DE19943813A1 - Inducing and detecting various functional states in cells, organelles, viruses, molecular aggregates and biomolecules, comprises using a surface or medium with different binding properties at different sites - Google Patents

Abstract

Inducing and detecting various functional states in cells, organelles, viruses, molecular aggregates and biomolecules on a surface, or in a medium, comprising using a surface or medium that has different binding properties at different sites, is new. Independent claims are also included for the following: (1) producing a fibronectin gradient on a surface, comprising: (a) immobilizing a material selected from gelatin, collagen and their fragments and biotinylated or digoxigenylated derivatives on the surface; (b) treating the surface with blood plasma or a fibronectin solution; and (c) treating the surface with a urea gradient; and (2) producing a laminin gradient on a surface, comprising: (a) immobilizing a material selected from gelatin, collagen and their fragments and biotinylated or digoxigenylated derivatives on the surface; (b) treating the surface with a laminin or laminin/nidogen solution; and (c) treating the surface with a urea gradient.

Description

It has been shown many times that the shape and functional activity of biomolecules, of Molecular aggregates and especially cells by binding partners in a characteri technical way can be influenced.

For example, molecules affect the extracellular matrix in animal cells the cell shape and intracellular arrangement of the cytoskeleton, mobility and the direction of movement of cells, the functional activity and also the development of cells.

For example, it has been known since the 1970s that transformed fibroblasts the cell surface protein fibronectin in much smaller amounts on their surface areas contained as normal fibroblasts. Add soluble fibronectin to trans formed cells can cause these cells to act like normal cells again hold. The rounded transformed cells take the flattened again In the form of normal fibroblasts and adhere firmly to documents. Also be like of the stress fibers that are missing in transformed cells.

It is also known that contacts between the extracellular matrix and cell surface Chen can influence the direction of migration of cells. For example, hike se neural crest cells in the direction of fibronectin fibrils placed on a substrate top are aligned.

And also the functional state such as the metabolism of cells influenced by the extracellular matrix. So the binding of different extracellu loosens lary matrix proteins to integral membrane proteins such as integrins signaling cas dig out from which even changes in gene expression can result (An overview of the interactions of extracellular matrix proteins with Cells and the resulting changes in the structure and function of the Cells give B. Alberts, D. Bray, J. Lewis, M. Raff, K. Roberts, J.D. Watson in Mole molecular cell biology, VCH publishing company, 3rd edition, Weinheim, 1995, Pp. 1119-1192).

That binding partners the form and function of biomolecules, cell organelles and Molecular aggregates such as liposomes or viruses can affect is common  known. For example, most enzymes can bind the substrate, one Inhibitors or activators influence the conformation and the enzyme activity. The same applies - albeit often in a modified form - to proteoliposomes, to Membrane components, viruses and virus components.

Previous methods for studying the interactions of cells, cell population share, molecular aggregates or biomolecules with substrates on surfaces or in A medium usually uses media with a uniform composition.

Possible attachment partners will therefore be almost the same at different locations Forms and functional states bound. It is therefore not easily possible conclusions can be drawn from the distribution of, for example, cells on a substrate surface on the functional state, the functional integrity and in morphologically very similar cells in their own way.

The present invention makes it possible to use media which are heterogeneous Have substance distribution, cells, cell components, viruses, molecular aggregates or Separate biomolecules and / or different functional states at different Locations of the medium.

The production of suitable media or surfaces can vary most methods. Some options with common techniques available and reagents can be realized are listed below.

So it is relatively easy to find concentration gradients of one or more binding partners to produce on a surface. A concentration gradient can be, for example be made by the surface such as a plastic sheet or Glass plate is immersed in a solution with a gradient of the binding partner. The binding partner can under suitable conditions by adsorption or by covalent bond can be immobilized.

