IE903404A1 - A method for cultivating cells in microporous beads - Google Patents

Abstract

The invention relates to a process for the culture of cells which are entrapped in hollow microbeads and are densely packed in a fixed bed and are supplied with nutrients discontinuously or continuously in a vessel by the medium flow. The suitable hollow microbeads have a membrane composed of a polysaccharide gel or of a polyelectrolyte.

Description

A method for cultivating cells in microporous beads The invention relates to a method for cultivating cells 5 in microporous beads which are located in a fixed bed and are supplied discontinuously or continuously with nutrients .

A number of different methods with which whole cells can be immobilized are known. Thus, cells can be entrapped by suitable measures in a crosslinked gel and continue to be viable and active therein (Klein, Wagner Methods for the Immobilisation of Microbial Cells in Appl. Biochem. Bioeng. 4: 11-51 (1983)). However, it has emerged in practice that live cells disrupt the gel matrix by growing and may then enter the surrounding nutrient solution. There they are able to grow further and thus greatly impede cell retention in continuous processes.

US Patent 2,958,517 discloses a device for the cultivation of free mammalian cells, in which the nutrient solution is mixed with a magnetically driven stirrer bar.

However, with this procedure there are always mutual collisions of the cells and, consequently, impairment of vital functions, which may lead to death of the cells.

US 706,872 describes a continuous cultivation of mam25 malian cells on porous sponge-like particles. About 1 to 5% of the cell population in the. reactor appear free in the nutrient solution, which may easily lead to the free cells settling on and blocking the retaining devices for the immobilizates, such as membranes or sintered glass disks.

It is very difficult to culture plant or animal cells in particular. These cells have very sensitive and fragile - 2 membranes which can easily be weakened or damaged by only slight mechanical effects. This greatly impairs the viability and the productivity of these cells.

Another method of immobilization is represented by the 5 technique of entrapment of cells in semipermeable membranes which are called microporous beads hereinafter.

Examples of this technique are specified in European Patent Application 0,173,915 or 0,280,155 and in German Offenlegungsschrift 3,529,203. It is also shown in the German Offenlegungsschrift how immobilized cells can be cultured in a stirred reactor. However, even in this reactor the mechanical stress on the cells from the stirrer is still too great. There may be damage to the capsules and release of the entrapped cells and, finally, damage to the cells.

It has now been found, surprisingly, that the abovementioned difficulties can be overcome by the closepacked arrangement of microporous beads in a fixed bed. Despite the close packing, the microporous beads retain their stability and thus permit optimal growth and production conditions for the cells contained therein. This close arrangement of the microporous beads in the reactor surprisingly also prevents the formation of channels between the microporous beads. This ensures a uniform and readily controllable supply of all the required nutrients to the cells. Hence this also makes it possible to cultivate the cells without interruption over a long period, covering many generations.

The invention thus relates to a method for cultivating cells in microporous beads whose membrane is composed of an anionic polysaccharide gel or of a polyelectrolyte membrane, wherein the microporous beads are located in a fixed bed.

The microporous beads lie in the fixed bed as close35 packed beads in the stream of nutrient solution. They - 3 experience only slight spatial changes in position and thus only slight mechanical friction, which might lead to damage, occurs on the membranes of the microporous beads.

The invention is described in detail hereinafter, espe5 cially in its preferred embodiments. The invention is furthermore defined in the claims.

The microporous beads suitable for the invention are composed of a biocompatible, non-toxic, semipermeable, water-insoluble membrane. The preparation of such mem10 branes is described, for example, in European Patent Application 0,173,915. For this purpose, the cells are suspended in, in particular, an aqueous solution of the core polymer. The core polymer increases the viscosity of the cell suspension so that, during the subsequent dropwise addition to the anionic polysaccharide solution, mixing of the solutions is prevented. Suitable core polymers are all neutral, water-soluble, biocompatible polymers which increase the viscosity. Examples of these are hydroxypropylmethylcellulose or hydroxymethyl20 cellulose. These core polymers are mixed with one or more divalent cations such as, for example, CaCl2. The mixture is then converted into the form of drops and introduced into an anionic polysaccharide solution composed of, for example, alginate, carrageenan, chitosan, pectinate or carboxymethylcellulose. Alginate is preferably used. A semipermeable membrane is formed at the phase boundary between core polymer and polysaccharide solution owing to the presence of one or more divalent cations, and then entraps the cells. The membrane is composed of the anionic polysaccharide which is converted by the divalent cations into the form of a gel. On the other hand, the core polymer in which the cells are suspended remains fluid.

