'fences' a Cell Migration Assay System for 24-Well Tissue Culture Plates

Literature concerning the 'fences' :
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• Migration assay for endothelial cells in multiwells. Application to studies on the effect of opioids.
  Fischer EG, Stingl A, Kirkpatrick CJ
  J.Immunol.Meth. (1990) 128: 235-239
      [Abstract]
  An assay system is described which permits rapid and effective evaluation of endothelial cell repair, using cells growing in a monolayer. With this method it was possible to obtain highly significant results. For example, endothelial growth factor and heparin, significantly enhanced cell migration and/or proliferation, whereas beta-endorphin, an endogenous opioid, had no effect on the migration and/or proliferation of human umbilical vein endothelial cells. This model may be used to study the cell migration of a variety of cell types which under certain experimental conditions (e.g., irradiation) do not proliferate.
• Measurement of cell adhesion and migration using phosphor-screen autoradiography
  Dalton BA, Dziegielewski M, Johnson G, Underwood PA, Steele JG
  Biotechniques (1996) 21,2:298-303
      [Abstract]
  This study demonstrates the rise of phosphor-screen autoradiography as a means of measuring cell adhesion and cell expansion on polymer surfaces. The method has particular merit in cases where a specific substrate may be opaque or biochemically incompatible with colorimetric assay methodologies. With the phosphor-screen autoradiography method. there was a linear relationship between cell number and quantitated radioactivity The technique has also been validated by comparison with a colorimetric assay of adhesion conducted for attachment to conventional culture substrata. The data supported the view that the use of phosphor-screen autoradiography was a valid method for detecting cell attachment, and it gave equivalent results to the colorimetric assay. Furthermore, a comparison between phosphor-screen autoradiography and a dye-staining method showed that this technique can be used as a means of quantifying cellular expansion over surfaces.
• Polymer surface chemistry and bone cell migration.
  Dalton BA, McFarland CD, Gengenbach TR, Griesser HJ, Steele JG
  J.Biomater.Sci.Polym.Ed. (1998) 9,8:781-799
      [Abstract]
  Implant devices for orthopaedic applications may be improved if the surface of the biomaterial provides for osteointegration. To understand the effect of hydrophilicity on colonisation by human bone derived (HBD) cells, we compared untreated polystyrene (PS) and a sulfuric acid-treated PS surface for mechanisms of cell migration. The chemical composition of the acid-treated PS surface was analysed by monochromatic X-ray photoelectron spectroscopy and found to contain various oxidatively produced groups and a minor amount of sulfonate groups. It was found that migration of HBD cells on both PS and acid-treated PS surface was dependent on the presence of vitronectin (Vn) and was higher on the hydrophilic acid-treated surface. Minimal migration of HBD cells occurred on either surface in the absence of Vn, even when fibronectin was present in the culture medium. Using radiolabelled protein, it was shown that Vn adsorption onto the acid-treated surface was two to three fold greater than that on the hydrophobic PS. When HBD cells were seeded onto a patterned surface in a medium containing Vn, the cells preferentially colonised the hydrophilic region and few, if any, cells traversed the haptotactic boundary from the hydrophilic to the hydrophobic side. Thus the enhanced HBD cell migration seen on the acid-treated PS compared with the untreated PS surface and the haptotactic boundary phenomenon, relate to Vn adsorption.
• Modulation of epithelial tissue and cell migration by microgrooves.
  Dalton BA, Walboomers XF, Dziegielewski M, Evans MDM, Taylor S, Jansen JA, Steele JG
  J.Biomed.Mater.Res. (2001) 56,2:195-207
      [Abstract]
  We used a polystyrene substratum to study the response of migrating epithelium to 1- or 5-m depth microgrooves with groove/ridge widths of 1, 2, 5, or 10 m. The migration of a tissue sheet was enhanced along the microgrooves, while migration across the microgrooves was inhibited. Changing the depth of the microgrooves had a greater effect on migration than alteration of the groove/ridge width. The migration of epithelial cells from a confluent monolayer culture followed a similar pattern to that of intact epithelial tissue. Cellular extensions generally followed the microgroove direction by tracking along the top of the ridges or following the ridge walls, as revealed by scanning electron microscopy. Actin filaments within the basal cell layer of the tissue were aligned with the microgrooves, unlike filaments in the superficial layers that did not appear to be affected by the presence of underlying microgrooves. The basal cell layer of the tissue conformed to the contours of the microgroove following migration. However, the ultrastructure of the tissue above the ridges resembled that of tissue on a flat surface. We concluded that surface microgrooves have the potential to direct the migration of immediately adjacent epithelial tissue, the effect of which is to guide epithelial tissue on the surface of implanted biomaterials.
• Migration mechanisms: corneal epithelial tissue and dissociated cells.
  Dalton BA, Steele JG
  Exp Eye Res. (2001) 73,6:797-814
      [Abstract]
  The migratory mechanism of intact bovine corneal epithelial tissue and individual corneal epithelial cells over synthetic surfaces in vitro were compared. In migrating tissue, adhesion between component cells was demonstrated by immunostaining for desmoplakin and identification of desmosomes by electron microscopy. The apparent intermeshing of microtubules within the tissue and interdigitation of cytoplasmic membranes showed the close association of cells. Portions of the advancing edge of the tissue contained actin filaments that were orientated parallel to the leading tissue front. These filaments appeared to span adjacent cells suggesting that migration partially involved the contraction of the actin cable, similar to the 'purse-string' mechanism originally identified in the closure of fetal skin wounds. Intact actin filaments and microtubules were necessary to maintain optimum migration rates for tissue and cells. However, tissue morphology was not dependent on microtubule integrity. During the migration of individual epithelial cells, no staining for desmoplakin was observed and there were clear divisions between the microtubules of adjacent cells. Actin filaments tended to be arranged parallel to the direction of cell movement.Therefore, migration of epithelial tissue sheets over synthetic surfaces occurs by mechanisms that differ from the migration of individual epithelial cells. Model systems based on the migration of intact tissue would give a more realistic assessment of the suitability of a material for biomaterial applications than the use of separate epithelial cells.