An alternative way of generating a concentration gradient is the bin formation or adsorption of one or more binding partners on a surface and the subsequent detachment with a suitable detachment reagent. As a replacement area genes come denaturants such as urea, guanidinium hydrochloride, Sodium dodecyl sulfate or chaotropic ions in question. By immersing the surface a gradient of the denaturing agent occurs with increasing concentrations of the denaturing agent in many cases to an increased detachment of a direct or indirectly bound binding partner. In addition to chemical and enzy  matic processes also physical methods for detachment such as Temperature, pressure or radiation gradients. Finally, specifics are also possible cal releasing reagents, for example, in the separation of interconnected moles cools or molecular complexes when the interaction between the complex components can be influenced by effectors.

Another way of producing surfaces or media with different binding properties creates media that are used, among other things, to separate Cells and used to generate different functional states of cells can be. This can be achieved by taking advantage of differences in the Cell-cell and cell-matrix recognition, which is caused by variations in carbohydrate composition of pure carbohydrates or glycoconjugates. At As is well known, cell-cell and cell-matrix recognition plays among other things the glyco portion of membrane proteins and membrane lipids play a major role. With the help of Chen or immobilized in media carbohydrates such. B. in the form of glycoproteins the extracellular matrix or in the form of pure carbohydrate residues such as mono-, di-, Oligosaccharides or polysaccharides can therefore correspond to cells if they are present of the receptors are specifically bound.

Through targeted gradual changes in the glycosylation pattern on the surface by adding additional monosaccharides or removing individual monosaccharis de, the recognition pattern can be changed more or less specifically.

Such a location-dependent change is with the help of glycosidases or glycosyl transerases z. B. possible in the presence of temperature, substrate, pH or enzyme concentration gradients. Alternatively, it can also be achieved by a wide variety of chemical modifications such as, for example, oxidation of vicinal OH groups with NaIO ₄ and subsequent reaction with ammonia, hydrazine, amines, hydrazines or hy drazides.

Finally, chemical new syntheses are also possible, depending on the location lead to different glycosylation patterns. The location-dependent different glyco Sylation can also here by substrate, cosubstrate, temperature, pH gradient or other gradients.

Bin provides another option for producing suitable surfaces or media partners who are responsible for the mutual recognition of peptide or protein components  need. Such binding partners can also on suitable surfaces or in suitable media are immobilized. A gradual change in attachment ability can be achieved through a targeted proteolytic degradation of the proteins or Peptides with specific or non-specific exo- or endopeptidases z. B. in Presence of a temperature gradient, a protease concentration gradient or a gradient of other substances that bring about the desired change can. For example, the effect and specificity of proteases can be affected are by protease inhibitors, metal ions, or by the pH. Further Modification reactions which lead to changes in the binding properties or Stabilization of the affinity media generated in this way can be connected. For example, a modification with cross-linking reagents such as glutar Dialdehyde may be required to prevent or prevent fragments from detaching hesitate. Through further selective or unspecific modifications of Seitenket ten or end groups may optionally have further properties on one Surface or changed in a medium. These changes can by choosing suitable gradients to very finely tuned location-dependent sub divide the affinity medium, with multidimensional gradients can be witnessed. The modification reactions mentioned above can of course even without prior proteolytic treatment with protei bound to surfaces or peptides or other binding partners.

Another possibility concerns the production of gradually changed surfaces by Culturing cells on surfaces or in a medium in the presence of gra served on glycosylation inhibitors such as tunicamycin or bacitracin. The latter change the glycosylation pattern and thus the capabilities of the cells, with coal hy interact with third-party binding partners (see e.g. L. Stryer in Biochemistry, Spektrum Akademischer Verlag Heidelberg, Berlin, Oxford, 4th edition 1995, p.996).

Numerous inhibiting or stimulating substances which have the properties of Influencing cells directly or indirectly can be used in a similar way. The production of a surface with different surface characteristics depending on the location can also be modified by chemical modification with one or more low moles molecular chemical reagents. For example, foils, slides  or culture dishes with reactive amines, acids or zwitterionic compo nents are treated to a different distribution of positive or negative To generate charges. A different phosphorylation leads, for example as well as the reaction with carboxylic acids, sulfonic acids or other acid groups differences in the distribution of negative charges. Biological rele Various modification reactions can be carried out. For example, that on certain surfaces by a different degree of phosphorylation different states are imitated by enzymes such as kinases or Phos phases are generated within living systems.

