Proteomic evaluation of cell preparation methods in primary hepatocyte cell culture

In vitro liver preparations are being used increasingly to study various aspects of chemical hepatotoxicity and thus have become powerful alternatives to in vivo toxicologic models. Primary hepatocyte culture systems are especially useful in screening cytotoxic and genotoxic compounds and assessing biochemical lesions associated with chemical exposure. We have begun to use this approach in combination with proteomic analysis to construct a molecular "toxicoproteomic" test system for a broad range of relevant and potentially toxic chemicals. Using a highly parallel two-dimensional electrophoretic (2-DE) protein separation system to analyze cells from culture systems, we previously observed significant variations in protein expression that were unrelated to chemical exposure. We hypothesized these artifactual protein alterations were the result of the variations in the culture conditions or cell manipulations, or both. Therefore, we conducted a study to assess the expression of hepatocyte proteins cultured on 6-well plates and recovered for analysis either by scraping/pelleting or direct in-well solubilization. Following incubation of 1.2 x 10(6) hepatocytes in six-well plate, recovery and solubilization of the cells and 2-DE of the solubilized lysates of 100 000 cells, we detected 1388 proteins in the in-well solubilized samples compared to 899 proteins in the washed/scraped/pelleted cell samples, a loss of 35%. Based on protein identification by peptide mass fingerprinting, the subcellular location of nearly all of the proteins whose abundance decreased were cytosolic and those few that increased were either microsomal, mitochondrial, or cytoskeletal proteins. These results emphasize the variation introduced by cell-handling during recovery of hepatocytes from culture plates and may explain at least some of the artifactual differences observed in earlier in vitro experiments.     

Hybrid cell adhesive material for instant dielectrophoretic cell trapping and long-term cell function assessment

Dielectrophoresis (DEP) for cell manipulation has focused, for the most part, on approaches for separation/enrichment of cells of interest. Advancements in cell positioning and immobilization onto substrates for cell culture, either as single cells or as cell aggregates, has benefited from the intensified research efforts in DEP (electrokinetic) manipulation. However, there has yet to be a DEP approach that provides the conditions for cell manipulation while promoting cell function processes such as cell differentiation. Here we present the first demonstration of a system that combines DEP with a hybrid cell adhesive material (hCAM) to allow for cell entrapment and cell function, as demonstrated by cell differentiation into neuronlike cells (NLCs). The hCAM, comprised of polyelectrolytes and fibronectin, was engineered to function as an instantaneous cell adhesive surface after DEP manipulation and to support long-term cell function (cell proliferation, induction, and differentiation). Pluripotent P19 mouse embryonal carcinoma cells flowing within a microchannel were attracted to the DEP electrode surface and remained adhered onto the hCAM coating under a fluid flow field after the DEP forces were removed. Cells remained viable after DEP manipulation for up to 8 d, during which time the P19 cells were induced to differentiate into NLCs. This approach could have further applications in areas such as cell-cell communication, three-dimensional cell aggregates to create cell microenvironments, and cell cocultures.     

Growth control by cell to cell contact.

Control of cell growth by cell to cell contact is reviewed with particular emphasis on two systems--contact inhibition of growth observed with Swiss 3T3 cells and the mitogenic stimulation of Schwann cells by dorsal root ganglia neurites. In both cases the biological effect can be reproduced by the addition of surface membranes to the corresponding cells. In the case of contact inhibition of 3T3 cells, biological activity appears to correlate with membrane binding to the cells. An octylglucoside extract of 3T3 plasma membranes retains the biological activity (growth inhibition) of the original membranes.     

Harmless glucose-modified ruthenium complexes suppressing cell migration of highly invasive cancer cell lines

Glucose-substituted ruthenium complexes [(η⁶-benzyl-glucose)RuCp*]⁺Cl⁻, where Cp* = η⁵-C₅Me₅; benzyl-glucose = peracetylated benzyl β-d -glucopyranoside ( 1 ), benzyl β-d -glucopyranoside ( 2 ), have been prepared and used as efficient antimigration and anti-invasive agents against metastatic breast cancer cells (MDA-MB-231) and cisplatin-resistant ovarian cancer cells (SK-OV-3). In addition, these complexes were found to be essentially non-toxic against non-cancerous human kidney cells (HEK293).     

Improvement of lactic cell production

The production ofLactobacillus brevis has been improved by changing the medium composition and the physiological conditions. The cellular concentration reaches 8.2 X 10¹⁰ cells/mL in 21 h with a fed-batch technique in MRS medium. Freeze-drying has been used as technology for drying the cells and survival rates have been improved with different additives such as glycerol, CaCO₃, and skimmed-milk powder up to 70%. A model has been developed to predict the stability of freeze-dried cultures during long-term conservation. This model, based on Arrhenius equation, has been confirmed by experimental data.

     

Direct linkage of thrombin to its cell surface receptors in different cell types

When 125I-thrombin was incubated with foreskin fibroblasts, cervical carcinoma cells or fibrosarcoma cells of human origin, or with secondary chick embryo cells or Chinese hamster lung cells, it became directly linked to its cell surface receptors. The thrombin-receptor complex (TH-R) was derived exclusively from a pool of 125I-thrombin that had become specifically bound to the cell surface. The linkage was probably covalent, since the complex was resistant to boiling in sodium dodecyl sulfate and 2-mercaptoethanol. Raising the pH to 12 disrupted TH-R, but did not affect a similar complex between epidermal growth factor and its receptor, suggesting that the linkage of these mitogens to their receptors was different. Mild trypsin treatment removed the ability of cells to form TH-R; however, after a 24-h incubation in serum-free medium, trypsin-treated cells recovered the capacity to form TH-R, suggesting that TH-R resulted from interaction of 125I-thrombin with a cellular rather than a serum component. The mitogenic response of cells to thrombin was inversely related to the fraction of specifically bound 125I-thrombin represented by TH-R. The role of TH-R in mitogenesis may be clarified in future studies by obtaining clones of Chinese hamster lung cells that vary in their capacities to form TH-R and to respond to the mitogenic action of thrombin.     

