| 1. |
- Bloch, K, et al.
(författare)
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Functional activity of insulinoma cells (INS-1E) and pancreatic islets cultured in agarose cryogel sponges
- 2005
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Ingår i: Journal of Biomedical Materials Research. Part A. - : Wiley. - 1552-4965 .- 1549-3296. ; 75A:4, s. 802-809
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Tidskriftsartikel (refereegranskat)abstract
- Here, we describe the preparation, structure, and properties of cryogel sponges, which represent a new type of macroporous biomaterial for tissue engineering. Cryogels were produced through freeze-thawing techniques, either from agarose alone or from agarose with grafted gelatin. The aim of this study was to evaluate agarose cryogel sponges as scaffolds for Culturing both isolated pancreatic islets and insulinoma cells (INS-IE). In order to evaluate the effect of cell entrapment in artificial scaffolds, cell function reflected by insulin secretion and content was studied in cells cultivated for a 2-week period either in Culture plastic plates or in cryogel sponge disks. Our results show that tumor-derived INS-1E cells grown either on plastic or on cryogels do not differ in their proliferation, morphology, insulin release, and intracellular insulin content. However, isolated pancreatic islets cultivated on cryogels sponge show 15-fold higher basal insulin secretion at 3.0 mM glucose than islets cultivated on plastic plates and fail to respond to stimulation with 16.7 mM glucose. In addition, these islets have about 2-fold lower insulin content compared to those grown in plastic plates. It is possible that the cell dysfunction noted in these in vitro experiments is due to the effect of the limited oxygen supply to the islets cultivated in cryogel sponge. Further in vivo Studies are needed to clarify the nature of such an observation since according to previous reports, agarose and gelatin induce new vessel formation supporting enhanced oxygen supply. (c) 2005 Wiley Periodicals, Inc.
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| 2. |
- Dainiak, Maria, et al.
(författare)
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Cell chromatography: Separation of different microbial cells using IMAC supermacroporous monolithic columns
- 2005
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Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 21:2, s. 644-649
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Tidskriftsartikel (refereegranskat)abstract
- Supermacroporous monolithic columns with CU2+-IDA ligands have been successfully used for chromatographic separation of different types of microbial cells. The bed of monolithic matrix is formed by a cryogel of poly(acrylamide) cross-linked with methylenebis(acrylamide) and has a network of large (10- 100 mu m) interconnected pores allowing unhindered passage of whole cells through the plain cryogel column containing no ligands. Two model systems have been studied: the mixtures of wild-type Escherichia coli (w.t. E. coli) and recombinant E. coli cells displaying poly-His peptides (His-tagged E. coli) and of w.t. E. coli and Bacillus halodurans cells. Wild-type E. coli and His-tagged E. coli were quantitatively captured from the feedstock containing equal amounts of both cell types and recovered by selective elution with imidazole and EDTA, with yields of 80% and 77%, respectively. The peak obtained after EDTA elution was 8-fold enriched with His-tagged E. coli cells as compared with the peak from imidazole elution, which contained mainly weakly bound w.t. E. coli cells. Haloalkalophilic B. halodurans cells had low affinity to the CU2+-IDA cryogel column and could be efficiently separated from a mixture with w.t. E. coli cells, which were retained and recovered in high yields from the column with imidazole gradient. All the cells maintained their viability after the chromatographic procedure. The results show that chromatography on affinity supermacroporous monolithic columns is a promising approach to efficient separations of individual cell types.
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| 3. |
- Dainiak, Maria, et al.
(författare)
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Improved methods for prepurification and detection of Staphylococcal Enterotoxin B from cell-free culture filtrate
- 2005
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Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 21:4, s. 1347-1351
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Tidskriftsartikel (refereegranskat)abstract
- An improved ELISA method for the detection of Staphylococcal. Enterotoxin B (SEB) in protein A preparations is presented. Fab fragments were obtained by digestion with papain of anti-SEB IgG bound to SEB immobilized on Sepharose 4B. Anti-SEB and peroxidase-labeled Fab fragments from secondary antibodies were successfully used in a modified ELISA of SEB in protein A preparations. SEB-Sepharose was used repeatedly for the production of anti-SEB Fab fragments by papain digestion without loss of affinity. In addition, for the purification of SEB from crude culture filtrates, an initial step utilizing a combined heat and pH treatment for the removal of significant amounts of contaminating proteins without losses of toxin activity is presented. This pretreatment step yielded positive effects in further downstream processing considering both shortened time and an increase in total recovery.
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| 4. |
- Galaev, Igor, et al.
