. The aim of the present study was to examine the effect of oryzalin on the encystation of E. invadens using an axenic encystation system in vitro. Oryzalin inhibited the encystation of E. invadens strain IP-1 in a dose-dependent manner. The addition of oryzalin after the induction of encystation was also inhibitory for encystation and cyst maturation. Trophozoites incubated for 1 day in encystation medium with oryzalin did not encyst after removal of the drug. Although trophozoites grown in the presence of 300 μM oryzalin for 2 days did not encyst after their transfer to encystation medium containing the same concentration of drug, a number of trophozoites survived for at least 3 days. In contrast, trophozoites grown in the absence of oryzalin neither survived nor encysted after their transfer to encystation medium supplemented with the drug, which suggests that pretreatment of trophozoites with oryzalin contributes to their continued survival as trophozoites, i.e., without their transforming into cysts, in encystation medium. Trophozoites grown with oryzalin did encyst after their transfer to encystation medium without the drug. Accumulation of trophozoites in the mitotic phase was observed after culture with oryzalin. When cysts prepared at day 1 of encystation, most of which were mononucleate, were reincubated in the presence of oryzalin for an additional 2 days, inhibition of their maturation was observed. Thus, oryzalin is a potent mitotic-phase inhibitor of E. invadens and may become a useful tool for studies on the relationship between the cell cycle and encystation of this parasite. Received: 29 November 1999 / Accepted: 28 January 2000     

On-chip testing device for electrochemotherapeutic effects on human breast cells

A microfabricated cell-based testing device for electrochemotherapy (ECT) has been developed by miniaturizing the widely used clinical electroporator with a two-needle array into two-dimensional planar electrodes while keeping the similarity of the electric field strength distribution. In this device, all the biological processes from cell culture to electroporation and final cell-based assays were carried out on a chip using a conventional 2D cell culture method, and the multiple electrochemotherapeutic assays could be realized by exploiting the six electroporation sites in a single device. With the proposed platform, the electroporation rate was evaluated with propidium iodide and cell proliferation after 48 h of electrochemotherapy with bleomycin was determined with T47D human breast ductal carcinoma cell line in various electric field strengths and drug concentrations. This microsystem has several advantages over conventional cuvette type electroporation assay, such as multiple assays on a chip, on-chip based operation from cell culture to final assay, and having similar electric field distribution as that of the clinical electroporator. As the clinical trials of electrochemotherapy are being carried out, this new platform is expected to have valuable applications in basic in vitro ECT studies, drug discovery, and development of clinical ECT equipment.     

Efficient Dielectrophoretic Patterning of Embryonic Stem Cells in Energy Landscapes Defined by Hydrogel Geometries

In this study, we have developed an integrated microfluidic platform for actively patterning mammalian cells, where poly(ethylene glycol) (PEG) hydrogels play two important roles as a non-fouling layer and a dielectric structure. The developed system has an embedded array of PEG microwells fabricated on a planar indium tin oxide (ITO) electrode. Due to its dielectric properties, the PEG microwells define electrical energy landscapes, effectively forming positive dielectrophoresis (DEP) traps in a low-conductivity environment. Distribution of DEP forces on a model cell was first estimated by computationally solving quasi-electrostatic Maxwell’s equations, followed by an experimental demonstration of cell and particle patterning without an external flow. Furthermore, efficient patterning of mouse embryonic stem (mES) cells was successfully achieved in combination with an external flow. With a seeding density of 10⁷ cells/mL and a flow rate of 3 μL/min, trapping of cells in the microwells was completed in tens of seconds after initiation of the DEP operation. Captured cells subsequently formed viable and homogeneous monolayer patterns. This simple approach could provide an efficient strategy for fabricating various cell microarrays for applications such as cell-based biosensors, drug discovery, and cell microenvironment studies.     

Cell-Culture System for Continuous Production of Toxoplasma gondii Tachyzoites

 The aim of this study was to identify a sustainable cell line and culture method that could continuously provide a sufficient quantity of Toxoplasma gondii tachyzoites to serve the needs of a general hospital laboratory. Three continuous cell lines (HeLa, LLC and Vero) and three cell-culture methods (culture in conventional flasks, culture in membrane-based flasks and an automated culture system) were investigated. In multiplicity-of-infection and time-course experiments, HeLa was the cell line of choice. Harvests from HeLa cells had significantly higher tachyzoite yields than those from LLC cells (P<0.00005) or Vero cells (P<0.05). Membrane-based flasks gave higher yields (6.15×10⁶ tachyzoites/ml) than conventional flasks (1–2×10⁶ tachyzoites/ml) initially, but these were not sustained. The automated cell-culture system was unsuitable for parasite culture. Continuous passage in 25 cm² flasks was successful, yielding 1×10⁶ tachyzoites/ml; viability exceeded 90% after 96–120 h of infection throughout 38 passes, during which time the viability improved and the time to harvest became more consistent. Toxoplasma gondii grown in continuous culture in HeLa cells can provide a regular supply of viable tachyzoites. Demonstration that HeLa-derived tachyzoites could be used for the dye test confirms the potential of this in vitro system for use in general hospital laboratories.     