• Stimulation of epithelial tissue migration by certain porous topographies is independent of fluid flux.
  Dalton BA, McFarland GA, Steele JG
  J.Biomed.Mater.Res. (2001) 56,1:83-92
      [Abstract]
  A surface with columnar pores 0.1 or 0.4 m in diameter is shown to have a novel effect on the migration of corneal epithelial tissue sheets; migration is stimulated in a nondirectional manner with respect to migration over a planar, nonporous surface (Dalton, Evans, McFarland, and Steele, J Biomed Mater Res 1999;45:384-394; Steele, Johnson, McLean, Beumer, and Griesser, J Biomed Mater Res 2000;50:475-482). By blind-ending the pores, we show that this increase in tissue migration is not dependent on fluid flux through the pores and so appears to occur as a result of surface topography. From transmission electron micrographs, the migrating tissue appears to form either close contacts or focal adhesions at the edge of some pore channels; we speculate that this may provide a fulcrum for the enhanced migration. Scanning electron micrographs suggest that within tissue that migrates over the surfaces that contain blind-ended pores, the cells are more extensively spread than those in tissue migrating on a planar surface. The migration of disaggregated epithelial cells is enhanced on surfaces that contain 0.1- or 0.4-m-diameter pores (compared with a planar surface), and this is similarly independent of fluid flux.
• Surface topography can interfere with epithelial tissue migration.
  Fitton JH, Dalton BA, Beumer G, Johnson G, Griesser HJ, Steele JG
  J.Biomed.Mater.Res. (1998) 42,2:245-257
      [Abstract]
  Corneal epithelial tissue migration over the surface of a synthetic polymer can be inhibited by pores in the substrate. The effects of this substrate topography upon epithelial tissue migration were studied in vitro. Membranes of different porosities and structures were used to provide two series of surfaces having a graded increase in discontinuities: cellulose nitrate/acetate membranes with a tortuous network of pores, and track-etched polycarbonate membranes with columnar pores. Corneal epithelial tissue outgrowth was inhibited by increased pore size, and for both series of membranes, outgrowth was completely halted on membranes with mean diameter of the pores 0.9 microm at the pore densities measured. On the track-etched membranes with pores of <0.9 microm diameter, tissue outgrowth could be partially 'rescued' by coating with fibronectin or collagen, but above this size, the inhibition predominated. The effect of porosity of the track-etched membranes upon the migration of dissociated epithelial cells was also examined. Although migration of these cells was reduced on membranes having pore sizes larger than 0.9 microm, it was not completely inhibited even on membranes of 2.3-microm pore diameter. Therefore, tissue movement of adult stratified epithelium may be inhibited by specific surface topographies, and in this assay system, epithelial tissue outgrowth was more affected than was the migration of dissociated epithelial cells.
• Effect of extracellular-matrix proteins on vascular smooth-muscle cell phenotype.
  Hayward IP, Bridle KR, Campbell GR, Underwood PA, Campbell JH
  Cell Biol.Internatl. (1995) ( 19,9:727-734 ) 19,10:839-846
      [Abstract]
  The effect on phenotypic expression of rabbit vascular smooth muscle cells (SMC) of the interstitial matrix proteins collagen I and fibronectin, the basal lamina proteins collagen IV and laminin, and the serum adhesion protein vitronectin was examined in culture. Experiments were performed in foetal calf serum stripped of fibronectin and vitronectin to eliminate their confounding effects. All the proteins promoted adhesion to the plastic culture dish (in a concentration dependent manner) of SMC freshly isolated from the artery wall. These cells had a high volume density of myofilaments (V(v)myo) in their cytoplasm. Laminin was best at maintaining SMC with a high V(v)myo (V(v)myo = 49.8%) followed by collagen IV (41.7%). Cells plated on vitronectin showed the lowest V(v)myo (31.3%). The results support the concept that the SMC basal lamina has a role in maintaining cells in the high V(v)myo phenotype.
• Hyperbaric oxygen stimulates epidermal reconstruction in human skin equivalents.
  Kairuz E, Upton Z, Dawson RA, Malda J .
  Wound Repair Regeneration (2007) 15,2:266-274
      [Abstract]
  The crucial role of oxygen during the complex process of wound healing has been extensively described. In chronic or nonhealing wounds, much evidence has been reported indicating that a lack of oxygen is a major contributing factor. Although still controversial, the therapeutic application of hyperbaric oxygen (HBO) therapy can aid the healing of chronic wounds. However, how HBO affects reepithelization, involving processes such as keratinocyte proliferation and differentiation, remains unclear. We therefore used a three-dimensional human skin-equivalent (HSE) model to investigate the effects of daily 90-minute HBO treatments on the reconstruction of an epidermis. Epidermal markers of proliferation, differentiation, and basement membrane components associated with a developing epidermis, including p63, collagen type IV, and cytokeratins 6, 10, and 14, were evaluated. Morphometric analysis of hematoxylin and eosin-stained cross sections revealed that HBO treatments significantly accelerated cornification of the stratum corneum compared with controls. Protein expression as determined by immunohistochemical analysis confirmed the accelerated epidermal maturation. In addition, early keratinocyte migration was enhanced by HBO. Thus, HBO treatments stimulate epidermal reconstruction in an HSE. These results further support the importance of oxygen during the process of wound healing and the potential role of HBO therapy in cutaneous wound healing.
• Differential expansion of human endothelial monolayers on basement membrane and interstitial collagens, laminin and fibrinectin in vitro.
