JPH0154992B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for culturing tether-dependent animal cells in a medium containing microcarriers in particulate form. In recent years, in vitro cell culture has become an essential research technique. Cultivation of such cells provides information on cell division, cell proliferation, differentiation, structure, metabolism and aging, and supports research on plant physiology, zoology, biochemistry, pathology, morphology, immunology and cancer research, among others. and it constitutes a basic technology in the common scientific control of cell biology. Furthermore, in vitro cell culture is facing a breakthrough for use on an industrial scale, where one limiting factor to date has been the ability to obtain sufficient yields of cells using limited material raw materials. or purely technical difficulties in obtaining secreted products by cells. This is relevant for example in the production of biochemical agents, pharmaceutical agents, hormones and viral vaccines. For cell culture methods to produce products for veterinary or medical applications, it is legally mandatory to conduct research on normal diploid cells.
The majority of the mammalian cells best studied in this context require a solid substrate in order to be able to grow. They are said to be mooring dependent. Mammalian cells that can grow in free suspension (e.g. human Hela cells or hamster kidney cells "BHK cells") are not completely normal from a genetic point of view and may give rise to tumors if injected. do not have. Classical tether-dependent cell culture methods utilize glass or plastic dishes or roller bottles or flasks into which a layer (monolayer) of cells can be obtained. Implementing such techniques on a large scale previously required increasing the number of culture vessels. Medium exchange, subculturing, cell harvesting and other intermediate steps required extensive human manipulation under sterile conditions. A clear improvement compared to the previously described methods was achieved with the introduction of so-called microcarrier cultures. According to this technique, it is possible to attach and grow cells on microparticles that can be kept in suspension in a nutrient medium by gradual agitation. These particles provide a much larger surface area per unit volume of culture vessel and thus enable the cultivation of large amounts of cells in a single tank. In terms of space input and direct operating costs, this technique offers considerable advantages for both laboratory work and large-scale operations. One method using such microcarrier culture technology is “Tissue Culture, Method and
Applications” (Academic press, 1973 edition)
It is described on pages 372-377. The carrier material used in this case consists of insoluble beads of dextran cross-linked with epichlorohydrin and substituted with diethylaminoethyl groups. The particulate microcarrier should have the following properties: In order to achieve a homogeneous suspension of the particles under careful stirring conditions, the density in the presence of water (i.e. swollen with water) is preferably significantly less than that of the aqueous environment in which the carrier material is used. They should not differ by a significant amount. That is, generally its density is 0.95 to 1.20 g/ml, preferably 1.00 to 1.10 g/ml.
ml, and especially should be in the range 1.02-1.05 g/ml. In order to obtain a uniform effective capacity and uniform attachment and proliferation of cells, the particles should constitute a very narrow fraction with respect to particle size. In general, 100 in the water-swollen state
Average particle sizes in the range ~500 μm, preferably in the range 100-300 μm, such as in the range 150-300 μm, are used. In order to obtain satisfactory cell adhesion and to minimize the chances of cell detachment if the particles collide with each other, the particles should be smooth (preferably including spherical or spheroidal shapes). )
and it should be able to withstand mechanical stress and strain when suspended so as not to disintegrate into smaller pieces. Transparency facilitates microscopic study of growing cultures. These particles must be non-toxic to primary cells, established cell lines and diploid cell lines from humans and animals. The ability of these particles to adsorb other components from the medium other than those involved in the attachment mechanism of cells to the particles should be negligible. However, known microcarriers have certain limitations. For example, it is known that in some cases it is difficult to culture human diploid cells on these carriers for certain unknown reasons. Bonding between cells and microcarriers with ionic groups, which is primarily electrically charged but secondarily by other types of bonding forces of mechanical and/or chemical nature. (enhanced) are not necessarily strong enough. For example, certain types of lymphocytes do not bind to these microcarriers. Cells can also become unbound during growth or maintenance in culture. This has been observed, for example, in the culture of a large number of normal fibroblasts. It has been found that other types of cells bind very strongly to existing microcarriers, resulting in problems when washing and releasing the cells for harvesting purposes. The usual method for this is tryptic digestion, ie treatment of the culture with proteolytic enzymes. Exhaustive tryptic digestion will result in cell death or damage. One object of the invention is therefore to meet the above-mentioned requirements