Lentivirus transduction of bone marrow hemopoietic precursor cells with Lin-c-kit<Superscript>+</Superscript> phenotype <Emphasis Type="Italic">Ex Vivo</Emphasis> using a genetic construct containing green fluorescent protein gene

We developed a technology of labeling bone marrow precursor cells with the Lin^—c-kit⁺ phenotype in culture by green fluorescent protein gene using a lentivirus vector. The proposed system provides effective transduction of bone marrow precursor cells and high transgene expression level in vitro (27%). The integration of the transgene into the transduced cell genome in vivo was verified by the method of splenic colonies. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 6, pp. 667–671, June, 2007     

Anin vitro traumatic injury model to examine the response of neurons to a hydrodynamically-induced deformation

A novelin vitro system was developed to examine the effects of traumatic mechanical loading on individual cells. The cell shearing injury device (CSID) is a parallel disk viscometer that applies fluid shear stress with variable onset rate. The CSID was used in conjunction with microscopy and biochemical techniques to obtain a quantitative expression of the deformation and functional response of neurons to injury. Analytical and numerical approximations of the shear stress at the bottom disk were compared to determine the contribution of secondary flows. A significant portion of the shear stress was directed in ther-direction during start-up, and therefore the full Navier-Stokes equation was necessary to accurately describe the transient shear stress. When shear stress was applied at a high rate (800 dyne cm⁻² sec⁻¹) to cultured neurons, a range of cell membrane strains (0.01 to 0.53) was obtained, suggesting inhomogeneity in cellular response. Functionally, cytosolic calcium and extracellular lactate dehydrogenase levels increased in response to high strain rate (>1 sec⁻¹) loading, compared with quasistatic (<1 sec⁻¹) loading. In addition, a subpopulation of the culture subjected to rapid deformation subsequently died. These strain rates are relevant to those shown to occur in traumatic injury, and as such, the CSID is an appropriate model for studying the biomechanics and pathophysiology of neuronal injury.     

Experimental–Computational Evaluation of Human Bone Marrow Stromal Cell Spreading on Trabecular Bone Structures

The clinical application of macro-porous scaffolds for bone regeneration is significantly affected by the problem of insufficient cell colonization. Given the wide variety of different scaffold structures used for tissue engineering it is essential to derive relationships for cell colonization independent of scaffold architecture. To study cell population spreading on 3D structures decoupled from nutrient limitations, an in vitro culture system was developed consisting of thin slices of human trabecular bone seeded with Human Bone Marrow Stromal Cells, combined with dedicated μCT imaging and computational modeling of cell population spreading. Only the first phase of in vitro scaffold colonization was addressed, in which cells migrate and proliferate up to the stage when the surface of the bone is covered as a monolayer, a critical prerequisite for further tissue formation. The results confirm the model’s ability to represent experimentally observed cell population spreading. The key advantage of the computational model was that by incorporating complex 3D structure, cell behavior can be characterized quantitatively in terms of intrinsic migration parameters, which could potentially be used for predictions on different macro-porous scaffolds subject to additional experimental validation. This type of modeling will prove useful in predicting cell colonization and improving strategies for skeletal tissue engineering.     

Enhanced effects of secreted soluble factor preserve better pluripotent state of embryonic stem cell culture in a membrane-based compartmentalized micro-bioreactor

Pluripotent stem cells are under the influence of soluble factors in a diffusion dominant in vivo microenvironment. In order to investigate the effects of secreted soluble factors on embryonic stem cell (ESC) behavior in a diffusion dominant microenvironment, we cultured mouse ESCs (mESCs) in a membrane-based two-chambered micro-bioreactor (MB). To avoid disturbing the cellular environment in the top chamber of the MB, only the culture medium of the bottom chamber was exchanged. Cell growth in the MB after 5 days of culture was similar to that in conventional 6-well plate (6-WP) and membrane-based Transwell insert (TW) cultures, indicating adequate nutrient supply in the MB. However, the cells retained higher expression of pluripotency markers (Oct4, Sox2 and Rex1) and secreted soluble factors (FGF4 and BMP4) in the MB. Inhibition of FGF4 activity in the MB and TW resulted in a similar cellular response. However, in contrast to the TW, inhibition of BMP4 activity revealed that autocrine action of the upregulated BMP4, which acted cooperatively with leukemia inhibitory factor (LIF), upregulated the pluripotency markers expression in the MB culture. We propose that BMP4 accumulated in the diffusion dominant microenvironment of the MB upregulated its own expression by a positive feedback mechanism—in contrast to the macro-scale culture systems—thereby enhancing the pluripotency of mESCs. The micro-scale culture platform developed in this study enables the investigation of the effects of soluble factors on ESCs in a diffusion dominant microenvironment, and is expected to be used to modulate the ESC fate choices.     

A silica–polymer composite nano system for tumor-targeted imaging and p53 gene therapy of lung cancer

In our study, a silica–polymer composite nano system (MB-NSi–p53–CS ternary complexes) composed of methylene blue-encapsulated amine-terminated silica nanoparticles (MB-NSi) and chondroitin sulfate (CS) were successfully developed for tumor-targeted imaging and p53 gene therapy of lung cancer. MB was employed as a NIR probe for in vivo imaging, MB-NSi nanoparticles were served as gene vector, while CS was applied to be a coating and targeting polymer. MB-NSi–p53–CS ternary complexes displayed nanosized diameter, effective p53 condensation ability, efficient p53 protection profile, and superior bovine serum albumin stability in vitro. Experiments on A549 cell line further revealed low cytotoxicity, high p53 transfection, and anticancer efficacy of MB-NSi–p53–CS ternary complexes. In vivo imaging and tumor targetability assays demonstrated that MB-NSi–p53–CS ternary complexes were a preferable system with desirable imaging and tumor-targeting properties.