Optimization of 3D hydrogel microenvironment for enhanced hepatic functionality of primary human hepatocytes

Although primary human hepatocytes (PHHs) are the gold standard in drug efficacy and metabolism studies, long-term survival of PHHs and maintenance of their hepatic function are still challenging. In this study, we focused on the effect of the initial microenvironment on upregulation and long-term preservation of hepatic function of PHHs encapsulated within biodegradable hydrogel systems. PHHs were encapsulated in RGD-functionalized hybrid hydrogels with various degrees of degradability, and their hepatic functionality was analyzed. Regardless of the hydrogel elastic modulus, the combination with nondegradable hydrogels had a predominantly negative effect on the prompt engraftment of PHHs, whereas a degradable hydrogel with intermediate initial degradability was most effective in maintaining hepatic function. Efficient network formation by PHHs and cocultured cells, along with the control of hydrogel degradation, governed the hepatic functionality at an early stage and upon long-term cultivation. Under optimized conditions, expression of genes involved in biological processes such as focal adhesions, cell survival, cytoskeleton formation, and extracellular matrix interactions was significantly higher than that in a control with relatively delayed initial degradation. Thus, we suggest that the orchestrated control of initial cellular remodeling may play an important role in the maintenance of hepatic function in a three-dimensional PHH culture.     

Methods and applications of antibody microarrays in cancer research

Antibody microarrays have great potential for significant value in biological research. Cancer research in particular could benefit from the unique experimental capabilities of this technology. This article examines the current state of antibody microarray technological developments and assay formats, along with a review of the demonstrated applications to cancer research. Work is ongoing in the refinement of various aspects of the protocols and the development of robust methods for routine use. Antibody microarray experimental formats can be broadly categorized into two classes: (1) direct labeling experiments, and (2) dual antibody sandwich assays. In the direct labeling method, the covalent labeling of all proteins in a complex mixture provides a means for detecting bound proteins after incubation on an antibody microarray. If proteins are labeled with a tag, such as biotin, the signal from bound proteins can be amplified. In the sandwich assay, proteins captured on an antibody microarray are detected by a cocktail of detection antibodies, each antibody matched to one of the spotted antibodies. Each format has distinct advantages and disadvantages. Several applications of antibody arrays to cancer research have been reported, including the analysis of proteins in blood serum, resected frozen tumors, cell lines, and on membranes of blood cells. These demonstrations clearly show the utility of antibody microarrays for cancer research and signal the imminent expansion of this platform to many areas of biological research.     

Preparation and characterization of polyvinyl alcohol-gelatin hydrogel membranes for biomedical applications

The purpose of this research was to design and develop hydrogels by esterification of polyvinyl alcohol (PVA) with gelatin. The membranes were characterized by Fourier Transform Infrared (FTIR) spectroscopy, x-ray diffraction (XRD), and differential scanning calorimetry. The viscosity of the esterified product (as solution) was compared with the mixture of PVA and gelatin of the same composition. The mechanical properties of the hydrogels were characterized by tensile tests. Swelling behavior and hemocompatibility of the membrane were also evaluated. The diffusion coefficient of salicylic acid (SA), when the receptor compartment contained Ringer's solution, through the membrane was determined. SA was used as a model drug. FTIR spectra of the membranes indicated complete esterification of the free carboxylic groups of gelatin. XRD studies indicated that the crystallinity of the membranes was mainly due to gelatin. The comparison of viscosity indicated an increase in segment density within the molecular coil. The membrane had sufficient strength and water-holding capacity. Hemocompatibility suggested that the hydrogel could be tried as wound dressing and as an implantable drug delivery system. The diffusion coefficient of SA through the membrane was found to be 1.32×10⁻⁵ cm²/s. The experimental results indicated that the hydrogel could be tried for various biomedical applications. Published: March 16, 2007 Formerly College of Pharmacy, University of Delhi, Pushp Vihar, New Delhi-110017 India     

Optimized normalization for antibody microarrays and application to serum-protein profiling