Further examples of implementations which lead to a different binding sponding binding partners are the production of biotin gradients, wel differences in the extent and / or strength of the binding of biotin Lead proteins. A different digoxigenyly can also be used in a corresponding manner degree of generation on a surface or in a medium, which leads to Un differs in the binding of digoxigenin binding binding partners.

Numerous haptens can also be immobilized in the form of gradients there are many other possibilities for the use of different binding partners conceivable. Suitable reagents for the implementation of surfaces with the mentioned bin There are numerous variants available to partner partners and they exist well Working instructions for their use (methods for the production of Affini Act media are for example by G. T. Hermanson, A. K. Mallia, P. K. Schmidt described in Immobilized Affmity Ligand Techniques, Academic Press, 1st edition, London 1992).

The different distribution of bound components and the resulting functions on states can be determined using various physical and chemical methods is sought and recognized.

Only a few examples are listed below:
Staining with suitable dyes, for example for the detection of a different distribution of proteins, carbohydrates or nucleic acids; Detection of the distribution of bound substances with specific labeled binding partners such as antibodies; Light microscopic or electron microscopic observation of the cell morphology of immobilized cells with and without staining methods; Immunofluorescence, e.g. B. view the cytoskeleton after fluorescent labeling to detect un different functional states of the cells; Measurement of enzyme activities on cell surfaces and in the cell interior using suitable substrates; Measurement of secreted enzyme activities, for example by adding chromogenic substrates; Radioactivity measurements such as autoradiography after adding radioactive binding partners or substrates;
In situ hybridization to detect differences in the structure and functional state of nucleic acids; In situ PCR (polymerase chain reaction);
Electrochemical methods also with the help of chips, which register a different distribution of bound components and possibly also give information about their functional state. In the case of cells, cell-cell contacts can be recognized under certain circumstances, such as gap junctions.
Modern optical methods such as the use of evanescent field absorption sensors, which register a different distribution and morphology of bound components;
An important prerequisite for the above-mentioned investigations is finding a suitable surface for the gradient immobilization, which allows an examination of the complex formation with the appropriate method. Surfaces can also be microchips, optical media or the like, on which the desired gradient can be generated.

There are numerous possible applications, some of which are listed below:
So the method can be used to separate and characterize cells with different affinities for molecules of the extracellular matrix such as. B. collagens, fibronectin, laminin, proteoglycans, polysaccharides.

If several binding partners are immobilized simultaneously, Va riation of the mixing ratios and variations of the gradient additional information tions about the importance of the individual components for attaching the Cells on matrix surfaces. Any gradient shapes are conceivable here.

The method can be used to detect and examine Tu mor cells and the metastatic potential of tumor cells. So it is known that at Tumor cell interactions between cells and also cell attachment Surfaces are disturbed. For example, gradually changing cell surface  Chen proteins, extracellular matrix proteins, lectins, or also compounds other than Binding partners used and their interactions with (potential) tumor cells to be examined. Resulting changes in the morphology, in the Ar architecture of the cytoskeleton and / or in the expression and presentation of surfaces antigens then allow further differentiation of the cells.

The method can also be used to separate subpopulations different cells with different affinities for one or more bin partners. For example, lymphocytes and other immune-competent Cells due to their different affinities for their corresponding antigen be recognized and separated. Since the corresponding cells are most likely to the best matching antigen areas, there are differences in Binding pattern of the cells and also in their displaceability by competitive bin partner.

The method can be used in the cultivation of cells to differentiate Liche, but still defined functional states to generate z. B. in in vitro Kon structure of tissues, organoids or organs. For example, it should be possible be to generate countercurrent principles by an opposite distribution of two or more cell types. This opposing distribution can be done with opposing gradients of binding proteins that match the cells, such as extracellular matrix proteins, Proteoglycans, carbohydrates or other binding partners become.