It is also possible in an analogous manner, as described in European Patent Application 0,280,155, for cells to be suspended in an aqueous solution of an anionic polymer - 4 (polyacid) such as, for example, alginate, carrageenan, hyaluronic acid, carboxymethylcellulose, xanthan or furcellaran, converted into the form of drops and subsequently introduced into an aqueous solution of a cationic polymer (polybase) such as, for example, of a copolymer of l-vinyl-3-methylimidazolium chloride and 1vinyl-2-pyrrolidone or of a polyallylamine/2-hydroxypropylene copolymer. A copolymer of l-vinyl-3-methylimidazolium chloride and l-vinyl-2-pyrrolidone is pre10 ferably used. A semipermeable polyelectrolyte membrane is formed at the phase boundary between polyacid and polybase and entraps the cells. In this case too the core polymer in which the cells are suspended remains fluid. The membrane prevents the passage of cells but is freely permeable to gases and constituents of the medium.

It is possible for all viable cells to be entrapped and cultivated in these microporous beads. These are bacteria, fungi or yeasts and, in particular, all cell lines of animal or plant origin, as well as, particular pre20 ferably, hybridoma cells. Under suitable conditions, the cells grow by cell division in the microporous beads. Supply with nutrients and transport away of formed products takes place by diffusion through the semipermeable membrane.

Cultivation is carried out in a vessel in which the microporous beads are closely packed. The vessel can be made of inert material such as, for example, metal, ceramic, glass or a plastic material which can be sterilized by chemical or physical methods. The vessel can be, in particular, 5n a form resembling a cylinder. The ratio of height to diameter can vary within a wide range. The ratio of height to width can vary within the range from 100:1 to 1:100, in particular from 2:1 to 20:1.

The cultivation vessel has at opposite ends inlet and outlet openings; between them are located the microporous beads. The openings are arranged so that the nutrient - 5 solution can flow through all the microporous beads located between them. Between the openings and the microporous beads are located retaining devices for the microporous beads, such as, for example, membranes or sintered glass disks, which are, however, easily permeated by the nutrient solution. It is possible with an arrangement of this type to allow nutrient solution to flow continuously or discontinuously at any desired rate through the vessel. The volume of liquid remains constant during this time. The nutrient solution can also be circulated in a loop by an appropriate device, so that better utilization of nutrient constituents, which are often costly, is possible.

All control, measurement and adjustment procedures can, if necessary, take place outside the cultivation vessel.

These are, in particular, the measurement of temperature, of pH and of oxygen partial pressure, but also of gas exchange of 02 and CO2, the control of the pH and the renewal of constituents of the nutrient solution.

It is possible, without impairing the encapsulated cells, to branch off a portion of the nutrient solution in order to remove and process the products which have been produced. It is also possible to replace the branched-off nutrient solution by fresh. The nutrient replacement and the product isolation can take place discontinuously or continuously from the device for recycling the nutrient stream.

The choice and adjustment of the chemical and physical parameters for the cultivation takes place in accordance with the requirements for growth and production of the cells entrapped in the microporous beads. The limits of these cultivation parameters can vary within a wide range. However, it will not be difficult for a person skilled in the art to discover the parameters which are optimal for the cells used in each case.

For hybridoma cells for example, the following physical and chemical cultivation parameters have proven suitable. The cultivation temperature is between 35 and 39°C, preferably at 37°C. Adequate growth is attained at a pH between 6.8 and 7.2, preferably at 7.0. The flow rate through the reactor can vary within a wide range depending on the stage of growth. Good results are obtained at between 0.1 and 50 liters of medium per hour and liter of reactor volume, preferably between 5 and 20 1/h.l.

It has been found, surprisingly, that the microporous beads retained their stability despite the close packing in the cultivation vessels. There was no channel formation and no clumping of the capsules. It was thus possible to carry out the cultivation over a long period.

The close packing of the microporous beads and the continuous recycling of nutrients make it possible to cultivate cells and produce products in a very small space with a simple apparatus under sterile conditions. In particular, the nutrient recycling results in effi20 cient and economic utilization of the required nutrients, which are often very costly. Regeneration of the nutrient solution is also possible owing to the selective removal of the final products of metabolism, such as, for example, lactic acid, C02 or ammonium ions, by ionic exchan25 gers or other separation methods in the cell-free nutrient stream.

The examples which are detailed below serve to illustrate the invention further.

Example 1 A suspension of hybridoma cells is diluted 1:2 (ratio by mass) with a 3% strength carrageenan solution (Sigma Chemie GmbH, Munich) in Dulbecco's medium (Dulbecco & Freeman, (1959), Virology 8, 396). The solution is converted into drops through a nozzle. The nozzle com35 prises a needle of internal diameter 0.2 mm and external diameter 0.4 mm. It is concentrically inserted into a hollow cylinder so that a tangential stream of air can be generated through the resulting annular space, which stream forces off the drops emerging from the needle. The diameter of the drops was 0.1 mm - 3 mm depending on the speed of the air. The drops fell into a solution of the polybase.

The polybase is prepared as follows: 1443 g (10 mole) of l-vinyl-3-methylimidazolium chloride 10 and 56 g (0.5 mole) of 1-vinyl-2-pyrrolidone are dissolved in 3.8 1 of water which contains 38 g of potassium peroxodisulfate as initiator in a 4 liter glass flask.

