AU592670B2 - Promoting cell adhesion and growth on a substrate - Google Patents

Description

AU-AI-78711/87 Prr WORLD INTELLECTUAL PR -Y AZriO J7 6 7 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 4 (11) International Publication Number: WO 88/ 01279 71 217/06,17/14, C12N 11/06 A (43) International Publication Date: 25 February 1988 (25.02.88) A61L 27/00 (21) International Application Number: PCT/AU87/00266 (74) Agents: MAXWELL, Peter, Francis et al.: Halford Maxwell, Level 20, National Mutual Centre, 44 Mar- (22) International Filing Date: 17 August 1987 (17.08.87) ket Street, Sydney, NSW 2000 (AU), (31) Priority Application Number: PH 7461 (81) Designated States: AT (European patent), AU, BE (European patent), CH (European patent), DE (Euro- (32) Priority Date: 15 August 1986 (15.08,86) pean patent), FR (European patent), GB (European patent), IT (European patent), JP, LU (European pa- (33) Priority Country: AU tent), NL (European patent), SE (European patent),

US.

(71) Applicants (for all designated States except US): COM- MONWEALTH SCIENTIFIC INDUSTRIAL RE- Published SEARCH ORGANISATION [AU/AU]; Limestone With international search report.

Avenue, Campbell, ACT 2601 TELECTRON- Before the expiration of the time limit for amending the ICS PTY, LIMITED [AU/AU]; 14 Mars Road, Lane claims and to be republished in the event of the receipt Cove, NSW 2066 of amendments, (72) Inventors; and A.O.J.P 3 1 MAR 1988 Inventors/Applicants (for US only) McAUSLAN, Brian, Richard [AU/AU]; 83 Hudson Parade, Clareville, AUSTRALIAN NSW 2107 HANNAN, Garry, Noel [AU/AU]; 32 Earl Street, Boronia Park, NSW 2111 8 MA 1988 PATENT OFFICE his documnnt L l i: (54) Title: PROMOTING CELL ADHESION AND GROWTH ON A SUBSTRATE cj(Ill 49 und IS c&r.ct iu ,ill ting.

(57) 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.

L ,WO 8801279 PCT/AU87/00266 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.

SHowever, 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 WO 88/01279 PCT/AU87/00,266 -2to 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 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 L

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WO 88/01279 PCT/AU87/00266 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 WO 88/01279 PCT/AU87/00266 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 WO 88/01279 PCT/AU87/00266 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 r

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WO 88/01279 PCT/AU87/00266 -6at 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-agc.rose 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 105 cells per ml into spinner flasks containing stirred suspensions of ligands immobilized on agarose beads in 10% FCS GMEN and grown at 37 0 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 nm. 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 WO 88/01279 PCT/AU87/00266 -7magnification 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 growt.. 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 fibronectinagarose. Following the growth of BHK cells and BAE cells further, it was found that by 48 hours BHK cells had grown to i WO 88/01279 PCT/AU87/00266 -8populate 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 fibronectinagarose. 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 serotoninagarose 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 fibronectinagarose beads. Comparable rates were achieved between the above two surfaces after 6 hours. Cells grown on tryptamineagarose and L-tryptophan-agarose beads also displayed an improved rate of attachment and growth over time, comparable SWO 88/01279 PCT/AU87/00266 -9to 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 'Che scope and ambit of the invention.

i; WO 88/01279 PCT/AU87/00266' TABLE 1 ATTACHMENT AND GROWTH OF CELLS TO IMMOBILIZED LIGANDS Percent adherent cells (a) BAE BSM BHK Ligand 24hrs 24hrs 4hrs 24hrs 1. Fibronectin 100 100 100 100 2. Serotonin 100 100 100 100 3. Tryptamine 96 95 95 100 4. L-Tryptophan 94 90 78 88 Ethanolamine 0 0 0 0 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:

BSM:

BHK:

Bovine aortal endothelial cells Bovine smooth muscle cells Baby hamster kidney cells

Claims (21)

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 of the indole-protein complex being immobilized to an appropriate substrate.

2. A surface according to claim 1 and further including cells adhered to the adhesive serum protein. *see*:

3. A surface according to claim 1 or claim 2 wherein said adhesive serum protein is selected from the group comprising 6: fibronectin, vitronectin and fibronectin-fragments.

