GB2059991A - Cultivating cells on particulate microcarriers - Google Patents

Abstract

The invention relates to a method of cultivating cells in a culture medium, containing particulate microcarriers. The microcarriers used comprise spherical particles of a water-insoluble, but water-swellable cross-linked organic macromolecular substance, said particles exhibiting at their surfaces covalently bound polypeptides capable of binding fibronectin biospecifically. In a water-swollen state the particles have a density within the range of 0.95 - 1.20 g/ml, preferably 1.00 - 1.10 g/ml.The mean size of the particles lies within the range of 100- 500 mu m.

Description

SPECIFICATION A method of cultivating cells in a culture medium containing particulate microcarriers The present invention relates to a method of cultivating cells in a culture medium containing microcarriers in particle form.

During recent years the cultivation of cells in vitro has become an indispensible technique within the research. Such cell cultivation provides information concerning cell division, cell growth, differentiation, structure, metabolism and ageing, and is of great significance in research pertaining, inter alia, to plant physiology, zoophysiology, biochemistry, pathology, histology, immunology and cancer research, and constitutes the basic technique in the cross scientific discipline of cell biology.

In addition, cell cultivation in vitro is facing a break-through with respect to use on an industrial scale, where one hitherto limiting factor has been the purely technical difficulties of obtaining with limited material resources sufficient yield of cells or products excreted by cells. This apertains, for example, to the preparation of biochemicals, pharmaceuticals, hormones and viral vaccines.

With respect to methods for cultivating cells which shall result in products for veterinary or medical use, one is legally forced to work with normal diploid cells. The great majority of mammalian cells, which are best studied in the present context, require a solid substrate in order to be able to proliferate; they are said to be anchorage-dependent. Mammalian cells which are able to grow in free suspension (for example human HeLa-cells or hamster-kidney cells, 'BHK-cells") are not fully normal from a genetic point of view, and may give rise to tumours when injected.

The classical methods for cultivating anchorage-dependent cells utilize glass or plastics dishes or roller bottles or flasks, in which it is possible to obtain a layer ("monolayer") of cells. When carrying out such a technique on a larger scale, it has previously been necessary to increase the number of culture vessels. The intermediary steps of changing the medium, subcultivation, harvest of cells etc., have necessitated a very large number of manual operations under sterile conditions.

A clear improvement in comparison with the aforementioned methods has been achieved by the introduction of so-called microcarrier culture. In accordance with this technique, cells are permitted to attach themselves to and to grow on minute particles which can be maintained suspended in the nutrient medium, by slowly stirring the same. These particles offer a much larger surface area per unit volume of culture vessel and thus make it possible to cultivate large quantities of cells in a single tank. In respect of space, man power and direct operational costs, this technique offers considerable advantages both in respect of work in the laboratories and work on a larger scale.

One method using such micocarrier culture techniques is described in the article "Microcarrier culture of animal cells" by A L van Wezel in Tissue Culture, Methods and Applications, P F Kruse Jr. and M K Patterson Jr., Eds., Academic Press (1973), pages 372-377. The carrier material used in this case consisted of insoluble beads of dextran cross-linked with epichlorohydrin, substituted with diethylaminoethyl groups.

Particulate microcarriers should have the following properties: The density in the presence of water (i.e. in water-swollen state) should preferably not differ to any marked extent from the density of the aqueous environment in which the carrier material is used to achieve a homogenous suspension of particles under careful stirring conditions, i.e. in general the density should lie within the range of 0.95 - 1.20 giml, preferably 1.00 - 1.10 giml, and particularly 1.02 - 1.05 g/ml.

In order to obtain a unitarily effective capacity and uniform adhesion and growth of the cells, the particles should comprise a very narrow fraction with regard to particle size. In general there is used a mean particle size in water-swollen state which lies within the range of 100 - 500 ,zim, preferably in the range of 100 - 300 um, e.g. in the range of 150 - 300 ttm.

The particles should be smooth (including preferably spherical or speroidal form) in order to obtain satisfactory cell adhesion and the least possible chance of cells being dislodged when particles collide with each other, and should be able to withstand mechanical stress and strain when suspended, so that they do not disintegrate into smaller parts.

