| 1. |
- Arvidsson, Pär, et al.
(författare)
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Chromatography of microbial cells using continuous supermacroporous affinity and ion-exchange columns
- 2002
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Ingår i: Journal of Chromatography A. - 0021-9673. ; 977:1, s. 27-38
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Tidskriftsartikel (refereegranskat)abstract
- Continuous supermacroporous chromatographic columns with anion-exchange ligands [2-(dimethylanlino)ethyl group] and immobilized metal affinity (IMA) ligands (Cu2+-loaded iminodiacetic acid) have been developed allowing binding of Escherichia coli cells and the elution of bound cells with high recoveries. These poly(acrylamide)-based continuous supermacroporous columns have been produced by radical co-polymerization of monomers in aqueous solution frozen inside a column (cryo-polymerization). After thawing, the column contains a continuous matrix (so-called cryogel) with interconnected pores of 10-100 mum in size. The large pore size of the matrix makes it possible for E. coli cells to pass unhindered through a plain column containing no ligands. E. coli cells bound to an ion-exchange column at low ionic strength were eluted with 70-80% recovery at NaCl concentrations of 0.35-0.40 M, while cells bound to an IMA-column were eluted with around 80% recovery using either 10 mM imidazole or 20 mM EDTA solutions, respectively. The cells maintain their viability after the binding/elution procedure. These preliminary results indicate that microbial cells can be handled in a chromatographic mode using supermacroporous continuous columns. These columns are easy to manufacture from cheap and readily available starting materials, which make the columns suitable for single-time use. (C) 2002 Published by Elsevier Science B.V.
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| 2. |
- Dainiak, Maria B, et al.
(författare)
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Gelatin-fibrinogen cryogel dermal matrices for wound repair: Preparation, optimisation and in vitro study.
- 2010
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Ingår i: Biomaterials. - : Elsevier BV. - 1878-5905 .- 0142-9612. ; 31, s. 67-76
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Tidskriftsartikel (refereegranskat)abstract
- Macroporous sponge-like gelatin-fibrinogen (Gl-Fg) scaffolds cross-linked with different concentrations (0.05-0.5%) of glutaraldehyde (GA) were produced using cryogelation technology, which allows for the preparation of highly porous scaffolds without compromising their mechanical properties, and is a more cost-efficient process than freeze-drying. The produced Gl-Fg-GA(X) scaffolds had a uniform interconnected open porous structure with a porosity of up to 90-92% and a pore size distribution of 10-120mum. All of the obtained cryogels were elastic and mechanically stable, except for the Gl-Fg-GA(0.05) scaffolds. Swelling kinetics and degradation rate, but not the porous structure of the cryogels, were strongly dependent on the degree of cross-linking. A ten-fold increase in the degree of cross-linking resulted in an almost 80-fold decrease in the rate of degradation in a solution of protease. Cryogels were seeded with primary dermal fibroblasts and the densities observed on the surface, plus the expression levels of collagen types I and III observed 5 days post-seeding, were similar to those observed on a control dermal substitute material, Integra((R)). Fibroblast proliferation and migration within the scaffolds were relative to the GA content. Glucose consumption rate was 3-fold higher on Gl-Fg-GA(0.1) than on Gl-Fg-GA(0.5) cryogels 10 days post-seeding. An enhanced cell motility on cryogels with reducing GA crosslinking was obtained after long time culture. Particularly marked cell infiltration was seen in gels using 0.1% GA as a crosslinker. The scaffold started to disintegrate after 42 days of in vitro culturing. The described in vitro studies demonstrated good potential of Gl-Fg-GA(0.1) scaffolds as matrices for wound healing.
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| 3. |
- Dainiak, Maria, et al.
(författare)
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Biomimetic macroporous hydrogel scaffolds in a high-throughput screening format for cell-based assays.
- 2008
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Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 24:6, s. 1373-1383
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Tidskriftsartikel (refereegranskat)abstract
- Macroporous hydrogels (MHs) hold great promise as scaffolds in tissue engineering and cell-based assays. In this study, the possibility of combination of three-dimensional (3D) cell culture with a miniaturized screening format was demonstrated on human colon cancer HCT116, human acute myeloid leukemia KG-1 cells, and embryonic fibroblasts cultured on MHs (12.5 mm x 7.1 mm I.D.) in a 96-minicolumn plate format. MHs were prepared by cryogelation technique and functionalized by coating with type I collagen and by copolymerization with agmatine-based mimetic of cell adhesive peptide RGD (abRGDm). Cancer cells formed multicellular aggregates while fibroblasts formed adhesions on abRGDm-containing and collagen-MHs but not on plain MHs, as was demonstrated by scanning electron microscopy. HCT116 and KG-1 cells grown as aggregates were more resistant to the treatment with cis-diaminedichloroplatinum (II) (cisplatin) and cytosine 1-beta-D-arabinofuranoside (Ara-C), respectively, during the first 18-24 h of incubation, than single cells grown on unmodified MH. HCT116 cells grown as 2D cultures in conventional 96-well tissue culture plates were 1.5- to 3.5-fold more sensitive to the treatment with 70 microM cisplatin than cells in 3D cultures in functionalized MHs. Further development of the described experimental system including matching of a specific cell type with appropriate extracellular matrix (ECM) components and 3D cocultures on ECM-modified MHs may provide a realistic in vitro experimental model for high-throughput toxicity tests.