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

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 V: ~v WO 88/01279 -12- PCT/AU87/00266 stabilized zirconium, carbon and silicon nitride.

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

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

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

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.

A process according to claim 14 wherein said covalent coupling is carried out in the presence of cyanogen bromide. 2 13-

16. A process according to claim 12 or claim 13 wherein said indoles ave 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 18 and claim 19 wherein the cells to be grown are selected from a group consisting of endothelial, epithelial, muscle and kidney cells.

21. A method according to claim 19 said method being substantially as hereinbefore described with reference to the a a example. i !li i INTERNATIONAL SEARCH REPORT l~IAWMationl Aaelicalg, He PCT/AU 87/00266 I. CLASSIFICArioap Of SUIJICT MATER :I :IS10"CISOr lweo@ I Paalj. Aoa:j: aJWI AgI*Mgtd to Interational Pgil.vm Cle..e(au.. (IpCi 0# 1 gmew NOIjqiAl C1iacah64tesn an. INC Int. C1. C07K 17/06, 17/14, C12N 11/06, A6:L 27/00 I. FIELDS 1ANCHIO lJAium Oatum~n~al~ei. DERWENT WPI AND WPIL DATA BASE ON LINE KEYWORD SEARCH 04wmeat4cer 5I41feied dien lher IA, mum Oaium.DlOM4t4I to i It M tha tucA OecumRatm are Included SR 0im t iel 486Cflhd AU C07K 17/06, 17/08, 17/14, C12N 11/06, 11/08, 11/14, A61L 27/00, C12M 3/00, 3/04, 3/02 Ill. DOCUMINTS CONSIDIAKID TO E RILIVAN41I Cter181 t Citation of Ocunient. MWA InOlct1on Aft* aeml .1 Ile r1levent P amOaga, '2 I NlowaG IO CtA-m 04. I X Biochimica et Biophysica Acta, Volume 801, published (1,3-6,12, 1984, G.N. Hannan et al, 'Similarity of the 14-15) Carbohydrate Moieties of Fibronectins deri'ved from Blood Plasma and synthesised by Cultured Endothelial Cells',. pages 396-402 A Chemical Abstracts, Volume 96, No 17, issued (1-19) 26 April 1982, (Columbus, Ohio, M.D. Gedevanishvili, 'Serotonin independently stimulates adhesion and propagation of fibroblasts in culture', Abstract No 136655g A US,A, 4373027 (BERNEMAN et al) 8 February 1983 (1-19) (08.02.83) See column 2 lines 41-48 A WO,A, 83/02954 (VENTREX LABORATORIES, INC) (1-19) 1 September 1983 (01.09.83) See page 19 lines 16-34 P,A EP,A, 205997 (DR. MULLER-LIERHEIM KG BIOLOGISCHE (11,17) LABORATORIEN) 30 December 1986 (30.12.86) See claim 4 toeciml csat en of citedg scuments: i sr l-t" o Cw nt OulISA 0 after thie ltmfIitA4tt11l lng dtto 'A 'ocjmenl diInig i gin*,rg i ml Ini n iCf nof pe IneIty data and not in COnIIAct In in* aomi,(at Gr SwI ICCDIMeef me meet eIn la e $fle(r Of to G oih.0l IMe thie of c tiOIn A O te C nvii eteid Is be of famc ii at rois ang s won I 41 4 atimer o eti etw osi t ee *ANS O &fat thle Mt*Onetlaen~ 'XI ueCVI"Avnn of ogIUItC relaveRCS: 1516 (teemed DA*I1o4tei Mling date Cannot oe (OM114414 e S6eg annot ento me seeg to Sdecufloinlt whiti wrly throtw double oR oftet, claffSI~s Of AivciwO an ,nvea A le 6140 *.Rchi to Cited to 64146l.448 In* muus len 46le of another gegmWI" ef SOMnCUler 0 eleVeRC: IRe C(lamed nPAVGAIeR C1itetio Ofi $soc ia tOeIII s n (as .eecafla4l CaRnot Of Coneeeeeo 0 1~t