Finally, the method should also have greater potential in the study of the Offer influence of external factors on the behavior of cells and cell functions than conventional cell culture experiments. So the influence of numerous substances on the behavior of cells in very different functions on a surface be examined.

Only a few examples of such substances are listed below:

• - Medicines
• - environmental chemicals
• - Signal substances such as B. hormones
• - nucleotides, oligonucleotides and nucleic acids.

In addition, one can in the cell culture itself and in the examination of the cell properties vary, such as the temperature, the  Gas composition, radiation with electromagnetic or radioactive rays and much more.

By using cell cultures in which the cells are in very different Functional states, you get more differentiated information about us of such substances than with conventional cell cultures in which the cells in have a similar functional state. The procedure could also be one Make a contribution to replacing animal experiments with cell culture experiments.

In certain cases, however, it is used to investigate certain cell properties be necessary to "freeze" the current properties. He can do that, for example may be necessary if cells produce substances in a long cell culture or in forms which falsify the results. For example, if cell binding expe riments with gradually changed extracellular matrix proteins are carried out and the cells themselves produce extracellular matrix proteins in large quantities and release to the outside, these can gradually change the effects of the given overlay the matrix molecules. Such "freezing" can take place, for example using standard histological fixation methods such as treatment with glutardialdehyde or other crosslinking agents, by embedding in suitable media, which a Prevent further changes in cell structures, or by lowering the temperature up to freezing.

Another certainly for different areas of chemistry, biochemistry or biology interesting area of application of the present invention is the investigation and also Differentiation of affinities and avidities of binding partners. Antibody, Lec tine, protein A, protein G, avidin and streptavidin and many other compounds zen several binding sites for the recognition and binding of their binding partners. The Probability that two or more binding sites correspond with the The corresponding binding partner on a surface interacts, the greater it is higher the local concentration of the binding partner on this surface. At a the distances of the immobilized binding partners are very low large that almost all bonds are made only via one binding site.

With increasing concentration or increasing density of the immobilized bin partners decrease their distances on the surface. This increases the truth Probability that a multi-point commitment occurs. Through the simultaneous connection the binding partner increases the bond strength by two over two or more bonds orders. So in the range of low concentrations it is primarily the affinity  of the individual binding sites is decisive for the binding strength. At higher concentrations The bond strength becomes extreme due to the multivalence of the binding partners greatly increased, and avidities are measured. By adding competent compounds additional information can be obtained.

The various functional states of cells, molecular aggregates and biomolecules can be produced on the surfaces or media described in the invention in various ways, such as

• - by direct binding / adsorption z. B. by immersing the surfaces or media in a solution with the binding partners corresponding to the surface.
• - by chromatographic separation on the surface (e.g. film) or in the Medium (e.g. gel) by thin layer or column chromatography.
• - by electrophoretic separation on the surface or in the medium
• - by magnetic separation for magnetically separable components
• - by culturing cells on the surface or in the medium.

example 1 Generation of a gelatin gradient on a nitrocellulose surface

A strip of nitrocellulose is soaked in water or a buffer for 10 min. With the help of a gradient mixer, an aqueous gradient solution of a gelatin and sucrose gradients generated in a beaker with a gelatin concentration from 0 mg / ml in 5% sucrose to 0.05 mg gelatin in 20% sucrose. Subsequently the solution is covered with a small amount of distilled water or buffer layers.

A suitably cut nitrocellulose strip is dipped into the gradient and 15 min. leave in it. Then the strip is carefully removed and washed thoroughly with distilled water or a buffer.

The quality of the gelatin gradient can be determined by suitable staining methods such as a Ponceau S staining, a gold staining with colloidal gold, or other suitable Staining method can be demonstrated. In most cases, this should be a separate one Strips are made and used because many of the staining methods are not reversible are. Free binding sites can be used with bovine serum albumin, Tween 20 or another blocking reagent.  