  Kirkpatrick CJ, Kampe M, Fischer EG, Rixen H, Richter H, Mittermayer C
  Pathobiology (1990) 58,4:221-225
      [Abstract]
  In this study the ability of a human endothelial cell monolayer to expand over specific components of the basement membrane and extracellular matrix was investigated over a 5-day period. The method was intended as a model to study the mechanisms of endothelial regeneration. All components were coated onto sterile coverslips at a concentration of 10 µg/ml. The highest expansion was obtained on fibronectin, laminin and collagen type III, all three being statistically significantly greater than on the uncoated control surface (0.002 > p > 0.0001). Collagens types I and IV and a high molecular weight fragment mixture of type IV (IV-F, consisting of 75, 120 and 140 kD fragments) elicited approximately similar expansion rates, significantly higher than the control (0.02 > p > 0.003), although significantly lower (approximately 15%) than collagen type III, fibronectin and laminin (p < 0.001). The high monolayer expansion on collagen type III is surprising, as it is a relatively minor biosynthetic product of the endothelial cell. It could, however, be of significance in wound healing, in which endothelial cells come into contact with this interstitial collagen. In addition, the similar results obtained with collagens IV and IV-F indicate that expansion of the endothelial monolayer is not dependent on the integrity of the tetrameric structure of type-IV collagen.
• In vitro studies on the expansion of endothelial cell monolayers on components of the basement membrane.
  Kirkpatrick CJ, Kampe M, Rixen H, Fischer EG, Ruchartz D, Mittermayer C
  Virchows Arch. Abt. B. Cell Pathol. (1990) 58:207-213
      [   -   ]
• Suppression of Corneal Neovascularization by Culture Supernatant of Human Amniotic Cells.
  Kobayashi N, Kabuyama Y, Sasaki S, Kato K-i, Homma Y
  Cornea (2002) 21,1:62-67   [Namie Kobayashi et al.]
      [Abstract]
  -Purpose- To examine the applicability of culture supernatant of human amniotic cells on basic fibroblast growth factor (bFGF)-induced corneal neovascularization. -Methods- Human amniotic epithelial and mesenchymal cells (AC) were obtained from human amniotic membranes by digesting with collagenase and maintained in serum-containing medium. The AC preparations predominantly contained cytokeratin-positive cells (91.2 +/- 3.1%, n = 4). The culture supernatant was prepared by cultivating AC in serum-free medium for 24 hours. Neovascularization was obtained by a micropocket assay with Hydron pellets containing bFGF. Migration assay was carried out by a double-chamber method using human umbilical vein endothelial cells (HUVEC). Cell growth assay was done by MTT assay using HUVEC. -Results- Basic fibroblast growth factor-induced corneal neovascularization was significantly reduced by administration of AC culture supernatant. When either control or AC culture supernatant was administered three times per day for 10 days, the area with neovascularization was 13.2 +/- 3.2 mm 2 and 4.0 +/- 1.4 mm 2 for the control and AC culture supernatant-treated eyes, respectively (n = 7, p = 0.021). AC culture supernatant strongly inhibited bFGF-induced migration and cell growth of HUVEC. -Conclusions- Amniotic cell culture supernatant contains potent inhibitors of neovascularization. This effect is explained in part by inhibition of migration and cell growth of vascular endothelial cells. AC culture supernatant may be applicable for treatment of corneal diseases with neovascularization.
• Signalling mechanisms of SDF-induced endothelial cell proliferation and migration.
  Kuhlmann CRW, Schaefer CA, Reinhold L, Tillmanns H, Erdogan A
  Biochemical Biophysical Res.Communications (2005) 335:1107-1114  [Christoph R.W. Kuhlmann et al.]
      [Abstract]
  The aim of our study was to investigate the effect of stromal-derived factor-1-alpha (SDF-1-alpha) on endothelial angiogenic effects. SDF-1-alpha (50 ng/ml) increased the number of cultured endothelial cells from 33,653 +/- 1183 to 55,398 +/- 2741, which significantly reduced by adding the BK(Ca)-inhibitor iberiotoxin, or the endothelial nitric oxide synthase-blocker, L-NMMA (n = 24, p < 0.05). Using the 'Fences'-assay a significant increase of HUVEC migration induced by SDF-1-alpha was reported, which was blocked by the addition of iberiotoxin or L-NMMA (n = 12, p < 0.05). BK(Ca) open-state probability (NPo) was analysed using the patch-clamp technique and NPo was increased from 0.003 (control) to 0.052 (SDF-1-alpha; n = 10, p < 0.05). NO synthesis was measured using a cGMP-radioimmunoassay. A significant increase of cGMP levels from 0.952 pmol/mg protein to 2.179 pmol/mg protein was observed, that was abolished by L-NMMA and significantly reduced by iberiotoxin (n=15, p<0.05). SDF-1-alpha increases endothelial proliferation and migration involving the activation of BK(Ca) and an increased production of NO.
• Discordant effects of nicotine on endothelial cell proliferation, migration, and the inward rectifier potassium current.
  Kuhlmann CRW, Scharbrodt W, Schaefer CA, Most AK, Backenköhler U, Neumann T, Tillmanns H, Waldecker B, Erdogan A, Wiecha J
