1. Field of the Invention
The present invention relates to patterned ultra-thin films (UTF) for the selective adhesion and outgrowth of cells and a method for producing such films. The present invention also relates to devices which contain such ultra-thin films, such as body implants or prosthetics, cell culture apparatus, and cell based sensors.
2. Discussion of the Background
It is well known that the morphological and functional development of adherent types of biological cells is critically dependent on, among other factors, the physical and geometrical properties of the underlying substrate. The effects of the substrate adhesivity, in particular, on these developmental processes have been vigorously investigated during recent years. However, substrates which are either unmodified, or completely remodified with only a single type of substrate coating have been used almost exclusively.
In many situations, the ability to influence and/or monitor a variety of intra- and intercellular processes using substrate geometry requires that the adhesive properties of that substrate be defined with a spatial resolution of cellular or subcellular dimensions (10 .mu.m to less than 1 .mu.m). For example, substrate patterns designed to spatially direct the adhesion and outgrowth of cells on the surfaces of sensor devices, prosthetic implants, and tissue repair templates are desired.
Several methods have been devised which might conceivably be used for the fabrication of substrate patterns used in the above-mentioned applications. The earliest reported methods rely on definition of a selectively adhesive substrate pattern by mechanically removing cell-repulsive phospholipid films or evaporated gold from cell-adhesive glass substrates (Ivanova et al, Nature, Vol. 242, p. 200 (1973)); and Cooper et al, Exp. Cell Res., Vol. 103, p. 435, (1976)) or by masked evaporation of cell-adhesive silicon monoxide onto polystyrene surfaces (Albrecht-Buehler, J. Cell Biol., Vol. 80, p. 53 (1980)). However, these methods suffer from drawbacks due to the instability of phospholipid films, the difficulty associated with the physical removal of thin coatings, and the limited number of materials which may be deposited by evaporation. More importantly, these methods are limited by their reliance on the native adhesive properties of unmodified regions of the substrates.
Other methods have been introduced to create substrate patterns which are based on molecular recognition between the cell surface and bulk protein films on the substrate. Hammarback et al. have shown that the outgrowth of dissociated chick embryo dorsal root ganglion neurons occurs on substrates which are defined using patterned UV irradiation to selectively denature cell-adhesive laminin films (Hammarback et al, Jour. Neurosci. Res., Vol. 13, p. 213 (1985)). An alternative method is to adhere neurons to laminin which has been selectively adhered to regions of albumin films which have become crosslinked by patterned UV exposure (Hammarback et al, Devel. Biol., Vol. 117, p. 655 (1986)). Although the development of most neurites is noticeably affected by the substrate patterns, a significant percentage (10-20%) of the plated cells initially adhere to and at least partially develop on the UV-denatured laminin regions.
Recently, pure UTFs of cell adhesion peptides (Arg-Gly-Asp and Try-Ile-Gly-Ser-Arg) have been formed by covalent linkage to silane-modified glass surfaces (Massia et al, Anal. Biochem., vol. 187, p. 292 (1990)), providing a much better defined system for cell adhesion. In this case, the adhesion is affected by known chemical functionalities which are present on the surface as a monolayer.
Silane films are anchored to the silicon substrate by chemical and physical adsorption, which may involve siloxane (Si-O-Si) bridges or van der Waals forces. Any substrate having a terminal ionizable hydroxyl group at the surface can provide an anchorage for the silane film. This procedure of using self-assembling films involves covalent bond formation between the monolayer and the substrate whereby the film adheres to the substrate more strongly than physisorbed Langmuir-Blodgett films.
The potential for producing high resolution patterns of silane-coupled UTFs has been demonstrated by Kleinfield et al., Jour. Neurosci., vol. 8, p. 4098 (1988). In this method, a conventional photoresist is photolithographically patterned and used to mask silicon and quartz substrate regions. The cell adhesivity of the exposed substrate is reduced by formation of a patterned UTF of covalently attached n-tetradecane. Removal of the photoresist and subsequent recoating of the previously masked regions with EDA produces high resolution (10 .mu.m line-space pairings) regions having completely different cell adhesivities.
Photoresist-defined UTFs have been used to very effectively to define both the initial adhesion and outgrowth of a heterogeneous mixture of cells (various types of glial cells and neurons) from the fetal rat cerebellum (Klienfeld et al, Jour. Neurosci., Vol. 8, p. 4098, (1988)). The photoresist-based UTF patterning process is important because it demonstrates that the entire substrate surface may be modified in the same molecular plane with high resolution, alternating UTF films having a desired two-dimensional architecture. However, the technique has significant drawbacks, the most notable of which is the number of steps required for fabrication due to the adhesion, polymerization, development, and stripping of the photoresist (18 steps are reported in Klienfeld et al, Jour. Neurosci., Vol. 8, p. 4098 (1988)).
Another method has been developed recently for the formation of orthogonal UTFs, (Laibinis et al, Science, Vol. 245, p. 845 (1989)). In this method, high resolution monolayer patterns are formed by the selective adsorption of alkanethiols on gold and alkane carboxylic acids on alumina. Selective cell adhesion has not been demonstrated on substrates prepared by this method. However, a large number of chemical functionalities should be compatible with this method, making it a possible fabrication technique for high resolution cell adhesive patterning. A significant limitation of the technique is that only hydrophobic UTF films may be formed on alumina (Laibinis et al, Science, Vol. 245, p. 845 (1989)).
U.S. Pat. No. 4,832,759 describes the use of "surface discontinuities" to at least partially define cell adhesion in zones having a width of between 0.2 and 20 .mu.m. U.S. Pat. Nos. 4,591,570 and 4,011,308 describe the use of patterns or arrays of antibody-coated spots for specific immunoabsorption of cells to optically-sensitive surfaces. U.S. Pat. No. 4,562,157 describes the photo-induced activation of adhered chemical species so that chemical functionalities and proteins may be covalently attached to "BIOCHEMFET" devices. However, this work does not address the problem of nonspecific absorption of proteins.
At least one biosensor has been developed which optically measures the metabolic activity of immobilized cells (Parce et al, Science, Vol. 246, p. 243 (1989)). However, groups of cells, not individual cells, are "immobilized" by gravitational sedimentation into micromachined silicon wells.
U.S. patent applications Ser. Nos. 07/022,439 filed Mar. 6, 1987 and 07/182,123 filed Apr. 14, 1988 disclose a method for preparing high resolution patterns of metals on solid substrates, by irradiation of an adherent thin film with deep ultraviolet (DUV) irradiation. However, there is no suggestion of patterned substrates for the selective adhesion and outgrowth of cells.
Thus, there remains a need for patterned ultra-thin films for the selective adhesion and outgrowth of cells which are free of the above-mentioned drawbacks. There also remains a need for a method producing such films and devices, such as body implants, cell culture apparatus, cell sensors, and neural prostheses, which utilize such ultra-thin films.