The measurements of coordinated patterns of protein abundance using antibody microarrays could be used to gain insight into disease biology and to probe the use of combinations of proteins for disease classification. The correct use and interpretation of antibody microarray data requires proper normalization of the data, which has not yet been systematically studied. Therefore we undertook a study to determine the optimal normalization of data from antibody microarray profiling of proteins in human serum specimens. Forty-three serum samples collected from patients with pancreatic cancer and from control subjects were probed in triplicate on microarrays containing 48 different antibodies, using a direct labeling, two-color comparative fluorescence detection format. Seven different normalization methods representing major classes of normalization for antibody microarray data were compared by their effects on reproducibility, accuracy, and trends in the data set. Normalization with ELISA-determined concentrations of IgM resulted in the most accurate, reproducible, and reliable data. The other normalization methods were deficient in at least one of the criteria. Multiparametric classification of the samples based on the combined measurement of seven of the proteins demonstrated the potential for increased classification accuracy compared with the use of individual measurements. This study establishes reliable normalization for antibody microarray data, criteria for assessing normalization performance, and the capability of antibody microarrays for serum-protein profiling and multiparametric sample classification.     

Fabrication and characterization of RNA aptamer microarrays for the study of protein-aptamer interactions with SPR imaging

RNA microarrays were created on chemically modified gold surfaces using a novel surface ligation methodology and employed in a series of surface plasmon resonance imaging (SPRI) measurements of DNA–RNA hybridization and RNA aptamer–protein binding. Various unmodified single-stranded RNA (ssRNA) oligonucleotides were ligated onto identical 5′-phosphate-terminated ssDNA microarray elements with a T4 RNA ligase surface reaction. A combination of ex situ polarization modulation FTIR measurements of the RNA monolayer and in situ SPRI measurements of DNA hybridization adsorption onto the surface were used to determine an ssRNA surface density of 4.0 × 10¹² molecules/cm² and a surface ligation efficiency of 85 ± 10%. The surface ligation methodology was then used to create a five-component RNA microarray of potential aptamers for the protein factor IXa (fIXa). The relative surface coverages of the different aptamers were determined through a novel enzymatic method that employed SPRI measurements of a surface RNase H hydrolysis reaction. SPRI measurements were then used to correctly identify the best aptamer to fIXa, which was previously determined from SELEX measurements. A Langmuir adsorption coefficient of 1.6 × 10⁷ M⁻¹ was determined for fIXa adsorption to this aptamer. Single-base variations from this sequence were shown to completely destroy the aptamer–fIXa binding interaction.     

Evaluation of surface chemistries for antibody microarrays

Antibody microarrays are an emerging technology that promises to be a powerful tool for the detection of disease biomarkers. The current technology for protein microarrays has been derived primarily from DNA microarrays and is not fully characterized for use with proteins. For example, there are a myriad of surface chemistries that are commercially available for antibody microarrays, but there are no rigorous studies that compare these different surfaces. Therefore, we have used a sandwich enzyme-linked immunosorbent assay (ELISA) microarray platform to analyze 17 different commercially available slide types. Full standard curves were generated for 23 different assays. We found that this approach provides a rigorous and quantitative system for comparing the different slide types based on spot size and morphology, slide noise, spot background, lower limit of detection, and reproducibility. These studies demonstrate that the properties of the slide surface affect the activity of immobilized antibodies and the quality of data produced. Although many slide types produce useful data, glass slides coated with aldehyde silane, poly-l-lysine, or aminosilane (with or without activation with a crosslinker) consistently produce superior results in the sandwich ELISA microarray analyses we performed.     

Green bioprinting: Fabrication of photosynthetic algae‐laden hydrogel scaffolds for biotechnological and medical applications

Embedding of mammalian cells into hydrogel scaffolds of predesigned architecture by rapid prototyping technologies has been intensively investigated with focus on tissue engineering and organ printing. The study demonstrates that such methods can be extended to cells originating from the plant kingdom. By using 3D plotting, microalgae of the species Chlamydomonas reinhardtii were embedded in 3D alginate‐based scaffolds. The algae survived the plotting process and were able to grow within the hydrogel matrix. Under illumination, the cell number increased as indicated by microscopic analyses and determination of the chlorophyll content which increased 16‐fold within 12 days of cultivation. Photosynthetic activity was evidenced by measurement of oxygen release: within the first 24 h, an oxygen production rate of 0.05 mg L^?1 h^?1 was detected which rapidly increased during further cultivation (0.25 mg L^?1 h^?1 between 24 and 48 h). Furthermore, multichannel plotting was applied to combine human cells and microalgae within one scaffold in a spatially organized manner and hence, to establish a patterned coculture system in which the algae are cultivated in close vicinity to human cells. This might encourage the development of new therapeutic concepts based on the delivery of oxygen or secondary metabolites as therapeutic agents by microalgae.     