Transparency facilitates microscopic studies of growing cultures.

The particles must be non-toxic to primary cells, established cell lines and strains of diploid cells from humans and animals.

The ability of the particles to absorb from the cultivation medium other components than those which take part in the mechanism for adherence of the cells to the particles should be negligible.

The known microcarriers, however, have certain limitations. For example, in certain instances, it has been found difficult to reasons unknown to cultivate human diploid cells on these carriers. The bond between cell and microcarrier having ionizable groups, which bond is initially electrovalent but is amplified secondarily with other types of binding forces of mechanical and/or chemical nature, is not always sufficiently strong. By way of example it can be mentioned that certain types of lymphoid cells do not fasten to these microcarriers.

The cells are also liable to loosen, either during growth or during the maintenance of the cultures. This has been observed, for example, when cultivating a number of normal fibroblasts.

Other types of cells have been found to bind to existing microcarriers with such strength as to cause problems when wishing to free the cells for harvesting purposes. The conventional method in this connection is trypsinisation, i.e. treatment of the culture with a proteolytic enzyme. Extensive trypsinisation results in the death or damage of the cells.

Consequently, one object of the present invention is to provide a method of cultivating cells in a culture medium containing spherical microcarriers which fulfil the aforementioned requirements placed on microcarriers and which, in addition, are not encumbered with the aforementioned disadvantages of known microcarriers, but which can also be used to cultivate cells in such systems where existing microcarriers give technical problems.

The invention is based on the use ofthe biospecific affinity which exists between on one hand certain polypeptides and on the other hand one or more proteins known under the designation fibronectins and existing in serum and in membranes of the majority of cell types. By attaching such polypeptides to a microcarrier there are obtained particles which in the presence of fibronectin are able to bind to cells.

In accordance herewith the method according to the invention is characterized in that the microcarriers used are spherical particles of a cross-linked organic macro-molecular substance which is insoluble, but swellable in water and which particles exhibit at their outer surfaces covalently bound polypeptide capable of binding biospecifically fibronectin, said particles having in a water-swollen state a density within the range of 0.95 - 1.20 giml, preferably 1.00 - 1.10 glml and a mean particle size within the range of 100 - 500 Flm.

(The expression polypeptide as used here and in the claims also includes proteins. According to another aspect, said expression is used to include one single polypeptide as well as mixtures of a number of polypeptides.) The macromolecular substance may, for example, be a polysaccharide or a protein or derivative thereof which has been cross-linked. In this respect examples of polysaccharides include dextran and agarose and derivatives thereof. Examples of proteins in this respect include collagen, collagen conformers or degradation products thereof, such as gelatin, whereat said polypeptide having the ability of biospecifically binding fibronectin can be the same substance as the macromolecular substance.The macromolecular substance may also be a cross-linked, synthetically produced polymer which is insoluble butswellable in water and which is not toxic against the cells.

The cross-linking of macromolecular substances containing e.g. amino groups or hydroxyl groups (e.g.