(författare)
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High throughput processing of particulate-containing samples using supermacroporous elastic monoliths in microtiter (multiwell) plate format
- 2005
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1065:2, s. 169-175
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Tidskriftsartikel (refereegranskat)abstract
- Two steps in parallel processing of multiple biosamples, namely, sample clarification and capture of the target protein, were integrated and combined with the direct assay of captured protein using a newly developed microtiter (96-well) plate system based on the monoliths of hydrophilic elastic supermacroporous material, cryogel. Cryogel monoliths have pore size large enough for microbial and mammalian cells to pass through unretained. Moreover, cryogel monoliths are elastic allowing them to be slightly compressed and easily introduced into the wells. When expanded, cryogel monoliths fill the well tightly with no risk of leakage in between the monolith and the walls of the well. The capillary forces keep the liquid inside the pores of the cryogel monolith making the monolith columns drainage protected. The application of a certain volume of liquid on top of a cryogel monolith column results in the displacement of exactly the same volume of liquid from the column. The concept of using supermacroporous gels in 96-well plate format offers new possibilities to the biotechnologist allowing separation of particulate matter, capturing of soluble material from particle containing media, and parallel assay of large number of non-clarified samples. (C) 2005 Elsevier B.V. All rights reserved.
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| 5. |
- Hanora, Amro, et al.
(författare)
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Capture of bacterial endotoxins using a supermacroporous monolithic matrix with immobilized polyethyleneimine, lysozyme or polymyxin B
- 2005
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Ingår i: Journal of Biotechnology. - : Elsevier BV. - 1873-4863 .- 0168-1656. ; 118:4, s. 421-433
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial endotoxins (BEs) are integrated part of Escherichia coli, a microorganism widely used for the production of recombinant proteins. BEs should be eliminated in the course of down stream processing of target protein produced by these bacteria. Supermacroporous monolith (continuous bed) columns, so called cryogel columns, with immobilized polyethyleneimine (PEI), polymyxin B (PMB) and lysozyme were employed for BEs capture. Due to the large interconnected pores it was possible to use cryogel columns at flow rates as high as 10 ml/min. The columns packed with Sepharose CL-4B with immobilized PEI, PMB and lysozyme were impossible to use at these high flow rates due to the collapse of the bed. The dynamic capacities of the cryogel columns were nearly independent of the flow rate. In the presence of EDTA, BEs were quantitatively captured from mixtures with a model protein, bovine serum albumin (BSA) at pH 7.2 with practically no protein losses. At pH 3.6 BEs were captured directly from non-clarified E. coli cell lysate resulting in more than 104 times BEs clearance. (C) 2005 Elsevier B.V. All rights reserved.
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| 6. |
- Hanora, Amro, et al.
(författare)
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Screening of peptide affinity tags using immobilised metal affinity chromatography in 96-well plate format
- 2005
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1087:1-2, s. 38-44
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Tidskriftsartikel (refereegranskat)abstract
- A method for high throughput screening of Green Fluorescent Proteins carrying metal binding tags in bacteria was developed. A random four amino acids tag-peptide library was successfully generated in E. coli. A 96-microtiter plate assembled with metal-iminodiacetic acid small cryogel columns was used for library screening. For the first time we were able to simultaneously screen a metal binding peptide tags library obtained from E. coli against different metal ions. From screening 25 different tags, three clones were able to bind to all metal ions studied (Ni2+, Zn2+, Co2+ and Cd2+). It was clearly demonstrated that the new construct could facilitate the screening of large peptide libraries. (c) 2005 Elsevier B.V. All rights reserved.
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| 7. |
- Hedström, Martin, et al.
(författare)
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Continuous measurements of a binding reaction using a capacitive biosensor
- 2005
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Ingår i: Biosensors & Bioelectronics. - : Elsevier BV. - 1873-4235 .- 0956-5663. ; 21:1, s. 41-48
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Tidskriftsartikel (refereegranskat)abstract
- A capacitive biosensor with polyclonal antibodies raised against human serum albumin (HSA) immobilized on a gold transducer has been developed for continuous measurement of HSA in the μM-range. A mathematical model has been refined to describe integral HSA-binding curves assuming that (i) binding is essentially irreversible under the conditions used, (ii) the signal is scaled as the number of non-occupied binding sites and (iii) the rate of disappearance of available binding sites is scaled as the number of available binding sites and analyte concentration in solution. Deconvolution of the curves using the mathematical model indicates clearly that it is possible to retrieve concentration profiles (isocratic, linearly or exponentially increasing gradients) of the analyte in the continuous sample flow from the normalized integral binding (NIB) curves. The data presented constitutes the theoretical background and the first step towards the development of an analytical system allowing on-line detection of the concentration profile of the analyte from NIB-curves. Since the system can be used for extended time periods between regeneration steps, a low frequency of regeneration steps can be expected.
