METHODS FOR IMMOBILIZING CELLS

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention relates to a method for immobilizing cells on a solid substrate, a novel peptide for cell immobilization, a microarray and a biosensor.

BACKGROUND OF TECHNIQUE

It is a fundamental and important factor to attach animal cells on the surface of solid materials in cell culture and tissue technologies. Cells immobilized by adequate attachment materials are generally proliferated [1, 2]. Extracellular matrix proteins such as collagen (gelatin), fibrin, elastin, fibronectin and laminin, and substances such as poly-L-lysine (PLL) have been used to coat the surface of cell culture vessel [3]. Particularly, gelatin obtained from partial hydrolysis of collagen has been used as a cell immobilization material in stem cell culture as one of intensively researched fields. Because cell death might occur where attachment- dependent animal cells are not bound to ECM, ECM components have been widely utilized in cell culture and tissue technologies. However, the utility of ECM components is associated with serious drawbacks: higher cost and infection likeness due to extraction from other animals [4]. PLL is a cost-effective substance but has the properties of non-specific cell attachment and cytotoxicity. In addition, PLL is considered not to be effective in cell adhesion due to induction of abnormal cell proliferation [3].

Recently, Spatz et a/, reported the formation of cellular microenvironment using a bioactive nanopattern [5]. The nano-unit cellular microenvironment is used in the preparation of a cell chip by regulating cellular activities such as cell migration, differentiation and proliferation. ECM components or PLL molecules could not be effective in enhancement of cell adhesion strength in nanostructures such as nano-

pattern due to their high molecular weight. Therefore, non-polymeric materials with low cytotoxicity and effective cell adhesion potential have been required for development of a nanostructure-fabricated cell chip.

The recent studies to develop cell chips for development of new drug using directly cells on a cell microarray or cellular metabolism-related experiments on chip

(a lab-on-a chip) have been carried out. In cell chips requiring an electrochemical measurement, gold plates are widely used and the adsorption of thiol groups and organic disulfide compounds is known to be generated by the following electrode reaction [12, 13]. M refers to a metal such as gold and silver to form M-S bonds on the surface.

RSH + M → RS-M + H ⁺ + e ^" (M) (M= Au, Ag)

RSSR + e ^" (M) → RS-M + RS ^"

Throughout this application, various publications and patents are referred and citations are provided in parentheses. The disclosures of these publications and patents in their entities are hereby incorporated by references into this application in order to fully describe this invention and the state of the art to which this invention pertains.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have made intensive studies to develop a method for immobilizing a cell, particularly an animal cell on a solid substrate. As results, we have discovered that sequences comprising a Arg-Gly-Asp (RGD) motif permitted cells to be specifically immobilized on the solid substrate without cytotoxicity and on a nano-pattern utilized in a cell chip with cellular microenvironments.

Accordingly, it is an object of this invention to provide a method for immobilizing a cell on a solid substrate.

It is another object of this invention to provide a novel peptide used in cell

immobilization.

It is still another object of this invention to provide a microarray or a biosensor coated by peptide linkers.

Other objects and advantages of the present invention will become apparent from the following detailed description together with the appended claims and drawings.

In one aspect of this invention, there is provided a method for immobilizing a cell on a solid substrate, comprising the steps of:

(a) coating on the solid substrate a peptide which comprises a Arg-Gly-Asp sequence and a cysteine residue at its terminal; and

(b) contacting the cell to the peptide coated on the solid substrate.

The present inventors have made intensive studies to develop a method for immobilizing a cell, particularly an animal cell on a solid substrate. As results, we have discovered that sequences comprising a Arg-Gly-Asp (RGD) motif permitted cells to be specifically immobilized on the solid substrate without cytotoxicity and on a nano-pattern utilized in a cell chip with cellular microenvironments. This invention relates to a method for immobilizing a cell on a solid substrate.

This invention is also described as a method for preparing a cell-immobilized solid substrate.

One of the prominent features of peptides suitable in the present invention is to comprise a RGD motif. According to a preferable embodiment, the present peptide is represented by the following general formula 1 and the cysteine residue is linked to its N-terminal or C-terminal:

(Arg-Gly-Asp) _n (1)

wherein n represents an integer of 1-100.

