EP0277993A4

EP0277993A4 - Promoting cell adhesion and growth on a substrate.

Info

Publication number: EP0277993A4
Application number: EP19870905567
Authority: EP
Inventors: Brian Richard Mcauslan; Garry Noel Hannan
Original assignee: Commonwealth Scientific and Industrial Research Organization CSIRO; Telectronics Pty Ltd
Current assignee: Commonwealth Scientific and Industrial Research Organization CSIRO; Telectronics Pty Ltd
Priority date: 1986-08-15
Filing date: 1987-08-17
Publication date: 1989-01-19
Also published as: EP0277993A1; JPH01500641A; WO1988001279A1

Abstract

A surface for the growth of cells is prepared by immobilizing an indole selected from the group containing serotonin, tryptamine, L-tryptophan and analogues thereof to an appropriate substrate and then exposing the indole to adhesive serum proteins whereby the indole adsorbs the proteins to form an immobilized indole-protein complex for the growth of cells. The surface may also be used as a human tissue implant.

Description

PROMOTING CELL ADHESION AND GROWTH ON A SUBSTRATE

FIELD OF INVENTION

This invention relates to a surface for the attachment and growth of cells, processes for preparing said surface, and methods of promoting cell adhesion and growth on said surface. BACKGROUND ART

The proliferation of many types of cells such as mammalian cells only takes place if there is a compatible surface on which the cells will adhere and spread. The compatibility of a culture surface is believed to be related to its ability to adsorb specific adhesive proteins from the serum component of the growth medium. A variety of chemically unrelated substances, including proteins, plastics, polysaccharides, metals and ceramics will support cell growth in culture, and in most cases it seems that cell attachment and growth is mediated by the adhesive protein, fibronectin, being adsorbed onto the substrate. Another serum protein, vitronectin or serum spreading factor, has also been proposed as a major promotor of cell adhesion in routine tissue culture.

However, adhesive proteins such as fibronectin, fibronectin-fragments and vitronectin are recognised as being relatively unstable when bound to substrate, due to their molecular complexity and structure-function specificity.

Careful matching of these proteins with the cells or tissue to be produced, is required to ensure successful attachment and growth of such cells on the prepared surface.

It has now been found by the present inventors that under appropriate conditions, indoles selected from the group containing serotonin, tryptamine, L-tryptophan and analogues thereof, adsorb from serum containing culture media adhesive proteins such as vitronectin, fibronectin or fibronectin fragments having a strong affinity for a variety of cells. Up to this discovery, serotonin (5-hydroxytryptamine) was believed to interact strongly with glycoconjugates only. In fact, when serotonin is immobilized by covalent coupling to agarose beads, it has a high affinity for glycoproteins and glycopeptides, and acts as an adhesive surface for growth of mammalian cells. The present inventors have now found that serotonin and other indole analogues adsorb serum proteins to provide a functionally stable complex, which complex has a strong affinity for a variety of cells. This property of the indole group to adsorb adhesive serum proteins was not recognised prior to the work of the present inventors.

DISCLOSURE OF INVENTION

Accordingly, it is an object of this invention to provide a surface for the growth of cells which surface has improved capacity for cell growth arising from enhanced serum protein-specific binding properties.

According to the invention there is provided a surface for the growth of cells, said surface comprising an adhesive serum protein adsorbed to an indole selected from the group containing serotonin, tryptamine, L-tryptophan and analogues thereof, said indole-protein complex being immobilized to an appropriate substrate. The surface may optionally include adherent cells, which effectively act as a starter culture to facilitate the further growth of cells on such surfaces.

The invention also provides a process for preparing a surface for the adhesion and growth of cells, said process comprising immobilizing an indole selected from the group containing serotonin, tryptamine, L-tryptophan and analogues thereof to an appropriate substrate and exposing adhesive serum proteins thereto, whereupon said indole adsorbs said proteins to form an immobilized indole-protein complex for the growth of cells. The surface so prepared may optionally be exposed to a predetermined concentration of cells to provide a starter culture for the subsequent adhesion and growth of cells.

According to another aspect of the invention there is provided a method of promoting cell adhesion and growth on a substrate, said method comprising exposing cells to a surface comprising an adhesive serum protein adsorbed to an indole selected from the group containing serotonin, tryptamine, L-tryptophan and analogues thereof, said indole-protein complex being immobilized to an appropriate substrate. Consistent with the common element of novelty of the invention, the invention also provides a method of promoting cell adhesion and growth on a surface, which method comprises a) providing a surface comprising an indole selected from the group containing serotonin, tryptamine, L-tryptophan and analogues thereof, immobilized to an appropriate substrate, and, b) exposing said surface to a medium containing . biological cells and adhesive proteins. It will be appreciated by persons skilled in the art that the surfaces and methods of the invention may be used both in vitro and in vivo. For instance, the surfaces and methods of the invention are likely to find application wherever surfaces need to be populated by adherent cells. The possibility of derivitizing the indoles of the invention onto agarose and other substrates opens the way to applying the invention to provide bioimplants such as vascular grafts, where there is a requirement for good surface repopulation by host or donor cells.