Q O4W R InVOAtIIV tS0U -AGA tIA OCtUMAI ete1eMAI t0 SR @f at guagieetde. Wee. 451116606A~ Of evwmeat is CofmOl~d .0"R *AS of mnere almor $wen OeCI* sto means maoft. SUgR Coameatinbeing eaVeOWe to A PeyseR, 61-144 110 d*CVMent atitiled SInne to tIeRS te6yn1Ate nGj IMI461 but i Id, ant. lotm i m then friefsty dala Claitled *v docuament membter of thle same taent 11511617 IV. CIATIPICAIO11 D tal o IA ACttal1 COMol tn AI ln t l*A i S0trtr I Dat a lt mlisng of th il trnaItin al Ci3# Rein 4 December 1987 (04.12.87) -e 3 r rC1414M *'T7 Inthrna.nio nau coirng Awtleinl y I 0inare et Authlertid Oltcmv Australian Patent Office D.Ei GLANVILLE p .,.v,.vCT113Aj21Q loo on @Mott) jj#Ay R 1642 1 '"1 -1 1 i -i Ilr I -7 liSln~ Aalc~o~ N.PCT/AU 87/00266 FUI TMJIR IDIVOAMAtION CONTINdU11 1FOM riii SWCONO S111T YV,i QUSR1411VATbOUS wmipta cipiTAIN CLAIMS WIIRE POUNO UNSIARMAU1LS This l11141 114110M4 6earCAivo looes Mat been goitolisfd in rescc of carlan Clai1ms under AllIC1 IIM? for ihfollow1ing reasons' ClAmr nullifiers *because the? 1el181e 10 sucoctd manor not reouired to tie searched by this Awltority, Aiamoly: *ClaiM AUtmtera b ecause Intl tolato sar.% of Iris international asslicstion trial do MCI comely .lift the Poscribed vequ. MIMI$ i0 SUCM 1A Walntia Me11n meaningful International sesJ cn Can 00 Carried out. WifiC~liy 0 C40a'" ne"Dim1... W-auM Oi1 acsd~lOtlams amd are mat arsflsa In 11Cd131r. Purim trio sacrc end 'Imard sairtooki of PaCT Awe 6,4c. Vt. 4 OBSEAVATIONS WHINE1 UNITY Of INVINTIOM IS LACK(ING Thias Informational Searchng Aulttent found mulilui. Invention$ in iris$ ilearsejonsi loolicallon 64 follows: IQAs all reouired additional search fead word timely paid by ifte spplicant. tmfu Informional $#Genf flooll covert 4ll searcmabllc Claims of te intefrisiionfil isallcation. 2-7AS emly some Of tilt resulteda dditional Searcht feet were Unfalr Paid by ftc aoeiiCAnt. Ithis lntfrnetioiif Search Frep ovet om.' nly tems clairms of te international aoolicaton for ssecn 100s wMe 0444, 81eCeAiC1iY claimsl: LoNO ?eaUirej additional aeerCra foes wet tilmely jiaid by Ifte 46slCIAnt. Conaei*AaelY, tis 11110netlHAI elarcht 19001 Is restricted ta tilt invention filet Mentioned In In* claims: ItlIs covered o by41 cam umbors: As sit soarnaole claims coulo be* Coatdhaid ,iftloul 6116M l~ealfyt~i an additional too. the Informational Seerdlinq Authtority did mots lysi Payment 0( any addeional foo. Nemcers an protsest f7 Trio 6,04,114111 aearfC? t of*e were OGI~ Ormmanc 1y aslCIAnt' 09014eL Cl NO Potest siCcom7011niei 1li. Payment Of Aidditldlscl IOAr load. Form PCTIISA/710 (140oe"W4ta SAeW 121 lJs'A I~ 111051 ANNEX T THE INERNATICNAL SEARCH REPCR' ON INTERNTIOCL APPLICATIM NO. PCT/AU 87/00266 This Annex lists the knwn publication level patent family members relating to the patent dc>xuments cited in the above-mentioned international search report. The Australian Patent Office is in no way liable for these particulars which are merely given for the purpose of information. Patent Document Cited in Search Patent Family Members Report US 4373027 CA 1167398 DK 5186/80 EP 30885 FR 2470794 JP 56095196 WO 8302954 EP 101724 EP 205997 DE 3521684 EP 205790 FI 862503 IL 79054 JP 62049856 JP 62051984 DE 3530440 END OF ANNEX