Design of acetylcholinesterases for biosensor applications

In recent years, the use of acetylcholinesterases (AChEs) in biosensor technology has gained enormous attention, in particular with respect to insecticide detection. The principle of biosensors using AChE as a biological recognition element is based on the inhibition of the enzyme's natural catalytic activity by the agent that is to be detected. The advanced understanding of the structure-function-relationship of AChEs serves as the basis for developing enzyme variants, which, compared to the wild type, show an increased inhibition efficiency at low insecticide concentrations and thus a higher sensitivity. This review describes different expression systems that have been used for the production of recombinant AChE. In addition, approaches to purify recombinant AChEs to a degree that is suitable for analytical applications will be elucidated as well as the various attempts that have been undertaken to increase the sensitivity of AChE to specified organophosphates and carbamates using side-directed mutagenesis and employing the enzyme in different assay formats.     

Signal stability of Cy3 and Cy5 on antibody microarrays

Background  

The antibody microarray technique is a newly emerging proteomics tool for differential protein expression analyses that uses fluorescent dyes Cy 3 and Cy 5. Environmental factors, such as light exposure, can affect the signal intensity of fluorescent dyes on microarray slides thus, it is logical to scan microarray slides immediately after the final wash and drying processes. However, no research data are available concerning time-dependent changes of fluorescent signals on antibody microarray slides to this date. In the present study, microarray slides were preserved at -20°C after regular microarray experiments and were rescanned at day 10, 20 and 30 to evaluate change in signal intensity.     

An investigation of antibody immobilization methods employing organosilanes on planar ZnO surfaces for biosensor applications

One critical aspect for the development of label-free immunosensors is the employment of highly uniform and repeatable antibody immobilization techniques. In this study, we investigated the use of two different silane molecules (3-glycidyloxypropyl)trimethoxysilane (GPS), and (3-mercaptopropyl)trimethoxysilane (MTS) for the immobilization of fluorescently labeled IgG antibodies on planar ZnO surfaces. The chemical modification of the surfaces was investigated using water contact angle measurements, AFM, and fluorescence microscopy. The results of the water contact angle measurements indicate increased surface hydrophobicity after treatment with GPS and MTS as compared to the control. Surface modification was further verified through AFM measurements which demonstrate an increased surface roughness and particle height after treatment with antibodies. The results of the fluorescence studies indicate that the immobilization protocol employing MTS produced 21% higher fluorescence on average with greater uniformity than the GPS-based protocol, which indicates a higher overall density in antibody coverage on the surface of the ZnO. Acoustic sensor tests were employed to confirm the functionality of sensors treated with the MTS protocol. The results indicate that the immobilization protocol imparts sensitivity and specificity to the ZnO-based devices.     

Computational design and biochemical characterization of maize nonspecific lipid transfer protein variants for biosensor applications

Lipid transfer proteins (LTPs) are a family of proteins that bind and transfer lipids. Utilizing the maize LTP, we have successfully engineered fluorescent reagentless biosensors for the natural ligand of LTPs; this was achieved by using computational protein design to remove a disulfide bridge and attaching a thio-reactive fluorophore. Conformational change induced by ligand titration is thought to affect the fluorescence of the fluorophore, allowing detection of ligand binding. Fluorescence measurements show that our LTP variants have affinity to palmitate that is consistent with wild-type LTP. These molecules have the potential to be utilized as scaffolds to design hydrophobic ligand biosensors or to serve as drug carriers.     

Adaptation of a manometric biosensor to measure glucose and lactose

A manometric sensor previously developed to measure urea was modified to measure glucose and lactose through enzymatic oxidation. Change in pressure in an enclosed cavity was correlated to the depletion of oxygen resulting from the enzymatic oxidation of glucose or lactose. The response of the sensor was linear and could be made adjustable over a large range by adjusting the amount of sample loaded into the fixed volume reactor. Because of the slow mutarotation of glucose, the oxidation of glucose was not allowed to proceed to completion. Therefore, the precision of the sensor (approximately 0.2 mM in a range from 0 to 5 mM) was limited by variations in the oxidation rate of glucose by glucose oxidase. Because the assay for lactose measured glucose subsequent to the hydrolysis of lactose by beta-galactosidase, the same degree of precision was observed in lactose. Milk lactose, typically at concentrations of about 150 mM, was estimated using the lactose assay after first diluting the samples. For many fluids such as milk, the use of manometric sensors for oxidizable substrates may be preferable to optical and electrochemical methods because they are robust and suffer a low degree of optical and chemical interferences. Glucose and lactose are representative of many important oxidizable substrates, which may be determined in this manner, many of which do not suffer from limitations caused by mutarotation. In theory, detection limits less than 1 microM may be achieved using these methods.     

