Chemistry: molecular biology and microbiology – Carrier-bound or immobilized enzyme or microbial cell;... – Enzyme or microbial cell is immobilized on or in an organic...
Reexamination Certificate
1997-01-15
2001-09-04
Ceperley, Mary E. (Department: 1641)
Chemistry: molecular biology and microbiology
Carrier-bound or immobilized enzyme or microbial cell;...
Enzyme or microbial cell is immobilized on or in an organic...
C435S007210, C435S007240, C435S029000, C435S034000, C435S040500, C436S531000, C436S532000, C436S533000, C436S811000, C436S823000, C530S391100, C530S816000
Reexamination Certificate
active
06284503
ABSTRACT:
2. FIELD OF THE INVENTION
The present invention is related to a composition and method for regulating the adhesion of cells, organisms, and molecules to hydrophobic surfaces. More specifically, the present invention is directed to a biomolecule, such as proteins, peptides amino acids, nucleic acids, lipids, and carbohydrates conjugated to end-group activated polymers (EGAPs) and uses thereof.
3. TECHNICAL BACKGROUND
Normal development and function in living organisms require interactions between cells and the molecules in the surrounding environment. One way cells communicate is via molecules that span the membrane of the cell called transmembrane proteins. When the portion of the transmembrane protein which is outside of the cell encounters specific molecules in the surrounding environment, it undergoes structural and conformational changes which triggers biological reactions inside the cell.
For example, in vivo, cells form complex multilayer structures which ultimately form tissues and organs. Tissue and organ formation, however, requires specific contacts with the environment. These cells are referred to as “anchorage-dependent” because they will not grow properly, if at all, unless they are anchored to others cells, an extracellular matrix (ECM), or other surface.
An ECM is a complex and variable array of molecules secreted by cells, such as collagens, glycosaminoglycans, proteoglycans, and glycoproteins. Together these cellular products form the basal lamina, bone, and cartilage which give tissues and organs their shape and strength. In fact, contact between anchorage-dependent cells and the ECM in many instances plays a dramatic role in determining the cells' shape, position, metabolism, differentiation and growth.
Cell contact is also important in other biological functions, such as the activation of an immune response. The immune system is a complex network of cells that have the ability to recognize and rid the body of foreign substances, such as viruses, bacteria and parasites. One mechanism used by the immune system to rid itself of foreign substances is a humoral response. A humoral response involves activation of specific cells called B cell lymphocytes. B-cells are activated when transmembrane proteins on their surface bind to foreign substances called antigens. Specifically, binding of B-cells to antigens stimulates B cells to proliferate and differentiate into immunoglobulin or antibody producing plasma cells.
The antibodies produced by plasma cells travel throughout the body binding to the pathogen or foreign substance. Binding of antibodies to foreign substances activates several other immunological pathways, including the “complement” pathway. The complement pathway is designed to destroy the foreign substance and to initiate an inflammatory response in the organism.
While cell contact with other cells and the environment is critical to the overall health and biological function of an organism, it creates unique problems in the art of biotechnology. Specifically, two areas where cell contact requirements create problems are: (1) cell culture; and (2) biomaterial transplantation.
Tissue or cell cultures comprise cells from a plant or animal which are grown outside the organism from which they originate. These cells are often grown, for example, in petri dishes under specific environmental conditions. Cell cultures are of great importance because they represent biological “factories” capable of producing large quantities of biological products such as growth factors, antibodies, and viruses. These products can then be isolated from the cell cultures and used, for example, to treat human disease. In addition, cell cultures are a potential source of tissue which could be used for transplantation into humans. For example, cell cultured skin cells could potentially be used in skin grafts to replace diseased or damaged skin. Finally, cell cultures usually comprise cells from only one or a few tissues or organs. Consequently, cell cultures provide scientists with a system for studying the properties of individual cell types without the complications and risk of working with the entire organism. For example, the effects of pharmaceutical drugs on certain cell types could be tested on cell cultures prior to clinical trials in order to assess the drug's health risks.
Like most cells in vivo, cells grown in culture are either anchored to an ECM or another cell. Only cells of the circulatory system (e.g., lymphocytes and red blood cells) grow unattached and suspended in solution in vitro. Many anchorage-dependent cells can grow on glass or plastic surfaces, such as polystyrene. These cells, however, often lose their natural architecture and do not function normally (e.g., the ability to differentiate and respond to hormones). Accordingly, these cells do not precisely mimic a cell's biological functions in vivo and thus have limited potential.
For this reason, glass and plastic cell culture dishes are often coated with an ECM protein such as collagen, fibronectin, laminin and the like. These proteins bind to surfaces such as polystyrene through a process known as adsorption. Although ECM coated cell culture surfaces have led to improved culture conditions, they are far from ideal.
First, biomolecules, such as proteins, often become inactivated upon adsorption to hydrophobic surfaces. The biological activity of proteins is conferred by their unique structure and their ability to undergo conformational changes upon binding to a substrate or other physiological event. In one study, the structure of proteins was measured using a technique called microcalorimetry. Microcalorimetric studies demonstrated that proteins which are bound to hydrophobic surfaces loose essentially all their cooperatively folded structure compared to the same protein in solution. Because a protein's structure and its ability to undergo conformational changes strongly correlates with biological activity, these data suggest that most proteins that are adsorbed by a hydrophobic surface loose there in vivo biological activity.
Second, the conformation and orientation of immobilized proteins have important effects on the nature of their interaction with cells. D. J. Juliano, S. S. Saaedra and G. A. Truskey, Journal of Biomedical Materials Research 2-7 1103-1113 (1993). Both are influenced by the chemistry and physical properties of the underlying substrate as well as by the method of immobilization. K. Lewandowska, E. Pergament, N. Sukenik and L. A. Culp, The Journal of Biomedical Materials Research 21 1343-1363 (1992).
Third, like in vivo, cells in culture release molecules such as serum proteins and growth factors into the culture media. As discussed above, the secretion and concentration of these molecules in the culture media are critical to the biological function of neighboring cells. Under current cell culture conditions, the careful balance and concentration of secreted molecules are disrupted because secreted molecules are adsorbed by the cell culture surface, Thus, the communication and biological function of cells grown under current cell culture techniques does not mimic in vivo environment.
Finally, the surface concentration of ECM components is a critical factor in the regulation of cell behavior. The ability to control and vary surface biomolecule concentration is therefore of upmost importance and depends on the method of immobilization and in some cases the physical nature of the base material. Simple ECM adsorption to cell culture substrates does not meet these requirements.
In short, to date there is no single method for conjugating proteins to potential cell culture substrates which addresses all these major concerns. Thus, current research is hindered by the fact that cell cultures do not accurately mimic an in vivo environment.
A second problem area created by cell contact is biocompatibility. It is generally acknowledged that artificial biomaterials, including fabricated biomedical polymers, are much less immunologically active than transplants or tissue-derived
Caldwell Karin D.
Neff Jennifer
Tresco Patrick A.
Ceperley Mary E.
Madson & Metcalf
University of Utah Research Foundation
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