The mixture is polymerized under nitrogen at 60'C for 6 h. A clear yellow-brown 40% strength solution of neutral pH is obtained. 100 ml of the 40% strength solution are made up to 2 1 with a 0.9% strength aqueous NaCl solution. About 100-120 ml of carrageenan/cell suspension are added dropwise to 1 liter of the diluted polymer solution.

After the dilution, the capsules are allowed to settle out, the solution is decanted off, suspension is carried out 3 times with 0.9% NaCl solution. It is subsequently possible to cultivate cells in the microporous beads.

Example 2 28.2 g of hydroxypropylmethylcellulose are dissolved in 675 ml of 0.9% strength NaCl solution. Then 75 ml of a 13.3% strength CaCl2 x 2H2O solution are added. This solution is mixed with the hybridcma cell suspension in the ratio of 3:1 (ratio by mass). This suspension is converted into drops as described in Example 1. The drops are collected in an alginate solution. The alginate solution is prepared as follows. 15 g of alginate are dissolved in 500 ml of 0.9% strength NaCl solution and then diluted to 2 1 with 0.9% strength NaCl solution. 80 ml of the above cell suspension are added dropwise to 1 of alginate solution. An alginate gel polymerizes out at the phase boundary between drops and alginate solution and holds the entrapped cells. The microporous beads are washed 3 times with 0.9% strength NaCl solution and then transferred into a 2% strength CaCl2 x 2H2O solution and stirred for 2 min. The solution is then decanted off, and the microporous beads are washed several times with 0.9% strength NaCl solution.

Example 3 The cells encapsulated as in Example 2 are transferred under sterile conditions into a sterile reactor column. The column is completely filled with capsules and then medium is continuously passed through in a closed system. The adjustment and control of the parameters takes place in a 5 1 reactor which is connected to the column via lines. The gas exchange is brought about in the reactor by tubular membranes. Cultivation is carried out under the following conditions: Medium Dulbecco's nutrient solution (Dulbecco (1959), Virology, 8, 296) 10% fetal calf serum Volume of the column 0.4 1 Medium flow rate 7 1/h pH 7.0 po2 60% air saturation T 37’C The p02 content is measured at the end of the column with an oxygen electrode. A peristaltic pump ensures the flowing over. The experiment lasts 25 days. The medium is changed 4 times. For this, 50% of the old medium was replaced by new each time. The timing of the medium change is determined by measuring metabolic products. A medium change is carried out at ammonium concentrations above 3 mmol/1 and glutamine amounts of less than 1.5 mmol/1. The cell count at the start is 20,500 cells per capsule and rises at the end of the cultivation to about 200,000 cells per capsule. The mean antibody production is 0.08 mg/ml of reactor volume and day.

The amount of ammonium and glutamine was determined with 5 an enzymatic standard determination supplied by Boehringer, Mannheim.

Example 4 The cells entrapped in microporous capsules are cultivated as indicated in Example 3. A cylindrical glass column with an effective volume of 400 ml is used as reactor. The height to diameter ratio is 3 to 1. Sintered glass disks with a pore diameter of 0.1 mm are installed to retain the microporous capsules of the entry and exit of the glass column. The area of the sintered glass disk at the exit is twice that at the entry.

Example 5 The cells encapsulated as in Example 1 are transferred under sterile conditions into a sterile reactor column as described in Example 3. The cultivation is carried out as indicated in Example 3 although in Iscove's nutrient solution (Iscove & Melchers (1978), J. Exp. Med., 147: 923). The mean antibody production is 0.06 mg/ml of reactor volume and day. The cultivation lasted 4 weeks.

Claims (11)

1. Patent claims:

1. A method for cultivating cells in microporous beads whose membrane is composed of an anionic polysaccharide gel or of a polyelectrolyte membrane, wherein the microporous beads are located in a fixed bed.

2. The method as claimed in claim 1, wherein cells of plant or animal origin are cultivated.

3. The method as claimed in claim 2, wherein hybridoma cells are cultivated.

4. The method as claimed in one or more of claims 1 to 3, wherein the membrane of the microporous beads is composed of an alginate gel.

5. The method as claimed in claim 4, wherein the alginate membrane is formed by complexation with a crosslinking agent from the drop phase.

6. The method as claimed in one or more of claims 1 to 3, wherein the membrane of the microporous beads is composed of a copolymer composed of l-vinyl-3-methylimidazolium chloride and l-vinyl-2-pyrrolidone.

7. The method as claimed in one or more of claims 1 to 6, wherein the nutrient supply is effected by a closed flow of nutrient solution.

8. The method as claimed in claim 7, wherein the nutrient supply takes place discontinuously or continuously.

9. The method as claimed in one or more of claims 1 to 7, wherein the nutrient replacement takes place discontinuously or continuously. - 11

10. A method as claimed in claim 1 for cultivating cells, substantially as hereinbefore described and exemplified.

11. Cells whenever cultivated by a method claimed in a preceding claim.