Functional peptide microarrays for specific and sensitive antibody diagnostics

Peptide microarrays displaying biologically active small synthetic peptides in a high-density format provide an attractive technology to probe complex samples for the presence and/or function of protein analytes. We present a new approach for manufacturing functional peptide microarrays for molecular immune diagnostics. Our method relies on the efficiency of site-specific solution-phase coupling of biotinylated synthetic peptides to NeutrAvidin (NA) and localized microdispensing of peptide-NA-complexes onto activated glass surfaces. Antibodies are captured in a sandwich manner between surface immobilized peptide probes and fluorescence-labeled secondary antibodies. Our work includes a total of 54 peptides derived from immunodominant linear epitopes of the T7 phage capsid protein, Herpes simplex virus glycoprotein D, c-myc protein, and three domains of the Human coronavirus polymerase polyprotein and their cognate mAbs. By using spacer molecules of different type and length for NA-mediated peptide presentation, we show that the incorporation of a minimum spacer length is imperative for antibody binding, whereas the peptide immobilization direction has only secondary importance for antibody affinity and binding. We further demonstrate that the peptide array is capable of detecting low-picomolar concentrations of mAbs in buffered solutions and diluted human serum with high specificity.     

Protein microarrays for gene expression and antibody screening.

Proteins translate genomic sequence information into function, enabling biological processes. As a complementary approach to gene expression profiling on cDNA microarrays, we have developed a technique for high-throughput gene expression and antibody screening on chip-size protein microarrays. Using a picking/spotting robot equipped with a new transfer stamp, protein solutions were gridded onto polyvinylidene difluoride filters at high density. Specific purified protein was detected on the filters with high sensitivity (250 amol or 10 pg of a test protein). On a microarray made from bacterial lysates of 92 human cDNA clones expressed in a microtiter plate, putative protein expressors could be reliably identified. The rate of false-positive clones, expressing proteins in incorrect reading frames, was low. Product specificity of selected clones was confirmed on identical microarrays using monoclonal antibodies. Cross-reactivities of some antibodies with unrelated proteins imply the use of protein microarrays for antibody specificity screening against whole libraries of proteins. Because this application would not be restricted to antigen-antibody systems, protein microarrays should provide a general resource for high-throughput screens of gene expression and receptor-ligand interactions.     

Assessment and optimisation of normalisation methods for dual-colour antibody microarrays

Background  

Recent advances in antibody microarray technology have made it possible to measure the expression of hundreds of proteins simultaneously in a competitive dual-colour approach similar to dual-colour gene expression microarrays. Thus, the established normalisation methods for gene expression microarrays, e.g. loess regression, can in principle be applied to protein microarrays. However, the typical assumptions of such normalisation methods might be violated due to a bias in the selection of the proteins to be measured. Due to high costs and limited availability of high quality antibodies, the current arrays usually focus on a high proportion of regulated targets. Housekeeping features could be used to circumvent this problem, but they are typically underrepresented on protein arrays. Therefore, it might be beneficial to select invariant features among the features already represented on available arrays for normalisation by a dedicated selection algorithm.     

Diagnostic and analytical applications of protein microarrays

DNA microarrays have changed the field of biomedical sciences over the past 10 years. For several reasons, antibody and other protein microarrays have not developed at the same rate. However, protein and antibody arrays have emerged as a powerful tool to complement DNA microarrays during the past 5 years. A genome-scale protein microarray has been demonstrated for identifying protein–protein interactions as well as for rapid identification of protein binding to a particular drug. Furthermore, protein microarrays have been shown as an efficient tool in cancer profiling, detection of bacteria and toxins, identification of allergen reactivity and autoantibodies. They have also demonstrated the ability to measure the absolute concentration of small molecules. Besides their capacity for parallel diagnostics, microarrays can be more sensitive than traditional methods such as enzyme-linked immunosorbent assay, mass spectrometry or high-performance liquid chromatography-based assays. However, for protein and antibody arrays to be successfully introduced into diagnostics, the biochemistry of immunomicroarrays must be better characterized and simplified, they must be validated in a clinical setting and be amenable to automation or integrated into easy-to-use systems, such as micrototal analysis systems or point-of-care devices.