stated with respect to microcarriers and furthermore not to be hampered by the above-mentioned disadvantages of known microcarriers and also to the extent that existing microcarriers pose technical problems. The object of the present invention is to provide a method for culturing cells in a medium containing spherical microcarriers, which can also be used for culturing cells in such a system. The invention relates to certain polypeptides on the one hand and to one or more proteins known under the name fibronectin and which are present in serum and in the membranes of most types of cells. based on the biologically specific affinity that exists between them. By attaching such polypeptides to microcarriers, particles are obtained that are capable of binding to cells in the presence of fibronectin. According to the present invention, the method of the invention provides that the microcarrier used is covalently bound, which is water-insoluble but water-swellable and capable of binding biologically specifically to fibronectin. on its outer surface and in the water-swollen state 0.95-1.20 g/ml, preferably
Densities ranging from 1.00 to 1.10g/ml and 100 to 500μ
They are characterized by being spherical particles of cross-linked organic polymeric material having an average particle size in the range of m. As used herein, the expression "polypeptide" also includes proteins. According to another aspect, the expression is used to encompass single polypeptides as well as mixtures of multiple polypeptides. This high molecular weight substance can be, for example, a polysaccharide or a protein or a derivative thereof with cross-linking. In this regard, examples of polysaccharides include dextran and agarose and their derivatives. Examples of proteins in this regard include collagen, collagen conformers or their degradation products such as gelatin. Here, the polypeptide having the ability to bind to fibronectin with biological specificity may be the same substance as the high molecular weight substance. The high molecular weight material may also be a cross-linked, synthetically produced polymer that is insoluble in water but swellable and non-toxic to cells. Cross-linking of e.g. amino- or hydroxyl-containing macromolecular substances (e.g. proteins and polysaccharides) by at least bifunctional cross-linking agents is well known in the art (e.g. British Patent No. 854,715;
(See the specifications of No. 974054 and No. 1013585). The high molecular weight material may also include a combination of two or more high molecular weight materials. Particles with spherical shape can be produced by bead polymerization techniques. In this case, the reaction mixture is dispersed in the form of droplets in an inert liquid immiscible with it, after which the cross-linked insoluble particles produced by the reaction in the droplets are recovered (e.g. (See Patent No. 974054). The desired particle size can be obtained by adjusting the size of the droplets and fractionating the particles (eg, screening them). Cross-linking agents for polymeric substances containing hydroxyl groups or amino groups are, for example, [wherein X, Y and Z are each a halogen atom, preferably chlorine or bromine, and A ₁ and A ₂ are each substituted with one or more hydroxyl groups (e.g. 1 to 3 hydroxyl groups) and preferably 3 to 20 carbon atoms,
Straight-chain or branched fatty acids containing, for example, 3 to 10 carbon atoms and optionally interrupted by one or more oxygen atoms (e.g. 1 to 3 oxygen atoms) saturated hydrocarbon chain] or the corresponding epoxide compound obtainable from the compound () or () by cleaving the hydrogen halide. Examples of difunctional substances having the formula X-A ₁ -Z and corresponding epoxide compounds obtainable from X-A ₁ -Z by cleavage of the hydrogen halide are e.g. dichlorohydrin,
epichlorohydrin and 1,4-butanediol diclyside ether. That is, the cross-linkage can be substituted, for example, by one or more hydroxyl groups (for example 1 to 3 hydroxyl groups), and 3 to 20
straight-chain or branched fatty acids containing for example 3 to 10 carbon atoms and optionally interrupted by one or more oxygen atoms (for example 1 to 3 oxygen atoms) Contains group saturated hydrocarbon chains. Many other types of crosslinkers are known, such as diisocyanates and diisothiocyanates. The swelling capacity and density of the particles in the water-swollen state can be varied by selecting different polymeric substances and cross-linking agents and by varying the degree of cross-linking. According to the invention, the polypeptide is so small that it largely remains on the surface of the particle and does not penetrate into the pores, yet high molecular weight substances formed during cell culture do not significantly enter the pores. Particular advantages are achieved by using particles with pores. According to one aspect of the invention, the polypeptide is applied in the form of a surface layer on the particles comprising a high molecular weight substance different from that of the polypeptide. A preferred polypeptide in this regard is collagen or its degradation products such as gelatin. Further according to this embodiment, the polypeptide is attached to the particulate material by a covalent bond that is resistant to normal culturing and washing conditions. Numerous methods for creating covalent bonds have been described in the literature for coupling polypeptides to polysaccharide or protein-based carriers. Thus, coupling with the aid of cyanogen halides is described, for example, in U.S. Pat.
