SE445116B - MAKE CULTURE CELLS ON MICROBATORS WITH FIBRONECTINE LAYERS - Google Patents

Description

7907573-5 The classical methods for culturing anchorage-dependent cells use glass or plastic bowls or roller bottles, in which one can obtain a layer ("monolayer") of cells, transfer of such a technique to a larger scale has previously only meant an increase of the number of culture vessels, which meant that medium change, subcultivation, harvesting of cells, etc. involved a very large number of manual operations under sterile conditions.

A clear improvement in comparison with the above-mentioned method has been achieved by the introduction of so-called microcarrier culture.

In this technique, cells are allowed to attach and grow on small particles, which can be kept suspended in the nutrient medium by means of slow stirring. These particles offer a radically much larger area per unit volume of culture vessels and thus provide opportunities for culturing large amounts of cells in a single culture tank. For both laboratory and large-scale work, this technology offers significant advantages in terms of space, manpower and direct operating costs.

A method that uses such a microcarrier culture is described in the essay "Microcarrier culture of animal cells" by AL van Wezel in Tissue Culture, Methods and Applications, PF Kruse Jr. and MK Patterson Jr., Eds., Academic Press (1973) p. 372 The carrier material used in this case consisted of beads of dextran crosslinked with epichlorohydrin, substituted by diethylaminoethyl groups.The suitability of this carrier material is based on the material meeting the following requirements, which are imposed on a good carrier material in addition to insolubility in water: of water (ie in the water-swollen state for materials swellable in water) should preferably be slightly different from the density of the aqueous environment in which the carrier material is used to achieve a homogeneous suspension of particles with gentle agitation, i.e. in In general, the density should be in the range 0.95 - 1.20 g / ml, preferably 1.00 - 1.10 g / ml, especially 1.02 - 1.05 g / ml. belong to a narrow fraction to achieve uniform effective capacity and even adhesion and growth of the cells. In general, an average particle size in the range 100 - 500 .mu.m is used, preferably in the range 150 - 300 .mu.m.

The particles should be smooth (including preferably a spherical or spheroidal shape) for good cell adhesion and the least possible abrasion in the event of collisions between the particles and withstand the mechanical stress of the suspension so that they do not decompose 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 uptake by the particles of components from the culture medium other than those involved in the mechanism of cell adhesion to the particles should be negligible.

However, there are some limitations with the known microcarriers. These limitations manifest themselves in such a way that in some cases for unknown reasons it has proved difficult to grow human diploid cells on said carrier. The bond between cell and microcarrier with ionizable groups, which bond is initially electrovalent but secondarily strengthened with other types of cohesive forces of a mechanical and / or chemical nature, is not always strong enough. As an example, certain types of lymphoid cells do not attach to the microcarriers.

Cells can also loosen either during growth or when the culture is maintained. This has been observed, for example, in the culture of some normal fibroblasts.

Other cell types have been shown to bind so well to existing microcarriers that problems have arisen when they wanted to free the cells for harvesting or transfer. The usual method in this context is trypsinization, ie. treatment 7907573-5 of the culture with a proteolytic enzyme. Extensive trypsinization leads to cell damage or death.

The use of existing microcarriers has also not brought any advantage in terms of the requirement for the presence of relatively high concentrations of serum in the culture medium in order to obtain optimal cell growth. Serum is by far the largest item of expenditure on the chemicals side in the cell culture context.

An object of the present invention is therefore to provide a method of culturing and / or attaching cells in a culture medium containing spherical microcarriers, which meet the above-mentioned requirements for microcarriers and moreover do not suffer from the above-mentioned disadvantages of the known microcarriers but can can be used for attachment and culture of cells even in such systems, where existing microcarriers present technical problems.

The invention is based on the utilization of the bio-specific affinity that exists between, on the one hand, certain polypeptides and, on the other hand, one or more proteins, which go under the name fibronectins and occur in serum and in membranes of most cell types. By attaching one of these components to a microcarrier, conjugates are obtained, which in the presence of the other component can bind to cells.

Accordingly, the method according to the invention is characterized in that spherical particles of a water-insoluble but swellable, crosslinked, organic macromolecular substance are used as microcarriers, which particles in their surface layer have covalently bound polypeptide capable of bio-specifically binding fibronectin and which particles in the water-swollen state have a density in the range 1.00-1.10 g / ml and an average particle size in the range 100-500 μm.

(The term polypeptide here and in the claims also includes proteins). The macromolecular substance may be, for example, a water-insoluble but preferably swellable polymer which is not toxic to the cells. The macromolecular substance may advantageously be an optionally crosslinked polysaccharide, which is insoluble but swellable in water.

According to the invention, it is particularly advantageous to use particles with such small pores that the substance predominantly remains on the surface of the particles and does not penetrate into the pores and that high-molecular substances formed from cell culture do not enter the pores to any appreciable extent either.