  J. Molecular Cellular Cardiol. (2005) 38,2:315-322  [Christoph R.W. Kuhlmann et al.]
      [Abstract]
  The inward rectifier K+ current (K(ir)) determines the resting membrane potential of endothelial cells. Basic fibroblast growth factor (bFGF) has been shown to activate K(ir) and acts as angiogenic factor and vasodilator. In contrast, nicotine has been demonstrated to reduce endothelium-dependent vasorelaxation by increasing radical formation. Aim of the present study was to investigate whether nicotine modulates K(ir) and if this plays a role in bFGF-mediated proliferation, migration and nitric oxide (NO)-formation of endothelial cells. Using the patch-clamp technique in cultured endothelial cells of human umbilical cord veins (HUVEC), we found characteristic K(ir), which were blocked by extracellular barium (100 micromol/l). Perfusion with nicotine (1 nmol/l-10 micromol/l) revealed a dose-dependent reduction of K(ir). The simultaneous perfusion with bFGF (50 ng/ml) and nicotine (10 micromol/l) still significantly reduced K(ir) (n = 8; P < 0.01). Cell counts revealed that bFGF-mediated proliferation of HUVEC was significantly inhibited when using 1-10 micromol/l nicotine (n = 8, P < 0.01). The bFGF-induced endothelial cell migration--examined using the 'Fences-Migration-Assay'--was significantly reduced by 10 mumol/l nicotine (n = 12; P < 0.05). NO-production was examined using a cGMP-Radioimmunoassay. The significant bFGF-induced increase of cGMP-levels was reduced by nicotine (n = 10; P < 0.05). Our data indicate that the modulation of K(ir) seems to be an essential pathway in the antagonistic effects of nicotine on bFGF-mediated endothelial cell growth, migration and NO-formation.
• Osteoblast alignment, elongation and migration on grooved polystyrene surfaces patterned by Langmuir-Blodgett lithography.
  Lenhert S, Meier M-B, Meyer U, Chi L, Wiesmann HP
  Biomaterials (2005) 26,5: 563-570
      [Abstract]
  Topographically patterned surfaces are known to influence cellular behavior in a controllable manner. However, the relatively large surface areas (several cm2) required for many biomaterial applications are beyond the practical limits of traditional lithography. Langmuir-Blodgett lithography, a recently developed method, was used to fabricate regularly spaced grooves of different depths (50 and 150 nm) with a periodicity of 500 nm over several square centimeter on silicon surfaces. These topographies were transferred into polystyrene surfaces by means of nanoimprinting. Primary osteoblasts were cultured on the patterned polymer surfaces. They were observed to align, elongate and migrate parallel to the grooves. The combination of Langmuir-Blodgett lithography with nanoimprinting enables the fabrication of large, nanostructured surface areas on a wide spectrum of different biomaterials. Osteoblasts show a significant anisotropic behavior to these surfaces, which can enhance cell settlement on the surface or be used to direct tissue generation on the biomaterial interface.
• Growth factor-induced cell migration: biology and methods of analysis.
  Manske M, Bade EG
  Int.Rev.Cytol. (1994) 155:49-96
      [   -   ]
• Albumin-binding surfaces: synthesis and characterization.
  McFarland CD, Jenkins M, Griesser HJ, Chatelier RC, Steele JG, Underwood PA
  J.Biomater.Sci.Polym-Ed. (1998) 9,11:1207-1225
      [Abstract]
  The nature of the proteinaceous film deposited on a biomaterial surface following implantation is a key determinant of the subsequent biological response. To achieve selectivity in the formation of this film, monoclonal antibodies have been coupled to a range of solid substrates using avidin-biotin technology. Antibody clones varied in their antigen-binding activity following insertion of biotin groups into lysine residues. Biotinylated antibodies coupled to solid substrates via an immobilized avidin bridge retained their biological activity. During immobilization of avidin a significant proportion of the protein molecules were passively adsorbed rather than covalently attached to the surface. This loosely bound material could be removed by stringent elution procedures which resulted in a surface density of 5.4 pmol avidin cm(-2). Although these conditions would be harsh enough to denature monoclonal antibodies, they did not destroy the biotin-binding activity of the residual surface-coupled avidin, enabling the subsequent immobilization of biotinylated antibodies. The two-step immobilization technique allowed the use of gentle protein modification procedures, reduced the risk of surface-induced denaturation and removed loosely bound material from the surface. The versatility of the technique encourages its application to a wide range of immobilization systems where retention of biological activity is a key requirement
• Albumin-binding surfaces: in vitro activity.
  McFarland CD, De Filippis C, Jenkins M, Tunstell A, Rhodes NP, Williams DF, Steele JG
  J.Biomater.Sci.Polym.Ed. (1998) 9,11:1227-1239
      [Abstract]
  Immobilized monoclonal antibodies (Mabs) have been used to attract specific molecules to a solid surface from complex mixtures such as blood, plasma or serum, thereby directing the response to the modified substrate, a key goal in rational biomaterial design. The nature of the Mab dictated the nature of the response: anti-albumin antibodies were used to prevent cell and platelet adhesion in vitro, whilst anti-fibronectin Mabs promoted attachment. Patterned surfaces could be formed, bearing Mabs that generated adhesive and non-adhesive regions. Fibrinogen adsorption from plasma showed a Vroman peak on unmodified control polymer, which was reduced by 64% in the presence of surface-bound anti-albumin Mab. Immobilization of a control Mab reduced fibrinogen adsorption only slightly, implying an albumin-mediated effect. In static tests, platelet adhesion from human platelet rich plasma was significantly reduced by the immobilization of anti-HSA Mab when compared to the untreated FEP surface (p < 0.0001). This effect was also seen with citrated blood flowing through Mab-treated polyurethane tubing at a shear rate of 132 s(-1) (p=0.034). Since platelets and proteins (as blood, plasma or serum) were introduced to the surface simultaneously, the generation of a defined protein film must have been sufficiently rapid as to shape the platelet or cell response.
• Protein adsorption and cell attachment to patterned surfaces.
  McFarland CD, Thomas CH, DeFilippis C, Steele JG, Healy KE
  J.Biomed.Mater.Res. (1999) 49,2:200-210
      [Abstract]
  To better understand the events involved in the generation of defined tissue architectures on biomaterials, we have examined the mechanism of attachment of human bone-derived cells (HBDC) to surfaces with patterned surface chemistry in vitro. Photolithography was used to generate alternating domains of N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (EDS) and dimethyldichlorosilane (DMS). At 90 min after seeding, HBDC were localized preferentially to the EDS regions of the pattern. Using sera specifically depleted of adhesive glycoproteins, this spatial organization was found to be mediated by adsorption of vitronectin (Vn) from serum onto the EDS domains. In contrast, fibronectin (Fn) was unable to adsorb in the face of competition from other serum components. These results were confirmed by immunostaining, which also revealed that both Vn and Fn were able to adsorb to EDS and DMS regions when coated from pure solution, i.e., in the absence of competition. in this situation, each protein was able to mediate cell adhesion across a range of surface densities. Cell spreading was constrained on the EDS domains, as indicated by cell morphology and the lack of integrin receptor clustering and focal adhesion formation. This spatial constraint may have implications for the subsequent expression of differentiated function.