Accordingly, in another aspect of this invention, there is provided a human tissue implant incorporating the novel surfaces of the invention. The substrate used in producing the surface may be any polymer, ceramic or metal considered appropriate by persons skilled in the art. For the sake of example, these polymers include agarose, collagen, polyvinyl alcohol, polyester, polytetrafluoroethylene, polyurethane, silicone rubber and polyhydroxyethyl methacrylate, and any other polymers having free -OH groups or free -SH groups which present sites for covalent coupling. Examples of ceramic substrates include alumina, partially stabilized zirconium, carbon and silicon nitride. Examples of metallic substrates include platinum, titanium and iridium. It is to be understood that the substrate is not to be restricted to the examples specified herein, and that other substrates may be used appropriately. Immobilization of indole onto substrate can occur by covalent coupling or radiation grafting techniques (including UV radiation grafting) commonly practiced in the art. Covalent coupling is preferably carried out in the presence of cyanogen bromide. The adhesive serum proteins most commonly used are fibronectin, vitronectin, and fibronectin-fragments. DESCRIPTION OF PREFERRED EMBODIMENTS

•The invention will now be described by reference to a specific example. EXAMPLE

CELL CULTURE AND LABELLING A clonal line of normal bovine aortal endothelial (BAE) cells and chick embryo fibroblasts (CEF) were grown and maintained as described by Hannan and McAuslan (Biochem. Int. 6 (1983) 375). Baby hamster kidney (BHK) cells were maintained in a Glasgow modification of minimal essential medium (GMEM) supplemented with 10% fetal calf serum (FCS). Bovine smooth muscle (BSM) cells were maintained as described by Hannan and McAuslan (Exp. Cell. Res. In press (1987)).

Cells were seeded in plastic tissue culture dishes or seeded in spinner flasks at 10⁵ cells/ml with modified agarose beads at 1 g/100 ml.

PREPARATION AND USE OF IMMOBILIZED LIGANDS Serotonin-agarose was prepared using the method described by Hannan, Redmon and McAuslan (Biochim. Biophys. Acta 801 (1984) 396) using either cyanogen bromide (CNBr) activated Sepharose 4B (Pharmacia) or activated CH-Sepharose 48 (Pharmacia). Essentially, the same procedures were used to couple tryptamine and L-tryptophan to cyanogen bromide activated Sepharose 4B. Ethanolamine-agarose and fibronectin-agarose were prepared by essentially the same procedures except that 1M ethanolamine or 3mg/ml purified bovine plasma fibronectin was substituted as the ligand.

Cells grown according to the above procedure were inoculated at 10 cells per ml into spinner flasks containing stirred suspensions of ligands immobilized on agarose beads in 10% FCS GMEN and grown at 37°C. Aliquots were ^" periodically removed to monitor cell attachment and growth on the beads by the following staining procedure. VITAL STAINING OF ADHERENT CELLS For the purposes of identifying cell morphology, BHK cells grown for 4 hours and BHK cells grown continuously to moderate density for 24 hours on serotonin-agarose beads were fixed and stained with acridine orange and examined by fluoroscence microscopy at 450 n . Cells were clearly delineated as bright red shapes, which is typical of cytoplasmic staining with this dye, against a pale green background of the uncovered bead surface. At low magnification serotonin-agarose beads could be observed to be in various stages of population by BHK cells, and at high magnification individual cells and those in contact with neighbours in the monolayer could be clearly delineated. This technique was used effectively to monitor cell attachment and growth of all the cell types investigated on the various surfaces. Additionally, other techniques such as phase contrast microscopy and scanning electron microscopy were used to monitor cell adhesion and growth. The results found by the above three techniques were comparable.

RESULTS

Cell Adhesion and Growth Kinetics on Immobilized Serotonin, Tryptamine and L-tryptophan

Early cell attachment and growth was investigated using both BAE cells and BHK cells inoculated into growth medium containing serotonin-agarose beads. It was found that these cells attached readily to the surface of these beads during the first few hours. In contrast, when the above cells were challenged with agarose beads to which the simple amine ethanolamine had been coupled, no cell attachment and growth occurred. By 24 hours BAE cells, BSM cells and BHK cells were observed to grow equally well on serotonin-agarose, tryptamine-agarose or L-tryptophan-agarose as on fibronectin- agarose. Following the growth of BHK cells and BAE cells further, it was found that by 48 hours BHK cells had grown to __fl_

populate the entire surface of the beads in a monolayer having the morphology characteristic of a fibroblast culture.

