Penny A. Bean

Hazards of the limiting-dilution method of cloning hybridomas

Three hybridoma clones, which were shown to change the characteristics of their antibody specificities when grown under different culturing conditions, are described in detail. This phenomenon was shown to be due to the persistence of mixed clones, even under conditions where standard statistical treatment indicated a high probability of monoclonality. Such mixed clones persisted, sometimes undetected, through repeated cycles of re-cloning. It was shown that the assumption that every viable clone has the same random chance of monoclonality, is invalid, and can lead to misleadingly high estimates for the probability of monoclonality. Verification of seeding of individual wells with single cells is recommended and the relative merits of this versus repeated limiting-dilution cloning are discussed.

Rate of endothelial expansion is controlled by cell:cell adhesion

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.

The influence of methods of production, purification and storage of monoclonal antibodies upon their observed specificities

A panel of 38 monoclonal antibodies was exposed to a variety of physical conditions commonly used in their purification and storage. Reactivity with the homologous (immunising) antigen was reduced in a significant proportion (42%) of antibodies following these physical treatments. Sensitivity to particular physical treatments correlated with antibody subclass. In 3 cases the relative specificity of the antibody (as measured by cross-reactivity with 16 closely related antigens) was also altered. Following passage of the cloned hybridomas as ascites, 3 antibodies exhibited marked differences in specificity, which suggested the selection of different genotypes. The importance of documenting the methods of production, purification and storage of particular monoclonal antibodies used in comparative assay is stressed.

Polymer coatings that display specific biological signals while preventing nonspecific interactions

Control over cell-material surface interactions is the key to many new and improved biomedical devices. It can only be achieved if interactions that are mediated by nonspecifically adsorbed serum proteins are minimized and if cells instead respond to specific ligand molecules presented on the surface. Here, we present a simple yet effective surface modification method that allows for the covalent coupling and presentation of specific biological signals on coatings which have significantly reduced nonspecific biointerfacial interactions. To achieve this we synthesized bottle brush type copolymers consisting of poly(ethylene glycol) methyl ether methacrylate and (meth)acrylates providing activated NHS ester groups as well as different spacer lengths between the NHS groups and the polymer backbone. Copolymers containing different molar ratios of these monomers were grafted to amine functionalized polystyrene cell culture substrates, followed by the covalent immobilization of the cyclic peptides cRGDfK and cRADfK using residual NHS groups. Polymers were characterized by GPC and NMR and surface modification steps were analyzed using XPS. The cellular response was evaluated using HeLa cell attachment experiments. The results showed strong correlations between the effectiveness of the control over biointerfacial interactions and the polymer architecture. They also demonstrate that optimized fully synthetic copolymer coatings, which can be applied to a wide range of substrate materials, provide excellent control over biointerfacial interactions.

Specific affinity depletion of cell adhesion molecules and growth factors from serum

Serum is a common component of most in vitro cell culture media, particularly of primary cells. Studies of cellular responses to particular adhesion molecules or growth factors are often confounded by the presence of these molecules in the serum supplement. We describe a combined affinity protocol for removing vitronectin and fibronectin from serum. This protocol can also be used to purify these molecules. We also describe the removal of growth-promoting elements using heparin-Sepharose. As vitronectin and fibronectin each bind to heparin, these molecules are removed first and the heparin-Sepharose depletion occurs last in the sequence. This protocol provides a detailed step-by-step guide to achieve quantitative depletion of serum in an optimised format, with additional information on pitfalls and problems. It should be of use to people who wish to accurately determine the relationship between cells, extracellular matrix molecules and growth factors.

Human endothelial cells grow poorly on vitronectin: Role of PAI‐1

The cell adhesive protein vitronectin is a common component of interstitial extracellular matrix and circulates in plasma. It competes effectively with other plasma proteins to adsorb to certain biomaterial surfaces, and is likely to represent an important cell adhesion mediator on the luminal surface of vascular grafts. It is also found associated with certain vascular pathologies. We have shown previously that human endothelial cells grow poorly on a vitronectin surface compared with other extracellular matrix molecules. In this paper we show that endothelial cells seeded on vitronectin and fibronectin produced substantially different profiles of extracellular matrix molecules. The most outstanding difference was in the amount of matrix‐localised plasminogen activator‐inhibitor‐1 which was high on vitronectin and negligible on fibronectin. This was correlated with a small but significant inhibition of cell adhesion to vitronectin compared with fibronectin, and very significant interference with dissociation of cell: extracellular matrix contacts, resulting either from direct inhibition of the proteolytic activity of urokinase, or from interference with urokinase‐receptor signaling and consequent focal adhesion turnover. Such interference would inhibit cell proliferation by disabling the cells from loosening their matrix contacts in order to proceed through mitosis. This would seriously compromise endothelial recovery in cases of damage to the vascular wall and placement of stents or grafts, where the presence of surface‐adsorbed vitronectin is likely to modulate the tissue response. J. Cell. Biochem. 82: 98–109, 2001.

Controlling cell-material interactions using coatings with advanced polymer architectures

Polymeric coatings which allow the effective control of biointerfacial interactions and cellular responses are of increasing interest in a range of biomedical applications in vitro and in vivo such as cell culture tools, biosensors and implantable medical devices. A variety of coating strategies have been developed to gain control over cell-surface interactions but many of them are limited with respect to their function and transferability between different substrate materials. Here, our aim was to establish an easily transferable coating that reduces non-specific cell-surface interactions to a minimum while at the same time presenting functional groups which allow for the subsequent immobilisation of bioactive signals. To achieve this, we have applied an allylamine plasma polymer coating followed by the covalent immobilisation of a macro-initiator providing iniferter functional groups. Subsequent controlled free radical graft polymerisation using the monomers acrylamide and acrylic acid in different molar ratios resulted in highly uniform polymer coatings. Non-specific cell attachment was significantly reduced on coatings representing molar ratios of less than 10% acylic acid. At the same time, we have demonstrated the suitability of these coatings for the subsequent covalent binding of bioactive compounds carrying amine functional groups using the label 2,2,2-trifluoroethylamine. Successful surface modifications were confirmed by X-ray photoelectron spectroscopy (XPS) and profilometry. The cellular response was evaluated using HeLa cell attachment experiments for up to 24 hours. We expect that the coating platform established in this study will be translated into a number of biomedical applications, including applications in implantable devices and regenerative medicine.