• Serum-free primary human fibroblast and keratinocyte coculture.
  Mujaj S, Manton K, Upton Z, Richards S
  Tissue Eng. Part A (2010) 16,4:1407-1420
      [Abstract]
  Research has shown that the inclusion of a fibroblast cell support layer is required for the isolation and expansion of primary keratinocytes. Recent advances have provided keratinocyte culture with fibroblast-free alternatives. However, these technologies are often undefined and rely on the incorporation of purified proteins/components. To address this problem we developed a medium that used recombinant proteins to support the serum-free isolation and expansion of human dermal fibroblasts and keratinocytes. The human dermal fibroblasts were able to be isolated serum free by adding recombinant human albumin to a collagenase solution. These fibroblasts were then expanded using a serum-free medium containing recombinant proteins: epidermal growth factor, basic fibroblast growth factor, chimeric vitronectin:insulin-like growth factor-I protein, and recombinant human albumin. These fibroblasts maintained a typical morphology and expressed fibroblast markers during their serum-free isolation, expansion, and freezing. Moreover, these fibroblasts were able to support the serum-free isolation and expansion of primary keratinocytes using these recombinant proteins. Real-time polymerase chain reaction and immunofluorescence analysis confirmed that there were no differences in expression levels of p63 or keratins 1, 6, and 10 when keratinocytes were grown in either serum-supplemented or serum-free medium. Using a three-dimensional human skin equivalent model we demonstrated that these keratinocytes also maintained their ability to reform an epidermal layer. In summary, the techniques described provide a valuable alternative for culturing fibroblasts and keratinocytes using recombinant proteins.
• Development of defined media for the serum-free expansion of primary keratinocytes and human embryonic stem cells.
  Richards S, Vesley DL, Topping G, Upton Z .
  Tissue Engineering C (2008) 14,3: 221-232  [Sean D. Richards et al.]
      [Abstract]
  Primary keratinocyte (Kc) cells and human embryonic stem (hES) cells are routinely propagated on a mouse fibroblast feeder layer in media containing fetal bovine serum or other nondefined factors. One disadvantage of using these nondefined factors is that they may inadvertently contaminate the culture system with infectious agents; thus, there remains a need to develop safe culture conditions free from poorly defined and/or animal products. Our laboratory has discovered that growth factors (GFs) and vitronectin (VN) can bind to each other resulting in synergistic short-term functional effects in several cell types. The aim of the current study was to determine whether primary Kc and hES cells can be established and serially propagated serum-free using medium containing VN, insulin-like growth factor-I, and insulinlike growth factor binding protein-3 (VN:GF). Here we demonstrate that primary Kc cells can be isolated, established, serially propagated, and re-form an epidermal layer using the VN:GF combination. Additionally, cell proliferation studies indicate that the Kcs proliferate using the VN:GF combination at a rate comparable to cells grown using serum. Similarly, we verified that this VN:GF combination could be employed for the serial propagation of hES cells. Importantly, both the Kc and hES cells retain their undifferentiated phenotype when cultured using the VN:GF combinations as a serum-free medium for up to 4 passages for Kc and at least 10 passages for hES cells as indicated by the expression of a range of cell surface markers. This study demonstrates that the novel, fully defined VN:GF medium is a viable alternative to media containing serum and highlights the potential of this technology for generating therapeutically viable cells and tissues.
• Towards feeder-free and serum-free growth of cells.
  Richards SD .
  Ph.D. thesis, Queensland University of Technology (June 2007)   [Sean D. Richards]
      [Abstract]
  The in-vitro culture of human embryonic stem and keratinocyte cells has great potential to revolutionise the therapeutics industry. Indeed it is hoped that these cells will provide a superior alternative to current tissue and organ transplantation. However, both of these cell types require animal and/or donor products for their successful maintenance in-vitro. This requirement results in a significant risk of cross contamination from the animal or donor products to either the primary keratinocyte or hES cells. These potentially transplantable cells therefore need to be cultured in an environment free from animal or donor products to remove the risk of contamination to the patient.
  The ideal growth conditions must comprise of two attributes; firstly they must be free from animal or donor products, and secondly the culture system must be fully defined. Recently, it was discovered that an extra-cellular matrix protein, vitronectin, could be used in conjuction with growth factors and growth factor-bindinq proteins (VN:GF combination), to promote enhanced cell migration and growth through the coactivation of inteqrin and growth factor receptors. Given that growth factors and serum are clearly important in supporting the in-vitro cultivation of mammalian cells, and that vitronectin is an abundant protein in serum, I hypothesised that these VN:GF combinations could be translated into a serum-free medium that would support the serial propagation and self renewal of primary keratinocytes and hES cells. As reported in this thesis I have developed a defined, serum-free media for the culture of these cells that incorporates the VN:GF combinations. While the two media differ slightly in their compositions, both support the serial, undifferentiated expansion of their respective cells types.
  Together, this represents a significant advance that will ultimately facilitate the therapeutic use of these cells. However, the in-vitro expansion of these cells in these new media still required the presence of a feeder cell layer. In view of this I aimed to explore the in-vitro micro-environment of primary keratinocytes using a novel proteomic approach in an attempt to find candidate factors that could be used in conjunction with the VN:GF media to replace both serum and the feeder cells. The proteomic approach adopted examined the secretion of proteins into the defined, minimal protein content VN:GF media when the feeder cells were cultured alone, as well as in co-culture with primary keratinocytes. This strategy allowed assessment of proteins/factors that are secreted in response to both autocrine and paracrine cellular interactions and revealed a number of candidate factors that warrant further investigation.
