# Patent

## Title
Use of nanopatterned surfaces and method for enriching or isolating cellular subpopulations

## Abstract

The invention relates to the use of nanopatterned surfaces. It also relates to a method for enriching or isolating cellular subpopulations. To create a simple, versatile and specific method for enriching or isolating cellular subpopulations from a complex mixture, the invention proposes the use of nanopatterned surfaces for isolating and enriching cellular subpopulations from a complex mixture.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is the National Stage of PCT/DE2008/001825 filed on Nov. 7, 2008, which claims priority under 35 U.S.C. §119 of German Application No. 10 2007 054 691.4 filed on Nov. 14, 2007. The international application under PCT article 21(2) was not published in English.
The invention relates to the use of nanopatterned surfaces. It also relates to a method for enriching or isolating cellular subpopulations.
Enriching or isolating cellular subpopulations from a complex mixture (e.g. T cells from blood) is an important step in pre-analytical sample preparation. It is currently done using antibodies directed against a specific surface protein and magnetic particles for separating the bound cells from the sample. This method, however, has the following drawbacks:

    
    
        The cells in question must express a specific surface protein.
        An antibody against this protein must be available.
        Storing antibodies is problematic.
        Antibodies are expensive.
        When the antibody binds to the surface protein, the cell physiology may be changed.
    
    


Instead of magnetic-particle-based isolation, it is also possible to use fluorescent antibodies together with a FACS (fluorescence associated cell sorter). Here too, there is the problem of cost and, in addition, complex apparatus.
The use of physical parameters offers a further alternative, for example cell density (HEXAL Oncoquick) or cell size (Am. J. Pathol., 2000, (1), 156, 57-63, Isolation by Size of Epithelial Tumor Cells, Giovanna Vona et. al.). However, as not every type of cell exhibits characteristic physical parameters, this approach is only possible if the cells in question do show characteristic physical parameters.
The object of the invention is to create a simple, versatile and specific method for enriching or isolating cellular subpopulations from a complex mixture.
This object is established according to the invention through use of nanopatterned surfaces for isolating and enriching cellular subpopulations from a complex mixture.
Surprisingly, it became apparent within the framework of the invention that specific cellular subpopulations adhere selectively and with hitherto unknown efficiency to nanopatterned surfaces of this kind—much better than to other substrates.
In a particularly preferred embodiment, these nanopatterned surfaces consist of one-dimensional nanowires of the kind described in DE 10 2006 013 484 A1. DE 10 2006 013 484 A1 relates to one-dimensional composite structures that contain at least one nanowire containing a metal core that is coated with a metal oxide. The metal oxide coating is preferably made of ceramics and the metal core is preferably a metal of the 3′ main group of the period system of the elements. The nanowires consist of a metallic core surrounded by a ceramic shell, and are particularly suited for enriching or isolating cellular subpopulations from a complex mixture.
It is within the scope of the invention that the branched nanowires are one-dimensional composite structures having two dimensions in the sub-micrometer range, and that these composite structures are made up of a core of one material, particularly metal, and a shell of another material, particularly ceramic, and can be produced, as described in DE 10 2006 013 484 A1, by thermolytic decomposition of compounds of the general formula El(OR)H2, where

    
    
        El stands for the elements Al, Ga, In and Tl
        R stands for an alkyl (C3-C10) or a cycloalkyl (C5-C8) group
    
    


It is within the scope of the invention that the cellular subpopulations are specific human or veterinary cells and that the complex mixture is plasma.
According to an embodiment of the invention, the plasma is human plasma.
A preferred embodiment of the invention consists in that the selectively adsorbed blood cells are intrinsically non-adherent cells, in particular Jurkat cells.
The term “intrinsically non-adherent cells” refers to cells that normally live in a suspension. As a rule, growing cells of this kind on a substrate is extremely difficult.
The scope of the invention also includes a method for enriching or isolating cellular subpopulations, the cellular subpopulations being enriched on nanopatterned surfaces.
In a particularly preferred embodiment, the nanopatterned surfaces are made up of one-dimensional nanowires of the kind described in DE 10 2006 013 484 A1 and consisting of a metallic core surrounded by a ceramic shell. Cells adhere selectively to these wires, thus becoming enriched.
It is within the scope of the invention that the cellular subpopulations are specific blood cells, and that the complex mixture is plasma.
It is within the scope of the invention that the plasma is human plasma.
According to the invention, ultimately, the cellular subpopulations are intrinsically non-adherent cells, in particular Jurkat cells.



The invention is described below in more detail and in non-restrictive manner on the basis of tests. The experimental results are shown in the drawings.
FIG. 1a and FIG. 1b show fluorescence micrographs of various cell cultures on different substrates and after trypsination;
FIG. 2 shows an EDX micrograph of the nanopatterned surface according to the invention.



