Method of making arranged cell structures

Technical field

The invention relates to the controlled organization of cells into patterns on the solid surfaces

Background art

Controlled organization of cells is a principle attractive not only for medicine or pharmacology in general, but also for nano-biotechnology or bio-electronics, which have been becoming more and more important fields in the recent years. Nanotechnologies often require controlled structuring and engineering of both material and chemical properties in the order of micro- and nanometers, which can be exploited for the fabrication of functional devices, for the selective growth and organization of structures as well as for the preparation of heterogeneous interfaces between organic and inorganic materials. Such hybrid devices can create a bridge between the organic and inorganic world, combining their advantages and being beneficial for meeting the today's high requirements on miniaturization, high performance, and new functions. The fabrication of microscopic templates for the attachment of carbon nanotubes, DNA molecules, cells and cellular membranes on a particular spot on an electronic chip could serve as a perfect example of such devices. Moreover, various ways of the organization of cells are being exploited in tissue engineering.

For the oriented attachment of cells onto a substrate, mainly ligand-receptor bonds are utilized. For example, peptides, containing an amino acid sequence RGD, distinguishable by various types of intengrin cell receptors, can be covalently bound to the surface. Subsequently, cell attached onto these peptides forms focal adhesions (or focal adhesion plaques composed from many structural and signal proteins, which the cell requires for the interaction with the substrate's surface). This type of interaction leads to the transfer of information between the cell and the substrate and the cell reacts by starting various life programs (proliferation, differentiation, death, etc.). Furthermore, "printing" of these RGD peptides in various distances and patterns leads to a regionally selective adhesion of cells. Other molecules able to mediate adhesion, such as P-selectin, whole colagen fibers or other extracellular matrix proteins which are commercially available can also be utilized. Other

possibilities include e.g. self-organized monolayers of organic chains which are covalently terminated with -CH ₃ , -NH ₂ and other groups.

Apart from the protein layer between cell and substrate, the substrate itself and its properties play a major role in cell adhesion. The substrate and its properties stimulate the cell reaction (response) to the entire environment, i.e. whether they can adhere, proliferate, differentiate or, on the other hand, whether they cannot adhere and die.

Various materials can be exploited as a substrate for the growth and organization of the cells. They can be, depending on the particular application, either flat with varying degrees of roughness or shaped into complex three-dimensional structures. From the point of view of their electronic properties, largely metals (titanium compounds) or dielectrics (insulators), such as inorganic oxides (SiO ₂ , TiO ₂ ) or various polymers (polystyrene - PS, or polymethylmatelcrylate - PMMA), are used. Recently, however, the use of semiconductors, whose conductive properties can be easily tuned to allow the fabrication of integrated circuits and biological structures based only on one type of material, has come to focus. In industry, silicon together with its oxides is the most widespread element. It has countless applications in microelectronics, optoelectronics, photovoltaics, xerography, sensors, etc. Emerging semiconductive materials include wide-band-gap semiconductors such as diamond or silicon carbide. Especially diamond offers a unique combination of semiconductive, mechanical, chemical and biological properties. Although diamond is a semiconductor with wide energy band gap (5.5 eV), which makes it an insulator in its intrinsic state, it can be doped with impurities (boron, phosphorus, etc.) in order to obtain p- or n-type conductivity. Moreover, a two-dimensional highly conductive layer can be generated on hydrogen-terminated diamond surfaces. Due to the wide band gap, diamond is optically transparent, which is important for the optical detection of biological properties. Furthermore, it is hard, and mechanically, chemically and physically stable. Its unusually wide electrochemical window (>3 V), in which the surface does not chemically react itself but supports chemical reactions, is highly advantageous for electrochemical measurements, such as impedance spectroscopy, a widely used technique in bio-sensors. Last but not least, diamond is considered highly biologically compatible, since it is actually made up of carbon. Consequently, its possible applications in prosthetics and bio-sensors are intensely studied. Diamond can be synthesized, either as bulk or in the form of thin films deposited on various substrates, using methane decomposition in a plasma discharge.

All in all, a material which firstly enables a controlled organization of cells into structures on surfaces, secondly can be prepared by simple procedures and at low cost, and

thirdly does not influence the biological behavior of adhered cells (and consequently ensures the right function and use of the prepared system) is highly desirable for biological applications. The most important parameter of any substrate is naturally its bio-compatibility, particularly the way it influences cell adhesion, growth and survival. Besides biocompatibility, mechanical and chemical resistance plays an important role. The use of special peptides and proteins for the selective adhesion and orientation of cells is relatively difficult and costly and, moreover, their bonding to and stability on the substrate needs to be considered.