No. 3,645,852, coupling with cyanates is described in US Pat. No. 3,788,948, and coupling by thiol disulfide exchange reaction in German Patent No. 2,808,515. According to another aspect of the invention, the microcarrier used is collagen or its degradation products which have been cross-linked to achieve the desired water insolubility. According to another aspect of the invention, after the cells have grown, they are optionally released from the carrier using a proteolytic enzyme (eg, collagenase if the polypeptide is collagen or gelatin).
Particularly in the case of collagenase, a good yield of living cells can be easily released. Collagenase is much more synchronous with cells than trypsin. The particles may be neutral or may exhibit ion-exchangeable groups in other surface layers of the polypeptide. Ion exchange groups can be anion or cation exchange groups known per se, such as amino groups or carboxyl groups. As mentioned above, fibronectin appears on the cell surface. For certain cell types, such as amebocytes, fibronectin content is low. When applying the method of the present invention to this type of cells,
Fibronectin can be added to this system in some suitable manner, for example by the addition of serum or by the use of microcarriers exhibiting on their surface fibronectin bound to polypeptides on the outer layer of said microcarriers. The invention will now be described with reference to a number of embodiments. Example 1 Growth of vero cells on microcarriers Vero is an established cell line derived from monkey (African green monkey) kidney cells and commercially available from Flow Laboratories. . An amount of autoclaved (Example B, see below) microcarriers corresponding to 30 mg of dry substance was placed in a Petri dish containing 4 mM glutamine and 10% fetal calf serum and at an initial cell concentration of 2 x 10 ⁵ cells/ml. 10ml Dulbecco with
[Virology, Vol. 12, pp. 185-196 (1960)]. Growth was carried out for 160 h at 37 °C in a _CO2 incubator and 0.1%
Proliferation was measured by staining cell nuclei with crystal violet and counting them under a microscope. In parallel with this experiment, the same test was performed using "Cytodex" 1 (a microcarrier based on cross-linked dextran derivatized with diethylaminoethyl groups, commercially available from Farussia Fine Chemicals). Cultivation was carried out under the following conditions. In both culture tests, approximately 2×10 ⁴ after 18 hours.
cells/ml were attached to the microcarriers. After 160 hours, cell numbers in both cultures were slightly greater than 10 ⁶ cells/ml. Example 2 Recovery of cells from microcarriers 0.5 ml of microcarrier suspension from Example 1 (Example B
and "Cytodex" 1) were transferred to a centrifuge tube. The supernatant was separated by light centrifugation (acceleration to 200×G and deceleration, 5 min total).
Puck the microcarrier twice in saline [Journal of Experimental Medicine, No. 108]
Vol. 945-956 (1958)] 0.03% EDTA/mg
twice and once with 1 ml of EDTA-free saline solution. After the final centrifugation, 1 ml of an enzyme solution of 0.05% collagenase (Boehringer Mannheim product) or 0.05% trypsin (Selva Fine Biotinica product) in Pacque saline was added. After culturing at 37°C for 5 and 10 minutes, the number of released cells was counted.
Table 1 below shows the release of Vero cells from the microcarriers. The measurements given are the average values of two parallel tests.

【table】

[Table] ○R Collagenasex 1
Patsuku saline solution 21 61
trypsin
Patsuku saline solution Not measured 1
Thus, when culturing according to the invention, considerably high free cell yields are obtained. This is especially the case when collagenase is used in its free state. Example 3 Culture of Vero cells on gelatin-Sephadex G50 and Cytodex 1 Packed microcarriers from Example B
Suspend 0.0375ml in the flask and add 1x
Culture of ¹⁰⁵ cells/ml (10% fetal calf serum,
4mM glutamine and 20mM HEPES (4-(2
-Hydroxyethyl)-1-piperazineethane-
Drubetzko's modified Eagle's medium) containing sulfonic acid) was added. Growth was carried out at 37° C. in the presence of water-filled air (5% CO ₂ ). The culture was stirred with a magnetic stirrer at a speed of 60 rpm. Cell culture was carried out on Cytodex 1 under the same conditions. Proliferation, measured as number of cells per ml of medium, is shown in Table 2.

[Table] It is interesting that the growth is much more rapid on Gelatin-Sephadex G50 at the beginning of the culture process. This difference between different microcarriers can be further increased if the cell culture is regenerated repeatedly after 2-4 days of culture. The microcarrier used in the examples was manufactured as follows. A. Preparation of activated microcarriers Dextran cross-linked with epichlorohydrin and in the form of spherical particles ("Sephadex" G50, Pharmacia Fines.