  Ultimately this proteomic information and the associated new insights into the keratinocyte in-vitro culture microenvironment may lead to the development of a culture system for these cells that is not reliant on either a feeder cell layer or serum for their successful propagation. Moreover, it is likely that this will also be relevant to the feeder cell-free propagation of hES cells. This has obvious advantages for the culture of primary keratinocytes and hES cells in that it will allow a safe defined culture system for the undifferentiated propagation of these cells. This will facilitate the generation of cells and tissues free from xenogeneic and allogeneic contaminants, thus ensuring any therapeutics developed from these cell types are approved for therapeutic applications and importantly, will minimise risks to patients.
• False-positive results with mtt assay.
  Rollino C, Borsa S, Bellone G, Piccoli G, Emanuelli G
  J.Immunol.Meth. (1995) 185,1:141-143
      [   -   ]
• Den diabetiske makroangiopati belyst ud fra en vaskulær biologisk synsvinkel. - Hyaluronsyre og dens receptors (CD44) dannelse og betydning for karvæggen.
  Schultz K
  Ph.D. thesis, Århus Universitet (12. March 2004) {[slides]}.
      [   -   ]
• Nanometer thickness laser ablation for spatial control of cell attachment.
  Thissen H, Hayes JP, Kingshott P, Johnson G, Harvey EC, Griesser HJ
  Smart Mater.Struct. (2002) 11: 792-799
      [Abstract]
  We demonstrate here a new method to control the location of cells on surfaces in two dimensions, which can be applied to a number of biomedical applications including diagnostic tests and tissue engineered medical devices. Two-dimensional control over cell attachment is achieved by generation of a spatially controlled surface chemistry that allows control over protein adsorption, a process which mediates cell attachment. Here, we describe the deposition of thin allylamine plasma polymer coatings on silicon wafer and perfluorinated poly(ethylene-co-propylene) substrates, followed by grafting of a protein resistant layer of poly(ethylene oxide). Spatially controlled patterning of the surface chemistry was achieved in a fast, one-step procedure by nanometer thickness controlled laser ablation using a 248 nm excimer laser. X-ray photoelectron spectroscopy and atomic force microscopy were used to confirm the production of surface chemistry patterns with a resolution of approximately 1 μm, which is significantly below the dimensions of a single mammalian cell. Subsequent adsorption of the extracellular matrix proteins collagen I and fibronectin followed by cell culture experiments using bovine corneal epithelial cells confirmed that cell attachment is controlled by the surface chemistry pattern. The method is an effective tool for use in a number of in vitro and in vivo applications.
• Development and characterisation of human skin equivalents and their potential application as a burn wound model.
  Topping G, Malda J, Dawson R, Upton Z .
  Primary Intention (2006) 14,1:14-21 [Gemma Topping et al.]
      [Abstract]
  Wound healing is a complex physiological process; hence a reproducible in vitro model of skin provides a valuable tool to further understand the biology of dermal wound repair and to investigate techniques to improve wound healing. Human skin equivalent models (HSEs) have been proposed to serve as an in vitro model for these purposes; however, there is currently no readily available HSE model in Australia.
  In this study, we describe the production of a HSE obtained by seeding human keratinocytes onto a de-epidermised dermis, (DED) which was then submerged in medium for 3 days and subsequently cultured at the air-liquid interface for up to 20 days. The model was characterised morphologically and biochemically over the 20 days of culture at the air-liquid interface and showed histological features similar to those observed in an in vivo epidermis. Immunohistochemistry of the epidermal markers keratin 6, keratin 14, keratins 1/10/11 and the basement membrane marker collagen type IV, revealed typical differentiation.
  The HSE was then examined for its potential as a burn wound healing model. Burn wounds were created in the model and the re-epithelialisation of the wounds was followed for 6 days by keratinocyte morphology; metabolic activity was analysed every 2 days. Keratinocytes began to migrate into the wound bed after 2 days and continued to migrate for the next 4 days, suggesting that the HSEs generated may be of great value for studies of the wound healing process and for the evaluation of new therapies.
• The effect of vitronectin and other extracellular matrix molecules on endothelial expansion and plasminogen activation.
  Underwood PA, Bean PA
  Cells Mater. (1996) 6:193-207
      [   -   ]
• Inhibition of endothelial cell adhesion and proliferation by extracellular matrix from vascular smooth muscle cells: role of type V collagen.
  Underwood PA, Bean PA, Whitelock JM
  Atherosclerosis (1998) 141(1):141-152
      [Abstract]
  Endothelial cells recovering from damage due to disease or surgical procedures come into close contact with extracellular matrix (ECM) secreted by intimal vascular smooth muscle cells (VSMCs). We have investigated these relationships using human umbilical artery endothelial cells (HUAECs) and human mammary artery VSMC in vitro. HUAEC adhesion and proliferation were significantly lower on ECM secreted by VSMC compared with HUAEC ECM or surface-coated fibronectin. Characterisation of the ECM of both cell types with monoclonal antibodies showed that the ECM secreted by VSMC contained significantly more elastin, chondroitin sulphate and collagen types I, III and V than that from HUAECs. HUAECs adhered poorly to collagen type V coated on plastic and not at all to elastin. When these proteins were co-coated with fibronectin, elastin did not inhibit migration or proliferation compared to the response on fibronectin but collagen type V significantly inhibited both. Treatment of VSMC ECM with enzymes which selectively depleted the matrix of collagen types I, III and IV, or chondroitin sulphate, had no effect on HUAEC responses to the ECM, suggesting that these molecules did not contribute to the inhibition of HUAECs. Treatment of VSMC ECM with a mixture of collagenases, selectively depleted the matrix of collagen type V, as well as types I, III and IV. Such depleted ECMs supported increased proliferation of HUAECs compared to buffer controls. Overall these results suggest that collagen V secreted into the ECM of VSMC may inhibit the recovery of adjacent endothelium
• Rate of endothelial expansion is controlled by cell:cell adhesion.
