Microstructured adhesion substrate for animal or human cells
AssigneesCentre National De La Recherche Scientifique, Universite Joseph Fourier
InventorsMartial Balland, Kalpana Mandal, Lionel Bureau
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Abstract
The present invention relates to an adhesion substrate for at least one animal or human cell on the surface of which is generated an adhesion pattern for at least one animal or human cell, said pattern including at least two areas adhering to said cell, which are spaced apart by an area not adhering to said cell on which a polymer not adhering to said cell is immobilized, characterized in that the polymer is selected so as to have a conformation which may be modified by varying a parameter of the environment in which the substrate is found, to a method for preparing such a substrate and to the cell analysis methods using such a substrate.
Claims27
1- A substrate for adhesion of at least one animal or human cell on the surface of which is generated an adhesion pattern for at least one animal or human cell, said pattern including at least two adhesive areas to said cell which are spaced from each other by a non-adhesive area to said cell on which a non-adhesive polymer to said cell is immobilized, characterized in that the adhesive areas of an adhesive pattern are placed so that the cell may adhere, under adhesion conditions, onto the adhesive areas by forming a bridge over the non-adhesive polymer, the polymer has a conformation which may be modified by changing a parameter of the environment in which the substrate is found and appears as a so-called compacted conformation, under adhesion conditions of the cell, in order to be deployed in a so-called deployed conformation by modification of a parameter of the environment in which the substrate is found.
2- The substrate according to claim 1, characterized in that the polymer defines a layer for which the thickness may be increased relatively to a reference thickness when the substrate is placed in pure water at 37° C. and in the absence of light, by modification of a parameter of the environment in which the substrate is found.
3- The substrate according to claim 1, characterized in that the ratio between the non-adhesive area and the total surface area of the pattern is between 50 and 90%.
4- The substrate according to claim 1, characterized in that the non-adhesive polymer forms a layer for which the average thickness may be increased by at least a factor of 2, relatively to its average reference thickness when the substrate is placed in pure water at 37° C. and in the absence of light, or relatively to its average thickness in its compacted conformation, by means of the modification of a parameter of the environment in which the substrate is found.
5- The substrate according to claim 1, characterized in that the polymer has a conformation which may be modified by varying the temperature and in particular by lowering the temperature.
6- The substrate according to claim 5, characterized in that the non-adhesive polymer is a heat-sensitive polymer having in water a lower critical solubility temperature which belongs to the range from 25 to 35° C.
7- The substrate according to claim 5, characterized in that the polymer is selected from poly(N-isopropylacrylamide), poly(oligo(ethylene glycol)methacrylate) and poly(N,N-dimethyl-aminoethyl methacrylate).
8- The substrate according to claim 1, characterized in that the polymer has a conformation which may be modified by varying the light illumination.
9- The substrate according to claim 8, characterized in that the polymer is poly{N,N-dimethylaminoethylmethacrylate-co-4-methyl-[7-(methacryloyl)oxyethyloxy]coumarin}.
10- The substrate according to claim 1, characterized in that the non-adhesive polymer is immobilized on the surface of the substrate through a covalent bond.
11- The substrate according to claim 1, characterized in that the non-adhesive polymer is immobilized with a density of polymeric chains belonging to the range from 0.1 to 1 chain per nm2.
12- The substrate according to claim 1, characterized in that the non-adhesive polymer is immobilized according to a brush of polymeric chains, each of the polymeric chains being bound to the surface of the substrate through one of its ends.
13- The substrate according to claim 1, characterized in that the non-adhesive polymer appears in the form of polymeric chains comprising from 50 to 10,000 monomer units.
14- The substrate according to claim 1, characterized in that the size of the pattern is adapted so as to allow adhesion of a single individual cell.
15- The substrate according to claim 1, characterized in that the maximum distance generated by the non-adhesive area separating two adhesive areas of a pattern, is less than 50 μm, preferably belongs to the range from 5 to 20 μm.
16- The substrate according to claim 1, characterized in that molecules selected from laminin, fibronectin and collagen are immobilized on the adhesive areas of the adhesive pattern.
17- The substrate according to claim 1, characterized in that the adhesive areas of the adhesive patterns are connected together in order to form an open or closed form or are isolated from each other.
18- The substrate according to claim 1, characterized in that the adhesive areas of the adhesive patterns form a broken line or curve which may be closed for example delimiting a polygon or a ring or open, for example with the shape of a V, U, C or L, or else correspond to two spaced apart lines or to a line and one or several spaced apart dots.
19- The substrate according to claim 1, characterized in that a network of identical adhesive patterns as defined in the preceding claims is present at its surface.
20- The substrate according to claim 19, characterized in that the non-adhesive polymer immobilized on the non-adhesive areas of the adhesive patterns is also immobilized on the regions separating two consecutive adhesive patterns.
21- The substrate according to claim 1, characterized in that the substrate consists of glass, silanized glass, silica or silicon oxidized at the surface.
22- A method for preparing a substrate according to claim 1, wherein the selected non-adhesive polymer is immobilized notably through a covalent bond, on the total surface of the substrate and photo-ablation of the non-adhesive polymer is then achieved by photolithography at the areas corresponding to the adhesive areas of the adhesive patterns.
23- The preparation method according to claim 22, characterized in that the immobilization of the selected non-adhesive polymer at the surface of the substrate is achieved by controlled radical polymerization of at least one monomer on an immobilized polymerization initiator, preferably through a covalent bond, at the surface of the substrate.
24- The preparation method according to claim 22, characterized in that molecules selected from laminin, fibronectin and collagen are immobilized at the adhesive areas after photo-ablation of the non-adhesive polymer.
25- A method for detachment in vitro of at least one cell having adhered beforehand on the adhesive pattern(s) of a substrate according to claim 1, characterized in that the detachment of the cell is achieved by varying an environmental parameter causing deployment of the non-adhesive polymer.
26- A method for in vitro analysis of at least one cell comprising the following steps:
- adhesion of a cell on an adhesive pattern of a substrate according to claim 1,
- cultivation of said cell,
- analysis by optical imaging of said cell adhered onto the substrate.
27- The analysis method according to claim 26, characterized in that it comprises, after the analysis step, detachment of the cell by varying an environmental parameter causing deployment of the non-adhesive polymer.