proteins and polysaccharides) with an at least bifunctional crosslinking agent is well-known to the art (see for example UK Patent Specifications Nos. 854715, 974054 and 1 013 585). The macromolecular substance may also comprise a combination of two or more macromolecular substances. Particles having a spherical form can be produced by bead polymerisation techniques. In this case, the reaction mixture is dispersed to droplet form in an inert liquid which is immiscible therewith, whereafter the cross-linked, insoluble particles formed by the reaction in the droplets are recovered. (See, for example, UK Patent Specification No. 974 054). The desired particle size can be obtained by adjustment of the size of the droplets and by fractionating the particles, e.g. by screening the same.The cross-linking agent for macromolecular substances containing hydroxyl groups or amino groups may, for example, be a compound of the type Y X - An Z(l)andXA2Z(ll) in which X, Y and Z are each a halogen atom, preferably chlorine or bromine, and A1 and A2 are each a straight or branched aliphatic, saturated hydrocarbon chain which is substituted with one or more hydroxyl groups (e.g. 1-3 hydroxyl groups) and which preferably contains 3-20 carbon atoms, e.g. 3-10 carbon atoms and which is optionally broken by one or more oxygen atoms (e.g. 1-3 oxygen atoms), or corresponding epoxide compounds obtainable from the compound (I) or (II) by splitting-off hydrogen halide.Examples of bifunctional substances having the formula X A, Z Z and corresponding epoxide compounds obtainable from X A1 Z by splitting-off hydrogen halide are e.g. dichlorohydrin, epichlorohydrin and 1 4-butanediol diglycide ether. Thus, the cross-links can, for example, contain straight or branched aliphatic saturated hydrocarbon chains which may be substituted by one or more hydroxyl groups (e.g. one to three hydroxyl groups) and which contain 3-20 carbon atoms, e.g. 3-10 carbon atoms, and which are optionally broken by one or more oxygen atoms (e.g. one to three oxygen atoms). Many other types of cross-linking agents are known, e.g. diisocyanates and diisothiocyanates.The swelling capacity and the density of the particles in water-swollen state can be varied by selecting different macromolecular substances and cross-linking agents, and by varying the degree of cross-linking.

In accordance with the invention particular advantage is achieved by using particles having pores which are so small that the polypeptide remains to a predominating extent on the surface of the particles and does not penetrate the pores, and that high molecular weight substances formed from the cell cultivation do not enter the pores to any appreciable extent.

In accordance with one embodiment of the invention the polypeptide is applied in the form of a surface layer on a particle comprising a macromolecular material different to that of the polypeptide. In this respect the polypeptide preferred is a collagen or degradation products thereof, such as gelatin. Further, in accordance with this embodiment, the polypeptide is bound to the particulate material with covalent bonds, which are able to resist normal culture and washing conditions. A large number of methods for producing covalent bonds have been described in the literature in conjunction with the coupling of polypeptides to carriers on a polysaccharide or protein basis.Thus, coupling with the aid of cyanogen halides has been described, for example, in the US Patent Specification No. 3 645 852; coupling with cyanates has been described in US Patent Specification No. 3788 948; and coupling with thiol-disulphide exchange reactions has been described in German Published Specification No. 2 808 515.

In accordance with another embodiment of the invention the microcarriers used as of collagen or degradation products thereof which have been cross-linked so as to achieve the desired insolubility in water.

In accordance with another aspect of the invention, the cells are released when desired from the carrier with a proteolytic enzym (e.g. collagenase, when the polypeptide is collagen or gelatin) subsequent to termination of the growth of the cells. Particularly in the case of collagenase, a good yield of viable cells can be readily released. (Collagenase is less detrimental to the cells than trypsin).

The particles may be neutral or may, in addition to said polypeptide, exhibit ion-exchange groups at the surface layer. The ion-exchange groups may be anion or cation exchange groups known per se, such as amino groups or carboxyl groups.

As previously mentioned, fibronectin appears on cell surfaces. With certain types of cells, for example with transformed cells, the fibronectin content is low. When applying the method according to the invention to this type of cells, the fibronectin can be added to the system in some suitable form, for example by adding serum or by using microcarriers which exhibit fibronectin which has bonded to the polypeptide on the outer layer of said microcarrier.

The invention will now be illustrated with reference to a number of examples.

EXAMPLE 1 Growth of Vero-cells on microcarriers (Vero is an established cell line which has originated from monkey-kidney cells /African Green monkey/ and is commercially available from Flow Laboratories, Ltd., Irwine, England.) Autoclaved microcarriers (from example BW, se below) in an amount corresponding to 30 mg of dry substance were suspended in a Petri dish in 10 ml of Dulbecco's medium (Smith, J.D., Freeman, G, Vogt, M.

and Dulbecco, R. (1960) Virology 12, 185-196), containing 4mM glutamine and 10% fetal calfserum, and with an initial cell concentration of 2.105 cells/ml. The growth took place in a CO2-incubator at 37"C for 160 hours, and was measured by staining the cell nuclei with 0.1 % crystal violet and by microscopic counting. In parallel with this experiment, cultivation was effected under identical conditions with Cytodex 91 (a microcarrier based on cross-linked dextran which had been derivatised with diethylaminoethyl groups, commercially available from Pharmacia Fine Chemicals AB, Uppsala, Sweden).