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| 4. |
- Hanora, Amro, et al.
(författare)
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Direct capture of plasmid DNA from non-clarified bacterial lysate using polycation-grafted monoliths
- 2006
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Ingår i: Journal of Biotechnology. - : Elsevier BV. - 1873-4863 .- 0168-1656. ; 123:3, s. 343-355
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Tidskriftsartikel (refereegranskat)abstract
- Monolith columns from macroporous polyacrylamide gel were grafted with polycations, poly(N,N-dimethylaminoethyl methacrylate) (polyDMAEMA), (2-(methacryloyloxy)ethyl)-trimethyl ammonium chloride (polyMETA) and partially quaternized polyDMAEMA prepared via treating polyDMAEMA-grafted columns with propylbromide. The polymer grafting degrees varied between 34 and 110%. The polycation-grafted monolithic columns are able to capture plasmid DNA directly from alkaline lysate of Escherichia coli cells. Due to the large pore size in macroporous monoliths the particulate material present in nonclarified feeds did not block the columns. The captured plasmid DNA was eluted with I M NaCl as particulate-free preparation with significantly reduced content of protein and RNA as compared to the applied lysate. (c) 2005 Elsevier B.V. All rights reserved.
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| 5. |
- Ivanov, Alexander, et al.
(författare)
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Evaluation of boronate-containing polymer brushes and gels as substrates for carbohydrate-mediated adhesion and cultivation of animal cells.
- 2010
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Ingår i: Colloids and Surfaces. B, Biointerfaces. - : Elsevier BV. - 1873-4367 .- 0927-7765. ; 75, s. 510-519
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Tidskriftsartikel (refereegranskat)abstract
- Boronate-containing thin polyacrylamide gels (B-Gel), polymer brushes (B-Brush) and chemisorbed organosilane layers (B-COSL) were prepared on the surface of glass slides and studied as substrates for carbohydrate-mediated cell adhesion. B-COSL- and B-Brush-modified glass samples exhibited multiple submicron structures densely and irregularly distributed on the glass surface, as found by scanning electron microscopy and atomic force microscopy. B-Gel was ca. 0.1mm thick and contained pores with effective size of 1-2mum in the middle and of 5-20mum on the edges of the gel sample as found by confocal laser scanning microscopy. Evidence for the presence of phenylboronic acid in the samples was given by time-of-flight secondary ion mass-spectrometry (ToF SIMS), contact angle measurements performed in the presence of fructose, and staining with Alizarin Red S dye capable of formation specific, fluorescent complexes with boronic acids. A comparative study of adhesion and cultivation of animal cells on the above substrates was carried out using murine hybridoma M2139 cell line as a model. M2139 cells adhered to the substrates in the culture medium without glucose or sodium pyruvate at pH 8.0, and then were cultivated in the same medium at pH 7.2 for 4 days. It was found that the substrates of B-Brush type were superior both regarding cell adhesion and viability of the adhered cells, among the substrates studied. MTT assay confirmed proliferation of M2139 cells on B-Brush substrates. Some cell adhesion was also registered in the macropores of B-Gel substrate. The effects of surface microstructure of the boronate-containing polymers on cell adhesion are discussed. Transparent glass substrates grafted with boronate-containing copolymers offer good prospects for cell adhesion studies and development of cell-based assays.
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| 6. |
- Lozinsky, Vladimir I, et al.
(författare)
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Polymeric Cryogels as Promising Materials of Biotechnological Interest.
- 2003
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Ingår i: Trends in Biotechnology. - : Elsevier BV. - 0167-7799. ; 21:10, s. 445-451
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Forskningsöversikt (refereegranskat)abstract
- Cryogels are gel matrices that are formed in moderately frozen solutions of monomeric or polymeric precursors. Cryogels typically have interconnected macropores (or supermacropores), allowing unhindered diffusion of solutes of practically any size, as well as mass transport of nano- and even microparticles. The unique structure of cryogels, in combination with their osmotic, chemical and mechanical stability, makes them attractive matrices for chromatography of biological nanoparticles (plasmids, viruses, cell organelles) and even whole cells. Polymeric cryogels are efficient carriers for the immobilization of biomolecules and cells.
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| 7. |
- Plieva, Fatima, et al.