According to one embodiment, the substance is applied in the form of a surface layer to a particle of another material. In this case, collagen or degradation products thereof such as gelatin are preferred as said substance. Furthermore, according to this embodiment, it is preferred that the substance has been bound to the particulate material with bonds that can withstand normal culture and washing conditions, especially covalent bonds. A large number of methods for effecting covalent bonds have been described in the literature in connection with the coupling of polypeptides to polysaccharide or protein-based carriers. Thus, for example, coupling by means of cyanogen halides has been described in U.S. Pat. No. 3,645,852, with cyanates in U.S. Pat. No. 3,788,948 and with thiol disulfide exchange reactions in German Offenlegungsschrift 2,808,515.

According to another embodiment of the invention, those of collagen or degradation products thereof are used as microcarriers, which may have been crosslinked to achieve the desired insolubility in water.

According to another aspect of the invention, the cells are released, when desired, from the carrier with proteolytic enzyme (eg collagenase, when the substance is collagen or gelatin) after the end of growth of the cells. Especially in the case of collagenase, one can easily release fully viable cells in good yield. (Collagenase is significantly gentler on the cells than trypsin.) The particles may be neutral or, in addition to said substance in the surface layer, may have ion-exchanging groups. The ion exchange groups may be per se known anion or cation exchange groups such as amino groups or carboxyl groups.

As previously mentioned, fibronectin is present on cell surfaces. In some cell types, for example in transformed cells, the content of fibronectin is low. When applying the method according to the invention to this type of cell, fibronectin can be added to the system in any suitable form, for example by adding serum or by using microcarriers which have fibronectin in their surface layer.

Polypeptides capable of biospecifically binding fibronectin, on the other hand, generally occur on cell surfaces in an amount sufficient for the method of the invention, so that no special measures need be taken to ensure their presence.

The invention will be further illustrated in the following by a number of examples.

EXAMPLE 1 Growth of Vero Cells on Microcarriers (Vero is an established cell line derived from monkey kidney cells / African Green monkey / and is commercially available from Flow Laboratories, Ltd., Irwine, England.) Autoclaved microcarriers (from Example BIV , see below) in an amount corresponding to 30 mg of dry matter was suspended in a petri dish in 10 ml of Dulbecco's medium (Smith, JD, Freeman, G., Vogt, M. and Dulbecco, R. (1960) Virology 12, 185-196.) containing 4 mM glutamine and 10% fetal, calf serum and with an initial cell concentration of 2'105 cells / ml.

Growth was allowed to occur in a CO 2 incubator at 37 ° C for 160 hours and was measured by staining cell nuclei with 0.1% crystal violet and microscope counting. In parallel, a culture was performed under identical conditions with Cytodexq1 7907573-5 (a microcarrier based on cross-linked dextran derivatized with diethylaminoethyl groups, commercially available from Pharmacia Fine Chemicals AB, Uppsala, Sweden).

In both cultures, after 18 hours, approximately 2'104 cells / ml had adhered to the microcarriers. After 160 hours, this number had increased to just over 106 / ml in both cultures.

EXAMPLE 2 Harvesting of microcarrier cells 0.5 ml of microcarrier suspension (from Example BV and Cytodex 1) from Example 1 was transferred to centrifuge tubes.

By light centrifugation (acceleration to 200xg and deceleration, a total of 5 minutes), the supernatant was separated.

The microcarriers were washed twice with 1 ml of 0.03% EDTA in Puck's saline (Puck, TT, Cieciura, SJ and Robinson, A. (1958) Journal of Experimental Medicine lgg, 945-956) and once with 1 ml of Puck's saline. containing EDTA.

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

TABLE 1 Release of Vero cells from microcarriers. The measured values are mean values of duplicate samples.

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

Gelatin microcarrier collagenase in Puck's saline solution 76 97 trypsin in Puck's saline solution 48 80 Puck's saline solution not best. 4 Cytodex () 1 collagenase in Puck's saline solution 9 38 trypsin in Puck's saline solution 21 61 Puck's saline solution not best. Thus, the culture of the present invention enabled significantly higher yields upon release of the bound cells, especially when using collagenase upon release.

EXAMPLE 3 Cultivation of Vero Cells on Gelatin-SephadexC> G50 and Cytodex -1 In a flask with magnetic stirring, 0.0375 ml of packed microcarriers from Example BII and 1 x 105 cells per ml of culture medium were suspended (Dulbecco's modified Eagle's medium with 7.5 % fetal calf serum, 4 mM glutamine and 20 mM HEPES (4- (2-hydroxyethyl) -1-piperazinethanesulfonic acid) buffer). The growth was allowed to take place at 37 ° C in the presence of water-saturated air (5% CO 2), whereby the bottle was rotated at a speed of 60 revolutions per minute.

Cultivation on Cytodex 1 was performed under identical conditions.

Growth measured in number of cells per ml of culture medium is reported in Table 2.