Disclosure of the invention

The present invention provides a method which both overcomes the above-mentioned limitations and meets the requirements by utilizing diamond as a substrate for the organization of cells. The main and unique advantage of diamond is that it combines excellent semiconductive, mechanical, chemical, and biological properties. From the viewpoint of semiconductors, its free-carrier mobility is higher than in silicon by several orders of magnitude. What is more, it is composed of carbon, a natural interface for organic materials. Last but not least, its both chemical and hard-radiation and high-temperatures resistance complements all the highly advantageous properties for biological and bio-electronic applications.

Diamond can be either natural or synthetic, prepared at high pressure and temperatures or by plasma-enhanced chemical vapor deposition or by the detonation of explosives.

Diamond can be either intrinsic or impurity (boron, phosphorus) doped, the concentration of the impurities is not limited.

At the same time, the surface of diamond can be terminated with carbon or oxygen or hydrogen or nitrogen or halogen atoms (fluorine, chlorine) or their compounds or with a combination of these species. The concentration of the terminating species is not limited.

The method of the preparation of organized cell patterns lies in the spontaneous adhesion of cells suspended in a cultivation medium onto a surface of a solid, preferably diamond. Prior to adhesion, hydrophilic areas separated with hydrophobic areas need to be fabricated on the surface of the solid. The hydrophobic areas are fabricated through their exposure to hydrogen plasma at at least 400 °C, while the exposure to oxygen radicals from plasma discharge or ultraviolet radiation leads to the formation of hydrophilic areas. The solid with such a chemically patterned surface is immersed in the cultivation medium with cells

with the ability to form bone or neural tissue, such as human bone-forming cells and mesenchymal (stem) cells able to differentiate into bone-forming cells or neurons.

In addition, the surface of the solid (diamond) can be structured morphologically. Diamond surfaces can be of different surface roughness depending on the selected regime of growth and at the same time three-dimensional structures can be fabricated.

Example

520-μm-thick boron-doped (conductivity 0.1 (ωcm) ^"1 ) silicon substrate is ultrasonically cleaned in isopropanol for 5 minutes, then it is dipped into deionized water and dried with a flow of dry nitrogen. Water-suspended nano-diamond powder with the nominal size of particles of 5 nm is then deposited onto the clean substrate in an ultrasonic bath. The deposition takes 40 minutes, leading to the formation of a 5- to 25-nm-thin and as much as from 80 % continual layer of nano-diamond powder.

A layer of nano-crystalline (NCD) diamond with the thickness in the order of several hundreds of nanometers is later deposited on this starting layer by microwave-plasma- enhanced chemical vapor deposition. 1 % methane dilution in hydrogen is used as the gas, other deposition parameters include the pressure of 3000 Pa, flow rate of 5xlO ^"6 m ³ /s, plasma generator output of 1200 W and substrate temperature of 800 °C. The resulting thickness of the NCD layer is around 150 nm.

After the deposition, the layer is boiled in a mixture of acids (H ₂ SO ₄ + KNO ₃ , ratio 3:1), rinsed with deionized water and dried with a flow of dry nitrogen. Afterwards, the layer is treated with oxygen r.f. plasma with the output of 300 W for 3 minutes and finally it is terminated with hydrogen in hydrogen plasma at 800 °C for 10 minutes. This procedure yields clean and well-defined surface of diamond, which is terminated with hydrogen and highly hydrophobic (contact angle ~ 90°).

The microscopic templates for the organization of cells on the NCD layer are fabricated using a lithographic mask (optical or electron lithography). The layers are exposed to high-frequency oxygen plasma (300 W) for 3 minutes with this mask, then the mask is rinsed from the surface of diamond using acetone and water, eventually other solvents. After subsequent drying the layer is prepared for cell-seeding.

Prior to seeding, the NCD layers are sterilized with UV irradiation or alcohol for 10 minutes. Silicon substrates with diamond layers are placed at the bottom a cultivation well- plates. Cells are seeded on diamond from a concentrated solution with typical concentrations

of 1000-10000 cells/cm ² and cultivated in McCoy's 5A medium, containing 0-15% fetal bovine serum (FBS) and antibiotics (penicillin, streptomycin). Cells are then cultivated under standard conditions for tissue cultures in an incubator at 37 °C in 5 % CO ₂ for two days.

Examples of structures fabricated using the above-described procedure are 30- to 200- μm-wide oxygen-terminated stripes separated by hydrophobic hydrogen-terminated areas. Cell line of transformed human bone-forming cells (osteoblasts) SAOS-2 was used for the demonstration of the cell organization. The cells preferentially adhered and proliferated on the oxygen-terminated diamond with sharp borders on the H/O interface. Moreover, cells adhered on narrow stripes were organized into chain-like patterns along the stripes.

Industrial applicability

The described method for the making of arranged cell structures can be applied in medicine, pharmacy, biology and electronics. It can also be exploited in tissue engineering, optical, electronic and chemical bio-sensors, bio-stimulators of growth and cell differentiation, bio-electronic signal transmitters, for the "micro-arrays" technology for modern proteomics and genomics, etc.