The exemplified peptide of this invention represented by the general formula 1 is a Cys-Arg-Gly-Asp peptide.

According to a preferable embodiment, the present peptide is represented by 5 the following general formula 2 and the cysteine residue is linked to its N-terminal or C-terminal:

(Arg-Gly-Asp-X _aal ) _n (2) wherein X _aai represents GIy or Ala and n represents an integer of 1-100.

In general formula 2, X _aa i represents an amino acid residue to confer 10 flexibility in the peptide sequence, suitably amino acid residues with small R groups. Preferably, X _aa i represents GIy.

According to a more preferable embodiment, the present peptide shows that the peptides represented by the general formula 1 or 2 are linked to each other by an amino acid linker and the number of the peptides is 2-20. In other words, the

I T) peptides of the present invention have the peptide map construction containing linked peptides.

In preparation of the peptide map, a linker used includes various linkers known to one of skill in the art. The amino acid sequences useful as linkers are described in Maratea et a/., Gene 40:39-46 (1985); Murphy et a/., Proc. Natl. Acad

20 Sc/. USA 83:8258-8562 (1986); US Pat. Nos. 4,935,233, 4,751,180 and 5,990,275.

The linker sequence may comprise amino acid residues of 1-50.

According to a preferable embodiment, the linker used is the amino acid containing an amino-group or a carboxyl group in its R-group, including Lys, Arg, Asp and GIu and most preferably Lys. 5 The size of linkers is preferably 1-5 amino acid residues, more preferably 1-3 and most preferably 1 amino acid residue.

According to a preferable embodiment, the present peptide is represented by the following Formula 3 or 4:

wherein X _aal represents GIy or Ala; nl-n8 independently represent an integer of 1-100.

More preferably, the present peptide is represented by the following sequences:

Most preferably, the present peptide is represented by the following sequence.

The peptides used in the present invention play a role as a linker to immobilize cells on the solid substrate and the peptide linkers form on the substrate a self-assembled monolayer (SAMs) to allow cells to be more conveniently immobilized on the solid substrate.

The solid substrate useful in the present invention includes various substrates known to those of skill in art. For example, the solid substrate includes, but not limited to, metal {e.g., gold, an alloy of gold and copper, aluminium), metal oxide, glass, ceramic product, quartz, silicon, semi-conductor, Si/SiO ₂ wafer, germanium, gallium arsenide, carbon, carbon nanotube, polymer {e.g., polystyrene, polyethylene, polypropylene, polyacrylamide), cephalose, agarose and colloid.

Most preferably, the solid substrate has a surface coated by gold.

Cells applied to the present invention include preferably animal cells containing the integrin proteins on its surface and most preferably human cells. In addition, it includes a primary cultured cell, an established cell, a cancer cell and a stem cell.

In another aspect to this invention, there is provided a peptide represented by the following Formula 3 or 4:

; or

wherein X _aa i represents GIy or Ala; nl-n8 independently represent an integer of 1-100. More preferably, the present peptide is represented by the following sequences:

In still another aspect of this invention, there is provided a microarray or a biosensor comprising the solid substrate coated by the present peptide.

The general description of the present microarray {i.e., cell chip) can be found in US Pat. Nos. 5,143,854, 5,242,974, 5,252,743, 5,324,633, 5,384,261, 5,405,783, 5,412,087, 5,424,186, 5,451,683, 5,482,867, 5,491,074, 5,527,681, 5,550,215, 5,571,639, 5,578,832, 5,593,839, 5,599,695, 5,624,711, 5,631,734, 5,795,716,

5,831,070, 5,837,832, 5,856,101, 5,858,659, 5,889,165, 5,936,324, 5,959,098, 5,968,740, 5,974,164, 5,981,185, 5,981,956, 6,025,601, 6,033,860, 6,040,193, 6,090,555, 6,136,269, 6,147,205, 6,262,216, 6,269,846, 6,310,189 and 6,428,752.

The general descriptions of the present biosensor can be found in Myska, J. MoI. Recognit, 12:279-284(1999); J. Dubendorfer et a/. Journal of Biomedical Optics, 2(4):391-400(1997); O'Brien eta/. Biosensors & Bioelectronics, 14:145-154(1999).