Design and fabrication of an integrated cell processor for single embryo cell manipulation

This paper presents an integrated cell processor for the automatic handling of individual embryo cells. The integrated processor can perform various functions such as cell transport, isolation, orientation, and immobilization. These functions are indispensable and frequently used for the manipulation of single cells, but can only be carried out by a skillful operator. The purpose of this study was the integration and automation of these functions for effective cell manipulation, using a MEMS approach. The isolation of a cell was performed using polypyrrole (PPy) valves in a microchannel into which cells were transported. The orientation of cells was controlled by electrorotation (ER), and the target cell was immobilized by suction from a microhole. All of these functions were seamlessly realized on a single chip. Excellent experimental results with mouse (B6CBA) embryo cells showed that this device could substitute for routine and cumbersome manual work. It is expected that the integrated chip will contribute significantly to faster and more reliable manipulation of cells.     

Rewiring cell adhesion

The adhesion of cells is mediated by the binding of several cell-surface receptors to ligands found in the extracellular matrix. These receptors often have overlapping specificities for the peptide ligands, making it difficult to understand the roles for discrete receptors in cell adhesion, migration, and differentiation as well as to direct the selective adhesion of cell types in tissue-engineering applications. To overcome these limitations, we developed a strategy to rewire the receptor-ligand interactions between a cell and substrate to ensure that adhesion is mediated by a single receptor with unique specificity. The strategy combines a genetic approach to engineer the cell surface with a chimeric integrin receptor having a unique ligand binding domain with a surface chemistry approach to prepare substrates that present ligands that are bound by the new binding domain. We show that Chinese hamster ovary cells that are engineered with a chimeric beta1 integrin adhere, signal, and even migrate on a synthetic matrix.     

Microfluidically-unified cell culture, sample preparation, imaging and flow cytometry for measurement of cell signaling pathways with single cell resolution

We have developed a microfluidic platform that enables, in one experiment, monitoring of signaling events spanning multiple time-scales and cellular locations through seamless integration of cell culture, stimulation and preparation with downstream analysis. A combination of two single-cell resolution techniques-on-chip multi-color flow cytometry and fluorescence imaging provides multiplexed and orthogonal data on cellular events. Automated, microfluidic operation allows quantitatively- and temporally-precise dosing leading to fine time-resolution and improved reproducibility of measurements. The platform was used to profile the toll-like receptor (TLR4) pathway in macrophages challenged with lipopolysaccharide (LPS)-beginning with TLR4 receptor activation by LPS, through intracellular MAPK signaling, RelA/p65 translocation in real time, to TNF-α cytokine production, all in one small macrophage population (< 5000 cells) while using minute reagent volume (540 nL/condition). The platform is easily adaptable to many cell types including primary cells and provides a generic platform for profiling signaling pathways.     

Red cell thermodynamics

A cohort of human red cells of the same age persists in the circulation for about 110 days without access to repair but disappears over the next 10 days. Hyperosmotic stress accelerates the process exponentially. The kinetics are Avrami in all cases we have examined, withn=2. We have previously modelled this as a stress failure in a viscoelastic cytoskeleton, but because of the two dimensional long range order in the cytoskeleton, the data can also be interpreted as a state change in a crystalline material.We thank H. T. Meryman for his continuing encouragement and support. Naval Medical Research and Development Command Work Unit 1462. The opinions and assertions contained herein are the private ones of the writers and are not to be construed as official at large.     

Inflammatory mimetic microfluidic chip by immobilization of cell adhesion molecules for T cell adhesion

Leukocyte adhesion to adhesion molecules on endothelial cells is important in immune function, cancer metastasis and inflammation. This cell-cell binding is mediated via cell adhesion molecules such as E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) found on endothelial cells. Because these adhesion molecules on endothelial cells vary significantly across several disease conditions such as autoimmune diseases, inflammation or cancer metastasis, investigations of therapeutic agents that down-regulate leukocyte-endothelial interactions have been based on in vitro models using endothelial cell lines. Here we report a new model, an inflammatory mimetic microfluidic chip, which emulates leukocyte binding to cell adhesion molecules (CAM) by controlling the types and ratio of adhesion molecules. In our model, E-selectin was essential for the synergic binding of Jurkat T cells. Immunosuppressive drugs, such as tacrolimus (FK506) and cyclosporine A (CsA), were used to inhibit T cell interactions under the physiologic model of T cell migration at a ratio of 5?:?4.3?:?3.9 (E-selectin?:?ICAM-1?:?VCAM-1). Our results support the potential usefulness of the inflammatory mimetic microfluidic chip as a T cell adhesion assay tool with modified adhesion molecules for applications such as immunosuppressive drug screening. The inflammatory mimetic microfluidic chip can also be used as a biosensor in clinical diagnostics, drug efficacy tests and high throughput drug screening due to the dynamic monitoring capability of the microfluidic chip.     

4T1 cell membrane-derived biodegradable nanosystem for comprehensive interruption of cancer cell metabolism

Glycolysis inhibition can effectively block the energy supply and interrupt tumorigenesis in many types of cancers. However, when glycolysis is inhibited, tumor cells will break down glutamine as the raw material for the replenishment pathway to maintain the tricarboxylic acid cycle ensuring energy supply, therefore inducing ineffective interruption of metabolic. Herein, we designed glutamine transporter antagonist L-γ-glutamyl-p-nitroanilide(GPNA) loaded and 4T1 cancer cell membrane coated irid...