Example 2 Generation of a fibronectin gradient on a gelatin surface

A strip of nitrocellulose is placed in a 1% gelatin solution overnight. Then it is washed with distilled water. After that it will be here made gelatin nitrocellulose strips or alternatively the gelatin nitrocellulose Strips from example 1 placed in human or animal plasma for 2 hours, to bind plasma fibronectin and possibly other proteins to the gelatin.

Alternatively, a self-prepared or purchased fibronectin solution can be used become. A gradient mixer is used in a beaker or a measuring cylinder which produces a urea gradient with a urea concentration of 0-8 mol / l in Water or a suitable buffer. The gradient is also low Amount of water or buffer overlaid. The nitrocellulose strip with the gelatin Bound proteins are immersed in the urea gradient and added for 15 min Leave room temperature in it. With increasing urea concentration, a increasing proportion of fibronectin and possibly other bound proteins from the Ni trocellulose detached so that a fibronection gradient on the nitrocellulose surface arises.

The immobilized gelatin gradient differs for the nitrocellulose those orientations of immersion possible, which different one or also generate two-dimensional gradients.

The strip is then removed from the urea solution and distilled with it Washed water or a suitable buffer.

The strips can then be used to examine fibronectin binding binding partner.

Claims (45)

1. Process for the production and determination of various functional states of Cells, cell organelles, viruses, molecular aggregates and biomolecules on one surface surface or in a medium in which the surface or the medium differs where locations have different binding properties. 2. The method of claim 1, wherein the surface is an artificial or natural manufactured film is. 3. The method according to claim 2, wherein the film of cellulose, nitrocellulose, one other cellulose derivative, from polyvinylidene fluoride, polypropylene, polystyrene, Ny lon, Perlon, glass fibers, siliconized glass fibers or derivatives thereof. 4. The method of claim 1, wherein the surface of glass, quartz, or deriva ten of them exist. 5. The method of claim 1, wherein the surface of electrically conductive mate rialien consists of or materials used for electrical detection can be. 6. The method of claim 1, wherein the medium consists of gels. 7. The method of claim 6, wherein the gels are polysaccharide gels, Polyacrylamide gels, protein gels, glycoprotein or proteoglycan gels, polynucleotides de or derivatives or mixtures thereof. 8. The method according to claims 1-7, characterized in that the differ Chen binding properties on the surface or in the medium due to a heterogeneous distribution of immobilized biomolecules or immobilized binding partner is caused by biomolecules. 9. The method according to claim 8, characterized in that the heterogeneous Ver division of the immobilized biomolecules is generated using gradients.   10. The method according to claim 9, characterized in that it is the gradient are linear gradients in any arrangement and design. 11. The method according to claim 9, characterized in that it is the gradient are step gradients in any arrangement and design. 12. The method according to claims 9-11, characterized in that it is in the Gradients around concentration gradients of the substances to be immobilized or of their activated forms. 13. The method according to claims 9-11, characterized in that it is in the Gradients are concentration gradients of substances that the upper Activate surface or the medium for the subsequent immobilization or inac tivier. 14. The method according to claims 9-11, characterized in that it is in the Gradients around gradients of substances or physical conditions delt which the activation or inactivation of the surface or the medium and / or influence the immobilization quantitatively or qualitatively. 15. The method according to claims 9-11, characterized in that it is in the Gradients around gradients of substances and / or physical conditions is a complex formation between immobilized components and influence possible binding partners, or immobilized components again replace. 16. The method according to claims 13-15, characterized in that it is in the Gradients around pH gradients, salt gradients, gradients of chaotropic ions, around Urea gradients or gradients of guanidinium salts or derivatives thereof acts. 17. The method according to claims 14-15, characterized in that it is in the physical gradients around temperature gradients, gradients electromagnetic sher radiation, radioactive radiation gradient or pressure gradient.   18. The method according to claims 1-17, in which on a surface or in a Medium one or more receptors, receptor binding components, fragments thereof, their biotinylated forms, their digoxigenylated forms and / or others Derivatives thereof are immobilized in the form of one or more gradients. 