In both of the cultivation tests, approximately 2 1 104 cells/ml I had attached themselves to the microcarriers after 18 hours. After 160 hours the number of cells in both cultures had increased to a little more than 106/ml.

EXAMPLE 2 Harvest of cells from microcarriers 0.5 ml suspension of microcarrier (from Example Brand Cytodex @1 ) from Example 1 was transferred to centrifugal tubes. The supernatantwas separated by light centrifugation (acceleration to 200 x gravity and retardation, total 5 minutes). The microcarriers were washed twice with 1 ml of 0.03% EDTA in Puck's saline (Puck, T.T., Cieciura, S.J. and Robinson, A. (1958) Journal of Experimental Medicine 108 945-956) and once with 1 ml of Puck's saline not containing EDTA.

After the last centrifugation there was added 1 ml of enzyme solution in Puck's saline, 0.05% collagenase (Boehringer-Mannheim, Mannheim, Germany) or 0.05% trypsin (Serva, Feinbiochemica GmbH & Co., Heidelberg, Germany). After incubation for 5 and 10 minutes at 37"C the number of released cells was counted (see table 1).

TABLE 1 The release of Vero-cells from microcarriers. The measurement values given are the mean values of two parallel tests.

Microcarrier Addition Number of cells released (%) After 5 min. After 10 min.

Gelatin collagenase in 76 97 microcarrier Puck's saline trypsin in 48 80 Puck's saline Puck's saline not determined 4 Cytodex@1 collagenasein 9 38 Puck's saline trypsin in 21 61 Puck's saline Puck's saline not determined 1 Thus, when cultivating in accordance with the present invention a considerably higher yield of released cells is obtained, particularly when using collagenase in the release.

EXAMPLE 3 Cultivation of Vero-cells on gelatin-SephadexG50 and Cytodex 1 0.0375 ml of packed microcarriers from Example Bwas suspended in a flask and 1 x 105 cells per millilitre of cultivating medium (Dulbecco's modified Eagle's medium with 10% fetal calf serum, 4 mM glutamine and 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethane-sulphonic acid) buffer) were added.

Growth was allowed to take place at 37"C in the presence of water-saturated air (5% CO2), the culture being stirred with a magnetic stirrer at at speed of 60 r.p.m. Cultivation of cells was effected on Cytodex1 under identical conditions. The growth measured as the number of cells per millilitre of culture medium, is shown in Table 2.

TABLE 2 Time hrs Gelatin - Sephadex Cytodex1 G50 cellsSml cells/ml 19 11.5 x 104 7.65 x 104 41 26.8x104 12.2 x104 65 37.6 x 104 22.8 x104 97 49 x104 34.5 x104 138 56 x104 57 x104 161 64.6 x 104 57 x104 It is of interest to note that the growth was much quicker on gelatin-SephadexeG50 at the beginning of the cultivation process. This difference between the different microcarriers can be further increased, if the cell cultures are reproduced repeatedly after from 2 to 4 days incubation.

The microcarriers used in the examples were produced in the following way: A. Preparation ofactivatedmicrocarriers 1. 10 grams of dextran cross-linked with epichlorohydrin and having the form of spherical particles (Sephadex & -50 from Pharmacia Fine Chemicals AB, Uppsala, Sweden) were washed on a glass filter with water and transferred to a 500 ml vessel with water to a total volume of water plus particles of 300 ml. 12 grams of sodium hydroxide dissolved in 15 ml of water and 0.1 g of sodium borohydride were added. The mixture was heated to 60"C. 38 ml of epichlorohydrin were added and the mixture was allowed to react for 2 hours, whereafter the particles were washed with water on a glass filter and the water was removed by suction.