The growth of eukaryotic cells was observed on watch glasses that are coated with one-dimensional composite structures in the submicrometer range and have a core made of one material and a shell made of another material.
Use was made of circular watch glasses (Menzel watch glasses, Germany, No. CB00120RA1, 12 mm diameter, 0.13-0.16 mm thickness, batch No. 4710486).
Some of these watch glasses were coated with a one-dimensional composite structure based on Al/Al2O3, as described in DE 10 2006 013 484 A1.
To remove impurities, both the coated and the uncoated watch glasses were treated as follows:

    
    
        The watch glasses (the coated ones with the coated side facing upwards) are placed in the wells of a 24-well cell culture plate.
        500 μl 70% ethanol are added and left for 5 min in the watch glasses.
        The watch glasses are rinsed 3 times with sterile water, using 500 μl water each time.
        The watch glasses are rinsed 3 times with sterile PBS (phosphate-buffered saline solution), using 500 μl of PBS each time.
    
    


Use was made of watch glasses coated with sterile polylysine (BD Sciences, 352085) and of uncoated watch glasses. The former were only washed. In addition, cells were cultivated directly in the 24-well cell culture plates, without watch glasses.
Cells of a suitable density (typically 4-8·104 cells per well) were introduced into 1 ml serum containing cell culture medium and cultivated at 37° C. with 5% CO2 and 100% relative humidity.
The cells were cultivated for up to 72 h and inspected daily with a light-optical microscope. Subsequently, non-adhering cells were washed off by rinsing the wells and watch glasses twice with 1000 μl PBS. The remaining, adherent cells were fixed by adding 500 μl 100% ethanol (5 minutes at RT). Following removal of the ethanol, the cell nuclei were stained by adding 500 μl Hoechst 33342 (Invitrogen, 0.1% in PBS). Micrographs were produced with a fluorescence microscope.
The following test series was carried out:
Test
In this test (07D.0031 pp 38; CK), Jurkat cells (human T cell line in suspension), Raji cells (human macrophage cell line in suspension), K562 cells (human B cell line in suspension) and MCF7 cells (adherent human breast cancer cell line) were cultivated on watch glasses made of plastic (designation in FIG. 1a: Plastic), on watch glasses coated with poly-L-lysine (designation in FIGS. 1a and 1b: Poly-L-lysine), on watch glasses made of glass and on watch glasses coated with Al/Al2O3 (designation in FIG. 1b: Al/AlO).
After 24 h, the cells were washed off and the cell nuclei stained without the cells having been fixed with ethanol. Micrographs were prepared and the cells detached by adding a trypsin/EDTA solution (designation in FIG. 1b: after T/E) (Invitrogen). The remaining cells were washed with PBS and photographed again. In this test, the enhanced adherence of Jurkat cells (as an example of intrinsically non-adherent cells) on the Al/AlOx coating was observed. It was possible to detach the adhering cells alive from the coating by means of trypsination, indicating that protein is involved in the adherence mechanism.
The fluorescence micrographs shown in FIGS. 1a and 1b show that the intensity of the fluorescence is somewhat less with the Al/AlOx-coated watch glasses because the coating absorbs light. Despite this, it is evident that intrinsically non-adherent cells, too, such as Jurkat cells, are enriched much more efficiently on Al/AlOx-coated watch glasses than on the other substrates, and, as shown in further tests, survive in fully functional form over longer periods (more than 72 h). The fact that these cells can be detached alive (and thus cultivated and processed further) means that even minute quantities of such cells can be cyclically enriched on this substrate. Furthermore, cell division is uniform and not too dense, making it easy to observe the cell morphology microscopically, for example, and thus to determine the stage.
Adherence and growth of intrinsically adherent cell lines such as MCF7 is not disturbed by the Al/AlOx coating.
The EDX micrograph (FIG. 2) of the nanopatterned surface according to the invention shows that it does not contain significant amounts of carbon and can thus be considered an inorganic substrate.

## Claims

1. A method for enriching or isolating Jurkat cells from a complex mixture of cellular subpopulations, the method comprising a step of:
enriching the cellular subpopulations cyclically by detaching cells alive and cultivating and further processing them on nanopatterned surfaces, said cellular subpopulations being intrinsically non-adherent cells and said nanopatterned surfaces being made up of one-dimensional nanowires comprising a metallic core surrounded by a ceramic shell, have two dimensions in the sub-micrometer range and are produced by thermolytic decomposition of compounds having the general formula El(OR)H2, where El stands for the elements Al, Ga, In and Ti and R for an alkyl (C3-C10) or a cycloalkyl (C5-C8) group.

2. The method according to claim 1, wherein the cellular subpopulations are blood cells;
wherein the enriching of the cellular subpopulations on the nanopatterned surfaces occurs from a complex mixture; and
wherein the complex mixture is plasma.

3. The method according to claim 2, wherein the plasma is human plasma.

4. The method according to claim 1, wherein the cellular subpopulations are Jurkat cells.