According to the present invention, cells particularly animal cells are effectively immobilized on the solid substrate without cytotoxicity in a more specific manner. Interestingly, the present invention permits cells to be immobilized on nano- patterns utilized in cell chips with cellular microenvironments.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 schematically represents the constructions of the RGD peptide maps for cell immobilization used in this invention.

Fig. 2 represents the analysis results of the surface of the gold substrates modified with the present peptides. The surface analysis was carried out using an atomic force microscope (AFM). The analysis range was 500 nm x 500 nm and the scanning was processed at scan speed of 1 Hz. (a) bare gold substrate, (b)-(d) C-

RGD, [C-RGD] _n -MAP and [RGD] _n -MAP-C-treated gold substrates, respectively.

Fig. 3 represents the cellular morphology of stem cells cultured on gold substrate (x40). Bare gold substrate (left), [RGD] _π -MAP-C-treated gold substrate (center) and gelatin-treated gold substrate (right).

Fig. 4 represents the effect of the present peptides on cell proliferation.

The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples.

EXAMPLES

EXAMPLE 1: Design of [RGD]n-MAP-C as Novel RGD Peptide Maps

As shown in Fig. 1, three RGD peptides were used in the present invention. The C-RGD in Fig. 1 is the structure with a single RGD sequence which was used as cell adhesion materials due to the properties of RGD motif enabling to combine cells with ECM proteins through recognition of integrins. The symbol "C" of C-RGD represents a cysteine residue with a thiol-functionalized group and additionally introduced into the RGD sequence for binding of materials on the gold substrate.

To prepare RGD peptides for immobilizing cells more effectively, the present inventors have designed the structure containing several RGD sequences in one peptide map.

Because the thiol-functionalized group was required to link the peptide map on the gold substrate, two types of an additional cysteine-introduction site were designed. [(C-RGD-G)n]n-MAP is a simple map structure composed of several C- RGD-Gs and stably immobilized onto the gold substrate by cysteine residues at the terminal of each RGD sequence. However, there was a probability that the RGD motif was hidden inside its structure. The [(RGD-G)n]n-MAP-C was prepared by linking several RGD motifs to each other in one MAP structure and additionally introducing a cysteine residue at the end of the linkage structure.

All peptides as used herein were synthesized in Peptron Inc. (Korea).

EXAMPLE 2: Analysis of Peptide Immobilization and Peptide-Modified Gold Surface

The gold substrate was prepared using a DC magnetron sputtering method and a silicon substrate. Cromium (Cr) was deposited as a mediator prior to deposition of gold. The thickness of gold and chromium depositions was 43 nm and 2 nm, respectively. To remove contaminants on the substrate before preparation of the peptide thin film, the gold surface was washed with piranha solution (70% vol H ₂ SO ₄ , 30% H ₂ O ₂ ). After the gold surface was treated with a peptide solution (0.1

mg/ml) for 12 hrs, it was washed with PBS (phosphate buffered saline) and dried using N ₂ gas, finally yielding a peptide thin film on the substrate. To analyze the surface of the peptide thin film, the gold substrate and its surface modified with C- RGD, [C-RGDJn-MAP or [RGDJn-MAP-C were observed under AFM (atomic force microscopy, NTEGRA spectra, NT-MDT, Russia). Their AFM images were obtained in a semi-contact mode and at scan speed of 1 Hz at room temperature.

The AFM analysis results showed that untreated bare substrates had an average height of 12 nm but the height of peptide-treated gold substrates were increased. Particularly, the modification with the peptides containing the map structures contributed to higher increase in heights of substrates than that of peptides comprising one C-RGD sequence. It could be appreciated that [(C-RGD- G)n]n-MAP was most stably linked to substrates in the peptides with map structures.