19. The method of claim 18, wherein the immobilized component is biotin Is a biotin derivative, a biotin-analogous substance or a binding partner of biotin. 20. The method according to claim 18, wherein the immobilized component Digoxi genin, a digoxigenin derivative or a binding partner of digoxigenin. 21. The method according to claim 18, in which the receptor-binding components or the receptors antigens, antigen receptors, antigen-recognizing molecular aggregates antigen-recognizing cells, parts or fragments thereof, their biotinylated forms, their digoxigenylated forms and / or other derivatives or any Gemi are like that. 22. The method according to claim 18, in which the receptor-binding components or Receptors one or more extracellular matrix proteins, fragments thereof, bio tinylated forms, digoxigenylated forms and / or other derivatives of these proteins or are peptides. 23. The method of claim 22, which is the extracellular matrix protein collagen, denatured collagen, fibronectin, laminin, nidogen, entactin, Tenascin, osteonectin, vitronectin, thrombospondin, to fragments of these proteins, for biotinylated forms, for digoxigenylated forms and / or other derivatives proteins or peptides or mixtures thereof. 24. The method according to claims 18, 22-23, in which collagen, degradation products of Collagen, denatured collagen or gelatin, fragments thereof, biotinylated form men, digoxigenylated forms or other derivatives or mixtures of these pro teins or peptides are bound to a surface or in a medium.   25. Methods in which binding partners of the immobilized components to the in the surfaces or media described claims 18-24 are bound. 26. The method according to claims 18-25, wherein a part of the bound binding part ner is replaced again. 27. The method of claim 26, wherein the detachment under denaturing conditions conditions. 28. The method according to claim 26-27, in which denaturing means for producing a uneven detachment in the form of one or more gradients the. 29. The method according to claims 27-28, which is denaturing means chaotropic ions. 30. The method according to claims 27-28, which is denaturing agents are urea or guanidinium salts or derivatives thereof. 31. Methods in which gelatin, a collagen, fragments thereof, are biotinylated or digoxigenylated forms or mixtures thereof bound to a surface, then the surface with blood plasma or a solution of fibronectin is treated, and by treatment with a urea gradient on the surface a fibronectin gradient is generated. 32. Methods in which one or more collagens or gelatin, fragments thereof, their biotinylated or digoxigenylated forms or mixtures thereof to one Surface bound, then the surface with a solution of laminin or a laminin / nidogen mixture, and by treatment with creates a lamination gradient on the surface of a urea gradient.   33. The method according to claims 25-32, wherein the surface is Ni trocellulose, glass surfaces, modified glass surfaces or culture vessels delt. 34. The method according to claims 27-28, which is denaturing means are physical denaturing agents. 35. The method of claim 34, which is physical denatury means of temperature, electromagnetic radiation, radioactive radiation, and / or pressure. 36. The method according to claims 1-7, characterized in that the differ Chen binding properties of the surface or the medium by a treatment development of compo already immobilized on the surface or in the medium takes place. 37. The method according to claims 1-7 and 36, in which gradients of substances and / or physical properties that are used for training of different binding properties. 38. The method according to claims 1-7 and 37, characterized in that it is the gradients are gradients of substances that are already immobilized modified components irreversibly or reversibly. 39. Process according to claims 1-7 and 36-38, characterized in that enzymes are involved in the formation of the different binding properties. 40. The method according to claim 39, characterized in that it is in the enzymes are proteases, glycosidases or glycosyltransferases. 41. The method according to claim 37, characterized in that it is in the physical gradients around temperature gradients, gradients electromagnetic shear or radioactive radiation or pressure gradients.   42. All methods in which the surfaces described in claims 1-41 or media for the binding of cells, cell organelles, viruses, molecular aggregates, Biomolecules or other molecules are used. 43. All methods in which the surfaces described in claims 1-41 or media for the cultivation of one or more cell types in any di dimensions and orientations are used. 44. All diagnostic and analytical procedures in which the claims 1-41 described surfaces or media can be used. 45. All preparative methods in which those described in claims 1-41 Surfaces or media are used.