  Underwood PA, Bean PA, Gamble JR
  Internatl. J. Biochem. Cell Biol. (2002) 34,1:55-69   [P. Anne Underwood et al.]
      [Abstract].
  Procedures used to alleviate blood vessel occlusion result in varying degrees of damage to the vascular wall and endothelial denudation. The presence of intact, functioning endothelium is thought to be important in controlling smooth muscle cell growth, and limiting the intimal thickening which results from damage to the vessel wall. Recovery of the endothelium is commonly slow and incomplete, due in part to endothelial lateral cell:cell adhesion, which limits cell migration and proliferation. We have investigated the effect of fibroblast growth factor 2 and vascular/endothelial growth factor on the relationship between the temporal distribution of the junctional adhesion proteins, platelet/endothelial cell adhesion molecule, vascular/endothelial cadherin and plakoglobin, and cellular migration and proliferation in an in vitro model of endothelial expansion. We found that whereas cell:cell junctions were initially disturbed to similar extents by single applications of the growth factors, outward cell migration and proliferation rates were inversely correlated with the speed at which cell:cell junctions were re-established. This occurred very rapidly with vascular/endothelial growth factor treatment and more slowly with fibroblast growth factor-2, resulting in more extensive outward migration and proliferation in response to the latter. Platelet/endothelial cell adhesion molecule and vascular/endothelial cadherin appeared to be associated with cell:cell junctional control of migration and proliferation, while plakoglobin did not contribute. It was concluded that the rate of endothelial expansion in response to growth factors, is limited by the rate of re-association of junctional complexes following initial disruption.
• Vitronectin: Growth Factor complexes hold potential as a wound therapy approach.
  Upton Z, Cuttle L, Noble A, Kempf M, Topping G, Malda J, Xie Y, Mill J, Harkin DG, Kravchuk O, Leavesley DI, Kimble RM
  J. Invest. Dermatology (2008) 128:1535-1544  [Zee Upton et al.]
      [Abstract]
  Topical administration of growth factors has displayed some potential in wound healing, but variable efficacy, high doses, and costs have hampered their implementation. Moreover, this approach ignores the fact that wound repair is driven by interactions between multiple growth factors and extracellular matrix (ECM) proteins. We report herein that complexes comprising IGF and IGF-binding proteins bound to the ECM protein vitronectin (VN) significantly enhance cellular functions relevant to wound repair in human skin keratinocytes in two- and three-dimensional in vitro cell models and are active, even in the presence of wound fluid. Moreover, these responses require activation of both the IGF receptor and the VN-binding α_v integrins. Further, we assessed the complexes as a topical agent in the treatment of deep dermal partial thickness burns in a porcine model. This pilot study revealed that the complexes may hold promise as a wound healing therapy. Critically, the significant responses observed in vitro and the encouraging preliminary data in vivo were obtained with nanogram doses of growth factors. This suggests that coupling delivery of growth factors to ECM proteins such as VN may ultimately prove to be a more effective strategy for developing a wound healing therapy.
• PTK787/ZK 222584, a Novel and Potent Inhibitor of Vascular Endothelial Growth Factor Receptor Tyrosine Kinases, Impairs Vascular Endothelial Growth Factor-induced Responses and Tumor Growth after Oral Administration
  Wood JM, Bold G, Buchdunger E, Cozens R, Ferrari S, Frei J, Hofmann F, Mestan J, Mett H, O'Reilly T, Persohn E, Rösel J, Schnell C, Stover D, Theuer A, Towbin H, Wenger F, Woods-Cook K, Menrad A, Siemeister G, Schirner M, Thierauch KH, Schneider MR, Drevs J, Martiny-Baron G, Totzke F, Marmé D
  Cancer Research (2000) 60:2178-2189   .
      [Abstract]
  PTK787/ZK 222584 (1-[4-chloroanilino]-4-[4-pyridylmethyl] phthalazine succinate) is a potent inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases, active in the submicromolar range. It also inhibits other class III kinases, such as the platelet-derived growth factor (PDGF) receptor ß tyrosine kinase, c-Kit, and c-Fms, but at higher concentrations. It is not active against kinases from other receptor families, such as epidermal growth factor receptor, fibroblast growth factor receptor-1, c-Met, and Tie-2, or intracellular kinases such as c-Src, c-Abl, and protein kinase C-{alpha}. PTK787/ZK 222584 inhibits VEGF-induced autophosphorylation of kinase insert domain-containing receptor (KDR), endothelial cell proliferation, migration, and survival in the nanomolar range in cell-based assays. In concentrations up to 1 µM, PTK787/ZK 222584 does not have any cytotoxic or antiproliferative effect on cells that do not express VEGF receptors. After oral dosing (50 mg/kg) to mice, plasma concentrations of PTK787/ZK 222584 remain above 1 µM for more than 8 h. PTK787/ZK 222584 induces dose-dependent inhibition of VEGF and PDGF-induced angiogenesis in a growth factor implant model, as well as a tumor cell-driven angiogenesis model after once-daily oral dosing (25–100 mg/kg). In the same dose range, it also inhibits the growth of several human carcinomas, grown s.c. in nude mice, as well as a murine renal carcinoma and its metastases in a syngeneic, orthotopic model. Histological examination of tumors revealed inhibition of microvessel formation in the interior of the tumor. PTK787/ZK 222584 is very well tolerated and does not impair wound healing. It also does not have any significant effects on circulating blood cells or bone marrow leukocytes as a single agent or impair hematopoetic recovery after concomitant cytotoxic anti-cancer agent challenge. This novel compound has therapeutic potential for the treatment of solid tumors and other diseases where angiogenesis plays an important role.