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| 8. |
- Hedström, Martin, et al.
(författare)
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Fast on-column protein digestion with subsequent peptide mapping using tandem mass spectrometry with information dependent acquisition
- 2005
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1080:2, s. 117-123
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Tidskriftsartikel (refereegranskat)abstract
- A platform for rapid on-line protein digestion of protein mixtures for direct infusion to a mass spectrometer is presented. A mixture of protein A, staphylococcal enterotoxin B and cytochrome c was used as a model mixture injected on a gel filtration column and a trypsin reactor which were connected in series to a micro liquid chromatography (mu LC) system. The peptides in the column eluate were analyzed with ESI tandem mass spectrometry, utilizing information dependent acquisition (IDA). In one step, the proteins in the mixture (mu M concentrations) were concomitantly desalted, separated, digested and identified with an overall analysis time of less than 40 min. Protein sequence coverage of 78-95% for the involved substances was achieved. (c) 2005 Elsevier B.V. All rights reserved.
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| 9. |
- Ivanov, Alexander, et al.
(författare)
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Binding of adenosine to pendant phenylboronate groups of thermoresponsive copolymer: a quantitative study
- 2005
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Ingår i: Macromolecular Bioscience. - : Wiley. - 1616-5195 .- 1616-5187. ; 5:8, s. 795-800
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Tidskriftsartikel (refereegranskat)abstract
- Binding of adenosine to the thermosensitive copolymer of N-isopropylacrylamide and 3-(acrylamido)aminophenylboronic acid (82:18, m ($) over bar (n)=47000 g center dot mol(-1)) was studied by equilibrium dialysis at 22 degrees C and 37 degrees C, in a 0.1 m glycine buffer containing 0.1 m NaCl at pH 9.2. The copolymer exhibited a the phase transition temperature (T-p) of 26.5 degrees C under the above conditions. At 22 degrees C the binding of adenosine to the water-soluble copolymer was well described by a Langmuir model, accounting for preferential ionisation of the boronate-nucleoside complexes and, therefore, restricted reactivity of the rest of boronates. At saturation, the copolymer contained 38% of its phenylboronic acid groups in the form of complexes, whereas the association constant was 1400 m(-1). At 37 degrees C no binding of adenosine to thermally precipitated copolymer was found, presumably owing to interaction of the phenylboronates with hydrophobic segments of polyNIPAM. At high loading of the copolymer by the reversibly bound adenosine the T (p) steeply increases with increasing fraction of the phenylboronate-adenosine complexes in the chains. The increase of the T-p observed above the saturating adenosine concentration (> 1 x 10(-3) m, 22 degrees C) very probably testifies to competition of the nucleoside with hydrophobic polyNIPAM segments for binding to the pendant phenylboronates.
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| 10. |
- Kumar, Ashok, et al.
(författare)
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Affinity binding of cells to cryogel adsorbents with immobilized specific ligands : effect of ligand coupling and matrix architecture
- 2005
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Ingår i: Journal of Molecular Recognition. - : Wiley. - 0952-3499 .- 1099-1352. ; 18:1, s. 84-93
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Tidskriftsartikel (refereegranskat)abstract
- The capture of human acute myeloid leukemia KG-1 cells expressing the CD34 surface antigen and the fractionation of human blood lymphocytes were evaluated on polyvinyl alcohol (PVA)-cryogel beads and dimethyl acrylamide (DMAAm) monolithic cryogel with immobilized protein A. The affinity ligand (protein A) was chemically coupled to the reactive PVA-cryogel beads and epoxy-derivatized monolithic cryogels through different immobilization techniques and the binding efficiency of the cell surface receptors specific antibody-labeled cells to the gels/beads was determined. The binding of cells to monolithic cryogel was higher (90-95%) compared with cryogel beads (76%). B-lymphocytes, which bound to the protein A-cryogel beads, were separated from T-lymphocytes with yields for the two cell types 74 and 85%, respectively. About 91% of the bound B-cells could be recovered without significantly impairing their viability. Our results show differences in the percentage of cell-binding to the immunosorbents caused by ligand density, flow shear forces and bond strength between the cells and the affinity surface once distinct chemical coupling of protein A, size of beads, sequence of antibody binding to protein A adsorbents, morphology and geometry of surface matrices were compared.
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