II. 40 ml of sedimented particles of dextran cross-linked with epichlorohydrin (SephadexG-50 from Pharmacia Fine Chemicals AB, Uppsala, Sweden) or particles of cross-linked agarose (SepharoseCL 4B from Pharmacia Fine Chemicals AB, Uppsala, Sweden) and 20 ml of water were transferred to a 250 ml vessel and admixed with 60 ml of cyanogen bromide solution (80 mg CNBr/ml H20). The mixture was allowed to react at a pH of 11.4 for 6 minutes at 4"C. The particles were then washed on a glass filter with 1000 ml of 0.1 M sodium hydrogencarbonate solution.

B. Coupling ofproteins or pep tides to activated micro carriers 1 gram of protein (polypeptide) was dissolved in 50 ml of 0.1 M NaHCO3 containing 0.5 M NaCI in water (pH 8.0) while heating when necessary, and the pH was adjusted to 9.5 - 10.5. The solution was added to microcarriers activated in accordance with A above, and the mixture was stirred for 24 hours at room temperature, whereafter the particles were washed, alternately with 0.2 M Na-acetate-buffer with 0.5 M NaCI in water (pH 4.5) and 0.2 M NaHCO3 with 0.5 M NaCI in water (pH 8.0) on a glass filter, and the liquid was removed by suction.

I. Collagen (Collagen type I from Sigma Chemical Company, Saint Louis, Missouri, USA) was coupled to activated Sephadex23G-50 from A I in the aforedescribed manner.

Yield: 11 mg of collagenig of dry product. The particle size was 160-200 item, and the density was 1.03 g/ml.

II. Gelatin (gelatin type Ill from Sigma Chemical Company, Saint Louis, Missouri, USA) was coupled in the aforedescribed manner to activated SephadexG-50 from Al.

Yield: 4 mg of gelatin of dry product. The particle size was 160-200 Flm and the density was 1.03 g/ml.

III. Collagen (the same as that recited in I above) was coupled in the aforedescribed manner to activated Sepharose CL 4B from A II.

Yield: 80 mg collagenig of dry product. The particle size was 100-150 tim, and the density was 1.02 g/ml.

IV. Gelatin (the same as that recited in II above) was coupled in the aforedescribed manner to activated Sepharoses CL 4B from A II.

Yield: 95 mg of gelatin/g of dry product. The particle size was 100-150 Flm, and the density was 1.02 g/ml.

V. Collagen (the same as that recited in I above) was coupled in the aforedescribed manner to activated Sephadex G-50 from A II.

Yield: 19 mg of collagen,g g of dry product. The particle size was 130-150 um, and the density was 1.04 g/ml.

VI. Gelatin (the same as that recited in II above) was coupled in the aforedescribed manner to SephadexG-50 from A II.

Yield: 12.5 mg of gelatin g of dry product. The particle size was 130-180 item, and the density was 1.04 g/ml.

The above values concerning particle size and density relate to particles in a water-swollen state.

Claims (9)

1. A method of cultivating cells in a culture medium containing particulate microcarriers, wherein the microcarriers are spherical particles of a water-insoluble, but water-swellable cross-linked organic macromolecular substance, said particles exhibiting at their surfaces a covalently bound polypeptide having the ability to bind biospecifically fibronectin, and said particles in a water-swollen state having a density within the range of 0.95-1.20 g ml, preferably 1.00-1.10 g ml and a mean particle size within the range of 100-500 ym.

2. A method according to claim 1, wherein the macromolecular substance is a water-insoluble but water-swellable polymer, which is not toxic to the cells.

3. A method according to claim 1 or claim 2, wherein the polypeptide is applied in the form of a surface layer on a particle of another macromolecular substance than the polypeptide.

4. A method according to any one of claims 1-3, wherein the polypeptide is collagen or degradation products thereof.

5. A method according to claim 1, wherein the microcarrier used is cross-linked collagen or degradation products thereof.

6. A method according to any one of claims 1-5, wherein the cells are released from the carrier with the aid of a proteolytic enzyme.

7. A method according to claim 6, wherein the polypeptide is collagen or gelatin, and wherein the cells are released from the carrier with the aid of collagenase.

8. A method of cultivating cells substantially as described in any one of the Examples herein.

9. Cells cultivated by a method according to any one of claims 1 to 8.