Quantitative assessment of the degree of attachment of BAE cells, BSM cells and BHK cells to each immobilized ligand over 24 hours of culture (see Table 1) confirmed that all these cell types seeded onto tryptamine-agarose, L-tryptophan-agarose or serotonin-agarose reached cell densities comparable with optimal growth on fibronectin- agarose. Results of the growth of CEF cells on the immobilized ligands of.Table 1 are not shown. BAE cells, BSM cells, CEF cells and BHK cells seeded onto agarose derivatized with ethanolamine did not attach or grow at all, even after 3 days.

The kinetics of cell growth was investigated using BHK cells grown on the various substrates. In contrast to the absence of cell attachment and growth on immobilized ethanolamine, BHK cells attached readily to both serotonin- agarose and fibronectin-agarose beads over the first few hours and grew well to reach a high density by three days. However, the initial rate of attachment of cells to serotonin-agarose beads was slower than for fibronectin- agarose beads. Comparable rates were achieved between the above two surfaces after 6 hours. Cells grown on tryptamine- agarose and L-tryptophan-agarose beads also displayed an improved rate of attachment and growth over time, comparable to that of serotonin, in keeping with the similarity of their structure as indole analogues.

By successfully growing a number of mammalian cell types on a variety of immobilized indole analogues in the presence of adhesive proteins in vitro, we have shown that these novel surfaces may be used to provide improved methods for the promotion of cell attachment and growth. This feature may be readily adapted for use in vivo and, as will be readily apparent to persons skilled in the art, will find particular application in the provision of bioimplants such as vascular grafts.

The foregoing describes only some embodiments of the present invention and modifications, obvious to those skilled in the art, may be made without departing from the scope and ambit of the invention.

TABLE 1

ATTACHMENT AND GROWTH OF CELLS TO IMMOBILIZED LIGANDS

(a) Numbers of cells attached were expressed as percentage values relative to numbers attached to fibronectin-agarose beads (5 x 10⁴ cells/ml at 4 hours and 3 x 10⁵ cells/ml at 24 hours). The results were the mean values of three determinations and errors were less than 8%.

BAE: Bovine aortal endothelial cells BSM: Bovine smooth muscle cells BHK: Baby hamster kidney cells

Claims

1. A surface for promoting cell adhesion and growth, said surface comprising an adhesive serum protein adsorbed to an indole selected from the group containing serotonin, tryptamine, L-tryptophan and analogues thereof, said indole- protein complex being immobilized to an appropriate substrate.

2. A surface according to claim 1 and further including adhered cells.

3. A surface according to claim 1 or claim 2 wherein said adhesive serum protein is selected from the group comprising fibronectin, vitronectin and fibronectin-fragments.

4. A- surface according to any one of claims 1 to 3 wherein said substrate is a polymer.

5. A surface according to claim 4 wherein said polymer is selected from the group of polymers having free -OH groups or free -SH groups.

6. A surface according to any one of claims 1 to 3 wherein said substrate is selected from the group comprising _.agarose, collagen, polyvinyl alcohol, polyester, polytetrafluoroethylene, polyurethane, silicone rubber and poly(hydroxyethyl methacrylate).

7. A surface according to any one of claims 1 to 3 wherein said substrate is a ceramic.

8. A surface according to claim 7 wherein said ceramic is selected from the group containing alumina, partially stabilized zirconium, carbon and silicon nitride.

9. A surface according to any one of claims 1 to 3 wherein said substrate is a metal.

10. A surface according to claim 9 wherein said metal is selected from the group containing platinum, titanium and iridiu .

11. A human tissue implant incorporating the surface of any one of claims 1 to 10.

12. A process for preparing a surface for the adhesion and growth of cells, said process comprising:- a) immobilizing an indole selected from the group containing serotonin, tryptamine, .L-tryptophan and analogues thereof to an appropriate substrate, and, b) exposing adhesive serum proteins thereto, whereupon said indole adsorbs said proteins to form an immobilized indole-protein complex for the growth of cells

13. A process according to claim 12 and further including the step of exposing said immobilized indole-protein complex to a predetermined concentration of cells, whereupon said cells adhere to said surface to form an adhesive surface for the growth of cells.

14. A process according to claim 12 or claim 13 wherein said indoles are immobilized by covalent coupling to an appropriate substrate.

15. A process according to claim 14 wherein said covalent coupling is carried out in the presence of cyanogen bromide. 16. A process according to claim 12 or claim 13 wherein said indoles are immobilized by radiation grafting to an appropriate substrate.

17. A human tissue implant whenever prepared by a process of any one of claims 12 to 16.

18. A method of promoting cell adhesion and growth on a surface, which method comprises exposing cells to a surface defined according to any one of claims 1 to 11.

19. A method of promoting cell adhesion and growth on a surface, which method comprises:- a) providing a surface comprising an indole selected from the group containing serotonin, tryptamine, L-tryptophan and analogues thereof immobilized to an appropriate substrate, and, b) exposing said surface to a medium containing biological cells and adhesive proteins.

20. A method according to claim 19 said method being substantially as hereinbefore described with reference to the example.