Chemicals Co., Ltd. product) was washed with water on a glass filter and transferred to a 500 ml container with water to make a total volume of water and particles of 300 ml. 15
12 g of sodium hydroxide and 0.1 g of sodium borohydride dissolved in ml of water were added. This mixture was heated to 60°C. Add 38 ml of epichlorohydrin and add 2 ml of this mixture.
After a period of reaction, the particles were washed with water on a glass filter and the water was removed by suction. Precipitated particles of dextran cross-linked with epichlorohydrin (“Sephadex” G-
50, a product of Pharmacia Huain Chemicals Co.) or cross-linked agarose particles ("Sepharose" CL 4B, a product of Pharmacia Huain Chemicals Co., Ltd.) and 40 ml of water.
Transferred to a 250ml container and mixed with 60ml of cyanogen bromide solution (80mgCNBr/ml _H2O ). This mixture was reacted at pH 11.4 for 6 minutes at 4°C. Then add 1000ml on a glass filter.
The particles were washed with 0.1M sodium bicarbonate solution. B. Coupling of proteins or peptides to activated microcarriers 1 g of protein (polypeptide) is added to 0.1 M in water containing 0.5 M NaCl, with heating if necessary.
Dissolve in 50ml of _NaHCO3 (PH8.0), and its PH
was adjusted to 9.5-10.5. This solution was added to the microcarriers activated according to A above, and the mixture was stirred for 24 hours at room temperature and the particles were then dissolved in 0.2M sodium acetate buffer with 0.5M NaCl in water (PH4. 5) and 0.5M NaCl in water
on the glass filter alternately with 0.2M NaHCO ₃ and the liquid was removed by aspiration. Collagen (Collagen type, Sigma・
Cefadex G- activated from A with chemical company products) as described above
I made it to 50. The yield was 11 mg collagen/g dry product. The particle size was 160-200 μm and the density was 1.03 g/ml. Add gelatin (gelatin type, Sigma Chemical Company product) to A as above.
Coupled with activated Cephadex G-50 from. The yield was 4 mg gelatin per g of dry product. The particle size was 160-200 μm and the density was 1.03 g/ml. Collagen (same as mentioned above)
was combined with activated Sepharose CL 4B from A as described above. The yield was 80 mg collagen/g dry product. The particle size was 100-150 μm and the density was 1.02 g/ml. Gelatin (same as above) was prepared from activated Cephalose CL from A as above.
I made a coupling to 4B. The yield was 95 mg of gelatin per g of dry product. The particle size was 100-150 μm and the density was 1.02 g/ml. Collagen (same as described above) was coupled to activated Cephadex G-50 from A as described above. The yield was 19 mg collagen/g dry product. The particle size was 130-150 μm and the density was 1.04 g/ml. Gelatin (same as listed above)
was coupled to Sephadex G-50 from A as described above. The yield was 12.5 mg gelatin per gram of dry product. The particle size was 130-180 μm and the density was 1.04 g/ml. The above values for particle size and density relate to the particles in the water-swollen state.

Claims (1)

[Claims] 1. Cultivating tether-dependent animal cells in a culture medium containing particulate microcarriers, wherein the microcarriers are insoluble in water but swellable with water; It has a covalently attached polypeptide on its surface that allows it to bind to fibronectin with biological specificity, and has a density in the range of 0.95-1.20 g/ml and 100-500 μm in the water-swollen state. A method for culturing tether-dependent animal cells, characterized in that they are spherical particles of cross-linked organic polymeric material having an average particle size in the range of . 2. The method according to claim 1, wherein the spherical particles of the organic polymeric substance have a density in the range of 1.00 to 1.10 g/ml in a water-swollen state. 3. The method of claim 1, wherein the polymeric material is a polymer that is insoluble in water but swellable in water and non-toxic to tether-dependent animal cells. 4. The method according to any one of claims 1 to 3, wherein the polypeptide is applied in the form of a surface layer on particles of a polymeric substance other than the polypeptide. 5. The method according to any one of claims 1 to 4, wherein the polypeptide is collagen or a degradation product thereof. 6. The method according to claim 1, wherein the microcarrier used is cross-linked collagen or a degradation product thereof. 7 Claims 1 to 7, wherein the tether-dependent animal cells are released from the carrier by a proteolytic enzyme.
The method described in any of Section 6. 8. The method of claim 7, wherein the polypeptide is collagen or gelatin and the tether-dependent animal cells are released from the carrier by collagenase.