EXAMPLE 3: Cell Culture and Cell Proliferation Analysis After each new-designed peptide was linked onto the gold substrate, animal cells [cancer cells (HeLa, purchased from ATCC) and stem cells (mouse embryonic cell, Jl ES cell, kindly provided by Professor Jung Ho Kim of Sogang University, 14)] were cultured. Gelatin coating has been generally used in animal cell dishes because the adhesion potential of stem cells was reduced in culture without feeder cells. Gelatin has been obtained from partial hydrolysis of an ECM protein (collagen) and not suitable in nano-unit microcellular environments due to its high molecular weight. For the preparation of a nano-pattem cell chip for stem cells, cell adhesion and cell proliferation after culture were compared to investigate whether the present RGD peptide maps were utilized as a substitutive material of gelatin. Fig. 3 represents images of mouse stem cells under a microscope observed on

3 day post-culture on the gold substrate. The stem cells cultured on the [RGDJn- MAP-C peptide-treated gold substrate exhibited proliferation potential at the same

level as those cultured according to a general gelatin coating method. The difference of cell morphology was also not shown in the two culture methods.

These results demonstrated that the present peptides as a substitutive of gelatin could be used in nanopatterns on gold substrates for cell chips. Fig. 4 represents the results of MTT assay. Cancer cells (HeLa) were cultured for 2 days either on bare gold substrates or on substrates modified with three types of RGD-series peptides and their cell proliferations were compared according to a MTT assay (Farokhzad, O. C; Cheng, 1; Teply, B. A.; Sherifi, L; Jon, S.; Kantoff, P. W.; Richie, J. P.; Langer, R. Proc. Natl. Acad. Sc/. USA 2004, 103 (16) 6315-6320). The cell proliferation potentials on substrates modified with peptides containing one CRGD sequence and [RGD]n-MAP-C peptide were analyzed to be 2.5- fold higher and over 4-fold higher, respectively than those on bare gold substrates. For the [C-RGDJn-MAP peptide elucidated to have uniform and stable linking abilities in the surface analysis, the cell proliferation was decreased compared to peptides containing one CRGD sequence. It would be expected that RGD sequences might be hidden inside the [C-RGDJn-MAP structure.

The RGD-series peptides of the present invention, particularly [RGDJn-MAP-C enables to be self-assembled on the gold substrate through a thiol group of a cysteine resiude and exhibit the enlarged effect of cell proliferation rate in animal cell (including stem cell) culture compared with a bare gold substrate. The low molecular weighted RGD-motif peptides of this invention to substitute for macromolecular ECM components can be utilized for the surface modifications of nano-patterns. The gold electrodes modified with the present peptides can be applied to an electrochemical or electrical detection system.

References

[IJ M. Tirrell, E. Kokkoli, M. Biesalski, Surf. ScL 500 (2002) 61.

[2] A. Bershadsky, A. Chausovsky, E. Becker, A. Lyubimova, B. Geiger, Curr. Biol. 6

(1996) 1279.

[3] U. Hersel, C. Dahmen, H. Kessler, Biomaterials Tλ (2003) 4385.

[4] CS. Chen, J. L. Alonso, E. Ostuni, G.M. Whitesides, D.E. Ingber, Biochem. Bioph. Res. Co. 307 (2003) 355.

[5] Spatz JP and Geiger B. Methods Cell Biol. 83 (2007) 89.

[6] S.M. Cutler, AJ. Garcia, Biomaterials 24 (2003) 1759.

[7] J. H. Jang, J. H. Hwang, CP. Chung, Biotechnol. Lett. 26 (2004) 1831.

[8] U. Staubli, D. Chun, G. Lynch, J. Neurosd. 18 (1998) 3460. [9] M. D. Pierschbacher, E. Ruoslahti, Nature 309 (1984) 30.

[10] R.A. Quirk, W.C Chan, M.C. Davies, SJ. B. Tendler, K.M. Shakessheff,

Biomaterials 22 (2001) 865

[11] Y.M. Bae, B.-K. Oh, W. Lee, W.H. Lee, J.-W. Choi, Materials Science and

Engineering C 24 (2004) 61 [12] S. Eu and W. Paik, Chem Lett. 337 (1998) 405

[13] W. Paik, SEu, K. Lee, S. Chon, and M. Kim, Langmuir 16 (2000) 10198

[14] Lee, J., Kim, H. K., Rho, J-Y., Han, Y-M., and J. Kim, J. Biol. Chem. 281(2006)

33554

Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.