(författare)
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Characterization of supermacroporous monolithic polyacrylamide based matrices designed for chromatography of bioparticles
- 2004
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Ingår i: Journal of Chromatography. B. - : Elsevier BV. - 1873-376X .- 1570-0232. ; 807:1, s. 129-137
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Tidskriftsartikel (refereegranskat)abstract
- Supermacroporous monolithic acrylamide (AAm)-based cryogels were prepared by radical cryo-polymerizaton (polymerization in the moderately frozen system) of AAm with functional monomers and cross-linker N,N'-methylene-bis-acrylamide (MBAAm). Electron microscopy studies revealed supermacroporous structure of the developed cryogels with pore size of 5-100 mum. Cryogel porosity depended on cryo-polymerization conditions. More than 90% of the monolithic bed volume is the interconnected supermacropores filled with water and less than 10% of the monolithic volume is pore walls. The total protein binding capacity (lysozyme in the case of immobilized metal affinity chromatography (IMAC) column and bovine serum albumin (BSA) in the case of anion-exchange (AE) column) was independent of the flow rates till 600 cm/h. Chromatographic behavior of E. coli cells when a cell suspension was applied to ion-exchange cryogel columns depended on both the density of functional ligand and the porosity of the cryogel. (C) 2004 Elsevier B.V. All rights reserved.
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| 8. |
- Savina, Irina, et al.
(författare)
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Analysis of polymer grafted inside the porous hydrogel using confocal laser scanning microscopy
- 2007
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Ingår i: Express Polymer Letters. - : Department of Polymer Engineering, Scientific Society of Mechanical Engineering. - 1788-618X. ; 1:4, s. 189-196
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Tidskriftsartikel (refereegranskat)abstract
- Graft polymerization of glycidyl methacrylate onto the pore surface of polyacrylamide macroporous gel was implemented in DMSO-aqueous solution using diperiodatocuprate(III) complexes as an initiator. The grafting densities up to 410% were achieved. The graft polymerization was confirmed by gravimetrical methods and FTIR. The graft polymerization of polymer inside the pores of the macroporous gel resulted in increased flow resistance through the gel matrix. The distribution of grafted polymer on the gel pore surface material was studied by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). CLSM is an alternative method for studying morphology of gel surface with grafted polymer having the advantages over the SEM allowing to investigate the distribution of grafted polymer inside the hydrogel in a native hydrated state. The microscopic techniques demonstrated uneven distribution of the grafted polymer inside the gel pores as a result of initiating the graft polymerization by insoluble initiator deposited on the pore surface.
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| 9. |
- Savina, Irina, et al.
(författare)
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Anion-exchange supermacroporous monolithic matrices with grafted polymer brushes of N,N-dimethylaminoethyl-methacrylate
- 2005
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1092:2, s. 199-205
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Tidskriftsartikel (refereegranskat)abstract
- Graft polymerization using potassium diperiodatocuprate as initiator was found to be an effective and convenient method for grafting functional polymer of N,N-dimethylaminoethyl methacrylate (DMAEMA) onto superporous polyacrylamide gels, so-called cryogels (pAAm cryogels). It was possible to achieve grafting degrees up to 110% (w/w). The two-step graft polymerization i.e. first activation of the matrix followed by displacement of initiator solution with the monomer solution, decreased pronouncedly the soluble homopolymer formation. The efficiency of graft polymerization using a two-step technique increased up to 50% (w/w) at a monomer conversion of 10%, compared to 10% graft efficiency with 60-70% monomer conversion for one-step direct graft polymerization. The pAAm cryogels grafted in one-step and two-step procedures, respectively, behaved similarly when binding low-molecular weight ligand but showed very different behavior for sorption of a high-molecular-weight ligand, bovine serum albumin (BSA). The differences in behavior were rationalized assuming different structure of the graft polymer layers and tentacle-type BSA binding to the grafted polymer. (c) 2005 Elsevier B.V. All rights reserved.
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| 10. |
- Savina, Irina, et al.
(författare)
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Cryogels from poly(2-hydroxyethyl methacrylate): macroporous, interconnected materials with potential as cell scaffolds
- 2007
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Ingår i: Soft Matter. - : Royal Society of Chemistry (RSC). - 1744-6848 .- 1744-683X. ; 3:9, s. 1176-1184
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Tidskriftsartikel (refereegranskat)abstract
- Macroporous hydrogels (MHs) have been prepared by cross-linking polymerization of hydroxyethyl methacrylate (HEMA) or cross-linking co-polymerization of HEMA with dimethylacrylamide (DMAA) in semi-frozen state. The MHs are elastic and have a unique structure of large interconnected pores with pore sizes up to 100 µm and total porosity of 94–97%, as demonstrated by micro-computed tomography. The stiffness of such MHs increased with total monomer concentration and with the ratio of DMAA in the composition. Pore-surface modification of the HEMA MHs was achieved by grafting a stimuli-responsive polymer, poly(N-isopropylacrylamide) (PNIPAM), with high density using atom transfer radical polymerization (ATRP). The effect of catalytic system, initiator content and the addition of sacrificial initiator on the ATRP process have been studied. The elasticity, possibility of drying and fast re-swelling, tunable mechanical properties and the macroporous, interconnected structure of HEMA MHs are of interest for biomedical applications such as cell culturing.
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