• Novel Antiangiogenic Effects of the Bisphosphonate Compound Zoledronic Acid
  Wood J, Bonjean K, Ruetz S, Bellahcène A, Devy L, Foidart JM, Castronovo V, Green JR
  Pharmacol.Exp.Ther. (2002) 302,3:1055-1061   [Jeanette Wood et al.]
      [Abstract]
  Bisphosphonate drugs inhibit osteoclastic bone resorption and are widely used to treat skeletal complications in patients with tumor-induced osteolysis. We now show that zoledronic acid, a new generation bisphosphonate with a heterocyclic imidazole substituent, is also a potent inhibitor of angiogenesis. In vitro, zoledronic acid inhibits proliferation of human endothelial cells stimulated with fetal calf serum, basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (IC50 values 4.1, 4.2, and 6.9 µM, respectively), and modulates endothelial cell adhesion and migration. In cultured aortic rings and in the chicken egg chorioallantoic membrane assay, zoledronic acid reduces vessel sprouting. When administered systemically to mice, zoledronic acid potently inhibits the angiogenesis induced by subcutaneous implants impregnated with bFGF [ED50, 3 µg/kg (7.5 nmol/kg) s.c.]. These findings indicate that zoledronic acid has marked antiangiogenic properties that could augment its efficacy in the treatment of malignant bone disease and extend its potential clinical use to other diseases with an angiogenic component.
• Ex-vivo investigation of novel wound healing therapies and development of a 3-D human skin equivalent wound model.
  Xie Y
  Ph.D. thesis, Queensland University of Technology (Nov. 2008)   [Yan Xie]
      [Abstract].
  It has previously been found that complexes comprised of vitronectin and growth factors (VN:GF) enhance keratinocyte protein synthesis and migration. more specifically, these complexes have been shown to significantly enhance the migration of dermal keratinocytes derived from human skin. In view of this, it was thought that these complexes may hold potential as a novel therapy for healing chronic wounds. However, there was no evidence indicating that the VN:GF complexes would retain their effect on keratinocytes in the presence of chronic wound fluid. The studies in this thesis demonstrate for the first time that the VN:GF complexes not only stimulate proliferation and migration of keratinocytes, but also these effects are maintained in the presence of chronic wound fluid in a 2-dimensional (2-D) cell culture model. Whilst the 2-D culture system provided insights into how the cells might respond to the VN:GF complexes, this investigative approach is not ideal as skin is a 3-dimensional (3-D) tissue. In view of this, a 3-D human skin equivalent (HSE) model, which reflects more closely the in vivo environment, was used to test the VN:GF complexes on epidermopoiesis. These studies revealed that the VN:GF complexes enable keratinocytes to migrate, proliferate and differentiate on a de-epidermalised dermis (DED), ultimately forming a fully stratified epidermis. In addition, fibroblasts were seeded on DID and shown to migrate into the DID in the presence of the VN:GF complexes and hyaluronic acid, another important biological factor in the wound healing cascade. This HSE model was then further developed to enable studies examining the potential of the VN:GF complexes in epidermal wound heading. Specifically, a reproducible partial-thickness HSE wound model was created in fully-defined media and monitored as it healed. In this situation, the VN:GF complexes were shown to significantly enhance keratinocyte migration and proliferation, as well as differentiation. This model was also subsequently utilized to assess the wound healing potential of a synthetic fibrin-like gel that had previously been demonstrated to bind growth factors. Of note, keratinocyte re-epitheliasation was shown to be markedly improved in the presence of this 3-D matrix, highlighting its future potential for use as a delivery vehicle for the VN:GF complexes. Furthermore, this synthetic fibrin-like gel was injected into a 4 am diameter full-thickness wound created in the HSE, both keratinocytes and fibroblasts were shown to migrate into this gel, as revealed by immunofluorescence. Interestingly, keratinocyte migration into this matrix was found to be dependent upon the presence of the fibroblasts. Taken together, these data indicate that reproducible wounds, as created in the HSEs, provide a relevant ex vivo tool to assess potential wound healing therapies. Moreover, the models will decrease our reliance on animals for scientific experimentation. Additionally, it is clear that these models will significantly assist in the development of novel treatments, such as the VN:GF complexes and the synthetic fibrin-like gel described herein, ultimately facilitating their clinical trial in the treatment of chronic wounds.
• A high-throughput cell migration assay using scratch wound healing, a comparison of image-based readout methods.
  Yarrow JC, Perlman ZE, Westwood NJ, Mitchison TJ
  BMC Biotechnology (2004) 4:21-29  [Justin C. Yarrow et al.]
      [Abstract]
  -Background- Cell migration is a complex phenomenon that requires the coordination of numerous cellular processes. Investigation of cell migration and its underlying biology is of interest to basic scientists and those in search of therapeutics. Current migration assays for screening small molecules, siRNAs, or other perturbations are difficult to perform in parallel at the scale required to screen large libraries. -Results- We have adapted the commonly used scratch wound healing assay of tissue-culture cell monolayers to a 384 well plate format. By mechanically scratching the cell substrate with a pin array, we are able to create characteristically sized wounds in all wells of a 384 well plate. Imaging of the healing wounds with an automated fluorescence microscope allows us to distinguish perturbations that affect cell migration, morphology, and division. Readout requires ~1 hr per plate but is high in information content i.e. high content. We compare readouts using different imaging technologies, automated microscopy, scanners and a fluorescence macroscope, and evaluate the trade-off between information content and data acquisition rate. -Conclusions- The adaptation of a wound healing assay to a 384 well format facilitates the study of aspects of cell migration, tissue reorganization, cell division, and other processes that underlie wound healing. This assay allows greater than 10,000 perturbations to be screened per day with a quantitative, high-content readout, and can also be used to characterize small numbers of perturbations in detail.