Method of forming a cell pattern on a surface

Details Method of forming a cell pattern on a surface Method of forming a cell pattern on a surface

2001-10-19

2004-09-07

Naff, David M. (Department: 1651)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Solid support and method of culturing cells on said solid...

C424S423000, C435S177000, C435S178000, C435S180000

Reexamination Certificate

active

06787358

ABSTRACT:

BACKGROUND OF THE INVENTION
In order to be able to study the functions of cells of various types so that their behaviour and spatial organisation in association with other cells of the same type can be better understood, it is necessary to be able to culture the cells under precisely controlled conditions. For a couple of years attempts have been undertaken to culture and grow cells on prepatterned substrates which guide the cell growth along the patterns on this substrate. This was done with the hindsight that, one day, one should be able to thereby build miniature biological electronic devices, incorporating live cells to make up a biological microcircuit. Another long-term goal of these studies is the capability of making artificial tissues suitable for implanting into an organism's body, thereby possibly replacing natural tissue of the same kind which is malfunctioning. A third aim of these studies is to be able to facilitate the integration of transplants and/or implants by “masking” the outer parts of these devices with a special array of cells which by their chemical and immunological nature as well as by their arrangement fit into the organism's body at the site in which the device is to be introduced.
One way of achieving cultures of cells to that extent, i.e. cultures which show a specific intended special pattern is to grow the cells along surfaces on which, previously, patterns of a “guiding” molecule which promotes cell growth have been created, and where there are regions which do not promote cell growth. Various cell growth promoting molecules have been used:
Mrksich et al. (1996, PNAS USA, 93, 10775-10778;1997, Exp. Cell Res., 235, 305-313) used alkanethiolate patterns on gold to control cell attachment to these substrates. By choosing an appropriately terminated alkanethiolate they succeeded in creating regions of cell growth promotion and cell growth inhibition. Corey et al. (1991, J. Neurosc. Res., 30, 300-307) managed to pattern neurons on polylysine-coated glass cover slips patterned by selective laser ablation so as to leave grids of polylysine with varying line widths, intrasection distances and nodal diameters.
Matsuzawa et al. (1996, J. Neurosci. Meth., 69, 189-196) chemically attached a synthetic peptide derived from a neurite-outgrowth-promoting domain of the B2 chain of laminin.
Others immobilised various other peptides (Matsuda et al., 1990, Trans. Am. Soc. Artif. Int. Organs; 36 (3): M559-63), or extracellular matrix proteins (ECM) such as laminin (Klein et al., 1999, J. Mat. Sci.: Mat. in Med.; 10: 721-727).
Various techniques for attaching and patterning biomolecules on a surface have been used, including crosslinkers (Clemence et al., 1985, Bioconjugated Chem. 6: 411-417), silane coupling agents (Plueddemann E. 2 edn New York Plenum Press, 1991: 1-250), amongst others. One recently and successfully applied technique to attach proteins in a specific pattern to a substrate is the so-called microcontact printing technique. It is comparatively simple and universal for patterning biomolecules (Kumar et al., 1993, Appl. Phys. Lett., 63 (14), 2002-2004). In this technique a stamp is produced by casting a silicon elastomer (polydimethyl siloxane, PDMS) in the desired pattern which is then coated with a solution of the biomolecule to be transferred. After contacting the “inked” stamp with the substrate surface the bio-molecules self-assemble in the pre-given pattern. Kumar et al. and Mrksich et al. developed this method of producing patterns by stamping alkane thiols on gold substrates (Mrksich et al. 1996, PNAS USA, 93, 10775-10778, Mrksich et al. 1997, Exp. Cell. Res. 235, 305-313). Poly-D-lysine and laminin have been immobilised using microcontact printing on amino silane derivatised glass substrates with glutaraldehyde as a cross linker (Branch et al. 1998, Med. Biol. Eng. Comput., 36, 135-141) and sulfo-GMBS (Wheeler et al. 1999, J. Biomech. Eng., 121, 73-78), and the technique of microcontact printing has been used in neuronal cell guidance (Wheeler et al. 1999, ibid.; Branch et al. 2000, IEEE Transact. Biomed. Eng., 47, 3, 290-300).
All of the aforementioned studies used dissociated cell cultures, mainly of neural origin and achieved successful pattern formation only in some cases. It is not clear, however, whether the patterns of cells thus formed do represent a true picture as it would appear in nature nor whether they are of any use, e.g. for bioelectronic devices. Therefore, the conclusions to be drawn from these studies, e.g. in respect of the spatial arrangement of cells within an organ or the interactions between cells within an organ are only of limited use. Likewise, if one looks at current bioelectronic interface devices and cell modified interfaces there is a problem of reproducibility in creating these devices. It is still not possible to fully control and guide cell attachment and growth on surfaces. Since with current bioelectronic interface devices and cell modified interfaces, the cells are being cultured directly onto the surface of these devices, there is no guarantee that growth on every device will be successful, and therefore a lot of devices and a lot of starter cultures are required just to ensure that some substrates, after culturing, may actually display a cellular network which is useful. A related problem concerning implants is that these are often only of limited biocompatibility due to their bad integration, a rejection by the host or simply the toxicity of the substrates. Lining them with a pattern of cells which mimic the spatial organisation of cells within an organ would certainly enhance the biocompatibility of implants.
OBJECTS AND SUMMARY OF THE INVENTION
Accordingly, one object of the present invention is to be able to control and guide cell growth on surfaces in a precise and hitherto unheard of manner. Another object of the present invention is to be able to limit the efforts in producing bioelectronic devices by reducing the number of starter cultures/substrates that will give a successful device. Another object of the present invention is to enhance that biocompatibility of implants and transplants.
The object is solved by
a method of forming a pattern of cells on a surface, said surface being prepatterned in having a pattern of cell-growth promoting molecules and/or cell-growth inhibiting molecules attached thereon, characterised in that cells are cultured on said prepatterned surface such that they form a pattern of cells on said surface, said cells being whole tissue.
Preferably said whole tissue is derived from an organism's body.
In one embodiment said whole tissue is derived from an organ selected from the group comprising brain, liver, kidney, muscle, skin, bone, lung and heart.
It is preferred that said cells are organ slices.
These organ slices are preferably organotypic in that they mimic the arrangement of cells within an organ.
Preferably said cells are brain slices.
In one embodiment said pattern of cell-growth promoting molecules and/or cell-growth inhibiting molecules attached on said prepatterned surface, allows for the guided growth and migration of cells, wherein preferably, said pattern of cell-growth promoting molecules and/or cell-growth inhibiting molecules mimics the arrangement of cells in an organ.
It is preferred that said pattern of cell-growth promoting molecules and/or cell-growth inhibiting molecules has a structure with lines and nodes, wherein preferably, said lines have a width in the range from 1-8 micrometers and said nodes have a diameter in the range from 1-30 micrometers, more preferably, said lines have a width in the range from 1-6 micrometers and said nodes have a diameter in the range from 8-16 micrometers, and most preferably, said lines have a width in the range from 2-4 micrometers and said nodes have a diameter in the range from 10-14 micrometers.
In one embodiment said pattern of cell-growth promoting molecules and/or cell-growth inhibiting molecules is formed by at least one layer of a substance selected from the group comprising polypeptide,

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