TABLE-2 time gelatin-Sephadex ® G50 Cytodex ® 1 hr cells / ml cells / ml 19 6 11.5 x 10: 7.65 x 10â 41 26.8 x 10 12.2 x 10 65 31.6 X 104 22 .0 X 104 4 4 97 49 x 10 34.5 x 10 138 56 x 104 57 X 104 161 64.6 X 104 57 x 104 Notable is the significantly faster growth of gelatin- Sephadex G50 at the beginning of the culture. This difference between the different microcarriers can be further increased if the cell cultures are reacted repeatedly after 2 - 4 days of incubation.

The microcarriers used in the above embodiments were prepared as follows: A. Preparation of activated microcarriers I. 10 g of epichlorohydrin crosslinked dextran in the form of spherical particles (Sephadex G50 from Pharmacia Fine Chemicals AB, Uppsala, Sweden) are washed on glass filters with water and transferred to a 500 ml vessel with water to a total volume of water plus particles of 300 ml. 12 g of sodium hydroxide dissolved in 15 ml of water and 0.1 g of sodium borohydride are added. The mixture is heated to 60 ° C. 38 ml of epichlorohydrin are added and the mixture is allowed to react for 2 hours, after which the particles are washed with water on a glass filter and filtered off with suction.

II. 40 ml of sedimented particles of dextran cross-linked with epichlorohydrin (Sephadex Chemicals AB, Uppsala, Sweden) or of cross-linked agarose CL4B from Pharmacia Fine Chemicals AB, Uppsala, G50 from Pharmacia Fine (Sepharose Sweden) and 20 ml of water are transferred to a 250 ml vessel and add 60 ml of bromocyanine solution (80 mg CNBr / ml H 2 O). The mixture is allowed to react at pH 11.4 and 4 ° C for 6 minutes. The particles are then washed on a glass filter with 1000 ml of 0.1 M sodium bicarbonate solution.

B. Coupling of proteins or peptides to activated microcarriers 1 q of protein (polypeptide) is dissolved in 50 ml of 0.1 M NaHCO 3 containing 0.5 M NaCl in water (pH 8.0) with heating if necessary and the pH is adjusted to 9, 5 - 10.5. The solution is added according to A. activated microcarriers above and the mixture is stirred for 24 hours at room temperature, after which the particles are washed alternately with 0.2 M Na-acetate buffer with 0.5 M NaCl in water (pH 4.5). And 0.2 M NaHCO 3 with 0.5 M NaCl in water (pH 8.0) on glass filter and aspirated.

I. Collagen (Collagen type I from Sigma Chemical Company, Saint Louis, Missouri, USA) was coupled in the above manner to activated Sephadex G50 from A I.

Yield: 11 mg collagen / g dry product. Particle size 160 - 200 .mu.m, density 1.03 g / ml.

II. Gelatin (type III gelatin from Sigma Chemical Company, Saint Louis, Missouri, USA) was coupled to the activated I I. Sephadex G50 activated above.

Yield: 4 mg gelatin / g dry product. Particle size 160 - 200 .mu.m, density 1.03 g / ml.

III. Collagen (same as in I ova? Š) was coupled in the above manner to activated Sepharose CL 43 from A [1_ Yield: 80 mg collagen / g dry product. Particle size 100 - 150 .mu.m, density 1.02 g / ml. 7907573-5 10 IV. Gelatin (same as in II was waxed in the above manner to activated Sepharose CL 43 from A 11, Yield: 95 mg gelatin / g dry product. Particle size 100 - 150 .mu.m, density 1.02 g / ml.

V. Collagen (same as in I above) was coupled as above to activated Sephadex G50 from A II.

Yield: 19 mg collagen / g dry product. Particle size 130-150 .mu.m, density 1.04 g / ml.

WE. Gelatin (same as in II above) was coupled as above to Sephadex G50 from A II.

Yield: 12.5 mg gelatin / g dry product. Particle size 130 - 180 .mu.m, density 1.04 g / ml.

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

Claims (7)

7907573-5 _ PATENT REQUIREMENTS A method of culturing cells in a culture medium containing particulate microcarriers, characterized in that spherical particles of a water-insoluble but swellable, cross-linked, organic macromolecular substance are used as microcarriers, which particles in their surface layer have a covalently bound polypeptide capable of to biospecifically bind fibronectin and which particles in the water-swollen state have a density in the range 1.00 - 1.10 g / ml and an average particle size in the range 100 - 500 / μm. 2. A method according to claim 1, characterized in that the water-insoluble but swellable macromolecular substance is a polymer which is not toxic to the cells. 3. A method according to claim 1 or 2, characterized in that the substance is applied in the form of a surface layer on a particle, said surface layer and particle being of different materials. 4. A method according to claim 3, characterized in that the substance is collagen or degradation products thereof. 5. A method according to claim 1, characterized in that as microcarriers those of collagen or degradation products thereof which have been crosslinked to achieve the desired insolubility in water are used. A method according to any one of claims 1 to 5, characterized in that the cells are released from the carrier with proteolytic enzyme. 7. A method according to claim 6, characterized in that the substance is collagen or gelatin and that the cells are released from the carrier with collagenase.